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J NeuroinflammationJ NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central 1742-2094-9-2232299963310.1186/1742-2094-9-223ResearchAlteration of astrocytes and Wnt/β-catenin signaling in the frontal cortex of autistic subjects Cao Fujiang [email protected] Ailan [email protected] Guang [email protected] Ashfaq M [email protected] Zujaja [email protected] Mazhar [email protected] Amenah [email protected] Michael [email protected] Frank [email protected] George [email protected] Shiqing [email protected] W Ted [email protected] Xiaohong [email protected] Department of Neurochemistry, NY State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island New York, USA2 Department of Developmental Neurobiology, NY State Institute for Basic Research in Developmental Disabilities, 10314, New York, NY, USA3 Digital Microscopy, NY State Institute for Basic Research in Developmental Disabilities, 10314, New York, NY, USA4 Department of Orthopaedics, General Hospital of Tianjin Medical Universtiy, Tianjin, China2012 21 9 2012 9 223 223 22 6 2012 27 8 2012 Copyright © 2012 Cao et al; licensee BioMed Central Ltd.2012Cao et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. To date the etiology of this disorder is poorly understood. Studies suggest that astrocytes play critical roles in neural plasticity by detecting neuronal activity and modulating neuronal networks. Recently, a number of studies suggested that an abnormal function of glia/astrocytes may be involved in the development of autism. However, there is yet no direct evidence showing how astrocytes develop in the brain of autistic individuals.
Methods
Study subjects include brain tissue from autistic subjects, BTBR T + tfJ (BTBR) and Neuroligin (NL)-3 knock-down mice. Western blot analysis, Immunohistochemistry and confocal microscopy studies have been used to examine the density and morphology of astrocytes, as well as Wnt and β-catenin protein expression.
Results
In this study, we demonstrate that the astrocytes in autisitc subjects exhibit significantly reduced branching processes, total branching length and cell body sizes. We also detected an astrocytosis in the frontal cortex of autistic subjects. In addition, we found that the astrocytes in the brain of an NL3 knockdown mouse exhibited similar alterations to what we found in the autistic brain. Furthermore, we detected that both Wnt and β-catenin proteins are decreased in the frontal cortex of autistic subjects. Wnt/β-catenin pathway has been suggested to be involved in the regulation of astrocyte development.
Conclusions
Our findings imply that defects in astrocytes could impair neuronal plasticity and partially contribute to the development of autistic-like behaviors in both humans and mice. The alteration of Wnt/β-catenin pathway in the brain of autistic subjects may contribute to the changes of astrocytes.
AutismAstrocytesMorphologyWnt/β-catenin pathwayNeural plasticity
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Background
Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. But the etiology of this disorder is poorly understood. Animal models offer opportunities for conducting biological studies to understand the mechanisms responsible for the phenotypes. The BTBR T + tfJ (BTBR) mice are currently used as a mouse model for understanding mechanisms that may be responsible for the pathogenesis of autism since they demonstrate the three core autistic symptoms [1-4]. In addition, the Neuroligin-3(NL3) knockdown mouse may be a useful model for studying autistic-related behaviors since this mouse model mimics autistic core symptoms [5].
The brain consists of two major cell types - neurons and glial cells. In the past, neurons have been extensively studied. However, research on glial cells has increased in the last decade. The development of the nervous system requires choreographed neuronal migration, axon guidance, target selection, dendritic growth and synapse formation.
Proper orchestration of each of these stages of neuronal development requires glial-derived factors. Glial cells in the central neural system (CNS) are categorized into four types, which include astrocytes, oligodendrocytes, microglia, and chondroitin sulfate proteoglycan NG2-positive cells. Astrocytes are a very heterogeneous population of cells, which interact with neurons and blood vessels. These cells detect neuronal activity and can modulate neuronal networks. Oligodendrocytes (or Schwann cells in the peripheral nervous system) form myelin and thus are prerequisites for the high conduction velocity of axons in vertebrates. Microglia cells are the immune cells of the CNS and respond to changes in the environment [6]. Together these cells play essential roles in nervous system development and function, from simple trophic support of neurons, and wrapping axons and allowing for rapid nerve impulse conduction, to modulating synaptic connectivity and efficacy. During nervous system development, the neural progenitor cells (NPCs) generate neurons first, followed by glia. The switch from neuron to glia at the proper time is critical for the establishment of normal brain function. The mechanisms regulating this transition and development of glia are complex, and currently are poorly understood [7]. However, a number of recent studies indicate that the fate switch is governed by both extrinsic environmental cues that promote astrogenesis in NPCs and NPC-intrinsic mechanisms that decrease neurogenic and increase astrogenic competence over developmental time. The Wnt(wingless-type MMTV integration site1) pathway has been shown to be required for the activation of the proneural genes neurogenin1 (ngn1) and neurogenin2 (ngn2) in NPCs, where, at an early stage, they act to promote neuronal differentiation [8-10]. Recently, Episcopo et al. demonstrated that Wnt1-regulated Frizzled-1/β-Catenin signaling pathway can also act as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk [11]. In addition, it was shown that Wnt/β-Catenin signaling increases in proliferating NG2+ progenitors and astrocytes during post-traumatic gliogenesis in the adult brain [12].
Recently, a number of studies have suggested that abnormal functioning of glia/astrocytes may play a role in the development of autism. Laurence and Fatemi reported that the glial fibrillary acidic protein (GFAP), a marker for astrocytes, is elevated in the superior frontal, parietal and cerebellar cortices of autistic subjects [13]. GFAP was also reported to be three times higher in the cerebrospinal fluid of autistic and autistic-like conditions as compared with a control group [14]. In addition, a study reported that the expression of the astrocytic markers aquaporin4 and connexin43 are altered in the brains of subjects with autism. Most recently, a role for glia in the progression of Rett syndrome (RTT), an X-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2), has been reported [15-17]. In these studies, it was found that mutant astrocytes from an RTT mouse model and their conditioned medium, fail to support normal dendritic morphology of either wild-type or mutant hippocampal neurons. It was also shown that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, a recent study demonstrated that astrocytes in the fragile × mouse model induced developmental delays in normal dendrites including maturation and synaptic protein expression, and implicated a role for astrocytes in the development of the fragile × syndrome [18]. Taken together, the evidence suggests that glia/astrocytes could develop or be regulated abnormally in the autistic brain and that alterations of glia/astrocytes could be critically involved in the pathogenesis of autism. However, as yet there is no study directly investigating how astrocytes develop in the brain of autistic individuals. The aim of this study was to examine the development and morphology of astrocytes in the brains of autistic subjects, as well as in the brains of BTBR mice and NL3 knockout murine models of autism.
Methods
Study subjects
Frozen human brain tissues of six autistic subjects (mean age ± SD, 8.3 ± 3.8 years) and six age-matched normal subjects (mean age 8.0 ± 3.7 years) were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders. Donors with autism fit the diagnostic criteria of the Diagnostic and Statistical Manual-IV, as confirmed by the Autism Diagnostic Interview-Revised. Participants were excluded from the study if they had a diagnosis of fragile × syndrome, epileptic seizures, obsessive-compulsive disorder, affective disorders, or any additional psychiatric or neurological diagnoses. This study was approved by the Institutional Review Board of the NY State Institute for Basic Research and the subjects’ information is summarized in Table 1.
Table 1 Study subject information
Case Age Sex Group PMI(h) Seizure Retardation Medication Cause of death
1 7 M Control 12 - - Concerta, Clonidone Drowning
2 8 M Control 36 - - - Drowning
3 4 F Control 21 - - - Lymphocytic myocarditis
4 9 F Control 20 - - Albuterol, Zirtec Asthma
5 6 M Control 18 - - - Multiple injuries
6 14 M Control 16 - - - Cardiac arrhythmia
7 7 M Autism 20 - - - Drowning
8 8 M Autism 16 - - - Drowning
9 4 F Autism 13 - - - Multiple injuries
10 9 F Autism 24 - - - Smoke inhalation
11 8 M Autism 12 - + - Drowning
12 14 M Autism 12 + + - Drowning
M, male; F, female; PMI (h) (Post-Mortem Intex).
Six BTBR T + tfJ (BTBR) mice and six age- and sex-matched B6 mice (7 weeks old) were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed for 24 hours with food and water ad libitumto ease the stress before sacrifice. Then the mice were rapidly sacrificed with cervical dislocation for removal of the brains. All procedures were conducted in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals and approved by the New York State Institute for Basic Research Institutional Animal Care and Use Committee.
The NL3 mouse was obtained by microinjection of neuroligin 3 RNAi into the fertilized CD-1 mouse and then transferring to the oviduct of surrogate CD-1 female mice. PCR analysis was conducted to confirm that it was a positive NL3 knockdown founder mouse. A number of behavioral tests including open field test, elevated plus maze, water maze, vocalization test and social behavior test were carried out to determine the mouse behavior. The NL3 mouse exhibited increased anxiety, impaired cognition, vocal communication deficits and decreased social interaction, compared with the age- and sex-matched littermate control mice (unpublished data).
Preparation of brain homogenates
The frontal cortex and cerebellum were dissected. The frozen frontal cortex and cerebellum tissues were homogenized (10% w/v) in cold buffer containing 50 mMTris–HCl (pH 7.4), 8.5% sucrose, 2 mM EDTA, 10 mM β-mercaptoethanol and a protease inhibitor cocktail (Sigma-Aldrich St. Louis, MO USA). The protein concentrations were assayed by the Bradford method [19].
Immunohistochemistry
Paraffin sections (6 μm)were deparaffinized with xylene (2×), ethanol of 100% (2×), 80%, 50%, and 25% concentration and washed in TBS, 5 minutes each time. The sections were then incubated with primary antibodies overnight at 4°C. After washing in TBS for 5 minutes, the sections were further incubated with secondary antibody (biotinylated horse anti-mouse IgG, or biotinylated horse anti-rabbit IgG, VectaStain Elite ABC Kit, Vector Lab Burlingame, CA, USA) for 30 minutes at room temperature, followed by incubation in Avidin-biotinylated peroxidase (VectaStain Elite ABC Kit) for 45 minutes at room temperature and in 0.0125 g DAB/25 ml 0.05 M TBS/1 drop 30% H2O2 for 10 minutes at room temperature. All sections were washed in sequence with TBS, 25%, 50%, 80%, and 100% ethanol (2×) and xylene (2X) before mounting for viewing under the microscope.
Western blot analysis
Brain homogenate samples in SDS sample buffer (20% glycerol, 100 mMTris, pH 6.8, 0.05% Bromophenol blue (w/v), 2.5% SDS (w/v), 250 mM DTT) were denatured by heating at 100°C for 5 minutes. Twenty to sixty micrograms of protein per lane per subject were loaded onto a 10% acryl-bisacrylamide gel and electrophoresed for 2 hours at 110 V at room temperature. The separated proteins were electroblotted onto a polyvinylidenedifluoridePVDF membrane for 1 hour at 100 V at room temperature. Protein blots were then blocked with 5% non-fat milk in PBS with 0.1% Tween-20 (PBST). After blocking, the blots were incubated with primary antibody overnight at 4°C followed by secondary antibody incubation for 1 hour at room temperature (goat ant-mouse IgG or goat anti-rabbit IgG, horse radish peroxidase (HRP)-conjugated, 1:5000, Sigma). After three washes in PBST (10 minutes each time), the blots were exposed to Hyper film ECL. Sample densities were analyzed with Image J software (NIH), an open domain Java image processing system. The densities of the protein expression bands, as well as the β-actin expression bands were quantified with background subtraction.
Confocal microscopy and data analysis
Immunostaining images were visualized using a laserscanning confocal microscope to obtain clear pictures (Nikon Eclipse 90I, 10 × 40 maglification, IBR-Microscopy Shared Research Facility). Image J analysis was used to calculate area and immunostaining density. Quantification of western blot analysis was performed by Image J analysis and the internal standard beta-actin was used throughout.
Statistical analysis
Statistical analysis was conducted using SPSS 13.0 software. Means, standard deviations and standard errors of the mean were determined in sets of study subjects versus control subjects. The unpaired t-test was used to compare each parameter measured and P values were determined. P < 0.05 was considered statistically significant.
Results
The density and morphology of astrocytes were significantly changed in the frontal cortex of autistic subjects
With immunohistochemistry studies using anti-GFAP antibody, and employing confocal microscopy, we observed that the number of astrocytes was clearly increased. Quantitative analysis conducted on the brain slices from six autistic subjects and six age-matched normal controls showed that the density of astrocytes was increased approximately 1.6-fold in the frontal cortex of autistic subjects compared with age-matched controls (P = 0.035, Figure 1A). We also observed that the astrocyte morphology was grossly changed in the autistic cortex. With Image J analysis, we showed that the number of astrocytic branch processes radiating from the cell body was significantly decreased by 60.2% in the autistic subjects compared with the controls (P = 0.027, Figure 1A). The total branching length was significantly decreased by 71% compared with the controls (P = 0.007, Figure 1A). We examined cell body sizes and found that the mean cell body size of the autistic subjects was decreased by 37% compared to controls (P = 0.0001, Figure 1A). We examined cerebellar astrocytes of both autistic and control subjects. Our results demonstrated that their density and morphology were not significantly different in the autistic cerebellum compared with the controls (P = 0.088 for density of astrocytes; P = 0.065 for branch processes; P = 0.349 for total branch length and P = 0.124 for cell body size, Figure 1B).
Figure 1 The morphology and density of astrocytes were significantly changed in the frontal cortex of autistic subjects. Immunohistochemistry studies were carried out on frontal cortex (A) and cerebellum sections (B) from six autistic subjects and six age-matched controls using an anti-glial fibrillary acidic protein (GFAP) antibody (dilution 1:1000). Pictures were taken under both low power (LP) and high power (HP). Immunostaining of GFAP (dark brown color) was present in all astrocytes. The density of astrocytes, number of astrocytic branch processes, cell body size and the total branch length were quantified using Image J analysis. *P < 0.05, ***P < 0.001. Scale bar: 20 μm. Data are shown as mean ± standard error (SE).
The density and morphology of astrocytes in the brain of BTBR mice
Since BTBR mice exhibit autistic-like behaviors and are currently used as a mouse model to study autism, we examined whether their astrocytes have similar changes. Our results analyzed from six BTBR mice and six age-matched B6 mice showed that the density of astrocytes in the brain of BTBR mice was not significantly changed compared with control B6 mice(P = 0.069, Figure 2). In addition, we also examined the morphology of astrocytes in both BTBR and control B6 mice. With Image J analysis, we showed that the number of astrocytic branch processes in BTBR mice was not significantly different compared with B6 mice (P = 0.11, Figure 2). Both the total branching length and cell body size also remained unchanged in BTBR mice compared with the B6 mice (P = 0.178 and P = 0.431 respectively, Figure 2). These results suggest that both the density and morphology of astrocytes in BTBR mice are not significantly altered compared with B6 mice.
Figure 2 The density and morphology of astrocytes in the brain of BTBR mice. Immunohistochemistry studies were carried out on whole brain sections from six BTBR mice and six age-matched control B6 mice using an anti-glial fibrillary acidic protein (GFAP) antibody (dilution 1:1000). Immunostaining of GFAP (dark brown color) was present in all astrocytes. The density of astrocytes, number of astrocytic branch processes, cell body size and the total branch length were quantified using Image J analysis. Scale bar: 5 μm. Data are shown as mean ± standard error (SE).
To further confirm the results, we examined the GFAP expression level in the brain of BTBR and B6 mice with western blot analyses. Our results demonstrated that there are no significant differences in the GFAP expression between the frontal cortex (P = 0.12), as well as the cerebellum (P = 0.36) of BTBR and B6 mice (Figure 3).
Figure 3 GFAP protein expression in the frontal cortex of BTBR and B6 mice. Western blot analyses were carried out on frontal cortex and cerebellum homogenates from six BTBR mice and six age-matched B6 mice using anti-glial fibrillary acidic protein (GFAP) antibody (dilution 1:1000). The blots on cortex and cerebellum were quantified respectively after being normalized by actin (lower left bar figure for cortex and lower right bar figure for cerebellum). Data are shown as mean ± standard error (SE).
The morphology of astrocytes was altered in the brain of a NL3 knockdown mouse
In this study, we only obtained one NL3 knockdown B6 mouse. These mice exhibit autism-like behaviors including increased anxiety, impaired cognition, vocal communication deficits and decreased social interactions. We found that the astrocytes in the frontal cortex of the NL3 knockdown mouse exhibited similar changes to those that we observed in the frontal cortex of autistic subjects. We found that the number of astrocytic branch processes was decreased by 41.3% in the NL3 knockdown mouse compared with the control B6 mouse (P = 0.01, Figure 4). The total branching length was also decreased by 52.6% compared with the control B6 mice (P = 0.022, Figure 4). In addition, we found that the mean value of the cell body size in the NL3 knockdown mouse was decreased by 43.7% compared with the control B6 mouse (P = 0.020, Figure 4). However, the density of astrocytes in the NL3 knockdown mouse was not significantly changed compared with the control B6 mouse (P = 0.11, Figure 4).
Figure 4 The morphology of astrocytes was altered in the frontal cortex of NL3 knockdown mice. Immunohistochemistry studies were carried out on the whole brain sections from one NL3 knockdown mouse and one age-matched control littermate using an anti-glial fibrillary acidic protein (GFAP) antibody (dilution 1:1000). Immunostaining of GFAP (dark brown color) was present in all astrocytes. The density of astrocytes from the frontal cortex, as well as the number of astrocytic branch processes, cell body size and the total branch length were quantified using Image J analysis. *P < 0.05. Scale bar: 10 μm. Data are shown as mean ± standard error (SE).
Wnt/β-catenin pathway signaling may be impaired in the brain of autistic subjects
Since the Wnt/β-catenin pathway has been suggested to play a role in controlling mesencephalic dopaminergic neuron-astrocyte crosstalk and is involved in the modulation of gliogenesis, we examined how the Wnt/β-catenin pathway is regulated in the autistic brain. By immunohistochemistry studies using anti-Wnt antibodies, we observed by confocal microscopy that the immunostaining was weaker in the neuronal cells of the frontal cortex of autistic subjects compared with the controls. Quantitative analysis showed that the immunoreactivity of Wnt was decreased in the autistic brain by 61% compared with the controls (P = 0.037, Figure 5). In addition, western blot studies were conducted to examine β-catenin protein expression in the brain of autistic subjects. Our results demonstrated that the mean value of β-catenin protein expression was decreased by 24.7% in the frontal cortex of autistic subjects (P = 0.002, Figure 5), but was not significantly changed in the autistic cerebellum compared with controls (data not shown). These results suggest that Wnt/β-catenin signaling activities may be down-regulated in the frontal cortex of autistic subjects.
Figure 5 Wnt and β-catenin protein expression in the brain of autistic subjects. Upper panel: western blot analyses were carried out on frontal cortex homogenates from six autistic subjects and six age- and sex-matched controls using an anti-β-catenin antibody (dilution 1:1000) (5A). The blots were quantified after being normalized by actin (5B). Data are shown as mean ± standard error (SE). **P < 0.01. Lower panel: immunohistochemistry studies were carried out on frontal cortex sections from six autistic subjects and six age-matched controls using an anti-Wnt antibody (dilution 1:100). Weaker immunostaining of Wnt protein (dark brown color as indicated by arrows) was present in the autistic subjects compared with the controls (5C). Immunostaining density was quantified using Image J analysis (5D). Data are shown as mean ± standard error (SE). *P < 0.05. Scale bar: 20 μm.
Discussion
Previously, a number of studies have suggested that abnormal functioning of glia and astrocytes may play a role in the development of autism. GFAP expression, a marker for astrocytes, has been reported to be significantly elevated in the cortex and cerebrospinal fluid of autistic subjects [13,14]. Other astrocyte markers such as aquaporin 4 and connexin 43 have also been shown to be altered in the brains of autistic individuals [20]. In particular, several recent studies have demonstrated that the abnormal functions of glia may also contribute to the progression of RTT, an X- linked autism spectrum disorder, and to the fragile X syndrome [15-18]. However, to date the information about glia/astrocyte development and function in the autistic brain is very limited. In this study, by employing western blotting and immunohistochemical approaches, we found that the morphology of astrocytes in the frontal cortex of autistic subjects was markedly altered compared with controls. The astrocytes in the autistic cortex exhibited significantly reduced branching processes, and the total branch length as well as the cell body size were significantly decreased. Further, the number of astrocytes was markedly increased compared with the controls. These results indicate that there is an astrocytosis in the autistic brain, and the structures of the astrocytes are altered.
Astrocytes are the most abundant cells in the CNS and have been suggested to detect neuronal activity and modulate neuronal networks. Thus their structural integrity and sustained function are essential for neuronal viability [21-23]. The astrocyte branching processes are important structures, which can interdigitate between and closely approximate adjacent neuronal elements, thereby facilitating the local homeostasis of a range of molecules, including glutamate [24-26]. Studies have shown that the neurons depend upon the physical proximity of the astrocyte processes for normal function [23]. Torres-Platas et al. also reported that changes in astrocyte structures including branching processes, and cell body sizes may be significantly involved in mood disorders [27]. Thus, we suggest that the interruption of the astrocyte structures in the autistic cortex could critically impair neuronal function and the homeostasis of certain molecules such as glutamate, which may lead to the development of autistic-like behaviors.
Recently, studies have also shown that astrocytes have a complex, dual role in the local regulation of immune reactivity. They form the glia limitans around blood vessels restricting the access of immune cells to the CNS parenchyma [28]. Astrocytes have also been shown to be important regulators of neuroinflammation. Previous studies have demonstrated that astrocytes carry a series of germline-encoded pattern-recognition receptors (PRRs), which are important for the primary recognition of infectious agents [29]. Several cytokines, including IL-1 and IL-6, have been implicated in the induction and modulation of reactive astrogliosis and pathological inflammatory responses [30-34]. In addition, astrocytes have been reported to secrete inflammatory cytokine IL-6 [35]. Recently, various studies have suggested that abnormal immunity and localized inflammation of the central nervous system may contribute to the pathogenesis of autism. A number of studies including ours have demonstrated that cytokines including IL-6, IL-1β TNF-α and IFN-γ are elevated in the serum and brain tissue of autistic individuals [35-41]. We reckon that the astrocytic changes could result from an inflammatory process.
It will be important to determine whether the observed changes in astrocyte structure, as well as the astrocytosis found in the autistic brain are associated with elevated inflammatory cytokines such as IL-6. In this study, we did not determine the IL6 concentration in the same sample used for examining the astrocytes. Further studies can be conducted to examine cytokines including IL-6 and astrocytes in the same brain region at the same time. We suggest that it is also possible that the increased cytokines, in particular IL-6 in the autistic brain, could result from the astrocytosis.
We next undertook to determine whether the alterations in the structure and density of astrocytes in the autistic brain also occurred in murine models of autism, including NL3 knockdown mice and BTBR mice. We found that the morphology of astrocytes in the NL3 knockdown mouse exhibited similar changes to that found in the autistic brain. They exhibit significantly reduced branching processes and total branch lengths, and as well the astrocytic cell body sizes were significantly decreased in comparison with the controls. Neuroligins are cell adhesion molecules localized postsynaptically in glutamatergic synapses, and interact with presynaptic neurexins to form heterophilic complexes, which likely play critical roles in synaptic transmission and differentiation of synaptic contacts [42-45]. A role of neuroligins in autism was implied by the discovery of deletions at Xp22.1 containing the NL4X gene in three female autistic individuals and a missense mutation (R451C) in NL3 in two Swedish families with autism [46,47]. NL3 knockdown mice have been shown to mimic certain human autistic behaviors [5]. Recent studies have demonstrated that NL3 is expressed in many types of glia during the development of the nervous system. In particular, NL3 is expressed in the olfactory ensheathing glia, retinal astrocytes, Schwann cells, and spinal cord astrocytes in the developing embryo [48]. The NL3 knock-down mouse in the current study was shown to exhibit autistic-like behaviors including increased anxiety, impaired cognition, vocal communication deficits and decreased social interactions (unpublished data). Thus, there is a possibility that alteration in astrocyte structure could be partially responsible for the development of autistic-like behavior in NL-3 knockdown mice. The mechanisms through which structural change in astrocytes could lead to behavioral changes remains to be further investigated. A limitation of this study was that we only had one NL3 knockdown mouse that could be analyzed. More studies are needed to further confirm our observations.
We did not detect a significant change in the morphology of astrocytes in either the cortex or cerebellum of the BTBR mice, another murine model of autism. There were no significant differences in the number of astrocyte branching processes, the total length of processes orcell body size between the BTBR and control B6 mice. Nor did we find that there was an astrocytosis in the brain of BTBR mice similar to that found in the autistic brain. The density of astrocytes remained unchanged compared with the control mouse. However, we have not examined the orientation of the glial fibers. Recently, it was reported that there is a misorientation of selected glial fibers present in the BTBR forebrain [49]. This study found that the astrocytic processes were oriented dorsoventrally rather than mediolaterally in the cingulum and alveus at the levels of the striatum and hippocampus. The misorientation of glial processes was only found in brain regions that normally receive corpus callosal innervations, indicating that these findings are likely to be a consequence of callosal agenesis [49]. We therefore reason that although there are no changes observed in the astrocyte density, as well as in the number of branching processes and cell body sizes in BTBR mice, a misorientation of glial processes could lead to impairments in the functions of astrocytes, and consequentially impair synaptic plasticity and various neural functions and might contribute to the development of autistic-like behaviors. It has been demonstrated that astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis [50]. Whether the change in astrocytes in autistic subjects, or NL3 knockdown and BTBR mice could impair the development of GABAergic axons, remains to be further studied.
Both NL3 knockdown and BTBR mice have been demonstrated to exhibit core autistic-like behaviors. Alterations found in the astrocytes of autistic subjects and the mice models imply that NL3 knockdown mice and BTBR mice could offer opportunities for conducting biological studies to understand the mechanisms responsible for autism.
More and more evidence suggests that astrocytes are intimately associated with synapses and govern key steps in synapse formation and plasticity. However, we understand little about the molecular underpinnings of astrocyte development. It is unclear how astrocytes are specified at the appropriate developmental time from NPCs and how their development and maturation are regulated. The Wnt/β-catenin signaling pathway has been intensely studied as a key regulator of cell proliferation and cell fate during development, including neural development [10,23,51-53]. Recently, several studies have reported a role of Wnt/β-catenin signaling in the development of astrocytes [12]. It has been shown that Wnt/β-catenin pathway signaling regulates post-traumatic gliogenesis. Wnt/β-catenin pathway has also been demonstrated to act as a candidate regulatory circuit that controls mesencephalic dopaminergic neuron-astrocyte crosstalk [11]. In this study, we found that both Wnt and β-catenin protein expression were decreased in the brains of autistic subjects, suggesting that Wnt/β-catenin signaling activities are down-regulated. There is some evidence for a direct genetic link between Wnt2 and autism spectrum disorders. Two studies have found correlations between mutations of the WNT2 locus and the incidence of autism in different populations [54,55]. Wnt2 has also been found to be expressed at lower levels in a mouse model of fragile X syndrome, a human disease strongly associated with autism [56]. Our findings imply that the decreased expression of Wnt and β-catenin may be associated with changes in astrocytes in the frontal cortex of autistic subjects. Further studies will be carried out to determine whether down-regulation of Wnt/β-catenin impairs the structure and density of astrocytes.
Conclusion
In summary, our study demonstrated that the morphology of astrocytes in the frontal cortex of autistic subjects was significantly altered compared with age- and sex-matched controls. The astrocytes in autisitc subjects exhibited significantly reduced branching processes, reduced total branching lengths and decreased cell body sizes. In addition, we detected astrocytosis present in the frontal cortex of autistic subjects. However these alterations of astrocytes are not detected in the autistic cerebellum. Interestingly, we found that astrocytes in the frontal cortex of the NL3 knockdown mouse also showed significantly reduced branching processes, reduced total branching length and decreased cell body sizes, which mimic the changes in the autistic brain. These findings imply that the defects in astrocytes could impair neuronal plasticity and functions, and may partially contribute to the development of autistic-like behaviors in both humans and mice. We did not detect significant alterations in the density and morphology of astrocytes from the frontal cortex or cerebellum of BTBR mice. Recently, one study reported that there is a misorientation of selected glial fibers present in the BTBR forebrain. We suggest that even small changes like misorientation of glial fibers could affect astrocyte functions and consequently neuronal networks and lead to behavioral changes. Finally, we found that both Wnt and β-catenin protein expression was decreased in the brains of autistic subjects, which suggests that the Wnt/β-catenin signaling activities may be down-regulated. We suggest that the alteration of the Wnt/β-catenin pathway in the frontal cortex of autistic subjects could be one of the underlying mechanisms responsible for the observed changes of astrocytes. Our findings indicate that astrocytes may play an important role in the development of autism and may further suggest new potential therapeutic targets and strategies for intervention in autism.
Abbreviations
CNS: central neural system;GFAP: glial fibrillary acidic protein;HRP: horse radish peroxidase;MeCP2: methyl-CpG-binding protein 2;NPC: neural progenitor cell;PCR: polymerase chain reaction;PRR: pattern-recognition receptor;PVDF: polyvinylidenedifluoride;RTT: Rett syndrome;SE: standard error
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS, GW, FC, AY, ZT, AN, MS, FS, GM participated in data collection; LX, MM, WTB designed the study, secured the research funding and wrote the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
This work was supported by the NYS Office for People with Developmental Disabilities, the Rural India Charitable Trust, Richmond County Savings Foundation and Northfield Bank Foundation.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23341893PONE-D-12-3061510.1371/journal.pone.0052184Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyLymphatic SystemMedicineGastroenterology and HepatologyOncologyBasic Cancer ResearchMetastasisCancers and NeoplasmsGastrointestinal TumorsEsophageal CancerHead and Neck TumorsHead and Neck Squamous Cell CarcinomaSDR9C7 Promotes Lymph Node Metastases in Patients with Esophageal Squamous Cell Carcinoma SDR9C7 and ESCCTang Shanhong
1
Gao Liucun
1
Bi Qian
1
Xu Guanghui
1
Wang Simeng
1
Zhao Guohong
1
Chen Zheng
1
Zheng Xiushan
1
Pan Yanglin
1
Zhao Lina
1
Kang Jianqin
1
Yang Guitao
1
Shi Yongquan
1
Wu Kaichun
1
Gong Taiqian
1
2
*
Fan Daiming
1
*
1
State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, People′s Republic of China
2
Department of Thoracic Surgery, Daping Hospital, Third Military Medical University, Chongqing, People′s Republic of China
St-Pierre Yves Editor
INRS, Canada
* E-mail: [email protected] (DF); [email protected] (TG)Competing Interests: The authors have declared that no competing interests exist
Conceived and designed the experiments: DF TG KW YS GY LG ST. Performed the experiments: TG JK LZ XZ GZ SW GX QB LG ST. Analyzed the data: YP QB ST. Contributed reagents/materials/analysis tools: ZC. Wrote the paper: TG ST.
2013 14 1 2013 8 1 e521849 10 2012 9 11 2012 © 2013 fan et al2013fan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The major reason for the poor prognosis of esophageal squamous cell carcinoma (ESCC) patients is lymph node (LN) metastases.
Methodology/Principal
In the present study, gene expression profiling assay (GEP) was performed to identify the differences in gene expression profiles between primary ESCC tumors that were with LN metastases (N+) and those without LN metastases (N-).
Conclusions/Significance
A total of 23 genes were identified as being significantly elevated, and 30 genes were sharply decreased in ESCC tumors that were N+ compared with N- tumors. Among these genes, two transcripts of the short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7) were observed 7 times more frequently in N+ compared with N- tumors. Immunohistochemical staining showed that SDR9C7 expression closely correlated with metastasis, and would be a prognostic marker for ESCC patients. To investigate the role of SDR9C7 in the ESCC metastasis, repeated transwell assays were adopted to establish highly and non-invasive ESCC sublines, and western blot showed that SDR9C7 expression was markedly higher in highly invasive cells compared with non-invasive ones. Down-regulation of SDR9C7 dramatically inhibited the metastatic abilities in vitro and in vivo, and repressed the expression of MMP11 in highly invasive cells, indicating that SDR9C7 promotes ESCC metastasis partly through regulation of MMP11, and might be a potential prognostic and therapeutic marker for ESCC patients.
This work was supported in part by grants from the China Postdoctoral Science Foundation 20090461447, National Natural Science Foundations of China 81172288, and 81101689. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Esophageal squamous cell carcinoma (ESCC) has been ranked as the sixth leading cause of cancer death worldwide [1]. Most patients with ESCC are diagnosed at an advanced stage with lymph node (LN) metastasis, subsequently resulting in a poor outcome [2]. The lymphatic system serves as the primary pathway for metastasis, which has been identified as a key prognostic factor for clinical outcome in ESCC patients [3]. Therefore, a better understanding of the gene expression involved in the development of LN metastasis may lead to therapeutic improvements for ESCC patients. The development of ESCC LN metastasis is regarded to arise from a multiple-step process including primary tumor formation, LN invasion, and distant metastasis. This process may be caused by the accumulation of genetic changes. An abnormal expression pattern of a single gene has been correlated with the metastasis in ESCC [4], [5], [6]. Our previous study demonstrated that the nuclear expression of twist promotes lymphatic metastasis in ESCC [7]. However, the diagnostic and prognostic biomarkers for ESCC metastasis remain obscure.
The gene expression profiling assay (GEP) is an important approach for identifying of individual molecules in the primary tumors at the time of diagnosis that are associated with LN metastasis. Differences in gene expression profiles have been identified in the primary tumors of oral squamous cell carcinoma [8], lung cancer [9] and cervical cancer [10] with and without LN metastasis, which has also been performed to evaluate the genetic signature of the primary tumors of esophageal adenocarcinoma patients with and without LN metastasis [11], and the expression patterns of primary ESCC and the matched metastatic LN has also been distinguished by GEP assay [12]. However, the differences in GEPs have not been indentified between primary ESCC tumors that were N+ and N- tumors.
The aims of this study were to identify the LN metastasis-associated genes in primary ESCC tumors, using GEP analyses. The short chain dehydrogenase/reductase family 9C, member 7 (SDR9C7), was used for further study, because 2 transcripts of this novel gene were present 7 times more frequently in N+ compared with N- tumors. To investigate the role of SDR9C7 in the metastasis of ESCC, repeated transwell assays were used to establish highly and non-invasive ESCC sublines. Then, we determined the influence of lentivirus-mediated SDR9C7 siRNAs on the ESCC metastatic potential. Overall, these data not only discovered a prognostic gene expression profile, but also identified SDR9C7 as a critical marker for ESCC metastasis.
Materials and Methods
Tissue Samples
The primary surgical tissues from 3 ESCC tumors that were N-, and 3 patients with strong N+ tumors, were collected for GEP assays from December 2009 to May 2010 (Table 1). A total of 104 paraffin-embedded tumor tissues (average age, 59.5 years; range, 37–77 years; 77 male and 27 female) were obtained from ESCC patients who underwent surgery during from April 2007 to April 2009. All patients were treated with three-field esophagectomy with extended lymphadenectomy at Xijing Hospital without preoperative anticancer treatment. All tumor tissues and LN metastasis were histologically confirmed, and the TNM stage was assessed according to the 7th edition of the TNM classification by the AJCC. No patients died of causes other than ESCC. The follow-up examinations were conducted from the date of discharge until death or the deadline date (April 31, 2012). All participants have provided their written informed consents to participate in this study, and this study was approved by the ethics committees of Xijing Hospital.
10.1371/journal.pone.0052184.t001Table 1 The clinical characteristics of patients included for gene expression profiling analysis.
Case Gender Age TN stage Differentiation Location
1 Man 68 T3N0 Mederate Middle
2 Man 59 T3N0 Well Lower
3 Man 62 T4aN0 Poor Middle
4 Man 63 T1bN3 Mederate Middle
5 Man 53 T2N3 Well Lower
6 Man 68 T2N3 Poor Middle
Gene Microarray and Data Acquisition
For GEP analysis, the total RNA from each sample was amplified and labeled by the Low RNA Input Linear Amplification kit (Agilent Technologies, Santa Clara, CA, USA), 5-(3-aminoallyl)-UTP (Ambion, Austin, TX, USA), and Cy3 NHS ester (GE Healthcare Biosciences, Pittsburgh, PA, USA) according to manufacturer’s protocol. The labeled cRNA was purified by the RNeasy mini kit (Qiagen, GmBH, Germany).
The kit was purchased from Agilent Technologies, Santa Clara, USA. Each slide was hybridized with 1.65 µg of Cy3-labeled cRNA using the Gene Expression Hybridization Kit in a hybridization oven. After 17 hours, the slides were washed in staining dishes (Thermo Shandon, Waltham, MA, USA) with the Gene Expression Wash Buffer Kit, and the stabilization and drying solution followed the manufacturer’s procedures. The samples were scanned by the Agilent Microarray Scanner and analyzed with the Feature Extraction software 10.7 using default settings. The raw data were normalized by Quantile Algorithm, Gene Spring Software 11.0 (Agilent Technologies, Santa Clara, CA, US).
Immunohistochemistical Staining
Immunohistochemistical analyses were performed as previously described [13]. The sections were incubated with SDR9C7 antibody (Sigma, 1∶100), and sections were incubated with PBS in place of the primary antibody performed for control staining. The results were examined by microscopy by two independent pathologists blinded to the clinical data. The intensity was scored as 0, 1, 2 or 3 based on the staining intensity. The immunoreactivity proportion was ranked as 0 (0%), 1(0–30%), 2(30–60%) or 3(>60%) respectively. The two scores were added to obtain the final results: Negative (−), 0∼2; Positive (+), 3∼6.
Highly and Non-invasive ESCC Sublines Construction
ESCC cell lines EC109 and EC9706 were purchased from the Chinese Academy of Medical Science (Beijing, China) [14] and were routinely maintained in our lab in 1640 medium (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37°C in humidified air containing 5% carbon dioxide. Highly and non-invasive EC9706 and EC109 sublines were constructed using repeated transwell assays as described previously [15]. After a ten-round selection and expansion, the highly invasive EC9706 and EC109 sublines were established and designated as EC9706-P and EC109-P, and the non-invasive cell lines were named EC109-N and EC9706-N.
Lentivirus-mediated siRNA Construction and Transfection
Lentivirus-mediated siRNAs with GFP were constructed by Shanghai Gene Chem Co, Ltd. The sequence for interfering with endogenous SDR9C7 expression was 5′-GCATGGAGCATGCTATTGTTT-3′, and the control Sequence (5′-CCAGAAGA GCAATCTGTAC-3′) targeting no known genes was used as a negative control. The SDR9C7 siRNA and control lentivirus were transfected into EC109-P and EC9706-P cells following the manufacturer’s protocol. Flow cytometry (FACScan; Becton Dickinson, San Jose, CA) was used to separate GFP-positive cells, and the purified cell lines were named Con-EC109-P, Si-EC109-P, Con-EC9706-P and Si-EC109-P.
Protein Preparation and Western Blot Analyses
Protein preparation and western blot analyses were performed according to previously published protocols [13]. The cell proteins were prepared and separated on SDS-PAGE gels. The expression of β-actin was used as loading controls. The following antibodies were used as followings: anti-SDR9C7 (Abcam, 1∶500); anti-VEGF (Abcam, 1∶500); anti-E-cadherin(Santa, 1∶100); anti-MMP11(Cell Signaling Technology, 1∶300), and anti-β-actin (Sigma, 1∶4000).
Proliferation Assay
The MTT assay was used to evaluate the proliferation of ESCC cell as previously described [13]. The absorbance values were determined by measuring the absorbance of the well at 490 nm using an ELISA reader (Bio-Rad Laboratories, CA). Each cell line was detected in triplicate.
Migration and Invasion Assays
Cells migration and invasion assays were performed as described in previous study [16] using transwells (8-µl pore size, Corning, USA). After 3-washes by PBS and air-dried, cells were counted under a microscope at ×200 magnification on 3 random fields in each well. Each experimental condition was repeated in triplicate.
Experimental Metastasis
The animal experimentation was performed according to the Institutional Animal Care and Use Committee guidelines of the Experiment Animal center of Fourth Military Medical University, and te approval ID as No 12015 by Experiment Animal Center of Fourth Military Medical University. Because EC9706-P cells demonstrated a more invasive phenotype in vitro, Con-EC9706-P and Si-EC9706-P cells were chosen for in vivo experiments with BALB/C-nu/nu nude mice (Shanghai Laboratory Animal Center of China) to induce metastases through caudal vein injection according to NIH Animal Care and Use Committee guidelines. Approximately 2×106 cells of each cell line in 200 µl of medium without serum were injected into the nude mouse caudal tail vein. Each group contained 5 mice, which were maintained in a sterile animal facility for 7 weeks before being sacrificed. The mice were killed by the cervical dislocation method, and their lung and liver tissues were examined for metastases and also made into serial sections before HE staining for microscopic examination.
Statistical Analyses
All statistical analyses were performed using SPSS 17.0. Student’s t-test was performed to analyze the results of gene expression profiling assays. The Kruskal–Wallis U or H test was used to analyze the significance of SDR9C7 expression as correlated with clinical factors, and the one-way ANOVA test was performed to evaluate the difference between three comparisons in cell proliferation, migration and invasion assays. The Kaplan-Meier method was used for univariate analysis, and a Cox regression model was used for multivariate analyses. A value of P<0.05 was considered significant.
Results
Identification of Differentially Expressed Genes
GEP assays were performed to identify the differentially expressed genes between the N- and N+ primary ESCC tissues. The up-regulated or down-regulated transcripts with a false discovery rate (FDR) <0.01 and FCA absoluteare >4 are shown in Table 2 and 3. A total of 26 transcripts were increasingly expressed in N+ tumors to 23 different genes, and a total of 32 transcripts with decreased expression in N+ tumors correspond to 30 different genes (Fig. 1).
10.1371/journal.pone.0052184.g001Figure 1 Gene expression profiling analysis of differential expression genes in ESCC with and without LN metastasis.
Red color represents the up-regulated genes, and green or blue color represents the down-regulated genes in ESCC with LN metastasis. a, Hierarchical clustering analysis of genes associated with LN metastasis, N+, positive lymph node metastasis; N-, negative lymph node metastasis. b, Volcano plot analysis of differential expressed genes in ESCC between with and without LN metastasis. The x axis represents the differential expression profiles with the fold-induction ratios in a log2 scale, and the y axis represents the P value of T-test in a log10 scale.
10.1371/journal.pone.0052184.t002Table 2 The probes of the up-regulated genes and their corresponding transcript names.
ProbeName P-Value Fold change Gene Symbol
A_23_P134946 0.005 4.01 LRRC14
A_23_P219072 0.006 4.28 SAMD9
A_23_P355244 0.007 4.50 SAMD9
A_23_P259692 <0.001 4.96 PSAT1
A_24_P191047 0.008 5.69 CRCT1
A_23_P103617 0.006 5.85 ANXA9
A_23_P68487 0.005 5.89 BMP7
A_23_P127663 0.010 5.95 PRRG4
A_23_P11025 0.008 6.17 ZNF185
A_24_P332314 0.003 6.41 FAM111B
A_24_P91566 0.003 6.47 BMP7
A_23_P104522 0.007 7.04 NEBL
A_23_P17814 0.004 7.27 PLA2G3
A_23_P115478 0.006 7.31 PADI1
A_23_P8253 0.004 8.38 RAET1E
A_23_P25086 0.009 8.70 SDR9C7
A_23_P371758 0.009 9.32 SDR9C7
A_32_P154053 0.002 9.71 ATG9B
A_23_P254654 0.005 10.15 CLIC3
A_23_P76743 0.006 10.58 ASPG
A_32_P536872 0.003 12.40 TDRD5
A_23_P27473 0.002 13.35 CNFN
A_24_P76558 0.003 14.57 PLAC4
A_24_P236935 0.002 14.78 KLK6
A_24_P411515 <0.001 18.54 TMPRSS11F
A_23_P144417 <0.001 33.33 TMPRSS11D
10.1371/journal.pone.0052184.t003Table 3 The probes of the down-regulated genes.
ProbeName P-Value Fold change Gene Symbol
A_32_P217750 0.009 4.12 IL3RA
A_23_P434398 0.002 4.13 TXLNB
A_23_P72651 0.009 4.14 ECSCR
A_23_P315451 0.009 4.21 KIRREL2
A_23_P39237 0.009 4.27 ZFP36
A_23_P62115 0.001 4.45 TIMP1
A_23_P374695 0.003 4.66 TEK
A_24_P648880 0.003 4.80 MEIS3P1
A_24_P237328 0.007 4.87 MCAT
A_32_P100439 0.007 4.94 C7orf41
A_24_P945113 0.008 4.98 ACVRL1
A_32_P32413 0.009 4.99 SETBP1
A_23_P4551 0.003 5.19 SETBP1
A_23_P119196 0.009 5.29 KLF2
A_23_P315320 0.006 5.37 IL27
A_23_P212105 0.001 5.39 DAZL
A_23_P1083 <0.001 5.48 GJA4
A_23_P44244 0.009 5.92 SMARCA1
A_24_P185854 0.006 6.35 DMD
A_23_P329321 <0.001 6.56 PLB1
A_24_P412734 0.004 6.57 PRSS36
A_23_P121813 0.005 6.96 ENPP6
A_23_P38712 0.007 7.022 ADCYAP1
A_32_P47754 0.009 7.23 SLC2A14
A_24_P335781 0.009 7.25 ADCYAP1
A_24_P148907 0.008 7.27 MAB21L2
A_23_P140384 0.007 7.36 CTSG
A_32_P55871 0.008 10.08 C3orf15
A_23_P421436 0.004 11.46 ADD2
A_23_P33356 0.009 14.68 ADAMTS9
A_24_P97342 0.002 31.18 PROK2
A_23_P381505 <0.001 31.41 VWDE
Up-regulated SDR9C7 Associated with Clinical Parameters
Two transcripts of the SDR9C7 gene were present 7 times more frequently in N+ tumors compared with N- tumors, indicating that SDR9C7 might be a significant prognostic signature for ESCC metastasis. The expression of SDR9C7 was further detected in 104 ESCC tissues by immunohistochemical staining, and presented positive staining in the cytoplasm of ESCC tissues (Fig. 2a–d). The positive rate was 64.4%(67/104). The relationship between SDR9C7 expression and the patients’ clinicopathological data including gender, age, TNM stage, differentiation, lymphatic invasion and LN metastasis are presented in Table 4. Positive expression of SDR9C7 was significantly correlated with lymphatic invasion and LN metastasis (P<0.001). However, SDR9C7 expression had no significant correlation with age, sex, differentiation and T stage (Table 4; P>0.05).
10.1371/journal.pone.0052184.g002Figure 2 Immunohistochemical staining of SDR9C7 expression in representative ESCC.
The staining of SDR9C7 occurred in cytoplasm of cancer cells. a and b, ESCC tissues with lymph node metastasis; c and d, ESCC tissues without lymph node metastasis original magnification, (SP×200). e, Kaplan–Meier survival curves for patients with ESCC according to the expression of SDR9C7. The survival rate for patientswith positive SDR9C7 expression was significantly lower than that for patients with negative SDR9C7 expression (P = 0.001).
10.1371/journal.pone.0052184.t004Table 4 SDR9C7 expression correlated with the clinical data of the patients.
Factor Total Negative Positive
P-value
Age 0.54
≤59 52 20 32
>59 52 17 35
Gender 0.78
Men 77 28 49
Women 27 9 18
Location 0.19
Upper 28 6 22
Middle 56 23 33
Lower 20 8 12
Differentiation 0.008
Well 49 23 26
Moderate 39 10 29
Poor 16 4 12
Lymphatic invasion <0.001
No 40 25 15
Yes 64 12 52
LN metastasis <0.001
N0 50 31 19
N1 35 6 29
N2 17 0 17
N3 2 0 2
T stage 0.11
1 13 7 6
2 42 18 24
3 47 12 35
4 2 0 2
The associations of SDR9C7 expression with clinical factors were detected by Kruskal-Wallis H or U test.
The Relationship between SDR9C7 Expression and Patient Prognosis
To further understand the clinical implications of SDR9C7 expression in ESCC patients, we analyzed the relationship between SDR9C7 expression levels and patient prognosis. The total survival rate of all patients with ESCC during the observation period was 41.3%. The mean follow-up time was 31.8 months with a median value of 24 months. The survival rate in patients with positive SDR9C7 expression was 29.9% (20/67), which was significantly lower that the survival rate in patients with negative SDR9C7 expression 62.2% (23/37). The Kaplan–Meier postoperative survival analyses showed that the following factors significantly correlated with postoperative survival: differentiation (P = 0.005), LN metastasis (P<0.001), lymphatic invasion (P<0.001), T stage (P<0.001) and SDR9C7 expression (Fig. 2 e; Table 5; P = 0.001). Multivariate regression analyses revealed that differentiation (P = 0.02), lymphatic invasion (P = 0.03) and T stage (P = 0.001) were independent prognostic factors, however, SDR9C7 expression was not an independent prognostic factor (Table 6; P = 0.31).
10.1371/journal.pone.0052184.t005Table 5 Univariate analysis of prognostic factors.
Factor n Survivalrate
P-value
Age 0.37
≤59 52 44.2% (23/52)
>59 52 38.5% (20/52)
Gender 0.38
Men 77 44.2% (34/77)
Women 27 33.3% (9/27)
Location 0.61
Upper 28 35.7% (10/28)
Middle 56 42.9% (24/56)
Lower 20 45% (9/20)
Differentiation 0.005
Well 49 55.1% (27/49)
Moderate 39 35.9% (14/39)
Poor 16 12.5% (2/16)
Lymphatic invasion <0.001
No 40 70.0% (28/40)
Yes 64 23.4% (15/64)
LN metastasis <0.001
N0 50 64.0% (32/55)
N1 35 28.6% (10/35)
N2 17 5.9% (1/17)
N3 2 0
T stage <0.001
1 13 76.9% (10/13)
2 42 52.4% (22/42)
3 47 23.4% (11/47)
4 2 0
SDR9C7 0.001
Negative 37 62.2% (23/37)
Positive 67 29.9% (20/67)
The univariate analysis reveals that differentiation, LN metastasis, lymphatic invasion, T stage and SDR9C7 expression significantly correlate with patient prognosis.
10.1371/journal.pone.0052184.t006Table 6 Multivariate analysis of prognostic factors.
Features 95%CI
P-value
Age 0.63–1.83 0.80
Gender 0.43–1.32 0.32
Location 0.69–1.45 0.90
Differentiation 1.10–2.16 0.02
LN metastasis 0.80–1.86 0.35
Lymphatic invasion 1.08–5.72 0.03
T Stage 1.41–3.87 0.001
SDR9C7 0.71–3. 0 0.31
Multivariate analysis shows that differentiation, lymphatic invasion and T stage are significantly correlated with the patient prognosis.
SDR9C7 Expression Correlated with Cell Invasive Potential
Repeated transwell assays were used to develop the highly invasive and the non-invasive ESCC cell lines. Statistical analyses showed that the levels of migration and invasion of the highly invasive cell lines EC109-P and EC9706-P were significantly stronger than the matched non-invasive cell lines EC109-N and EC9706-N (Fig. 3 a–b). The western blot analyses showed that SDR9C7 expression was markedly higher in EC109-P and EC9706-P cells compared with the matched non-invasive cell lines (Fig. 3c), indicating that SDR9C7 might be associated with the invasive phenotype of the ESCC cells.
10.1371/journal.pone.0052184.g003Figure 3 Expression of SDR9C7 correlated with the invasive potential of ESCC cell lines.
a and b, both migration and invasion assays showed that the migration and invasion ability of highly invasive ESCC cells were significantly higher than matched non-invasive cells. c, Western blot revealed that the expression of SDR9C7 was obviously higher in highly invasive ESCC cells compared with matched non-invasive cells. * Statistical significance (a
P<0.05 versus matched non-invasive cells).
Knockdown of the SDR9C7 Inhibited ESCC Cell Metastasis in vitro
To identify the influence of SDR9C7 on ESCC proliferation, migration and invasion, lentivirus-mediated SDR9C7 siRNA and control siRNA were transfected into EC9706-P and EC109-P cells. Western blot analyses confirmed that SDR9C7 protein was significantly down-regulated by lentivirus-mediated SDR9C7 siRNA transfection in both EC109-P and EC9706-P cells (Fig. 4 a–b). As shown in Fig. 4 c and d, there was no significant difference in the growth rate between SDR9C7 knockdown cells compared with the controls (P>0.05). The results of the transwell assays showed that the migration and invasion of SDR9C7 siRNA-transfected EC109-P and EC9706-P cells were notably reduced compared with untreated cells or cells transfected with a control siRNA (Fig. 4 e–h). These results indicated that SDR9C7 overexpression played an important role in ESCC cell invasion in vitro.
10.1371/journal.pone.0052184.g004Figure 4 Lentivirus-mediated siRNA targeting SDR9C7 Inhibited the metastasis of ESCC in vitro and in vivo.
a and b, Western blot analysis showed that SDR9C7 expression was significantly down-regulated by lentivirus-mediated siRNA targeting SDR9C7 compared with mathced controls. β-actin was used as loading control. c and d, MTT showed that SDR9C7 knockdown couldn’t significantly influence the growth of ESCC cells. e and f, Repressing SDR9C7 expression decreased the migration and invasion of EC109-P cells. g and h, Inhibiting SDR9C7 decreased the migration and invasion of EC9706-P cells. i, Representative HE staining of lungs and livers isolated from mice that received injections of Con-EC9706-P or Si-EC9706-P cells. j, Incidence of metastasis in lungs and livers of mice. *Statistical significance (P<0.05, Si-EC109-P or Si-EC9706-P versus matched controls).
Down-regulated SDR9C7 Inhibited ESCC Cell Metastasis in vivo
To further study the influence of SDR9C7 on ESCC metastasis in vivo, the highly invasive ESCC cells and the siRNA-transfected cells were used to induce experimental metastases in mice. Consistent with the in vitro results, the animal experiments showed that liver and lung metastases were apparently recognized in mice injected with Con-EC9706-P cells, but few metastases were observed in mice injected with SDR9C7-siRNA transfected cells (Fig. 4i). Histological analyses revealed that the number and the size of metastatic nodules in the lungs and livers of mice were significantly smaller in the controls (Fig. 4j; P<0.01). Thus, down regulated SDR9C7 inhibited the metastasis of ESCC in vivo.
Molecular Mechanisms of SDR9C7 Involved in the Metastasis of ESCC
To explore the potential mechanisms of SDR9C7 involved in the metastasis of ESCC, we examined the expression of metastasis-related molecules, including MMP11, VEGF and E-cadherin, in highly invasive cells transfected with SDR9C7 siRNA and control lentivirus (Fig. 5). The results showed that inhibiting SDR9C7 expression can markedly repress the expression of MMP11, but no obvious alteration observed on VEGF or E-cadherin expression. These data indicated that SDR9C7 might influence ESCC cell metastasis partially through regulating MMP11 expression level. Moreover, other molecular mechanisms are supposed to be further studied in future work.
10.1371/journal.pone.0052184.g005Figure 5 Effects of SDR9C7 siRNA on the expression of metastasis-related molecules by western blot analysis.
The expression of MMP11 was markedly inhibited in cells transfected with siRNA compared with controls. β-actin was used as an internal control. The pictures shown are representatives of 3 independent experiments.
Discussion
Lymphatic metastasis is a critical prognostic factor for the clinical outcome of ESCC patients, and may be involved in models of operation and chemotherapy program selection. The patients without lymphatic dissemination can benefit from a more limited transhiatal surgery or organ-preserving endoscopic resection, as opposed to these patients with lymphatic dissemination, who require a more extensive therapy. However, the methods for determining the status of LN metastasis including EUS, CT and PET/CT x-ray examination in ESCC patients are not always accurate. Interestingly, in our clinical work, we discovered that individuals showed a variable potential for lymphatic metastasis. Some T1 patients presented with a highly metastatic potential, but some T3 and T4 patients were found without lymphatic metastasis, which may be attributed to the gene expression signatures of the primary tumor tissues. Therefore, the identification of reliable molecular prognostic markers for LN metastasis of ESCC is critical for the improvement of therapeutic strategies for ESCC patients.
In the present study, we identified molecular prognostic markers for LN metastasis in primary ESCC tissues by gene expression microarray analyses. A total of 23 genes were found to reveal significantly higher expression levels, and 32 genes had significantly lower expression in N+ tumors compared with the N- ones. Of the 23 identified genes, a novel gene SDR9C7 is of particular interest because two transcripts of this gene were shown 7 times more frequently in N+ tissues compared with the N- ones. In addition, we checked the protein expression of SDR9C7 in 104 paraffin-embedded ESCC tissues by immunohistochemical analyses. Our study showed that over-expression of SDR9C7 was associated with lymphatic invasion and LN metastasis in these ESCC patients. Univariate analyses showed that evaluation factors including poor differentiation, lymphatic invasion, LN metastasis, advanced T stage and SDR9C7 expression correlated with poor prognosis. However, those factors like age and gender exhibited no prognostic value.
SDR9C7, also named RDHS, SDR-O, a retinol dehydrogenase similar protein, was localized in 12q13.3, which was cloned in 2002 [17]. The SDR superfamily is one of the largest enzyme superfamilies with over 46,000 members in the sequence databases [18]. These enzymes were found to be involved in multiple physiological roles including steroid hormone, prostaglandin and retinoid metabolism and are therefore involved in signaling [19], and the metabolization of lipids and xenobiotics [20]. A growing number of single-nucleotide polymorphisms of SDR genes have been identified, and abnormalities of SDR genes cause a variety of inherited metabolic diseases [21]. SCDR10B was found to be up-regulated in human lung cancer [22]. As far as we have concerned, there is no reported data regarding SDR9C7 expression in human cancer. Therefore it is necessary to investigate the expression and the role of SDR9C7 expression in ESCCs.
To investigate the effects of SDR9C7 on ESCC metastasis, highly invasive and non-invasive EC109 and EC9706 cell subpopulations were constructed using a repeated transwell approach. Migration and invasion assays showed that the migration and invasion capabilities of EC109-P and EC9706-P cells were significantly stronger than the matched non-invasive cell lines, indicating that the cell models are suitable to study ESCC metastasis. Consistent with the results of the gene expression profiles, western blot analyses confirmed that the expression of SDR9C7 was significantly higher in the highly invasive lines compared with the matched non-invasion cells. Then lentivirus-mediated siRNA targeting SDR9C7 was transfected into EC109-P and EC9706-P cells. The MTT assays showed that SDR9C7 knockdown did not markedly influence the cell proliferation, but significantly inhibited the cell migration and invasion, indicating that SDR9C7 is an important factor for ESCC metastases. In addition, western blot assays revealed that inhibiting SDR9C7 expression could markedly repress the expression of MMP11, but not VEGF or E-cadherin. Thus, decreasing SDR9C7 repressed the metastasis of ESCC might be partially by regulating MMP11 expression.
In conclusion, for the first time, the present study identified LN metastasis-related genes by comparing the expression profiles of primary ESCC tumors with and without LN metastasis. Within our observation, SDR9C7 expression correlated with LN metastasis, lymphatic invasion and poor patient prognosis. Knockdown of SDR9C7 could significantly inhibit the metastasis of ESCC cells. These findings suggest that SDR9C7 plays an important role in the metastasis of ESCC. In addition, the present study provides valuable information for further exploration of identifying the molecular mechanisms of SDR9C7-involved ESCC metastasis.
We thank Professor Zengshan Li (Department of Pathology in Xijing hospital) for pathological analyses, Mr Guocai Wang (Shanghai Biotechnolgy Corporation) for gene microarray analyses, and Guangbo Tang and Jianhua Dou in our lab for excellent technical assistance.
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56 : 279 –289 .19436836 | 23341893 | PMC3544840 | CC BY | 2021-01-05 17:10:56 | yes | PLoS One. 2013 Jan 14; 8(1):e52184 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23341874PONE-D-12-2799210.1371/journal.pone.0050598Research ArticleBiologyEvolutionary BiologyGeneticsHistologyMicrobiologyVirologyMolecular Cell BiologyVeterinary ScienceAnimal ManagementAnimal TypesVeterinary DiseasesVeterinary VirologyVeterinary EpidemiologyVeterinary MedicineVeterinary MicrobiologyVeterinary PathologyMutations in the Fusion Protein Cleavage Site of Avian Paramyxovirus Serotype 4 Confer Increased Replication and Syncytium Formation In Vitro but Not Increased Replication and Pathogenicity in Chickens and Ducks Pathogenicity and Replication OF APMV-4Kim Shin-Hee
1
Xiao Sa
1
Shive Heather
2
Collins Peter L.
3
Samal Siba K.
1
*
1
Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
2
Experimental Transplantation and Immunology Branch, National Cancer Institute/National Institute of Health, Bethesda, Maryland, United States of America
3
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, United States of America
Velayudhan Binu T. Editor
Texas A&M Veterinary Medical DIagnostic Laboratory, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: SHK PLC SKS. Performed the experiments: SHK SX. Analyzed the data: SHK SX HS. Contributed reagents/materials/analysis tools: PLC SKS. Wrote the paper: SHK PLC SKS.
2013 14 1 2013 8 1 e5059812 9 2012 26 10 2012 2013This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.To evaluate the role of the F protein cleavage site in the replication and pathogenicity of avian paramyxoviruses (APMVs), we constructed a reverse genetics system for recovery of infectious recombinant APMV-4 from cloned cDNA. The recovered recombinant APMV-4 resembled the biological virus in growth characteristics in vitro and in pathogenicity in vivo. The F cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, at the -1 position. Six mutant APMV-4 viruses were recovered in which the F protein cleavage site was mutated to contain increased numbers of basic amino acids or to mimic the naturally occurring cleavage sites of several paramyxoviruses, including neurovirulent and avirulent strains of NDV. The presence of a glutamine residue at the -3 position was found to be important for mutant virus recovery. In addition, cleavage sites containing the furin protease motif conferred increased replication and syncytium formation in vitro. However, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old and 2-week-old chickens, and 3-week-old ducks showed that none the F protein cleavage site mutations altered the replication, tropism, and pathogenicity of APMV-4, and no significant differences were observed among the parental and mutant APMV-4 viruses in vivo. Although parental and mutant viruses replicated somewhat better in ducks than in chickens, they all were highly restricted and avirulent in both species. These results suggested that the cleavage site sequence of the F protein is not a limiting determinant of APMV-4 pathogenicity in chickens and ducks.
This research was supported by National Institute of Allergy and Infection Diseases (NIAID) contract no N01A060009 (85% support) and NIAID, National Institutes of Health (NIH) Intramural Research Program (15% support). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The family Paramyxoviridae consists of enveloped viruses with a nonsegmented, single-stranded, negative-sense RNA genome [1]. The family is divided into two subfamilies: Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae comprises five genera: Rubulavirus, Respirovirus, Morbillivirus, Henipavirus, and Avulavirus. Subfamily Pneumovirinae is divided into two genera: Pneumovirus and Metapneumovirus. Nearly all of the paramyxoviruses that have been isolated from avian species are classified into the genus Avulavirus and are called avian paramyxoviruses (APMVs); the only exceptions are the avian metapneumoviruses, which are classified in the genus Metapneumovirus. The APMVs of genus Avulavirus have been divided into nine different serotypes (APMV 1 to 9) based on hemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays [2], and more recently a tenth serotype has been provisionally identified [3]. APMV-1, which comprises all strains of Newcastle disease virus (NDV), has been extensively characterized because virulent NDV strains cause severe disease in chickens and have a major economic impact. Complete genome sequences and reverse genetics systems are available for several NDV strains [4]–[8]. As an initial step towards characterizing other APMV serotypes, complete genome sequences of one or more representative strains of APMV serotypes 2 to 9 have been determined [9]–[16].
The APMVs are frequently isolated from a wide variety of avian species worldwide. APMV-1 has been isolated from most species of birds and, as noted, can be highly pathogenic and has a major agricultural impact. APMV-2 and -3 also have been reported to cause significant disease in poultry [17]–[18], whereas the pathogenic potential of serotypes 4–9 is generally unknown [19]. APMV-4 strains have been isolated predominantly from feral birds of the order Anseriformes
[20] and from domestic ducks, chickens, and geese [21]–[23]. APMV-4 has been reported to cause an increase in white-shelled eggs but did not affect the egg production in laying hens [2]. The genome of the prototype APMV-4 strain duck/Hong Kong/D3/75 consists of 15,054 nt [10], which follows the “rule of six” common to other members of subfamily Paramyxovirinae. APMV-4 encodes a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion glycoprotein (F), a hemagglutinin-neuraminidase glycoprotein (HN), and a large polymerase protein (L). The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 9 to 42 nt. The genome contains a 55 nt leader region at the 3′ end. The 5′ trailer region is 17 nt, which is the shortest in the family Paramyxoviridae.
Our understanding of the viral factors responsible for tissue tropism and virulence of APMVs is incomplete and is based mainly on studies with NDV in chickens. For NDV, the amino acid sequence at the F protein cleavage site has been identified as the primary determinant of NDV pathogenicity in chickens [8], [24]. The NDV F protein, like that of all paramyxoviruses, is synthesized as a precursor (F0) that is activated by cleavage by host cell protease into two disulfide-linked subunits F2-F1 [25]. Cleavage of the F protein is necessary for virus entry and cell-to-cell fusion. The F protein of virulent NDV strains typically contains a polybasic cleavage site [(R/K)RQ(R/K)R↓F] that includes the preferred cleavage site for furin [RX(R/K)R↓], which is an intracellular protease present in a wide range of cells and tissues [26]. The F protein of these strains can be cleaved in most tissues, thus providing the potential for systemic spread. In contrast, avirulent NDV strains typically contain basic residues at the -1 and -4 positions in the cleavage site [(G/E)(K/R)Q(G/E)R↓L], lack the furin site, and depend on secreted protease (or added trypsin in cell culture) for cleavage, thus limiting their replication to the respiratory and enteric tracts where appropriate secreted protease is available. In addition, the residue at the +1 position that immediately follows the cleavage site can affect the efficiency of cleavage (i.e., phenylalanine in virulent NDV; leucine in avirulent NDV) [11], [27].
The cleavage site sequence of APMV-4 (DIQPR↓F) contains a single basic amino acid, present at the -1 position. This resembles the cleavage site sequence of avirulent NDV strains. However, unlike avirulent NDV strains, APMV-4 replicates in vitro without added protease and its replication is not augmented by added protease [10]. Interestingly, APMV-4 produces single-cell infections and does not cause syncytium formation that typically is a hallmark of paramyxovirus cytopathic effect (CPE). Thus, the importance of the F protein cleavage site in APMV-4 infectivity and pathogenicity was unclear. To investigate this, we developed a reverse genetics system for APMV-4 and used it to generate 6 APMV-4 mutants whose F protein cleavage sites contained various numbers of basic residues and in some cases were derived directly from paramyxoviruses, including NDV. All the mutants exhibited protease independence, but syncytium formation and enhanced replication in vitro were observed only when a furin site was present. However, none of the mutations altered the avirulent nature of APMV-4, as we determined pathogenicity of these viruses using well-established methods for APMV-1, such as mean death time (MDT) in 9-day-old embryonated chicken eggs, intracerebral pathogenicity index (ICPI) in 1-day-old chicks, and using natural route of infection in 2-week-old chickens and in 3-week-old mallard ducks. The parental and mutant viruses replicated better in the natural host ducks than in chickens, but were avirulent in both species. These results suggest that the cleavage site sequence is not the limiting factor for APMV-4 replication and pathogenesis in chickens and ducks.
Materials and Methods
2.1. Cells and Viruses
The chicken embryo fibroblast cell line (DF1), human epidermoid carcinoma cell line (HEp-2), and African green monkey kidney cell line (Vero) (ATCC, Manassas, VA, USA) were grown in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS. Primary chicken neuronal cells were grown in Neurobasal medium with B-27 supplement (Invitrogen, Carlsbad, CA).
The modified vaccinia virus strain Ankara (MVA) expressing T7 RNA polymerase was kindly provided by Dr. Bernard Moss (NIAID, NIH) and propagated in primary chicken embryo fibroblast cells in DMEM with 2% FBS. APMV-4 strain duck/Hong Kong/D3/75 was obtained from the National Veterinary Services Laboratory (Ames, Iowa). This biologically-derived APMV-4 virus and all of the generated recombinant viruses were grown in the allantoic cavities of 9-day-old specific pathogen free (SPF) embryonated chicken eggs. Virus stocks were quantified by plaque titration on DF1 cells [10] and by hemagglutination (HA) assay with chicken erythrocytes. All experiments involving experimental animals were approved by the committee of IACUC, University of Maryland (protocol number R-09-81) and conducted following the guidelines. All animal care and handling, including euthanasia were conducted according to the procedures of Animal Care and Use committee at the University of Maryland, College Park and guideline of the American Veterinary Medical Association. All efforts were made to minimize discomfort and pain. The personnel conducting this experiment examined infected birds three times a day for clinical symptoms following the well-established scoring system. Birds that show a total score of 0 were considered “normal” while birds showing scores of 1 to 8 were considered “sick.” If a bird presents a score of 2 or 3 in any of the categories above, we increased the monitoring frequency to 3 times daily. Supportive care was provided (soft food) for animals that show scores of 2 or 3. If the condition worsens or does not improve after it has reached a score of 3, it was euthanized as it is considered that the bird has reached a moribund state. If necessary, Dr. Yanjin Zhang, facility veterinarian in the department was contacted to determine whether the bird needs to be euthanized or requires supportive care. Supportive care (soft food, subcutaneous fluids) was provided if it does not interfere with the objective of this study. Birds were anesthetized and killed by an overdose of isoflurane, the inhalant anesthetic. Briefly, sterile cotton gauze was placed in the bottom of a sterile bell jar and was covered with a wire mesh. Approximately, 1 to 2 ml of isoflurane was added into the cotton gauze. The bird was placed in the jar and the lid was closed quickly. The bird was removed from the jar after cessation of breathing.
2.2. Construction of Plasmid Bearing the Full-Length Antigenomic cDNA of APMV type 4 and Support Plasmids
A full-length antigenomic cDNA of APMV-4 was constructed based on the complete consensus sequence of APMV-4 strain duck/Hong Kong/D3/75 (GenBank accession no. FJ177514). Based on this sequence, gene-specific primers were designed for RT-PCR using as template RNA from APMV-4 virions. Six fragments were generated and were inserted into a modified pBR322 [6] using five restriction sites (SbfI, SnaBI, MluI, NotI, and PvuI) that were introduced into untranslated regions (UTRs) (Fig. 1). Fragments bearing the N, P, M, F, HN, and L genes were consecutively inserted into pBR322. To construct support plasmids, cDNA fragments containing the ORFs of the N, P, and L genes of APMV-4 were generated by RT-PCR. The N and P ORFs were cloned individually into plasmid pTM-1 between NcoI and XhoI sites. The L gene ORF was cloned into plasmid pGEM between AatII and SacI sites by a two-step cloning procedure using XmaI site as the third restriction site. The full-length cDNA of APMV-4 in pBR322, the N and P ORFs in pTM-1, and the L ORF in pGEM were sequenced in their entirety.
10.1371/journal.pone.0050598.g001Figure 1 Construction of a cDNA clone encoding a full-length antigenomic RNA of APMV-4, and the introduction of modified F genes.
The APMV-4 cDNA was assembled between the T7 promoter (to the left) and the hepatitis delta virus antigenomic ribozyme sequence followed by a T7 RNA polymerase transcription-termination signal (to the right). Assembly of the cDNA employed SbfI, SnaBI, MluI, NotI, and PvuII sites that were introduced into untranslated regions. To generate F protein cleavage site mutant viruses, the cDNA fragment containing the F gene was swapped using the MluI and NotI sites.
Infectious APMV-4 was generated as previously described for NDV [6]. Briefly, HEp-2 cells were transfected with three plasmids individually encoding the N, P, and L proteins (2.0 µg, 1.0 µg, and 0.5 µg per single well of a six-well dish, respectively) and a fourth plasmid encoding the full-length antigenome (5.0 µg) using Lipofectamine (Invitrogen) and simultaneously infected with vaccinia MVA expressing T7 RNA polymerase at a multiplicity of infection (MOI) of 1 PFU/cell. Two days after transfection, supernatant was inoculated into the allantoic cavity of 9-day-old embryonated chicken eggs. Recovery of the virus was confirmed by hemagglutination assay using 1% chicken red blood cells (RBCs). The presence of unique restriction enzyme sites in the recovered virus was confirmed by RT-PCR analysis.
2.3. Generation of rAPMV-4 with Mutations at Cleavage Site Sequence of the F Protein
Initially, the wild-type APMV-4 F protein cleavage site (DIQPR↓F) was changed to the naturally occurring cleavage sites of mesogenic NDV strain Beaudette C (BC) (RRQKR↓F), APMV-3 (RPRGR↓L), and APMV-5 (KRKKR↓F). Mutagenesis was performed by overlapping PCR on the MluI-NotI fragment bearing the F gene (Fig. 1), which was then cloned into the full-length antigenomic APMV-4 cDNA. Sequence analysis confirmed the absence of adventitious mutations in the MluI-NotI fragment. Transfection for virus recovery was conducted as described above. We readily recovered the mutant bearing the NDV BC cleavage site (rAPMV-4/Fc BC) but failed to recover the other two mutants in five separate transfection experiments in which the wild type rAPMV-4 virus was readily recovered each time. We noted that the F protein cleavage site sequence of APMV-4 has a glutamine residue at the -3 position, and we previously had observed that the glutamine residue at the -3 position in the cleavage site of NDV is important in virus replication and pathogenicity [28]. Therefore, we replaced the arginine and lysine residues at the -3 position in the cleavage site sequences of APMV-3 and APMV-5, respectively, with glutamine to yield the cleavage sites RPQGR↓F and KRQKR↓F, respectively, which were then introduced into rAPMV-4 (Table 1). We also prepared rAPMV-4 mutants containing the cleavage site from the avirulent NDV strain LaSota (GRQGR↓L), Sendai virus (SV) (VPQSR↓F), and human parainfluenza virus type 1 (PIV1) (NPQTR↓F) (Table 1). These manipulations involved the MluI-NotI fragment noted above, and sequence analysis confirmed the absence of adventitious mutations in each mutated fragment. Each of these mutant viruses was readily recovered, and the sequences of the F protein cleavage sites were confirmed by RT-PCR analysis of recovered virus.
10.1371/journal.pone.0050598.t001Table 1 Cleavage site sequence of parental and recombinant APMV-4 viruses and pathogenicity in MDT and ICPI assays.
Virus Cleavage site MDTa
ICPIb
Sequence Furin motif
wtAPMV-4 DIQPR ↓F - >168 0.00
rAPMV-4 DIQPR ↓F - >168 0.00
rAPMV-4/Fc type 3-Q
RPQGR↓F - >168 0.00
rAPMV-4/Fc type 5-Q
KRQKR↓F + >168 0.00
rAMPV-4/Fc BC
RRQKR↓F + >168 0.00
rAMPV-4/Fc LaSota GRQGR ↓L - >168 0.00
rAMPV-4/Fc SV VPQSR↓F - >168 0.00
rAMPV-4/Fc PIV 1 NPQTR↓F - >168 0.00
a Mean embryo death time (MDT): the mean time (h) for the minimum lethal dose of virus to kill all of the inoculated embryos. Pathotype definition: virulent strains, <60 h; intermediate virulent strains, 60 to 90 h; avirulent strains, >90 h.
b Intracerebral pathogenicity index (ICPI): pathogenicity of NDV in 1-day-old SPF chicks following intracerebral inoculation. Pathotype definition: virulent strains, 1.5–2.0; intermediate virulent strains, 0.7–1.5; and avirulent strains, 0.0–0.7.
2.4. Expression and Cleavage Efficiency of the F Protein
Incorporation of wild-type and mutated F proteins into the virions was analyzed by Western blot using an antiserum raised against a synthetic peptide from the F protein of APMV-4. The parental and APMV-4 mutant viruses were grown in embryonated eggs and subjected to discontinuous sucrose gradients to obtain partially purified stocks [29]. In addition, cleavage efficiency of wild-type and mutated F proteins were evaluated in virus-infected DF1 cells at an MOI of 0.1, and cell lysates collected at 24 h post-infection (hpi) were subjected to Western blot analysis with anti-APMV-4 F rabbit polyclonal antiserum [13], [30]. Surface expression of the F protein was evaluated on virus-infected DF1 cells at an MOI of 0.1. At 24 h post-infection (hpi), the cells were labeled with the anti-peptide antiserum against the F protein of APMV-4 followed by anti-Alexa Fluor 488 antibody, fixed with 4% paraformaldehyde, and analyzed by flow cytometry (AriaII, BD Bioscience) with Flowjo program (Tree Star, Inc.).
2.5. Growth Characteristics of Parental and Cleavage site Mutant APMV-4 Viruses in DF1 Cells
The growth characteristics of the parental and recombinant viruses were evaluated in DF1 and Vero cells. The plaques were fixed and immunostained using a previously-described antiserum raised against gel-purified N protein of partially-purified APMV-4 virions [31]. The requirement for supplementation of exogenous protease for cleavage of the F protein was determined by addition of either 1 µg/ml of acetyl trypsin or 5% chicken egg allantoic fluid [32] in preliminary experiments. Protease supplementation was not used in subsequent experiments. The growth kinetics of parent and mutant viruses was evaluated in DF1 cells at a multiplicity of infection (MOI) of 1 or 0.01 [33]. Virus titers in the collected supernatants were quantified in DF1 cells by limiting dilution and immunostaining with N-specific antiserum and expressed as 50% tissue culture infectious dose (TCID50/ml) by the end-point method of Reed and Muench [34].
2.6. Mean Death Time and Intracerebral Pathogenicity Index Tests
The pathogenicity of parental and mutant APMV-4 viruses was determined by the mean death time (MDT) test in 9-day-old SPF embryonated chicken eggs and by the intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks [35]. Briefly, for the MDT test, a series of 10-fold dilutions of infected allantoic fluid (0.1 ml) was inoculated into the allantoic cavities of five 9-day-old eggs per dilution and incubated at 37°C. The eggs were examined once every 8 h for 7 days, and the time of embryo death was recorded. The MDT was determined as mean time (h) for the minimum lethal dose of virus to kill all the inoculated embryos. The criteria for classifying the virulence of NDV isolates are: <60 h, virulent strains; 60 to 90 h, intermediate virulent strains; and >90 h, avirulent strains. For the ICPI test, 0.05 ml of a 1∶10 dilution of fresh infective allantoic fluid for each virus was inoculated into group of 10 1-day-old SPF chicks via the intracerebral route. At each observation, the birds were scored as follows: 0 if normal; 1 if sick; and 2 if dead. The ICPI is the mean of the score per bird per observation over the 8-day period. Highly virulent velogenic viruses give values approaching 2, and avirulent or lentogenic strains give values at or close to 0.
2.7. Neurotropism of Parental and Cleavage Site Mutant APMV-4 Viruses
To evaluate neurotropism of parental and mutant APMV-4 in vitro, primary chicken neuronal cells were prepared from 9-day-old chicken embryos for virus infection [27], [36]. After 72 h of infection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton, stained with antiserum against the N protein and polyclonal antibodies against a neuronal marker (anti-neuron specific beta III tubulin antibody, abcam®, Cambridge, MA) followed by anti-Alexa Fluor 488 and 594 antibodies, and then analyzed by confocal microscopy. In addition, during the infection period, virus titers in the collected supernatants at 12-h intervals were quantified in DF1 cells by immunostaining with N-specific antiserum as described above.
To determine the ability of parental and mutant viruses to replicate in chicken brains, 1-day-old SPF chicks in groups of ten were inoculated with 0.05 ml of a 1∶10 dilution of fresh infective allantoic fluid for each virus via the intracerebral route. Two birds per group were sacrificed daily until 5 days post-infection (dpi) and analyzed for infectious virus by immunostaining with N-specific antiserum in DF1 cells.
2.8. Replication of Parental and Mutant APMV-4 Viruses in Chickens and Ducks Following Inoculation of the Respiratory Tract
Replication was evaluated in 1-day-old chicks, 2-week-old chickens, and 3-week-old mallard ducks following respiratory tract inoculation. One-day-old chicks in groups of 8 were inoculated with 100 µl of each virus (256 HA units/bird) via the intranasal route. At 3 dpi, one half of the chicks from each group were sacrificed and tissue samples (lung, trachea, spleen, and brain) were collected for virus titration by limiting dilution and immunoperoxidase assay. The remaining three chicks per group were observed daily for 14 days for any clinical signs and then sacrificed for virus titration of various tissues as described above. Sera were collected from the chicks on 14 dpi and evaluated for seroconversion by HI assay [35]. Two-week-old SPF chickens in groups of six were inoculated with 200 µl of each virus (256 HA units/bird) by the intranasal route. Three birds from each group were sacrificed at 4 dpi and tissues samples (lung, trachea, spleen, and brain) were collected for virus titration. Virus titers in DF1 cells were determined by limiting dilution and immunostaining as described above. To obtain more sensitive detection of replication, tissue samples from the 2-week-old chickens also were inoculated into 9-day-old embryonated chicken eggs [36]. At 3 dpi, the allantoic fluids were tested for virus growth by HA assay. The remaining three birds per group were observed daily for 14 days for any clinical signs and then sacrificed for virus titration of various tissues as described above. Sera were collected from the chickens on 14 dpi and evaluated for seroconversion by HI assay [35]. Three-week-old mallard ducks in groups of six were infected with 500 µl of each virus (256 HA units/bird) via the combined intranasal and intratracheal routes. For virus titrations, three ducks per group were sacrificed at 4 dpi and used for collection of tissue samples (lung, trachea, spleen, and brain). The remaining ducks were observed daily for 10 days for any clinical signs.
2.9. Histopathology and Immunohistochemistry
The various tissue samples harvested from infected 2-week-old chickens and 3-week-old ducks were fixed in phosphate-buffered formalin (10%), embedded in paraffin, and sectioned (Histoserv, Inc., Germantown, MD). Sections from mock-infected birds were used as controls. The tissues were deparaffinized, rehydrated, and subsequently, immunostained to detect viral N protein [36].
Results
3.1. Development of an APMV-4 Reverse Genetics System
A cDNA clone of the antigenomic RNA of APMV-4 was constructed from six cDNA segments that were synthesized by RT-PCR from virion-derived genomic RNA (Fig. 1). The cDNA segments were cloned in a sequential manner into the low-copy-number plasmid pBR322/dr [6] between a T7 promoter (TAATACGACTCACTATAGG) and the hepatitis delta virus ribozyme sequence. The resulting APMV-4 cDNA in the plasmid pBR322/dr/APMV-4 is a faithful copy of the published consensus sequence for the APMV-4 antigenome [10] except for 10 silent nucleotide changes that were introduced to create five new unique restriction enzyme sites used in the construction (SbfI, SnaBI, MluI, NotI, and PvuI, Fig. 1). This construct contains a T7 promoter that initiates the encoded antigenomic RNA with three extra G residues at its 5′ end, which was previously shown for NDV to increase the efficiency of T7 polymerase transcription without interference with viral recovery [6]. Three support plasmids were constructed that express the N, P, and L ORFs of APMV-4 under the control of the T7 promoter. Recombinant APMV-4 (rAPMV-4) was readily recovered by transfection of the antigenome plasmid into HEp-2 cells together with plasmids encoding the N, P, and L proteins, necessary for viral RNA replication and transcription. Recovery of the virus was facilitated by inoculating the transfection mixture into 9-day-old embryonated chicken eggs, since APMV-4 replicates to high titer in embryonated chicken eggs than in cell culture. Furthermore, the recombinant virus did not require exogenous protease during transfection and passage, which is consistent with the previous observation that replication of biologically-derived wild-type APMV-4 is unaffected by added protease [10].
3.2. Generation of Cleavage Site Mutant APMV-4 Viruses
The F protein cleavage site of APMV-4 has a single basic amino acid residue (DIQPR↓F), and thus lacks the preferred cleavage site for the protease furin (RX[K/R]R↓). Initially, we constructed and attempted to recover three mutant rAPMV-4 viruses bearing the naturally occurring F protein cleavage sites of three different viruses: namely, the neurovirulent NDV strain BC (RRQKR↓F), which contains four basic amino acids as well as the furin motif; APMV-3 (RPRGR↓L), which has three arginine residues but lacks the furin motif; and APMV-5 (KRKKR↓F), which has five tandem basic residue and contains the furin motif [13]. Somewhat surprisingly, virus recovery was successful only with the mutant containing the cleavage site sequence of NDV BC (rAPMV-4/Fc BC, Table 1); the other two mutants could not be recovered in five different attempts in which positive controls were readily recovered.
We had previously found that the presence of a glutamine residue at the -3 position in the cleavage site of NDV is important in virus replication and pathogenicity [28]. We also noted that the F protein cleavage site sequence of unmodified APMV-4 has glutamine residue at the -3 position. Glutamine also is present at position -3 in the NDV BC F cleavage site, but not in the cleavage sites of APMV-3 or -5. Therefore, we modified these latter two sites to contain a glutamine at position -3 (RPQGR↓F and KRQKR↓F, respectively) and inserted these sites into rAPMV-4. Virus was readily recovered from these cDNAs (rAPMV-4/Fc type 3-Q and rAPMV-4/Fc type 5-Q, respectively, Table 1), suggesting an important role for glutamine at position -3 in F protein function and virus recovery. We also prepared rAPMV-4 mutants bearing the naturally-occurring F cleavage site sequences of three other viruses: namely, avirulent NDV strain LaSota (GRQGR↓L), SV (VPQSR↓F), and human PIV-1 (NPQTR↓F). Each of these contained a glutamine residue in the -3 position. Each of these additional mutants was readily recovered (yielding viruses rAPMV-4/Fc LaSota, rAPMV-4/Fc SV, and rAPMV-4/Fc PIV1, Table 1). Added protease was not necessary for any of these recoveries. To evaluate genetic stability, the viruses were passaged five times in 9-day-old embryonated chicken eggs, and the sequence of the F gene was confirmed, showing that the introduced mutations were maintained without any adventitious mutations. Out of the six mutant viruses that were successfully recovered, only two had furin motifs in the F protein cleavage site: namely, rAPMV-4/Fc BC and rAPMV-4/Fc type 5 Q (Table 1)
The viral proteins in partially purified parental and chimeric viruses were analyzed by Western blot using antiserum raised against a synthetic peptide representing amino acids 358 to 372 of the APMV-4 F protein. The protein composition of the wild-type and recombinant viruses was verified by Coomassie blue staining of the purified viral proteins (Fig. 2A). This confirmed that all of the recombinant viruses contained a similar pattern of the major structural proteins, suggesting that the various mutations of the F protein cleavage site did not affect virion assembly (Fig. 2B).
10.1371/journal.pone.0050598.g002Figure 2 Analysis of proteins present in virions of parental and F protein cleavage site mutant APMV-4 viruses, and cell-surface expression of the viral F protein.
(A) Virus that was partially purified from infected chicken egg allantoic fluids by sucrose step gradients was separated by electrophoresis, and the gel was stained with Coomassie brilliant blue. Lanes: 1. Biologically-derived APMV-4, 2. rAPMV-4, 3. rAPMV-4/Fc type 3-Q, 4. rAPMV-4/Fc type 5-Q, 5. rAMPV-4/Fc BC, 6. rAMPV-4/Fc Las, 7. rAMPV-4/Fc SV, and 8. rAMPV-4/Fc PIV-1 (B) Incorporation of wild-type and mutated F proteins into the virions was further analyzed by Western blot. The separated proteins in the gel (8%) under reducing condition (in panel A) were transferred into a membrane, and the F protein was detected by using an antiserum raised against a synthetic peptide from the F protein of APMV-4. (C) Surface expression of the F protein on infected DF1 cells. DF1 cells were infected with each virus (MOI of 0.1) and, at 24 h post-infection, were stained with anti-peptide antiserum against the F protein followed by anti-Alexa Fluor 488 antibody, and were analyzed by flow cytometry.
The effect of the cleavage site mutations on the surface expression of the F protein in DF1 cells was further evaluated by flow cytometry analysis. This showed that wild type biologically-derived APMV and its recombinant version rAPMV-4 were indistinguishable with regard to the level of surface expression of the F protein (Fig. 2C). Most of mutant viruses also had similar levels of surface expression to those of the biological and recombinant parental viruses. There was one exception: the level of expression of F protein by the rAPMV-4/Fc BC virus was 275% that of the parental viruses, indicating that expression was substantially increased by introduction of the NDV BC cleavage site.
3.3. Cytopathic Effect and Growth Characteristics of APMV-4 Mutant Viruses in Vero and DF1 Cells
We previously reported that biologically-derived wild-type APMV-4 replicates in Vero and DF1 cells without supplementation by exogenous protease, and causes single-cell infections in cell culture, with cell rounding and detachment but without the formation of syncytia. Furthermore, replication and syncytium formation were not enhanced by the addition of protease [10]. To investigate the effects of the mutant cleavage site sequences on syncytium formation by APMV-4, DF1 cells were infected with the parental biologically-derived wild-type APMV-4, its recombinant version rAPMV-4, and the six different mutant viruses. The cells were visualized 72 hpi by photomicroscopy directly (Fig. 3A) and following immunostaining with antiserum against the APMV-4 N protein (Fig. 3B). In parallel, the ability of the viruses to produce plaques was tested on DF1 cells under 0.8% methylcellulose overlay (not shown). Biologically-derived and recombinant wild-type APMV-4 produced similar CPE (i.e., single-cell infections) in DF1 cells (Fig. 3). The two viruses did not produce plaques under methylcellulose overlay in the presence or absence of exogenous protease (i.e., acetyl trypsin or chicken egg allantoic fluid, data not shown) up to 72 hpi. Among the F protein cleavage site mutant viruses, only rAPMV-4/Fc BC produced distinctive syncytia (Fig. 3) and plaques (not shown) that resembled those of NDV strain BC. rAPMV-4/Fc type 5-Q showed sporadic syncytium formation both in DF1 cells, but failed to produce distinguishable plaques. In contrast, the other four mutant viruses produced CPE that was characterized by single-cell infections, cell rounding, and detachment of virus-infected cells, similar to that of the wild-type APMV-4 parents, indicating that these mutations in the F protein cleavage site did not affect viral CPE. Furthermore, the addition of acetyl trypsin or chicken egg allantoic fluid to the cell cultures as a source of exogenous protease did not result in increased virus replication, syncytium, and plaque formation for any virus (data not shown). The two APMV-4 mutants that were able to produce syncytia in cell culture, rAPMV-4/Fc BC and rAPMV-4/Fc type 5-Q, were also the two that contained a furin motif in the F protein cleavage site. Western blot analysis showed that the F protein of these two mutants was efficiently cleaved in DF1 cells, whereas other mutants that did not produce syncytia showed incomplete cleavage of F protein (Fig. 3C). Our results suggest that efficient cleavage of APMV-4 F protein is required for syncytium formation. Supplementation of 10% allantoic fluid did not change the status of wt and mutant F proteins (data not shown).
10.1371/journal.pone.0050598.g003Figure 3 Production of cytopathic effect and proteolytic cleavage of the F0 proteins of parental and F protein cleavage site mutant APMV-4 viruses.
(A) DF1 cells in six-well plates were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.1 PFU/cell and incubated for 72 h. (B) The viral plaques in the infected cells were visualized by immunoperoxidase staining using antiserum against the N protein of APMV-4; viral antigen is stained red. (C) Proteolytic cleavage of the F0 proteins of parental and mutant viruses in infected DF1 cells was analyzed by Western blot (I) in triplicate. The positions of the precursor protein F0 and the cleavage product F1 are indicated. The relative levels of the F0 and F1 proteins in the Western blot images were measured by Bio-Rad Gel Image analysis, and the efficiency of cleavage was determined by dividing the amount of F1 by the amount of F1 plus F0 (II).
We evaluated the replication kinetics of parental and F protein cleavage site mutant viruses in DF1 cells (Fig. 4). In one experiment, cells were infected at a low MOI of 0.01 in order to evaluate multi-cycle replication. Biological wild-type APMV-4 and its recombinant version rAPMV-4 did not exhibit efficient multi-cycle replication in DF1 cells, yielding very low titers (<102 TCID50/ml, Fig. 4A). However, two of the mutant viruses exhibited substantial increases in the kinetics and magnitude of replication. rAPMV-4/Fc BC grew much better than any other APMV-4 mutants, reaching a maximum titer of 5.0×105 TCID50/ml at 84 hpi. rAPMV-4/Fc type 5-Q also replicated substantially better than other mutant viruses, reaching a maximum titer of 102 TCID50/ml at 84 hpi. Efficient replication of the other four APMV-4 mutant viruses was not detected, which is similar to that of the biological and recombinant wild-type viruses (<102 TCID50/ml). We further evaluated virus replication in DF1 cells by infecting at an MOI of 1. Under these conditions, biologically-derived wild-type APMV-4 and its recombinant version rAPMV-4 reached a maximum titer of 103 TCID50/ml at 72 hpi. Among the mutant viruses, rAPMV-4/Fc BC and rAPMV-4/Fc type 5-Q grew to titers of 5.0×105 TCID50/ml at 24 hpi and 8.0×104 TCID50/ml at 72 hpi, respectively, thus replicating to a 100-fold higher titer compared to the parental viruses. The other four mutant viruses replicated to maximum titers similar to those of the parental viruses. The increased in vitro replication of rAPMV-4/Fc BC and rAPMV-4/Fc type 5-Q (Fig. 4) correlated with the ability to form syncytia (Fig. 3), enhanced surface expression of the F protein (Fig. 2C), and the presence of a furin motif (Table 1). On the contrary, APMV-4 wild-type and mutant viruses grew to high titer (28 HAU/ml) in embryonated eggs, indicating that probably, there is a protease in the cells of allantoic membrane that efficiently cleaves the F protein of APMV-4.
10.1371/journal.pone.0050598.g004Figure 4 Replication of parental and F protein cleavage site mutant APMV-4 viruses in DF1 cells.
The growth kinetics of parental and recombinant APMV-4 viruses was determined by infecting DF1 cells with each virus at an MOI of 0.01 (A) and 1 (B). The viral titers were determined by limiting dilution on DF1 cells and immunostaining with antiserum raised against the N protein of APMV-4.
3.4. Evaluation of the Pathogenicity of Wild-Type and Cleavage Site Mutant APMV-4 Viruses in Chickens by the MDT and ICPI Tests
The pathogenicity of the wild-type and mutant APMV-4 viruses was evaluated by the standard, internationally-accepted pathogenicity tests for NDV, namely the MDT assay in embryonated chicken eggs and the ICPI assay in 1-day-old chicks (Table 1). Biologically-derived wild-type APMV-4 and its recombinant version rAPMV-4 were identical with regard to the values of MDT and ICPI, which were >168 h and 0.00, respectively, indicating their avirulence in chickens. Interestingly, the MDT and ICPI values of each of the six mutant viruses were identical to those of the parental APMV-4 viruses. None of the viruses caused the death of any of the chicken embryos within the standard 7-day time limit for assay. Chicks infected with each mutant virus had no apparent clinical signs during the 8-day period of the ICPI test. Thus, all of the APMV-4 viruses in this study were highly attenuated, and none of the various mutations in the cleavage site of the F protein had any discernable effect on the virulence of APMV-4.
3.5. Replication of Parental and Mutant APMV-4 in Neuronal Cells in vitro and in the Brains of 1-day-old Chicks
To evaluate possible neurotropism of the parental and mutant APMV-4 viruses, we assayed their ability to replicate in primary chicken neuronal cells in vitro. First, neuronal cell cultures were infected with the panel of viruses at an MOI of 0.1 PFU. The cells were fixed at 72 hpi, immunostained with antiserum against the N protein of APMV-4, and visualized by confocal microscopy (Fig. 5). Neurovirulent NDV strain BC was used as a control, and the expression of N protein by this virus was clearly detected in dendrites and axons. In contrast, N protein expression could not be detected in the case of biologically-derived wild-type APMV-4 or its recombinant version. Among the F protein cleavage site mutant viruses, N protein expression was detected sporadically in the case of rAPMV-4/Fc BC, but was not detected with any other mutant. We also examined replication in the neuronal cell cultures by collecting supernatants from the cultures at 12 h intervals and assaying for infectious virus by titration in DF1 cells (not shown). Although neurovirulent NDV strain BC was able to grow to 5.5 TCID50/ml at 72 hpi, none of the parental or mutant APMV-4 viruses, including rAPMV-4/Fc BC, were detected, indicating their inability to replicate in these cultures of primary chicken neuronal cells (data not shown).
10.1371/journal.pone.0050598.g005Figure 5 Replication of parental and F protein cleavage site mutant APMV-4 viruses in primary chicken neuronal cells.
Neuronal cells were infected with APMVs at an MOI of 0.1. At 48 hpi, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with a neuronal marker (anti-neuron specific beta III tubulin polyclonal antibodies) and antiserum against the N protein of APMV-4 followed by anti-Alexa Fluor 488 and 596, and then analyzed by confocal microscopy. The neuronal marker and viral N protein stained red and green, respectively.
We investigated the ability of the parental and mutant viruses to replicate in chicken brains by inoculating 1-day-old chicks via the intracerebral route. The infected chickens were sacrificed daily and brain tissue was collected for virus titration (Fig. 6). We included neurovirulent NDV strain BC and avirulent NDV strain LaSota as controls. In chicks infected with NDV strain BC, virus replication reached a peak titer of >6.0 log10 TCID50/g at 2 dpi, and all of the infected chicks died on 3 dpi. The LaSota strain was detected at low titer on day 1, but was not detected on subsequent days and did not cause disease or death during the 5 days of observation. Replication of the parental and mutant APMV-4 viruses was not detectable on any day, and there was no detectable disease or death, indicating that wild type APMV-4 does not replicate detectably in chicken brains, and that none of the cleavage site mutations gained this ability.
10.1371/journal.pone.0050598.g006Figure 6 Growth kinetics of parental and F protein cleavage site mutant APMV-4 viruses in the brains of 1-day-old chicks.
Ten 1-day-old SPF chicks were inoculated with the indicated parental or mutant viruses via the intracerebral route. Two birds in each group were sacrificed daily until 5 dpi. Brain tissue samples were harvested and virus titers were determined by limiting dilution in DF1 cells and immunostaining with antiserum against the N protein of APMV-4. Each bar represents mean and standard error of the mean of duplicate samples.
3.6. Replication of Parental and Mutant APMV-4 Viruses in 1-day-old and 2-week-old Chickens
We next evaluated replication and tissue tropism of the parental and mutant APMV-4 viruses in 1-day-old and 2-week-old chickens following intranasal inoculation. One-day-old chicks were infected, and one half of each group was sacrificed at 3 dpi and tissue samples (trachea, lung, spleen, and brain) were collected for virus titration by limiting dilution. Replication of parental and mutant APMV-4 in 1-day-old chicks was detected only in the trachea, with titers that were low and ranged from 1.5 to 2.2 log10 TCID50/g (data not shown). The viruses were not detected in other collected tissue samples (data not shown). The remaining birds in each group were observed daily for a total of 14 days for any clinical signs and then sacrificed for virus titration of various tissues as described above. Virus was not detected in any of the 14 dpi samples (data not shown). Clinical signs of illness were not observed in any of the infected groups.
Two-week old chickens were inoculated by the intranasal route with the various viruses, and one half of the birds in each group were sacrificed at 4 dpi and tissue samples (trachea, lungs, spleen, and brain) were collected for virus titration by limiting dilution. Replication of parental and mutant APMV-4 was not detected in any the harvested tissue samples from 4 dpi (data not shown). Since APMV-4 does not replicate efficiently in cell culture (Fig. 4), but replicates efficiently in embryonated eggs, the tissue homogenates from the experiment with the 2-week-old birds also were inoculated into eggs to as a more sensitive assay to detect possible in the various tissues. For the samples from 4 dpi, replication of parental and mutant APMV-4 viruses were detected sporadically (in 1 out of 3 birds in each case) in the trachea from chickens infected with biologically-derived wild-type APMV-4, rAPMV-4/Fc type 5-Q, rAMPV-4/Fc BC, rAMPV-4/Fc LaSota, and rAMPV-4/Fc SV (Table 2). Virus was not detected in samples from chickens infected with rAPMV-4, rAPMV-4/Fc type 3-Q, or rAPMV-4/Fc PIV1. This sporadic pattern indicated that replication in 2-week-old chickens was not significantly different between the parental and the various mutant APMV-4 viruses. The remaining birds in each group were observed daily for a total of 14 days for any clinical signs and then sacrificed for virus titration of various tissues by limiting dilution and by inoculation of embyonated eggs as described above. Virus was not detected in any of the 14 dpi samples (data not shown). Clinical signs of illness were not observed in any of the infected groups.
10.1371/journal.pone.0050598.t002Table 2 Replication of parental and F protein cleavage site mutant APMV-4 viruses in 2-week-old chickens.
Virus Virus replication in embryonated eggsa
Brain Trachea Lung Spleen
wtAPMV-4 0/3 1/3 0/3 0/3
rAPMV-4 0/3 0/3 0/3 0/3
rAPMV-4/Fc type 3-Q 0/3 0/3 0/3 0/3
rAPMV-4/Fc type 5-Q 0/3 1/3 0/3 0/3
rAMPV-4/Fc BC 0/3 1/3 0/3 0/3
rAMPV-4/Fc LaSota 0/3 1/3 0/3 0/3
rAMPV-4/Fc SV 0/3 1/3 0/3 0/3
rAMPV-4/Fc PIV 1 0/3 0/3 0/3 0/3
a Groups of 2-week-old chickens were inoculated with each virus by the intranasal route. Three birds from each group were sacrificed, and tissues samples (brain, trachea, lung, and spleen) were collected and homogenized. To investigate virus replication, aliquots (100 µl each) of the collected samples on 4 dpi was inoculated into three eggs, and allantoic fluids were collected on 3 dpi. Virus replication was determined by hemagglutination assay.
Tissue samples collected on 4 dpi from the 2-week-old chickens also were evaluated for histopatholgy (data not shown). This revealed similar microscopic findings for the parental and cleavage site mutant viruses. Specifically, the trachea infected with parental and all of the mutant APMV-4 viruses except for rAPMV-4/Fc BC exhibited minimal lymphocytic tracheitis and mild multifocal mucosal attenuation, whereas the trachea infected with rAPMV-4/Fc BC showed mild lymphocytic tracheitis and mild to moderate multifocal mucosal attenuation (data not shown). In general, lung sections exhibited mild to moderate, multifocal, lymphocytic to lymphohistiocytic bronchitis with minimal perivascular and parabronchial interstitial inflammation. Significant lesions were not found in any of the brain or spleen tissues. These results suggested that parental and cleavage site mutant APMV-4 viruses were avirulent in chickens, and their replication was mostly restricted to the trachea.
Virus replication in 1-day-old and 2-week-old chickens infected with parental and mutant APMV-4 viruses was further investigated by measuring seroconversion at 14 dpi in the studies described above (Fig. 7). Sera were analyzed by HI assay using chicken erythrocytes. All of the infected chickens from each age group seroconverted at 14 dpi. Although parental and mutant APMV-4 viruses replicated with low systemic spread, all viruses were able to induce a good humoral immune response. In general, sera collected from chickens infected as 1-day-old chicks showed higher HI titers than those infected as 2-week-old-chickens. Also, certain mutant viruses, such as rAMPV-4/Fc BC and rAMPV-4/Fc SV, induced slightly better immune response than did parental APMV-4.
10.1371/journal.pone.0050598.g007Figure 7 Induction of serum antibodies in response to infection of 1-day-old and 2-week-old chickens with parental and F protein cleavage site mutant APMV-4 viruses.
Chickens were inoculated with each virus (256 HA units) by the intranasal route in the same experiment as Table 2. Sera were collected at 14 dpi and evaluated for virus-specific antibodies by a hemagglutination inhibition assay using chicken erythrocytes.
3.7. Replication of APMV-4 Parental and Mutant Viruses in 3-week-old Ducks
Since biologically-derived wild-type APMV-4 was isolated originally from a mallard duck, we evaluated the wild-type and mutant APMV-4 viruses in this species. Three-week-old ducks (six each) were inoculated by the combined intranasal and intratracheal routes with the various APMV-4 viruses. One half of each group was sacrificed at 4 dpi and tissue samples (trachea, lungs, spleen, and brain) were collected for virus titration by limiting dilution in DF1 cells. Replication of parental and mutant APMV-4 viruses was not detected by this assay in any of the tissue samples (data not shown). However, using the more sensitive assay of inoculating tissue homogenates into embryonated eggs, virus was detected in most of the samples of trachea, lungs, and spleens, but not in the brain (Table 3). For example, both biologically-derived wild-type AMPV-4 and its recombinant version rAPMV-4 were detected in the trachea and lung of one or two each out of three ducks in each group, and rAPMV-4 was detected in the spleen of a single duck. All the F protein cleavage site mutant viruses were also detected in the trachea and lungs of one or two ducks per group. Among the mutant viruses, rAMPV-4/Fc type 3-Q, rAMPV-4/Fc BC, and rAMPV-4/Fc SV were each detected in the spleen of a single duck.
10.1371/journal.pone.0050598.t003Table 3 Replication of parental and F protein cleavage site mutant APMV-4 viruses in 3-week-old ducks.
Virus Virus replication in embryonated eggsa
Brain Trachea Lung Spleen
wtAPMV-4 0/3 2/3 1/3 0/3
rAPMV-4 0/3 1/3 1/3 1/3
rAPMV-4/Fc type 3-Q 0/3 2/3 1/3 1/3
rAPMV-4/Fc type 5-Q 0/3 2/3 1/3 0/3
rAMPV-4/Fc BC 0/3 2/3 2/3 1/3
rAMPV-4/Fc LaSota 0/3 2/3 2/3 0/3
rAMPV-4/Fc SV 0/3 2/3 2/3 1/3
rAMPV-4/Fc PIV 1 0/3 1/3 1/3 0/3
a Groups of 3-week-old ducks were inoculated with each virus by the combined intranasal and intratracheal routes. Three birds from each group were sacrificed, and tissues samples (brain, trachea, lung, and spleen) were collected on 4 dpi and homogenized. To confirm the virus replication, aliquots (100 µl each) of the collected samples were inoculated into three eggs, and allantoic fluids were collected on 3 dpi. Virus replication was determined by hemagglutination assay.
Histopathological examinations of tissue samples collected on 4 dpi also showed similar patterns between parental and mutant viruses (Fig. 8A). In the trachea, there was mild to moderate lymphocytic tracheitis with mild to moderate multifocal mucosal attenuation and reduction of tracheal mucous glands. Lung sections exhibited moderate to marked multifocal, lymphohistiocytic bronchointerstitial pneumonia with moderate perivascular cuffing in the pulmonary interstitium. Spleen sections showed moderate reactive lymphoid hyperplasia characterized by expansion of the white pulp by reactive lymphocytes and increased size and cell density of lymphoid follicles. However, microscopic lesions were not found in the brain tissues. The presence of viral antigens in various tissues was investigated by immunohistochemistry analysis. Deparaffinized sections of the virus-infected and uninfected control tissue were immunostained using antiserum against the N protein of APMV-4. Viral antigens were detected in tissue samples that were also positive by virus isolation, confirming that the detection of virus in harvested tissue indeed was associated with infection of the organ. This is illustrated with representative virus, rAPMV-4, in Fig. 8B. In the trachea, the presence of antigens for most APMV-4 was detected in the epithelial lining of trachea. In the lungs, viral antigens were mostly localized in the epithelium surrounding the medium and small bronchi. Sporadic distribution of viral antigens was found in the spleen, indicating that replication of APMV-4 viruses was not extensive in this tissue. Although all the APMV-4 viruses replicated better in ducks than chickens, there was no clear relationship between virus replication and sequence of the F protein cleavage site.
10.1371/journal.pone.0050598.g008Figure 8 Histopathology and immunohistochemistry in sections of collected tissues from 3-week-old ducks infected with parental and F protein cleavage site mutant APMV-4 viruses.
As described in Table 3, ducks were inoculated with each virus (256 HA units) by the combined intranasal and intratracheal routes, and tissue samples were harvested on 4 dpi. The tissues were fixed with formalin and sections were prepared and stained with hematoxylin and eosin for histopathology (A) or with antiserum against the N protein of APMV-4 for immunohistochemistry (B). (A) Histopathological examination of tissue samples revealed similar microscopic findings in parent and mutant APMV-4 viruses. This is illustrated with representative virus, rAPMV-4. The trachea showed mild to moderate lymphocytic tracheitis with mild to moderate multifocal mucosal attenuation and reduction of tracheal mucous glands. Lung sections exhibited moderate to marked multifocal, lymphohistiocytic bronchointerstitial pneumonia with mild to moderate perivascular cuffing in the pulmonary interstitium. Spleen sections showed moderate reactive lymphoid hyperplasia characterized by expansion of the white pulp by reactive lymphocytes and increase size and cell density of lymphoid follicles. (B) The presence of antigen (stained red) was detected for parental and mutant APMV-4 in the epithelial lining of trachea, in the epithelium surrounding the medium and small bronchi of the lungs and in the spleen.
Discussion
Paramyxovirus infectivity depends on activation of the F protein by proteolytic cleavage by host protease. In the case of NDV, the cleavage site sequence is a major determinant of viral pathogenicity [8], [24], [26]. Virulent NDV strains contain a multibasic cleavage site with a furin motif [RX(R/K)R↓]. In cell culture, this provides for intracellular cleavage without the need for exogenous protease [26]. In vivo, these viruses replicate in most cell types and can cause systemic infection. In contrast, the cleavage site sequences of avirulent NDV strains characteristically have one or a few basic residues immediately upstream of the cleavage site in place of a furin cleavage site, thus requiring exogenous protease in cell culture [8], [26]. Replication of avirulent NDV strains is restricted mostly to the respiratory and gastrointestinal tracts, where these extracellular proteases are present. However, some of the other APMV serotypes have incongruities with the NDV paradigm. For example, for a number of the other eight serotypes, the sequence of the cleavage site does not predict whether exogenous protease is needed for growth in vitro. For example, the F protein cleavage sites of APMV-2 (PASR↓F), APMV-4 (IQPR↓F) and APMV-7 (PSSR↓F) all contain a single basic residue (R) immediately upstream of the putative cleavage site, but supplementation of exogenous protease (i.e., 10% allantoic fluid) did not enhance virus growth [10], [14], [15]. Thus, for a number of the APMV serotypes, the F cleavage site sequence does not predict the cleavage phenotype in vitro. Its contributions to virus replication and pathogenicity in vivo remain largely unknown.
In this study, a reverse genetics system for APMV-4 was developed to investigate the role of the F protein cleavage site in APMV-4 replication and pathogenesis. We constructed a series of mutants with increasing numbers of basic residues, including examples with a furin cleavage site, and evaluated their effects on replication and formation of syncytia in vitro as well as replication, tissue tropism, and pathogenicity in chickens and mallard ducks. We were unable to recover mutants containing the cleavage sites of APMV-3 and APMV-5 (RPRGR↓L and KRKKR↓F, respectively), even though the latter was multi-basic and contained the furin motif. However, we readily recovered a mutant containing the cleavage site of NDV strain BC (RRQKR↓F). We noted that this last sequence had a glutamine residue in the -3 position, which typically is the case for NDV strains in general and also is the case for APMV-4. Furthermore, we had previously found that changing this glutamine residue to a basic residue, arginine, in NDV strain BC resulted in virus attenuation in chickens [28]. This indicated that this residue can be important for F protein function in some situations, although it is found in some but not all naturally occurring paramyxovirus F cleavage sequences. We therefore modified the cleavage site sequences of APMV-3 and APMV-5 to contain a glutamine in the -3 positions (RPQGR↓F and KRQKR↓F, respectively) and inserted these sequences into rAPMV-4. These viruses were readily recovered, indicating that this residue indeed was important for APMV-4. This was further confirmed by recovery of three additional mutants containing a glutamine residue in the -3 position.
The six mutant viruses contained 1 to 4 basic residues at their F protein cleavage sites, and in only two cases was a furin motif present (rAPMV-4/Fc type5-Q and rAPMV-4/Fc BC). Nonetheless, none of the mutants required added protease for replication in cell culture, and their replication was not enhanced by added protease. Thus, although a number of these cleavage sites are associated with protease dependence in their respective native viruses (i.e., NDV strain LaSota, SV, and PIV1), all of them became protease independent when transferred into the APMV-4 backbone. However, the ability to form syncytia in cell culture depended on the presence of a furin motif. This was observed most prominently with the mutant containing the cleavage sequence from NDV strain BC (rAPMV-4/Fc BC), and to a lesser extent with the modified site from APMV-5 (rAPMV-4/Fc type5-Q). In contrast, the other cleavage site sequences did not confer syncytium formation in infected cells even after supplementation with allantoic fluid as a source of protease. Furthermore, we confirmed that the presence of a furin site (rAPMV-4/Fc BC and rAPMV-4/Fc type5-Q) can efficiently enhance the F protein cleavage of APMV-4, thus increasing virus replication in vitro. This suggests that the enhanced growth was due to increased cell-to-cell spread of the virus associated with the observed syncytium formation. In contrast, the other cleavage site sequences did not confer increased cleavage efficiency and replication in vitro. In contrast, evaluation of the pathogenicity of the wild-type and mutant APMV-4 viruses using the ICPI and MDT assays indicated avirulence of APMV-4 [10], [36] without any increase in pathogenicity by any of the mutations. These findings are incongruent with the well-known NDV paradigm, in which the presence of a furin site is a major determinant of virulence [11], [26]. Since the natural host of APMV-4 is not known, it is possible that the F cleavage site of APMV-4 (IQPR↓F), which is not a furin motif, is cleaved efficiently by a protease that is only found in its natural host.
We investigated whether the avirulence of APMV-4 was due to its inability to replicate in neuronal cells, as assayed using primary neuronal cell cultures as well as inoculation into the brains of 1-day-old chicks followed by quantitation of viral replication. Sporadic expression of APMV-4 antigen in neuronal cells in vitro was only observed with the APMV-4 mutant bearing the cleavage site from the neurovirulent NDV strain BC, and compared to the abundant expression of viral antigen observed with the NDV strain BC positive control. Furthermore, all of the parental and mutant APMV-4 viruses, including the one with the NDV BC cleavage site, failed to detectably replicate in neuronal cell cultures in vitro or in the brains of chicks inoculated intracerebrally at 1 day of age, in contrast to the abundant replication observed with the NDV strain BC positive control. These results indicate that the avirulence of parental and mutant APMV-4 may be determined, at least in part, by their inability to infect neuronal cells.
For NDV, virus neurotropism depends on the presence of a furin cleavage site, since secretory proteases are unavailable in the brain [37]. However, the present results show that the introduction of a furin cleavage site into the F protein of APMV-4 did not confer neurotropism apart from the limited antigen expression by the rAPMV-4/Fc BC mutant. The general lack of neurotropism by the present rAPMV-4 mutants is reminiscent of our previous study in which the F protein cleavage site sequence of APMV-2 or APMV-7 was changed by reverse genetics into mutants that included ones with furin cleavage sites. In those studies, we made the similar observation that the changes did not increase the neurotropism of APMV-2 or APMV-7 in 1-day-old chicks or 2-week-old chickens [30], [32]. This indicated that, for these three APMV serotypes, the sequence at the F protein cleavage site is not a major determinant of neurotropism. However, in another study, replacement of the complete APMV-2 F protein with that of neurovirulent NDV strain BC was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes to APMV-2 [33]. This previous result suggested that the F protein plays an important role in neurotropism, but the present study indicates that this effect does not appear to be determined primarily by the sequence at the F protein cleavage site.
We further evaluated APMV-4 replication in 1-day-old chicks and 2-week-old chickens following inoculation of the respiratory tract. In 1-day-old chicks, the parental viruses and all the mutant APMV-4 viruses were restricted to the trachea and replicated only to low titers. In the 2-week-old chickens, virus replication was detected only sporadically and only in the trachea. However, all of the birds exhibited seroconversion, suggesting that viral infection occurred in each case, but at a low level that often was undetectable by assays for infectious virus. Our recent pathogenicity study with the nine APMV serotypes showed inefficient replication of APMV-4 in chickens [36]. The present results confirm the highly restricted nature of replication of APMV-4, and show that this high level of restriction was not alleviated by the introduction of additional basic residues, including a furin motif, into the F protein cleavage site.
We also evaluated infection of mallard ducks, representing a natural host. The parental and mutant viruses replicated somewhat better in ducks than in chickens. Detection was sporadic for every virus, with virus being detected in the trachea and lungs, and in some cases the spleen, but without inducing any disease. Thus, ducks were more permissive than chickens, but the parental and mutant APMV-4 viruses remained highly restricted and avirulent even in ducks. Detection in the spleen was suggestive of systemic spread, but this was seen sporadically with wild-type rAPMV-4 and several mutants and did not correlate with the presence of a furin cleavage site.
The present results indicate that, while the introduction of a furin motif into the cleavage site of APMV-4 increased F protein cleavage efficiency, viral replication in vitro and conferred the ability to induce syncytia, it did not confer increased replication, tropism, or pathogenesis in vivo. Thus, APMV-4 remained highly restricted and avirulent even with the presence of the F protein cleavage site from the neurotropic NDV strain BC. These observations are consistent with previous findings that modification of the F protein cleavage site sequence of APMV-2 and APMV-7 to contain multibasic residues, including furin motifs, did not increase virus pathogenicity in chickens despite their gain of in vitro replication and syncytium formation [30], [32]. This indicated that the F protein cleavage site is not a primary determinant or limiting factor to viral virulence for these three serotypes of APMV, indicating incongruity with the well-known NDV paradigm. These results suggest that, for these APMV serotypes to be virulent, changes in other regions of the F protein or in some other viral proteins are necessary. Further characterization of virus replication and pathogenicity using reverse genetics may enhance our understanding of overall APMV pathogenesis.
We thank Daniel Rockemann, Girmay Gebreluul, Yonas Araya, and our laboratory members for excellent technical assistance. We thank Dr. Bernard Moss (NIAID, NIH) for providing the vaccinia T7 recombinant virus and the pTM1 plasmid. The views expressed herein do not necessarily reflect the official policies of the Department of Health and Human Services, nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23335978PONE-D-12-2929110.1371/journal.pone.0053834Research ArticleBiologyBiochemistryProteinsImmune System ProteinsMedicineClinical ImmunologyImmunologic SubspecialtiesTumor ImmunologyDiagnostic MedicinePathologyGeneral PathologyBiomarkersOncologyBasic Cancer ResearchImmune EvasionMetastasisTumor PhysiologyCancers and NeoplasmsGynecological TumorsCervical CancerCancer TreatmentTim-3 Expression in Cervical Cancer Promotes Tumor Metastasis Tim-3 Promotes Metastasis of Cervical CancerCao Yang Zhou Xiaoxi Huang Xiaoyuan Li Qinlu Gao Lili Jiang Lijun Huang Mei
*
Zhou Jianfeng
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, P.R. China
Busson Pierre Editor
Institute of Cancerology Gustave Roussy, France
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YC MH JZ. Performed the experiments: YC XZ QL LG. Analyzed the data: XH. Contributed reagents/materials/analysis tools: LJ. Wrote the paper: MH YC.
2013 15 1 2013 8 1 e5383427 9 2012 6 12 2012 © 2013 Cao et al2013Cao et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
T cell immunoglobulin mucin-3 (Tim-3) has been identified as a negative regulator of anti-tumor immunity. Recent studies highlight the important role of Tim-3 in the CD8+ T cell exhaustion that takes place in both human and animal cancer models. However, the nature of Tim-3 expression in the tumor cell and the mechanism by which it inhibits anti-tumor immunity are unclear. This present study aims to determine Tim-3 is expressed in cervical cancer cells and to evaluate the role of Tim-3 in cervical cancer progression.
Methodology
A total of 85 cervical tissue specimens including 43 human cervical cancer, 22 cervical intraepithelial neoplasia (CIN) and 20 chronic cervicitis were involved. Tim-3 expression in tumor cells was detected and was found to correlate with clinicopathological parameters. Meanwhile, expression of Tim-3 was assessed by RT-PCR, Western Blot and confocal microscopy in cervical cancer cell lines, HeLa and SiHa. The migration and invasion potential of Hela cells was evaluated after inhibiting Tim-3 expression by ADV-antisense Tim-3.
Conclusions
We found that Tim-3 was expressed at a higher level in the clinical cervical cancer cells compared to the CIN and chronic cervicitis controls. We supported this finding by confirming the presence of Tim-3 mRNA and protein in the cervical cell lines. Tim-3 expression in tumor cells correlated with clinicopathological parameters. Patients with high expression of Tim-3 had a significant metastatic potential, advanced cancer grades and shorter overall survival than those with lower expression. Multivariate analysis showed that Tim-3 expression was an independent factor for predicting the prognosis of cervical cancer. Significantly, down-regulating the expression of Tim-3 protein inhibited migration and invasion of Hela cells. Our study suggests that the expression of Tim-3 in tumor cells may be an independent prognostic factor for patients with cervical cancer. Moreover, Tim-3 expression may promote metastatic potential in cervical cancers.
This article was supported by National Science Foundation of China (No. 81001049) and National Science Fund for Distinguished Young Scholars of China (No. 81025011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Current studies continue to demonstrate a strong correlation between Tim-3 expression and tumor-associated immune suppression [1]–[3]. Sakuishi et al [4] found that Tim-3 was expressed on CD8+ tumor-infiltrating lymphocytes (TILs) in mice bearing solid tumors. All Tim-3+ TILs coexpress PD-1, and this kind of TILs represents the predominant fraction of T cells infiltrating tumors. Tim-3+PD-1+ TILs exhibit the most severe exhausted phenotype as defined by failure to proliferate and produce IL-2, TNF and IFN-γ. Combined targeting of the Tim-3 and PD-1 pathways is more effective in restoring anti-tumor immunity than targeting either pathway alone. Zhou et al [5] detected the same phenomenon in mice with disseminated acute myelogenous leukemia. Even in melanoma patients, upregulation of Tim-3 and PD-1 expression is also found to be associated with tumor antigen-specific CD8+ T cell dysfunction [6]. In our previous study, we found that Tim-3 was preferentially expressed in lymphoma-derived endothelial cells and suppressed activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway. Tim-3 also facilitated the establishment of lymphoma immune tolerance [7]. However, whether Tim-3 is also expressed on cancer cells in other nonhematologic cancers remain an open question.
Cervical cancer is a tumor that possesses distinct Tumor-associated antigens (TAAs). Human papillomaviruses (HPVs) have been shown to cause progressive changes in the cervical epithelium, leading to cervical cancer. Greater than 99% of cervical malignancies harbor HPV (mainly HPV-16 and HPV-18). E6 and E7 are the two proteins expressed by the virus that are necessary for the initiation and progression of cancer [8]. Several studies [9], [10] show that there are other potential roles such as Fas-FasL system and loss of HLA class I for immune escape in cervical cancer. Because many pathways are potentially involved in cervical cancer, we wondered if Tim-3, an antigen that has been implicated in the development of various cancers, might also play a role in the development of cervical cancer. In addition, our previous study found that Tim-3 was overexpressed in epithelial cancers. These reasons combined made us interested in investigating the role of Tim-3 in cervical cancer.
Two recent studies [11], [12] have identified Tim-3 expression on leukemic stem cells (LSC) in patients with acute myeloid leukemia (AML). Tim-3+ AML cells were able to reconstitute AML and anti-human Tim-3 antibody blocked AML engraftment in a xenotransplant model. Although anti-Tim-3 antibodies seem to reduce the metastatic potential of cancer cells, the mechanism of action is not well understood. In this study, we used ADV-antisense Tim-3 to down-regulate Tim-3 in the cervical cancer Hela cell line and then assessed the ability of the cancer cells to migrate and invade.
Our results from this study confirmed the mRNA and protein level expression of Tim-3 in cervical cancer cell lines and revealed for the first time that Tim-3 was preferentially expressed in the clinical primary cervical cancer cells when compared to the CIN and chronic cervicitis. In addition, patients with high expression of Tim-3 had a significantly greater metastatic potential and advanced cancer grades and shorter overall survival than those with lower Tim-3 expression. Therefore, Tim-3 may potentially be an independent prognostic factor for patients with cervical cancer. We also found that ADV-antisense Tim-3 can inhibit migration and invasion of Hela cells. Combined with our previous study, in which we found that Tim-3 activates the IL-6–STAT3 pathway to suppress the activation of CD4+ T lymphocytes, our study opens the possibility that Tim-3 may promote metastasis through the activation of IL-6-STAT3 pathway.
Materials and Methods
Patients
Samples of 43 cervical cancer tissues, 22 CIN tissues and 20 chronic cervicitis tissues were derived from patients that underwent primary surgery for cervical diseases at the Department of Gynecologic Oncology in Tongji Hospital (Huazhong University of Science and Technology, Wuhan, China) from 2004–2006. All of the selected cervical cancer tissues met the following inclusion criteria: no history of any other type of malignant tumor, without neoadjuvant therapy prior to surgery. All patients gave informed written consent for analysis of their tissue for research purposes. This study was approved by the ethics committee of the Tongji Hospital for analysis of human tissues. The patients ranged in age from 27 to 67 years (median age, 39 years). Histological examination of the excised cervical tissues were carried out following hematoxylin & eosin (H & E) staining of paraffin-embedded sections. 31 patients of invasive cervical cancers were classified as grade I, IIa without metastasis, while 12 patients were grade IIb, III and IV with metastasis.
Follow-up
Follow-up data retrieved from the clinical record ranged from 5–60 months post-surgery (median, 45.2 months). Each patient’s overall survival (OS) is calculated as the period from the date of surgery until the date of death.
Immunohistochemical Detection of Tim-3 in Cervical Tissues
Paraffin-embedded tissue sections were dewaxed in xylene and subjected to immunohistochemical analysis as previously described [13]. Anti-Tim-3 goat polyclonal antibody (Santa Cruz Biotechnology, Inc.) and biotinylated secondary antibody were used in the present study. For semiquantitative evaluation, an immunoreactivity-scoring (IRS) system was applied. Intensity of staining was designated as either nonexistent (0), weak (1), moderate (2), or strong (3). The percentage of positive cells was termed as the expression score. The IRS was calculated by multiplying the expression score with the intensity score, and may range from 0–3. The sample with IRS scores of 0–1 points was considered as negative expression of Tim-3, otherwise was designated as positive staining. These data were analyzed along a continuum, and the objective was to use this semiquantitative method to assess differences between various experimental groups. The subcellular localization of the staining (cytoplasmic and/or nuclear) was also observed.
Cell Lines
Cervical cancer cell lines used in the current study were obtained from American Type Culture Collection (ATCC) (HeLa, SiHa). These two cells were cultured in DMEM medium with 10% fetal bovine serum.
Reverse Transcriptase-Polymerase Chain Reaction Detection of Tim-3
Briefly, total RNA was extracted from two cervical cancer cell lines. A 5′ sense primer (5′-CGGAGGTCGGTCAGAATGCCTATC-3′) and a 3′ antisense primer (5′-GGGCTCCTCCA CTTCATATACGTTC-3′) were used to amplify Tim-3 transcripts. The expected product for full-length Tim-3 is 749 bp. A 5′ sense primer (5′-CTCACGAAACTGGAATAAGC-3′) and a 3′ antisense primer (5′-AAGCCACACGTACTAAAGGT-3′) were used to amplify a 180-bp β-actin internal control. Total RNA extracted from PBL was used as positive control. The primers used for RT-PCR detection of Tim-3 were designed to span introns to avoid false positive amplifications resulting from DNA amplifications. Meanwhile we use total RNA product without reverse transcription as templates for PCR as negative control to be sure free of genomic DNA contamination.
Western Blot Detection of the Expression of Tim-3 Protein
Two kinds of cervical cancer cells were lysed for 30 min at 4°C in a lysis buffer composed of 150 mmol/L NaCl, 50 mmol/L Tris (pH 8.0), 5 mmol/L EDTA, 1%(v/v) NP40, 1 mmol/L phenyl-methylsulfonyl fluoride, 20 µg/ml aprotinin, and 25 µg/ml Leupeptin. Equal amounts of protein extracts (10 µg) were resolved by SDS-PAGE. Following transfer to a nitrocellulose filter, it was blocked for 1 h at room temperature with buffer containing 20 mmol/L Tris-Hcl (pH7.5), 500 mmol/L NaCl, and 5% nonfat milk; incubated with Tim-3 antibody (1∶1000, Santa Cruz Biotechnology, Inc.) for overnight at 4°C; washed; and incubated with a horseradish peroxidase-labeled secondary antibody donkey anti-goat IgG (1∶5000) for 1 h at room temperature. Finally, the blots were developed using an enhanced chemiluminescence detection system (Amersham Life Science). In the preliminary experiment, we found NK-92 cell line predominantly expressed Tim-3 protein, while THP-1 cell line did not express Tim-3 protein. Total protein extracted from NK-92 cell line was used as positive control of Tim-3 protein, while total protein extracted from THP-1 cell line was used as negative control.
Immunofluorescent Detection of HeLa Cell Line
HeLa cells were harvested with a combination of trypsin and ethylenediamine tetraacetic acid (EDTA) and washed in PBS, and grown on coverslips. cells then were fixed in 95% ethnol and blocked with 1% BSA for 1 h, and then incubated with primary antibody at 4°C overnight. The primary antibody used were goat anti-Tim-3 (Santa Cruz Biotechnology, Inc.) at a dilution of 1∶100. The specimens then were washed in PBS for 5 minutes 3 times. Negative control slides were incubated without the primary antibody. Donkey anti-goat IgG conjugated with FITC diluted with 2% BSA/PBS to a dilution of 1∶100 was incubated for 2 h at room temperature, cell nuclear was stained by PI at a dilution of 1∶1000, checked by confocal microscope.
Adenoviral Mutants
The AdEasy system (MP Biomedicals) was used in this study to construct a recombinant replication-deficient adenovirus vector named ADV-antisense Tim-3 which contained a fragment of reverse Tim-3 cDNA (bp 198–1312). Standard protocols were followed as described previously [14]. ADV-GFP containing a GFP gene under the control of a Rous sarcoma virus long terminal repeat promoter in the region of the excised E1 adenoviral genes was used as a control in this study. Hela cells were infected with ADV-antisense Tim-3 or ADV-GFP at a proper MOI determined by Apoptosis assay for 2 h, then cultured for another 24 h and subjected to subsequent experiments.
Apoptosis Assay
Briefly, Hela cells infected with different titers of adenovirus mutants or treated with PBS for 72 h. Then cells were fixed with 70% ethanol for 1 h, washed in PBS and, treated with RNase for 15 minutes at 37°C and then stained with 50 µg/ml propidium iodide. Cells were collected and subjected to FACS analysis of sub-G1 population.
Wound Healing Assay
The cells were grown to confluence in a 6-well culture plate. A linear wound was made by scratching the monolayer with a sterile 10-ul pipette tip. The wounded monolayers were washed 3 times with regular medium and incubated in fresh serum-free medium. Photographs were taken at 0 h, 24 h and 48 h after wounding by phase contrast microscopy.
Transwell Invasion Assay
Cell invasion was assayed using Transwell chambers (Costar, Cambridge, MA, USA) with 8-µm pore polycarbonate filters that were coated with Matrigel™ (BD Biosciences, Franklin Lakes, NJ). Cells infected with adenoviral mutants for 24 h were seeded into the upper chambers in serum-free medium at a density of 2.0×104 per well, and 500 µl of 10% fetal bovine serum-containing medium was placed in the lower chamber as a chemo-attractant. After 48 h at 37°C in 5% CO2, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. Cells on the upper surface of the filter were removed with cotton buds. Invaded cells on the underside of the filter were photographed and counted by phase contrast microscopy (×200 magnifications). The experiments were performed in triplicate.
Statistical Analysis
The SPSS statistical software program was used to test for correlations between quantitative variables by the establishment of nonparametric linear regression. Data are presented as means ± SD of at least three experiments and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. The Kaplan-Meier method was used to estimate the overall survival rate as a function of time. Survival differences were analyzed using the log-rank test. The Cox proportional hazard model was used in the univariate and multivariate analysis of prognostic factors. All p-values were two-sided, and P<0.05 was considered significant. SPSS software (version 11.5) was used for all statistical procedures.
Results
Tim-3 is Preferentially Expressed in Cervical Cancer Tissue
We analysed sections of tumor tissue from 43 patients that were operated on for cervical cancer, from 22 patients with CIN and from 20 patients with chronic cervicitis. Positive staining for Tim-3 protein was seen in only 15.0% (3 out of 20) of chronic cervicitis, but 50.0% (11 out of 22) of the CIN and 65.1% (28 out of 43) of cervical cancer stained positively for Tim-3 protein (Fig. 1A–D). When the expression of Tim-3 protein was further compared by semiquantitative immunoreactivity H-scoring, cervical cancer and CIN displayed a much higher Tim-3 score than chronic cervicitis tissue. (0.905±0.584, 0.558±0.123 vs 0.102±0.075; P<0.01) (Table 1).
10.1371/journal.pone.0053834.g001Figure 1 Representative immunohistochemical staining for Tim-3 in cervical tissues.
(A) cervical squamous carcinoma, (B) cervical adenocarcinoma, (C) cervical intraepithelial neoplasia, and (D) chronic cervicitis tissue. Original magnification ×200.
10.1371/journal.pone.0053834.t001Table 1 The expression of Tim-3 in cervical tissues.
Group Cases Tim-3
positive cases(%) scorea
Cervicitis 20 3 (15.0) 0.152±0.075b,d
CIN 22 11 (50.0) 0.558±0.123c
Cervical cancer 43 28 (65.1) 0.905±0.584
a Expression score (mean ± SE). The expression score represents the expression level of Tim-3 protein in cervical cancer tissue as calculated by the immunoreactivity-scoring system.
b Cervical cancer group versus Cervicitis group P = 0.002;
c Cervical cancer group versus CIN group P = 0.006;
d CIN group versus Cervicitis group P = 0.002.
Tim-3 Expression Correlated with Clinicopathologic Parameters
We correlated the Tim-3 expression data to clinicopathologic characteristics such as age, histological type, clinical grade, histological grade, metastasis and overall survival. High immunoreactivity of Tim-3 was found to be significantly correlated with clinical grade (p = 0.018), histological grade (p = 0.038) and metastasis (p = 0.004). There were no significant correlations with age (p = 0.178) and histological type (p = 0.773) (Table 2).
10.1371/journal.pone.0053834.t002Table 2 The expression of Tim-3 in cervical cancer correlates with clinical features.
Variable Cases Tim-3
scorea
F
P
Age <39 21 0.781±0.457 1.882 0.178
>39 22 1.023±0.673
Clinical stage I,IIa 31 0.776±0.553 6.066 0.018*
IIb,III,IV 12 1.238±0.547
Type Adenocarcinoma 8 0.850±0.657 0.084 0.773
Squamous cellcarcinoma 35 0.917±0.575
Metastasis No 31 0.748±0.526 9.59 0.004**
Yes 12 1.308±0.547
Histology grade I 13 0.784±0.603 4.589 0.038*
II,III 30 1.183±0.438
a Expression score (mean ± SE). The expression score represents the expression level of Tim-3 protein in cervical cancer tissue as calculated by the immunoreactivity-scoring system. Corresponding F and P values are displayed for each cross tabulation.
*
P<0.05;
**
P<0.01.
At the end of our follow-up period, 15 patients died in Tim-3 positive group while 3 in Tim-3 negative group, the 5-year survival rate was 46.4% vs 80% respectively (P = 0.006) (Fig. 2).
10.1371/journal.pone.0053834.g002Figure 2 The correlation between Tim-3 expression and survival rate.
A comparison of five-year cumulative-survival curve of cervical cancer patients between Tim-3 positive expression (broken line) and Tim-3 negative expression (thick line) is shown.
Tim-3 is Expressed in Cervical Cancer Cell Lines
RT-PCR and western blot analysis were used to detect Tim-3 mRNA and protein levels in two human cervical cancer cell lines Hela and SiHa. As expected, a 749-bp product was found to be present in these two cell lines, thereby confirming the presence of Tim-3 mRNA expression. β-actin oligonucleotides were used to detected a 180-bp RNA band and were used as a control (Fig. 3A). A 33 kd band was found confirming Tim-3 protein expression (Fig. 3B). Confocal microscopy was used to test subcellular localization of Tim-3. Tim-3 was distributed in the whole cytoplasm (green fluorescence) of the Hela cell line, with no distribution in the nucleus (red) (Fig. 3C).
10.1371/journal.pone.0053834.g003Figure 3 Expressions of Tim-3 in cervical cancer cell lines.
(A) Tim-3 transcripts were detected in Hela and SiHa cell lines. The templates of different lanes as follows: Lane 1, total RNA of PBL; Lane 2, cDNA of PBL; lane 3, total RNA of SiHa; lane 4, cDNA of SiHa; lane 5,total RNA of Hela; lane 6, cDNA of Hela. (B) Tim-3 protein was determined by Western blotting in Hela and SiHa cell lines. (C) Hela cells stained with immunofluorescent with anti-Tim-3 antibody and observed with a confocal laser scanning microscope. Tim-3 protein (green, arrowheads) is in the cytoplasm of Hela. Cell nuclei (red) were visualized by staining with PI.
Repressing Tim-3 Expression Inhibited Migration and Invasion of Hela Cells
To further understand the correlation of Tim-3 and tumor metastasis, we infected Hela cells with ADV-antisense Tim-3. At a Multiplicity of Infection (MOI) of 1, ADV-antisense Tim-3 infected Hela cells showed significantly decreased Tim-3 expression with a minor cell death response; thus, this concentration was used in the following experiments (Fig. 4A–B). Since cancer cell migration and invasion are directly related to metastasis, a wound healing assay and a cell invasion assay were performed to determine whether repression of Tim-3 expression inhibits Hela cell migration and invasion. Hela cells infected with either ADV-antisense Tim-3 or with ADV-GFP as a control were evaluated for 24 h and 48 h. As shown in Figure 5A–B, at 24 h and 48 h respectively, ADV-antisense Tim-3 infected cells showed 40% and 70% wound closure, while ADV-GFP infected cells showed 60% and 100%, suggesting that inhibition of Tim-3 expression decreased the migration of Hela cells. As shown in Figure 5C–D, the number of Hela cells that passed through the filter in the ADV-antisense Tim-3 group (151±33) was markedly less than that in the ADV-GFP group (545±48), which shows that inhibition of Tim-3 expression suppressed Hela cell invasion in vitro.
10.1371/journal.pone.0053834.g004Figure 4 Effect of ADV- antisense Tim-3 on Hela cell line.
(A) Western blotting was performed to detect Tim-3 expression in Hela cells infected with ADV-GFP and ADV- antisense Tim-3 or treated with PBS. (B) Typical result of cell apoptosis determined by flow cytometry in Hela cells infected with ADV-antisense Tim-3 and ADV-GFP or treated with PBS. Data are represented as the mean ± SD of triplicates.
10.1371/journal.pone.0053834.g005Figure 5 Effect of Tim-3 inhibition on Hela cell migration and invasion in vitro.
(A) Cell migration capability was determined with a wound healing assay. Photographs were taken immediately (0 h), at 24 h and 48 h after wounding. (B) Quantification of wound closure. The data present the mean distance of cell migration to the wound area at 24 h and 48 h after wounding in three independent wound sites per group. (C) The ability of the cells to invade Matrigel was analyzed by the transwell invasion assay through a gel matrix. Hela cells were either infected with ADV-GFP or with ADV-antisense Tim-3, After 10 h viable invasive cells were fixed and counted. Values and error bars shown in this graph represent the averages and standard deviations respectively, of three independent experiments. (D) Representative images of the transwell invasion assay.
Discussion
In our previous work, we found that Tim-3 was preferentially expressed in lymphoma-derived endothelial cells (ECs), and that the level of Tim-3 in B cell lymphoma endothelium was closely correlated to both dissemination and poor prognosis. Tim-3+ ECs modulated T cell response to lymphoma surrogate antigens by suppressing activation of CD4+ T lymphocytes through the activation of the interleukin-6–STAT3 pathway, inhibiting Th1 polarization, providing protective immunity, and facilitating the establishment of lymphoma immune tolerance. Although studies suggest that Tim-3 is involved in the immune regulation of tumors, its direct expression in the tumor cell and its function in tumor metastasis were still unknown.
In the present study, we found that Tim-3 was preferentially expressed in cervical cancer tissues, and its expression was significantly correlated with advanced cancer grades (p = 0.018), histological grades (p = 0.038), metastasis (p = 0.004) and shorter survival (p = 0.006). The presence of Tim-3 mRNA and protein in two cervical cancer cell lines (Hela and Siha) in addition to the localization of Tim-3 to the cytoplasm of the Hela cell confirmed that Tim-3 was indeed expressed in the cervical cancer cell. To clarify the link between Tim-3 and tumor metastasis, we used ADV-antisense Tim-3 to do a wound healing assay and transwell invasion assay. We found that ADV-antisense Tim-3 infected cells showed 40% and 70% wound closure at 24 h and 48 h respectively, compared to the 60% and 100% seen in ADV-GFP infected cells. In addition, markedly fewer Hela cells passed through the filter in the ADV-antisense Tim-3 group (151±33) than that in the ADV-GFP group (545±48). These results strongly suggest that down-regulating the expression of Tim-3 decreases the migration and invasion of Hela cells significantly.
In line with our study, Wiener et al [15] also found that Tim-3 was expressed not only in mast cells around melanomas, but also in tumor cells in tissue sections and human melanoma cell lines WM35 and HT168-M1. Meanwhile, Kikushige and Jan [11], [12] identified Tim-3 expression on leukemia stem cells (LSC) in patients with acute myeloid leukemia. To determine whether Tim-3 is universally expressed in tumor cells, additional studies on other cancer cells are required.
Despite the progress made in understanding the involvement of Tim-3 in tumor immunity, the link between Tim-3 expression and tumor cell itself has not yet been defined. One of our most striking findings is that when we used ADV-antisense Tim-3 to down-regulate the expression of Tim-3 in HeLa cells, both the migration and invasion of HeLa cells were inhibited significantly. Indeed, high score expression of Tim-3 was significantly correlated with metastasis (p = 0.004) in cervical cancer patients. The next step would be to determine how Tim-3 expression is correlated with tumor metastasis and which pathways are involved? In our previous work, we have verified that Tim-3 can activate the IL-6-STAT3 pathway. Tim-3 expression increased the EC-derived production of IL-6 by almost 10-fold, and the addition of IL-6 significantly increased the level of phosphorylated STAT3 (p-STAT3). According to published studies [16]–[19], the IL-6-STAT3 pathway plays an important role in tumor metastasis. STAT3 can promote premetastatic niche formation and metastatic cells can escape the pro-apoptotic effects of TNF-α through increased autocrine IL-6-STAT3 signalling. In addition, inhibition of p-STAT3 enhances IFN-α efficacy against metastatic melanoma in a murine model. Based on these previous findings and our results from this study, we hypothesize that Tim-3 might facilitate tumor metastasis through the IL-6-STAT3 pathway. Because distant metastasis is a major factor in the survival of individuals with cervical cancer, Tim-3 may be a critical prognostic marker for cervical cancer.
Taken together, our study suggests that Tim-3 not only negatively regulates anti-tumor immunity, but also influences cancer development directly via its expression in cancer cells. For the first time, we have associated the expression of Tim-3 in tumor cells with worse clinical pathological parameters in cervical cancer. In addition, we found that the inhibition of Tim-3 protein expression can prevent tumor metastasis. Thus, it is rational for future experiments to explore Tim-3 as a target for anti-cancer therapy, tumor immunotherapy, and in the control of metastatic diseases.
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Nutr JNutr JNutrition Journal1475-2891BioMed Central 1475-2891-12-32328629510.1186/1475-2891-12-3ResearchHigh bioavailablilty iron maize (Zea mays L.) developed through molecular breeding provides more absorbable iron in vitro (Caco-2 model) and in vivo (Gallus gallus) Tako Elad [email protected] Owen A [email protected] Leon V [email protected] Raymond P [email protected] USDA-ARS Robert W. Holley Center for Agriculture and Health, 538 Tower Road, Cornell University, Ithaca, NY, 14853, USA2013 4 1 2013 12 3 3 27 8 2012 30 12 2012 Copyright ©2013 Tako et al.; licensee BioMed Central Ltd.2013Tako et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Iron (Fe) deficiency is the most common micronutrient deficiency worldwide. Iron biofortification is a preventative strategy that alleviates Fe deficiency by improving the amount of absorbable Fe in crops. In the present study, we used an in vitro digestion/Caco 2 cell culture model as the guiding tool for breeding and development of two maize (Zea mays L.) lines with contrasting Fe bioavailability (ie. Low and High). Our objective was to confirm and validate the in vitro results and approach. Also, to compare the capacities of our two maize hybrid varieties to deliver Fe for hemoglobin (Hb) synthesis and to improve the Fe status of Fe deficient broiler chickens.
Methods
We compared the Fe-bioavailability between these two maize varieties with the presence or absence of added Fe in the maize based-diets. Diets were made with 75% (w/w) maize of either low or high Fe-bioavailability maize, with or without Fe (ferric citrate). Chicks (Gallus gallus) were fed the diets for 6 wk. Hb, liver ferritin and Fe related transporter/enzyme gene-expression were measured. Hemoglobin maintenance efficiency (HME) and total body Hb Fe values were used to estimate Fe bioavailability from the diets.
Results
DMT-1, DcytB and ferroportin expressions were higher (P < 0.05) in the "Low Fe" group than in the "High Fe" group (no added Fe), indicating lower Fe status and adaptation to less Fe-bioavailability. At times, Hb concentrations (d 21,28,35), HME (d 21), Hb-Fe (as from d 14) and liver ferritin were higher in the "High Fe" than in the "Low Fe" groups (P < 0.05), indicating greater Fe absorption from the diet and improved Fe status.
Conclusions
We conclude that the High Fe-bioavailability maize contains more bioavailable Fe than the Low Fe-bioavailability maize, presumably due to a more favorable matrix for absorption. Maize shows promise for Fe biofortification; therefore, human trials should be conducted to determine the efficacy of consuming the high bioavailable Fe maize to reduce Fe deficiency.
MaizeBiofortificationIron bioavailabilityIn vitro digestion/Caco- 2 cell modelBroiler chickenIntestine
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Introduction
Iron (Fe) deficiency affects one-third of the world's population
[1]. Iron is vital for oxygen transport and energy metabolism
[2]. The consequences of Fe deficiency anemia include impaired growth, retarded psychomotor and cognitive development, damaged immune mechanisms with increased morbidity and mortality rates
[1,3].
Efforts to decrease dietary Fe deficiency utilize fortification, supplementation and diversification of diets. These strategies had limited success in resource-limited environments and poor countries due to cost, limited health care, and availability of food processing facilities
[4-7]. Hence, genetic improvement (biofortification) of staple crops is an attractive alternative to dietary fortification or diversification, as delivery of the Fe-rich staple is achieved through the development and promotion of new plant varieties that are aimed to alleviate dietary Fe deficiency and anemia
[7].
Maize (Zea mays L.) is widely consumed in developing countries and provides energy, vitamins and minerals
[8-15]. However, a major cause of Fe deficiency is poor intake of Fe, due to low bioavailability from plant-based diets containing mineral absorption inhibitors as polyphenols and phytates. In the most maize-dependent countries, where maize provides ≥ 20% of dietary protein, Fe deficiency and anemia are prevalent
[1,16-18]. Hence, maize is an attractive candidate for Fe biofortification.
Increased Fe concentration in staple food crops may not necessarily translate into a proportional increase in absorbed Fe, because crop varieties with high Fe concentrations may have increased (or decreased) concentrations of Fe absorption inhibitors or enhancers. It is necessary to measure the amount of Fe concentration and bioavailability in new Fe-enhanced crops. The in vitro screening employs a simulated gastric and intestinal digestion of food coupled with culture of human intestinal cells
[19]. This bioassay is necessary to pinpoint genetic markers for Fe bioavailability.
Research into the genetic basis for Fe nutritional quality in maize has established the potential for Fe biofortification, as Fe concentration and bioavailability are under genetic control and have demonstrated potential for improvement
[8,9]. Previously, we utilized quantitative trait locus (QTL) mapping to characterize the genetic complexity of Fe concentration and bioavailability in maize
[9,20,21]. New varieties were developed using members of the mapping population, that were largely identical except in the chromosomal regions surrounding the 3 QTL with largest effect on Fe bioavailability. These derivatives were selected to create a maximal degree of contrast in predicted Fe bioavailability. With High and Low varieties in both parental backgrounds, these High-Fe and Low-Fe bioavailability hybrids are essentially identical for all parts of their genomes (all features of grain quality would be expected to be the same) except the 3 QTL-containing regions on maize chromosomes 3, 6 and 9
[8,9]. Preliminary in vivo study indicated that the predictions made with the Caco-2 bioassay were valid for predicting Fe bioavailability
[8]. The equivalence of the High-Fe and Low-Fe bioavailability varieties for grain Fe concentration, flowering time, and other characteristics except Fe bioavailability suggests that our strategy of creating these hybrids and the focus on the effect of the 3 major QTL was successful
[8,9].
The poultry model have been used for nutritional research and was shown to be an excellent animal to model Fe bioavailability, as chicks respond quickly to malnutrition, and their micronutrient deficient phenotypes include poor Fe status, growth stunting, and organ hypertrophy
[22-24]. Also, this model agrees well with human cell line in vitro results
[22-25]. Hence, the objective of the current study was to compare the capacities of our two new maize hybrid varieties to deliver Fe for hemoglobin synthesis and to improve the Fe status of Fe deficient broiler chickens.
Materials and methods
Creation of high-Fe bioavailability and low-Fe bioavailability maize varieties
QTL-mapping is the process of utilizing genetically mapped varieties coupled with a biological measurement (as Fe bioavailability) and then utilizing statistics to correlate that measurement with genetic markers. QTL-mapping revealed that Fe concentration in maize grain was under the control of at least 10 regulatory factors on 6 of the 10 chromosomes of maize
[9]. However, Fe-bioavailability was regulated by fewer, larger QTL, which suggested that this trait might be easier to manipulate. Furthermore, Fe concentration and bioavailability had only a small positive association between them indicating that Fe concentration differences between members of the mapping population were not driving the differences in Fe bioavailability
[9]. Derivation of the High-Fe and Low-Fe bioavailability maize hybrids was previously described
[8,9]. Briefly, The Caco-2 bioassay was the guiding tool for the measure of Fe bioavailability in the maize grain
[9]. Statistical analysis was used to identify molecular markers (i.e. QTL) associated with Fe bioavailability. These markers were used to select sister lines that contrasted for the 3 largest effect QTL in order to create new varieties that were highly genetically similar but different (high or low) for Fe-bioavailability. As sister lines were created in both of the parental genetic backgrounds used in the mapping population, nearly isogenic hybrids were made by crossing the parents lines (high with high and low with low). These hybrids were heterozygous everywhere except the 3 Fe-bioavailability QTL
[9] and were similar except for bioavailable-Fe in the whole grain
[8] (Figure
1). The High-Fe and Low-Fe maize were produced using standard agronomic practices at the Cornell University Research Farm (Poplar Ridge, NY) in the summer of 2009. Plots were mechanically planted and harvested. Grain was dried to ~12% moisture, processed in bulk (~ 800 Kg of each variety), and stored at 4°C until the feeding study began. In preparation for the in vivo trial, maize grains were thoroughly washed in ddH2O prior to cooking and freeze drying. Maize varieties were ground prior to mixing the diets.
Figure 1 Description of maize (Zea mays L.) varieties used in this study.
Animals, diets and study design
One hundred and twenty fertile Cornish cross broiler eggs were obtained from a commercial hatchery (Moyer’s Chicks, Quakertown, PA). Eggs were incubated under optimal conditions at the Cornell University Animal Science poultry farm incubator. Upon hatching (92% hatchability), chicks were allocated into 4 treatment groups on the basis of body weight, gender and blood hemoglobin concentration (aimed to ensure equal distribution between groups, n=10): 1. "High + Fe": 75% High-Fe bioavailability cooked maize with added Fe based diet (65 μg/g Fe). 2. "High": 75% High-Fe bioavailability cooked maize with no Fe added based diet (24 μg/g Fe). 3. "Low + Fe": 75% Low-Fe bioavailability cooked maize with added Fe based diet (66 μg/g Fe). 4. "Low": 75% Low-Fe bioavailability cooked maize with no Fe added based diet (23 μg/g Fe) (Table
1). Cooked/raw maize were compared as in vitro pilot studies indicated that cooking may increase the difference in Fe bioavailability between the two lines. Chicks were housed in a total-confinement building (1 chick per 0.5 m2 cage). Birds were under indoor controlled temperatures and were provided 16 h of light. Cages were equipped with an automatic nipple drinker and manual self feeder. All birds were given ad libitum access to water (Fe concentration was 0.379±0.012 μg/g). Iron concentrations in the water and diets were determined by an inductively-coupled argon-plasma/atomic emission spectrophotometer (ICAP 61E Thermal Jarrell Ash Trace Analyzer, Jarrell Ash Co. Franklin, MA) following wet ashing. Feed intakes were measured daily (from day 1). Iron intakes were calculated from feed intakes and Fe concentration in the diets.
Table 1 Composition of experimental diets
Ingredient "High+Fe" Diet "High" Diet "Low+Fe" Diet "Low" Diet
g/Kgdiet (by formulation)
High-Fe bioavailability Maize (21 μg Fe/g) 750 750 - -
Low-Fe bioavailability Maize (20 μg Fe/g) - - 750 750
Dry skim milk 100 100 100 100
DL-Methionine 2.5 2.5 2.5 2.5
Corn oil 30 30 30 30
Corn starch 46.50 46.75 46.50 46.75
Choline Chloride 0.75 0.75 0.75 0.75
Vit/Min1 (no Fe) 70 70 70 70
Ferric citrate 0.25 - 0.25 -
Total 1000 1000 1000 1000
Concentrations of selected components means±SEM, n=10 (by analysis)4
Fe, μgFe/g diet2 65.3±0.9a 24.5±1.0b 66.1±2.4a 23.6±0.2b
Phytate, μmol/g diet3 10.2 ± 0.2a 10.1 ±0.2a 10.1 ± 0.2a 10.0 ±0.2a
1Vitamin and mineral premix provided/kg diet (330002 Chick vitamin mixture; 230000 Salt mix for chick diet; Dyets Inc. Bethlehem, PA).
2Dietary iron concentrations analysis is described in the materials and methods section.
3Method for determining phytate contents are described in the materials and methods section.
4Values are means±SEM. a,bWithin a row, means without a common letter are significantly different, P < 0.05.
Blood analysis and hemoglobin (Hb) measurements
Blood samples were collected from the wing vein (n=10,~100 μL) using micro-hematocrit heparinized capillary tubesa (Fisher, Pittsburgh, PA). Samples were collected following an 8 h overnight feed deprivation. Samples were analyzed for Hb concentration (see below). Body weights (BW) and Hb concentrations were measured weekly.
Fe-bioavailability was calculated as hemoglobin maintenance efficiency (HME)
[23-29]:
(1) HME=HbFe,mgfinal−HbFe,mginitialTotalFeIntake,mg×100
Where Hb-Fe (index of Fe absorption) = total body hemoglobin Fe. Hb-Fe was calculated from hemoglobin concentrations and estimates of blood volume based on BW (a blood volume of 85 mL per kg body weight is assumed)
[23-25,28]:
(2) Hb−Femg=BWkg×0.085Lblood/kg×Hbg/L×3.35mgFe/gHb.
Fe intakes were calculated from feed intake data and Fe concentrations in the feed.
Blood Hb concentrations were determined spectrophotometrically using the cyanmethemoglobin method (H7506-STD, Pointe Scientific Inc. Canton, MI) following the kit manufacturer’s instructions.
At the end of the experiment (day 42), birds were euthanized by carbon-dioxide exposure. The digestive tracts and livers were quickly removed and separated. Tissue samples were taken from the small intestine and liver (~ 1–2 cm; ~2-3 g, respectively). The samples were immediately frozen in liquid nitrogen, and then stored in a -80°C freezer until analysis.
All animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee.
Isolation of total RNA
Total RNA was extracted from 30 mg of the proximal duodenal tissue (n=10) using Qiagen RNeasy Mini Kit (RNeasy Mini Kit, Qiagen Inc.,Valencia, CA) according to the manufacturer’s protocol. Total RNA was eluted in 50 μL of RNase free water. All steps were carried out under RNase free conditions. RNA was quantified by absorbance at A260/280. Integrity of the 28S and 18S ribosomal RNAs was verified by 1.5% agarose gel electrophoresis followed by ethidium-bromide staining. DNA contamination was removed using TURBO DNase treatment and removal kit from AMBION (Austin, TX, USA).
DMT1, DcytB and ferroprtin gene expression analysis
As previously described
[23-25,27,30], Divalent metal transporter-1 (DMT1); Duodenal cytochrome-B (DcytB) and Ferroprtin mRNA levels in duodenal mucosa were analyzed by quantitative real-time RT-PCR (20 μL reactions); values were normalized to 18S expression. The total RNA was reverse-transcribed to complementary DNA in a 25 μL volume containing 1 μg of extracted RNA. Reverse-transcription was carried out using the Superscript-First Strand Synthesis Kit for reverse-transcription PCR according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Gene-specific primers were designed using Primer Express software (Applied Biosystems, Carlsbad, CA) chosen from the fragment of the chicken (Gallus gallus) duodenal DMT1 gene (GeneBank database; GI 206597489) (forward: 5’-AGC CGT TCA CCA CTT ATT TCG-3’; reverse: 5’-GGT CCA AAT AGG CGA TGC TC-3’), DcytB gene (GI 20380692) (forward: 5’-GGC CGT GTT TGA GAA CCA CAA TGT T-3’; reverse: 5’-CGT TTG CAA TCA CGT TTC CAA AGA T-3’) and Ferroportin gene (GI 61098365) (forward: 5’-GAT GCA TTC TGA ACA ACC AAG GA’; reverse: 5’-GGA GAC TGG GTG GAC AAG AAC TC-3’). Ribosomal 18S was used to normalize the results (GI 7262899) (forward: 5’- CGA TGC TCT TAA CTG AGT-3’; reverse: 5’-CAG CTT TGC AAC CAT ACT C-3’). Real-time PCR was performed in a 7500 Real-Time PCR system instrument (Applied Biosystem, Carlsbad, CA). The 20 μL PCR mixture consisted of 10 μL of POWER SYBR Green PCR Master Mix (Applied Biosystem, Carlsbad, CA), 5 μL of water, and 1 μL of each primer that was added to 3 μL of the cDNA diluted 1:25. All reactions were performed in duplicates and under the following conditions: 50°C for 2 min, 95°C for 2 min, 42 cycles of 95°C for 30 s, and 60°C for 1 min. Also, to ensure amplification of a single product, a dissociation curve was determined under the following conditions: 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s. Specificity of the product was also confirmed by running samples on a 1.5% agarose gel, excising for purification using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). Calculations of threshold cycles, amplification efficiencies, and R0 values (the starting fluorescence value that is proportional to the relative starting template concentration) were performed using the data analysis for real-time PCR Excel workbook and as previously described
[31].
Ferritin and Fe in the liver
We followed previously described procedures
[23,24,32,33]. Briefly, 1 g of sample was diluted into 1 mL of 50 mM Hepes buffer, pH 7.4, and homogenized on ice for 2 min (5000 g). One mL of each homogenate was subjected to heat treatment for 10 min at 75°C to aid isolation of ferritin (other proteins are not stable at that temperature). Subsequently, samples were immediately cooled down on ice for 30 min. Thereafter, samples were centrifuged for 30 min (13000 g) at 4°C until a clear supernatant was obtained and the pellet containing most of the insoluble denaturated proteins was discarded. Iron concentrations in the liver samples were determined by an inductively-coupled argon-plasma/atomic emission spectrophotometer (ICAP 61E Thermal Jarrell Ash Trace Analyzer, Jarrell Ash Co. Franklin, MA) following wet ashing.
Electrophoresis, staining and measurement of gels
Native polyacrylamide gel electrophoresis was conducted using a 6% separating gel and a 5% stacking gel. Samples were run at a constant voltage of 100 V. Thereafter, gels were treated with either of the two stains: Coomasie blue G-250 stain, specific for proteins, or potassium ferricyanide (K3Fe(CN)6) stain, specific for Fe. The corresponding band found in the protein and Fe stained gel was considered to be ferritin
[23,24,32,33].
Measurements of the bands were conducted using the Quantity-One-1-D analysis program (Bio-Rad, Hercules, CA). The local background was subtracted from each sample. Horse spleen ferritin (Sigma Aldrich Co., St. Louis, MO) was used as a standard for calibrating ferritin protein and Fe concentrations of the samples. Dilutions of the horse spleen ferritin were made and treated similarly to the liver supernatant samples in order to create a reference line for both protein and Fe-stained gels
[23,24,32,33].
In-vitro iron bioavailability assessment
An in vitro digestion/Caco-2 cell culture model
[19,23-28,34,35] was used to assess Fe-bioavailability. The maize only samples (High- Fe bioavailability maize; Low-Fe bioavailability maize and control-commercial maize) and the diets (High diet; Low diet; High + Fe diet; Low + Fe diet) were subjected to simulated gastric and intestinal digestion. Briefly, the intestinal digestion is carried out in cylindrical inserts closed on the bottom by a semipermeable membrane and placed in wells containing Caco-2 cell monolayers bathed in culture medium. The upper chamber was formed by fitting the bottom of Transwell insert ring (Corning) with a 15000 Da molecular weight cut off (MWCO) membrane (Spectra/Por 2.1, Spectrum Medical, Gardena, CA). The dialysis membrane was held in place using a silicone ring (Web Seal, Rochester, NY).
Iron uptake by the Caco-2 cell monolayers was assessed by measuring ferritin concentrations in the cells. Six replicates of each Fe bioavailability measurement were performed. In terms of materials for the study, Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 17 and used in experiments at passage 29. Cells were seeded at densities of 50,000 cells/cm2 in collagen-treated 6 well plates (Costar Corp., Cambridge, MA). The integrity of the monolayer was verified by optical microscopy. The cells were cultured at 37°C in an incubator with 5% CO2 and 95% air atmosphere at constant humidity, and the medium was changed every 48 h.
The cells were maintained in Dulbecco’s modified Eagle medium plus 1% antibiotic/antimycotic solution, 25 mmol/L HEPES, and 10% fetal bovine serum. 48 h prior the experiment, the growth medium was removed from culture wells, the cell layer was washed, and the growth medium was replaced with minimum essential media (MEM) at pH 7.0. The MEM was supplemented with 10 mmol/L PIPES, 1% antibiotic/antimycotic solution, 4 mg/L hydrocortisone, 5 mg/L insulin, 5 μg/L selenium, 34 μg/L triiodothyronine, and 20 μg/L epidermal growth factor. This enriched MEM contained less than 80 μg Fe/L.
All ingredients and supplements for cell culture media were obtained from GIBCO (Rockville, MD). The cells were used in the Fe uptake experiment at 13 days post seeding. In these conditions, the amount of cell protein measured in each well was highly consistent between wells. On experiment day, 1.5 mL of the digested sample was added to the insert’s upper chamber and incubated for 2 h. Then, inserts were removed and 1 mL of MEM was added. Cell cultures were incubated for 22 h at 37°C.
It was previously shown that intracellular ascorbic acid status might influence ferritin formation (i.e. cellular Fe uptake), and Fe related transporters and enzyme expression in Caco-2 cells
[23,24,34]. In the current study, samples were not added with ascorbic acid when Fe bioavailability was tested in vitro.
Harvesting of caco-2 cells for ferritin analysis
The ferritin and total protein contents analyses protocols were previously described
[19,23,24,35]. Briefly, growth medium was removed from the culture well by aspiration and the cells were washed twice with a solution containing 140 mmol/L NaCl, 5 mmol/L KCl, and 10 mmol/L PIPES at pH 7.0. The cells were harvested by adding an aliquot of deionized water and placing them in a sonicator (Lab-Line instruments, Melrose Park, IL).
The ferritin and total protein concentrations were determined on an aliquot of the harvested cell suspension with a one-stage sandwich immunoradiometric assay (FER-IRON II Ferritin assay, Ramco laboratories, Houston, TX) and a colorimetric assay (Bio-Rad DC Protein assay, Bio-Rad, Hercules, CA), respectively. Caco-2 cells synthesize ferritin in response to increases in intracellular Fe concentration. Therefore, we used the ratio of ferritin/total protein (expressed as ng ferritin/mg protein) as an index of the cellular Fe-uptake.
Phytate content in diets
A Dionex liquid (Dionex Corp. Sunnyvale, CA) chromatograph system (AS50 autosampler), equipped with conductivity detector model ED50, and gradient pump GS50 were used along with an IonPac AG11 guard column and IonPac AS11 column (4×250 mm) to quantify phytate. PeakNet 6.40 software was used to process chromatographic data. The mobile phases were (A) 200 mmol/L NaOH (carbonate-free) and (B) deionized water, using a flow rate of 1 mL/min. Phytate was extracted from 250 mg of dry, lyophilized diet sample, in 10mL of a 1.25% H2SO4 solution; the extraction process was 2 h, after which the samples were centrifuged at 3660 g for 10 min. Subsamples were diluted 1:10 with deionized water, and 10 μL was injected and analyzed (n=10).
Statistical analyses
Results were analyzed by ANOVA using the general linear models procedure of SAS software (SAS Institute Inc. Cary, NC). Differences between treatments were compared by Tukey’s test were considered statisticant at P < 0.050. Values in the text are means ± SEM.
Results
Hemoglobin (Hb), Hb Fe and Hb maintenance efficiency (HME)
No significant differences were measured in body weights between treatment groups (P > 0.05). However, as from day 21 of the study, hemoglobin (Hb) concentrations were higher (P < 0.05) in the "High" group than in the "Low" group. In addition, as from day 14, Hb-Fe values were higher in the "High" group than in the "Low" group; the increase in total body Hb-Fe from the beginning of the study to the end of the 6th wk was significantly greater in the "High" group than in the "Low" group (12.8 ± 0.5 mg vs. 9.7 ± 0.3 mg, respectively, P < 0.05, Table
2). Significant differences in HME (P < 0.05) were measured between the "High" group and "Low" group on day 21 (P < 0.05). Also, significant differences in HME (P < 0.05) were measured between the "High + Fe" and "Low + Fe" groups on days 28 and 42 (P < 0.05, Table
2).
Table 2 Hemoglobin (Hb, g/L), Total body Hb-Fe content (mg) and hemoglobin maintenance efficiency1(HME, %) in chicken fed the tested diets from d 0 to d 422
Treatment3 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42
"High + Fe" Hb 92a±9.0 88a±5.0 104a ±11 102a ±6.0 102a ±5.0 97a ±13 97a ±7.0
Hb Fe 1.02a ±0.1 2.52a±0.2 4.35a ±0.4 5.80a ±0.4 8.35a ±0.6 9.62a ±0.6 16.49a ±0.7
HME - 22.7b ±2.9 22.5bc ±2.9 19.8bc±2.5 20.1b ±2.6 15.8b ±2.0 20.2b ±2.6
"Low + Fe" Hb 92a±9.0 86a±8.0 100a ±13 94ab ±14 94ab ±6.0 88a ±6.0 87a ±3.0
Hb Fe 1.02a ±0.1 2.30a±0.2 4.28a ±0.3 5.53a ±0.4 7.72a ±0.5 8.84ab±0.6 14.52ab±0.7
HME - 18.5b ±2.4 19.2c±2.5 15.4c ±2.0 14.4c ±1.9 11.3bc±1.5 13.8c ±1.7
"High" Hb 92a±9.0 88a±6.0 76b ±3.0 81b ±3.0 81b ±4.0 82a ±7.0 82ab ±9.0
Hb Fe 1.02a ±0.1 2.37a±0.1 3.04b ±0.2 4.72b ±0.2 6.30b ±0.4 8.07b ±0.5 13.79b ±1.0
HME - 58.4a ±7.6 37.8a ±4.9 40.3a ±5.2 37.7a ±4.9 35.3a ±4.6 44.9a ±5.8
"Low" Hb 92a±9.0 82a±5.0 70b ±3.0 66c ±7.0 66c ±5.0 68b ±4.0 67b ±8.0
Hb Fe 1.02a ±0.1 2.21a±0.1 2.54c ±0.2 3.41c ±0.2 4.62c ±0.3 6.29c ±0.4 10.73c ±0.6
HME - 52.1a ±6.7 29.7ab±3.8 28.0b ±3.6 27.1ab ±3.5 27.7a ±3.6 35.8a ±4.6
a,b,cWithin a column and for each parameter (i.e. Hb, Hb Fe, HME), treatment group means without a common letter differ, P < 0.05.
1Calculations are described in the materials and methods section.
2Values are means±SEM, n=10.
3The experimental diets are described in the materials and methods section.
Ferritin and iron in the liver
Avian ferritins corresponded to a weight of approximately 470 to 500 kDa
[23,24,32,33,36]. Liver Fe and ferritin concentrations were higher in the "High" group than in the "Low" group (n=10, P < 0.05, Table
3).
Table 3 Liver ferritin protein and liver iron1concentration in chicken given the treatment diets
Treatment Liver ferritin1,μg/gwet weight Liver iron2,μg/gtissue
"High + Fe" 650±18a 64.3±3.8a
"Low + Fe" 645±22a 39.6±2.3c
"High" 435±13b 52.2±3.1b
"Low" 355±10c 43.3±2.5c
a,b,cWithin a column and for each parameter (i.e. liver ferritin or liver Fe), treatment group means without a common letter differ, P< 0.05 (values are mean±SEM, n=10).
1Atomic mass for iron is 55.8 g/mol.
2Liver tissue iron concentrations analysis is described in the materials and methods section.
Gene expression of iron transporters (DMT-1, Ferroportin) and iron reductase (DcytB) in the duodenum
Gene expression analysis of duodenal DMT-1, Ferroportin and DcytB, with results reported relative to 18S rRNA, revealed greater mRNA levels for DMT1, DcytB and Ferroportin in the "Low" group compared to the "High" group (mean±SEM) (n=10, P < 0.05, Figure
2).
Figure 2 Duodenum mRNA expression of DMT1, divalent metal transporter 1; DcytB, duodenal cytochrome b reductase; and ferroportin in chickens at the age 6 weeks. Changes in mRNA expression are shown relative to expression of 18S rRNA in arbitrary units (AU). Values are means ± SEM, n = 10, P < 0.05.
Caco-2 cell ferritin protein formation
An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe bioavailability from the tested maize only and maize based diets by measuring ferritin formation in the cells (ie. a measure of cell Fe uptake) following exposure to digests of the samples. The amount of bioavailable iron in vitro was significantly higher (P < 0.05) in the "High" and "High + Fe" diets than in the "Low" and "Low + Fe" diets (mean±SEM) (n=6, P < 0.05, Table
4).
Table 4 Ferritin concentrations in Caco-2 cells exposed to samples of maize only and maize-based diet digests; and Fe concentrations in samples of maize only and maize-based diet digests1
Tested sample Caco-2 Cell Ferritin2, ng/mg of total protein Fe concentration3, μg/g sample
High Fe maize only 22.51±0.9c 20.9 ±0.2c
Low Fe maize only 13.40 ±0.6d 20.0 ±0.9c
"High + Fe" diet 74.36 ±1.6a 65.3 ±0.9a
"Low + Fe" diet 56.89 ±1.1b 66.1 ±2.4a
"High" diet 6.55 ±0.5e 24.5 ±1.0b
"Low" Diet 1.31 ±0.4f 23.6 ±0.2bc
1Values are means ± SEM, n = 6.
a,b,c,d,e,f Within a column (ferritin or Fe concentrations), means without a common letter differ, P < 0.05.
2Caco-2 bioassay procedures and preparation of the digested samples are described in the materials and methods section.
3Dietary iron concentrations analysis is described in the materials and methods section.
Phytate concentration in the diet samples
No significant differences in phytate concentration (IP6) were measured between treatments diets (n=5; P > 0.05, Table
1).
Discussion
Maize is an important component of the human food supply, especially in Eastern and Southern Africa, the Caribbean, and the Andean region of South America
[1]. In these regions where dietary Fe deficiency and anemia are common and are a critical health concern, maize is often a component of every meal
[1,37-40]. Hence, increasing Fe bioavailability in maize has potential to alleviate dietary Fe deficiency.
Biofortification is the process of enriching the nutrient quality of staple food crops via plant breeding
[38,40], as a nutritional agricultural intervention it can provide a sustainable source of micronutrients to at risk populations
[41]. Iron biofortifcation and bioavailability from plant foods is influenced by many factors, especially polyphenols and phytic acid
[42]. Iron biofortification can be done via enhancement of concentration and or bioavailability, and recent studies indicate that both factors have a genetic basis but are also greatly influenced by environment and genotype by environment interactions
[43,44]. Given the generally low Fe bioavailability in staple crops, enhancing the bioavailable fraction of Fe rather than merely increasing the total concentration may represent an improved path for Fe biofortification
[8,9,44,45]. Additionally, the correlation between bioavailable-Fe and total-Fe is not always robust while both traits may have similar genetic complexity
[9].
Crop improvement via conventional breeding can produce vast numbers of varieties
[46]. Only a fraction of these genetically distinct individuals will have the desired gain in quality to justify being released as a new variety. The selection process is a key issue. One option could be the target of selection in order to biofortify maize. Hence, Fe concentration is an obvious choice, as its evaluation is amenable to high-throughput screening methods
[47]. For maize and wheat, Fe concentration is not well correlated with Fe bioavailability, while these traits are correlated in beans
[9,23,24].
The mechanisms that modulate Fe bioavailability are unclear, therefore, estimating Fe bioavailability is important. We employed the Caco-2 bioassay as part of a recursive process to create maize varieties with different levels of bioavailable-Fe
[9,19,23-28,48,49]. The bioassay was used to evaluate 145 members of a maize mapping population, where neither Fe concentration nor phytate levels were well correlated with bioavailable-Fe
[9]. Also, molecular genetic markers were used to evaluate nearly 700 genetically distinct individuals from our breeding program in order to create the 4 varieties that were selected to differ in bioavailable-Fe. Molecular breeding approaches with these 4 inbred varieties were used to create the 2 hybrids evaluated in this study and our preliminary study
[8].
The observation that bioavailable-Fe was being modulated through the course of our breeding strategy needed verification beyond the Caco-2 bioassay. This assay also indicated that Fe bioavailability could be reliably modified across several years in NY and other sites in North America
[9]. The current results demonstrate that Caco-2/QTL approach can be used to enhance maize Fe bioavailability. Also, if adequate mapping populations are available, this approach can be extended to other crops.
In this study the maize lines were grown under standard agronomic conditions on a research farm, similar to other varieties of maize grown that summer. This demonstrates that the High and Low Fe bioavailability varieties can be grown using production scale agriculture. Current study followed a previous study, where similar results were obtained with smaller amounts of maize (~30 kg), where all plants were hand pollinated and harvested
[8]. Thus we have demonstrated that the nutritional differential between the High and Low Fe bioavailability varieties can be created and maintained in consecutive years using different field practices. This benefit was confirmed as birds receiving the High-Fe bioavailability maize diets had improved Fe status as their liver Fe and ferritin concentrations (Table
3), and body Hb-Fe (Table
2) were higher (P < 0.05) than birds receiving the Low-Fe bioavailability maize diets. The low-Fe bioavailability maize-fed birds had elevated expression of DMT1, DcytB and Ferroportin, which indicates adaptation to the low Fe bioavailability (Figure
2).
Iron biofortification of crops can be accomplished via an increase in concentration or an increase in bioavailability. Either way, the net result is that more Fe is delivered for absorption. Increased Fe concentrations in beans
[24,26] and rice
[38,50] have a beneficial effect on the Fe status in vivo; in a human study
[50] Fe-biofortified rice improved Fe stores in Fe-deficient (not anemic) women, even though Fe concentrations in the rice were low (3.2 μg/g and 0.57 μg/g for the high Fe and control rice, respectively). Recently, the effects of high-Fe (71 μg/g) and standard-Fe (49 μg/g) red mottled Andean beans, on Fe status of chickens were investigated. Final body Hb-Fe contents were different between the standard (12.58±1.0 mg) and high Fe (15.04 ± 0.65 mg) bean groups (P < 0.05). DMT-1, DcytB and ferroportin expression were higher and liver ferritin was lower (P < 0.05) in the standard group vs. the biofortified group, indicating a physiological effort to compensate for lower dietary-Fe. In vitro analysis showed lower Fe bioavailability in cells exposed to standard bean based diet. It was concluded that the higher Fe beans provided more bioavailable-Fe than standard beans
[24]. These studies showed that the higher Fe concentration improved Fe status, as no difference in percent bioavailability was apparent. However, in the present study, Fe concentration was similar yet the amount that was bioavailable from the High-Fe bioavailability maize was higher.
Many cereal grains as maize are rich with phytate that may decrease mineral bioavailability
[8,9,51-53]. Our study suggests that it is possible to counteract the Fe absorption inhibitory effect of phytate and possibly other inhibitors by increasing Fe bioavailability (not necessarily concentration). This knowledge is vital for developing plant breeding strategies as part of the continuing battle with dietary Fe deficiency.
Iron deficiency is a worldwide, endemic public health problem. Food system-based interventions such as biofortification are a practical and sustainable solution for at risk populations
[7]. An efficacy trial comparing biofortified and standard maize in human populations is now warranted.
Conclusions
Based on the data shown here, we conclude that the enhanced bioavailable-Fe maize we have generated via a molecular plant breeding strategy is a promising vehicle for alleviating Fe deficiency in human populations where maize is a major dietary staple.
The results presented in this study show that breeding can improve the Fe quality in maize. These findings demonstrate the potential for Fe biofortification in maize.
Endnote
aMention of a trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the United states Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
Abbreviations
Fe: Iron; Hb: Hemoglobin; Hb-Fe: Hemoglobin-Iron; HME: Hemoglobin maintenance efficiency; DMT-1: Divalent metal transporter 1; DcytB: Duodenal cytochrome B; QTL: Quantitative trait locus.
Competing interests
The authors declare no conflict of interest.
Authors’ contributions
ET designed research, conducted research, collected and analyzed data and wrote the paper. OAH designed research, created and provided the High Fe and Low Fe maize varieties, and co-authored the paper. LVK co-authored the paper. RPG designed research, and co-authored the paper. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to recognize the technical contributions of Mary Bodis, Yongpei Chang, Zhiqiang Cheng, Meghan den Bakker, Jon Hart, Larry Heller, Paul Stachowski, Michael Rutzke, and Alan Westra.
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Environ Health PerspectEnviron. Health PerspectEHPEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Sciences 23010656ehp.120518810.1289/ehp.1205188ArticleRetracted: Hyaluronan Activation of the Nlrp3 Inflammasome Contributes to the Development of Airway Hyperresponsiveness Feng Feifei 12Li Zhuowei 1Potts-Kant Erin N. 1Wu Yiming 2Foster W. Michael 1Williams Kristi L. 13*Hollingsworth John W. 14*1 Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, School of Medicine, Duke University Medical Center, Durham, North Carolina, USA2 Department of Occupational and Environmental Health, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China3 School of Nursing, Duke University Medical Center, Durham, North Carolina, USA4 Department of Immunology, School of Medicine, Duke University Medical Center, Durham, North Carolina, USAAddress correspondence to J.W. Hollingsworth, Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Duke University School of Medicine, DUMC 103004, 106 Research Dr., Durham, NC 27710 USA. Telephone: (919) 684-4588. Fax: (919) 684-5266. E-mail: [email protected]* These authors contributed equally to the content of this manuscript.
24 9 2012 12 2012 120 12 1692 1698 08 3 2012 24 9 2012 2012This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use,
properly cited.Background: The role of the Nlrp3 inflammasome in nonallergic airway hyperresponsiveness (AHR) has not previously been reported. Recent evidence supports both interleukin (IL) 1β and short fragments of hyaluronan (HA) as contributors to the biological response to inhaled ozone.
Objective: Because extracellular secretion of IL-1β requires activation of the inflammasome, we investigated the role of the inflammasome proteins ASC, caspase1, and Nlrp3 in the biological response to ozone and HA.
Methods: C57BL/6J wild-type mice and mice deficient in ASC, caspase1, or Nlrp3 were exposed to ozone (1 ppm for 3 hr) or HA followed by analysis of airway resistance, cellular inflammation, and total protein and cytokines in bronchoalveolar lavage fluid (BALF). Transcription levels of IL-1β and IL-18 were determined in two populations of lung macrophages. In addition, we examined levels of cleaved caspase1 and cleaved IL-1β as markers of inflammasome activation in isolated alveolar macrophages harvested from BALF from HA-treated mice.
Results: We observed that genes of the Nlrp3 inflammasome were required for development of AHR following exposure to either ozone or HA fragments. These genes are partially required for the cellular inflammatory response to ozone. The expression of IL-1β mRNA in alveolar macrophages was up-regulated after either ozone or HA challenge and was not dependent on the Nlrp3 inflammasome. However, soluble levels of IL-1β protein were dependent on the inflammasome after challenge with either ozone or HA. HA challenge resulted in cleavage of macrophage-derived caspase1 and IL-1β, suggesting a role for alveolar macrophages in Nlrp3-dependent AHR.
Conclusions: The Nlrp3 inflammasome is required for the development of ozone-induced reactive airways disease.
asthmaenvironmentextracellular matrixinnate immunityozonetoll-like receptor
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Ambient ozone is a highly reactive oxidant associated with increased morbidity and mortality in human populations (Bell et al. 2004, 2006; Ito et al. 2005). Despite effective regulatory oversight to reduce ambient levels of ozone (Samet 2011), current evidence suggests that adverse health effects may occur at exposures below current regulatory standards (Berman et al. 2012; Kim et al. 2011). Furthermore, changes in global climate are anticipated to increase both ambient levels of ozone and associated adverse health effects (Chang et al. 2010). Inhalation of ozone can immediately cause lung injury, inflammation, and decrements in lung function (Foster et al. 2000; Kehrl et al. 1987; Koren et al. 1989). The delayed responses to ozone include induction of asthma-like phenotypes with compromises in epithelial barrier function and development of airway hyperresponsiveness (AHR) (Que et al. 2011). For this reason, we believe that fundamental insight into the mechanisms that regulate the biological response to ozone could have broad implications to our understanding of the pathogenesis of airways diseases, including asthma and chronic obstructive pulmonary disease (COPD).
The biological response to ozone appears to be dependent on activation of the innate immune system (reviewed by Al-Hegelan et al. 2011; Peden 2011). Components of the response to ozone requires activation of the surface receptor toll-like receptor 4 (TLR4) (Hollingsworth et al. 2004; Kleeberger et al. 2001; Williams et al. 2007). More recent data have demonstrated that ozone-induced production of short fragments of hyaluronan (HA) (Garantziotis et al. 2009) activates the TLR4–CD44 surface receptor complex and contributes to AHR (Garantziotis et al. 2010; Taylor et al. 2007). Interestingly, short fragments of HA are also detected in bronchoalveolar lavage (BAL) fluid (BALF) from patients with COPD (Dentener et al. 2005) and allergic asthma (Liang et al. 2011; Sahu and Lynn 1978). In our previous studies (Garantziotis et al. 2010; Li et al. 2011), the response to either ozone or HA fragments were also closely associated with the levels of interleukin (IL)-1β in the BALF. Previous work supports a central role for IL-1–dependent signaling in the biological response to ozone (Johnston et al. 2007; Park et al. 2004; Verhein et al. 2008; Wu et al. 2008). Although the specific mechanism by which IL-1β contributes to AHR remains unknown, IL-1β is recognized to increase both cyclooxygenase (COX)-2 expression and prostaglandin E2, which results in decreased β-adrenergic responsiveness of smooth muscle cells, providing a link to AHR (Moore et al. 2001). However, the mechanisms leading to IL-1β activation in the context of either ozone exposure or reactive airways disease remains poorly understood.
The inflammasome protein complex is an important regulator of IL-1β and IL-18 maturation and release into the extracellular space (reviewed by Hoffman and Brydges 2011; Jin and Flavell 2010). The inflammasome complex consists of the protein pro-caspase1 and may include the adaptor molecule ASC, as well as specific members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) family. Inflammasome activation leads to caspase1-mediated cleavage and activation of the proinflammatory cytokines pro-IL-1β and pro-IL-18. Yamasaki et al. (2009) recently demonstrated that HA-induced IL-1β release is dependent on the Nlrp3 inflammasome in a model of sterile skin injury. Recent work in macrophages showed that oxidant stress can prime the Nlrp3 inflammasome (Bauernfeind et al. 2011). On the basis of these observations and the recognized role of HA in the response to ozone, we hypothesized that HA activation of the Nlrp3 inflammasome would contribute to the airway response to ozone.
Materials and Methods
Mice. Wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice deficient in caspase1 (caspase1–/–) were generously provided by F. Sutterwala and R. Flavell (Sutterwala et al. 2006). ASC-deficient (ASC–/–) and Nlrp3-deficient (Nlrp3–/–) mice were generously provided by Genentech Inc. (San Francisco, CA, USA). Male mice, 6–8 weeks of age, were used in experiments. Animals were treated humanely with due consideration to alleviation of distress and discomfort. Experimental protocols were approved by the Institutional Animal Care Committee at Duke University Medical Center and performed in accordance with established guidelines (National Research Council 1996).
Ozone exposure. Mice were exposed to either filtered air or ozone (1 ppm × 3 hr) in a Hinner-style chamber as previously described (Hollingsworth et al. 2004). The ozone concentrations used are based on similar biological responses observed in human exposure studies and in published deposition fraction data for ozone in rodent models (Hatch et al. 1994; Wiester et al. 1988). The ozone concentration in the chamber was monitored continuously by an ultraviolet light photometer (model 400E; Teledyne Technologies Inc., Thousand Oaks, CA, USA). WT mice were phenotyped 3, 6, 12, 24, 48, and 72 hr after ozone exposure. Twenty-four hours after exposure, we observed the phenotype of C57BL/6J and inflammasome-deficient mice based on the level of cellular inflammation, HA in BALF, and the transcription level of IL-1β. In blocking experiments, C57BL/6J mice were anesthetized with isoflurane; then 0.22 mg hyaluronic acid-binding peptide (HABP) or scrambled-binding peptide (GenScript, Piscataway, NJ, USA) were oropharyngeally instilled into the lung immediately before ozone exposure.
HA challenge. Sterile, endotoxin-free high-molecular-weight HA (Healon GV; Abbott Medical Optics, Santa Ana, CA, USA) was reconstituted to 0.5 mg/mL in 0.02 M acetate, 0.15 M sodium chloride, pH 6.0. For the production of low-molecular-weight HA, high-molecular-weight HA was sonicated 3 sec three times on ice. Sizes of HA fragments were confirmed by agarose gel electrophoresis. For in vivo experiments, 50 µL HA (25 µg/mouse) or vehicle were instilled intratracheally into isoflurane-anesthetized mice. This HA dose was sufficient to significantly induce AHR (Garantziotis et al. 2009). AHR was measured invasively 2-hr later. The selection of HA treatment time point was based on HA time course experiments.
Airway physiology. We performed direct measurements of airway response to methacholine as previously reported (Garantziotis et al. 2009). Briefly, mice were ventilated with a computer-controlled small animal ventilator (FlexiVent; SCIREQ, Montreal, Quebec, Canada), and measurements of respiratory mechanics were made by the forced oscillation technique. Mice were challenged with aerosolized methacholine at 0, 10, 25, or 100 mg/mL, and airway response was determined by resistance measurements every 30 sec for 5 min. Total lung resistance (RT) measurements (given in centimeters of water per milliliter of gas per second; RT cm H2O/mL/sec) were averaged at each dose and graphed along with the initial baseline measurement.
BAL. BAL was performed as described previously (Garantziotis et al. 2009). Briefly, immediately after pulmonary function measurements, mice were euthanized with CO2, and the lungs were exposed and inflated with 0.9% NaCl three times to a pressure of 25 cm H2O. Cell counting was performed using a hemocytometer, and differentials were performed using hematoxylin and eosin–stained cytospins. Cytokines/chemokines [IL-1α, IL-1β, IL-6, KC (cytokine-induced neutrophil chemoattractant), MCP-1 (monocyte chemoattractant protein-1), IL-17, and TNF-α (tumor necrosis factor α)] were determined by Luminex (Bio-Rad, Hercules, CA, USA) using reagents from Millipore (Billerica, MA, USA). IL-18 and C3a were detected by ELISA (Bender MedSystems, Vienna, Austria; Kamiya Biomedical Company, Seattle, WA, USA). Total protein concentration in BALF was detected by Lowry Assay (Bio-Rad), and HA level in BALF was measured by ELISA (Echelon, Salt Lake City, UT, USA).
Alveolar macrophage isolation. Alveolar macrophages were harvested by BAL with 0.9% NaCl/0.5 mM EDTA buffer. Briefly, a total of 10 fills of lavage fluid was collected. After centrifugation at 1,500 rpm for 10 min, the cell pellet obtained was used for RNA analysis. We achieved 98% macrophages with this technique, as detected by cytospin.
Whole-lung macrophage isolation. After taking airway physiology measurements and collecting BAL, the lungs were removed from the mouse and placed into a tissue culture dish. They were minced and digested in Hank’s balanced salt solution with 1 mg/mL collagenase A and 0.2 mg/mL of DNase1 (both from Sigma Chemical Co., St. Louis, MO, USA) for 1 hr at 37°C. The digestion solution was passed through a 70-μm mesh strainer and centrifuged at 1,600 rpm for 20 min at room temperature over an 18% nycodenz (Accurate Chemical Co.) cushion. After red blood cell lysis, low density cells were collected and then washed twice. We achieved 87% macrophages by cytospin using this technique. The cell pellet was retained for RNA analysis.
Real-time polymerase chain reaction (PCR). Total RNA was isolated using the RNAeasy kit (Invitrogen, Carlsbad, CA, USA). Samples were treated with DNase treatment and removal reagents to remove DNA contamination (Ambion Inc., Austin, TX, USA). RNA samples were then reverse-transcribed into cDNA using SuperScript II RT (Invitrogen) following the manufacturer’s protocol. Real-time PCR was performed using the SYBR green detection system (Applied Biosystems, Foster City, CA, USA). Primer sequences used for IL-1 and IL-18 amplification are as follows: IL-1β forward: 5´-CTGTGTCTT TCCCGTGGACC-3´, reverse 5´-CAGCTCATATGGGTCCGACA-3´; IL-18 forward: 5´-CACATGCGCCTTGTGATGAC-3´, reverse 5´-TGCAGCCTCGGGTATTCTG-3´; 18S forward: 5´-GCTGCTGGCACCAGACTT-3´, reverse 5´-CGGCTACCACATCCAAGG-3´. After ΔΔCt calculation, all results were normalized to 18S ribosomal RNA internal controls and expressed as fold increase of RNA expression compared with the vehicle control.
Immunoblot analysis. BALF from five mice per group was pooled into one sample and centrifuged to form an alveolar macrophage cell pellet, which was then centrifuged and dissolved in RIPA lysis buffer containing proteinase inhibitors. The protein concentration was determined using the DC Protein Assay (Bio-Rad), and 20 µg protein per sample was mixed with sample loading buffer and boiled for 5 min. Samples were separated by 4–20% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Bio-Rad). Immunoblots were blocked with 5% milk in Tris-buffered saline (TBS)/0.1% Tween 20 for 1 hr at room temperature and incubated overnight at 4°C with rabbit polyclonal anti-mouse caspase1 p10 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat polyclonal anti-mouse IL-1β (1:2,000; R&D Systems, Minneapolis, MN, USA), or goat polyclonal anti-mouse GAPDH (1:1,000; Santa Cruz Biotechnology) in 3% milk/TBS/0.1% Tween 20. Membranes were incubated with the secondary antibody— goat anti-rabbit IgG (1:2,000) or donkey anti-goat IgG (1:2,000)—in 3% milk/TBS/0.1% Tween 20 at room temperature for 1 hr. Bands were detected using the ECL Plus Western Blotting Detection Kit (GE Healthcare, Piscataway, NJ, USA).
Statistics. Data are representative of at least two replicate experiments and are expressed as mean ± SE. We used GraphPad software (San Diego, CA, USA) for statistical analyses. We used analysis of variance to determine significant differences among multiple groups with one variant, and every two groups were then compared by the Neuman–Keuls method. The Student t-test was used for comparisons between two groups. A two-tailed p-value < 0.05 was considered statistically significant.
Results
Significant biological response in the lung was observed 24 hr after ozone inhalation. We examined the airway response to ozone (1 ppm for 3 hr) in WT mice over a 72-hr time course. We observed a significant increase in the total number of inflammatory cells 24–72 hr after ozone exposure (Figure 1A); BALF differential cell counts revealed an increase in the numbers of macrophages and neutrophils (Figure 1B,C). Cellular inflammation peaked 24 hr after ozone exposure and maintained a high level until 72 hr. The level of BALF protein peaked at 12–48 hr and declined to near baseline at 72 hr (Figure 1D). We detected BALF HA levels to examine extracellular matrix injury during ozone-induced acute airway inflammation. The level of HA in BALF increased at 24 hr, peaked at 48 hr, and declined by 72 hr after ozone exposure (Figure 1E). We examined the transcription level of IL-1β and IL-18 and found that IL-1β mRNA in alveolar macrophages was up-regulated at 6 hr, and this level remained at a similar level throughout the 72-hr time course (Figure 1F). Expression of IL-18 mRNA in alveolar macrophages was increased at 24 hr and peaked at 72 hr (Figure 1G). On the basis of previous epidemiological data and this time course, we selected the 24-hr time point to conduct subsequent studies.
Figure 1 Biological response measured by cellular inflammation (A–C), total protein (D), HA (E), and cytokines (F,G) in BALF from WT mice exposed to ozone (1 ppm for 3 hr) and followed over a 72‑hr time course. (A) Total number of inflammatory cells. (B) Number of macrophages. (C) Number of neutrophils. (D) Total protein. (E) HA production. Significant increases were observed at 24, 48, and 72 hr after exposure to ozone compared with naive (unexposed mice). No increases in the number of total cells and macrophages or the level of HA were detected at 3, 6, or 12 hr after exposure; however, the number of neutrophils began to increase at 12 hr and reached its highest level at 72 hr. (F, G) Expression of IL‑1β mRNA (F) and IL‑18 mRNA (G) in alveolar macrophages. (F)IL‑1β mRNA was up‑regulated at 6 hr and remained at that level throughout the 72‑hr time course. (G) IL‑18 mRNA increased beginning at 24 hr and peaked at 72 hr. Data are presented as mean ± SE (n = 5–8) and are representative of two similar experiments. *p < 0.05 vs. naive. **p< 0.05 vs. 12 hr. †p < 0.05 vs. 24 hr. ††p< 0.05 vs. 72 hr.
The Nlrp3 inflammasome regulates the inflammatory response and AHR after ozone exposure. We exposed WT, ASC–/–, caspase1–/–, and Nlrp3–/– mice to either filtered air or ozone (1 ppm for 3 hr). In a methacholine challenge 24 hr after exposure, we observed that ozone-exposed WT mice developed enhanced sensitivity to methacholine compared with air-exposed controls. ASC–/–, caspase1–/–, and Nlrp3–/– mice were protected from enhanced AHR after ozone exposure, with values for RT similar to those of air-exposed WT animals (Figure 2A). We characterized the level of inflammatory cell influx into the airspace after ozone exposure in each mouse strain (Figure 2B). As we anticipated, the number of total cells, macrophages, and neutrophils in BALF from ozone-exposed WT mice were higher than those from air-exposed animals. However, we observed significantly reduced numbers of macrophages and neutrophils in ASC–/–, caspase1–/–, and Nlrp3–/– mice compared with ozone-exposed WT animals.
Figure 2 The role of Nlrp3 inflammasome in response to ozone. WT mice and inflammasome-deficient mice exposed to ozone (1 ppm × 3 hr) and phenotyped for airway responsiveness after methacholine challenge (A–C), cell infiltration (B), and BAL protein level (C) measured at 24 hr after exposure. (A–C) caspase1 (A), ASC (B), and Nlrp3 (C) are necessary for the development of ozone-induced AHR. (B) Total cells, macrophages, and neutrophils in BALF from ozone-exposed WT mice were higher than those from caspase1-, ASC-, and Nlrp3-deficient mice. (C) The level of BAL protein was higher after ozone exposure and was completely dependent on caspase1 and ASC, and partially dependent on Nlrp3. Data are presented as mean ± SE (n = 5) and are representative of two similar experiments. *p < 0.05.
When we measured the level of total protein in BALF as a marker of lung injury after ozone inhalation, we found that inflammasome-deficient animals had significantly lower levels of total protein in the BALF than ozone-exposed WT mice (Figure 2C). To measure cytokine expression directly regulated by the Nlrp3 inflammasome in response to ozone inhalation, we measured transcription levels of IL-1β and IL-18 in alveolar macrophages and soluble levels of IL-1β and IL-18 in BALF at 24 hr after exposure. We observed up-regulation of IL-1β and IL-18 mRNA in alveolar macrophages in ozone-exposed mice compared with air-exposed controls; this respose was not dependent on the Nlrp3 inflammasome (Figure 3A,B). In addition, we observed no differences in mRNA expression of IL-1β and IL-18 in whole lung macrophages after ozone exposure [see Supplemental Material, Figure S1A,B (http://dx.doi.org/10.1289/ehp.1205188)]. The levels of secreted IL-1β and IL-18 protein in BALF from ozone-exposed WT mice were significantly higher than those in BALF from ozone-exposed ASC–/–, caspase1–/–, and Nlrp3–/– mice (Figure 3C,D). We also observed a broad impact of inflammasome activation on additional proinflammatory cytokines and mediators previously associated with the biological response to ozone (see Supplemental Material, Figure S2A–G). Ozone-exposed animals deficient in ASC and caspase1, and to a lesser extent in Nlrp3, had reduced levels of IL-1α, IL-6, TNF-α, and KC in BALF compared with WT animals. MCP-1 was completely dependent on Nlrp3; IL-17 was completely dependent on caspase1, Nlrp3, and partially dependent on ASC; and C3a was partially dependent on Nlrp3 inflammasome.
Figure 3 IL‑1β, IL‑18, and HA in BALF from WT mice and inflammasome-deficient mice 24 hr after exposure to ozone (1 ppm × 3 hr). (A–B) The transcription levels of IL‑1β (A) and IL‑18 (B) in alveolar macrophages were up‑regulated in ozone-exposed mice compared with air-exposed mice, and were not dependent on the Nlrp3 inflammasome. (C–D) Increases in IL‑1β (C) and IL‑18 (D) proteins were completely dependent on ASC and caspase-1, IL‑1β was partially dependent on Nlrp3, and IL‑18 was completely dependent on Nlrp3. (E) Ozone exposure enhanced the level of soluble HA in BAL; the level of HA was not dependent on Nlrp3 inflammasomes. (F) The level of secreted IL‑1β in BAL was increased after ozone exposure, but was partially decreased by HABP compared with air, saline, and scrambled-binding peptide (SBP) controls. Data are presented as mean ± SE (n= 5) and are representative of two similar experiments. *p < 0.05.
HA contributes to the increased level of secreted IL-1β in BALF after ozone exposure. Similar to observations in WT animals, the levels of soluble HA in BALF from ASC-, caspase1-, and Nlrp3-deficient animals were increased 24 hr after inhalation of ozone, but values were not significantly different compared with ozone-exposed WT animals (Figure 3E). To further identify the role of HA in ozone-induced inflammasome activation, we pretreated WT animals with HABP, which reduces the level of HA in the lungs (Garantziotis et al. 2009), immediately before ozone exposure and then measured the level of secreted IL-1β in BALF. We found that HABP pretreatment partially blocked the production of BALF IL-1β (Figure 3F); this supports the contribution of HA to ozone-induced release of soluble IL-1β.
Significant biological response in the lung is observed after direct HA challenge. Because HA was previously reported to be functionally relevant (Garantziotis et al. 2009), we instilled HA (25 µg/mouse) or vehicle intratracheally into isoflurane-anesthetized WT mice. We then characterized the biological responses at 1, 2, and 6 hr after HA challenge. We found no increase in numbers of total cells, macrophages, or neutrophils in BALF until 6 hr after the challenge (Figure 4A–C). No increase in BALF protein was detected after HA challenge at any of the time points (Figure 4D). However, IL-1β mRNA in alveolar macrophages was up-regulated at both 1 hr and 2 hr after HA challenge compared with vehicle controls, but the level decreased to near baseline at 6 hr (Figure 4E). We observed no significant difference in the transcription level of IL-18 after HA challenge compared with vehicle at any of the three time points (Figure 4F). Based on the data of IL-1β mRNA and the physiological response (Garantziotis et al. 2009, 2010; Li et al. 2011), we selected 2 hr after HA challenge for subsequent experiments.
Figure 4 Time course of biological responses to HA (50 µL; 25 µg/mouse) or vehicle instilled intratracheally into isoflurane-anesthetized WT mice; responses were observed BALF at 1, 2, and 6 hr after HA treatment. (A–C) No increases were observed in total cells (A), macrophages (B), or neutrophils (B) until 6 hr after treatment. (D) No increases were detected in protein after HA treatment compared with vehicle at these time points. (E) IL‑1β mRNA in alveolar macrophages was up‑regulated 1 hr and 2 hr after HA treatment compared with vehicle, but values decreased to near baseline at 6 hr. (F) The transcription level of IL‑18 was not significantly different after HA treatment at the three time points. Data are presented as mean ± SE (n = 5) and are representative of two similar experiments. *p < 0.05.
The Nlrp3 inflammasome is required for pulmonary response to HA fragments. To determine the role of HA activation of the inflammasome in reactive airways disease, we directly challenged animals to short fragments of HA. As previously reported (Garantziotis et al. 2009, 2010; Li et al. 2011), WT animals developed AHR 2 hr after challenge with HA but not with vehicle (Figure 5). The physiological response, as measured by methacholine sensitivity after treatment with HA fragments, was completely dependent on ASC, caspase1, and Nlrp3. However, similar to WT animals, HA challenge in ASC–/–, caspase1–/–, and Nlrp3–/– mice had no effect on either the number of cells [see Supplemental Material, Figure S3A (http://dx.doi.org/10.1289/ehp.1205188)] or the level of total protein (see Supplemental Material, Figure S3B) in BALF at this time point. After HA treatment, IL-1β mRNA in alveolar macrophages was up-regulated in a manner independent of the Nlrp3 inflammasome (Figure 6A), similar to that observed after ozone exposure; the release of soluble IL-1β was completely dependent on ASC and caspase1, and partially dependent on Nlrp3 (Figure 6C). However, we observed no detectible increases in IL-18 mRNA expression in alveolar macrophages or soluble IL-18 in BALF after direct challenge with HA (Figure 6B,D). The increases of IL-1β and IL-18 mRNA observed after HA challenge was not detected in whole-lung macrophages (see Supplemental Material, Figure S1C–D). Moreover, the release of IL-1α, IL-6, MCP-1, TNF-α, and KC were dependent on both ASC and caspase1 and partially dependent on Nlrp3; IL-17 was partially dependent on ASC and caspase1 but not on Nlrp3. C3a was dependent on ASC and caspase1 and partially dependent on Nlrp3 after HA fragment challenge (see Supplemental Material, Figure S4A–G).
Figure 5 The role of the Nlrp3 inflammasome in airway response to HA in WT and inflammasome-deficient mice 2 hr after challenge with short-fragment HA (25 µg/mouse). Compared with WT mice, mice deficient in caspase1 (A), ASC (B), or Nlrp3 (C) were protected from HA-induced AHR. Data are presented as mean ± SE (n = 5) and are representative of two similar experiments. *p < 0.05.
Figure 6 IL‑1β and IL‑18 mRNA (A,B) and protein (C,D), cleaved caspase1 (E), and cleaved IL‑1β (F) in alveolar macrophages from WT and inflammasome-deficient mice 2 hr after challenge with short-fragment HA (25 µg/mouse). (A) IL‑1β transcription was increased in alveolar macrophages from HA-treated mice compared with vehicle controls and was not dependent on the Nlrp3 inflammasome; however, IL‑18 mRNA expression (B) was not affected by HA. (C) After HA treatment, IL‑1β in BALF from WT mice was higher than that from Nlrp3 inflammasome-deficient mice. (D) The IL‑18 protein level was not increased. HA challenge results in cleavage of pro-caspase1 and cleavage of pro-IL‑1β in WT mice. (E,F) Western blot analysis for cleaved caspase1 (E) and cleaved IL‑1β (F) identified cleavage only in HA-exposed WT mice but not in ASC–/–, Nlrp3–/–, or caspase1–/– mice. Data are presented as mean ± SE (n = 5) and are representative of two similar experiments. *p< 0.05.
HA activation of the Nlrp3 inflammasome results in cleavage of macrophage-derived caspase1 and IL-1β. Activation of the inflammasome requires cleavage of pro-caspase1 to an active form that can subsequently cleave pro-IL-1β to an active soluble form. We therefore determined whether in vivo challenge with HA fragments resulted in cleavage of pro-caspase1 in alveolar macrophages. HA resulted in cleavage of pro-caspase1 in WT mice but not in ASC–/–, Nlrp3–/–, or caspase1–/– mice (Figure 6E). The cleavage of pro-IL-1β is a result of Nlrp3 inflammasome activation. We also observed cleavage of pro-IL-1β in alveolar macrophages from WT mice but not in those from Nlrp3 inflammasome-deficient mice (Figure 6F).
Discussion
We found a dominant role of the Nlrp3 inflammasome in nonallergic reactive airways disease after inhalation of ozone. Development of ozone-induced AHR requires several components of the inflammasome complex, including ASC, caspase1, and Nlrp3. Ozone inhalation results in an increased level of soluble HA, which we previously reported to contribute to AHR (Garantziotis et al. 2009). In the present study, we found that HA-induced cleavage of alveolar macrophage-derived pro-IL-1β is dependent on the Nlrp3 inflammasome. In addition, the levels of several proinflammatory factors (IL-1α, IL-6, KC, TNF-α, MCP-1, IL-17, and C3a) are partially dependent on genes of the Nlrp3 inflammasome after challenge with either ozone or HA. Together these findings strongly support biological and functional roles of the Nlrp3 inflammasome in the development of ozone-induced reactive airways disease.
Current evidence supports an important role for IL-1–dependent signaling in the pulmonary response to ozone (Johnston et al. 2007; Park et al. 2004; Verhein et al. 2008; Wu et al. 2008). Our previous work demonstrated that ozone exposure induced the release of HA fragments (Garantziotis et al. 2009, 2010) and led to an increase in soluble IL-1β within the airways (Garantziotis et al. 2010; Li et al. 2011). In a dermal injury model, activation of pro-IL-1β was shown to be regulated by the Nlrp3 inflammasome in response to HA (Yamasaki et al. 2009). In the present study, we found that ASC, caspase1, and Nlrp3 contribute to ozone-induced release of soluble IL-1β.
Although immunostimulatory short fragments of HA can contribute to the production of many macrophage-derived proinflammatory cytokines (Garantziotis et al. 2009, 2010), we have identified the relationship with genes of the Nlrp3 inflammasome. The levels of soluble HA in the airspace and macrophage-derived transcript of pro-IL-1β were not dependent on ASC, caspase1, or Nlrp3. However, soluble HA functionally contributed to the generation of active soluble IL-1β after exposure to ozone, as observed in HA-binding experiments. The functional response to direct HA fragment challenge in the lung was dependent on genes of the inflammasome. We observed a dominant role for both ASC and caspase1 in induction of many proinflammatory factors previously associated with the biological response to ozone, including IL-1β, IL-18, IL-1α, IL-6, MCP-1, TNF-α, KC, IL-17, and C3a. These data support that the inflammasome components not only activate IL-1β and IL-18 but also can regulate the release of additional proinflammatory cytokines (Barker et al. 2011). The near complete dependence on caspase1 supports activation of the inflammasome and essentially excludes the contribution of pyronecrosis, which is caspase1 independent but Nlrp3 and ASC dependent (Ting et al. 2008). However, we did not observe consistent complete reduction in any single soluble factor to explain the complete dependence of the Nlrp3 inflammasome in AHR. This observation suggests that factors other than measured cytokines may contribute to AHR in the Nlrp3-deficient mouse, such as additional soluble factors (Backus et al. 2010; Williams et al. 2008), neural responses (Caceres et al. 2009), or smooth muscle function. Furthermore, the partial reduction in proinflammatory cytokines observed in Nlrp3-deficient mice suggests that additional proteins in the NLR family may contribute to the response to both ozone and HA. Although ASC and caspase1 are ubiquitously expressed in many cell types, Nlrp3 is primarily expressed in myeloid cells (Guarda et al. 2011). NLR proteins expressed in non-myeloid cells may prove important. However, the partial dependence on Nlrp3 does suggest an important contribution of myeloid-derived activation of the inflammasome for the complete response to either ozone or HA. These findings demonstrate that both ASC and caspase1 play a dominant role in airway hyperresponsiveness and suggest a functional role for the alveolar macrophages as an important source of proinflammatory cytokines.
Current evidence supports an emerging paradigm in which inhalation of ozone results in oxidant-induced fragmentation of HA in the extracellular matrix (Li et al. 2010). These immune-stimulatory fragments of HA interact with a surface receptor complex of CD44–TLR4 (Garantziotis et al. 2010; Taylor et al. 2007) to activate an intracellular signaling cascade that includes MyD88, TIRAP, and NF-κB (Garantziotis et al. 2010; Li et al. 2011), which in turn results in increased mRNA expression of pro-IL-1β in alveolar macrophages. Either ozone or HA results in cleavage and activation of pro-caspase1 resulting in activation of the Nlrp3 inflammasome complex. Finally, the activated inflammasome complex results in cleavage of pro-IL-1β into active soluble IL-1β. Our results suggest that active IL-1β contributes to the complex signaling network required for proinflammatory signaling and development of AHR. Together, these new findings provide additional support for a dominant role of genes of innate immunity in nonallergic reactive airways disease. Understanding the mechanisms that contribute to ozone-induced functional phenotypes in the airway can provide fundamental insight into the pathogenesis of AHR, a defining phenotype of clinical asthma. Our results establish a central role for the Nlrp3 inflammasome in both the induction of AHR and production of proinflammatory cytokines. Our observations support that airway therapy targeted toward local deactivation of the Nlrp3 inflammasome may provide benefit to some patients with reactive airways disease.
Supplemental Material
(262 KB) PDF Click here for additional data file.
This work was supported by National Institutes of Health grants ES016126, AI081672, ES020350, and ES020426 (to J.W.H.); AI089756 (to K.L.W.); and an unrestricted educational grant from the China Scholarship Council (to F.F.).
The authors declare they have no actual or potential competing financial interests.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23349848PONE-D-12-2966010.1371/journal.pone.0054303Research ArticleBiologyDevelopmental BiologyMorphogenesisCell MigrationModel OrganismsAnimal ModelsRatMolecular Cell BiologyCellular TypesEndothelial CellsCell GrowthMedicineCardiovascularMyocardial InfarctionRehmannia Glutinosa Extract Activates Endothelial Progenitor Cells in a Rat Model of Myocardial Infarction through a SDF-1 α/CXCR4 Cascade Rehmannia Glutinosa Protected Infarcted MyoccardimWang Ying-Bin
1
2
Liu Yun-Fang
3
Lu Xiao-Ting
1
Yan Fang-Fang
2
Wang Bo
2
Bai Wen-Wu
1
Zhao Yu-Xia
2
*
1
Key Laboratory of Cardiovascular Remodeling and Function Research, Shandong University, Jinan, Shandong, China
2
Department of Traditional Chinese Medicine, Qilu Hospital, Shandong University, Jinan, Shandong, China
3
Department of Diagnosis, College of Medicine, Shandong University, Jinan, Shandong, China
Singh Shree Ram Editor
National Cancer Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YBW YXZ YFL. Performed the experiments: YBW. Analyzed the data: YBW XTL. Contributed reagents/materials/analysis tools: YBW YXZ XTL BW FFY WWB. Wrote the paper: YBW.
2013 18 1 2013 8 1 e5430325 9 2012 10 12 2012 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Objectives
Endothelial progenitor cells (EPCs) can be used to repair tissues after myocardial infarction (MI) but EPC activators have adverse reactions. Rehmannia glutinosa is a herb used in traditional Chinese medicine, which can promote bone-marrow proliferation and protect the ischemic myocardium. We investigated the effects of Rehmannia glutinosa extract (RGE) on EPCs in a rat model of MI.
Methods
A total of 120 male Wistar rats were randomized to 2 groups (n = 60 each) for treatment: high-dose RGE (1.5 g·kg−1·day−1 orally) for 8 weeks, then left anterior descending coronary artery ligation, mock surgery or no treatment, then RGE orally for 4 weeks; or normal saline (NS) as the above protocol. The infarct region of the left ventricle was assessed by serial sectioning and morphology. EPCs were evaluated by number and function. Protein and mRNA levels of CD133, vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-X-C motif receptor 4 (CXCR4), stromal cell–derived factor-1α (SDF-1α) were measured by immunohistochemistry, Western blot and quantitative PCR analysis.
Results
RGE significantly improved left ventricular function, decreased the ischemic area and the apoptotic index in the infarct myocardium, also decreased the concentration of serum cardiac troponin T and brain natriuretic peptide at the chronic stage after MI (from week 2 to week 4). RGE increased EPC number, proliferation, migration and tube-formation capacity. It was able to up-regulate the expression of angiogenesis-associated ligand/receptor, including CD133, VEGFR2 and SDF-1α/CXCR4. In vitro, the effect of RGE on SDF-1α/CXCR4 cascade was reversed by the CXCR4 specific antagonist AMD3100.
Conclusion
RGE may enhance the mobilization, migration and therapeutic angiogenesis of EPCs after MI by activating the SDF-1α/CXCR4 cascade.
This study was partially supported by grants from the National 973 Basic Research Program of China (No. 2012CB518603), National Natural Science Foundation of China (Nos. 30873325, 81100103, 81173251), Natural Science Foundation of Shandong Province (Nos. ZR2011HQ020, ZR2009CM049), and postdoctoral special foundation for innovative projects of Shandong Province (No. 201103049). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Myocardial infarction (MI) occurs with the deprivation of coronary blood and is usually caused by stenosis or occlusion of the coronary artery. The culminating event is necrosis of myocardial tissue and dysfunction of the left ventricle. Bone-marrow-derived stem cells, including endothelial progenitor cells (EPCs), are attractive targets for repair of the ischemic myocardium [1]–[3]. EPCs can home to ischemic tissues and contribute to therapeutic angiogenesis [4]–[5]. Many EPCs agonists such as granulocyte-colony stimulating factor, vascular endothelial growth factor (VEGF) and statins, can mobilize EPCs in bone marrow [6]–[7]. However adverse reactions, such as increased vascular permeability and high ratio of restenosis and liver damage, limit their use for MI [8]. A safe EPC activator is needed for MI therapy.
Activated EPCs first migrate to the ischemic tissue for their roles. Stromal-derived factor-1 (SDF-1, or CXCL12) is the only known chemokine capable of migration of hematopoietic stem cells (HSCs), as the fluctuations in SDF-1 expression controlled the fluctuated steady-state of HSCs and their progenitors in peripheral blood [9]. Among these, the SDF-1α and its receptor 4 (CXCR4) play a key role in mobilization and migration of EPCs [10]. After MI, SDF-1α/CXCR4 interaction plays a crucial role in recruiting EPCs to the ischemic myocardium, the increased CXCR4 expression lead to increased EPCs homing to the ischemic zone and participated in therapeutic angiogenesis [11]–[13]. These suggest that the SDF-1α/CXCR4 cascade is critical for the regulation of EPCs, and it might be an important therapeutic target for cardiovascular diseases especially in MI [14].
Rehmannia glutinosa, belongs to the family of Scrophulariaceae, is a widely used traditional Chinese medicinal herb. It has been used to treat hypodynamia caused by many kinds of diseases for thousands of years in China, Japan, Korea and many other Asian countries. It has been effective and safe, but the involved mechanism has not been verified. Recently, Rehmannia glutinosa extract (RGE) has been used in modern medicine studies [15]. RGE can stimulate the proliferation and differentiation of hematopoietic stem cells in bone marrow [16] and increase the numbers of leucocytes, thrombocytes, reticulocytes and DNA content of bone marrow [17]. Furthermore, RGE can antagonize myocardial cell death induced by caspase-3 activation, thus protecting the ischemic myocardium [18].
Our preliminary experiments in rat showed an increase in number of EPCs in blood and bone marrow after oral administration of RGE (Table S1). These suggested that RGE had effect on EPCs.
In this study, we used the rat MI model to imitate the pathological changes after MI and observed the effects of RGE on preserving the left ventricle, up-regulating the number and function of EPCs and increasing therapeutic angiogenesis. Thus, we could examine whether the RGE is a EPCs activator or not. We further analyzed the alteration of the SDF-1α/CXCR4 cascade with RGE in vivo and in vitro, to investigate the possible mechanism of RGE on EPCs after MI.
Materials and Methods
Preparations of Rehmannia Glutinosa Extract (RGE)
Rehmannia glutinosa (purity>96%) was from the National Institutes for Food and Drug Control (Beijing). RGE was prepared by alcohol extraction. Briefly, dried Rehmannia glutinosa was soaked with distilled water in a 1∶10 volume ratio for 24 h, then heated to 80°C for 12 h. The supernatant was collected and ethanol was added to a 3∶4 volume ratio. The extracts were stored at room temperature for 24 h and centrifuged at 3000 rpm for 10 min, and the supernatant was mixed with ethanol in a 1∶4 volume ratio. Then extracts were incubated at room temperature for an additional 24 h and centrifuged at 3000 rpm for 10 min. Ethanol was evaporated from the supernatant. The extract was diluted in H2O and stored at 20°C overnight. This process was repeated 3 times. The final extracts were concentrated under reduced pressure and filtered, lyophilized, and serially stored at 4°C. The yield of dried extract (RGE) from starting crude materials was 22.5% (w/w) [19]. The RGE was dissolved in saline or phosphate-buffered saline (PBS) for experiments.
Animal Models and Treatment
All animal surgeries were performed under isoflurane, and all efforts were made to minimize suffering.
A total of 120 male Wistar rats (Laboratory Animal Services Centre, College of Medicine, Shandong University; 8 weeks old; body weight 180 - 200 g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. All animal studies were carried out at the Animal Care Center of Key Laboratory of Cardiovascular Remodeling and Function Research, Shandong University. The experiment complied with the Animal Management Rule of the Ministry of Public Health, People’s Republic of China (document No. 55, 2001), and the experimental protocol was approved by the Animal Care Committee of Shandong University. The rats were randomly divided into 2 groups (n = 60 each) for treatment: control group, normal saline (NS) orally; treated group, high-dose RGE-NS solution of 1.5 g·kg−1·day−1 orally (in preliminary experiments, high-dose RGE, 1.5 g·kg−1·day−1 as compared with 0.38 and 0.75 g·kg−1·day−1, which was converted from human oral administration doses, effectively increased bone marrow mobilization of EPCs and migration to peripheral blood; TableS1). At the end of week 8, 20 rats from both groups underwent ligation of the left anterior descending coronary artery under endotracheal intubation and mechanical ventilation by use of the Harvard Appratus Mini-Vent (Type B-90218, China) [20]. Another 20 rats from both groups underwent mock surgery with a silk suture across the coronary artery without ligation. The other 20 rats in both groups were blank controls. The rats continued to be oral-fed RGE or NS, then rats were sacrificed, 5 each on the 3rd day and the end of 1, 2, 4 weeks, recording as day 3, week 1, week 2 and week 4 respectively.
Electrocardiography and Ultrasonic Cardiography
Before and after surgery, rats underwent electrocardiography (ECG) by use of a Micromaxx P04224 system (SonoSite, China) and ultrasonic cardiography (UCG) by a high-frequency duplex ultrasonic cardiogram system (Visual Sonics Vevo 770, Germany) and a transducer (RMV™ Scan Head 710B-048, Germany). Rats underwent ultrasonic cardiography at day 3, weeks 1, 2 and 4 before sacrificed. The transducer for ultrasonic cardiography was placed at the left thoraces between the 3rd and 4th ribs to obtain B-mode tracings of the heart from just below the level of the papillary muscles of the mitral valve. We obtained left-ventricular end-diastolic diameters (LVD-d) and end-systolic diameters (LVD-s) with M-mode tracings between the anterior and posterior walls. The time of end-diastole and end-systole was defined as time of maximum and minimum diameter of the left ventricle, respectively, in one heart cycle. Following the American Society of Echocardiology leading-edge method, we obtained 3 images, on average, in each view, which were averaged over three consecutive cycles [21].The system calculated the left-ventricular end-diastolic volume (LV-d), left-ventricular end-systolic volume (LV-s), mass of the left ventricle (LV-mass), left-ventricular fractional shortening (LVFS) and left-ventricular ejection fraction (LVEF).
EPC Identification and Assessment of Function
Isolation and cultivation of EPCs
10 ml peripheral blood was obtained from rats by aspiration of the heart. Bone-marrow cells were obtained by flushing the cavity of femurs, tibias, and humerus with growth medium EBM-2 (Lonza Walkersville, USA, basal medium with 8 factors and 5% fetal bovine serum [FBS]). Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll (Ficoll-Paque PLUS, GE Healthcare Bio-Sciences AB, USA) density-gradient centrifugation [22]. 10 million isolated cells were resuspended in growth medium EBM-2 and plated in 25-cm2 culture flasks. After 48 h, non-adherent cells were discarded and growth medium was changed every 2 days.
Identification of EPCs
Direct fluorescent staining was used to detect dual binding of fluorescein isothiocyanate -labeled ulex europaeus agglutinin(FITC-UEA-1; Sigma, USA) and dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-acLDL; Invitrogen Molecular Probes, USA). Briefly, cells were incubated with Dil-acLDL (2 µg/ml) at 37°C for 2 h then fixed with 2% paraformaldehyde for 10 min. After a washing with PBS, cells were treated with FITC-UEA-1 (10 µg/ml) for 1 h. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for viewing by laser scanning confocal microscope (LSMT10, ZEISS, Germany). Cells double stained for Dil-Ac-LDL and FITC-UEA-1 were considered EPCs [23]. Immunocytochemistry followed standard protocols.
Determination of EPC number
Fluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200 µl) was incubated with the monoclonal antibodies: anti-VEGFR2 (Abcam, USA, 1 mg/ml, 1∶100), anti-CD133 (Abcam, USA, 0.5 mg/ml, 1∶100) and anti-CD34-PerCP-Cy5.5 (Santa Cruz Biotechnology, Santa Cruz, CA; 0.2 mg/ml, 1∶10) for 20 min at room temperature, then with 2 ml Lysing solution (BD, USA) for 10 min and washed with PBS twice by centrifugation. The cells were resuspended with 200 µl PBS, then incubated with the secondary antibodies: goat polyclonal rabbit IgG-FITC (Abcam, USA, 2 mg/ml, 1∶80) and goat polyclonal mouse IgG-F(ab)2 fragment PE (Abcam, USA, 0.5 mg/ml, 1∶40) for 30 min at room temperature. Cells were washed with PBS and resuspended in 400 µl PBS. Flow cytometry involved use of a FACS calibur flow cytometer and Cell-Quest software (BD Biosciences, USA). Each analysis included at least 10,000 cells.
EPC proliferation, migration and tube formation
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to evaluate EPC proliferation. Cells were cultivated for 4 days, then plated at 1×104 per well in a 96-well plate with EBM-2 (200 µl) for 24 h. A filter-sterilized MTT solution was added for final concentration of MTT of 0.5 µg/ml for 4 h. The supernatant was discarded, and wells were washed twice with PBS. The EPC preparation was shaken at 90-100 rpm with 200 µl dimethyl sulfoxide for 10 min at room temperature. Optical density (OD) was measured at 490 nm. Each test was repeated 4 times [24].
EPC migration was evaluated by use of a transwell chamber (6.5 mm diameter inserts, 8.0 mm pore size; Corning, USA). Transwell inserts were placed in a 24-well plate containing EBM-2. EPCs (5×104) were added to the upper chamber of the well, without FBS. Cells were allowed to migrate from the upper to the lower chamber for 12 h at 37°C. Non-migratory cells were removed from the upper chamber by wiping the upper surface with use of an absorbent tip. The number of migrating cells was counted in 5 different high-power fields per insert [25].
Capillary-like tube formation was analyzed by use of Matrigel Matrix (BD Biosciences, USA). EPCs (8×104 cells in 100 µl EBM-2) were placed in 96-well plates pre-coated with solidified Matrigel Matrix (100 µl) and cultured at 37°C for 24 h. Capillary-like tubular structures were photographed, and the number of incorporated EPCs in tubules was determined in 5 random fields. A tube was defined as a straight cellular segment connecting 2 cell masses (nodes) [26].
ELISA
ELISA was used to measure cardiac troponin T (Tn-T) and brain natriuretic peptide (BNP) concentration in serum for left ventricular function evaluation, by use of a BNP kit (Rat-45, Abcam, USA) and Tn-T kit (TSZ ELISA, USA). Briefly, standards and diluted serum of rats were added into the pre-coated 96-well plates and incubated for 30 min in 37°C. After a washing with PBS, the horseradish peroxidase-conjugated anti-body was added for 30 min incubation at 37°C. After a washing by PBS, the tetramethylbenzibine substrate was added. After reaching the desired color density, the reaction was terminated by stop solution. OD450 was determined by use of an ELISA plate reader (Varioskan Flash, Thermo Fisher, Germany). Each samples repeated in 3 wells.
Histology and Immunechemistry
Myocardial tissues (approximately 2 mm thick) in the left ventricle of rats were removed and fixed in 4% pre-cooled paraformaldehyde for 72 h, then embedded in paraffin, and sectioned into slices 5 µm thick. Poley’s stain was used to assess the ischemic myocardial area. Images were visualized under an optical microscope at ×200 magnification.
Myocardial tissue sections underwent the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using an in situ detection kit (Roche, Germany) following the manufacturer’s instructions. The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total myocardial cells.
Immunohistochemical staining involved standard techniques as described [27]. Briefly, endogenous peroxidase activity was inhibited by incubation with 3% H2O2. Sections were blocked with 5% calf serum in PBS and incubated overnight at 4°C with the monoclonal antibodies: anti-VEGFR2 (Abcam, USA, 1 mg/ml, 1∶100), anti-CD133 (Abcam, USA, 0.5 mg/ml, 1∶50) and anti-CXCR4 (Abcam, USA, 1 mg/ml, 1∶100). After a washing with PBS, sections were incubated with secondary antibody at 37°C for 30 min. Immunohistochemical staining was visualized by use of a diaminobenzidine kit (Zhongshan Goldenbridge Biotechnology, Beijing). Samples were counter stained with hematoxylin for nuclei.
RT-PCR
Tissue samples were frozen with the use of liquid nitrogen. Total RNA was extracted by use of TRIZOL reagent (Invitrogen, USA), quantified by spectrophotometry and reverse transcribed by use of the M-MLV Reverse Transcriptase System (Osaka, Japan) with oligo-dT primers. The mRNA expression of VEGFR2, CD133, and CXCR4 in myocardium was examined by real-time RT-PCR with SYBR Green Real-time PCR Master Mix (TOYOBO, Life Science Department, Japan) and an MYIQ™ Single Color Real-Time PCR Detection System (Bio-Rad, Germany). The mRNA sequences were obtained from Gene-bank (NCBI, Bethesda, MD; Table 1). Actin level was an internal control. Experiments were performed in triplicate, and data were analyzed by the 2−△△CT method [28].
10.1371/journal.pone.0054303.t001Table 1 Primers for RT-PCR.
Molecules MW Locus Primer sequence Tm °C
VEGFR2 123 NM_013062 F 5′-ATCGGTGAGAAAGCCTTGATCTC-3′
53
R 5′-TTCTAGCTGCCAGTACCATTGGA-3′
CD133 97 NM_001110137 F 5′-CTGCAAACCCATGATTACAGCAA-3′
57
R 5′-CCCTATGCCGAACCAGAACAG-3′
SDF-1α 240 NM_022177 F 5′-TCTTTGGCCTCCTGTAGAATGG-3′
56
R 5′-TCACGGCAAGATTCTGGCTTA-3′
CXCR4 177 NM_001033882 F 5′-CGTGAATGAGTGTCTAGGCAGG-3′
55
R 5′-GGCTTTGGTTTTAAGTGCCATC-3′
ACTIN 94 NM_0010822 F 5′-AGACCTTCAACACCCCAG-3′
55
R 5′-CACGATTTCCCTCTCAGC-3′
VEGFR2, vascular endothelial growth factor receptor 2; SDF-1α, stromal cell–derived factor-1α; CXCR4, chemokine (C-X-C motif) receptor 4; MW, molecular weight; Tm, melting temperature.
Western Blot Analysis
Total protein was isolated in lysis buffer (Beyotime, China) and was resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, which was blocked in 5% skimmed milk in PBS containing 0.1% Tween-20 for 1 h at room temperature and incubated with monoclonal antibodies: anti-CXCR4 (Abcam, USA, 1 mg/ml, 1∶1000) and anti-SDF-1α(Abcam, USA, 0.5 mg/ml, 1∶500) overnight at 4°C. The bands were visualized with use of an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA), photographed by use of Epson Perfection (V700 Photo, Japan) and analyzed by use of Quantity One software. Experiments were performed in triplicate, and data were normalized to level of β-actin.
In vitro Cellular Experiments
To explore the molecular mechanism probably involved in the RGE effect on EPCs, EPCs from peripheral blood and bone marrow of normal rats were incubated in EBM-2 without FBS for 24 h. RGE was dissolved in PBS and filtered by millex (Millipore, USA, 0.22 µm), then added to EPCs at 10, 25, 50, 100, 500 and 1000 µg/ml. The inhibiter was CXCR4-specific antagonist AMD3100 (Abcam, USA; 5 µg/ml, purity >99%) for 1 h before RGE [29]. The blank control was cultivated with EBM-2.
MTT was used to test proliferation of EPCs after stimulation for determining the proper concentration of RGE for EPCs. Western blot and real-time PCR analysis were used to examine the expression of SDF-1α/CXCR4 cascade in EPCs stimulated by RGE at different concentrations and for different time. Capillary-like tube formation was performed to test the function of RGE-stimulated EPCs.
Statistical Analysis
Data are expressed as mean ± SD and were assessed by one-sample Kolmogorov–Smirnov test to check for normal distribution. Differences between 2 groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS 11.5 (SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.
Results
During the experiment, 7 rats died: 2 each in the NS mock, NS MI and RGE MI groups and 1 in the RGE mock group.
Model Evaluation and RGE’s Effect on the Improvement of Ischemic Myocardium
After surgery induction, the ECG revealed elevated ST segment and pathologic waveforms (Fig. 1-A), the UCG revealed changes in left ventricular wall mobility, blood flow at the mitral valve (Fig. 1-B) and the increased LV-d, LV-s, LV-mass, the decreased LVEF, LVFS, the reversed E/A ratio (P<0.01 to 0.05, Table 2 to 5). These revealed the successful establishment of the MI model. After MI, the function of left ventricular was reflected by LVEF of UCG. In acute stage (day 3 to week 1), the MI groups with both treatment showed almost no difference in LVEF, while as to the chronic stage (week 2 and week 4), the recovery of LVEF was greater with RGE than NS (P<0.05; Fig. 1-C). These revealed that RGE systemic delivery protected the function of left ventricular in chronic stage after MI.
10.1371/journal.pone.0054303.g001Figure 1 Heart function and serum myocardium markers level of rats.
(A) Electrocardiography (ECG) before and after myocardial infarction (MI). Leads I, II, III, aVR, aVL and aVF of normal (N) and MI rats. (B) Ultrasonic cardiography before and after MI. Images are M-mold (m) ultrasound and hemorheologic (h) ultrasound in normal and MI rats. (C) Left ventricular ejection fraction (LVEF) after MI. NS-b, normal saline (NS)-blank; NS-m, NS-mock; RGE-b, Rehmannia glutinosa extract (RGE)-blank; RGE-m, RGE-mock. (D) The levels of myocardium markers: cardiac troponin T (Tn-T) and brain natriuretic peptide (BNP). Data are mean ± SD. *P<0.05, **P<0.01 vs. NS group at the same time; #P<0.05, ##P<0.01 vs. the same group at day 3.
10.1371/journal.pone.0054303.t002Table 2 UCE measurement before and 3 days after myocardium infarction (MI).
Group LV,d (µl) LV,s (µl) LV,mass (mg) LVEF (%) LVFS (%) E/A
NS-b prior 55.89±12.35 6.25±1.79 60.6±8.65 88.17±7.02 58.51±8.80 1.22±0.056
after 104.40±12.78 9.96±1.11 76.0±16.06 90.23±2.93 62.47±4.71 1.19±0.068
NS-m prior 105.00±42.34 8.44±2.11 80.10±13.62 90.82±5.61 63.41±9.38 1.21±0.096
after 185.60±33.83e 59.58±5.96 142.90±19.93e 67.76±3.22f 38.44±2.46f 1.10±0.058
NS-MI prior 98.98±5.59 10.38±0.82 72.00±9.38 89.52±1.68 61.05±2.28 1.22±0.059
after 440.30±51.03f 322.50±28.04f 315.20±60.45f 13.29±4.67f 26.97±8.93f 0.91±0.027f
RGE-b prior 108.50±28.67 13.25±1.05 74.14±7.59 87.52±1.58 57.49±2.46 1.14±0.045
after 119.30±10.59 22.56±3.22 65.45±8.55 81.12±5.82 50.38±5.72 1.18±0.056
RGE-m prior 94.63±17.94 11.62±2.61 67.66±7.48 87.99±5.19 58.67±7.46 1.16±0.071
after 109.80±5.50 29.36±2.54 115.10±31.00 73.32±2.95f 42.44±2.60e 1.16±0.048
RGE-MI prior 112.20±15.71 19.09±1.50 65.54±8.96 83.03±0.89 52.02±0.93 1.15±0.038
after 367.00±84.96f 234.30±38.74f 307.80±54.53f 19.01±6.86f 37.09±11.63e 0.81±0.147f
10.1371/journal.pone.0054303.t003Table 3 UCE measurement before and 1 week after MI.
Group LV,d (µl) LV,s (µl) LV,mass (mg) LVEF (%) LVFS (%) E/A
NS-b prior 101.90±8.24 14.08±1.54 78.37±5.59 86.13±3.64 55.83±4.55 1.18±0.052
after 124.10±20.36 17.92±3.14 97.58±16.87 85.81±4.03 55.85±5.25 1.18±0.079
NS-m prior 100.31±21.60 12.27±3.04 62.76±6.81 88.34±4.61 59.14±6.79 1.23±0.038
after 122.38±40.57 35.80±7.38 127.21±12.02 72.11±6.47f 41.73±5.59e 1.20±0.028
NS-MI prior 109.57±11.57 11.87±1.04 103.78±14.13 89.19±1.47 59.94±2.21 1.19±0.032
after 353.45±79.49f 106.80±34.05f 317.49±21.58f 21.78±6.88f 42.06±11.53e 0.90±0.106f
RGE-b prior 121.52±18.87 10.16±0.59 112.13±11.78 91.39±2.08 63.65±3.86 1.21±0.039
after 142.19±17.56 22.23±2.07 80.6±4.46 84.08±4.29 53.88±5.31 1.20±0.025
RGE-m prior 131.47±15.97 23.27±1.44 100.52±8.95 82.29±1.45 51.44±1.63 1.24±0.084
after 108.9±37.43 32.59±4.62 174.89±22.25 69.55±5.76e 39.33±4.73 1.22±0.027
RGE-MIprior 117.11±29.94 21.65±7.55 100.00±19.54 83.29±9.09 53.55±9.83 1.23±0.147
after 338.28±47.84f 189.02±6.87f 260.61±32.38f 22.42±3.91f 43.08±6.28 0.89±0.086f
10.1371/journal.pone.0054303.t004Table 4 UCE measurement before and 2 weeks after MI.
Group LV,d (µl) LV,s (µl) LV,mass (mg) LVEF (%) LVFS (%) E/A
NS-b prior 95.92±15.34 14.56±2.11 69.11±5.75 85.05±4.13 54.49±5.23 1.15±0.099
after 151.31±27.67 38.19±5.99 88.72±9.09 75.32±5.01 44.82±4.62 1.19±0.044
NS-m prior 106.00±8.63 15.48±1.29 71.18±6.89 85.32±2.80 54.76±3.60 1.19±0.067
after 127.51±22.59 32.56±8.79 100.00±24.94 75.67±9.87 45.51±9.14 1.13±0.041
NS-MI prior 83.21±31.23 9.06±4.14 60.87±4.86 90.26±5.37 61.91±7.77 1.27±0.048
after 342.60±28.62f 181.10±13.61f 301.18±21.07f 24.69±4.79f 46.94±7.82 0.83±0.076f
RGE-b prior 101.21±28.24 14.64±4.29 63.27±3.08 86.64±5.78 56.85±6.88 1.18±0.073
after 125.61±27.61 25.99±5.02 87.45±10.49 78.45±9.01 48.58±11.21 1.18±0.050
RGE-m prior 117.82±22.83 16.91±3.55 82.49±8.46 86.21±4.11 56.23±5.08 1.22±0.106
after 241.89±97.95f,g 88.00±21.25f,g 223.57±32.48f,h 65.66±7.43f 37.23±5.49e 1.17±0.086
RGE-MIprior 99.32±14.82 8.97±3.23 90.89±24.56 91.53±5.10 64.52±8.64 1.19±0.060
after 185.45±38.65h 98.34±16.13f,h 165.82±21.75h 47.18±9.97f,h 24.34±6.13f,g 0.91±0.026f
10.1371/journal.pone.0054303.t005Table 5 UCE measurement before and 4 weeks after MI.
Group LV,d (µl) LV,s (µl) LV,mass (mg) LVEF (%) LVFS (%) E/A
NS-b prior 104.31±38.15 16.59±4.70 87.28±11.29 85.57±5.65 55.52±6.91 1.21±0.048
after 161.26±30.61 41.51±6.66e 153.31±11.38 74.89±5.52e 44.57±5.24 1.15±0.046
NS-m prior 96.78±31.16 13.34±3.80 62.63±2.86 87.28±4.92 57.55±6.56 1.21±0.025
after 305.81±21.93e 116.11±5.58f 384.26±39.28f 62.03±3.06f 34.62±2.28f 1.19±0.050
NS-MI prior 89.44±15.05 9.06±1.95 64.68±8.50 89.98±3.58 61.19±5.97 1.20±0.053
after 347.4±23.18f 207.89±7.45f 264.27±23.91f 24.02±3.76f 35.52±14.58f 0.94±0.084f
RGE-b prior 99.67±44.15 10.21±2.10 76.88±10.05 89.11±3.46 59.88±6.10 1.19±0.027
after 125.21±18.79 30.26±4.55 84.37±8.84 76.30±6.34e 45.66±6.29e 1.18±0.042
RGE-m prior 93.36±7.21 9.32±0.95 69.33±4.20 90.04±1.78 61.96±1.88 1.19±0.038
after 184.4±41.21e,h 58.21±5.78f,h 143.40±14.32h 67.73±3.28e 38.41±2.83f 1.18±0.039
RGE-MI prior 100.60±41.53 7.51±1.30 80.78±11.92 92.23±1.65 64.67±3.40 1.20±0.079
after 169.03±22.4h 81.52±7.53f,h 135.40±10.19h 51.61±8.14f,h 26.98±5.04f 0.73±0.053f,h
LV-d, left-ventricular end-diastolic volume; LV-s, left-ventricular end-systolic volume; LV-mass, relative mass of left ventricle; LVEF, the left-ventricular ejection fraction; LVFS, the Left-ventricular fractional shortening; E/A, the radio of peak E velocity and peak A velocity at mitral valve protocol; NS-b, NS blank; NS-m, NS mock; RGE-b, RGE blank; RGE-m, RGE mock; prior, before MI; after, after MI and before being killed. Data are mean ± SD.
e P<0.05,
f P<0.01 vs. the same animal before MI;
g P<0.05,
h P<0.01 vs. blank group.
The significantly up-regulated serum levels of Tn-T and BNP in NS-MI and GRE-MI groups also showed the successful establishment of mouse MI model (P<0.01). After MI, the high level of Tn-T decreased in RGE group (from week 1, P<0.01 to 0.05) earlier than that in NS group (from week 4, P<0.01). As for BNP, the level increased from day 3 to week 4 with NS (P<0.01 to 0.05), while had no changes with RGE treatment after MI (Fig. 1-D). These revealed that RGE systemic delivery decreased myocardial damage and protected them from further inflammatory reaction after MI.
Poley’s stain showed the ischemic myocardial zone (stained red) in normal myocardium (stained blue, Fig. 2-A). In chronic stage after MI, the relative ischemic area was lower with RGE than NS (P<0.05, Fig. 2-C). TUNEL stain and quantitative analysis showed that the apoptotic myocardium was less with RGE than NS in chronic stage after MI (P<0.01 to 0.05, Fig. 2-B, 2-C). These showed RGE’s function on improving ischemic myocardium and decreasing myocardial apoptosis.
10.1371/journal.pone.0054303.g002Figure 2 Poley’s stain and TUNEL stain of ischemic myocardium.
(A) Poley’s stain of NS-b, RGE-b, NS-MI, RGE-MI groups at week 4. The ischemic areas were stained red indicated by red arrow and non-ischemic area were stained blue. (B) TUNEL stain of NS-b, RGE-b, NS-MI, RGE-MI groups at week 4. The apoptotic myocardial cells were green under fluorescent and brown under Immunohistochemistry stain indicated by red arrow. (C) Quantitative analysis of mean relative ischemic area (percentage of the total transverse sections area) and myocardium apoptotic index (ratio to total myocardial cells). Data are mean ± SD. *P<0.05, **P<0.01 vs. NS group at the same time, #P<0.05, ##P<0.01 vs. the same group at day 3, $P<0.05, vs. the same group at week 1.
RGE’s Function on Activating EPCs
EPCs were identified as Dil-acLDL and FITC-UEA-1 double-stained cells with the nuclei stained by DAPI (Fig. 3-A). FACS was used to analyze the quantity of EPCs marked by CD34, VEGFR2 and CD133 in blood and bone marrow (Fig. 3-B).After MI, the quantity of EPCs in peripheral blood increased (P<0.01) and it decreased in bone marrow (P<0.05) with both RGE and NS. These suggested that the EPCs in bone marrow were mobilized to peripheral blood as the injury of myocardium. In chronic stage after MI, the increase of EPCs in peripheral blood was more significant with RGE compared to NS (P<0.01), and the decrease of EPCs in bone was not so much with RGE as it with NS (Fig. 3-C). These suggested that in chronic stage after MI, RGE was able to increase EPCs mobilizing to ischemic myocardium and maintain the quantity of EPCs stored in bone marrow. With the increased EPC population in both bone marrow and peripheral blood, the total number of EPCs in vivo was much more with RGE than NS.
10.1371/journal.pone.0054303.g003Figure 3 Effect of RGE on endothelial progenitor cells (EPCs) number.
(A) EPCs were identified by Dil-acLDL (red), lectin-FITC (green) and DAPI (blue) staining. (B) FACS analysis of peripheral blood and bone marrow levels of EPCs with 3 surface markers: CD34-PE, VEGFR2-FITC, CD133-PerCP-Cy5.5 at week 4. (C) Quantification of percentage of triple-marked cells from the initial monocytes in gate. Data are mean ± SD. *P<0.05, **P<0.01 vs. NS group at the same time, #P<0.05, ##P<0.01 vs. the same group at day 3.
By MTT assay, we tested the proliferation of EPCs in each group. The MI surgery made the proliferated activity of EPCs up-regulated with both GRE and NS (P<0.01). However, it maintained at a high level with RGE compared to it decreased in week 2 and 4 with NS (P<0.05, Fig. 4-A). These showed that RGE was able to up-regulated the proliferation of EPCs after MI, especially in chronic stage. At week 4 after MI, the migration of EPCs was more active with RGE than NS (P<0.05, Fig. 4-B, 4-D). From week 2 after MI, EPCs participated in capillary-like tube formation were increased with RGE compared to NS (P<0.05), and a similar increase occurred in normal rats with RGE relative to NS (P<0.05, Fig. 4-C, 4-D). These suggested REG’s function on motivating EPCs tube-formation capacity after MI as well as in normal physiological status.
10.1371/journal.pone.0054303.g004Figure 4 Effect of RGE on EPC function.
(A) MTT assay of proliferation of EPCs. (B) Transwell-chamber migration assay of EPCs at week 4. The activated EPC (marked by red arrow) migrated from upper chamber to the lower chamber. (C) Assessment of angiogenesis with EPCs placed on Martrigel and forming tubular-like structures at week 2. EPCs integrated in tubular-like structures are marked by red @; the formed tube was marked by red arrow. (D) Quantification of migration and angiogenesis of EPCs. For migration, data are percentage of EPCs migrating to the lower chamber as compared with those in the upper chamber. For angiogenesis, data are the ratio of EPCs integrated in tubular-like structures and the tubes involved. Each test was repeated 4 times, and data are mean ± SD. *P<0.05 vs. NS group at the same time, #P<0.05 vs. the same group at day 3.
RGE’s Function on Therapeutic Angiogenesis
Immunohistochemistry showed the fluctuant expression of VEGFR2 and CD133, which were signals of new-born capillary. After MI, the expression of VEGFR2 increased from week 1 until week 4 with RGE (P<0.05 to 0.01), and was much more significant than that with NS (P<0.05 to 0.01). In chronic stage after MI, the expression of CD133 also increased more with RGE than NS (P<0.01, Fig. 5-A, 5-B). The expression fluctuation at the level of mRNA was almost the same (Fig. 5-C). These revealed that RGE was able to promote the newborn of capillary at the chronic stage of MI.
10.1371/journal.pone.0054303.g005Figure 5 Effect of RGE on angiogenesis at the MI area.
(A) Immunohistochemistry of VEGFR2 and CD133 in NS-b, NS-MI, RGE-b and RGE-MI groups at week 4. The vessels are indicated by red @, and positive colorations are indicated by red arrows. (B) Quantification of VEGFR2 and CD133 immunoreactivity (percentage of positive area) in each group. (C) Quantitative PCR analysis of VEGFR2 and CD133 in myocardial tissue. For real-time PCR, the NS-b group was considered the baseline, actin was the internal reference. Data are mean ± SD. *P<0.05, **P<0.01 vs. NS group at the same time, #P<0.05, ##P<0.01 vs. the same group at day 3, $P<0.05, $$P<0.01 vs. the same group at week 1.
RGE’s Function on SDF-1α/CXCR4 Cascade
To investigate the mechanism involved in effect of RGE on EPCs, we detected the change in SDF-1α/CXCR4 cascade expression with MI in vivo, and with cellular experiments in vitro.
Immunohistochemistry revealed the increased protein expression of CXCR4 in MI groups compared to control groups (P<0.01) and the increase was more significant with RGE than NS in chronic stage after MI (P<0.01, Fig. 6-A, 6-B). Western blots analysis revealed the same findings for CXCR4 protein expression in ischemic myocardial tissue. As for SDF-1α, the expression increased with RGE but not with NS at week 1 compared to day 3 (P<0.01). And there was no statistical difference between the two treatments in chronic stage after MI (Fig. 6-C, 6-D). RT-PCR showed the changes in mRNA level of SDF-1α/CXCR4 cascade. The fluctuation of mRNA expression was just like what it showed in protein expression by Western blot (Fig. 6-E). These suggested that RGE might activate SDF-1α/CXCR4 cascade mainly by up-regulating transcription and translation of CXCR4.
10.1371/journal.pone.0054303.g006Figure 6 The expression of the stromal-derived factor-1α (SDF-1α) and its receptor 4 (CXCR4) in tissue.
(A) Immunohistochemistry of CXCR4 in NS-b, NS-MI, RGE-b and RGE-MI groups at week 4. The vessels are indicated by red @, and positive colorations are indicated by red arrows. (B) Western blot analysis of protein expression of CXCR4 and SDF-1α. β-actin was an internal reference protein for Western blot. (C) Quantitative of CXCR4 immunoreactivity (percentage of positive area) in each group. (D) Quantitative Western blot analysis of CXCR4 and SDF-1α in myocardial tissue. (E) Quantitative PCR analysis of CXCR4 and SDF-1α in myocardial tissue. Data are mean ± SD. *P<0.05, **P<0.01 vs. NS group at the same time, #P<0.05, ##P<0.01 vs. the same group at day 3, $P<0.05, $$P<0.01 vs. the same group at week 1.
To further certify the effect of RGE on SDF-1α/CXCR4 cascade, EPCs were stimulated with RGE-PBS solutions in vitro. The proliferation of EPCs decreased with 500 and 1000 µg/ml RGE solution (Fig. 7-A), so we chose other 4 kinds of solutions (10, 25, 50, 100 µg/ml) in subsequent experiments. Through Western blot and RT-PCR, we chose the optimal RGE-PBS solution concentration and duration for SDF-1α/CXCR4 cascade (Fig. 7-B to 7-E). For CXCR4, the optimal concentration is 25 µg/ml (P<0.01) and the optimal duration is 48 h (P<0.05 to 0.01). For SDF-1α, the optimal concentration is 50 µg/ml (P<0.01) and the optimal duration is 24 h (P<0.01). The optimal concentration and duration were used to stimulate EPCs and the tube-formation capacity of EPCs was tested. The inhibitor group, blocking the SDF-1α/CXCR4 cascade with AMD3100, showed poor functional EPCs that barely participated in capillary-like tube formation, CXCR4 showed poor expression and its ligand SDF-1α showed over-expression (P<0.01). The CXCR4 optimal concentration and duration (RGE = 25 µg/ml, 48 h) most significantly increased EPCs participating in capillary-like tube formation as compared with the other groups (P<0.05 to 0.01). The optimal concentration and duration for SDF-1α (RGE = 50 µg/ml, 24 h) had a similar but not as obvious an effect as for CXCR4 (P<0.05 to 0.01). These revealed that RGE was able to increase EPCs participating in capillary-like tube formation by up-regulating SDF-1α/CXCR4 cascade expression in vitro. And this effect of RGE could be reversed by CXCR4 specific inhibitor AMD3100.
10.1371/journal.pone.0054303.g007Figure 7 Effect of RGE on SDF-1α/CXCR4 cascade in vitro.
(A) MTT assay for proliferation of EPCs after stimulation with RGE-phosphate buffered saline (PBS) solutions. The final concentration of RGE-PBS solution was 10 µg/ml (RGE = 10), 25 µg/ml (RGE = 25), 50 µg/ml (RGE = 50), 100 µg/ml (RGE = 100), 500 µg/ml (RGE = 500) and 1000 µg/ml (RGE = 1000). (B) Western blot analysis of protein expression of SDF-1α/CXCR4 cascade with EPCs treated by different concentrations of RGE-PBS solutions. (C) Western blot analysis of protein expression of SDF-1α/CXCR4 cascade with EPCs stimulated for different times. (D) Expression of SDF-1α/CXCR4 cascade in EPCs with RGE treatment. The final concentration of RGE-PBS solution was 100, 50, 25 and 10 µg/ml. The duration of stimulation was 72 h. Control, culture with only EBM-2. Inhibitor, pre-stimulated with specific CXCR4 antagonist AMD3100 (5 µg/ml) for 1 h, then with RGE (100 µg/ml, 72 h). (E) Expression of SDF-1α/CXCR4 cascade with EPCs stimulated for different times. The final concentration of RGE used to stimulate EPCs was 25 µg/ml for CXCR4 and 50 µg/ml for SDF-1α. Control, culture with EBM-2 without RGE for 72 h. RGE groups, stimulation for 6, 12, 24, 48 and 72 h. (F) EPCs’ tube-formation capacity in vitro. C, control group culture with only EBM-2. AMD, pre-stimulation with AMD3100 (5 µg/ml) for 1 h and then RGE at 25 µg/ml for 48 h. RGE25-48 h, RGE at 25 µg/ml for 48 h. RGE50-24 h, RGE at 50 µg/ml for 24 h. Data are ratio of EPCs integrated in tubular-like structure (marked by red @) and the tubes involved (marked by red arrow). Each test was repeated 3 times and every group had 3 repetitive wells on one 6-well plate. Data are mean ± SD. *’P<0.05, *P<0.01 vs. all the other groups.
Discussion
The traditional Chinese herb Rehmannia glutinosa can promote bone-marrow proliferation and protect the ischemic myocardium without mechanism studied [15], [16]. And the EPCs are attractive targets for repair of the ischemic myocardium [1]–[3]. Therefore, we investigated the effect of RGE on EPCs in a rat model of MI. In preliminary experiments, 3 different oral doses of RGE given to normal rats could increase the number of EPCs in peripheral blood and bone marrow at 8th to 16th weeks. Among these, the high dose had the most significant effect at 8th to 12th weeks (P<0.01; TableS1). The preliminary experiment showed that the RGE made effect on EPCs needed a relatively long time (about 12 weeks). And the mortality of rats after MI will increase with the time goes by. So we orally fed high-dose RGE (1.5 g·kg-1·day-1) to rats 8 weeks before MI induction and 4 weeks after MI. RGE significantly improved ischemic myocardium and protect left ventricular function after MI. As well, RGE activated EPCs by promoting their proliferation, mobilization, migration and participating in therapeutic angiogenesis at the ischemic region. It also up-regulated the expression of angiogenesis-associated ligand/receptor CD133, VEGFR2, SDF-1α and CXCR4. As these effects of RGE almost occurred at the chronic stage after MI (systemic delivered for 10 to 12 weeks), we suggested that patients with MI might benefit from RGE in chronic stage rather than acute stage. RGE could be an EPC activator mediated by SDF-1α/CXCR4 cascade activation, thus preserving the ischemic myocardium in rats.
The changes showed in ECG, UCG and significant increased Tn-T, BNP level revealed the successful establishment of the MI model, with no difference between NS and RGE in 3 days after MI. Thus, RGE might not produce effects in a relatively short time. RGE began to have effects in the chronic stage of MI (from weeks 2 to 4). In the chronic stage after MI, the LV-mass was lower with RGE than NS because of the decreased LV-d and LV-s, the LVEF and LVFS were higher with RGE than NS, although still less than those in the blank and mock groups. RGE ameliorated the MI-induced Tn-T increasing before the NS effected and prevented the BNP level from increasing as with NS. The relative ischemic area and myocardial apoptotic index in the infarcted myocardium was decreased with RGE than with NS. Therefore, at the chronic stage of MI, RGE could preserve the ischemic myocardium by enhancing the function of the left ventricle, decrease the risks of acute coronary syndromes associated with increased BNP [30] and apoptosis in the myocardium.
To determine whether the RGE’s effects on ischemic myocardium are associated with its effects on EPCs showed in preliminary experiments, we examined the number and function of EPCs obtained from peripheral blood and bone marrow of rats. Stem cells, including EPCs, can be mobilized from the bone marrow and other niches, homing to the area of injured tissue and trans-differentiating into functional cardiomyocytes [4]–[5], [31]–[32]. Here we defined EPCs as cells that can absorb ac-LDL and UEA-1 [23] and counted the number of cells positive for CD34, CD133 and VEGFR2, widely accepted markers of EPCs [33]–[35]. In peripheral blood, the number of EPCs increased in acute stage after MI and it went on increasing with RGE but not NS in the chronic stage. In bone marrow, the EPC number decreased with NS because of EPCs mobilizing from bone marrow to blood, while it maintained high with RGE. The increase in RGE-b and RGE-m groups compared with respective NS groups coincided with our preliminary study. In the chronic stage of MI, RGE statistically activated EPC function as compared with NS: in increased EPCs proliferation, migration and tube-formation capacity. These effects occurred later after MI, especially for migration made effect until week 4, might attribute to multitudinous EPCs migration occurred at the terminal stage of acute cardiovascular event and the slow-release of RGE. In general, RGE could increase the number of EPCs in normal and MI rats by increasing the storage in bone marrow and increasing the mobilization to peripheral blood, then migration to injured tissue. Besides increasing cellular number, RGE also activated the function of EPCs, which made them available for the injured myocardium.
Angiogenesis is the most important way to improve the supply of blood to the infarcted myocardium and an important potential role for EPCs, especially for development of new capillaries in adults [36]–[38]. The detection of new capillaries may be an effective way to explain the relationship between the effect of RGE on the infarcted myocardium and on EPCs. CD133 and VEGFR2, markers of EPCs which participated in new-born capillary, are also effective markers of early stage angiogenesis [33]–[36]. We tested the levels of thm in infarcted tissue to reflect the level of angiogenesis. The expressions of CD133 and VEGFR2 were greater with RGE than NS at the chronic stage of MI. From these we suggested that RGE activated the EPCs with CD133 and VEGFR2 migrating to ischemic region, then increased them participated in capillary-like tube formation, and further developed to new-born capillary which highly expressed CD133, VEGFR2. Therefore, RGE is able to increase new-born capillary formation. Combined with RGE’s effect on increasing the number and function of EPCs, RGE protect the myocardium after MI through angiogenesis mediated by EPCs.
The expression of the SDF-1α/CXCR4 cascade was increased with RGE after MI. SDF-1α–CXCR4 interaction plays a crucial role in recruiting EPCs to the heart after MI and could increase homing, thus inducing border-zone angiogenesis and preserving ventricular function [12]–[13]. We observed this effect of RGE on the SDF-1/CXCR4 cascade after MI, the expression of CXCR4 was up-regulated while there was no statistic different in SDF-1α expression. When SDF-1α reactive with CXCR4, Arg8 and Arg12 of SDF-1α bind with Glu15 and Asp20 of CXCR4 firstly, and make the disruption of the salt bridge between Arg188 and Glu277 in CXCR4, then Lys1 of SDF-1α bind with Asp262 which was exposed from the disrupted salt bridge in CXCR4, in this way activate SDF-1α/CXCR4 cascade and signal transduction down-stream [39], [40]. Thus, we suggested that RGE activated SDF-1α/CXCR4 cascade mainly through increasing the expression of CXCR4 and activating SDF-1α/CXCR4 interaction mediated by CXCR4, then the EPCs were mobilized and homing to the injured region [9], [13]. In this way, the SDF-1α/CXCR4 cascade was involved in mediating RGE’s effects. To confirm our finding and search for the therapeutic theory of RGE, we used RGE-PBS solution to stimulate EPCs in vitro. The expressions of both SDF-1α and CXCR4 were higher with RGE than the control group. RGE was able to up-regulate tube-formation capacity of EPCs at its optimal actuation concentration and duration. When stimulated with RGE at its optimal actuation concentration and duration for CXCR4 and SDF-1α, the tube-formation capacity of EPCs was up-regulated. When SDF-1α/CXCR4 was blockade by specific CXCR4 inhibitor AMD3100, RGE had no effect on EPCs, CXCR4 showed poor expression and its ligand SDF-1α showed over-expression. Therefore, RGE promoted EPCs function by up-regulating the expression of the SDF-1α/CXCR4 cascade, and with the SDF-1α/CXCR4 cascade blocked, the effects of RGE were eliminated. RGE may mobilize EPCs in bone marrow and for migration to the injured myocardium, thus enhancing local angiogenesis after MI, with the SDF-1α/CXCR4 cascade involved in mediating RGE’s effects on EPCs after MI.
Although RGE was alcohol extracted from the herb Rehmannia glutinosa, the specific structures and molecular formulas of RGE remain to be clarified. As well, we certified that the activation of SDF-1α/CXCR4 cascade was involved in mediating RGE-associated EPC activation after MI, but the detailed genetic loci underlying require further investigation.
In summary, we demonstrated that in rats with MI, extracts of the herb Rehmannia glutinosa promoted the mobilization of EPCs in bone marrow, enhanced their migration to the local ischemic region and participation in angiogenesis, thus preserving the ischemic myocardium. The mechanism may involve mediation by the SDF-1α/CXCR4 cascade.
Supporting Information
Table S1
Effect of RGE on endothelial progenitor cell (EPC) number. Control, rats oral-treated with normal saline (NS); RGE-L, rats oral-treated with RGE at 0.38 g·kg−1·day−1; RGE-M, rats oral-treated with RGE at 0.75 g·kg−1·day−1; RGE-H, rats oral-treated with RGE at 1.5 g·kg−1·day−1. Data are mean ± SD. ∧P<0.05, *P<0.01 vs. control group.
(DOCX)
Click here for additional data file.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23349727PONE-D-12-1692410.1371/journal.pone.0053654Research ArticleBiologyGeneticsCancer GeneticsMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesOncogenic SignalingMedicineOncologyBasic Cancer ResearchTumor PhysiologyCancer TreatmentGene TherapyThe Different Role of Notch1 and Notch2 in Astrocytic Gliomas Notch1 and Notch2 in GliomasXu Peng
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Zhang Anling
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Jiang Rongcai
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Qiu Mingzhe
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Kang Chunsheng
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2
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Jia Zhifan
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Wang Guangxiu
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Han Lei
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Fan Xing
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Pu Peiyu
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Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, People’s Republic of China
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Tianjin Neurological Institute, Tianjin, People’s Republic of China
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Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin, People’s Republic of China
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Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin, People’s Republic of China
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Assistant Professor of Neurosurgery and Cell & Developmental Biology, University of Michigan Medical School, Department of Neurosurgery, Ann Arbor, Michigan, United States of America
Cheng Jin Q. Editor
H. Lee Moffitt Cancer Center & Research Institute, United States of America
* E-mail: [email protected] (XF); [email protected] (PYP)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: PYP XF. Performed the experiments: PX ALZ. Analyzed the data: RCJ MZQ CSK. Contributed reagents/materials/analysis tools: GXW ZFJ LH. Wrote the paper: PX.
2013 21 1 2013 8 1 e5365412 6 2012 4 12 2012 © 2013 Xu et al2013Xu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.
This work was supported by The China National Natural Scientific Fund (30300365). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Notch signaling plays a pivotal role in the regulation of many fundamental cellular processes such as proliferation, stem cell maintenance, differentiation during embryonic and adult development and homeostasis of adult self-renewing organs [1]. Accumulating evidences have shown that alteration of Notch signaling plays an important role in a wide range of human neoplasms including brain tumors [2]–[13]. In central nerve system (CNS), Notch signaling is thought to maintain a pool of undifferentiated progenitors by inhibiting neuronal commitment and differentiation into neurons [14]. However, in some scenario, Notch activation promotes a particular cell fate, especially in the differentiation of certain types of glia such as radial glia and astrocytes [15], [16]. Gliomas which may arise from tumorigenic events within all steps of maturation from neural stem cell (NSC) to neurons or glia display diverse expression profiles of the Notch signaling, reflecting the cell of origin. Recent studies imply that Notch signaling plays different roles in the tumorigenesis of low-grade astrocytomas and secondary GBM when compared with primary GBM, possibly indicating that these tumors originate from different precursor cells [17]–[19].
In our previous study, we found that Notch family members were differentially expressed in astrocytic gliomas and medulloblastoma, Notch1 was highly expressed but Notch2 nearly not expressed or barely detectable in astrocytic gliomas. Fan et al also found that the percentage of immunopositive tumor cells and expression level of Notch1 were increased with tumor grade [13]. On the other hand, overexpression of Notch2 was detected in medulloblastomas in contrast with low or no expression of Notch1 [13], [20]. The different outcome of Notch signaling in cancer may be attributed to its intricate roles in cell/organ development process.
We had studied the effect of downregulation of Notch1 expression by siRNA on glioblastoma (GBM) cells with Notch1 overexpression and found a significant growth inhibition of GBM cells in vitro and in vivo
[21]. For further elucidating the distinct roles of Notch 1 and Notch 2 in the development and progression of astrocytic gliomas, in the present study, the effect of enforced expression of Notch2 with transfection of Notch2 plasmid in cultured malignant glioma cells and xenograft gliomas in nude mice was studied as compared to that of knocking down Notch1. The activity of EGFR/PI3k/AKT pathway was detected to explore whether Notch signaling cooperated with the major aberrant EGFR/PI3K/AKT signaling pathway of gliomas participating in the progression of astrocytoma.
Materials and Methods
Cell Culture and Transfection
Human glioblastoma U251 and A172 cell lines were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science. Cells were cultured in DMEM supplemented with 10% fetal calf serum. Twenty four hours after plating in 60 mm plates and washing with DMEM, cells were transfected with empty vectors (pcDNA3) and Notch 2 plasmid (Notch2- pcDNA3, kindly provided by Dr Xin Fan, Johns Hopkins University, USA). Briefly, 2 µg plasmid DNA (pcDNA3 or Notch2-PCDNA3) in 100 µl serum-free DMEM mixed with 100 µl DMEM containing 10 µl lipofectamine 2000 (Invitrogen, USA). after standing for 45 min, they were added into cell plates together with 800 µl DMEM. Six hours later, serum-free medium was replaced with DMEM supplemented with 10% fetal calf serum and cells were incubated at 37°C in 5% CO2. After 48 hours, cells were passaged with 1∶10 ratio and screened with G418 (SIGMA, USA). Medium was replaced every 3–5 days and G418 resistant clones were selected and expanded.
Western Blot Analysis
Total proteins were extracted from U251 and A172 glioma cells, cells were transfected with empty vector and Notch2 plasmid, respectively. The protein concentration was determined by Lowry method. Forty microgram of protein lysates from each sample was subjected to SDS-PAGE on 10% SDS-polyacrylamide gel. The separated proteins were transferred to a PVDF membrane and the membrane was incubated with primary antibodies (1∶500 dilution), followed by HRP-conjugated secondary protein (1∶1000 dilution, Zymed, USA). The specific protein was detected using a SuperSignal protein detection kit (Pierce, USA). After washing, the membrane was rehybridized with a primary antibody against β-actin (1∶500 dilution), using the same procedures described above. The band density of specific proteins was quantified after normalization with the density of β-actin. EGFR, phosphorylated AKT (p-AKT), PI3K, NF-κB, PCNA, Bcl-2, Caspase-3, CyclinD1, MMP2 and MMP9 were detected (all the antibodys ordered from Santa Cruz Biotech Corp, USA).
MTT [3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium Bromide] Assay
The growth rate of control and transfected U251 and A172 cells was measured by MTT assay. Briefly, 4×103 cells per well were plated into a 96-well plate. On each day of consecutive 6 days after plating, 20 µl (5 mg MTT/ml) was added to each well and the cells were incubated at 37°C for additional 4 h. The reaction was then terminated by lysing the cells with 200 µl of DMSO for 5 min. Optical density was measured in triplicate at 570 nm and expressed as percentage of control.
Flow Cytometric Analysis of Cell Cycle Kinetics
Parental and transfected U251 and A172 cells in the log phase of growth were harvested and incubated with RNase at 37°C for 30 min. The cell nuclei were stained with propidium iodide for an additional 30 min. A total of 10,000 nuclei were analyzed by FacsCalibur flow cytometer and the DNA histograms were generated by Modifit software (Becton Dickinson, USA).
Measurement of Apoptosis by Annexin V and TUNEL Staining
Annexin V-cy3-labeled Apoptosis Detection Kit 1 (Abcam, USA) was used for detection of apoptotic cells by flow cytometry. Data were analyzed by Cell Quest software (Becton Dickinson, USA). The extent of apoptosis in the tumor specimens of mouse models from in vivo study was evaluated by TUNEL method using an in situ Cell Death Kit (Roche, USA). Cell nuclei were counterstained with Hoechst 33342 and visualized by fluorescent microscopy and analyzed by IPP5.1 (Olympus, Japan).
Transwell Assay
Transwell filters (Costar, USA) were coated with Matrigel (3.9 µg/µL, 60–80 µL) on the upper surface of the polycarbonic membrane (diameter 6.5 mm, pore size 8 µm). After incubation at 37°C for 30 min, Matrigel became solidified and simulated the major components of extracellular matrix (ECM) for tumor cell invasion. Transfected and control cells (1×105) suspended in 200 µL of serum-free DMEM were added to the upper chamber and conditional medium of tumor cells was placed into the lower chamber as a chemo-attractant. After 24 hr of incubation at 37°C in 5% CO2, the medium was removed from the upper chamber. The non-invaded cells on the upper surface of the inserted filter were gently scraped off with a wet cotton swab. The cells that had invaded the lower surface of the filter were fixed with 4% paraformaldehyde and stained with hematoxylin. The migrated cells were counted by light microscopy (200× magnification) and the average number of cells of at least five fields from each well was calculated.
Establishment of Subcutaneous Xenograft Glioma Model and Treatment with Notch1 siRNA and Notch2 Plasmid
Six week-old female immune-deficient nude mice (BALB/C-nu) were purchased from the animal center of the Cancer Institute, Chinese Academy of Sciences, bred at the facility of laboratory animals, Tianjin Medical University, and housed in microisolator individually ventilated cages with water and food. All experiments were carried out according to the regulations and internal biosafety and bioethics guidelines of Tianjin Medical University and Tianjin Municipal Science and Technology Commission.
A subcutaneous U251 glioma xenograft model was established as described previously [22]. Once the tumor size reached approximately 5 mm in diameter, the mice were randomly divided into six groups: 1) Control group with tumors untreated; 2) Scramble Notch1 siRNA (scr-siRNA) treated group; 3) Notch1 siRNA treated group, sequence as previous study used. (5′-UGGCGGGAAGUGUGAAGCG-3′, Gima Biol Engineering, Shanghai, China) 4) pcDNA3 empty vector treated group; 5) Notch2 plasmid treated group and 6) Combined Notch1 siRNA with Notch2 plasmid treated group. Each group consisted of eight mice. Mice were injected intratumorally with 25 µl siRNA/oligofectamine mixture containing 400 pmol siRNA or/and 10 µg Notch 2 plasmid every four days; the same dosage of scramble siRNA or pcDNA3 empty vector were used. During 32 days of observation period, the tumor volume was measured with a caliper every three days using the following formula: volume = length × width2/2.
At the end of observation period, the mice were sacrificed and the removed tumor specimens were prepared as paraffin embedded sections for detection of the expression of Notch1, Notch2, AKT, p-AKT, PI3K, p53, MMP9, cyclinD1 and PCNA by immunohistochemical staining. Apoptosis in the tumor specimens was determined by TUNEL method as previously described.
Results
Expression of Notch in Control and Transfected U251/A172 Cells
After transfection with Notch1 siRNA and Notch2 plasmid, the expression of Notch1 and Notch2 was detected by Western blot. As shown in Figure 1, Notch1 was remarkably downregulated while Notch2 was upregulated to a high level in U251 and A172 cells.
10.1371/journal.pone.0053654.g001Figure 1 Expression of Notch1 and Notch2 in U251/A172 cells transfected with Notch1 siRNA or Notch2 construct by Western blot.
Notch1/2 expression of U251/A172 cells transfected with Notch1 siRNA or Notch2 construct as shown by western blot.
Effect of Notch2 Upregulation on U251/A172 Cell Growth, Apoptosis and Invasion
The cell growth rate of parental and transfected U251/A172 cells was examined by MTT assay. As compared with the parental cells, the growth rate of cells transfected with Notch2 plasmid was inhibited since 24 h following transfection and the suppressive effect tended to be steadily increased during six days of observation, whereas the cells transfected with empty vector was not affected (Fig. 2A).
10.1371/journal.pone.0053654.g002Figure 2
In vitro study of proliferation, apoptosis and invasive ability in U251 cells transfected with Notch1 siRNA.
A. Proliferation rate of U251/A172 cells transfected with Notch2 construct determined by MTT assay. B. Cell cycle analysis of U251/A172 cells transfected with Notch2 construct examined with flow cytometry. C. Apoptosis of U251/A172 cells transfected with Notch2 construct detected with Annexin V staining by flow cytometry. D. Invasive ability of U251/A172 cells transfected with Notch2 construct examined by transwell assay. *: p<0.05.
Cell cycle kinetics examined by flow cytometry showed that Notch2 upregulation of U251/A172 cells resulted in the decrease of S phase fraction and arrest of cells in G0/G1 phase (Fig. 2B), whereas the number of apoptotic cells evaluated by Annexin V labeling was significantly increased (Fig. 2C).
The invasive ability of parental and transfected cells was assessed by transwell assay. For both U251 and A172 cells, the number of invasive cells in Notch2 plasmid transfected groups decreased to 50% of that in the control groups (F = 20.343, p = 0.000 in U251 cells; F = 19.265, p = 0.001 in A172 cells). This result suggests that enforced expression of Notch2 attenuates the aggressive capability of malignant glioma cells (Fig. 2D).
Expression of Proteins Involved in EGFR/PI3K/AKT Core Signaling Pathway of Gliomagenesis in Control and Notch 2 Plasmid Transfected U251/A172 Cells
The upregulation of Notch2 altered the activity of EGFR/PI3K/AKT core signaling pathway significantly. The results of western blot showed us that the expression of EGFR, PI3K, p-AKT and NF-κB proteins decreased, while PTEN, suppressor of this pathway, was upregulated. Other indexes for cell biological behavior, including PCNA for cell proliferation, Bcl-2 and Caspase-3 for cell apoptosis, CyclinD1 for cell cycle and MMP2/9 for cell invasion, all altered obviously following Notch2 ectopic overexpression (Fig. 3).
10.1371/journal.pone.0053654.g003Figure 3 Expression of EGFR/PI3K/AKT pathway relative genes in U251/A172 cells transfected with Notch2 construct by Western blot.
Western blot analysis of EGFR, phosphorylated AKT (p-AKT), PI3K, NF-κB, PCNA, Bcl-2, Caspase-3, CyclinD1, MMP2 and MMP9 expression in U251/A172 cells transfected with Notch2 construct.
Effect of Notch1 siRNA and Enforced Expression of Notch2 on the Xenograft Tumor Growth
Effect of Notch1 siRNA and enforced expression of Notch2 in vivo was investigated using U251 subcutaneous xenograft glioma model. Nude mice bearing the largest and smallest tumors were eliminated from the study. The mean volume of the tumors was 81.55±22.86 mm3 before treatment. During the first 2 weeks of observation period, the tumors in either control or treated groups grew slowly and revealed no difference in tumor size. As shown in Figure 4, since day 16 after implantation, especially from day 24, the tumors in the control, scr-siRNA and pcDNA3 empty vector treated mice had been growing rapidly until to the end of observation period on day 25. However, the tumors in Notch1 siRNA, Notch2 plasmid and combined Notch1 siRNA and Notch2 plasmid treated groups still maintained the slower growth rate and had been shown significant difference of tumor volume in the last half of observation period (p<0.01), but there were no superimposed effect in combination of Notch1 siRNA and Notch2 plasmid treated group. The tumors removed from the control, scr-siRNA and pcDNA3 empty vector treated mice were large and exhibited hemorrhage, liquidation and necrosis macroscopically, whereas the tumors resected from the Notch1 and Notch2 treated mice were small, solid and few necrotic foci.
10.1371/journal.pone.0053654.g004Figure 4
In vivo study of nude mice treated with Notch1 siRNA and Notch2 plasmid.
Tumor growth in nude mice treated with Notch1 siRNA and Notch2 construct compared to that in control, scr-siRNA and empty vector treated mice. *: p<0.05.
Expression of Proteins Related to EGFR/PI3K/AKT Signaling Pathway in Xenograft Tumors Treated with Notch1 siRNA and Notch2 Plasmid
Similar to the results obtained from in
vitro studies, the expression of Notch1, AKT, p-AKT, PI3K, Bcl-2 MMP9, and cyclinD1 in tumor specimens from Notch1 siRNA, Notch2 plasmid and those two combined treated mice was decreased. Meanwhile, the expression of Notch2, PTEN and p53 were increased. In addition, the PCNA expression, a marker of cell proliferation activity, was reduced (Fig. 5).
10.1371/journal.pone.0053654.g005Figure 5 Expression of EGFR/PI3K/AKT pathway relative genes in xenograft tumors was detected by Immunohistochemistry.
The expression of Notch1, PCNA, P53, MMP9, PI3K, p-AKT, Bcl-2 and Cyclin D1 in Notch1 siRNA,Notch2 construct and those two plus group treated mice treated tumors compared with that in control, scr-siRNA and empty vector treated tumors (×200).
Detection of Apoptosis in Xenograft Tumor Samples
The apoptosis in tumor samples obtained from control and treated mice were examined by TUNEL staining. The number of apoptotic cells was significantly increased in the tumors treated with Notch1 siRNA, Notch2 plasmid and those two combined group as compared to that in control, scr-siRNA and pcDNA3 empty vector treated mice (Fig. 6).
10.1371/journal.pone.0053654.g006Figure 6 Apoptosis cells in xenograft tumors was detected by TUNEL.
Apoptosis cells in xenograft tumors, tumors treated with scr-siRNA, empty vector, Notch1 siRNA, Notch2 construct and those two plus group detected by TUNEL method (×200).
Discussion
Astrocytic gliomas, particularly glioblastomas (GBMs), are the most common and highly aggressive primary brain tumor. Current standard of care therapy results in medium survival only 12–15 months [23]. Treatment of GBM is a considerable therapeutic challenge. Thus, it is imperative to understand its molecular pathology for development of novel therapeutic strategies [24]. In the past several years, Notch deregulation has been shown to be involved in a wide range of tumors. Notch plays an oncogenic activity or tumor suppressive role in various tumors that depends on the cellular context [25] or may be a matter of Notch expression level, as observed in neural stem cells [26]. Notch signaling, a major player in normal development of the central nervous system, is often dysregulated in brain tumors [27]. Fan et al has reported that Notch1 expression is rarely detected or undetectable while Notch2 is highly expressed in medulloblastomas [13]. Moreover, in nonneoplastic meninges and meningiomas, Notch2 and Jagged1 are the main components expressed, whereas the Notch1 homologue is expressed at much lower levels [12]. In our previous study, we identified the differential expressions of Notch family members between astrocytic gliomas and medulloblastomas. Contrary to meduuloblastomas, Notch1, 3, 4 were highly expressed but Notch2 was reduced or lack of expression in astrocytic gliomas. These findings indicate that there are different expression patterns of Notch members among various intracranial neoplasms. Whether the differential expression of Notch1 and Notch2 in different types of brain tumors is attributed to the different cellular context and the role they play in the development of the tumor progenitor cells need to be further explored.
Our previous study had shown that knocking down Notch1 overexpression in U251 glioblastoma cells with siRNA significantly suppressed the cell growth and invasion, and induced cell apoptosis in vitro and in vivo. In the present study, We tried to explore the role of Notch2 in astrocytic gliomas by upregulation of Notch2 expression with transfection of Notch2 plasmid, and found that enforced overexpression of Notch2 in glioma cells was identically associated with the inhibition of cell proliferation, arrest of cell cycle, reduction of invasiveness of tumor cells and induction of cell apoptosis as shown by in vitro study. Furthermore, the tumor growth in vivo was decelerated after treatment with Notch2 plasmid in established subcutaneous xenografts of nude mice or treatment with Notch1 siRNA, but combination of these two treatments did not show more efficient than using them singly, indicating that simultaneous downregulation of Notch1 and upregulation of Notch2 had no superimposed effects on biological behaviour of GBM cells. These evidences imply that Notch1 may play an oncogenic role while Notch2 maybe function as tumor suppressor in the development and progression of astrocytic gliomas. Moreover, Fan et al have found that transfection with constitutively active form of Notch1 or Notch2 has antagonistic effects on cell growth in medulloblastoma cell line DAOY. Overexpression of Notch2 promotes while overexpression of Notch1 inhibits the cell proliferation, soft agar colony formation and xenograft growth. These findings have been further confirmed by knocking down Notch1 and Notch2 with siRNA [13], and strongly indicate that the effect of Notch1 and Notch2 in these embryonic brain tumors is quite different in the oncogenic role which is similar to what we have observed in the astrocytic gliomas.
Since the Notch signaling is versatile in different events occurred during embryogenesis, it is possible that the consequence of Notch alteration is depending on biological background in a given tissue. Notch1 has been shown recently to promote the differentiation of various glial cell types, including Schwann cells in the peripheral nervous system, radial glia cells in developing central nervous system and Muller cells in retina. It has been proposed that many of the radial glial cells are developed into astrocytes. On the contrary, Notch1 is not expressed in proliferating cerebellar precursors, but expressed in differentiated internal granular layer neurons, whereas Notch2 is expressed in external granule cell layer of developing cerebellum (rodent cerebellar granule cell precursors) and acts as a mitogen, and its expression negatively correlates with glial differentiation in mammalian brain development [28]–[31]. These results seem to contradict to what we have found in astrocytic glioma cell. However, chaotic genomic defects may contribute to the disordered response of glioma cell to Notch signaling, after all Notch signaling pathway involved both in maintaining a pool of undifferentiated progenitors cells and determining proper differentiation of progeny cells. Accordingly, the differential expression and function of Notch1 and Notch2 in different types of brain tumors should be studied in depth.
The aberrant Notch signaling pathway in tumorigenesis has been widely discussed. In addition to the activation of its downstream target genes such as HES, c-myc, cyclin D1, there are many clues to show interaction between Notch with other signaling pathways, such as p53, Ras, NF-κB, Wnt, Shh, TGF-β, PI3K and EGFR [32]–[42]. EGFR/PI3K/AKT signaling pathway plays a major oncogenic role in GBM, we measured the activity of EGF pathway after intervening the Notch signaling pathway, and found that Notch2 ectopic expression downregulated the EGFR expression as well as its downstream signaling proteins, including PI3K, p-AKT, which were essential to cell survival and proliferation in gliomas, also, affected the expression of protein relevant to cell invasion and apoptosis, such as MMP2, MMP9, Bcl-2 and Caspase-3. It revealed that there was a crosstalk existing between Notch and EGFR/PI3K/AKT pathway and might contribute to the effect of Notch signaling on glioma cell growth, apoptosis and invasion processes. Several lines of evidences also indicate that the Notch pathway is intimately coupled to signaling through EGFR, or downstream targets, in both normal development and in the onset and maintenance of cancer [43], [44]. Physical interactions between Notch target gene products HES1 and HEY1 with Stat3 point to crosstalks between Notch and Stat3-activating pathways such as Gp130/Jak2/stat3 and Sonic hedgehog (Shh) [45], [46]. In parallel, Shh is also capable of stimulating HES1 transcription [47]. Besides, β-catenin has been shown to interact with Notch and RBPJk to induce HES1 transcription, making crosstalk between Wnt and Notch pathways [48]. Thus, it can be supposed that all these pathways interconnect as a network contributing to the glioma formation and progression.
In conclusion, our findings in the present study suggest the activated Notch1 and suppressed Notch2 activity in astrocytic gliomas have an important impact on the biological behavior of astrocytic glioma. These effects to some degree may attribute to the modulation of EGFR/PI3K/AKT signaling pathway caused by Notch. The disparate expression and effect of Notch1 and Notch2 in different type of brain tumors is hypothesized as response of different cellular context to Notch signaling. The exact underlying mechanisms should be further investigated.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23372666PONE-D-12-3001910.1371/journal.pone.0053725Research ArticleBiologyImmunologyImmunityInflammationMicrobiologyImmunityInflammationChemistryAnalytical ChemistryChromatographyMedicineClinical ImmunologyImmunityInflammationGastroenterology and HepatologyInflammatory Bowel DiseaseNutritionApple Peel Polyphenols and Their Beneficial Actions on Oxidative Stress and Inflammation Antioxidant and Antiinflammatory Effects of ApplesDenis Marie Claude
1
2
Furtos Alexandra
3
Dudonné Stéphanie
4
Montoudis Alain
1
Garofalo Carole
1
Desjardins Yves
4
Delvin Edgard
1
3
Levy Emile
1
2
4
*
1
Research Centre, Sainte-Justine Hospital, Montreal, Quebec, Canada
2
Department of Nutrition, Faculty of Medicine, Université de Montréal, Montreal, Quebec, Canada
3
Department of Biochemistry, Faculty of Medicine, Université de Montréal, Montreal, Quebec, Canada
4
Institute of Nutraceuticals and Functional foods, Université Laval, Quebec, Quebec, Canada
Kaveri Srinivas Editor
Cordelier Research Center, France
* E-mail: [email protected] Interests: The authors received funding from Leahy Orchards Inc. and Appleboost Products Inc. However, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: MCD AF EL. Performed the experiments: CG AM YD SD. Analyzed the data: MCD ED EL. Contributed reagents/materials/analysis tools: CG AM YD SD. Wrote the paper: EL.
2013 23 1 2013 8 1 e5372529 9 2012 4 12 2012 © 2013 Denis et al2013Denis et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Since gastrointestinal mucosa is constantly exposed to reactive oxygen species from various sources, the presence of antioxidants may contribute to the body’s natural defenses against inflammatory diseases.
Hypothesis
To define the polyphenols extracted from dried apple peels (DAPP) and determine their antioxidant and anti-inflammatory potential in the intestine. Caco-2/15 cells were used to study the role of DAPP preventive actions against oxidative stress (OxS) and inflammation induced by iron-ascorbate (Fe/Asc) and lipopolysaccharide (LPS), respectively.
Results
The combination of HPLC with fluorescence detection, HPLC-ESI-MS TOF and UPLC-ESI-MS/MS QQQ allowed us to characterize the phenolic compounds present in the DAPP (phenolic acids, flavonol glycosides, flavan-3-ols, procyanidins). The addition of Fe/Asc to Caco-2/15 cells induced OxS as demonstrated by the rise in malondialdehyde, depletion of n-3 polyunsaturated fatty acids, and alterations in the activity of endogenous antioxidants (SOD, GPx, G-Red). However, preincubation with DAPP prevented Fe/Asc-mediated lipid peroxidation and counteracted LPS-mediated inflammation as evidenced by the down-regulation of cytokines (TNF-α and IL-6), and prostaglandin E2. The mechanisms of action triggered by DAPP induced also a down-regulation of cyclooxygenase-2 and nuclear factor-κB, respectively. These actions were accompanied by the induction of Nrf2 (orchestrating cellular antioxidant defenses and maintaining redox homeostasis), and PGC-1α (the “master controller” of mitochondrial biogenesis).
Conclusion
Our findings provide evidence of the capacity of DAPP to reduce OxS and inflammation, two pivotal processes involved in inflammatory bowel diseases.
This study was supported by the J. A. DeSève Research Chair in Nutrition, the Leader Canadian Foundation of Innovation (EL) and scholarship award from Fonds de recherche du Québec-Nature et technologies (MCD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Gastrointestinal mucosa is constantly exposed to luminal oxidants from ingested nutrients, such as alcohol, cholesterol oxides, and key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules [1], [2]. Moreover, local microbes or infections, ischemia/reperfusion, gastric acid production and nonsteroidal anti-inflammatory drugs may promote the formation of reactive radicals [3]–[5]. Additionally, the intestinal mucosa is subject to prolonged oxidative stress (OxS) from reactive oxygen species (ROS) generated during aerobic metabolism [6], [7]. The influx of neutrophils and monocytes associated with inflammation can further generate ROS via respiratory burst enzymes as well as those involved in prostaglandin and leukotriene metabolism [8]. Even if the etiology of inflammatory bowel diseases (IBD) has yet to be fully elucidated, a close relationship has been noted between ROS and the mucosal inflammatory process [9]–[13]. Although the specific events by which oxidants contribute to inflammation are not entirely elucidated, potential mechanisms include the activation of cyclooxygenage-2 (COX-2) and the transcription factor nuclear factor-kappa B (NF-κB) by pro-oxidants, thereby resulting in the initiation of the expression of genes controlling several aspects of the inflammatory, immune and acute phase responses [14]–[18].
Current epidemiological and experimental studies support a beneficial role of dietary polyphenols in several gastrointestinal diseases, including IBD [19]–[21]. Polyphenols are the most abundant antioxidants in the diet, (i.e. fruit, vegetables, beverages, herbs and spices) [22]–[26]. However, their poor intestinal absorption is responsible for luminal concentrations of phenolic compounds up to several hundred µmols in the gastrointestinal tract [27]. Most of these polyphenols exhibit powerful antioxidant activity by acting as free radical scavengers, hydrogen donating compounds, singlet oxygen quenchers and metal ion chelators, while they are also able to induce cellular antioxidant defense modulating protein and gene expressions [22], [24], [25], [28]. In the present investigation, we hypothesize that apple peel-derived polyphenols act in the gut as powerful antioxidants and anti-inflammatory agents capable of exerting protective effects against harmful intraluminal components in the gut, which may maintain the body’s natural defenses against a variety of intestinal diseases, including IBD.
Materials and Methods
Chemical and Reagents
HPLC-grade acetonitrile, methanol, acetone and Optima grade water were from Fisher Scientific (New Jersey, USA). Formic acid was purchased from Fluka (Steinheim, Germany). MTT was from Sigma (MO, USA). Apple peel crude extract (AB powder) and a purified polyphenolic fraction (JC-047) derived from dried apple peel powder (DAPP) were supplied from Leahy Orchards Inc. and AppleBoost Products Inc.
DAPP Extraction
The phenolic compounds of apples (80% McIntosh and 20% a blend of Northern Spy, Cortland, Empire, Ida Red, Jonagold and Spartan) were extracted by a method similar to that reported previously by Liu’s laboratory [22], [29], [30]. Briefly, 25 g apple peels were blended with 200 g chilled 80% acetone solution in a Waring blender for 5 min. The sample was then homogenized for 3 min using a Virtis 45 homogenizer. The slurry was filtered through Whatman No. 1 filter paper in a Buchner funnel under vacuum. The solids were scraped into 150 g of 80% acetone and homogenized again for 3 min before refiltering. The filtrate was recovered and evaporated using a rotary evaporator at 45°C. This residue represented the apple peel crude extract (AB powder) while the purified polyphenolic fraction (JC-047) was isolated by preparative HPLC.
LC-MS Analysis of DAPP Crude Extract and Purified Fraction
A reversed phase LC-MS method has been developed to separate and identify the mass and chemical structure of phenolic compounds derived from crude extract and purified fraction. Separations were performed on HPLC with fluorescence detection and HPLC-ESI-MS TOF (Agilent Technologies, Santa Clara, CA). The chromatographic column was a Halo C18, 3.0×100 mm, 2.7 µm particle sizes (Advanced Materials Technology Inc., Wilmington, DE) maintained at 50°C and operated at 0.3 mL/min. A two-step linear acetonitrile gradient was used for elution. The acetonitrile concentration was increased from 2 to 40% over 20 min then from 40 to 90% over the next 15 min followed by an equilibration step with the initial mobile phase composition for a total run time of 40 minutes. The mass spectrometer was operated in negative electrospray mode with a dual spray configuration allowing for internal calibration and therefore for a very good mass accuracy. This allowed us to extract narrow mass range peaks for quantitation purposes and increase the selectivity of the method. Mass spectra were acquired from m/z 100 to 2000 with an acquisition cycle of 0.89 s and a resolution greater than 10 000. The electrospray voltage was set at 3.5 kV, the fragmentor at 200 V and the source temperature at 300°C. Major phenolic compounds identified by HPLC-ESI-MS TOF were quantified by ultra-performance liquid chromatography system (UPLC) coupled to a tandem quadrupole mass spectrometer (MS/MS QQQ) equipped with an ESI source (UPLC-ESI-MS/MS QQQ). The UPLC-ESI-MS/MS QQQ system consisting of a Waters-ACQUITY UPLC with an AQUITY TDQ mass spectrometer (Waters, MA, USA). An Agilent Plus C18 column (2.1×100 mm, 1.8 µm particle sizes) (CA, USA) was used, and column temperature was maintained at 30°C. The phenolic compounds were separated using a gradient mobile phase consisted of 0.1% formic acid in ultrapure water and acetonitrile (solvent A and B respectively) with the flow rate of 0.4 µL/min. The following gradient was used: 0–8 min, 3–35% B; 8–9 min, 35–60% B; 9–10 min, 60–85% B; 10–11 min, 85% B; 11–11.10 min, 85–3% B; 11.10–14 min, 3% B. Data were acquired by MassLynx V4.1 software and processed for quantification with QuanLynx V4.1 (Waters, MA, USA). The UPLC-ESI-MS/MS QQQ system was operated with an ESI interface in negative ionization mode. Cone and collision gas flow rates, obtained from a nitrogen generator N2 were 80 L/h and 900 L/h, respectively. The mass spectrometer parameters were defined with Waters IntelliStart software (automatic tuning and calibration of the AQUITY TQD), and manually optimized as follow: capillary voltage of 3 kV, source temperature at 130°C and desolvation temperature at 400°C. Cone voltage was 30 V, and collision energy was 18 eV for all phenolic compounds. Quantification was determined using multiple reactions monitoring mode for all transitions of phenolic acids, flavonols, flavan-3-ols, procyanidins and dihydrochalcones.
Determination of Total Phenolic Content of DAPP Crude Extract and Purified Fraction
The total phenolic content of AB powder or JC-047 fraction was determined using the Folin-Ciocalteu method [31], with gallic acid as a main standard. Briefly, 100 µL Folin-Ciocalteu reagent (diluted 10-fold in ultrapure water) and 80 µL sodium carbonate solution (7.5% in ultrapure water) were added to 20 µL MeOH (50% solution of extracts) in a 96-well plate. A blank sample and five calibration solutions of gallic acid (12.5 to 200 µg/mL) were analyzed under the same conditions. After 1 h-incubation at room temperature, the absorbance was measured at 765 nm using a Fisher Scientific Multiskan GO microplate reader (MA, USA). All determinations were carried out in triplicate and results were expressed as percentage of extract weight ± SEM.
Heterogeneity of Fractionated Oligomers and Polymers of DAPP on Normal-phase HPLC
The procyanidin composition of AB powder and purified JC-047 fraction was analyzed as previously described [32] by normal phase analytical HPLC using an Agilent 1260/1290 Infinity system. Samples (5 µL of 25 mg/mL solutions in acetone/ultrapure water/acetic acid, 70∶29.5∶0.5) were injected into the HPLC system, and the separation was performed at 35°C with a flow rate of 0.8 mL/min using a Develosil Diol column (250 mm × 4.6 mm, 5 µm particle size), protected with a Cyano SecurityGuard column (Phenomenex, CA, USA). The elution was performed using a solvent system comprising solvents A (acetonitrile/acetic acid, 98∶2) and B (methanol/water/acetic acid, 95∶3∶2) mixed using a linear gradient from 0% to 40% B in 35 min, 40% to 100% B in 40 min, 100% isocratic B in 45 min and 100% to 0% B in 50 min. The column was re-equilibrated for 5 min between samples. Fluorescence of the procyanidins was monitored at excitation and emission wavelengths of 230 and 321 nm with the fluorescence detector, set to low sensitivity with a gain of 7X for the entire run. Individual procyanidins with DP from DP1 to DP>10 were quantified using an external calibration curve of (-)-epicatechin, taking into account their relative response factors in fluorescence [33]. The results were expressed as percentage of extract weight ± SEM.
Intestinal Caco-2/15 Cell Culture
The human epithelial colorectal adenocarcinoma Caco-2/15 cell line, a stable clone of the parent Caco-2 cells (American Type Culture Collection, Rockville, MD), was obtained from Dr. JF Beaulieu (Department of Cellular Biology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada). Intestinal Caco-2/15 cells were cultured as described previously [34]–[42]. Briefly, they were grown in MEM supplemented with 10% decomplemented fetal bovine serum, 1% Penicillin-Streptomycin and 1% non-essential amino acids (all reagents from GIBCO-BRL, Grand Island, NY) at 37°C, 95% humidity and 5% CO2 as described previously [34]–[42]. Caco-2/15 cells were maintained in T-75 cm2 flasks (Corning Glass Works, Corning, NY) and were split (1∶5) when they reached 90% confluence using 0.05% trypsin-0.5 mM EDTA (GIBCO-BRL). For individual experiments, cells were plated at a density of 1×106 cells/well on six-well culture plates, and were cultured for 10 days postconfluence, a period at which they are highly differentiated and appropriate for experimental treatments [34]–[42]. The medium was refreshed every second day.
Caco-2/15 Cell Integrity
After various treatments, cell integrity was estimated by viability, morphology and differentiation assays. Briefly, cell differentiation was assessed by determination of villin protein expression. Monolayer intactness and physical barrier function were tested by evaluating morphology, transepithelial electric resistance and occludin protein expression. Finally, cell viability was appraised with 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl Tetrazolium Bromid (MTT).
Induction of Oxidative Stress and Inflammation
Differentiated intestinal Caco-2/15 cells were used to study the effects of the aforementioned polyphenols in OxS (Fe, 200 µM/Asc, 2 mM) and inflammation (LPS, 200 µg/mL) [41]. Crude extract (AB powder, 250 µg/mL) and purified (JC-047, 250 µg/mL) fraction were added to the apical compartment of Caco-2/15 cells for 24 h before incubation with iron/ascorbate (Fe/Asc) and/or lipopolysaccharide (LPS) for 6 h at 37°C. In order to distinguish between acute and chronic inflammation, Caco-2/15 cells were also incubated with LPS for a 24-h period. To highlight the mechanisms behind the beneficial actions of DAPP against OxS and inflammation, some experiments were carried out with 50 µM caffeic acid phenethyl ester (CAPE; Sigma, MO, USA) and 0.4 µM indomethacin heptyl esters (Cayman Chemical, Ann Arbor, MI) to inhibit NF-κB and COX-2, respectively.
Lipid Peroxidation
Estimation of lipid peroxidation was assessed by measuring the release of malondialdehyde (MDA) from Caco-2/15 cells exposed to Fe/Asc (200 µM/2 mM) by HPLC. Briefly, proteins were precipitated with 8% sodium tungstate (Na2WO4) (Aldrich, Milwaukee, WI). The protein-free supernatants were then reacted with an equivalent volume of 0.5% (wt/vol) thiobarbituric acid solution (TBA; Sigma, MO, USA) at 95°C for 60 min. After cooling to room temperature, the pink chromogene [MDA-(TBA)2] was extracted with 1-butanol and dried over a stream of nitrogen at 50°C for 3 hours. The dry extract was then resuspended in 100% MeOH before MDA determination by HPLC with a fluorescence detection (Jasco Corporation, Tokyo, Japan) set at 515 nm excitation and 550 nm emission.
Fatty Acid Analysis
Following differentiation, Caco-2/15 cells were incubated for 6 h at 37°C in the absence or presence of Fe/Asc (200 µM/2 mM), LPS (200 µg/mL) or both following pre-incubation with 250 µg/mL AB powder or JC-047 fraction, cells were then homogenized in PBS containing 0.005% (w/v) 2,6-Di-tert-butyl-4-methylphenol (Sigma, St-Louis, MO). Samples were subjected to transesterification and injected into a gas chromatograph using a 90 m×0.32 mm WCOT-fused silica capillary column VF-23 ms coated with 0.25 µm film thickness (Varian, Canada) according to the method described previously [43].
Endogenous Antioxidant Enzyme Activities
Differentiated Caco-2/15 cells were harvested in hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF). Total superoxide dismutase (SOD) activity was determined as described by McCord et al. [44]. Briefly, superoxide radicals (O2
-) were generated by the addition of xanthine and xanthine oxidase, and the oxidation of the SOD assay cocktail was followed using a spectrophotometer at 550 nm for 5 min. The same reaction was then repeated with the addition of the sample, and the SOD assay cocktail was less oxidized because of the SOD activity in the sample. The total SOD activity was then calculated. For glutathione peroxidase (GPx) activity, aliquots of cell homogenates were added to a PBS buffer containing 10 mM GSH, 0.1 U G-Red and 2 mM NADPH with 1.5% H2O2 to initiate the reaction. Absorbance was monitored every 30 sec at 340 nm for 5 min [41]. For G-Red activity, cell homogenates were added to a PBS buffer containing 2 mM NADPH and 10 mM of GSSG to initiate the reaction. Absorbance was monitored every 30 sec at 340 nm for 5 min [41].
Immunoblot Analysis
Following the incubation with the various stimuli, differentiated Caco-2/15 cells were sonificated and the Bradford assay (Bio-Rad, Mississauga, Ontario) was used to determine the protein concentration of each sample. Proteins were denatured in sample buffer containing SDS and ß-mercaptoethanol, separated on a 7.5% SDS-PAGE and electroblotted onto Hybond nitrocellulose membranes (Amersham, Baie D’Urfé, Quebec, Canada). Signals were detected with an enhanced chemiluminescence system for antigen-antibody complexes. No specific binding sites of the membranes were blocked using defatted milk proteins followed by the addition of one of the following primary antibodies: 1/1000 polyclonal anti-villin (94 kDa; BD Biosciences, Mississauga, Ontario); 1/1000 polyclonal anti-occludin (59 kDa; Abcam, Campbridge, MA); 1∶1000 polyclonal anti-COX-2 (70 kDa; Novus, Oakville, ON); 1∶10000 polyclonal anti-NF-κB (65 kDa; Santa Cruz Biotechnology, Santa Cruz, CA); 1∶5000 polyclonal anti-IκB (39 kDa; Cell Signaling, Beverly MA); 1/5000 polyclonal anti- tumor necrosis factor (TNF)-α (26 kDa; R&D, Canada); 1/5000 monoclonal anti- interleukin (IL)-6 (25 kDa; R&D, Canada), 1/1000 polyclonal anti-Nrf2 (68 kDa; Abcam, MA, USA) and 1/1000 polyclonal anti-PGC-1α (92 kDa; Abcam, MA, USA), and 1∶40000 monoclonal anti-β-actin (42 kDa; Sigma, MO, USA).
The relative amount of primary antibody was detected with specie-specific horseradish peroxidase-conjugated secondary antibody (Jackson Laboratory, Bar Harbor, Maine). The β-actin protein expression was determined to confirm equal loading. Molecular size markers (Fermentas, Glen Burie, Maryland) were simultaneously loaded on gels. Blots were developed and the protein mass was quantitated by densitometry using an HP Scanjet scanner equipped with a transparency adapter and the UN-SCAN-IT gel 6.1 software.
Prostaglandin E2 Determination
Cellular prostaglandin E2 (PGE2) was measured by enzyme-linked immunosorbent assay (Arbor Assay, Michigan, USA). After a short incubation, the reaction was stopped and the intensity of the generated color was detected in a microtiter plate reader (EnVision Multilabel Plate Readers, PerkinElmer) capable of measuring 450 nm wavelengths.
Nuclear Extraction for Immunoblot Analysis of NF-κB, Nrf2 and PGC-1α
Differentiated Caco-2/15 cells were washed twice with PBS and left on ice for 4 min in a lysis buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 2 mM DTT, and 0.4% Nonidet and antiproteases. Cells were then scraped and centrifuged for 5 min at 1,500 g at 4°C. Pellets were then washed with the same buffer, but without the Nonidet, and centrifuged again under the same conditions. The resulting pellets were then resuspended in 50 µL of final hypertonic lysis buffer (20 mM HEPES, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2 1,4-dithio-DL-treitol, and 20% glycerol and antiproteases) and left on ice for 1 h with vortexing. They were then centrifuged for 10 min at 10,000 g at 4°C, and the supernatants were collected for protein and Western blotting to analyze NF-κB, nuclear factor erythroid-2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) protein expression.
Statistical Analysis
All values are expressed as mean ± SEM. Data were analyzed by using a one-way analysis variance and the two-tailed Student’s t test using the Prism 5.01 (GraphPad Software) and the differences between the means were assessed post-hoc using Tukey’s test. Statistical significance was defined as P<0.05.
Results
Profile of Phenolic Compounds of Crude and Purified DAPP
A reversed phase LC-MS method has been developed in order to separate and identify masses and chemical structures of polyphenolic compounds contained in the crude extract (AB powder) and purified polyphenol fraction (JC-047) derived from DAPP. Flavonoids figured among the major polyphenol classes: they were identified on the basis of their common structure consisting of two aromatic rings bound together by three carbon atoms that form an oxygenated heterocycle. Representative extracted ion chromatograms of identified polyphenolic compounds (using accurate mass measurement) are shown in Figures 1A and 2A. In the crude extract, flavonols constituted the dominant subclass of flavonoids and were present as a mixture of aglycone and glycosylated quercetin and dihydrochalcone (Figure 1B). Negative electrospray mass spectra of the deprotonated species [M-H]- were observed [Table 1 and Figure 1C and at m/z 463,090 for quercetin 3-O-glucoside (1A); quercetin 3-O-galactoside (1B); m/z 433,079 for quercetin 3-O-arabinoside (1C); m/z 447,079 for quercetin 3-O-xyloside (1D); m/z 447,095 for quercetin 3-O-rhamnoside (1E) and m/z 435,131 for phloridzin (1F)]. On the other hand, the purified fraction was mainly composed of catechin, epicatechin and their oligomers eluting between 15 to 18 min (Figure 2B). Extracted ion chromatograms at m/z 289.076 are shown (+)-catechin eluting at 15.4 min and (−)-epicatechin eluting at 16.9 min (Figure 2B, left). The trimeric oligomers as proanthocyanidin trimer C1–C4 (Figure 2B, right) share the same m/z of 865.199 (Figure 2C). Colorimetric methods, including the Folin-Ciocalteu, were used for quantifying total phenolics content. The purified fraction contains a higher proportion (26%, P<0.01) of total phenolic compounds (1900±160 mg of gallic acid equivalents/100 g of extract weight) compared to the crude extract (1410±120 mg of gallic acid equivalents/100 g of extract weight). Furthermore, the combination of high performance liquid chromatography system (HPLC), coupled to a time-of-flight mass spectrometer (TOF) equipped with an HPLC-ESI-MS TOF; and UPLC-ESI-MS/MS QQQ analysis revealed higher amounts (mg/100 g) of flavonols+procyanidins and phenolic acids in purified JC-047 fraction compared to controls (137±9 vs. 369±10 and 74±1 vs. 43.0±6, respectively). However, the crude extract (AB powder) contained more flavonols and dihydrochalcones than the purified JC-047 fraction (346±14 vs. 207±16 and 261±6 vs. 255±11, respectively).
10.1371/journal.pone.0053725.g001Figure 1 Separation and identification of polyphenolic compounds in DAPP crude AB powder.
Extracted ion chromatograms of some identified polyphenolic compounds in AB powder using accurate mass measurement; (A). Extracted ion chromatograms of quercetin glycosides and dihydrochalcone (B) and their related mass spectra (C): quercetin 3-O-glucoside, m/z 463.0905 (1A); quercetin 3-O-galactoside, m/z 463.09018 (1B); quercetin 3-O-arabinoside, m/z 433.07914 (1C); quercetin 3-O-xyloside, m/z 447.07977 (1D); quercetin 3-O- rhamnoside, m/z 447.09506 (1E); and phloridzin, m/z 435.13171 (1F) obtained with negative ion electrospray ionisation. These polyphenols are provided from a mixture of 250 µg of crude extract DAPP in 1 mL Optima grade water.
10.1371/journal.pone.0053725.g002Figure 2 Separation and identification of polyphenolic compounds in DAPP purified JC-047 fraction.
Extracted ion chromatograms of some identified polyphenolic compounds in JC-047 using accurate mass measurement (A). Extracted ion chromatograms of catechin, epicatechin and trimeric flavan-3-ol oligomers C1 to C4 (B) and their related mass spectra (C): (+)-catechin, m/z 289.07541 (1A) and (-)-epicatechin, m/z 289.07659 (1B) and that trimeric flavan-3-ol oligomers C1 to C4 (2A to 2D) obtained with negative ion electrospray ionisation. These polyphenols provided of a mixture of 250 µg of purified fraction DAPP in 1 mL Optima grade water.
10.1371/journal.pone.0053725.t001Table 1 Identification of procyanidins in DAPP.
AB powder Formula Ion formula[M-H]-
Experimentalmass (m/z) Theoreticalmass (m/z) Diff. ppm(<5 ppm)
Flavonols Quercetin 3-O-glucoside C21H20O12
C21H19O12
463,09050 463,08820 4.96
Quercetin 3-O-galactoside C21H20O12
C21H19O12
463,09018 463,08820 4.27
Quercetin 3-O-arabinoside C20H18O11
C20H17O11
433,07914 433,07763 3.47
Quercetin 3-O-xyloside C20H18O11
C20H17O11
433,07977 433,07763 4.92
Quercetin 3-O-rhamnoside C21H20O11
C21H19O11
447,09506 447,09329 3.96
Dihydrochalcone Phloridzin C21H24O10
C21H23O10
435,13171 435,12967 4.68
Experimental mass measurement and empirical formula calculation for quercetin glycosides and dihydrochalcone. A good agreement between the theoretical and the experimental m/z values was obtained for all compounds examined (<5 ppm). Separations were performed on an 1100 LC system coupled to an ESI-MSD-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA).
The distribution of oligomers in the AB powder and JC-047 fraction was from degrees of polymerization (DP1 to DP10) [Table 2 and Figure 3]. However, the determination of total procyanidins content was 3 times higher in the JC-047 compared to the AB powder extract (Table 2). No procyanidin oligomers higher than decamers were detected in the polymeric procyanidin signal.
10.1371/journal.pone.0053725.g003Figure 3 Identification of procyanidins in DAPP.
Representative chromatograms of DAPP (25 mg/mL) from AB powder (A) or JC-047 (B) were obtained by normal phase analytical HPLC using an Agilent 1260/1290 Infinity system coupled to a fluorescence detector.
10.1371/journal.pone.0053725.t002Table 2 Heterogeneity of fractionated procyanidin oligomers and polymers of DAPP on normal-Phase HPLC.
Procyanidin content DAPP (mg/100 g extract weight)
Crude extract (AB powder) Purified fraction (JC-047)
Monomers 25.0±0.4 42.0±1.0***
Dimers 38.0±3.0 97.0±2.0***
Trimers 19.0±2.0 53.0±2.0***
Tetramers 13.0±1.0 46.0±1.0***
Pentamers 10.0±0.3 34.0±2.0***
Hexamers 9.0±1.0 29.0±2.0***
Heptamers 4.0±0.3 13.0±1.0*
Octamers 2.0±0.1 6.0±1.0
Nonamers 2.0±0.5 7.0±1.0
Decamers 1.0±1.0 2.0±0.5
Polymers DP>10 13.0±5.0 40.0±4.0***
Total
137.0±9.0
369.0±10.0***
The procyanidin composition of DAPP from 25 mg/mL crude extract (AB powder) and purified fraction (JC-047) was analyzed by normal phase analytical HPLC using an Agilent 1260/1290 Infinity system coupled to a fluorescence detector. Individual procyanidins with degrees of polymerization (DP) from DP1 to DP>10 were quantified using external calibration curve of (−)-epicatechin, taking into account their relative response factors in fluorescence. The results were expressed as mg/100 g of extract weight ± SEM. *P<0.05, ***P<0.001 vs. AB powder.
Cell Integrity following Various Treatments
The effects of Fe/Asc and LPS on Caco-2/15 cells integrity were examined by morphology assessment, protein content quantification and MTT assay after incubation periods of 6 and 24 h. The morphology and the protein content remained unchanged with the administration of Fe/Asc, LPS and their combination, as well as following treatment with the AB powder or JC-047 (data not shown). Similarly, Caco-2/15 cell viability was not affected by the addition of the various treatments (Figure 4). Interestingly, an enhancement of villin protein mass was observed when Caco-2/15 cells were cultured in the presence of AB powder. Finally, there was no impact on Caco-2/15 cell monolayer transepithelial resistance (an indicator of cell confluence and monolayer integrity) (Figure 4) and on occludin protein mass (a biomarker for tight junction and mucosal barrier functions) (Figure 4). Therefore, it could be concluded that our experimental conditions, including the use of DAPP, did not exert any cytotoxic effects on Caco-2/15 cells.
10.1371/journal.pone.0053725.g004Figure 4 Effects of DAPP on cell integrity in Caco-2/15 cells.
Integrity of the monolayer was determined by cell viability, morphology (data not shown), differentiation and tight junction assays using fully differentiated Caco-2/15 cells. Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. MTT (A), villin protein mass (B), transepithelial resistance (C) and occludin protein expression (D) were assessed. Results represent the means ± SEM of n = 3 independent experiments. *P<0.05 vs. Ctrl; ##
P<0.01 vs. Fe/Asc.
Effects of DAPP on Lipid Peroxidation
The extent of lipid peroxidation following the treatment of Caco-2/15 cells with Fe/Asc during 6 h was assessed by determining cellular levels of MDA. HPLC analyses indicated a four-fold increase in MDA (P<0,001) following the administration of the oxygen free radical-generating system Fe/Asc compared to controls (Figure 5A). The presence of the AB powder or JC-047 fraction counteracted Fe/Asc-mediated lipid peroxidation with a more favorable impact of the former.
10.1371/journal.pone.0053725.g005Figure 5 Effects of DAPP on lipid peroxidation and regulatory endogenous antioxidant activities in Caco-2/15 cells.
Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. Estimation of lipid peroxidation was assessed by measuring the MDA by HPLC (A). The activity of SOD (B), GPx (C) and G-Red (D) was then measured. Results represent the means ± SEM of n = 3 independent experiments. **P<0.01, ***P<0.001 vs. Ctrl; #
P<0.05, ##
P<0.01, ###
P<0.001 vs. Fe/Asc; $$
P<0.01, $$$
P<0.001 vs. Fe/Asc+LPS.
Since OxS markedly altered the composition and properties of the bilayer lipid environment, we determined the profile of fatty acids (FA). In fact, the addition of Fe/Asc resulted in substantial differences in FA following the 6 h-period of cell incubation (Table 3). In particular, a significant decrease was noted in n-3 and n-6 polyunsaturated fatty acids (PUFA) (EPA, 20∶5n-3; DHA, 22∶6n-3; AA 20∶4n-6) as well as in monounsaturated FAs (18∶1n-9) (Table 2). As a consequence, the calculated total n-3, n-6 and n-9 was reduced by 3-fold, 0.5-fold and 2-fold compared to controls (Table 3). As n-3 FAs were more affected by OxS than n-6 FAs, a decline was recorded in the ratio n-6/n-3, which indicates an inflammatory state. Nevertheless, preincubation with the AB powder or JC-047 fraction restored the levels and composition of PUFAs.
10.1371/journal.pone.0053725.t003Table 3 Effects of DAPP on fatty acid composition in Caco-2/15 cells.
Fatty acids Ctl (ug/mg protein) Fe/Asc (ug/mg protein) AB powder+Fe/Asc(ug/mg protein) JC-047+ Fe/Asc (ug/mg protein)
14∶0
6,93±0,76 4,36±0,08 5,46±0,66 7,78±0,41
16∶0
61,70±5,20 41,85±0,164*** 55,27±7,71 70,43±6,04###
18∶0
56,14±2,04 49,57±0,41 54,7±6,36 62,88±9,35##
20∶0
1,97±0,27 1,24±0,01 2,08±0,44 2,32±0,54
22∶0
1,33±0,14 0,80±0,02 1,37±0,32 1,51±0,39
24∶0
2,50±0,30 1,32±0,01 2,55±0,78 3,08±0,79
ALA:18∶3n-3
0,12±0,02 0,14±0,01 0,17±0,03 0,15±0,02
20∶3n-3
0,07±0,02 0,12±0,03 0,13±0,04 0,16±0,09
EPA:20∶5n-3
3,40±0,26 1,10±0,12*** 3,06±0,72##
3,38±1,04###
22∶5n-3
1,66±0,23 0,550±0,05 1,55±0,58 1,58±0,75
DHA:22∶6n-3
4,62±0,45 1,24±0,18*** 4,04±1,09##
4,61±1,67###
AL:18∶2n-6
3,92±0,17 5,72±0,19** 4,20±0,57 4,00±0,46#
18∶3n-6
0,71±0,13 0,33±0,01 0,72±0,13 0,87±0,22
20∶2n-6
0,10±0,03 0,01±0,01 0,23±0,12 0,19±0,09
20∶3n-6
2,05±0,20 1,44±0,06 1,81±0,36 2,17±0,62
AA:20∶4n-6
9,55±0,81 5,76±0,66*** 8,97±2,22###
9,42±3,13###
22∶2n-6
0,35±0,09 0,11±0,04 0,37±0,23 0,51±0,31
22∶4n-6
0,20±0,08 0,02±0,00 0,26±0,16 0,30±0,17
16∶1n-7
14,17±1,06 4,67±0,26*** 12,50±1,86 15,21±0,54
18∶1n-7
32,09±3,47 10,83±0,31*** 29,07±4,92###
37,10±7,47###
18∶1n-9
64,69±5,67 29,50±0,50*** 60,81±10,81###
72,99±12,11###
20∶1n-9
4,27±0,75 1,59±0,01 4,46±1,26 5,15±1,46
20∶3n-9
0,63±0,03 0,33±0,05 0,56±0,09 0,58±0,09
22∶1n-9
10,69±3,43 3,65±0,10 4,58±0,72 4,97±1,16
24∶1n-9
2,59±0,43 1,03±0,04 2,86±1,35 4,16±1,81
Total
300.78±21.86 177.44±2.56*** 274.48±43.71###
332.52±50.49###
Total n-3
9,87±1,32 3,15±0,484** 8,93±2,39#
9,88±3,51##
Total n-6
16,9±1,97 13,4±1,22 16,6±3,70 17,5±4,80
Total n-7
49,7±6,20 17,1±0,797*** 44,5±6,90###
56,0±8,11###
Total n-9
82,9±13,3 36,1±0,669*** 73,3±13,9###
87,8±15,9###
Saturated FA
137±9,58 105±0,270*** 127±16,2###
156±18,0###
Mono-unsaturated
135±19,5 54,4±1,70*** 120±21,3###
146±24,3###
PUFA
27,4±2,32 16,0±1,26*** 26,1±6#
27,9±8,39#
DHA/AA
0,48±0,02 0,21±0,01** 0,44±0,02#
0,47±0,03##
ALA/LA
0,031±0,004 0,024±0,002** 0,039±0,003###
0,037±0,003###
n-6/n-3
1,73±0,04 4,29±0,19*** 1,95±0,14###
0,17±0,04###
After 10 days differentiation, Caco-2/15 cells were incubated for 6 h at 37°C in the absence or presence of Fe/Asc (200 µM/2 mM) with DAPP from 250 µg/mL AB powder or JC-047 and collected for fatty acid (FA) composition. Data represent the means ± SEM of two experiments, each done in duplicate (n = 4). Student’s t test (two-tailed) was used to compare differences between means (X±SEM). *P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; #
P<0.05, ##
P<0.01, ###
P<0.001 vs. Fe/Asc.
AA: arachidonic acid, ALA: alpha-linolenic acid, DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid, LA: linoleic acid, PUFA: polyunsaturated fatty acids.
Mechanisms for the Action of DAPP on Oxidative Stress
As failure of antioxidant defense may explain the induction of OxS, we examined various endogenous antioxidant enzymes in Caco-2/15 cell line. Treatment with Fe/Asc alone or in combination with LPS caused a significant augmentation in the SOD activity, but preincubation of Caco-2/15 cells with the AB powder or JC-047 fraction blunted the effects of OxS and inflammation (Figure 5B). Under these conditions, GPx activity was down-regulated by Fe/Asc and LPS, and restored by treatment with the AB powder or JC-047 (Figure 5C). On the other hand, G-Red (Figure 5D) showed a trend of increase with the polyphenol treatments.
Effects of DAPP on Inflammatory Markers
Cytokines and eicosanoids are pro-inflammatory compounds produced by the cells in response to injury. We therefore assessed the production of TNF-α and IL-6, two powerful inflammatory biomarkers, in Caco-2/15 cells incubated with Fe/Asc, LPS or their combination for 6 h. Analysis by Western Blot disclosed an elevation of protein mass of TNF-α (1.5 to 2.0-fold) and IL-6 (1.5 to 1.8-fold) in the presence of Fe/Asc and LPS, respectively, compared to control cells (Figure 6). Pre-treatment with the AB powder or JC-047 fraction abolished the increase in TNF-α and IL-6 protein expression in Caco-2/15 cell line.
10.1371/journal.pone.0053725.g006Figure 6 Effects of DAPP on oxidative stress or LPS-induced inflammation on inflammatory markers in Caco-2/15 cells.
Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. Protein expression of the inflammatory markers TNF-α (A to C) and IL-6 (D to F) was determined by Western blot, respectively. Results represent the means ± SEM of n = 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; ###
P<0.001 vs. Fe/Asc; †
P<0.05, †††
P<0.001 vs. LPS; $$$
P<0.001 vs. Fe/Asc+LPS.
We next turned to the formation of inflammatory eicosanoids such as PGE2 that is synthesized from arachidonic acid by COX-2. Our experiments showed that Fe/Asc and LPS elicited exaggerated synthesis of PGE2 whereas preincubation with the AB powder displayed high ability to prevent PGE2 accumulation in response to LPS but not Fe/Asc (Figure 6).
Mechanisms for the Action of DAPP on Inflammation
Since the COX-2 enzyme may be behind the elevation of Fe/Asc- and LPS-induced PGE2, we determined its protein expression. Both stimuli raised its protein mass as evidenced by Western blot (Figure 7). Pre-incubation of Caco-2/15 cells with the AB powder or JC-047 fraction averted the positive action of the oxidative and inflammatory stimuli on COX-2 protein expression. Importantly, the polyphenol antioxidants were as effective as indomethacin heptyl ester, a selective COX-2 inhibitor [45] in preventing the elevation of PGE2. In addition, their combination provided a more substantial synergetic effect, which is indicative of different mechanisms of action for LPS-induced inflammation (Figure 8).
10.1371/journal.pone.0053725.g007Figure 7 Effects of DAPP on oxidative stress and LPS-induced inflammation on prostaglandin E2 in Caco-2/15 cells.
Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) (A), as well as indomethacin heptyl ester (0.4 µM) (B), as a selective cyclooxygenase (COX)-2 inhibitor, for 6 h at 37°C as described in Materials and Methods. PGE2 was determined by enzymatic immunoassay. Results represent the means ± SEM of N = 3 independent experiments. ***P<0.001 vs. Ctrl; †††
P<0.001 vs. LPS; $$$
P<0.001 vs Fe/Asc+LPS.
10.1371/journal.pone.0053725.g008Figure 8 Effects of DAPP on oxidative stress or LPS-induced inflammation on cyclooxygenase 2 modulation in Caco-2/15 cells.
Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) (A to C), as well as indomethacin heptyl ester (0.4 µM) (D), as a selective cyclooxygenase (COX)-2 inhibitor, for 6 h at 37°C as described in Materials and Methods. Protein expression of COX-2 was determined by Western blotting. Results represent the means ± SEM of n = 3 independent experiments. **P<0.01, ***P<0.001 vs. Ctrl; ###
P<0.001 vs. Fe/Asc; ††
P<0.01, †††
P<0.001 vs. LPS; $$$
P<0.001 vs Fe/Asc+LPS.
Mechanisms for the Action of DAPP on Transcription Factors
NF-κB signaling pathway plays a crucial role in the initiation and amplification of inflammation via the modulation of multiple inflammatory mediators. Figure 9 shows that Caco-2/15 cells exposed to Fe/Asc or LPS displayed a high NF-κB signal in the nucleus along with a low level of IκB protein expression in the cytoplasm, which suggests that the inhibitory protein is degraded by the proteasome, leaving NF-κB free to enter the nucleus and activate the transcription of its target genes. As a consequence, the NF-κB/IκB ratio was increased under the presence of Fe/Asc (Figure 9A) and LPS (Figure 9B and 7C). Importantly, the AB powder or JC-047 fraction displayed their great potential to neutralize IκB degradation and NF-κB mobilization to the nucleus compared to CAPE, the NF-κB inhibitor, with LPS at 6 h (Figure 9B) and LPS at 24 h (Figure 9C) to mimic an acute and a long inflammation, respectively. The combined administration of CAPE and AB powder or JC-047 fraction did not produce significant changes, thereby indicating the same mechanisms of action.
10.1371/journal.pone.0053725.g009Figure 9 Effects of DAPP on oxidative stress or LPS-induced inflammation on NF-κB in Caco-2/15 cells.
Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) in the presence or absence of 50 µM caffeic acid (CAPE, a specific NF-κB inhibitor) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) (A) and LPS (200 µg/mL) (B) for 6 h at 37°C, and LPS (200 µg/mL) (C) for 24 h at 37°C to mimic a chronic inflammation as described in Materials and Methods. Results represent the means ± SEM of n = 3 independent experiments. **P<0.01, ***P<0.001 vs. Ctrl; #
P<0.05, ##
P<0.01, ###
P<0.001 vs. Fe/Asc; †††
P<0.001 vs. LPS.
To decipher the mechanisms of action of the AB powder or JC-047 fraction, we examined the transcription factors that are involved in the regulation of antioxidant genes expression. The protein mass of Nrf2 in homogenates (Figure 10A) and nuclei (Figure 10B) was down-regulated by Fe/Asc- or LPS-induced OxS and inflammation, respectively. However, treatment with the AB powder or JC-047 fraction restored Nrf2 protein expression to the basal level. We also assessed the protein expression of PGC-1α a powerful transcriptional co-activator that up-regulates Nrf2. PGC-1α protein mass was down-regulated in response to OxS and inflammation in homogenates (Figure 10C) and nuclei (Figure 10D) in Caco-2/15 cells. However, the effect was reestablished when Caco-2/15 cells were pre-incubated with the AB powder or JC-047 fraction.
10.1371/journal.pone.0053725.g010Figure 10 Effects of DAPP oxidative stress or LPS-induced inflammation on transcription factors in Caco-2/15 cells.
AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. Protein expression of the transcription factors Nrf2 in homogenates (A) and in nucleus (B), as well as PGC-1α in homogenates (C) and in nucleus (D) was determined by Western blot. Results represent the means ± SEM of n = 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; #
P<0.05, ##
P<0.01, ###
P<0.001 vs. Fe/Asc; $
P<0.05, $$
P<0.01, $$$
P<0.001 vs. Fe/Asc+LPS.
Discussion
Growing evidence suggests important roles of dietary factors in preserving health and even reversing the progression of chronic diseases, with anti-inflammatory effects as important underlying mechanisms. In the present study, we first characterize the polyphenol compounds of DAPP by HPLC-ESI-MS TOF and then tested their impact on cell integrity and viability. After we excluded any possible toxicity of this natural DAPP (crude extract) and its purified fraction, which has frequently been detected in various chemical drugs, we could subsequently document their remarkable capacity in scavenging ROS and neutralizing inflammation in intestinal absorptive cells. By dissecting the mechanisms of action, our in vitro experiments highlighted the ability of apple peel polyphenols to increase the antioxidant/anti-inflammatory defense by (i) preventing LPS-induced inflammation via limitation of the pro-inflammatory expression and activity of COX-2; (ii) ruling out LPS-mediated cytokine production through downregulation of NF-κB, an essential transcription factor for numerous cytokines and (iii) up-regulating the expression of transcription factors (Nrf2 and PGC-1α), key redox-sensitive transcription factors and crucial elements for mitochondrial biogenesis.
The results of our comprehensive study provide fundamental information on the apple peel polyphenols. The high-resolution HPLC-ESI-MS TOF delivers the composition of the different biomolecules in DAPP (AB powder or JC-047 fraction). In the former, flavonols (composed of aglycone and glycosylated quercetin and dihydrochalcone) are the major subclasses of flavonoids present, while in the purified fraction, we mostly found the flavan-3-ols and their oligomers. Noteworthy, quercetin represents the preponderant flavonol in DAPP and, according to previous studies; it has exhibited anti-inflammatory and antioxidant activities, prevented platelet aggregation and promoted relaxation of cardiovascular smooth muscle [46]. As a matter of fact, flavan-3-ols are a family of bioactive compounds and potent antioxidants as has been described in in vitro and in vivo studies. Importantly, in the current work, we have evaluated the antioxidant and anti-inflammation power of both the crude extract (AB powder) and purified polyphenol fraction (JC-047) derived from DAPP since there was a need to prove that the beneficial effects are derived from the polyphenos contained in apple peels.
In the present work, we used the Caco-2/15 cell line that undergoes a process of spontaneous differentiation leading to the formation of a monolayer of cells expressing several morphological and functional characteristics of the mature enterocyte. This remarkable intestinal model is regarded as the most appropriate for the investigation of gut absorption and interactions, nutrition, toxicology food microbiology, bioavailability tests, and screening of drug permeability in discovery programs. Multiple studies from our laboratory have shown that Caco-2/15 cell monolayers are fully appropriate for the study of OxS and inflammation [38], [39], [41], [47].
To produce OxS, we employed the Fe/Asc complex, a widely used oxygen-radical generating system [34], [36], [39], [41], [42] since our laboratory reported the ability of iron to initiate strong lipid peroxidation, whereas ascorbic acid can amplify iron-oxidative potential by promoting metal ion-induced lipid peroxidation [34]. The data of the present study clearly indicate that the Fe/Asc system functioned as a producer of lipid peroxidation given the production of MDA and the degradation of PUFAs and the production of pro-inflammatory eicosanoids. Additionally, with the Fe/Asc complex, the antioxidant/oxidative balance deteriorated the endogenous antioxidant enzymes. In this context, co-supplementation of iron and vitamin C worsens OxS in the gastrointestinal tract, thereby leading to ulceration in healthy individuals, and exacerbates chronic gastrointestinal inflammatory diseases, which may result in the development of cancers [48]. Importantly, supplementation of DAPP by crude extract or its purified fraction significantly prevented lipid peroxidation and restored the depletion of some n-3 PUFA, likely by strengthening the endogenous antioxidant defense as illustrated, in our results, through SOD down-regulation and GPx up-regulation activities.
For the induction of inflammation, we used LPS that has been extensively studied for the past two decades. This is a ubiquitous endotoxin mediator of gram-negative bacteria, which facilitates microbial translocation by a mechanism implicating physical perturbation of the gut mucosal barrier [1], [49]. LPS is also a potent inducer of the host’s immune response via its capacity to stimulate the pro-inflammatory cytokine cascade. In our studies, LPS led to amplification of the inflammatory response in Caco-2/15 cells given the enhanced production of PGE2 and the raised protein expression of TNF-α and IL-6, probably due to elevated COX-2 and NF-κB, respectively. DAPP was effective in preventing the elevation of PGE2, TNF-α and IL-6 via the down-regulation of COX-2 and NF-κB, as evidenced by the co-administration of their specific inhibitors indomethacin heptyl ester and CAPE, respectively. The combination of CAPE and DAPP (either as crude extract or its purified JC-047 fraction) did not further anti-inflammatory benefits, which suggests a common mechanism of action. On the other hand, compounding indomethacin heptyl ester and DAPP resulted in amplified anti-inflammatory effects, which argues in favor of synergetic mechanisms.
Since the Keap1-Nrf2-antioxidant response element (ARE) is an integrated redox sensitive signaling system that regulates from 1% to 10% of our genes [50], [51], we assessed the protein expression of Nrf2 and could document its significant increase. It is therefore possible that, upon exposure to AB powder or JC-047 fraction, Nrf2 was able to escape Keap1-mediated ubiquitin-dependent proteasomal degradation, translocate to the nucleus, and activate ARE-dependent gene expression of a series of antioxidative and cytoprotective proteins that include SOD and GPx. Our study went even further since it revealed the positive modulation of PGC-1α by DAPP. PGC-1α controls many aspects of oxidative metabolism, including mitochondrial biogenesis and respiration through the coactivation of many nuclear receptors [52], [53]. As an example, Nrf2 is a key target of the PGC-1α in mitochondrial biogenesis and important protective molecules against ROS generation and damage. It is therefore possible that PGC-1α activates NRF2 to induce the SOD and GPx that were altered by Fe/Asc-mediated lipid peroxidation. However, additional efforts are needed to understand the role of DAPP in PGC-1α and Nrf2 cross-talk.
Noteworthy, in some experiments, Caco-2/15 cells were serum-starved for 24 h prior to the addition OxS or inflammation. The serum-depleted media were used to minimize the formation of adducts between DAPP and serum proteins, and to exclude the interferences originating from available factors present in fetal bovine serum, as described in previous studies with other types of antioxidants [54]. The pre-incubation time of 24 h with DAPP was used to maximally strengthen the antioxidant and anti-inflammatory defense before the addition of the iron-ascorbate oxygen radical-generating system or LPS that triggers inflammation. By allocating this period of time, we allow Caco-2/15 cells to deploy various powerful protection mechanisms via transcription factors and signaling pathways. The transport and processing of DAPP have been elaborated in the Discussion section.
Following their consumption, polyphenols are extensively metabolized by hydrolyzing and conjugating enzymes [55], [56]. They are first conjugated in the small intestine to form O-glucuronides, sulphate esters and O-methyl ether [57] before reaching the liver for further metabolism [58]. The formation of anionic derivatives by conjugation with glucuronides and sulphate groups facilitates their urinary and biliary excretion and explains their rapid elimination. Non-absorbed polyphenols and the fraction re-excreted by the bile are extensively metabolized and transformed by the microbiota before absorption [59], [60]. The transformation by commensal bacteria via esterase, glucosidase, demethylation, dehydroxylation, and decarboxylation is often essential for absorption and modulates the biological activity of these polyphenols [60]. In our intestinal model, no flora is present, which suggests an absorption via paracellular route of transport as suggested previously [61]. However, additional studies are still needed to highlight the contribution of trans-membrane vs. intercellular absorption as well as the influence of polyphenols of enterocyte metabolism just by adherence to the brush border membrane.
Previous studies investigated the preventive effectiveness of polyphenolic content of flesh apple in cultured gastric mucous cells under conditions independent of acid secretion or systemic factors [62]. They identified the composition of phenolic compounds (chlorogenic acid, caffeic acid, catechin, epicatechin, rutin and phloridizin) in apple flesh extracts, which prevented OxS-induced injury to gastric epithelial cells by permeating cell membranes, increasing intracellular antioxidant activity, and inhibiting ROS-dependent lipid peroxidation. In further studies, the same apple flesh extracts demonstrated prevention of aspirin-induced damage to the rat gastric mucosa [63] and an anti-inflammatory effect on colonic injury in rats with trinitrobenzensulphonic acid-induced colitis [64]. Even though these reports with apple flesh extracts, and ours with DAPP show anti-inflammatory and antioxidant effects, it is not possible to compare their effectiveness given the differences in the apple species, extraction methodology, experimental models and techniques.
In conclusion, a plethora of studies demonstrates significant health benefits of nutrient rich fruits. If various studies have shown this relationship by indirect evidences, the present work demonstrated the presence of a nonpolar bioactivity in extracts of DAPP and their direct beneficial actions, which negated operational OxS and inflammation, both elicited by state-of-the-art techniques. Our results suggest that DAPP may represent a new strategy for the prevention of OxS and inflammation associated with IBD. Further studies are needed to investigate this hypothesis.
The authors thank Leahy Orchards Inc. and Appleboost Products Inc. for supplying DAPP and Mrs Schohraya Spahis is acknowledged for excellent technical assistance.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23365678PONE-D-11-2283210.1371/journal.pone.0054692Research ArticleBiologyMolecular cell biologySignal transductionSignaling cascadesApoptotic signaling cascadeMAPK signaling cascadesSignaling in Cellular ProcessesApoptotic SignalingCell DeathNeuroscienceMolecular NeuroscienceSignaling PathwaysNeurobiology of Disease and RegenerationMedicineAnatomy and PhysiologyEndocrine SystemDiabetic EndocrinologyDrugs and DevicesAdverse ReactionsNeurologyNeurodegenerative DiseasesNeuro-OphthalmologyOphthalmologyRetinal DisordersDiabetes and Overexpression of proNGF Cause Retinal Neurodegeneration via Activation of RhoA Pathway ProNGF-Induced Retinal NeurodegenerationAl-Gayyar Mohammed M. H.
1
5
6
¤a
Mysona Barbara A.
1
5
6
Matragoon Suraporn
1
5
6
Abdelsaid Mohammed A.
1
5
6
El-Azab Mona F.
1
5
6
¤b
Shanab Ahmed Y.
1
5
6
Ha Yonju
3
5
Smith Sylvia B.
3
4
5
Bollinger Kathryn E.
5
El-Remessy Azza B.
1
2
4
5
6
*
1
Program in Clinical and Experimental Therapeutics. College of Pharmacy, University of Georgia, Athens, Georgia, United States of America
2
Department of Pharmacology and Toxicology, Georgia Health Sciences University, Augusta, Georgia, United States of America
3
Department of Cell Biology and Anatomy, Georgia Health Sciences University, Augusta, Georgia, United States of America
4
Department of Ophthalmology, Georgia Health Sciences University, Augusta, Georgia, United States of America
5
Vision Discovery Institute, Georgia Health Sciences University, Augusta, Georgia, United States of America
6
Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia
Linden Rafael Editor
Universidade Federal do Rio de Janeiro, Brazil
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ABE MMA BAM. Performed the experiments: MMG BAM MAA MFE SM AYS YH. Analyzed the data: MMA BAM SM ABE. Contributed reagents/materials/analysis tools: SBS KEB. Wrote the paper: MMA BAM ABE.
¤a Current address: Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura City, Egypt
¤b Current address: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Suez Canal University, Ismaileya, Egypt
2013 24 1 2013 16 4 2014 8 1 e5469215 11 2011 17 12 2012 © 2013 Al-Gayyar et al2013Al-Gayyar et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotosin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75NTR, cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75NTR and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.
This work was supported by a career development award from Juvenile Diabetes Research Foundation grant (2-2008-149) to ABE and NIH RO1 EY022408 to ABE and a Grant from Vision Discovery Institute, Georgia Health Science University to ABE and KEB, NIH R01 EY014560 to SBS and post-doctoral fellowship from AHA to BAM and a post-doctoral fellowship from Islamic Development Bank to MFE. Funding entities had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Our recent studies showed significant accumulation of proNGF that was positively correlated with accelerated retinal neurodegeneration in models of diabetes [1], [2]. Our studies demonstrated a mechanism by which diabetes-induced peroxynitrite impairs the activity and expression of MMP-7, an extracellular enzyme involved in maturation of NGF leading to accumulation of the proNGF [2]. While mature NGF mediates neuronal cell survival through binding TrkA and p75NTR receptors, proNGF can promote neuronal apoptosis because of its high affinity to p75NTR
[3], [4]. It has been shown that the outcome of the neurotrophin signaling, neurotrophic or apoptotic can be dependent upon relative levels of its receptors [5], [6]. Our studies in diabetic human and rat retina demonstrated tyrosine nitration and inhibition of the survival receptor TrkA and upregulation of the proapoptotic receptor p75NTR
[1], [7].
In non-diabetic models, overexpression of p75NTR has been shown to constitutively activate RhoA leading to neuronal death via activation of p38MAPK pathway [8]–[12]. Rho family GTPases are monomeric G-proteins that act as key transducers of integrin signaling [13] and growth factor signaling [2], [14], [15]. RhoA is a major small GTP-binding protein that acts as a molecular switch to play either a pro-death or pro-survival role in the nervous system depending on both the type of neuron and the particular neurodegenerative insult involved (for review [16]). Prior report showed that activation of RhoA can directly induce neuronal death in excitotoxic model [9]. Yet, whether RhoA activation can induce retinal neurodegeneration in response to proNGF or diabetic insult remains unexplored.
The MAPK family includes four groups: extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), p38 and ERK5. MAPKs are activated in response to a wide range of extracellular stimuli including growth factors, serum, hormones, cytokines and others more related to stress responses (i.e. UV radiation, X-rays, heat and osmotic shock), and they are involved in many different cellular processes such as embryogenesis, proliferation, differentiation, transformation and apoptosis (for review [17]). In particular, JNK/SAPK and p38 MAPK pathways have been shown to play an important role in neuronal apoptosis in various models including neurotoxicity, diabetes, neurotrophic deprivation or excessive proNGF in vitro
[1], [18]–[21] and in vivo
[2], [7], [22].
In this study, we aimed to elucidate molecular events by which proNGF contributes to diabetes-induced retinal neurodegeneration. In particular, we examined the specific role of RhoA kinase activation as downstream signaling pathway in response to proNGF. Here, we demonstrate the first evidence of the neuroprotective effects of inhibiting RhoA kinase in models of diabetes as well as overexpression of proNGF in healthy rat retina and cultures of primary retinal ganglion cells.
Materials and Methods
Animal preparation
All procedures with animals were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the Charlie Norwood VA Medical Center Animal Care and Use Committee. Three sets, a total of 60 Male Sprague-Dawley rats (∼250 g body weight) from Harlan laboratories (Indianapolis, IN) were housed in a 12 h light/dark cycle at a controlled temperature and humidity with free access to food and water.
Induction of diabetes in rats
Rats (24) were randomly assigned to control, treated control, diabetic or treated diabetic groups. Diabetes was induced by intravenous injection of streptozotocin (60 mg/kg). Detection of glucose in urine and blood glucose levels >13.9 mmol/l indicated diabetes. Treatment was initiated 2-weeks after confirmation of diabetic status and continued for additional 3 weeks for various endpoints. Treated control and diabetic rats were injected intravitreally with the specific RhoA kinase inhibitor Y27632 (100 nmole/eye) once/week. Treatment with Y27632 did not alter body weight in control (360±4 vs 370±22) or diabetic animals (295±20 vs 286±14). Treatment with Y27632 did not alter blood glucose levels in control (120±8 vs 125±10) or diabetic animals (421±20 vs 418±22).
ProNGF overexpression studies
Rats (36) were randomly assigned to receive intravitreal injection with pGFP plasmid (5 µg/eye), pGFP-proNGF123 construct (5 µg/eye) or combination of pGFP-proNGF123 plasmid and the specific RhoA kinase inhibitor Y27632 (100 nmole/eye) from Cayman (Ann Arbor, MI). Electroporation was performed as described previously by our group [23]. Rats were sacrificed after 1-week and eyes were enucleated and processed for further analyses.
Tissue culture studies
Primary retinal ganglion (RGC) cells
RGC were isolated from retinas of 2-day-old mice by immunopanning techniques as previously described by our group [24]. Cells were maintained in culture for 2 days in neurobasal medium containing 50 µg/ml BDNF, 10 µg/ml CNTF, 10 µg/ml Forskolin and 10 µg/ml bFGF, in the 30 mm dishes coated with poly D-lysine and laminin. RGC were rinsed with neurobasal medium without growth factors and equilibrated in the same medium for treatment. RGC were treated with mouse mutant proNGF (50 ng/ml) from Alomon (Israel) in the presence of Y27632 (1 µM) for overnight. For G-LISA studies, cells were harvested after 6-hours.
Adult mouse retinal mixed neuronal culture
Adult mouse retinal mixed neuronal culture was prepared as previously described [25]. 20 retinas from 6week-old mice were dissected in Hank's buffered-saline solution (HBSS) and incubated at 37°C for 10 min in a digestion solution containing papain (10 U/mL; Worthington, Lakewood, NJ) and L-cysteine (0.3 mg/mL; Sigma) in HBSS. Retinas were rinsed and triturated in HBSS containing bovine serum albumin (1 mg/mL; Sigma) and DNase (0.2 mg/mL; Sigma). Dissociated cells were passed through a strainer (40-µm nylon net; Falcon, Bedford, MA) and collected by centrifugation. Cells were resuspended in a 1 mL neurobasal medium (Invitrogen) with B27 supplement (Invitrogen). Cells were seeded into slide chamber and used characterized p75NTR expression in RGC cells using immunolocalization techniques.
Retinal ganglion cell line (RGC-5)
Retinal ganglion cell line (RGC-5), was a kind gift from Dr. N. Agarwal (Department of Cell Biology UT Health Science Center, Fort Worth, TX) and have been previously characterized [26]. Cells were grown to confluence in complete medium (DMEM with 6% FBS and 10% penicillin/streptomycin) then switched to serum free media. Cells were treated with mouse mutant proNGF (50 ng/ml) from Alomon (Israel) in the presence or absence of Y27632 (1 µM) for overnight.
Immunolocalization studies
OCT-frozen sections (10 µm) of eyes were fixed using 2%PFA in PBS and reacted with polyclonal p75NTR (Millipore, Billerica, MA), monoclonal anti-Brn-3a antibody (specific RGC marker, Santa Cruz) or monoclonal anti-Thy-1, a neuronal marker, antibody (Santa Cruz) overnight followed by Texas-red or Oregon-green-conjugated goat anti-mouse or goat anti-rabbit antibodies (Invitrogen, Carlsbad, CA). Images (n = 4–6 in each group) were collected using AxioObserver.Z1 and confocal Microscope (Zeiss, Germany).
Evaluation of retinal cell death in vivo
TUNEL assay was performed to detect retinal cell death by using immunoperoxidase staining (ApopTag-Peroxidase), in whole-mount retina as described previously by our group [2], [7]. Briefly, formalin-fixed retinas were flat-mounted, dehydrated in ethanol, defatted by xylenes and rehydrated. After permeabilization, TUNEL-HRP staining with 3-amino-9-ethylcarbazole was performed following the manufacturer's instructions.
Determination of RGC death in vitro
TUNEL-positive cells were determined using TUNEL fluorescence (ApopTag-Fluorescein) and counterstained with DAPI (blue) or Propidium Iodide (red). The total number of TUNEL-positive cells was counted and expressed as percentage of TUNEL positive cells/total number of cells in various groups.
Counting number of total retinal neuronal and ganglion cells
Total cells in ganglion cell layer (GCL) was counted as described previously by our group [27]. Briefly, OCT frozen retinal sections were stained with Hematoxylin and Eosin (H/E) for light microscopy. The nuclei in the GCL, not including nuclei in the vessels, were counted in four locations in the retina including both sides of the optic nerve (posterior) and mid-retina (central) in a masked manner. Since neuronal cells in GCL contain RGC and amacrine cells, ganglion cells were labeled using Brn3a monoclonal antibody (Santa Cruez) and total number of cells were counted using DAPI. Cells in the GCL were counted from ora serrata to ora serrate (retina length). RGCs were identified as cells positive for Brn3a, a specific RGC marker and DAPI positive in the GCL. Number of RGC was normalized to retina length (mm) in each section. For each animal two sections were counted, one near the optic nerve and one located more peripherally. Four to six animals from each group and two fields for each location were used. Retinas were imaged by AxioObserver.Z1 Microscope (Zeiss, Germany).
Retinal protein extraction and Western blot (WB) analysis
Retinas were isolated and homogenized in RIPA buffer as described previously [7]. Samples (50 µg protein) were separated by SDS-PAGE and electroblotted to nitrocellulose membrane. Antibodies for p75NTR (Millipore, Billerica, MA), JNK, p-JNK (Santa Cruz Biotechnology, Santa Cruz, CA), p-p38, p38, cleaved caspase-3 (Cell Signaling, Danvers, MA) and cleaved PARP (BD Bioscience Pharmingen, San Diego, CA) were used. Membranes were reprobed with β-actin (Sigma-Aldrich, St. Louis, MO) to confirm equal loading. The primary antibody was detected using a horseradish peroxidase-conjugated sheep anti-rabbit antibody (GE Healthcare, Piscataway, NJ) and enhanced chemiluminescence. The films were scanned and the band intensity was quantified using densitometry software (alphEaseFC) and expressed as relative optical density (ROD).
Quantitative real time PCR
Retinal mRNA was prepared according to the manufacturer's instructions as described in our previous study [7]. The One-Step qRT-PCR Invitrogen kit was used to amplify 10 ng retinal mRNA from each sample. PCR primers were designed to amplify TNF-α: 5′GGGTGATCGGTCCCAACA′ and reverse primer 5′TGGGCTACGGGCTTGTCA. Primers were designed to amplify p75NTR: 5′-GCA GCT CCC AGC CTG TAG TG-3′ and reverse primer 5′-TAG GCC ACA AGG CCC ACA AC-3′. Amplification of 18S rRNA was used as an internal control. Quantitative PCR was performed using a Realplex Master cycler (Eppendorf, Germany). TNF-α expression was normalized to the 18S level in each sample and expressed as relative expression to control.
Retinal RhoA kinase activity
GTPase activity was assessed by pull down assay. As previously described, retinas or retinal ganglion cells were homogenized in assay buffer [28]. Homogenates were incubated with agarose conjugated rhotekin-RBD (Millipore, Billerica, MA,) for 45 min at 4°C and washed three times with lysis buffer. Agarose beads were boiled in Laemmli reducing sample buffer to release active RhoA. Bound RhoA was detected by Western blot using anti-RhoA monoclonal antibody (Millipore, Billerica, MA).
G-LISA detection of RhoA GTPase activity in primary RGC cultures
G-LISA detection of RhoA GTPase activity in primary RGC cultures was performed using a G-LISA kit from (Cytoskeleton, Denver, CO) according to manufacture protocol. RGC cells were washed with PBS, resuspended in lysis buffer from the kit and harvested from the dishes with cell scraper. Total protein concentration in each lysate was determined by protein assay reagent from the kit. The G-LISA's contains a RhoA-GTP-binding protein immobilized on microplates. Bound active RhoA was detected with a specific antibody and luminescence, which was quantified using a microplate reader (BioTek Instruments) after removing the reaction mixture.
Data analysis
The results were expressed as mean ± SEM. Differences among experimental groups were evaluated by ANOVA followed by Tukey-Kramer Multiple comparison test. Significance was defined as P<0.05.
Results
Inhibiting RhoA blocked diabetes- and proNGF-induced neuronal death in vivo
Our previous studies in STZ-diabetic model showed accumulation of proNGF and neuronal cell death that were positively correlated with activation of RhoA [1], [2]. Here we tested the direct neuroprotective effects of inhibiting Rho kinase in response to diabetes (5-weeks) or proNGF overexpression (1-week). As shown in Figure 1A, diabetic rat flat-mounted retina showed ∼9-fold increase in the TUNEL-HRP-positive cells counted in each retina as compared with non-diabetic controls (Fig. 1A, 1B). Intravitreal treatment with the RhoA kinase inhibitor Y26732 blocked apoptotic effects of diabetes in the rat retina. To dissect the role of proNGF in retinal neurodegeneration apart from the complex diabetic milieu, overexpression of the cleavage-resistant proNGF construct was used as described previously [23]. As shown in Figure 1C, overexpression of proNGF caused ∼6-fold increase in TUNEL-positive cells compared to the GFP-control. Overexpression of proNGF also caused 20 and 30% reduction of total neuronal cells in ganglion cell layer (GCL) in both central and posterior retina, respectively (Fig. 2A, 2C). Since GCL contains a mixed population of retinal ganglion cells and displaced amacrine cells, RGCs were labeled and counted using Brn3a antibody (Fig. 2B) and normalized to retina length. As shown in Figure 2D, overexpression of proNGF caused significant reduction (60%) of RGC count compared to pGFP controls. Co-treatment with the RhoA kinase inhibitor Y26732 blocked proNGF effects and protected retinal ganglion cells from cell death.
10.1371/journal.pone.0054692.g001Figure 1 Inhibiting Rho kinase blocked proNGF and diabetes-induced retinal neurodegeneration.
A,B. Representative images and statistical analysis of TUNEL-HRP-positive cells counted in each retina flat-mount showing ∼9-fold increased number of cell death in retinas from 5 weeks diabetic rats as compared with the controls (n = 4–5). C. Statistical analysis of total number of TUNEL-HRP-positive cells counted in each retina showing ∼6-fold increase of cell death in retinas that overexpress proNGF as compared with the GFP controls (n = 4–5). Treatment with the selective Rho kinase inhibitor Y27632 blocked these effects in diabetic and proNGF overexpression and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control; D, diabetic; Y, Y27632.
10.1371/journal.pone.0054692.g002Figure 2 Inhibiting Rho kinase blocked proNGF-induced neuronal cell death.
A,C. Representative images and statistical analysis of rat retina sections stained with H/E showing a reduction in total number of neuronal cells in the GCL in rats injected with proNGF as compared with pGFP-controls in central and posterior retina (n = 4, 200× magnification). B,D. Representative images and statistical analysis of rat retina sections stained with anti-Brn3, specific RGC marker, showing a reduction in number of RGC in proNGF as compared with GFP-controls in central and posterior retina (n = 6, 400× magnification). Treatment with Y27632 blocked these effects in proNGF injected rats. * = significant difference as compared with the rest of the groups at p<0.05. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer.
Inhibiting RhoA blocked proNGF-induced death in primary RGC cultures
We next investigated the neuroprotective effects of Y27632 in RGC cultures. We compared the apoptotic effects of proNGF on freshly isolated primary RGC to the cell line RGC-5. Our studies in RGC-5 showed ∼5-fold increase in the number of TUNEL-positive cells in response to mutant proNGF (50 ng/ml) compared to controls (Fig. 3A, 3B). Studies using primary RGC cells showed higher sensitivity (>10-fold) to apoptotic effects of proNGF (Fig. 3C, 3D) compared to controls. Co-treatment with Y27632 (1 µM) protected RGC in proNGF-treated group but had no effect on control cells.
10.1371/journal.pone.0054692.g003Figure 3 Inhibiting RhoA blocked proNGF-induced death in primary RGC cultures.
A,B. Representative images and statistical analysis showing ∼5-fold increase in TUNEL-positive cells in RGC-5 cells in response to mutant proNGF (50 ng/ml) (200× magnification). C,D. Representative images and statistical analysis showing ∼10-fold increase in TUNEL-positive cells in primary RGC cultures in response to mutant proNGF (50 ng/ml) (200× magnification). Co-treatment with Y27632 (1 µM) protected RGC in proNGF-treated group but had no effect on control cells. * = significant difference as compared with the rest of the groups at p<0.05 (n = 4). C, control; Y, Y27632.
Diabetes and overexpression of proNGF induced expression of p75NTR in vivo and in vitro
Our previous analyses showed that diabetes and proNGF overexpression induce p75NTR expression [1], [2], [7], [23]. We examined the effects of inhibiting Rho kinase on p75NTR expression. As shown in Figure 4A–B, diabetes caused 2-fold and proNGF caused 1.5-fold increase in p75NTR expression compared to controls. These effects were partially but significantly reduced by treatment with Y27632. In addition, prominent immunolocalization of p75NTR was observed in GCL and inner retinal layers in proNGF group as compared with pGFP-controls (Fig. 4C). Colocalization of p75NTR with both Brn-3a, specific RGC marker, and Thy-1, neuronal marker, in the ganglion cell layer (Fig. 4D) confirmed the upregulation of p75NTR within retinal ganglion cells. In vitro, in comparison to controls, proNGF induced 1.6-fold increase in p75NTR expression in RGC-5 cell line (Fig. 4E) as well as 2.5-fold increase in p75NTR mRNA expression in freshly isolated primary RGC (Fig. 4F). Treatment with Y27632 significantly reduced p75NTR expression in RGC-5. To further confirm p75NTR expression in adult RGC cultures, we isolated mixed neuronal cultures and immunolocalized p75NTR (red) with the specific RGC marker Brn3a (green). As shown in Figure 4G, numerous co-localized RGC cells (yellow) that express p75NTR were detected in the field.
10.1371/journal.pone.0054692.g004Figure 4 Diabetes and overexpression of proNGF induced expression of p75NTR in vivo and in vitro.
A. WB analysis showing 1.9-fold increase in the expression of p75NTR in diabetic rats as compared with the controls (n = 4–6). B. WB analysis of rat retinal lysate showed significant increase in p75NTR expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 4). C. Representative images of rat retina sections showing prominent immunolocalization of p75NTR in GCL and INL in proNGF overexpressing retinas as compared with GFP-controls (400× magnification). D. Representative images of rat retina sections showing colocalization between p75NTR in the ganglion cell layer (green) and the specific neuronal marker Thy-1 (red) in the upper pannel or with the specific RGC marker Brn-3a (red)in the lower pannel (400× magnification). E. Western blot analysis shwoing 1.6-fold increase in the expression of p75NTR in RGC-5 cells treated with proNGF as compared with the controls (n = 4). F. Real-time PCR analysis showing that proNGF induced p75NTR mRNA expression in freshly isolated primary RGC as compared with the control group. Samples of primary RGC cultures were pooled from 4-different cultures. Treatment with Y27632 significantly reduced p75NTR expression in vivo and in vitro. G. Representative images showing colocalization (yellow arrow heads) of RGC that expressed p75NTR (red) and the specific RGC marker Brn3a (green) in isolated mixed neuronal cultures from adult mice. * = significant difference as compared with the rest of the groups at p<0.05. C, control; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. C, control.
ProNGF selectively causes RhoA kinase activation in vivo and in RGC cultures
Our studies in the diabetic retina showed positive association between increased proNGF and activation of RhoA [7]. Indeed, diabetic rat retina showed significant increase (2.3-fold) in active RhoA compared to controls (Fig. 5A). Next, we examined the effect of overexpressing proNGF on activating RhoA kinase in vivo and in vitro. As shown in Figure 5B and 5C, proNGF induced 1.7- and 1.6-fold increase in active RhoA in vivo and in RGC-5 cells, respectively as compared with the control groups. Recent findings demonstrated proinflammatory effects of proNGF/p75NTR via stimulating TNF-α expression, which can induce RGC death [29], [30]. As shown in Figure 5E, overexpression of proNGF in healthy retina induced TNF-α mRNA expression (3-fold) as compared with the control group. This effect was partially and significantly reduced (1.9-fold) by treatment with Y27632. We next compared the direct effects of proNGF versus TNF-α on activating RhoA in retinal ganglion cells. Cultures of primary retinal ganglion cells or RGC-5 were stimulated with proNGF (50 ng/ml) or TNF-α (10 ng/ml). Mutant proNGF induced activation (1.6-fold) of RhoA in RGC-5 cells (Fig. 5D) as detected by pull down assay and activation (1.75-fold) of RhoA in primary RGC cultures (Fig. 5F) as detected by G-LISA. In contrast, TNF-α caused modest increase in RhoA activation (20%) in both RGC-5 (Fig. 5D) and primary cultures as compared with controls (Fig. 5F).These results were further confirmed with complete blockade of active RhoA in groups treated with the selective Rho kinase inhibitor Y27632 in vivo and in vitro.
10.1371/journal.pone.0054692.g005Figure 5 ProNGF selectively activates RhoA kinase activation in vivo and in RGC cultures.
A. Pull-down assay of rat retinal lysate showed 2.3-fold increase in the expression of active Rho in diabetic rats as compared with the controls (n = 4–5). B. Pull-down assay of rat retinal lysate showed 1.7-fold increase in active RhoA expression in rats electroporated with proNGF as compared with those electroporated with GFP (n = 5). C. Pull-down assay of RGC-5 lysate showed 1.6-fold increase in RhoA expression in RGC-5 cells treated with proNGF as compared with the controls (n = 4). Treatment of rats or RGC-5 with Y27632 blocked RhoA activation proNGF-treated samples but not the control groups. D. Pull-down assay of RGC-5 showing that treatment of RGC-5 cells with TNF-α did not increase RhoA activation as compared with the control group (n = 4). E. Statistical analysis showing overexpression of proNGF in healthy retina induced 3-fold increase in TNF-α mRNA expression as compared with the control group. F. Statistical analysis of G-LISA showing 1.7-fold increase of RhoA in primary RGC cultures treated with proNGF as compared with the control. TNF-α caused modest increase in RhoA activation (20%) as compared with the controls. These effects were reduced by treatment with Y27632. * = significant difference as compared with the control group at p<0.05.
Inhibiting RhoA kinase blocked proNGF-induced p38/JNK MAPK activation
We next evaluated the activation of p38/JNK MAPK as a common signaling pathway implicated in neuronal death [2]. WB analysis of rat retinal lysate showed 1.5- and 1.8-fold increase in the phosphorylation of p38MAPK and JNK, respectively in proNGF group compared with GFP group (Fig. 6A, 6B). Furthermore, WB analysis of RGC-5 lysate showed 2.4 and 1.9-fold increase in the phosphorylation of p38MAPK and JNK, respectively in RGC-5 cells treated with mutant proNGF compared with the control (Fig. 6C, 6D).
10.1371/journal.pone.0054692.g006Figure 6 Inhibiting Rho kinase blocked proNGF-induced p38/JNK MAPK activation.
A,B. WB analysis of rat retinal lysate showed 1.6- and 1.8-fold increase in the phosphorylation of p38MAPK and JNK in rats electroporated with proNGF as compared with those electroporated with GFP (n = 4–6). C,D. WB analysis of RGC-5 lysate showed 2.4 and 1.9-fold increase in the phosphorylation of p38MAPK and JNK, respectively in RGC-5 cells treated with proNGF as compared with the controls (n = 4). Treatment of rats or RGC-5 with Y27632 blocked all these effects in rats and media treated with proNGF and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control.
Inhibiting RhoA kinase blocked diabetes- and proNGF-induced apoptotic markers expression
The expression of apoptotic markers was examined in vivo and in vitro. As shown in Figure 7A, 7C, diabetic rat retinal lysate showed 1.7-fold and 2.3-fold in the expression of cleaved-PARP and caspase-3 as compared with the controls. In parallel, there was 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in proNGF group compared with GFP-control (Fig. 7B, 7D). Treatment of rats with Y27632 blocked these effects. In addition, treatment of RGC-5 cells with mutant proNGF caused 2.1- and 1.6-fold increase in the expression of cleaved PARP and caspase-3 as compared with the controls (Fig. 7E). Co-treatment of RGC-5 with Y27632 blocked these effects.
10.1371/journal.pone.0054692.g007Figure 7 Inhibiting Rho kinase blocked diabetes- and proNGF-induced apoptotic markers expression.
A,C. WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in rats electroporated with proNGF as compared with the controls (n = 4–5). B,D. WB analysis showing 1.9- and 2.2-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 cells treated with proNGF as compared with the controls (n = 4). E. WB analysis showing 2.1- and 1.6-fold increase in the expression of cleaved PARP and caspase-3 in RGC-5 treated with proNGF as compared with the controls. Treatment of rats or RGC-5 with Y27632 blocked all these effects in rats and media treated with proNGF and did not affect the control groups. * = significant difference as compared with the rest of the groups at p<0.05. C, control.
Discussion
The main findings of the current study are: 1) Overexpression of the proNGF mimics diabetes action resulting in retinal neurodegeneration in vivo and in vitro, 2) Inhibiting Rho kinase exerted neuroprotective effects by inhibiting p75NTR expression, inhibiting inflammation and activation of JNK/p38MAPK in response to proNGF or diabetes (Fig. 8). We believe that this is the first study to demonstrate a direct apoptotic effect of proNGF on retinal ganglion cells and delineate the apoptotic role of RhoA activation in retinal neurodegeneration. Together, these results support inhibition of RhoA kinase as a potential effective therapeutic target for the treatment of diabetic retinopathy.
10.1371/journal.pone.0054692.g008Figure 8 Diagram depicting the proposed role of proNGF/p75NTR in diabetic retinopathy.
Retinal neurodegeneration is thought to be induced via direct activation of RhoA kinase in RGC and paracrine inflammatory action in response to increases in proNGF.
Diabetes and proNGF overexpression caused significant increases in number of TUNEL-positive cells (Fig. 1) and loss of total neuronal cells and RGC in the ganglion cell layer (Fig. 2). Interestingly, while loss of total neuronal cells in GCL was 20–30%, specific loss of RGC identified by Brn3a, the selective RGC marker was 60%, suggesting that RGC cells are more sensitive to death signals in response to proNGF. These results lend further support to previous reports showing retinal neurodegeneration in response to diabetes or proNGF overexpression [1], [7], [23], [29]. Although it has been documented that proNGF can promote neuronal apoptosis through binding p75NTR (reviewed in [3], [4]), the exact molecular events by which proNGF mediates its apoptotic action in retinal neurons are not fully understood. Whether RGCs express p75NTR remains a controversy. We and others have demonstrated p75NTR expression in both Müller cells and RGCs [1], [31]–[34], while other groups have reported p75NTR expression only in Müller cells [29], [35], [36]. Findings from the current study support a direct apoptotic effect of proNGF on RGC (Fig. 3) as well as demonstrates p75NTR expression in primary RGC cultures isolated from neonatal retinas (Fig. 4F) and mixed neuronal cultures isolated from adult retina (Fig. 4G). One possible explanation of these seemingly contradictory findings is that p75NTR expression in RGC varies depending on the health and age whether developing or adult retina.
Overexpression of p75NTR constitutively activates endogenous RhoA [8], [9] leading to neuronal death. Therefore, we hypothesized that inhibition of Rho kinase is neuroprotective. In agreement, our results showed significant activation of RhoA in diabetic retina, proNGF overexpressing retina and RGC cultures using pull down assay (Fig. 5A–C). Due to sample limitation, activity of Rho GTPase was detected using G-LISA technique in primary RGC cultures. These results also showed that mutant proNGF markedly (1.75-fold) causes Rho GTPase activity (5.E). Therefore, we examined the neuroprotective effects of RhoA kinase inhibitor Y27632, the first identified specific inhibitor of the ROCK family of protein kinases, in diabetic retinas as well as in response to proNGF in vivo and in vitro. Treatment with Y27632 showed significant neuroprotective effects both in diabetic animals (Fig. 1) and proNGF overexpressing retina (Figs. 1 and 2). In vitro, Y27632 completely blocked proNGF-induced cell death in primary RGC cultures and RGC-5 cell line. Of note, primary RGC cultures were far more sensitive to the apoptotic effects of proNGF compared to RGC-5 cell line (Fig. 3). Our results lend further support to previous reports showing neuroprotective of Y27632 in cultured cortical neuronal cells [37] and in models of cerebral ischemia and transient retinal ischemia [38], [39]. Although inhibitors of both Rho kinase and Rho GTPase have been shown to enhance ocular blood flow, retinal ganglion cell survival (reviewed in [40]), we believe that this is the first study to document the neuroprotective effects of Y27632 in diabetic retina or proNGF overexpression models.
In addition to the direct apoptotic effect of proNGF/p75NTR in neurons, a proinflammatory role of proNGF/p75NTR has been proposed in Müller glial cells. We and others have shown that proNGF overexpression can induce marked retinal neuronal death via p75NTR-mediated TNF-α production in Müller glia cells [29], [30]. Inhibition or genetic deletion of p75NTR exerted neuroprotective effects [29], [30], [41]. To investigate the effects of inhibiting Rho kinase activity on proNGF proinflammatory effects, we assessed TNF-α expression using rtPCR. The results showed that proNGF induced 3-fold increase in TNF-α expression that was partially but significantly reduced by Y27632 (Fig. 5E). These results support a proinflammatory role of proNGF in the retina and indicate that the neuroprotective effects of inhibiting RhoA could be attributed, at least in part to inhibiting inflammatory mediators including TNF-α. Accordingly, previous reports have shown that Y-27632 inhibited production of TNF-α via modulation of NFκB in non-diabetic models [42], [43]. Interestingly, our analyses showed that proNGF could activate RhoA in primary RGC cultures (1.75-fold) and RGC-5 cell line (1.6-fold) while TNF-α caused modest activation (20%) in both primary RGC and cell line (Fig. 5).These results support the direct and unique pathway proposed for proNGF activating RhoA apoptotic signal in RGC.
Activation of p38 and JNK in sensory neurons has been reported in early diabetes in rats and in diabetic patients [44]. In parallel, studies have also shown that activation of the RhoA/p38 MAPK pathway causes neuronal death [8]–[12]. In agreement, our results showed significant increases in phosphorylation of JNK and p38 MAPK in response to overexpression of proNGF in rat retina or RGC-5 cells. Treatment of the diabetic animals, proNGF-overexpressing animals or RGC-5 cultures with Y27632 prevented neuronal cell death and blocked expression of apoptotic markers including cleaved PARP. These results demonstrate a novel pathway by which increased expression of proNGF leads to retinal neurodegeneration directly via activation of p75NTR/RhoA in RGC. Inhibition of Rho/ROCKs might be an attractive therapeutic target in the treatment of diabetic retinopathy; however further studies are warranted to determine the role of ROCK inhibitors in clinical practice [45].
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DaruDaruDARU Journal of Pharmaceutical Sciences1560-81152008-2231BioMed Central 2008-2231-20-2910.1186/2008-2231-20-29Research ArticleDrying of a plasmid containing formulation: chitosan as a protecting agent Mohajel Nasir [email protected] Abdolhossein R [email protected] Kayhan [email protected] Alireza [email protected] Mohsen [email protected] Esmail [email protected] Amirabbas [email protected] Kambiz [email protected] Aerosol Research Laboratory, Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran2 Department of Pharmaceutics, Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran3 Department of Virology, Pasteur Institute of Iran, Tehran, Iran4 Department of Medicinal Chemistry, School of Pharmacy, Tehran University of Medical Science, Tehran, Iran5 Department of Nanobiotechnology, Pasteur Institute of Iran, Tehran, Iran2012 10 9 2012 20 1 29 29 6 7 2012 10 7 2012 Copyright ©2012 Mohajel et al.; licensee BioMed Central Ltd.2012Mohajel et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The purpose of the study. Along with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients.
Method
Molecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones.
Results and major conclusion
Size distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.
NanocomplexPolymeric gene deliveryGene therapySpray-dryingFreeze-drying
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Introduction
While viral gene delivery systems are more efficient and targeted than non-viral gene delivery systems in transducing cells, there are serious concerns about their safety
[1]. In addition, they have limited capacity for DNA packing and are not easy to produce. These major shortcomings have become a motive force for research on safe and efficient non-viral gene delivery systems. Polymers have received more attention because their ease of production and modification can be applied to overcome major defects of non-viral gene delivery systems such as low transfection, targeting efficiency and toxicity
[2].
Alongside efforts to make more safe and efficient gene carriers, it is necessary to make them stable during storage. Stability of non-viral gene delivery systems has been a major concern since the first days of gene therapy clinical trials
[3]. Presence of water can alter the stability of non-viral gene delivery systems in short term storage by facilitating their aggregation
[4] and in long term by increasing the likelihood of water catalyzed chemical reactions, which may alter the stability of nucleic acids
[5]. On the other hand, low efficiency of non-viral gene delivery systems calls for application of high doses of DNA in a limited volume in-vivo, which cannot be achieved during preparation of polymeric gene delivery systems. Because polyplexes aggregate in high concentrations
[6]. Removing water from polyplex suspensions, results in pharmaceutically reasonable stability profile at room temperature and simultaneously increases concentration of therapeutic gene in the formulation. These formulations can be applied as dry powder (in respiratory gene delivery) or reconstituted with water before application.
In solid state, polymer based gene delivery systems have an advantage over lipid based ones because they are less vulnerable to oxidative reactions
[7]. Although freeze-drying is a well-established industrial approach, conventional freeze-drying can exacerbate aggregation in its freezing step by concentrating nanosuspension in unfrozen phase. Therefore, special modifications have been considered to increase cooling rate to overcome this defect
[8]. On the other hand, it has been shown that spray-drying of insulin loaded chitosan/tripolyphosphate nanoparticles did not change their size and zeta potential significantly
[9]. This was attributed to the high drying speed
[10]. However, it has been reported that excessive shear rate can destroy supercoiled structure of plasmid
[11]. Appropriate design of a gene delivery system is of critical importance not only for protecting nucleic acids in-vivo but also for preserving them against the process stresses like temperature and shear.
The ability of a lipid-protein gene delivery system to protect DNA in a spray-drying process and the effects of various excipients on transfection efficiency and aerodynamic properties of the resulted powder had been thoroughly studied by Li et al.
[12,13]. In vivo gene transfer performance of a chitosan-pDNA complex processed by supercritical technology have been studied by Okamuto et al.
[14]. Regarding DNA stability in supercritical fluid technology, the main destabilizing factor is dissolved carbon dioxide in water, which builds up an acidic pH and promotes DNA hydrolysis, while in spray-drying, the shear stress and temperature are main destabilizers.
Availability, biocompatibility, inherent positive charge and permeability enhancing capability make chitosan an attractive non-viral gene delivery system
[15]. Polyion complex associates of chitosan and DNA are strong and have been suggested to be further stabilized by hydrogen and hydrophobic bonds. It has also been hypothesized that these bonds are strong enough to prevent DNA liberation from complexes and thus decrease free plasmid content after endosomal escape and detract from transfection efficiency of the polymer
[16]. Molecular weight reduction has been applied as a successful strategy to increase the transfection efficiency of chitosan, with a suggested mechanism of loosening the bond between DNA and the polymer
[17]. So, Chitosan can be applied as a good representative to study protection of plasmid DNA by polymeric gene delivery systems against the process stresses.
The aim of this study was to evaluate the ability of low molecular weight chitosan to protect bioactivity of pDNA against shear and thermal stresses generated during spray-drying and high freezing rate freeze-drying processes and study the effect of leucine as a commonly used excipient in preparation of spray dried formulations on this protection.
Materials and methods
Materials
Medium viscose chitosan was obtained from Primex (Siglufjordur, Iceland). Sodium nitrite, sodium hydroxide, glacial acetic acid and L-leucine were purchased from Merck KGaA (Frankfurt, Germany). Lactose monohydrate free sample was provided by DMV-Fonterra Excipients (Foxhol, Netherlands). All cell culture materials were obtained from Invitrogen (CA, USA).
Amplification of plasmid
pEGFP-N1 plasmid (Clontech Laboratories, CA, USA) encoding the enhanced green fluorescent protein as a reporter was amplified in Escherichia coli DH5α by overnight incubation of the bacteria and then purification of the plasmid by a Giga plasmid purification kit (Nucleobond™ PC 10000, Macherey-Nagel GmbH &CO.KG, Düren, Germany). The resulted plasmid was analyzed for its purity and concentration by gel electrophoresis and UV absorption at 260 and 280 nm (Picodrop™, Saffron Walden, UK).
Preparation of low molecular weight chitosan (LMWC)
Low molecular weight chitosan was prepared by chemical reaction between chitosan and nitrous acid. Two grams of medium viscose chitosan Primex (Siglufjordur, Iceland) slowly dispersed into 78 ml deionized water. Then, 6 ml glassial acetic acid was added to the dispersion to dissolve chitosan. Nitrogen bubbled into this solution and then 16 ml of a freshly prepared sodium nitrite solution with a concentration of 5 mg/ml was added. Reaction mixture was sealed by a cap and stirred in room temperature for 90 minutes then pH was adjusted to 9 by adding 4 M sodium hydroxide to terminate the reaction. The resulting suspension was centrifuged at 6000 rpm, and the sediment was washed by ice-cold water thoroughly over a buchner funnel, redissolved in a minimum amount of 1% acetic acid solution and dialyzed against two liters of deionized water, which was replaced every 12 hours, for 48 hours. The resulted solution was freeze-dried, and the powder was kept at 2-8°C in the dark until use.
Molecular weight (Mw, Mn) and polydispersity (Mw/Mn) of LMWC were determined by gel permeation chromatography. The polymer dissolved at the concentration of 0.1% in 0.3 M acetate buffer with a pH value of 4.6. The resulted solution filtered through a 0.22 μ filter and chromatographed at 25°C using the same eluent at a flow rate of 4 ml/min through a PL aquagel-OH Mixed –H 25 mm ID 8 μm column on a Knuer chromatographic system (Knuer, Berlin, Germany), which was equipped with an intrinsic viscosity detector. Pullulan standards in five molecular weights were used for calibration using the same conditions.
Particle formation
To prepare a stock solution of LMWC, the polymer was dissolved in 0.005 M acetate buffer with a pH value of 5 and its concentration was adjusted to 7 mg/ml, then this solution was centrifuged at 30000 Relative Centrifugal Force (RCF) for 60 minutes in 4°C (Sigma Laborzentrifugen GmbH, Osterode, Germany) and the supernatant was filtered through a 0.22 μ filter. This solution was stored at 2-8°C in the dark and used within a week from the date of preparation.
Nanocomplexes of LMWC and pEGFP-N1 were prepared using the ionic gelation method. For preparation of each nanocomplex formulation predetermined volumes of LMWC stock solution were diluted with the same solvent to obtain target concentration and was added to the same volume of pDNA solution with concentration of 40 ng/μl, which was prepared in Milli-Q water, in three increments at room temperature and under the vortex. After addition of LMWC to pDNA solution, the mixture was incubated in room temperature for 20 minutes for the particle formation before further experiments.
Spray-drying
Nanocomplex formulations spray dried immediately after formation (Buchi B-191 mini-spray dryer™; Buchi Labortechnik AG, Switzerland) to make powder formulations given in Table
1. The spray-drying conditions were the same as reported by Li et al.
[13], which were: inlet temperature 150°C, spray flow rate 600 liter/h, aspirator adjusted to 35 m3/h, pump adjusted to 30% to give a flow rate of 7.5 ml/min. These conditions resulted in an outlet temperature of 70-85°C. The spray-drying yield was calculated, and the recovered powders stored in moist resistant containers at 2-8°C until next experiments.
Table 1 Theoretical composition of freeze dried and spray dried powders
Components Formulations
100L 100N 70L 70N 50L 50N 20L 20N 10L 10N 7L 7N 5L 5N 2L 2N
DNA 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
LMWC 33.7 33.7 23.6 23.6 16.8 16.8 6.7 6.7 3.4 3.4 2.36 2.36 1.68 1.68 0.67 0.67
Lactose 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000 2000
Leucine - 200 - 200 - 200 - 200 - 200 - 200 - 200 - 200
All measurements are in milligrams.
Freeze-drying
Nanocomplex formulations were freeze-dried immediately after preparation by a modified freeze-drying technique. Briefly, prepared nanosuspensions were transferred to cryotubes and immersed in liquid nitrogen for 10 minutes to snap freeze the nanosuspensions, then these tubes were transferred to a pre-cooled freeze-dryer chamber (Alpha 1–4 LD plus™, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) after loosening their caps. Freeze-drying conditions were: 48 h in 0.04 mbar to give an ice condenser temperature of −50°C and 12 h in 0.001 mbar for ice condenser temperature of −80°C.
Particle size and zeta potential analyses
Particle size and zeta potential of the nanocomplexes were measured before and after freeze or spray-drying by a Zetasizer Nano ZS™ (Malvern Instruments, Malvern, Worcestershire, UK). Freshly prepared nanocomplexes and dry powders were diluted in Milli-Q water to give a count rate of 100–200 KC/S. All measurements were performed at 25°C in triplicate. Viscosity and refractive index of water in this temperature (0.8905 mPa s, 1.333) were used for data analysis.
Stability of pDNA
To analyze the integrity of DNA after spray and freeze-drying, plasmid DNA was electrophoresed after dissociation from LMWC. To dissociate DNA, an amount of each nanocomplex formulation equivalent to 1.5 μg of DNA was dispersed in 30 μl water. This suspension was added to 1.5 mg of heparin, which was dissolved in the same volume of water and incubated for five hours at room temperature. After the incubation time, 20 μl of the resulted suspension was mixed with 4 μl of loading dye and was loaded into each well of a 0.5% agarose gel, which were stained with EtBr, and electrophoresed at a voltage of 100 V, in TBE buffer (pH 8) for 60 minutes. Gel images were acquired by a BioDoc-It™ imaging system (UVP, Upland, CA, USA), while uncomplexed pDNA was used as control. The acquired images were processed to reduce EtBr background fluorescence and analyzed by Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,
http://rsb.info.nih.gov/ij/, 1997–2009).
In-vitro transfections
The day before transfection, each well of a 24 well cell culture plate, containing 1 ml of DMEM media supplemented with 10% FBS and 1% penicillin-streptomycin, was seeded with 70000 low passage 293 T cells. Transfection was conducted when confluency was about 80%. Before transfection, the media was replaced with 500 μl of fresh serum free media (at pH 6.9). After replacement of media 100 μl of nanocomplex formulation or reconstituted powder (equal to 2 μg of pEGFP-N1) were transferred to each well. Cells were incubated at 37°C, 5% CO2, with nanocomplexes for 4 h and then the medium replaced by 1 ml of fresh serum containing media.
Seventy-two hours later, cells were washed with warm PBS buffer twice and trypsinized for 5 minutes. Then 500 μL PBS buffer, supplemented with 10% FBS was added to neutralize trypsin and cells were analyzed by flow cytometry (CyFlow™, Partec GmbH, Münster, Germany) to assess GFP expression.
Statistical analysis
To compare particle size distribution curves two different techniques were applied (i.e. Kolmogorov-Smirnov (K-S test) and similarity factor (f2)) K-S test was selected as a non-parametric method to compare distribution curves of the processed nanocomplexes by freshly prepared ones. Similarity factors were calculated using the following equation as a parallel test:
(1) f2=50×log1+1n∑j=1nwj|Rj−Tj|−0.5×100.
In which Rj and Tj were the average intensity percent given by the instrument for a particular size (j) and wj is a weight factor for each size point, which here was set to be 1. These tests were selected because in the case of particle size the assumption for parametric tests (i.e. t-test) that sample is normally distributed is not correct and these distribution profiles are mostly right-skewed and bimodal, which cannot be fully represented by mean and standard deviation.
To study the effect of process, addition of leucine and N/P ratio on stability of pDNA and transfection efficiency a three-way analysis of variance was used.
Results
Molecular weight
Chemical reaction with NaNO2 has been used before for depolymerization of chitosan. The molecular weight of the polymer was demonstrated to reduce linearly with the ratio of chitosan/NaNO2[18]. The molecular weight of chitosan was determined by gel permeation chromatography coupled with refractive index detector. The number and weight molecular weight of LMWC were 9.8 kDa and 16 kDa respectively, which resulted in a polydispersity index of 1.63.
Size and zeta potential of nanoparticles
Size, polydispersity index (PdI) and zeta potential data of the nanocomplex formulations before and after processing by spray-drying or freeze-drying are presented in Figure
1. Reduction of N/P ratio increased the Z-average particle size and polydispersity index and decreased the zeta potential.
Figure 1 Size, polydispersity index and zeta potential of freshly prepared and reconstituted nanocomplexes from freeze dried or spray dried powders. a, z-average size, b, polydispersity index and c, zeta potential, Points are averages of three measurements and error bars are standard deviation.
To determine success of the two processing techniques in preservation of the particle size of nanocomplexes, Kolmogorov-Smirnov (K-S test) test, a nonparametric statistical test, was used. Similarity factor was also used to compare average particle size distribution curves of the freshly prepared nanocomplex formulations with processed ones. Results of these two tests are presented in Tables
2 and
3. The p-Values for K-S test (Table
2), showed that all formulations prepared by freeze-drying and 13 out of 16 spray-dried formulations successfully preserved the original size distribution profile of nanocomplexes over a broad range of N/P ratios. Results of K-S test for particle size comparison were in consistent with polydispersity data (Figure
1B). Calculated similarity factors showed a reduction as N/P ratio decreased and the statistical difference between formulations became significant when the similarity factor was below 82.
Table 2 p-Values of K-S tests for comparison of size distribution profiles of processed nanocomplexes with freshly prepared ones
Formulations
100L 100N 70L 70N 50L 50N 20L 20N 10L 10N 7L 7N 5L 5N 2L 2N
Freeze dried 0.860 0.508 0.822 0.713 0.943 0.877 0.84 0.806 0.205 0.08 0.109 0.518 0.681 0.448 0.244 0.595
Spray dried 0.84 0.777 0.240 0.38 0.748 0.174 0.799 0.79 0.115 0.167 0.799 0.343 0.392 0.02* 0.02* 0.045*
.*: statistically meaningful difference from original size distribution profile.
Table 3 Calculated similarity factors between size distribution profiles of freeze dried and spray dried nanocomplexes with those of freshly prepared ones
Formulations
100L 100N 70L 70N 50L 50N 20L 20N 10L 10N 7L 7N 5L 5N 2L 2N
Freeze dried 93.73 93.13 97.31 97.39 94.36 98.79 97.5 99.35 90.96 87.32 88.48 83.19 87.49 86.15 93.67 90.31
Spray dried 95.84 94.41 95.14 89.33 88.93 86.85 89.56 90.38 85.62 83.79 94.25 93.35 86.57 81.24 74.41 81.61
The data indicated that the average zeta potential decreased from around 30 for N/P equal to 100 and 70 to around 20 for N/P of 5 and 2.
Comparing the processed nanocomplexes with similar N/P ratios, the K-S test did not show significant differences between particle size distribution curves of the formulations prepared in presence or absence of leucine (all p-values were above 0.05).
Stability of pDNA
Stability of plasmid bounded with LMWC after freeze-drying and spray-drying processes was determined by electrophoresis. Figure
2a, shows images of two sample gels and Figure
2b presents percent of preserved supercoiled structure in the both processes. Nanocomplex formulations prepared with N/P ratios of 2 and 5 showed lowest stability of DNA during both processes, with approximately 70 percent of the supercoiled pDNA remaining intact.
Figure 2 Relative stability of supercoiled structure during freeze-drying and spray-drying processes. a, Electrophoresis images of dissociated pDNA from reconstituted nanocomplexes processed by freeze-drying (upper) and spray-drying (lower) pure pDNA was used as control. b, relative intensity of supercoiled pDNA band of freeze dried and spray dried nanocomplexes. Error bars are standard deviation (n = 3).
A three-way ANOVA test used to study the effect of process, addition of leucine and N/P ratio on supercoiled structure stability. There was a significant difference between the effect of freeze-drying and spray-drying processes on preserving the supercoiled structure (p <0.001). In addition, it was shown that the stability of supercoiled structure was increased significantly with the increase in N/P ratio (p < 0.001). The test did not show a significant destabilization effect for leucine on supercoiled structure (p = 0.184).
In-vitro transfection
The relative transfection efficiency of the nanocomplexes is presented in Figure
3. Except for N/P ratios of 2 and 5, all N/P ratios in both kinds of processed powders could retain approximately their original transfection efficiency. Even spray dried powders showed 100% relative transfection, although their supercoiled pDNA content was lower than the freeze-dried powders. To analyze the effect of the process, the addition of leucine and the N/P ratios a three-way analysis of variance was conducted. The test did not show a meaningful effect for the type of process or addition of leucine (p = 0.827 and p = 0.476, respectively), while the effect of N/P ratio was reemphasized (p < 0.001).
Figure 3 Relative transfection efficiency of reconstituted freeze dried or spray dried powders. Error bars are standard deviation (n = 3).
Discussion
Effects of three different variables (i.e. processing technique, N/P ratio and presence of leucine) on physicochemical and biological properties of nanocomplexes were tested by statistical analysis. These effects have been discussed below.
Effect of N/P ratio
The decrease in the number of positively charged amine groups with respect to negatively charged phosphate groups led to a reduction in zeta potential and in turn, made the nanocomplexes more vulnerable to aggregate. It has been shown that chitosan-DNA nanocomplexes aggregate quickly in Hank’s buffered salt solution at the amine per phosphate ratio of 5
[19]. Increased aggregation rate in lower N/P ratios makes stabilization of the nanocomplexes more challenging
[20]. This effect can be seen in the difference of particle size distribution of three spray-dried formulations with lower N/P ratios form freshly prepared ones.
According to our results N/P ratio had a significant effect on stability of supercoiled structure and preservation of biological activity during both tested processes. It has also been demonstrated in previous researches that increased N/P ratio can increase physical stability of DNA containing nanocomplexes against shear stress
[21]. The level of protection also depends on the type of complexing agent. Lipoplexes are shown to be more vulnerable against shear stress generated during nebulization
[22].
Effect of processing technique
Kuo et al.
[21] compared Z-average particle sizes of poly(ethyleneimine)-pDNA polyplexes before and after a spray-freeze-drying process and observed no statistically meaningful difference between the two groups. However, to our knowledge there are not more reports, comparing size distribution of nanocomplexes prepared by pDNA and a polymeric carrier before and after a drying process.
The high rate of aggregation of LMWC-pDNA nanocomplexes necessitates instant immobilization of the system to prevent changes in particle size. Although both techniques applied in this work have such a capability, but spray-drying of liquid formulations took more time than freezing in liquid nitrogen to immobilize the nanocomplexes. Therefore, it seemed that in low N/P ratios, the rate of aggregation of the nanocomplexes was higher than the rate of drying of the nanocomplexes during the spray-drying process.
Stability of the supercoiled structure can also be altered by processing technique. Seville et al. freeze dried a lipid :polycation :pDNA vector with a similar freeze-drying technique
[11]. Their results showed 100% stability of supercoiled DNA during the process. Mohri et al., showed that chitosan can protect pDNA in N/P ratios over 5 during a spray freeze-drying process
[22]. It was shown that a freeze drying process with increased cooling rate can improve preservation of biological activity of non-viral gene delivery systems (8), despite the fact that rapid freezing of naked DNA can extensively damage its structure
[23,24]. Our results also indicated that by applying a similar freeze-drying condition, in N/P ratios > 10, a 100% preservation of supercoiled plasmid could be obtained.
On the other hand, spray-drying is known to be a harsher technique compared to freeze-drying. Seville et al. recovered 70-80% of original supercoil band strength after processing of a lipid-protein carrier by spray-drying technique
[13], but here we achieved over 90% of the band strength in N/P ratios of 10, 20 and 50 and even 100% at N/P ratios of 70 and 100.
While powders processed by spray-drying showed lower stability profile in nanocomplex size and supercoiled DNA content, compared to freeze-dried ones, their transfection efficiencies were not significantly affected. Our data is in accordance with Seville and coworkers, who has reported an enhancement in transfection efficiency of spray dried powders despite lower supercoiled DNA content compared to freeze dried formulations. They did not compare size distribution of nanocomplexes before, and after processing but our data showed an increase in polydispersity of spray-dried formulations, which along with reduction in content of supercoiled structure should have led to a decrease in biological activity of the formulations. These findings suggest even enhancement of transfection activity in spray-drying or reduction of this ability in freeze-drying via unknown mechanisms.
There are some reports indicating enhanced gene expression after freeze-drying or spray-drying of gene delivery systems. Seville et al. witnessed a 50% increase in gene expression using powders processed by spray-drying technique but freeze-dried powders did not show the same result
[11]. Talsma et al. observed enhanced transfection efficiency after freeze-drying of an adenovirus-enhanced AVET system, but in the case of pCMVL:transferrin-poly(ethlenimine) complexes, they showed 100% of original transfection efficiency after freeze-drying in a 10% sucrose solution
[25]. Furthermore, Kuo and Hwang did not report an enhancement in transfection ability of poly (ethyleneimine)-DNA complexes processed by spray-freeze-drying
[21]. In this work, we did not see a meaningful increase in transfection efficiency after spay drying and freeze-drying.
Effect of leucine
Leucine is a common excipient in the formulation of dry powder inhalers to enhance dispersibility of the powders. A previous report indicated that co-processing of a lipid-polycation-pDNA with leucine and lactose led to the reduction of the biological activity of the plasmid, compared to those which formulated with just lactose in transfecting A549 cells
[26]. Therefore, it has been suggested that leucine acts as a destabilizer for complex between DNA and a positively charged material. We added leucine to our powder formulations to assess the ability of chitosan in maintaining original size distribution, DNA structure and transfection in the presence of this excipient. Our results did not show a meaningful effect for leucine on any of the mentioned indicators. This effect can be attributed to higher resistance of chitosan bond with DNA against the effect of leucine. Köping-Höggård and coworkers found that incubation with sodium chloride, 3.5 M, sodium dodecyl sulfate, 0.5 M or heparin, with a 10-fold excess of negative charges could not liberate DNA from its complex by ultrapure chitosan. Based on these works higher stability of chitosan nanocomplexes with plasmid is plausible.
Conclusion
In this work, we tried to assess the ability of LMWC as a carrier to protect supercoiled structure and thus transfection ability of plasmid DNA, and maintain the original size of the freshly formed nanocomplexes during the harsh conditions of spray-drying and freeze-drying processes. Our indicators were size, zeta potential, polydispersity index, intensity of supercoiled bond on electrophoresis gel and transfection efficiency in in-vitro conditions.
LMWC could efficiently protect plasmid supercoiled structure and retain its transfection capability against stresses imposed by freeze-drying and spray-drying processes. The stability of supercoiled structure was also retained in the presence of leucine, which has been reported to destabilize the complex between DNA and a positively charged material. Our finding suggests that LMWC carrier is resistant to the drying process stresses imposed to gene delivery systems.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Concept and design: KG, NM, KA, MA Acquisition of data: NM, EM, AR Analysis and interpretation of data: NM, KG, KA, AV, ARN Drafting and revising: NM, KG, KA, ARN. All authors read and approved the final manuscript.
Acknowledgment
Financial support for this work was provided by research grant NO. 427 from Pasteur Institute of Iran.
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Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 10.1155/2013/369180Research ArticleThe Effect of Elephantopus scaber L. on Liver Regeneration after Partial Hepatectomy Tsai Chin-Chuan
1
2
Wu Jia-Ping
3
Lin Yueh-Min
4
5
Yeh Yu-Lan
4
5
Ho Tsung-Jung
6
Kuo Chia-Hua
7
Tzang Bor-Show
8
9
Tsai Fuu-Jen
10
Tsai Chang-Hai
11
Huang Chih-Yang
3
10
12
*1School of Chinese Medicine for Post-Baccalaureate, I-Shou University, No 91. Hsueh-Shih Road, Taichung, R.O.C., Kaohsiung 40402, Taiwan2Chinese Medicine Department, E-Da Hospital, Kaohsiung, Taiwan3Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan4Department of Pathology, Changhua Christian Hospital, Changhua, Taiwan5Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan6Chinese Medicine Department, China Medical University Beigang Hospital, Taiwan7Laboratory of Exercise Biochemistry, Taipei Physical Education College, Taipei, Taiwan8Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan9Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan10Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan11Department of Healthcare Administration, Asia University, Taichung 40402, Taiwan12Department of Health and Nutrition Biotechnology, Asia University, Taichung 40402, Taiwan*Chih-Yang Huang: [email protected] Editor: R. Govindarajan
2013 10 1 2013 2013 36918015 6 2012 4 12 2012 4 12 2012 Copyright © 2013 Chin-Chuan Tsai et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Liver regeneration after partial hepatectomy (PHx) is a physiological response for maintaining homeostasis. The aim of this study is to investigate effects of Elephantopus scaber L.- (ESL-) induced liver regeneration on growth factors (HGF and IGF-1), cell cycle regulation, and apoptosis suppressed. In this study, we fed five Chinese medicinal herbs (1 g/kg/day), Codonopsis pilosula (CP, Dangshen), Salvia miltiorrhiza Bunge (SMB, Danshen,), Bupleurum kasi (BK, Chaihu), Elephantopus scaber L. (ESL, Teng-Khia-U), and Silymarin (Sm, 25 mg/kg) for 7 days to male Spraue-Dawley rats. Then surgical 2/3 PHx was conducted and liver regeneration mechanisms were estimated on the following 24 hrs and 72 hrs. The activities of growth factors (HGF and IGF-I) and cell cycle proteins were measured by Western blot and RT-PCR. Histological analysis and apoptosis were detected by H&E stain and TUNEL. The results showed that extraction of Elephantopus scaber L. (ESL) and Silymarin (Sm, positive control) were increased protein expression levels of HGF and IGF-1 which leads into cell cycle. These results suggest that the ESL plays a crucial role in cell cycle-induced liver regeneration and apoptosis. These results suggested that the ESL plays a crucial role in cell cycle-induced liver regeneration and suppressed hepatocytes apoptosis.
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1. Introduction
The liver is an excellent tissue when it is underwent surgical resection for growth regulation. Hepatocytes are ability to regenerate by a process of compensatory growth and then return to quiescent state [1–3]. Much of the investigation on the mechanisms of hepatic growth has been done in partial hepatectomy (PHx). Most liver cancer patients have to section partial liver by surgery. After surgery, hepatocytes need to regeneration and increase cell numbers. The native hepatocyte function cannot maintain the integrated whole liver function. Therefore, we suggested that Chinese herbal medicines may act as cell cycle progression agents. On the other hand, Silymarin has been used to protect the liver agent as a cytoprotection for treatment of liver disease. Several mechanisms of cytoprotection have been identified, but liver resection has not been reported. In vitro and animal studies have suggested that milk thistle's active ingredient, Silymarin, promoted hepatocyte regeneration and survival [4]. In this study, we suggested Silymarin as a positive control. Almost immediately after PHx, there are major changes in the complete mitogens expression for hepatocytes and in the expression of a relatively large number of genes. TGF beta1 is a potent antagonist to the mitogenic effects of terminating the proliferative response of hepatocytes during liver regeneration [5–7].
In this study, we detected traditional Chinese medicines, such as Codonopsis pilosula (CP), Salvia miltiorrhiza Bunge (SMB), Bupleurum kasi (BK), Elephantopus scaber L. (ESL), and Silymarin (Sm) effects on liver regeneration. Codonopsis pilosula is an widely edible traditional Chinese medicine (TCM) in China [8]. Some paper studies evaluate that CP would be even stimulated the survival signaling [9], control cell cycle [10], and antiscar formation. Salvia miltorrhiza Bunge root is also a traditional Chinese medicine, which is considered to promote blood flow and remove blood stasis. Some studies show that SMB has protective effects on human kidney possibly through inhibition of inflammatory cytokines and has long been used for treating liver and heart disease in China [11, 12]. Recent papers have indicated that SMB plays an adjuvant role in inhibited the proliferation and anticarcinogenesis. Bupleurum kasi is one of the most important traditional Chinese crude drug [13, 14] for treating hepatitis malaria and intermittent fever. It has the function of soothing the liver. BK was observed in resisting the level of cytokines and antifibrosis [15]. Elephantopus scaber L. is a folk medicine of Taiwan derived from the entire plants of Elephantopus scaber L. E mollis H.B.K and Pseudeelephantopus spicatus (Jass) Rohr. However, some studies elucidated Taiwan folk as a medicine. ESL has hepatoprotective effects [16]. There are some studies showed that ESL exerted anticancer effects on various cancer cells and induced cancer cells apoptosis from cell cycle arrest [17, 18].
As it is well known, partial patients with hepatocellular carcinoma need section partial liver. At the same time, liver needs proliferation to maintain original liver mass. We detected possible molecular mechanisms for these traditional Chinese medicines by examining the levels of external and intrinsic signal mechanisms. The liver can precisely regulate its growth and mass after surgical resection of hepatic lobes or hepatocytes loss caused. Hepatocyte replication while enlarged liver mass is corrected by apoptosis. Regeneration requires the cytokines TGF-beta1 to prevent cytotoxicity. In addition, extensive remodeling of the hepatic extracellular matrix occurs shortly after PHx. Several growth factors have been suggested to play a crucial role in liver regeneration after treatment TCM. HGF is believed to play a primary role in liver regeneration and promotes cell proliferation, survival, and morphogenesis through regulated DNA synthesis. Downstream of hepatocyte growth factor receptor activation is FAK (focal adhesion kinase), an important mediator of integrin signaling in the regulation of cell cycle, survival and regulates cell cycle progression [19, 20]. However, we also detected cell apoptosis expression by examining the levels of cytochrome c and bad from mitochondrial to find cell lost.
2. Materials and Methods
2.1. Animals
Male Sprague-Dawley rats weighing 180 to 220 g were obtained from the Animal House of National Science Council in Taiwan, and house five to a cage in a room with a controlled temperature of 22 ± 5°C, relative humidity of about 60% and free access to standard food in pellets and tapwater. Two or three cages were randomly assigned into the same group. All rats were acclimatised for 1 week prior to the beginning of all experiments.
2.2. Preparation of Hot-Water Extract from Chinese Medical Herbs
The hot-water extract was prepared by boiling the dried roots with distilled water for 1 h. The extract was filtered, freeze-dried, and kept at 4°C. The yield of extraction which Codonopsis pilosula (CP, Dangshen)was 21.34% [21], Salvia miltiorrhizae Bunge (SMB, Danshen) was 16.95% [22], Buplearum kaoi (BK, Chaihu) was 23.24% [23], Elephantopus scaber L. (ESL, Teng-Khia-U) was 11.84% [20], and Silymarin (Sm) was 16.73% [24]. The dried extract was dissolved in distilled water before use.
2.3. Experimental Partial Hepatectomy (PHx) and Sham (0 hr)
Three randomly selected animals were used for each time point. After injecting ketamine subcutaneously at a dose of 30 mg/kg, liver resections consisting of 2/3 of the liver mass were performed in partial hepatectomy group. Animals underwent the same operative anesthesia with the partial hepatectomy (PHx) group [25]. All the surgical operations were done the same as PHx, except the liver lobes were not resected. All the operations were performed between 8:00 AM and 12:00 PM to minimize diurnal effects. After completion of the procedure, the animals were placed under a lamp to prevent hypotermy and then put into cages (five animals per cage) with continuous supply of food and water. The animals in the PHx and corresponding were sacrificed at 6 hrs, 24 hrs, 72 hrs, and 168 hrs after the operation. The group of animals in which no surgery was performed, was used as control liver group and mentioned time “0” in quantitated groups. After all animals were sacrificed by cervical dislocation, the remnant liver lobes were excised and washed in PBS, then immediately frozen in liquid nitrogen.
2.4. Histological Analysis
Rats of all groups from different parts of time at 0 hr, 6 hrs, 24 hrs, and 72 hrs were sacrificed. The liver sections were taken out and fixed in 10% formalin and embedded in paraffin. Paraffin blocks were cut into 5-mm sections and stained with Hematoxylin-eosin (H&E) solution stain [26]. Silymarin (Sm, 25 mg/kg) oral gavages after PHx at 0 hr, 6 hrs, 24 hrs, and 72 hrs were also sacrificed and fixed and stained with H&E solution stain.
2.5. Transferase-Mediated dUTP Nick End Labeling (TUNEL)
Left ventricular sections were deparaffinized by immersing in xylene, rehydrated, and incubated in 2% H2O2 to inactivate endogenous peroxidases. The sections were then incubated with proteinase K (20 μg/mL), Protein K, working solution: [10-20 ug/ml in 10 mM Tris/HCl, pH 7.4-8]. Use Proteinase K from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results [27, 28]. Wash in phosphate-buffered saline, and incubated with terminal deoxynucleotidyl transferase for 90 min and fluorescein isothiocyanate-Dutp for 30 min at 37°C using an apoptosis detection kit [29]. Silymarin (S, 25 mg/kg) and Elephantopus scaber L. (ESL) oral gavages after PHx at 6 hrs, 24 hrs, and 72 hrs were also fixed and stained with kit. Samples were analyzed in a drop of PBS under a fluorescence and UV light microscope at this state by an excitation wavelength in the range of 450–500 nm.
2.6. Western Blot
Proteins were separated by 12% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked in 5% milk (diluted in Tris-buffered saline and 0.1% Tween 20) and incubated with the appropriate primary antibodies (TGFβ1, HGF, IGF-I, Cyclin D1, Cyclin E, pRb, cytochrome c, Bad, and E2F) at 4°C overnight and HRP anti-IgG was used as secondary reagent. After extensive washing, the targeted proteins were detected by enhanced chemiluminescence (ECL) [30].
2.7. Reverse Transcriptase PCR (RT-PCR)
0.5 μg of total RNA derived from liver plus primers by RT-PCR. The first-strand synthesis kit was applied according to the manufacturer's instructions of PCR. The primer pairs used for each gene were as follows. Cyclin D1: F:5′AGGAGACCATTCCCCTGACT3′
R:5′TTCTTCCTCCACTTCCCCTT3′
pRb: F:5′AGGAGGACTGTTCTCTAAGG3′
R:5′GAGTGAGGTGTGTCTTCTGA3′
E2F: F:5′AACATCCAGAACATCCAGTGGGTAGGC-AG3′
R:5′GGCTGTCAGTAGCCTCCAAG3′
Cyclin E:F: 5′CACCCCTGGCATCTTCTCCTT3′
R:5′AGCGTCTTCAGAGACAGCCAG3′
Cytochrome c: F:5′ACAGCACGCTTGTGGAT3′
R:5′GTCTTCAAGCAAGAGGACCA3′
Bad: F:5′TAAGACTCACCTGGGTACTG3
R:5′GCATGTAGTCACTCTTCACC3′
GAPDH: F:5′GGGTGTGAACCACGAGAAAT3′
R:5′CCACAGTCTTCTGAGTGGCA3′
The RT-PCR results were analyzed based on the assessment of product sizes upon ethidium bromide agarose gel electrophoresis. For each gene, we determined the cycle number of PCR reactions in which the PCR reaction was not saturated [31]. Based on this, we used the following PCR conditions, The initial denaturation step was at 95°C, then at annealing temperature and extension at 72°C. The final extension at 72°C for 10 min was applied to all the reactions and the PCR products were electrophoresed on a 1.2% agarose gel.
2.8. Quantification of Western Blot and RT-PCR
The intensity (area × density) of the individual bands on western blots and RT-PCR were measured by densitometry [32]. The background was subtracted from the calculated area.
2.9. Statistical Analysis
All data examined were expressed as mean ± S.E. For Western blot and RT-PCR analysis, quantitation was carried out by scanning and analyzing the intensity of the hybridization signals using FUJIFILM Imagine program. Statistical analysis of the data was performed using SigmaStat software. Comparison between group was made using one way ANOVA test [32]. A P value of less than 0.05 and 0.01 was considered to be statistically significant.
3. Results
3.1. Establishment of Liver Regeneration Animal Model Partial Hepatectomy
During liver regeneration after 2/3 hepatectomy, hepatocytes divide once or twice and return to quiescence. We detected the role of Chinese medicinal herbs in the process of liver regenerating after PHx. We suggest that Chinese herbal medicines may act as cell progression agent to make cell progress. Several mechanisms of cytoprotection have been identified, but the mechanisms of liver resection have not been reported. Surgical resection to remove a tumor together with surrounding liver tissue while preserving enough liver remnant for normal body function. After PHx, we found liver regeneration was started at 24 hrs and increases liver mass (Figure 1(b)), until 72 hrs and 168 hrs. However, Liver regeneration (%) was increased at 24 hrs, 72 hrs and 168 hrs PHx (Figures 1(a), 1(b), and 1(c)). More commonly, during liver regeneration the liver is injured and it attempts to repair the injured site referred to as internal scar tissue as quickly as possible. Cytokine, TGFβ1, increased in the plasma very shortly time kinetics and then decreased, but increased at the long time (Figure 1(f)). TGFβ1 increased reaching plateau amounts at 72 hrs PHx. Hepatocyte proliferation and apoptosis are coordinately regulated by TGFβ1. TGFβ1 protein expression were increased by treatment of SMB, CP, ESL, and Sm at 24 hrs PHx. However, at 72 hrs TGFβ1 was increased only by CP, ESL, and Sm (Figure 1(d)). Silymarin induced TGFβ1 decreased at 6 hrs PHx (*P < 0.05 versus Sham), but increased at 72 hrs (**P < 0.01 versus Sham; ##
P < 0.01 versus PHx). Silymarin mitigated regeneration and made cell normal. At long time, we did not find apoptotic body in regeneration liver (Figure 1(e)).
3.2. Elephantopus scaber L.-Induced Growth Factors Immediately Increased after 2/3 PHx
Growth factor signals (HGF and IGF-I) play a role in initiating regeneration of hepatocytes after 2/3 PHx. We suggested that Chinese herbal medicines may act as a cell cycle progression agents to make primed cells progress through the cycle and undergo DNA synthesis. However, progression through the cell cycle beyond the initiation phase requires growth factors. Starting with expression of a large number of immediate growth factors in the regenerating stage, hepatocytes can fully respond to the growth factors (HGF and IGF-I) to stimulate cell cycle from G1 phase to S phase to increase DNA synthesis and rebuild the lost hepatic tissue. ESL and Sm were increased HGF and IGF-I protein expression (Figure 2) (*P < 0.05, **P < 0.01 versusSham) at 24 hrs PHx and 72 hrs PHx. In addition, Silymarin (Sm) was induced HGF increased compared with Sham or PHx in spite of 6 hrs, 24 hrs, and 72 hrs PHx (*P < 0.01 versus Sham; #
P < 0.01 versus PHx) (Figure 3(a)).
3.3. Elephantopus scaber L. Accelerated Cell Cycle in Liver Regeneration
Cyclin D1/pRb and Cyclin E/E2F are key regulators of G1-to-S phase progression of the cell cycle. We found Cyclin D1 was increased at 24 hrs and 72 hrs PHx by ESL and Sm (*P < 0.05, **P < 0.01 versusSham); however, pRb was only increased in Sm treatment (Figures 2(a) and 2(c)). The positive control, Silymarin, was permission increased at 6 hrs, 24 hrs, and 72 hrs PHx compared with Sham (*P < 0.05, **P < 0.01 versus Sham) and PHx (#
P < 0.05, ##
P < 0.01 versus PHx (Figure 3(b)). Moreover, Cyclin D1, Cyclin E, pRb, and E2F mRNA expression levels were increased at 72 hrs PHx by ESL or Sm treatment (*P < 0.05, **P < 0.01 versus Sham). The same result we found Sm also increased compared with Sham and PHx (Figure 3(c)).
3.4. Effects of Elephantopus scaber L. on Cell Death after PHx
During liver regeneration after liver injury, hepatocytes were lost. Cell death or apoptosis was a physiological process to regulate hepatocyte development and maintain liver mass. We detected apoptosis protein bad and cytochrome c at 24 hrs and 72 hrs (Figures 5(a) and 5(b)). Apoptosis occurs rapid cellular divisions after PHx, resulting in fine-tuning of the liver size and tissue remodeling. Therefore, the results showed us that Elephantopus scaber L. (ESL) and Silymarin (Sm) induced bad and cytochrome c protein and mRNA expression downregulated (*P < 0.05, **P < 0.01 versus Sham). Moreover, TUNEL assay showed apoptotic body only at long time 72 hrs PHx including Silymarin treatment (Figure 4). In contract, we also observed apoptotic body in traditional Chinese medicines. We did not find apoptotic body in ESL and Sm treatment at long time 72 hrs PHx. We did not found any apoptotic body in treatment TCM at 24 hrs PHx.
4. Discussion
The liver is one of the most complex organs, and the regeneration induced by surgical injury is an orchestrated response. In order to set in the optimal mass in relationship to its body size, the liver induced its compensatory hyperplasia mechanisms. Herbal medicines have been used to treat liver disorders for thousands of years in the East and have now become a promising therapy internationally for pathological liver conditions. Growth factors (HGF and IGF-I) and cytokine (TGFβ1) are triggering cell cycle progression from G0 phase to G1 phase. Hepatocyte growth factor, also known as scatter factor, is believed to play a primary role in liver regeneration. Growth factors may play a role in initiating the proliferation of hepatocytes after PHx in the rat were investigated immediately after surgical resection of the liver. In this paper, we presumed that Chinese medicines including Codonopsis pilosula (CP, Dangshen), Salvia miltiorrhiza Bunge (SMB, Tanshinone), Bupleurum Kasi (BK, Chaihu), Elephantopus scaber L. (ESL, Teng-Khia-U), and Silymarin (Sm) may promote the function of liver regeneration after PHx.
We found that ESL (Teng-Khia-U) and Silymarin (Sm) have the best effects on liver regeneration. In the present study, ESL from the toxicity study they were observed that the root extract are nontoxic and caused no death up to a dose of 3.2 g/kg orally [24]. It is safe and was used in doses for the this study. Two known compounds, isodeoxyelephantopin and deoxyelephantopin [33, 34], were isolated from the whole plant of Elephantopus scaber L. (ESL, Teng-Khia-U) [35]. The whole plant of ESL is rich in novel antitumor substances-sesquiterpene lactones. The plant of ESL extracts has the ability to influence programmed cell death or arrest proliferation of tumor cells. We find that ESL and Sm stimulated several growth factors to regulate cell cycle and DNA synthesis. Growth factors are paracrine-regulated hepatic regenerative response [36]. The active form of HGF is a powerful stimulator of DNA synthesis and cell motility [37, 38]. PHx triggers the entry of rat liver cells into the cell cycle. We found ESL induced growth-regulated genes (HGF and IGF-I) to express later and persist longer, paralleling the rapid growth phase of the liver after PHx [39, 40]. The maximal expression after 24 to 72 hrs when the maximal growth period ends and are thought to be involved in re-establishing quiescence. Therefore, we can find that ESL mediated growth factors (HGF and IGF-I) and cytokines (TGFβ1) to remodel hepatic at 24 hrs PHx, but fail to at 72 hrs PHx. However, the other TCMs are also enhanced cytokines expression during this time [41–45]. Thus, PHx is a cell cycle-dependent regulation and a potential physiological role in G1 progression. Liver growth after PHx does not involve cell death and is a purely proliferative event. In summary, our data suggest liver regeneration may regulate the kinetics of cell cycle progression at the G1 to S phase transition [46, 47]. However, we found ESL induced growth factors and cell cycle expression at 24 hrs, until 72 hrs. Because ESL maybe delay apoptosis [48, 49].
Overall, the information thus derived should enhance our knowledge on the liver regeneration functions of treatment of TCMs as well as the basic mechanisms of cell cycle and apoptosis [50, 51].
Authors' Contributions
C.-C. Tsai and J.-P. Wu equally contributed to this work.
Acknowledgments
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interests with respect to this paper. This study is supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004).
Abbreviations
HGF:Hepatocyte growth factor
PHx:Partial hepatectomy
IGF-I:Insulin-like growth factor I
CP: Codonopsis pilosula
BK: Bupleurum kasi
ESL:SMB: Salvia miltiorrhiza Bunge
Sm:Silymarin
UPA: Urokinase plasminogen activator
TCMs:Traditional Chinese medicines.
Figure 1 Traditional Chinese medicine improves liver regeneration after liver toxicity injury. (a) Body weight was decreased at 168 hrs PHx. (b) Partial liver weight was increased at 72 hrs PHx. (c) Liver regeneration (%) was increased at 24 hrs, 72 hrs, and 168 hrs PHx. (d) Cytokines, TGF-β1, was increased in SMB, CP, ESL, and Sm at 24 hrs PHx, but CP, ESL, and Sm was at 72 hrs PHx. (e) Histology of PHx section and after Sm section during liver regeneration. (f) TGF-β1 expression was decreased at 6 hrs PHx after Silymarin, but increased at 72 hrs PHx. (g) Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, *P < 0.05 significant difference compared with Sham. ##
P < 0.01 significant difference compared with PHx.
Figure 2 Effects of Elephantopus scaber L. (ESL) and Silymarin (Sm, positive control) on liver regeneration after PHx. (a), (c) Equal amounts of protein lysate were separated by 12% SDS-PAGE by western blotting with antibodies to HGF, IGF-I, cyclin D1, and pRb. Protein expression levels are increased in Elephantopus scaber L. (ESL) and Silymarin (Sm) at 24 hrs and 72 hrs PHx during liver regeneration. (b), (d). Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, *P < 0.05, **P < 0.01 significant difference compared with Sham group. (e). Expression mRNA of Cyclin D1, Cyclin E, pRb, and E2F were increased in ESL and Sm after 72 hrs PHx. (f). Quantification of densitometry analysis of mRNA levels. All data are presented as means ± SEM *P < 0.05, **P < 0.01 significant difference compared with Sham group.
Figure 3 Expression of cell cycle proteins in G1 to S Phase. Western blot analysis of HGF, Cyclin D1, Cyclin E, and E2F expression were increased in Elephantopus scaber L. (ESL) and Silymarin (Sm) at 24 hrs and 72 hrs PHx. Tubulin was used as a loading control for western blotting. Quantification of densitometry analysis of protein expression levels. All data are presented as means ± SEM *P < 0.05, **P < 0.01 significant difference compared with Sham group. #
P < 0.05, ##
P < 0.01 significant difference compared with PHx group.
Figure 4 TUNEL assay in liver regeneration after PHx. To detect cell apoptosis during liver regeneration at different times. At 6 hrs PHx and 24 hrs PHx was not found, but at 72 hrs PHx appeared. Also not observed after silymarin treatment PHx 72 hrs.
Figure 5
Elephantopus scaber L. suppressed apoptosis during liver regeneration. (a) Expression protein levels of Bad and cytochrome c were decreased protein expression in Elephantopus scaber L. (ESL) and Silymarin (Sm) after 24 hrs and 72 hrs PHx by western blot. However, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm) at after 24 hrs PHx, but no changes at 72 hrs PHx. (b) mRNA expression levels of Bad and cytochrome c decreased apoptosis in ESL and Sm treatment at 24 hrs PHx; however, we can observed a little elevated expression at 72 hrs PHx. In contrast, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm) at 24 hrs, but no changes at 72 hrs. (c) TUNEL assay after traditional Chinese medicines in liver regeneration at 24 hrs and 72 hrs PHx. Only ESL and Sm have suppressed apoptosis function.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23383034PONE-D-12-2307810.1371/journal.pone.0055008Research ArticleBiologyBiochemistryNucleic acidsRNARNA processingBiophysicsNucleic acidsRNARNA processingGeneticsGene expressionRNA processingMolecular cell biologyGene expressionRNA processingNucleic acidsRNARNA processingMedicineOncologyCancers and NeoplasmsNeurological TumorsGliomaMiR-30a-5p Antisense Oligonucleotide Suppresses Glioma Cell Growth by Targeting SEPT7 AS-MiR-30a-5p Suppresses Glioma Cell GrowthJia Zhifan
1
Wang Kun
2
Wang Guangxiu
1
Zhang Anling
1
Pu Peiyu
1
*
1
Department of Neurosurgery, Tianjin Medical University, General Hospital, Tianjin Neurolgical Institute, Laboratory of Neuro-Oncology, Key Laboratory of Post-Trauma Neuro-Repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Tianjin, People’s Republic of China
2
Department of Neurosurgery, Hangzhou Xiasha Hospital, Sir Run Run Shaw Hospital, Medical College, Zhejiang University, Hangzhou, People’s Republic of China
Najbauer Joseph Editor
City of Hope National Medical Center and Beckman Research Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ZFJ PYP. Performed the experiments: ZFJ KW GXW ALZ. Analyzed the data: ZFJ KW. Contributed reagents/materials/analysis tools: ZFJ PYP. Wrote the paper: ZFJ KW.
2013 28 1 2013 8 1 e5500831 7 2012 17 12 2012 © 2013 Jia et al2013Jia et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting the mRNAs of hundreds of human genes. Variations in miRNA expression levels were shown to be associated with glioma. We have previously found miR-30a-5p overexpression in glioma cell lines and specimens. Bioinformatics analyses predict that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7. SEPT7 is a member of the septin family, which is a highly conserved subfamily of GTPases implicated in exocytosis, apoptosis, synaptogenesis, neurodegeneration and tumorigenesis. Our previous study has also demonstrated that SEPT7 expression is decreased in astrocytic gliomas with different grades and plays a tumor suppressor role. In the present study, we knocked down miR-30a-5p with antisense oligonucleotide (miR-30a-5p AS) in LN229 and SNB19 glioblastoma(GBM) cells, and found that cell growth and invasion were inhibited, while apoptosis was induced. miR-30a-5p AS treated cells showed upregulation of SEPT7 and downregulation of PCNA, cyclin D1, Bcl2, MMP2 and MMP9. In contrast, when miR-30a-5p mimics were transfected into LN229 and SNB19 GBM cells, cell growth and invasion were promoted and the expression of relevant proteins increased. Meanwhile, the effect of miR-30a-5p mimics on glioma cells can be reversed by transfection of SEPT7 construct. Additionaly, miR-30a-5p directly targeting SEPT7 was identified by the reporter gene assay. Our study demonstrates,for the first time, that miR-30a-5p is a bona fide negative regulator of SEPT7 and the oncogenic activity of miR-30a-5p in human gliomas is at least in part through the repression of SEPT7.
This work was supported by the China National Natural Scientific Fund (30872985). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and aggressive malignant primary brain tumors. Despite the advances in surgery, radiation therapy, and chemotherapy, the prognosis of patients with GBM has not been improved significantly over the past 20 years [1]. It is imperative to have a detailed and comprehensive understanding of the molecular pathogenesis of the gliomas for developing novel strategies in treatment.
miRNAs are small, evolutionarilly conserved noncoding RNA molecules, Recent studies have shown that the expression of many miRNAs are deregulated in a variety of cancers, including lymphoma, colorectal cancer, lung cancer, breast cancer, papillary thyroid carcinoma, hepatocellular carcinoma and glioblastoma [2]–[8]. Regarding the role of miRNAs in cancer, there is no doubt that miRNAs play a key role in the initiation and progression of cancer. Specific miRNAs have been demonstrated to regulate known oncogenes or tumor suppressor genes or act as so called onco-miRs or tumor suppressor-miRs by directly targeting other genes involved in cell differentiation, proliferation, invasion, apoptosis and angiogenesis in various cancers.
Because miR-30a-5p expression is up-regulated in glioma cell lines and glioma specimens (data not shown), miR-30a-5p may contribute to gliomagenesis.
Bioinformatics analyses with HuMiTar, a sequence-based method for prediction of human microRNA targets [9], have predicted that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7.
SEPT7 is a member of the septin family, which is a highly evolutionarily conserved subfamily of GTPases, consisting of at least 13 human septin genes that play important roles in cytokinesis, vesicle trafficking, polarity determination, microtubule and actin dynamics and can form membrane diffusion barriers. Septins have also recently been shown to be involved in oncogenesis [10]. SEPT7 has an open reading frame of 1254 nucleotides encoding 418 amino acids, including a GTP-binding motif, located on chromosome 7p14.4-14.1 [11]. Our previous study on the expression of SEPT7 in glioma cell lines and samples by mircroarray, RT-PCR, Western blotting and immunohistochemical staining demonstrated that SEPT7 gene expression was negatively correlated with the ascending order of glioma grades [12]–[14]. Moreover, we observed that enforced overexpression of SEPT7 inhibited cell proliferation, arrested the cell cycle at G0/G1 phase, and induced cell apoptosis in vitro and in vivo
[14]. In addition, we showed that SEPT7 overexpression suppressed the invasion and migration ability of human gliomas, reversed the imbalanced state of MMPs/TIMPs, downregulated the expression of integrin alpha(v)beta(3) and altered the structure of tubulin-alpha [15]. These data strongly supported that SEPT7 is an important molecule in gliomagenesis, and should be considered as a tumor suppressor.
In the current study, we demonstrate that miR-30a-5p regulates the tumor-suppressive gene-SEPT7 as target gene, and suppression of miR-30a-5p directly lead to up-regulation of SEPT7. The effect of miR-30a-5p mimics promoting cell proliferation and inhibition of apoptosis can be reversed by enforced overexpression of SEPT7 in glioma cell lines.
Materials and Methods
Cell Culture
Human glioblastoma cell lines SNB19 and LN229 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science. All cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) at 37°C in 5% CO2, and subcultured every 2–3 days.
Oligonucleotides and Cell Transfection
The miR-30a-5p antisense oligonucleotides, mimics as well as scramble miRNA were obtained from Gene Pharma Co, Ltd (Shanghai, China), Their sequences were listed as follows: antisense-miR-30a-5p(30a-5p AS):5′-CUUCCAGUCGAGGAUGUUUACA-3′,miR-30a-5p mimics(30a-5p m):5′-UGUAAACAUCCUCGACUGGAAG-3′ and scramble miRNA(scr ODN) : 5′-CAGUACUUUUGUGUAGUACAA-3′. Oligonucleotides (20 pmol/µL) were transfected into glioblastoma cells at 70% confluence using Lipofectamine 2000 according to the manufacturer’s instruction (Invitrogen, USA). SEPT7 recombinant adenovirus (Ad-S7) was preserved by our laboratory.
qRT-PCR Analysis of miRNA Expression
Total RNA from glioblastoma cells was isolated using Trizol reagent (Invitrogen, USA) for mRNA and miRNA analysis. For analysis of miRNA expression, real-time RT-PCR analysis was carried out. Amplification reaction was performed with MJ-real-time PCR (Bio-Rad, Hercules, CA, USA) using Hairpin-itTM miRNAs qPCR Quantitation Kit according to the manual. Relative gene expression was calculated using the 2ΔΔCT method [16] and analyzed initially using Opticon Monitor Analysis Software V2.02 software (MJ Research, Waltham, MA, USA), normalized to the expression of U6. All qRT-PCRs assays were performed in triplicates, and the data are presented as means ± standard errors of the means (SEM).
In situ Hybridization
Using antisense locked nucleic acid (LNA)-modified oligonucleotides probe, in situ hybridization was performed with In situ hybridization kit (Boster Biol Sci Co, Wuhan, China). The sequence of LNA-miR-30a-5p was 5′-CTTCCAGTCGAGGATGTTTACA-3′, scr ODN sequence was GTGTAACACGTCTATACGCCCA, and U6 ODN sequence was CACGAATTTGCGTGTCATCCTT. At 48 h after transfection, glioma cells were fixed with freshly prepared 4% paraformaldehyde (containing 0.1% DEPC). In situ hybridization detection of miR-30a-5p in GBM cells was conducted according to the protocol of the manufacturer. The fixed glioma cells were incubated with 20 µL LNA-miR-hybridization solution at 42°C for 16 h, and Cy3-avidin was used to label miR-30a-5p at a concentration of 0.5 mg/mL. Nuclei were counterstained with DAPI karyotyping kit (Genmed, Boston, MA, USA) and visualized using FluoView Confocal Laser Scanning Microscopes-FV1000 (Olympus, Tokyo, Japan) and analyzed using IPP5.1 (Olympus).
Plasmid Constructs and Luciferase Reporter Assay
The human 3′-UTR of the SEPT7 gene which contains the putative binding site for miR-30a-5p, was amplified by PCR using the following primers carrying XbaI sequence: forward: 5′-CCTCTAGATTTTTTATT AAA-3′ and reverse: 5′-CCTCTAGAATTGTAATTATC-3′. The product was digested using XbaI enzyme and cloned into the XbaI treated PGL3-control vector (Promega, USA), then the 3′-UTR of the SEPT7 just located at the region immediately downstream of the luciferase gene in the reporter gene vector. The ligated product was transduced into E. coli JM109 and colony PCRs were used to screen for the clones harboring the forward-oriented insert, The desired construct was subsequently sequenced to obtain the vector PGL3-SEPT7-3′-UTR(PGL3-S7).
For the luciferase reporter assay, the human glioblastoma cell lines SNB19, LN229 were cultured in 96-well plates. They were transfected with 0.2 µg each of the PGL3 or PGL3-S7 plasmids and 5 pmol of the 30a-5p AS using Lipofectamine 2000. At 48 h after transfection, luciferase activity were measured using the Luciferase Assay System (Promega, USA).
Cell Proliferation Assay
SNB19 and LN229 cells were seeded into 96-well plates at 4000 cells per well. After transfection as described previously, on each day of consecutive 7 days, 20µL MTT (5 mg/mL) was added to each well and the cells were incubated at 37°C for additional 4 h. and the supernatant was discarded. The reaction was then terminated by lysing the cells with 200µl of DMSO. Optical density was measured at wavelength of 570 nm and expressed as percentage of control. The data are presented as the mean ± SEM, which were derived from triplicate samples of at least three independent experiments.
Flow Cytometric Analysis of Cell Cycle Kinetics
For analysis of cell cycle kinetics by flow cytometry, transfected and control cells in the log phase of growth were harvested, washed with PBS, fixed with 90% ethanol overnight at 4°C, and then incubated with RNase at 37°C for 30 min. Nuclei of cells were stained with propidium iodide for additional 30 min. A total of 10,000 nuclei were examined in a FACS Calibur flow cytometer (Becton-Dickinson, USA) and DNA histograms were analyzed by Modifit software.
Measurement of Apoptosis by Annexin V staining
Parental and transfected cells were harvested at 48 h post-transfection. The apoptosis was analyzed by using AnnexinV FITC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s instruction. Annexin V-FITC and propidium iodide double staining cells detected by FACSCalibur were evaluated as the apoptotic cells [17]. The data obtained were analyzed with CellQuest software.
Cell Invasion Detected with Transwell Assay
For analyzing the invasive activity of GBM cells, the upper surface of the transwell filters was coated with matrigel. Cells in the logarithmic growth phase were harvested. Cells(1×105) in 200 µL of serum-free DMEM were added into the upper chamber. A total of 600 µL of conditioned medium derived from tumor cell culture was used as a source of chemoattractant and placed in the lower chamber. After 24-hour incubation at 37°C, the filters were gently taken out, the medium was removed from the upper chamber. The noninvaded cells on the upper surface of the inserted filter were scraped off with a cotton swab. The cells that had migrated into the lower surface of the inserted filter were fixed with methanol. The number of cells invading through the matrigel was counted using three randomLy selected visual fields under inverted microscope.
Western Blot Analysis
At 48 h after transfection with oligonucleotides, total proteins from the parental and transfected cells were extracted and the protein concentration was determined by Lowry method. A total of 40 µg protein lysates from each sample was subjected to SDS-PAGE on 10%SDS-polyacrylamide gel. Separated proteins were transferred to a PVDF membrane (Millipore, USA). The membrane was incubated with primary antibodies against SEPT7, PCNA, Cyclin D1, Bcl2 and MMP9 (1∶1000 dilution, Santa Cruz, USA) followed by incubation with HRP-conjugated secondary antibodies(1∶1000 dilution, Zymed, USA). The specific protein was detected using a SuperSignal protein detection kit (Pierce, USA). After washing with stripping buffer, the membrane was reprobed with antibody against β-actin (1∶1000 dilution, Santa Cruz, USA). The band density of specific proteins was quantified after normalization with the density of β-actin.
Immunofluorescence Staining
For immunofluorescence staining, control and transfected cells were seeded on coverslips and fixed with 4% paraformaldehyde (Sigma), treated with 3% H2O2 for 10 min and incubated with the primary antibodies described above overnight at 4°C. FITC- or TRITC-labeled secondary antibody (1∶200 dilutions) was added for 2 h at 37°C. DAPI reagent was used to stain the cell nuclei and the cells was visualized using FV-1000 laser scanning confocal microscopes and analyzed using IPP5.1 (Olympus).
Statistical Analysis
Data were expressed as means ± SEM. Statistics was determined by ANOVA and χ2 test using SPSS11.0 (Windows). Statistical significance was determined as P<0.05.
Results
30a-5p AS Specifically Knocks Down miR-30a-5p Expression
To evaluate the significance of miR-30a-5p overexpression in glioma cells, we used a loss-of-function antisense approach. miR-30a-5p antisense oligonucleotide was used to knock down miR-30a-5p expression in glioma cells. qRT-PCR results showed that the relative expression level of miR-30a-5p in 30a-5p AS treated SNB19 cells was 24.12±0.06% (P<0.05), and 19.02±0.04% in LN229 cells (P<0.05) compared with that in their control cells, respectively (Figure 1A). Furthermore, in situ hybridization demonstrated that the Cy3 red fluorescence signal in 30a-5p AS transfected SNB19 and LN229 cells was lower in contrast to the signal in control cells and cells transfected with scr ODN (Figure 1B). These data suggested that 30a-5p AS was able to inhibit specifically the endogenous miR-30a-5p expression in SNB19 and LN229 cells.
10.1371/journal.pone.0055008.g001Figure 1 MiR-30a-5p expression was suppressed by 30a-5p AS in SNB19 and LN229 cells.
MiR-30a-5p expression was knocked down in SNB19 and LN229 glioma cells compared with control by real time PCR (A) and In Situ Hybridization. (B).
30a-5p AS Suppresses Cell Proliferation
The proliferation of glioma cells in vitro was determined by MTT assay. As shown in Figure 2A1 and 2A2, 30a-5p AS treated SNB19 and LN229 cells showed a significant decrease in viability compared with the control and scr ODN transfected cells (P<0.05, from day 2 to 7). We found that the growth-inhibitory effect of 30a-5p AS reached maximum at 7th day, the end of observation period. The lowest survival rate was 50.88±6.28% for SNB19 cells, and 59.23±2.97% for LN229 cells.
10.1371/journal.pone.0055008.g002Figure 2 30a-5p AS suppressed glioma cell growth in SNB19 and LN229 cells.
(A) The viability of glioma cells transfected with 30a-5p AS was decreased gradually during the 7-day observation period compared with control group and scramble groups determined by MTT assay in SNB19 cells and LN229 cells. (B) Flow cytometry data represented that S-phase fraction was much lowered and more cells were arrested in G0/G1 phase in the 30a-5p AS group compared with control and scramble groups. (C) Apoptotic Index (AI) of control and transfected SNB19 and LN229 cells was examined by Annexin V staining. As compared with the control and scramble group, the AI of the 30a-5p AS group cells was increased. (D) Invasive capability of parent and treated SNB19 and LN229 cells was examined by Transwell assay. Cell invasion was decreased in 30a-5p AS group compared with the control and scramble groups as assessed by the number of cells invading into the lower surface of the polycarbonic membrane via the matrigel.
30a-5p AS Arrests Cell Cycle in the G0/G1 Phase
To further analyze whether decreased viability effect of 30a-5p AS on SNB19 and LN229 was a result of cell-cycle arrest, the cell-cycle kinetics was analyzed using flow cytometry. Treatment with 30a-5p AS resulted in a decrease in the population of cells that were in S phase (Figure 2B1 and 2B2). A representative experiment was shown in SNB19 cells that S phase cell population accounted for 27.7±4.2% and 28.1±1.2% in control and scr ODN treatd groups, respectively, in contrast to 19.5±5.2% in 30a-5p AS treated cells. Similarly, the percentage of S phase cells decreased from 31.4±3.1% of control and 34.1±4.1% of scr ODN groups to 21.4±4.8% of 30a-5p AS group in LN229 cells. Compared with control and scramble ODN cells, the 30a-5p AS treated cells substantially and consistently increased the G0/G1 cell population from 50.9±3.4% of control group and 51.2±6.3% of scr ODN group to 61.1±3.2% in 30a-5p AS treated SNB19 cells. LN229 cells exposed to 30a-5p AS also arrested in the G0/G1 phase (60.2±5.5%) as compared to the control and scr ODN treated cells (49.1±3.1% and 48.2±3.2%, respectively), indicating that 30a-5p AS functions as a negative regulator of the cell cycle from G1-to-S transition.
30a-5p AS Induces Apoptosis
The effect of decreased miR-30a-5p on apoptosis was analyzed by conducting Annexin V and PI double staining. Untreated cells served as a negative control. Percentages of apoptotic cells are shown in the histogram (Figure 2C1 and 2C2). Compared with the apoptotic cells in control group (3.24±0.56% and 2.52±0.43%) and scr ODN treated group (3.87±0.21% and 2.38±0.78%) in SNB19 and LN229 cells, respectively, the downregulation of miR-30a-5p resulted in a significant (p<0.05) increase of apoptotic cells in SNB 19 and LN229 cells (14.94±2.15% and 12.76±1.78%, respectively), indicating an induction of apoptosis in SNB19 and LN229 cells by transfection of the 30a-5p AS.
30a-5p AS Inhibits Glioma Cell Invasion
The inhibitory effect of 30a-5p AS on glioma cells invasion was assessed by the transwell assay. Representative micrographs of transwell cell invasion are shown in Figure 2D1 and 2D2. The number of cells invading through the matrigel in the 30a-5p AS group (21.4±10.3) was significantly decreased from those of the Control group (87.2±8.7) and the scr ODN treated group (85.6±9.4) in SNB19 cells. In LN229 cells, invasive activity were also inhibited in the 30a-5p AS group (18.4±8.9) compared with that of the control group (93.5±10.5) and scr ODN treated group (95.7±5.8).
30a-5p AS Affects the Expression of Relevant Proteins Examined by Immunofluorescence Staining and Western Blot Analysis
When miR-30a-5p was knocked down, the expression of SEPT7 was upregulated while the expression of PCNA, Cyclin D1, Bcl2, MMP2 and MMP9 downregulated in SNB19 cells and in LN229 cells (Figure 3A, 3B), which coincided with the results determined by MTT, cell cycle kinetics, apoptosis and transwell assay.
10.1371/journal.pone.0055008.g003Figure 3 The expression of SEPT7, PCNA, CyclinD1, Bcl2, MMP2, MMP9 in parent and 30a-5p AS treated SNB19 and LN229 cells detected by Immunofluorescence and Western blotting.
SEPT7(49kDa) was up-regulated while expression of PCNA(36kDa), CyclinD1(37kDa), Bcl2(26kDa), MMP2(63kDa), MMP9(92kDa) significantly down-regulated when the miR-30a-5p expression was inhibited.
SEPT7 is a Target of miR-30a-5p
To explore the mechanism by which miR-30a-5p regulates cell proliferation, invasion and apoptosis, miRNA targets prediction databases were searched, including miRanda, Targetscan and HuMiTar [9], and found 3′UTR of SEPT7 containing the highly conserved putative binding sites of miR-30a-5p (Figure 4A). To verify SEPT7 is one of the target genes of miR-30a-5p, we constructed the PGL3-S7 plasmid containing 3′UTR of SEPT7 with miR-30a-5p putative binding site and conducted a reporter gene assay. As shown in Figure 4B, reporter assay revealed that reduction of miR-30a-5p led to a remarkable increase of luciferase activity in PGL3-S7 combined with 30a-5p AS transfected cells(4.73±1.16 fold for SNB19, 5.14±0.87 fold for LN229), whereas no change of luciferase activity was found in PGL3-S7 with scr ODN transfected cells(0.95±0.32 fold for SNB19, 0.97±0.25 fold for LN229) and PGL3-S7 transfected cells(1.00±0.00 fold for SNB19 and LN229). Moreover, Western blot analysis showed that SEPT7 expression was up-regulated in glioma cells treated with 30a-5p AS compared to the cells treated with scr ODN or control cells, whereas no difference at the mRNA level of SEPT7 expression was observed among 30a-5p AS, control and scr ODN groups (Figure 4C). These evidences indicate that miR-30a-5p directly modulates SEPT7 expression at the translational level by binding to 3′UTR of SEPT7.
10.1371/journal.pone.0055008.g004Figure 4 SEPT7 was a direct target gene of miR-30a-5p.
(A) Schematic representation of the putative binding sites in SEPT7 mRNA 3′UTR for miR-30a-5p. (B) PGL3-S7-Luc vector was transfected into SNB19 and LN229 cells with 30a-5p AS transfection and luciferase activity was significantly increased. The ratio of normalized sensor to control luciferase activity was shown. * P<0.05 compared with control group. (C) SEPT7 protein and mRNA level was detected by Western blot assay and RT-PCR in SNB19 cells and LN229 cells transfected with 30a-5p AS, SEPT7 protein expression was upregulated while SEPT7 mRNA expression without change.β-actin protein and mRNA expression were regarded as endogenous normalizer.
Downregulation of miR-30a-5p Results in Reduction of Glioma Cell Growth that is Partly Reversed by Tranfection with Ad-SEPT7
Having established that glioblastoma-derived cell lines displayed decreased proliferation and invasiveness in vitro when miR-30a-5p expression was decreased or SEPT7 was increased, we sought to further identify the role of miR-30a-5p and its target SEPT7 in gliomagenesis. We examined the proliferation, cell cycle progression, apoptosis and invasiveness of glioma cells after being treated with miR-30a-5p mimics (30a-5p m), SEPT7 recombinant adenovirus(Ad-S7) and 30a-5p m combined with transfection of Ad-S7(30a-5p+Ad-S7).
The expression of SEPT7 was downregulated in SNB19 and LN229 cells tranfected with 30a-5p m and upregulated when transfected with Ad-SEPT7 as detected by Western blotting, and SEPT7 expression was moderately increased in the cells transfected with 30a-5p m+Ad-SEPT7(Figure 5).
10.1371/journal.pone.0055008.g005Figure 5 The expression of SEPT7, PCNA, CyclinD1, Bcl2, MMP2, MMP9 detected by Western blotting.
SEPT7 is up-regulatd in group treated with Ad-SEPT7, down-regulated in 30a-5p m group and moderate expression in 30a-5p m+Ad-S7 group. The expression of PCNA, Cyclin D1, Bcl2, MMP2 and MMP9 was downregulated in Ad-SEPT7 group while up-regulated in 30a-5p m group, and the increased expression of relevant proteins was partly reversed in 30a-5p m+Ad-S7 group in SNB19 cells and LN229 cells.
The miR-30a-5p m cells proliferated at the highest level while Ad-S7 cells at the lowest and 30a-5p m+Ad-S7 cells grew at the intermediate level(Figure 6A). Similarly, As shown in Figure 6B and Table 1, compared to the cell cycle analysis of control cells, the S phase fraction of the SNB19 and LN229 cells treated with 30a-5p m was markedly increased while G0/G1 fraction decreased, in contrast to the cells transfected with Ad-S7 in which S phase fraction was significantly decreased while G0/G1 fraction increased. In the cells treated with 30a-5p m+Ad-S7 the decrease of S phase fraction or increase of G0/G1 fraction was moderate. The overexpression of SEPT7 led to a increased apoptotic index(15.48±2.34% and 17.34±3.94%) in SNB19 and LN229 cells respectively (Figure 6C). The 30a-5p m+Ad-S7 group showed a result (2.77±0.67 and 2.19±0.48) between the 30a-5p m (1.23±0.34% and 1.25±0.28%) and control group (3.03±0.75% and 2.67±0.53%). Transwell assay demonstrated that the SNB19 and LN229 cells transfected with 30a-5p m migrated through matrigel were increased, 124.7±16.3 and 135.6±17.8, respectively. These figures were higher than those in control groups. The migrating cells in control SNB19 and LN229 cells were 82.2±10.7 and 90.3±9.5, respectively. Enforced overexpression of SEPT7 obviously attenuated the cell invasion, migrating cells in Ad-SEPT7 group were 25.6±7.3 in SNB19 and 19.6±7.7 in LN229 cells, respectively, whereas the migrating cells in the 30a-5p m+Ad-S7 group were moderately increased, i.e. 100.5±12.5 in SNB19 cells and 107.4±12.6 in LN229 cells, respectively (Figure 6D). The expression of PCNA, Cyclin D1, Bcl2, MMP2 and MMP9 was downregulated in Ad-SEPT7 group while up-regulated in 30a-5p m group in SNB19 cells and in LN229 cells (Figure 5). The protein expression coincided with the results mentioned above in cell proliferation, invasion and apoptosis.
10.1371/journal.pone.0055008.g006Figure 6 Upregulation of miR-30a-5p resulted in promotion of glioblastoma cell growth that was reversed partially by Ad-S7.
(A) The figure showed that 30a-5p m increased the cell survival rate at a significantly higher rate in SNB19 cells and LN229 cells, (B) accelerated the glioma cells from G0/G1 to S phase in SNB19 cells and LN229 cells, (C) decreased the apoptotic cells in the 30a-5p m group, (D) promoted the cell invasive ability. And Ad-S7 inhibited cell proliferation(A), arrested the cell cycle in the G0/G1 phase(B), induced cell apoptosis(C) and suppressed the cell invasive ability(D). While the results of 30a-5p m+Ad-S7 group showed that 30a-5p m promoted the glioma cell growth that was partially reversed by overexpression of SEPT7.
10.1371/journal.pone.0055008.t001Table 1 30a-5p m promotes cell cycle progression.
Group G0/G1 S G2/M
SNB19 Control 51.3±3.4 22.1±2.7 26.6±3.1
30a-5p m 36.7±2.1 52.6±2.3 10.7±1.8
Ad-S7 64.2±3.1 15.5±2.1 20.3±2.6
30a-5p m+Ad-S7 45.8±4.1 25.3±6.2 28.9±2.5
LN229 Control 49.4±2.6 20.3±2.8 30.3±3.1
30a-5p m 38.8±6.4 38.1±2.7 23.1±3.3
Ad-S7 65.2±4.2 12.2±1.8 22.6±1.9
30a-5p m+Ad-S7 43.8±6.5 26.4±5.2 29.8±2.8
Glioma cell lines transfected with 30a-5p m demonstrated increased cell viability and invasion, promotion of cell cycle and decreased apoptosis in vitro (Figure 6A, B, C, D), whereas transfected with Ad-SEPT7 showed the inhibition of cell proliferation, invasion, cell cycle progression, and induction of cell apoptosis, just the same as we observed previously [14]. Combined transfection with 30a-5p m and Ad-SEPT attenuate the effect of 30a-5p m. Taking together, all these results indicate that the effect of 30a-5p m on promoting cell growth, invasion and induction of apoptosis was able to be reverted by SEPT7 to a considerable degree. More importantly, this evidence identifiy that miR-30a-5p affects the cell biological behavior is partially through the negative regulation of SEPT7.
Discussion
GBM is a highly invasive tumor of the central nervous system. Currently available combined therapies offer only limited benefits for patients with glioblastoma. It is imperative to develop novel therapeutic approaches by better understanding the molecular mechanism of gliomagenesis.
Recent studies indicate that various miRNAs play important roles in the initiation and progression of cancer. miRNAs may function as tumor suppressors by down-regulating the expression of tumor-promoting genes, or may have oncogenic role by inhibiting the expression of tumor suppressor genes. Regarding to the role of miR-30a-5p in cancers, there are so far only very limited data available. There has been reported that miR-30a-5p is aberrantly expressed in thyroid cancer [18], [19], gastric cancer [20], colon cancer [21], [22] and squamous cell carcinoma of human head and neck (HNSCC) and esophagus (ECC) [23]. Moreover, the expression of miR-30a-5p in these cancers are not inconsistent. For example, miR-30a-5p is upregulated in HNSCC and ESC while downregulated in colon cancer. Even in thyroid carcinoma its expression is different between papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma(ATC). It is upregulated in PTC and dowregulated in ATC. The underlying mechanism of the different expression in various type of cancer is not yet clear. As to the expression of miR-30a-5p in glioma, that has not been reported before. Our previous study on microRNA expression profiles in glioma cell lines with microarray has demonstrated that miR-30a-5p is highly expressed, subsequently we confirmed this finding in seven glioma cell lines and forty three freshly resected glioma samples with different grades. According to bioinformatic analyses, SEPT7 is one of the predicted targets of miR-30a-5p. SEPT7 is reduced expression and plays a tumor suppressor role in gliomagenesis as we demonstrated before[12]–[14]. So in the present study, we are attempting to further identify SEPT7 is regulated by miR-30a-5p and miR-30a-5p may exert its oncogenic role through regulation of SEPT7.
The luciferase reporter assay validated that miR-30a-5p directly regulates SEPT7 expression by the presence of a binding site for miR-30a-5p in the 3′UTR of SEPT7. Moreover, Western Blot showed obvious upregulation of SEPT7 in the 30a-5p AS group compared to control and scr ODN groups, whereas no differences at the mRNA expression of SEPT7 were observed among 30a-5p AS, control and scr ODN groups. These results indicate the inverse correlation between the expression of SEPT7 and miR-30a-5p in glioma cells and miR-30a-5p negatively regulates SEPT7 expression at the translational level. Therefore, SEPT7 is a direct target gene of miR-30a-5p.
Our previous study has demonstrated that SEPT7 is downregulated in human gliomas. Forced overexpression of SEPT7 was able to inhibit cell proliferation and invasion, arrested cell cycle in the G0/G1 phase and induced apoptosis both in vitro and in vivo.
In this study, it has shown that when miR-30a-5p is knocked down with antisense oligonucleotide in SNB19 and LN229 cell lines, cell proliferation, invasion and cell cycle progression are inhibited, and apoptosis is induced. Meanwhile, SEPT7 expression is up-regulated. So that SEPT7 seems to be one of regulatory mechanism involved in the role of miR-30a-5p contributing to gliomagenesis.
Because miR-30a-5p can target multiple genes, we sought to explore whether SEPT7 is the major effector of miR-30a-5p for the change of biological behavior in glioma cells. Following transfection with miR-30a-5p mimics, we observed that glioblastoma-derived cell lines in which miR-30a-5p expression had been up-regulated, cell proliferation and invasion was enhanced compared to untreated control cell lines. More importantly, we found that the increased glioma cell growth can be reverted to a considerable degree by transfection of SEPT7. These results suggest that SEPT7 plays a major role in miR-30a-5p affecting biological behaviors of glioma cells.
Our results also showed that after inhibition of miR-30a-5p with 30a-5p AS, not only the expression level of SEPT7 was upregulated dramatically, but also the expression of proteins involved in cell proliferation, cell cycle progression, invasion and apoptosis, including PCNA, CyclinD1, Bcl2, MMP2, MMP-9 were decreased while BCL2 increased. On the contrary, When miR-30a-5p was up-regulated by transfection with miR-30a-5p mimics, PCNA, CyclinD1, Bcl2, MMP2, MMP-9 were increased while BCL2 decreased, and these results was also able to be partially reversed by transfection of Ad-SEPT7.
As miRNAs have a key role in the development of cancer, it is conceivable that miR-mimetics (for downregulated miRNAs) or anti-miRs (for upregulated miRNAs) could emerge as new class of molecular targeted therapeutic intervention [24]. On the basis of the 30a-5p AS-mediated upregulation of SEPT7, it is logical to predict that anti-miR-30a-5p could function as an potential effective, alternative therapeutic regimen against glioma. In addition to SEPT7, there may be other targets of miR-30a-5p that may contribute to the gliomagenesis which should be further explored.
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15 : 2070 –2079 .17878899 | 23383034 | PMC3557229 | CC BY | 2021-01-05 17:11:53 | yes | PLoS One. 2013 Jan 28; 8(1):e55008 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23382911PONE-D-12-3166310.1371/journal.pone.0054540Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionGeneticsGene ExpressionMolecular Cell BiologySignal TransductionSignaling CascadesStress Signaling CascadeCellular Stress ResponsesGene ExpressionMedicineCardiovascularHypertensionObstetrics and GynecologyLabor and DeliveryPregnancyWomen's HealthCardiovascular Diseases in WomenHeat Shock Protein 70 Expression Is Spatially Distributed in Human Placenta and Selectively Upregulated during Labor and Preeclampsia HSP70 is Upregulated in Labor and PreeclampsiaAbdulsid Akrem
1
Hanretty Kevin
2
Lyall Fiona
1
*
1
University of Glasgow, Institute of Medical Genetics, Yorkhill Hospital, Glasgow, United Kingdom
2
Maternity Hospital, Southern General Hospital, Glasgow, United Kingdom
Buratti Emanuele Editor
International Centre for Genetic Engineering and Biotechnology, Italy
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: FL AA KH. Performed the experiments: AA. Analyzed the data: FL AA. Contributed reagents/materials/analysis tools: AA FL. Wrote the paper: FL AA.
2013 28 1 2013 8 1 e545404 10 2012 13 12 2012 © 2013 Abdulsid et al2013Abdulsid et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Placental oxidative stress is a feature of both human labor and the pregnancy syndrome preeclampsia. Heat shock proteins (HSPs) can be induced in cells as a protective mechanism to cope with cellular stress. We hypothesized that HSP 70 would increase during labor and preeclampsia and that expression would vary in different placental zones. Samples were obtained from 12 sites within each placenta: 4 equally spaced apart pieces were sampled from the inner, middle and outer placental regions. Non-labor, labor and preeclampsia were studied. HSP 70 expression was investigated by Western blot analysis. HSP 70 protein expression was increased in the middle compared with the outer area (p = 0.03) in non-labor and in both the inner and middle areas compared with the outer area (p = 0.01 and p = 0.02 respectively) in labor. HSP 70 was increased in the preeclampsia non-labor group compared to the control non-labor group in the inner region (p = 0.003) and in the control labor group compared with the preeclampsia labor group at the middle area (p = 0.001). In conclusion HSP 70 is expressed in a spatial manner in the placenta. Changes in HSP 70 expression occur during labor and preeclampsia but at different zones within the placenta. The physiological and pathological significance of these remains to be elucidated but the results have important implications for how data obtained from studies in placental disease (and other organs) can be influenced by sampling methods.
The authors are grateful to the Libyan Ministry of Higher Education and Scientific Research for funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The mechanisms that are involved in maintaining a human pregnancy to term, and the switches that lead to a normal labor and pregnancy outcome or indeed an adverse outcome such as miscarriage, preeclampsia, fetal growth restriction or preterm labor, are complex but the role of the placenta is crucial to them all [1]–[4]. During a healthy pregnancy maternal spiral arteries are dramatically remodeled. They become widely dilated and lose their responsiveness to vasoconstrictive stimuli. Thus blood enters the intervillous space in a non-pulsatile manner and under low pressure [5].
Preeclampsia affects about 2 to 3% of all pregnancies but this can be much higher in underdeveloped countries. It is an important cause of maternal death worldwide and a leading cause of iatrogenic prematurity and fetal growth restriction [6]. In preeclampsia spiral artery remodeling is partial or incomplete [5]. The ensuing high pressure flow results in hydrostatic damage to the placental villi. Furthermore perfusion by intermittent pulses of fully oxygenated arterial blood is thought to lead to fluctuations in oxygen delivery resulting in oxidative stress [4], [7]. The maternal syndrome is, at least in part, due to the maternal response to this damaged placenta. This is known as the two-stage model of preeclampsia [7].
Oxidative stress occurs when the production of reactive oxygen species overwhelms the intrinsic anti-oxidant defenses. It may induce a range of cellular responses depending upon the severity of the insult and the compartment in which reactive oxidative species are generated [4], [7]. There is irrefutable evidence of placental oxidative stress in preeclampsia, including increased concentrations of protein carbonyls, lipid peroxides, nitrotryosine residues and DNA oxidation [4], [8].
Uterine contractions during labor are also associated with intermittent utero-placental perfusion providing the basis for ischemia-reperfusion type injury to the placenta. Doppler ultrasound studies have demonstrated a linear inverse relationship between uterine artery resistance and the intensity of the uterine contractions during labor [9]. Labor is also associated with placental alterations in several pathways linked to oxidative stress [10].
Heat-shock proteins (HSPs) are expressed by all cells and organisms. They have many important physiological functions as well as helping cells to cope with stressful situations. Some HSPs are expressed constitutively while others are induced by a range of damaging insults including heat shock, ischemia, hypoxia, oxidative stress and physical injury [11]. HSPs are named according to their molecular weight. The inducible HSP 70 is one of the best studied HSPs [12].
The aim of this study was to examine the spatial expression of inducible HSP 70 in placentae obtained from women who delivered by cesarean section and were not in labor, by defining precise sampling zones, and then to compare the expression of each zone with the equivalent zone of placentas obtained from women who delivered vaginally following an uncomplicated labor. The second aim was to determine the expression of HSP 70 in normal pregnancy with preeclampsia, both labor and non-labor.
Materials and Methods
Subjects
Human term placentae were collected from pregnant women at the Southern General Hospital, Glasgow. The study was approved by the local ethics committee. Placentae were collected from: (i) women who had uncomplicated pregnancies and delivered at term either vaginally (labor group) or by caesarean section (non-labor group) and (ii) women who had pregnancies complicated by preeclampsia. The number of patients recruited is shown in Table 1. Caesarean sections were performed for obstetric reasons such as breach presentation, previous caesarean section or maternal request. Patient consent was obtained prior to delivery. Preeclampsia was defined as a blood pressure of >140/90 mm Hg on at least 2 occasions at least 6 hours apart occurring after 20 weeks’ gestation and accompanied by proteinuria (>300 mg/L in a 24 hour urine collection) with no other underlying clinical problems.
10.1371/journal.pone.0054540.t001Table 1 Shows the demographics of patients used for placenta collection.
Category Normotensivenonlabour n = 6 Normotensivelabour n = 6 Pre-eclampsia n = 9 p value
Maternal age (years) 28.33±5.7 26±2.28 31±6.98 ANOVA p = 0.27
Placenta weight (g) 594.7±110.5 589.5±75.0 463.3±139.0 ANOVA p = 0.07
Birth weight (g) 3443±537 3719±347 2545±900* ANOVA p = 0.01L v NL (p = 0.32)NL v PE (p = 0.04)L v PE (0.001)
No. primigravid 1 4 6
Gestation age at delivery (weeks) 39.3±1.0 40.31±1.4 35.86±4.5* Kruskal Wallis (p = 0.01)L v NL (p = 0.22)NL v PE (p = 0.03)L v PE (p = 0.02)
No. Smokers 2 0 0
Sample Collection
For each patient (6 patients per group), placental samples (∼1 cm3) were obtained from three sites by taking measurements from the cord insertion point: 0–2 cm (inner position), 2–4 cm (middle position) and 4–6 cm (outer position) of placenta. Within each zone four separate samples were obtained representing the four quadrants (Figure 1). Samples were rinsed and immediately flash frozen in liquid nitrogen. For this study we had performed a power analysis using G*Power 3.1 for Macintosh.
10.1371/journal.pone.0054540.g001Figure 1 Drawing showing areas where samples were taken from in each individual placenta.
Materials
All chemicals were purchased from Sigma-Aldrich (U.K.) unless stated otherwise.
Tissue Homogenizing for Western Blot
Samples were recovered from storage at −70°C and ground in liquid nitrogen to a fine powder using a mortar and pestle. Tissues was homogenised in the presence of protease inhibitors as described previously [13]. Placenta homogenates were spun at 5000 g for 10 minutes at 4°C to remove debris then supernatants were collected and divided into aliquots and stored at −70°C. Protein concentrations were determined using bovine serum albumin as a standard.
Western Blotting
Western blotting was performed as described previously [13] with some modifications. A volume corresponding to 50 µg of each sample was separated by SDS-PAGE electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide resolving gels. Pre-stained low range molecular weight markers (BioRad) were loaded onto each gel. Transfer of proteins to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) was carried out at 22 V and 200 mA for 30 min. Membranes were blocked in 5% donkey serum (Serotec) in TBSTB buffer (20 mM TRIS pH 7.5, 0.5 M NaCl, 0.4% Tween and 0.25% bovine serum albumin) for 1 h at room temperature (RT). Primary antibodies were pre-absorbed in 5% human serum in TBSTB at RT during the blocking process. Membranes were incubated for 1 h at RT with primary antibody solution. The HSP 70 (rabbit polyclonal antibody) was obtained from Enzo Life Sciences (ABI-SPA-812, lot: 09061120) and used at concentration of 1∶1000. Membranes were washed and then incubated for 1 h at RT with horseradish peroxidase conjugated donkey anti-rabbit secondary antibody (Abcam (ab7083, lot: gr35152-1) diluted 1∶3000 in TBSTB. Membranes were rinsed with TBSTB (2×5 min) and once with distilled water. Filters were re-probed with a β-actin antibody (Sigma) to ensure even protein loading. Immunologically reactive proteins were visualised and quantified as described previously [13]. Statistical analysis was performed using MiniTab on a PC using analysis of variance (Kruskal Wallis for non-parametric data and ANOVA for normally distributed data). Comparison of groups was performed by the Mann Whitney test or student’s t-test as appropriate.
Quantitative RT-PCR
Total RNA was isolated using the RNeasy® Midi Kit (Qiagen, 75142). RNA (100 ng) was reverse transcribed into cDNA. Buffers and primers were obtained from the QuantiTect® Kit (Qiagen, 205310) and GoScript™ reverse transcriptase from Promega (A501C). HSP 70 (ID:NCBI 3303) expression (was analyzed by RT-PCR using validated TaqMan® Gene Expression assays with StepOnePlus (Applied Biosystems). b-actin was used as an endogenous control. A positive control human placenta cDNA (Primer design) was used. The relative target gene levels were calculated by comparative CT (ΔΔCT). Statistical analysis was performed as above.
Results
Table 1 shows the demographics of the patients.
Western Blotting
The first set of experiments was designed to test whether there was a difference in HSP 70 expression within individual placentae in both labor or non-labor. Figure 2 shows representative blots of HSP 70 expression in the area sampled 0–2 cm, 2–4 cm and 4–6 cm from the cord insertion point. The upper panel shows a placenta obtained from non-laboring caesarean section delivery. The bottom panel shows a placenta obtained from a women who was in labor and delivered vaginally. Figure 3 shows the results for the mean optical densities for HSP 70 expression for this set of experiments (6 patients in each group). The upper panel shows non-labor and the lower panel shows labor. Overall there was a significant difference between the 3 areas of the placenta for the non-labor group (ANOVA p = 0.008). There was significantly more HSP 70 expression in the 2–4 cm (middle) compared with the 4–6 cm (outer) area (student’s t-test, p = 0.03). No other differences were found (0–2 v 2–4, p = 0.14) and (0–2 v 4–6, p = 0.06). Overall there was also a significant difference between the 3 areas of the placenta in the labor group (ANOVA p = 0.002). There was significantly more HSP 70 expression in both the 0–2 cm and 2–4 cm areas compared with the 4–6 cm areas (p = 0.01 and p = 0.02 respectively). There was no difference between the 0–2 cm and 2–4 cm areas (p = 0.3).
10.1371/journal.pone.0054540.g002Figure 2 Shows a representative Western blot analysis of HSP 70 expression in placenta of a patient (non-labor) and a patient in labor (n = 6 patients in each group for entire study).
Samples are grouped according to distance sampled from cord insertion point. Four samples were obtained within each zone (see Figure 1). Molecular weight markers (kDa) are indicated by arrows. Also shown is a representative β-actin loading control for the gel above showing equal protein loading.
10.1371/journal.pone.0054540.g003Figure 3 Shows the optical densities for HSP 70 expression in three different placenta zones for all patients.
The upper panel shows non-labor (n = 6 patients) and the lower panel shows labor (n = 6 patients).
The second set of experiments was designed to test whether there was a difference in HSP 70 expression between labor and non-labor groups for each of the three sites. Figure 4 shows representative blots of non-labor versus labor for the three different areas of the placenta (upper panel 0–2 cm, middle panel 2–4 cm and lower panel 4–6 cm). Figure 5 shows an interaction plot for HSP 70 showing the relationship between the means of the 3 different areas of the placenta sampled (0–2, 2–4 and 4–6 cm) and the two patient groups (Non-labor solid line (n = 6 patients); labor broken line (n = 6 patients)). Individual groups were then compared using the student’s t test. HSP 70 was significantly increased in the labor group when compared to the non-labor group at the 2–4 cm site (p<0.005). There was no significant difference in HSP 70 expression between non-labor and labor at the 0–2 cm (p = 0.99) or the 4–6 cm (p = 0.06) sites.
10.1371/journal.pone.0054540.g004Figure 4 Shows a representative Western blot analysis of HSP 70 expression in labor versus non-labor measured at three distances from the cord insertion point of the placenta: 0–2 cm (top panel), 2–4 cm (middle panel) and 4–6 cm (bottom panel).
10.1371/journal.pone.0054540.g005Figure 5 Shows an interaction plot for HSP 70 showing the relationship between the means of the 3 different areas of the placenta sampled (0–2, 2–4 and 4–6 cm) and the two patient groups.
Non-labor solid line(n = 6 patients); labor broken line (n = 6 patients).
The third set of experiments was designed to test the difference between HSP70 expression in normotensive pregnancies and pregnancies complicated by preeclampsia. Sample representative blots are shown in Figure 6 for some of the patients. The data is summarised in Table 2. There was a significant increase in HSP 70 expression in the preeclampsia non-labor group (n = 4 patients) compared to the control non-labor group (n = 6) in the 0–2 cm site (p = 0.003). This difference was not seen for at the 2–4 cm site. Next the labor groups were compared. There was no significant difference between the control labor (n = 6) and preeclampsia labor groups (n = 5) at the 0–2 cm sites (p = 0.31) however there was a significant increase in HSP 70 expression in the control labor group (n = 6) compared with the preeclampsia labor group at the 2–4 cm site (n = 6) (p = 0.001).
10.1371/journal.pone.0054540.g006Figure 6 Shows a representative Western blot analysis of HSP 70 expression in labor versus non-labor normotensive and preeclampsia cases measured at 0–2 cm and 2–4 cm from the cord insertion point.
Statistical analysis for all gels is shown in Table 2.
10.1371/journal.pone.0054540.t002Table 2 Shows the median optical density for each group of patients and p value for each comparison from all patients combined for Western blot analysis of HSP 70 expression in non labor control versus non-labor PE at 0–2 cm site, non labor control versus non-labor PE at 2–4 cm site, labor control verus labor PE at 0–2 cm site and labor control versus labor PE at 2–4 cm site.
Group Group Sampling site p value C.I.
Non Labor group controlMedian 12.6 Non Labor group PE*Median 20 0–2 cm 0.003 95%
Non Labor group controlMedian 5.83 Non Labor group PEMedian 6.25 2–4 cm 0.41 95%
Labor controlMedian 12.1 Labor PEMedian 16.4 0–2 cm 0.31 95%
Labor control*Median 17.6 Labor PEMedian 12.7 2–4 cm 0.001 95%
The representative blot is shown in Figure 6.
The next of experiments (Figure 7) was designed to determine if there was any difference in HSP 70 expression in second versus third trimester preeclampsia cases. For all cases combined there were no significant differences noted for either the 0–2 cm sites (median optical density second trimester 24.8), (median optical density third trimester 26) (p = 0.47, 95% C.I. ) or the 2–4 cm sites (median optical density second trimester 19.9), (median optical density third trimester 19.3) (p = 0.72, 95% C.I.).
10.1371/journal.pone.0054540.g007Figure 7 Shows a representative Western blot analysis of placental HSP 70 expression in 2nd trimester preeclampsia cases versus 3rd trimester preeclampsia cases measured at 0–2 cm and 2–4 cm from the cord insertion point.
The final experiment was performed to confirm that the scanning densitometry provided similar results to other quantitative methods. To do this confirmatory experiments were performed as follows. The labour group samples used in experiment one were repeated as above however this time the signals were quantified using the BioRad gel documentation ECL imager system, removing the need for autoradiographs. As for experiment one there was more HSP70 in the inner compared to the outer region and in the middle compared to the outer region (Figure 8 lower panel). A second experiment was performed where a single protein sample was serially diluted (90–10 µg) and HSP70 expression determined. As shown in Figure 8 (upper panel) there was a linear relationship between protein loading and signal intensity which levelled off after 70 µg. This confirmed that the original experiments performed herein (50 µg loaded) were performed with samples within the linear area.
10.1371/journal.pone.0054540.g008Figure 8 Shows HSP 70 expression in three different placenta zones for all patients in the labor group (n = 6 patients) (upper panel).
Quantification was performed using the BioRad documentation ECL imager system. The lower panel shows the relationship between protein loading and signal obtained.
Real Time PCR
There was no differences in any groups except one. The labor control group was increased compared to the labor preeclampsia group (p = 0.03) matching the protein findings.
Discussion
This study shows for the first time that HSP 70 is expressed in a spatial manner in the placenta with the highest expression being in the 2–4 cm (middle) area in both labour and non-labour groups. It also shows the importance of using a systematic method to sample the placenta. Most previous reports of placental protein expression do not take this into account. Taking a single or a few samples or averaging protein expression of several samples may well mask possible changes in expression. Apart from the reported changes and their link to placental pathology the results have important implications for how results in placental disease (and perhaps other organs) can be influenced by sampling methods.The increase in HSP 70 in labor and preeclampsia at precise zones suggests that there is a controlled spatial change in HSP 70 expression. The physiological and pathological significance of this remains to be elucidated but oxidative stress is the common link. Oxidative stress occurs when the production of reactive oxygen species overwhelms the intrinsic anti-oxidant defenses.
The main components of the HSP 70 family are HSP 72 (HSP 70i) (induced during cell stress) and HSP 73 (HSC 70) which is constitutively expressed in all cells. Both have very similar amino acid sequences. Both are involved in translocation of proteins from the cytosol into the endoplasmic reticulum and mitochondria and in protein folding during and after synthesis [14], [15]. Under non-stressful conditions constitutively expressed members of each HSP family are found in almost all organelles including the nucleus, cytoplasm, endoplasmic reticulum and mitochondria. By interacting with proteins and peptides they play an important role in cell and organ survival. HSPs are induced in response to cell stresses including heat shock, oxidative stress, ultraviolet radiation, ischemia-reperfusion injury, viral infections, nutrient deprivation, hypoxia, physical damage, ischemia and chemicals. Two mechanisms counteract protein misfolding: (i) the molecular chaperones (including HSPs) that facilitate assembly, folding and translocation of proteins as well as the refolding of denatured proteins and (ii) the ubiquitin-proteasome system which regulates the degradation of misfolded proteins which cannot be renatured [16].
Although originally thought to bind directly to the signalling receptors TLR2, TLR4, CD40, or CD91 it is now known that HSP 70 binds to scavenging receptors LOX-1, SREC-1, and FEEL-1. On binding to the receptor it is thought that HSP 70 then signals to the TLR2 receptor which in turn signals MyD88 activation leading to the phosphorylation of ERK which can trigger the activation of an undetermined transcription factor that will bind the IL-10 gene promoter leading to IL-10 production [16]. Interestingly IL-10 can be pro-inflammatory at the end of labor and it has been proposed that this inflammatory action of a usually anti-inflammatory cytokine might accelerate parturition and delivery [17].
Apoptosis has been implicated in both preeclampsia and labor. In the apoptotic pathway, HSPs act at several stages to prevent cell death initiated by stress-induced damage. For example HSP 70 inhibits caspase 3 and 9. Thus it is possible HSP 70 acts to keep the rate of apoptosis in check [14], [15], [16].
Secreted HSPs, including HSP 70, can take part in immune surveillance. They can capture antigens and interact with receptors on antigen presenting cells. HSP 70 can bind to, and activate, human monocytes, inhibiting the secretion of inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and IL-10 [18].
Previous publications of HSP 70 expression in the placenta and changes during adverse pregnancy have not controlled for sampling and the confounding effects of labor. These studies can be summarized as follows and unless stated otherwise controlling for labor or sampling site was not done.
Shah et al [19] used immunohistochemistry to assess HSP 70 expression in paraffin sections of placentae from normal term pregnancies and reported immunostaining on cell types, both in cytoplasm and nucleus. Site of sampling or labor was not assessed. An immunohistochemical study of HSP 70 expression in pre-term labor, term labor, term non-labor and pre-term cesarean section for preeclampsia or intra uterine growth retardation found no changes in HSP 70 expression on amniochorion and basal plate [20]. The placenta was not examined and controlled sampling was not performed. Increased expression of HSP 70 was reported in placenta of what was termed “placental vascular disease” (preeclampsia, preeclampsia, preeclampsia plus IUGR all combined in one group) compared with term non-diseased placentae [21]. All were delivered by caesarean section. Labor was not studied.
One study reported that HSP 70 was expressed in placenta and reported no difference between labor and non-labor however no data or p values were shown to support this statement and no systematic sampling was performed [22]. Similarly Li et al [21] found no difference between labor and non-labor but similar issues applied. Several years ago we examined HSP70 expression in placentae from normal and preeclampsia with our without IUGR [23]. Others have preformed immunofluorescence on paraffin sections. HSP 70 expression was reported to be increased in preeclampsia [24].
The presence of a uterine artery notch in a mixed group of normal pregnant, preeclampsia and preeclampsia plus IUGR was associated with increased eNOS and HSP 70 in basal plate samples taken from patients who underwent caesarean section. Placental villous tissue was not studied [25].
Some studies have examined HSP 70 expression in early pregnancy. HSP 70 temporarily increases during 8–9 weeks of gestation when blood flow to the placenta is initiated leading to an oxidative stress insult [26]. HSP 70 immunostaining also increased in early pregnancy miscarriage [27]. Janiaux et al [28] examined HSP 70 and nitrotyrosine expression in placentae obtained from surgically terminated pregnancies between 8–13 weeks of gestation. They sampled the inner and outer third. Immunoreactivity for HSP 70 and nitrotyrosine residues was greater in samples from peripheral than from central regions of normal placentas and from missed miscarriages compared to controls. They proposed that oxidative damage to the trophoblast, induced by premature onset of the maternal placental circulation is a key factor in early pregnancy loss.
HSP 70 was reported to be reduced in purified cytotrophoblast cells from preeclampsia cases compared to controls however labor and site of sampling was not studied. The shock of enzyme digestion and cell purification are also confounding factors [29].
Since intracellular HSP 70 binds to the progesterone receptor and functions as a co-repressor of this receptor [30] this may in part explain our results providing a mechanism linking HSP 70 to labor.
HSF-1 is the stress responsive transcriptional activator responsible for the inducible transcription of genes encoding HSPs [31]. Padmini et al [32] reported increased HSP 70 and HSF-1 in placentae from preeclampsia cases compare with uncomplicated pregnancies.
Malyshev et al (1995) [33] showed that oxidative stress increases NFκB which in turn activates nitric oxide synthase, nitric oxide release and subsequently HSP 70 induction in several organs. Blocking nitric oxide synthase activity inhibited HSP 70 induction. We have previously shown that villous eNOS [34], peroxynitrite production [35] and lipid peroxidation [23] are increased in preeclampsia.
HSPs can be detected in the circulation. The few reported studies of HSP 70 serum concentrations in preeclampsia and labor are conflicting [30], [36].
In summary spatial changes in HSP 70 expression occur during labor and preeclampsia. The physiological and pathological significance of this remains to be elucidated.
We are grateful to Dr George Baillie for the use of the ECL imager system.
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31 : 72 –77 .15669997 | 23382911 | PMC3557260 | CC BY | 2021-01-05 17:11:55 | yes | PLoS One. 2013 Jan 28; 8(1):e54540 |
==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23383203PONE-D-12-2460310.1371/journal.pone.0055487Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyCytokinesDevelopmental BiologyMolecular DevelopmentCytokinesStem CellsMesenchymal Stem CellsImmunologyImmune SystemCytokinesMolecular cell biologyCell DivisionCytokinesisCellular TypesStem CellsMesenchymal Stem CellsSignal transductionSignaling in cellular processesSTAT signaling familyMedicineAnatomy and PhysiologyImmune PhysiologyCytokinesClinical ImmunologyImmune SystemCytokinesNovel Mechanism of Inhibition of Dendritic Cells Maturation by Mesenchymal Stem Cells via Interleukin-10 and the JAK1/STAT3 Signaling Pathway Inhibition of Dendritic Cell Maturation by MSCsLiu Wen-hua
1
Liu Jing-jin
2
Wu Jian
2
Zhang Lu-lu
2
Liu Fang
2
Yin Li
2
Zhang Mao-mao
2
Yu Bo
2
*
1
Intensive Care Unit (ICU) Department, Second Affiliated Hospital of Harbin Medical University, Harbin, Province Heilongjiang, China
2
Cardiology Department, Second Affiliated Hospital of Harbin Medical University, Harbin, Province Heilongjiang, China
Shi Xing-Ming Editor
Georgia Health Sciences University, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: WHL JW BY. Performed the experiments: WHL JJL LLZ. Analyzed the data: WHL JJL FL. Contributed reagents/materials/analysis tools: FL LY MMZ. Wrote the paper: WHL BY.
2013 30 1 2013 8 1 e5548714 8 2012 27 12 2012 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Mesenchymal stem cells (MSCs) can suppress dendritic cells (DCs) maturation and function, mediated by soluble factors, such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and nitric oxide (NO). Interleukin-10 (IL-10) is a common immunosuppressive cytokine, and the downstream signaling of the JAK-STAT pathway has been shown to be involved with DCs differentiation and maturation in the context of cancer. Whether IL-10 and/or the JAK-STAT pathway play a role in the inhibitory effect of MSCs on DCs maturation remains controversial. In our study, we cultured MSCs and DCs derived from rat bone marrow under different culturing conditions. Using Transwell plates, we detected by ELISA that the level of IL-10 significantly increased in the supernatants of MSC-DC co-cultures at 48 hours. The cell immunofluorescence assay suggested that the MSCs secreted more IL-10 than the DCs in the co-cultures. Adding exogenous IL-10 to the DCs monoculture or MSC-DC co-cultures stimulated IL-10 and led to a decrease in IL-12, and lower expression of the DCs surface markers CD80, CD86, OX62, MHC-II and CD11b/c. Supplementing the culture with an IL-10 neutralizing antibody (IL-10NA) showed precisely the opposite effect of adding IL-10. Moreover, we demonstrated that the JAK-STAT signaling pathway is involved in inhibiting DCs maturation. Both JAK1 and STAT3 expression and IL-10 secretion decreased markedly after adding a JAK inhibitor (AG490) to the co-culture plate. We propose that there is an IL-10 positive feedback loop, which may explain our observations of elevated IL-10 and enhanced JAK1 and STAT3 expression. Overall, we demonstrated that MSCs inhibit the maturation of DCs through the stimulation of IL-10 secretion, and by activating the JAK1 and STAT3 signaling pathway.
This project was supported by research grants from Heilongjiang Provincial Health Department issued in 2011 (2011-087), Heilongjiang Provincial Education Department issued in 2012 (12521265), China.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into various lineages, including adipogenic, osteogenic, and chondrogenic [1]. MSCs are characterized by their expression of surface markers, such as CD29, CD90, and CD44, and the absence of the hematopoietic lineage markers CD34 and CD45 [2]–[4]. MSCs have unique characteristics, such as the previously reported low immunogenicity and immunoregulatory properties [5]–[9]. Recently, MSCs gained attention because of their immunosuppressive abilities against T cells [5], DCs [6]–[8], B cells [9], and natural killer (NK) cells [10]. Thus, MSCs are currently being used to reduce immunological rejection and prolong graft survival. Because dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), they play a pivotal role in initiating immune response. Thus, it is important to study the mechanism(s) involved in the activity of the MSCs and the maturation of DCs. A few studies have demonstrated that IL-6, prostaglandin E2 (PGE2) and indoleamine-2,3-dioxygenase (IDO) may be implicated in MSCs-mediated inhibition of DCs function. Djouad et al.
[11] observed that MSCs secrete higher levels of IL-6 which may be involved in reversing the maturation of DCs into a less mature phenotype, and in the partial inhibition of bone marrow progenitor differentiation into DCs. Chen et al.
[12] demonstrated that blocking PGE2 synthesis in MSCs could revert most of the inhibitory effects on differentiation and function of DCs. In short, MSCs disrupt the transition of DCs from immature to mature states by secreting inhibitory soluble factors.
It is known that DCs play a key role in the initiation of primary immune responses and the induction of tolerance [13]. Normally, the life of DCs can be divided into two major phases [14] – an immature stage, and a mature stage [13], [15] that is associated with a high expression of molecules involved in antigen presentation (i.e., CD80, CD86, OX62, MHC-II, and CD11b/c). DCs maturation is a prerequisite for induction of immunogenic T cell responses. Recent studies have focused on the influence of bone marrow MSCs on the activity of DCs via specific signaling pathways [16], [17]. Therefore, we may conclude that MSCs modulate the differentiation, maturation, and function of DCs via the secretion of cytokines and /or activation of defined signaling pathways.
IL-10 is a potent immunosuppressive cytokine, produced primarily by Th2 cells, macrophages, and activated B cells [18].This cytokine has a wide range of biological activities, including immunosuppressive, anti-inflammatory and immunomodulatory properties, which regulate a variety of immune cell differentiation and proliferation events [19]. Corinti et al.
[20] found that immature monocyte-derived DCs released sizeable amounts of IL-10. After stimulation with LPS, mDCs secreted high levels of IL-10 [21], which is known to inhibit DCs maturation and function. Thus, inhibiting the ability of DCs to produce IL-12, which is essential for driving Th1 cell differentiation. IL-10 can also down-regulate the major histocompatibility complex I (MHC-I) and growth and differentiation of B cells, T cells, DCs and other cells involved in inflammatory responses. Kim et al. [22] concluded that interleukin (IL)-10, induced by CD11b (+) cells and IL-10-activated regulatory T cells, play a role in the immune modulation of mesenchymal stem cells in rat islet allografts.
We herein formulate, and test, the hypothesis that MSCs may express IL-10 to influence the activity and maturation of DCs through a similar mechanism as regulatory T cells. Furthermore, we also propose a mechanism of action of IL-10-mediated inhibition of DCs that involves MSCs. Conzelmann et al. [23] reported that the JAK/STAT signaling pathway is strictly complementary for the induction of a pro-inflammatory cytokine profile in human antigen-presenting cells (APCs). We postulate that the downstream signaling pathway of JAK-STAT may be implicated in this specific inhibitory effect.
The JAK-STAT signaling pathway is one of the most important signal transduction cascades and is essential for the regulation of cytokine receptor signaling. When combined, the IL-10 and the IL-10 receptor activate the JAK1-STAT3 pathway [24]. Several studies [25], [26] have shown that the members of the STAT family, and more specifically STAT3, could be responsible for abnormal DCs differentiation and function in cancer. Hirata et al.
[26] examined the role of JAKs in the regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional DCs. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by DCs. JAKi completely blocked TLR-mediated STATs activation. In summary, these studies [24]–[26] have demonstrated that IL-10 and the JAK / STAT signaling pathway play important roles in DCs maturation.
Previous studies have suggested that several soluble factors, such as IL-6 [11], IDO [10], [27], PGE2 [6], [10], and NO [28], are involved in this process. However, the mechanism(s) by which MSCs exert their inhibitory effects on the maturation of DCs is still poorly understood. In the following experiments, we cultured MSCs and DCs within different culture media and under various conditions. Then, we established co-cultures of the two cell types using Transwell plates. In some experiments, we added IL-10, IL-10NA and AG490 to the co-culture medium to probe the role of the IL-10 and JAK1/STAT3 signaling pathways in the inhibitory mechanisms of MSCs on DCs. FACS, ELISA and Western blotting were used to characterize the cell cycle, cytokine production and protein expression, respectively. Based on our findings, we propose an IL-10 feedback loop is involved in the inhibitory effect of MSCs on DCs. In the co-culture, IL-10 secretion increased significantly, compared with MSCs or DCs alone. Furthermore, co-cultures activated the JAK1 / STAT3 signaling pathway, inhibiting the maturation of DCs, and affecting IL-12 secretion from DCs. Moreover, we report for the first time that IL-10 and the JAK1 / STAT3 pathway play important roles in MSCs-mediated inhibition of DCs maturation.
Methods
Animals
Male Sprague Dawley (SD) rats weighing 60–80 g were cared for in accordance with US National Institutes of Health published guidelines published by the National Institutes of Health. All of the study procedures were approved by the Harbin Medical University Institutional Animal Care and Use Committee. The study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy Press (NIH, revised in 1996).
Isolation, Culture and Characterization of MSCs
The MSCs were isolated and cultured from the rat bone marrow of the femur and tibia [6]. Bone marrow cells were plated in culture flasks at a concentration of 106 cells/mL in DMEM/F-12 (1∶1) (Hyclone, Logan, UT), supplemented with 10% fetal calf serum (FCS; Hyclone) and 1% penicillin/streptomycin, at 3×105/cm2 at 37°C in 5% CO2 humidified atmosphere. At 80–90% confluence, MSCs were treated with trypsin and further expanded at a ratio of 1∶2. MSCs were used in the experiments only after 3–5 expansion passages, to ensure depletion of monocytes/macrophages. MSCs were characterized by flow cytometric analysis for the expression of the typical markers, CD29, CD90, and CD44, and the absence of the hematopoietic markers CD45 and CD34. All data are expressed as percentages from FACS measurements.
Culture, Generation and Characterization of DCs
We cultured DCs in accordance with our protocol previously described in detail elsewhere [29]. Like the MSCs, DCs were also derived from the rat femur and tibia bone marrow. In the course of the isolation, we added 5 mL red blood cell lysate (Beyotime, China) to the cell suspension. After cleavage at room temperature for 5 min, the cell mixture was centrifuged (1000 rpm, 5 min). Cells were then cultured in 6-well plates using RPMI 1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin, recombinant rat GM-CSF (50 ng/mL), and IL-4 (50 ng/mL) (PeproTech, Rocky Hill, NJ). After 5 days, cultured cells were harvested by gentle aspiration and analyzed by flow cytometry to assess the immature DCs (iDCs) phenotype. DCs maturation was induced by stimulation with lipopolysaccharide (LPS, Sigma). After LPS (200 ng/mL) stimulation for 48 h, DCs were analyzed by flow cytometry for CD80, CD86, OX62, MHC-II and CD11b/c to assess whether the mature phenotype (mDCs) was successfully induced.
Transwell Co-cultures of MSC-DC
A Transwell system (0.8-µm pore size membrane, Corning, Acton, MA) was used to prevent MSCs from contacting directly the DCs. MSCs and iDCs were placed in the upper and lower layers of the Transwell plate, respectively, at various ratios (1∶1, 1∶10, and 1∶100). Each well in a 6-well Transwell co-culture plate contained 5×105 DCs in RPMI 1640 medium, supplemented with 10% FCS, 1% penicillin / streptomycin, recombinant rat GM-CSF (50 ng/mL), and IL-4 (50 ng/mL). LPS (200 ng/mL) was added to the DCs layer for 2 additional days (48 h) to stimulate iDCs maturation in the presence or absence of MSCs. In some experiments, IL-10 (500 ng/ml; PeproTech) was added to the MSC layer for 48 h. To investigate whether MSCs exerted their inhibitory effect on DCs differentiation by IL-10 production, IL-10NA (5 µg/mL; Abcam, Cambridge, UK) was added to the co-cultures for 48 h. To distinguish the exogenous IL-10 added to the culture from the endogenous IL-10 produced by the cells, IL-10 (500 ng/ml) and LPS (200 ng/mL) was added to iDCs monoculture, or IL-10NA (5 µg/mL) followed by IL-10 was added to iDCs-LPS. In another group, AG490 (40 µmol/L; Beyotime, China) was added to the co-culture for 48 h to investigate the role of the JAK1 / STAT3 signaling pathway in the effects of MSCs on DCs.
FACS
For the analysis of the cell surface-marker expression, cells were digested by trypsin, washed twice with phosphate-buffered saline (PBS), and kept at 4°C in the dark until analysis. For MSCs, the following rat antibodies (Abs) were used: Phycoerythrin-cy5 (PE-cy5)-labeled anti-CD45, PE-anti-CD90, Fluorescein Isothiocyanate (FITC)-labeled anti-CD44, anti-CD29 (BD Pharmingen, USA) and Alexa Flour 647-labeled anti-CD34 (eBioscience, San Diego, CA). Like the MSCs, DCs were harvested by incubation in ice-cold PBS for 30 min. Cells were stained with antibodies against CD80, OX62 and CD11b/c (PE-labeled), and CD86 and MHC-II (FITC-labeled; eBioscience). As a control, cells were stained with mouse IgG1 isotype-control antibodies. DCs were combined with MSCs in various ratios, and we measured changes in CD80, OX62, CD86, MHC-II and CD11b/c. Cells were analyzed using a FACScan (BD Biosciences, Franklin Lakes, NJ, USA) with the CellQuest Analysis (BD Biosciences) and FlowJo software (TreeStar). Results are expressed by the percentage of positively stained cells relative to total cell number.
ELISA
The supernatants of MSCs and DCs cultures, and also of the co-cultures were collected respectively in 1.5 ml micro-tubes and kept at −20°C. IL-10 quantification was performed in the supernatants of the co-cultures at 48 h. To measure IL-12 production by mDCs, culture supernatants were collected at day 7 (5 d + 48 h after the LPS stimulation). The measurement was conducted according to the manufacturer’s protocol. Detection limits were 7.8 pg/mL for IL-12 and 7.7 pg/mL for IL-10. All determinations were made in triplicates. IL-10 and IL-12 ELISA kits were purchased from the Xitang Company (Shanghai, China). ELISA plates were read at OD450 on a Microplate ELISA reader (Autobio Diagnostics Co. Ltd, China).
Immunofluorescence
To investigate the secretion of IL-10 from MSCs and DCs in Transwell co-culture plates, we designed a cell immunofluorescence study of monocultures of MSCs and of mDCs, and co-cultures of MSCs and iDCs (1∶10) plus LPS. Both cell types were grown on glass coverslips and fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.3% Trition-X100, blocked with goat serum and incubated with anti-IL-10 antibody (1∶200, ab9969, Abcam) overnight at 4°C. After washing, the cells were incubated with the Alexa Fluor 555 goat anti-rabbit IgG (1∶500, Invitrogen Technology, USA) for 2 h at 37°C. The nuclei of cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml, Sigma). The fluorescence images were acquired with a confocal laser-scanning microscope (Olympus FluoView V5.0 FV1000).
Western Blotting
DCs were washed with ice-cold PBS. After at least 30 min on ice, insoluble components were removed by centrifugation (12,000 rpm, 4°C, 15 min). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes. Total protein concentration was measured using the bicinchoninic acid assay, with bovine serum albumin (BSA) used to construct the standard curve. Nonspecific binding was blocked by incubating the membranes with 5% nonfat dry milk and TBST (0.05% Tween 20 in Tris-buffered saline, TBS). Membranes were incubated overnight at 4°C with anti-JAK1 mAb ( monoclonal antibody,3344,Cell Signaling Technology, Danvers, MA, USA), anti-STAT3 (9132, Cell Signaling Technology), anti-phospho-JAK1 mAb (ab5493, Abcam), or anti-phosphor STAT3 mAb (Tyr705, Cell Signaling Technology) at an appropriate dilution(1∶1000 in each Ab). Subsequently, the membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Specific protein bands were visualized on film by enhanced chemiluminescence (ECL) (Applygen Technologies Inc., China) following the protocol of the manufacturer. The bands were quantified by scanning densitometry using a GS-710 Imaging Densitometer (Bio-Rad, Hercules, CA) and normalized to that of β-actin. All data was obtained in triplicate independent experiments.
Statistical Analysis
Descriptive and analytical statistics were performed using SPSS software (ver. 15; Chicago, IL). Values for all measurements are presented as mean ± standard deviation (SD). Comparisons for two pairs were performed using the Student’s t-test. Values of p< 0.05 were considered to indicate statistically significant differences.
Results
MSCs Assessed by Morphology and FACS
The plastic-adherent cells under standard culture conditions obtained from rat bone marrow (MSCs) showed the typical spindle-shape. The MSCs were expanded for 3–5 passages in a plastic dish, and exhibited a typical fibroblastic morphology. The percentage of the MSCs obtained from culturing bone marrow cells was 99.04%. MSCs at passage 5 were analyzed for the expression of cell surface molecules by flow cytometry. As reported in other studies [3], [4], MSCs express CD29 and CD90, partly express CD44, but show negative expression of CD34 and CD45. We detected the expression of CD29 (99.04%), CD90 (88.27%), CD44 (35.65%), CD34 (0.84%), and CD45 (2.13%) in MSCs, respectively (Fig. 1). Thus, these cells have the typical expression profile of MSCs.
10.1371/journal.pone.0055487.g001Figure 1 Cell surface markers of MSCs.
The surface marker molecules expressed by the Passage 5 culture of MSCs were analyzed by flow cytometry. (B) Quantification by image analysis of the positive cells. The numbers in the histograms indicate the mean fluorescence of each MSC markers. (C) Percent of MSCs obtained from culturing bone marrow cells.
MSCs Inhibition of DCs Maturation Stimulated by LPS
Myeloid cells purified from Sprague Dawley rat bone marrow were cultured in medium with GM-CSF and IL-4 to induce DCs differentiation. After 5 days, DCs differentiation was assessed by analyzing the expression of OX62, MHC-II, CD11b/c and of the co-stimulatory molecules, CD80 and CD86. iDCs at 5 days were positive for CD80 (64.02%), CD86 (69.55%), OX62 (86.62%), MHC-II (61.05%) and CD11b/c (60.50 %), (Fig. 2A). These iDCs were stimulated by LPS for 48 h to acquire the mature phenotype (mDCs). mDCs showed significantly increased expression levels of the same panel of markers CD80 (91.38%), CD86 (88.04%), OX62 (93.33%), MHC-II (91.06%) and CD11b/c (79.84%) (Fig. 2B). However, when mDCs are co-cultured with MSCs, and stimulated with LPS, the cells displayed a phenotype similar to those of iDCs. For example, at a ratio of MSC: DC of 1∶10, the expression of CD80, CD86, OX62, MHC-II and CD11b/c was 65.46%, 55.05%, 70.19%, 66.63%, and 66.64%, respectively (Fig. 2C). A maximal inhibitory effect could be observed at a 1∶1 DC: MSC ratio. The inhibitory action was decreased at a 100∶1 ratio. Thus, the effect of MSCs on the maturation of DCs features a dose-dependent relationship.
10.1371/journal.pone.0055487.g002Figure 2 Phenotype analysis of different DCs.
(A) FACS histograms showed the expression of cell surface markers on iDCs, which expressed low levels of CD80, MHCII, OX62, CD86 and CD11b/c. OX62 expressed much higher than other makers, because OX62 is an important marker of rat DCs. (B) LPS- stimulated mature DCs (mDCs) showed about equally up-regulated levels of all markers. Except CD86, three makers showed above 90 percent expression. (C) MSC-DC-LPS-activated DCs in co-culture expressed lower levels of the four markers compared with mDCs, but the expression was slightly higher when compared with iDCs. This result suggests that MSCs inhibit DCs phenotype expression (MSC:DC = 1∶10). Data represent mean fluorescence intensity for the surface density of markers. (D) Quantification by image analysis of the positive cells. The numbers in the histograms indicate the mean fluorescence of each DCs markers. *P<0.05(mDC group versus iDC group), #P<0.05(MSC-DC group versus mDC group).
MSCs Inhibition of IL-12 Secretion by DCs in Co-culture
iDCs were cultured in the presence and absence of MSCs after 48 h, and mDCs supernatants were collected for cytokine quantification. Using ELISA, we analyzed the amount of IL-12 secreted by these cells. As shown in Figure 3A, MSCs produced a small amount of IL-12 (13.67±4.34 pg/mL). iDCs secreted significantly more IL-12 (56.07±14.83 pg/mL) compared with MSCs alone. Larger amounts of IL-12 (413.3±93.99 pg/mL) were released by mDCs cultured alone (P < 0.05; Fig. 3A), while much lower amounts were produced by DCs co-cultured with MSCs (72.1±21.41 pg/mL), which are comparable to levels of IL-12 secreted by iDCs in monoculture.
10.1371/journal.pone.0055487.g003Figure 3 IL-10 and IL-12 secretion as assessed by ELISA.
(A) IL-12 secreted in monocultures of MSCs, iDCs and mDCs and in the co-culture of MSC-DC (MSC:DC = 1∶10). (B) IL-10 secreted in monocultures of MSCs, iDCs and mDCs and in the co-culture of MSC-DC (MSC:DC = 1∶10). *P<0.05(mDC group versus iDC group), #P<0.05(MSC-DC group versus MSCs group).
MSCs Secretion of IL-10 in Co-culture
Co-culture supernatants were collected for analysis of IL-10 expression via ELISA. The supernatants of MSCs cultured alone were associated with a small amount of IL-10 (8.57±1.26 pg/mL). The DCs in monoculture, both iDCs and mDCs, also secreted low amounts of IL-10 (16.53±3.25 and 54.77±12.42 pg/mL, respectively). However, the IL-10 expression level in MSCs was increased significantly (583.37±100.81 pg/mL) when DCs were present in the co-culture (in a ratio of 1∶10) (Fig. 3B).
Immunofluorescence Assay
The expression of IL-10 was very weak in the monoculture of MSCs. The monoculture of DCs showed higher expression of IL-10 than the monoculture of MSCs. In the co-culture of MSCs and DCs (ratio 1∶10), the MSCs showed a clear increase in IL-10 expression when compared with the monoculture. When compared with DCs in monoculture, the DCs in co-culture plate do not overexpress IL-10 significantly. It is clear that in the co-culture condition the MSCs expressed significantly more IL-10 than DCs (Fig. 4 A, B, C, D).
10.1371/journal.pone.0055487.g004Figure 4 Immunofluorescence staining of MSCs, DCs and both cells in co-culture plates with IL-10.
(A) Monoculture of MSCs. (B) MSCs in Transwell co-culture with DCs. (C) Monoculture of iDCs-LPS. (D) iDCs-LPS in Transwell co-culture with MSCs (MSC:DC = 1∶10). Red staining shows IL-10 expression; DAPI blue staining of cell nuclei. Scale bar 251658240 = 251658240100 µm.
DCs Expression of (Phospho-) JAK1 and (Phospho-) STAT3 in the Presence of MSCs
We collected DCs, iDCs, mDCs, and DCs co-cultured with MSCs. From all of these cells, the protein was extracted and analyzed using Western Blotting to detect the expression of JAK1, STAT3, phospho-JAK1, and STAT3 (P-JAK1 and P-STAT3). We found that MSCs could enhance the expression of JAK1, STAT3, phospho-JAK1, and STAT3 in DCs (eg.P-JAK1 2.367 in the MSC-DC group, 1.125 and 1.253 in iDCs and mDCs, respectively, p<0.05; Fig. 5 A, B).
10.1371/journal.pone.0055487.g005Figure 5 Protein expression of JAK1, P-JAK1, STAT3 and P-STAT3 in monocultures of iDCs, mDCs and in co-cultures of MSC-DC (MSC:DC ratio of 1∶10).
(A) Western Blot; (B) Quantification by image analysis of the protein expression. * P<0.05 (MSC-DC group versus mDC group).
IL-10 and IL-12 Secretion in the Presence of IL-10, IL-10NA and AG490
To examine the effect of IL-10 and JAK1/STAT3 signaling pathways on DCs activity, we added IL-10, IL-10NA and AG490 to the MSCs layer in co-culture plates for 48 h, respectively. Cell supernatants were collected and analyzed by ELISA to characterize the secretion of IL-10 and IL-12. The results showed that IL-10 levels in the IL-10 group (630.97±110.38 pg/mL) increased even more than in the MSC-DC co-culture group (583.37±100.81 pg/mL). IL-10NA added into the co-culture system successfully reduced the IL-10 level (126.4±29 pg/mL). In the presence of AG490, a JAK inhibitor, the level of IL-10 secretion also decreased, to a degree that was similar to the effect of IL-10NA. (Fig. 6A). IL-12 levels decreased in the IL-10 (64.43±15.59 pg/mL) and in the IL-10NA group (98.83±32 pg/mL) by comparison to mDCs (413.3±93.99 pg/mL, Fig. 3A). When AG490 was added to the system, IL-12 levels (149.7±78.42 pg/mL) increased compared to that in IL-10 group, but still remained much lower than levels of IL-12 released from mDCs (Fig. 6B). In DC-IL-10 group, IL-10 and IL-12 secreted 548.22± 86.45 and 68.49±10.64 pg/mL, respectively, while in DC -IL-10-IL-10NA group data were 78.86±23.09 and 377.38±102.29 pg/mL, respectively (Fig. 6A, B).
10.1371/journal.pone.0055487.g006Figure 6 ELISA analysis of co-cultures of MSC-DC supplementated with exogenous IL-10, IL-10NA and JAK inhibitor (AG490).
IL-10 and IL-12 levels in DC–IL-10 and DCs -IL-10 -IL-10NA group by ELISA. (A) IL-12; (B) IL-10.* P<0.05 (IL-10NA group versus MSC-DC group), # P<0.05 (AG490 group versus MSC-DC group), & P<0.05 (DC- IL-10 group versus MSC-DC group), § P<0.05 (DC- IL-10-IL-10NA group versus IL-10NA group),$ P<0.05 (DC- IL-10-IL-10NA group versus DC- IL-10 group).
Phenotype of DCs in the Presence of Exogenous IL-10, IL-10NA and AG490
To investigate the mechanism(s) involved in the MSC-mediated inhibition of DC differentiation, we added IL-10, IL-10NA and AG490 to the co-cultures. Using this approach allowed us to elucidate whether IL-10 and/or the JAK1/STAT3 signaling pathways play a role in this mechanism. As shown in figure 6, CD80, CD86, OX62, MHC-II and CD11b/c showed reduced expression in the IL-10 group. When IL-10NA was added to the co-culture medium, as expected, IL-10NA strengthened the expression of these markers. AG490 reduced CD80, CD86, OX62, MHC-II and CD11b/c expression levels compared with the MSC-DC co-culture (Fig. 7A, B, C, F).
10.1371/journal.pone.0055487.g007Figure 7 DCs surface markers (CD80, CD86, OX62, MHC-II and CD11b/c) in the MSC-DC co-culture system (ratio MSC:DC of 1∶10) supplemented with exogenous: (A) IL-10; (B) IL-10NA and (C) AG490.
(D, E) These makers expression in DC-IL-10 and DC - IL-10 - IL-10NA group by FACS. (F, G) Quantification by image analysis of the positive cells. Numbers in the histograms indicate the mean fluorescence of each DCs markers. *P<0.05(IL-10 group versus MSC-DC group), # P<0.05(IL-10NA group versus MSC-DC group), $ P<0.05(AG490 group versus MSC-DC group).
In DC-IL-10 group, the expression of CD80, CD86, OX62, MHC-II and CD11b/c was 69.62%, 62.56%, 76.03%, 57.16%, and 57.99%, respectively. However, in DC-IL-10 -IL-10NA group, the makers of CD80 (88.11%), CD86 (65.65%), OX62 (81.94%), MHC-II (78.75%) and CD11b/c (72.97%) expressed higher than in DC-IL-10 group (Fig. 7 D, E, G).
Expression of (Phospho-) JAK1 / STAT3 in the Presence of IL-10, IL-10NA and AG490
We aimed to test the hypothesis that the IL-10 and JAK1/STAT3 pathway have a major role in MSC-mediated inhibition of DCs maturation. To address this hypothesis we studied the effect of exogenous IL-10, IL-10NA and AG490 on the expression of (phospho-) JAK1 / STAT3 in co-cultures of MSC-DC. Surprisingly, we observed that by adding IL-10 to the co-culture, the expression of total JAK1/STAT3 and phospho-JAK1 / STAT3 was enhanced. (Fig. 8A, B). As expected, in the presence of IL-10NA, the expression of all these markers (total JAK1/STAT3 and phospho-JAK1/STAT3) was reduced. In the DC-IL-10 group, total JAK1/STAT3 and phospho-JAK1/STAT3 were overexpressed when compared with the monoculture of DCs, but showed weaker expression than those of MSC-DC-IL-10 in the co-culture. When IL-10NA was added to the DC-IL-10 plates, the expression of those antibodies showed a decrease. (Fig. 8 C, D) Similar to the effect of IL-10NA, AG490 also reduced the expression of total JAK1/STAT3 and phospho-JAK1/STAT3 (Fig. 8 E, F). The expression levels of phospho-JAK1/STAT3 are significantly weaker than total JAK1/STAT3.
10.1371/journal.pone.0055487.g008Figure 8 Protein expression of JAK1, P-JAK1, STAT3 and P-STAT3 of DCs in co-cultures of MSC-DC (MSC:DC ratio of 1∶10).
(A) Western Blotting of the co-culture, without supplementation, or supplemented with, IL-10 or with IL-10NA (P<0.01); (B) Quantification by image analysis of the protein expression in the same conditions as (A); (C) Western Blotting of the co-culture, without supplementation, or supplemented withAG490 (P<0.01); (D) Quantification by image analysis of the protein expression in the same conditions as in (C). (E) Western Blotting of DC -IL-10 and DC- IL-10 -IL-10NA (P<0.01); (F) Quantification by image analysis of the protein expression in the same conditions as in (E). * P<0.05(IL-10 group versus MSC-DC group), # P<0.05(IL-10NA group versus MSC-DC group), $ P<0.05 (AG490 group versus MSC-DC group), & P<0.05 (DC- IL-10 group versus DC group), § P<0.05 (DCs- IL-10-IL-10NA group versus DC group).
Discussion
MSCs have been shown to potently suppress immunological activity by acting on various cells of the immune system. Additionally, many reports have documented a potent inhibitory effect of MSCs on myeloid or monocyte DCs maturation [6]–[7]. These inhibitory mechanisms involve two aspects: cell-to-cell contact, and secretion of specific cytokines. Several studies have previously demonstrated that the expression of IL-10 is significantly increased when MSCs and T cells are co-cultured in a Transwell system. Thus, we hypothesized that MSCs may inhibit DCs maturation through the action of the cytokine IL-10. It is well known that IL-10 is a suppressive cytokine being implicated in the proliferation and on the cytokine production of T cells. IL-10 has also been shown to have a role in inducing T cell anergy (a tolerance mechanism in which the lymphocyte is intrinsically functionally inactivated, in a hyporesponsive state) [22]. Thus, IL-10 may also inhibit DCs maturation and function. However, in MSC-DC co-cultures, and in the presence of IL-10, we observed that the expression of cytokines is increased markedly. In addition, we provide evidence by cell immunofluorescence that the MSCs secreted more IL-10 than the DCs in the Transwell co-cultures (Figure 4). This result shows that the mechanisms underlying the MSCs and the IL-10 inhibitory effect are still poorly understood, being necessary to study them in greater detail.
In the current study, we cultured and characterized MSCs and DCs in monoculture, and in co-cultures using Transwell plates. We aimed to use this culturing platform (DCs and MSCs co-cultured in the Transwell two-chamber system) to exclude the effect of cell-to-cell contact from our experiments. Under these culture conditions, a significant inhibition of DCs differentiation was observed at day 5 or 7, suggesting that soluble factors may be involved in this inhibitory mechanism. We found that there was a robust increase in the expression of IL-10 in the supernatants of co-cultures (583.37±100.81 pg/mL) compared with monocultures of MSCs (8.57±1.26 pg/mL) or DCs (either iDC: 16.53±3.25 pg/mL or mDC: 54.77±12.42 pg/mL).
The JAK-STAT signaling pathway was also analyzed in detail to evaluate the mechanism of action of IL-10. Using Western blotting, our results showed that JAK1, P-JAK1, STAT3, and P-STAT3 were all overexpressed when either MSCs or IL-10 was added to the co-culture medium. The expression levels are also much higher than those observed in monocultures of DCs (iDCs or mDCs). Additionally, we noticed that the MSCs group (MSC:DC = 1∶10) had greater cytokine expression than other groups using different proportions of the two cell types (1∶1 or 1∶100). The MSCs, particularly at the highest ratio in the co-culture (1∶10), could reproduce the inhibition patterns without cell-cell contact in the Transwell system. In the IL-10 group, the expression of JAK1 and STAT3 was higher compared with the MSCs group. A possible reason for this effect is that IL-10, when associated with MSCs [30] in the co-culture system, may enhance the expression of JAK1 and STAT3, which synergistically contribute to inhibition of DCs maturation.
The MSC-mediated inhibition of DCs maturation may also affect the expression of DCs surface markers, and on the development of DCs function, such as IL-12 production. Our data suggests that MSCs can suppress the secretion of IL-12 by DCs. Additionally, we analyzed the expression of CD80, CD86, OX62, MHC-II, and CD11b/c as these are considered to be markers of DCs maturation. We observed that the expression of these markers declined in the presence of MSCs. This result suggests that the presence of MSCs in co-culture directly interferes with the maturation of DCs.
We analyzed the involvement of IL-10 on the inhibitory effect of MSCs on the maturation of DCs. To assess this effect, IL-10 or IL-10 neutralizing antibody were added into the DCs monoculture or MSC-DC co-cultures in the Transwell system. ELISA results show that IL-10 was overexpressed in the IL-10 supplemented group, and markedly decreased in the IL-10NA group, as expected. We also observed that the expression of CD80, CD86, OX62, MHC-II and CD11b/c (panel of positive markers for DCs maturation) declined in the IL-10 group, while the expression of those markers was restored to baseline levels in the IL-10NA group. This result confirms that in the presence of MSCs, or in the presence of IL-10, a similar inhibitory pattern is observed. Exogenous IL-10 and the presence of MSCs seem to have a synergistic effect. Furthermore, the presence of IL-10NA in the co-culture reverses the inhibition.
Based in our results, we propose the concept of an IL-10 feedback loop mechanism of control for the maturation of DCs (Fig. 9). This feedback loop, helps in explaining the mechanism of increase of IL-10 expression that we have reported. MSCs or DCs in monoculture secrete low amounts of IL-10. When these cells are in co-culture, IL-10 combines with the IL-10 receptor and further activates the JAK-STAT signal transduction pathway [26]. Once activated (by LPS), this signaling pathway led to decreased IL-12 secretion. Simultaneously, DCs maturation was inhibited; the number of iDCs increased, and conversely the number of mDCs decrease. That is, the pool of immature DCs is enlarged at the expense of mature DCs. Our data suggest that mDCs can secrete more IL-10 than iDCs, which is consistent with previous results reported in the literature [18]. Thus, in the supernatants of co-cultures, fewer iDCs secrete lower amounts of IL-10. Moreover, more IL-10 may be produced through a positive loop. Accordingly, we observed a marked increase on the levels of IL-10 in MSC-DC co-culture medium supernatants.
10.1371/journal.pone.0055487.g009Figure 9 Newly proposed IL-10 feedback loop.
The figure illustrates the interplay between IL-10 and the JAK1/STAT3 signaling pathway involved in mechanism inhibition of DCs maturation by MSCs. The MSCs facilitate the combination of the cytokine IL-10 with its IL-10 receptor. This complex activates the JAK1/STAT3 signaling pathway that suppresses the DCs maturation. This inhibition of maturation causes a decrease in the expression of IL-10. Thus, the levels of the cytokine IL-10 command this positive feedback loop.
Our results showed that MSCs, IL-10, JAK-STAT, and DCs are all connected to each other. In previous reports, differing soluble factors (PGE2, IDO, IL-6 [6], [10], [11], [27]) and signaling pathways (Notch [31], toll-like receptor (TLR) [32]) have been suggested to mediate the inhibitory effect exerted by MSCs on the activity of DCs. However, to the best of our knowledge, this is the first report establishing a link between the levels of the cytokine IL-10, and the JAK-STAT signaling pathway with respect to the mechanism of inhibition of MSCs on DCs. We postulate that the MSCs specifically inhibit DCs function and maturation through IL-10 and the JAK-STAT signaling pathway.
In this study, we investigated the JAK-STAT signaling pathway with regard to the inhibitory mechanism of action of MSCs on the maturation of DCs. Our study did not focus on effects at the protein level, for example, the implication of the suppressor of cytokine signaling (SOCS) [33]. Additionally, this study was only performed in vitro. Our data needs to be confirmed by in vivo data. To date, our studies only discuss IL-10 and JAK-STAT, without involvement of other cytokines or signal transduction pathways. It is possible that other cytokines and signaling pathways may also be implicated in the inhibitory mechanism of action of MSCs on DCs.
In summary, our present study provides novel information on the molecular mechanisms and on the timing of MSCs-mediated inhibition of DCs maturation and function. Our data suggests that MSCs may modulate the immune system, not only through acting directly on T cells, but also in the first step of the immune response through the inhibition of DCs differentiation and maturation. We report data supporting the existence of a link between the cytokine IL-10 and the JAK-STAT pathway. This inhibition may have interesting implications, for example in the transplantation of MSCs to inhibit the maturation of DCs and reduce the incidence and degree of severity of graft-versus-host disease (GVHD). This study may open new, clinically-relevant, research avenues. A pioneering clinical study by Le Blanc et al
[34] revealed that the infusion of MSCs could successfully treat severe acute GVHD. Because DCs play a central role in the induction of T cell-mediated transplant rejection or GVHD [35], we studied the inhibitory mechanism of MSCs activity on DCs. These findings may enhance the future prospects of the clinical application of MSCs in new and broader immune-related applications. An understanding of the mechanism(s) of action may allow the translation of basic knowledge of MSCs biology into the design of new clinical therapies. Ongoing and future clinical trials with MSCs will provide a rich source of bedside information that can be extended in the laboratory to further advance this important field of research.
We thank Ren-ke Li and Hu-lun Li for assistance with the experimental design and guidance, Wei Liu and Zhen Liu of Harbin Medical University Research Laboratory Center for technical assistance, and Jing-bo Li for the assistance with the FACS analysis.
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26(10) : 2531 –2541 .18635870 | 23383203 | PMC3559548 | CC BY | 2021-01-05 17:12:00 | yes | PLoS One. 2013 Jan 30; 8(1):e55487 |
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BMC Complement Altern MedBMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central 1472-6882-13-102330511410.1186/1472-6882-13-10Research ArticleImproved glycemic control, pancreas protective and hepatoprotective effect by traditional poly-herbal formulation “Qurs Tabasheer” in streptozotocin induced diabetic rats Ahmed Danish [email protected] Manju [email protected] Alok [email protected] Pramod W [email protected] Vikas [email protected] Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS)-Deemed University, Allahabad, Uttar Pradesh, India2 Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India3 United Institute of Pharmacy, UCER, Allahabad, Naini, India4 Department of Biological Sciences, Sam Higginbottom Institute of Agriculture, Technology & Sciences (SHIATS)-Deemed University, Allahabad, Uttar Pradesh, India2013 10 1 2013 13 10 10 8 8 2012 8 1 2013 Copyright ©2013 Ahmed et al.; licensee BioMed Central Ltd.2013Ahmed et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The present study was undertaken to evaluate the antihyperglycemic, antihyperlipidemic and hepatoprotective effect of a traditional unani formulation “Qurs Tabasheer” in streptozotocin (STZ) induced diabetic wistar rats. Up till now no study was undertaken to appraise the efficacy of “Qurs Tabasheer” in the diabetic rats. Qurs Tabasheer is a unani formulation restraining preparations from five various herbs namely Tukhme Khurfa (Portulaca oleracea seed), Gule Surkh (Rosa damascena flower), Gulnar (Punica granatum flower), Tabasheer (Bambusa arundinasia dried exudate on node), Tukhme Kahu (Lactuca sativa Linn seed).
Methods
Effect of Qurs Tabasheer was assessed in STZ (60 mg/kg, i.p single shot) induced diabetic wistar rats. STZ produced a marked increase in the serum glucose, Total Cholesterol, LDL cholesterol, VLDL Cholesterol, Triglycerides and trim down the HDL level. We have weighed up the effect of Qurs Tabasheer on hepatic activity through estimating levels of various liver enzymes viz. Hexokinase, Glucose-6-Phosphatase and Fructose-1-6-biphosphatase in STZ diabetic wistar rats.
Results
In STZ-induced diabetic wistar rats level of Hexokinase, and Glucose-6-Phosphatase was decreased to a significant level while the level of fructose-1-6-biphophatase was augmented. Therapy with Qurs Tabasheer for 28 days to STZ-induced diabetic rats significantly reduces the level of serum glucose, total cholesterol, triglycerides, glucose-6-phosphatase and fructose-1-6-biphosphatase, while magnitude of HDL cholesterol and hexokinase was amplified.
Conclusion
Antihyperglycemic, antihyperlipidemic activity of Qurs Tabasheer extract in STZ- induced wistar rats was found to be more effective than standard oral hypoglycemic drug Glimepiride.
Diabetes mellitusHepatoprotectiveHyperlipidemiaPolyherbalQurs TabasheerUnani formulation
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Background
Diabetes mellitus is rapidly reaching epidemic proportions in many areas of the world. According to WHO an estimated 80 million people in India will suffer from diabetes by the year 2030 [1]. The purported Indian Phenotype proposed to have inimitable biochemical as well as clinical idiosyncrasy in the Indians of Asia. This assemblage of abnormalities is well thought-out to be one of the foremost factors contributing to raise pervasiveness of type 2 diabetes in Indians of Asia.
Diabetes mellitus is linked with prejudice glucose metabolism that escorts to a rise in free radical production and augmentation in the lipoprotein and triglyceride levels. Experimental diabetes in animals has endowed with extensive approach into the physiologic and biochemical clutter of the diabetic state. Many of the disorder have been characterized in hyperglycemic animals. Significant changes in lipid metabolism also crop up in diabetes [2]. Deregulation of hepatic enzymes such as hexokinase, glucose-6-phosphatase, fructose-1-6-biphosphatase occurs in diabetic rats [3,4].
Alternative and traditional medicines have scores of advantages over the conventional medicines. Despite many conventional therapies are present in the market to curtail the diabetes and its complications, traditional medicines such as Unani formulations has unambiguous advantage of being almost free from adverse effects. Diversity, flexibility, easy accessibility, broad continuing acceptance in developing countries and increasing popularity in developed countries, relative low cost, low levels of technological input, relative low side effects and growing economic importance are some of the positive features of traditional medicine (WHO 2002).
Polyherbal formulations more willingly than monotherapeutic herbal formulation are frequently used because of the synergistic effect. Many polyherbal formulation such as Okudiabet [5] Diashis [6], Diasulin [7] etc. have revealed their efficacy and potency against diabetes.
Qurs Tabasheer is composed of 5 (five) medicinal plants (Table 1). Till now no research has been reported on Qurs Tabasheer’s hypoglycemic, antihyperlipidemic and hepatoprotective activity on STZ- induced diabetic rats. The present exploration was undertaken to study the effect of Qurs Tabasheer, a polyherbal unani formulation on alterations in plasma glucose, glycated heamoglobin (A1c), total cholesterol, triglycerides, hexokinase, glucose-6-phosphatase, fructose-1-6-biphosphatase along with weight variation in STZ-induced diabetic wistar rats. The results obtained from Qurs Tabasheer were weighed against standard drug Glimepiride.
Table 1 Qurs Tabasheer (Composition & concentration)
S.No. Botanical name Hindi name (common name) Family Part used Composition* (%)
1 Portulaca oleracea Tukhme Khurfa Portulacaceae Seed 10 ≈ (500 mg/kg) [8]
2 Rosa damascena Gule Surkh Rosaceae Flower 10 ≈ (500 mg/kg) [9]
3 Punica granatum Gulnar Lythraceae Flower 10 ≈ (500 mg/kg) [10]
4 Bambusa arundinacea Tabasheer Poaceae Dried exudate on node) 50 ≈ (500 mg/kg) [11]
5 Lactuca sativa Linn Tukhme Kahu Asteraceae Seed 10 ≈ (500 mg/kg) [12]
*Stock sample used in the experiment.
Criteria for selection of herbs
In order to support and to select the preeminent composition/ratio of the five herbs: Tukhme Khurfa (Portulaca oleracea seed), Gule Surkh (Rosa damascena flower), Gulnar (Punica granatum flower), Tabasheer (Bambusa arundinasia dried exudate on node), Tukhme Kahu (Lactuca sativa Linn seed utilized to prepare Qurs Tabasheer, a polyherbal formulation, we have executed the in-vitro antidiabetic assays with the various selected doses of the polyherbal formulation:
α – amylase inhibition assay
The α-amylase inhibition assay was carried out according to the procedure reported by Subashini Devarajan et al. [13]. Each test tube containing 500 μL of concentrations of Portulaca Oleracea (100 mg.kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Rosa damascena(100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Punica granatum (100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Bambusa arundinacea (100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1) and Lactuca sativa Linn(100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-, 400 mg kg-1 L-1,500 mg kg-1 L-1of methanol) of Polyherbal formulation and 500 μL of 0.02 mol.L-1 sodium phosphate buffer (pH 6.9 with 0.006 mol.L-1 NaCl) containing α- amylase solution (0.5 mg. mL-1) were incubated for 10 min at 25°C. After pre-incubation, 500 μL of 1% starch solution in 0.02 mol.L-1 sodium phosphate buffer (pH 6.9 with 0.006 mol.L-1 NaCl) was added to each tube. The reaction mixtures were then incubated at 25°C for 10 min. The reaction was stopped with 1.0 mL of dinitrosalicylic acid color reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 10 mL of distilled water and absorbance was measured at 540 nm; α-amylase inhibition assay was calculated using the formula:
(1) %=A540control−A540extract/A540control×100
Natural α -amylase inhibitors from herbal sources offer an attractive therapeutic approach to the treatment of postprandial hyperglycemia by decreasing glucose release from starch and have potential for the treatment of diabetes mellitus and obesity [14,15]. The inhibitory activity of methanolic extract of various ingredients of Qurs Tabasheer against pancreatic amylase is shown in Table 2. As evident from the results shown in Table 2, the maximum inhibition of α – amylase has been achieved at 500 mg/kg L-1 of each constituent. Concentration dependent inhibitory activity of α – amylase was observed at 100, 200, 300, 400, 500 mg/kg L-1. As a result, we have chosen the unsurpassed concentration according to the propensity to inhibit α –amylase. Results are expressed as the percentage sample absorbance decrease relative to the absorbance of control solution in the absence of extract ingredients at 540 nm.
Table 2 α – amylase inhibition of methanolic extracts of various ingredients of Qurs Tabasheer (±SE, n = 3)
S.No. Qurs Tabasheer (Polyherbal formulation) ingredients and α – amylase inhibition activity (%)
Portulaca oleracea c/(mg.kg L-1) α – amylase inhibition activity (%) Rosa damascena c/(mg.kg L-1) α – amylase inhibition activity (%) Punica granatum c/(mg.kg L-1) α – amylase inhibition activity (%) Bambusa arundinacea c/(mg.kg L-1) α – amylase inhibition activity (%) Lactuca sativa Linn c/(mg.kg L-1) α – amylase inhibition activity (%)
1 100 37.41 ± 0.58 100 28.81 ± 0.61 100 34.82 ± 0.28 100 38.09 ± 0.72 100 32.81 ± 0.96
2 200 41.82 ± 1.83 200 44.76 ±0.92 200 40.03 ±1.61 200 45.18 ±0.51 200 49.58 ±0.59
3 300 68.38 ±2.81 300 52.92 ±1.29 300 60.18 ±0.69 300 68.71 ±1.59 300 66.75 ±1.61
4 400 71.19 ±1.97 400 67.71 ±1.58 400 69.71 ±0.21 400 74.49 ±2.57 400 76.07 ±1.97
5 500 79.09 ±0.82 500 72.82 ± 1.09 500 76.70 ±1.08 500 93.59 ±1.88 500 81.10 ±0.48
α – glucosidase inhibition assay
α – glucosidase inhibition assay was performed according to Dong et al. [16]. The inhibitory activity was determined by incubating a volume of 60 μL of each of sample solution containing Portulaca Oleracea (100 mg.kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Rosa damascena(100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Punica granatum (100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1), Bambusa arundinacea (100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-1, 400 mg kg-1 L-1,500 mg kg-1 L-1) and Lactuca sativa Linn(100 mg kg-1 L-1, 200 mg kg-1 L-1, 300 mg kg-1 L-, 400 mg kg-1 L-1,500 mg kg-1 L-1of methanol and 50 μL of 0.1 M phosphate buffer (pH 6.8) containing α –glucosidase solution (0.2 U/ml) was incubated in 96 well plates at 37°C for 10 min. After pre-incubation, 50 μL of 5 mM p-nitrophenyl-a-D-glucopyranoside (PNPG) solution in 0.1 M phosphate buffer (pH 6.8) was added to each well and incubated at 37°C for another 20 min. Then the reaction was stopped by adding 160 μL of 0.2 M NaCO3 into each well, and absorbance readings (A) were recorded at 405 nm. α –glucosidase inhibitory activity was expressed as inhibition % and was calculated as follows:
(2) Inhibition%=Acontrol−Asample/Acontrolx100
Therefore, as it is evident from the above Table 3 exhibiting the α – amylase and α – glucosidase inhibition. The maximum percentage inhibition in both the cases was achieved by the extract of Bambusa arundinacea ingredient of Qurs Tabasheer. For this reason, we have selected the 50% composition of Bambusa arundinacea as compared to the others in order to maximize the glycemic control in STZ-diabetic rats.
Table 3 α – glucosidase inhibition of methanolic extracts of various ingredients of Qurs Tabasheer (±SE, n = 3)
S.No. Qurs Tabasheer (Polyherbal formulation) ingredients and α – glucosidase inhibition activity (%)
Portulaca oleracea c/(mg.kg L-1) α – glucosidase inhibition activity (%) Rosa damascena c/(mg.kg L-1) α – glucosidase inhibition activity (%) Punica granatum c/(mg.kg L-1) α – glucosidase inhibition activity (%) Bambusa arundinacea c/(mg.kg L-1) α – glucosidase inhibition activity (%) Lactuca sativa Linn c/(mg.kg L-1) α – glucosidase inhibition activity (%)
1 100 15 ± 1.61 100 17 ± 0.85 100 12 ± 1.66 100 19 ± 1.72 100 14 ± 2.98
2 200 26 ± 0.51 200 24 ±1.42 200 29 ±0.96 200 21 ±1.61 200 19 ±0.77
3 300 38 ±1.84 300 31 ±2.66 300 34 ±1.07 300 29 ±1.53 300 26 ±1.90
4 400 51 ±2.77 400 50 ±0.87 400 56 ±1.95 400 48 ±1.43 400 41 ±0.18
5 500 67 ±0.19 500 61 ± 1.82 500 60 ±0.17 500 71 ±0.60 500 56 ±1.07
Accelerated stability testing of qurs tabasheer
Herbal preparations are thought to be degraded if stored for a longer period of time. Therefore, we have performed accelerated stability testing of the Qurs Tabasheer, a polyherbal formulation. To establish the stability of the Qurs Tabasheer, we have prepared five samples of polyherbal formulation and the parameters like pH, Viscosity, Refractive Index (R.I), Surface tension, Specific gravity and microbiological load was assessed at an interval of 0, 24, 48, 72, 96 and 120 Hrs, maintaining the packs of formulations at 30 ± 2°C and at 65% Relative humidity [17].
1. Determination of pH: The pH of Qurs Tabasheer at an interval of 0, 24, 48, 72, 96 and 120 Hrs was determined using pH meter (Orion digital pH meter).
2. Determination of Viscosity: Ostwald viscometer (Sigma Aldrich, M.O. USA) was used to determine the viscosity of all the samples of Qurs Tabasheer at an interval of 0, 24, 48, 72, 96 and 120 Hrs.
3. Determination of Refractive Index (R.I): Abee’s refractometer (Cole-Parmer, India) was used to determine the refractive index of the formulation at an interval of 0, 24, 48, 72, 96 and 120 Hrs. as per the procedure.
4. Determination of Surface Tension: The samples of Qurs Tabasheer were assessed by Stalagmometer (Kocour, US) at an interval of 0, 24, 48, 72, 96 and 120 Hrs.
5. Determination of Specific Gravity: All the samples of Qurs Tabasheer were determined by using Pycnometer (Chemkind, India) at an interval of 0, 24, 48, 72, 96 and 120 Hrs.
6. Microbiological Load: Bioburden level [18,19] The basis of Bioburden level is the determination of microbial contamination limits in medicinal plant materials. It indicates the quality of an herbal formulation. The total viable aerobic count of the polyherbal formulation being examined by utilizing plate count method. Polyherbal formulation, Qurs Tabasheer after treatment with sodium chloride-peptone buffer solution (pH = 7.0) was inoculated on liquefied casein-soybean digest agar. The samples were incubated at 30-35°C at an interval of 0, 24, 48, 72, 96 and 120 Hrs. The numbers of colonies formed were counted after the specified time interval.
It is apparent from the Table 4 that accelerated stability data follows a linear pattern throughout the stability testing. Physical parameters such as color, odor etc. does not produce significant changes. Furthermore, the harmful microorganism were absent throughout the accelerated stability studies. The above stability studies indicate that Qurs Tabasheer is stable at room temperature for quite a longer period of time. However, real time stability studies are underway to confirm these findings.
Table 4 Accelerated stability data of Polyherbal formulation, Qurs Tabasheer
S. No. Parameters Observations and time interval
Times (hrs) (0) Times (hrs) (24) Times (hrs) (48) Times (hrs) (72) Times (hrs) (96) Times (hrs) (120) Mean ± SD
1 Colour Greenish Greenish Greenish Greenish Greenish Greenish
2 Odor Pleasant Pleasant Pleasant Pleasant Pleasant Pleasant
3 External Appearance Clear Liquid Clear Liquid Clear Liquid Clear Liquid Clear Liquid Clear Liquid
4 pH 4.1 4.1 4.2 4.2 4.3 4.2 4.18 ± 0.72
5 Viscosity 1.02 1.04 1.02 1.07 1.05 1.03 1.038 ± 1.07
6 Surface Tension 110.26 112.71 112.09 114.83 111.05 113.29 112.37 ± 0.28
7 Specific Gravity 1.51 1.49 1.50 1.43 1.58 1.54 1.508 ± 1.70
8 Refractive Index (RI) 1.429 1.448 1.584 1.461 1.502 1.490 1.485 ± 0.69
9 Microbiological Load (Bioburden level)
9.1 Total Aerobic plate count 4900 CFU/g 4891 CFU/g 4740 CFU/g 4684 CFU/g 4591 CFU/g 4410 CFU/g 4702 ± 2.05
9.2 E.coli Absent Absent Absent Absent Absent Absent
9.3 Salmonella Absent Absent Absent Absent Absent Absent
9.4 S. aureus Absent Absent Absent Absent Absent Absent
9.5 Klebsiella Absent Absent Absent Absent Absent Absent
9.6 Clostridium botulinum Absent Absent Absent Absent Absent Absent
CFU = Colony Forming Unit.
Methods
Preparation of qurs tabasheer extract
The five medicinal plants stated above were obtained from different sources viz. Bio India Biologicals (BIB) Corporation, Hyderabad, India, Green Earth Products Pvt. Ltd. New Delhi, India, & Raj Hans Products, Mumbai, India. The plants were confirmed by experts from Department of Botany, Sam Higginbottom Institute of Agriculture, Technology & Sciences. The preferred parts of the five medicinal plants were kept and dried in an incubator for about 24 hours at 37°C. The dried parts were then crushed and minced in the ratio specified in Table 1. This polyherbal formulation was prepared according to the procedure specified by Pandy et al. [20].
Reagents and chemicals
Streptozotocin solution was prepared by dissolution in 0.1 M citrate buffer (pH = 4.5).
Streptozotocin (STZ) was procured from Sisco Research Laboratory, Pvt. Ltd. Mumbai, India. Glimepiride was generous gift from Ranbaxy Laboratories, Gurgaon, India. Chemical including ethyl alcohol, trichloro acetic acid, diethyl ether, and citric acid was purchased from CDH, Mumabi, India. All other chemicals and bioassay kits were purchased from Sigma Chemical Company Inc. (St. Louis, MO, USA) and Span Diagnostics, Surat, India.
Animals
Male Wistar rats, weighing between 190-230 g, were selected. All animals were provided with standard pellets and drinking water ad libitum. All experiments and protocols described in the current study are in accordance with guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). This study has been duly approved by the IAEC (Institutional Animal Ethical Committee, Jamia Hamdard) and CPCSEA. Water used for the solution preparation and glassware washing was passed through an Easy Pure UF water purification unit (Thermolyne Barnstead, NH, USA).
Induction of diabetes
Wistar rats were injected intraperitoneally with STZ dissolved in 0.1 M citrate buffer (pH = 6.5) at 60 mg/kg. Animals of control group were received equal volume of vehicle. After 48 hours of STZ injection, blood glucose of the induced rats was estimated. The rats depicting FBG ≥ 230 mg/dL considered to be diabetic.
Statistical analysis
Data was put across as the mean ± SEM. For statistical analysis of the data, group means were compared by one-way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test, which was used to identify difference between groups. P value <0.05 was considered significant.
Experimental design
In our experiment, rats were randomized into six groups comprising of five animals each group as discussed below:
Group I. Normal control rats received citrate buffer (pH = 4.5) for 28 days. (1 mL/kg p.o.)
Group II. Normal control rats received Qurs Tabasheer (200 mg/kg p.o.) and continued for 28 days
Group III. STZ-diabetic rats received STZ (intraperitoneally, 60 mg/kg, single shot)
Group IV. Qurs Tabasheer treated diabetic rats received Qurs Tabasheer (50 mg/kg p.o.) and continued for 28 days.
Group V. Qurs Tabasheer treated diabetic rat received Qurs Tabasheer (100 mg/kg p.o) and continued for 28 days.
Group VI. Qurs Tabasheer treated diabetic rat received Qurs Tabasheer (200 mg/kg p.o) and continued for 28 days.
Group VII. Glimepiride treated diabetic rats received Glimepiride (1 mg/kg p.o.) and continued for 28 days.
Drug was given to the rats with the help of oral catheter every morning. At the finish of the drug treatment all the animals was faster overnight but allow free access to water. Rats were divided into the above seven groups for 28 days of study. The duration of drug treatment was set to be 28 days for the reason that 28 days were the threshold in our pilot experiments.
Results
To evaluate the effect of Qurs Tabasheer on STZ-induced diabetes mellitus rats, several biochemical estimations were carried out in all groups of experimentally induced diabetes rats for the estimation of plasma glucose, serum cholesterol, serum triglycerides, glycated heamoglobin (A1c), hexokinase, glucose-6-phosphatase and fructose-1-6-biphophatase (Table 5). The following pharmacological effects were observed:
Table 5 Biochemical parameters at the end of study
S.No Biochemical parameter Normal control Normal control + Qurs Tabasheer (200 mg/kg) STZ-diabetic control STZ-diabetic + Qurs Tabasheer (50 mg/kg) STZ-diabetic + Qurs Tabasheer (100 mg/kg) STZ-diabetic + Qurs Tabasheer (200 mg/kg) STZ-diabetic + Glimepiride
1. Fasting plasma glucose (mg/dL) 84.64 ± 3.634 78.64 ± 3.091 301.1 ± 5.345 194.2 ± 2.873* 133.8 ± 4.149* 88.52 ± 3.923*** 101.1 ± 4.106
2 Fasting Plasma Insulin (μU/mL) 11.22 ± 0.2080 11.80 ± 0.3041 2.708 ± 0.2008 4.866 ± 0.3105 6.890 ± 0.1796* 9.674 ± 0.2214** 7.430 ± 0.2577
3. Glycated Heamoglobin (A1c) (%) 1.594 ± 0.07737 1.600 ± 0.08961 3.444 ± 0.2352 1.718 ± 0.09896 1.874 ± 0.09239** 2.594 ± 0.2068*** 1.878 ± 0.04271
4. Total Cholesterol (mg/dl) 77.98 ± 4.946 85.60 ± 3.832 166.8 ± 3.133 152.6 ± 3.320 133.9 ± 3.762* 118.9 ± 5.337** 164.2 ± 5.620
5. Triglycerides (mg/dl) 82.52 ± 5.211 77.54 ± 2.119 124.3 ± 3.229 118.9 ± 3.214 102.0 ± 1.360** 100.9 ± 3.313** 129.0 ± 3.316
6. Hexokinase (μg/mg of tissue) 148.4 ± 1.606 142.5 ± 1.888 102.7 ± 1.732 107.3 ± 1.875 128.2 ± 3.487** 137.6 ± 3.432*** 121.2 ± 1.511
7. Glucose-6-Phosphatase (unit/mg of tissue) 10.27 ± 0.1574 10.22 ± 0.3006 15.79 ± 0.6483 14.45 ± 0.5288 12.99 ± 0.5063* 10.06 ± 0.2851*** 15.08 ± 0.5064
8. Fructose-1-6-biphosphatase (unit/mg of tissue) 30.30 ± 0.7938 30.04 ± 0.8185 51.19 ± 1.223 48.20 ± 1.272 38.19 ± 1.389* 34.67 ± 1.700** 41.02 ± 1.236
9. Weight Variation (g) 201.8 ± 4.664 208.0 ± 4.713 134.5 ± 3.681 137.2 ± 3.374 144.9 ± 4.532* 150.8 ± 2.453** 155.3 ± 2.409
The data are expressed in mean ± SEM) (n = number of animals in each group = 5). The comparisons were made by ANOVA followed by Dunnett’s test.
ns-non-significant; STZ-streptozotocin.
*P < 0.05 is considered as significant.
**P < 0.01 is considered as very significant.
***P < 0.001 is considered as extremely significant.
Effect on glycemic control
The mean blood glucose level in rats fed on normal diet (normal control wistar rats, group I) was almost invariable throughout the experimental study. In unison, the blood glucose level of normal control rats treated with Qurs Tabasheer kept on normal diet (group II) was close to the normal control rats. On the contrary, the blood glucose level of STZ- treated wistar rats (STZ-diabetic control) was increased to a significant level (P < 0.01). When STZ-induced diabetic rats (FBG ≥ 230 mg/dL) was treated with Qurs Tabasheer with dose of 200 mg/kg (group VI), lowering in blood glucose was observed to maximum as compared to the dose of 50 mg/kg p.o (group IV),100 mg/kg p.o (group V), 200 mg/kg p.o and standard drug Glimpepiride (1 mg/kg p.o) respectively (Figure 1).
Figure 1 Effect of Qurs Tabasheer on glycemic control at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of plasma insulin
Plasma insulin levels of STZ-induced diabetic rats were significantly lowered as compared to the normal control (group I) and Qurs Tabasheer treated normal control (group II) rats. Qurs Tabasheer boosts the level of plasma insulin in dose dependent manner and exhibited the maximum threshold at a dose of 200 mg/kg p.o (for 28 days) when compared to the other doses of 50, 100 mg/kg p.o of Qurs Tabasheer and 1 mg/kg p.o of Glimepiride (Figure 2).
Figure 2 Effect of Qurs Tabasheer on level of plasma insulin at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of glycated heamoglobin (A1c)
Glycated heamoglobin (A1c) of STZ-induced treated diabetic rats was increased to a momentous level. Level of A1c was normal in the wistar rats fed with normal diet (group I) in conjunction with the normal control rats received Qurs Tabasheer with dose of 200 mg/kg (group II). When STZ-induced diabetic rats were treated with Qurs Tabasheer with dose viz. (200 mg/kg), level of glycated heamoglobin (A1c) was significantly reduced, compared to the groups received 50 mg/kg p.o (group IV), 100 mg/kg p.o (group V) 200 mg/kg p.o of Qurs Tabasheer and 1 mg/kg p.o of Glimepiride correspondingly (Figure 3).
Figure 3 Effect of Qurs Tabasheer on glycated heamoglobin (A1c) (%) at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of total cholesterol
It is perceptible from figure 3 that serum cholesterol levels of untreated diabetic rats was significantly higher than those in normal rats (group I) as well as in normal control rats receiving Qurs Tabasheer (group II). Upon administration of unani herbal formulation Qurs Tabasheer (50 mg/kg p.o, 100 mg/kg p.o and 200 mg/kg p.o for 28 days, group IV, V & VI) in the STZ-induced diabetic rats the level of serum cholesterol lowered to a considerable level with maximum effect seen in the group administered with 200 mg/kg of Qurs Tabasheer. While the group received only Glimepiride (1 mg/kg p.o for 28 days) (group VII) shows no significant changes in the serum cholesterol (Figure 4).
Figure 4 Effect of Qurs Tabasheer on total cholesterol at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of serum triglycerides
The administration of Qurs Tabasheer in normal control rats shows a slight decrease in the serum triglyceride level. On contrary, level of serum triglycerides significantly increased in STZ-induced diabetic rats (group III). Upon administration of different doses of Qurs Tabasheer (50 mg/kg, 100 mg/kg & 200 mg/kg) the level of serum triglycerides subordinate to a good extent. The maximum lowering of serum triglycerides was appeared in group received Qurs Tabasheer at a dose of 200 mg/kg (Figure 5).
Figure 5 Effect of Qurs Tabasheer on serum triglycerides at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of hexokinase
To evaluate the effect of Qurs Tabasheer on distressed hepatic activity, we administered Qurs Tabasheer to normal as well as in STZ-induced diabetic rats. Hexokinase level decreased in a considerable in STZ-treated diabetic rats. Administration of Qurs Tabasheer in normal rats shows little or no significant changes in the level of hepatic hexokinase. STZ-induced diabetic rats received Qurs Tabasheer shows exponential increase in the level of hepatic hexokinase (Figure 5). Diabetic rats treated with Qurs Tabasheer with a dose of 200 mg/kg p.o (for 28 days) showed maximum augmentation in the level of hexokinase as compared to other groups received different doses of Qurs Tabasheer. While the Group received Glimepiride (1 mg/kg p.o) develop slight increase in the level of hepatic hexokinase (group VII) (Figure 6).
Figure 6 Effect of Qurs Tabasheer on level of hexokinase at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of glucose-6-phosphatase
It is evident from figure that upon administration of STZ to wistar rats the level of glucose-6-phosphatase was declined to a considerable level. Qurs Tabasheer when administered to normal control rats shows little or no changes in the levels of glucose-6-phosphatase. STZ-induced diabetic rats received Qurs Tabasheer with the dose of 200 mg/kg (group VI) shows remarkable increase in the level of glucose-6-phosphatase when weighed against the dose of 50 mg/kg p.o (group IV), 100 mg/kg p.o (group V) and 200 mg/kg p.o (group VI). STZ-induced diabetic rats’ administered with Glimepiride (1 mg/kg) shows a trivial boost in the level of glucose-6-phosphatase (Figure 7).
Figure 7 Effect of Qurs Tabasheer on level of glucose-6-phosphatase at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on the levels of fructose-1-6-biphosphatase
STZ-induced diabetic rats develop high levels of Fructose-1-6-biphosphatase. Upon administration of Qurs Tabasheer to normal control rats the level of Fructose-1-6-biphosphatase does not change much. When STZ-induced diabetic rats received Qurs Tabasheer, shows significant decrease in the level of Fructose-1-6-biphosphatase with the dose of 200 mg/kg (group VI). Effect of 50 mg/kg p.o (group IV) and 100 mg/kg p.o (group V) of Qurs Tabasheer was subordinate as compared to 200 mg/kg p.o (Figure 8).
Figure 8 Effect of Qurs Tabasheer on level of fructose-1-6-biphosphatase at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Effect on weight variation
Administration of Qurs Tabasheer demonstrates weight gain in STZ-induced diabetic rats. Weight of STZ-diabetic rats increases to a remarkable extent with dose of 200 mg/kg p.o of Qurst Tabasheer as compared to the other doses of 50, and 100 mg/kg p.o of Qurs Tabasheer and 1 mg/kg p.o of Glimpepiride (Figure 9).
Figure 9 Effect of Qurs Tabasheer on body weight at different concentrations on normal and STZ induced diabetic rats, compared to standard drug Glimepiride; values are mean ± SEM; n = 6; *P < 0.05; **P < 0.01; ***P < 0.001; P > 0.05 is considered as non-significant (ns).
Histopathological studies
Animals were sacrificed with administration of overdose of anesthetic ether. The liver and pancreas were immediately expurgated. Liver and whole pancreas were removed and washed with ice cold 0.9% sodium chloride solution. The tissues were conserved in buffered 10% neutral formalin and stored at −20°C until processed [21].
Histopathological studies on liver and pancreas of normal and STZ-diabetic rats shows relative more degree of hepatoprotection and retardation of pancreatic degradation with increasing dose of herbal medicine Qurs Tabasheer as compared to the standard oral hypoglycemic Glimepiride. The section of rat pancreas from normal control group exhibits normal pancreatic acini (Figure 10 PN). STZ diabetic rats demonstrates degenerative and lytic changes in the islet of langerhans of pancreas (Figure 10 PST). Normal control rats treated with herbal medicine Qurs Tabasheer showed no signs of degenerative changes in islet of langerhans and in contrast shows normal acini and islets of langerhans (Figure 10- PQ). Pancreatic sections of STZ diabetic rat treated with different doses of Qurs Tabasheer viz 50 mg/kg p.o., 100 mg/kg p.o and 200 mg/kg p.o (Figure 10-PQ50; Figure 10-PQ100; Figure 10-PQ200) showed a marked improvement in the morphology of islet of langerhans and acini of pancreas with greatest improvement being showed in the dose of 200 mg/kg p.o of Qurs Tabasheer as compared to the standard drug Glimepiride (Figure 10-PGL). While the section of rat liver of normal control group showed normal lobular pattern with a centrilobular vein and scorching irregular anastomosing plates of hepatocytes with intervening sinusoids (Figure 11-LN). Liver of normal rat treated with Qurs Tabasheer also shows normal hepatocytes and sinusoids (Figure 11-LQ). Sections of the diabetic rat liver cells shows accumulation of droplets with distorted morphology of hepatocytes, centrilobular vein and sinusoids (Figure 11-LS). The photomicrograph of STZ-diabetic rats treated with different doses of Qurs Tabasheer viz 50 mg/kg p.o., 100 mg/kg p.o and 200 mg/kg p.o (Figure 11-LQ 50; 11-LQ 100; 11-LQ-200) showed marked enhancement of morphology of liver hepatocytes with normal sinusoids with the greatest effect exhibited in the dose of 200 mg/kg/p.o (Figure 11-LQ200) when compared to the standard drug Glimepiride (Figure 11-LG).
Figure 10 PN = Photomicrograph of section of normal pancreas (150x), showing normal lobules of pancreatic acini. PQ = Photomicrograph of section of pancreas (150x) of normal rat administered with 200 mg/kg/p.o of Qurs Tabasheer, showing normal lobules and pancreatic acini. PST = Photomicrograph of section of pancreas of STZ treated diabetic Wistar rat, 150x, yellow arrows showing lobules of pancreatic acini with areas of fibrosis. PGL = Photomicrograph of section of pancreas of diabetic rat treated with Glimepiride alone for 28 days, (150x), yellow arrows showing mild fibrosis of pancreatic acini. PQ 50 = Section of pancreas of diabetic Wistar rat treated with Qurs Tabasheer (50 mg/kg p.o) for 28 days, (150x), yellow arrows showing mild fibrosis of pancreatic acini and normal islet of langerhans. PQ 100 = Photomicrograph of section of pancreas of diabetic Wistar rat treated with Qurs Tabasheer (100 mg/kg p.o) for 28 days (150x), yellow arrows showing very mild fibrosis of pancreatic acini. PQ 200 = Photomicrograph of section of pancreas of diabetic Wistar rat treated with with Qurs Tabasheer (200 mg/kg p.o) for 28 days (150x), yellow arrows showing normal pancreatic acini and islet of langerhans.
Figure 11 LN = Photomicrograph of section of liver of normal control rat (150x), yellow arrows showing lobular pattern with a centrilobular vein and scorching irregular anastomosing plates of hepatocytes with intervening sinusoids. LQ = Photomicrograph of section of liver of normal control rat received 200 mg/kg p.o of Qurs Tabasheer (150x), yellow arrows showing normal lobular pattern and hepatocytes. LS = Photomicrograph of section of liver of STZ-diabetic rat (150 x) yellow arrow demonstrate accumulation of droplets with distorted morphology of hepatocytes, centrilobular vein and sinusoids. LG = Photomicrograph of section of liver of STZ-diabetic rat (150 x) administered with 1 mg/kg p.o of Glimepiride, yellow arrow portrayed no signs of normal hepatocytes and normal lobular pattern. LQ 50 = Photomicrograph of section of liver of STZ-diabetic rat (150 x) administered with 50 mg/kg p.o of Qurs Tabasheer, yellow arrow exhibits docile hepatocytes and slightly distorted cetrilobular vein. LQ 100 = Photomicrograph of section of liver of STZ-diabetic rat (150 x) administered with 100 mg/kg p.o of Qurs Tabasheer, yellow arrow revealed slightly normal hepatocytes and sinusoids. LQ 200 = Photomicrograph of section of liver of STZ-diabetic rat (150 x) administered with 200 mg/kg p.o of Qurs Tabasheer, yellow arrow divulged the marked improvement in the distorted cetrilobular vein and hapatocytes.
Discussion
The cytotoxic action of Streptozotocin (STZ) is mediated by reactive oxygen species (ROS). Streptozotocin (STZ) penetrates the β-cells via glucose transporter (GLUT2) and causes alkylation of the DNA [14]. The alkylating activity of STZ is related to its nitrosourea motiety [22]. According to West et al. [23] Streptozotocin action in β-cells is being an adjunct to distinctive amendment in blood insulin and glucose concentrations. Two hours after STZ administration, hyperglycemia develops with concomitant plunge in insulin level. After six hours, hyperglycemia develops with high levels of insulin. Finally, severe hyperglycemia develops with decrease in insulin levels [23].
In the present research exertion, the administration of Qurs Tabasheer revealed the balanced decrease in the blood glucose, serum cholesterol, serum triglycerides, & fructose-1-6-biphosphatase while showed a significant decrease in body weight, hepatic hexokinase, & glucose-6-phosphatase (Table 5).
Many scientists have reported that Portulaca oleracea, Rosa damascene, Punica granatum, Bambusa arundinacea, and Lactuca sativa Linn. have noteworthy anti-hyperglycemic and glucose tolerance effect in the experimentally induced diabetic rats. The plausible mechanism of action of Qurs Tabasheer could be unswerving with the evocative effect of sulfonylureas which bolster the insulin secretion by closure of the K+ -ATPase channels, membrane depolarization and increase in Ca++ ions influx.
In this perspective, various medicinal plants of Qurs Tabasheer viz. Portulaca oleracea[8]Rosa damasceneI[9], Punica granatum[24], Bambusa arundinacea,[11]Lactuca sativa Linn[12] (ingredients of Qurs Tabasheer) have been pragmatic to show analogous effects. Body weight of Qurs-Tabasheer administered STZ-induced diabetic rats was significantly increased (Table 5, Figure 9). This effect may be due to the competence of Qurs Tabasheer to abridged hyperglycemia. Administration of Qurs Tabasheer to STZ-induced diabetic rats decreases the plasma glucose level (Table 5, Figure 1), perhaps due to the augmented quantity of insulin in diabetic rats. Additionally, Qurs Tabasheer might improve the utilization of glucose and crafts the adipose tissues more sensitive towards the insulin by enhancing the PPAR-γ dependent mRNA expression, to reduce the case of insulin resistance. In this framework, other researchers [25] have reported that Punica Granatum flower extract (one of the ingredients of Qurs Tabasheer) targets the PPAR-γ for plummeting insulin resistance. Li et al., [26] described that Punica Granatum flower (PGF) extract targets the PPAR-γ as one of the mechanism of targeting the type-II diabetes mellitus. It has been recently researched that PGF may thwart the decrease in glucose metabolism in diabetic cardio-myocytes by triggering the cardiac PPAR-γ [26].
Earlier researchers have observed that Portulaca oleracea extract showed marked decrease in the blood glucose level and increased insulin concentration in alloxan induced diabetic rats by closure of K+ ATP channels, membrane depolarization and stimulation of Ca++ influx[8]. Furthermore, many scientists have established the efficacy of Bambusa arundinacea to curtail the hyperglycemia. Bambusa arundinacea may inhibit the cohort of free radicals accountable for destruction of pancreatic β-cell [14] and may thus prevent the hyperglycemia in diabetic rats.
Gholamhoseinian et al. [9] investigated that extract of Rosa damascene flowers inhibits α –glucosidase (enzyme that is responsible for carbohydrate digestion and elevation of fasting blood glucose) in diabetic rats to facilitate the decrease in blood glucose levels.
Consequently, the antihyperglycemic effect of Qurs Tabasheer may be due to the synergistic effects of the Portulaca oleracea, Rosa damascene, Punica granatum, Bambusa arundinacea, and Lactuca sativa Linn. The plausible mechanism of action of the polyherbal formulation may either be due to the activation of PPAR-γ receptor or increased insulin secretion from pancreatic β-cells due to closure of K+ATP channels or may be attributable to free radical scavenging property to shield β- cell from destruction or perhaps as a consequence of inhibition of α – glucosidase enzyme in diabetic rats. As a result, it could be possible the mechanism of action of Qurs Tabasheer may be the amalgamation of all the probable mechanism described.
The enhanced level of glycated heamoglobin (A1c) in STZ-induced diabetic rats is primarily due to the excessive production of glucose in the blood which further reacts with blood heamoglobin to construct glycated heamoglobin [27]. Qurs Tabasheer lowers the glycated heamoglobin (A1c) in STZ-induced diabetic rats (Table 5, Figure 3). The plausible cause of reduced glycated heamoglobin is the diminution of blood glucose level.
In consequence, we have reported in our present research that Qurs Tabasheer also amends the imperative glucose metabolizing enzymes in liver (Table 5). Hepatic hexokinase is a prime enzyme that converts glucose into glucose-6-phosphate. Decreased level of hexokinase STZ-induced diabetic rats can be accountable for diminished glycolysis which results in decreased utilization of glucose for energy production [28]. The Qurs Tabasheer administered STZ-induced diabetic rats significantly amplify the level of hepatic hexokinase. (Table 5, Figure 6). Increased level of hepatic hexokinase cause increased glycolysis and consequently improves the utilization of glucose. Another vital enzyme of liver that regulates the glucose metabolizing enzyme is glucose-6-phosphatase. Other scientists depicted the enhanced activity of gluconeogenetic enzyme in diabetic states [29,30]. Diabetes increases the activity of glucose-6-phosphatase [31]. The increased activity of glucose-6-phosphatase was depicted in the STZ-induced diabetes mellitus rats (Table 5). Raised amount of Administration of glucose-6-phosphatase enhances the production of fats from carbohydrates [32]. Qurs Tabasheer significantly reduces the level of glucose-6-phosphatase (Figure 7). Activity of Fructose-1-6-biphosphate was considerably raised in STZ-induced diabetic rats (Table 5). Qurs Tabasheer lowers the activity of this gluconeogenetic enzyme to a considerable extent (Figure 8).
Plasma insulin levels in STZ-induced diabetic rats were diminished significantly (Table 5) Plasma insulin levels were found to be increased a substantial level in Qurs Tabasheer treated diabetic rats (Figure 2). This increase may be a corollary to the decreased level of the glucose-6-phosphatase and fructose-1-6-biphosphatase.
Earlier researches have demonstrated that in STZ-induced diabetic rats, insulin paucity is coupled with hypercholesterolemia and hypertriglyceridemia. As HMG Co-A reductase enzyme is accountable for the synthesis of cholesterol and insulin has an inhibitory effect on HMG-Co-A reductase. It is obvious that deficiency of insulin will improve the generation of cholesterol and triglycerides [33]. Administration of Qurs Tabasheer to STZ-induced diabetic rats decreased the level of total cholesterol and triglycerides (Table 5, Figures 4 &5). As the levels insulin has been increased in Qurs Tabasheer treated diabetic rats, which may be the outcome of decreased cholesterol and triglycerides level.
Conclusion
It is worth mentioning that Qurs Tabasheer efficiently trims down the levels of blood glucose, total cholesterol, triglycerides and gluconeogenetic enzymes without producing any adverse effect viz. hypoglycemia. The results from the present study and histological analysis indicate the administration of Qurs Tabasheer, has significantly protective effects against STZ-induced diabetic state. This significant protection of Qurs Tabasheer may be due to synergistic effect of the constituents of the drug. The antidiabetic effect of Qurs Tabasheer was more effectual than Glimepiride. These finding strengthen the observation that naturally occurring compounds of plant origin are much more effective in controlling diabetes than synthetic oral hypoglycemics. Further, biochemical and pharmacological investigations are in progress in our laboratory to explicate the mechanism of action of the Qurs Tabasheer.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DA premeditated and carried out the extraction of the Qurs Tabasheer. VK, PWR and AM carried out the biochemical estimations. MS analyses the statistical data and interpretation of histological analysis. All the authors are involved in the critical evaluation of the manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6882/13/10/prepub
Acknowledgement
The present research was supported by a grant from UGC (University Grants Commission). Authors are thankful to Prof. (Dr.) Mohd. Ali for his valuable phytochemical and pharmacognostical suggestions.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23393583PONE-D-12-2692210.1371/journal.pone.0055482Research ArticleBiologyGeneticsEpigeneticsHistone ModificationChromatinGene ExpressionMolecular Cell BiologySignal TransductionSignaling CascadesStress Signaling CascadePlant SciencePlant BiochemistryPlant GeneticsPlant PhysiologyIdentification of a Novel Jasmonate-Responsive Element in the AtJMT Promoter and Its Binding Protein for AtJMT Repression JA-Responsive Element and Trans-Acting FactorSeo Jun Sung
1
Koo Yeon Jong
1
Jung Choonkyun
1
Yeu Song Yion
1
Song Jong Tae
2
Kim Ju-Kon
3
Choi Yeonhee
4
Lee Jong Seob
4
Do Choi Yang
1
*
1
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea
2
School of Applied Biosciences, Kyungpook National University, Daegu, Korea
3
School of Biotechnology and Environmental Engineering, Myongji University, Yongin, Korea
4
School of Biological Sciences, Seoul National University, Seoul, Korea
Somers David Edward Editor
Ohio State University, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: JSS JTS YDC. Performed the experiments: JSS YJK SYY CJ. Wrote the paper: JKK YC JSL YDC.
2013 5 2 2013 8 2 e554824 9 2012 24 12 2012 © 2013 Seo et al2013Seo et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Jasmonates (JAs) are important regulators of plant biotic and abiotic stress responses and development. AtJMT in Arabidopsis thaliana and BcNTR1 in Brassica campestris encode jasmonic acid carboxyl methyltransferases, which catalyze methyl jasmonate (MeJA) biosynthesis and are involved in JA signaling. Their expression is induced by MeJA application. To understand its regulatory mechanism, here we define a novel JA-responsive cis-element (JARE), G(C)TCCTGA, in the AtJMT and BcNTR1 promoters, by promoter deletion analysis and Yeast 1-Hybrid (Y1H) assays; the JARE is distinct from other JA-responsive cis-elements previously reported. We also used Y1H screening to identify a trans-acting factor, AtBBD1, which binds to the JARE and interacts with AtJAZ1 and AtJAZ4. Knockout and overexpression analyses showed that AtBBD1 and its close homologue AtBBD2 are functionally redundant and act as negative regulators of AtJMT expression. However, AtBBD1 positively regulated the JA-responsive expression of JR2. Chromatin immunoprecipitation from knockout and overexpression plants revealed that repression of AtJMT is associated with reduced histone acetylation in the promoter region containing the JARE. These results show that AtBBD1 interacts with JAZ proteins, binds to the JARE and represses AtJMT expression.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (project nos. PJ008053 to YDC and PJ007971 to JKK), Rural Development Administration, Republic of Korea through the National Center for GM Crops. A graduate research assistantship to JSS from the Brain Korea 21 project of the MEST is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Jasmonic acid and its derivatives, collectively referred to as jasmonates (JAs), act as important regulators in plant biotic and abiotic stress responses [1], [2], [3]. JA also plays important roles in physiological and developmental processes, including root growth, senescence, trichome formation, cell cycle progression, and flower development [4], [5].
The molecular mechanisms by which JA regulates gene expression were illuminated by the discovery of jasmonate ZIM-domain proteins (JAZs) and the finding that the SCFCOI1 complex-mediated 26S proteasome degrades JAZs [6], [7]. In the absence of JA, JAZ proteins bind to transcription factors (TFs) and prevent their activity by recruiting the general co-repressor TOPLESS (TPL), through interaction with the adaptor protein Novel Interactor of JAZ (NINJA) [8], or by directly recruiting histone-modifying proteins, such as histone deacetylases (HDACs) [9], [10]. In the presence of the signal, JA is converted into jasmonoyl-isoleucine (JA-Ile) by JAR1 in Arabidopsis
[11], [12]. JA-Ile then promotes the interaction between JAZ proteins and the F-box protein Coronatine insensitive1 (COI1), in the SCF complex, resulting in proteolytic degradation of JAZ proteins by the 26S proteasome [6], [7]. Degradation of JAZ proteins liberates TFs from NINJA, TPL or HDACs, and initiates transcriptional reprogramming in response to JA [13], [14].
Recent reports on JA-responsive TFs have further improved our understanding of JA-responsive regulatory mechanisms. AtMYC2, a bHLH TF, is a primary target in the JA signaling pathway and interacts with some members of the AtJAZ family to regulate various JA-responsive target genes [15], [16], [17]. Other TFs also have been shown to interact with specific JAZ proteins [18], [19], [20]. It has been speculated that the specific interactions between TFs and JAZs could be largely responsible for the specificity and diversity of JA responses to different stimuli [21], [22].
These TFs bind to specific promoter elements of downstream genes and propagate JA signaling. One well-defined JA-responsive element, which is bound by MYC2, is the G-box (CACGTG) or G-box like motif (core ACGT) [17]. The G-box has been found in the promoters of many JA-responsive genes, such as VSP1 in Arabidopsis
[23], PIN2 in potato [24], VSPB in soybean [25], and ORCA3 in Catharanthus
[26]. Another JA-responsive element is the GCC-motif in PDF1.2 in Arabidopsis
[27], PMT in tobacco [28] and STR in Catharanthus
[29]. Other JA-responsive sequence motifs have also been reported [30], [31]. Transcriptome shifts of gene clusters responding to hormonal signals closely corresponded with the set of cis-elements in the genes’ promoters [32]. Some elements are involved in signal transduction in response to a specific hormone; others respond to two or more hormonal signals [33]. Therefore JA-responsive cis-elements are key to understanding both JA-specific signal transduction and inter-hormonal cross-talk.
Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play key roles in regulating gene expression through histone modification. The addition of acetyl groups to conserved lysine residues neutralizes the positive charge of histone tails and decreases their affinity for DNA [34], [35]. Hypoacetylation mediated by HDACs has the opposite effect on chromatin, enabling the histones to bind more tightly to the negatively-charged DNA, and is associated with the repression of gene expression [36], [37]. HATs and HDACs interact with co-activator and co-repressor complexes, respectively, to regulate expression of target genes [38], [39]. There are reports that transcription levels of some JA-responsive genes are altered in Arabidopsis HDAC6 or HDAC19 knockout mutants and overexpression plants [40], [41].
One key aspect of JA signaling is feedback regulation of JA synthesis. In Arabidopsis, the expression of AtJMT, which encodes a jasmonic acid carboxyl methyltransferase responsible for MeJA formation, is developmentally regulated and induced upon wounding or JA application [42]. BcNTR1 encodes the orthologous JA carboxyl methyltransferase in Brassica campestris
[43] and its expression pattern is similar to the pattern of AtJMT expression. In this study, we identified a novel JA-responsive cis-element (JARE) in the AtJMT and BcNTR1 promoters and isolated a trans-acting factor, AtBBD1, which binds to the JARE and interacts with AtJAZ1 and AtJAZ4. We also showed that AtBBD1 regulates transcription of AtJMT and another JA-regulated gene.
Materials and Methods
Plant Materials and Treatments
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild type for all experiments. The Brassica campestris variety and source were described in Song et al. (2000) [44]. Plants were grown on soil or one-half-strength Murashige and Skoog agar medium (Duchefa) in a growth chamber maintained at 22°C and 60% relative humidity under long-day conditions (16-h-light/8-h-dark cycle). Arabidopsis was transformed with Agrobacterium tumefaciens (strain C58C1) using the floral dip method [45]. A construct list of transgenic plants used in this study is provided in Supplemental Table 1 online. Transformed lines (T1 generation) were selected on MS plates containing kanamycin (30 µg/ml) or hygromycin (20 µg/ml). At least 40 independent T1 plants per genotype were tested for GUS expression in response to JA. We identified homozygous lines by testing T3 progeny for resistance to antibiotics. The basal level and MeJA responsive induction of reporter gene were variable among transformants. A line showing medium level of expression was selected from each construct by RT-PCR analysis. Several lines showing extremely high or low level of basal expression were excluded. For chemical treatment, solutions of 100 µM MeJA (Aldrich), 100 µM (±)-JA (Duchefa), 100 µM (±)-ABA (Duchefa), 50 µM SA (Sigma), or 5 mM ethephon were applied to soil-grown 4-week-old plants by spraying.
Y1H and Y2H Assays
The yeast one-hybrid screening was performed using MATCHMAKER One-Hybrid Library Construction and Screening Kit (Clontech). To isolate JARE-binding proteins, a cDNA library was prepared by RT-PCR from MeJA-treated seedlings of Arabidopsis Col-0 into pGADT7-Rec2. Bait DNA (−3518 to −3390 bp) containing JARE was cloned into the pHIS2 reporter vector. Positive clones were identified by nucleotide sequencing with AD sequencing primers. To identify the AtBBD1 binding sequence, various promoter fragments were cloned into the pHIS2 vector. The full length CDS or specific domains of AtBBD1 were cloned into pGADT7-Rec2.
Y2H assays were carried out using the MATCHMAKER Two-Hybrid System (Clontech). Full-length cDNAs for 12 AtJAZ genes were amplified by RT-PCR from 14-day-old seedlings of Arabidopsis Col-0 (Table S1). Each gene was cloned into the Y2H prey vector, pGADT7, to get the prey gene construct. The full-length coding region of AtBBD1 was amplified by RT-PCR and cloned into the Y2H bait vector, pGBKT7. All constructs used in Y1H and Y2H are shown in Table S1.
Electrophoretic Mobility Shift Assay
Full length CDS or DNA binding domain (a.a. residue 257 to 325) of AtBBD1 were fused in frame with the maltose-binding protein (MBP) at the C-terminus and expressed in Escherichia coli. A soluble crude extract of recombinant protein was used for EMSA. DNA fragments labeled with [γ-32P]dCTP were incubated with MBP-AtBBD1 or MBP-AtBBD1DB in the binding buffer [20 mM HEPES, pH 7.9, 50 mM KCl, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 5 mM MgCl2, 0.01% Triton X-100, and 100 ng poly(dI-dC)] for 1 hour. For competition analysis, unlabeled DNA fragments were included in the binding reactions as competitors in 10-fold molar excess relative to the labeled probes in each step. The reaction mixture was analyzed by 10% polyacrylamide gel electrophoresis and the wet gel was exposed and detected by BAS reader (BAS-2010, Fujifilm).
Northern Blot and RT-PCR Analysis
Northern blot analysis and RT-PCR were carried out as described by Seo et al. (2011) [46]. Primer pairs used to amplify cDNA probes are listed in Table S1. For RT-PCR analysis, first-strand cDNA was synthesized from 2 ug of total RNA with oligo(dT)15 using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR was performed using sequence-specific primers described in Table S1 with 24 cycles, which was optimized to be within the linear range of amplification.
In Vitro Pull-down Assay
In vitro pull-down assays were carried out according to the procedure of Seo et al. (2011) [46]. The full length CDS of AtJAZ1 was fused in frame with the maltose-binding protein (MBP) at the C-terminus in pMAL-c2E vector, expressed in Escherichia coli and purified using amylose resin. Pulled-down mixtures were separated by SDS–PAGE, transferred to nitrocellulose membrane (Whatman), and detected using anti-c-myc antibody (Santa Cruz Biotechnology).
Agroinfiltration and Transient Protein Expression
Agroinfiltration for transient protein expression in tobacco leaves was performed according to the method described by Voinnet et al. [47], with minor modifications. Briefly, Agrobacterium tumefaciens strains C58C1 carrying either the 6xMYC-BBD1 or 3xHA-AtJAZ1 construct under the control of the 35S promoter were grown at 28°C in Luria-Bertani medium and resuspended in infiltration media. For cotransfections, Agrobacterium cultures carrying each construct were mixed in equal proportion. Leaves of 4-week-old Nicotiana benthamiana plants were infiltrated with a needleless syringe carrying bacterial cultures through the abaxial air spaces.
Chromatin Immunoprecipitation
ChIP-PCR was carried out as described by Saleh et al. (2008) [48]. Anti-acetylated H4K12 and anti-acetylated H3K14 antibodies were purchased from Upstate. After immobilization using protein A agarose (Pierce), bound DNA was eluted and amplified by primers corresponding to sequences neighboring the AtBBD1 binding sites in the promoter of AtJMT. PCR products were separated on ethidium bromide-stained agarose gel or real-time PCR was used to quantify the amplification. For real-time PCR, amplification of PJMT (near the JARE) was normalized to that of Actin [49]. Chromatin precipitated without antibody was used as a negative control, and the chromatin before precipitation was used as the input control. ChIP assays were independently repeated twice with the same result. Real-time qRT-PCR was performed using a LightCycler 480 II machine (Roche Diagnostics) with a SYBR Premix EX Taq Kit (TaKaRa). Primers used for qRT-PCR are listed in Table S1. Each qRT-PCR was independently repeated three times with the same expression pattern.
Results
The Promoter Regions of BcNTR1 and AtJMT Contain a JA-responsive Element
AtJMT and BcNTR1 are orthologues, which are both induced by MeJA treatment [42], [43], [44]. To understand the regulatory mechanism of BcNTR1 and AtJMT JA-responsive expression, we first defined the promoter regions that mediate induction by JA. Promoter regions 4.4 kb upstream of BcNTR1 and 4.5 kb upstream of AtJMT, respectively, were combined with the GUS coding sequence and transformed into Arabidopsis. Transcription of GUS was induced within 1 hour after MeJA treatment in both sets of transgenic plants (Figure 1), showing that these promoter regions could recapitulate JA-responsive induction. These results indicate that both fragments contain putative JA-responsive cis-acting elements (JARE). Also, these results showed that the BcNTR1 promoter works in Arabidopsis although it originated from Brassica.
10.1371/journal.pone.0055482.g001Figure 1
AtJMT and BcNTR1 promoters contain JA-responsive transcriptional regulatory elements.
Northern blot analysis of the recombinant GUS gene after MeJA treatment of transgenic Arabidopsis. BcNTR1 (4.4 kb length, A) and AtJMT (4.5 kb length, B) promoters fused to the GUS gene are shown. Nucleotide sequence of the promoter is numbered from translation initiation site.
Identification of a JARE in the AtJMT Promoter
We next used promoter-deletion analysis to locate the JARE(s) present in the AtJMT promoter. A series of 5′-deleted promoters was fused to the GUS coding region and transformed into Arabidopsis. Each construct contains from 4.5 kb to 500 bp of the promoter region (Figure 2A). GUS expression in response to MeJA treatment was examined by RT-PCR. When MeJA was applied, transgenic plants containing promoter fragments longer than 2.0 kb (JP4.5∼JP2.5) showed GUS induction within 1 hour; however, those containing promoter fragments shorter than 2.0 kb (JP2.0∼JP0.5) did not show GUS induction (Figure 2A). These results indicate that a putative JARE is located in the 500 bp region between position −2500 and −2000 bp in the AtJMT promoter.
10.1371/journal.pone.0055482.g002Figure 2 Localization of the JA-responsive cis-element (JARE) in the AtJMT promoter.
(A) A series of 5’ deleted promoters (closed bar) was cloned upstream of GUS coding region (open bar) and transformed into Arabidopsis. RT-PCR analysis of each transgenic plant was carried out after 1 hour of MeJA treatment. The JARE is located in the region between −2500 and −2000. (B) Additional promoter deletion constructs between −2500 and −2000 are shown and their GUS gene expression in response to MeJA treatment is shown. The putative JARE is located in the region between −2294 and −2280 (gray bar). The positions of the G-boxes are shown at −2529, −2406 and −2342 (▾).
To narrow down the position of the JARE, we generated additional 5′ deletion constructs subdividing the −2,400 to −2,000 bp region of the AtJMT promoter. As before, promoter regions were fused to GUS and transformed into Arabidopsis. When GUS mRNA levels were analyzed by RT-PCR after MeJA treatment, constructs containing 2,400 and 2,294 bp of the AtJMT promoter were responsive to MeJA, but constructs containing regions shorter than 2,294 bp were not responsive to MeJA (Figure 2B). Therefore, the putative JARE is located in the 15 bp region between the positions −2,294 and −2,280 bp of the AtJMT promoter.
Identification of a JARE in the BcNTR1 Promoter
In parallel, a series of BcNTR1 promoter deletion constructs was made and transformed into Arabidopsis to identify the JARE in BcNTR1. GUS mRNA levels were examined by RT-PCR and were induced rapidly, within 1 hour after MeJA treatment, in NP4.0 but not in NP3.0 and NP2.0 lines. Another construct, NPfr1, containing a 3,807 bp fragment of the BcNTR1 promoter with a deletion between −3,108 and −446, showed a similar response to NP4.0 (Figure 3A). These results showed that the region between −3,807 and −3,256 in the BcNTR1 promoter also contains a JARE. To test this hypothesis, an additional deletion construct, NP4-A, was made, in which the −3,518 to −3,480 region was deleted. When transgenic Arabidopsis plants containing the NP4-A construct were treated with MeJA, these lines showed no induction of GUS (Figure 3B). These results show that the JARE is localized in the 39 bp region, between −3,518 and −3,480 bp of the BcNTR1 promoter.
10.1371/journal.pone.0055482.g003Figure 3 Localization of JARE in the BcNTR1 promoter.
(A) Structures of promoter deletion constructs of BcNTR1 for JA response tests (left) and RT-PCR analysis of transgenic Arabidopsis after MeJA treatment (right). The JARE is located in the region between −3807 and −3256. (B) The BcNTR1 promoter has a region A (−3518 to −3480) of sequence identity with the AtJMT promoter in NPfr1. In the NP4-A construct, the A region was deleted from NP4.0. GUS was analyzed by RT-PCR in transgenic plants after MeJA treatment. JARE resides in the A region, −3518 to −3480. (C) Sequence alignment between putative JARE-containing regions in JP2294 of Fig. 2B and A region in NPfr1. Sequence elements (putative JARE) that are identical between JP2294 and NP4-A. are shown in bold.
Sequence alignment between the JARE-containing regions of the AtJMT promoter (15 bp) and the BcNTR1 promoter (39 bp) showed a highly conserved sequence motif, TCCTGA (Figure 3C). We hypothesized that this conserved sequence element is a putative JARE (TCCTGA) that could play a critical role in the JA responsiveness of AtJMT and BcNTR1 expression.
A Multimerized JARE-containing Construct Responds to MeJA
To show regulation of JA responses by the JARE, we next made a construct containing multimers of the JARE-containing promoter region linked to a minimal promoter and tested whether it could mediate JA-responsive induction of transcription. A region containing the putative JARE, between −2305 and −2278 of the AtJMT promoter, was duplicated 4 times and fused with the TATA-box sequence (−46 to +8) of the CaMV 35S promoter and a GUS coding sequence. Also a mutant version, in which the core 6 nucleotides, TCCTGA, were mutated to TTTTTT, was constructed in the same manner to determine the role of this core element in response to JA (Figure 4A). All the constructs were transformed into Arabidopsis (Col-0) and transgenic lines were treated with MeJA. Histochemical staining of transgenic plants showed that JA-responsive GUS activity was present only in 4xJARE:GUS lines, but not in 4xmJARE:GUS lines (Figure 4B). RT-PCR analysis also showed that GUS transcript was induced within 1 hour in 4xJARE:GUS lines (#17 and # 25) in response to MeJA, but was not induced in 4xmJARE lines (#12 and #31) (Figure 4C). Taking these data together, we concluded that the conserved 6-nucleotide element (TCCTGA) in the AtJMT and BcNTR1 promoters is indeed a JARE.
10.1371/journal.pone.0055482.g004Figure 4 JARE-containing transgenic plants show MeJA response.
(A) Schematic representation of multimerized JARE- containing construct (4xJARE:GUS), and its mutant version (4xmJARE:GUS). The DNA fragment from the AtJMT promoter (−2305 to −2278) containing the JARE was repeated 4 times and recombined to the GUS reporter containing a minimal promoter (TATA) from CaMV 35S. JARE(TCCTGA) and its mutant version are shown in bold. (B) Histochemical staining of 4xJARE:GUS (#17) and 4xmJARE:GUS (#12) transgenic plants with (+) or without (−) MeJA treatment for 4h. (C) RT-PCR analysis of GUS in each transgenic line was carried out after MeJA treatment.
Identification of a JARE-binding Protein
We next carried out yeast one-hybrid (Y1H) screening to isolate protein factors that bind to the JARE. A segment (−3,518 to −3,390) of the BcNTR1 promoter containing the JARE was employed as bait in the reporter construct. The yeast cells were co-transformed with activator constructs incorporating cDNA libraries prepared from MeJA treated Arabidopsis. Clones were sequenced from positive colonies and sequence analysis showed that multiple positive clones corresponded to AtBBD1 (Figure S1). AtBBD1 is an Arabidopsis homologue of the Oryza minuta bifunctional nuclease in basal defense response (OmBBD1), which acts in abscisic-acid (ABA)-dependent callose deposition [50]. The Arabidopsis thaliana genome also contains an AtBBD1 homologue, AtBBD2, with 81% amino acid sequence identity to AtBBD1 (Figure S2C).
To determine whether AtBBD1 binds to the putative JARE core sequence of GTCCTGA in the BcNTR1 promoter fragment, or to another cis-element, the bait region (−3518 to −3390) of the BcNTR1 promoter was divided into three segments; a (−3518 to −3471), b (−3471 to −3430) and c (−3430 to −3390) (Figure 5A). Each segment was tested for interaction with AtBBD1 by Y1H assay. AtBBD1 was fused with the activation domain of GAL4 (AD) in the activator construct. These assays showed that AtBBD1 bound only to the segment (−3518 to −3471) that contains the GTCCTGA core sequence.
10.1371/journal.pone.0055482.g005Figure 5 Identification of sequence element in the BcNTR1 promoter region to which AtBBD1 binds.
(A) Structures of reporter and activator genes used in Y1H assays. The promoter region of BcNTR1, −3518 to −3390, was divided into 3 segments and each segment was used as bait for Y1H assays. The control does not contain any of those segments. AtBBD1 was fused with the GAL4 activating domain (AD) as an activator. The position of the putative JARE is shown (▾). (B) The segment a was divided further into 8 subsegments (6 nt each) and each subsegment, a1 to a8, was mutated into 6 adenines. Each mutant segment was tested as bait in Y1H assays. (C) Subsegments a6 and a7 to which AtBBD1 bound, were dissected further by mutation in overlapping frames. In each mutant, 6 nucleotides were mutated into 6 adenines. Each mutant subsegment, M0–M5, was tested by Y1H assays. The sequence motif to which AtBBD1 binds is shown in bold. (D) Mutation analysis of the AtBBD1 binding element. Mutant series (CM1 to CMR) of JARE was created by changing a single nucleotide from purine to pyrimidine, or vice versa, in the fragment −2305 to −2278 as shown in Fig. 4A as a bait and Y1H assays were carried out with AD-AtBBD1. CMR is a JARE in reverse orientation.
To further narrow down the binding sequences within this segment (−3518 to −3471), a series of mutated bait segments (a1 to a8) was designed by changing 6 nucleotides of each subsegment into 6 adenines (Figure 5B) and testing by Y1H whether these changes affected AtBBD1 binding. AtBBD1 did not bind mutant segments a6 and a7 in yeast; therefore, those 12 nucleotides include sequences necessary for AtBBD1 binding (Figure 5C). Another series of six overlapping mutant constructs, M0–M5, in which 6 nucleotides were mutated into 6 adenines, revealed that the nucleotide element, GTCCTGA, is necessary for AtBBD1 interaction (Figure 5C).
Additional point mutation experiments showed that adenine and thymine in the first nucleotide eliminated AtBBD1 binding (Figure 5D). However, cytosine was acceptable as in PJMT (see Figure 6). The 7th adenine, which was not tested in the M0–M5 constructs above, was also necessary. The orientation of the heptameric element was also important in the Y1H assay, as the CMR construct, which has the JARE sequence in reverse orientation, did not show AtBBD1 binding in yeast (Figure 5D). In conclusion, the heptameric nucleotide element, G(C)TCCTGA, is critical for AtBBD1 to interact with these DNA sequences (Figure 5D). This result is consistent with the promoter deletion experiments and the multimerized JARE analysis (Figure 4).
10.1371/journal.pone.0055482.g006Figure 6 AtBBD1 and AtBBD2 bind to promoter sequences containing the JARE.
Promoter segments, PNTR1, (−3497 to −3470 of BcNTR1 promoter) or PJMT (−2305 to −2278 of AtJMT promoter) were used as bait in Y1H assays. A mutated segment, PG-box, which contains G-box sequence in PNTR1 was used as a bait and an empty vector (pHIS2) was used as a control. AtBBD1 and AtBBD2 were fused with AD. G-box is AtMYC2 binding element (CACGTG) (Boter et al., 2004).
JARE is Distinct from the G-box
To test the specificity of AtBBD1 binding, Y1H assays were carried out with promoter segments containing different JA-responsive cis-elements, including the G-box, placed into the same sequence context as the JARE. For JARE-containing constructs, PNTR1 (−3497 to −3470 of the BcNTR1 promoter) or PJMT (−2305 to −2278 of the AtJMT promoter), which contain the JARE, were used as bait in the Y1H assay. For the G-box, PG-box is a mutant version of PNTR1 in which the JARE was replaced with a G-box. The G-box is a typical JA-responsive element and is bound by AtMYC2 [15]. Y1H results showed that AtBBD1 interacted with PJMT and PNTR1 but did not interact with PG-box (Figure 6). Therefore, the JARE ((G/C)TCCTGA) of PNTR1 and PJMT, is a distinct cis-element in the AtJMT and BcNTR1 promoters for JA-responsive gene expression. AtBBD1 could regulate expression of BcNTR1 and AtJMT in response to JA through binding to the JARE. AtBBD2, a homologue of AtBBD1, also bound to the same DNA sequences as JARE in Y1H assays (Figure 6).
The C-terminal Region of AtBBD1 has DNA Binding Activity
Sequence analysis of the AtBBD1 protein family had previously shown that the AtBBD1 proteins contain several conserved domains, including a highly conserved region (HCR), a domain of unknown function 151 (DUF151), and a UV responsive (UVR) domain at the C-terminus [50]. However, this analysis did not identify a known DNA-binding motif; therefore, we tested whether different domains of AtBBD1 had DNA-binding activity. We made five truncated protein constructs (BBD1A-E) each containing one or two domains of AtBBD1 and fused these with AD for Y1H assays to determine their DNA binding activity (Figure 7A). Each construct was co-transformed into yeast with a bait DNA sequence (PNTR1) that is known to interact with full-length AtBBD1. Constructs BBD1B (116–325) and BBD1E (257–325), which both contain the C-terminal predicted UVR domain, showed DNA binding activity in yeast, but the other constructs showed no DNA binding activity (Figure 7A). This result suggests that the AtBBD1 DNA binding domain resides in the C-terminal region.
10.1371/journal.pone.0055482.g007Figure 7 The DNA binding domain of AtBBD1 resides in the C-terminal region.
(A) A schematic representation of truncation mutants of AtBBD1. Numbers indicates amino acid residues, and putative domains are represented (HCR, Highly Conserved Region; DUF151, Domain Unknown Function 151; UVR, putative UV-Response domain) [50]. Each truncated protein was fused with AD as shown in Fig. 5A. PNTR1 (Figure 6) was used as a bait DNA sequence (bottom). (B) Electrophoretic mobility shift assays were carried out using fusion protein (MBP-BBD1) and a 70 bp fragment containing JARE was used as a probe.
To confirm the DNA binding activity of BBD1E (257–325) by electrophoretic mobility shift assays (EMSA), we made a construct, MBP-BBD1E, which fused amino acids 257–325 with Maltose Binding Protein (MBP) and expressed this fusion protein in E. coli. Crude extracts containing MBP-BBD1E bound to the 70 bp DNA fragment containing the JARE of the BcNTR1 promoter. Competition assays with unlabeled probe showed the specificity of binding (Figure 7B). These results show that amino acid residues from 257 to 325 at the C-terminus are involved in DNA binding by AtBBD1.
AtBBD1 Interacts with the ZIM/TIFY Domain of AtJAZ1 through its HCR Domain
Because JAZ proteins interact with various transcription factors involved in JA-responsive gene expression, we used yeast-two-hybrid (Y2H) assays to test whether AtBBD1 interacts with Arabidopsis JAZ proteins. Full-length AtBBD1 was fused to the GAL4 DNA binding domain (BD) and the full-length protein for each of of 12 AtJAZs was fused to the AD. AtBBD1 showed strong interactions with AtJAZ1 and AtJAZ4 in Y2H assays (Figure 8A).
10.1371/journal.pone.0055482.g008Figure 8 AtBBD1 interacts with AtJAZ proteins.
(A) Y2H assay between AtBBD1 and each of 12 AtJAZs. Full length CDS of AtBBD1 was fused to GAL4 DNA binding domain (BD) and each full length CDSs of 12 AtJAZs was fused to AD. (B) The pull-down assay between AtBBD1 and AtJAZ1. 35S:6xmyc-AtBBD1 plant extract (input) was incubated with amylose resin bound recombinant MBP-AtJAZ1 protein. Pulled-down protein complex was detected by immunoblotting using anti-MYC antibody (left). MBP protein was used as a pull-down control. The panel on the right shows input recombinant MBP and MBP-AtJAZ1 proteins in the pull-down assay. (C) Immunodetection of the AtBBD1 and AtJAZ1 complex in vivo. 35S:6xMYC-AtBBD1 and 35S:3xHA-AtJAZ1 constructs were transiently coexpressed in tobacco leaves by agroinfiltration. The expressed proteins were immunoprecipitated (IP) using anti-HA antibody (+/+) and immunoblotting was carried out with anti-myc antibody. Left lane (−/−) is control leaf extract that was not agroinfiltrated. MYC-AtBBD1 and HA-AtJAZ1 proteins were detected in input coexpressed leaf extracts by each antibody (right). (D) Each truncated AtBBD1 protein was fused to AD as a prey for Y2H assay with AtJAZ1. AtJAZ1 was fused to BD as bait. Numbers indicates amino acid residues, and putative domains were represented. (E) Each truncated AtJAZ1 protein was fused to AD as a prey for Y2H assay with AtBBD1 protein. AtBBD1 was fused to BD as bait.
To confirm the results of the Y2H assay, in vitro pull-down assays were carried out. Recombinant MBP-AtJAZ1 bound to amylose resin was incubated with plant extracts prepared from a 35S::6xMYC-AtBBD1 transgenic plant and pulled-down proteins were analyzed by Western blotting with anti-MYC antibody. Recombinant 6xMYC-AtBBD1 was pulled down by recombinant MBP-AtJAZ1 (Figure 8B).
To confirm the interaction between AtBBD1 and AtJAZ1 in vivo, 35S:6xMYC-AtBBD1 and 35S:3xHA-AtJAZ1 constructs were transiently coexpressed in tobacco leaves by agroinfiltration. Leaf extract was immunoprecipitated with anti-HA antibody and then immunoblotted with anti-MYC antibody (Figure 8C). These results showed that AtBBD1 directly interacts with AtJAZ1.
To identify the domain of AtBBD1 that mediates interaction with AtJAZs, truncated AtBBD1 proteins were fused with the AD and full length AtJAZ1 was fused with BD for Y2H assays. Y2H results showed that BBD1A (1–116), and H (81–116) interacted with AtJAZ1 in yeast, indicating that the HCR domain between amino acid residues 81 and 116 of AtBBD1 interacts with AtJAZ1 (Figure 8D).
Reciprocally, to identify the domain of AtJAZ1 that mediates interaction with AtBBD1, 5 truncated protein constructs containing the ZIM/TIFY or Jas domains of AtJAZ1 were designed for Y2H assays. JAZ1A (1–204) and JAZ1B (100–181) fragments as well as JAZ1F (full length JAZ1) interact with AtBBD1. These results indicate that the amino acid sequence from 100 to 181 of AtJAZ1, which contains the ZIM/TIFY domain, is responsible for interaction with AtBBD1 (Figure 8E). Therefore these results lead us to conclude that the N-terminal region containing the HCR domain of AtBBD1 interacts with the ZIM/TIFY domain of AtJAZ1 in Arabidopsis.
AtBBD1 Negatively Regulates AtJMT
To investigate the in vivo function of AtBBD1 in regulating AtJMT gene expression, a T-DNA insertion knockout mutant, atbbd1, was examined. When treated with MeJA, the atbbd1 mutant showed no difference from wild type plants (Col-0) in AtJMT gene expression (Figure 9A). However, AtBBD2, which has 81% amino acid sequence identity to AtBBD1 (Figure S2C), may have overlapping functions. To test whether these two genes act redundantly, the double knockout mutant, atbbd1 atbbd2, was made by crossing the atbbd1 and atbbd2 single mutant plants (Figure S2A–B). When the double knockout plants were treated with MeJA, AtJMT expression was induced to a higher level and the induction lasted longer than in wild type. In wild type, induction of AtJMT transcription by JA was short-lived and transcript levels began to decline after 3 hours of MeJA induction, but in double knockout plants, AtJMT transcript levels continued to increase, even 6 hours after MeJA treatment (Figure 9A). The atbbd1 atbbd2 plants however, showed reduced expression levels of the JA-regulated gene JR2 in response to MeJA treatment. These results showed that AtBBD1 and AtBBD2 have redundant functions as negative regulators of AtJMT gene expression in response to MeJA, but may act as positive regulators of JR2.
10.1371/journal.pone.0055482.g009Figure 9 Gene expression pattern in mutants of AtBBD1 and AtBBD2.
(A) MeJA response of AtJMT in Col-0, atbbd1, and atbbd1 atbbd2 mutants after MeJA treatment. AtBBD1, AtBBD2 and JR2 were analyzed by Northern blot, and AtJMT was analyzed with RT-PCR. (B) Basal levels of AtJMT expression in Col-0, OX-4, and OX-13. (C) MeJA response of AtJMT expression between Col-0, OX-4, and OX-13. AtBBD1 and JR2 was analyzed by Northern blot and AtJMT was analyzed by RT-PCR.
To understand AtBBD1 function further, AtBBD1 was overexpressed under the control of the CaMV 35S promoter in transformed lines (Figure S3). Transgenic lines OX4 and OX13, single copy transformants, were selected for further analysis. Consistent with the atbbd1 atbbd2 mutant phenotype, the basal and JA-induced levels of AtJMT expression were lower in OX4 and OX13 compared to wild type (Col-0) (Figure 9B-C). Also, JA-regulated JR2 gene expression was enhanced in overexpression plants. These results further support the hypothesis that AtBBD1 functions as a repressor of AtJMT gene expression in vivo by binding to JARE but acts as a positive regulator of JR2.
Chromatin Immunoprecipitation Reveals that AtBBD1 Repression of AtJMT is Associated with Histone Deacetylation
To understand the mechanism by which AtBBD1 represses AtJMT gene expression, we next examined the level of histone acetylation in the promoter region of AtJMT. It has been reported that AtJAZ1 interacts with HDA6 directly and contributes to histone deacetylation [10]. Chromatin immunoprecipitation was carried out with antibodies against modified histones. Fragmented chromatin DNA was incubated with anti-AcH3K14 or anti-AcH4K12 antiserum and isolated DNA was amplified with sets of primers specific to the AtJMT promoter region neighboring the JARE (PJMT ). Upon MeJA treatment, the levels of histone H3 and H4 acetylation in the promoter region of AtJMT were enhanced in atbbd1 atbbd2 mutant plants but reduced in OX-4 compared to wild type plants (Figure 10A). The basal level of histone acetylation in the promoter region of AtJMT was also higher in atbbd1 atbbd2 double knockout plants and lower in OX-4 compared to wild type. qPCR data showed that the histone acetylation level of AtJMT was significantly different from wild type, double knockout and OX-4 plants at a confidence level of P<0.05 (Figure 10B). These results showed that AtBBD1 repression of AtJMT is associated with histone deacetylation; this deacetylation may occur through the AtBBD1 interaction with AtJAZ1, which was reported to interact with HDA6 [10].
10.1371/journal.pone.0055482.g010Figure 10 Acetylation of chromatin histones associated with PJMT is enhanced by MeJA.
(A) Chromatin immunoprecipitation was carried out with antibodies recognizing acetylated histone H3 (AcH3K14) or H4 (AcH4K12). Precipitated DNA was amplified by primers corresponding to sequences adjacent to the AtBBD1 binding sites in the AtJMT promoter (PJMT). PCR product was analyzed by agarose gel electrophoresis. Actin was used as a control. Input indicates samples before immunoprecipitation. (B) qPCR analysis of ChIP assay with Col-0, atbbd1 atbbd2, and OX-4. Open bar is without MeJA treatment and closed bar is with MeJA treatment for 3 hours. Relative fold difference is represented. Statistical significance of the measurements was determined using a t-test (P≤0.05) by comparison with the value for Col-0 (*). Comparison between indicated values is also shown by (**). Data represent the mean values of 3 independent experiments and error bars represent standard deviation.
Discussion
Identification of a JARE in the AtJMT and BcNTR1 Promoters
Here we have identified a novel cis-element, the JARE, which regulates JA-responsive gene expression and contains a heptanucleotide sequence motif (G/C)TCCTGA. The JARE was identified in two orthologous genes encoding an enzyme involved in JA biosynthesis, −3480 bp upstream of BcNTR1 and –2290 bp upstream of AtJMT, respectively. The same JARE sequence is present in both genes, although at slightly different positions in each promoter. Considering that the two genes are orthologous, encoding the same enzymatic activities in the same plant family, the two loci could have conserved mechanisms of transcriptional regulation in which they share homologous cis-acting elements and trans-acting factors. The JARE is also found in the promoters of other Arabidopsis JA-responsive genes, including LOX2, COI1, JAZs (JAZ6, 7,and 8), WRKY70, PDF1.2, VSP1, and MYBs (MYB24 and 44), and in other plants, including the promoter of the rice JA-responsive gene OsbHLH148. Therefore, JA-responsive regulation through the JARE may affect many Arabidopsis genes and may also be conserved beyond the Brassicaceae.
The JARE is distinct from other JA-responsive elements previously reported. For example, G-box (CACGTG) and GCC motifs (GCCGCC) are known JA-responsive elements in plants [17], [51]. There are several G-boxes or G-box like elements in the BcNTR1 and AtJMT promoters, but tests of promoter deletions and multimerized JARE constructs showed that these G-box elements are not necessary for the JA response of AtJMT and BcNTR1 expression (Figure 4). Moreover, MYC2 is a TF that interacts with the G-box to regulate JA-responsive genes [15] and AtJMT gene expression in response to MeJA treatment was not affected in myc2 knockout plants (jin1–7 and jin1–8) (Figure S4). These results therefore indicate that JA-responsive regulation of AtJMT and BcNTR1 occurs through the JARE and not through G-box elements and suggest that the transcription factors binding to JARE could be different from bHLH transcription factors like MYC2.
The JARE is also distinct from other reported JA-responsive cis-elements. For example, the GCC motif was initially defined as an ethylene (ET)-responsive element in EREBPs [52], but it also plays a role in conferring JA- and ET-responsive expression of the PDF1.2 gene [27]. Also, TGACG sequences were found to be essential for the JA response in promoters of tobacco nopaline synthase (nos) and barley lipoxygenase 1 (LOX1) genes [53], [54], [30]. JASE1 (CGTCAATGAA) and JASE2 (CATACGTCGTCAA) of Arabidopsis OPR1 are also reported to be JA-responsive motifs [31]. All of these motifs reported as JA-responsive elements are different from the JARE, which is therefore a novel cis-element controlling JA-responsive gene expression.
AtBBD1 Binds to JARE
To understand the mechanism of AtJMT gene expression regulation by the JARE cis-acting element, we identified a trans-acting factor, AtBBD1, which binds to the JARE. AtBBD1 is an Arabidopsis homologue of OmBBD1, which is involved in ABA dependent callose deposition [50]. AtBBD1 expression was induced by various plant hormones such as MeJA, SA, ABA, and ETP (Figure S5A). The complex regulation of AtBBD1 indicates that it may act in additional hormonal responses, or in cross-talk among hormonal signaling pathways.
AtBBD1 specifically binds to the JARE sequence, which we defined by mutational analysis as (G/C)TCCTGA, and does not bind to other sequences such as the G-box (Figure 6). Sequence analysis of AtBBD1 did not identify a known DNA binding motif, but Y1H assays revealed that that the AtBBD1 C-terminal domain, containing the UVR domain, binds to JARE. The DNA binding motif of AtBBD1 is also similar to the C-terminal region of DELLA proteins (GAI, and RGA/RGLs) although the DNA binding domain of DELLA proteins has not yet been clearly defined. Consistent with the ability of AtBBD1 to bind DNA, AtBBD1-sGFP fusion proteins were localized in the nuclei of the transformed Arabidopsis plants (Figure S5B). Therefore, the characteristics of AtBBD1 are consistent with a role as a nuclear transcription factor.
AtBBD1 and AtBBD2 Repress AtJMT gene Expression
Unlike other JA-dependent transcription factors, which act as transcriptional activators after release from JAZ interaction, AtBBD1 acts to repress expression of the JARE-regulated target gene AtJMT. This repression is shown by induction of AtJMT expression in mutants lacking both AtBBD1 and its close homologue AtBBD2 (Figure 9 and S6). Y1H assays showed that AtBBD2 binds to JARE (Figure 6) and Y2H assays also showed that, similar to AtBBD1, AtBBD2 interacts with AtJAZ1 and 4 (Figure S7). These results also suggest that AtBBD2 is functionally redundant with AtBBD1. Similar to other JAZ-interacting proteins, the JAZ-AtBBD1 complex could repress AtJMT gene expression by recruitment of co-repressors, and by histone deacetylation through interaction between JAZ proteins and HDACs. For example, EIN3/EIL1 directly interact with AtJAZ1 and HDA6 to repress ERF1 in JA- and ethylene (ET)-responses through histone deacetylation [10].
AtBBD1/2 can also act, directly or indirectly, as positive activator, as shown by increased AtJR2 expression in the atbbd1 atbbd2 mutant (Figure 9). Also, Arabidopsis overexpressing OmBBD1 showed enhanced expression of the JA-related gene PDF 1.2 and the ABA-related genes ABA1, RD29. Considering their narrow spectrum of interaction with JAZs, AtBBD1 and 2 are expected to be involved in a specific subset of JA-related defense signaling, rather than global JA-responses. AtMYC2 interacts with most of the JAZ proteins and is involved in most JA-related phenotypes [21]. By contrast, other TFs involved in specific JA-responses, including TFs such as MYB21/24 and EGL3/GL3/TT8, interact with a small set of JAZ proteins [19], [20]. AtBBD1 might be involved in blocking a MeJA metabolic sink and thus may contribute to increasing the local concentration of JA-Ile, an active form of JA [55]. AtBBD1 could function as a positive regulator responding to JA by post-translational modification or interaction with other proteins [56]. For example, bifunctional TFs, APETALA2 and WUSCHEL, act as activators or repressors on different target genes in plant flower development [57].
Moreover, in addition to, or instead of, acting in initial JA responses, AtBBD1 may act during the recovery after JA induction. Slow induction of AtBBD1 by JA could reflect its role in the recovery phase. Indeed, the double knockout plant atbbd1 atbbd2 shows a higher level of AtJMT transcript at a later time (Figure 9), indicating a failure of recovery from JA stimulation.
Our results also suggest the existence of a positive regulator or activator responding to JA. For example, induced expression of the multimerized JARE reporter construct (Figure 4) and increased expression of AtJMT in the atbbd1 atbbd2 mutant are consistent with the presence of an activator that also binds the JARE. Although we did not find such an activator by Y1H hybrid screening, it is possible that the specific activator could compete with AtBBD1 for binding to the JARE. The postulated positive regulator may also be subject to JAZ-dependent regulation, as AtJMT expression is still strongly induced by JA in the atbbd1 atbbd2 mutant plants.
Regulation by competing positive and negative transcription factors has substantial precedent. In plants, a family of transcription factors, such as the auxin-responsive element binding factors (ARFs) or ET-responsive element binding factors (ERFs), can share the same DNA binding domain and the same cis-element, but many of the members have opposite functions in target gene regulation [58], [59], [60]. For example, ERFs bind to the same cis-element (GCC-box) but regulate target gene expression in the opposite manner. ERF1, 2, and 5 function as activators and ERF3, 4, and 7–12 function as repressors [58], [61]. Their repression activity is conferred by the EAR motif in ERF3 and 4 [61], [62]. The EAR motif interacts with the corepressor TPL. ERF-TPL complexes repress target genes by modification of chromatin structure through histone deacetylase [39]. In the case of ARF family TFs, ARF5–8 and 19 activate target gene expression and the others repress target gene expression although they bind to the same auxin-responsive cis-element [59]. ARFs require the association with Aux/IAA repressors for an auxin response [59], [60]. Many Aux/IAA proteins are degraded by the SCFTIR1 complex in an auxin-dependent manner [63]. These examples are reminiscent of the mechanism of JA signal transduction through JARE. Therefore, it is possible that AtBBD1 could bind to the JARE as a repressor and that other TFs with a similar DNA binding domain could compete with AtBBD1 and act as activators.
In conclusion, the mechanism of AtBBD1 negative regulation of AtJMT could be postulated to function as follows (Figure 11); AtBBD1 recognizes the JARE in the AtJMT promoter and interacts with AtJAZs. When the JA signal is absent, the AtBBD1-AtJAZ complex recruits co-repressors or HDACs. Putative activator also competes with AtBBD1 for binding to the JARE; in the absence of JA, this activator may be bound by JAZs. When the JA signal comes in, JAZs are degraded by the 26S proteasome pathway through SCFCOI, and AtBBD1 is then released from JAZs. At the same time, the activator is also released from JAZs and activates AtJMT. The AtJMT expression level is regulated by the balance between activator and AtBBD1. In knockout plants, the activator occupies the JARE and AtJMT gene expression is activated to higher levels than wild type because the AtBBD1 repressor is absent. In AtBBD1-overexpressing plants, AtBBD1 occupies the JARE dominantly over the activator and AtJMT gene expression is repressed more than in wild type. Identification of putative activator protein interacting with JARE would fill out the model more precisely.
10.1371/journal.pone.0055482.g011Figure 11 Proposed model for transcription repression by AtBBD1.
In the absence of signal, AtBBD1 represses AtJMT gene expression by recruiting corepressor or HDAc through AtJAZ. In the presence of signal, JA-Ile is released and the SCFCOI1 complex degrades JAZ proteins. A putative activator (+) that binds to the JARE competes with AtBBD1 (repressor). In knockout plants, the putative activator dominantly occupies the JARE and AtJMT gene expression is activated higher than wild type. In the AtBBD1-overexpressing plant, AtBBD1(repressor) competes with the putative activator and dominantly occupies the JARE; therefore, AtJMT gene expression is repressed more than in wild type. Size of each circle represents relative abundance.
Supporting Information
Figure S1
DNA binding ability of AtBBD1.
(TIF)
Click here for additional data file.
Figure S2
Analysis of
atbbd1
and
atbbd1 atbbd2
knockout lines.
(TIF)
Click here for additional data file.
Figure S3
Analyses of transgenic
Arabidopsis
overexpressing
AtBBD1.
(TIF)
Click here for additional data file.
Figure S4
AtJMT
expression pattern in
myc2
mutants.
(TIF)
Click here for additional data file.
Figure S5
Expression pattern and localization of AtBBD1.
(TIF)
Click here for additional data file.
Figure S6
Gene expression pattern in
atbbd2
mutant.
(TIF)
Click here for additional data file.
Figure S7
Interaction of AtBBD2 with AtJAZ1 and AtJAZ4 in yeast.
(TIF)
Click here for additional data file.
Table S1
List of constructs and primer sequences used in this study.
(PDF)
Click here for additional data file.
Procedure S1 (PDF)
Click here for additional data file.
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23405087PONE-D-12-3365410.1371/journal.pone.0054679Research ArticleBiologyMicrobiologyBacteriologyEmerging Infectious DiseasesMicrobial ControlPopulation BiologyEpidemiologyInfectious Disease EpidemiologyMedicineEpidemiologyInfectious Disease EpidemiologyMolecular EpidemiologyInfectious DiseasesBacterial DiseasesRickettsiaVeterinary ScienceAnimal TypesWildlifeMolecular Evidence for the Presence of Rickettsia Felis in the Feces of Wild-living African Apes Rickettsia Felis in African Wild-Living Apes FecesKeita Alpha Kabinet
1
2
Socolovschi Cristina
1
Ahuka-Mundeke Steve
2
Ratmanov Pavel
1
Butel Christelle
2
Ayouba Ahidjo
2
Inogwabini Bila-Isia
3
Muyembe-Tamfum Jean-Jacques
4
Mpoudi-Ngole Eitel
5
Delaporte Eric
2
Peeters Martine
2
Fenollar Florence
1
Raoult Didier
1
*
1
Aix-Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France
2
UMI 233, TransVIHMI, IRD (Institut de Recherche pour le Développement) and University of Montpellier 1, Montpellier, France
3
Projet Lac Tumba, WWF, Mbandaka, Democratic Republic of Congo
4
Institut National de Recherche Biomédicales and Service de Microbiologie, Cliniques Universitaires de Kinshasa, Kinshasa, Democratic Republic of Congo
5
Projet PRESICA and Virology Laboratory IMPM/IRD, Yaoundé, Cameroon
Bereswill Stefan Editor
Charité-University Medicine Berlin, Germany
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: DR FF MP ED BII JJMT EMN. Performed the experiments: AKK SAM CS CB. Analyzed the data: AKK CS AA SAM PR MP FF DR. Contributed reagents/materials/analysis tools: DR FF MP ED BII JJMT EMN. Wrote the paper: AKK DR FF AA MP.
2013 6 2 2013 8 2 e5467922 10 2012 13 12 2012 © 2013 Keita et al2013Keita et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.
Methods
We conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.
Results
Overall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.
Conclusion
For the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.
The authors have no support or funding to report.
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Introduction
Rickettsia felis is an obligate intracellular bacterium; it is the causative agent of a widely distributed infection throughout the world. It was described first in fleas [1], [2]. Considered until recently a rare disease, it is emerging in sub-Saharan Africa [2]. Studies conducted with robust methods in western and eastern sub-Saharan Africa by two different teams reported a very high incidence of this bacterium in patients with a fever of unknown origin, including 6 out of 163 (3.7%) patients in Kenya and 8 out of 134 (6%) patients in Senegal [3]–[5]. In many respects, R. felis infection in sub-Saharan Africa is comparable to malaria, that is, it has a very high incidence in febrile patients and may be associated with relapses [5]. Based on these similarities, it has been speculated that these patients may have been exposed to mosquitoes [3], [5], [6]. Arthropod cell lines capable of supporting R. felis growth include those of toad tadpoles (Xenopus laevis), ticks (Ixodes scapularis) and mosquitoes (Aedes albopictus and Anopheles gambiae) [6]–[9]. R. felis DNA has been recently found in mosquitoes, such as A. albopictus (GenBank JQ674484) [6] and A. gambiae (GenBank JQ354961) [8]. The majority of emerging infectious diseases have their origin in wildlife [10]. Apes are the closest relatives to humans with 98–99% genomic similarity and may suffer from the same diseases [10]–[13]. In recent years, some pathogens that were thought to strictly infect humans have been shown to have an ape origin [14]–[16]. Recently, R. felis DNA was detected in Ctenocephalides felis from the Cercopithecus cephus monkey in Gabon (sub-Saharan Africa), suggesting that nonhuman primates may be infected, as well as humans, and may represent a reservoir for R. felis
[4]. Apes’ blood is difficult or impossible to obtain in the wild. A major step in the identification of microorganisms associated with apes was achieved when stools were shown to contain DNA from bloodborne pathogens [10], [16]–[18]. The analysis of gorilla stools has revealed the presence of human immunodeficiency virus (HIV) and plasmodium in the collected samples [16], [17]. Based on these facts, we hypothesized that apes may be commonly infected by R. felis and release the pathogen’s DNA in their stool.
Materials and Methods
Ethics Statement
We sampled feces collected primarily near night nests or feeding sites. This non invasive collection method has not presented any threat to apes. The study was approved by the national ethics committee of Cameroon (agreement number N°259/CNE/SE/2011) dated November 10, 2011. No invasive sampling was done on any animals. Also the agreement covers all aspects of the study in both Cameroon and in DRC (relationships with forests residents until laboratory analysis via the collection of feces).
Sample Collection and Transportation
Fecal samples were collected from wild-living apes (chimpanzees, gorillas and bonobos) in central Africa (Cameroon and the Democratic Republic of Congo; Figure 1). The Cameroon samples from chimpanzees and gorillas were obtained at 16 forest sites located in the southern part of the country (Figure 1A). The fecal samples from the Democratic Republic of Congo (DRC) were obtained at one site (Malebo) from bonobos located in the western part of the country (Figure 1B). Overall, fecal samples were collected primarily near night nests or feeding sites. The GPS position and estimated time of deposition were recorded for almost all of the samples, and the species origin was defined in the field according to the nesting sites, prints, vocalizations and morphological and physical aspects of the samples. At some sites both gorilla and chimpanzee samples were collected. Approximately 20 mg of dung was collected in a 50 ml tube containing 20 ml of RNAlater (Applied Biosystems/Ambion, Austin, TX). These tubes were kept at base camps at room temperature for a maximum of 3 weeks and subsequently transported to a central laboratory for storage at −20°C or −80°C before the analysis was completed.
10.1371/journal.pone.0054679.g001Figure 1 Ape feces collection sites in Cameroon (A) and the DRC (B).
MB, AL BQ, BB, BM, CP, DJ, DP, EK, GB, LB, MS, LM, MM, MF, MP, ML: Sites of chimpanzee, bonobo and gorilla feces collection (more details in Table 1). The map shown is from Google© 2012. Image was generated using Quantum GIS 1.7.4-Wroclaw software.
Molecular Assays
DNA was extracted using Qiamp DNA Mini Kit (QIAGEN, Valencia, CA, USA), in accordance with the manufacturer’s recommendations and protocols. Two quantitative real-time PCR (qPCR) assays were used to screen for rickettsial nucleic acids in the ape fecal samples. This approach was previously described [5], [6], [19]. Each sample was tested with an ABI 7500 qPCR machine (Applied Biosystems®) with the QuantiTect Probe PCR Kit according to the manufacturer’s protocol. For Rickettsia spp. detection, the specimens were tested with primers and a specific TaqMan probe targeting a partial sequence of the citrate synthase gltA gene. When a specimen was positive in this assay, the result was confirmed by a second qPCR using primers and a probe targeting a chromosomal gene specific for a R. felis (biotin synthase, bioB) gene. Human stools were used as negative controls in our analysis. These controls were consistently negative. To analyze the results, positive controls of R. felis DNA were included in each test. An evaluation of the bacterial load detected from the cycle threshold (Ct) was performed based on previous studies [5], [6].
Species Determinations
For all fecal samples that were positive for the Rickettsia-specific gltA gene based on the RKND03 system in qPCR, we confirmed the ape species by DNA analysis by amplifying a 386-bp fragment of the 12S gene with traditional PCR followed by sequencing. Phylogenetic analysis of these sequences allowed identification of all ape species (Gorilla gorilla, Pan troglodytes troglodytes and Pan paniscus). With this approach, we also control the quality of the amplification products after DNA extraction [18], [20].
Rickettsia Characterization: Traditional PCR and Sequencing
Ape fecal samples that were positive by qPCR were subjected to a traditional PCR analysis targeting the citrate synthase (gltA) gene and the outer membrane protein A (ompA) gene. The first PCR was performed using primers CS.409D and CS.1258R, which amplify a 750 bp fragment of the gltA gene of Rickettsia. The second PCR was performed using primers 190.70, 190.180 and 190.701, which amplify a 629–632 bp fragment of the ompA gene, as previously described [5]. For these assays, we did not use nested PCR. DNA extracts from human stools were used as negative controls. These controls were consistently negative. Positive controls of Rickettsia montanensis DNA were included in each test to avoid contamination by R. felis DNA. The sequencing was performed as previously described [5], [6]. For phylogenetic analysis, all of the sequences were analyzed and compared to those of the rickettsiae sequences present in GenBank using the BLAST search tool. The obtained sequences were aligned using the multi-sequence alignment ClustalX program. The data were examined using maximum likelihood methods MEGA version 5 [21] and TOPALi v2.5 [22]. We have summarized, in a diagram, the different steps of our samples analysis (Figure 2).
10.1371/journal.pone.0054679.g002Figure 2 Analysis procedure.
Statistical Analysis
The data were analyzed using PASW statistics 17 software (SPSS, Chicago, IL, USA). Non-parametric values were compared using two tests. The corrected chi-squared test or the Fisher’s exact test was used where indicated. Statistical significance was defined as P<0.05.
Results
Rickettsia Detection in Ape Fecal Samples by Real-time PCR
A total of 1,028 fecal samples from wild-living apes in central Africa were analyzed in our study; 743 fecal samples (72.3%) were collected in Cameroon in 16 forest sites, and 285 samples were from one site of the DRC (Table 1). Included in our study were 313 samples from chimpanzees, 430 from gorillas and 285 from bonobos. Overall, 113 (11%, 95% confidence interval CI 9.2%–13.2%) samples were positive for the Rickettsia-specific gltA gene-based RKND03 qPCR system. The Ct value mean and standard deviation [SD] in these samples was 33.98±3.18. Among the positive samples for the Rickettsia-specific gltA gene-based RKND03 qPCR system, gorillas were most affected with 74 out of 430 gorilla fecal samples (17.2%), followed by 31 out of 313 chimpanzees (9.9%) and 8 out of 285 bonobos (2.8%). The difference between gorillas versus chimpanzees and bonobos was statistically significant (p<10−6, OR = 1.68).
10.1371/journal.pone.0054679.t001Table 1 Distribution of ape fecal samples according to the country and ape species.
Chimpanzee (Pan) Gorilla (Gorilla) Total
Country Forest Site Subspecies Nch Nch positive PCR Subspecies Ngor Ngor positive PCR Positive Nch+Ngor
Cameroon
Mambélé MB
P.t.t
169 21(9)
G.g.g
60 2 (0) 23 (9)
Alpicam AL
–
–
–
G.g.g
7 2 (2) 2 (2)
Belgique BQ
P.t.t
10 0
G.g.g
62 21 (3) 21 (3)
Boumba-Bek BB
P.t.t
14 1 (1)
G.g.g
7 2 (0) 03 (1)
Bouamir BM
P.t.t
20 1 (0)
G.g.g
1 0 0
Campo Ma’an CP
P.t.t
10 1 (0)
G.g.g
74 11 (4) 12 (4)
DJOUM DJ
P.t.t
2 1 (0)
G.g.g
9 2 (0) 0
Doumbo Pierre DP
P.t.t
9 0
G.g.g
10 2 (0) 0
Ekom EK
P.t.t
24 4 (0)
G.g.g
2 0 0
Gribi GB
P.t.t
2 0
G.g.g
18 1 (0) 0
Lobéké LB
P.t.t
21 2 (0)
G.g.g
5 0 0
Messok MS
–
–
–
G.g.g
142 31 (2) 31 (2)
Lomié LM
P.t.t
6 0
G.g.g
13 0 0
Mengamé MM
–
–
–
G.g.g
20 0 0
Mamfé MF
P.t.t
20 0
–
–
–
0
Metep MP
P.t.t
6 0
–
–
–
0
DRC
Malebo ML
P.p
285 8 (4)
–
–
–
8 (4)
Total
598
39 (14)
430
74 (11)
113 (25)
Legend: Forest sites of feces collection (See Figure 1). Nch: number of chimpanzee samples, Ngor: number of gorilla samples.
N positive PCR: number of samples found to be positive for Rickettsia spp. The number of samples positive for Rickettsia felis is in parentheses.
P.t.t
: Pan troglodytes troglodytes;
P.p: Pan paniscus; G.g.g
: Gorilla gorilla gorilla/.
DRC: Democratic Republic of Congo.
Rickettsia felis Detection in Ape Samples
All positives samples for the Rickettsia-specific gltA gene-based RKND03 qPCR system were confirmed by R. felis species-specific qPCR. Overall, 25 (22.1%, 95% CI 15.2%–30.5%) samples were positive for R. felis-specific qPCR (Table 1). Among these, 12 samples were from gorillas versus 9 samples from chimpanzees and 4 samples from bonobos. The difference between gorillas, chimpanzees and bonobos was not statistically significant (p = 0.77, OR = 0.68). The Ct value mean and standard deviation [SD] in these samples was 34.48±2.7.
Rickettsia Characterization
One hundred and thirteen out of 1,028 ape samples were positive for the Rickettsia-specific gltA qPCR. Sequencing of a 165-bp fragment of these samples revealed that the closest match to a validated bacterium was with R. felis (GenBank, CP000053 and AF210692) at 100% (124/124) similarity with a BLAST search. Among these samples, 25 were positive for R. felis specific qPCR and 88 were negative. All samples were also subjected to a traditional PCR analysis targeting the gltA gene and outer membrane protein A (ompA) gene [5], [6], [8]. For the positive samples for R. felis specific qPCR (n = 25), we had valid and interpretable sequences for 19 samples (7 chimpanzee, 10 gorillas and 2 bonobos) that were 99% homologous at the nucleotide level (661/670 for gltA gene and 500/503 for ompA gene) to R. felis sequences (GenBank CP000053 and AF210692). For gltA sequence, 19 samples (7 chimpanzee, 10 gorillas and 2 bonobos) demonstrated the same level of similarity (99%) to R. felis detected in A. albopictus (JQ674484) from Gabon. It had also 98% identity to Rickettsia spp. detected in A. gambiae voucher 101731(JN620082) from the Ivory Coast, Rickettsia spp. SGL01 detected in tsetse flies (GQ255903) from Senegal, and Rickettsia spp. Rf31 detected in Ctenocephalides canis (AF516331) from Thailand. A sequence analysis of the outer membrane protein A (ompA) gene indicated for 17 samples (7 chimpanzee, 9 gorillas and 1 bonobos) a 99% (500/503) homology to R. felis URRWXCal2 (CP000053), R. felis strain LSU-Lb (HM636635) from Liposcelis bostrychophila, R. felis (EU012496) in a dog from Mexico, R. felis (DQ408668, AY727036) from C. felis and R. felis scc50 (DQ102710) that was detected in Carios capensis from the USA. We have not found ompA sequence data for Rickettsia spp. RF31 in GenBank but for Rickettsia spp. SGL01 we found a sequence of 484 nucleotides long (Rickettsia sp. SGL01 OmpA pseudogene, partial sequence, GQ255904). However, our sequences had only 9% (49/484) coverage with a similarity of 98% (49/50). This low coverage did not allow us to include it in our analysis.
In maximum likelihood phylogenetic analysis based on the alignment of 468 bp of the gltA gene from 30 Rickettsia spp. (Figure 3A) and 402 bp of the ompA gene from 33 Rickettsia species (Figure 3B), including our samples and those from GenBank, the Rickettsia spp. detected in our study clustered with R. felis and R. felis-like organisms and demonstrate high bootstrap values (Figure 3).
10.1371/journal.pone.0054679.g003Figure 3 Maximum likelihood phylogenetic tree constructed from 30 Rickettsia spp.
based on the alignment of 468 bp of the gltA gene (
Figure 3A
) and 402 bp of the ompA gene from 33 Rickettsia samples (
Figure 3B
), including our samples. On these trees, we see the relationship between the Rickettsia spp. that has been previously described (black) and the Rickettsia spp. detected in our study (red).
Detection of other Rickettsia spp
Of the 113 positive samples with the Rickettsia-specific gltA gene-based RKND03 system, 88 samples (78%) were negative by the R. felis-specific qPCR. We performed specific qPCR for R. africae and R. conorii with these samples; all were negative. We then conducted a PCR analysis targeting gltA gene and the ompA gene (Eurogentec, Seraing, Belgium) followed by sequencing. We obtained sequences for the gltA gene, and generally, sequencing data were available when high DNA loads were found, which occurred in 30 specimens representing 17 chimpanzees, 12 gorillas and 1 bonobo. The additional rounds of sequencing allowed the retention of the same sequence each time, confirming the robustness of our data. All sequences obtained revealed that the closest match to a validated bacterium was with R. felis (GenBank CP000053 and AF210692) at 97% (701/720). In addition, these sequences demonstrated the same level of similarity to five Rickettsia spp. detected in A. gambiae voucher 101731(JN620082) from the Ivory Coast, A. albopictus (JQ674484) from Gabon, Canis lupus familiaris (JQ284386) from Australia, Glossina morsitans submorsitans (tsetse flies) from Senegal (GQ255903) [23] and Coccidula rufa from Iran (FJ666771). It had also 96% similarity to six Rickettsia spp. detected in horse and dogs (HM582437) from Brazil, Aulogymnus balani (FJ666770) from United Kingdom, C. canis from Thailand (AF516333), Synosternus pallidus (JF966774) from Senegal, C. canis (JN315968) from Kenya, and Haemaphysalis sulcata (FJ67737) from Croatia.
Discussion
The findings reported in this work were confirmed by several methods and establish the presence of R. felis and closely related bacteria in stools collected from wild apes in Africa. The validity of the data is based on strict experimental procedures that are commonly used in the WHO Reference Center for Rickettsial Diseases, including rigorous positive and negative controls to validate the test. Indeed, each positive PCR result was confirmed with the successful amplification of an additional DNA sequence, all negative controls were negative and the bacteria were sequenced. Therefore, we are confident in the results presented here.
The results show that R. felis was detected in 22% (25/113) of ape fecal specimens. R felis is permanently identified, and the other bacteria appear to be particularly close to R. felis as this has already been found in humans and in mosquitoes in Africa [3]–[6]. R. felis has also been detected in fleas from Ethiopia [19], Algeria [24], the Congo [25] and the Ivory Coast [26]. Human cases have been reported to be a common cause of fever in Kenya and Senegal [3], [5].
In endemic malaria areas, especially in western and eastern sub-Saharan Africa, studies conducted with two different teams reported a very high incidence of R. felis infection in patients with a fever of unknown origin, including patients in Kenya and Senegal [3], [5]. Indeed, in the same areas R. felis and related bacteria were found in An. gambiae which is also the vector for malaria [8] and in A. albopictus, which which has a notably large distribution in the world and can be a vector for Chikungunya and Dengue [6]. This work was initiated because we thought that the apes were at risk for infective bites by mosquitoes in this region. In light of data found in our study which corroborate with those published previously [4]–[6], [8], [23], we believe it is possible that arthropods (mosquitoes) could play a role as vectors for transmission of this bacterium. Given that, in this study we found 25 samples (22%) that were positive for R. felis out of 113 samples positive for the Rickettsia-specific gltA gene. The real infection rates are likely to be higher still, since R. felis detection in fecal samples can be expected to be less than detection in blood. For R. felis, the reservoir and source of bacteria responsible for bacteremia in Africa is unknown [5]. It is possible that, like malaria and HIV, R. felis appears in apes [10], [17], [27], [28].
In the recent study conducted by another team [17], the prevalence of Plasmodium spp. found in the feces of gorillas, chimpanzees in central Africa is comparable to the Rickettsia spp. prevalence that was found in our study (Table 2). The differences are not significant when comparing gorillas (p = 0.7) and bonobos (p = 0.18) in both studies, although in their study the prevalence of Plasmodium spp. was equal to zero in bonobos. Surprising when we comparing the prevalence found in chimpanzees in the two studies, the difference is significant (p = 0.0007). But it seems that this difference may be related to sample size in both groups.
10.1371/journal.pone.0054679.t002Table 2 Prevalence of Plasmodium spp.
[17] and Rickettsia spp. in chimpanzees, gorillas and bonobos found in Cameroon and DRC.
Fecal samples tested Fecal samples positive
Liu W and al. study [17]
Our study Liu W and al. study Our study
Plasmodium spp.
Rickettsia spp.
Plasmodium spp.
Rickettsia spp.
P value
Cameroon
Chimpanzee (Pan troglodytes troglodytes)
612 313 147 39
0.0007
Gorilla (Gorilla gorilla gorilla)
659 430 120 74 0.7
DRC
Bonobo (Pan paniscus)
107 285 0 8 0.18
Future work, in particular phylogenetic studies, integrating the different sequences of R. felis (particularly ompB, Sca4, which amplify respectively 4346-bp and 2783-bp), will allow us to determine the greater heterogeneity of R. felis (and R. felis like organism) in apes. This would argue in favor of the fact that R. felis infection, which is common in Africa, would originate from a strain for which the reservoir would be the ape.
In conclusion, this study showed that apes can be infected and carry R. felis and other bacteria close to R. felis in their stools. It also confirms that stool from apes are a particularly useful tool to help identify the pathogenic community in humans and apes.
We wish to thank Amandine Esteban and Tahar Kernif for technical help and all field staff who collected the feces in Cameroon and the DRC.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23408929PONE-D-12-1841510.1371/journal.pone.0051309Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesImmunological SignalingMedicineDrugs and DevicesAdverse ReactionsOncologyCancer TreatmentRadiation TherapyCancers and NeoplasmsBone and Soft Tissue SarcomasPediatricsPediatric OncologySurgeryPediatric SurgeryCurcumin Potentiates Rhabdomyosarcoma Radiosensitivity by Suppressing NF-κB Activity Curcumin Radiosensitizes RhabdomyosarcomaOrr W. Shannon
1
2
Denbo Jason W.
1
2
Saab Karim R.
2
Ng Catherine Y.
2
Wu Jianrong
3
Li Kui
4
Garner Jo Meagan
5
Morton Christopher L.
2
Du Ziyun
5
Pfeffer Lawrence M.
5
Davidoff Andrew M.
1
2
5
*
1
University of Tennessee Health Science Center, Department of Surgery, Memphis, Tennessee, United States of America
2
St. Jude Children's Research Hospital, Department of Surgery, Memphis, Tennessee, United States of America
3
St. Jude Children's Research Hospital, Department of Biostatistics, Memphis, Tennessee, United States of America
4
University of Tennessee Health Science Center, Department of Microbiology, Immunology and Biochemistry, Memphis, Tennessee, United States of America
5
University of Tennessee Health Science Center, Department of Pathology and the Center for Cancer Research, Memphis, Tennessee, United States of America
Loeb David Editor
Johns Hopkins University, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: WSO JWD KRS CYN JMG CLM ZD LMP AMD. Performed the experiments: WSO JWD KS CYN JMG. Analyzed the data: WSO CYN JW CLM LMP AMD. Contributed reagents/materials/analysis tools: WSO JW KL JMG CLM. Wrote the paper: WSO LMP AMD.
2013 7 2 2013 8 2 e5130925 6 2012 31 10 2012 © 2013 Orr et al2013Orr et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Ionizing radiation (IR) is an essential component of therapy for alveolar rhabdomyosarcoma. Nuclear factor-kappaB (NF-κΒ) transcription factors are upregulated by IR and have been implicated in radioresistance. We evaluated the ability of curcumin, a putative NF-κΒ inhibitor, and cells expressing genetic NF- κΒ inhibitors (IκBα and p100 super-repressor constructs) to function as a radiosensitizer. Ionizing radiation induced NF-κΒ activity in the ARMS cells in vitro in a dose- and time-dependent manner, and upregulated expression of NF-κΒ target proteins. Pretreatment of the cells with curcumin inhibited radiation-induced NF-κΒ activity and target protein expression. In vivo, the combination of curcumin and IR had synergistic antitumor activity against Rh30 and Rh41 ARMS xenografts. The greatest effect occurred when tumor-bearing mice were treated with curcumin prior to IR. Immunohistochemistry revealed that combination therapy significantly decreased tumor cell proliferation and endothelial cell count, and increased tumor cell apoptosis. Stable expression of the super-repressor, SR-IκBα, that blocks the classical NF-κB pathway, increased sensitivity to IR, while expression of SR-p100, that blocks the alternative pathway, did not. Our results demonstrate that curcumin can potentiate the antitumor activity of IR in ARMS xenografts by suppressing a classical NF-κΒ activation pathway induced by ionizing radiation. These data support testing of curcumin as a radiosensitizer for the clinical treatment of alveolar rhabdomyosarcoma.
Impact of work
The NF-κΒ protein complex has been linked to radioresistance in several cancers. In this study, we have demonstrated that inhibiting radiation-induced NF-κΒ activity by either pharmacologic (curcumin) or genetic (SR-IκBα) means significantly enhanced the efficacy of radiation therapy in the treatment of alveolar rhabdomyosarcoma cells and xenografts. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of ionizing radiation and warrants clinical trials.
This work was supported by the Assisi Foundation of Memphis, the US Public Health Service Childhood Solid Tumor Program Project Grant No. CA23099, the Cancer Center Support Grant No. 21766 from the National Cancer Institute, Grant No. CA133322 from National Cancer Institute, Grant No. AI069285 from United States National Institutes of Health, and by the American Lebanese Syrian Associated Charities (ALSAC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood [1]. There are two main histologic subtypes of RMS, embryonal (ERMS) and alveolar (ARMS). ARMS accounts for only 20–30% of newly diagnosed RMS in children but is associated with a much poorer prognosis [2]. An analysis of patients with nonmetastatic RMS from the Intergroup Rhabdomyosarcoma Studies (IRS) indicates a 5-year failure-free survival of only 65% for ARMS, despite intensive therapy; the outcome for patients presenting with metastatic ARMS is much worse [2]. Multimodal treatment of ARMS consists of surgical resection, systemic chemotherapy and ionizing radiation (IR). Findings from the IRS support the use of IR for all ARMS, regardless of stage and grade [3].
Although ionizing radiation is a critical component of RMS therapy, it is associated with significant early and late complications. Early complications such as inflammation can occur within hours to several weeks after therapy. Changes in cell membrane permeability and histamine release subsequently lead to cell loss, causing mucositis and epidermal desquamation. Late effects of ionizing radiation include cognitive defects, hypothyroidism, musculoskeletal deformities, pulmonary fibrosis, cardiac dysfunction and second malignancies [4]–[8]. Since the adverse effects of radiation are dose-dependent, increasing the efficacy of ionizing radiation on a per dose basis could limit its toxic side effects.
The nuclear factor-kappaB (NF-κΒ) family of transcription factors regulate the expression of genes involved in immune and inflammatory responses, developmental processes, and cellular proliferation and apoptosis [9]. Inhibitory IκB proteins tightly regulate NF-κΒ activity [10]. The classical NF-κΒ pathway involves serine phosphorylation and degradation of IκB proteins leading to the dissociation of the cytosolic inactive NF-κΒ/IκB complexes, and subsequent NF-κΒ translocation into the nucleus for DNA binding. However, a growing number of NF-κB inducers activate an alternative NF-κB pathway that does not involve IκB degradation, but rather involves the ubiquitination and proteolytic processing of p100/NFKB2 protein and nuclear translocation of p52:RelB dimers to regulate specific NF-κB target genes [9], [11]. Induction of NF-κB has been linked to tumor cell proliferation, angiogenesis, and inhibition of apoptosis, metastasis and radioresistance [12], [13].
Curcumin (diferuloylmethane), a yellow-colored polyphenol, is an active component of Curcuma longa, commonly known as turmeric. Curcumin has been shown to suppress NF-κΒ activation and down-regulate expression of NF-κΒ gene targets [13]–[17]. Curcumin has also been shown to sensitize colorectal cancer cells and neuroblastoma cells to ionizing radiation in vitro by inhibiting induction of NF-κΒ activity [18], [19]. Curcumin has been shown to be to be nontoxic in clinical trials with doses as high as 12 gm/day [20].
Despite advances in multi-modality therapy, many children with ARMS face a poor prognosis and so new treatment strategies are desperately needed. In this study, we tested the ability of curcumin to inhibit NF-κΒ activity and to act as a radiosensitizer. Our studies demonstrate that curcumin can potentiate the antitumor effects of ionizing radiation in vitro and in orthotopic alveolar rhabdomyosarcoma xenografts in mice.
Methods
Cell lines
The human alveolar rhabdomyosarcoma-derived cell lines, Rh30 and Rh41, were established at St. Jude Children's Research Hospital, Memphis, TN by Dr. Peter Houghton (Columbus, OH). The handling of these two cell lines in this manner has been supported by the St. Jude Children's Research Hospital IRB. Cells that stably express the IκBα and p100 super-repressors were made by selecting blastocidin-resistant cell populations following transduction with retroviral vectors encoding either an Iκβα mutant (SR-IκBα), a p100 mutant (SR-p100), or empty vector (EV). The constructs encoding the IκBα and p100 mutants were kindly provided by Dr. Jiandong Li (Atlanta, GA) and Dr. Shao-Cong Sun (Houston, TX), respectively.
Curcumin preparation
A liposomal formulation of curcumin was prepared to improve the delivery of the drug. 1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dimyristoylsn-glycero-3-[phosphor-rac- (1-glycerol)] (DMPG) were obtained from Avanti Polar Lipids (Alabaster, AL). Curcumin (ACROS, Morris Plains, NJ), dimethylsulfoxide (DMSO), and tert-butanol were obtained from Sigma Chemical Company (St. Louis, MO). Lyophilization of curcumin involved several steps. A 10∶1 total lipid to curcumin ratio (weight/weight) was used [21]. Curcumin was dissolved in DMSO at 50 mg/ml and the lipid (9∶1, DMPC:DMPG) was dissolved at 20 mg/ml in tert-butanol. Aliquots of this solution were then lyophilized to remove all DMSO and tert-butanol. The lyophilized powder was stored at −20°C and then reconstituted in normal saline for use.
Cell viability analysis
The effects of curcumin and IR on cell viability were determined by an Alamar Blue viability assay (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. Cells were irradiated with a cesium-137 source and/or treated with liposomal curcumin. Alamar blue was added to treated cells and after 4 hours the absorbance was read on a Synergy 2 Multi-Mode Microplate Reader (Biotek, Winooski, VT). These experiments were performed in triplicate.
Apoptosis
To analyze the effects of curcumin on apoptosis, cells were treated with 10 μmol of liposomal curcumin for 24 hours before irradiation. Cells were analyzed by flow cytometry for apoptosis (Annexin V staining) at 48 hours following irradiation.
NF-κB DNA binding assays
Nuclear extracts were prepared using the Affymetrix Nuclear Extraction Kit (Affymetrix, Santa Clara, CA) and stored at −80°C. To determine NF-κΒ DNA binding activity, an electrophoretic mobility shift assay (EMSA) was performed on nuclear extracts following the protocol by Promega Gel Shift Assay Systems. In brief, nuclear extracts were incubated with a 32P-end-labeled double-stranded NF-κΒ oligonucleotide containing a tandem repeat consensus sequence of 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, and separated by electrophoresis on 4% polyacrylamide gels. Bands were visualized on PhosphorImager and quantified with ImageQuant 5.2 software (Molecular Dynamics, Sunnyvale, CA).
Western blot analysis
Antibodies for matrix metalloproteinase-9 (MMP-9), IκBα, and pIκBα were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies for bcl-2 and XIAP were obtained from Cell Signaling (Danvers, MA). Antibodies for Cox-2 were obtained from Cayman Chemical (Ann Arbor, MI). Protein expression of MMP-9, Cox-2, bcl-2, XIAP, IκBα and pIκBα was determined by western blot analysis as previously described [22].
Murine ARMS tumor model
All murine experiments were done in accordance with a protocol approved by the Institutional Animal Care and Use Committee of St. Jude Children's Research Hospital. Orthotopic intramuscular (IM) ARMS xenografts were established in male CB-17 severe combined immunodeficient mice (Taconic, Hudson, NY), as previously described [23]. The size of the IM tumors was estimated by measuring the volume of the normal left calf and subtracting that from the volume of the tumor-bearing right calf. Measurements were done weekly using handheld calipers, and volumes calculated as width2 x length x 0.5. Mice with size-matched Rh30 or Rh41 xenografts were placed into one of four groups approximately 4 weeks after tumor cell injection and then treated with empty liposome given intraperitoneally (IP), 50 mg/kg liposomal curcumin IP daily until sacrifice, 10 Gy IR, or combination therapy. A single dose of 10 Gy IR was administered using an Orthovoltage D3000 x-ray tube (Gulmay Medical Ltd, Surrey, UK) [23]. A lead shield with a 2 cm hole was used to deliver radiation to the tumor while protecting the mice from radiation. Four mice in each group were sacrificed for tumor analysis 4 hr after radiation. The remaining mice were sacrificed approximately 3 weeks following the start of treatment.
In addition, we examined the response in vivo of Rh30 cell lines that express the SR-IκBα or SR-p100 super-repressor construct. Xenografts of Rh30-EV, Rh30 SR-IκBα, and Rh30 SR-p100 were established in the right calf of mice. For each xenograft, mice were placed into 2 groups of equivalent tumor burden to receive 10 Gy IR or to serve as controls.
Immunohistochemistry
Formalin-fixed, paraffin-embedded, 4-μm thick tumor sections were stained with rat anti-mouse CD34 (RAM 34; PharMingen, San Diego, CA) antibodies as previously described [24]. Apoptosis in tumors was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling using a commercially available in situ apoptosis detection kit (Serologicals, Billerica, MA) as previously described [23]. Tumor cell proliferation was analyzed by staining with an anti-Ki67 antibody (rabbit monoclonal clone SP6; Neomarkers, Fremont, CA). Results were expressed as the percentage of Ki-67 positive cells ± SE per 400x magnification as previously described [23].
Statistical analysis
Isobolograms were used to assess the in vitro synergy between curcumin and radiation [25], [26]. A weighed nonlinear dose-response model [26] using SAS NLIN procedure was used to fit fluorescence emission intensity data. Observed three-dimensional dose-response plots, fitted dose-response plotsand isobolograms were generated. Dose-independent synergy of combined curcumin and radiation was estimated from the model by the non-additivity parameter, α, which indicates synergism (α>0), antagonism (α<0), or no interaction (α = 0). For in vivo experiments, Bliss's independent joint action principle was used to define a synergy index (SI). Continuous variables are reported as mean ± SEM and were compared using an unpaired Student's t test with a p-value <.05 considered significant. Data were analyzed and graphed using Sigma Plot (Version 9; SPSS Inc, Chicago, IL).
Results
Ionizing radiation induces NF-κB activation in alveolar rhabdomyosarcoma cells in a dose- and time-dependent manner through the canonical pathway
To determine NF-κΒ DNA binding activity in Rh30 and Rh41 cells exposed to ionizing radiation (10 Gy), nuclear extracts were prepared at various times after irradiation and analyzed by electrophoretic mobility shift assay (EMSA). Both cell lines showed a time-dependent induction of NF-κΒ DNA binding activity, with activity evident within 1 hr after radiation exposure, and reaching a maximum activity at ∼2 hr (Fig. 1A). To further characterize the induction of NF-κΒ DNA binding activity, Rh30 and Rh41 cell lines were exposed to varying radiation doses (0–10 Gy) for 2 hr and nuclear extracts were prepared. In both cell lines, ionizing radiation activated NF-κΒ in a dose-dependent manner, with marked induction occurring at 10 Gy (Fig. 1B). Supershift assays were then performed with antibodies directed to specific NF-κΒ subunits to determine which NF-κΒ proteins comprised the radiation-induced complex. These assays showed that the p50 and p65 subunits comprise the radiation-induced NF-κΒ complex (Fig. 1C).
10.1371/journal.pone.0051309.g001Figure 1 NF-κB induction by IR is dose- and time-dependent via the canonical pathway.
(A) Ionizing radiation induces NF-κB in a time dependent manner. Rhabdomyosarcoma cell lines (Rh30 and Rh41) were exposed to 10 Gy radiation and nuclear extracts were harvested at various time points. In both cell lines, NF-κB activation, judged by EMSA showed maximal activity at 2 hrs (B) Ionizing radiation induction of NF-κB is dose dependent. In both cell lines, the maximum induction of NF-κB was seen at 10 Gy. (C) Ionizing radiation induces NF-κB activation via the classical pathway. Antibody super-shifts were performed with antibodies directed to specific NF-κB subunits. In both cell lines, the p50 and p65 subunits were the principal components of NF-κB activation. (D) IκBα super-repressor blocks NF-κB induction. Rh30 and Rh41 cell lines were transduced to express IκBα and p100 super-repressors. Radiation induced NF-κB activity in the empty vector and SR-p100 cell lines, but not in the SR- IκBα cell line. (E) IκBα and p100 super-repressor cell lines. TNFα (10 ng/ml for 1 hr) and LIGHT, also known as TNSF14, (10 ng/ml for 24 hr) were used to assess the activity of the super-repressor constructs on the classical and alternative NF-κB pathways, respectively. Expression of the SR- IκBα blocked TNFα stimulated NF-κB activity, expression of SR-p100 blocked LIGHT stimulated NF-κB activity.
The p50:p65 complex of NF-κΒ is induced through activation of the classical NF-κΒ pathway, which involves IκBα serine phosphorylation, polyubiqitination and subsequent proteosomal degradation. Therefore, expression of a mutant IκBα construct, in which the inducible serine phosphorylation sites have been mutated to alanine, should block classical NF-κΒ activation. In the alternative pathway, activation of the NF-κΒ-inducing kinase leads to proteolysis of p100 and liberation of the p52 complexes. A mutant p100 construct, in which the inducible serine phosphorylation sites have been mutated to alanine, should block the activation of the alternative NF-κΒ pathway. To determine whether radiation-induced NF-κΒ activation in ARMS cells involved the classical or alternative pathway, EMSA was performed on nuclear extracts prepared from ARMS cells that expressed IκBα and p100 super-repressors, respectively. As shown in Figure 1D, while radiation induced NF-κΒ activation in both Rh30 and Rh41 cell lines that express the empty vector or the SR-p100, expression of the SR-IκBα blocked the radiation-induced activation of NF-κΒ. The function of the IκBα and p100 constructs in the Rh30 cell line was confirmed by treatment with TNF-α (inducer of the classical NF-κΒ pathway) and by LIGHT (homologous to Lymphotoxin; inducer of alternative NF-κΒ activity) (Fig. 1E). [9], [11], [27] Taken together these results indicate that radiation-induced activation of NF-κΒ is both time- and dose-dependent, and involves the classical NF-κΒ pathway.
Curcumin suppresses radiation-induced NF-κB activation in alveolar rhabdomyosarcoma cells and sensitizes them to ionizing radiation by potentiating the apoptotic and cell cycle effects of IR
ARMS cells were treated with curcumin, a pharmacological inhibitor of NF-κΒ activity, to determine whether curcumin could abrogate the radiation-induced activation of NF-κΒ. Rh30 and Rh41 cells were pretreated with varying doses of liposomal curcumin (10, 25 and 50 µM) for varying lengths of time (0, 3 and 24 hr) prior to receiving a single dose of 10 Gy IR. Nuclear extracts were prepared 2 hr following radiation and assayed for NF-κΒ DNA binding activity by EMSA. As shown in Fig. 2, pretreatment for 24 hr with curcumin at all concentrations tested inhibited radiation-induced NF-κΒ activity in Rh30 and Rh41 cells. In addition, induction of NF-κΒ activity was also blocked when Rh41 cells were pretreated with 50 µM of curcumin for 3 hr prior to radiation.
10.1371/journal.pone.0051309.g002Figure 2 Curcumin blocks radiation induced NF-κB induction in rhabdomyosarcomas.
Rh30 and Rh41 cell lines were pretreated with varying doses of curcumin (10, 25 and 50 µM) for various lengths of time (0, 3 and 24 hrs) before exposure to radiation. EMSA was performed 2 hours following exposure to radiation to determine NF-κB activity. Pretreatment with curcumin for 24 hrs blocked the induction of NF-κB activity in both cell lines. In the Rh41 cell lines, NF-κB activity was blocked when 50 µM of curcumin was added 3 hrs before radiation treatment.
We next sought to determine the effects of curcumin and IR on the proliferation of the ARMS cell lines. Liposomal curcumin inhibited the proliferation of Rh30 and Rh41 cells in a dose-dependent manner. The IC50 for inhibition of cell proliferation by curcumin was similar in both cell lines (17.5±1.92 µM for Rh30 to 19.3±1.89 µM for Rh41) (Fig. S1A). The anti-proliferative effects of liposomal curcumin were equivalent to that of free curcumin (IC50 12.4±1.83 μmol and 15.32±1.67 µM for Rh30 and Rh41, respectively), confirming that there was no loss in the potency of curcumin during liposomal formulation. Treatment with empty liposomes alone had no effect on the proliferation of the rhabdomyosarcoma cell lines (data not shown). IR inhibited proliferation of Rh30 and Rh41 cells, in a dose dependent manner as well. The IC50 doses varied from 18.3±1.3Gy for Rh30 to 18.9±1.2Gy for Rh41 (Fig. S1B)
To test the effects of combination therapy, cells were pretreated with varying doses of curcumin for 24 hr prior to exposure to varying doses of radiation and cell viability was assessed at 96 hr following curcumin administration. The potential synergy between curcumin and radiation, on cell viability was assessed by an isobologram based-approach (Fig. S1C). (25, 26) A weighted non-linear dose-response model was fitted and the synergy of curcumin and radiation used in combination was estimated by the model non-additivity parameter (α). Curcumin and radiation showed synergy against the Rh30 and Rh41 cell lines. Treatment of Rh30 cells resulted in a positive non-additivity parameter of 5.54 with a 95% confidence interval (3.87, 7.20) that does not include 0. Treatment of Rh41 cells resulted in a non-additivity parameter of 5.86 with a 95% confidence interval (4.55, 7.17), which also does not include 0. Thus, we conclude that there was synergism between curcumin and radiation treatments in the Rh30 and Rh41 cell lines.
Cells were next treated with 10 µM liposomal curcumin (24 hr) prior to radiation exposure (10 Gy) and then analyzed by FACS for apoptosis by Annexin V-staining 48 hr after irradiation. The combination of curcumin and radiation significantly increased apoptosis compared to control in both cell lines. Apoptosis in control Rh30 cells was 4.6%±0.41 as compared to 8.67%±0.64 or 7.5%±0.3 in cells treated with radiation, or curcumin alone, respectively. In contrast apoptosis in Rh30 cells treated with a combination of radiation and curcumin was 19.5%±0.54 (p = .00003). A similar trend was observed in Rh41 cells. Apoptosis in control cells was 6.63%±0.66 as compared to 10.1%±1.6, or 9.9%±0.8 in cells treated with radiation or curcumin alone, while the combination of radiation and curcumin resulted in 22.9%±1.5 apoptosis (p = .0001) (Fig. S2A).
Curcumin inhibits degradation of IκBα in vitro
Since the classical NF-κΒ pathway involves the degradation of IκB, we investigated whether radiation induced IκBα degradation in IR-treated rhabdomyosarcoma cells. Whole cell extracts were prepared from Rh30 and R41 cells at various times after radiation exposure (10 Gy). Western blot showed IR induced a decrease in cellular IκBα levels within 1 hr and IκBα levels returned to control levels by 4 hr (Fig 3A). Treatment with curcumin suppressed the radiation-induced decrease in IκBα levels and phosphorylation of IκBα in Rh30 and Rh41 cells (Fig. 3B). These data demonstrate that curcumin inhibits radiation-induced NF-κΒ activation by suppressing the phosphorylation and degradation of IκBα in rhabdomyosarcomas.
10.1371/journal.pone.0051309.g003Figure 3 Curcumin inhibits radiation induced degradation of IκBα in vitro.
(A) Western blot showing radiation induces degradation of Iκβα in a time dependent manner in both Rh30 and Rh41 cell lines. (B) Curcumin pretreatment suppresses radiation-induced degradation of IκBα and phosphorylation of IκBα in Rh30 an Rh41.
Curcumin inhibits radiation-induced NF-κB dependent genes involved in proliferation, metastasis, and anti-apoptosis
NF-κΒ has been shown to regulate the expression of target genes involved in proliferation (cyclin D1), inflammation (COX-2), metastasis (MMP-9), and anti-apoptosis (XIAP and Bcl-2). We next performed western blot analysis to determine whether curcumin modulated these target proteins in rhabdomyosarcoma cells treated with IR. This analysis revealed that, while radiation induced the expression of cyclin D1, COX-2, MMP-9, XIAP, and Bcl-2, curcumin treatment suppressed their induction by radiation (Fig. S3).
SR-IκBα potentiates the antiproliferative and apoptotic effects of radiation
To further demonstrate the functional role of the radiation-induced canonical NF-κΒ pathway, the effect of IR on proliferation of Rh30 and Rh41cells expressing the IκBα super-repressor was analyzed. The Rh30 and Rh41 cells expressing the IκBα super-repressor were significantly more sensitive to the antiproliferative effects of 10 Gy IR when compared to the control (empty-vector expressing) cells and cells expressing the p100 super-repressor. The proliferation of irradiated Rh30 EV and Rh30 SR-p100 cell lines was 85.73±1.38% and 84.62±0.9% of control non-irradiated cells, respectively, as compared to 71.48±0.9% in the irradiated Rh30 cells expressing SR- IκBα (p = 0.0002). The proliferation of irradiated Rh41 EV and Rh41 SR-p100 cell lines was 81.66±0.53% and 80.63±0.44% of control nonirradiated cells, respectively, as compared to 68.93±1.7% in the Rh41 SR- IκBα cell line (p = 0.0002) (Fig. S4A).
The effects of the NF-κΒ super-repressors were also analyzed by Annexin V-staining 48 hr after irradiation. Both Rh30 and Rh41 SR-IκBα cells had a significantly increased number of apoptotic cells compared to empty vector after exposure to 10 Gy radiation. The Rh30 EV and SR-p100 cell line had 10.87±0.9% and 11.73±1.5% Annexin V positive cells, respectively, as compared to 24.56±2.2% for the IκBα super-repressor cell line. The Rh41 EV and SR-p100 cell line had 11.66±0.9% and 12.8±0.5% Annexin V positive cells, respectively, as compared to 21.3±1.5% Annexin V positive cells in the IκBα super-repressor cell line (p = 0.0007) (Fig. S4B).
Curcumin and radiation have synergistic antitumor activity against ARMS xenografts
We next determined whether curcumin sensitized rhabdomyosarcoma to IR in vivo. An orthotopic model of Rh30 and Rh41 ARMS was established in the right calf of CB-17 severe combined immunodeficient mice. After ∼1 month, mice with established xenografts were size-matched by tumor volume into 4 groups (n = 9/group). Mice received lyophilized vehicle IP daily, 50 mg/kg lyophilized curcumin IP daily, a single dose of 10 Gy IR, or combination therapy. A single dose of 10 Gy IR was given on day 7 of curcumin treatment for the group receiving combination therapy. Curcumin treatment was continued until mice were sacrificed. Following 3 weeks of treatment, the combination of curcumin and radiation had synergistic antitumor activity in the Rh30 xenografts (synergistic index = −0.940, SE 0.072, 95% confidence interval −1.080, −0.799). The combination group had a tumor burden of 201.8±19.33 mm3 compared to 786±44.4 mm3, 679±51.46 mm3, and 1033.4±89.71 mm3 for the curcumin, radiation and control groups, respectively. Combination therapy in the Rh41 xenografts was also synergistic (synergistic index = −0.915, SE 0.141, 95% confidence interval −1.192, −0.638). The combination group had a tumor burden of 275.1±55.9 mm3 compared to 927.4±98.7 mm3, 904.9±94.2 mm3and 1221.7±232.4 mm3 for the curcumin, radiation and control groups, respectively (Fig. 4A).
10.1371/journal.pone.0051309.g004Figure 4 Curcumin and Radiation have synergistic antitumor activity against ARMS xenografts.
(A) The combination of curcumin and radiation had synergistic antitumor activity in the Rh30 xenografts (synergistic index = −0.940, SE 0.072, 95% confidence interval −1.080, −0.799). In the Rh41 cell line, the combination of curcumin and radiation was also synergistic (synergistic index = −0.915, SE 0.141, 95% confidence interval (−1.192, −0.638). (B) The antitumor activity when curcumin was given either concomitantly with or prior to radiation.
We next confirmed the importance of the timing of curcumin administration on the effectiveness of radiation exposure. Rh30 and Rh41 tumors were established and mice were size-matched into 3 groups (n = 5). Mice received lyophilized vehicle IP daily, 10 Gy radiation followed immediately by 50 mg/kg liposomal curcumin IP daily, and 10 Gy radiation 7 days after the start of liposomal curcumin. Curcumin was continued until the animals were sacrificed. In both rhabdomyosarcoma xenografts, the effect of the timing of curcumin treatment prior to irradiation was found to be critical. In the Rh30 cell line, the group that received radiation before curcumin administration had a tumor volume of 620±68.5 mm3 compared to 242±27.5 mm3 for the group that was pretreated with curcumin before radiation (p = 0.0017). In the Rh41 cell line, the group that received radiation before curcumin administration had a tumor volume of 756±92.4 mm3 compared to 227±26.4 mm3 for the group that was pretreated with curcumin before radiation (p = 0.0003) (Fig. 4B). These results confirm that timing of curcumin administration is critical when attempting to maximize the anti-tumor effect of combination therapy.
Curcumin suppresses radiation-induced NF-κB activation in vivo
We performed EMSA to determine NF-κΒ activity in tumors harvested 4 hours after radiation therapy. EMSA confirmed that radiation induced NF-κΒ activation in both Rh30 and Rh41 xenografts and that pre-treatment with curcumin inhibited this radiation-induced NF-κΒ activation (Fig. 5A). As was done previously in our in vitro studies, we performed western blot analysis for IκBα from tumor tissues harvested post-IR to determine the mechanism whereby curcumin inhibits NF-κΒ activation in vivo. This analysis showed that in both rhabdomyosarcoma xenografts, radiation-induced NF-κΒ activation was mediated by IκBα degradation (Fig. 5B), and IκBα degradation was abrogated by curcumin.
10.1371/journal.pone.0051309.g005Figure 5 Curcumin treatment suppresses radiation-induced NF-κB activation in vivo.
Xenograft tumors were harvested 4 hr after radiation therapy. (A) EMSA revealed that in both the Rh30 and Rh41 curcumin suppressed radiation-induced activation of NF-κβ. (B) Western blot analysis revealed radiation-induced degradation and increased phosphorylation of IκBα which was suppressed by curcumin.
Curcumin enhances the effect of radiation on tumor cell proliferation and apoptosis
To investigate the mechanism of synergy, we examined the effects of curcumin and radiation on tumor cell proliferation and apoptosis by Ki67 and TUNEL immunohistochemical staining, respectively. In both rhabdomyosarcoma xenografts, the percentage of proliferating cells was significantly decreased in the combination therapy. In the Rh30 cell line, the combination therapy group had a proliferation index of 63.29±1.33% compared to 80.51±1.08%, 76.56±1.59%, and 86.47±1.02% for the curcumin group, radiation group and control groups, respectively (p<0.0001). In the Rh41 cell line, the combination therapy group had a proliferation index of 66.8±0.98% compared to 81.44±1.7%, 80.77±1.6% and 86.61±0.84% for the curcumin group, radiation group and control group, respectively (p<0.0001) (Fig. S5A).
Next, we examined tumor cell apoptosis by TUNEL staining. In both cell lines, the greatest increase in apoptotic cells was found with combination therapy. In the Rh30 cell line the combination group had 18.46±1.8 apoptotic cells/1000 cells compared to 12.15±1.1, 12.14±1.4 and 8.98±1.1 apoptotic cells/1000 cells for the curcumin group, radiation group and the control groups, respectively (p<0.0001). In the RH41 cell line the combination group had 17.11±1.1 apoptotic cells/1000 cells compared to 11.67±1.09, 10.90±.85 and 9.52±0.5 apoptotic cells/1000 cells for the curcumin group, radiation group and the control group (p<0.0004) (Fig. S5B).
Curcumin enhances the effect of radiation on tumor cell angiogenesis
We next examined tumor microvessel density by assessing CD34 immunohistochemistry. The combination therapy resulted in the greatest decrease in tumor microvessel density in both rhabdomyosarcoma xenografts. In the Rh30 cell line, combination therapy group had 20,022±2,225 CD34 pixels/hpf compared to 33,363±2,055, 37,481±2,514 and 41,245±3,225 CD34 pixels/hpf for the curcumin group, radiation group and the control group, respectively (p<0.0001) In the Rh41 cell line, the combination therapy group had 14,353±1,740 CD34 pixels/hpf compared to 34,827±2,357, 38,994±2,955 and 46,121±4,393 CD34 pixels/hpf for the curcumin group, radiation group and the control group, respectively (p<0.0007) (Fig. S5C).
IκBα super repression sensitizes rhabdomyosarcoma tumors to radiation
Next, we established orthotopic Rh30 EV, SR-p100 and SR-IκBα xenografts. Approximately 1 month after injection, mice bearing tumors of each cell line were size-matched into 2 groups, one group was subjected to IR (n = 10) while the other served as control. Five mice in each group were sacrificed 4 hr after exposure to 10 Gy radiation to confirm suppression of NF-κΒ activity. In the control tumors and tumors derived from the p100 super-repressor expressing cells exposed to IR, there was an increase in the NF-κΒ activity, specifically in complexes containing the p50 and p65 subunits. There was no increase in the NF-κΒ activity upon radiation exposure in tumors derived from SR-IκBα expressing cells (Fig. 6A), confirming that expression of the IκBα super-repressor inhibits radiation-induced NF-κΒ activation.
10.1371/journal.pone.0051309.g006Figure 6 Effects of radiation on xenografts stably expressing p100 and IκBα super-repressors.
(A) EMSA revealed that radiation treatment induced NF-κB activity in tumors with empty vector and expressing p100 super-repressor, but there was no induction of NF-κB activity in tumors that expressed IκBα super-repressors. (B) Tumors expressing SR-IκBα were more sensitivity to radiation therapy than tumors with empty vector or SR-p100.
At the end of two weeks, the remaining animals were sacrificed. Tumors derived from IκBα-SR cells were 94% smaller when IR was given, compared untreated xenografts (mean tumor volume = 62.4±10.8 mm3 vs. 1,019±61 mm3, p<0.0003). Tumors with empty vector treated with IR had a mean tumor volume of 967±64.6 mm3 that was only 27% less than the mean tumor volume of 1,331±133 mm3 in mice that did not receive radiation. Similarly, tumors derived from the p100-SR expressing cells when treated with IR, had a volume of 790±133mm3, which was only 30% less than the mean tumor volume of 1,125±109 mm3 in mice not receiving IR (Fig. 6B).
Discussion
Radiation therapy is a critical component of the multimodality approach to the treatment of many solid tumors, including rhabdomyosarcoma. However, evidence suggests that activation of NF-κΒ, a nuclear factor involved in critical cell survival signaling pathways, can not only contribute to cancer development and progression but also mediates resistance to radiation therapy. Thus, inhibition of NF-κΒ activity has the potential to sensitize tumor cells to IR.
In the two alveolar rhabdomyosarcoma cell lines used in our study, IR upregulated NF-κΒ activity in both a time (maximally at 2 hours) and dose (10 Gy) dependent manner. Supershift assays performed with antibodies directed to specific NF-κΒ subunits showed that the p50 and p65 subunits comprised the radiation-induced NF-κΒ complex. We confirmed that IR-mediated induction of NF-κΒ activity occurred via the classical pathway in experiments with enforced expression of super repressor constructs which showed abrogation of radiation-mediated NF-κΒ activation in cell lines transduced with an SR-IκBα mutant (canonical) but not an SR-p100 (non-canonical) mutant.
Treatment with curcumin, a naturally occurring inhibitor of NF-κΒ activity, prior to the delivery of ionizing radiation, inhibited the upregulation of NF-κΒ activity in the ARMS cell lines and xenografts in response to IR, as demonstrated by EMSA. This in turn led to increased IR-mediated tumor cell apoptosis and antitumor efficacy. Activation of the classical NF-κΒ pathway is normally achieved by phosphorylation and subsequent degradation of IκBα, a cytoplasmic protein that binds and inactivates NF-κΒ by preventing its translocation into the nucleus. In our study of ARMS xenografts treated with curcumin prior to ionizing radiation, curcumin prevented the degradation of IκBα, by inhibiting the formation of the activated form of IκBα (phospho-IκBα).
The timing of curcumin administration was critical to its radiosensitization effect as its ability to effect NF-κΒ inhibition prior to the administration of ionizing radiation was essential. We found maximal induction of NF-κΒ activation two hours after the delivery of ionizing radiation to ARMS cells in vitro. We did not perform time course experiments in vivo in our xenograft model but did find improved antitumor activity when mice were treated with curcumin for one week prior to the delivery of ionizing radiation as compared to when both were given concomitantly.
In addition to demonstrating that blocking activation of NF-κΒ by curcumin resulted in IR-mediated induction of NF-κΒ-responsive genes involved in cell proliferation (cyclin D1) and survival (XIAP and bcl-2), we showed that the combination of curcumin and IR caused a profound decrease in microvessel density in rhabdomyosarcoma xenografts. Thus, the mechanism of synergy between curcumin and IR is likely to be multifactorial and involves direct tumor cell cytotoxicity, inhibition of angiogenesis as well as radiosensitization.
One of the disadvantages of curcumin for use in the clinical setting is its limited solubility. We have overcome this problem by using liposomal curcumin. Our in vivo study with this formulation showed it to be well-tolerated and therapeutic doses should be easily achieved.
This is the first study, to our knowledge, that demonstrates the adjuvant antitumor activity of curcumin in vivo against rhabdomyosarcoma tumors treated with radiation therapy. In addition, we have shown that this in vivo radiosensitization is due to the abrogation of the increase in the NF-κB activity that occurs in these solid tumors in response to ionizing radiation via the classical pathway. We further confirmed these findings through suppressing NF-κB activation by genetic means. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of adjuvant ionizing radiation and warrants study in clinical trials.
Supporting Information
Figure S1
Effects of curcumin, radiation and combination therapy on rhabdomyosarcoma tumor cell proliferation. (A) Curcumin treatment resulted in a dose-dependent decrease in proliferation in both Rh30 and Rh41 cell lines. IC50 concentrations determined at 96 hr after treatment varied from 17.5 µM±1.92 for Rh30 and 19.3 µM±1.89 for Rh41 (B) Radiation therapy resulted in a dose-dependent decrease in proliferation in both the Rh30 and Rh41 cell lines. IC50 doses determined at 72 hr after treatment varied from 18.3±1.3Gy for Rh30 to 18.9±1.2Gy for Rh41. (C) The effects of combination therapy was synergistic in both cell lines. The Rh30 treatment resulted in a positive α non-additivity parameter of 5.54 with a 95% confidence interval (3.87, 7.20). The Rh41 treatment resulted in an α of 5.86 with a 95% confidence interval (4.55, 7.17).
(TIF)
Click here for additional data file.
Figure S2
Effects of curcumin, radiation and combination therapy on rhabdomyosarcoma apoptosis. Cells were treated with 10 µM liposomal curcumin (24 hr) prior to radiation exposure (10 Gy) and then analyzed for apoptosis by Annexin V staining. The combination therapy of curcumin and irradiation significantly increased apoptosis compared to control in both the Rh30 and Rh41 cell lines (p = .00003 and p = .0001, respectively).
(TIF)
Click here for additional data file.
Figure S3
Curcumin inhibits radiation-induced upregulation of NF-κΒ dependent genes. Western blot analysis revealed that radiation induced NF-κΒ dependent genes (MMP-9, XIAP, Bcl-2, Cox-2, Cyclin D1), which was suppressed by curcumin pretreatment in Rh30 and Rh41.
(TIF)
Click here for additional data file.
Figure S4
IκBα super-repression sensitizes rhabdomyosarcoma to radiation in vitro. (A) Rhabdomyosarcoma cell lines expressing the IκBα super-repressor were more sensitive to radiation compared to the cell lines expressing empty vector or p100 super-repressor. (B) The Rh30 and Rh41 SR-IκBα cell lines showed a significant increase in Annexin V positive cells compared to the cell lines expressing empty vector and SR-p100.
(TIF)
Click here for additional data file.
Figure S5
Curcumin enhances the effect of radiation on tumor cell proliferation, apoptosis and angiogenesis. Ki67, TUNEL, CD34 immunohistochemistry staining was used to analyze Rh30 and Rh41 tumors for tumor cell proliferation, apoptosis and angiogenesis, respectively (A) Combination therapy resulted in a significant decrease in tumor cell proliferation (p<0.0001 and p<0.0001, respectively), shown is representative Ki-67 staining (400X) (B) Combination therapy resulted in a significant increase in the TUNEL positive cells compared to control (p<0.0001 and p<0.0004, respectively), shown is representative TUNEL staining (400X) (C) Combination therapy resulted in a significant decrease in tumor microvessel density compared to control tumors (p<0.0001 and p<0.0007, respectively), shown is representative CD34 staining (400X).
(TIF)
Click here for additional data file.
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278 : 166 –74 .12169272 | 23408929 | PMC3567084 | CC BY | 2021-01-05 17:18:22 | yes | PLoS One. 2013 Feb 7; 8(2):e51309 |
==== Front
PLoS One
PLoS ONE
plos
plosone
PLoS ONE
1932-6203
Public Library of Science San Francisco, USA
23409045
PONE-D-12-27856
10.1371/journal.pone.0055790
Research Article
Medicine
Oncology
Cancer Risk Factors
Hormonal Causes of Cancer
Cancers and Neoplasms
Genitourinary Tract Tumors
Prostate Cancer
Basic Cancer Research
Cancer Treatment
Urology
Prostate Diseases
Prostate Cancer
Genitourinary Cancers
Somatostatin Derivative (smsDX) Targets Cellular Metabolism in Prostate Cancer Cells after Androgen Deprivation Therapy
smsDX Regulates Prostate Cancer Cell Metabolism
Yan Lei 1
Xing Zhaoquan 1
Guo Zhaoxin 1
Fang Zhiqing 1
Jiao Wei 1
Guo Xiaoyu 2
Xu Zhonghua 1 *
Fang Zhenghui 3
Holmberg Anders 4
Nilsson Sten 4
Liu Zhaoxu 1 2 4 *
1 Department of Urology, Qilu Hospital, Shandong University, Jinan, China
2 Aging and Health Center, School of Nursing, Shandong University, Jinan, China
3 Department of Obstetrics and Gynecology, Jinan Central Hospital, Shandong University, Jinan, China
4 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden
Ling Ming Tat Editor
Queensland University of Technology, Australia
* E-mail: [email protected] (ZXL); [email protected] (ZHX)
Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ZL SN. Performed the experiments: LY Z. Xing ZL ZG Zhiqing Fang. Analyzed the data: Z. Xu AH Zhenghui Fang WJ XG. Contributed reagents/materials/analysis tools: AH SN. Wrote the paper: ZL.
2013
7 2 2013
8 2 e5579012 9 2012
31 12 2012
© 2013 Yan et al
2013
Yan et al
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Cancer cell metabolism responsive to androgen deprivation therapy (ADT) may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-deprivation therapy. To investigate the metabolism regulation effects on androgen-independent growth of prostate cancer, an established LNCaP-s cell model that resembles the clinical scenario of castration-resistant prostate cancer (CRPC), was used in this current study. This cell line was cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition, resembling clinical androgen deprivation therapy. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay. We evaluated differentially expressed proteins of the parental LNCaP cells and LNCaP-s cells after ADT by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle. 56 proteins were found differentially expressed in LNCaP-s cells compared to LNCaP cells, majority of them were down-regulated after ADT treatment. 104 proteins of LNCaP cells and 86 in LNCaP-s cells, separately, were found differentially expressed after treatment with smsDX, When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins were found to be involved in the cellular metabolism and mitochondrial function regulation. LNCaP-s as potential metastatic androgen-independent cancer cells, its metabolism and mitochondrial functions could be altered by a new somatostatin derivative smsDX, the smsDX regulatory effects on metabolism in LNCaP-s deliver more therapeutic information with the treatment of CRPC.
Grant Sponsor: The National Natural Science Foundation of China (No. 81172435) and Shandong Natural Science Foundation (No. ZR2010HM026). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Prostate cancer is the most common malignancy in men, and the leading cause of cancer-related mortality in US and Europe males [1]. The tumor progression to CRPC stage is a complex process that may be involving both clonal selection and adaptive mechanisms in heterogeneous tumors composed of cells that respond differently to androgen deprivation therapy (ADT). However, the mechanisms by which tumors acquire androgen independence remain unclear and need to be addressed before effective treatment strategies can be developed.
ADT is commonly employed in the treatment of advanced prostate cancer. But androgen deprivation therapy is not curative [2], so the lethal CRPC is inevitable. Signs of vascular degeneration, hypoxia, and metabolic stress in the prostate tumor tissue are exacerbated following surgical or medical castration [3]. After a short remission period, the majority of prostate cancer becomes androgen-independent. CRPC cells after ADT are able to survive the low oxygen and nutrient environment and emerge with a different phenotype. Androgen deprivation is known to induce neuroendocrine (NE) differentiation in LNCaP cells, and involves in the transition to androgen independence [4], [5]. NE tumors have been proven to overexpress somatostatin receptors (SSTRs) [6]. The SSTR1-5 expression could be regulated by somatostatin and its derivative smsDX possible via the regulation of the mitochondria of LNCaP that eventually could trigger mitochondrial-mediated apoptosis [7]. Somatostatin analogs bind to SSTRs and are believed to have dual antitumor activity, both direct (anti-proliferative) and indirect (inhibition of various peptide hormones secreted by the tumor cells) [8], [9]. Somatostatin analog, lanreotide has been demonstrated to have considerable antineoplastic effect in various tumors, including CRPC [10]. But the regulation of somatostatin analog on prostate cancer cellular metabolism has not been clearly addressed.
We argue that inhibition of androgen receptor (AR) expression is in itself sufficient to induce cell death in AR-positive cells. But when these AR-positive cells gradually lost AR expression or in a lower AR expression in prostate cancer cells, those CRPC cells could get energy supply via mitochondrial activities. According to the findings of Sotgia F group [11], epithelial cancer cells could take up energy-rich metabolites from neighboring stromal fibroblasts which provide the necessary energy-rich microenvironment for facilitating tumor growth and angiogenesis. These starved cells stripped of androgen could use these metabolites in the mitochondrial tricarboxylic acid cycle (TCA), resulting in a higher proliferative capacity. For CRPC cells emerging after ADT, up-regulate enzymes that convert adrenal androgens to testosterone and DHT (in particular AKR1C3) further enhancing their intratumoral androgen synthesis and reactivating AR transcriptional activity [12], AR reactivation is increased intratumoral synthesis of testosterone and DHT from weak androgens produced by the possibly de novo from cholesterol in host stroma and extracellular metabolites.
The principal problem arising from prostate cancer is its propensity to metastasize. Local invasion is one of the fundamental early steps in metastasis, as without it tumor spread cannot occur. The multistep process of invasion and metastasis has been schematized as a sequence of discrete steps, often termed the invasion-metastasis cascade [13], [14]. During this cascade the metabolic reprogramming to support synthesis of the new proteins, lipids, and nucleic acids is critical for cell growth and division [15]. We speculate that the cellular metabolic alterations would accompany the prostate cancer progress to lethal CRPC status. Our study will focus on the smsDX inhibitory effects on the invasiveness of prostate cancer and regulation of relating cellular metabolism after inhibition of AR activity, mediated by androgen depletion. Using LNCaP and LNCaP-s cells to examine the proteins involving in the cell metabolism and energy functions in prostate cancer and to determine the regulatory effects caused by somatostatin derivative smsDX, we demonstrated ADT down-regulates most of mitochondrial proteins, possibly results in the activation of mitochondrial-mediated apoptotic pathway. The smsDX effects on ER leads to overexpression of GRP78, together with other two ER proteins PDIA1 and PDIA3. smsDX exerts its effects by dysregulating of metabolic enzymes at multiple levels, which involves in the process of CRPC cell invasiveness and survival. In conclusion, these results suggest that ADT regulates mitochondrial-mediated and ER stress signalling in LNCaP cells and leads to reprogramming of metabolism in CRPC cells, smsDX regulatory effects on the CRPC cells provides useful information for the treatment of CRPC.
Materials and Methods
Cell culture and Reagents
LNCaP human prostate cancer cell line (American Type Culture Collection, Rockville, MD, USA) were routinely maintained in the regular medium (Phenol Red-positive RPMI 1640 medium supplemented with 5% FBS, 1% glutamine, and 0.5% gentamicin) as described previously at 37°C in a humidified atmosphere of 5% CO2. The medium was changed two times by week and the cells were trypsinized and subcultivated once a week. For experiments, androgen sensitive LNCaP cells with passage numbers 28–33 were utilized. To establish an androgen-independent prostate cancer cell line, we cultured a LNCaP-s cell line from a parental prostate cancer cell line LNCaP in a androgen-deprivation condition. After 8–12 passages, LNCaP-s cells were treated with smsDX. Somatostatin was from Ferring, Kiel, Germany. The cell culture was treated with smsDX (from Professor Sten Nilsson Lab) or with somatostatin for three days, 1 nM per day, as described by Liu Z [7]. Anti-human AR, CgA and NSE, anti-TOM40 antibody (sc11414), β-actin (rabbit anti-actin antibody R-22) were purchased from Santa Cruz Biotecnology. TCTP (#5128), STMN1 (#3352) and VDAC2 (#9412) antibodies were purchased from Cell Signaling commercially. CBX3 (HPA004902) and GRP78 (G9043) antibodies were purchased from Sigma commercially.
Wound healing assay
Cells were grown to confluence on 6-well tissue culture plates and a wound was made by scraping in the middle of the cell monolayer with a P200 pipette tip. After floating cells were removed by extensive washing with ice-cold phosphate buffered saline fresh complete medium containing 10 nM smsDX or the corresponding amount of PBS was added. Migration and cell movement throughout the wound area were examined after 24 hours.
Matrigel Invasion Assay
Invasion assay was performed using Matrigel coated Transwell inserts (BD™) with 8 µm pores in 24-well plates, as per manufacturer's instructions. Briefly, a suspension of 1×106 cells in 100 µl serum-free medium was added to the insert and 500 µl of RPMI 1640 medium containing 20% FBS supplemented with 1–10 nM smsDX or the corresponding amount of saline were added to the bottom of the well. After the plates were incubated for 48 hours at 37°C the inserts were fixed in methanol, the filters were stained with 0.1% crystal violet and the number of cells that invaded through the Matrigel coated Transwell inserts were counted at 40× magnification. The number of cells was counted in independent triplicate experiments in at least 10 fields per well. The assays were repeated 3 times.
Western blot analysis
In androgen-dependent LNCaP cells after ADT treatment, we cultured a stable LNCaP-s cell line. The androgen independent prostate cancer cell biomarkers and proteins affected by smsDX were tested. Western blotting was performed to validate several selected differentially expressed proteins identified by 2-DE based MS. Primary antibodies were used at the following dilutions: goat anti-human AR, CgA and NSE, 1∶1000; β-actin, 1∶1500; anti-TOM40 antibody, 1∶800. TCTP, 1∶1000; STMN1, 1∶1000; VDAC2, 1∶1000; CBX3, 1∶1000; GRP78, 1∶1000. Western blots were performed as described [16]. In short, cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1% NP-40 and 5 mM EGTA, 50 mM sodium flu-oride, 60 mM β-glycerol-phosphate, 0.5 mM sodium-vanadate, 0.1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin. Protein samples (35 µg) were subjected to a 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were first incubated with 5% nonfat milk in Tris-buffered saline (TBS). After washing three times in 0.1% Tween 20-TBS (TBST), the membranes were incubated with different primary antibodies separately at 4°C overnight, followed with the corresponding secondary antibodies separately (1∶2000) for 1 h at room temperature and the antibody-bound proteins were detected by the ECL system (Amersham Biosciences, Little Chalfont Buckinghamshire, UK).
Sample preparation and protein extraction and concentration
The cellular extraction from LNCaP and LNCaP-s cells and the preparation of the total cell lysate were performed as previously described by [17]. Protein determination was made using Pierce BCA protein assay reagent (Rockford, IL, U.S.A.).
IEF and SDS-PAGE
The samples were diluted to a total volume of 250 µl, 0.2% Pharmalyte, 8 M urea, 0.3% DTT, 2 M CHAPS and a trace of bromphenol blue (Sigma). An amount of 100 µg of protein was loaded on each strip via rehydration using non-linear pH 3–10 Ready Strip IPG, strips (Bio-Rad, Hercules, Ca, USA). Focusing was carried out for a total of 45,500 Vh in a PROTEAN IEF cell (Bio-Rad). Precast gels (12.5% homogenous Tris-HCL Criterion) SDS-PAGE (Bio-Rad) were run using a Criterion Dodeca cell gel apparatus (Bio-Rad). A total of 4 gels were run per sample group. The electrode running buffer was 25 mM Tris, 192 mM Glycine, 0.1% w/v SDS. Gels were run at 250 V for approximately 1 hour until the bromophenol blue marker had reached the bottom of the gel at a temperature of approximately 15°C. Proteins were visualized by silver staining as described by [18].
Gel scanning and image analysis
2-DE gels were scanned at 100 µm resolution (12-bits/pixel) using a GS-710 laser densitometer (Bio-Rad). Data was analyzed using PDQuest™ software Version 7 (Bio-Rad). After auto-detection of all protein spots, gel-images were carefully edited. The individual proteins quantities were expressed as ppm of the total integrated OD. All 2-DE maps were matched and evaluated independently. The methodological reproducibility of the 2-DE analysis was determined using group correlation analysis. Briefly, the total optical density is directly correlated to the total protein concentration. Minor differences in gel loading, running conditions, and silver staining may affect sample comparisons and affecting the 2-D gel reproducibility. Four gels were run from each treatment group and comparisons of the intensity of matched spots between 2-DE gels were performed using the correlation coefficient analysis. A correlation coefficient was measured between two gels based on the optical densities of the same spots in the two gels being compared. A correlation coefficient of 1 will imply that the two samples being compared are identical. In a group consisting of 4 samples, a maximum of six pair wise comparisons are possible. The average correlation coefficient among the smsDX samples was 0.85 (n = 6 gel pairs, range 0.80–0.92).
Mass spectrometry analysis
Proteins were identified with a vMALDI-LTQ instrument (Thermo Electron, San José, CA, USA). The spot picking, destaining, digestion, extraction, sample preparation and spotting on MALDI target plates were carried out using a spothandling workstation (ETTAN Spothandling workstation, GE Healthcare) and a standard protocol provided by GE Healthcare. The plate containing the combined extracts was evaporated to dryness. Each sample was prepared by constituting the dried peptides in 2.5 µl of matrix solution (2.5 mg/ml of α-cyano-4-hydroxy-cinnamic acid (Sigma) in 50% acetonitrile containing 0.05% TFA). 2.0 µl sample was spotted on a clean MALDI target slide surface and allowed to dry. The samples were analysed with a vMALDI-LTQ (Thermo Electron, San José, CA, USA). The analysis was done using Xcalibur 1.4 software in data dependent mode. A survey scan (MS) was followed by MS/MS scans on the 5 most abundant ions. This string of 6 scan events was repeated six times for each sample spot. Dynamic Exclusion™ ensured that in total 30 different peptides were selected and fragmented for each sample. The MS spectra were collected in the 900–2000 Da mass range while the mass range for the MS/MS spectra were automatically selected by the system based on a Q value of 0.25. The standard collision energy of 38 was set for all the analysis. A time limit of 5 minutes/sample was selected, whether or not the 30 MS/MS spectra could be acquired. Database searches were done using both the MASCOT and Sequest search algorithm against the human session of the IPI protein database (version 2.38). The two searches were compared in the in house developed software Promiscuous MS/MS. A minimum of two peptides and A Mascot score of 45 were required for a protein to be accepted as identified.
Results
Establishment of androgen-independent cell line, LNCaP-s and detection of AR, CgA and NSE expression in LNCaP-s cells
To address the natural progression of prostate cancer from androgen sensitive to androgen independent state, a prostate cancer cell model is very important for in vitro study. So we cultured a LNCaP-s cell line from a parental prostate cancer cell line LNCaP in a androgen-deprivation condition. NE cells are characterized by a neuronal-like phenotype which produce and secrete a series of neuropeptides involved in tumor proliferation, transformation and metastasis [19]. The morphologic characteristics showed a neuronal morphology with compactly rounded cell bodies, having extended and fine branched processes. Thus androgen-sensitive LNCaP cells acquired a NE-like phenotype (Figure 1) LNCaP-s in an androgen-reducing condition. The NE-like cells, characteristics were evaluated by testing the markers of NED, chromogranin A (CgA) and neuron-specific enolase (NSE). After culturing in the androgen-reduced condition, the LNCaP cells showed a decreased expression level of AR, an increased NSE expression level, but CgA with no significant expression. Figure 1 showed different expressions of AR, CgA and NSE in LNCaP-s cells.
10.1371/journal.pone.0055790.g001 Figure 1 Characterizition of LNCaP-s cells.
Parental LNCaP cells have an epitheial morphology and LNCaP-s cells showed a neuronal morphology. AR, CgA and NSE expressions in LNCaP and LNCaP-s cells examined by RT-PCR and Western Blotting.
smsDX inhibited the invasiveness of prostate cancer cells
It is well established that somatostatin and its analogue effects on the proliferation of prostate cancer cells [20]. But the somatostatin effects on invasiveness had not been investigated. To assess the effects of smsDX on the invasiveness of prostate cancer cells we used wound healing assay and Matrigel™ invasion assay, The covered area in the wound and the number of cells invading through a Matrigel chamber were significantly smaller for cells treated with smsDX than they were for control cells treated with vehicle in LNCaP and LNCaP-s cells (Figure 2 and 3). The inhibition of smsDX shows a dose-dependent manner with the cell number invading ability in both LNCaP and LNCaP-s cells. These results suggest that smsDX decreased the invasiveness of prostate cancer cells.
10.1371/journal.pone.0055790.g002 Figure 2 Inhibition of cell migration of prostate cancer cells LNCaP/LNCaP-s by smsDX.
Representative photomicrographs demonstrate wound closure in LNCaP cells (A) and LNCaP-s cells (B). Monolayers of LNCaP/LNCaP-s cells were disrupted with sterile pipette tip to create uniformand treated with PBS or 1–10 nM smsDX for 24 hours.
10.1371/journal.pone.0055790.g003 Figure 3 Inhibition of cell invasiveness of prostate cancer cells LNCaP/LNCaP-s by smsDX.
Matrigel invasion data when LNCap/LNCaP-s cells in upper well were incubated in serum-free medium and lower well was filled with serum-free medium and 1–10 nM smsDX or PBS. After 24 hours, number of cells that invaded through Matrigel was counted in at least 10 fields per well. Representative photographs reveal LNCaP (A) and LNCaP-s (B) cells that invaded through Matrigel. Reduced from ×100.
2D gel electrophoresis analysis
Figure 4 shows representative 2D gels of control and smsDX samples in LNCaP and LNCaP-s cells. Three replicates were run for each group, control and smsDX. The smsDX effects on LNCaP and LNCaP-s cells were compared separately with the control group using a Mann-Whitney test.
10.1371/journal.pone.0055790.g004 Figure 4 Representative examples of 2-DE gels.
Whole-cell lysate was subjected to 2-DE using IPG strips pH 3–10 in the first and 12.5% SDS polyacrylamide gel in the second dimension. A, Parental LNCaP cells, B, derivative LNCaP-s cells. C, LNCaP+smsDX , D, LNCaP-s+smsDX.
Proteins differentially expressed between LNCaP and LNCaP-s cells
A total of 222 spots were successfully identified using 2DE-based MS. In the first analysis, smsDX-treated LNCaP cells were compared to LNCaP control group using a Mann-Whitney test. 56 proteins were found to be differentially expressed in LNCaP and LNCaP-s (Table S1). In the second analysis, 104 and 86 proteins, separately, were found differentially expressed in LNCaP cells and LNCaP-s cells following smsDX treatment (Table S2A and Table S2B). The expression of specific isoforms of metabolic enzymes has been showed to be crucial for the adaptation of tumor cells to changes in nutrient availability, especially for glycolytic enzymes [21]. So isoforms of proteins were also listed in this table. The same protein was sometimes found in multiple closing spots on 2DE gels, it is possible due to posttranslational modification of protein after stress.
Fifty-six proteins were found down-expressed (more than 1.2-fold change) in LNCaP-s cells compared to the parental LNCaP cells, as listed in table S1.
Proteins both in LNCaP and LNCaP-s cells affected by smsDX incubation with a fold change ≥1.2 were listed in table S2A and table S2B.
Sorted functions of proteins in the effect of smsDX on prostate cancer (LNCaP-s) cells after ADT treatment
The different functions of proteins with accession code refer to the website, http://www.uniprot.org/uniprot/. The proteins identified were categorized on the basis of their known biochemical functions including metabolism, signalling transduction, maintenance of cell structure, transcriptional regulation and cell cycle regulation. Most of androgen deprivation stressed proteins were down-regulated after ADT treatment. When we explored these protein functions within the website UniProtKB/Swiss-Prot, surprisingly, most of the proteins affected by ADT therapy were found to be involved in the cell metabolism and mitochondrial function regulation. Based on the putative functions in the KEGG pathway database (http://www.genome.jp/kegg/pathway.html), Table S3(A,B,C) were derived from table S2A and table S2B to highlight the similarities/differences in the effect of smsDX between LNCaP and LNCaP-s cell lines. Table S3A listed forty-eight common proteins between LNCaP and LNCaP-s cells after exposure to smsDX. Table S3B, S3C listed the differentially affected proteins in LNCaP and LNCaP-s cells, separately.
Androgen deprivation could affect mitochondrial protein expression, for example, HSPD1, ETFA, GLUD1, PMPCB and et al. These proteins were found belonging to mitochondrial proteins involving in glucose metabolism, the production of reactive oxygen species (ROS) and intrinsic mitochondrial-mediated apoptotic function. The proteins with important roles in cell metabolic process, including energy (APRT, ATP5B, CKB, TUBB), lipid (ACAT2, ACADM, PRDX6), glucose (ENO1, TPI1) and amino acid and protein biosynthesis (PHGDH, EIF5A, EIF1AY) , were found down-regulated in LNCaP-s cells. Some metabolic enzymes involved in the TCA were also identified. ADT treatment leads to a decline in the protein expression of several ER chaperones, including GRP78, ERP29, PDIA1 and PDIA3 protein.
Validation of proteins affected by smsDX and ADT treatment in prostate cancer cells with Western Blotting
Proteins in LNCaP-s cells involving in different signaling pathways were identified in this current study, these proteins could be regulated by smsDX at different change fold as listed in table S2. Validation of proteins affected by smsDX and ADT treatment in prostate cancer cells were carried out by Western Blotting. Several mitochondrial proteins, ER proteins and proteins involving metabolism were selected to detect for validation with immunoblotting. Figure 5 showed that TCTP, STMN1, and CXB3 in LNCaP cells after ADT were down-regulated and TOM40, GRP78 and VDAC2 which involving in the mitochondrial activity in LNCaP-s cells were up-regulated by when treated with smsDX.
10.1371/journal.pone.0055790.g005 Figure 5 Validation of selected proteins by using western blotting.
A, Lower expressions of protein TCTP, STMN1, and CXB3 in LNCaP cells after ADT in LNCaP-s cells. B, Down regulated expressions of TOM40, GRP78 and VDAC2 in LNCaP cells by androgen-deprivation, up regulated expressions in LNCaP-s cells by smsDX treatment.
Metabolic pathways regulated by somatostatin derivative (smsDX) in prostate cancer cells
Based on the KEGG pathway database (http://www.genome.jp/kegg/pathway.html), we presented a diagram of metabolic pathways regulated by smsDX in prostate cancer from androgen dependent to CRPC status (Figure 6). The enzymes in circle were identified by 2-DE MS in this current study.
10.1371/journal.pone.0055790.g006 Figure 6 Metabolic pathways regulated by somatostatin dervivative (smsDX) in prostate cancer cells.
Enzymes and metabolites that are part of glycosis, fatty acide synthesis and the TCA cycle are shown. G6PD, Glucose-6-phosphate 1-dehydrogenase. ENO1, Alpha enolase. PPP, pentose phosphate pathway. ACAT2, Acetyl-CoA acetyltransferase. ACAMD, Acyl-CoA dehydrogenase. IDH2, Isocitrate dehydrogenase [NADP]. MDHM, Malate dehydrogenase. GLUD1, Glutamate dehydrogenase1. α-KG, α-Ketoglutarate. This simplified diagram is based on the KEGG pathway database (http://www.genome.jp/kegg/pathway.html). The enzymes in circle were identified by 2-DE MS in this current study.
Discussion
Androgen plays an essential role in prostate cancer growth, so androgen deprivation and the blockade of AR signaling axis is currently the main treatment for prostate cancer and its progression. It is well documented that clinical androgen-deprivation therapy is associated with increased NE differentiation in prostate carcinomas, and involved in the transition to androgen independence [22], NE cells are characterized by a neuronal-like phenotype: they are non-proliferative and express neuronal-like proteins, such as NSE and CgA. Androgen deprivation can influence the serum CgA levels to different extents in prostate cancer [23]. The overexpression of NSE in LNCaP-s in our study was found in the progression of NED from androgen dependent LNCaP cells. In this current study, after up to 10 passages of LNCaP cells in a reduced androgen condition, the NE-like characteristics of LNCaP-s were identified by a neuronal morphology and altered expressions of AR and NSE.
The effects of somatostatin inhibition on cancer cell viability and proliferation have been well examined but to our knowledge, its effects on invasiveness had not been investigated. Using wound healing and Matrigel invasion assay we observed that smsDX inhibited the invasiveness of both LNCaP and LNCaP-s cells. Although the exact mechanisms of smsDX induced suppression of invasiveness are not currently clear, it might be due to responses to damaged mitochondrial functions. In our previous studies [7], [24], smsDX regulated mitochondrial and related proteins both in androgen dependent and independent prostate cancer cells, so the cancer-associated alterations in metabolism are possibly responsible for the cell proliferation and survival signals in recurrent prostate cancer and CRPC.
To determine the metabolism regulation effects of smsDX on progression of prostate cancer from androgen-dependent to androgen independent status, we performed two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis. After comparison of protein expression in LNCaP-s and its parental LNCaP cells, fifty-six proteins were found to be differentially expressed up to 1.2 fold change as showed in table S1. These proteins were sorted according to their different functions in cell metabolism, including sugar, energy, lipid and amino acid process. The results suggest that androgen-regulated metabolic alteration is the main differences during the development of prostate cancer from androgen-sensitive to androgen-resistant status. The similar and different effects caused by smsDX were found between LNCaP and LNCaP-s cells. The stress caused by androgen deprivation and smsDX to prostate cancer cell environment could possibly caused mitochondrial function damage and activate AMPK pathway in the prostate cancer cells. Mitochondrial processes play an important role in tumor initiation and progression. Increased metabolic activity is a hallmark of proliferating cancer cells [25]. So suppressing the cancer cell metabolic activity provides an alternative strategy for CRPC stage. ADT for prostate cancer caused abnormal microenvironment condition elicit responses from tumor cell to affect metabolic activity. These adaptations optimize tumor cell metabolism to get energy and nutrients from tumor microenvironment. Several reports have shown that androgen biosynthesis and AR signaling in prostate cancer cells are intimately affected by lipogenesis [26]–[28]. So targeting the possible underlying molecular mechanisms linking metabolism could facilitate further development of promising therapeutic approaches for CRPC status.
Based on the data we collected here, we found the smsDX regulated PI3K/AKt/mTORC1 pathway and TCA cycle via up and down regulating different metabolic proteins listed in tbale S2A and table S2B. In prostate cancer AKt is activated via the PI3K pathway that has emerged as a critical pathway for cell survival. Removing androgen support from LNCaP cells triggers a series of events, including cell cycle arrest and increased PI3K/Akt activity, culminating in the eventual acquisition of the androgen- independent phenotype [29]. Activated PI3K/AKt leads to enhanced glucose uptake and glycolysis [30].This pathway also promotes glucose carbon flux into biosynthetic pathways that rely upon functional mitochondrial metabolism, including fatty acid, cholesterol and isopronoid synthesis all require acetyl-CoA. AKt also activates ATP-citratelyase (ACL) promoting the conversion of mitochondrial-derived citrate to acetyl-CoA for lipid synthesis. mTORC1 is a well-characterized cell growth regulator acting as downstream of PI3K/AKt, has many effects interwined with mitochondrial metabolism. mTORC1 is best known for enhancing protein synthesis [25]. Alpha enolase (ENO1), also known as pyruvate dehydrogenase phosphatase, it is critical for cellular energy metabolism [31]. ENO1, as a key glycolytic enzyme, plays a critical role in anaerobic glycolysis. In this current study, ENO1 could be up regulated by smsDX, which showed that smsDX has a regulatory effect on glycolysis in prostate cancer cells.
The TCA cycle is a central pathway in the metabolism of sugars, lipids and amino acid, TCA cycle metablites result in reduced cellular differentiation. [32] Glutamine is a non-essential amino acid that is metabolized to glutamate and enters the TCA cycle as alpha-ketoglutarate, resulting in high ATP generation via oxidative phosphorylation [33]. Glutamine is the primary mitochondrial substrate and is required to maintain mitochondrial membrane potential and integrity as well as support of the NADPH production needed for redox control and macromolecular synthesis [34]. Mitochondrial enzyme glutaminase converted glutamine→glutamate→α-ketoglutarate (α-KG) by glutamate dehydrogenase, α-KG involves in TCA and can provide carbon backbones for cellular anabolic reaction [35]. NADPH functions as a cofactor and critical antioxidant provides the reducing power for both the glutathione (GSH) and thioredoxin (TRX) that scavenge ROS and repair ROS-induced damage [36]. NADP-dependent isocitrate dehydrogenase IDH1 and IDH2 convert isocitrate to α-KG. The metabolism of amino acid and fatty acids, like glucose, is reprogrammed to provide the building block for cancer cell growth and proliferation. DJ-1 and SOD1 identified in the current study were also found to have antioxidant properties [37]. Apparently, enzymes affected by smsDX, for example, glutamate dehydrogenase1, isocitrate dehydrogenase, glutathione synthetase, isocitrate dehydrogenase [NADP], Glucose-6-phosphate 1-dehydrogenase, were involved in the regulation of prostate cancer cell metabolism via different metabolic pathways. These adaptations altered tumor cell metabolism for proliferation by regulating multiple levels of energy in the form of ATP, biosynthetic capacity and the maintenance of balanced redox status. Here we demonstrated a simplified regulatory pathways (Figure 6) [38], [39] altered by smsDX during the development of prostate cancer. These metabolites and enzymes in this diagram, identified by 2-DE based MS analysis, are involving in the multiple pathways supporting cancer cells: glycolysis, TCA cycle, pentose phosphate pathway, glutaminolysis and lipid and nucleotide synthesis. For example, the acetyl-CoA groups (ACAT, ACADM and ACDSB) identified in our study could be activated by smsDX treatment which means the perturbation effect of smsDX on TCA cycle in mitochondrion in prostate cancer cells. Acetyl-CoA, a central metabolite at the intersection of carbohydrate, fatty acid, and acid oxidation, exerts tremendous influence on cell signaling.
Currently, very little information is currently available on the mitochondrial and ER proteins expression linked profile of clinical prostate cancer. The ER and mitochondria are physically and functionally linked, and there is increasing evidence of the GRPs influencing ER and mitochondrial cross-talk to maintain mitochondrial function. GRP78 is traditionally regarded as a major ER (endoplasmic reticulum) chaperone facilitating protein folding and assembly, protein quality control and regulating ER stress signaling [40]. The cytoplasmic GRP78 isoform is a newly identified regulator of the ER stress signalling pathway [41], in addition to the function of canonical GRP78 in the cytoplasm. Beyond the ER, the mitochondrial, nuclear and secreted forms of GRP78 have been linked to cellular homoeostasis and therapeutic resistance. The partial reduction in GRP78 in white adipose tissue leads to the elevated expression of GRP75, suggesting increased energy expenditure in the mitochondria probably as a compensatory measure. It is possible that GRP78 might physically interact with GRP75 in the mitochondria, since it has been reported that GRP78 is also localized in the mitochondria under ER stress [42]. GRP78 is expressed on the cell surface of prostate cancer cells and appears to mediate the signal transduction of Beta2-M [43]. It correlates with the development of androgen-independent disease and shorter overall survival in prostate cancer patients [44]. As a receptor, ligation of surface GRP78 with its ligands, such as α2-M and GRP78 auto-Abs, also activates the PI3K/Akt pathway. The activated PI3K/Akt pathway can further enhance the stability of GRP78 [45]. Thus, GRP78 and PI3K/Akt pathway may thereby constitute a positive feedback loop, which is involved in protecting tumors against hypoxia and nutrient starvation and regulating cell metabolism in the microenvironment. Arap et al. [46] has recently identified GRP78 as a potential molecular target that may prove useful for translation into clinical applications.
Our data show that: (i) proteins analysis of human prostate cancer cell lines reveals that proteins involving cancer cell metabolism could be alerted by smsDX. (ii) LNCaP-s was a derivative cell line from LNCaP cell lines, so it has the most same characteristics with LNCaP cells. After ADT treatment, most prostate cancer cells will die from starvation/energy stress, LNCaP cells become to LNCaP-s cells with NE-like phenotype which mimic the clinical progression from androgen dependent to androgen independent CRPC stage. Data presented in this article are collected from the study of established AR-positive, androgen-dependent and lower AR expression human prostate cancer cells (LNCaP/LNCaP-s), additional study may be warranted to define the links with the host microenvironment, for example, cancer-associated fibroblast cell interaction which was proposed by Sotgia F in his “two-compartment tumor metabolism” model, these catabolic host cells could fuel anabolic cancer cell growth and metastasis via mitochondrial metabolism. A study by Nieman KM et al., [47] shows that triglyceride catabolism in adipocytes drives ovarian cancer metastasis by providing fatty acids as mitochondrial fuels. In CRPC, however, somatostatin analogues were found ineffective when given as monotherapy. More promising results were obtained when lanreotide or octreotide were administered in combination with other agents within a novel concept of “antisurvival factor therapy” [48]. This concept aims to target not only the neoplastic cells but also various factors secreted in their microenvironment that confer protection from apoptosis. The combination of ethinyl estradiol and somatostatin analogue lanreotide offered a median overall survival that was superior to the 10-month median survival in patients with hormone refractory disease [23].
SmsDX could regulate the different metabolic enzymes and proteins which involving metabolic pathways supporting cancer cells: glycolysis, TCA cycle, pentose phosphate, glutaminolysis and oxidative phosphorylation in LNCaP-s cells. So we speculate that inhibition of invasiveness of LNCaP-s is possible due to regulation of metabolism of prostate cancer cells. So it's tempting to speculate the possible role of smsDX in the treatment of CRPC, but the complex crosstalk at multiple levels among them were not fully understood, so further validating investigations are needed.
Taken together, alterations of AR affected by androgen-deprivation could change prostate cancer cell metabolism via multiple intra- and extra-cellular signaling pathways, smsDX effects on the cancer cell metabolism regulating energy, lipid, amino acid and protein biosynthesis provide in-depth information to improve the response to therapy and result in a positive clinical outcomes. The smsDX targeting effects on cellular metabolism in androgen-dependent prostate cancer after ADT therapy improve our understanding of somatostatin's anti-cancer mechanism(s) and possibly lead to the introduction of a novel therapeutic approach for CRPC.
Supporting Information
Table S1 Fifty -six lower expressed proteins in LNCaP-s compared to LNCaP cells (≥1.2 in fold change). LNCaP cells were cultured from androgen-sensitive LNCaP parental cells, in an androgen-reduced condition. Proteins of the parental LNCaP cells after ADT were identified by means of two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF mass spectrometric analysis.
(XLSX)
Click here for additional data file.
Table S2 Proteins affected by smsDX incubation with a fold change ≥1.2 both in LNCaP (table S2A) and LNCaP-s (table S2B) cells. Protein accession numbers from SwissProt/TrEMBL are given and “trend” indicates the direction of the change in expression level. Ctrl: control, SD: smsDX.
(XLS)
Click here for additional data file.
Table S3 Proteins highlighting the similarities/differences in the effect of smsDX between LNCaP and LNCaP-s cell lines. Sorted function descriptions were included in these tables. Table S3A listed forty-eight common proteins between LNCaP and LNCaP-s cells after exposure to smsDX. Table S3B, S3C listed the differentially affected proteins in LNCaP and LNCaP-s cells, separately. Proteins with mark#indicate the common proteins in Table S1.
(XLS)
Click here for additional data file.
We thank Cheng Liu for his writing assistance and helpful discussion. The authors thank the reviewers for providing helpful suggestions that improved the merit and organization of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23409001PONE-D-12-3261010.1371/journal.pone.0055563Research ArticleBiologyBiochemistryEnzymesEnzyme ClassesDehydrogenasesGeneticsGene ExpressionImmunologyImmunologic SubspecialtiesPulmonary ImmunologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCell DeathChemistryMetallurgyMetal AlloysTitanium AlloysMolecular Mechanisms of Nanosized Titanium Dioxide–Induced Pulmonary Injury in Mice Gene Expression in Nano-TiO2-Induced Lung DamagesLi Bing
1
Ze Yuguan
1
Sun Qingqing
1
Zhang Ting
2
3
Sang Xuezi
1
Cui Yaling
1
Wang Xiaochun
1
Gui Suxin
1
Tan Danlin
1
Zhu Min
1
Zhao Xiaoyang
1
Sheng Lei
1
Wang Ling
1
Hong Fashui
1
*
Tang Meng
2
3
*
1
Medical College of Soochow University, Suzhou, China
2
Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, China
3
Jiangsu key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China
Wu Min Editor
University of North Dakota, United States of America
* E-mail: [email protected] (FH); [email protected] (MT)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: FH BL YZ QS. Performed the experiments: FH BL YZ QS YZ TZ. Analyzed the data: FH BL YZ QS TZ XS YC XW SG DT MZ XZ LS LW MT. Contributed reagents/materials/analysis tools: YZ QS TZ XS YC XW SG. Wrote the paper: FH BL YZ QS.
2013 7 2 2013 8 2 e5556325 10 2012 27 12 2012 © 2013 Li et al2013Li et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The pulmonary damage induced by nanosized titanium dioxide (nano-TiO2) is of great concern, but the mechanism of how this damage may be incurred has yet to be elucidated. Here, we examined how multiple genes may be affected by nano-TiO2 exposure to contribute to the observed damage. The results suggest that long-term exposure to nano-TiO2 led to significant increases in inflammatory cells, and levels of lactate dehydrogenase, alkaline phosphate, and total protein, and promoted production of reactive oxygen species and peroxidation of lipid, protein and DNA in mouse lung tissue. We also observed nano-TiO2 deposition in lung tissue via light and confocal Raman microscopy, which in turn led to severe pulmonary inflammation and pneumonocytic apoptosis in mice. Specifically, microarray analysis showed significant alterations in the expression of 847 genes in the nano-TiO2-exposed lung tissues. Of 521 genes with known functions, 361 were up-regulated and 160 down-regulated, which were associated with the immune/inflammatory responses, apoptosis, oxidative stress, the cell cycle, stress responses, cell proliferation, the cytoskeleton, signal transduction, and metabolic processes. Therefore, the application of nano-TiO2 should be carried out cautiously, especially in humans.
This work was supported by the National Natural Science Foundation of China (grant numbers 81273036, 30901218, 81172697), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Major State Basic Research Development Program of China (973 Program) (grant number 2006CB705602), National Important Project on Scientific Research of China (grant number 2011CB933404), National Natural Science Foundation of China (grant numbers 30671782, 30972504) and the National Ideas Foundation of Student of Soochow University (grant number 111028534). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Nanosized titanium dioxide (nano-TiO2) particles, due to their high surface area to particle mass ratio, have been increasingly used as catalysts and are now being commercially manufactured for use in medical, diagnostic, energy, component, and cosmetic applications as opposed to bulk TiO2 (micrometer-sized) [1], [2]. However, concerns have been raised over the safety of nano-TiO2 particles, as the toxicological effects of nano-TiO2 have been demonstrated through several exposure routes, including dermal, oral, and pulmonary. Especially, following inhalation, nano-TiO2 particles are internalized by clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis by both phagocytic and non-phagocytic cells [3]. Reportedly, industrial nano-TiO2 production, which includes a process that produces heavy nano-TiO2 dust, increased the risk of pneumoconiosis to workers. Several reports have shown that human exposure to nano-TiO2 occurs through different pathways, including inhalation and exposure of the integumentary system. The pulmonary responses induced by inhaled nanoparticles (NPs) are considered to be greater than those produced by micron-sized particles because of the increased surface area to particle mass ratio [4], [5]. In vitro studies have demonstrated that both rutile and anatase nano-TiO2 impaired cellular function of human dermal fibroblasts and decreased cellular area, proliferation, mobility, and ability to contract collagen, with the latter being more potent in inducing damage [6]. Animal experiments arrived at the same results regarding the relationship between nano-TiO2 exposure and lung inflammation. Moreover, inhaled NPs, after deposition in the lungs, largely escaped the alveolar macrophage surveillance system and gained greater access to the pulmonary interstitium by translocation from alveolar spaces through the epithelium [7]. Liu et al. [8] reported that intratracheal administration of nano-TiO2 (5 nm) led to significant increases in lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities, infiltration of inflammatory cells, and interstitial thickening in the rat lung.
Our previous in vivo studies demonstrated that exposure to nano-TiO2 induced pulmonary inflammation and apoptosis in mice, which were associated with expression levels of nuclear factor–κB, tumor necrosis factor-α, cyclooxygenase-2, nuclear factor erythroid 2-related factor 2, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, interleukin (IL)-2, -4, -6, -8, -10, -18, and -1β, cytochrome P450 1A1, NF-κB-inhibiting factor, and heat shock protein 70 in the mouse lung [9], [10]. Although the above-mentioned studies clarified the toxicological effects of nano-TiO2, further studies are needed to elucidate the synergistic molecular mechanisms of multiple genes activated by nano-TiO2-induced pulmonary inflammation and apoptosis in animals and humans.
DNA microarrays have been used to identify gene clusters involved in the progression of pulmonary fibrosis and lung injury [11]–[14]. Furthermore, gene expression profiling has been performed to elucidate the toxicological effects of single-walled carbon nanotubes, nano-TiO2, and C60 fullerene particles [15]–[17]. In the present study, we investigated gene expression profiles of the murine lung to explore mechanisms of immune/inflammation responses, apoptosis, and oxidative stress induced by exposure to nano-TiO2 for 90 consecutive days to serve as a reference for future mechanistic studies on the effects of nano-TiO2 and other NPs in pulmonary toxicity to animals and humans.
Materials and Methods
Preparation and Characterization of TiO2 NPs
Nanoparticulated anatase TiO2 was prepared via controlled hydrolysis of titanium tetrabutoxide. The details of the synthesis and characterization of nano-TiO2 have been previously described by our group [18], [19]. TiO2 powder was dispersed on the surface of 0.5% (w/v) hydroxypropyl methylcellulose (HPMC) K4M solution, treated ultrasonically for 15–20 min, and then mechanically vibrated for 2 or 3 min. X-ray-diffraction (XRD) patterns of TiO2 NPs were obtained at room temperature with a charge-coupled device (CCD) diffractometer (Mercury 3 Versatile CCD Detector; Rigaku Corporation, Tokyo, Japan) using Ni-filtered Cu Kα radiation. The particle sizes of both the powder and the NPs suspended in 0.5% (w/v) HPMC solution after incubation for 24 h (5 mg/mL) were determined using transmission electron microscopy (TEM) (Tecnai G220; FEI Co., Hillsboro, OR, USA) operating at 100 kV. The mean particle size was determined by measuring >100 randomly sampled individual particles. XRD measurements showed that TiO2 NPs exhibited an anatase structure with an average grain size of ∼ 6 nm, as calculated from the broadening of the (101) XRD peak of anatase using the Scherrer’s equation. TEM demonstrated that the average size of the particles suspended in HPMC solvent for 24 h was 5–6 nm. The surface area of the sample was 174.8 m2/g. The average aggregate or agglomerate size of the nano-TiO2 after incubation in 0.5% w/v HPMC solution for 24 h (5 mg/mL) was measured by dynamic light scattering using a Zeta PALS+BI-90 Plus zeta potential analyzer for nanoparticles (Brookhaven Instruments Corp., Holtsville, NY, USA) at a wavelength of 659 nm. The mean hydrodynamic diameter of nano-TiO2 in HPMC solvent was 294 nm (range, 208–330 nm) and the zeta potential after 12 and 24 h of incubation was 7.57 and 9.28 mV, respectively [19].
Ethics Statement
All experiments were approved by the Animal Experimental Committee of the Soochow University (grant no.: 2111270) and performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Animals and Treatments
One hundred and twenty female CD-1 (Imprinting Control Region) mice aged 5 weeks with an average body weight (BW) of 23±2 g were purchased from the Animal Center of Soochow University (Jiangsu, China). All mice were housed in stainless steel cages in a ventilated animal facility with a temperature maintained at 24±2°C and relative humidity of 60±10% under a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Prior to dosing, the mice were acclimated to the environment for 5 days.
Nano-TiO2 powder was dispersed onto the surface of 0.5% w/v HPMC, treated ultrasonically for 30 min, and mechanically vibrated for 5 min. For the experiment, the mice were randomly divided into four groups (n = 30 each), including a control group (treated with 0.5% w/v HPMC) and three experimental groups (treated with 2.5, 5, and 10 mg/kg BW TiO2 NPs, respectively). The mice were weighed and then the nano-TiO2 suspensions were administered by nasal instillation every day for 90 days. All symptoms and deaths were carefully recorded daily. After the 90-day period, all mice were weighed, anesthetized with ether, and then sacrificed. Blood samples were collected from the eye vein by rapidly removing the eyeball and serum was collected by centrifuging the blood samples at 1,200×g for 10 min. The lungs were quickly removed and placed on ice and then dissected and frozen at −80°C.
Coefficients of Lung
After weighing the body and lungs, the coefficients of lung mass to BW were calculated as the ratio of lung (wet weight, mg) to BW (g).
Bronchoalveolar Lavage (BAL) Analysis
After blood collection, the mice were euthanized and the lungs from the control and treated groups were immediately lavaged twice with phosphate buffer saline (PBS). An average of >90% of the total instilled PBS volume was retrieved both times and the amounts did not differ among the groups. The resulting fluid was centrifuged at 400×g for 10 min at 4°C to separate the cells from the supernatant containing various surfactants and enzymes. The cell pellet was used for enumeration of total and differential cell counts as described by AshaRani et al. [20]. Macrophages, lymphocytes, neutrophils, and eosinophils recovered from the BALF were counted using dark field microscopy to assess the extent of phagocytosis. LDH, ALP, and total protein (TP) in the cell-free lavage fluid were analyzed using an automated clinical chemical analyzer (Type 7170A; Hitachi, Ltd., Tokyo, Japan).
Lung Titanium Content Analysis
The frozen lung tissues were thawed and ∼ 0.1 g samples were weighed, digested, and analyzed for titanium content. Briefly, prior to elemental analysis, the lung tissues were digested overnight with nitric acid (ultrapure grade). After adding 0.5 mL of H2O2, the mixed solutions were incubated at 160°C in high-pressure reaction containers in an oven until the samples were completely digested. Then, the solutions were incubated at 120°C to remove any remaining nitric acid until the solutions were colorless and clear. Finally, the remaining solutions were diluted to 3 mL with 2% nitric acid. Inductively coupled plasma-mass spectrometry (Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA) was used to determine the titanium concentration in the samples. Indium (20 ng/mL) was chosen as an internal standard element. The detection limitation of titanium was 0.074 ng/mL and data are expressed as ng/g of fresh tissue.
Histopathological Analysis
For pathological studies, all histopathological examinations were performed using standard laboratory procedures. The lungs were embedded in paraffin blocks, then sliced (5-µm thickness), and placed on glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments using light microscopy (U-III Multi-point Sensor System; Nikon, Tokyo, Japan).
Observation of Pulmonary Ultrastructure
Lungs were fixed in fresh 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4°C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate, then the specimens were dehydrated in a graded series of ethanol (75, 85, 95, and 100%) and embedded in Epon 812 resin. Ultrathin sections were made, contrasted with uranyl acetate and lead citrate, and observed by TEM (model H600; Hitachi, Ltd., Tokyo, Japan). Lung apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation).
Confocal Raman Microscopy of Lung Sections
Raman analysis of pulmonary glass or TEM slides was performed using backscattering geometry in a confocal configuration at room temperature with an HR-800 Raman microscope system equipped with a 632.817 nm He-Ne laser (JY Co., Fort De, France). Laser power and resolution were approximately 20 mW and 0.3 cm−1, respectively, while the integration time was adjusted to 1 s.
Oxidative Stress Assay
Reactive oxygen species (ROS) (O2
− and H2O2) production and levels of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxy deoxyguanosine (8-OHdG) in the lung tissues were assayed using commercial enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer's instructions.
Microarray and Data Analysis
Gene expression profiles of the lung tissues isolated from control and nano-TiO2-treated mice were compared by microarray analysis using Illumina BeadChip technology (Affymetrix, Santa Clara, CA, USA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no.1755; Ambion, Inc., Austin, TX, USA) according to the manufacturer’s protocol and stored at −80°C. RNA amplification has become the standard method for preparing RNA samples for array analysis [21]. Total RNA was then submitted to Biostar Genechip, Inc. (Shanghai, China) to analyze RNA quality using a bioanalyzer and complementary RNA (cRNA) was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized to a single array according to standard Illumina RNA Amplification Kit protocols for all arrays.
Illumina BeadStudio data analysis software (Illumina, Inc., San Diego, CA, USA) was used to analyze the data generated in this study. This program identifies differentially expressed genes and establishes the biological significance based on the Gene Ontology Consortium database (http://www.geneontology.org/GO.doc.html). Differentially expressed genes were identified using the Student’s t-test (two-tailed, unpaired) with a threshold of 13.0 to limit the data set to genes up-regulated or down-regulated (DiffScore >13).
Quantitative Real-time PCR
Expression levels of coagulation factor VII, hydroxymethylglutaryl CoA synthase 2, plasminogen activator - urokinase receptor, tubulin folding cofactor B, and adenosine deaminase (Ada) mRNA in the mouse lung tissues were determined using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) [22]–[24]. Synthesized complimentary DNA was generated by qRT-PCR with primers designed with Primer Express Software (Applied Biosystems, Foster City, CA, USA) according to the software guidelines. PCR primer sequences are available upon request.
Statistical Analysis
All results are expressed as means ± standard error. Significant differences were examined using the Dunnett’s pair-wise multiple comparison t-test using SPSS version 19 software (SPSS, Inc., Chicago, IL, USA). A p-value <0.05 was considered statistically significant.
Results
BW, Relative Lung Weight, and Titanium Accumulation
Titanium accumulation, BW, and relative lung weight of the mice are listed in Table 1. As shown, an increased nano-TiO2 dose led to a gradual decrease in BW, whereas the relative lung weight and titanium content were significantly increased (p<0.05), indicating growth inhibition and lung damage in the mice. These findings were confirmed by subsequent pulmonary histological and ultrastructural observations and oxidative stress assays.
10.1371/journal.pone.0055563.t001Table 1 Body weight, relative weight of lung and titanium accumulation in the mouse lung after nasal administration with nano-TiO2 for 90 consecutive days.
Index Nano-TiO2 (mg/kg BW)
0 2.5 5 10
Net increase of body weight (g)
20±1a 16±0.8b 11±0.55c 5±0.25d
Relative weight of lung (mg/g)
9.27±0.47a 9.67±0.48a 11.31±0.57b 14.28±0.71c
Ti content (ng/g tissue)
Not detected 65±3.25a 113±5.65b 207±10.35c
Letters indicate significant differences between groups (p<0.05). Values represent means ± SE(N = 10).
Histopathological Lung Evaluation
The histological changes in the lung specimens are shown in Fig. 1. Unexposed lung samples did not exhibit any histological changes (Fig. 1a), while those exposed to increasing nano-TiO2 concentrations exhibited severe pathological changes, including infiltration of inflammatory cells, thickening of the pulmonary interstitium, and edema (Fig. 1b–d). In addition, we also observed significant black agglomerates in the lung samples exposed to 10 mg/kg of nano-TiO2 (Fig. 1d). Confocal Raman microscopy further showed a characteristic nano-TiO2 peak in the black agglomerate (148 cm−1), which further confirmed the deposition of nano-TiO2 in the lungs (see spectrum B in the Raman insets in Fig. 1d).
10.1371/journal.pone.0055563.g001Figure 1 Histopathology of the lung tissue in ICR mice caused by nasal administration of nano-TiO2 for 90 consecutive days.
(a) Control group; (b) 2.5 mg/kg BW nano-TiO2 group indicates inflammatory cell infiltration (green cycles) and thickening of pulmonary interstitium (green arrows); (c) 5 mg/kg BW nano-TiO2 group indicates severs inflammatory cell infiltration (green circles), and great thickening of pulmonary interstitium (green arrows) and pulmonary edema (yellow arrows); (d) 10 mg/kg BW nano-TiO2 group indicates severe inflammatory cell infiltration (green arrows) and great thickening of pulmonary interstitium (green arrows), yellow circles show black deposition in the lung. Arrow A spot is a representative cell that not engulfed the nano-TiO2, while arrow B spot denotes a representative cell that loaded with nano-TiO2. The right panels show the corresponding Raman spectra identifying the specific peak at about 148 cm-1.
Ultrastructural Changes of the Lung
Changes to the pneumonocytic ultrastructure in the mouse lung samples are presented in Fig. 2. As shown, the untreated mouse pneumonocytes (control) had no abnormal changes (Fig. 2a), whereas the pneumonocytic ultrastructure from the nano-TiO2-treated groups indicated a classical morphology characteristic of apoptosis, including mitochondrial swelling, nuclear shrinkage, chromatin condensation, and evacuation of the pneumonocytic lamellar bodies (Fig. 2b–d). In addition, black deposits were observed in the pneumonocytes exposed to 10 mg/kg of nano-TiO2 via TEM (Fig. 2d) and Raman signals of nano-TiO2 were also exhibited via confocal Raman microscopy (Fig. 2d).
10.1371/journal.pone.0055563.g002Figure 2 Ultrastructure of pneumonocyte in female mice lung caused by nasal administration of nano-TiO2 for 90 consecutive days.
(a) Control: chromatin is well distributed, normal lamellar bodies; (b) 2.5 mg/kg BW nano-TiO2 indicates a significant shrinkage and chromatin marginalization of the nucleus (yellow arrows), mitochondria swelling(red arrows); (c) 5 mg/kg BW nano-TiO2 indicates a significant nucleus pyknosis (green arrows); (d) 10 mg/kg BW nano-TiO2 indicate a significant nucleus pyknosis (yellow arrows), mitochondria swelling(red arrows) as well as evacuation of lamellar bodies (green arrows), circles show black deposition. Arrow A spot is a representative cell that not engulfed the nano-TiO2, while arrow B spot denotes a representative cell that loaded with nano-TiO2. (c) The right panels show the corresponding Raman spectra identifying the specific peak at about 148 cm−1.
Inflammatory Cells and Biochemical Assessments in BALF
To further determine whether long-term nano-TiO2 exposure induces lung inflammation, we analyzed inflammatory cell content and biochemical changes in BALF. As shown in Table 2, the numbers of macrophages, lymphocytes, neutrophils, and eosinophils, and LDH, ALP, and TP contents in the nano-TiO2-exposed mice showed obvious increases with increasing nano-TiO2 dose (p<0.05), indicating that nano-TiO2 exposure caused severe inflammation and biochemical dysfunction in mice.
10.1371/journal.pone.0055563.t002Table 2 Numbers of inflammatory cells and biochemical changes in BALF of mice after nasal administration with nano-TiO2 for 90 consecutive days.
Parameter Nano-TiO2 (mg/kg BW)
0 2.5 5 10
Macrophages (104/per mouse)
11±0.55a 20±1.0b 36±1.80c 50±2.95d
Lymphocytes (104/per mouse)
3±0.15a 6±0.30b 11±0.55c 19±0.95d
Neutrophils (104/per mouse)
6±0.31a 14±0.70b 22±1.10c 36±1.80d
Eosinophils (104/per mouse)
5±0.25a 10±0.50b 17±0.85c 28±1.40d
LDH (unit/L)
582±29a 689±34b 778±39c 986±49d
ALP (unit/L)
100±5a 136±7b 188±9c 225±11d
TP (g/L)
26.68±1.34a 33.49±1.67b 41.96±2.10c 48.21±2.41d
Letters indicate significant differences between groups (p<0.05). Values represent means ± SE(N = 10).
Oxidative Stress Analysis
The effects of nano-TiO2 on the production of O2
− and H2O2 in mouse lung tissues are shown in Table 3. With increasing nano-TiO2 dose, the rate of ROS generation in the nano-TiO2-exposed groups was significantly elevated (p<0.05), suggesting that exposure to nano-TiO2 accelerated ROS production in lung tissues.
10.1371/journal.pone.0055563.t003Table 3 Oxidative stress in the mouse lung after nasal administration with nano-TiO2 for 90 consecutive days.
Oxidativestress TiO2 NPs (mg/kg BW)
0 2.5 5 10
O2.− (nmol/mg
prot. min)
23±1.15a 30.27±1.51b 39.18±1.96c 50±2.50d
H2O2 (nmol/mg
prot. min)
43±2.15a 61.22±3.06b 78.96±3.95c 110±5.50d
MDA
(µmol/
mg prot)
1.08±0.05a 1.59±0.08b 2.89±0.15c 5.15±0.26d
Carbonyl
(µmol/mg
prot)
0.54±0.03a 0.98±0.05b 1.85±0.09c 3.04±0.15d
8-OhdG
(mg/g tissue)
0.42±0.02a 2.26±0.11b 4.25±0.21c 7.12±0.36d
Letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
To further demonstrate the effects of nano-TiO2 on ROS generation in mouse lung tissue, the levels of lipid peroxidation (MDA), protein peroxidation (PC), and DNA damage (8-OHdG) were examined. As shown in Table 3, levels of MDA, PC, and 8-OHdG in tissues from the nano-TiO2-exposed groups were markedly elevated (p<0.05), suggesting that nano-TiO2–induced ROS accumulation led to lipid, protein, and DNA peroxidation in the lung.
Change in Gene Expression Profiles
Treatment with 10 mg/kg BW of nano-TiO2 resulted in the most severe pulmonary damage and these tissues were used to detect gene expression profiles to further explore the mechanisms of pulmonary damage induced by nano-TiO2. Whole-genome expression profiling using mRNAs from pulmonary tissues of vehicle control groups and those treated with 10 mg/kg BW of nano-TiO2 for 90 consecutive days were analyzed with the Illumina Bead Chip. The nano-TiO2-treated group was compared with the vehicle control under these criteria: DiffScore ≥13 or ≤ −13 and p≤0.05. The results showed that ∼ 1.16% of the total genes (521/45,000 genes with known functions) were significantly changed following nano-TiO2 exposure. Of these 521 genes, 361 were up-regulated and 160 were down-regulated. The gene expression profile of the lung tissues from the TiO2 NPs-treated mice was classified using the ontology-driven clustering algorithm included with the PANTHER Gene Expression Analysis Software (www.pantherdb.org/). The 521 genes were closely involved in immune responses, inflammatory responses, apoptosis, oxidative stress, metabolic processes, stress responses, signal transduction, cell proliferation, the cytoskeleton, cell differentiation, cell cycling, and so on (Fig. 3), whereas the functions of another 327 genes were unknown. Genes related to immune responses, inflammatory responses, apoptosis, oxidative stress, and the cell cycle are listed in Table 4 (representative genes) and Table S1 (all data).
10.1371/journal.pone.0055563.g003Figure 3 Functional categorization of 521 genes.
Genes were functionally classied based on the ontology-driven clustering approach of PANTHER.
10.1371/journal.pone.0055563.t004Table 4 Significant alteration of representative genes after nasal administration of 10 mg/kg BW TiO2 NPs for 90 consecutive days.
Symbol Gene ID Ontology DiffScore Pval
Immune response
Defb4 NM_019728 defense response to bacterium 121.33 0
H2-Oa NM_008206 regulation of T cell differentiation −26.34 0
Inflammatory response
Chi3l3 NM_009892 inflammatory response 93.12 0
Alox5ap NM_009663 leukotriene production involved in inflammatory response 20.37 0
Il1b NM_008361 inflammatory response 14.56 0
Apoptosis
Pdia2 NM_001081070 apoptosis 73.16 0
Niacr1 NM_030701 apoptosis 67.22 0
Ada NM_007398 negative regulation of thymocyte apoptosis 28.04 0.18
Sphk2 NM_203280 anti-apoptosis −13.20 0
Erbb2 NM_001003817 negative regulation of apoptosis −14.43 0
Cell cycle
Cdkn1a NM_001111099 cell cycle arrest 15.26 0
Cdkn1c NM_001161624 Cell cycle −15.89 0
Oxidative stress
Cryab NM_009964 oxygen and reactive oxygen species metabolic process 25.36 0
Alkbh7 NM_025538 oxidoreductase activity −19.72 0
qRT-PCR
To verify the accuracy of the microarray analysis, five genes that demonstrated significantly different expression patterns were further evaluated by qRT-PCR due to their association with apoptosis, cell differentiation, blood coagulation, and the cytoskeleton. The qRT-PCR analysis of all five genes displayed expression patterns comparable with the microarray data (i.e., either up- or down-regulation; Table 5).
10.1371/journal.pone.0055563.t005Table 5 Comparison of fold-difference between the control and 90 day 10 mg/kg BW dosage.
Gene △△Ct Fold Microarray
F7
−1.786201 3.4490546778 2.458522
Hmgcs2
1.163294 0.44649192843 0.523989
Plaur
−1.536868 2.9016389168 1.98002
Tbcb
−0.004397 1.0030524173 1.562935
Ada
−2.280629 4.8588975045 6.867184
Discussion
The results of the present study indicated that nasal administration of 2.5, 5, and 10 mg/kg of nano-TiO2 for 90 consecutive days induced BW reduction, increased relative lung mass, nano-TiO2 deposition, (Table 1, Figs 1d, 2d), pulmonary inflammation, thickening of pulmonary interstitium, edema (Fig. 1), and pneumonocytic apoptosis (Fig. 2) in mouse lung tissues coupled with biochemical dysfunction, marked by increased LDH, ALP, and TP levels in the BALF (Table 2), and severe oxidative stress, marked by significant production of O2
.
− and H2O2, and peroxidation of lipids, proteins, and DNA (Table 3). Furthermore, nano-TiO2 exposure significantly increased the influx of inflammatory cells, including macrophages, lymphocytes, neutrophils, and eosinophils, in the BALF (Table 2), further supporting the assertion that nano-TiO2 exposure induced pulmonary inflammation. The pulmonary injuries and oxidative stress caused by nano-TiO2 exposure may be involved in impaired immune function and antioxidant capacity in mice and, thus, may be associated with altered gene expression in lung tissue. To elucidate the molecular mechanisms of lung damage and identify specific biomarkers induced by nano-TiO2 exposure, RNA microarray analysis of mouse lung tissue was performed to establish a global gene expression profile and identify toxicity-response genes in mice following exposure to 10 mg/kg BW of nano-TiO2 for 90 consecutive days. Our analysis indicated that the expression levels of 847 genes were significantly changed and 521 of these genes were involved in immune responses, inflammatory responses, apoptosis, oxidative stress, metabolic processes, stress responses, signal transduction, cell proliferation, the cytoskeleton, cell differentiation, and the cell cycle.
In the present study, severe inflammatory responses in the lung tissue occurred due to nano-TiO2-induced toxicity (Table 2, Fig. 2). Some studies have demonstrated that ultrafine particle exposure to the respiratory tract can induce pulmonary inflammation [9], [10], [25]–[27]. Latex nanomaterials instilled intratracheally enhanced neutrophilic lung inflammation with pulmonary vascular permeability related to LPS resulting from the activation of innate immune responses [28]. The present study was performed to assess pulmonary immune responses and toxicity in response to nasal administration of nano-TiO2 and found that 38 genes (4.49% of 847 genes) involved in immune and inflammatory responses were significantly changed as shown by the microarray data (Table S1). Of these 38 genes, 31 were up-regulated and seven down-regulated. Beta-defensins contribute to the innate and adaptive immune responses in a role as chemoattractants, of which, beta-defensin 4 (Defb4), an antibiotic peptide which is locally regulated by inflammation [29], and the presence of H2-Oa (histocompatibility 2, O region alpha locus) in B cells may serve to focus presentation of antigens internalized by membrane immunoglobulins to increase the specificity of the immune response and avoid reactivity to self antigens [30]. Our data showed that Defb4 expression was increased by 121.13-fold and H2-Oa expression was decreased by 2.69-fold in the nano-TiO2-exposed group (Table 4), suggesting that nano-TiO2 induced Defb4 expression and suppressed H2-Oa expression, which are both closely related to immune system impairment and inflammation generation (marked by significantly increased levels of macrophages, lymphocytes, neutrophils and eosinophils) in the mouse lung following nano-TiO2-induced toxicity. Therefore, we suggest that Defb4 and H2-Oa may be potential biomarkers of nano-TiO2 exposure in the lung. In addition, the gene for chitinase 3-like 3 (Chi3l3) is characteristically expressed by alternatively activated macrophages. Previous studies have demonstrated innate Chi3l3 expression in the lungs of infected severe combined immunodeficiency (SCID) mice [31] and eosinophils [32]. Increased Chi3l3 protein expression has been associated with inflammatory diseases, in particular with eosinophilic chemotaxis and promotion of cytokine production [33]–[35]. The arachidonate 5-lipoxygenase-activating protein (ALOX5AP) is involved in inflammation by mediating the activity of 5-lipoxygenase, which is a regulator of leukotriene biosynthesis, which are pro-inflammatory lipid mediators secreted by inflammatory cells [36], [37]. IL-1 induces pro- and anti-inflammatory response of macrophages. The IL-1 gene cluster contains three related genes (IL-1A, IL-1B, and IL1-RN), which encode the proinflammatory cytokines IL-1α, IL-1β, as well as their endogenous receptor antagonist IL-1ra, respectively [38]. In the current study, nano-TiO2 exposure resulted in significantly increased expression of the Chi3l3, Alox5ap, and IL1b genes with the DiffScores of 93.12, 20.37, and 14.56 (Table 4), respectively, indicating a pulmonary inflammatory response, which was closely related to excessive increases of inflammatory cells in the lung (Table 2). Taken together, Chi3l3, Alox5ap, and Il1b may be potential biomarkers of nano-TiO2-induced pulmonary toxicity.
In the present study, classic morphological characteristics of apoptosis, such as mitochondrial swelling and nuclear chromatin condensation in the pneumonocytes was observed following exposure to 10 mg/kg BW of nano-TiO2 (Fig. 1d). To further clarify the apoptotic molecular mechanisms, we analyzed microarray data and found that 31 genes (25 up-regulated and six down-regulated) were altered significantly by exposure to nano-TiO2 (Table S1). The expression of several apoptotic mRNAs, including protein disulfide isomerase associated 2, niacin receptor 1, and Ada were significantly up-regulated, of which DiffScores were 73.16, 67.22, and 28.04, respectively; whereas sphingosine kinase 2 and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 were down-regulated with DiffScores of −13.20 and −14.43, respectively (Table 4). As shown in Fig. 4, specifically, the apoptotic pathway analysis showed that nano-TiO2 regulated toxicological pathways by increasing the expression of a key factor, Ada, which is an essential enzyme of purine catabolism that is responsible for the hydrolytic deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. These biochemical pathways are essential for maintaining homeostasis, as both Ada substrates have substantial signaling properties. Adenosine engages G protein–coupled receptors on the surface of target cells to evoke a variety of cellular responses, whereas 2'-deoxyadenosine is cytotoxic via mechanisms that interfere with cellular growth and differentiation or the promotion of apoptosis and inflammation [39]. Ada deficiency is a fatal autosomal recessive form of SCID, of which failure to thrive, impaired immune responses, and recurrent infections are characteristics [40], [41]. Adenosine is generated in response to lung hypoxia and injury, and several studies have suggested that this signaling pathway might play an important role in chronic lung diseases, such as asthma and chronic obstructive pulmonary disease [42]–[44]. Therefore, increased Ada expression due to nano-TiO2 exposure may reduce the accumulation of adenosine and 2'-deoxyadenosine in lung tissue, which in turn can cause cytoprotective or anti-inflammatory responses. Ada may be a potential biomarker of lung toxicity caused by nano-TiO2 exposure. Since apoptosis is accompanied by altered cell cycle progression, our data suggest that 10 genes involved in the cell cycle were also significantly altered (Fig. 3 and Table S1). Of these 10 genes, seven were up-regulated and three down-regulated. For instance, cyclin-dependent kinase inhibitor (Cdkn)1a was increased with a DiffScore of 15.26, whereas Cdkn1c was reduced with a DiffScore of −15.89 (Table 4). Among the cell cycle regulatory proteins that are activated following DNA damage, CDKN1A plays essential roles in the DNA damage response by inducing cell cycle arrest, direct inhibition of DNA replication, as well as regulation of fundamental processes, such as apoptosis and transcription [45]. Excessive Cdkn1a expression following nano-TiO2 exposure may affect DNA damage repair and promote apoptosis in the mouse lung. Since Cdkn1c is a cell cycle inhibitor, its role has been largely implicated as a tumor suppressor gene whose loss of function promotes tumor growth and progression [46]. Thus, inhibition of nano-TiO2-induced Cdkn1c expression is speculated to contribute to apoptotic progression in lung tissue.
10.1371/journal.pone.0055563.g004Figure 4 Ada network pathway obtained from network analysis of differentially expressed genes.
Gene Spring software was used to construct and visualize molecular interaction networks.
The present study suggested that nano-TiO2 exposure promoted ROS production (such as O2
− and H2O2) and led to peroxidation of lipids, proteins, and DNA in mouse lung tissue, indicating oxidative stress, which may be associated with alterations of oxidative stress-related gene expression. Our microarray analysis showed that approximately 22 genes involved in oxidative stress were significantly changed in the nano-TiO2-exposed lung (Fig. 3 and Table S1). Of these 22 genes, 11 were up-regulated and 11 down-regulated (Fig. 3 and Table S1). In this study, crystallin-alpha B (Cryab) was highly expressed following nano-TiO2 exposure, with a DiffScore of 25.36, whereas alkylation repair homolog 7 (Alkbh7) was significantly suppressed, with a DiffScore of −19.72 (Table 4). Reportedly, Cryab expression in the retina is increased in response to oxidative stress and it has been postulated that this represents a protective mechanism against oxidative stress-induced apoptosis [47]. Elevated Cryab expression may increase in response to oxidative stress following nano-TiO2-induced pulmonary damage. Alkbh7 is an oxidoreductase, which plays an important role in cardioprotection during ischemia/reperfusion by reducing oxidative stress [48]. In the current study, reduced Alkbh7 expression induced by nano-TiO2 exposure may cause pulmonary peroxisomal disorders and decrease antioxidative capacity or detoxification. Therefore, Cryab and Alkbh7 may be potential biomarkers of nano-TiO2–induced pulmonary toxicity.
In regard to the dose selection in this study, we consulted a 1969 study from the World Health Organization, which reported a median lethal dose of TiO2 of >12,000 mg/kg BW orally administered to rats. In the present study, we selected 2.5, 5, and 10 mg/kg BW of nano-TiO2 and exposed mice to these concentrations every day for 90 days, which was equal to approximately 0.15–0.7 g of nano-TiO2 in a human weighing 60–70 kg following such exposure. Although these doses were relatively safe, we recommend using caution for the long-term application of products containing nano-TiO2 in humans.
Conclusion
After exposing mice to nano-TiO2 for 90 consecutive days, depositions of nano-TiO2 in pulmonary tissues and even in pneumonocytes were observed, which in turn resulted in significant infiltration of inflammatory cells, biochemical dysfunction, oxidative stress, and pneumonocytic apoptosis in mouse lung tissue. The pulmonary injuries following long-term nano-TiO2 exposure may be closely associated with significant changes in the expression of genes involved in immune responses, inflammatory responses, apoptosis, oxidative stress, metabolic process, stress responses, signal transduction, cell proliferation, the cytoskeleton, cell differentiation, and cell cycle, specifically, with an increase in Ada expression. The obvious elevation in Ada expression following nano-TiO2 exposure may trigger signaling cascades associated with inflammatory or apoptotic pathways. Therefore, the application of nano-TiO2 should be carried out cautiously, especially in humans.
Supporting Information
Table S1 Genes of known function altered significantly after nasal administration of 10 mg/kg BW TiO2 NPs for 90 consecutive days.
(DOC)
Click here for additional data file.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23409026PONE-D-12-2770210.1371/journal.pone.0055717Research ArticleBiologyGeneticsCytogeneticsCytogenetic AnalysisCytogenetic TechniquesGenetic MutationMutation TypesCancer GeneticsGenetics of DiseaseGenomicsGenomic MedicineGenetic TestingPharmacogenomicsGenome SequencingMedicineClinical GeneticsChromosomal DisordersTranslocationsHematologyHematologic Cancers and Related DisordersLeukemiasChronic Myeloid LeukemiaOncologyCancer TreatmentChemotherapy and Drug TreatmentSensitive Detection of Pre-Existing BCR-ABL Kinase Domain Mutations in CD34+ Cells of Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia Patients Is Associated with Imatinib Resistance: Implications in the Post-Imatinib Era Natural BCR-ABL Mutants in Newly Diagnosed CP-CMLIqbal Zafar
1
9
12
15
*
Aleem Aamer
2
Iqbal Mudassar
3
15
Naqvi Mubashar Iqbal
4
15
Gill Ammara
5
15
Taj Abid Sohail
6
Qayyum Abdul
7
ur-Rehman Najeeb
8
Khalid Ahmad Mukhtar
9
Shah Ijaz Hussain
10
Khalid Muhammad
10
Haq Riazul
11
12
Khan Mahwish
12
15
Baig Shahid Mahmood
13
Jamil Abid
14
Abbas Muhammad Naeem
15
Absar Muhammad
15
Mahmood Amer
16
Rasool Mahmood
17
Akhtar Tanveer
15
1
College of Applied Medical Sciences, King Saud Bin Abdulaziz University for Health Sciences (KSAU-HS), National Guards Health Affairs, Riyadh, Kingdom of Saudi Arabia
2
Department of Medicine, Division of Hematology/Oncology, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi Arabia
3
Foreign Faculty, Asian Medical Institute, Kant City, National Surgical Centre, Bishkek, Kyrgyzstan
4
Computer Sciences and Bio-informatics Laboratory, Government. Elementary School Chak 19 S.B., Sargodha, Pakistan
5
Springfield, Missouri, United States of America
6
Institute of Radiotherapy and Nuclear Medicine, Peshawar, Pakistan
7
Department of Oncology, Pakistan Institute of Medical Sciences, Islamabad, Pakistan
8
Medilaser, Lahore, Pakistan
9
School of Biological Sciences, University of Sargodha, Sargodha, Pakistan
10
Department of Oncology, Allied Hospital and Punjab Medical College, Faisalabad, Pakistan
11
Health Centre, University of Texas San Antonio, San Antonio, Texas, United States of America
12
Institute of Molecular Biology and Biotechnology and Centre for Research in Molecular Medicine, the University of Lahore, Lahore, Pakistan
13
Human Molecular Genetics Group, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
14
Post-graduate Medical Institute, Hayatabad Medical Complex, Peshawar, Pakistan
15
Higher Education Commission Program in “Hematology Oncology and Pharmacogenetic Engineering Sciences (HOPES)”, HOPES Group, Health Sciences Research Laboratories, Department of Zoology, University of the Punjab, Lahore, Pakistan
16
Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia
17
Centre of Excellence in Genomic Medicine Research, King Abdulaziz University Jeddah, Kingdom of Saudi Arabia
Ellis Steven R. Editor
University of Louisville, United States of America
* E-mail: [email protected] Interests: The authors have the following interest: Najeeb ur-Rehman is employed by Medilaser, Lahore, Pakistan. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Conceived and designed the experiments: ZI. Performed the experiments: ZI MI RH MNA. Analyzed the data: ZI MI AA MIN AHT AST AQ NUR IHS M. Khalid SMB AMK AJ MNA MA TA AM M. Khan MR. Contributed reagents/materials/analysis tools: ZI MI RH M. Khan MNA. Wrote the paper: ZI AA MA AM.
2013 8 2 2013 8 2 e5571710 9 2012 29 12 2012 © 2013 Iqbal et al2013Iqbal et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients.
Methods
We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain.
Results
Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8–48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation.
Conclusion
Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.
This work was partially supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia. Research funding provided by Higher Education Commission Pakistan is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the t (9; 22) chromosomal translocation. This translocation results in the formation of BCR-ABL fusion gene, which is central to the pathogenesis of CML. The BCR-ABL gene exhibits constitutive tyrosine kinase activity, resulting in myeloid proliferation [1]. Imatinib mesylate, a tyrosine kinase inhibitor (TKI), induces durable responses in the majority of CML patients and is currently the standard of care for CML [2], [3]. However, imatinib resistance, usually due to BCR-ABL kinase domain (KD) point mutations, remains a significant problem in the management of CML patients [4], [6]. BCR-ABL mutations have varying effects on the patient’s sensitivity to imatinib and other TKIs, and may cause partial or complete resistance depending upon the nature and location of the mutations [5], [7]–[10]. The presence of KD mutations has been studied mostly in the advanced phase of CML (accelerated phase and blast crisis), in chronic phase (CP) patients who develop resistance to imatinib, and in Philadelphia-positive (Ph+) acute lymphoblastic leukemia [5], [10]–[13].
BCR-ABL KD mutations can exist in the newly diagnosed CP-CML patients and may affect the outcome of imatinib treatment [14]–[18]. There are limited data available from imatinib-naive patients in CP-CML regarding the incidence of KD mutations, and the correlation of these mutations with the therapeutic response in unselected patients has not been established [14], [17]–[18]. Although KD mutations are infrequently detected in newly diagnosed CP-CML patients [18], KD mutations were found in a substantial number of patients when CD34+ stem cell were analyzed [19], [20]. Previous studies indicated that a small population of CD34+ CML (stem/progenitor) cells are less responsive to imatinib and other TKIs, and act as a reservoir for the emergence of imatinib-resistant subclones [19], [21]–[23]. Thus, the detection of pre-existing mutations (PEMs) in primitive stem/progenitor (CD34+) cells may have therapeutic and prognostic implications and is likely to be helpful in optimizing the management of CML patients, specifically after availability of three tyrosine kinase inhibitors as first-line treatment of CML which vary in their effectiveness against different BCR-ABL mutants as well as after FDA approval of ponatinib for TKI-resistant CML, particularly the most aggressive T315I-mutant CML [19]–[23]. Large-scale studies to assess the role of BCR-ABL PEMs in CD34+ cells and their correlation with imatinib therapy in CP-CML are lacking. To address this issue, we analyzed 100 newly diagnosed CP-CML patients for BCR-ABL PEMs in CD34+ CML cells using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and sequencing, and studied the outcome of these patients after imatinib treatment.
Design and Methods
Ethics Statement
Patients’ samples were collected from the following centers. 1) Department of Oncology, Allied Hospital and Punjab Medical College Faisalabad, Pakistan. 2) Pakistan Institute of medical Sciences Hospital, Islamabad, Pakistan. 3) Khyber Teaching Hospital & Hayatabad Medical Complex, PGMI Peshawar, Pakistan. 4) Institute of Radiotherapy and Nuclear Medicine, Peshawar, Pakistan, & 5) HOPES, Department of Zoology, University of the Punjab, Lahore, Pakistan. All patients gave written informed consent, and the institutional ethics committees of the participating centers approved the study as well as contents of the written consent.
Patients and Definitions
One hundred newly diagnosed CP-CML patients were included in the study. The study was conducted from March 2006 until February 2010 and 4 centers participated in the study while experiments were carried out at HOPES, Department of Zoology, University of the Punjab, Lahore, Pakistan. All patients had newly diagnosed CP-CML at the time of sample collection, and patients with accelerated-phase or blast-crisis CML were excluded. Patients’ clinical characteristics are given in Table 1.
10.1371/journal.pone.0055717.t001Table 1 Patients’ characteristics.
S. No. Patients Demographics Subcategory Newly diagnosedCP-CML Patients(n = 100) Patients with PEM(n = 32) Patients without PEM (68)
1 Gender Male 69 22 47
– – Female 31 10 21
2 Age (years) Median 35 38 34.2
– – Range 12–70 22–70 12–67
3 Splenic enlargement – 87 29 58
4 Hemoglobin <10.0 g/dl – 55 20 35
5 WBC count (mm3) 50–100 15 7 21
– – >100 72 25 47
6 Platelet count (mm3) 100–450 15 7 21
– >450 19 6 13
8 Mode of diagnosis (Ph+) 99 32 67
– – BCR-ABL fusion oncogene + 100 32 68
9 BCR-ABL splice variants b2a2 37 9 28
– – b3a2 63 23 40
Ph+ = Philadelphia chromosome positive.
PEM = pre existing mutations.
CP was defined by the presence of less than 15% blasts, less than 20% basophils, and less than 30% blasts and promyelocytes in the peripheral blood and bone marrow (BM) and no extramedullary blastic disease [24]. Complete hematologic response (CHR), complete cytogenetic response (CCyR), and a partial cytogenetic response were defined according to previously published response criteria [24]. Briefly, CHR required the normalization of blood counts; leucocytes counts <10,000/mm3; normal differential counts without blasts, promyelocytes, or myelocytes; platelet counts from 150,000/mm3 to 450,000/mm3; and no evidence of extramedullary disease. CCyR was defined as 0% Ph+ cells in metaphase BM samples, and a Major (partial) cytogenetic response (PCyR) was defined as the presence of 1–35% Ph+ cells in BM. Other categories included minor cytogenetic response (36–65% Ph+ cells in BM) and minimal cytogenetic response (66–95% Ph+ cells in BM). Molecular response was defined as BCR-ABL fusion transcript negativity according to nested reverse-transcriptase PCR. We could not record the major molecular response (MMR) due to the non-availability of real-time quantitative PCR during the study, but a real-time quantitative PCR was performed on archived samples preserved in 10% DMSO and 90% FBS stored at −80°C using IPSOGEN BCR-ABL Mbcr FusionQuant® Kit (Catalogue FQPP-10-CE) at the end of the study after the availability of real-time PCR (AB1 7500 real-time PCR, Applied Biosystems, USA). A 3-log reduction in BCR-ABL transcripts was considered an MMR.
Resistance patterns were adopted as defined by the LeukemiaNet guidelines [24]. Primary or intrinsic resistance was defined by the failure to achieve CHR by 3 months, any cytogenetic response by 6 months, partial cytogenetic response by 12 months, and complete cytogenetic response by 18 months. Acquired or secondary resistance was defined as the loss of previous hematological, cytogenetic, or molecular responses, sustained CHR that was followed by transformation to the accelerated or blastic phase, Ph+ clonal evolution, or the emergence of clinically relevant BCR-ABL KD mutations predicted to confer resistance [25].
Isolation of CD34+ CML Stem/Progenitor Cells
BM mononuclear cells were isolated by Ficoll-Hypaque (Sigma Diagnostics, St Louis, MO) density gradient separation (specific gravity, 1.077) for 30 min at 400×g. The cells were then suspended in a solution of 10% dimethylsulfoxide (DMSO) in fetal calf serum (FCS) and cryopreserved in liquid nitrogen until required [26]. Before use, cells were thawed and stained with antibodies to CD34 directly conjugated to fluorescein isothiocyanate (Becton Dickinson Immunocytometry System, San Jose, CA). After staining for 30 min at 4°C, the cells were washed twice in phosphate-buffered saline containing 2% FCS (Stem Cell Technologies Inc.) and resuspended in 2 µg/mL propidium iodide (Sigma). CD34+ cells were collected by fluorescence-activated cell sorting (FACS) using a FACSVantage cell sorter (Becton Dickinson, San Jose, CA) [27].
Detection of Pre-existing BCR-ABL Mutations
RNA and DNA were extracted from FACS-sorted CD34+ cells using TriZol and DNAzol (Invitrogen Life Technologies, Carlsbad, CA) methods, respectively [28]. RNA and DNA quality was checked by spectrophotometry, gel electrophoresis, and by the amplification of the ABL gene [14], [18]. As BCR-ABL PEMs are known to be rare among wild-type BCR-ABL and thus cannot be detected by sequencing the whole BCR-ABL KD, we employed a very sensitive ASO-PCR assay for this purpose which has already been optimized and clinically validated using appropriate positive and negative controls elsewhere [31]. This assay can detect 18 of the most clinically relevant and common BCR-ABL mutations [14], [29]–[30]. PCR amplifications were performed exactly as reported, without changing any of the reagents, PCR mix formulations and thermal profile [29]. The sequences of ASO primers specific for each mutation with the corresponding annealing temperatures are given in Table 2. HL60 cell line (ATCC # CCL-240™) was used as a negative control in ASO-PCR reactions. Although we used pre-validated ASO-PCR assays and reproduced those assays using exactly same reaction conditions, reagents and PCR mix formulation, to eliminate the possibility of false-positive results ASO-PCR products were sequenced on both strands using an automated ABI377 sequencer (Applied Biosystems). Sequences were analyzed with Sequence Analysis software V3.3 and Sequence Navigator software V1.0.1 (Applied Biosystems). A mutation was considered present only if it was detected in both strands in two or more independent ASO-PCR amplified products [14], [29]–[31].
10.1371/journal.pone.0055717.t002Table 2 Sequences of ASO primers and corresponding annealing temperatures (bold nucleotides in the primers denote nucleotide changes corresponding to mutations).
*Primer name Primer polarity
**Nucleotide change 5′–3′ sequence
$L(bp)
#A Tm(°C)
1. M244V-F F A1094G
GAACGCACGGACATCACCG
19 65.7
2. L248V F C1106G
ACCATGAAGCACAAGG
16 55
3. G250E F G1113A
GAAGCACAAGCTGGGCGA
18 56
4. Q252H(a) F G1120C
AGCTGGGCGGGGGCCAC
17 62
5. Q252H(b) F G1120T
AGCTGGGCGGGGGCCAT
17 62
6. Y253H F T1121C
GCTGGGCGGGGGCCAGC
17 62
7. Y253F F A1122T
CTGGGCGGGGGCCAGTT
17 55
8. E255K F G1127A
GCGGGGGCCAGTACGGGA
18 68
9. E255V F A1128T
GCGGGGGCCAGTACGGGGT
19 58
244 R R(1–9) –
GCCAATGAAGCCCTCGGAC
19
10. F311L F T932C
CACCCGGGAGCCCCCGC
17 62
– R(10) –
CCCCTACCTGTGGATGAAGT
20
11. T315I F C1308T
GCCCCCGTTCTATATCATCAT
21 63.4
12. F317L F C1315G
CCGTTCTATATCATCACTGAGTTG
24 54
315 R R(11–12)
GGATGAAGTTTTTCTTCTCCAG
22
13. M343T F T1392C
GTGGTGCTGCTGTACAC
17 62
14. M351T F T1416C
CCACTCAGATCTCGTCAGCCAC
22 70
351 R1 R (13–14)
GCCCTGAGACCTCCTAGGCT
20
15. E355G – A1428G
GTCAGCCATGGAGTACCTAGG
21 56
16. F359V F T1439G
GAGTACCTAGAGAAGAAAAACG
22 50
351 R2 R (15–16)
ATGCCCAAAGCTGGCTTTG
19
17. H396R F A1551G
GGACACCTACACAGCCCG
18 62.5
369 R R(17)
GGACACCTACACAGCCCG
18
18. F486S F T1821C
TCTGACCGGCCCTCCTC
17 62
486 R R(18)
AGCTTTCTGGTCTCAGGA
18
* Substitutions of amino acids; positions according to GenBank no. AAB60394for ABL type 1a.
** Changes of nucleotide; positions according to GenBank no.M14752.
$ L (bp) = Primer length in base pairs.
# A Tm = Annealing temperature in degree Celsius.
Imatinib Treatment and Response Monitoring
All patients were treated with 400 mg of imatinib/day. Clinical studies were performed in collaboration with CML treatment centers. Patients were monitored every 2 weeks for hematological response and every 3 months for cytogenetic and molecular response during imatinib treatment and follow-up. Secondary resistance, as described previously, was also monitored. For imatinib-resistant patients, second-generation TKIs were not available due to financial constraints. However, imatinib-resistant patients were treated with 600–800 mg of imatinib/day, irrespective of presence or absence of PEMs [7]. Patients were monitored regularly every 2 weeks for hematological response and every 12 weeks for cytogenetic and molecular responses after imatinib dose escalation.
Detection of Mutations After the Manifestation of Imatinib Resistance
All imatinib-resistant patients, irrespective of their PEM status, were investigated for BCR-ABL mutations using ASO-PCR [14], [29]–[30], as well as by DNA sequencing of the RT-PCR–amplified whole BCR-ABL KD. For RT-PCR and DNA sequencing of the BCR-ABL KD, we adopted the protocol described by Branford and Hughes [31] using an automated ABI377 sequencer (Applied Biosystems). HL60 cell line (ATCC # CCL-240™) was used as a negative control in PCR and sequencing while KCL22 cell line (DSMZ # ACC 519) was used as a positive control. Sequences were analyzed with Sequence Analysis software V3.3 and Sequence Navigator software V1.0.1 (Applied Biosystems). To confirm mutation detection by sequencing, the opposite strand of the PCR product was sequenced. Moreover, the whole procedure of RNA extraction, RT-PCR, and sequencing was repeated once. Detection of the mutation was confirmed only if the same mutation was detected in both DNA strands as well as in the repeat analysis [15], [29], [31].
Statistical Analysis
Various clinical parameters, frequencies of imatinib resistance, and clinical response rates were compared in the two subgroups of patients with and without PEMs by Chi-square test using “Statistical Package for Social Sciences (SPSS)” software, version 17. A p-value of <0.05 was considered significant.
Results
Pre-existing and Post-resistance BCR-ABL Mutations
BCR-ABL PEMs were detected in 32 out of 100 (32%) patients (Table 3). We found three mutations, namely T315I, F311L, and M351T, either alone or in combination, as PEMs in this group of CML patients. The frequencies of the M351T, F311L, and T315I mutations were 87.5%, 50%, and 37.5%, respectively, either alone or in combination. Thus, M351T was the most common PEM, whereas T315I was the least common PEM detected (Figure 2). After a median follow-up of 30 months (range 8–48), patients with BCR-ABL PEMs exhibited imatinib resistance (32/32, 100%). Upon re-investigation of BCR-ABL mutations in these patients using ASO-PCR and DNA sequencing, all patients had the same PEMs (Figure 1 and 2). Regarding the 68 patients without PEMs, imatinib resistance developed in 24 (24/68, 35.3%) patients. BCR-ABL mutations (alone or in combination) were found in 22 of these patients (Table 3; Figure 2). By DNA sequencing, we were able to detect Y253F mutation in one of the patients as an acquired mutation (not as a PEM). T315I (12/22, 54.5%) and F311L (15/22, 68.2%) were the most common mutations in this group of patients, whereas M351T was detected in 9/21 (42.8%) patients.
10.1371/journal.pone.0055717.g001Figure 1 Detection of BCR-ABL mutations by ASO-PCR and DNA sequencing.
(-ve control = negative control, bp = base pair, PEM = pre-existing BCR-ABL mutations, C = Cytosine, T = Thymine, A = Adenine, G = Guanine). HL60 cell line was used as a negative control in ASO-PCR and sequencing while KCL 22 was used as positive control in RT-PCR and DNA sequencing).
10.1371/journal.pone.0055717.g002Figure 2 Comparison of the frequencies of pre-existing BCR-ABL KD mutations and mutations detected after manifestation of imatinib resistance in CML patients.
10.1371/journal.pone.0055717.t003Table 3 Clinical, cytogenetic, and molecular follow-up studies of CML patients with and without BCR-ABL PEMs who received imatinib treatment.
Group (s) Characteristics N (%) Hematological response N (%) Cytogenetic response N (%) MMR N (%)
CHR PHR No HR CCyR PCyR Minor CyR Minimal CyR
Group 1
Patients with PEM (A) 32 (100) 23 (71.9) 9 (28.1) – 17 (53.1) 7 (21.9) 3 (9.4) 5 (15.6) –
Group 2
Patients without PEM (B = C+D) 68 (100) 62 (91.2) 3 (4.4) 3 (4.4) 38 (55.9) 19 (27.9) 5 (7.4) 6 (8.8) 28 (63.6)
Patients without PEM, resistant to imatinib (C) 24 (68) 19 (79.2) 2 (8.3) 3 (12.5) 7 (29.2) 11 (45.8) 2 (8.3) 4 (16.7) –
Patients without PEM, susceptible to imatinib (D) 44 (68) 43 (97.7) 1 (2.3) – 31 (70.5) 8 (18.2) 3 (6.8) 2 (4.5) 28 (63.6)
N: number of patients, PEMs: pre-existing mutations; IM: imatinib; CHR: complete hematological response; PHR: partial hematological response; CCyR: complete cytogenetic response; MCyR: major cytogenetic response; minor CyR: minor cytogenetic response; MMR: major molecular response.
Association of Mutations with Clinical Parameters
No significant association was found between BCR-ABL KD PEMs and clinical parameters such as age, gender, type of BCR-ABL splice variant, white blood cell count, hemoglobin level, and platelet count (data not shown). Imatinib-resistant CML patients with and without PEMs significantly differed with respect to frequency of imatinib resistance (100% vs. 35.3%, p = 0.01), CHR (71.9% vs. 91.2%, p = 0.05), partial hematological response (28.1% vs. 4.4%, p = 0.01), minor cytogenetic response (15.6% vs. 8.8%, p = 0.05), and CMR (0% vs. 41.2%, p = 0.001), while no significant difference was found in terms of CCyR (53.1% vs. 55.9%), time to development of resistance (16.5 vs. 18.2 months), and time to progression of disease (27.6 vs. 32.3 months) in the two groups. All CCRs correlated with MMRs on archived samples.
Management of Resistant Patients
Resistant patients were treated with 600–800 mg of imatinib/day irrespective of PEM status. Patients harboring the T315I mutation (alone or in combination with F311L/M351T) did not exhibit any response, and progressed to accelerated-phase or blast-crises (12/32, 37.5%). In this group of patients with F311L/M351T PEMs (20/32, 62.5%), 16 patients (16/20, 80%) exhibited complete hematological, cytogenetic, and molecular responses to dose escalation, whereas four patients had partial cytogenetic responses (4/20, 20%). Fifteen CML patients without PEMs harboring a T315I mutation (alone or in combination with F311L/M351T/Y253F) did not respond to imatinib dose escalation and progressed to an advanced phase, whereas 7 out of 9 (77.8%) patients harboring F311L/M351T mutations responded to dose escalation with complete hematological, cytogenetic, and molecular responses.
Discussion
CP-CML comprises of two types of cells. The majority of cells of the leukemic clone comprise a more mature type that is sensitive to TKIs. A small population of stem/progenitor (CD34+) cells is less sensitive to TKIs and is usually responsible for the development of resistance to therapy [21], [22]. The BCR-ABL fusion gene is highly unstable in these primitive CML cells, and it is associated with frequent genetic alterations and mutations in BCR-ABL itself as well as in other genes such as p53 even in the absence of imatinib exposure [30]. These naturally occurring genetic variants of BCR-ABL are known as pre-existing BCR-ABL mutations (PEMs) [7]. Although the mechanism of clinical resistance to imatinib in CML varies widely, BCR-ABL KD point mutations are the leading cause of imatinib resistance [1], [11]. If these mutations are present in critical regions of BCR-ABL, they can affect the binding of BCR-ABL protein with TKIs. The impaired binding of imatinib to these BCR-ABL mutants results in an inadequate response or loss of response. The mutant strains proliferate under selective pressure of TKIs after treatment initiation, leading to drug resistance [4], [11]. These mutations are likely to be present at an early stage of disease evolution and become clinically manifested due to selective overgrowth after imatinib treatment [20], [23].
Our study demonstrated that BCR-ABL PEMs might be found in a substantial number of newly diagnosed CP-CML patients if sensitive techniques such as ASO-PCR are used to assess CD34+ stem/progenitor cells, and these PEMs can significantly affect the outcome of imatinib therapy. BCR-ABL PEMs have been reported previously in newly diagnosed CP-CML patients in some studies [14]–[15], [17], [20], [31], whereas others failed to detect any mutations in CP-CML patients before treatment initiation despite using sensitive techniques [18]. Most of these studies were limited by a small sample size and CD34+ cell population was not specifically targeted for mutation detection. Ours is the largest study to date on the incidence of naturally occurring BCR-ABL KD mutations using CD34+ cells and their association with imatinib resistance. Although more than 50 BCR-ABL mutations have been reported, we analyzed for the 18 most common mutations as: 1) they cover more than 90% of the mutations responsible for imatinib resistance and not all the mutations are clinically relevant [18], [20], 2) these 18 mutations can be detected by ASO-PCR which is the most sensitive technique to detect low copy number mutations like pre-existing BCR-ABL mutations. Furthermore, detection of PEMs using ASO-PCR in a group of CML patients and detection of the same mutations after a period of time after manifestation of imatinib resistance in that group using ASO-PCR as well as sequencing, is an indirect proof of validity of ASO-PCR for PEM detection with a minimal possibility of false positive results, which was done in our study.
The reasons for the presence of PEMs in almost one-third of our CP-CML patients are not entirely clear. We selected CD34+ cells to detect KD mutations because this compartment of primitive cells is likely to be the source of many of these mutations [14] and this, in combination with the use of a sensitive technique such as ASO-PCR in a larger number of patients, may explain our findings [19]. Furthermore, it is also known that patients with advanced CP-CML are more likely to exhibit various KD mutations and primary resistance [5], [33]. Many patients in our area present late due to poor knowledge, lack of education, and the use of traditional remedies before seeking medical advice. Therefore, we cannot rule out the possibility that our patient population may be skewed toward a higher-risk group of CP-CML patients [19]. This could explain the higher mutation detection rate in some of these patients.
To the best of our knowledge, there are no studies of KD mutation detection in CML patients comparing whole mononuclear cells (MNC) with CD34+ cells, so we can only hypothesize that by selecting CD34+ cells, the likelihood of detection of low level mutations is increased. Our findings are also supported by the work performed by Chu et al, who reported that KD mutations, when studied in CD34+ cells, were present even during complete cytogenetic remission in 5 of 13 CML patients treated with imatinib [19]. Recent studies indicate that residual BCR/ABL+ progenitors persist despite undetectable molecular disease in CML patients responsive to imatinib [32]. Furthermore, Jiang et al recently showed that KD mutations were present even in very primitive (CD34+CD38−) stem cells [20]. Overall, these data suggest that CD34+ progenitor/stem cell compartment usually harbors KD mutations in CP-CML, and these low level mutations are more likely to be detected in CD34+ cell population, as compared to MNC. These findings also support the idea that primitive CML cells have an intrinsic tendency to continuously acquire new mutations independent of therapy. Some of the mutations would be expected to confer imatinib resistance; others could lead to disease progression. It is the nature and timing of these mutations at diagnosis and during imatinib treatment that may explain the variable clinical responses in different patients [6], [15]–[16], [20], [33]–[34]. Thus, the CML patients labeled clinically as imatinib responders and non-responders display significant differences in the frequencies of mutant BCR-ABL transcripts present in their pretreatment CD34+ cells [20], [35].
Different BCR-ABL mutations have prognostic significance and vary in their effects on the sensitivity to standard doses and dose escalations of imatinib and as well as to other TKIs [3], [6]–[7], [13], [36]–[39]. All of our resistant patients were treated with imatinib dose escalation to 600–800 mg daily irrespective of their PEM status. We did not have an opportunity to treat imatinib-resistant patients with second-line TKIs because these agents were not obtainable due to the high cost and lack of funding (only imatinib was supplied free of cost to these patients by a non-governmental organization). Twelve patients with T315I PEM (alone or in combination with F311L and/or M351T) did not respond to imatinib dose escalation, and they progressed to accelerated-phase or blast-crisis. In the group of patients with PEM, 16 of 20 patients with F311L and/or M351T mutations exhibited complete hematological, cytogenetic, and molecular responses to dose escalation, whereas the other four patients exhibited partial cytogenetic responses. Fifteen CML patients without PEMs harboring T315I mutation (alone or in combination with F311L, M351T, and/or T253L mutations) did not respond to imatinib dose escalation, as expected, and progressed; whereas 7 out of 9 patients harboring F311L and/or M351T mutations responded to dose escalation, achieving complete hematological and cytogenetic responses. Overall, 31 CML patients remained resistant to imatinib even after dose escalation.
We detected more than one mutation in some of the patients. These mutations could be either in the same clone or two different clones, and cannot be confirmed by direct ASO-PCR or sequencing techniques. In order to know if such multiple mutations in the same patient are present in the same or different clones, one need to sub-clone the PCR fragments, select at least 20 or more different clones and perform sequencing of each clone. Although it may be interesting to study the clone specificity and the response to treatment in patients with “multiple mutations in two or more different clones” and “multiple mutations in the same clone”, the clinical value of determining this clone specificity of multiple mutations remains to be established.
Currently, screening for BCR-ABL mutations is not recommended in newly diagnosed CP-CML patients [24] because the frequency of mutations in these patients was found to be low in previous studies, these mutations may not necessarily correlate with response, and the screening costs are prohibitive [18], [24], [36], [39]–[40]. According to the European LeukemiaNet guidelines for CML management, mutation analysis of CP-CML patients treated with imatinib should be performed when there is evidence of inadequate response or loss of response [24]. Our study revealed that using sensitive techniques and CD34+ cell population, BCR-ABL KD mutations may be found in a substantial number of patients and correlate with response to imatinib therapy and pre-treatment mutation detection may have important clinical implications in the post-imatinib era. FDA has approved two second-line TKIs–dasatinib and nilotinib–for the frontline treatment of CML. BCR-ABL mutations respond differently to three tyrosine kinase inhibitors approved for first-line treatment of CML, e.g., Y253F and G250E mutations resistant to imatinib can respond to nilotinib or/and dasatinib, T315A shows better response to imatinib though resistant to Dasatinib and Nilotinib, while some mutations are less sensitive to nilotinib (E255K/V and F359V/C) or dasatinib (F317L and V299L) [41]. In this scenario, knowledge about the presence and type of mutations may facilitate timely decision making regarding the choice of first-line therapy at the time of diagnosis. Patients with mutations known to confer resistance to standard or high doses of imatinib can benefit from an upfront treatment with a second-line TKIs and vice versa. For patients with mutations such as T315I which is known to confer resistance to all TKIs currently approved for first-line treatment of CML, one of the newer agents such as ponatinib (AP24534) which is effective against this mutation [41] and very recently been approved by FDA for TKI-resistant CML [42], or allogeneic transplantation must be considered.
Second-generation TKIs induce cytogenetic responses in around 50% of patients with CP-CML in whom imatinib treatment has failed. Although two of the second lines TKIs have been approved for first line therapy of CML, we still find the applicability of this study for the future because of the cost issues. Imatinib patent is about to finish in the near future and with the availability of generic forms of imatinib, the cost difference between imatinib and 2nd line TKIs is going to be substantial. Stratification of patients based on mutations before the start of therapy may have significant cost savings.
We acknowledge the fact that there is high incidence of imatinib resistance in our study patients. Patients with CML vary in their response to treatment and although the basis for this variation is not known, it has been attributed to the biologic heterogeneity of the disease. Some of the factors implicated in poor response to CML therapy include low level of expression of molecular transporter hOCT1 and multidrug resistance gene (MDR1) polymorphisms [43]–[44]. Population based studies have shown lower efficacy of imatinib in CML patients when compared to the clinical trial results. Lucas et el reported 49% imatinib failure by 24 months and suggested caution in extrapolating clinical trial data to the general CML population [45]. Possible causes of inferior results in the community setting include less strict conditions than in the clinical trials, lesser motivation and poorer compliance with the treatment. Marin et al recently showed that lack of adherence to treatment was an important factor leading to poor results in CML patients [46]. Poor compliance, inclusion of patients in the late chronic phase and genetic variability are the possible explanations for high resistance in our study.
In summary, we found that by using sensitive techniques like ASO-PCR in CD34+ cells, BCR-ABL KD mutations could be detected in almost one-third of CP-CML patients at the time of diagnosis and were found to be associated with the outcome of imatinib therapy. Therefore, testing for BCR-ABL mutations in CD34+ CML stem/progenitor cells using validated sensitive assays like allele-specific PCR may be cost-effective and should be considered before the start of TKI therapy, particularly in patients who present in the late CP. Larger population-based studies with longer follow-up times are needed to estimate the true incidence of KD mutations in this group of patients and determine whether screening is useful in management planning in present scenario of availability of second generation and third generation TKIs for different resistant forms of CML.
We acknowledge the help and collaboration of Professor Moustapha Kassem working at Department of Endocrinology Molecular Endocrinology Laboratory (KMEB), Department of Endocrinology, Odense University Hospital and University of Southern Denmark, and visiting professor at Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University and King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia, for CML stem/progenitor cells isolation and characterization. Help and collaboration of Professor Sai-Juan Chen & Prof Zhu Chen, Directors Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, China for giving us an opportunity of training in leukemia stem cell characterization, BCR-ABL mutation detection and functional characterization of leukemia genes. This work was partially supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia. Research funding provided by Higher Education Commission Pakistan is also acknowledged.
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Exp Ther MedExp Ther MedETMExperimental and Therapeutic Medicine1792-09811792-1015D.A. Spandidos 10.3892/etm.2013.887etm-05-03-0701ArticlesEffects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocytes HU LI *ZHAO JIAJIA *LIU JIARONG GONG NIYA CHEN LILI Department of Stomatology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR
ChinaCorrespondence to: Professor Lili Chen, Department of Stomatology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei 430030, P.R. China, E-mail: [email protected]* Contributed equally
3 2013 07 1 2013 07 1 2013 5 3 701 706 02 9 2012 11 12 2012 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.Adipose stem cell-conditioned medium (ASC-CM) has been successfully used to treat multiple types of tissue and organ defects, including skin wounds both in vitro and in vivo. However, the mechanisms through which ASC-CM promotes wound healing remain unclear. We hypothesized that the wound healing effect of ASC-CM is mediated in part by the promotion of the migration of vascular endothelial cells, fibroblasts and keratinocytes, the three cell types essential for wound healing. We reported that ASC-CM stimulated the migration of these cells sequentially, and endothelial cells were the first cell type to respond to ASC-CM stimulation (4 h), followed by fibroblasts (12 h) and then keratinocytes (24 h). We also determined the optimal concentration of ASC-CM in stimulating these cells (50% dilution) in addition to the optimal time to intervene in order to maximize the wound healing activity of ASC-CM. Our data suggest an important role for ASC-CM in wound healing, possibly through the synthetic action of multiple adipose stem cell-derived cytokines that in turn promote cell migration. Thus, ASC-CM appears to have significant potential in wound healing applications.
adipose stem cell-conditioned mediumvascular endothelial cellsfibroblastskeratinocytesmigration
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Introduction
Adipose-derived stem cells (ASCs) were first isolated by Zuk et al(1) in 2001 from adipose tissues. These cells are able to differentiate into multiple cell lineages including adipocytes, chondrocytes, osteoblasts, muscle cells, endothelial cells and neurocytes (2,3). The yield of mesenchymal stem cells from adipose tissues is much higher than that from bone marrow tissues, adipose tissues are more readily available and the derived stem cells are easier to culture, therefore ASCs are considered to be an ideal source for tissue engineering and have a significant application and research value (4–6). Previous studies have confirmed that adipose stem cell-conditioned medium (ASC-CM) has a marked promoting effect on wound healing (7).
In the skin wound healing process, keratinocytes, fibroblasts and vascular endothelial cells all play important roles and they are the first cell types activated by trauma. Activated cells participate in wound covering, granulation, scar tissue formation, wound remodeling and angiogenesis via a series of cellular activities, including migration and proliferation (8). It is known that the migration of these cells is a key step in the early wound healing process. However, the majority of previous studies have focused on the effect of ASCs on cell proliferation (9) rather than cell migration.
Our previous data have confirmed that ASCs promote the migration of these three types of cells in vitro. As ASCs are known to promote wound healing mainly through a paracrine mechanism, it is plausible that ASCs may exert their effect by secreting cytokines and growth factors that act on neighboring cells to repair the damaged tissues (10–11). In terms of the potential clinical application of ASCs, a few issues have to be resolved such as the selection of an appropriate scaffold (12) and the integration of various cytokines into other tissues. However, ASC-CM has distinct advantages, including that it may be applied locally or via intravenous injection. More importantly, the levels of major cytokines in the ASC-CM may be precisely quantitated. Thus, ASC-CM may be more feasible and practical to use in wound healing than ASCs themselves. However, it is unknown whether ASC-CM influences cell migration, and if so, what the optimal concentrations and intervention times for different cells are. We therefore investigated the effect of ASC-CM on the migration of human keratinocytes, fibroblasts and vascular endothelial cells.
Materials and methods
Isolation and culture of primary human keratinocytes and fibroblasts
Human foreskins were obtained from donors (16–30 years old) undergoing circumcision after giving their informed consent. All procedures were approved by the ethics committee of Wuhan Union Hospital (Wuhan, China). The foreskins were washed several times with sterile phosphate-buffered saline (Thermo Scientific Hyclone, Rockford, IL, USA) and digested as described by Häkkinen et al(13) for isolation of keratinocytes and fibroblasts. The EA.hy926 cell line was used as an alternative for human umbilical vein endothelial cells (HUVECs). These cells were cultured at 37°C in 5.0% CO2. The media were replaced every 2–3 days.
Isolation, characterization and multi-differentiation assay of human adipose-derived stem cells (ASCs)
Human subcutaneous adipose tissues were obtained from female patients (18–35 years old) undergoing lipoaspiration surgery after informed consent was obtained from the patient and approval provided by the ethics committee of Wuhan Union Hospital. The procedures described by Bunnell et al(2) were followed. Cells of passages 3–7 were used in the present study. Surface markers CD13, CD14, CD44, CD90, CD105 and CD34 were detected using a fluorescence-activated cell sorter. Following the differentiation of ASCs in various directions, such as adipogenesis and osteogenesis, the adipogenic lineage was detected by Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) staining and the osteogenic lineage was detected by Alizarin red (Sigma-Aldrich) staining.
Preparation of ASC-CM and protein microarray analysis
ASCs were cultured in DMEM/F-12 containing 10% fetal bovine serum until the cells reached 80% confluence. The culture medium was then replaced by serum-free DMEM/F-12 and incubated for an additional 48 h. The conditioned medium was collected, centrifuged at 165 g for 5 min and filtered through a 0.22-μm syringe filter. The ASC-CM was stored at −20°C and 5 ml medium was used for protein array analysis with the RayBio® Biotin Label-based Human Antibody Array I (AAH-BLM-1-2; RayBiotech, Norcross, GA, USA) which contains antibodies for 507 human proteins.
Migration assays
The effect of ASC-CM on cell migration was determined using a modified Boyden Chamber assay. Briefly, 1×105 HUVECs, fibroblasts or keratinocytes were seeded into the upper chambers, with 300 μl culture medium in the upper chambers and 600 μl culture medium in the lower chambers. After the cells adhered to the bottom of the upper chambers, the medium in the upper chambers was replaced by serum-free DMEM/F-12. The medium in the lower chambers was replaced with medium containing different concentrations of ASC-CM (0, 10, 25, 50, 75 and 100%). In our preliminary studies, we observed that HUVECs clearly migrated within a few hours. However, fibroblasts began to migrate after ten hours, while keratinocytes began to migrate within one or two days. Therefore, we chose to evaluate HUVEC migration within the period of 4–20 h, fibroblasts at 12–36 h and keratinocytes at 24–72 h. Cells on the upper surface of the inserts were removed using a cotton swab and those that had migrated through the filter were stained with crystal violet. Cells in 16 microscopic fields at ×200 magnification were counted. The experiments were performed in triplicate.
Statistical analysis
The values are expressed as mean ± standard deviation. Comparisons between two groups were analyzed by Student’s t-test and comparisons among more than two groups were obtained by ANOVA. P<0.05 was considered to indicate a statistically significant result. The analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA).
Results
Morphology, flow cytometry and multi-differentiation analysis
HUVECs were flat and polygonal-shaped, arranged in short spindles or a cobblestone morphology (Fig. 1A). The primary skin keratinocytes also had cobblestone morphology, a characteristic of epithelial cells in an undifferentiated stage (Fig. 1B). The primary fibroblasts were spindle-shaped and distributed in a radial or swirl shape (Fig. 1C). In the primary and first passage, ASCs proliferated slowly and generated a homogeneous population of flat and fibroblast-like cells after 3 passages (Fig. 2A–D). Flow cytometry showed that the ASCs were positive for CD13 (99.49%), CD44 (92.13%), CD90 (97.78%) and CD105 (96.82%) but negative for CD14 (1.14%) and CD34 (2.71%; Fig. 2E). In order to determine the multipotency of the ASCs, the cells were cultured in adipogenic and osteogenic differentiation medium and the multi-differentiation potential was confirmed by lipid vacuoles positive for Oil Red O staining (Fig. 2F) and colonies positive for Alizarin red staining (Fig. 2G).
Protein microarray analysis of ASC-CM
The amounts of cytokines secreted by ASCs into the medium were analyzed by protein microarrays of ASC-CM. As shown in Fig. 2H, a total of 268 cytokines had a signal that exceeded 300 times that of the background following normalization against the internal control (IC). Among them were 57 common cytokines that have known properties that have the potential to influence cell migration (Table I).
Determination of the optimal concentration of ASC-CM to promote the migration of HUVECs, fibroblasts and keratinocytes
In order to investigate whether ASC-CM impacts the migration of HUVECs, fibroblasts and keratinocytes and to determine the optimal ASC-CM concentration, we performed a dose-response experiment, in which serially diluted ASC-CM (0, 10, 25, 50, 75 and 100%) was added to the lower chambers and its ability to induce cell migration was measured. The stained cells are shown in Fig. 1 [(D) HUVECs, (E) keratinocytes, (F) fibroblasts]. Fig. 3 shows that the migratory effects of 50% ASC-CM on HUVEC, fibroblast and keratinocyte migration were significantly higher than those of either lower concentrations (0, 10 and 25%) or higher concentrations (75 and 100%; P<0.05; Fig. 3A–C). The average numbers of HUVECs, fibroblasts and keratinocytes that migrated to the other side of the chamber in the 50% ASC-CM treated group were 122.69±22.02, 90.88±16.52 and 46.00±10.59, respectively.
Migration assay of HUVECs, fibroblasts and keratinocytes stimulated by 50% ASC-CM for different time periods
To further characterize the effect of ASC-CM on different types of cells and determine the cell types most sensitive in responding to ASC-CM, we examined the responsiveness of HUVECs, fibroblasts and keratinocytes toward 50% ASC-CM for different time periods. The results shown in Fig. 3D–F indicate that the migration of HUVECs occurred the fastest. ASC-CM-stimulated HUVEC migration started within 4 h and peaked at 12 h. Fibroblasts were the second fastest to respond. Fibroblasts started to migrate at 12 h and reached a maximum at 24 h. Keratinocytes appeared to be the slowest to respond to ASC-CM stimulation with the first appearance of migration at 24 h and reaching a maximum at 60 h (P<0.05; Fig. 3D–F, Fig. 4). The net increase in the number of migrated cells was greatest in the period of 4–8 h for HUVECs and 18–24 h for fibroblasts, while keratinocytes kept a constant rate of migration over the time period of this study (Fig. 4 and Table II).
Discussion
Epithelial keratinocytes, dermal fibroblasts and local vascular endothelial cells play significant roles in the skin wound healing process. Previous studies have reported that ASCs are able to accelerate wound healing, possibly through a paracrine mechanism. ASC-CM contains a number of cytokines secreted by ASCs. The effect of these cytokines on cell proliferation has been extensively studied. However, it is less clear whether ASC-CM also influences cell migration and if so, whether it is dose-dependent and what is the optimal intervention timing for different cells. We therefore addressed these unanswered questions in the current study and the results reported in this paper provide a more comprehensive understanding of the effect of ASC-derived cytokines on wound healing.
Previous studies have shown that cytokines including VEGF, bFGF, Ang-1, Ang-2, CDK-5, CD44 and PECAM-A (14–17) are important in promoting the migration of endothelial cells. Kanazawa et al(18) also reported that bFGF may activate RhoA, Rac1, PI3-kinase and JNK in cultured fibroblasts, and promote fibroblast migration. In addition, Maheshwari et al(19) observed that epidermal growth factor (EGF) and fibronectin had a synergistic effect on fibroblast migration. Concerning the migration of keratinocytes, Bae et al(20) reported that keratinocytes could be induced by TGF-β to express the extracellular matrix protein βig-h3 that supported keratinocyte migration by interacting with α3β1 integrin. The results of the protein microarray analysis in the current study demonstrated that ASCs are able to secret multiple cytokines including VEGF, HGF, TGF-β, EGF, FGF, SDF-1 and Ang-1. In addition, flow cytometry revealed high expression levels of CD44 on ASCs. These results suggest an important role for ASCs in wound healing, likely through the secretion of multiple cytokines that in turn promote cell migration.
In particular we studied the effect of ASC-CM on the migration of endothelial cells, fibroblasts and keratinocytes. The results showed that cell migration increased with increasing concentrations of ASC-CM and reached a maximum with 50% of ASC-CM (Fig. 3). Further increases of ASC-CM concentration did not result in any further increase in cell migration but instead diminished cell migration. The low migratory activity at low ASC-CM concentration is likely to be due to the low concentration of cytokines. However, it is currently unknown why a high concentration of ASC-CM is inhibitory. One possible reason is the existence of inhibitory factors in the conditioned medium. The optimal dose of stimulatory and inhibitory cytokines may be different. At the same concentration of ASC-CM, HUVECs were the first to migrate (Fig. 4), followed by fibroblasts and then keratinocytes. These results are consistent with a recent study which suggested that during the tissue remodeling stage of wound healing, dermal fibroblasts along with microvascular endothelial cells may migrate into the wound area prior to keratinocytes (21). Under the optimal concentration of ASC-CM (50%), the increase of HUVEC migration was greatest in the period of 4–8 h and that of fibroblasts was greatest in the period of 18–24 h, while the speed of keratinocyte migration remained constant over the 72 h. Therefore, the optimal intervention timing for vascular endothelial cell migration and fibroblast migration were within 8 and 24 h, respectively. The intervention point for keratinocyte migration was not time-sensitive. Notably these results were generated from in vitro studies. Wound healing in vivo is a much more complex process, so further in vivo studies are required to fully understand the effect of ASC-CM on the migration of different cells in a more physiologically relevant setting.
This study was supported supported by a major grant from the National Natural Science Foundation of China (no. 31110103905).
Figure 1. (A) Human umbilical vein endothelial cells (HUVECs), (B) keratinocytes and (C) fibroblasts from skin, ×100 magnification. Cells stained with crystal violet after migration, (D) HUVECs, (E) keratinocytes and (F) fibroblasts, ×200 magnification.
Figure 2. Isolation and culture of human adipose-derived stem cells (hASCs) in vitro [(A) P0 (B) P1 (C) P2 (D) P3, ×100 magnification]. (Ea-d) Flow cytometry performed on the hASCs in the 3rd passage showed that isolated hASCs positively expressed CD13, CD44, CD90, CD105 and negatively expressed CD14, CD34. (F) Adipogenic differentiated ASCs were positively stained by Oil Red O. (G) Osteogenic differentiated adipose-derived stem cells (ASCs) were positively stained by Alizarin red. (H) Protein microarray analysis of ASC-CM. ASC-CM, ASC-conditioned medium. P0, primary ASCs; P1, passage 1; P2, passage 2; P3, passage 3.
Figure 3. The effect of different concentrations of adipose stem cell-conditioned medium (ASC-CM) on migration of (A) human umbilical vein endothelial cells (HUVECs), (B) fibroblasts and (C) keratinocytes. The migratory effects of 50% ASC-CM were significantly greater than those of either lower concentrations (0, 10 and 25%) or higher concentrations (75 and 100%). Migration assay of (D) HUVECs, (E) fibroblasts and (F) keratinocytes stimulated by 50% ASC-CM at different time periods. Results showed that HUVEC migration started at 4 h, and peaked at 12 h. Fibroblasts started to migrate at 12 h and reached a maximum in 24 h. Keratinocytes appeared to be the slowest to respond to ASC-CM stimulation. Control, 0% ASC-CM; *P<0.05.
Figure 4. Comparison of the migration of human umbilical vein endothelial cells (HUVECs), fibroblasts and keratinocytes stimulated by 50% adipose stem cell-conditioned medium (ASC-CM). Results showed 50% ASC-CM had a clear effect on cell migration, particularly on HUVEC and fibroblast migration, but the effect on keratinocyte migration was less marked, with 0% ASC-CM as the control.
Table I. Common cytokines whose internal control normalization without background exceeded 300 in ASC-CM.
Cytokine Internal control
EDA-A2 10,056.00
IGFBP-7 6,651.00
TSP 4,544.50
RTIMP-1 3,221.50
SPARC 2,607.00
GDF3 1,400.50
NRG3 1,328.50
HCR/CRAM-A/B 1,261.00
MSP α chain 1,253.50
MMP-20 1,085.00
TGF-β5 839.00
IL-22 811.50
FGF-11 800.50
CNTF 704.00
FGF R4 697.00
Angiopoietin-like 1 627.50
MMP-7 566.50
Insulin R 541.00
Endothelin 540.00
CTGF/CCN2 524.00
CCR4 523.50
CXCR2/IL-8 503.50
MMP-1 501.00
BMP-8 493.00
IGF-II 486.00
BMP-5 476.50
VEGF R2 (KDR) 469.50
MMP-15 467.50
G-CSF R/CD114 464.00
HB-EGF 458.00
PF4/CXCL4 456.00
MMP-3 455.00
CCR5 450.50
CXCR6 450.50
IGF-ISR 449.50
HGF 444.00
FGF-16 425.00
Angiopoietin-like 2 419.50
MMP-13 416.50
FGF-10/KGF-2 413.00
FGF-9 410.50
BMP-4 405.50
TGF-β2 402.50
SDF-1/CXCL12 400.00
VEGF R3 396.00
VEGF-D 395.00
FGF Basic 389.50
MMP-8 371.50
PDGF-AA 363.50
Angiopoietin-like factor 361.50
VEGF 360.50
MMP-2 359.00
Angiopoietin-1 352.50
Angiopoietin-4 342.50
IL-1β 333.00
PDGF-BB 312.50
TNF-β 309.00
ASC-CM, adipose stem cell-conditioned medium.
Table II. Effects of 50% ASC-CM on the migration of HUVECs, fibroblasts and keratinocytes over different time periods.
Migration cells/field
Cell Time (h) 50% ASC-CM Control M1 M2
HUVEC 4 80.63±15.82 33.63±14.80 47.00 -
8 122.69±22.02 43.13±21.86 79.56 32.56
12 151.69±57.74 60.25±14.99 91.44 11.88
16 141.56±29.14 58.38±11.54 83.19 −8.25
20 130.19±22.36 49.19±14.57 81.00 −2.19
Fibroblast 12 62.19±27.46 23.19±10.42 39.00 -
18 90.88±16.52 26.94±13.40 63.94 24.94
24 136.69±10.20 44.81±11.31 91.88 27.94
30 125.44±23.55 51.13±9.27 74.31 −17.56
36 118.94±21.66 48.81±7.33 70.13 −4.19
Keratinocyte 24 32.94±12.17 18.00±8.70 14.94 -
36 39.56±13.49 21.25±9.50 18.31 3.38
48 46.00±10.59 24.25±11.13 21.75 3.44
60 54.44±11.55 29.50±10.52 24.94 3.19
72 53.81±11.73 32.13±10.59 21.69 −3.25
At each time point, cells stimulated by 50% ASC-CM migrated more than control cells. The net increase of migrated cells was greatest between 4–8 h for HUVECs and 18–24 h for fibroblasts, while keratinocytes demonstrated a constant migration over the entire time period. (M1=M(50% ASC−CM)−M(control), M2=Mn−Mn−1). ASC-CM, adipose stem cell-conditioned medium; HUVEC, human umbilical vein endothelial cell.
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Exp Ther MedExp Ther MedETMExperimental and Therapeutic Medicine1792-09811792-1015D.A. Spandidos 10.3892/etm.2012.868etm-05-03-0711ArticlesThe correlation between microvessel pathological changes of the endplate and degeneration of the intervertebral disc in diabetic rats CHEN SEN 12*LIAO MEIMEI 2*LI JIANPING 1PENG HAO 1XIONG MIN 21 Department of Orthopedics, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060;2 Dongfeng General Hospital, Hubei University of Medicine, Shiyan, Hubei 442008,
P.R. ChinaCorrespondence to: Dr Hao Peng, Department of Orthopedics, Renmin Hospital of Wuhan University, 99 Zhangzhidong Road, Wuhan, Hubei 430060, P.R. China, E-mail: [email protected]* Contributed equally
3 2013 20 12 2012 20 12 2012 5 3 711 717 29 10 2012 26 11 2012 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.In this study, the pathological microvessel changes to the endplate and the degeneration of the intervertebral disc of diabetic rats were examined in order to identify the possible mechanism by which diabetes mellitus (DM) induces degeneration of the intervertebral disc. A total of 30 Sprague-Dawley rats were randomly divided into two groups. DM was induced in one of the groups by streptozotocin (STZ) administration. The rats were sacrificed 4, 8 and 12 weeks later. Five rats from each group were sacrificed at each time interval and lumbar disc and endplate tissue were obtained from each rat. The histological changes, collagen expression, microvessel density (MVD) and apoptosis of the disc were investigated by different methods. The expression of collagen I in the diabetic DM group was higher compared to the control group at the three time points (P<0.01), in contrast to the expression of collagen II. The factor VIII-related antigen (FVIII RAg) was expressed in the control and DM groups, while its expression was relatively low in the DM group. The MVD of the DM group was smaller compared to that of the control group at the three time points (P<0.01). The apoptotic index (AI) in the diabetic group was significantly higher compared to that of the control group at the three time points (P<0.01). A negative correlation was observed between the MVD of the endplates and the notochordal cell AI in the two groups. Compared to the control group, the endplate MVD decreased and the cavity became smaller or disappeared in the diabetic rats. In conclusion, there was a negative correlation between MVD and degenerative changes of the intervertebral disc in diabetic rats.
diabetes mellitusintervertebral discmicrovessel
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Introduction
Diabetes mellitus (DM) is a systemic endocrine and metabolic disease with chronic complications that damages almost every organ and system of the body (1–5). One of the characteristic complications of DM is microangiopathy, which constitutes the pathophysiological basis of a wide range of organ damage. In addition to causing diabetic retinopathy, nephropathy and cardiomyopathy, diabetic microangiopathy damages the inter-vertebral disc (6–11). Although the disc is avascular, it depends on diffusion from microvessels at the endplate of the disc to supply the nutrients essential for cell activity and viability and to remove metabolic wastes (12–14). Changes in blood supply cause a deficiency of nutrient supply (10,15–18). This study examined the pathological microvessel changes to the endplate and the degeneration of the intervertebral disc in diabetic rats in order to identify the possible mechanism by which DM induces degeneration of intervertebral discs.
Materials and methods
Animals
A total of 30, three-month-old male adult Sprague-Dawley (SD) rats, obtained from the Experimental Animal Center of Wuhan University, Wuhan, China, and weighing 231–263 g, were used in this study. The rats were housed 5/cage under standard laboratory conditions (12/12-h light/dark cycle, at a temperature of 24–25°C and humidity of 50–55%) and allowed free access to food and water during the study. The rats were fed on a normal pellet diet. All the experimental protocols adhered to the Guidelines for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, revised 1996). The study was approved by the Ethics Committee for Animal Research of Wuhan University, (Wuhan, China).
Grouping and treatment
The rats were randomly divided into the streptozotocin (STZ)-induced diabetes group (DM) and the control group (n=15 rats/group). The fasting blood glucose was measured by examining blood samples from the tail vein using a rapid blood glucose meter (One Touch II; Johnson & Johnson, New Brunswick, NJ, USA). DM was then induced by a single intraperitoneal (i.p.) injection of STZ solution (50 mg/kg). In the control group, the rats were administered the same volume of sodium citrate buffer. Fasting blood glucose levels were measured 3 days later. The blood glucose was examined ≥3 times following STZ injection and every two weeks thereafter. The standard glucose measurement for the diagnosis of DM was >13.8 mmol/l. The rats were sacrificed with a lethal dose of sodium pentobarbital (60 mg/kg i.p.) at intervals of 4, 8 and 12 weeks later. Five rats in each group were sacrificed at each time interval and lumbar disc tissue and endplate was obtained from each rat.
Histopathology
The lumbar 5/6 disc was fixed in 4% para-formaldehyde-0.1 M phosphate buffer (pH 7.4), followed by decalcification with 10% ethylenediaminetetraacetic acid (EDTA)-0.1 M phosphate buffer (pH 7.4). Following decalcification, the tissues were dehydrated in graded ethanol, embedded in paraffin, cut into 6-μm sections in the coronal plane and processed for routine hematoxylin and eosin staining for the evaluation of degeneration under a light microscope. The sections were assessed blindly by two independent authors.
Ultrastructure observation
At 12 weeks, two rats were randomly selected from each group for ultrastructure observation. The lumbar 2/3 disc was fixed with 3% glutaraldehyde solution and then sent to the Microscope Center of Wuhan University for ultrastructure observation.
Immunohistochemistry
Immunohistochemical staining was conducted using the streptavidin-peroxidase complex method. Rabbit anti-mouse collagen I and II, and the factor VIII- related antigen (FVIII RAg) (dilution, 1:200) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) polyclonal antibody was used as the primary antibody. Color development was achieved with 3,3′-diaminobenzidine (DAB), which stained positive cells brown. Cells with brown particles in the plasma were evaluated as positive results. Five views of each slice were randomly selected for analysis. Images were captured on a Zeiss Axioskop 40 microscope equipped with a Canon Eos 10D digital camera (Canon, New York, NY, USA). The optical density of collagen I and II was analyzed by a high resolution graphic analysis system and the average optical density of the five views was recorded as the expression of collagen in the sample.
Microvessel density (MVD)
Microvessels were immunohistochemically marked by FVIII RAg staining. Positive results were determined by Weidner’s method (19). After screening the areas with microvessel spots at low-power field (magnification, ×100), microvessels in the area were counted in a ×400 field. Five separate areas were assessed and the mean was calculated to determine the MVD of each section.
Appotosis of notochordal cells
Apoptotic notochordal cells were detected using the TUNEL method, with an in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany). The assay was performed according to the manufacturer’s instructions. Briefly, following routine deparaffinization and treatment with H2O2 (3%), sections were digested with proteinase K (20 μg/ml, pH 7.4, 12 min) at 25°C and incubated with the reaction mixture (1:40, 60 min) at 37°C. Incorporated fluorescein was detected with horseradish peroxidase following a 30-min incubation at 37°C and subsequently dyed with DAB. Brown nuclei were assessed as positive apoptotic cells. The apoptotic index (AI) was evaluated for one section of 5 high-power fields.
Statistical analysis
Data were presented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used to examine differences between groups. The correlation between the endplate MVD and the nucleus pulposus cell AI was examined by Pearson’s correlation analysis. P<0.01 was considered to indicate a statistically significant difference.
Results
Body weight and serum glucose levels
No accidental deaths occurred during the experiment. The STZ-induced diabetic rats showed significantly smaller body weights compared to the control animals. The typical symptoms of DM in the rats were polydypsia, polyphagia, polyuria and emaciation. The data presented in this study are the blood glucose measurement obtained the third time after the STZ injection. Following STZ injection, the serum glucose levels in the DM group were significantly higher compared to those in the control group (P<0.01) (Table I).
Histological investigation
The histological structure of the disc appeared to be normal at 4, 8 and 12 weeks in the control group (Fig. 1A, C and E). Numerous microvessels were evident in the endplate. The discs of the controls consisted of a large amount of extracellular matrix interspersed with a small number of cells comprising ∼1% of the total volume, where cell morphology varied. Those in the annulus fibrosus and cartilage endplate were more elongated and fibroblast-like compared to those of the nucleus pulposus, which were more rounded or oval and chondrocyte-like, sometimes with a capsule around them. Misalignment was not observed in the inner or intermediate layers of the annulus fibrosus. Cracks and tears were also not observed.
By contrast, in the DM group, the histological structure of the discs gradually exhibited more degeneration at 4, 8 and 12 weeks (Fig. 1B, D and F). Fewer microvessels were evident in the endplate compared to the control group. Decreases in notochordal cells and increases in fibroblasts were observed. Enlarged chondrocytes were observed in the inner and intermediate layers of the annulus fibrosus. The annulus fibrosus demonstrated disrupted alignment and formation. There was also a reduction in the nucleus pulposus matrix accompanied by fibrosis and hyalinization, suggesting depletion of the extra-cellular matrix.
Electron microscopic findings
In the control group, organ-elles such as the golgi apparatus, mitochondria, lysosomes and the cell membrane were intact and orderly with abundant glycogenosome and sparse lipid droplets in the plasma (Fig. 2A and C). Collagenous fibers were scattered neatly and closely in the plasma, while the mesh between the fibers was relatively small. The ultrastructure of the notochordal cell was different between the control and DM groups (Fig. 2B and D). The membrane of the cell and organelles was disrupted, while the organelles swelled and vacuolized and the nucleus structure was destroyed. Collagenous fibers were scattered loosely and messily in the plasma, while the meshes between the fibers were relatively large.
Expression of collagen I and II
The optical density of collagen I and II was used to perform a semi-quantitative analysis (Figs. 3 and 4). It was found that the collagen composition changed greatly in the nucleus pulposus, such that the proportion of collagen I increased, while the proportion of collagen II decreased. The results showed that the expression of collagen I in the diabetic group was higher compared to the control at the three time points (P<0.01) (Table II). However, the expression of collagen II in the DM group was lower compared to the control group at the three time points (P<0.01) (Table III).
MVD of the endplates
FVIII RAg was selected as a marker of vascular endothelial cells to reveal the microvessels of the endplates. FVIII Rag was expressed in the control and DM groups, although the expression in the DM group was relatively low. MVD decreased in the two groups over time. The MVD of the DM group was smaller compared to that of the control group at the three time points (P<0.01) (Table IV).
AI of notochordal cells
AI was measured by TUNEL assay (Fig. 5). A brown nucleus was assessed to be a positive apoptotic cell. Cell apoptosis occurred in the two groups. There were more apoptotic cells/high-power field in the DM group compared to the control group. AI increased over time in the two groups. AI in the diabetic group was significantly higher compared to the control group at the three time points (P<0.01) (Table V).
Correlation analysis between MVD and notochordal cell AI
A decrease in MVD occurred while AI of the disc increased over time in the two groups. A negative correlation was observed between the endplate MVD and the notochordal cell AI in the control group (Pearson’s correlation coefficient, r=−0.953, P<0.01) (Fig. 6A). The same was observed in the DM group, with a correlation coefficient of −0.936 (P<0.01) (Fig. 6B).
Discussion
There are two major nutrient transportation pathways in the intervertebral disc. One is the endplate pathway by which nutrients pass through the bone marrow cavity-blood sinus-cartilage endplate route to support the nucleus pulposus. The other is the annulus pathway. Previous studies have suggested that the endplate route is the major pathway for nutrient transfer to the intervertebral disc (20,21). There is a significant decrease in cortex thickness over the central portion of endplates and shells, with a mean minimum thickness of 0.40 mm, a mean maximum thickness of 0.86 mm and an overall mean of 0.64±0.41 mm. Increased porosity is also observed along the central regions of the cortical shells (22). The porous structure accounts for ∼7–10% of the endplate area (23), providing direct contact between the vertebral blood sinus and the endplate. There is a continuous capillary bed that is most dense in the area adjacent to the nucleus pulposus. There are a number of microvessel plexuses in the center with less MVD at the boundary (24). The vascular branches issuing from the microvessel plexuses pass through the porous structure and form microvessel buds in the endplate (25). These vessels drain either into the subchondral post-capillary venous network or directly into the veins of the marrow spaces in the vertebral bodies (26). The decrease in nutrients constituent in the disc is considered a key factor in disc degeneration (27–29). Loss of nutrient supply may lead to cell death, loss of matrix production and increase in matrix degradation, eventually leading to disc degeneration (10).
The changes of collagen, proteoglycan and water content are the pathological characteristics of intervertebral disc degeneration caused by the dysfunction and quantitative reduction of the disc cells (7,23,29). The collagen ingredient changes that occur during the degeneration process include the decrease of collagen II and the increase of collagen I. A positive correlation reportedly exists between the amount of collagen reduction and the degree of intervertebral disc degeneration (30,31). The reason for the decrease in cell density is cell apoptosis, which is thought to be the principal cause of the extracellular matrix degradation (32,33). However, a high inverse correlation between the density of openings in the osseous endplate (in particular the size of the capillary buds) and the morphologic degeneration grade of the disc, supports the hypothesis that occlusion of these openings may deprive the cells of nutrients, leading to insufficient maintenance of the extracellular matrix and disc degeneration (15,34,35).
In this study, the expression of collagen I increased along with the progression of DM. When compared to the control, the difference was significant (P<0.01). The expression of collagen II follows the opposite pattern, decreasing with the progression of DM. In this instance, the difference between the experimental and the control groups was also significant (P<0.01). In addition, this study has demonstrated that the disc cell AI in the DM group was significantly higher compared to the control group (P<0.01).
Microangiopathy is one of the characteristic complications of DM and the pathophysiological basis of multi-organ damage. In this study, the pathological changes of microvessels in the endplate increased over time in the DM group. The cavity of the microvessels became smaller and less dense. When compared to the control, the difference was significant. The AI of the DM group was also significantly higher compared to the control group, resulting in changes in the extracellular matrix that included the decrease of collagen II and the increase of collagen I. The difference was statistically significant compared to the control (P <0.01). The histological findings confirmed the observations that the disc degeneration of the DM rats was greater compared to that of the control. The statistical analysis demonstrated a negative correlation between the endplate MVD and the notochordal cell AI in the DM and control groups.
It can be hypothesized that hyperglycemia, low oxidative stress and advanced glycosylation end products in DM rats would cause microvessel endothelial cell injury of the disc endplates, thereby leading to either occlusion or a decrease in the number of microvessels. In the immediate aftermath, the blood supply of the disc would decline sharply, causing damage to the disc in two major ways. First, the disc nutrition would be decreased or lost entirely. Second, cellular metabolic wastes and toxins would not be able to be excreted and would accumulate in the disc leading to ischemia, hypoxia, acidosis and eventually to cell necrosis and apoptosis. Cell products would also change. The contents of proteglycan, collagen II and water would decrease, while the contents of collagen I would increase. Calcification and disc fissures would occur and the mechanical properties of the disc would degrade.
Limitations in using a rat lumbar disc model include a smaller disc size and a different cell composition of the rat lumbar disc compared to a human disc. In addition, unlike the human disc, the rat disc is subjected to less mechanical load as it stands on four legs. Due to the limited applicability of the rat model, experiments are necessary to determine the pathological changes of the disc in human beings suffering from DM.
In conclusion, compared to the control, the endplate MVD decreased and the cavity became small or disappeared in the DM rat. DM accelerated the degeneration process of the disc. The results of this study have shown that a negative correlation exists between the mircrovessel density and the degenerative changes of the intervertebral disc within diabetic rats. The aforementioned limitations should be taken into consideration when extrapolating the results to humans.
Figure 1. Photomicrographs showing histological analysis of the transected disc samples. Hematoxylin and eosin (H&E) staining: (A), (C) and (E) show the apparently normal histological structure of the disc at 4, 8 and 12 weeks in the control group. (B), (D) and (F) show the histological structure of the discs exhibiting a gradual degeneration process at the different time points. Arrows in (B) and (D) show a reduction in notochordal cells. The arrow in (F) shows the disruption of the annulus fibrosus. Original magnification, ×100.
Figure 2. The ultrastructure of the disc by scanning electron microscopy (SEM). (A) and (C) show the ultrastructure of the apparently normal disc at 12 weeks in the control group. (B) and (D) show the poor the performance of the discs in the DM group at 12 weeks. The arrow in (B) shows swelled organelles and incomplete nucleus. The arrow in (D) shows that the fibers were in disarray. Original magnification, ×6,000.
Figure 3. Photomicrographs showing the expression of collagen I in the disc samples. Immunohistochemical staining was used to show collagen I in the disc: (A), (C) and (E) show that the expression of collagen I was not significantly changed. (B), (D) and (F) show that the expression of collagen I significantly increased over time. Original magnification, ×400.
Figure 4. Photomicrographs showing the expression of collagen II in the disc samples. Immunohistochemical staining was used to show collagen I in the disc: (A), (C) and (E) show that the expression of collagen II was not significantly changed. (B), (D) and (F) show that the expression of collagen II significantly decreased over time. Original magnification, ×400.
Figure 5. Photomicrographs showing cell apoptosis in the disc samples. TUNEL staining was used to investigate the cell apoptosis of the disc at 12 weeks. The arrow indicates an apoptotic cell. Original magnification, ×400.
Figure 6. Images showing the correlation between the microvessel density (MVD) of the endplates and the notochordal cell apoptotic index (AI). There was a negative correlation between the MVD of the endplates and the notochordal cell AI in the (A) control and (B) DM groups.
Table I. Blood glucose of the rats of each group.
Group Before injection After injection
Control 5.21±0.696 5.13±0.464
DM 5.31±0.519a 20.27±2.600b
a P>0.01 and
b P<0.01 compared to the corresponding value in the control group. DM, diabetes mellitus.
Table II. Optical density of collagen I in the disc of each group.
Group 4 weeks 8 weeks 12 weeks
Control 0.1547±0.00765 0.1710±0.01117 0.1799±0.01395
DM 0.2783±0.01258a 0.4535±0.07003a 0.6078±0.02440a
a P<0.01 compared to the corresponding value in the control group. DM, diabetes mellitus.
Table III. Optical density of collagen II in the disc of each group.
Group 4 weeks 8 weeks 12 weeks
Control 0.6473±0.02904 0.6039±0.03206 0.5774±0.01169
DM 0.4951±0.01540 0.3609±0.04598 0.2594±0.02365
DM, diabetes mellitus.
Table IV. MVD of the disc in each group.
Group 4 weeks 8 weeks 12 weeks
Control 20.80±1.048 18.96±0.910 17.28±0.716
DM 18.36±0.684a 15.24±1.479a 10.72±0.460a
a P<0.01 compared to the corresponding value in the control group. MVD, microvessel density; DM, diabetes mellitus.
Table V. AI of the notochordal cells in the disc of each group.
Group 4 weeks 8 weeks 12 weeks
Control 4.42±0.653 6.06±1.064 8.86±0.559
DM 15.92±1.403a 17.72±0.890a 23.38±1.798a
a P<0.01 compared to the corresponding value in the control. AI, apoptotic index; DM, diabetes mellitus.
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BMC BiochemBMC BiochemBMC Biochemistry1471-2091BioMed Central 1471-2091-13-252315722810.1186/1471-2091-13-25Research ArticleProteomic analysis on N, N′-dinitrosopiperazine-mediated metastasis of nasopharyngeal carcinoma 6-10B cells Li Yuejin [email protected] Na [email protected] Damao [email protected] Zhenlin [email protected] Zhengke [email protected] Chaojun [email protected] Xiaowei [email protected] Gongjun [email protected] Guangrong [email protected] Wenhua [email protected] Faqing [email protected] Medical Research Center and Clinical Laboratory, Zhuhai Hospital, Jinan University, Zhuhai, 519000, Guangdong, China2 Medical Research Center and Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, China3 Metallurgical Science and Engineering, Central South University, Changsha, 410008, People's Republic of China4 Institute of Life and Health Engineering, and National Engineering and Research Center for Genetic Medicine, Jinan University, Guangzhou, 510632, China2012 19 11 2012 13 25 25 5 11 2012 16 11 2012 Copyright ©2012 Li et al.; licensee BioMed Central Ltd.2012Li et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Nasopharyngeal carcinoma (NPC) has a high metastatic feature. N,N′-Dinitrosopiperazine (DNP) is involved in NPC metastasis, but its mechanism is not clear. The aim of this study is to reveal the pathogenesis of DNP-involved metastasis. 6-10B cells with low metastasis are from NPC cell line SUNE-1, were used to investigate the mechanism of DNP-mediated NPC metastasis.
Results
6-10B cells were grown in DMEM containing 2H4-L-lysine and 13C 6 15 N4-L-arginine or conventional L-lysine and L-arginine, and identified the incorporation of amino acid by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Labeled 6-10B cells were treated with DNP at 0 -18 μM to establish the non-cytotoxic concentration (NCC) range. NCC was 0 -10 μM. Following treatment with DNP at this range, the motility and invasion of cells were detected in vitro, and DNP-mediated metastasis was confirmed in the nude mice. DNP increased 6-10B cell metastasis in vitro and vivo. DNP-induced protein expression was investigated using a quantitative proteomic. The SILAC-based approach quantified 2698 proteins, 371 of which showed significant change after DNP treatment (172 up-regulated and 199 down-regulated proteins). DNP induced the change in abundance of mitochondrial proteins, mediated the status of oxidative stress and the imbalance of redox state, increased cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression. DNP also increased the expression of secretory AKR1B10, cathepsin B and clusterin 6-10B cells. Gene Ontology and Ingenuity Pathway analysis showed that DNP may regulate protein synthesis, cellular movement, lipid metabolism, molecular transport, cellular growth and proliferation signaling pathways.
Conclusion
DNP may regulate cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression, increase NPC cells motility and invasion, is involved NPC metastasis.
DinitrosopiperazineCarcinogenNasopharyngeal carcinomaMetastasisQuantitative proteomics
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Background
Nasopharyngeal carcinoma (NPC) is a common malignant cancer in southern China [1]. Epidemiological investigations have revealed that the incidence of NPC has remained high in endemic regions, particularly in southern China with an incidence of 30–80 per 100,000 people per year [2]. NPC has the feature of high invasion and metastasis, and cervical lymphadenopathy is often the only clinical manifestation at initial diagnosis of NPC patients [3]. Therapeutic failure in advanced NPC has resulted from both high rates of local recurrence and distant metastasis.
In Chinese populations in high-incidence regions, the relative risk of NPC is related to their eating habits of the region, particularly with dietary intake of salt-preserved fish [2,4-6]. The process of salt preservation is inefficient and foods can become partially putrefied , consequently, these foods accumulate significant levels of nitrosamines [7,8], which are known carcinogens [7,9,10]. NN ′-Dinitrosopiperazine (DNP) is a predominant volatile nitrosamine in salted fish [11,12]. The carcinogenic potential of DNP in salt-preserved fish is supported by experiments in rats, which develop malignant nasal and NPC [13-15]. Furthermore, DNP can induce malignant transformation of human embryonic nasopharyngeal epithelial cells [16]. Our previous works have shown that DNP induces rat NPC and shows organ specificity for nasopharyngeal epithelium, and found that DNP triggers over-expression of hot shock protein 70 and mucin 5B [17]. Additionally, DNP induces ezrin phosphorylation at Thr567 through activating Rho kinase and protein kinase C, and increases motility and invasion of NPC cells [18]. In the present study, to fully understand the mechanism of DNP-mediated NPC invasion and metastasis, we used a stable isotope labeling with amino acids in cell culture (SILAC) to further analyze the proteomic changes caused by DNP. We found that 371 proteins were regulated by DNP, most of which were not previously reported to be involved in NPC metastasis. Analysis of this vast information provides us with better understanding of the complex regulatory mechanism of NPC high metastasis. Using bioinformatics analysis, we detected many novel signaling components in DNP-regulated signaling pathways.
Methods
Cell culture and stable isotope labeling
NPC cell line 6-10B was derived from the cell line SUNE-1, and has a low metastatic ability [19]. Thus, 6-10B cells were used in the present study to investigate DNP-mediated NPC metastasis. DNP was a carcinogens specially for NPC and its chemical structure is shown in Figure 2A. Additionally, heavy lysine and arginine (2H4-L-lysine and 13C6 15N4-L-arginine) were purchased from Sigma-Aldrich. 6-10B cells were grown in DMEM containing 2H4-L-lysine and 13C6 15N4-L-arginine (“heavy”) or conventional L-lysine and L-arginine (“light”) supplemented with dialyzed fetal bovine serum. After six cell doublings, we assessed the labeled amino acids in cells, and then identified whether cells were completely incorporated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an Autoflex MALDI-TOF-MS instrument (Bruker Dalton). Mass spectra were searched against a database of human proteins and subsequently quantified using Mascot Server (http://www.matrixscience.com).
Figure 1 Non-cytotoxic concentration of DNP in stable isotope-labeled 6-10B cells.A, Structure of DNP, an N-nitroso compound. B, Stable isotope-labeled 6-10B cells were treated with the indicated DNP concentration and then subjected to MTT cell viability assay as described in the Materials and Methods. “OD” indicates the relative optical density at 492 nm. C, After stable isotope-labeled 6-10B cells were treated with the indicated DNP concentration, and cell media were subjected to the LDH assay. LDH activity is per 1 L of media. Data are presented as the means ± standard deviation from three independent experiments, statistically analyzed using Student’s t- test (*, p < 0.05; α=0.05).
3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxyme -thoxyphenyl)-2-(4-ulfophenyl) -2H-tetrazolium assay
To determine the non-cytotoxic concentration of DNP, 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxyme -thoxyphenyl)- 2-(4-ulfophenyl) -2H-tetrazolium (MTT) assay was performed to determine the viability of “heavy” labeled 6-10B cells. Briefly, “heavy” labeled 6-10B cells were seeded in 96-well plates at a density of 5 × 103 cells / well and treated with DNP at a concentration between 0 and 18 μM at 37°C for 24 h. Thereafter, 20 μl MTT (5 mg/ml, 0.5% MTT) was added per well for 4 h. The viable cell number per dish is directly proportional to formazan production, which can be measured spectrophotometrically at 492 nm following solubilization with isopropanol.
Lactate dehydrogenase assay
To further evaluate the non-cytotoxic concentration of DNP in “heavy” labeled 6-10B cells, lactate dehydrogenase (LDH) activity in cell culture media was detected after DNP treatment. Briefly, “heavy” labeled 6-10B cells were seeded in 6-well plates at a density of 2 × 104 cells/well and treated with DNP at a concentration between 0 and 18 μM at 37°C for 24 h. After the exposure period, media were collected for LDH activity measurement using the LDH assay kit (Autec Diagnostica).
DNP treatment and protein preparation
DNP crystals were dissolved in DMSO. Appropriate amounts of the DNP stock solution were added into the culture medium to achieve the indicated concentrations (DMSO concentration, 0.1%) and then incubated with cells for the indicated time periods. At approximately 60% confluence, the “heavy” labeled 6-10B cells were treated with 10 μM DNP for 24 h according to MTT assay data, while the “light” labeled 6-10B cells were treated with only 0.1% DMSO, served as the control. The treated cells were then harvested and suspended with lysis buffer. Lysate was centrifuged at 13,200 rpm at 4°C for 30 min. Supernatant fractions were collected and protein concentrations were determined using BCA assay kit (Pierce).
Cell invasion and motility assay
Cell invasion and motility were assayed according to methods described previously with minor modifications [18]. For the invasion assay, “heavy” labeled 6-10B cells were treated with the indicated concentrations of DNP for the indicated times. The treated cells were seeded into Boyden chamber with Matrigel (Neuro Probe, Cabin John, MD) at the upper part at a density of 1.5 × 104 cells/well in 50 μl serum-free medium and incubated for 12 h at 37°C. The bottom chamber also contained standard medium with 20% fetal bovine serum. The cells invaded to the lower surface of chamber membrane were fixed with methanol and stained with hematoxylin and eosin. The invaded cell numbers were counted under a light microscope. The motility assay was performed as described in the invasion assay without Matrigel coating.
Evaluation of the effect of DNP on NPC metastasis in nude mice
Nude mice experiments were performed as previously described [18]. Twenty BABL/c nude mice (approximately 5–6 weeks old) were purchased from the Animal Center of Central South University. All animal studies were conducted according to the standards established by the Guidelines for the Care and Use of Laboratory by Animals of Central South University. Additionally, the present study protocols were approved by the ethical committee at Central South University. Briefly, 100-μl aliquots of 6-10B cell suspensions (1 × 104 cells) were mixed with Matrigel and injected respectively into the tail veins of the 20 nude mice. They were then randomly divided into two groups, DNP-treated and control groups, containing 10 mice per group. The DNP-treated group was abdominally injected with DNP at a dose of 40 mg/kg (body weight) twice a week for 60 days using a 1-ml sterile syringe. The control group was treated with 0.1% DMSO. After DNP treatment, the metastasis of 6-10B cells to the lung, liver, and lymph nodes was observed. Their metastatic abilities were evaluated by counting tumor metastatic foci on day 60 after the injection.
Gel electrophoresis and in-gel trypsin digestion
Prior to gel electrophoresis, equal amounts of DNP-treated and untreated cell proteins were mixed, separated using 10% SDS-PAGE (4 – 12% Bis-Tris Novex minigel, Invitrogen), and stained silver solution to visualize the gel bands. The entire protein gel lanes were horizontally excised and cut into 48 slices each, and then destained, reduced, alkylated and digested with gold-trypsin at 37°C overnight as described previously [20]. The resulting tryptic peptides were extracted by 90% acetonitrile (Fisher) and 2.5% trifluoroacetic acid (Promega), lyophilized in a SpeedVac, and dissolved in 1% formic acid and 2% acetonitrile before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS analysis
The peptide mixtures were separated using Finnigan Surveyor high-performance liquid chromatography system (Thermo Electron, San Jose, CA) on a C18 reverse phase column, which was coupled online to a linear ion trap/Orbitrap (LTQ-Orbitrap) mass spectrometer (Thermo Electron, San Jose, CA). Briefly, the peptide mixtures were first loaded onto a C18-reversed phase column (100-μm inner diameter, 10-cm long, 3-μm resin from Michrom Bioresources, Auburn, CA), and then separated at a maximal flow rate of 300 nl/min controlled by IntelliFlow technology. The peptide mixtures were separated using the following parameters: 1) mobile phase A: 0.1% formic acid, 2% the acetonitrile, Dissolved in water; 2) mobile phase B: 0.1% formic acid, dissolved in acetonitrile; 3) flow rate: 300nl/min; 4) gradient: B-phase increased from 5% to 35%, 120min. Next, the eluate was online analyzed online in LTQ-Orbitrap mass spectrometer operated in a data-dependent mode, the temperature of the heated capillary was set to 200°C, and the spray voltage was set to 1.85 kV. Full-scan MS survey spectra (m/z 400–2,000) in the profile mode were acquired in Orbitrap with a resolution of 60,000 at m/z 400 after the accumulation of 1,000,000 ions, and followed by five MS/MS scans in LTQ with the following Dynamic Exclusion settings: a repeat count of 2, a repeat duration of 30 s, and an exclusion duration of 90 s. The lock mass option was enabled for survey scans to improve mass accuracy [21]. The data were acquired using Xcalibur (Thermo Electron, version 2.0.7).
Protein identification, quantification and bioinformatics analysis
Protein identification and quantification were performed as previously described with minor modifications [22,23]. Briefly, the mass spectrometric raw data were analyzed using MaxQuant 1.0.13.13 software and the derived peak lists were searched using the Mascot search engine (Matrix Science, version 2.2.04, London, UK) against a concatenated real and false International Protein Index human protein database (V3.52). Mascot search results were further processed by MaxQuant 1.0.13.13 at the false discovery rate of 1% at both the protein, peptide, and site levels. The normalized heavy versus light (H/L) ratios, significance, and variability (%) were automatically produced by MaxQuant 1.0.13.13 software. The final reported protein ratio represents a normalized ratio of H/L SILAC obtained in all technological repeats where the same protein was identified. International Protein Index numbers of all significantly regulated proteins and some unaltered proteins were imported into the Ingenuity Pathway Analysis software tool (http://www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as Gene Ontology, Uniport, and TrEMBL.
Western blotting analysis
Western blotting was used to validate the expression levels of eight dysregulated proteins in DNP-treated and untreated 6-10B cells as described above. 6-10B cells were treated with 5, 10, 20 μM for dose-course and treated with 10 μM for 6, 12, 18, 24, 36, 48 h for time-course. After treatment, supernatants were centrifuged at 300 × g for 4 min and 2000 × g for 8 min to remove dead cells and cell fragments, and proteins were concentrated by centrifugal ultrafiltration using Microcon YM-3 Centrifugal filters (Millipore, Billerica, MA, USA). The treated cells were disrupted with 0.6 ml lysis buffer [1 × PBS, 1% Nonidet P-40, 0.1% SDS, and freshly added 100 μg/ml PMSF, 10 μg/ml aprotinin, 1 mM sodium orthovanadate]. Cell lysates were then subjected to centrifugation of 10000 × g for 10 min at 4°C. Equal protein amounts of cell lysates and culture supernatants were separated by 10% polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (Bio-rad). The membranes were subsequently incubated with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h to block non-specific binding and then overnight with antibodies against aldo-keto reductase (AKR) 1B10, S100P, cathepsin B, cathepsin D, ferritin, α-E-catenin (Cell Signaling Technologies), or clusterin, AGR2, and GAPDH (Santa Cruz.), then incubated with the secondary antibody for 1 h at room temperature. The band signal was developed using 4-chloro −1-napthol/3,3-o-diaminobenzidine, and relative photographic density was quantitated using a gel documentation and analysis system (Pierce, Rockford, USA).
Gene transfect and wound-healing assays
Wound-healing assay was performed as previously described with minor modifications [24]. 6-10B cells (2 × 106) were seeded in 10-mm plates at 37°C for 24 h, and transiently transfected with si-AGR2 or si-mock (Dharmacon) [25] using Lipofectamine 2000 reagent (Life Technologies, Inc.) following the manufacturer’s suggested protocol, and then confluent monolayer of the transfected cell was wounded using a plastic tip. Cells were treated with DNP at 10 μM, and then photographed after 48 h. The cells moving cross the boundaries lines were counted. The transfect cell samples were harvested, and total proteins were extracted. These protein samples were subjected to Western blotting analysis.
Results and discussion
In this study, quantitative proteomics with SILAC were used to identify the different protein of 6-10B cells with or without DNP treatment. As the first step 6-10B cells were labeled with amino acid, and then we assessed the incorporation efficiency of 2H4-L-lysine and 13C6 15N4-L-arginine in 6-10B cells for full incorporation in all proteins after six cell doublings. Three peptides, VEVTEFEDIK (Figure 1A), GHYTEGAELVDSVLDVVR (Figure 1B) and LRQPFFQK (Figure 1C) were separated by 4 Da, 10 Da, and 14 Da corresponding to the mass difference between the above light and heavy isotopes. The entire signal corresponded to the heavy peptide, indicating that incorporation of 2H4-L-lysine or 13C6 15N4-L-arginine was complete. To illustrate the quality of the protein identifications reported, we present MS and MS/MS spectra of clusterin and AKR1B10 from the data obtained from the LTQ-Orbitrap mass spectrometer (Figure 1D, E).
DNP is a carcinogenic N-nitroso compound, and its chemical structure is shown in Figure 2A. Although it is known that the non-cytotoxic concentration of DNP to 6-10B cells is 0–4 μM, even up to 6 μM [18], we determined the non-cytotoxic concentration of DNP in stable isotope-labeled 6-10B cells, cell viability was not significantly altered at 0–10 μM DNP compared with control cells (0.1% DMSO; Figure 2B, *, p < 0.05). To further confirm that 0–10 μM DNP was non-cytotoxic, LDH activity in the cell culture media was detected after DNP treatment. The data revealed that LDH activity was not significantly altered by treatment with DNP concentrations between 0 and 10 μM (Figure 2C, *, p < 0.05). Thus, in all subsequent experiments, the concentration of 10 μM DNP was used.
Figure 2 Labeling of 6-10B cells with2H4-L-lysine and13C6 15N4-L-arginine. 6-10B cells were cultured in DMEM containing 2H4-L-lysine and 13C6 15N4-L-arginine or conventional L-lysine and L-arginine as described in the Materials and Methods. A, the peptide VEVTEFEDIK has a charge state of 1+ and contains one lysine; B, the peptide GHYTEGAELVDSVLDVVR has a charge state of 1+ and contains one arginine. C, the peptide LRQPFFQK has a charge state of 1+ and contains one lysine and one arginine. The peaks marked with blue and red represent the light isotope signal and the heavy isotope signal, respectively. Representative MS/MS and MS spectra from clusterin (D) and AKR1B10 (E). The inset shows the relative ratio of heavy to light versions of each precursor ion.
Although previous work has shown that DNP is involved in NPC metastasis, we first confirmed here that DNP mediated NPC metastasis. A Matrigel-coated Boyden chamber was used to measure invasion. 6-10B cells were treated with DNP at 0, 2, 4, 6, 8, and 10 μM for 24 h and then seeded into the Boyden chamber. The cells that invaded the lower chamber were counted. The invaded cells increased dose-dependently after DNP treatment (Figure 3A-c, lanes 4 to 6 vs lane 1; *, p < 0.05). Compared with the control, the increase was 4.1-fold with 8 μM DNP (Figure 3A-c, lane 5). For detecting 6-10B cell motility with DNP treatment, the treated cells were seeded into a Boyden Chamber uncoated with Matrigel, and motile cells were counted. A similar effect was observed for the motility of DNP-treated cells (Figure 3B-c, lanes 4 to 6 vs lane 1; *, p < 0.05). The cell motility increased by 5.6-fold after treatment with 8 μM DNP (Figure 3B-c, lane 5). To further confirm DNP –involved metastasis in vivo, the treated 6-10B cells were mixed with Matrigel, and then were injected into the tail veins of BABL/c mice. Tumor metastatic nodes of 6-10B cells in the lungs, livers and lymph nodes were detected. Metastatic foci in mice lungs were significantly observed in nude the mice with DNP treatment (Figure 3C, left panel vs right upper line), and pathologically confirmed under microscope (Figure 3C, left panel vs right down line). These data indicated that DNP mediates NPC metastasis in vitro and in vivo.
Figure 3 DNP-mediated 6-10B cell invasion and motility in vitro or metastasis in vivo. A, stable isotope-labeled 6-10B cells were treated with the indicated DNP concentration for 24 h. The treated cells were subjected to analyses for motility and invasion as described in the Materials and Methods. A, invasion of 6-10B cells at various concentrations. Arrow, invaded cell. B, motility of 6-10B cells at various concentrations; Arrow, motile cell. The data were statistically analyzed by one-way analysis of variance with post hoc Dunnett’s test (*, p < 0.05). Scale bar, 5 μM. C, twenty nude mice were injected with 6-10B cells in Matrigel through the tail vein (1 × 104 cells/mouse), and then randomly divided into two groups with 10 mice per group. The DNP group was abdominally injected with DNP using a stumped needle at a dosage of 40 mg/kg (bodyweight), twice a week for 60 days. The control was injected with 0.1% DMSO. After 60 days, tumor metastatic foci of 6-10B cells were observed. Scale bar, 10 μM. Arrow, tumor metastatic foci.
To fully investigate the mechanism of DNP-mediated NPC metastasis, SILAC coupled with LC-MS/MS was used to identify and quantify the proteomic differences. A total 2853 proteins were detected, and 2698 (94.57%) proteins could be quantified. Of these 2698 protein, 172 were calculated to be highly up-regulated, and 199 were significantly down-regulated at a ratio H/L >2.0 or ratio H/L <0.5 and p < 0.05 (Figure 4A). To gain functional insight into the cellular proteome, the 172 up-regulated and 199 down-regulated proteins were respectively assigned to different molecular functional classes and subcellular annotations according to the underlying biological evidence from the Gene Ontology database (false discovery rate < 0.05). Because some proteins generally have more than one component annotation or function annotation, the sum of each category may be higher than 100%. The 15 most abundant terms are shown in Figure 4B, C, with additional data shown in Additional file 1: Table S1. Mitochondrion proteins and proteins related to junctional mechanisms were highlighted in up-regulated and down-regulated proteins individually (Figure 4D), suggesting that further exploration at subcellular levels is necessary. Functional analysis of these differential proteins showed that DNP-treated high metastatic 6-10B cells demonstrated significant changes in oxidoreductase activity, cofactor binding, and cytoskeletal protein binding (Figure 4E).
Figure 4 Proteome quantitation, significance, and classification analysis.A, signal intensities of all quantified proteins after DNP treatment are shown as a function of their fold change. Ratios of most proteins distributed around 1.0 (Log10 (ratio) = 0), indicating that whole proteins of the two groups of cells were mixed equally and 6-10B cells were fully labeled. The spread of the cloud was lower at high abundance, indicating that quantification is more precise, and their fold change level is indicated in blue, red, and green, respectively. To reduce test error, proteins at more than 2.0-fold or less than 0.5-fold and p < 0.05 were deemed to indicate significantly changed proteins induced by DNP treatment. Together, this gives quantifiable results for 371 proteins being significantly altered upon DNP, with 172 and 199 proteins being up- and down-regulated, respectively. The 15 most abundant terms of component (B) and function annotation in disregulated proteins (C) are shown. As each protein is generally assigned to more than one term, the percentage of proteins in each term is shown instead of the total number to avoid redundancy. Number distribution of the 15 most abundant terms for up-regulated or down-regulated proteins (D). Oxidoreductase activity-1, 2, 3, and 4 represent respectively oxidoreductase activity, oxidoreductase activity acting on NADH or NADPH, oxidoreductase activity acting on the CH-CH group of donors, and oxidoreductase activity acting on the CH-CH group of donors, NAD or NADP as acceptor (E).
Proteins that changed significantly in DNP-treated cells were mapped to 15 specific functional networks with each network containing 11 or more “focus” members (Figure 5A, Additional file 2: Table S2). The four networks of interest correspond to the following: (A) Cancer, Renal and Urological Disease, Cell Death (Figure 5B); (B) Cancer, Reproductive System Disease, Cell Death (Figure 5C); (C) Cellular Movement, Lipid Metabolism, Molecular Transport (Figure 5D), and (D) Protein Synthesis, Cell Death, Cellular Growth and Proliferation (Figure 5E). Proteins that are present in these pathways and that were identified in our analysis as up-regulated are depicted in red, and proteins that were identified as down-regulated are shown in green. Proteins known to be in the network but that were not identified in our study are depicted in white. The shade of the color indicates the magnitude of the change in protein expression level.
Figure 5 Ingenuity Pathway Analysis of proteins induced by DNP.A, overview of 15 specific functional networks, each of which contains 11 or more “focus” proteins (proteins that were significantly up- or down-regulated). Each box contains an arbitrary network number. B, cancer, renal and urological disease, and cell death. C, cancer, reproductive system disease, and cell death. D, cellular movement, lipid metabolism, and molecular transport. E, protein synthesis, cell death, and cellular growth and proliferation. Red, up-regulated proteins; Green, significantly down-regulated proteins; White, proteins known to be in the network but were not identified in our study. The color depth indicates the magnitude of the change in the protein expression level. Lines connecting the molecules indicate molecular relationships. Dashed lines indicate indirect interactions, and solid lines indicate direct interactions. The arrow styles indicate specific molecular relationships and directionality of the interaction. Abbreviations are shown in Additional file 2: Table S2.
To confirm the SILAC results, eight proteins with different fold changes, AKR1B10, clusterin, cathepsin B, cathepsin D, ferritin, α-E-catenin, AGR2, and S100P were chosen to validate SILAC results. Western blotting results showed that the ratios of eight representative proteins between treated and untreated cells showed either close-degrees or similar fold changes consistent with those obtained from SILAC (Figure 6A). Quantification results by SILAC of the eight proteins are shown in Additional file 3: Table S3. Some of them are secretory proteins, to determine whether DNP also induces these secretory proteins, AKR1B10, cathepsin B and clusterin in the culture supernatants of DNP treated 6-10B cells were detected. The results showed that AKR1B10, cathepsin B and clusterin dramatically increased in the culture supernatants after DNP treatment (Figure 6B). These findings imply that DNP may induce 6-10B cells to secrete AKR1B10, cathepsin B and clusterin.
Figure 6 Differential protein expression confirmed using Western blotting.A, eight representative proteins, aldo-keto reductase (AKR) 1B10, clusterin, cathepsin B, cathepsin D, ferritin, α-E-catenin, AGR2, and S100P were detected in 6-10B cells with or without DNP treatment using Western blotting. GAPDH served as a loading control. The relative photographic density was quantitated using a gel documentation and analysis system. B, AKR1B10, Cathepsin B and Clusterin were detected in the cell culture supernatant of 6-10B cells with or without DNP treatment using Western blotting. WB, Western blotting. SILAC, stable isotope labeling with amino acids in cell culture.
To further confirm whether the different proteins involve DNP-mediated NPC metastasis, we chose high-expressed protein AGR2 as target. DNP induced AGR2 expression at dose- and time- course (Figure 7A). As a metastasis-associated protein, AGR2 may play an important role in DNP-mediated metastasis. The next step is to observe DNP-mediated metastasis when AGR2 blocked. We used si-AGR2 to knockdown AGR2 (Figure 7B), and then used wound-healing assays to detect the cell motility of 6-10B-siAGR2 with DNP treatment. Following si-AGR2 transfect, DNP-mediated motility decreased, and consequently the cells were unable to migrate into the wound (Figure 7C, panel b vs. d and Figure 7D, lane 2 vs. 4). Hence, we concluded that AGR2 plays an important role in DNP-mediated metastasis.
Figure 7 DNP-mediated 6-10B cell motility through AGR2.A, 6-10B cells were treated with the indicated concentration DNP for dose-course, and treated with 10 μM DNP for the indicated time for time-course. AGR2 expression in the DNP-treated cells was detected using Western-blotting. 6-10B cells (2 × 106) were transiently transfected with si-AGR2 or si-mock, incubated at 37°C for 16–24 h. Confluent monolayer of the transfect cells was then wounded using a plastic tip, and then treated with 10 μM DNP. (a) 6-10B cells treated with 0.1% DMSO. (b) 6-10B cells treated with 10 μM DNP. (c) The transfect with AGR2 si-RNA treated with DMSO. (d) The transfect treated with DNP. B, AGR2 expression in the above transfect cells with DMSO or DNP treatment using western-blotting. C, The treated cells were observed under microscope, and the cells migrating cross the boundaries lines in the center of the wells were counted. D, Numbers of cells that moved cross the lines (10 fields). The data are represented as the mean ± SD from three independent experiments. Results were statistically analyzed using one-way analysis of variance (ANOVA) with a post hoc Dunnett’s test (* p < 0.05). The error bars represent SDs. Scale bar, 10 μM. Arrow, cells moving cross the boundary.
Conclusion
In clinic, NPC has the features of high invasion and metastasis, but its mechanism has been unclear. As one of three carcinogen factors for NPC, the Epstein–Barr virus (EBV) has been proven to be involved in NPC metastasis through latent membrane protein 2A inducing epithelial-mesenchymal transition (EMT), however latent membrane protein is positive at only a 56.7% rate [26]. Recently, another important carcinogen factor, DNP was also found to be involved in NPC metastasis [18]. In the present study, using SILAC and a systematic data analysis method, we obtained unbiased interpretation of NPC cell metastasis induced by DNP. Approximately 2698 proteins were quantified and 371 of these proteins showed apparent alterations in expression levels after DNP treatment, involving the regulation of biosynthesis and energy metabolism, as well as cell adhesion or invasion. We speculated that biosynthesis, energy metabolism and invasion are associated with NPC metastasis mediated by DNP. Based on subcellular and biological function analysis, many differential proteins in the present study were located in mitochondrion, such as mitochondrial membrane part, and mitochondrial respiratory chain. Additionally, tumor cells with mitochondria damage or dysfunction were reported to enhance anti-apoptosis ability and invasion [27,28]. This suggests that mitochondrial dysfunction may be linked to metastasis of DNP-treated 6-10B cells.
In the differential proteins mediated by DNP, oxidoreductase activity and oxidoreductase activity acting on NADH or NADPH, the CH-CH group of donors, and the CH-CH group of donors, NAD or NADP as the acceptor related to proteins accounted for a large proportion. Peroxiredoxins 3, NADH-dehydrogenase ubiquinone iron-sulfur protein 3 (NDUFS3), NADH-dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NDUFB8), pirin, ferritin heavy chain, and AKR1 were significantly up-regulated in the high metastatic 6-10B cells with DNP treatment. Oxidative stress have been shown to play important roles in tumorigenesis and progression of tumors [29], in which there is aberrant or improper regulation of the redox status. The balance of redox state affects many physiological and pathophysiological processes of cells, its mechanisms include gene transcription, cell signal transduction, activity of enzymes and biological macromolecules, cell proliferation, adhesion, and apoptosis. These findings suggest that the significant change of oxidoreductase activity in high metastatic 6-10B cells with DNP treatment is correlated with the status of oxidative stress and imbalance of the redox state.
Cytoskeleton has been identified as a major target for destruction during apoptosis and is important under pathological conditions such as cancers [30]. The differential proteins were distributed in the cytoskeleton, including N-myc downstream-regulated gene 1 protein, paxillin, and syntenin-1. Conversely, some proteins associated with the cytoskeleton were up-regulated, such as catenin alpha-1, radixin, macrophage-capping protein, integrin beta-5, tubulin-specific chaperone D, tubulin beta 2C (TUBB2C), tubulin beta 2A, and tubulin 5 beta. And subcellular localization of these differential proteins is related to junctional mechanisms. Based on these data, we speculate that in high metastatic 6-10B cells with DNP treatment, dynamic modifications and remodeling in the cytoskeleton exist, and the dynamic alteration affects endocytosis, cell shape, cell motility, cell adhesion and invasion.
Additionally, some important proteins directly related to metastasis were discovered in our study, such as, annexin A6, S100P, S100A4, hot shock protein 90B1, ferritin heavy chain, TUBB2A, and anterior gradient-2 (AGR2, Additional file 3: Table S3). Cathepsin B, AKR1B10 and custerin were not only up-regulated in 6-10B cells with DNP treatment, but also in the cell culture supernatant. Cathepsins, initially described as intracellular peptide hydrolases, play a role in invasion and metastasis of cancer [31]. In the present study, cathepsins B and D were respectively up-regulated 7.9-fold (Additional file 3: Table S3) and 4.6-fold (Additional file 3: Table S3), respectively. Cathepsin B is a key enzyme in invasion and metastasis of malignant tumors. It is up-regulated in laryngeal cancer [32], cervical cancer [33,34], and bladder cancer [35], and its expression level is correlated with metastatic potential. Cathepsin D, a lysosomal aspartate proteolytic enzyme that is similar to cathepsin B, also plays an important role in invasion and metastasis of cancer. It is up-regulated in metastasis of some malignant tumors, including primary laryngeal cancers correlated with neck lymph node involvement [36], gastric cancer with lymphatic and/or blood vessel invasion [37], and breast cancer. Furthermore, Cheng et al. [38] found that significant cathepsin D expression occurred in lymph node metastasis versus primary NPC and was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. AGR2 was reported to be linked with several human cancers and induced metastasis [39]. Additionally, Dumartin, et al. [25] found that cathepsins B and D are downstream functional molecules of the proinvasive AGR2 in vitro, and AGR2, cathepsins B and D were considered to be essential for dissemination of pancreatic cancer cells in vivo. High expressed-cathepsins B and D in DNP-treated 6-10B may be mediated by AGR2, but it is also possible that DNP directly mediated cathepsins B and D. Additionally, DNP-induced 6-10B motility decreased when AGR2 blocked (Figure 7). We speculated that cathepsins B, D and AGR2 expression mediated by DNP and AGR2 regulating cathepsins B, D are involved in NPC metastasis.
Significantly, AKR1 proteins were predominantly up-regulated in high metastatic DNP-treated 6-10B cells, including AKR1C1, AKR1B10, AKR1C3, and AKR1B1 (Additional file 3: Table S3). Family members of AKR1C play a pivotal role in maintaining steroid homeostasis and catalyzing reductive detoxification of reactive aldehydes and ketones, which are produced as a result of oxidative stress [40,41]. AKR1B10 is also correlated positively with tumor size and lymph node metastasis [42]. These findings suggest that DNP would affect oxidative stress and steroid homeostasis in 6-10B cells through the above aldo-keto reductase family 1 proteins, thereby increasing 6-10B cell metastasis.
Higher clusterin levels were expressed in various malignant tumors with metastasis including ovarian [43], breast [44], and gastric cancers [45]. An emerging query, clusterins enhanced cell invasion and metastasis of tumors through EMT. Lee, et al. [46] found that clusterin was involved in Smad2/3 stability at the protein level, and believed that clusterin regulates transforming growth factor-beta signaling pathway by modulating the stability of Smad2/3 proteins and mediates EMT. Lenferink et al. [47] also found that clusterin gene expression was highly up-regulated throughout transforming growth factor-beta, and speculated that secreted clusterin served as an important extracellular promoter of EMT. In the present study, proteins related to EMT and cell adhesion were also dysregulated, including clusterin myosin-VI, catenin alpha-1 (CTNNA1), fibronectin type III domain-containing protein 3B (FNDC3B, 2.1-fold), L1 cell adhesion molecule (L1CAM), desmoplakin, plakophilin-3 (Additional file 3: Table 3), implying that the mobility of DNP-induced 6-10B cells is probably related to EMT and cell adhesion.
The SILAC technique was used to conduct a comparison of the proteomes of 6-10B cell metastasis induced by DNP. A cooperative response, including many proteins, and a group of pathways were identified and some interesting clues were provided. DNP may induce a change in abundance of mitochondrial proteins, mediate the status of oxidative stress and the imbalance of the redox state, and increase cytoskeletal protein, cathepsin, AGR2, and clusterin expression, and finally promote cell metastasis. DNP may be involved in NPC metastasis through regulation of cancer protein synthesis, cellular movement, lipid metabolism, molecular transport, cell death, and cellular growth and proliferation signaling pathways. DNP may also induce 6-10B cells to secrete AKR1B10, cathepsin B and clusterin. These dataset provide important clues for investigation on high metastatic NPC.
Competing interests
The authors declare that they have no competing interests
Authors’ contribution
YJL performed cell culture, stable isotope labeling, Gel electrophoresis, bioinformatics analysis, and wrote the paper. NL performed MTT, LDH, cell invasion and motility assay, and metastasis in nude mice. DMH performed cell invasion and motility assay. ZLZ performed cell culture, 6-10B cells labeling. ZKP performed Western-blotting. CJD designed experiments and revised the manuscript. XWT performed nude mice breeding and DNP preparation. GJT performed protein preparation. GRY performed Gel electrophoresis and bioinformatics analysis. WHM revised the paper. FQT coordinated the study and revised the paper. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Table S1. Gene Ontology analysis.
Click here for file
Additional file 2
Table S2. Ingenuity Pathways analysis.
Click here for file
Additional file 3
Table S3. Information for up- and down- regulated proteins identified in DNP-induced cell.
Click here for file
Acknowledgments
This work was in part supported by the National Natural Science Foundation of China (81071718,81000881, 30973400), Foundation of State Key Laboratory of Oncology in South China (HN2011-04), Fundamental Research Funds for the Central Universities (21611612).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23424616PONE-D-12-1673210.1371/journal.pone.0054592Research ArticleBiologyBiophysicsCell MotilityMolecular cell biologySignal transductionSignaling cascadesERK signaling cascadeMedicineOncologyCancers and NeoplasmsGenitourinary Tract TumorsProstate CancerHuman Chorionic Gonadotropin β Induces Migration and Invasion via Activating ERK1/2 and MMP-2 in Human Prostate Cancer DU145 Cells HCGβ Signaling in Cell Migration and InvasionLi Zongwen
1
Li Chunliu
1
Du Lianlian
1
Zhou Yan
2
Wu Wei
1
2
*
1
Department of Epidemiology and Health Statistics, School of Public Health and Family Medicine, Capital Medical University, Beijing, China
2
Department of Biochemistry, School of Basic Medical Sciences, Capital Medical University, Beijing, China
Pizzo Salvatore V. Editor
Duke University Medical Center, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Contributed reagents/materials/analysis tools: YZ. Conceived and designed the experiments: WW. Performed the experiments: ZL CL. Analyzed the data: LD. Wrote the paper: ZL CL WW.
2013 12 2 2013 8 2 e5459211 6 2012 14 12 2012 © 2013 Li et al2013Li et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.We previously demonstrated that human chorionic gonadotropin β (hCGβ) induced migration and invasion in human prostate cancer cells. However, the involved molecular mechanisms are unclear. Here, we established a stable prostate cancer cell line overexpressing hCGβ and tested hCGβ-triggered signaling pathways causing cell migration and invasion. ELISA showed that the hCGβ amount secreted into medium increased with culture time after the hCGβ-transfected cells were incubated for 3, 6, 9, 12 and 24 h. More, hCGβ standards promoted MAPK (ERK1/2) phosphorylation and increased MMP-2 expression and activity in both dose- and time-dependent manners in hCGβ non-transfected cells. In addition, hCGβ promoted ERK1/2 phosphorylation and increased MMP-2 expression and activity significantly in hCGβ transfected DU145 cells. Whereas ERK1/2 blocker PD98059 (25 µM) significantly downregulated phosphorylated ERK1/2 and MMP-2. Particularly, hCGβ promoted cell migration and invasion, yet the PD98059 diminished the hCGβ-induced cell motility under those conditions. These results indicated that hCGβ induced cell motility via promoting ERK1/2 phosphorylation and MMP-2 upregulation in human prostate cancer DU145 cells.
This study was funded by Science and Development Plan of Beijing Education Committee (grant number: KM200910025008) and National Natural Science Foundation of China (grant number: 81272843). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Prostate cancer is one of the most commonly diagnosed cancers and the sixth leading cause of death in the males in the world [1]. Currently, the main methods for the treatment of prostate cancer are radical prostatectomy, external beam radiation therapy, brachytherapy, systemic androgen deprivation therapy and chemotherapy, etc. [2]–[6]. But the therapeutic efficacy is unsatisfactory. Development of new therapy such as molecular therapy is necessary for treating prostate cancer. But the characterization of the molecular mechanisms causing prostate cancer is the basis for establishing a new therapy. If we determine the therapeutic molecular targets that contributed to prostate cancer first, then we can treat patients via targeting those tumor markers to inhibit prostate cancer, further improve the prognosis and lower the mortality.
Human chorionic gonadotropins (hCGs) are heterodimeric glycoproteins secreted by trophoblastic cells in normal pregnancy. HCGs have a few isoforms containing intact hCG, hCGα, hCGβ, hyperglycosylated (hCGh), nicked (hCGn) and core fragment of hCGβ (hCGβcf) [7]. HCGβ is a molecule with independent function. It has been shown that free hCGβ is a potential tumor marker produced by a variety of tumors [8]–[10]. We previously reported that hCGβ decreased E-cadherin expression leading to migration and invasion in prostate cancer cells [11]. However, the involved whole mechanisms are not clear.
Extracellular signal-regulated kinase1/2 (ERK1/2) activation has been implicated in carcinogenesis and cancer progression. Increased ERK1/2 activity promotes cancer cell proliferation and metastasis in various cancer cell lines [12]–[15]. ERK1/2 blocker, PD98059 resulted in a reduction of cell growth and invasiveness in prostate cancer. In MA-10 Leydig cells, hCG triggered transient ERK1/2 activation via upstream protein kinase A (PKA) [12]. In addition, hCG also induces ERK1/2 phosphorylation in a PKA-independent manner in endometrium and is involved in carcinogenesis [13], [14]. Besides, there is evidence that matrix metalloproteinases (MMPs) expression was regulated by ERK1/2 in invasive carcinomas. Studies showed that activated ERK1/2 regulated the activity of MMPs leading to extracelluar matrix (ECM) degradation and cell motility [13]–[15]. Many MMPs are considered as essential proteases in the ECM degradation and remodeling [16]. Report showed that hCG stimulated the secretion of MMP-2 and MMP-9 in a dose-dependent manner in cytotrophoblastic cells [17]. Further, hCG upregulated MMP-2 activity and promoted cell motility in SGHPL-5 cell lines [18]. In human prostate cancer, enhanced MMP-2 and MMP-9 activity contributed to tumor invasion and metastasis [19]–[21]. Studies showed that vitamin D and Vitamin D analog ZK191784 downregulated MMPs to inhibit invasion in prostate cancer [22]–[24]. Undoubtedly, MMPs are the important anti-invasion targets. Hence, we propose that hCGβ might increase ERK1/2 phosphorylation and further upregulate MMPs to promote cell motility.
Consequently, here we will investigate hCGβ-triggered signaling pathways and the linkage between hCGβ expression and cell motility. Through this study, we hope that we will find new clues in molecular therapy to treat prostate cancer.
10.1371/journal.pone.0054592.g001Figure 1 HCGβ expression and secretion after the cells were grown for 24 hours.
(A) Quantitative analysis of hCGβ mRNA and β-actin mRNA transcription in DV and DH cells by Real-time PCR. DV: DU145 cell transfected with empty vector; DH: DU145 cells transfected with hCGβ cDNA construct. Note that hCGβ mRNA was highly expressed versus control. (B) Analysis of hCGβ protein expression by Western blot. β-actin was used for equal loading and normalization. The results indicated that hCGβ was highly expressed in DH cells. *, indicates P<0.05 versus control DV cells. Data were shown as means ± SEM from three separate tests.
Materials and Methods
Materials
HCGβ standards were purchased from Abcam (Hong Kong). The construct pVSneo-hCGβ containing hCGβ cDNA was purchased from Stratagene (La Jolla, CA). Restriction enzymes SalI, XhoI, EcoRI, BamHI, HindIII and T4 DNA ligase, competent cells are the products of Invitrogen (Carlsbad, CA). G418 and crystal violet were purchased from Sigma (St. Louis, MO). Human prostate cancer DU145 and PC3 (to be a backup) cells were the products of American Typical Culture Collection (Rockville, MD). Cells were cultured in DMEM medium (Hyclone) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C, 95% air and 5% CO2. Transwell plates with inner inserts or artificial basement membranes were bought from BD Biosciences (Bedford, MA). Anti-hCGβ was from BioDesign International (Saco, ME); anti-ERK1/2, anti-phospho-ERK1/2 and anti-MMP-2 were purchased from Cell Signaling Technology, Inc (Shanghai, China).
Transfection
Via transfection, we established stable cell line overexpressing hCGβ in DU145 cells. The control vector pVSneo–vector was made by cutting hCGβ cDNA by restriction enzymes first and then autoligation as described previously [11]. Cells were seeded in six-well plates with DMEM growing medium. When the cells come to 90% confluency, the constructs containing the pVSneo–hCGβ or pVSneo–Vector were transfected into DU145 cells following FuGene HD transfection reagent (Roche, USA) and the manufacturer’s instructions. After the cells were incubated at 37°C for 48 hours, the cells were digested by trypsin-EDTA and grown in the selection medium containing G418 (1.2 mg/mL). Further, cells were incubated for two more weeks. The cells without integration of hCGβ gene were dead and floating in the medium and the single colonies which stably express hCGβ were collected. The screened cells were cultured in selection medium (600 µg/mL G418) for two more weeks until no dead cells were found. The selected cells were maintained in medium containing 600 µg/mL G418 for further test. We succeeded in establishing the stable cell lines in the previous work [11]; here we used a new transfection reagent to enhance transfection efficiency.
Real-time PCR
Cells were grown as the routine procedures in the culture medium. Then, total RNA was isolated using TriZol Reagent (Invitrogen, USA). HCGβ cDNA was made using Reverse Transcriptase Kit (TaKaRa, China) with 1.5 µg of total RNA following the manufacturer’s instruction. Real-time PCR was performed on a Stratagene Mx3000P instrument. For real-time thermal cycling, triplicate aliquots of cDNA were used in a reaction mixture containing 250 nM of each primer in a reaction volume of 25 µl by the PrimeScriptTM RT reagent Kit (TaKaRa, China). The PCR cycling program was run with an initial predenaturation step at 94°C for 60 s, then with a 40 cycle of amplification steps, at 94°C for 30 s, 57°C for 30 s, 72°C for 20 s. The primers were as follows:
hCGβ (forward): 5′-TCTGTGCCGGCTACTGCCCC-3′;
hCGβ (reverse): 5′-TTGGGACCCCCGCAGTCAGT-3′;
β-actin (forward): 5′-AACTCCATCATGAAGTGTGACG -3′; β.
β-actin (reverse): 5′-GATCCACATCTGCTGGAAGG-3′.
Data was collected and analyzed following the manufacturer’s manual instruction.
ELISA
HCGβ is a secreted protein, which might interact with some receptors, such as luteinizing hormone receptor, etc. to play a role in triggering the signaling pathways. Therefore we need to make sure that expressed hCGβ was secreted into cell medium. After the stable DU145 cells were grown to 70% confluency in the growing medium, the cells were washed for three times with serum-free DMEM medium. Then the cells were cultured in serum-free DMEM medium at 3, 6, 9, 12 and 24 h. The medium was collected at the indicated time points and the ELISA was performed to test secreted hCGβ via a β-hCG ELISA kit (DRG Diagnostics, New Jersey, USA) following the manufacturer's instruction. In the previous study, we succeeded in detecting the hCGβ secretion in hCGβ transfected cells via an hCGβ detection Kit, F-hCGβ ccubind ELISA, from Monobind (Costa Mesa, CA) [11]. In order to reproduce and confirm these results, we used a β-hCG ELISA kit to determine hCGβ secretion.
Immunoblotting
After the DU145 cells were cultured for the required time, cells were collected and lysed with lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate and protease inhibitors, Thermo Scientific, USA). Cell lysate was centrifuged at 18,000 g for 20 min. Total protein concentrations were tested by BCA Protein Assay Kit (invitrogen). Eighty microgram of protein was loaded on 10% SDS-PAGE gels and run for required time depending on the molecular weight, then transferred to nitrocellulose membranes via the semi-dry transfer. The membranes were blocked in 1.5% BSA in TBS-T buffer for 1 h at room temperature with gentle shaking, then were incubated with primary antibodies separately, at 4°C, overnight. After washing with TBST 3×10 minutes each, the membranes were probed with the fluorescence-labeled secondary antibody (LI-COR Bioscience, Lincoln, NE) for 1 h at room temperature. After three washes, the membranes were scanned in the 700 or 800 channels using the Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, NB, USA). β-actin was used for equal loading and normalization. Antibodies were diluted appropriately.
Gelatin Zymography
After the ERK1/2 blocker PD98059 (25 µM) was applied to treat DU145 cells for 2 h, we changed the 10% fetal bovine serum DMEM medium into serum-free medium, and cultured for 24 hours. Medium with secreted hCGβ was collected from an equal number of cells and mixed with equal amounts of non-reduced sample buffer. The equal volumes of medium were electrophoresed on 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin as a protease substrate. The gel was washed in 2.5% Triton X-100 solution at room temperature with gentle agitation and was soaked in the buffer (50 mM Tris–HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl2·2 H2O, and 0.02% Brij-35, pH7.6) at 37°C for 42 hours. After incubation, the gel was stained for 30 min with staining solution (0.5% Coomassie Brilliant Blue, 25% isopropanol, and 10% acetic acid). Then the stained gel was destained with an appropriate Coomassie R-250 destaining solution (50% methanol, 10% acetic acid). In the area with matrix metalloproteinases, clear bands against a dark blue background will show up. To show equal loading, a parallel SDS gel was run to test MMP-2 and β-actin via Western blot.
Cell Motility Assays
Prostate cancer cell migration was done in a 24-well plate with inner chamber; the chamber bottom has 8 µm pores. Total 1×105 cells were seeded in the upper chamber with 500 µl serum-free medium, and 1 ml DMEM medium with 10% fetal bovine serum was added in the lower chamber. After the cells were grown for 6 hours, the cells on the upper chamber were removed with a cotton swab. The migrated cells (or pseudopodia) on the bottom of the chamber were fixed with 100% methanol for 10 min at –20°C, then stained with 0.5% crystal violet solution at room temperature for 10 min. After moving away the crystal violet solution, the cells were rinsed with distilled water until no excess dye was viewed. The migrated cells or pseudopodia were photographed and counted from 5 randomly selected areas; the images were photographed with camera with a Leica DM IRB microscope at ×200 magnification. Invasion assay was performed by the Tumor Invasion System (8 µm pore, BD BioCoat). The bottom of cell culture insert was coated with artificial basement membrane coated with matrigel. Basement membrane is thin extracellular matrix underlying epithelial cells. Matrigel is a commercial product extracted from a mouse sarcoma rich in extracellular matrix proteins. The major component is laminin, followed by collagen IV and heparin sulfate proteoglycans. The other procedures are the same as in the migration assay.
Statistical Analysis
Data were subjected to analysis of variance with posttests for comparison among specific groups. Data were expressed as means ± SEM and analyzed for statistical significance using analysis of variance (ANOVA). Bonferroni corrections for multiple comparisons against a single group were used. P<0.05 was considered statistically significant. The minimum number of repetitive experiments was 3.
Results
HCGβ Expression
First we constructed a control vector as previously described; then DU145 cells were transfected with constructs either with or without hCGβ cDNA. After establishment of the stable cell lines, cells were maintained in the medium containing 600 µg/mL G418 for further studies. To test hCGβ expression in transfected DU145 cells, both real-time PCR and Western blot were used to determine mRNA and protein expression after the cells were incubated for 24 hours, respectively (Figure1A, Figure1B). These results showed that hCGβ was highly expressed in both mRNA and protein levels versus empty-vector transfected DU145 (DV) cells. Either hCGβ mRNA or protein amount was little in the control cells under these experimental conditions.
HCG Secretion
ELISA showed that hCGβ was secreted into medium tremendously in 24 hours; the amount was up to 150 ng/ml (Figure 2). The amount of hCGβ secretion was increased by incubation time. Note that we established a stable cell line overexpressing hCGβ. HCGβ might play an important role in the signaling between extracellular and intracellular communications.
10.1371/journal.pone.0054592.g002Figure 2 HCG detection in the culture medium via ELISA.
Data showed that there was hCGβ secreted into cell medium. Furthermore, the level of hCGβ secreted into medium increased with time after the hCGβ transfected cells were incubated for 3, 6, 9, 12 and 24 h.
HCGβ Activated ERK1/2
HCGβ standards was applied to treat non-transfected DU145 cells at doses of 25, 50, 100 and 200 ng/ml for 24 h, Western blot showed that ERK1/2 expression was increased following a dose-dependent trend, and ERK1/2 phosphorylation came to peak with a treatment 200 ng/ml hCGβ (Figure 3A). Consequently, we treated cells at 0 (CON, control), 5, 15, 30 and 60 min to determine ERK1/2 phosphorylation. The results showed that ERK1/2 phosphorylation followed a time dependent manner and came to peak at 30 min of treatment with 200 ng/ml hCGβ (Figure 3B). In addition, ERK1/2 phosphorylation was found significantly higher in the hCGβ transfected DU145 (DH) cells than control cells. However, PD98059 (25 µM), a specific blocker, prevented ERK1/2 phosphorylation significantly versus untreated cells (Figure 3C). Note that hCGβ caused ERK1/2 activation following dose- and time-dependent manners.
10.1371/journal.pone.0054592.g003Figure 3 HCG phosphorylated ERK1/2.
(A)We have demonstrated that the concentration of 200 ng/ml hCGβ standards induced cell invasion [11]. Thus, here we treated non-transfected DU145 cells with different doses of 0, 25, 50, 100, and 200 ng/ml hCGβ for 1 h. Western blot showed that hCGβ standards phosphorylated ERK1/2 in a dose-dependent trend. At the concentration of 200 ng/ml, ERK1/2 activation reached the peak. (B) Non-transfected DU145 cells in serum-free medium were treated with 200 ng/ml hCGβ for 0, 5, 15, 30 and 60 min. Western blot showed that hCGβ activated ERK1/2 in a time-dependent manner. At the time point of 30 min, ERK1/2 phosphorylation reached the peak and then went down. (C) The cells were pretreated with PD98059 (25 µM) 30 min and then incubated for 24 h in serum-free medium, Western blot showed that hCGβ activated ERK1/2, but PD98059 decreased ERK1/2 activation. *, indicates P<0.05 versus DV. Data were shown as means ± SEM from three separate tests.
HCGβ Induced MMP-2 Expression via Activation of ERK1/2
MMP-2 was demonstrated to be involved in cancer cell migration and invasion. Here we investigated hCGβ-induced MMP-2 expression in the hCGβ non-transfected DU145 cells. HCGβ standards was added into the serum-free medium at the doses of 0 (CON, control), 25, 50, 100 and 200 ng/ml. Western blot showed that hCGβ increased MMP-2 expression in a dose dependent manner, MMP-2 expression was increased to peak when treated with 200 ng/ml hCGβ (Figure 4A). Furthermore, we treated the cells with PD98059 (25 µM), the results showed that MMP-2 expression in DH cells was remarkably downregulated (Figure 4B), indicating that MMP-2 upregulation resulted from ERK1/2 phosphorylation.
10.1371/journal.pone.0054592.g004Figure 4 HCGβ increased MMP-2 expression and via ERK1/2.
(A) We treated non-transfected DU145 cells with 0, 25, 50, 100, and 200 ng/ml hCGβ for 1 h, Western blot indicated that hCGβ standards upregulated MMP-2 in a dose-dependent manner, at the concentration of 200 ng/ml, MMP-2 expression reached the peak. (B) We treated DH and DV cells with or without PD98059 (25 µM) 30 min and then incubated for 24 h with serum-free medium, Western blot showed that hCGβ upregulated MMP-2 expression and PD98059 abolished those increase. The sizes for pro-MMP-2 and active-MMP-2 are 72 Kd and 64 Kd, respectively. *, indicates P<0.05 versus control. Data were shown as means ± SEM from three separate tests.
HCGβ Increased MMP-2 Activity via Activation of ERK1/2
Gelatin zymography assay showed that MMP-2 activity was reduced by PD98059 (25 µM) (Figure 5), indicating that hCGβ activated MMP-2 via ERK1/2 phosphorylation.
10.1371/journal.pone.0054592.g005Figure 5 HCGβ increased MMP-2 activity via ERK1/2.
We collected the conditional medium from the above treatment was for geltin zymography assay as the Methods. The results showed that hCGβ increased MMP-2 activity and PD98059 reduced those effects. *, indicates P<0.05 versus control. Data were shown as means ± SEM from three separate tests. The lower panel of gel pictures showed an equal loading that a parallel SDS gel was run to test β-actin via Western blot.
HCGβ Increased Cell Motility via ERK1/2 Phosphorylation
In the previous study, we demonstrated that hCGβ induced prostate cancer cell migration and invasion. In the present study we further confirmed how hCGβ induced cell migratory and invasive behaviors. To know whether hCGβ induced cell motility resulted from ERK1/2 phosphorylation, the migration and invasion assays were conducted either with or without PD98059 (25 µM). The exact methods were as described in the Methods. Results showed that hCGβ significantly increased both cell migration and invasion; but PD98059 significantly decreased those effects (Figure 6A, Figure 6B). Note that hCGβ exactly accelerated cell migration and invasion via ERK1/2 phosphorylation.
10.1371/journal.pone.0054592.g006Figure 6 HCGβ promotes cell migration and invasion.
(A) We seeded 1×105 cells in a 24-well plate with cell inserts, the cells were added with/without PD98059(25 µM) for 6 h to detect cell migration, the results showed that hCGβ promotes cell migration significantly versus control. (B) In the same conditions we incubated the cells in the invasion chamber with artificial basement membrane, the cells were added with/without PD98059(25 µM) for 12 h to detect cell invasion, the results showed that hCGβ promotes cell invasion significantly. All procedures were performed as described in Methods. *, indicates P<0.05 versus control. Data were shown as means ± SEM from three separate tests.
Discussion
Prostate cancer accounts for 29% of all cancers in men [25]. Prostate cancer cells have a striking tendency to metastasis, and metastasis is the major cause of mortality for cancer patients [26]. Therefore, it is necessary to find and characterize the molecular targets to inhibit invasion and metastasis via gene targeting.
HCGβ is a significant marker of malignant transformation. Almost every human cancer produces hCGβ to some extent [27]. Studies showed that hCGβ promotes cancer cell proliferation and might also promote metastasis. For instance, Laurence A Cole discovered that hCGβ can compete with a TGFβ to bind a TGFβ receptor, as a TGFβ receptor antagonism to control apoptosis and promote invasion by activating metalloproteinases [28]. Also, another report showed that hCGβ and VEGF play a co-ordinated role through their angiogenic and invasive properties in the development of Barrett’s adenocarcinomas [29]. Through the above results we can infer that hCGβ might play an important role in cancer-promoting via multiple signaling pathways. Thus, it is essential to investigate hCGβ-triggered signaling pathways in tumor migration and invasion.
In the previous study we demonstrated that hCGβ changed cancer cell morphology, accelerating cell motility, downregulating migration-inhibiting protein E-cadherin, promoting human prostate cancer migration and invasion [11]. However, the further mechanisms to cause migration and invasion keep unknown. In the present study, we found that some signaling pathways were related to migration and invasion. Here we demonstrated that hCGβ upregulated ERK1/2 and MMP-2 leading to cell migration and invasion. Using the ERK1/2 phosphorylation blocker PD98059, we discovered that MMP-2 upregulation resulted from ERK1/2 phosphorylation. ERK1/2 have been proved to contribute to tumor proliferation, migration and metastasis, and several studies reported that hCG promoted the ERK1/2 activation in some cell types [30], [31]. Obviously, these results are consistent with our results that hCG induced ERK1/2 phosphorylation in a dose- and time-dependent manners in DU145 cells. The ERK related pathway is one of the most critical signaling pathways in tumor occurrence, development and clonal expansion. Particularly, MMP-2 is the key molecule in tumor invasion. Our findings that hCGβ activated MMP-2 via ERK1/2 phosphorylation in DU145 cells are very important. These results not only revealed the relationship between hCGβ and cancer, but also indicated that we found a novel key pathway to inhibit cancer. Obviously the hCGβ/ERK1/2/MMP-2 pathway is vital in tumor invasion, at least in prostate cancer. In other words, we found more useful approaches to inhibit tumor invasion, and further we can develop molecular target drugs.
hCG is composed of two subunits, hCGα and hCGβ. These two subunits make a complex, which binds to luteinizing hormone receptor and triggers signaling pathways. However, we do not know if hCGβ also binds to luteinizing hormone receptor separately. This study gave us the new clue and will push us to characterize hCGβ specific receptor.
Besides, MMPs were regarded to be metastasis-promoting molecules with many kinds of isoforms. They have potentials to degrade the extracellular matrix and basement membrane. MMP-2 has been implicated to be a key member in MMPs. More, ERK1/2 might translocate to the nucleus and activate some transcriptional factor AP-1 to regulate gene transcription [32]. AP-1 locates in the MMP-2 promoter region of MMP-2 gene, thus, ERK1/2 might promote MMP-2 transcription and release [33], [34]. Hence, in order to investigate the role of ERK1/2 in regulating the cell migration and invasion, we successfully determined MMP-2 expression and activity. We found that hCGβ significantly increased MMP-2 expression and activity. These effects were remarkably repressed by PD98059 in hCGβ transfected DU145 cells. These results showed a crosstalk between ERK1/2 and MMP-2. Coincidently, we also found that hCGβ promoted ERK1/2 phosphorylation and MMP-2 expression following the same dose and time patterns. These results indicated that hCGβ triggered ERK1/2 activation resulted in MMP-2 upregulation and increased MMP-2 activity. Consequently, hCGβ-caused migration and invasion resulted from ERK1/2 activation. However, we have no sufficient evidence whether hCGβ-caused MMP-2 upregulation plays a major role in migration and invasion. Transfection with MMP-2 construct and knock out of MMP-2 study might help to answer these questions. Further characterization of hCGβ signaling will assist us to find more metastasis markers to meet the therapeutic requirements.
Actually, some experts have started the research on hCGβ related antibodies in the field of cancer [35], [36]. Here we uncovered that hCGβ phosphorylated ERK1/2 and further upregulated MMP-2 to increase cancer motility in prostate cancer cells. Our results may give new insights into the molecular mechanisms of hCGβ regulation, and provide a stronger basis for the hCGβ related research on the tumor suppressor agent.
We found a few new molecular targets to inhibit invasion and metastasis of prostate cancer. Effective treatment of prostate cancer is of great significance. Blocking hCGβ signaling is a potential therapeutic strategy to treat prostate cancer and other cancers. Further work needs to characterize hCGβ receptor; development of hCGβ signaling blockers will be a prospective field to lower invasion and metastasis.
We thank Dr. Ameae Walker, University of California, Riverside for her kind direction in the original project design. We also thank Dr. Tao Jiang, Tiantan Hospital, Capital Medical University for his assistance in the experiments.
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23418439PONE-D-12-2365210.1371/journal.pone.0055313Research ArticleBiologyBiochemistryProteinsRegulatory ProteinsModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCellular TypesMedicineGastroenterology and HepatologyOncologyBasic Cancer ResearchCancer TreatmentCancers and NeoplasmsOncology AgentsRadiotherapyRadiologySilencing of APE1 Enhances Sensitivity of Human Hepatocellular Carcinoma Cells to Radiotherapy In Vitro and in a Xenograft Model Silencing of APE1 Enhances RadiosensitivityCun Yanping
1
Dai Nan
1
Xiong Chengjie
2
Li Mengxia
1
Sui Jiangdong
1
Qian Chengyuan
1
Li Zheng
1
Wang Dong
1
*
1
Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China
2
Department of Orthopedics, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic of China
Vooijs Marc Editor
University of Maastricht (UM), The Netherlands
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ML DW. Performed the experiments: YC ND CX. Analyzed the data: YC ND. Contributed reagents/materials/analysis tools: JS CQ ZL. Wrote the paper: YC CX ML.
2013 13 2 2013 8 2 e553136 8 2012 21 12 2012 © 2013 Cun et al2013Cun et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy in vitro and in vivo.
Grant support was provided by the National Natural Science Foundation of China (No.30872975). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Hepatocellular carcinoma (HCC) is one of the most prevalent malignant diseases in the world [1]–[3]. Radiotherapy represents a major therapeutic option for HCC patients [4], but the efficacy of this therapy is limited by intrinsic radioresistance of the tumor cells. Ionizing radiation (IR) can result in lethal cell damage, which is correlated with DNA damage induction and repair [5]. The activity of the DNA damage repair pathway is the major factor leading to radioresistance in tumors, including hepatoma.
DNA-repair systems play an important role in protecting the genomic stabilization and integrity. However, an elevated DNA repair capacity in tumor cells is associated with drug or radiation resistance. The human apurinic/apyrimidinic endonuclease (hereafter, APE1) is a key enzyme in the DNA base excision repair (BER) pathway, which plays a critical role in repairing DNA damaged by irradiation [6], [7]. In addition to its DNA repair function, APE1 maintains a number of transcriptional factors including p53 by both redox-dependent and –independent mechanisms in their reduced and active state, thereby regulating their DNA-binding activity, influencing gene expression and maintaining genomic stability [8], [9].
In fact, the tumor suppressor p53 gene is activated in response to DNA damage and encodes a transcription regulatory protein that acts as a brake by inducing either cell cycle arrest or apoptosis, thereby preventing the propagation of genetically damaged cells and then maintaining genomic stability by its participation in stress-response pathways and DNA repair pathways [10]. If p53 is mutated, however, the cell with DNA damage can escape from apoptosis and turn into cancer cells [11]. To date, some studies have documented that p53 alterations are correlated with the sensitivity to radiotherapy in human HCC cells [12], [13]. As known that, the p53 gene is mutated in approximately 50% of hepatoma cells [14], and the mutant p53 (mutp53) proteins not only lose their tumor suppressive activities but often gain additional oncogenic functions that endow cells with growth and survival advantages, differences in radio-sensitivity [15], [16]. The transversion in codon 249 of p53 gene, which causes an arginine to serine (R→S) substitution is most commonly present in human HCC patients [17]. Mutated R249S p53 protein expression may induce cell proliferation and apoptosis inhibition [18], [19].
Several studies demonstrated that APE1 was overexpressed in several human tumors, such as osteosarcoma, colorectal cancer, ovarian cancer, cervical cancer, and non-small cell lung cancer [20]–[24]. In normal hepatocytes and endothelial and biliary duct cells, APE1 was detected only in nucleus of cells, and the shift of APE1 from nucleus to cytoplasm was observed in HCC cells. The expression of nuclear and cytoplasmic APE1 was significantly higher in HCC tissue than in the surrounding cirrhosis [25].Furthermore, more recent analysis showed that increased APE1 expression was associated with radioresistance. A decrease in APE1 levels led to enhanced cell sensitization to ionizing radiation in human osteogenic sarcoma cells and lung carcinoma cells as well as in colorectal cancer cells [22], [23], [26], [27].We have previously shown that APE1 was overexpression in human colorectal cancer, and chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1) potentiates radiosensitivity of human colorectal cancer cells [23].
In this study, we explored the radiosensitivity profiles of human HCC cell lines by measuring cell survival and apoptosis in MHCC97L, HepG2 and Hep3B cells, and investigated the correlation existing between APE1 deficiency and the sensitivity of HCC cells to radiotherapy. Additionally, we tested whether the downregulation of APE1 protein could potentiate the inhibition of tumor growth by irradiation in vivo. The results provided by our study demonstrate that MHCC97L showed strongly resistance to irradiation, and Ad5/F35-siAPE1 could inhibit irradiation-induced APE1 and p53 expression. More importantly, the present study firstly demonstrated that Ad5/F35-siAPE1 enhanced sensitivity of human HCC cells to radiotherapy in vitro and in vivo.
Materials and Methods
Materials
DMEM and fetal bovine serum were from Invitrogen (Grand Island, NY, USA). Polyvinylidene difluoride (PVDF) transfer membrane was purchased from Sigma-Aldrich (St. Louis, MO, USA). The monoclonal antibody against hAPE1 was from Novus Biological (Littleton, CO, USA). All of the antibodies directed against p53 (DO-1), p21 and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell Lines
HepG2 (wtp53, carrying wild-type p53), Hep3B (p53 null, lacking p53) and MHCC97L (mutp53, harboring mutant p53) human hepatoma cell lines [28], [29] were obtained from the Cell Institute of Shanghai (Academia Sinica, Shanghai, China). Cells were maintained at 37°C in a humidified incubator under 5% CO2 and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 50 mg/ml penicillin/streptomycin.
Irradiation
For X-ray treatment, cells were cultured in six-well plates until they reached 75% confluence and then irradiated at 200 cGy/min, at room temperature, with an Elekta Precise Linear Accelerator operating at 8 MV.
MTT Assay
Cells(1×105 cells/ml) were immediately inoculated into 96-well plates (200 µl/well) in triplicate post-irradiation. After 72 h, 15 μ l MTT (5 g/l) was added to each well and incubated for 4 h, and then the culture medium was discarded followed by addition of 200 μ l DMSO and vibrated for 10 min. The OD value at 492 nm was determined using a microplate reader. Cell viability (%) = OD value of the treatment group/OD value of the control group×100%.
Colony Formation Assay
Following irradiation, cells were counted, and plated in triplicate at a density to give between 30 and 300 colonies/10-cm dish. Cells were allowed to proliferate in culture media for 10∼14 days, with fresh media replacement every 3 days. Colonies were fixed and stained in 0.1% crystal violet in absolute ethanol for cell counting. Clones of at least 50 cells were counted as one colony. Survival curves were plotted as the log of survival fraction versus radiation dose.
Assessment of Apoptosis by Annexin-V and PI Double-staining Flow Cytometry
Cells were treated with X-ray radiation in different doses as described above. At 48 h post-irradiation, the cells were harvested and then stained with annexin V-FITC and PI (Invitrogen). Then samples were analyzed by flow cytometry.
Infections
Adenovirus vector Ad5/F35-siAPE1 carrying human APE1 siRNA sequence was constructed and purified as described previously [23]. The control adenovirus, Ad5/F35-EGFP, was purchased from Vector Gene Technology Company Limited (Beijing, China). HepG2, Hep3B and MHCC97L cell lines were infected with Ad5/F35-EGFP or Ad5/F35-siAPE1 for 2 hours and replaced with fresh medium. Cells were cultured for another 48 hours and then analyzed by Western blot and MTT assay or prepared for following experiments.
Western Blot Analysis
Equal amounts of protein from nuclear or cytosolic extract or whole-cell lysate, were electrophoresed by 10% SDS–PAGE. Proteins were then transferred onto PVDF membranes. After being blocked in Tris-Buffered Saline and Tween 20 (TBST) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.1% [volume/volume] Tween 20) containing 5% (weight/volume) defatted milk for 1 h at 37°C, membranes were incubated with the specific primary antibody. After three washes with TBST, the membranes were incubated for 1 h at 37°C with the appropriate peroxidase-conjugated secondary antibodies. Then, the membranes were washed three times with TBST and the blots were reacted with chemiluminescence reagents and revealed with Biomax-Light films (Kodak, Rochester, NY, USA). Band intensities were analyzed using the Gel Doc 2000 apparatus and software (Quantity One; Bio-Rad). Suppliers of incubation conditions for antibodies used for Western blot were as follows: anti-APE1 monoclonal (Novus), 1 h at 37°C, dilution 1∶5000; anti-p21 monoclonal antibody (Santa Cruz), overnight at 4°C, dilution 1∶500; anti-p53 monoclonal antibody (DO-1), overnight at 4°C, dilution 1∶500; anti-β-actin monoclonal (Santa Cruz), 1 h at 37°C, dilution 1∶2000.
Quantitative RT-PCR
Total RNA was extracted by using Trizol (Invitrogen) and chloroform/isoamyl alcohol precipitation following the manufacturer’s instructions. RNA concentrations were determined by spectrophotometer (Eppendorf AG, Hamburg, Germany). Subsequently, 1 µg of total RNA were reverse transcribed into single-stranded DNA using SuperScript II (Invitrogen). Quantitative RT-PCR was performed using SYBR Premix Ex Taq (TaKaRa) in a LightCycler 480 real-time PCR system (Roche, Indianapolis, IN, USA). Primer sequences were: 5′-TTAGATTGGGTAAAGGAAGAAGC-3′ (forward), and 5′-CTGACCAGTATTGATGAGAGAGT-3′ (reverse) for APE1; 5′-GGAGGGGCGATAAATACC-3′ (forward) and 5′-AACTGTAACTCCTCAGGCAGGC-3′ (reverse) for p53; 5′-CTGGAGACTCTCAGGGTCGAAA-3′ (forward) and 5′-TTCTCCAAGAGGAAGCCCTAATC-3′ (reverse) for p21. Control PCR was performed using β-actin primer: 5′-GATCATTGCTCCTCCTGAGC-3′ (forward) and 5′-TGTGGACTTGGGAGAGGACT-3′ (reverse). Primer pairs for all detected genes were designed to yield 100- to 300-bp amplicons, which are suitable for real-time quantitation. Gene expression was determined by normalization against β-actin expression.
In Vivo Experiments
All experiments were carried out in accordance with China Animal Welfare Legislation and were approved by the Third Military Medical University Committee on Ethics in the Care and Use of Laboratory Animals. HepG2 or MHCC97L cells (5×106) in 100 µl phosphate-buffered saline (PBS) were injected subcutaneously into the right flank of nude mice, respectively. When the tumors grew to approximately 50 mm3 on day 7 after cell injection, 28 tumor-bearing mice were randomized into the following four treatment groups (n = 7 animals per group): (a) Ad5/F35-EGFP; (b) Ad5/F35-siAPE1; (c) Ad5/F35-EGFP+IR; (d) Ad5/F35-siAPE1+IR. Mice were injected directly into the tumors with Ad5/F35-EGFP or Ad5/F35-siAPE1. Two days later, tumors in groups (c) and (d) were irradiated with 6 Gy of X-ray. On day 16, xenografts from each group were completed isolated and tumor volumes were then examined exactly.
The maximum diameters (Dmax) and minimum diameters (Dmin) of xenografts were measured before each treatment and after mice killed, and tumor size was calculated according to the formula: tumor size (mm3) = (Dmax×Dmin
2)/2. From the tumor growth curves, tumor-doubling time (DT) was determined for each individual tumor. Tumor growth delay was calculated by subtracting the mean tumor volume doubling time of the untreated tumors (control) from the mean tumor volume doubling time of each experimental group. Specific growth delay (SGD) was calculated according to the formula: SGD = (T2−T1)/T1; where T1 is the time taken in days for control tumors and T2 is the time for treated tumors to double in volume. Inhibition ratio, expressed in percent, was calculated at day 16 after mice killed by the formula [1-(treated tumor average volume/untreated tumor average volume)]×100%.
Immunohistochemical Analysis of Tumors for Ki67
Sections from paraffin-embedded tumors were incubated with Ki67 antibody (clone MIB-1; Dako) overnight at 4°C. Sections were rinsed with PBS and incubated with goat anti-mouse secondary antibody. Sections were rinsed with PBS and developed with diaminobenzidine substrate, and then counterstained with diluted Harris haematoxylin. Ki67 staining was quantified using computer-assisted image analysis with Image Pro Plus software (Media Cybernetics). The image analysis was done on four random fields (magnification,×100) per section from a total of five sections per group.
In situ Apoptosis Detection by TUNEL Staining
The formalin-fixed and paraffin-embedded 5 µm-thick sections of all tumor samples were analyzed for apoptosis by terminal dUTP nick end labeling (TUNEL) staining using the Apoptag Kit (Intergen, Purchase, NY, USA). The extent of apoptosis was evaluated by counting the positive brown-stained cells as well as the total number of cells at 10 arbitrarily selected × 100 microscope fields in a blinded manner.
Statistical Analysis
All quantitative data were obtained from three independent experiments and expressed as mean ± standard deviation values. The statistical significance of differences was determined by one-way analysis of the variance (ANOVA) using computer SPSS software SPSS 10.0 (SPSS, Chicago, IL, USA). A value of P<0.05 was considered statistically significant.
Results
Radiosensitivity of Human HCC Cell Lines
To examine the sensitivity of human HCC cell lines to radiotherapy, MTT and colony formation assays were performed on HepG2, Hep3B and MHCC97L cells treated with various doses (0∼10 Gy) of radiation at 48 h post-irradiation. MTT assay showed that a significantly increased cell survival was observed in MHCC97L cells, compared with HepG2 or Hep3B cells (Fig. 1A). In addition, Fig. 1A showed that the cell survival in Hep3B cells after irradiation at 4, 6, 8 or 10 Gy dose was higher than that in HepG2 cells. Because MTT assay was an overall measure of cell viability and was not necessarily indicative of long-term cell survival and proliferation, a clonogenic survival assay also was used. Fig. 1B reveals that cell colonies at all tested doses of radiation significantly increased in MHCC97L cells, compared with HepG2 or Hep3B cells. Moreover, the cell colonies in Hep3B cells after irradiation at 4, 6, 8 or 10 Gy dose significantly increased, compared with HepG2 cells (Fig. 1B).
10.1371/journal.pone.0055313.g001Figure 1 Dose-dependent growth inhibition of three human HCC cells with different p53 status.
Cell survival following exposure to various doses of X-ray was evaluated by MTT assay (A) and colony formation assay (B). The values are expressed as the mean±standard deviation from three independent experiments.
To investigate the apoptosis induction effect by radiation in vitro in human HCC cells, cells were treated with different doses (0, 4 or 10 Gy) of radiation, stained with annexin V-FITC and PI, and analyzed by flow cytometry at 48 h post-irradiation. Significantly decreased apoptotic cells at 10 Gy of radiation were observed in MHCC97L cells, compared with HepG2 and Hep3B cells, but there are no significant differences of apoptotic cells between MHCC97L and Hep3B cells at 4 Gy (Fig. 2). In addition, the apoptotic cells in Hep3B after 4 or 10 Gy irradiation were lower than that in HepG2 cells (Fig. 2). Therefore, these results meant that the sensitivity of MHCC97L cells to radiotherapy was lower than that of HepG2 and Hep3B cells, and Hep3B cells were much more radioresistant compared to HepG2 cells.
10.1371/journal.pone.0055313.g002Figure 2 Dose-dependent apoptosis induction in three human HCC cell lines irradiated with X-rays.
Each data point represents the mean±standard deviation of three independent determinations. *P<0.05, **P<0.01 vs HepG2 cells; #P<0.05 vs Hep3B cells.
APE1 is Induced by Irradiation in a Dose-dependent Manner in MHCC97L Cells
We have determined that MHCC97L cells were less sensitive to irradiation compared to HepG2 and Hep3B cells. To investigate the role of APE1 in sensitivity of MHCC97L cells to radiotherapy, we analyzed the protein expression of APE1 at 48 h post-irradiation by western blotting. A dose-dependent increase in APE1 protein expression in MHCC97L cells was observed post irradiation (Fig. 3A and B), possibly promoting radioresistance. The APE1 protein expression in 6 Gy irradiation group was much higher than that in high dose (8 or 10 Gy) X-ray irradiation group, which may due to the high-dose induced cell death in MHCC97L cells.
10.1371/journal.pone.0055313.g003Figure 3 APE1 is induced by X-ray radiation in a dose-dependent manner in MHCC97L cells.
Western blot analysis of cell lysates for the protein expression of APE1 at 48 h post irradiation. Normalized APE1 protein levels after adjusting for loading. *P<0.05, **P<0.01 vs control.
Ad5/F35-siAPE1 Suppresses APE1-target Gene Expression and Inhibits Irradiation-induced APE1 Expression
To determine the combined effect of APE1 silence and irradiation, western blotting and qRT-PCR were performed on MHCC97L cells. A significant decrease in APE1 and P53 protein expressions was observed at 48 h after Ad5/F35-siAPE1 infection in MHCC97L cells, whereas there was no P21 protein expression in all tested groups (Fig. 4A). Interestingly, we also found that irradiation-induced APE1 and P53 expressions were almost completely inhibited by the pretreatment of cells with Ad5/F35-siAPE1 (Fig. 4A). The mRNA expressions of APE1, P53 and P21 decreased in Ad5/F35-siAPE1 group, and irradiation-induced expressions of APE1 and APE1-target genes mRNA were significantly suppressed by Ad5/F35-siAPE1 pretreatment (Fig. 4B).
10.1371/journal.pone.0055313.g004Figure 4 Ad5/F35-siAPE1 inhibits irradiation-induced APE1 expression.
(A) MHCC97L cells were treated with Ad5/F35-siAPE1 or Ad5/F35-EGFP; cells were irradiated with 6 Gy of X-ray at 48 h post-infection, and the APE1, p53 and p21 protein expressions were determined at 48 h post irradiation by western blot. Normalized APE1, P53 and P21 protein levels after adjusting for loading. (B) Quantitative RT-PCR reaction target gene analysis was similarly performed in MHCC97L cells. Each data point represents the mean ± standard deviation of three independent determinations. *P<0.01 vs Ad5/F35-EGFP; #P<0.01 vs Ad5/F35-EGFP+IR.
Ad5/F35-siAPE1 Enhances Cell Growth Inhibition and Apoptosis Induction by Irradiation In Vitro
We investigated the effect of Ad5/F35-siAPE1 combined with radiotherapy on human hepatoma cell lines, MTT and colony formation assays were employed. HepG2, Hep3B and MHCC97L cells were treated with an empty adenoviral vector (Ad5/F35-EGFP) or Ad5/F35-siAPE1, and then irradiated with various doses (0∼10 Gy) of radiation. Significantly decreased cell survival was observed in Ad5/F35-siAPE1+IR group compared to Ad5/F35-EGFP+IR group at all tested doses of radiation in HCC cells, respectively (Fig. 5A). As shown in Fig. 5B, significantly decreased cell colonies at all tested doses of irradiation in HCC cells were observed in Ad5/F35-siAPE1 group when compared with Ad5/F35-EGFP group, which indicates a protective effect of APE1 on IR-induced apoptosis. Although there were no significant differences in Ad5/F35-siAPE1+IR induced cell growth inhibition in HCC cell lines, the results suggest that Ad5/F35-siAPE1 enhanced sensitivity of mutp53 cells to radiotherapy as well as wtp53 and p53 null cells.
10.1371/journal.pone.0055313.g005Figure 5 Ad5/F35-siAPE1 enhances cell killing following irradiation in human HCC cell lines.
Cells were infected with Ad5/F35-EGFP or Ad5/F35-siAPE1. At 48 h post-infection, samples were treated with different doses of irradiation. Cell survival following exposure to various doses of irradiation was evaluated by MTT (A) and colony formation assay (B). Each data point represents the mean±standard deviation of three independent determinations. Significant differences existed at all doses levels at the P<0.05 level.
To examine the effects of Ad5/F35-siAPE1 on apoptosis induction by irradiation in vitro, cells were stained with annexin V-FITC antibody and PI, and analyzed by flow cytometry. As shown in Fig. 6, the number of apoptotic cells in Ad5/F35- siAPE1-transfected group was much more than that of Ad5/F35-EGFP-transfected group at 4 and 10 Gy doses radiation in all tested HCC cell lines (HepG2∶17.45 vs 12.16, 28.11 vs 20.08; Hep3B: 15.22 vs 10.65, 23.35 vs 16.32; MHCC97L: 13.75 vs 9.09, 20.54 vs 12.59). Compared to Ad5/F35-EGFP control group, the percentage of apoptotic cells in Ad5/F35-siAPE1 group increased by 63.15% at 10 Gy of radiation in MHCC97L cells, which was much higher than that in HepG2 (39.99%) and Hep3B (43.08%) cells. Thus, these results demonstrate that silencing of APE1 by Ad5/F35-siAPE1 enhanced the apoptosis induction by irradiation in HCC cells.
10.1371/journal.pone.0055313.g006Figure 6 Cell apoptosis following combined treatment with Ad5/F35-siAPE1 and irradiation in vitro.
HepG2, Hep3B and MHCC97L cells were treated with Ad5/F35-EGFP or Ad5/F35-siAPE1.Samples were collected at 48 h for a range of X-ray irradiation (0, 4 and 10 Gy). At another 48 h post-irradiation, the cells were harvested and then stained with annexin V-FITC and PI. Bar graphs represent the mean values of triplicate determinations ± standard deviation. *P<0.01 vs Ad5/F35-EGFP.
Ad5/F35-siAPE1 Potentiates the Inhibition of Tumor Growth by Irradiation In Vivo
We have shown that Ad5/F35-siAPE1 enhances cell growth inhibition by irradiation in human hepatoma cells in vitro. To investigate whether Ad5/F35-siAPE1 could potentiate the inhibition of tumor growth by irradiation in vivo, tumor-bearing mice were injected intratumorally with Ad5/F35-EGFP or Ad5/F35-siAPE1, and two days later tumors were irradiated with 6 Gy of X-ray. Fig. 7A shows that Ad5/F35-siAPE1 combined with irradiation caused a significant inhibition of tumor growth compared with Ad5/F35-EGFP+IR or Ad5/F35-siAPE1 alone in HepG2 xenografts. On day 16, the tumor-inhibition rates of Ad5/F35-siAPE1 group, Ad5/F35-EGFP+IR group and Ad5/F35-siAPE1+IR group were 30.5, 28 and 74.65% (Fig. 7A and Table 1), respectively (P<0.05). As shown in Fig. 7B and Table 1, the results showed that the tumor-inhibition rates of Ad5/F35-siAPE1 group, Ad5/F35-EGFP+IR group and Ad5/F35-siAPE1+IR group at day 16 were 29.56, 25.76 and 72.15% in MHCC97L xenografts, respectively (P<0.05). As shown in Table 1, treatment with Ad5/F35-siAPE1 or irradiation alone significantly delayed HepG2 xenografts regrowth, however, only Ad5/F35-siAPE1 alone significantly delayed MHCC97L xenografts regrowth, whereas treatment with irradiation alone did not delay MHCC97L xenografts regrowth. More importantly, the combination of Ad5/F35-siAPE1 and irradiation significantly increased the delay of HepG2 and MHCC97L xenografts regrowth compared with that produced by Ad5/F35-siAPE1 or irradiation alone (Table 1).
10.1371/journal.pone.0055313.g007Figure 7
In vivo evaluation of tumor growth in human hepatoma wtp53 and mutp53 xenografts.
Tumor-bearing mice were injected directly into the tumors with Ad5/F35-siAPE1 or Ad5/F35-EGFP at the 1,6,11th day. Two days later, tumors were irradiated with 6 Gy of X-ray. Tumors were assessed for growth by measuring the volume of xenografts. (A) Tumor volume of HepG2 xenograft. (B) Tumor volume of MHCC97L xenograft.
10.1371/journal.pone.0055313.t001Table 1 Tumor growth after treatment with single factor (Ad5/F35-siAPE1 or irradiation) or combination on hepatoma tumor model.
HepG2 xenografts MHCC97L xenografts
Experimental groups N DT (days) Mean±SD SGD Mean±SD Inhibitionratio (%) N DT (days) Mean±SD SGD Mean±SD Inhibitionratio (%)
Ad5/F35-EGFP 4 3.48±0.13 4 3.56±0.17
Ad5/F35-siAPE1 4 3.9±0.1 0.12±0.07 30.5 4 4.09±0.06 0.15±0.07 29.56
Ad5/F35-EGFP+IR 4 3.86±0.14 0.11±0.07 28 4 3.9±0.11 0.1±0.07 25.76
Ad5/F35-siAPE1+IR 4 6.1±0.47 0.75±0.08 74.65 4 5.73±0.16 0.61±0.07 72.15
IR = irradiation; N = number of mice in the experimental group; DT = tumor doubling time; SGD = specific growth delay.
To further investigate cell growth inhibition of Ad5/F35-siAPE1 by irradiation, Ki67 immunohistochemistry was performed on human hepatoma xenografts. As shown in Fig. 8A, the Ki67+ nuclei in Ad5/F35-siAPE1+IR group were lower than that in Ad5/F35-siAPE1 or Ad5/F35-EGFP+IR group alone. As in MHCC97L xenografts, Fig. 8B also shows that Ad5/F35-siAPE1 in combination with irradiation caused a significant inhibition of proliferating cell numbers compared with Ad5/F35-siAPE1 or Ad5/F35-EGFP+IR alone. However, no significant difference was observed between Ad5/F35-siAPE1 and Ad5/F35-EGFP+IR groups. The results further reveal that Ad5/F35-siAPE1 enhanced the cell growth inhibition by irradiation in human hepatoma xenografts.
10.1371/journal.pone.0055313.g008Figure 8 Combined treatment with Ad5/F35-siAPE1 and irradiation in tumor cell proliferation in vivo.
HepG2 or MHCC97L cells were treated with Ad5/F35-siAPE1 or Ad5/F35-EGFP; 48 h after infection, cells were irradiated (6 Gy), and tumor cell proliferation was assessed by Ki67 immunohistochemistry. (A) Immunohistochemistry of HepG2 xenograft. (B) Immunohistochemistry of MHCC97L xenograft. Each data point represents the mean±standard deviation of three independent determinations. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-siAPE1; lane 3,Ad5/F35-EGFP+IR; lane 4, Ad5/F35-siAPE1+ IR. *P<0.01 vs Ad5/F35-EGFP; #P<0.01 vs Ad5/F35-siAPE1; $P<0.01 vs Ad5/F35-EGFP+IR.
Ad5/F35-siAPE1 Enhances the Apoptosis Induction by Irradiation In Vivo
To investigate the effect of Ad5/F35-siAPE1 on apoptosis induction by irradiation in HepG2 and MHCC97L xenografts, we measured apoptotic cells by TUNEL assay. We found that a much higher apoptosis index was observed in the Ad5/F35-siAPE1+IR group compared with Ad5/F35-EGFP control group, Ad5/F35-EGFP+IR group or Ad5/F35-siAPE1 group in HepG2 xenografts (Fig. 9A). Then, we investigated whether silencing of APE1 could potentiate the apoptosis induction by irradiation in MHCC97L xenografts, and found that Ad5/F35-siAPE1 increased significantly cell apoptosis induction by irradiation (Fig. 9B).
10.1371/journal.pone.0055313.g009Figure 9 Combined treatment with Ad5/F35-siAPE1 and irradiation induces apoptosis in vivo.
HepG2 or MHCC97L cells were treated with Ad5/F35-siAPE1 or Ad5/F35-EGFP; 48 h after infection, cells were irradiated (6 Gy), and apoptosis was determined at 24 h post irradiation by terminal dUTP nick end labeling (TUNEL) staining. (A) TUNEL staining of HepG2 xenograft. (B) TUNEL staining of MHCC97L xenograft. Data are expressed as percentage of apoptosis-positive cells examined with TUNEL. Bar graphs represent the mean values of triplicate determinations ± standard deviation. Lane 1, Ad5/F35-EGFP; lane 2, Ad5/F35-siAPE1; lane 3,Ad5/F35-EGFP+IR; lane 4, Ad5/F35-siAPE1+ IR. *P<0.01 vs Ad5/F35-EGFP; #P<0.01 vs Ad5/F35-siAPE1; $P<0.01 vs Ad5/F35-EGFP+IR.
Discussion
Human hepatocellular carcinoma (HCC),one of the most common lethal malignant diseases worldwide, is responsible for a large number of deaths annually [30]. Surgery and radiotherapy are two commonly used treatment modalities for HCC. Although surgical resection offers a good therapeutic effect and low risk of complication, only 15% of the patients are eligible for optimal resection at diagnosis. Radiotherapy represents a major therapeutic option for HCC patients, but its efficacy is limited by the inherent tumor radioresistance and low radiation tolerance of the surrounding normal liver [31]. Therefore, researches that overcome tumor resistance to radiotherapy and reduce normal tissue complications are urgently needed. To improve the therapeutic efficacy, there has been much interest in using of radiosensitizers in combination with radiotherapy [22], [23], [32].
DNA-repair systems are important in maintaining genomic stabilization and integrity. However, an elevated DNA repair capacity in cancer cells leads to drug or radiation resistance. Therefore, reducing the DNA repair capability of cancer cells could enhance the efficacy of these agents. DNA repair proteins are becoming fundamental targets for enhancing cancer therapy [33]–[35]. In particular, APE1 is becoming a leading target due to its central involvement in DNA base excision repair (BER) pathway, which accounts for nearly all of the abasic site cleavage activity in most cultured human cell lines [36]. APE1 is also thought to interact with several proteins including 8-oxoguanine DNA glycosylase, X-ray cross-complementing-1, DNA polymerase β, proliferating cell nuclear antigen and flap endonuclease 1 [37]. Moreover, APE1 has 3′-repair diesterase or phosphatase activity, which is important in repairing DNA that has been damaged by radiation. In addition to its DNA repair functions, APE1 exerts its reduction–oxidation (redox) modification activity on some transcriptional factors, and regulates their DNA-binding activity, and thereby, regulate gene expression [38]. p53 is an important tumor suppressor that helps maintain genomic stability by its participation in many DNA repair pathway [39], [40]. The previous studies showed that APE1 was responsible for reducing p53, thus enhancing its DNA-binding activity [8], [9]. Since APE1’s redox constitutively influences on p53, APE1 contributes to p53’s DNA repair activities.
The present study found that the radiosensitivity of MHCC97L mutp53 cells was lower than that of Hep3B p53 null and HepG2 wtp53 cells, which is in accord with the previous studies [12], [13]. Given that the mutp53 proteins not only lose wtp53 tumor suppressor activities, but also gain new oncogenic properties favoring cancer development [41], our observations suggest a key role of mutp53 involved in the cellular response to irradiation. Furthermore, our investigations provide a dose-dependent growth inhibition and apoptosis induction by irradiation for hepatoma cell lines and mutp53 cells provide much more resistance to radiotherapy than p53 null and wtp53 cells, which giving far more detailed information. Moreover, our results also showed that the radiosensitivity of HepG2 cells was higher than that of Hep3B cells. Taken together, the loss or mutation of p53 proteins produced radioresistance, which is in line with other studies showing that p53 is essential in regulating the radiosensitivity of mammalian cells [13], [42].
APE1 expression has been found to be associated with radioresistance in tumors [22], [23], [26], [27]. However, very few data exist on APE1 expression and response to irradiation in HCC. The present study found that APE1 was strongly expressed in MHCC97L cells and irradiation resulted in APE1 accumulation, probably representing an early event in the cell response to irradiation because of its role in DNA BER. Furthermore, Ad5/F35-siAPE1 inhibited APE1 expression and AP endonuclease activity (Figure S1), which suggests that Ad5/F35-siAPE1 would potentiate irradiation-induced DNA damage and suppress DNA repair. Loss or inhibition of p53 provided resistance to radiotherapy, which shows that p53 has been linked to radioresistance in tumor cells [13], [42]. p53 not only promotes the repair of minor DNA damage induced by radiation but also induces apoptosis of cells with severe DNA damage [43]. Failure of this p53-dependent apoptosis procedure may result in genomic instability after irradiation [44]. In this study, the radioresistance of mutp53 cells was higher than that of wtp53 cells, which is in line with the previous research [13], indicating that mutp53 gain new properties related to radiotherapy.
Several studies demonstrated a high expression in several human tumors, such as cervical cancer [20], ovarian cancer [21] and osteosarcoma [22]. Furthermore, more recent study shows that APE1 expression levels are correlated with sensitivity of cancer cells to radiotherapy and chemotherapy, and APE1 inhibitor could enhance the efficacy of conventional cancer treatment such as radiotherapy. Our previous study has demonstrated that vector-based APE1 siRNA decreased APE1 protein expression, and enhanced the chemosensitivity of multiple myeloma to melphalan [45] and radiosensitivity of human colorectal cancer [23]. In this study, combined treatment with Ad5/F35-siAPE1 and irradiation not only enhanced the cell growth inhibition and apoptosis induction, but also increased the tumor-doubling time, specific growth delay and tumor-inhibition ratio (%). The present study is the first to confirm that silencing of APE1 by adenoviral vector Ad5/F35-mediated APE1 siRNA enhanced sensitivity of human HCC cells to radiotherapy in vitro and in vivo. Therefore, inhibition of APE1 protein by APE1-specific siRNA may be a strategy to overcome radioresistance and then improve its therapeutic efficacy for hepatoma. Furthermore, the clinical use of Ad5/F35-APE1 siRNA in combination with radiotherapy is unexplored to date and yet important to investigate in human HCC patients.
Supporting Information
Figure S1
Effects of combined Ad5/F35-siAPE1 and irradiation on AP endonuclease activity. HepG2 and MHCC97L cells were treated with Ad5/F35-EGFP or Ad5/F35-siAPE1; 48 h after infection, cells were irradiated (6 Gy) and AP endonuclease activity of cell lysates was examined at 48 h post irradiation by oligonucleotide cleavage assay. The data indicated that the AP endonuclease activity of cell lysates was drastically decreased in Ad5/F35-siAPE1 group. Meanwhile, Ad5/F35-siAPE1 inhibited irradiation-induced AP endonuclease activity.
(TIF)
Click here for additional data file.
Text S1
Supporting information materials and methods.
(DOCX)
Click here for additional data file.
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BMC Health Serv ResBMC Health Serv ResBMC Health Services Research1472-6963BioMed Central 1472-6963-12-4732325654310.1186/1472-6963-12-473Research ArticleProfiling the different needs and expectations of patients for population-based medicine: a case study using segmentation analysis Lega Federico [email protected] Alessandro [email protected] Department of Policy Analysis and Public Management, Cergas and Area PMP SDA Bocconi, Bocconi University, Via Rontgen 1, 20136, Milan, Italy2 ASUR Marche, Via caduti del lavoro 40, 60100, Ancona, Italy2012 21 12 2012 12 473 473 31 1 2012 4 12 2012 Copyright ©2012 Lega and Mengoni; licensee BioMed Central Ltd.2012Lega and Mengoni; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
This study illustrates an evidence-based method for the segmentation analysis of patients that could greatly improve the approach to population-based medicine, by filling a gap in the empirical analysis of this topic. Segmentation facilitates individual patient care in the context of the culture, health status, and the health needs of the entire population to which that patient belongs. Because many health systems are engaged in developing better chronic care management initiatives, patient profiles are critical to understanding whether some patients can move toward effective self-management and can play a central role in determining their own care, which fosters a sense of responsibility for their own health. A review of the literature on patient segmentation provided the background for this research.
Method
First, we conducted a literature review on patient satisfaction and segmentation to build a survey. Then, we performed 3,461 surveys of outpatient services users. The key structures on which the subjects’ perception of outpatient services was based were extrapolated using principal component factor analysis with varimax rotation. After the factor analysis, segmentation was performed through cluster analysis to better analyze the influence of individual attitudes on the results.
Results
Four segments were identified through factor and cluster analysis: the “unpretentious,” the “informed and supported,” the “experts” and the “advanced” patients. Their policies and managerial implications are outlined.
Conclusions
With this research, we provide the following:
– a method for profiling patients based on common patient satisfaction surveys that is easily replicable in all health systems and contexts;
– a proposal for segments based on the results of a broad-based analysis conducted in the Italian National Health System (INHS).
Segments represent profiles of patients requiring different strategies for delivering health services. Their knowledge and analysis might support an effort to build an effective population-based medicine approach.
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Context and scope
This research investigates the segmentation of patients for planning and management purposes. Specifically, we identified segments corresponding to different patient profiles that could be used for three main goals: First, these profiles could improve the basis for developing effective prospective and population-based medicine within managed care. a In general, a prospective and population-based approach to medicine involves (a) assessing the health needs of a specific population; (b) implementing and evaluating interventions designed to improve the health of that population; and (c) providing care for individual patients considering the culture, health status, and health needs of the population to which that patient belongs
[1-14].b Our profiles contribute significantly to point (c). Because many health systems are engaged in developing chronic care model initiatives
[15-19], the profiles are important to understanding whether some patients can move toward effective self-management and can play a central role in determining their care, which fosters a sense of responsibility for their own healthc.
Second, within single healthcare organization—whether private or public, for-profit or not-for-profit—profiles are fundamental for developing targets and increasing understanding of the forces that drive healthcare consumption. Customer relations could also benefit from a better understanding of the different patient clusters.
Third, in all types of systems and organizations, profiles and their corresponding segments are important for building empowerment strategies to facilitate a shift from simple compliance to a “concordance”d approach
[20-23].
We have noticed a lack of both empirical analysis within health contexts and research on market segments and targets within health systems. Most of the analysis is inferred from general surveys of consumer behavior. In this research, we provide the following:
– a method for profiling based on common patient satisfaction surveys, which is easily replicable in all health systems and contexts;
– a proposal for segments based on the results of a broad-based analysis conducted in the Italian National Health System (INHS).
A review of the literature on patient segmentation provided the background for this research. After presenting our analyses and findings, we draw some preliminary conclusions that could have implications for healthcare policy.
Background
Several studies highlight a process for differentiating the populations of developed countries
[24-31]. This process is often referred to as a polarization process, and studies found in the literature can be divided into two interpretations.
From the perspective of the health and social conditions of a potential patient, many studies introduce a dichotomous scheme with the two poles represented as follows:
– frail, vulnerable elderly patients who lack family support, have multiple chronic conditions, are not self-sufficient, have cognitive disorders, are financially distressed, and are unable to express an appropriate demand for health and social services;
– healthy and wealthy elderly patients who are educated and pursue well-being through recurring access to an extended range of health services (preventive, curative, and aesthetic) and are willing to pay out-of-pocket or premium prices for high-quality and additional services.
From the perspective of the role of behaviors, some studies have theorized that patient polarization may occur among different dominant profiles
[32-34] Researchers at the Institute for the Future have identified the concept of ‘Personal Health Ecologies’ (PHEs), which reflects a consumer’s unique approach to managing their health. The principal PHEs proposed are illustrated in Table
1.
Table 1 Personal Health Ecologies (PHEs)
- Mainstreamers: the traditional patient;
- Allopathic self-care: prefer over-the-counter products or toughing it out rather than seeing a physician;
- Maximizers: highly engaged with their physicians and try to get the most out of their health care plans;
- Nutritionists: rely on food and diet to prevent illness;
- Naturalists: rely on complementary and alternative medicine and their bodies’ natural healing process and dislike using the health care system;
- Integrators: those who rely on the health care system for medical diagnoses but also dabble in complementary and alternative medicine (CAM);
- Holistics: use the health care delivery system and CAM for the things each modality excels in;
- Healthy Lifestylers: dramatically change their lives to maximize their health and look for health benefits across a wide range of products and services.
Although both efforts to categorize patients into clusters are effective to some extent, they are either too narrow or too broad and abstract to provide useful information to health care managers and policy makers. A context-embedded and evidence-based segmentation of patient populations could help to explain the driving forces necessary to improve service delivery (appropriateness, access, timeliness) and to engage and empower the patient in the care process
[35]. This approach contributes to the transformation of the health care system from one that is essentially reactive, primarily responding when a person becomes ill, to one that is proactive and focused on keeping a person as healthy as possible. In this respect, we describe a new analysis of patient segmentation derived from the combination of patient characteristics and evidence gathered from survey data. The analysis is easily replicable and can be contextualized to specific subpopulations or geographical areas.
Methodology
Survey definition
In the first phase of research, a literature review was conducted to identify published patient satisfaction questionnaires and surveys of patient opinions regarding outpatient services between January 1990 and January 2009 to identify various issues that patients may consider in their assessment of such services. The bibliographic databases used were Medline, Scopus, Social Science Citation Index and EconLit. Search terms included “satisfaction,” “evaluation,” “assessment,” “judgement,” “opinions,” “perceptions,” “questionnaire,” “patient,” “user,” “people,” “outpatient,” “primary care,” “out of hours,” and “care continuity.” A bibliographical search was supplemented by reviewing references from identified articles and by an Internet search of relevant web sites. Studies were included if they were experimental and if they were written in English. The search methodology generated 497 possible references. By reading the complete articles or abstracts, possible evaluation schemes—such as those related to outpatient services
[36-39], after-hours services
[40] and walk-in clinics
[41]—were identified. In the second phase, to ensure valid content, a preliminary list of issues and statements produced from the literature review was submitted to two focus groups of 5 patients who had recently used outpatient services. The method was chosen to generate a limited number of relevant variables and items related to patient satisfaction and experiences with outpatient services. The patients were asked to select the most important aspects affecting their satisfaction, the significant features influencing their utilization of the services and to suggest additional issues or questions. Both groups were examined by a trained interviewer and the examinations followed a similar structure. The groups were audiotaped and coded separately by two researchers. Analysis revealed 4 recurrent domains that characterized patient responses: quality of healthcare services, quality of administrative services, access to out-of-hour care and interpersonal aspects. These dimensions formed the basis for developing the questionnaire. In the third phase, an expert panel of primary care managers and the district managers of local health authorities (LHAs)e reviewed the questionnaire to ensure relevance and clarity of the items. Questions that were confusing or ambiguous were removed, replaced or rewritten with the appropriate terminology. Moreover, two additional items regarding home care and vaccinations were included. Therefore, the final set of evaluation characteristics included specialist visits, diagnostic services, administrative services, home care, advisories, vaccinations, and the coordination of continuity of care. The professionalism and kindness of the health care and the administrative staff working in the outpatient clinics and the professionalism of the after-hours doctors (AHDs) were also rated for satisfaction. These 12 variables were fed into a 12-question questionnaire using a five-point rating scale. The variables were integrated with another 10 multiple choice or yes/no questions designed to examine the subjects’ experiences with these services. The questionnaire also included sociodemographic data (see Additional file
1: Appendix 1). Four questions, unrelated to the outpatient services assessment, were incorporated in the final section of the survey to evaluate communication initiatives of the LHAs within the Tuscan region. The results of these 4 variables are not reported in this paper.
Data collection
The reference population consisted of Tuscan citizens over 18 years of age (3,168,955 in 2009). We selected Tuscan citizens because they are the target population for the Tuscany Regional Health System (TRHS). During 2008–2010, the TRHS introduced a regional health plan
[42] with a strategic priority to develop a proactive and chronic care model for population-based medicine. All Tuscan LHAs and their primary care and district managers are engaged in this strategic goal f. Therefore, the result of this research would be of great interest to the TRHS. The sample was stratified into the 34 health districts in the region. In each health district, a sample size of approximately 196 subjects was required, assuming a 50% satisfaction at a 95% confidence level with a margin of error of ± 7%. Assuming a response rate of approximately 24%, which is in line with previous studies in that area, oversampling was performed to ensure that the minimum sample size was obtained. The calculated sample size was then multiplied by 34 to obtain the total sample size of 27,300, representing 0.9% of the reference population. The sample used was randomly selected from an updated regional phone directory, containing all listed residential telephone numbers. A pilot test was performed on a sample of 34 individuals of differing ages and geographical locations to verify whether the subjects understood the questionnaire and to determine if other relevant issues had been omitted from the survey. Based on the respondents’ feedback, response rates and item response rates, the pilot program indicated that no topics other than those already included in the questionnaire were considered relevant. Some changes were made to the wording of the questions and the instructions to eliminate ambiguous phrasing. Interviews were conducted during the summer of 2009 with a computer-aided telephone interview system during both working and non-working hours to reach a wide variety of patients g. The use of a sample list from the telephone directory created intrinsic variation in the sociodemographic composition of the interviewees. Women and elderly subjects were overrepresented with respect to the actual composition of the population. However, a non-response bias test to identify possible distortions among the opinions of the interviewees classified in the dataset
[43-48] provided reassuring results. The testing compared the responses from the first 200 and the last 200 respondents for each of the 12 questions to determine the degree of satisfaction. A chi-square test indicated no significant statistical differences (at the 5%leve) between the scores of the two groups for the different variables.
Statistical analyses
Questions used to investigate more specific services with a percentage of missing values greater than 10% (home care, advisories and vaccinations) were eliminated from the satisfaction assessment, leaving nine variables. “I don’t know” and similar answers were considered missing values and were replaced with an “expectation-maximization” algorithm
[49]. Principal component factor analysis via varimax rotation was used to extrapolate the key structures on which the subjects’ perceptions of outpatient services were based. After the factor analysis, segmentation was performed using cluster analysis to better analyze the influence of individual attitudes on the results
[44-46]. The “scores” given to various factors were employed for hierarchical cluster analysis using Ward’s method to identify the correct number of clusters and their respective centers
[47]h while accounting for any a priori expectations concerning the data structure. Using the previously identified cluster centers, K-means cluster analysis was performedi, and the results were validated through linear discriminant analysis
[48]. All statistical analyses were performed using SPSS 16.0.
Results
The interviewees evaluated the analyzed characteristics, which are listed in Table
2. When analyzing single variables, neither the effectiveness of the outpatient services nor the professionalism of their staff appears to be objectionable. However, questions regarding the administrative services of the outpatient clinics and the continuity of care received lower scores. The results also indicate a degree of variability in the average judgments, especially those focused on variables related to diagnostics, administrative services and the service staff.
Table 2 Mean and standard deviation of the evaluations of the characteristics analyzed
Mean Std. Dev. % Missing
Specialist visits 3.69 0.68 6.76
Diagnostic tests 3.76 0.79 2.34
Home care (Removed) 3.96 1.27 27.69
Advisories (Removed) 4.15 0.81 31.40
Vaccinations (Removed) 4.04 0.76 16.98
Administrative services 3.59 0.76 9.45
Kindness of administrative staff 3.98 0.69 5.38
Professionalism of administrative staff 3.95 0.69 5.68
Kindness of health care staff 4.15 0.76 1.91
Professionalism of health care staff 4.15 0.73 1.93
Coordination of continuity of care service 3.63 0.50 8.22
Professionalism of after-hours doctors (AHDs)* 3.85 0.52 8.17
5=Totally satisfied, 1=Totally unsatisfied.
* AHDs substitute for general practitioners during night shifts. They are employees or contracted by the local health authority.
Factor identification
Factor analysis was performed to reduce the nine characteristics to a more condensed set of dimensions and to test the construct validity of the questionnaire. The analysis met the Kaiser-Meyer-Olkin measure of sampling adequacy equal to 0.8, and the application of Bartlett’s test of sphericity yielded a highly significant chi-square value. During the extraction, all communalities exhibited values greater than 0.7. Based on the explained variance and the scree plot results, a three-factor solution (with 87.5% of the total variance explained) was considered appropriate. Table
3 shows the rotated structure matrix used to identify the extracted factors and the variables related to each factor. The first factor identified was the outpatient clinic staff, which is correlated to variables assessing the professionalism and kindness of the staff in the outpatient district structures that perform health and administrative duties. As this dimension exhibited the greatest percentage of variance explained (37.3%), the 4 correlated items were reanalyzed using principal component analysis with varimax rotation and eigenvalues greater than 1 to determine whether this factor could be divided into 2 components representing, for example, the characteristics of the health staff and that of the administrative staff. The analysis revealed loading values greater than 0.8 for all variables and no statistically significant loadings for the other factors, suggesting the homogeneity of this domain. The second factor (28.5% of variance explained) includes the other aspects of the outpatient clinics and indicates a close connection between all attributes of the services provided, including diagnostic tests, specialist visits and administrative services. The high correlation coefficients for all variables and the absence of large variations in them allow one to define this factor as representative of all the scores given to the outpatient clinic services.
Table 3 Rotated structure matrix (correlation coefficients between the variables and the extracted factors)
Outpatient clinics’ staff Outpatient clinics’ services Continuity of care
Kindness of administrative staff 0.863
Professionalism of administrative staff 0.841
Kindness of health care staff 0.809
Professionalism of health care staff 0.775
Diagnostic tests 0.910
Specialistic visits 0.820
Administrative services 0.788
Organization of continuity of care service 0.922
Professionalism of AHDs 0.835
The third factor (21.7% of variance explained) was continuity of care. Although this factor consists of only two variables, it was strong because it did not change with modifications to the factors used for the analysis or the methods of calculation. Continuity of careis closely associated with organizationand professionalism of the after-hours doctors (AHDs). The item internal consistency was satisfactory for all dimensions as the correlation level of each item with its scale achieved the 0.40 standard
[50]. The item discriminant validity was also adequate
[51], indicating that all items correlate more highly with the dimension in which they fit than with the other dimensions (0.18–0.52 for the first factor, 0.20–0.32 for the second factor and 0.17–0.31 for the third factor). Cronbach's alpha coefficient (that should exceed 0.7
[52]) was 0.92 for the first dimension, 0.95 for the second dimension and 0.89 for the third, indicating a high internal reliability for each factor. The discriminant validity was tested by comparing the mean dimension scores across the patient groups (age, gender and education). As in the literature
[53-55], we found that older patients and those with lower education levels are more satisfied with respect to all dimensions; furthermore, gender does not have a significant effect on the satisfaction scores for any dimensions.
Group creation
After reducing the district service assessments to three macroelements, cluster analysis was used to investigate how they varied within the interview samples
[56-62]. According to the Ward method, the results of the first hierarchical cluster indicated the presence of four groups. The cluster’s final centers were obtained using the K-means method. Statistical tests confirmed the robustness of the analysis. The high values of the F-test for each of the factors used in the analysis demonstrate that the differences in the means of the groups in each factor were statistically significant (p < .001). To validate the results, discriminant analysis was performed using the original composition of the different groups as a grouping variable. The discriminant functions were significant (p < .001) and support the existence of four different clusters. Based on the values of the three factors, the confusion matrix (Table
4) indicates the success of the prediction algorithm, confirming that the three discriminant functions correctly sorted 99.6% of the cases into the four groups. The reliability of the clusters obtained was also investigated using cluster analysis on a random sample of 50% of the cases and a second analysis on the remaining cases. For both samples, the composition of the cluster was the same.
Table 4 The discriminant analysis confusion matrix
Actual group Predicted group membership
1 2 3 4
CLUSTER 1 97.7% 0.6% 1.7% 0.0%
CLUSTER 2 0.1% 99.6% 0.3% 0.0%
CLUSTER 3 0.0% 0.0% 100.0% 0.0%
CLUSTER 4 0.0% 0.0% 0.0% 100.0%
99,6% of cases classified correctly.
For each segment, Table
5 provides the mean scores of the three factors in the various groups, the sociodemographic characteristics and the principal variables for access to and use of the outpatient services. Due to the high number of missing values, 4 variables related to patient experiences were not included in the analysis. With the exception of the sex variable, the values from the chi-square test indicated that the sociodemographic and behavioral differences between clusters were statistically significant. Therefore, identification of each group is potentially useful for developing policies aimed specifically at that group. Finally, the size of each group could also lead to better prioritization of decision-making processes when coping with allocation of scarce resources.
Table 5 Segment characteristics (mean factor scores, sociodemographic conditions and past experience with healthcare services for the identified groups)
Unpretentious Informed & supported Experts Advanced Total
SIZE 2070 779 531 81 3461
FACTORS*
Outpatient clinic staff 0.29 0.30 −1.65 0.66
Outpatient clinic services 0.48 −1.37 0.03 0.74
Continuity of care 0.22 −0.07 −0.11 −4.25
GENDER (%)
Males 22.4 20.9 21.3 18.5 21.8
AGE (%)
18–45 23.7 28.7 30.7 40.5 26.3
46–65 36.7 43.1 42.1 44.3 39.1
Over 65 39.6 28.3 27.1 15.2 34.5
EDUCATION (%)
None / Primary school 38.8 31.4 28.1 16.3 34.9
Middle school 24.4 25.3 28.5 32.5 25.4
High school 28.6 33.1 32.3 37.5 30.4
Degree and post degree 8.2 10.1 11.2 13.8 9.2
JOB (%)
Legislator, executives and entrepreneurs 1.3 0.6 0.8 2.5 1.1
Intellectual, scientific and highly skilled professions 3.3 5.2 5.7 12.7 4.3
Technical professions 4.0 4.4 4.4 8.9 4.3
Clerks 7.1 9.3 9.4 8.9 8.0
Skilled activity in commerce and services 5.4 6.2 7.6 5.1 5.9
Artisans, skilled labor and farmers 4.6 3.2 5.2 5.1 4.4
Semi-skilled labor 1.3 0.9 0.8 3.8 1.2
Unskilled labor 1.3 1.6 1.7 1.3 1.4
Students 2.8 3.9 2.7 1.3 3.0
Housewives 18.9 22.1 21.2 24.1 20.1
Unemployed 1.4 2.5 1.3 2.5 1.7
Retired 48.4 39.7 39.0 24.1 44.4
FAMILY SITUATION (%)
1 (live alone) 13.4 9.2 9.0 7.5 11.6
2 35.3 34.0 30.3 18.8 33.9
3 23.4 28.5 26.7 27.5 25.1
More than 3 27.9 28.3 34.0 46.3 29.4
CHRONIC DISEASES (%)
Yes 46.3 43.2 39.1 38.8 44.3
No. OF VISITS IN OUTPATIENT C. IN THE LAST YEAR (%)
1 32.0 27.2 26.4 23.5 29.8
2 27.1 26.7 31.6 23.5 27.6
3–4 22.1 27.7 25.6 23.5 24.0
Over 4 18.8 18.4 16.4 29.6 18.6
WHO REFERRED TO OUTPATIENT CLINIC (%)
Personal initiative 18.9 15.1 18.5 16.0 17.9
Relative/Friend 0.6 0.3 0.6 0.0 0.5
GP/PD 65.3 74.2 70.8 77.8 68.4
Hospital physician 4.5 3.2 2.4 1.2 3.8
Private specialist 2.1 2.2 1.1 1.2 2.0
Social services worker 0.1 0.0 0.0 0.0 0.1
Clinic invitation letters 8.5 5.0 6.6 3.7 7.3
SERVICES UTILIZED IN THE OUTPATIENT CLINIC (%)a
Specialist visits 20.9 23.5 21.5 23.9 21.7
Diagnostic tests 68.5 69.2 67.7 62.5 68.4
Home care 0.6 0.2 1.2 2.3 0.7
Administrative services 6.4 6.1 7.3 6.8 6.5
Advisory 0.9 0.0 0.2 0.0 0.6
Vaccinations 2.6 1.0 2.1 4.5 2.2
TYPE OF STAFF CONSULTED IN THE OUTPATIENT C. (%)a
Administrative staff 30.3 28.8 33.9 37.8 30.7
Health care staff 69.7 71.2 66.1 62.2 69.3
AHD CONSULTATION (%)
Yes 11.5 14.2 15.1 100.0 14.8
METHOD OF AHD CONSULTATION (%)
Telephone consultation 17.6 12.6 11.3 38.3 18.8
Home visit 64.4 62.2 60.0 39.5 59.3
Ambulatory visit 18.0 25.2 28.8 22.2 21.9
A&ED VISIT AFTER AHD CONSULTATION (%)
Yes 17.6 20.7 23.8 45.7 23.7
REASON FOR A&ED VISIT AFTER AHD CONSULTATION (%)
AHD referral 85.7 73.9 78.9 45.2 71.3
Unsatisfied with AHD consultation 7.1 17.4 21.1 48.4 22.6
Further information on diagnosis/therapy proposed by AHD 7.1 8.7 0.0 6.5 6.1
GP: General practitioner; PD: Pediatrician; AHD: After-hours doctor; A&ED: Accident & emergency department.
a Percentages are based on responses.
p < .001 for age, education, job, family situation, no. of visits in outpatient c. in the last year, AHD consultation, method of AHD consultation, A&ED visit after AHD consultation, reason for A&ED visit after AHD consultation; p = .005 for those referred to outpatient clinic; p = .025 for chronic diseases.
* Higher factor scores indicate that the respondents are more satisfied with the items in the factor or have rated the items in the factor more positively.
Discussion
Segment 1: The unpretentious patients
This segment includes the highest number of interviewees who gave positive evaluations for all three factors and comprises the highest percentages of the elderly, those who only completed elementary school or had no qualifications, retired people, the chronically sick, and those living alone or, at most, with one other person. This segment utilizes outpatient clinics less frequently. In general, the users included in this segment appear to be fragile, with no clear ideas regarding available health care and appear to be incapable of turning their needs into demands. These characteristics lead to the hypothesis that the positive opinions of the considered services derive from the fact that the group members received better results than expected (hence, the name “unpretentious,” given their low expectations). Due to their poorer health and social conditions compared with the other groups, this segment uses home visits more frequently to consult with their AHD and reports a high level of satisfaction with the AHD. The frequent use of AHDs explains the low use of the accident and emergency department (A&ED). Although not completely comparable due to differences in the methodological approach and the subject of analysis, this group’s characteristics are similar to those of the “easy to please” cluster identified by Morrison et al.
[43] in their segmentation analysis of GP services, which indicated that the subjects in this segment exhibit a laissez-faire attitude toward factors such as communication, relationships and empowerment and are quite satisfied with their GPs.
Segment 2: The informed and supported patients
The subjects in this segment gave positive evaluations of the outpatient clinic staff, but they were not satisfied with the services received. In addition, they were confused by the continuity of care. The health needs of this group, though less complex, do not differ greatly from those in the previous segment. In fact, when compared with the other groups, the percentage of chronically ill patients is high due to the high number of individuals over 50 years of age. This segment differs from the first because of their greater awareness of their health needs (due to a higher level of education and a higher number of qualified professionals) and the availability of a better network of health care facilitators (support from a larger family group and a deeper knowledge of the system, which leads to a greater use of GPs) Therefore, these patients utilize outpatient services more frequently than the previous group defining this group “informed and supported.” These patients tend to blame the system more than the workers for failing to deliver processes, which could explain the positive perception of the district staff. This group may be related to the “engagement needed” segment described by Morrison et al.
[43] in which people are particularly interested in the caring qualities of the GPs yet have low health status.
Segment 3: The expert patients
This segment includes all patients who consider the staff the weak element in the services. Although these patients are not enthusiastic, they are generally satisfied with the services. However, they have a slightly negative opinion of the continuity of care, not unlike second group. From a sociodemographic and epidemiological point of view, this segment does not differ significantly from the others and can be defined as an “evolution” of segment 2. The sociocultural level of this segment and its frequent use of the services indicate that these subjects are knowledgeable of the system (hence, the name “expert patients”). They expect the staff to solve problems and to improve the system. These patients are experts because they can self-manage their symptoms. With regards to continuity of care, the same considerations apply as for segment 2. After interactions with the AHDs, these patients understand the nature of their problems, which confirms a high capacity to interpret their symptoms correctly.
Segment 4: The advanced patients
Although the previously identified cluster is rather limited in number, its members provided such distinctive scores that a fourth segment was required. These subjects provided positive evaluations for both the services and the staff of the outpatient clinics, but gave the continuity of care strongly negative evaluations. This group is characterized by a higher percentage of young users with the highest level of education and includes highly skilled professionals and technicians (twice as many as in the other groups). The proportion of retired persons is 50% lower than that in the other groups. Its patients are the least burdened by chronic diseases, and nearly 50% of them live in a family of more than three members. One can assume that these characteristics entail a good capacity to interpret health needs, a deep knowledge of the health system and the ability to independently identify the best offers available. As a result, this group is the greatest user of the district services, primarily responding to personal situations and needs (low compliance with letters of invitation from the clinics, but high use of GPs and high individual initiative). Given these characteristics and their significant experience with the previously mentioned services, this segment represents “advanced” patients. This group provides an extremely negative evaluation of the continuity of care, which corresponds to the hypotheses formulated for segments 2 and 3. Furthermore, this segment has common characteristics with the “generation X” and “service users” groups described by Morrison et al.
[43] and Gabbott and Hogg
[63], respectively. Both groups include young people with high socio-economic and health status. In the former, the subjects demonstrated preferences for quality communication and an aversion to GP advice regarding their treatment options. In the latter, individuals that are frequent users of general practice services are concerned with the overall health care experience, including empathy from the staff and the responsiveness of the service.
Conclusion: preliminary implications for a policy and research agenda
Some early conclusions can be drawn from the segmentation. Actions can be taken to address specific priorities and alter the driving forces for each segment and the direction and change for health organizations and managerial practices.
Better knowledge of the patient segments could be useful on three levels:
– First, this knowledge would aid in the design of more effective communication tools and relationship processes. Interactive web design provides an example. How should health organizations use the internet to respond to the expectations and capabilities of different segments? Access processes are another example. Should health organizations diversify channels of access to meet different patient profiles? For example, could some segments have direct access to secondary care, or should everything originate with the GPs?
– Second, strategies for empowering patients might differ. For example, some segments could have more control over their health budgets and could be targets for a policy of healthcare vouchers with more responsibility and the freedom to choose their own providers, thus making them more engaged in appraising their medical services.
– Third, segmentation could become a mechanism to address cultural issues and could provide a good excuse to engage clinicians and health staff to review their patient relationship practices. Do they recognize and pay attention to differences? Different segments might require different language, information, and individual approaches (paternalistic, autocratic, democratic, etc.). In contrast, the segments could be used to cause patients to consider their attitudes toward health issues and clinicians. For example, patients could be asked to identify the segment to which they believe they belong and to discuss the implications with their GP.
The results for the “unpretentious” patients could be used to prioritize preventive actions, such as the creation of medical records for chronic illnesses (hypertension, diabetes, etc.) to more accurately monitor the clinical evolution of more serious patients. This strategy should be reinforced, whenever possible, by specific incentives to aid the outpatient services staff (in collaboration with the primary health services) in implementing proactive attitudes for contacting and guiding patients who do not thrive and who can neither interpret the nature and dynamics of their pathology nor manage the necessary stages of their diagnostic/therapeutic procedures. For the “informed and supported” patients, integrated medical records could be helpful but not a priority because of the patients’ greater awareness of their health needs and their better use of the healthcare network. In this case, the role of the GP should be stressed. These patients expect “customized” diagnostic/therapeutic paths or direction toward the best possible paths. The GPs should work as “mentors” and supervisors to patients who, given the proper health care procedures and “activated” by empowerment, could make more autonomous use of the services they need. Quick access to information seems to be critical for “expert” patients. This group could benefit from more exhaustive and rapid information on specific services (including other providers who can offer these services) and ways to make the most appropriate use of this information. The process for determining the most adequate provider could be greatly simplified, and the patient would have greater responsibility for the correct use of all available resources. Information should be provided from a variety of sourcesj because these patients often do not consider GPs a primary source of advice. Finally, communication and marketing (or demarketing) initiatives are central for the “advanced” patients as a way to direct them toward a more appropriate use of general and specialist services. This segment appears to be independent in its decision-making and is expected, due to its members’ relatively young age, to respond to informative materials and educational initiatives.
Limitations
Some limitations must be noted and should be addressed in future research. First, the questionnaire used in this study has proven reliable and valid; however, further tests are needed to assess the stability of our findings in other samples. Second, the profiles developed in this work are the result of an analysis conducted in one region of Italy, thus have some path dependency and are strongly influenced by the dominant cultural traits in Tuscany. Therefore, the segments are not universally valid, although we can expect some agreement with similar analyses conducted in other developed countries. However, the method is universally valid and can be used by managers and policy makers to investigate their own systems and to develop their own profiles. Third, a comparison of the mean age of the subjects sampled with the Tuscan population data revealed that, to a small extent, younger females and older adults were less likely to respond, while individuals aged 46–65 were more likely to respond
[64] (see Additional file
2: Appendix 2). This result highlights a possible bias in the sampling that suggests caution when generalizing these findings to the wider community. The telephone directory sampling may yield biased samples (for example with younger persons less likely to be listed in the sampling frame)
[65], but it is still considered a reasonable approach because the selection bias is sufficiently small, particularly for health-related variables
[66]. In future studies, the potential for this bias must be addressed by expending additional resources on the recruitment of subjects.
Endnotes
aFor example, a national or public health system, an integrated delivery system, and insurance or other third-party payer.
bAs Snyderman and Williams stated, “The ability to identify those individuals most at risk for developing chronic diseases and to provide a customized means to prevent or slow that progression are emerging competencies and provide the foundation for prospective care”
[37].
cIf we consider that the most recent data indicate that almost half of all US citizens live with a chronic condition
[33] and the rate of increase is estimated at more than one percent per year by 2030, resulting in an estimated chronically ill population of 171 million, we can understand the urgent need for information and tools for improved population-based medicine. Almost half of all patients suffering chronic illness have multiple conditions, and their treatment suffers from deficiencies, such as the following:
– hurried practitioners who do not follow established guidelines;
a lack of care coordination;
– a lack of active follow-up to ensure the best outcomes;
– patients who are inadequately trained to manage their illnesses.
d“Concordance” refers to the explicit participation of the patient in the decision-making process. We do not refer to the definition of concordance as the similarity, or shared identity, between the physician and patient based on a demographic attributes, such as race, sex, or age
[16].
eLHAs are integrated delivery systems, or “umbrella” organizations that manage the entire spectrum of services and levels of care. LHAs might involve different combinations, including community services with hospitals, home care schemes, rehabilitation facilities, nursing homes, mental health centers, etc. More specifically, LHAs regroup facilities providing care at different levels: prevention and environmental health services, primary care (GPs), secondary care (outpatient services), tertiary care (general or community hospitals), quaternary care (academic medical centers and specialty hospitals), rehabilitation (nursing homes, rehabilitation centers), and long-term care (long-stay inpatient centers, home care units). In short, an LHA provides or aims to provide a coordinated continuum of services to a defined population and is willing to be held clinically and fiscally accountable for the outcomes and the health status of the populations served.
fThe Tuscan Regional Health System serves a population of roughly 3.7 million and is organized with 12 LHAs and 4 independent teaching hospitals. The LHAs are accountable for the residents of a provincial geographical area and are sub-organized into health districts run by managers responsible for planning and governing the delivery network of primary care and continuity of care.
gParticipants that had not used outpatient services in the previous 12 months were only asked to answer questions regarding communication initiatives and sociodemographics.
hOne of the most widespread hierarchical clustering methods is Ward’s method
[56,57], which attempts to generate clusters to minimize the within-cluster variance. Starting from with t clusters, each containing one object, at each step, Ward’s method combines the two clusters that will result in the smallest increase in the sum-of-square index (or variance), and repeats the process until one cluster remains containing all the objects. At each stage, the mean of each cluster, or the average of the variable values for the objects in the given cluster, is first calculated. Then, the sum of the squared differences between each object in a given cluster and its cluster mean are computed
[58].
The method has been shown to perform better than the other hierarchical procedures
[59] because it tends to produce robust, dense, spherical clusters with distinct characteristics
[60]. However, the solutions it provides tend to be distorted by outliers
[59] and produce poorer results than the K-means non-hierarchical partitioning
[61,62] if a nonrandom starting point is specified
[47]. Hence, a two-stage clustering approach has been suggested. In the first step, a hierarchical method should determine a candidate number of clusters, a starting point for the iterative partitioning analysis and should identify outliers that may be eliminated from further analysis. Then, non-hierarchical approach should be performed to refine the clusters
[47].
iSuch clustering procedures yield solutions for discrete optimization problems, as opposed to model-based clustering methods that posit an underlying statistical model, producing for each object a probability of membership in each group. In general, insufficient evidence exists to recommend the model-based over more deterministic methods for clustering applications
[67].
jA survey conducted in the US
[68] analyzed a variety of sources for health-related information used by patients:
– 64% of those sampled consider a GP or a specialist doctor the primary source;
– 54% use the family network;
– 47% use specialized web sites;
– 32% use specific mailing lists;
26% use media programs (especially television).
Competing interests
The authors declare that they have no competing interests (either financial or otherwise) in this manuscript.
Authors’ contributions
All authors contributed equally to the conception, design and drafting of this manuscript. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6963/12/473/prepub
Supplementary Material
Additional file 1
Appendix 1. Questionnaire.
Click here for file
Additional file 2
Appendix 2. Mean ages and standard deviations of respondents compared to Tuscany region population.
Click here for file
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23441162PONE-D-12-2725910.1371/journal.pone.0056123Research ArticleBiologyImmunologyImmunologic TechniquesImmunoassaysMajor Histocompatibility ComplexModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCellular TypesEndothelial CellsGene ExpressionSignal TransductionMedicineCardiovascularVascular BiologyRheumatologySclerodermaHLA-B35 and dsRNA Induce Endothelin-1 via Activation of ATF4 in Human Microvascular Endothelial Cells ATF4 Activates ET-1 GeneLenna Stefania
1
Chrobak Izabela
1
Farina G. Alessandra
1
Rodriguez-Pascual Fernando
2
Lamas Santiago
2
Lafyatis Robert
1
Scorza Raffaella
3
Trojanowska Maria
1
*
1
Arthritis Center, Boston University School of Medicine, Boston, Massachusetts, United States of America
2
Centro de Biología Molecular “Severo Ochoa” (CSIC/UAM), Madrid, Spain
3
Referral Center for Systemic Autoimmune Diseases, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico and University of Milan, Milan, Italy
Feghali-Bostwick Carol Editor
University of Pittsburgh, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interest exist.
Conceived and designed the experiments: S. Lenna GAF FR-P SL RL RS MT. Performed the experiments: S. Lenna IC GAF. Analyzed the data: S. Lenna IC GAF FR-P S. Lamas RL RS MT. Contributed reagents/materials/analysis tools: FR-P S. Lamas. Wrote the paper: S. Lenna MT.
2013 18 2 2013 8 2 e561235 9 2012 5 1 2013 © 2013 Lenna et al2013Lenna et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.
This work was supported by the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) P50 AR060780 to MT and RL, Scleroderma Foundation grants to MT and AF, and by a grant from Gruppo Italiano per la Lotta alla Sclerodermia (GILS) to SL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Endothelin-1 (ET-1) is a potent vasoconstrictor and one of the key regulators of vascular homeostasis. ET-1 dysfunction is associated with a number of pathological conditions including hypertension, atherosclerosis, cardiovascular disorders, and cancer [1], [2], [3]. Under physiological conditions, ET-1 is produced in small amounts mainly in endothelial cells (ECs). However in pathophysiological conditions, its production is stimulated in a large number of different cell types, including endothelial cells, vascular smooth muscle cells, cardiac myocytes, and inflammatory cells such as macrophages and leukocytes. In addition to its main role as a vasoconstrictor, ET-1 also contributes to inflammation, as well as fibrosis during various pathophysiological processes.
Extensive studies of ET-1 gene expression have led to characterization of the signaling pathways and transcription factors involved in its regulation [4], [5]. A complex network consisting of the common and tissue specific transcription factors responding in the coordinated fashion to physiological and pathological stimuli have been shown to regulate ET-1 expression in a cell type and context specific manner. One of the main regulatory factors is a FOS/JUN complex that binds to an activator protein 1 (AP-1) response element located at a -108 bp in the ET-1 promoter region. This site mediates upregulation of the ET-1 gene by phorbol esters, Angiotensin II, Thrombin, and High-density lipoprotein (HDL), which stimulate AP-1 in a Protein kinase C (PKC)-dependent manner. On the other hand, Leptin activates AP-1 through the Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinases 1/2 (Erk1/2) pathways. AP-1 in cooperation with GATA-binding factor 2 (GATA2) is also required for the basal transcription of the ET-1 gene in endothelial cells, while other members of the GATA family regulate ET-1 expression in other cell types. Additional important transcription binding sites include hypoxia response element, Hypoxia-inducible factors (HIF-1), transforming growth factor β (TGF-β)/Smad response element, which have been also described to cooperate with AP-1 to induce ET-1 [6], as well as the Nuclear factor κB (NF-κB) binding site that mediates responses to inflammatory cytokines. Other cell type specific response elements have also been characterized [7].
Endoplasmic Reticulum (ER) stress is defined as accumulation of unfolded or misfolded proteins in the ER, triggering an adaptive program called the unfolded protein response (UPR). The UPR alleviates ER stress by suppression of protein synthesis, facilitation of protein folding via induction of ER chaperones, and reinforced degradation of unfolded proteins. Three major transmembrane transducers of ER stress have been identified in the ER. Those are the RNA-dependent protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α). Activation of PERK leads to phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), causing general inhibition of protein synthesis. In response to ER stress, ATF6 transits to the Golgi where it is cleaved by the proteases Site-1 protease (S1P) and Site-2 protease (S2P), yielding a free cytoplasmic domain which functions as an active transcription factor. Similarly, activated IRE1α catalyzes removal of a small intron from an X-box-binding protein 1 (XBP1) mRNA. This splicing event produces an active transcription factor XBP1. If the cell fails to deal with the protein-folding defect and restore homeostasis, a pro-apoptotic CCAAT/−enhancer-binding protein homologous protein (CHOP)-mediated pathway is initiated [8].
There is extensive evidence that ER stress/UPR is closely linked to the inflammatory pathways through activation of the two key inflammatory mediators, JNK and NF-κB [8], [9] Recent studies have also revealed that UPR and innate immune pathways share common mediators [10], [11]. It was shown that stimulation of the Toll-like receptor (TLR) 2 or TLR4 leads to selective activation of the IRE1a/XBP1 pathway, contributing to the optimal and sustained production of proinflammatory cytokines in macrophages [12]. However, activation of the IRE1α/XBP1 pathway by these TLR agonists did not lead to expression of the genes typically regulated by these mediators during ER stress, suggesting an alternative utilization of the components of the UPR pathways. The specific mechanisms involved in these atypical responses are still not well understood and require further investigation.
We have recently shown that ectopic expression of HLA-B35, an antigen associated with SSc in Choctow Indians [13] and SSc-PAH in Italian patients [14], [15], led to a significant increase of ET-1 and a decrease of eNOS in cultured endothelial cells (ECs) [16]. In addition to ET-1, we have also observed upregulation of interferon-regulated genes and other inflammatory genes in ECs expressing HLA-B35. Furthermore, expression of HLA-B35, but not a control antigen HLA-B8, potently upregulated several cellular chaperones including BiP, HSP70 and HSP40, suggesting an activation of ER stress/UPR in these cells. However, other UPR genes such as ERO1 (ER oxidoreductin 1), and PDI (protein disulphide isomerase), which are involved in oxidative protein folding, as well as a pro-apoptotic UPR mediator, CHOP were not upregulated, consistent with activation of an adaptive phase of the UPR. Relevant to these findings Farina et al have reported upregulation of ET-1 in response to a synthetic analog of dsRNA, Poly(I:C), in dermal endothelial cells and fibroblasts [17]. Given the recently uncovered cross-talk between the UPR and the innate immune pathways, the goal of this study was to further investigate whether common mediators are involved in ET-1 gene regulation in response to these stimuli. Here we report that induction of ER stress or stimulation with Poly(I:C) activate the eIF2α-ATF4 pathway and promote formation of the ATF4/c-JUN complexes. This protein complex in concert with the NF-κB pathway activates ET-1 gene transcription in endothelial cells.
Materials and Methods
Reagents
Thapsigargin (TG) was purchased by Sigma-Aldrich (St. Louis, MO). Poly(I:C) was purchased by InvivoGen (San Diego, CA). Tissue culture reagents, EBM kit by Lonza (Walkersville, MD). The protease inhibitor cocktail set III and phosphatase inhibitor cocktail set II were purchased from Calbiochem (San Diego, CA). Enhanced chemiluminescence reagent and bicinchoninic acid protein assay reagent were obtained from Pierce Chemical Co. (Rockford, IL). TRI Reagent was purchased from the Molecular Research Center Inc. (Cincinnati, OH).
For western blot, antibodies were used as followed: goat ATF4 and rabbit ATF6 (Santa Cruz Biotechnology, Santa Clara, CA) at a 1∶500 dilution; rabbit pPERK and PERK (Santa Cruz Biotechnology, Santa Clara, CA) at a 1∶500 dilution, rabbit p-eIF2α and mouse eIF2α Ab (Santa Cruz Biotechnology, Santa Clara, CA) at 1∶500 dilution; rabbit cJun and rabbit NF-κBp65 Ab (Santa Cruz Biotechnology, Santa Clara, CA) at a 1∶500 dilution; monoclonal β-actin Ab (Sigma-Aldrich) at 1∶5000 dilution and mouse Lamin A/C at 1∶1000 dilution.
Cell Culture
Human dermal microvascular endothelial cells (HDMECs) were isolated from human foreskins using the protocol of Richard et al [18]. Upon informed consent and in compliance with the Institutional Review Board of Human studies, written approval was obtained from Perinatal Committee (IRB number H-29190) of Boston University Medical School. Briefly, primary cultures of human foreskins were established after the removal of epidermis. Such cultures consist of a mixture of HDMECs, dermal fibroblasts, and some keratinocytes. Subconfluent cultures were treated with tumor necrosis factor-α for 6 h to selectively induce the expression of E-selectin in HDMECs. HDMECs were then purified using magnetic beads coupled to an anti-E-selectin monoclonal antibody. First passage cultures usually consist of >99% HDMECs. A second immunomagnetic purification step ensures homogenous population of HDMECs suitable for long term culturing. Purity of the HDMEC cultures was evaluated using anti-CD31 and anti-von Willebrand factor antibodies. These cells were cultured on collagen-coated 6-well plate in EBM medium supplemented with 10% FBS, EC growth supplement mix at 37°C under 5% CO2 in air. The culture medium was changed every other day. HDMECs harvested between passage 2 and 6 were used for experiments.
Adenoviral Constructs
An adenoviral vector expressing HLA-B35 (or Ad-B8) and control green fluorescent protein (Ad-Go) were generated as described earlier [18]. The dose used to transduce human dermal microvascular endothelial cells was 10 multiplicities of infection of the adenovirus (MOI). ECs grown in a 6-well dish were transduced with Ad (Ad-B35/GFP, -B8/GFP, and -GFP), after 48 h cells were collected for RNA analyses or for Western blot.
Real-time PCR
Total RNA was extracted using the guanidiniumthiocyanate-phenol-chloroform method, concentration and purity was determined by measuring OD at 260 and 280 nm using a spectrophotometer. RNA was reversibly transcribed by aid of the first-strand cDNA Synthesis Kit for RT-PCR (Roche Applied Science, Indianapolis, IN). To avoid amplification from traces of possible DNA contamination in the RNA isolation, PCR primers were designed to span introns. All primers were checked for specificity by Blast search. Real-time RT-PCR was performed using IQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad). The amount of template used in the PCR reactions was cDNA corresponding to 200 ng reverse-transcribed total RNA. DNA polymerase was first activated at 95°C for 3 min, denatured at 95°C for 30 s, and annealed/extended at 61°C for 30 s, for 40 cycles according to the manufacturer's protocol. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. The primers are listed in
Table 1
.
10.1371/journal.pone.0056123.t001Table 1 Primer sequences for quantitative PCR.
Forward Reverse
Human-PPET-1
5′-gctcgtccctgatggataaa-3′ 5′-ccatacggaacaacgtgct-3′
Human-ATF4
5′-tggctggctgtggatgg-3′ 5′-tcccggagaaggcatcct-3′
Human-ATF6
5′-ttttagcccgggactctttc-3′ 5′-tcagcaaagagagcagaatcc-3′
Human-XBP1u
5′-ccttgtagttgagaaccagg-3′ 5′-gggcttggtatatatgtgg-3′
Human-XBP1s
5′-ggtctgctgagtccgcagcagg-3′ 5′-gggcttggtatatatgtgg-3′
Human-PKR
5′-tgttgggatggatttgattatg-3′ 5′-gaaaaggcacttagtctttgacct-3′
Human-βactin
5′-aatgtcgcggaggacctttgattgc-3′ 5′-aggatggcaagggacttcctgtaa-3′
Mouse-ATF4
5′-gagcttcctgaacagcgaagtg-3′ 5′-tggccacctccagatagtcatc-3′
Mouse-βactin
5′-ctaaggccaaccgtgaaaag-3′ 5′-accagaggcatacagggaca-3′
Immunofluorescence Staining on Adherent Cell Cultures
Cultured HDMECs grown on collagen-coated cover slips were transduced with Adenovirus carrying HLA-B35 (Ad-B35/GFP), Ad-B8/GFP and Ad-G0/GFP (virus control) for 48 hours. Treated cells were fixed with 4% paraformaldehyde for 15 minutes followed by incubation with 0.15 M Glycine for 30 min. Non-specific protein binding was blocked with 3% BSA for 1 h. Next, cells were incubated at 4°C overnight with primary mouse monoclonal MHC class I Ab (W6/32) (Santa Cruz Biotechnology, Santa Clara, CA) at a 1∶500 dilution. After washing, cell cultures were incubated with Alexa fluor 594 donkey anti-mouse (Invitrogen, Grand Island, NY) antibody for 1.5 h. Cells were mounted on slides using Vectashield with DAPI (Vector Laboratories, Burlingame, CA) and examined using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA) at 488 nm (green), 594 nm (red) and 405 nm (blue).
Western Blot Analysis
Cells were collected and washed with PBS. Cell pellets were suspended in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 15 mM NaCl,1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, and 1 mM glycerophosphate with freshly added phosphatase inhibitors (5 mM sodium fluoride and 1 mM Na3VO4) and a protease inhibitor mixture (Sigma-Aldrich). Protein concentration was quantified using the BCA Protein Assay kit (Pierce). Equal amounts of total proteins per sample were separated via SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in milk in TBST overnight at 4°C and probed with primary Ab overnight at 4°C. After TBST washes, membranes were probed with HRP-conjugated secondary Ab against the appropriate species for 1–2 h at room temperature. Protein levels were visualized using ECL reagents (Amersham Biosciences, Piscataway, NJ).
ET-1 Bio-assay
The ET-1 bioassay was performed according to the protocol supplied with the kit from Assay Designs (cat no. 900-020A). Standards and samples were incubated in supplied pre-coated 96-well plate, washed, incubated with horse radish peroxidase labeled anti- ET-1 antibody and washed again before adding the provided TMB substrate and measuring the absorbance.
siRNA Experiments
HDMECs were trasfected with either siRNA specific to human ATF4 (ON-TARGET plus SMARTpool, Dharmacon RNA Technologies, CO), PKR (Santa Cruz biotechnology, CA) or negative-control siRNA (Qiagen, Chatsworth, CA) at concentration of 20 nM using HiPerfect reagent (Qiagen) according to the manufacturer's protocol. After 48 hours, RNA was extracted and Real-time PCR was performed.
Inhibitor Experiment
HDMECs were incubated in the presence of the 25 nM of JNK SP600125 or NF-κB SN50 inhibitor (Enzo Life Sciences, Farmingdale, NY) for 3 hours before treatment. After 48 hours, RNA and protein were extracted.
Co-Immunoprecipitation
Cell lysates were prepared after appropriate treatment in radioimmune precipitation buffer. For immunoprecipitation of cJUN (or NF-κB p65), antibody was added to 300 µg of precleared cell lysate, and complex formation was carried out at 4°C overnight. The protein-antibody complexes were recovered using protein G-Sepharose beads for 2 h at 4°C. The immunoprecipitates were washed four times in radioimmune precipitation buffer, eluted by boiling for 5 min in 2× SDS sample buffer, and analyzed by Western blot with anti-ATF4 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Plasmids, Transient Transfections and Luciferase Assay
Luciferase reporters driven by −650-bp and −193-bp fragments (representing the wildtype and mutated AP-1 site) of the human ET-1 promoter described previously 1. Transient transfections of promoters were performed in HDMECs seeded into 6-well plates using Fugene6 (Roche Applied Science) according to the manufacturer's instructions. After overnight incubation, cells were treated and then further incubated for 24 h. The cells were harvested and assayed for luciferase reporter activity using the Promega luciferase assay kit according to the manufacturer's instructions. Promoter/reporter plasmids were cotransfected with pCMV-βGal (Clontech), which was used to adjust for differences in transfection efficiencies between samples. Cells were harvested and Luciferase activity of the promoter was assayed using Promega Luciferase assay kit. Values given are means ± standard errors of triplicate assays from three individual experiments.
In vivo Administration of Poly(I:C)
C57Bl/6 WT and C57Bl/6 TICAM/TRIF −/− mice were obtained from The Jackson Laboratory; C57Bl/6 IFNAR1−/− mice were provided by Dr John Sprent. All of the experiments were performed under the guidelines of the Boston University Institutional Animal Care and Use Committee. Osmotic pumps designed to deliver Poly(I:C) (0.5 mg/ml in PBS, 0.1 mg total dose in 200 µl released over 7 days, Alzet) or PBS were implanted subcutaneously on the back in 6–10-weekold mice. After 7 days mice were killed and skin (∼1 cm2) surrounding the pump outlet was homogenised in Trizol for preparation of RNA, then minced and disrupted using a Polytron homogeniser and processed according to the manufacturer's protocol.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Boston University (Permit Number: AN-14942.2012.01). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.”.
Immunostaining
Skin sections biopsies from healthy individuals and limited cutaneous systemic sclerosis (lcSSc) patients including 8 patients with pulmonary arterial hypertension (lcSSc-PAH) based on echoradiography and right heart catheterization and 11 patients without PAH (lcSSc-noPAH) and 5 healthy controls were provided by the Boston University Core Centers (http://www.bu.edu/sscores/); IRB: H31–506. Patient information is included in Table 2. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Briefly, 8-micrometer-thick sections were mounted on APES (amino-propyl-triethoxy-silane)-coated slides, deparaffinized with Histo-Clear (National diagnostic, Atlanta GA), and rehydrated through a graded series of ethanol. Endogenous peroxidase was blocked by incubation in 0.3% hydrogen peroxide for 15 minutes and incubated with blocking buffer for 1 hour. The sections were then incubated overnight at 4°C with antibody against ATF4 (Abcam, Cambridge, MA) diluted 1∶500 in blocking serum, followed by the incubation with secondary antibody. The concentration of primary antibody was first tested to determine the optimal sensitivity range. The immunoreactivity was visualized with diaminobenzidine (Vector laboratories, Burlingame, CA) and the sections were counterstained with hematoxylin.
10.1371/journal.pone.0056123.t002Table 2 Clinical and hemodynamic data of study subjects.
Sample Age Gender DiseaseDuration PAP PCWP PASP Treatment ATF4 staining
Normal 11-6 30 M ++
Normal 11-7 24 M +
Normal 11-8 47 F ++
Normal 12-1 25 M −
Normal 12-2 25 F +
lcSSc PAH 12-8 65 F 16 years 1 month 49 30 44 no medications −
lcSSc PAH 12-7 58 M 16 years 30 12 65 no medications ++
lcSSc PAH 12-6 69 F 16 years 54 5 71 no medications ++
lcSSc PAH 12-3 75 F 25 years 48 9–10 62 sildenafil +
lcSSc PAH 12–17 30 F 1 years 8 months 25 2 N/A cell cept revatio +
lcSSc PAH 12–20 60 F 3 years 25 2 N/A prednisone +++
lcSSc PAH 12–18 59 F 15 years N/A N/A N/A none +
lcSSc PAH 12–19 52 F 25 years N/A N/A N/A viagra ++
lcSSc NoPAH 12–27 59 M 45 none
+
lcSSc NoPAH 12-2 65 F 10 years myocophenolate +
lcSSc NoPAH 12-10 40 F 2 years none
++
lcSSc NoPAH 12-1 58 F 2 years prednisone, mycophenolate −
lcSSc NoPAH 12–35 56 F 8 years 33 none +
lcSSc NoPAH 12–31 61 F 14 years hydroxychloroquine +
lcSSc NoPAH 12–28 65 F 6 months 25 none −
lcSSc NoPAH 12–25 67 M 7 years 25 none +
lcSSc NoPAH 12–10 40 F 2 years cyclophosphamide ++
lcSSc NoPAH 12–13 39 F 1 year cytoxan +
lcSSc NoPAH 12–21 20 F 6 years none ++
PAP = pulmonary artery pressure. PCWP = pulmonary capillary wedge pressure. PASP = pulmonary artery systolic pressure.
− indicates no staining or little staining in <10% of the cells.
+ indicates faint, partial staining in >20% of the cells.
++ indicates light to moderate stain in >50% of the cells.
+++ indicates bright staining in >50% of the cells.
Statistical Analysis
Student's t test analysis was performed to determine statistical significance. Values less than or equal to 0.05 were considered statistically significant. All experiments were repeated at least three times using independently isolated endothelial cell cultures.
Results
ER Stress and Poly(I:C) Activate Common ER Stress/UPR Pathways in HDMECs
In the initial experiment we compared the effects of HLA-B35 with a known ER stress inducer, thapsigargin (TG), and a TLR3 agonist, Poly(I:C), on the expression of ET-1 mRNA and protein in primary dermal microvascular endothelial cells (HDMECs). HLA-B35 was expressed using adenoviral delivery as previously described [16]. To control for the presence of adenoviral genes we used adenovirus expressing a closely related antigen, HLA-B8, as well as an empty virus. As shown in Fig. 1a, HLA-B35 and Poly(I:C) upregulated (pre-pro-endothelin-1) PPET-1 mRNA levels with a similar potency 7-fold ±0.58, p = 0.05 vs 8-fold±0.25, p = 0.05, respectively, while TG was a stronger inducer of PPET-1 (26-fold ±0.75, p = 0.001). Ad-HLA-B8, as well as an empty virus vector (data not shown) did not affect PPET1 mRNA expression. To verify that the increase in PPET-1 mRNA corresponds to an increase of the bioactive 21-aa ET-1 peptide, we measured levels of ET-1 protein in supernatants of Ad-B35 (and Ad-B8), TG and TLR ligand-treated ECs. Consistent with the mRNA measurements ER stress inducers increased ET-1 protein levels (Ad-B35, 3.5-fold±0.6, p = 0.05 and TG, 5.3-fold±0.80, p = 0.05). Similarly, Poly(I:C) induced ET-1 protein by 3.7-fold±0.98 (Fig. 1b). PPET-1 mRNA expression was further enhanced by a combination of HLA-B35, and to a lesser degree TG, and Poly(I:C) (Fig. 1c).
10.1371/journal.pone.0056123.g001Figure 1 HLA-B35, TG, and Poly(I:C) upregulate ET-1 mRNA and protein in HDMECs.
Upregulation of PPET-1 mRNA after HLA-B35 (or HLA-B8), TG, or Poly(I:C) treatments alone (a) or in combination (c) in HDMECs. Confluent dishes of HDMECs were transduced with 10 MOI of Adenovirus encoding HLA-B35/GFP (Ad-HLA-B8/GFP) for 48 h or treated with 10pM TG, or 2.5 µg/µl Poly(I:C) for 24 hours. Total RNA was extracted and mRNA levels of PPET-1 were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. (b) Bioactive 21-aa ET-1 peptide in HDMECs after Ad-B35/GFP (Ad-B8/GFP), TG, or Poly(I:C) treatment. ET-1 protein was measured by ELISA in the supernatants. The average protein concentration for each group is represented as a bar ± SE. *p = 0.05; **p = 0.001 (d) Immunofluorescence was performed using mouse monoclonal MHC class I Ab (W6/32) in HDMECs transduced with HLA-B35 (Ad-B35/GFP), Ad-B8/GFP and Ad- G0/GFP (virus control). Left column DAPI, middle column GFP, right column HLA. Bar: 100 µm.
To further determine if HLA-B35 mediates its effects via peptide binding to the antigen-binding groove we utilized mouse monoclonal anti-MHC class I blocking antibody (W6/32). HDMECs transduced with Ad-HLA-B35/GFP and HLA-B8/GFP for 24 hours were treated with increasing dose of W6/32 (0.1–10 µg/ml) for the following 24 hours. Treatment with antibody did not affect ET-1 mRNA levels, suggesting that HLA-B35 does not modulate ET-1 through peptide binding to the antigen-binding groove (supplemental data, Fig. S1). To determine subcellular localization of ectopic HLA-B35 we performed immunofluorescence staining using HDMECs transduced with Ad-HLA-B35/GFP, Ad-HLAB8/GFP and Ad-G0/GFP (virus control) for 48 hours. Spotty positive staining was observed in the cytoplasm primarily around the nucleus, suggesting ER retention of the HLA-B35 and HLA-B8. Together, these data suggest that HLA-B35 is primarily retained in the ER, where it might induce ER stress and UPR. Interestingly, although HLA-B8 displayed a similar cellular distribution, expression of HLA-B8 was not associated with upregulation of BiP and other heat shock proteins [18]. The specific structural determinants of HLA-B35 that may explain its biological effects are currently not known.
To further characterize the nature of the HLA-B35-mediated ER stress, we examined the effect of HLA-B35 (or HLA-B8), TG, and Poly(I:C) on the mRNA expression of the three main UPR mediators, transcription factors ATF4, ATF6 and XBP1. ATF4 mRNA levels were significantly increased in response to the HLA-B35 and Poly(I:C) treatment, while TG was a less potent inducer of ATF4 mRNA under this experimental conditions (Fig. 2a). When Poly(I:C) was used in combination with the ER stress inducers, a further upregulation of ATF4 mRNA was observed (Fig. 2b). Furthermore, both HLA-B35 and TG moderately increased spliced (active) form of the transcription factor XBP1 (XBP1s), while TLR3 had no effect on the XBP1 splicing (Fig. 2c). In contrast, the expression level of ATF6 was not responsive to any of these treatments in HDMECs (Fig. 2d).
10.1371/journal.pone.0056123.g002Figure 2 ER stress and Poly(I:C) activate selected ER stress/UPR pathways.
ATF4 (a, b), XBP1 splicing (c) and ATF6 (d) mRNA levels in HDMECs treated with HLA B35 (HLA-B8), TG, and Poly(I:C) alone or in combination. Confluent dishes of HDMECs were transduced with 10 MOI of Ad-B35/GFP (or Ad- B8/GFP) for 48 h treated with 10pM TG, or 2.5 µg/µl Poly(I:C) for 24 hours. Total RNA was extracted and mRNA levels of transcription factors were examined by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. *p = 0.05; **p = 0.001.
Since nuclear translocation of ATF4 is indicative of its activation status, we examined nuclear extracts for the presence of ATF4 by western blot. Nuclear ATF4 was examined at various time points (15 min. to 6 hours) after TG and Poly(I:C) treatments and 24 hours post infection with HLA-B35 or HLA-B8 adenoviruses. Nuclear ATF4 was rapidly increased (15–30 min) after TG and Poly(I:C) treatments (Fig. 3a and Fig. 3b). Likewise, HLA-B35 markedly increased nuclear presence of ATF4 (Fig. 3c). Furthermore, increased phosphorylation of the upstream activators of ATF4, PERK and eIF2α, was observed in response to these treatments (Fig. 3, right panels). Consistent with the mRNA data, nuclear levels of ATF6 remained unchanged. Together, these results demonstrate that ATF4 is activated in a similar manner by ER stress/UPR and Poly(I:C) in endothelial cells.
10.1371/journal.pone.0056123.g003Figure 3 ER stress and Poly(I:C) activate ATF4 nuclear translocation and enhance phosphorylation of PERK and eIF2α.
ATF4, pPERK/PERK, and p-eIF2α/eIF2α protein levels in HDMECs after treatment with 10pM TG (a), 2.5 µg/µl Poly(I:C) (b), or transduction with 10 MOI of HLA-B35 or HLA-B8 Ads for 48 hours (c). 20 µg of nuclear extract were separated via 15% SDS-PAGE for ATF4 and 10% SDS-PAGE for ATF6. 20 µg of total cellular proteins were separated via 15% SDS-PAGE for pPERK/PERK and 10% SDS-PAGE for peIF2α/eIF2α, then transferred to a nitrocellulose membrane. The blots were probed overnight with primary Abs at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for Lamin A/C or β-actin. Representative blots from at least three independent experiments are shown.
PKR Mediates Activation of the ET-1 Gene in Response to Poly(I:C)
Protein kinase RNA-activated (PKR) is activated by dsRNA and plays an important role in the IFN signaling [19]. Similar to PERK, activated PKR induces phosphorylation of eIF2α and a subsequent ATF4 nuclear translocation. To clarify whether activation of PKR is involved in the ET-1 upregulation by Poly(I:C) treatment, we used siRNA approach to knock down PKR. Initial experiments have established an optimal dose and time for PKR siRNA to achieve maximal inhibition of the PKR mRNA level. Treatment of HDMECs with 20 nM of PKR siRNA for 48 hours resulted in depletion of PKR mRNA levels (around 50%) (Fig. 4a). Following 24 hour incubation with siRNA, cells were treated with Poly(I:C) for additional 24 hours. Under these conditions basal PPET-1 gene expression was downregulated by the siRNA treatment, suggesting that PKR contributes, in part, to the constitutive expression of ET-1 (Fig. 4b). Furthermore, depletion of PKR almost completely abrogated Poly(I:C)-induced stimulation of PPET-1 (Fig. 4b). Interestingly, depletion of PKR upregulated phosphorylated eIF2α (Fig. 4c), as well as nuclear ATF4 level (Fig. 4d) in unstimulated cells, suggesting that without activation, PKR may negatively regulate this pathway. However, in the Poly(I:C)-stimulated cells phosphorylation of eIF2α was abrogated and the ATF4 nuclear level was markedly decreased. These results suggest that PKR mediates ET-1 upregulation in response to Poly(I:C) in HDMECs.
10.1371/journal.pone.0056123.g004Figure 4 Poly(I:C) induces ET-1 gene through the PKR-dependent activation of the eIF2α/ATF4 pathway.
80% confluency, HDMECs were treated with 20 nM PKR siRNA (siSCR) with or without Poly(I:C) treatment (a and b). Total RNA was extracted and mRNA level of PKR (a) and PPET1 (b) were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. 20 µg of nuclear extract were separated via 15% SDS-PAGE for ATF4 (c), 20 µg of total cell lysate were separated via 10% SDS-PAGE for peIF2α/eIF2α (d), then transferred to a nitrocellulose membrane. The blots were probed overnight with primary Abs at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for Lamin A/C or β-actin. Representative blots of at least three experiments are shown. (e) Expression of ATF4 by real-time PCR analysis of skin mRNA from C57B1/6 WT (n = 10), C57B1/6 IFNAR1−/− (n = 8) and C57B1/6 TRIF/TICAM −/− (n = 6) mice 1 week after subcutaneous insertion of osmotic pumps containing Poly(I:C). Fold-change shown in the graphs is normalized to mRNA expression by one of the control mice. *p = 0.05; **p = 0.001 (f) ATF4 protein expression in dermal biopsies obtained from C57Bl/6 WT mice 1 week after subcutaneous insertion of osmotic pumps containing Poly(I:C) or PBS (as control), and processed for immunohistochemistry as described under Methods. Representative images of microvessels from PBS and Poly(I:C) skin is shown. Bar: 20 µm, 10 µm.
We next investigated whether ATF4 is involved in regulation of ET-1 in the Poly(I:C)-treated mice. It has been previously reported that mice receiving continuous Poly(I:C) injection through the osmotic pump for 7 days showed highly increased expression of ET-1 [17]. In addition, this response was abrogated in the TIR-domain-containing adapter-inducing interferon β/Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule-1 (TRIF/TICAM) deficient mice, and only partially inhibited in the interferon-α/β receptor (IFNAR)-1 deficient mice, indicating that ET-1 induction following Poly(I:C) stimulation is mediated by TLR3. As shown in Fig. 4e, expression of ATF4 closely correlated with the previously shown ET-1 expression in the Poly(I:C) treated mice [16]. Increased expression of ATF4 mRNA was also observed in the Poly(I:C) treated WT mice, but this response was significantly decreased in the TRIF/TICAM−/− mice [WT Poly(I:C) vs TRIF/TICAM−/− Poly(I:C) p = 0.001]. On the other hand, depletion of IFNAR1 had only a modest, non-significant inhibitory effect on the Poly(I:C)-induced ATF4 mRNA level in comparison to WT mice. We next examined the distribution of ATF4 protein in the mouse skin by immunohistochemistry. Analyses of skin showed ATF4 staining in vascular endothelial cells in mouse skin after 1 week of Poly(I:C) infusion, and no staining in PBS infused skin. Unfortunately there were only few visible vessels in the sections analyzed (Fig. 4f).
ATF4 is Required for the Upregulation of ET-1 in Response to HLA-B35/ER Stress and Poly(I:C) in HDMECs
In order to determine whether ATF4 is directly involved in the regulation of ET-1 gene we employed a siRNA approach to knock down ATF4. Initial experiments established an optimal dose and time for the ATF4 siRNA to achieve maximal inhibition of endogenous ATF4 mRNA level. Treatment of HDMECs with 20 nM ATF4 siRNA for 48 hours resulted in depletion of ATF4 mRNA levels (up to 50–60%) (Fig. 5a). Under these conditions basal expression levels of ET-1 mRNA and protein were also consistently decreased by ∼30% (Fig. 5c and d). Following 24 hour incubation with siRNA, cells were treated with Ad-B35/GFP (or Ad-B8), TG and Poly(I:C) for additional 24 hours. Depletion of ATF4 completely abolished upregulation of ET-1 mRNA (Fig. 5b) and protein (Fig. 5c) in response to these treatments, suggesting that ATF4 is required for these responses.
10.1371/journal.pone.0056123.g005Figure 5 ER stress and Poly(I:C) upregulate ET1 via ATF4.
80% confluency, HDMECs were treated with 20 nM ATF4 siRNA (or siSCR) prior to treatment with HLA-B35 (HLA-B8), TG, or Poly(I:C). Total RNA was extracted and mRNA level of ATF4 (a) and PPET-1 (b) were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. (c) ET-1 protein was measured by ELISA in the supernatants (n = 2). The average ET-1 protein concentration for each group is represented as a bar ± SE. *p = 0.05; **p = 0.001. (d) ATF4 protein expression in human skin microvessels. Lesional skin biopsies were obtained from patients with lcSSc (with and without PAH) and healthy controls, and processed for immunohistochemistry as described under Methods. Representative images of microvessels from healthy control and lcSSc patients are shown; similar immunostaining pattern was observed in control and lcSSc skin biopsies. Bar: 50 µm, 10 µm.
We next examined the distribution of ATF4 protein in the human skin by immunohistochemistry. Analyses of skin microvessels showed heteregoneus distribution of ATF4, with some vessels exhibiting strong endothelial cell nuclear staining, while other vessels were negative for ATF4 (Fig. 5d). The number of positive vessels also varied between different individuals. Because patients with SSc have elevated circulating levels of ET-1 [20] we also analyzed skin samples obtained from 19 patients with limited cutaneous SSc (lcSSc), including 8 patients with PAH (lcSSc-PAH), and 11 lcSSc-noPAH. Similar to healthy control skin, endothelial cell expression of ATF4 varied between the patients, however there was no overall difference in the intensity or staining pattern between lcSSc and healthy individual biopsies. We did not have information regarding the level of circulating ET-1 or the presence of HLA-B35 antigen in this group of patients. Together, these data indicate that ATF4 is highly expressed in a subset of dermal microvessels, where it is likely involved in responses to various environmental stimuli.
JNK and NF-κB Contribute to the ER Stress and Poly(I:C) Induction of ET-1
JNK and NF-κB pathways have been previously reported to contribute to the ET-1 gene expression in response to various stimuli [4], [5]. To determine if JNK and NF-κB contribute to the upregulation of ET-1 in response to ER stress and TLR3 agonists, cells were treated with HLA-B35, TG, and Poly(I:C) in the presence or absence of the pharmacological inhibitors of these pathways. Treatment with JNK inhibitor (SP6001, 25 nM) resulted in down regulation of the basal and agonist-induced PPET-1 mRNA levels (Fig. 6a, top panel). On the other hand, basal expression of PPET-1 mRNA was not affected by the NF-κB inhibitor (SN50, 25 nM), however stimulation of PPET1 by HLA-B35, TG, and Poly(I:C) was completely inhibited (Fig. 6b, top panel http://www.nature.com/jid/journal/v128/n8/full/jid200839a.html - fig. 1). Similar results were observed at the protein levels (Fig. 6a and b, bottom panels). Interestingly, while stimulation of ET-1 by HLA-B35 or TG was similarly affected by the inhibitors of the JNK and NF-κB pathways, Poly(I:C) stimulation of ET-1 was particularly sensitive to depletion of the NF-κB pathway, suggesting that NF-κB plays a predominant role in activation of the ET-1 gene by Poly(I:C) in HDMECs.
10.1371/journal.pone.0056123.g006Figure 6 JNK and NF-κB contribute to the ER stress and Poly(I:C) induction of ET-1.
Cells were treated with 25 nM of JNK (a) or NF-κB (b) inhibitors for 3 hours before HLA-B35 (HLA-B8), TG, or Poly(I:C) treatments. Total RNA was extracted and mRNA levels of PPET-1 were quantified by quantitative RT-PCR (top panel). ET-1 protein was measured by ELISA in the supernatants (bottom panel). The average protein concentration for each group (n = 2) is represented as a bar ± SE *p = 0.05; **p = 0.001.
ATF4/c-JUN Complexes Mediate ET-1 Induction by ER Stress and Poly(I:C)
ET-1 gene promoter contains binding sites for a number of transcription factors, however using bioinformatics tools we were unable to locate consensus ATF4 binding site within the promoter region. Based on the previous report identifying ATF4 as a partner of c-JUN in a two-hybrid screen [21], we asked whether ATF4 could form protein complexes with c-JUN in HDMECs. As shown in Fig. 7a, ATF4/c-JUN complexes were present in unstimulated cells and were increased upon stimulation with HLA-B35, TG, and Poly(I:C). While, we could also detect formation of the ATF/NF-κB complexes in unstimulated cells, formation of these complexes was not affected by the agonists (Fig. 7b), suggesting that formation of the ATF4/c-JUN complexes is not simply driven by the elevated levels of ATF4 in stimulated cells.
10.1371/journal.pone.0056123.g007Figure 7 Transcriptional upregulation of ET-1 by ER stress and Poly(I:C) is mediated through the ATF4/cJUN complex.
Cell lysates from the HLA-B35 (HLA-B8), TG, or Poly(I:C) treated HDMECs were immunoprecipitated with cJUN (a) or NF-κB p65 antibodies (b) and then analyzed for ATF4 by western blot. Cells were transfected with the luciferase reporter driven by the −650/+172–bp fragment of the human ET-1 promoter (c) or with the −193-bp ppET-1-prom-luc construct (wild type) or constructs with specific mutations in the AP-1 binding site (d). 24 hours post transfection with the indicated plasmids, cells were stimulated with HLA-B35 (HLA-B8) Ads, TG and Poly(I:C) for an additional 24 h. Transfections were normalized using pSVgalactosidase control vector. Basal and induced luciferase activity was measured by luminometry. The graph represents fold change in promoter activity in response to various treatments in comparison with control promoter, which was arbitrarily set at 1. (e) Schematic diagram showing PKR and PERK induced activation of the eIF2α-ATF4 axis followed by the protein complex formation with c-JUN and induction of the ET-1 gene transcription through the AP1 response element.
We next utilized human ET-1 promoter constructs consisting of the −650/+172–bp fragment fused to the luciferase reporter gene to confirm functional role of ATF4 in regulation of the ET-1 gene. Transcriptional activation of the ET-1 promoter was observed after treatment with HLA-B35, TG, and Poly(I:C) (Fig. 7c). To analyze whether AP1 binding site was required for the regulation of ET-1 transcription, cells were transfected with the 193-bp ET-1-prom-luc construct (wild type) or the same construct carrying mutated AP-1 binding site. As shown in Fig. 7d, mutation in the AP1 binding site reduced the ER stress and Poly(I:C) induction of the ET-1 promoter. These results support the functional role of the AP1 complex in the ER stress and Poly(I:C)-mediated induction of the ET-1 gene expression.
Discussion
In this study we show for the first time that ATF4 is a novel regulator of the ET-1 gene in endothelial cells. ATF4 contributes to the basal expression of ET-1 and is required for the induction of ET-1 in response to both ER stress and dsRNA. Our results strongly suggest that activation of the eIF2α/ATF4 pathway leads to increased formation of the ATF4 protein complexes with c-JUN, which, in turn, activate ET-1 transcription through the AP1 response element. ER stress inducers, including HLA-B35 and TG, as well as dsRNA, also upregulate mRNA and protein expression of ATF4, thus further amplifying this signaling pathway (see diagram, Fig. 7e). Additional experiments show that NF-κB, which is also activated by the ER stress and dsRNA in HDMECs, contributes to the activation of ET-1 gene expression. Interestingly, although, ATF4 forms protein complexes with NF-κB in HDMECs, formation of these complexes was not increased by the stimuli used in our study. Since NF-κB plays a key role in activation of the ET-1 gene by cytokines, it is possible that the ATF4/NF-κB complexes are involved in those responses. Together, this study identifies ATF4 as a key mediator of ET-1 gene activation in response to cellular stress.
ATF4 is a short-live, basic region-leucine zipper (bZip) protein that belongs to a family of the ATF/CREB transcription factors [22]. Under normal physiological conditions translation of the ATF4 protein is inefficient due to the presence of a short open reading frame in its 5′ untranslated region; however ATF4 protein translation is facilitated by various stress conditions that trigger global inhibition of protein synthesis [22]. Such conditions, including ER stress, viral infection, nutrient starvation, and low levels of heme induce activation of distinct protein kinases that in turn lead to phosphorylation of a common downstream mediator, eIF2αβresulting in translational repression. The known kinases that phosphorylate eIF2α include ER stress induced PERK, dsRNA induced PKR, as well as GCN2 (general control non repressed 2) and heme regulated inhibitor, HRI [22]. Here we show that stimulation of ET-1 in response to Poly(I:C) is mediated by PKR. In a related study Gargalovic et al have reported activation of the eIF2α-ATF4 in human atherosclerotic lesions and in cultured aortic endothelial cells exposed to oxidized phospholipids [10]. The authors demonstrated that ATF4 contributed to the upregulation of several inflammatory cytokines in cultured aortic endothelial cells. ATF4 was also upregulated by herpesvirus 8 infection and contributed to proangiogenic response via MCP1 upregulation [23]. Furthermore, rapid induction of ATF4 has been observed in smooth muscle cells (SMCs) in the medial compartment of balloon injured rat carotid arteries [24]. Additional studies with cultured SMCs have demonstrated that Fibroblast growth factors (FGF)-2 and mechanical injury stimulate ATF4 levels, and that ATF4 is required for the FGF-2 mediated upregulation of Vascular endothelial growth factor (VEGF)-A [24]. Our study indicates that ET-1 is among the target genes positively regulated by the eIF2α-ATF4 axis in response to ER stress in endothelial cells. Collectively, these studies support a key role for the eIF2α-ATF4 pathway in response to vascular injury [25].
TLR3 is recognized by viral double-stranded RNA (dsRNA) or its synthetic analog, Poly(I:C), and is expressed on the cell membrane or in the intracellular vesicles, depending on the cell type [26], [27]. Ligand binding to the dimerized TLR3, leads to recruitment of an adaptor protein TRIF/TICAM, which functions as a platform for binding of additional signaling molecules and activation of type I interferon and NF-κB pathways [27]. Previous studies have shown that activation of TLR3 signaling is harmful to endothelial cells by promoting inflammatory and atherogenic response in vitro and causing impairment of vessel regeneration in vivo [28]. Elevated levels of TLR3 were found on fibroblasts, immune and endothelial cells in SSc skin biopsies, thus implicating this pathway in the pathogenesis of SSc [29], [30]. This study extends previous work from our group that demonstrated activation of the markers of vascular injury by dsRNA/Poly(I:C) and potential role of the TLR3 signaling to the pathogenesis of SSc [16]. Several studies have shown that activation of the TLR3 signaling pathway leads to impairment of the endothelial cell function including activation of proinflammatory and pro-atherosclerotic mechanisms [31], [32]. ER stress/UPR, as well as the activation of the innate immunity pathways has been implicated in the pathogenesis of several inflammatory diseases [9], [12], however the role of these pathways in PAH and in SSc-related vasculopathy has not yet been explored.
Endothelial cells constitute a first line of defense protecting tissues from injury. Elevated production of ET-1 is a common characteristic associated with endothelial cell dysfunction in various pathological conditions, including pulmonary arterial hypertension [33]. Previous studies have shown that HLA-B35 is associated with an increased risk for developing PAH in patients with scleroderma (SSc) [15]. The current study further supports the potential pathogenic role of HLA-B35 in upregulating ET-1 production and clarifies the molecular mechanism involved in this process in endothelial cells. In addition, this study raises an intriguing possibility that chronic activation of the eIF2α/ATF4 pathway could contribute to the disease pathogenesis. Although, we were able to demonstrate, activation of ATF4 in selected skin biopsies of patients with lcSSc, absence of full clinical data, including levels of circulating ET-1 and presence of HLA-B35 antigen, precluded proper analyses of these samples. This limitation may be addressed in future studies with a larger set of fully characterized samples from patients with lcSSc.
Supporting Information
Figure S1
Treatment with mouse monoclonal anti-MHC class I blocking antibody did not affect ET-1 and ATF4 mRNA levels in HDMECs transfected with HLA-B35. PPET-1 (right panel) and ATF4 (left panel) mRNA after HLA-B35 (or HLA-B8) treatments with increasing dose of mouse monoclonal anti-MHC class I blocking antibody (W6/32) in HDMECs. Confluent dishes of HDMECs were transduced with 10 MOI of Adenovirus encoding HLA-B35/GFP (Ad-HLA-B8/GFP) for 24 h and then with increasing dose of W6/32 (0.1–10 µg/ml) for the following 24 hours. Total RNA was extracted and mRNA levels of PPET-1 and ATF4 were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value.
(TIF)
Click here for additional data file.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23441155PONE-D-12-3261710.1371/journal.pone.0055727Research ArticleBiologyGeneticsHuman GeneticsGenetic Association StudiesPopulation GeneticsGenetic PolymorphismPopulation BiologyPopulation GeneticsGenetic PolymorphismMedicineClinical Research DesignMeta-AnalysesEndocrinologyDiabetic EndocrinologyDiabetes Mellitus Type 2EpidemiologyGenetic EpidemiologyGenetic Polymorphism of Glucokinase on the Risk of Type 2 Diabetes and Impaired Glucose Regulation: Evidence Based on 298, 468 Subjects Type 2 Diabetes GeneticsFu Da
1
Cong Xianling
2
Ma Yushui
1
Cai Haidong
1
Cai Mingxiang
1
Li Dan
1
Lv Mingli
1
Yuan Xueyu
1
Huang Yinghui
2
*
Lv Zhongwei
1
*
1
Department of Nuclear Medicine, Shanghai 10th People’s Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China
2
China Japan Union Hospital, Jilin University, Changchun, People’s Republic of China
Baroni Marco Giorgio Editor
University of Cagliari, Italy
* E-mail: [email protected] (YH); [email protected] (ZL)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: DF YH ZL. Performed the experiments: DF XC YM HC MC DL ZL. Analyzed the data: YM DL ML XY. Contributed reagents/materials/analysis tools: XC YM MC XY YH. Wrote the paper: DF XC YM YH ZL.
2013 18 2 2013 8 2 e5572719 10 2012 29 12 2012 © 2013 Fu et al2013Fu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Glucokinase (GCK) is the key glucose phosphorylation enzyme which has attracted considerable attention as a candidate gene for type 2 diabetes (T2D) based on its enzyme function as the first rate-limiting step in the glycolysis pathway and regulates glucose-stimulated insulin secretion. In the past decade, the relationship between GCK and T2D has been reported in various ethnic groups. To derive a more precise estimation of the relationship and the effect of factors that might modify the risk, we performed this meta-analysis.
Methods
Databases including Pubmed, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI) were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.
Results
A total of 24 articles involving 88, 229 cases and 210, 239 controls were included. An overall random-effects per-allele OR of 1.06 (95% CI: 1.03–1.09; P<10−4) was found for the GCK −30G>A polymorphism. Significant results were also observed using dominant or recessive genetic models. In the subgroup analyses by ethnicity, significant results were found in Caucasians; whereas no significant associations were found among Asians. In addition, we found that the −30G>A polymorphism is a risk factor associated with increased impaired glucose regulation susceptibility. Besides, −30G>A homozygous was found to be significantly associated with increased fasting plasma glucose level with weighted mean difference (WMD) of 0.15 (95%: 0.05–0.24, P = 0.001) compared with G/G genotype.
Conclusions
This meta-analysis demonstrated that the −30G>A polymorphism of GCK is a risk factor associated with increased T2D susceptibility, but these associations vary in different ethnic populations.
This study was supported by National Nature Scientific Foundation of China (81201535, 81071662), Nature Scientific Foundation of Shanghai (12ZR1436000), Scientific Research Foundation of Jilin Province (200905169, 20100945, 2011756), the Knowledge Innovation Program of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (2012KIP203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Type 2 diabetes (T2D) is a complex metabolic disease characterised by hyperglycemia, insulin resistance, impaired insulin secretion due to pancreatic β-cell defects and increased hepatic glucose production. Despite much investigation, the causes underlying the development and progression of T2D have not been fully elucidated, accumulated evidence suggests that multiple genetic and environmental factors, as well as the interplay between these factors, determine the phenotype. Although the genetic contribution to T2D is well recognized, the current set of 56 established susceptibility loci, identified primarily through large-scale genome-wide association studies (GWAS), captures at best 10% of familial aggregation of the disease [1]–[3]. This has maintained interest in other biochemical and genetic factors that might contribute to the underlying pathophysiology of the disease.
Glucokinase (GCK) is the key glucose phosphorylation enzyme responsible for the first rate-limiting step in the glycolysis pathway and regulates glucose-stimulated insulin secretion from pancreatic β-cells and glucose metabolism in the liver [4]. Inactivating GCK mutations lead to maturity-onset diabetes of the young and neonatal diabetes [5]–[7], whereas activating GCK mutations cause persistent hyperinsulinaemic hypoglycaemia [8]–[11]. Moreover, a common variant (−30G>A, rs1799884) in the pancreatic beta cell-specific promoter of GCK has been shown to be associated with increased risk of type 2 diabetes, hyperglycaemia and impaired beta cell function [12]–[16]. Furthermore, GCK −30 A>G has been conclusively associated with fasting glucose in European populations [17].
To date, many case–control studies have been carried out to investigate the role of the GCK −30G>A polymorphism in the development of T2D among various populations. Genetic association studies can be problematic to reproduce due to insufficient power, multiple hypothesis testing, population stratification, source of controls, publication bias, and phenotypic heterogeneity. In addition, with the increased studies in recent years among Asian, and other populations, there is a need to reconcile these data. We therefore conducted a comprehensive meta-analysis to quantify the overall risk of GCK −30G>A polymorphism on developing T2D.
Materials and Methods
Literature Search Strategy
Genetic association studies published before the end of Sep. 2012 on T2D and polymorphisms within GCK gene were identified through a search of PubMed, ISI Web of Science, EMBASE and CNKI (Chinese National Knowledge Infrastructure) without language restrictions. Search term combinations were keywords relating to the glucokinase gene (e.g., “glucokinase”, “GCK”, and “MODY 2”) in combination with words related to T2D (e.g., “type 2 diabetes mellitus”, “T2DM”, “type 2 diabetes”, “T2D”, “non-insulin-dependent diabetes mellitus” and “NIDDM”) and polymorphism or variation. The search was supplemented by reviews of reference lists for all relevant studies and review articles. The major inclusion criteria were (a) original papers containing independent data, (b) case–control or cohort studies and (c) genotype distribution information or odds ratio (OR) with its 95% confidence interval (CI) and P-value. The major reasons for exclusion of studies were (a) overlapping data and (b) case-only studies, family-based studies and review articles.
Data Extraction
Data extraction was performed independently by two reviewers, and differences were resolved by further discussion among all authors. For each included study, the following information was extracted from each report according to a fixed protocol: first author’s surname, publication year, definition and numbers of cases and controls, diagnostic criterion, frequency of genotypes, source of controls, age, gender, body mass index (BMI), Hardy–Weinberg equilibrium status, ethnicity and genotyping method. Not all researchers use the same SNP, we report herein 2 common SNPs (rs1799884 and rs4607517), as these SNPs are in complete disequilibrium (r2 = 1) [18].
Statistical Methods
The strength of association between −30G>A polymorphism of GCK and T2D risk was assessed by odds ratio (OR) with the corresponding 95% confidence interval (CI). We first used the chi square test to check if there was significant deviation from Hardy–Weinberg equilibrium among the control subjects in each study. The meta-analysis examined the association between each polymorphism and the risk of T2D for the: (i) allele contrast, (ii) dominant, and (iii) recessive model. For continuity variable, weighted mean difference (WMD) was used to pool results from studies.
Heterogeneity across individual studies was calculated using the Cochran chi-square Q test followed by subsidiary analysis or by random-effects regression models with restricted maximum likelihood estimation [19]–[21]. Random-effects and fixed-effect summary measures were calculated as inverse variance-weighted average of the log OR. The results of random-effects summary were reported in the text because it takes into account the variation between studies. In addition, we investigated potential sources of identified heterogeneity among studies by stratification according to the number of T2D cases (≥1000 and <1000), ethnic group and diagnostic criteria (WHO, ADA or other criterion). Ethnic group was defined as Asians, Caucasians (i.e. people of European origin) and others (e.g. Tunisian and African–American). The Z test was used to determine the significance of the pooled OR. Gender distribution in cases and controls, genotyping method and mean age of cases and controls were analysed as covariates in meta-regression. The transcript expression level trends by genotypes were evaluated by using general linear model.
We assessed publication bias by using an ancillary procedure attributed to Egger et al. [22], which uses a linear regression approach to measure funnel plot asymmetry on the natural logarithm of the OR. Sensitivity analysis was performed by removing each individual study in turn from the total and re-analysing the remainder. This procedure was used to ensure that no individual study was entirely responsible for the combined results. All statistical analyses were carried out with the Stata software version 10.0 (Stata Corporation, College Station, TX, USA). The type I error rate was set at 0.05. All the P-values were for two-sided analysis.
Results
Characteristics of Studies
Study selection process was shown in Figure S1. In all, we included 36 data points from 24 studies in this meta-analysis, with a total of 88, 229 cases and 210, 239 controls [12]–[14], [16], [18], [23]–[41]. The distribution of genotypes in the controls was consistent with Hardy–Weinberg equilibrium in all studies. Of the cases, 75% were Caucasians 21% were Asians and 4% were of other ethnic origins. The main study characteristics were summarized in Table 1.
10.1371/journal.pone.0055727.t001Table 1 Characteristics of the studies included in the meta-analysis.
Reference Year Ethnicity Case Control No. of case No. of control Genotyping method
Cauchi [26]
2012 Arab T2D per WHO criteria Healthy 2639 1997 TaqMan
Cho [27]
2011 Asian T2D patients Non-diabetic participants 6952 11865 Genechip
Kooner [28]
2011 Asian T2D patients Non-diabetic participants 5561 14458 Genechip
Hu [29]
2010 Chinese T2D per WHO criteria Normal glucose tolerance 3410 3412 MassArray
Murad [30]
2010 British T2D patients Non-diabetic participants 1551 2993 TaqMan
Tam [31]
2010 Chinese T2D per WHO criteria Normal fasting glucose 1320 1595 MassArray
Dupuis [32]
2010 European, American, Austrian T2D per WHO/ADA criteria Non-diabetic participants 40655 87022 SNPstream, Genechip, TaqMan, MassArray
Ezzidi [33]
2009 Tunisian T2D per ADA criteria Normoglycemic participants 865 505 TaqMan
Prokopenko [18]
2009 European T2D per WHO criteria; T2Dpatients Normal glucose tolerant; Non-diabetic participants 11785 49799 Genechip
Reiling [34]
2009 Dutch T2D per WHO criteria Normal glucose tolerance 2498 1912 TaqMan
Qi [35]
2009 Chinese T2D per WHO criteria Normal fasting glucose 416 1877 SNPstream
Ma [36]
2009 Chinese T2D per WHO criteria Non-diabetic participants 279 110 RFLP
Cauchi [37]
2008 French T2D per ADA criteria Normoglycemic participants 2637 4159 TaqMan
Vaxillaire [12]
2008 French T2D per ADA criteria Normoglycemic participants 292 2752 TaqMan
Holmkvist [38]
2008 Swedish T2D per ADA criteria Non-diabetic participants 1988 15019 TaqMan
Winckler [39]
2007 British T2D per WHO criteria Non-diabetic participants 2248 3561 MassArray
Bonnycastle [40]
2006 Finnish T2D per WHO criteria Normal glucose tolerance 784 617 MassArray
Rose [14]
2005 Dane T2D per WHO criteria Normal glucose tolerance 1408 4773 MassArray
März [13]
2004 Austrian T2D per ADA criteria Non-diabetic participants 463 830 RFLP
Rissanen [41]
1998 Finnish T2D patients Normal glucose tolerance 36 294 SSCP
Yamada [16]
1997 Japanese T2D per WHO criteria Normal glucose tolerance 94 321 RFLP
Lotfi [42]
1997 Swedish T2D per WHO criteria Healthy 31 158 SSCP
Shimokawa [43]
1994 Japanese T2D patients Non-diabetic participants 240 111 SSCP
Chiu [44]
1994 American Blacks T2D per NDDG criteria Non-diabetic participants 77 99 SSCP
Association of GCK −30G>A Variant with T2D
For T2D risk and the −30G>A polymorphism of GCK, our meta-analysis gave an overall OR of 1.06 (95% CI: 1.03–1.09; P<10−4; Fig. 1). Significantly increased T2D risks were also found under dominant (OR = 1.08; 95% CI: 1.01–1.19; P = 0.003) and recessive genetic models (OR = 1.12; 95% CI: 1.01–1.25; P = 0.008). This analysis is based on pooling of data from a number of different ethnic populations. When stratifying for ethnicity, an OR of 1.08 (95% CI: 1.04–1.12; P<10−4), 1.01 (95% CI: 0.98–1.05; P = 0.53) and 1.13 (95% CI: 1.04–1.24; P = 0.006) resulted for the A allele, among Caucasian, Asian and other ethnic population, respectively. Similar results were also detected using dominant and recessive genetic models (Table 2). When studies were stratified for sample size, significant risks were found among studies with small sample size in all genetic model (A allele: OR = 1.14, 95% CI: 1.04–1.25; dominant model: OR = 1.17, 95% CI: 1.06–1.28; recessive model: OR = 1.23, 95% CI: 1.05–1.43). Positive results still maintained for large sample size studies in all genetic models. Subsidiary analyses of diagnostic criterion yielded a per-allele OR for WHO criterion of 1.06 (95% CI: 1.01–1.11; P = 0.02), ADA criterion of 1.13 (95% CI: 0.99–1.28; P = 0.06) and for other criterion of 1.05 (95% CI: 0.99–1.11; P = 0.12).
10.1371/journal.pone.0055727.g001Figure 1 Forest plot for the overall association between the GCK−30G>A polymorphism and type 2 diabetes risk.
10.1371/journal.pone.0055727.t002Table 2 Meta-analysis of the GCK −30G>A polymorphism on type 2 diabetes risk.
Sub-group analysis No. of data sets No. of case/control A allele Dominant model Recessive model
OR (95%CI) P(Z) P(Q)a
P(Q)b
OR (95%CI) P(Z) P(Q)a
P(Q)b
OR (95%CI) P(Z) P(Q)a
P(Q)b
Overall 36 88229/210239 1.06 (1.03–1.09) <10−4
0.003 1.08 (1.01–1.19) 0.003 0.009 1.12 (1.01–1.25) 0.008 0.001
Ethnicity 0.01 0.006 0.003
Caucasian 23 66376/173889 1.08 (1.04–1.12) <10−4
0.002 1.13 (1.04–1.21) 0.0007 0.0006 1.17 (1.06–1.29) 0.002 <10−4
Asian 9 18272/33749 1.01 (0.98–1.05) 0.53 0.77 0.96 (0.84–1.10) 0.57 0.82 1.05 (0.93–1.18) 0.76 0.34
Others 4 3581/2601 1.13 (1.04–1.24) 0.006 0.49 1.06 (1.03–1.13) 0.008 0.63 1.12 (1.05–1.24) 0.03 0.52
Sample size 0.001 0.006 <10−4
Small 15 4640/12804 1.14 (1.04–1.25) 0.006 0.09 1.17 (1.06–1.28) 0.009 0.21 1.23 (1.05–1.43) 0.01 0.10
large 21 83589/197435 1.04 (1.01–1.07) 0.004 0.03 1.09 (1.02–1.23) 0.006 0.13 1.11 (1.07–1.35) 0.002 0.18
Diagnostic criterion 0.44 0.53 0.14
WHO criterion 19 72385/169960 1.06 (1.01–1.11) 0.02 0.12 1.07 (1.02–1.19) 0.01 0.31 1.09 (1.05–1.17) 0.003 0.29
ADA criterion 7 6247/23265 1.13 (0.99–1.28) 0.06 <10−4
1.06 (0.97–1.16) 0.13 <10−4
1.18 (0.94–1.47) 0.20 <10−5
Other criterion 10 9597/17014 1.05 (0.99–1.11) 0.12 0.24 1.07 (0.97–1.19) 0.16 0.35 1.02 (0.87–1.19) 0.76 0.09
a Cochran’s chi-square Q statistic test used to assess the heterogeneity in subgroups.
b Cochran’s chi-square Q statistic test used to assess the heterogeneity between subgroups.
Significant heterogeneity was present among the included studies (P<0.05). In meta-regression analysis, mean age of cases (P = 0.31) and controls (P = 0.24) and genotyping method (P = 0.96) did not significantly explain such heterogeneity. By contrast, ethnicity (P = 0.02) and the sample size in cases (P = 0.01) was significantly correlated with the magnitude of the genetic effect, explaining 11% and 16% of the heterogeneity, respectively. Since significant between-study heterogeneity still maintained in Caucasian subgroup, hence studies on Caucasian populations is the main source of heterogeneity.
Association of GCK −30G>A Variant with Impaired Glucose Regulation
To investigate how glucose metabolism was related to glucokinase, we analyzed individuals with impaired glucose regulation (impaired glucose tolerance and/or impaired fasting glucose). The data on genotypes of the −30G>A polymorphism among impaired glucose regulation cases and controls were available in 5 (including 3177 cases and 8970 controls) studies. In the overall analysis, the −30G>A polymorphism of GCK was significantly associated with elevated impaired glucose regulation risk with a per-allele OR of 1.23 [95% CI: 1.14–1.32; P(Z) <10−5; P(Q) = 0.73; Fig. 2]. Significant associations were also found under dominant [OR = 1.24; 95% CI: 1.13–1.35; P(Z) <10−5; P(Q) = 0.56] and recessive [OR = 1.52; 95% CI: 1.20–1.91; P(Z) = 0.004; P(Q) = 0.77] genetic model.
10.1371/journal.pone.0055727.g002Figure 2 Forest plot for the overall association between the GCK−30G>A polymorphism and impaired glucose regulation risk.
Association of GCK −30G>A Variant with Fasting Plasma Glucose Level
The data on fasting plasma glucose (FPG) level among subjects stratified by genotype of −30G>A polymorphism were available in 6 studies, including 31,616 subjects. Significant increases of fasting plasma glucose level were observed in A allele carriers compared with non-carriers. Using the random-effects model, compared with G/G genotype, the WMD for heterozygous were 0.07 [95%: 0.05–0.08, P(Z) <10−5, P(Q) <10−5; Figure 3] and homozygous 0.15 [95%:0.05–0.24, P(Z) = 0.001, P(Q)<10−5], respectively.
10.1371/journal.pone.0055727.g003Figure 3 Meta-analysis of weighted mean differences (WMD) of fasting plasma glucose levels between GG and GA genotype of −30G>A polymorphism.
Sensitivity Analyses and Publication Bias
Sensitivity analysis indicated that no single study influenced the pooled OR qualitatively, suggesting that the results of this meta-analysis are stable (data not shown). The shape of the funnel plots was symmetrical (Figure S2). The statistical results still did not show publication bias in these studies (Begg test, P = 0.21; Egger test, P = 0.60).
Discussion
Large sample and unbiased epidemiological studies of predisposition genes polymorphisms could provide insight into the in vivo relationship between candidate genes and diseases. This is the most comprehensive meta-analysis examining the GCK −30G>A polymorphism and the relationship to T2D risk. Its strength was based on the accumulation of published data giving greater information to detect significant differences. In total, the present meta-analysis combined 24 studies for T2D including 88, 229 cases and 210, 239 controls. Our results demonstrated that a modest association existed between the −30G>A variant of GCK and T2D risk.
In meta-analysis, heterogeneity evaluation was always conducted in statistical analysis. Thus, several subgroup meta-analyses were performed. In the stratified analysis by ethnicity, significant associations were found in Caucasians for the polymorphism in all genetic models; while no associations were detected among Asians. There are several possible reasons for such differences. First, the distribution of the A allele varies extensively between different races, with a prevalence of ∼23% among Asians and ∼17% among Caucasians. Therefore, additional studies are warranted to further validate ethnic difference in the effect of this polymorphism on T2D risk. In addition, different populations usually have different linkage disequilibrium patterns. A polymorphism may be in close linkage with another nearby causal variant in one ethnic population but not in another. GCK −30G>A polymorphism may be in close linkage with different nearby causal variants in different populations. Moreover, clinical heterogeneity like age, sex ratio, dietary, years from onset and disease severity may also explain the discrepancy. Finally, such different results could also be explained by study design or sample size. As significant between-study heterogeneity was found among Caucasian subgroup, so the result must be interpreted with caution since the Caucasian population reports in the subgroup analysis include a mixture of populations from very distant countries.
The present study also provides evidence that the −30G>A polymorphism of GCK influences susceptibility to phenotypes of impaired glucose regulation. In terms of genetic versus environmental influences on T2D susceptibility, this finding supports previous heritability studies, including a Danish twin study, generating considerably higher heritability estimates for the impaired glucose tolerance state compared with manifest T2D [42].
We also observed a significant effect of the −30G>A variant on FPG levels. This confirms a recent observation in a French study [12]. Therefore, this variant may have a non-negligible impact on human health. Indeed, there is strong evidence that even small changes in FPG, well below the impaired fasting glucose threshold of 6.1 mmol/l, may be associated with risk of cardiovascular morbidity and mortality [43], [44]. In this context, the GCK (−30A) allele was previously shown to be associated with type 2 diabetes and increased risk for coronary heart diseases in both diabetic and nondiabetic samples [13]. The exact mechanism by which the GCK −30A allele causes hyperglycemia is uncertain, but its effect seems constant throughout the lifespan, although insulin secretion is known to decrease with age in the general population. This is in accordance with the constant effect of the GCK −30A allele on fasting glucose reported in several groups of normoglycemic subjects whose median age varied from 8 to 72 years [12].
A number of factors predict T2D; however, detailed pathogenesis mechanisms of T2D remain a matter of speculation. GCK is a key regulatory enzyme in the pancreatic β-cell, and it plays a crucial role in determining the threshold for glucose-stimulated insulin secretion. Heterozygous inactivating mutations in GCK cause maturity-onset diabetes of the young subtype 2, in which hyperglycemia is present from birth. The decreased expression of functional GCK seems to be the cause of the observed hyperglycemia among maturity-onset diabetes of the young subtype 2 patients. The mechanism by which the −30G>A polymorphism causes hyperglycemia is uncertain. Previously studies suggested that the minor A allele or a genetic variant with which it is in linkage disequilibrium may alters the expression of GCK
[14].
In interpreting the results, some limitations of this meta-analysis should be addressed. Firstly, in the subgroup analyses, different ethnicities were pooled in the other ethnic group which may bring in some heterogeneity. As studies among the other ethnic group are currently limited, further studies including a wider spectrum of subjects should be carried to investigate the role of these variants in different populations. Secondly, our results were based on unadjusted estimates, while a more precise analysis should be conducted if all individual raw data were available, which would allow for the adjustment by other co-variants including age, drinking status, obesity, cigarette consumption, and other lifestyle. Thirdly, only published studies were included in this meta-analysis. Therefore, publication bias may have occurred, even though the use of a statistical test did not show it.
Despite these limitations, this meta-analysis suggests that GCK −30G>A polymorphism was significantly associated with increased risk of T2D, particularly in Caucasian population. In addition, GCK –30A allele is a true risk factor for the development of impaired glucose regulation, having a significant impact on FPG level.
Supporting Information
Figure S1 Study selection process.
(TIF)
Click here for additional data file.
Figure S2 Begg’s funnel plot of GCK −30G>A polymorphism and T2D risk.
(TIF)
Click here for additional data file.
Checklist S1 (DOC)
Click here for additional data file.
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Oncol LettOncol LettOLOncology Letters1792-10741792-1082D.A. Spandidos 10.3892/ol.2013.1123ol-05-03-0829ArticlesmicroRNA-125b inhibits cell migration and invasion by targeting matrix metallopeptidase 13 in bladder cancer WU DEYAO 1DING JINGJING 2WANG LINMAO 3PAN HUIXING 1ZHOU ZHENGDONG 1ZHOU JIAN 1QU PING 11 Department of Urology, The Fourth Affiliated Hospital of Nantong Medical College, Yancheng City No. 1 People’s Hospital, Yancheng 224001;2 School of Basic Medical Science, Nanjing Medical University, Manjing 210029;3 Department of Thoracic Surgery, The Fourth Affiliated Hospital of Nantong Medical College, Yancheng City No. 1 People’s Hospital, Yancheng 224001,
P.R. ChinaCorrespondence to: Professor Ping Qu, Department of Urology, The Fourth Affiliated Hospital of Nantong Medical College, Yancheng City No. 1 People’s Hospital, 15 Yuehe Road, Yancheng 224001, P.R. China, E-mail: [email protected] 2013 10 1 2013 10 1 2013 5 3 829 834 12 10 2012 04 1 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.The expression of microRNA-125b (miR-125b) has been investigated in many human cancers. It has been demonstrated to be downregulated in certain types of cancer, such as bladder cancer, thyroid anaplastic carcinomas, squamous cell carcinoma of the tongue, hepatocellular carcinoma, ovarian and breast cancer. In the present study, we examined the effects of miR-125b on bladder cancer cell migration and invasion. Following transfection of miR-125b, the expression of miR-125b was analyzed in T24 and EJ bladder cancer cells. Additionally, cell migration, cell invasion and luciferase assays, as well as western blot analysis were conducted in the bladder cancer cells. In this study, we demonstrated that miR-125b inhibited cell migration and invasion in T24 and EJ cells. We also provided the first evidence that miR-125b may directly target matrix metalloproteinase 13 (MMP13) in bladder cancer. Our study provided evidence that miR-125b suppresses cell migration and invasion by targeting MMP13 in bladder cancer cell lines. These results suggested that miR-125b could be used for the development of new molecular markers and therapeutic approaches to inhibit bladder cancer metastasis.
bladder cancerMMP13miR-125b
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Introduction
Bladder cancer is one of the most common malignancies worldwide. In the western world it is the seventh most common malignancy among males, following lung, prostate, colon, stomach, liver and esophageal cancer. In addition, bladder cancer represents the second most common cause of mortality among individuals with genitourinary tumors. It was estimated that there would be 70,530 new cases and 14,680 deaths due to bladder cancer in 2010 (1–3). Bladder cancer is comprised of tumors that exhibit a wide spectrum of clinical behavior. Approximately 90% of patients with bladder cancer have transitional cell carcinoma (TCC), whereas 5% exhibit squamous cell carcinomas and 1–2% have adenocarcinomas (3,4). Currently, there are many therapeutic modalities available for use depending on the extent of the disease. The treatment methods include surgery, intravesical chemotherapy, radiation therapy and systemic chemotherapy (5). The majority (50–80%) of patients with superficial TCC who solely undergo transurethral resection of bladder (TURB) suffer from recurrence. In 16–25% of these cases, superficial tumors recur with a higher grade, mostly within the first year following TURB (6). Therefore, for an improved prognosis, new therapeutic targets and approaches should be sought in order to suppress cancer recurrence.
microRNA (miRNA) belongs to a class of endogenously expressed, non-coding small RNA and contains ∼22 nucleotides (7). miRNAs are transcribed as hairpin pri-miRNAs and processed into pre-miRNAs by Drosha, an RNAse III endonuclease complexed with DGCR8. Pre-miRNAs are exported into the cystoplasm by Exportin 5 prior to cleavage by Dicer into mature miRNAs (8). Mature miRNAs are important in cell growth, proliferation, differentiation and cell death (9–11). It has also been proposed that miRNAs regulate the expression of ∼1/3 of human genes (12,13). miRNAs regulate gene expression at the post-transcriptional level through imperfect base pairing with the 3′-untranslated regions (3′-UTR) of target mRNAs (7). A growing body of evidence indicates that miRNAs are aberrantly expressed in numerous human cancers, and they may function as oncogenes and tumor suppressors. Upregulated miRNAs in cancer may function as oncogenes by negatively regulating tumor suppressors. By contrast, downregulated miRNAs may normally function as tumor suppressor genes and inhibit cancer by regulating oncogenes (14). It has been suggested that miRNA may be a target for cancer therapy.
The expression of miRNA-125b (miR-125b) has been investigated in numerous human cancers. It has been demonstrated to be downregulated in certain types of cancer, such as bladder cancer, thyroid anaplastic carcinomas, squamous cell carcinoma of the tongue, hepatocellular carcinoma, ovarian and breast cancer, functioning as a tumor suppressor (15,16). However, miR-125b was found to be upregulated in pancreatic cancer, oligodendroglial tumors, prostate cancer, myelodysplastic syndromes and acute myeloid leukemia (17,18). In this study, we demonstrated that miR-125b was capable of inhibiting bladder cancer cell migration and invasion by targeting matrix metalloproteinase 13 (MMP13). These results enhance our understanding of the mechanisms of metastases, thus aiding the identification of new targets that may be used for the development of novel molecular markers and therapeutic approaches to inhibit bladder cancer metastasis.
Material and methods
Cells and culture conditions
The human bladder cancer cell lines T24 and EJ were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/l streptomycin, at 37°C in a humidified atmosphere containing 5% CO2. Cells were subcultured every 2 days using trypsin/ethylenedinitrilotetraacetic acid (EDTA) solution (saline containing 0.05% trypsin, 0.01 M sodium phosphate and 0.53 μM EDTA; pH 7.4). The study was approved by the faculty/institutional research committee of Yancheng City No. 1 People’s Hospital, Yancheng, China.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) for miR-125b after transfection with miR-125b mimics
Total RNA was extracted from cells using TRIzol reagent (Invitrogen Life Technologies; Carlsbad, CA, USA). Real-time qRT-PCR for miR-125b was performed with SYBR-Green miRNA assay (Genepharma; Shanghai, China) according to the manufacturer’s instructions. The primer sequences for miR-125b were as follows: Forward: ACTGATAAATCCCTGAGACCCTAAC and reverse: TATGGTTGTTCTGCTCTCTGTCAC. Briefly, a total of 500 ng RNA was used for the initial reverse transcription reaction with the gene specific stem-loop RT primer available in the kit. Real-time PCR was performed in an AB7300 thermal cycler (Applied Biosystems; Foster City, CA, USA) using the miR-125b primer set and the double-stranded DNA binding dye SYBR-Green. GAPDH was used as the internal control. The primers for GAPDH were as follows: Forward: GAAATCCCATCACCATCTTCCAGG and reverse: GAGCCCCAGCCTTCTCCATG. Every sample was replicated three times with no RT or template control included. Data were analyzed by comparing the Ct values.
Transfection of miR-125b mimics, NC and luciferase reporter plasmid
Mature miR-125b mimics and scrambled control (NC) were designed and synthesized by Genepharma. The sequence of miR-125b mimics and scrambled control are listed in Table I. The insertion fragment was confirmed by DNA sequencing. Cell transfection and co-transfection were performed using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions.
Cell migration and invasion assay
Cell motility was measured using an 8-μm-pore polycarbonate membrane Boyden chamber insert in a Transwell apparatus (Costar; Cambridge, MA, USA). The transfected cells (miR-125b mimics and NC) growing in the log phase were treated with trypsin/EDTA solution, washed once with serum-containing RPMI-1640 medium, centrifuged and re-suspended as single-cell solutions. A total of 1×105 cells in 0.2 ml serum-free RPMI-1640 medium were seeded on a Transwell apparatus. RPMI-1640 containing 20% FBS (600 μl) was added to the lower chamber. An invasion assay was conducted following the same procedure, with the exception that the filters of the Transwell chambers were coated with 30 μg Matrigel (BD Biosciences; San Jose, CA, USA). Following incubation of the cells for 12–24 h at 37°C in a 5% CO2 incubator, cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed in 100% methanol for 2 min, stained in 0.5% crystal violet for 2 min, rinsed in PBS and then subjected to microscopic inspection (×200). Values for invasion and migration were obtained by counting five fields per membrane and represented the average of three independent experiments.
Luciferase assay
Firefly luciferase reporter plasmids containing 3′UTR of the MMP13 gene were obtained from SwitchGear Genomics (Menlo Park, CA, USA). The mutations were generated with the predicted target site of MMP13 3′UTR using the QuickChange XL sitedirected mutagenesis kit (Stratagene, La Jolla, CA, USA). Cells were plated in a 12-well plate at ∼90% confluence and transfected with 0.5 μg reporter plasmid, 50 nmol miR-125b mimics or miR-Ctrl using Lipofectamine 2000. Each sample was also co-transfected with 0.05 μg pRL-CMV plasmid expressing Renilla luciferase as an internal control for transfection efficiency. Twenty-four hours after transfection, cells were harvested with passive lysis buffer, according to the manufacturer’s instructions. Firefly luciferase activity and Renilla luciferase activity were measured with a luminometer (Tecan; Theale, UK). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Each assay was replicated three times.
Western blot analysis
Primary antibodies used in this study, including MMP13 and β-actin, were purchased from Bioworld Technology (Louis Park, MN, USA). Total protein of cells was prepared using radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentration in the resulting lysate was determined using the bicinchoninic acid protein assay. Equal amounts of protein were loaded onto a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride (PVDF) membrane. Following blocking with 5% degreased milk in Tris-buffered saline and Tween-20 (TBST; containing 0.1% Tween-20), the membranes were incubated overnight with the appropriate primary antibody. Subsequently, the membranes were washed and incubated with the corresponding horse-radish peroxidase conjugated secondary antibody at 1:1000 dilution in TBST. The blot was developed with enhanced chemiluminescence (ECL) solution (Pierce; Rockford, IL, USA) and photographed by the FluorChem imaging system (Alpha Innotech; San Leandro, CA, USA). The intensity of each spot was read and analyzed with the AlphaEaseFC software. β-actin was used as the loading control.
Statistical analysis
Data were presented as mean ± standard deviation, and compared using the Student’s t-test in Stata 10.0 (StataCorp.; College Station, Texas, USA). A two-tailed P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of miR-125b following transfection of miR-125b in T24 and EJ cells
We assessed the endogenous levels of miR-125b in T24 and EJ cells, as well as the expression following transfection of miR-125b every 24 h, as demonstrated in Fig. 1. The basal expression of miR-125b was barely detectable, and therefore not shown in Fig. 1. After transfection of miR-125b, the expression level was dramatically increased, then gradually decreased between 24 h and 144 h after transfection.
miR-125b suppresses cell migration and invasion in T24 and EJ cells
To measure the effect of miR-125b on tumor cell migration and invasion, the Transwell apparatus assay was performed (Fig. 2). In the migration assay, we found that miR-125b groups exhibited a 47.61±8.25% decrease in cell migration in T24 cells and a 54.17±6.73% decrease in that of EJ cells, compared with the controls. In the invasion assay, we found that miR-133a groups demonstrated a 48.45±7.22% decrease in cell migration in T24 cells and a 51.17±6.34% decrease in that of EJ cells, compared with the controls. These results indicated that miR-125b reduced the migration and invasion in bladder cancer T24 and EJ cells.
MMP13 is downregulated following the overexpression of miR-125b in T24 and EJ cells
We performed western blot analysis to investigate whether MMP13 expression was decreased following the transfection of miR-125b mimics in bladder cancer cell lines T24 and EJ. As demonstrated in Fig. 3, MMP13 was significantly downregulated in bladder cancer T24 and EJ cell lines following the overexpression of miR-125b (P<0.05). These results indicated that miR-125b reduced the protein level of MMP13 in bladder cancer cells.
MMP13 is a direct target gene of miR-125b in bladder cancer
According to a computational prediction, there is an evolutionarily conserved putative binding site of miR-125b in MMP13 3′UTR. Luciferase reporter assays were performed to evaluate whether MMP13 is a true target of miR-125b. As demonstrated in Fig. 4, overexpression of miR-125b suppressed MMP13 3′UTR-luciferase activity by 41% in T24 cells and by 43% in EJ cells, compared with the controls (P<0.05). Mutation of four nucleotides within the seed-matching sequence of the predicted miR-125b binding site abolished the inhibitory effect of miR-125b on luciferase activity. Therefore, MMP13 may be a direct target of miR-125b in vitro.
Discussion
In humans, miR-125 has two mature isoforms, miR-125a and miR-125b, encoded by three distinct genes: miR-125a, miR-125b-1 and miR-125b-2. miR-125b-1 and miR-125b-2 map to 11q24.1 and 21q21.1, and their precursors are processed to form the same mature miRNA, miR125b (19). miR-125b is a well-characterized miRNA. Although dysregulation of miR-125b has been demonstrated to occur in multiple human cancer types, its role in disease is not completely understood (20), as in certain cell types it is observed to have an oncogenic role, while in others it exhibits a tumor suppressive role. For example, miR-125b expression is upregulated in prostate cancer, and it stimulates androgen-independent growth of prostate cells (21). By contrast, miR-125b is down-regulated in breast cancer, osteosarcoma and bladder cancer, and it suppresses tumor growth in vitro and in vivo(22–24). In a previous study, it was demonstrated that miR-125b was downregulated in the keratinocytes of psoriasis, which is an inflammatory skin disease characterized by non-malignant hyperproliferation of keratinocytes, and the miR-125b inhibited cell proliferation in human primary keratinocytes (25). The seemingly paradoxical findings indicate that the biological function of miR-125b is complex and highly cell-type dependent, which may result from the varied expression contexts of miR-125b target genes in each tumor.
Identification of miR-125b target genes is critical for understanding the role of miR-125b in tumorigenesis, and is important for defining novel therapeutic targets. To date, ERBB2/ERBB3, Bak1, CYP24, NR2A, TNF-a Bmf, Smo and p53 have been identified as targets of miR-125b (26). In bladder cancer, miR-125b was able to inhibit the proliferation and suppress the bladder cancer cells, to form colonies in vitro and to develop tumors in vivo by targeting E2F3 (23). In the present study, we demonstrated that miR-125b transfection resulted in decreased cell migration and invasion in bladder cancer T24 and EJ cells by targeting MMP13. It is concordant with the recent finding in human cutaneous squamous cell carcinoma (27). Our finding suggested that miR-125b may be used for the development of new molecular markers and therapeutic approaches to inhibit bladder cancer metastasis.
MMPs are a family of structurally related zinc-dependent endopeptidases, which, as a group, are capable of degrading essentially all extracellular matrix (ECM) components. There are 24 soluble and membrane-anchored members of the MMP family in humans that demonstrate extensive sequence homology and overlap, but distinct substrate specificities (28).MMPs are found in both normal and pathological tissue in which matrix remodeling is involved, including embryonic development, wound healing, arthritis and angiogenesis, as well as tumor invasion and metastasis (29,30). Therefore, elevated levels of MMPs have been detected in the serum and urine of patients with numerous different types of cancer, including cancer of the bladder, breast, lung, colon, head and neck, as well as melanoma (31). Proteolytic activity of the MMPs is regulated at several levels, most notably via gene transcription, activation via proteolysis of a propeptide, cell compartmentalization and inhibition by the endogenous tissue inhibitors of metalloproteinases (TIMPs) (32). Although they have pro-invasive properties, the functions of MMPs have been demonstrated to be significantly more widespread than simply facilitating migration and invasion. They are also involved in processes such as tumor initiation and progression, activation of chemokines and growth factors, angiogenesis and apoptosis induction. Therefore, it is not surprising that numerous MMPs have been identified in cancer tissue (33).
MMP13 was first identified in breast carcinoma (34). Compared with the other MMPs, MMP13 has wide substrate specificity and a limited expression pattern (35). Physiological expression of MMP13 is observed to be limited to tissues undergoing rapid connective tissue remodeling, such as during fetal bone development, post-natal bone remodeling and gingival wound repair (36). However, MMP13 is expressed in various diseases involving degradation of collagenous ECM and in malignant tumors, such as squamous cell carcinomas of both the head and neck, and the vulva, cutaneous basal-cell carcinomas, chondrosarcomas and melanomas. In bladder cancer, it was demonstrated that MMP13 was expressed in tumor cells, particularly at the invading edges. In a previous study, it was demonstrated that there was no MMP13 expression in normal urothelium (37). This suggested that MMP13 may serve as a marker for transformation and invasion in TCC, otherwise, it may be a target for cancer therapy in order to inhibit metastasis from TCC. Our results suggested that miR-125b suppressed bladder cancer cell migration and invasion through downregulation of MMP13. This could be investigated as a predictive value for early detection of tumor recurrence and target therapy drugs to prevent bladder cancer from becoming invasive.
In summary, to our knowledge, this is the first study to demonstrate that miR-125b regulates MMP13, and contributes to cell migration and invasion in bladder cancer. These findings have therapeutic implications and may be exploited for further treatment of bladder cancer. miRNA-based therapy is expected to be more efficient than the traditional single target therapy, as miRNAs regulate multiple target genes simultaneously. Thus, the likelihood of tumor cells developing resistance by accumulating mutations is smaller. Future studies are required to address whether the potential of miR-125b may be fully realized in cancer treatment. If so, this may be beneficial for the treatment of bladder cancer.
This study was supported by the Program of Key Medical Departments of Jiangsu Province (the Department of General Surgery and the Department of Urology of Jiangsu Province Hospital).
Figure 1 Expression of miR-125b in T24 and EJ cells. The basal expression of miR-125b in T24 and EJ cells was barely detectable. The expression of miR-125b increased for 144 h following transfection of miR-125b. miR-125b, microRNA-125b.
Figure 2 miR-125b inhibition of cell migration and invasion. Representative photos and statistical plots of migration and invasion assays in bladder cancer T24 and EJ cells. Following 12 h incubation, the number of T24 and EJ cells that transversed the Transwell membrane decreased after the transfection of miR-125b, compared with the controls (Student’s t-test, P<0.05). Following 24 h of incubation, the number of T24 and EJ cells that transversed the Transwell membrane pre-coated with Matrigel also decreased after the transfection of miR-125b, compared with the controls (Student’s t-test, P<0.05). miR-125b, microRNA-125b; NC, scrambled control.
Figure 3 MMP13 expression significantly decreased in T24 and EJ cells following transfection of miR-125b, compared with the controls (P<0.05). MMP13, matrix metalloproteinase 13; miR-125b, microRNA-125b; NC, scrambled control.
Figure 4 MMP13 is a direct target of miR-125b. Luciferase activity significantly decreased following co-transfection with miR-125b and reporter plasmid in T24 and EJ cells. Overexpression of miR-125b suppressed MMP13 3′UTR-luciferase activity by 41% in T24 cells and 43% in EJ cells compared with the controls (P<0.05). Three independent experiments were performed and data are presented as mean ± standard deviation. MMP13, matrix metalloproteinase 13; miR-Ctrl, miRNA-control; miR-125, microRNA-125b.
Table I Sequence of the miR-125b mimic and scrambled control.
Sequence (5′-3′)
hsa-miR-125b UCCCUGAGACCCUAACUUGUGA
Scrambled control UUCUCCGAACGUGUCACGUTT
miRNA-125b, microRNA-125b.
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Int J Mol Med
Int J Mol Med
IJMM
International Journal of Molecular Medicine
1107-3756
1791-244X
D.A. Spandidos
22200784
10.3892/ijmm.2011.868
ijmm-29-04-0564
Articles
Inhibition airway remodeling and transforming growth factor-β1/Smad signaling pathway by astragalus extract in asthmatic mice
QU ZHENG-HAI 1
YANG ZHAO-CHUAN 2
CHEN LEI 1
LV ZHI-DONG 3
YI MING-JI 2
RAN NI 2
1 Department of Pediatrics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, P.R. China
2 Department of Child Health Care, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, P.R. China
3 Department of Breast Surgery, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, P.R. China
Correspondence to: Dr Zheng-Hai Qu, Department of Pediatrics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, P.R. China, E-mail: [email protected]
23 12 2011
4 2012
23 12 2011
29 4 564568
27 10 2011
02 12 2011
Copyright © 2012, Spandidos Publications
2012
https://creativecommons.org/licenses/by/3.0/ This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
Airway remodeling is characterized by airway wall thickening, subepithelial fibrosis, increased smooth muscle mass, angiogenesis and increased mucous glands, which can lead to a chronic and obstinate asthma with pulmonary function depression. In the present study, we investigated whether the astragalus extract inhibits airway remodeling in a mouse asthma model and observed the effects of astragalus extract on the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway in ovalbumin-sensitized mice. Mice were sensitized and challenged by ovalbumin to establish a model of asthma. Treatments included the astragalus extract and budesonide. Lung tissues were obtained for hematoxylin and eosin staining and Periodic acid-Schiff staining after the final ovalbumin challenge. Levels of TGF-β1 were assessed by immunohistology and ELISA, levels of TGF-β1 mRNA were measured by RT-PCR, and levels of P-Smad2/3 and T-Smad2/3 were assessed by western blotting. Astragalus extract and budesonide reduced allergen-induced increases in the thickness of bronchial airway and mucous gland hypertrophy, goblet cell hyperplasia and collagen deposition. Levels of lung TGF-β1, TGF-β1 mRNA and P-Smad2/3 were significantly reduced in mice treated with astragalus extract and budesonide. Astragalus extract improved asthma airway remodeling by inhibiting the expression of the TGF-β1/Smad signaling pathway, and may be a potential drug for the treatment of patients with a severe asthma airway.
astragalus plant
airway remodeling
asthma
trans-forming growth factor-β1/Smad signaling pathway
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pmcIntroduction
The morbidity and mortality of asthma have increased sharply worldwide and it has become a severe global public health problem (1). The frequent occurrence of injury and repair initiated by chronic inflammation could lead to structural changes in the airway, collectively termed airway remodeling. Airway remodeling is characterized by airway wall thickening, subepithelial fibrosis, increased smooth muscle mass, angiogenesis and increased mucous glands (2,3). The direct consequence of the airway remodeling is persistent airway hyper-responsiveness and irreversible airway obstruction leading to a chronic and obstinate asthma with pulmonary function depression (4).
Astragalus membranaceus, a is traditional chinese herbal medicine used for the treatment of the common cold, diarrhea, fatigue anorexia and cardiac diseases (5,6). It has also been used as an immunomodulating agent in treating immunodeficiency diseases and to alleviate the adverse effects of chemotherapeutic drugs. Research studies have been performed to investigate the usefulness of astragalus extract in the treatment of asthma, which can efficiently relieve symptoms and reduce the frequency of asthma attacks (7,8). However, little is known about the underlying mechanisms that regulate this activity.
Transforming growth factor-β1 (TGF-β1) is a pro-fibrotic cytokine thought to play an important role in promoting the structural changes of airway remodeling in asthma (9–11). Recently, the TGF-β1/Smad signaling pathway was found to be one of the important mechanisms involved in the development of airway remodeling in asthma (7,12,13). As important members of the TGF-β signal transduction system, Smad proteins directly transport signals from the cell membrane to the nucleus, and mediate intracellular TGF-β signal transduction regulating cell proliferation, transformation, synthesis, secretion and apoptosis. After being phosphorylated by the activated TGF-β1 receptor type I, Smad2 and Smad3 form a heterocomplex with co-Smad (Smad4), and transfer into the cellular nucleus activating DNA transcription to regulate the target gene expression. On the other hand, Smad6 and Smad7 can block the transcription induced by TGF-β through inhibiting its signaling pathway (14,15). Therefore, studies on signaling mechanisms, cytokines and their receptors in asthma could shed light on the mechanisms involved in airway remodeling and the treatment of asthma.
In this study, we report that astragalus extract inhibits airway remodeling in a mouse asthma model and regulates the TGF-β1/Smad signaling pathway in ovalbumin-sensitized mice, providing a novel mechanism for the astragalus extract inhibitory effect on airway remodeling in animal models of asthma.
Materials and methods
Reagents
Astragalus extract (formononetin and calycosin) were obtained from Haerbin Shengtai Botanical Development Co., Ltd. China; their chemical structures are shown in Fig. 1. Chicken egg ovalbumin (OVA) was purchased from Sigma (USA); TRIzol was purchased from Gibco-BRL (USA); the PCR kit was obtained from Promega (USA); the TGF-β1 ELISA-kit was purchased from R&D Systems (USA); Total Smad-2/3, phosphorylated-Smad2/3 antibodies, as well as secondary antibodies (P-Smad2 and P-Smad3) were purchased from Santa Cruz Biotechnology, Inc. (USA). Other laboratory reagents were obtained from Sigma.
Sensitization and antigen challenge
Forty-eight healthy female BABL/c mice, weighing 18–24 g were randomly divided into 4 groups, with 12 mice in each group: normal control group (A), asthma group (B), astragalus extract group (C), budesonide group (D). The asthmatic model were established by OVA. The mice were sensitized on Days 0, 7 and 14 by intraperitoneal injection of 20 μg OVA emulsified in 1 mg of aluminum hydroxide in a total volume of 0.2 ml in groups B, C and D. Seven days after the last sensitization, the mice were exposed to 1% OVA aerosol for up to 30 min every other day for 8 weeks. The 1% OVA aerosol was generated by a compressed air atomizer driven by filling a Perspex cylinder chamber (diameter 50 cm, height 50 cm) with a nebulized solution. Saline was used in group A instead of OVA. At the same time, mice in group C were treated with 0.4 ml (0.2 g/ml) astragalus extract by gavage before stimulation. Mice in group were treated with 4 ml (0.25 mg/ml) budesonide by atomization 15 min before stimulation and used as a drug control. All the experiments described below were performed in accordance with the regulations of the Centre of Animal Experiments of Qingdao University.
Enzyme linked immunosorbent assay (ELISA)
At 24 h after the last challenge, bronchoalveolar lavage fluid was obtained from the mice under anaesthesia using 1 ml sterile isotonic saline. Lavage was performed four times in each mouse and the total volume was collected separately. The lavage fluid sample was immediately centrifuged at 2,000 rpm for 10 min at room temperature, and stored at -80°C until use. The TGF-β1 levels were then assayed with a TGF-β1 ELISA kit according to the manufacturer’s instructions. The data on the TGF-β1 protein levels were summarized as mean ± SE of each sample.
Tissue samples
Lungs were removed from the mice after sacrificing 24 h after the last challenge. The tissues from the left lung were directly obtained from the surgical suite and immediately fixed in 10% buffered formalin and then embedded in paraffin. Sections (5 μm) were prepared and stained with hematoxylin and eosin (H&E). Additionally, Periodic acid-Schiff (PAS) staining was performed to identify mucus production in epithelial cells and the number of positive cells per unit length of basement membrane perimeter was determined. The thickness of the submesothelial extracellular matrix was determined after the tissue sections were H&E stained. The average of 10 independent measurements was calculated for each section and then the data were summarized.
Reverse transcription polymerase chain reaction (RT-PCR)
Total-RNA was isolated from the right lung tissue using the TRIzol reagent according to the manufacturer’s instructions. One microgram of the total cellular RNA was then reverse-transcribed into cDNA for PCR amplification using a kit from Sigma. The primer sequences used for PCR are listed in Table I. Amplification consisted of an initial 5 min incubation at 95°C and then 30 cycles of amplification using 30 sec of denaturation at 95°C, 30 sec at 57°C, and 60 sec at 72°C. The final extension was set for 10 min at 72°C. All data were expressed as the relative differences between control and treated cells after normalization to β-actin expression.
Immunohistochemistry
The expression of TGF-β1 was assessed by semi-quantitative immunohistochemistry. After being deparaffinized, the sections were incubated in 0.01 mol/l citric acid buffer (pH 6.0) for 15 min in a microwave for antigen retrieval. After cooling, the sections were incubated in 3 g/l H2O2 for 30 min, to inactivate endogenous peroxidase. After blocking by 1:10 normal horse serum for 30 min, the supernatant was discarded. Primary anti-mouse TGF-β1 (1:300 dilution) was added overnight at 4°C. Then, biotinylated goat anti-rat secondary antibody and streptavidin horseradish peroxidase were added to the slides and incubated for 30 min at room temperature. Staining was completed by incubation with diaminobenzidine chromogen solution at room temperature. The stained cells were mounted and viewed under light microscopy.
Westen blotting
Total protein was isolated from the right lung using a lysis buffer and quantified using protein quantification reagents from Bio-Rad. Next, 100 μg of the protein were suspended in 5X reducing sample buffer, boiled for 5 min, electrophoresed on 10% SDS-PAGE gels, and then transferred to polyvinylidene difluoride membranes by electroblotting. The membrane was blocked in 1% BSA/0.05% Tween-20/PBS solution overnight at 4°C, followed by incubation with the primary antibody for 24 h. A horseradish peroxidase-labeled IgG was used as the secondary antibody. The blots were then developed by incubation in a chemiluminescence substrate and exposed to X-ray films.
Statistical analysis
Data are expressed as mean ± SD. Statistical comparisons of the data from the various groups were performed using the Student’s t-test. Differences between groups were considered statistically significant at P<0.05.
Results
We have developed a mouse model of airway remodeling through repetitive OVA challenge. Mice were subjected to OVA challenge three times a week for 8 weeks and developed significant eosinophilic inflammation and airway remodeling similar to that observed in human chronic asthma.
Influence of astragalus extract on collagen deposition in asthma airway remodeling
We first stained and examined the histology of the airway wall in the four groups of mouse lung tissue. There was a little collagen deposition in the airway wall surrounding the normal mice, and the deposition increased significantly with an extensive distribution in the airway wall surrounding the asthma model mice (Fig. 2). Compared with the model group, collagen deposition in the mice treated with astragalus extract or budesonide was found to be significantly decreased (P<0.05).
The effects of astragalus extract on goblet cell hyperplasia and mucus plugging of the airways
To identify the degree of goblet cells hyperplasia and mucus plugging of the airways, lung tissue sections obtained from mice 24 h after the last OVA challenge were stained with PAS staining. Compared to the control, goblet cells hyperplasia and mucus plugging in the OVA groups were significantly greater. However, the difference of goblet cells hyperplasia and mucus plugging between the astragalus extract and the budesonide groups was not significant (P>0.05) (Fig. 3).
Effects of astragalus extract on TGF-β1 mRNA in mouse lung tissue
We next determined whether the astragalus extract can affect TGF-β1 mRNA production in the four groups of mouse lung tissue. Our data showed that, after an 8-week OVA-challenge, TGF-β1 mRNA expression in the OVA group was increased compared with the control group, whereas TGF-β1 mRNA expression in the astragalus extract and budesonide groups was decreased compared with that in the OVA group. There was no significant difference in TGF-β1 mRNA expressions among mice treated with astragalus extract and budesonide (P>0.05) (Fig. 4).
Detection of TGF-β1 levels in the bronchoalveolar lavage fluid
We assayed TGF-β1 protein levels in the bronchoalveolar lavage fluid and found that TGF-β1 levels were significantly higher in asthmatic mice than those in the control group. Levels were even lower in washes from the astragalus extract-treated group and budesonide-treated group than in the asthmatic group (Fig. 5).
Influence of astragalus extract on TGF-β1 expression in mouse lung tissue
TGF-β1 protein was found to be expressed in various cells of the lung including airway epithelial cells, fibroblasts, smooth muscle cells, vascular endothelial cells as well as the infiltrative inflammatory cells in model mice, while there was low expression of TGF-β1 protein in normal mice (Fig. 6). There was no significant difference in TGF-β1 expression in mice treated with astragalus extract and budesonide (P>0.05).
Effects of astragalus extract on Smad expression in mouse lung tissue
In order to investigate the expression of active TGF-β1 signaling in situ, we examined the expression of the intracellular effectors, Smads. An increase in the expression of P-Smad2/3 was observed during prolonged allergen challenge, whereas administration of astragalus extract and dexamethasone both considerably decreased P-Smad2/3 expression (Fig. 7). In contrast with P-Smad2/3, total Smad 2/3 (T-Smad2/3) expression levels remained unchanged. There was no significant difference of P-Smad2/3 in mice treated with astragalus extract and budesonide (P>0.05).
Discussion
In the current study, we investigated the role of astragalus extract in the development of airway remodeling in asthma by immunohistochemistry and morphometric analysis of lung tissue in vivo. We identified an important role for astragalus extract in the progression of airway remodeling changes. Furthermore, we confirmed that the astragalus extract could modulate the expression of signaling molecules of the TGF-β1/Smad pathway, which may involved in modulating airway remodeling.
Airway remodeling is one of the pathophysiological characteristics of asthma, and its main pathological changes include subepithelial fibrosis formation and increased collagen deposition on the airway wall (16). Our study demonstrated the therapeutic effect of astragalus extract on airway remodeling in allergic airways disease. Astragalus membranaceus extract includes formononetin and calycosin, which have been identified as the major components responsible for the immunosuppressive and anti-inflammatory effects of this herb (17). The astragalus extract inhibits several pro-inflammatory cytokines and adhesion molecules that are important mediators of some autoimmune diseases, such as rheumatoid arthritis and asthma, and has been shown to be safe and clinically beneficial in these diseases (18). In the present study, we observed that the astragalus extract reduced collagen deposition and airway wall thickening involving the reticular basement membrane, smooth muscle layer and epithelial hyperplasia in the mouse model.
Steroids have been administered widely for their anti-proliferative activity in asthma airway remodeling, but they are not free of adverse effects (19). Such adverse reactions may be avoided if astragalus extract proves effective for the treatment of asthma airway remodeling. The present study indicated that astragalus extract could be a potential therapeutic agent for asthma by its anti-proliferative and anti-inflammatory properties. Compared with budesonide, they have equal ability to prevent asthma airway remodeling in our study. These findings further encourage the use of this small molecule in the treatment of asthma airway remodeling.
How does the astragalus extract inhibit asthma airway remodeling? To use astragalus extract for clinical development effectively, it is essential to understand its mechanism. TGF-β1 is a potent fibrotic factor responsible for the synthesis of extracellular matrix. In recent years, a large number of studies demonstrated that TGF-β1 is an important cytokine in airway remodeling (20–22). Smads are the group of intracellular proteins that are critical for transmitting the TGF-β1 signals from the cell surface to the nucleus to promote transcription of target genes (23,24). In our study, we investigated the expression of active TGF-β1 signaling by detecting the expression of the intracellular effectors, Smads. Treatment with astragalus extract reduced the expression of TGF-β1 and TGF-β1 mRNA and modulated active TGF-β1 signaling in the airways, as demonstrated by a decrease in P-Smad2/3 expression. From our study, we can deduce that decrease of TGF-β1 levels and modulation of the activity of the TGF-β1 signaling pathway is a possible mechanism by which the astragalus extract inhibits airway remodeling in asthma.
In conclusion, our study demonstrated that the astragalus extract inhibited asthma airway wall remodeling through mechanisms involving a decrease in the production of TGF-β1 mRNA and TGF-β1 as well as modulation of active TGF-β1 signaling in the lung. It suggests the possibility of further developing astragalus extract as a candidate for the systemic therapy of asthma airway remodeling.
Acknowledgements
This study was supported by the Natural Science Foundation of the Shandong Province (no. Y2007C113) and Science and Technique Foundation of the Shandong Province (no. 2010GWZ20216). None of the authors have any financial and/or personal relationships with other people or organizations that could inappropriately influence or bias the study.
Figure 1 Chemical structures of formononetin, calycosin.
Figure 2 Hematoxylin and eosin (H&E) staining of airway tissues in asthmatic mice. (A) Lung tissue sections obtained from mice 24 h after the last OVA challenge were stained with H&E, and all photos were captured at ×100 magnification. (a) Control group; (b) asthmatic group; (c) astragalus extract-treated group; (d) budesonide-treated group. (B) Morphometric parameters of airway tissues in four groups. Data are expressed as the mean ± standard error of the mean of at least 3 separate experiments.
Figure 3 Periodic acid-Schiff (PAS) staining of airway tissues in asthmatic mice. To identify the degree of goblet cells hyperplasia and mucus plugging of the airways, lung tissue sections obtained from mice on 24 h after the last OVA challenge were stained with PAS staining, and all photos were captured at ×100 magnification. (A) Control group; (B) asthmatic group; (C) astragalus extract-treated group; (D) budesonide-treated group.
Figure 4 RT-PCR analysis of TGF-β1 mRNA in lung tissue. Mice were sacrificed 24 h after the final OVA challenge, and mRNA was then isolated and subjected to semi-quantitative RT-PCR analysis of TGF-β1. Expression of β-actin was used as a loading control. Lane M, marker; lane 1, control group; lane 2, asthmatic group; lane 3, astragalus extract-treated group; lane 4, budesonide-treated group.
Figure 5 ELISA analysis of TGF-β1 protein levels in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid was obtained from the mice under anaesthesia using 1 ml sterile isotonic saline and subjected to ELISA analysis. The data were summarized as mean ± standard error of the mean from at least 3 separate experiments. *P<0.05 in comparison with the OVA group.
Figure 6 Effects of astragalus extract on the expression of TGF-β1 protein in the four groups of mouse lung tissue. Expression of TGF-β1 in lung tissue was determined by immunohistochemical staining. Positive staining is depicted in brown. Positive staining showed TGF-β1 expression in the epithelium, macrophage leukocytes and in smooth muscle. (A) Control group; (B) asthmatic group; (C) astragalus extract-treated group; (D) budesonide-treated group.
Figure 7 Effects of astragalus extract on Smad2 and 3 phosphorylation in lung tissue. Mice were sacrificed 24 h after the final OVA challenge. Total cellular protein was then extracted, and expression of P-Smad2/3 and T-Smad2/3 in lung tissue was determined by western blotting. Expression of GAPDH was used as a loading control.
Table I Primers used for semi-quantitative RT-PCR.
Primer Sequence Length (bp)
TGF-β1-F 5′-GAAGTGGATCCACGAGCCCAAG-3′ 247
TGF-β1-R 5′-GCTGCACTTGCAGGAGCGCAC-3′
β-actin-F 5′-TTGATGTCACGCACGATTT-3′ 222
β-actin-R 5′-GCTGTCCCTGTATGCCTCT-3′
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23437282PONE-D-12-2018310.1371/journal.pone.0056966Research ArticleBiologyImmunologyImmunologic TechniquesImmunoassaysImmunofluorescenceMicrobiologyVirologyVirulence Factors and MechanismsPathogenesisMolecular cell biologyCellular TypesEukaryotic CellsCytometryFlow CytometryGene expressionRNA interferenceSignal TransductionSignaling CascadesApoptotic Signaling CascadeSignaling in Cellular ProcessesApoptotic SignalingCell DeathMedicineInfectious DiseasesViral DiseasesHand, Foot, and Mouth DiseaseThe Interplays between Autophagy and Apoptosis Induced by Enterovirus 71 The Interplay of Autophagy and Apoptosis in EV71Xi Xueyan
1
Zhang Xiaoyan
1
2
Wang Bei
1
Wang Tao
1
Wang Ji
1
Huang He
1
Wang Jianwei
1
Jin Qi
1
Zhao Zhendong
1
*
1
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
2
Department of Medical Laboratory Science, Fengyang College Shanxi Medical University, Shanxi, China
Poojary Venuprasad K. Editor
Karmanos Cancer Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: XX ZZ. Performed the experiments: XX XZ BW TW Ji Wang HH. Analyzed the data: XX XZ. Contributed reagents/materials/analysis tools: Jianwei Wang QJ. Wrote the paper: XX ZZ.
2013 20 2 2013 8 2 e5696611 7 2012 17 1 2013 © 2013 Xi et al2013Xi et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear.
Methodology/Principal Findings
In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles.
Conclusions/Significance
In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.
This work was supported by grants from National Natural Science Foundation of China (NSFC 31270200), National Basic Research Program of China (973 Project, 2011CB504903), Eleven-fifth Mega-Scientific project on “prevention and treatment of AIDS, viral hepatitis and other infectious diseases” (2009ZX10004-303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Enterovirus 71 (EV71) was first identified and isolated from the feces of an infant suffering from encephalitis in 1969 in California [1]. Subsequently, EV71 was reported as the agent involved in severe neurological diseases such as meningitis, encephalitis, monoplegia and acute flaccid paralysis [2]. The virus was also associated with non-neurological diseases such as hand, foot and mouth disease (HFMD), herpangina and pulmonary edema [3]–[6]. Among young children, EV71 is a notable cause of central nervous system (CNS) disease that usually results in rapid clinical deterioration and death. However, due to the lack of understanding of its viral pathogenesis, there are no effective vaccines or antiviral therapies currently available for the control and prevention of its fatal outbreaks.
After the cells are infected with EV71, the cells go through the disease process until death. According to morphological changes during the process of cell death, the programmed cell death (PCD) was divided into three types including apoptosis, autophagy and programmed necrosis. Recently, more researches have been focused on the relationship of autophagy and apoptosis [7]–[11]. The functional relationship between autophagy and apoptosis is complex under certain circumstances. Autophagy constitutes a stress adaptation that avoids cell death, whereas in other cellular settings, it constitutes an alternative cell-death pathway [12]–[14]. Autophagy and apoptosis might be triggered by common upstream signals, which sometimes results in a combined autophagy and apoptosis. In other instances, the cell switches between the two responses in a mutually exclusive manner [15]. Apoptotic cell death is induced by inhibiting the accumulation of autophagosomes in various carcinoma cells [16], which suggests that the autophagic process prevents apoptotic cell death. However, some studies have demonstrated that the autophagy process can also induce apoptotic cell death [17]. As previously reported, EV71 virus induced the autophagy [18] as well as apoptosis [19]–[21]; however, the relationship between EV71 virus-induced autophagy and apoptosis remains unclear. Thus, unraveling this relationship could provide new clues to elucidate the pathogenesis of EV71 infection.
Reducing the viral particle release is an effective strategy to control virus infection. Wang et al. [22] demonstrated that when apoptosis was inhibited by Phyllaemblicin B, the viral virulence of coxsackie virus B3 was markedly inhibited. However, Ahn et al. [23] suggested that the caspase inhibitor significantly inhibited apoptosis with no influence on coxsackie virus production and cell death in Hela cells. In addition, Jackson et al. [24] demonstrated that the stimulation of autophagy increased poliovirus yield while its inhibition decreased it. Therefore, the influence of autophagy and apoptosis on EV71 viral particle release was studied by our group. Also, a process to decrease the EV71 viral particles by regulating the autophagy or apoptosis was evaluated, which could provide a new strategy for prevention and control of EV71 infection.
RD-A cells are a subset of cells that are often used for the proliferation and amplification of EV71. Anhui strain of EV71 is isolated from the throat swab of a child with severe HFMD, when HFMD broke out in Fuyang, Anhui Province, China in May 2008. The cells and the strain were selected in this study as a model to understand the impact of autophagy and apoptosis on viral replication. The autophagy and apoptosis induced by Anhui strain of EV71 in RD-A cells was examined. The relationship between the autophagy and apoptosis induced by EV71 was studied by either using chemical inhibitors or RNA interference against essential autophagic or apoptotic genes. Finally, the effect of autophagy and apoptosis on EV71 viral particle release was investigated.
Results
EV71 induced autophagy in RD-A cells
To determine if autophagy was induced by EV71 virus in RD-A cells, confocal microscopy was performed in EV71-infected cells (Fig. 1A). The RD-A cells were initially transfected with a green fluorescent protein (GFP) and microtubule-associated protein light chain 3 (LC3) fusion protein, a specific marker of autophagosomes, for 24 hours. These cells were then infected with EV71 virus (Anhui strain) at the Multiplicity of Infection (MOI) of 10. In contrast to the diffused expression pattern of GFP-LC3 in control cells, GFP-LC3 showed punctuated accumulation in the EV71 infected cells (Fig. 1A). The number of punctated GFP-LC3 in each cell was counted (at least 50 cells were included for each group). The EV71-infected cells showed an increased punctate staining of GFP-LC3 (Fig. 1A) (P<0.01).
10.1371/journal.pone.0056966.g001Figure 1 EV71 induced autophagy in RD-A cells.
(A) The LC3 punctate were detected by confocal microscopy. After transfected with GFP-LC3 plasmid for 24 h, the RD-A cells were infected with EV71 virus (Anhui strain) at MOI of 10. After 12 hours of infection, cells grown on coverslips were fixed with freshly prepared 4% paraformaldehyde for 15 minutes at room temperature. The anti-EV71 monoclonal antibody and TRITC-conjugant anti mouse IgG antibody were added. The coverslips with fixed cells were mounted into microscopy chamber and directly visualized in phosphate buffer. The fluorescence of GFP-LC3 was viewed, images were acquired through GFP channel and the dots of GFP-LC3 were counted. Data are shown as the mean of three independent experiments. The double asterisks denote significant difference (p<0.01). (B) The RD-A cells were infected by EV71 at different MOI for 12 hours. The cells were lyzed and Western blot were performed. (C) The RD-A cells were infected by EV71 at multiplicity of infection of 10, lyzed at different time points post infection, and then Western blot were performed. Results are representative of three independent experiments. (D) The RD-A cells were infected by EV71 at multiplicity of infection of 1, 5 and 10, lyzed at different time points (6 h, 9 h, 12 h and 24 h) post infection, and then Western blot were performed. Results are shown as histogram. The results are representative of two independent experiments.
The conversion of LC3-I to LC3-II is assumed to be an accurate indicator of autophagy [18]. Therefore, the expression of LC3 in RD-A cells that were exposed to EV71 virus was investigated. At the fixed infection time of 12 hours, EV71 induced the conversion of LC3-I to LC3-II in a dose dependent fashion (Fig. 1B). To detect the impact of infected EV71 on autophagic flux, the expression of sequestosome 1 (SQSTM1/p62), a selective substrate of autophagy, was measured. The level of P62 was decreased after the cells were infected with EV71, suggesting an enhanced autophagic flux (Fig. 1B). Meanwhile, at the fixed infection dosage (MOI = 10), EV71 virus increased the conversion of LC3-I to LC3-II as well as P62 degradation in a time-dependent manner (Fig. 1C). The change of LC3 and P62 at different infection dosages and infection time points was also detected. With an increase in the dosage and time course of infection, the conversion of LC3-I to LC3-II and the degradation of P62 increased accordingly (Fig. 1D). Based on these results, EV71 induced autophagy in RD-A cells.
EV71 induced caspase-dependent apoptosis in RD-A cells
In this study, EV71-induced cell death was examined by ImageStream Multispectral Flow Cytometer. The data was analyzed using the ImageStream Data Analysis and Exploration Software (IDEAS). In the software, the apoptosis wizard provided the guide through the process of creating the features and graphs to measure apoptosis using the images of the nuclear dye and the bright field. The cells in R4 region presented in Fig. 2A were identified as apoptotic cells. The percentage of apoptotic cells was also shown in Fig. 2A. Visual confirmation of morphology was performed by clicking on the dots in the region in the appropriate locations. The EV71 induced apoptosis was further confirmed by Flow cytometry (Fig. 2B). The sum of the percentage in second quadrant and fourth quadrant was considered to be the percentage of apoptotic cells. These results demonstrated that EV71 infection caused an apparent apoptotic cell death in RD-A cells.
10.1371/journal.pone.0056966.g002Figure 2 EV71 induced caspase-dependent apoptosis in RD-A cells.
(A) RD-A cells infected with EV71 virus at the MOI of 0, 1, 5 and 10 for 12 h were trypsinized and harvested. The cells were washed with PBS and incubated with 7-AAD at 4°C for 30 minutes, detected by ImageStream multispectral flow cytometer and analyzed using the ImageStream Data Analysis and Exploration Software. Apoptotic cells are identified in R4 region. The percentage of apoptosis cells was listed in histogram. Visual confirmation of morphology was performed by clicking on dots in the region in the appropriate locations. (B) RD-A cells infected with EV71 virus at MOI of 0, 1, 5 and 10 were trypsinized and harvested. The cells were washed with PBS and incubated with a FITC-labeled Annexin V and stained with propidium iodide (PI) at room temperature for 15 minutes. The cells were analyzed on flow cytometer. The Annexin V-positive and PI-negative cells were considered to be apoptotic cells in early phase. The annexin V-positive and PI-positive cells were considered to be apoptotic cells in later phase. The percentage of apoptotic cells was also presented in histogram. Data are shown as the mean of three independent experiments. (C) The RD-A cells were infected by EV71 at different MOI for 12 hours. The cells were lyzed and Western blot were performed. (D) The RD-A cells were infected by EV71 at MOI of 10. The cells were lyzed at different time points post infection and Western blot were performed. Results are representative of three independent experiments.(E) The RD-A cells were infected by EV71 at multiplicity of infection of 1, 5 and 10, lyzed at different time points (6 h, 9 h, 12 h and 24 h) post infection, and then Western blot were performed. Results are shown as histogram. The results are representative of two independent experiments.
The ploy (ADP-Ribose) polymerase (PARP) and caspase-3 are believed as apoptosis hallmarks [25] and the cleavage of PARP and caspase-3 in RD-A cells that were exposed to EV71 virus was investigated. At a fixed infection time (12 hours), EV71 virus induced the cleavage of PARP and caspase-3 in a dose dependent manner (Fig. 2C). Meanwhile, at the fixed infection dosage (MOI = 10), EV71 virus induced the cleavage of PARP and caspase-3 in a time-dependent fashion (Fig. 2D). In addition, with the increase of the dosage and time course of EV71 infection, the cleavage of PARP and caspase-3 was increased accordingly (Fig. 2E). These results further demonstrated the occurrence of apoptosis in the RD-A cells that were infected with EV71.
At MOI of 10, both autophagy and apoptosis took place in the RD-A cells at 9 hours after infection with EV71. The most outstanding timing of both autophagy and apoptosis was 12 hours after infection. At 24 hours, cytopathic effect was remarkable and the cells were likely to undergo necrosis (data not shown). Therefore, in the rest of the studies, to explore the relationship between autophagy and apoptosis, the infection dose and infection time were set at 10 MOI and 12 hours, respectively.
Inhibition of autophagosome formation decreased the apoptosis induced by EV71
To verify the relationship between the autophagy and apoptosis induced by EV71 in the early stages of autophagy, the cleavage of PARP and caspase-3 were detected after the infected cells were treated with chemical autophagy inhibitor or siRNA inference against autophagy related gene (Atg) 5 and Beclin1 gene. The cells were infected with or without EV71 and were co-cultured with different dosage of Wortmannin (an inhibitor of phosphoinositide 3-kinase (PI3K), which blocked autophagy at autophagosome formation stage). The cells were then collected and were applied to Western blot and flow cytometry analysis with fluorescein isothiocyanate (FITC)-Annexin V and Propidium iodide (PI). Upon EV71 infection, the inhibition of autophagy with Wortmannin decreased the cleavage of PARP and caspase-3 in a dose-dependent manner (Fig. 3A). Meanwhile, the inhibition of autophagy with Wortmannin decreased the number of apoptotic cells (Fig. 3B). In addition, using the RNA interference approach, it was found that the knockdown of Atg5 and Beclin1 remarkably reduced the cleavage of PARP and caspase-3 (Fig. 3C) along with decreasing the number of apoptotic cells (Fig. 3D).
10.1371/journal.pone.0056966.g003Figure 3.The inhibition of autophagosome formation decreased the apoptosis induced by EV71.
(A) When RD-A cells infected with EV71 or without EV71 were co-cultured with different dosage of Wortmannin for 12 hours, the cells were lyzed and Western blot were performed. The results of western are representatives of the three independent experiments. The statistical analysis of PARP/Actin and caspase-3/Actin are the mean of three independent experiments. Mock value is set on 100% each experiment. (B) The RD-A cells treated as (A) were collected and stained with Annexin V-FITC and PI. The flow cytometry analysis was performed. The percentage of apoptotic cells were shown in the form of histogram. (C) RD-A cells were transfected with control, Atg5 and beclin1 siRNA for 36 hours, the cells were treated with EV71 at the MOI of 10 for 12 h before Western blot with the indicated antibodies. (D) RD-A cells treated as (C) were collected and stained with Annexin V-FITC and PI. The flow cytometry analysis was performed. The percentage of apoptotic cells were shown in the form of scatter plots.
Some previous reports [26], [27] suggested that UV radiation resistance-associated gene (UVRAG) formed two different complexes (UVRAG-Beclin1 and UVRAG-Bax), which regulated the balance between autophagy and apoptosis. So, the expression of UVRAG was detected in RD-A cells treated with Wortmannin upon EV71 infection. The results showed that the degradation of UVRAG induced by EV71 was dose- and time-dependent (Fig. 4A, 4B) and it could be inhibited by Wortmannin (Fig. 4C). In addition, one report [27] suggested that UVRAG interacted with Bax, which inhibited apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome release and activation of caspase-9 and -3. Our results demonstrated that with the degradation of UVRAG that could be inhibited by Wortmannin, the expression of Bax increased correspondingly (Fig. 4C).
10.1371/journal.pone.0056966.g004Figure 4 The inhibition of autophagy at the autophagosome formation inhibited the apoptosis through the interaction of UVRAG and BAX.
The RD-A cells were infected by EV71 at different MOI for 12 hours (A) and at multiplicity of infection of 10, lyzed at different time points post infection (B). The western blot for UVRAG was performed. (C) When RD-A cells infected with EV71 or without EV71 were co-cultured with different dosage of Wortmannin for 12 hours, the cells were lyzed and Western blot were performed. The UVRAG and BAX were detected using corresponding monoclonal antibody. Results are representative of three independent experiments.
Inhibition of autophagosome and lysosome fusion promoted the EV71-induced apoptosis
Chloroquine (CQ) has been reported to inhibit lysosomal acidification and therefore prevent autophagy by blocking autophagosome fusion and degradation [28]. The CQ was used in this study to detect the effect of autophagy on EV71-induced apoptosis. The cells were infected with or without EV71 and were co-cultured with different dosage of CQ. The cells were then collected and submitted to Western blot and flow cytometry. It was found that the inhibition of autophagy with CQ promoted the cleavage of PARP and caspase-3 in a dose-dependent manner (Fig. 5A). Also, the inhibition of autophagy with CQ increased the number of apoptotic cells (Fig. 5B).
10.1371/journal.pone.0056966.g005Figure 5 Inhibition of the fusion of autophagosome with lysosome promoted the apoptosis induced by EV71.
(A) When RD-A cells infected with EV71 or without EV71 were co-cultured with different dosage of CQ for 12 hours, the cells were lyzed and Western blot were performed. The results of western are representatives of the three independent experiments. The statistical analysis of PARP/Actin and caspase-3/Actin are the mean of three independent experiments. Mock value is set on 100% each experiment. (B) RD-A cells treated as (A) were collected and stained with Annexin V-FITC and PI. The flow cytometry analysis was performed. The percentage of apoptotic cells were shown in the form of histogram. Data are shown as the mean of three independent experiments.
Inhibition of the lysosomal protease inhibited the EV71-induced apoptosis
Pepstatin A and E64d, the inhibitors of lysosomal protease, were used to detect the effect of autophagy on apoptosis induced by EV71. The cells infected with or without EV71 and were co-cultured with different dosage of E64d and pepstatin A. The cells were then collected for Western blot and flow cytometry analysis. The inhibition of autophagy with E64d and pepstatin A decreased the cleavage of caspase-3 in a dose-dependent manner (Fig. 6A). Likewise, the inhibition of autophagy with E64d and pepstatin A decreased the number of apoptotic cells (Fig. 6B).
10.1371/journal.pone.0056966.g006Figure 6 Inhibition of the lysosomal protease decreased the apoptosis induced by EV71.
(A) When RD-A cells infected with EV71 or without EV71 were co-cultured with different dosage of E64d and pepstatin A for 12 hours, the cells were lyzed and Western blot were performed. The results of western are representatives of the three independent experiments. The statistical analysis of PARP/Actin and caspase-3/Actin are the mean of three independent experiments. Mock value is set on 100% each experiment. (B) RD-A cells treated as (A) were collected and stained with Annexin V-FITC and PI. The flow cytometry analysis was performed. The percentage of apoptotic cells were shown in the form of histogram. Data are shown as the mean of three independent experiments.
Inhibition of apoptosis increased the conversion of LC3-I to LC3-II and degradation of P62
Z-VAD-FMK, a pan-caspase inhibitor, has been reported to block the caspase-dependent apoptosis [29]. In this study, the LC3 type conversion and P62 degradation were examined after inhibition of the EV71-induced apoptosis by Z-VAD-FMK. The results showed that inhibition of apoptosis by Z-VAD-FMK increased the conversion of LC3-I to LC3-II and the degradation of P62 in a dose-dependent fashion (Fig. 7A). Meanwhile, the inhibition of apoptosis by Z-DEVD-FMK, a caspase-3 inhibitor, also increased the conversion of LC3-I to LC3-II and the degradation of P62 in a dose dependent fashion (Fig. 7B). Using the RNA interference approach, it was found that knockdown of caspase-3 significantly increased the conversion of LC3 and P62 degradation (Fig. 7C). Zalckvar's report [10] suggested that the connection of Atg5 and caspase-3 played an important role in the autophagy and apoptosis. In order to clarify the impact of Atg5 to apoptosis, we detected the expression of Atg5 when casapase-3 was inhibited or knocked down. The results demonstrated that with the increase of Z-DEVD-FMK, the expression of Atg5 was also increased (Fig. 8A). When caspase-3 was knocked down, the expression of Atg5 also increased (Fig. 8B).
10.1371/journal.pone.0056966.g007Figure 7 Inhibition of apoptosis increased the conversion of LC3-I to LC3-II and degradation of P62.
When RD-A cells infected with EV71 or without EV71 were co-cultured with different dosage of Z-VAD-FMK (A) and Z-DEVD-FMK (B) for 12 hours, the cells were lyzed and Western blot were performed. (C) RD-A cells were transfected with control and caspase-3 siRNA for 36 hours, the cells were treated with EV71 at the MOI of 10 for 12 h before Western blot with the indicated antibodies. The results of western are representatives of the three independent experiments. The statistical analysis of LC3II/LC3I and P62/Actin are the mean of three independent experiments. Mock value is set on 100% each experiment.
10.1371/journal.pone.0056966.g008Figure 8 Inhibition of apoptosis promoted the autophagy through the ATG5.
(A) When RD-A cells infected with EV71 were co-cultured with different dosage of Z-DEVD-FMK for 12 hours, the cells were lyzed and Western blot were performed. (B) RD-A cells were transfected with control and caspase-3 siRNA for 36 hours, the cells were treated with EV71 at the MOI of 10 for 12 h for Western blot with ATG5 antibody. Results are representative of three independent experiments.
Inhibition of autophagy in the autophagosome formation stage and apoptosis decreased EV71 viral particle release
To check the influence of autophagy and apoptosis on the viral particle release, cultural supernatants from EV71-infected RD-A cells under the action of different dosage of Wortmannin and Z-VAD-FMK were collected and subjected to virus titeration by using the TCID50 method. The results showed that with the increase of the dosage of Wortmannin (Fig. 9A) and Z-VAD-FMK (Fig. 9B), the viral particles released into the cultural supernatants decreased gradually. By taking advantage of RNA inference of Atg5 and caspase-3, it was also shown that the virus titer in the cultural supernatant was decreased as compared to that of the supernatant of the cells treated with control siRNA (Fig. 9C).
10.1371/journal.pone.0056966.g009Figure 9 The inhibition of autophagy and apoptosis prevented EV71 viral particle from release.
RD-A cells were infected with EV71 in the presence or absence of Wortmannin (A) and Z-VAD-FMK (B) for 24 hours. The culture supernatant was collected and TCID50 examination was performed. (C) RD-A cells were transfected with control and Atg5 and caspase-3 siRNA for 36 hours, the cells were treated with EV71 at the MOI of 10 for 12 h. The culture supernatant was collected and TCID50 examination was performed. Data are shown as the mean of two independent experiments.
Cathepsins might participate in the interplay of autophagy and apoptosis in the fusion stage of autophagosome with lysosome and autophagy execution stage
Previous reports demonstrated that treatment with CQ led to a dramatic increase in cathepsin D [30], which promoted apoptosis through activating caspase pathway and modifying pro-apoptotic molecular BAX and BAK [31]. In order to check the role of cathepsin D in EV71 infected cells, the expression of cathepsin D was detected in RD-A cells that were infected with EV71 upon being treated with different dosage of CQ. The results showed that with the increase of CQ dosage, the expression of cathepsin D increased (Fig. 10A). In autophagy execution stage, pepstatin A, an inhibitor of cathepsins D [32], [33], and E64d, a cell permeable inhibitor of cathepsins B, H and L [34] were used to evaluate the effect of autophagy to apoptosis induced by EV71. We found that with the increase of E64d and pepstatin A dosage, the expression of cathepsin D and cathepsin B in RD-A cells infected by EV71 decreased (Fig. 10B). Based on these results and that from Fig. 5 and Fig. 6, it was suggested that the cathepsins might participate in the interplay of autophagy and apoptosis in the fusion stage of autophagosome with lysosome and autophagy execution stage.
10.1371/journal.pone.0056966.g010Figure 10 The cathepsins might participate in the interplay of autophagy and apoptosis in the fusion of autophagosome with lysosome and autophagy execution stage.
When RD-A cells infected with EV71 were co-cultured with different dosage of chloriquine (A) and E64d and pepstatin A (B) for 12 hours, the cells were lyzed and Western blot was performed. Results are representative of three independent experiments.
Discussion
Many studies have shown multiple connections between the autophagic and apoptotic processes, and the two phenomena jointly sealing the fate of the cells. Autophagy is a catabolic pathway that is often characterized by the formation of double-membrane vesicles (autophagosomes) that engulf cytoplasmic organelles and proteins and fuse with lysosomes in the end, which degrade their luminal content. Autophagy acts as a cytoprotective mechanism, favoring stress adaptation to avoid cell death [35], [36]. Apoptosis is programmed cell death and leads to the rapid demolition of cellular structures and organelles. When this process culminates in cellular shrinkage with nuclear chromatin condensation and nuclear fragmentation, it complies with the morphological definition of apoptosis [37]. Shimizuet al. [38] demonstrated that autophagy related to apoptosis by acting as a partner, an antagonist or an enabler of apoptosis depending on the cell type, stimulus and environment. In this study, in order to clarify the interplay of autophagy and apoptosis in EV71 infection in RD-A cells, apoptosis and autophagy were detected when the autophagy and apoptosis were inhibited by either a chemical inhibitor at different stages or by a single knockdown of essential genes through RNA interference; respectively. We ruled out the role of various single inhibitors on RD-A cells and provided the interaction possibility between autophagy and apoptosis under the action of various inhibitors in EV71 infection. During the course of EV71 infection in RD-A cells, the crosstalk between autophagy and apoptosis was very complex. In this study, it was demonstrated that the inhibition of EV71 induced autophagy at autophagosome formation and autophagy execution stages that might inhibit apoptosis. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion might promote the apoptosis and the inhibition of EV71-induced apoptosis contributed to the autophagy. We summarized these findings in a brief chart about the relationship of autophagy and apoptosis in Fig. 11. In addition, the inhibition of autophagy or apoptosis by either chemical inhibitors or RNA interference decreased the release of EV71 viral particles.
10.1371/journal.pone.0056966.g011Figure 11 The brief chart about the interplays of autophagy and apoptosis.
EV71 is a single, positive-stranded RNA virus that belongs to the Picornaviridae family [39]. The 7.4-kb genome of EV71 encodes a single polyprotein that is proteolytically cleaved to various structural and nonstructural viral proteins. In order to illustrate the contribution of the different viral components to both host autophagy and apoptosis pathway, we transfected the structural proteins of VP1, VP2, VP3 and VP4 and non-structural proteins or protein intermediate of 2A, 2B, 2C, 3A, 3AB, 3C and 3D into 293T cells. The cells were then subjected to autophagy and apoptosis detection by checking the LC3 conversion, p62 degradation and PARP cleavage. Due to the low transfection efficiency in RD-A cells (data not shown), these experiments were carried out in 293T cells. The results were shown in Fig. S1. It was found that none of the structural and non-structural proteins of EV71 separately induced a significant autophagy and apoptosis like the viral particles. This observation suggested that the induction of both autophagy and apoptosis might depend on a comprehensive interaction among the structural and non-structural proteins of EV71 in its life cycle in the host cells.
In the study, we also explored the possible mechanism of the interplay between autophagy and apoptosis according to the current results. The preliminary mechanism was presented in Fig. 11.
Wortmannin has been proposed to suppress the autophagosome formation by inhibiting the class III PI3K to block the production of PI3P, which is essential for the initiation of autophagy via recruitment of other ATG proteins at the isolation membrane or phagophore [40]. To explore the mechanism that the inhibition of autophagy by Wortmannin inhibited apoptosis, we detected the UVRAG, which regulated the balance between autophagy and apoptosis by two different complexes (UVRAG-Beclin1 and UVRAG-Bax) [26]. UVRAG interacted with Bax [27], which inhibited apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome release and activation of caspase-9 and -3. The results in our study also showed that the degradation of UVRAG was inhibited by Wortmannin, and the expression of Bax increased correspondingly (Fig. 4C). It provided a potential mechanism for the Wortmannin-inhibited autophagy that leads to an increased UVRAG and Bax expression in order to inhibit the apoptosis (Fig. 11). Definitely, an in-depth study is needed next to address the specific mechanisms of UVRAG and Bax in the interaction between autophagy and apoptosis.
Our results also showed that when the EV71-induced apoptosis was inhibited by Z-VAD-FMK (pan-caspase inhibitor), the RD-A cells treads to autophagy (Fig. 7A). This was consistent with the previous report of Sirois [41] highlighting that ZVAD-FMK negatively impacted autophagic vacuole (AV) maturation, possibly through preventing the release of AV components in the extracellular milieu and increasing the mean distance between AV and the cell membrane. Meanwhile, these results were corroborated caspase-3 inhibitor (Z-DEVD-FMK) caspase-3-deficient mice. It was observed that caspase-3 was an important regulator in autophagosome maturation. This encouraged us to detect the role of caspase-3 in the relationship of autophagy and apoptosis. As shown in Fig. 7B and 7C, the RD-A cells tend to undergo autophagy when they were treated with caspase-3 inhibitor (Z-DEVD-FMK) or caspase-3 was knocked down. In addition, by applying computational tools that are based on mining the protein–protein interaction database, a novel biochemical pathway between Atg5 and caspase-3 was obtained [10]. In order to clarify the impact of Atg5 on apoptosis in this study, the expression of Atg5 was detected when caspase-3 was inhibited or knocked down. The results demonstrated that with an increase in Z-DEVD-FMK, there was an increased expression of Atg5 (Fig. 8). Taken together, the co-relation of autophagy (in the autophagosome formation stage) and apoptosis was assumed to be related to ATG5 and caspase-3. When ATG5 was inhibited, the caspase-3 was also inhibited, thus inhibiting the apoptosis. Likewise, when caspase-3 was inhibited, ATG5 was activated, thus promoting the autophagy (Fig. 11). We are conducting an in-depth study of the role of the interaction between ATG5 and caspase-3 on the autophagy and apoptosis.
It was found that the inhibition of autophagosomes and lysosome fusion of by CQ promoted the apoptosis (Fig. 5). CQ is a weak alkali that can inhibit lysosomal acidification, which prevents the fusion of autophagosomes with lysosomes and subsequently leads to autophagic degradation. Carew et al. demonstrated that the treatment with CQ led to a dramatic increase in cathepsin D [30], which was active at physiologcal pH and promoted apoptosis through two aspect of action including activating caspase pathway and modifying pro-apoptotic molecular BAX and BAK [31]. Our results were consistent with the above report, where CQ inhibited EV71-induced autophagy and promoted the apoptosis of RD-A cells by activating cathepsin D (Fig. 10A). Another possibility was that the inhibition of autophagy might have resulted in a bioenergetic shortage which triggered apoptosis [42]. In a more subtle fashion, inhibition of autophagosome and lysosome fusion might subvert the capacity of cells to remove damaged organelles or misfolded proteins, which in turn would favor apoptosis.
In autophagy execution stage, pepstatin A (an inhibitor of cathepsins D) [32], [33] and E64d (a cell permeable inhibitor of cathepsins B, H and L) [34] were used to evaluate the effect of autophagy to apoptosis induced by EV71. Motyl et al. [43] suggested that inhibition of cathepsins by E64d significantly reduced the apoptotic cell number. Cathepsins, particularly cathepsin B, could be involved in the molecular switch between autophagy and apoptosis. Furthermore, it was found that E64d and pepstatin A inhibited EV71-induced autophagy and decreased the RD-A cells apoptosis by inhibiting cathepsin D and B (Fig. 10B). In the late stage of autophagy (the fusion of autophagosome with lysosome and autophagy execution stage), the role of CQ, E64d and pepstatin A seems to be linked through cathepsins.
Taken together, the inhibition of autophagy by Wortmannin inhibited the EV71-induced apoptosis possibly through increasing the expression of UVRAG thus impact on UVRAG and BAK interaction. The inhibition of apoptosis by Z-DEVD-FMK promoted the EV71-induced autophagy possibly through activating ATG5. In the fusion stage of autophagosome with lysosome, the inhibition of autophagy promoted the apoptosis in RD-A cells by activating cathepsins D. And the inhibition of autophagy in the execution stage inhibited the apoptosis by inhibiting cathepsin D and cathepsin B.
In this study, we also explored the role of the interplay between autophagy and apoptosis in viral particle release. It seems that influence of autophagy at different stages of autophagy development on EV71 viral particles release was achieved through the effect on the apoptosis The autophagy at the stage of autophagosome formation was inhibited by Wortmannin, the viral particle released was also inhibited (Fig. 9). Although autophagy during the stage of autophagosome formation might not have a direct impact on the virus release, it probably had an inhibitory effect on virus release through apoptosis inhibition. We did not check into the viral particles release at the fusion stage of autophagosome with lysosome and execution stage. But we might speculate that EV71 might have prompted the release of viral particles through concomitantly induced apoptosis at the fusion stage. And during the execution stage, viral particles release might reduce due to the inhibition of apoptosis. In addition, the apoptosis was inhibited by Z-VAD-FMK, the viral particle released was also inhibited (Fig. 9). Therefore, the information should be potentially helpful for the selection of inhibitors used to control EV71 viral particle release as a potential strategy for prevention and control of EV71 infection.
Materials and Methods
Cell culture and virus propagation
Human rhabdomyosarcoma (RD-A; ATCC, CCL-136) cells were maintained in L-glutamine containing Dulbecco's modified Eagle's medium (DMEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Gibco) plus penicillin/streptomycin (200 U/ml), and cultured at 37°C in a 5% CO2 incubator. At 80% confluence, cells were trypsinized with 0.25% trypsin (Solarbio) and sub-cultured in the complete medium. Virus infection was carried out as follows. Briefly, RD-A cells were infected with EV71 at the indicated multiplicity of infection (MOI). Viruses were washed away after 2 hours, cells were then cultured with fresh medium supplemented with 2% FBS. At the indicated time points post infection, the cells and culture supernatant were harvested.
Chemicals and antibodies
The antibodies to Pepstatin A (Sigma-Aldrich, P5318), E64d (Sigma-Aldrich, E8640), β–actin (Sigma-Aldrich, A5316), ATG 5 (Sigma-Aldrich, A0731) and polyclonal antibodies of LC3 (Sigma-Aldrich, L7543) were purchased from Sigma-Aldrich; Z-VAD-FMK (R&D, FMK001) was purchased from R&D systems; antibodies against UVRAG (CST, 5320), caspase-3 (CST, 9662) and PARP (CST, 9542) were obtained from Cell Signaling; antibodies to P62 (Santa Cruz, sc-28359) and beclin 1 (Santa Cruz, sc-11427) were purchased from Santa Cruz Biotechnology; anti-EV71 (Millipore, MAB979) antibody was got from Millipore; Chloroquine and Z-DEVD-FMK were purchased form MBL Company.
Plasmids and siRNAs
GFP-LC3 plasmid was a kind gift of Dr. Xuejun Jiang (Biological Resource Center of Institute of Microbiology; Chinese Academy of Sciences). To construct plasmids VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3AB, 3C, and 3CD, fragments of EV71 cDNA were cloned into the HindIII and SalI sites of pEGFPC1 vector, resulting in GFP fusion proteins. The siRNA specific for Caspase-3 (Santa Cruz, sc-29437), Atg5 (Santa Cruz, sc-41445) were purchased from Santa Cruz Biotechnology along with the control siRNA. siGENOME SMART pool of BECN1 (8678) targeting Beclin 1 was bought from Dharmacon.
Confocal microscopy
RD-A cells were transfected with GFP-LC3 expressing plasmid, after 24 hours of transfection, cells were infected with EV71 virus at indicated MOI. After 12 hours, cells grown on coverslips were fixed with freshly prepared 4% paraformaldehyde for 15 minutes at room temperature. The anti-EV71 antibody (Millipore, MAB979) and TRITC-conjugant anti mouse IgG antibody (Zhongshan, ZF-0313) was incubated. The coverslips with fixed cells were mounted into confocal microscopy (Leica, TCSSP5) and directly visualized in phosphate buffer. The fluorescence of GFP-LC3 was viewed, imaged and the dots of GFP-LC3 were counted.
Western blot
Cells were lyzed in buffer containing 150 mMNaCl, 25 mMTris (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA as well as a proteinase inhibitor cocktail (Roche). Lysed cells were electrophoresed on 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (BioRad). The membranes were blocked with 5% dried skimmed milk and then incubated with corresponding antibodies at 4°C overnight. This was followed by incubation with the corresponding IRDye Fluor 800-labeled IgG or IRDye Fluor 680-labeled IgG secondary antibody (Li-Cor Bioscience). After the membranes were washed with 0.1% Tween20 in PBS, the signal were scanned by using an Odyssey Infrared Imaging System (Li-Cor Bioscience) at a wavelength of 700 nm to 800 nm and analyzed with Odyssey software. The molecular sizes of the developed proteins were determined by comparison with pre-stained protein markers (New England Biolabs). Several western bands were analyzed to verify the linear range of the chemiluminescence signals and the quantifications were carried out using Quantity One software.
ImageStream multispectral flow cytometer
RD-A cells infected with EV71 virus at the MOI of 0, 1, 5 and 10 for 12 h were trypsinized and harvested. The cells were washed with PBS and incubated with 7-AAD (Invitrogen) at 4°C for 30 minutes and then analyzed on ImageStream multispectral flow cytometer (Amnis). An ImageStream multispectral imaging flow cytometer works similar to classical flow cytometer except that it also allows visualization of individual cells passing through its flow chamber. The snapshots of each cell provided additional information of cellular morphology and spatial distribution. The data was analyzed using the ImageStream Data Analysis and Exploration Software (IDEAS).
Flow cytometry
For flow cytometry analysis, RD-A cells that were infected with EV71 or without EV71 co-cultured with autophagy and apoptosis inhibitor. The cells were collected, washed with PBS and incubated with a FITC-labeled annexin V and stained with PI (Baosai Biotech) at room temperature for 15 minutes and then analyzed on flow cytometer (BD CantoII). The annexin V-positive/PI-negative cells were considered to be apoptotic cells at the early period, annexin V-positive/PI-positive cells were considered to be apoptotic cells at the later period, whereas PI-single positive cells were considered necrotic.
TCID50 (50% tissue culture infective dose)
The titer of virus was calculated with TCID50. The virus particle in cell culture supernatant were collected by centrifugation at 4000 rpm for 5 minutes at 4°C. 5000 cells/well were put in 96 wells plate in DMEM medium plus 2% FBS before 1 day. The determined virus suspension were diluted with DMEM medium plus 2% FBS by 10-fold of serial dilutions from 10−1 to 10−8. 100 µl of each dilution was transfered to the plate with cells to make total of 200 µl per wells. The cells were cultured at 37°C for 5 days and cells were observed daily. The TCID50 was calculated according to the Behrens-Kärber formula: log TCID50 = L-d (S −0.5), where: L = lowest log dilution values used in the experiment; d = log value of the dilution gradient; S = the sum of the positive parts.
Statistics analysis
The statistical comparisons were performed by using Student's t test. Value of P<0.05 was considered statistically significant.
Supporting Information
Figure S1
None of the structural and non-structural proteins of EV71 separately induced a significant autophagy and apoptosis. The plasmid pEGFPC1 containing VP1,VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3AB, 3C, and 3CD were transfected into 293T cells for 36 h, the cells were lyzed and Western blot for LC3, P62, PARP, GFP and β-actin was performed.
(TIF)
Click here for additional data file.
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Crit CareCrit CareCritical Care1364-85351466-609XBioMed Central cc114782289782110.1186/cc11478ResearchDiacerhein attenuates the inflammatory response and improves survival in a model of severe sepsis Calisto Kelly L [email protected] Angélica C [email protected] Francine C [email protected] Bruno M [email protected] Dioze [email protected] José B [email protected] Mario J [email protected] Department of Internal Medicine, FCM, State University of Campinas (UNICAMP), Cidade Universitária Zeferino Vaz, Campinas, SP, Brazil2012 16 8 2012 16 4 R158 R158 20 3 2012 21 6 2012 13 8 2012 Copyright ©2012 Callisto et al.; licensee BioMed Central Ltd.2012Callisto et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction
Hyperglycemia and insulin resistance have been associated with a worse outcome in sepsis. Although tight glycemic control through insulin therapy has been shown to reduce morbidity and mortality rates, the effect of intensive insulin therapy in patients with severe sepsis is controversial because of the increased risk of serious adverse events related to hypoglycemia. Recently, knowledge about diacerhein, an anthraquinone drug with powerful antiinflammatory properties, revealed that this drug improves insulin sensitivity, mediated by the reversal of chronic subclinical inflammation. The aim of the present study was to evaluate whether the antiinflammatory effects of diacerhein after onset of sepsis-induced glycemic alterations is beneficial and whether the survival rate is prolonged in this situation.
Methods
Diffuse sepsis was induced by cecal ligation and puncture surgery (CLP) in male Wistar rats. Blood glucose and inflammatory cytokine levels were assessed 24 hours after CLP. The effect of diacerhein on survival of septic animals was investigated in parallel with insulin signaling and its modulators in liver, muscle, and adipose tissue.
Results
Here we demonstrated that diacerhein treatment improves survival during peritoneal-induced sepsis and inhibits sepsis-induced insulin resistance by improving insulin signaling via increased insulin-receptor substrate-1-associated phosphatidylinositol 3-kinase activity and Akt phosphorylation. Diacerhein also decreases the activation of endoplasmic reticulum stress signaling that involves upregulation of proinflammatory pathways, such as the I kappa B kinase and c-Jun NH2-terminal kinase, which blunts insulin-induced insulin signaling in liver, muscle, and adipose tissue. Additionally, our data show that this drug promoted downregulation of proinflammatory signaling cascades that culminate in transcription of immunomodulatory factors such interleukin (IL)-1β, IL-6, and tumor necrosis factor-α.
Conclusions
This study demonstrated that diacerhein treatment increases survival and attenuates the inflammatory response with a significant effect on insulin sensitivity. On the basis of efficacy and safety profile, diacerhein represents a novel antiinflammatory therapy for management of insulin resistance in sepsis and a potential approach for future clinical trials.
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Introduction
Sepsis is defined as a systemic inflammatory response syndrome caused by the body's response to an infection [1]. During the onset of sepsis, the inflammatory system becomes hyperactive, leading to a production of proinflammatory molecules and cytokine release [2], which contribute to septic shock, multiple organ failure, and death. Hyperglycemia and insulin resistance occur as a consequence of the metabolic effects of stress hormones and the overproduction of proinflammatory mediators in sepsis [3,4]. In this regard, tissue insulin resistance may be used as an important indicator of the resultant actions of the proinflammatory cytokines, being a tissue marker of the severity of sepsis before organ failure. In addition, this hyperglycemia may lead to inflammatory stress [5,6] and aggravate sepsis, as previously described [7,8].
Both bacterial components such as lipopolysaccharide (LPS), and proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, resulting from the immune response to sepsis, may activate intracellular mechanisms associated with insulin resistance, such as the IKKβ/NF-κB, and JNK pathways. JNK has been shown to promote insulin resistance through serine phosphorylation of IRS-1, preventing signaling from the insulin receptor. Furthermore, IKKβ induces insulin resistance through activation of NF-κB, which in turn induces the transcription of several genes related to proinflammatory cytokine release (IL-1β, TNF-α, IL-6, and IL-8) [9]. IKK activation leads to phosphorylation, ubiquitination, and degradation of IκB, which releases NF-κB, allowing it to translocate to the nucleus and activate transcription of target genes [10].
In this regard, we believe that the evaluation of insulin signaling pathways through PI3K/Akt in liver, muscle, and adipose tissue may be important indicators of this overreaction at the tissue level, and the improvement in this signaling pathway, induced by some treatments, in parallel with a decrease in tissue inflammation, may predict the effectiveness of this treatment. Substantial resources have been invested in developing and evaluating potential therapies for sepsis and in increasing knowledge of systemic inflammation and multiple-system organ failure [11,12], although pharmacologic interventions available now are not effective in decreasing the high mortality rates.
Diacerhein (4,5-diacetoxy-9,10-dihydro-9,10-dioco-2-anthracenecarboxylic acid) is an anthraquinone that shows antiinflammatory properties, in addition to moderate analgesic and antipyretic characteristics [11], and is used in the treatment of osteoarthritis. Clinical studies have suggested that diacerhein can exert beneficial effects on the symptoms of osteoarthritis, including antiarthritic and chondroprotective effects [13]. Rhein, the active metabolite of diacerhein, has been demonstrated to inhibit the synthesis and activity of proinflammatory cytokines such as TNF-α, IL-6, and IL-1β [14-17]. The compound also acts directly on inflammatory cells, inhibiting superoxide anion production by human neutrophils, release of lysosomal enzymes, chemotaxis, and phagocytic activity of neutrophils and macrophages [18-22].
Further studies have suggested that diacerhein inhibits nitric oxide production induced by the reduction of IL-1β [23,24]. Besides its inhibitory effects on proinflammatory genes, rhein has been shown to have antitumor activity on several cancer cell lines [25,26]. Moreover, studies suggest that diacerhein and rhein inhibit NF-κB activation and expression of NF-κB-dependent genes [24]. In this regard, diacerhein has the potential to improve insulin resistance in sepsis and to reduce the overreaction of the inflammatory response, without inducing hypoglycemia.
The aim of the present study was to investigate whether diacerhein, by reducing tissue activation of inflammatory pathways, can improve insulin signaling and survival in sepsis.
Materials and methods
Anti-IR-β (α-IR), anti-IRS-1, anti-Akt, anti-caspase-3 anti-p-IR, anti-p-IRS-1, anti-p-IKKβ, anti-p-IκBa, anti-NF-κB, anti-p-JNK, anti-JNK1, anti-p-PERK, and anti-IRE1 antibodies were obtained from Santa Cruz Technology (Santa Cruz, CA, USA). Anti-p-Akt was from Cell Signaling Technology (Beverly, MA, USA). Anti-p-IRS-1ser307 was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Anti-p-eIF2α was from Abcam (Cambridge, MA, USA). Diacerhein was kindly provided by TRB-Pharma (Campinas, Brazil). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, IN, USA). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise specified.
Animal care and experimental procedures
All experiments were approved by the Ethics Committee at the University of Campinas, CEEA/Unicamp 1267-1. Male Wistar Hannover rats (8 weeks old) were maintained in a room with 12-hour day/night cycles and room temperature of 21°C, with food and water ad libitum.
Polymicrobial sepsis was induced by subjecting rats to CLP, as previously described [27], and is a commonly used surgical technique in rodents and thought to be a clinically relevant animal model of sepsis. Anesthesia was induced by i.p. administration of ketamine (80 mg/kg BW) and xylazine (15 mg/kg). Through a 1-cm abdominal midline incision, the cecum was ligated below the ileocecal valve, with careful attention to avoid obstruction of the ileum or colon. The cecum was subjected to four ''through-and through'' perforations (20-gauge needle). The abdominal incision was closed in layers. Sham-operated rats underwent the same procedure, except for ligation and perforation of the cecum. All procedures were performed under sterile conditions. Wistar rats were randomly divided into four groups; diacerhein-treated sepsis (Sepsis/Dia), vehicle-treated sepsis (Sepsis/Veh), diacerhein-treated sham (Sham), and vehicle-treated sham (Control).
Diacerhein-administration protocol
Dried diacerhein was diluted in 0.01 M PBS 3% DMSO, to a final concentration of 18 mg/ml. Three hours after the induction of sepsis and every 24 hours, rats received diacerhein (100 mg/kg/day) or an equivalent volume of vehicle by oral gavage.
Preliminary experiments using lower doses of diacerhein (10 and 20 mg/kg/day) showed no beneficial effects on survival curves, nor even on insulin sensitivity in septic animals. The chosen dose (100 mg/kg/day) was based on a previous study [28]. This dose is safe and lower than the LD50 for rats (980 mg/kg). The only adverse effect observed was softening of fecal contents, given the anthraquinone nature of diacerhein. The oral route of administration is indicated for this drug, which is promptly deacetylated to rhein, its active metabolite, soon after its administration.
Sepsis-survival studies
For survival studies, Wistar rats (n = 15 per group) were induced to sepsis, allowed to recover, and then treated with diacerhein (100 mg/kg/day) or placebo 3 hours after surgery and then once per day, and were then observed twice per day. To avoid interference, these animals were not submitted to any other experimental procedures. The overall difference in survival rate was determined with the Kaplan-Meier test followed by a log-rank test.
Homeostasis model assessment
To perform the Insulin Tolerance Test (ITT), a group of overnight-fasted rats (n = 8 per group) were treated with diacerhein or vehicle only once, 3 hours after sepsis or sham surgery. Insulin (1.5 U/kg) was administered by i.p. injection at 24 hours after surgery, and blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 minutes. Blood glucose was measured by the glucose oxidase method (Optium Xceed; Abbott, Libertyville, IL, USA) as previously described [29]. The constant rate for glucose disappearance (Kitt) was calculated by using the formula 0.693/t1/2. Glucose t1/2 was calculated from the slope of the least-squares analysis of plasma glucose concentrations during the linear decay phase [30].
Tissue extraction and immunoblotting
To perform the tissue analysis 24 hours after CLP or sham surgery, other groups (n = 8 per group) were used, and the animals were treated with diacerhein or vehicle 3 hours after CLP and were then anesthetized with intraperitoneal injection of sodium thiopental and were used 10 to 15 minutes later (that is, as soon as anesthesia was assured by the loss of pedal and corneal reflexes). Five minutes after saline (0.2 ml) or insulin injection (3.8 U/kg i.p.), liver, muscle, and adipose tissue were removed, minced coarsely, and homogenized immediately in extraction buffer, as described elsewhere. NF-κB p50 activation was determined in nuclear extracts from liver, muscle, and adipose tissue. The whole-tissue extracts were subjected to SDS-PAGE and immunoblotting, as previously described [31].
ELISA assays
Blood was collected 24 hours after CLP or sham surgery from other groups (n = 8 per group), and IL-1β, IL-6, TNF-α and NF-κB were determined by using commercially available ELISA kits (Pierce Biotechnology Inc., Rockford, IL, USA), following the instructions of the manufacturer.
Statistical analysis
Specific protein bands presented in the blots were quantified with digital densitometry (ScionCorp Inc., Frederick, MD, USA). Means ± SEMs obtained from densitometric scans, area measurements, and the values for blood cytokines and glucose were compared with ANOVA with post hoc test (Bonferroni). A value of P < 0.05 was accepted as statistically significant.
Results
Diacerhein improves survival in septic rats
To test the hypothesis that diacerhein decreases sepsis mortality, we monitored the survival in animals in which sepsis was induced via CLP. Diacerhein (100 mg/kg/day) or placebo was administered by gavage 3 hours after surgery and in sham-operated animals. No deaths occurred in the sham-operated animals, whether or not they had been treated with diacerhein. The survival curves showed a significantly improved survival (P < 0.0001) after diacerhein treatment (Figure 1A).
Figure 1 Effect of diacerhein on survival in CLP sepsis model. (A) Male Wistar rats, 8 weeks old, were given vehicle (Sepsis/Veh (■), n = 15) or diacerhein, 100 mg/kg (Sepsis/Dia (◆), n = 15), 3 hours and once a day after CLP. Survival of the rats was monitored at intervals of 12 hours for 15 days. The overall difference in survival rate between the groups with and without diacerhein was significant (P < 0.05).
As shown in Figure 2A, septic animals were more insulin resistant than sham-operated rats, fasting plasma glucose was higher in septic rats than in the control group, and diacerhein treatment reduced both of these levels. As depicted in Figure 2B, the plasma glucose disappearance rates measured during the insulin tolerance test (Kitt), were lower in septic animals, and diacerhein treatment attenuated this alteration. Diacerhein treatment had no effect on glucose tolerance in the sham group. Taken together, these data suggest that diacerhein improves sepsis-induced insulin resistance.
Figure 2 Fasting blood glucose (A) and glucose disappearance rate (B). Data are presented as mean and SD of six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). C, Sham/Vehicle; ShD, Sham/Diacerhein; VEH, Sepsis/Vehicle; DIA, Sepsis/Diacerhein.
Effect of diacerhein on serum levels of IL-1β, IL-6, and TNF-α
IL-1β, IL-6, and TNF-α serum levels were examined in the four groups studied. As expected, cytokine levels in the septic rats were higher than in the sham-operated rats. After diacerhein treatment, a significant decrease was found in IL-1β (Figure 3A), IL-6 (Figure 3B), and TNF-α (Figure 3C) circulating levels.
Figure 3 Serum levels of IL-6 (A), IL-6 (B), and TNF-α (C). Data are presented as mean and SD of six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). C, Sham/Vehicle; ShD, Sham/Diacerhein; VEH, Sepsis/Vehicle; DIA, Sepsis/Diacerhein. Representative blots show total and insulin-induced protein expression.
Diacerhein improves insulin signaling in septic animals
We then examined the effects of diacerhein administration on the insulin signaling pathway in its main target tissues. In the sepsis group, insulin-induced IR and IRS-1 tyrosine phosphorylation were decreased in liver, muscle, and adipose tissue when compared with those in sham rats, and these alterations were attenuated by diacerhein (Figure 4A to 4C). Also, a decrease was found in insulin-induced Akt serine phosphorylation in liver, muscle, and adipose tissue of septic animals when compared with sham rats, and diacerhein was able to increase Akt phosphorylation (Figure 4A to 4C). The modulation in IR, IRS-1 and Akt phosphorylation induced by sepsis was independent of changes in tissue protein expression (Figure 4A to 4C). The protein concentrations of IR, IRS-1, and Akt did not change among the groups. Equal protein loading in the gels was confirmed by repeated probing of the membranes with an anti-β-actin antibody (lower panels).
Figure 4 Effects of diacerhein treatment on insulin signaling in the CLP rat. Representative blots show total protein expression and insulin-induced tyrosine phosphorylation of IRβ, IRS1, and serine phosphorylation of Akt in liver (A), adipose tissue (B), and muscle (C) of sham and septic rats. Data are presented as mean ± SEM from six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). IB, immunoblot; Sham, Sham/Vehicle; Sham+Dia, Sham/Diacerhein; Sepsis, Sepsis/Vehicle; Sepsis+Dia: Sepsis/Diacerhein.
Diacerhein attenuates sepsis-induced inflammatory changes
During sepsis, the activation of proinflammatory signaling involves upregulation of intracellular inflammatory pathways, such as the IKKβ and the JNK pathways. We examined the antiinflammatory effects of diacerhein on the IKK/NF-κB pathway by monitoring the main function of IKK phosphorylation and degradation of the NF-κB inhibitor (IκBα) [32]. NF-κB was monitored through analysis of NF-κB p65 nuclear expression. As expected, IKKβ and IκB phosphorylation were increased in liver, muscle and adipose tissue of septic animals. When treated with diacerhein, septic rats showed a reduction in IKKβ and IκB phosphorylation in all tissues studied (Figure 5A through C). Likewise, we assessed the nuclear translocation of NF-κB p65 and, as expected, in nuclear tissue extracts from treated septic rats, we detected reduced expression of NF-κB p65 compared with the nontreated group (Figure 5A through C).
Figure 5 Effects of diacerhein treatment on the IKK/NF-κB pathway signaling in the CLP rat. Representative blots show the IKKβ, IκB phosphorylation, and protein expression of NF-κB in liver (A), adipose tissue (B), and muscle (C) of sham and septic rats. Blots were stripped and reprobed with β-actin (lower panels) to confirm equal loading of proteins. Data are presented as mean ± SEM from six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). IB, immunoblot; Sham, Sham/Vehicle; Sham+Dia, Sham/Diacerhein; Sepsis, Sepsis/Vehicle; Sepsis+Dia, Sepsis/Diacerhein.
JNK activation was determined by monitoring phosphorylation of JNK1 and total expression of this protein. JNK phosphorylation in liver, muscle, and adipose tissue was increased in septic animals, and diacerhein induced a downmodulation in the phosphorylation of this serine kinase in liver and adipose tissue (Figure 6A through C). We also investigated Ser307 phosphorylation of IRS-1 in the three tissues from the four groups of rats. Ser307 phosphorylation was induced by sepsis, and the diacerhein treatment attenuated this alteration (Figure 6A through C).
Figure 6 Effects of diacerhein treatment on the JNK pathway signaling in the CLP rat. Representative blots show the JNK phosphorylation, total protein expression of JNK, and serine 307 phosphorylation of IRS1 in liver (A), adipose tissue (B), and muscle (C) of sham and septic rats. Blots were stripped and reprobed with β-actin (lower panels) to confirm equal loading of proteins. Data are presented as mean ± SEM from six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). IB, immunoblot; Sham, Sham/Vehicle; Sham+Dia, Sham/Diacerhein; Sepsis, Sepsis/Vehicle; Sepsis+Dia, Sepsis/Diacerhein.
Previous studies showed that sepsis is also characterized by endoplasmic reticulum (ER) stress. It is clear that ER stress can also induce activation of JNK and IKKβ. We therefore investigated the effect of sepsis (treated or nontreated with diacerhein) on proteins that reflect ER stress. Our data showed that sepsis induced ER stress, with activation of the membrane kinase PERK (PKR-like endoplasmic reticulum kinase) and its substrate eIF2α (eukaryotic translation initiation factor 2α), and increased the expression of IRE1 (Figure 7A through C). Treatment with diacerhein significantly reduced the activation of IRE1, PERK, and its substrate eIF2α (Figure 7A through C). Also, we measured caspase 3, a critical effector molecule of cell death, and observed that diacerhein inhibited caspase 3 activation in liver and adipose tissue (Figure 7A through C).
Figure 7 Effects of diacerhein treatment on proteins that reflect ER stress in the CLP rat. Representative blots show the PERK, eIF2α phosphorylation, and IRE-1 expression in liver (A), adipose tissue (B), and muscle (C) of sham and septic rats. Blots were stripped and reprobed with β-actin (lower panels) to confirm equal loading of proteins. Data are presented as mean ± SEM from six to eight rats per group. *P < 0.05 versus Sham/Vehicle; #P < 0.05 (Sepsis/Veh versus Sepsis/Dia). IB, immunoblot; Sham, Sham/Vehicle; Sham+Dia, Sham/Diacerhein; Sepsis, Sepsis/Vehicle; Sepsis+Dia, Sepsis/Diacerhein.
Discussion
In the present study, we demonstrated that administration of the antiinflammatory diacerhein improved survival during peritoneal-induced sepsis, with a significant effect on insulin sensitivity. In addition, this drug promoted downregulation of proinflammatory signaling cascades that culminate in the transcription of immunomodulatory factors such as interleukins and TNF-α. Our data show that diacerhein is able to attenuate increased levels of IL-1β, IL-6, and TNF-α, and reduce insulin resistance, as demonstrated by the insulin tolerance test. The improvement in insulin sensitivity was probably due to the increased IRS-1-associated PI3-kinase activity and Akt phosphorylation.
In recent years, studies have shown a direct link between metabolic and immune signaling systems in different conditions of insulin resistance [9,33-35]. We and others previously demonstrated that activation of inflammatory signaling through IKKβ and JNK is triggered in metabolic disorders and that this activation culminates in an increase in proinflammatory gene expression, which may play critical roles in insulin resistance [31,35,36].
Our data show that diacerhein decreases activation of the IKK/IκB/NF-κB pathway, a modulation that may play a role in the attenuated expression of inflammatory mediators in response to a septic insult. IKK pathway activation increases serine phosphorylation of IR and IRS-1, inducing insulin resistance [37,38]. It has also been proposed that increased IKK activity can inhibit insulin-stimulated PI3-kinase activity [39]. In this respect, the capacity of insulin to stimulate PI3-kinase activity and Akt phosphorylation was highly improved in septic animals treated with diacerhein.
Enhanced NF-κB activation is associated with a poorer outcome in sepsis [40-42]. NF-κB nuclear translocation induces transcription of IL-1β, IL-6, and TNF-α [32,43]. The picture of hyperglycemia and insulin resistance observed in sepsis, often referred to as "stress diabetes," reflects the activation of signaling pathways and hyperexpression of inflammatory mediators that inhibit insulin action [44]. In this regard, the inhibition of NF-κB activation explains the reduced serum levels of TNF-α, IL-1β, and IL-6 in diacerhein-treated animals and, consequently, the improvement in the sepsis-induced insulin-resistance process.
Another mechanism involved in the host response to sepsis is activation of the proinflammatory JNK pathway. Several studies suggest that JNK contributes to insulin resistance. Our data show that diacerhein inhibits JNK phosphorylation in septic rats and indicate that the beneficial effects of this drug in improving survival and reducing insulin resistance are mediated by different pathways. Because many inflammatory pathways are triggered in sepsis, merely blocking a single component is likely to be insufficient to halt the process [45,46]. Indeed, therapies modulating entire families of mediators seem to be more efficacious [45,47].
Here we observed that sepsis led to serine phosphorylation of IRS-1, and diacerhein reduced this phenomenon in three insulin-target tissues. Thus, negative modulators of the intracellular cascade triggered by insulin, such as JNK and IKK, are partly responsible for the establishment of insulin resistance and represent potential therapeutic targets for sepsis-induced insulin resistance.
One mechanism that, based on newly emerging data, appears to have a central role in the activation of inflammatory pathways is endoplasmic reticulum (ER) stress [48]. If ER stress continues for a certain period, then programmed cell death is triggered. This response has a close relation to sepsis, because sepsis generates conditions that increase demands on the ER. In the present study, we showed that diacerhein strongly inhibited phosphorylation of PERK and its substrate eIF2α, as well as IRE1α expression, suggesting that this drug can attenuate ER stress induced by sepsis.
ER stress plays a central role in the activation of inflammatory signaling. In both in vitro and in vivo studies, ER stress leads to activation of JNK and thus contributes to insulin resistance [49-51]. Interestingly, ER stress also activates IKK, and thus may represent a common mechanism for the activation of these two important signaling pathways [52]. Acute inflammation, oxidative stress, and ER stress seem to contribute to the association of sepsis with insulin resistance. The pharmacologic attenuation of all the aforementioned stresses leads to improved insulin sensitivity and consequently to favorable sepsis outcomes.
An initial investigation by Van den Berghe and colleagues [53] suggested that controlling blood glucose levels by intensive insulin therapy decreased mortality and morbidity after surgery on critically ill patients. Moreover, intensive insulin therapy halved the prevalence of bloodstream infections and prolonged inflammation, showing the antiinflammatory action of insulin. These findings were supported by further studies demonstrating that insulin has antiinflammatory effects via activation of the PI3K-Akt pathway [54,55]. In addition, insulin has potent acute antiinflammatory effects, including reductions in intranuclear NF-κB [56] and several key mediators of oxidative stress [57].
It is important to mention that the reduced inflammatory response induced by diacerhein probably played a central role in improving insulin signaling, but the improvement in this pathway may also have a role in the improved survival. It is possible that reduced insulin signaling through the IRSs/PI3K/Akt pathways in sepsis may contribute to multiorgan failure by activation of apoptosis [58]. Additionally, studies have shown that prevention of apoptosis may be a potential treatment for sepsis in humans [59-61]. Diacerhein, by improving insulin-induced PI3K and Akt, may play a critical role in protection from apoptosis in sepsis. In accordance with this, our data showed that caspase 3, which was increased in tissues of septic animals, decreased after diacerhein treatment.
Growing evidence suggests that the PI3K/Akt pathway plays an important role as a negative regulator of innate immune response by counteracting excessive production of proinflammatory mediators. The PI3K-Akt pathway has been shown to regulate negatively NF-κB and the expression of inflammatory genes [62-64]. Inhibition of the PI3K-Akt pathway enhances LPS-induced TNF-α and TF gene expression [55], and activates the mitogen-activated protein kinase pathways (ERK1/2, p38, and JNK) as well as the downstream target AP-1 [54]. Also relevant in this regard is a report showing that PI3K-knockout mice fail to respond to LPS [65]. In this context, it is possible that restoration of this pathway, induced by diacerhein, may contribute to the antiinflammatory effect of this drug.
Conversely, the role of intensive insulin therapy in patients with severe sepsis is uncertain, because the beneficial effects of insulin may be overcome by the increased risk of serious adverse events related to hypoglycemia [66]. We propose that drugs capable of reversing sepsis-induced insulin resistance, in the context of maintenance of adequate glycemic control, may be a potential therapy for sepsis. In this regard, diacerhein may be a potential therapeutic strategy for sepsis, with a significant effect on insulin sensitivity and insulin signaling in peripheral tissues.
Our data show that administration of the antiinflammatory diacerhein in septic animals increased survival, with significant effects on insulin sensitivity and insulin signaling in peripheral tissues. The treatment also reduced NF-κB activation, in association with upstream JNK and IKK activation, decreased serum levels of cytokines, and improved ER stress. Our results indicate that diacerhein treatment attenuates insulin resistance in sepsis, and in parallel modulates inflammatory pathways.
Conclusions
This is the first report demonstrating that diacerhein improves survival and insulin signaling, which is blunted in sepsis, and in parallel, attenuates the inflammatory response. On the basis of its efficacy, safety profile, and rare side effects, this drug may be an alternative therapy for management of insulin resistance in sepsis.
Key messages
• Hyperglycemia and insulin resistance have been associated with poorer outcomes in sepsis, and tight glycemic control through insulin therapy has been shown to reduce morbidity and mortality rates.
• However, insulin-induced hypoglycemia may counteract the beneficial effects of aggressive insulin therapy in patients with severe sepsis.
• These data suggest that the ideal drug to improve survival in sepsis should reduce overreaction by the inflammatory response and, in parallel, should improve the insulin signaling pathway, without inducing hypoglycemia.
• Diacerhein improved survival during peritoneal-induced sepsis, with a significant effect on insulin sensitivity. In addition, this drug promoted downregulation of proinflammatory signaling cascades, which culminate in the transcription of immunomodulatory factors such as interleukins and TNF-α.
• The effect of diacerhein on suppression of the inflammatory response may play a central role in the regulation of insulin signaling and survival in septic insult. Diacerhein may represent a potential new approach to sepsis treatment.
Abbreviations
Akt: protein kinase B (PKB); AP-1: activator protein 1; CLP: cecal ligation and puncture; DIA: diacerhein; eIF2α: eukaryotic translation initiation factor 2α; ELISA: enzyme-linked immunosorbent assay; IKK: I kappa B kinase; IL1β: interleukin 1β; IR: insulin receptor; IRE1: inositol-requiring enzyme 1; IRS1: insulin receptor substrate 1; ITT: insulin tolerance test; JNK: c-Jun NH2-terminal kinase; KITT: glucose disappearance rate; LPS: lipopolysaccharide; NF-κB: nuclear factor kappa B; PERK: PKR-like endoplasmic reticulum kinase; PI3K: phosphatidylinositol 3-kinase; SDS-PAGE: sodium dodecylsulfate polyacrylamide gel; ShD: sham/diacerhein; TNF-α: tumor necrosis factor-α; UPR: unfolded protein response; Veh: vehicle.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KLC carried out the studies, analyzed data, and drafted the manuscript; ACAC carried out the studies and drafted the manuscript; FCM performed the experiments; BMC performed the experiments and drafted the manuscript; and DG carried out ELISA measurements. JBCC participated in the design of the study, MJAS designed studies, analyzed data, and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We acknowledge the excellent technical assistance of Luis Janieri, Jósimo Pinheiro, and Ramon Zorzeto. This study was supported by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo) and INCT-CNPq (Instituto Nacional de Ciência e Tecnologia-Conselho Nacional de Desenvolvimento Científico e Tecnológico).
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Oncol RepOncol. RepOROncology Reports1021-335X1791-2431D.A. Spandidos 2215981610.3892/or.2011.1580or-27-04-1090ArticlesAlpinetin suppresses proliferation of human hepatoma cells by the activation of MKK7 and elevates sensitization to cis-diammined dichloridoplatium TANG BO 1*DU JIAN 2*WANG JINGWEN 3TAN GUANG 2GAO ZHENMING 1WANG ZHONGYU 2WANG LIMING 11 Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, Dalian 1160272 Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, P.R. China3 Department of Cell and Molecular Biology, Uppsala University, Uppsala, SwedenCorrespondence to: Dr Liming Wang, Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, No. 467 Zhongshan Road, Dalian 116027, P.R. China, E-mail: [email protected]. Dr Zhongyu Wang, Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, P.R. China, E-mail: [email protected]* Contributed equally
4 2012 6 12 2011 4 2012 6 12 2011 27 4 1090 1096 13 10 2011 17 11 2011 Copyright © 2012, Spandidos Publications2012This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.Alpinetin is a type of novel plant flavonoid derived from Alpinia katsumadai Hayata, found to possess strong anti-hepatoma effects. However, the detailed antitumor mechanism of Alpinetin remains unclear. Mitogen-activated protein kinase kinase-7 (MKK7) can regulate cellular growth, differentiation and apoptosis. The aim of this study was to investigate the role of MKK7 in the anti-hepatoma effect mediated by Alpinetin. HepG2 cells were treated with Alpinetin at various doses and for different times, and the levels of phosphorylated MKK7 (p-MKK7) and total MKK7 were tested by RT-PCR and Western blotting. Following transient transfection with RNA interference, cell viability and cell cycle stage were determined using methyl thiazolyl tetrazolium assay and flow cytometry, in order to assess the antitumor action of Alpinetin. In addition, chemosensitization to cis-diammined dichloridoplatium (CDDP) by Alpinetin was assessed by cell counting array and the cell growth inhibitory rate was calculated. The results showed that Alpinetin suppressed HepG2 cell proliferation and arrested cells in the G0/G1 phase by up-regulating the expression levels of p-MKK7. On the contrary, inhibiting the expression of MKK7 reversed the antitumor effect of Alpinetin. Moreover, Alpinetin enhanced the sensitivity of HepG2 hepatoma cells to the chemotherapeutic agent CDDP. Taken together, our studies indicate that activation of MKK7 mediates the anti-hepatoma effect of Alpinetin. MKK7 may be a putative target for molecular therapy against hepatoma and Alpinetin could serve as a potential agent for the development of hepatoma therapy.
alpinetinhepatocellular carcinomaproliferationMKK7cis-diammined dichloridoplatium
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Introduction
Hepatocellular carcinoma (HCC) represents the fifth most prevalent cancer in terms of incidence. In addition, HCC is the third most common cause of cancer-related death in the world, resulting in more than 600,000 deaths per year. Like other solid tumors, surgical treatment is the main treatment option, but only 10–30% patients are eligible for radical treatment because of difficult early diagnosis and chronic liver disease, and it is also hard to efficiently treat live cancer by chemotherapy and radiotherapy (1–5).
Alpinia katsumadai Hayata, as a traditional medicine with low toxicity, has been shown to have antitumor and anti-oxidation effects (6,7). Alpinetin, (7-hydroxy-5-methoxyflavanone, molecular formula C16H14O4, molecular weight 270.28) a kind of novel plant-derived flavonoid, is the major active ingredient of Alpinia katsumadai Hayata (8,9). Previous studies have proved that Alpinetin has a strong antitumor effect by suppressing proliferation of tumor cells. The anti-cancer capability of Alpinetin has also been confirmed in the treatment of various tumors, such as breast cancer, hepatoma, leukemia, carcinoma of colon and pulmonary cancer (7,10–12). However, the detailed antitumor mechanisms of Alpinetin remain largely unknown.
c-Jun N-terminal kinase (JNK) signal pathway is one of three paralleled pathways at the center of the mitogen-activated protein kinase (MAPK) pathways and plays an important role in regulating organized cellular responses, such as proliferation, differentiation or apoptosis (13–16). MKK4 and MKK7, which is also called c-jun N-terminal kinase kinase 2 (JNKK2) or stress-activated protein kinase/extracellular signal-regulated protein kinase kinase 2 (SEK2), are two upstream kinases of JNK pathway and directly activate the JNKs by phosphorylating the Tyr and Thr residue (17). Unlike other MAPK subfamilies, the monophosphorylation of MKK7 on the Thr residue is sufficient and specific to activate JNK pathway which, in turn, activates substrates like transcription factors or pro-apoptotic proteins (18). In addition, studies on pro-inflammatory cytokines also showed that only MKK7 is essential for JNK activation (19,20). Given its important role in JNK activity, it is necessary to illustrate the role of MKK7 in the anti-hepatoma of Alpinetin.
The aim of this study was to determine the action of Alpinetin in the anti-hepatoma proliferation effect and its influence on cell cycle in vitro. We also investigated whether Alpinetin can sensitize HepG2 hepatoma cells to CDDP. The possible signal transduction pathway involved in Alpinetin-induced inhibition of human hepatoma cell proliferation was also studied.
Materials and methods
Cell culture, antibodies and reagents
Human HepG2 hepatic cancer cell line and rat N1-S1 hepatic cancer cell line were purchased from American Type Culture Collection (ATCC), cultured in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal bovine serum (FBS) and maintained at 37˚C in 5% CO2. Alpinetin (≥98% purity) was obtained from the National Institute for Food and Drug Control (Beijing, China). Phospho-MKK4, MKK4, phospho-MKK7, MKK7 and GAPDH antibodies were from Cell Signaling Technology, Inc. (USA). Lipofectamine 2000 was from Invitrogen Corp. (USA). Propidium iodide (PI) was from Sigma-Aldrigh (USA). Reverse transcription polymerase chain reaction (RT-PCR) kit and primers were from Takara (Japan).
Cell proliferation assay
Cell viability was determined using methyl thiazolyl terazolium (Sigma) assay. Cells in logarithmic phase were seeded in the 96-well plate and then treated with Alpinetin. MTT (20 μl) (0.5 mg/ml) was added to each well and the cells were incubated at 37°C for 4 h to allow the yellow dye to be transformed into blue crystals. The medium was removed and 200 μl of dimethyl sulfoxide (DMSO) (Sigma) was added to each well to dissolve the dark blue crystals. Finally, the optical density was measured with a microtiter plate reader at 570 nm. Six replicates were prepared for each condition.
RNA extraction and RT-PCR assay
Total RNA from hepatic cancer cells was prepared using RNAisoTM Plus (Takara) according to the routine method. The concentration of total RNA samples was valuated with spectrophotometer (Beckman Coulter, Inc., USA). The specific primers for GAPDH and MKK7 were designed and synthesized by Guangzhou Ribobio Co., Ltd. (China). The primers for amplification were as follows: GAPDH, forward primer, 5′-GAACGGGAAGCT CACTGG-3′, reverse primer, 5′-GCCTGCTTCACCACCT TCT-3′; MKK7, forward primer, 5′-CCCCGTAAAATCAC AAAGAAAATCC-3′, reverse primer, 5′-GGCGGACACA CACTCATAAAACAGA-3′. The RT-PCR was performed using an RT-PCR kit according to the protocols of the manufacturer.
Small interfering RNA (siRNA) transfection
Cells (5×105 cells/2 ml/well) were plated at 60% confluence in a 6-well plate in RPMI-1640 without antibiotics. After 24 h, siRNA or negative control oligonucleotide was transfected into cells with Lipofectamine 2000 according to the instructions of the manufacturer’s. After 4–6 h of incubation in the CO2 incubator, the medium containing siRNA-Lipofectamine 2000 complexes was replaced with fresh RPMI-1640 containing 10% FCS and the cells were cultured for further experiment. All siRNAs were obtained from Guangzhou Ribobio Co., Ltd. and the three specific sequences for silencing were: human MKK7 siRNA-1, sense 5′-GGAAGAGACCAAA GUAUAAdTdT-3′, and anti-sense 3′-dTdTCCUUCUCUGG UUUCAUAUU-5′; siRNA-2, sense 5′-CCUACAUCGUG CAGUGCUUdTdT-3′, and anti-sense 3′-dTdTGGAUGUA GCACGUCACGAA-5′; siRNA-3, sense 5′-GCAUUGAGAU UGACCAGAAdTdT-3′, and anti-sense 3′-dTdTCGUAACU CUAACUGGUCUU-5′. The effect of RNA interference was checked by RT-PCR and Western blot analysis.
Western blot assay
Cells were washed once with ice-cold phosphate-buffered saline (PBS) containing 100 mM sodium orthovanadate and solubilized in lysis buffer [50 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 100 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% Nonidet P-40, 5 mM cocktail; pH 7.4]. After centrifugation at 12,000 × g for 20 min, the supernatant was collected. After determination of the protein concentration using BCA kit assay (Pierce, USA). β-mercaptoethanol and bromophenol blue were added to the sample buffer for electrophoresis. The protein was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and trans-blotted to polyvinylidene difluoride membranes (Bio-Rad Laboratories, USA). The blots were incubated at 4˚C overnight with antibodies, and the resulting bands were detected using enhanced chemiluminescence. Intensities of the bands were semi-quantified using an image-analysis system.
Analysis of cell cycle by flow cytometry
HepG2 cells were grown at 48 h confluence in 6-well plates. Transfection was done for 24 h with Lipofectamine 2000 follow by treatment with Alpinetin (60 μg/ml) for 24 h. The cells were then pelleted by centrifugation and washed twice with PBS. Then, the cell pellets were suspended in 5 ml ice-cold 70% ethanol at 4˚C. After 1 h, the fixed cells were spun by centrifugation and the pellets were washed with PBS. After resuspension with 1 ml PI integration staining solution, the cells were incubated with RNase A (10 mg/lt), PI (50 mg/lt), 1% Triton X-100 and sodium citrate (1 g/lt) shaken for 30 min at 37˚C in the dark. The stained cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, USA).
Examination of transfection efficiency by fluorescence microscopy
HepG2 cells (5×105) were plated in 6-well plates with poly-lysine-coated cover slips and cultured for 24 h, and then cells were transfected with Lipofectamine 2000. After 12 h, cells on cover slips were washed twice with PBS. Cell nuclei were stained with DAPI (Sigma), and fluorescent images were checked using a fluorescent microscope (Leica Microsystems, Germany).
Statistical analysis
SPSS 16.0 statistical software was used for statistical analysis. Values were shown as mean ± SD. Statistical analysis was carried out using Student's t-test. Differences between groups were identified as statistically significant at p<0.05.
Results
Alpinetin inhibits growth of both human HepG2 and rat N1-S1 hepatic cancer cells
To investigate the anti-hepatoma effect, the two cell lines were treated with Alpinetin for different doses and times and MTT assay was performed to determine cell viability. Results showed that the viability of Alpinetin-treated cells greatly decreased with increased drug dose or treatment time (Fig. 1). Furthermore, the effect of inhibition in hepatoma cells increased proportionately when treated with Alpinetin at a range from 20–80 μg/ml and the effective dosage of inhibition is 60 μg/ml. Our data indicate that proliferation of hepatoma cells were suppressed in a dose- and time-dependent manner by Alpinetin.
Alpinetin increases phosphorylation of MKK7 in human hepatic cancer cells
To explore the role of MKK7 in the anti-hepatoma effect of Alpinetin, we checked the levels of MKK7 and p-MKK7 in HepG2 hepatoma cells treated with different concentration of Alpinetin for 24 h by RT-PCR and Western blot assay (Fig. 2A and B). Furthermore, as MKK4 is also able to regulate the JNK pathway, the expressions of MKK4 and p-MKK4 were examined by Western blot assay simultaneously (Fig. 2B). Then the results of Western blot assay were further semi-quantitatively estimated using Gel-Pro Analyzer 4.0 software (Fig. 2C). Our results demonstrated that the expression of total MKK4/7and p-MKK4 remained relatively unchanged for cells treated at different concentrations of Alpinetin. The level of p-MKK7 increased in a dose-dependent manner and phosphorylation increased evidently when treated with Alpinetin (60 μg/ml) for 24 h. The results indicated that Alpinetin elevated the expression level of p-MKK7 (but not total MKK7 or MKK4) which may be responsible for its ability to suppress proliferation of HepG2 cells.
MKK7 siRNA-3 is optimal for silencing the expression of MKK7
Three siRNAs (siRNA-1, -2 and -3) were planned to silence the expression of MKK7 in HepG2 hepatoma cells. Three siRNAs and FAM-negative control oligonucleotide were transfected into HepG2 cells at 57 nM for 24 h and transfection efficiency was examined by fluorescence microscopy (Fig. 3), RT-PCR and Western blot assays (Fig. 4). To explore the optimal interference conditions, a variety of doses of siRNA-3 were transfected into HepG2 cells for various durations. The results of our study showed that MKK7 siRNA-3 was more efficient in silencing the expression of MKK7 than others (Fig. 4A). Furthermore, we found that transfection with 57 nM siRNA-3 for 24 h notably declined the expression of MKK7, and this silencing effect lasted no less than 72 h (Fig. 4B and C). The result suggested that transfecting with 57 nM siRNA-3 for 24 h was the most favorable condition for MKK7 silencing.
Inhibition of MKK7 reduced the ability of Alpinetin to anti-proliferation in vitro
To further confirm the role of MKK7 in the anti-hepatoma effect of Alpinetin, we transfected HepG2 cells with siRNA-3 for 24 h to silence the expression of MKK7. After treatment with Alpinetin for 24 h at 60 μg/ml, Western blot assay was performed to determine the change in the expression of MKK7 and p-MKK7 (Fig. 5) and HepG2 cell growth was calculated by an MTT assay (Fig. 6). Our data found that cell viability in the MKK7 siRNA-3 + Alpinetin group was higher than that in the control siRNA + Alpinetin group. This result revealed that down-regulation of MKK7 by siRNA-3 attenuated the anti-proliferative effect of Alpinetin in vitro.
Down-regulation of MKK7 by siRNA suppresses Alpinetin-induced G0/G1-phase arrest in human hepatoma cells
To further investigate the mechanism by which Alpinetin suppressed hepatoma cells proliferation, HepG2 cells were transfected with siRNA or siRNA negative control and treated with Alpinetin (60 μg/ml) for 24 h, before cell cycle progression was assessed using flow cytometry. The percentage of different treatment groups in G0/G1 phase are shown by histograms (Fig. 7E). The percentage of hepatoma cells in the G0/G1 phase was higher in Alpinetin-treated group than the untreated cells (Fig. 7A and B). The fraction of hepatoma cells in the G0/G1 phase were lower in siRNA transfected group treated by Alpinetin than in Alpinetin-treated group (Fig. 7B and D). Our data imply that the anti-proliferation effect induced by Alpinetin is possibly through the activation of MKK7 pathway, thereby causing G0/G1 phase arrest.
Alpinetin enhances chemosensitivity of HepG2 hepatoma cells to cis-diammined dichloridoplatium (CDDP)
Previous study has reported that activation of JNK and P38/MARK pathway was associated with enhanced chemosensitivity to CDDP in HepG2 hepatoma cells (21). To investigate whether treatment with Alpinetin sensitized HepG2 cells to CDDP, cells were plated in 6-well plates (5×105/well) and cultured for 24 h. Cells were treated in the following groups: control group (untreated, Fig. 8Aa), Alpinetin group (treated with 60 μg/ml Alpinetin for 24 h, Fig. 8Ab), CDDP group (treated with 20 μg/ml CDDP for 24 h, Fig. 8Ac) and Alpinetin + CDDP group (treated with 60 μg/ml Alpinetin and 20 μg/ml CDDP for 24 h, Fig. 8Ad). After above treatment, surviving cells were measured by cell counting (Beckman Coulter, Inc., USA). The cell growth inhibitory rate (GIR) was calculated as the ratio of (number of cells in the control group - number of cells in the treated group) to (number of cells in the control group) × 100% (Fig. 8B). The results demonstrated that the GIR was higher in Alpinetin + CDDP group than that in CDDP group and Alpinetin group (Fig. 8B). In addition, the effect of combined treatment was stronger than the presumed additive effect of Alpinetin and CDDP treatments. Our result indicated that Alpinetin enhances chemosensitivity of HepG2 hepatoma cells to CDDP.
Discussion
In vitro, Alpinetin exerts anti-proliferative activity against various types of tumors such as hepatoma, breast carcinoma and leukemia. Some studies have reported that the antitumor effect of Alpinetin is connected to inhibition of NF-kappaB (11). Our present study also found that Alpinetin showed strong antitumor activity in hepatoma cell lines from both human and rat. However, less is known regarding defined signaling pathways involved in these processes.
The mitogen-activated protein kinase (MAPK) signaling pathways are composed of a large family of protein kinases which allow the cells to respond to exogenous and endogenous stimulus (22–24). These protein kinases are part of cascade reaction of a three-tiered signaling module which consist of MAPKKKs (MKKKs)-MAPKKs (MKKs)-MAPKs. JNK, p38 MAPK and extracellular signal-regulated kinase (ERK) are three major MAPKs and play important roles in regulating organized cellular responses. JNK1 and JNK2 are widely expressed in the tissues and are connected with the development of various cancers (25,26).
As two important members of a three-tiered cascade reaction, MKK4 and MKK7 can phosphorylate distinct JNK activation sites to activate the JNK pathway and regulate cellular growth, differentiation and apoptosis (27). MKK4/7 can be activated through phosphorylation by MKKKs. MKK4/7 form complexes with their upstream kinases via the DVD domain specificity. For instance, mixed linage kinase 3 (MLK3), MEKK1 and TAK1 can interact with MKK4 and MKK7, while DLK specifically binds to MKK7 and MEKK4 to MKK4 (28–32). Apart from this, MKK7 is independent and specific to trigger JNK signal pathway activity while the additional phosphorylation by MKK4 ensures optimal JNK activation (18). It has been reported that MKK7 frequently mediates the antitumor effects of various agents, such as Withanolide D, and Phenethyl isothiocyanate (33,34). Thus, MKK7 is the pivotal factor in our study of the anti-hepatoma mechanisms of Alpinetin. Previous studies have found that Alpinetin suppress the activity of NF-kappaB in various malignant tumors (7). Meanwhile, inhibition of NF-κB activity can induce MKK7/JNK1-dependent apoptosis in human acute myeloid leukaemia cells (35). These studies indicate that the antitumor effect of Alpinetin is related to the activation of MKK7-JNK signaling pathway. The present study showed that Alpinetin suppresses the proliferation of hepatoma cells through the activation of the MKK7-JNK signaling pathway. In addition, a down-regulation of MKK7 expression by RNA interference reduced the phosphorylation level of MKK7 and reversed the anti-hepatoma effect of Alpinetin. Therefore, in view of its key function in inhibiting the proliferation of human hepatoma cells, activation of MKK7 by Alpinetin offers a significant strategy for molecular therapy against hepatoma.
Direct phosphorylation of target proteins by p38 arrests the cells in a G0/G1 phase while ERK1/2 activation has the reverse effect (36). Accumulating evidence suggests that JNK pathway is also a physiologic activator of p38 under certain conditions, resulting in cell cycle arrest (37). In our study, we found that Alpinetin arrested hepatoma cells in G0/G1 through activating MKK7 phosphorylation. In addition, when MKK7 level was down-regulated by siRNA, the inhibitory effect of Alpinetin decreased. Most likely the activation of JNK pathway in our study leads to p38 activation, thereby arresting the cell cycle, but this mechanism needs to be validated with further experiments.
CDDP is a common clinical chemotherapeutic agent, used to treat many malignant solid tumors including hepatocellular carcinoma. Current study has found that the chemosensitivity to CDDP in HepG2 cells can be improved by JNK signal pathway (21). Therefore, we tested the sensitivity of Alpinetin-treated HepG2 cells to CDDP-induced cytotoxicity. Our data indicate that Alpinetin and CDDP have a synergistic inhibitory effect on HepG2 cell growth and proliferation. Our research suggests that the augmentation of CDDP's efficacy by Alpinetin is connected with the activation of the MKK7-JNK signaling pathway. Furthermore, as either a promising chemosensitizer or adjuvant, Alpinetin is worth further investigation, which may bring about the development of a therapeutic regimen combining Alpinetin with CDDP or other chemotherapeutic drugs to treat malignant tumors.
In summary, we have found that activation of MKK7, a specific upstream regulator of JNK signal pathway, mediates the anti-proliferative effect of Alpinetin. Furthermore, the antitumor effect of Alpinetin is found to be responsible for the arrest of hepatoma cell cycle. Taken together, our study suggests that MKK7 is a novel molecular target and combination chemotherapy in hepatoma, while Alpinetin may be a potential traditional Chinese medicine for the future development of hepatoma therapy.
Acknowledgements
This research was supported by National High Technology Research and Development Program (863 Program) funding (2006AA02A309) and the Natural Science Foundation of China (no. 30870719). We also thank Lin Gen (EMBL) for advice on the manuscript, particularly regarding English expressions.
Figure 1 Alpinetin inhibits HepG2 and N1-S1 cell proliferation. (A) HepG2 and N1-S1 cells were treated with Alpinetin at different dosage (0, 20, 40, 60 and 80 μg/ml) for 24 h, and then MTT assay was done to determine cell viability. (B) HepG2 and N1-S1 cells were treated with Alpinetin (60 μg/ml) for 0, 12, 24, 36 and 48 h. The treated cell viability was examined by MTT assay.
Figure 2 Alpinetin increases phosphorylation level of MKK7 in human hepatic cancer cells. (A) HepG2 cells were treated with different concentrations of Alpinetin for 24 h, and then the level of MKK7 mRNA was assessed using RT-PCR assay. (B) p-MKK4/7 and total MKK4/7 levels were determined respectively by Western blot analysis after treatment with various concentrations of Alpinetin for 24 h. (C) The protein expressions of p-MKK4/7 and MKK4/7 were further analyzed using Gel-Pro Analyzer 4.0 software. The changes in the expression level of p-MKK7 and MKK7 were estimated by a polygram.
Figure 3 After transfection with siRNA, HepG2 cells were stained with DAPI. A high efficiency of transfection was verified using a fluorescence microscope.
Figure 4 MKK7 siRNA-3 is optimal in silencing the expression level of MKK7. (A) siRNA-1, -2 and -3 were transfected into HepG2 cells at 57 nM for 24 h, and then the mRNA level of MKK7 was determined by RT-PCR. Results show that siRNA-3 was more efficient in inhibiting the expression of MKK7 than siRNA-1 or -2. (B) HepG2 cells were transfected with siRNA-3 at various doses for different durations. RT-PCR assays were done to determine the interference efficiency. Transfection with 57 nM siRNA-3 for 24 h remarkable decreased the mRNA expression of MKK7, and this effect of interference lasted at least 72 h. (C) Western blot analysis was performed to further verify the interference efficiency. The protein expression of MKK7 was effectively inhibited by treatment with siRNA-3 at 57 nM for 24 h.
Figure 5 MKK7 siRNA-3 inhibits the increased phosphorylation level of MKK7 induced by Alpinetin. (A) The changes in the expression of MKK7 and p-MKK7 were determined using Western blot assay. (B) The results of (A) were analyzed with Gel-Pro Analyzer 4.0 software. The level of p-MKK7 in control siRNA + Alpinetin treated group is higher than that in control siRNA group (*P<0.05); the expression of MKK7 and p-MKK7 were lower in MKK7 siRNA-3 + Alpinetin treated group than that in control siRNA + Alpinetin treated group (**,#P<0.05).
Figure 6 Silencing of MKK7 by siRNA-3 blocks the anti-proliferative effect of Alpinetin. siRNA-3 was tranfected into HepG2 cells for 24 h to down-regulate the expression of MKK7. After treating with Alpinetin (60 μg/ml) for 24 h, HepG2 cell viability was confirmed using the MTT assay. Cell viability in the MKK7 siRNA-3 + Alpinetin group was higher than that in the control siRNA + Alpinetin group (*P<0.05).
Figure 7 Transfection with MKK7 siRNA reverses G0/G1-phase arrest induced by Alpinetin in HepG2 cells. (A-D) After transfection with siRNA for 24 h, HepG2 cells were treated with Alpinetin (60 μg/ml) for 24 h, and then the distribution of cell cycle was determined by flow cytometry. (E) The percentages of G0/G1 phrase cell in different groups are shown by columns. The percentage of HepG2 cells in the G0/G1 phase was higher in Alpinetin-treated group (B) than in the control group (A) (*P<0.05). Compared with Alpinetin-treated group, the percentage in the siRNA transfected plus Alpinetin-treated group (D) was lower than the Alpinetin-treated group (#P<0.05).
Figure 8 Treatment with Alpinetin increases the sensitivity of HepaG2 cells to CDDP. (A) HepaG2 cells were plated in 6-well plates (5×105 cells/well) and cultured for 24 h. Cells were treated as following four groups: (a) control group (untreated); (b) Alpinetin group (treated with 60 μg/ml Alpinetin for 24 h); (c) CDDP group (treated with 20 μg/ml CDDP for 24 h); (d) Alpinetin + CDDP group (treated with 60 μg/ml Alpinetin and 20 μg/ml CDDP for 24 h). (B) The cell growth inhibitory rate (GIR) = [(number of cells in the control group - number of cells in the treated group)/(number of cells in the control group)] × 100%. The GIR in Alpinetin + CDDP group was higher than that in the respective CDDP (*P<0.01) and Alpinetin (*P<0.01) groups; furthermore, the effect of drug combination was higher than the supposed additive effect of Alpinetin and CDDP treatments.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23469073PONE-D-12-2787310.1371/journal.pone.0057818Research ArticleBiologyGeneticsCancer GeneticsMolecular Cell BiologyCell DeathCell GrowthMedicineGastroenterology and HepatologyPancreasOncologyCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerBasic Cancer ResearchNet Expression Inhibits the Growth of Pancreatic Ductal Adenocarcinoma Cell PL45 In Vitro and In Vivo
Net Inhibits the Growth of Pancreatic Cancer CellLi Baiwen
1
Wan Xinjian
1
Zhu Qi
2
Li Lei
1
Zeng Yue
1
Hu Duanmin
2
Qian Yueqin
1
Lu Lungen
1
Wang Xingpeng
1
*
Meng Xiangjun
1
*
1
Department of Gastroenterology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
2
Department of Gastroenterology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
Nie Daotai Editor
Southern Illinois University School of Medicine, United States of America
* E-mail: [email protected] (XW); [email protected] (XM)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: BL X. Wang XM. Performed the experiments: BL LL YZ YQ DH. Analyzed the data: X. Wang X. Wan XM QZ. Contributed reagents/materials/analysis tools: X. Wang XM. Wrote the paper: BL XM X. Wang LL.
2013 28 2 2013 8 2 e5781812 9 2012 26 1 2013 © 2013 Li et al2013Li et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Pancreatic ductal adenocarcinoma has a poor prognosis due to late diagnosis and a lack of effective therapeutic options. Thus, it is important to better understand its molecular mechanisms and to develop more effective treatments for the disease. The ternary complex factor Net, which exerts its strong inhibitory function on transcription of proto-oncogene gene c-fos by forming ternary complexes with a second transcription factor, has been suspected of being involved in pancreatic cancer and other tumors biology. In this study, we found that the majority of pancreatic ductal adenocarcinoma tissues and cell lines had weak or no expression of Net, whereas significantly high level of Net expression occurred in paired adjacent normal tissues we studied. Furthermore, using in vitro and in vivo model systems, we found that overexpression of Net inhibited cell growth and survival and induced cell apoptosis in human pancreatic ductal adenocarcinoma cell PL45; the mechanisms by which Net inhibited the cell cycle progression were mainly through P21-Cyclin D1/CDK4 Pathway. Our data thus suggested that Net might play an important role in pancreatic carcinogenesis, possibly by acting as a tumor suppressor gene.
This work is supported by Natural Science Fund of Shanghai of China (#11ZR1429000,http://www.stcsm.gov.cn) and Pujiang Talent Program Fund (#10PJ1408500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Pancreatic cancer is a devastating life threatening causes in the world [1], [2]. The 5-year survival rate is only 3% to 5% and has remained unchanged for the past three decades, although research progresses have been made in early diagnosis and treatments [3], [4], [5]. Therefore further understanding of its biological properties at a molecular level and identifying novel molecular targets for early diagnosis and therapeutic intervention is urgently required.
Net is a member of the ternary complex factor (TCF) subfamily of ETS-domain transcription factors [6], [7], [8]. The important function of the TCFs is to activate the immediate early genes by forming ternary complexes with a second transcription factor, a serum response factor (SRF) at serum response elements (SREs) found in the promoters of target genes [9], [10], [11]. Proto-oncogene c-fos which is upregulated rapidly upon stimulution from cells in the setting of various mitogens is fully characterized as a target of TCF. SRF has been found to be constitutively bound to the SRE in the c-fos promoter and hence be able to recruit Net [8], [12], [13], [14], [15].
Studies have suggested that the activation of the mitogen activated protein kinases (MAPKs) pathway leads to activation of Net as well as other TCFs [10], [16]. Moreover, Net is different from other TCFs by exerting its strong inhibitory function on transcription of proto-oncogene gene c-fos [17], [18], [19], [20]. C-fos, as an important component of the transcription factor activating protein 1 (AP-1), is involved in a wide variety of cellular processes, including cell proliferation, gene expression, differentiation, cell death, survival and tumorigenesis [21], [22], [23]. The transcription of c-fos is tightly controlled in normal conditions and it’s abnormal expression contributes to various phenotypical changes. Previous study suggested impaired serum inducibility of c-fos is a feature of senescent human fibroblasts [24], and its overexpression is a suggestive marker for progression of skin tumors tumorigenicity [25], [26]. However, the role of Net in pancreatic ductal adenocarcinoma is poorly understood to data.
Our previous preliminary study found that Net overexpression inhibited synthesis of the proto-oncogene c-fos in pancreatic carcinoma BxPC-3 cell [27]. In the present study, we examined the expression of Net in human pancreatic ductal adenocarcinoma and paired normal adjacent tissues, as well as in pancreatic cancer cell lines, and investigated the effect of Net expression on pancreatic ductal adenocarcinoma cell growth, proliferation and apoptosis in vitro and in vivo assays.
Materials and Methods
Tumour Samples and Cell Lines
The study group consisted of 21 men and 15 women with pancreatic ductal adenocarcinoma (median age 64; range 42–78 years) who underwent surgical treatment in the Department of Surgery of Shanghai First People’s Hospital and Pancreatic Disease Center of Shanghai Jiaotong University from January 2009 to March 2011. The expression of Net was evaluated on fresh pancreatic ductal adenocarcinoma tissues (PDATs) and paired normal adjacent tissues (NATs, >2 cm from tumor tissues). All the patients examined in this study had not received preoperative chemoradiation therapy. Surgical staging of tumors was performed according to the American Joint Committee on Cancer tumor-nodes metastasis system and International Union Against Cancer (2002). The clinicopathologic characteristics of patients were obtained from clinical notes and hospital computer database.
All studies were performed after written consent was obtained, according to the guidelines of the Institutional Review Board and the ethical committee of Shanghai First People’s Hospital (the approval number code: SYXK(Hu) 2009-0086). Human pancreatic ductal epithelial cell line hTERT-HPNE, pancreatic adenocarcinoma cell lines PL45, SW1990 and Panc-1 were commercially obtained from Institute of biochemistry and cell biology, SIBS, Chinese Academy of Science, and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin.
Immunohistochemistry
Immunohistochemistry studies were performed according to previously described with a slight modification [28]. Briefly, formalin-fixed, paraffin- embedded tissues were de-waxed in xylene and rehydrated in graded concentration of alcohols. Following antigen retrieval and slide section blocking, tissue sections were incubated with antibodies at 4°C degree for overnight. A combined sigling nintensity and percentage of positive scoring method was used as reported previously [29]. Strong intensity staining was scored as 3, moderate as 2, weak as 1, and negative as 0. For each intensity score, the percentage of cells with that score was estimated visually. A combined weighted score consisting of the sum of the percentage of cells staining at each intensity level was calculated for each sample, e.g., a case with 70% strong staining, 10% moderate staining, and 20% weak staining would receive a score as follows: (70×3+10×2+20×1) = 250. Net immunolabeling was categorized as negative (score<30) or positive (score≥30).
Plasmid Construction and Transfection
Net expression plasmids were constructed as previously described [28]. The full-length cDNA of Net was amplified by polymerase chain reaction (PCR). The primers were 5′-gat ctc gag atg gag agt gca atc acg ct-3′ (forward) and 5′-gag ggt acc gga ttt ctg aga gtt tga aga-3′ (reverse), which provided with the XhoI and KpnI restriction site (underlined), respectively. PCR products were cloned into pDC316 to generate a pDC316-Net plasmid. The pDC316- Net and the skeleton plasmid pBHG-fiber5/35 were subsequently co-transfected into 293 cells using Polyfect (Qiagen, Hilden, Germany) yielding the recombinant Ad5/F35-Net plasmid. Successful recombination was confirmed by observation of cytotoxicity and sequencing. The control recombinant plasmid Ad5/F35-GFP (green fluorescent protein) was prepared by the same method. PL45 or hTERT-HPNE cells were transfected with Ad5/F35-Net or Ad5/F35-GFP plasmid.
Reverse Transcription-polymerase Chain Reaction (RT-PCR)
Briefly, total RNA was extracted from cultured cells or human pancreatic tissue using the RNeasy RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA was quantitated and PCR reactions were performed: initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 40 s, 55°C for 40 s, 72°C for 90 s and an extension for 10 min at 72°C. The PCR products were electrophoresed on 1.2% agarose gels. Primers used for RT-PCR as showed in Table 1.
10.1371/journal.pone.0057818.t001Table 1 List of primer pairs.
Net Forward 5′- acg ctg cca gta ttt cat cc -3′ 387 bp
Reverse 5′-gac taa ggc tgc tcc aga aat c-3′
P21 Forward 5′-cag ggg aca gca gag gaa ga-3′ 335 bp
Reverse 5′-ggg cgg cca ggg tat gta c-3′
P27 Forward 5′-cgg tgg acc acg aag agt ta -3′ 320 bp
Reverse 5′-tct ggc tgt ccg acg gat ca -3′
CDK2 Forward 5′-aaa ttc atg gat gcc tct gc-3′ 332 bp
Reverse 5′-cga gtc acc atc tca gca aa-3′
CDK4 Forward 5′-ccc gaa gtt ctt ctg cat tc-3′ 313 bp
Reverse 5′-agg cag aga ttc gct tgt gt-3′
Cyclin D1 Forward 5′-att gtg atc agg tgt cca ca- 3′ 380 bp
Reverse 5′-tac ttg acg gcc acg gac at-3′
Cyclin E Forward 5′-act caa cgt gca agc ctc gg-3′ 400 bp
Reverse 5′-gaa caa gct cca tct gtc ac-3′
c-Fos Forward 5′- tgt caa cgc gca gga ctt ct -3′ 443 bp
Reverse 5′- cct tct cct tca gca ggt tg -3′
c-Jun Forward 5′-agc tgg aga gaa tcg ccc ggc tg-3′ 356 bp
Reverse 5′-cca agt cct tcc cac tcg tgc aca ct-3′
β-actin Forward 5′-tga cgg ggt cac cca cac tgt gcc cta cta -3′ 658 bp
Reverse 5′-cta gaa gca ttt gcg gtg gac gat gga ggg
Western Blot
Cells at 70∼80% confluency were washed with PBS and then lysised on ice for 20 min. The protein was quantified using a BCA protein assay kit (Pierce, Rockford, Illinois, USA). Cell lysate (30 µg) was separated on 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane using a Bio-Rad transfer system and then visualized with ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Cell Viability Assessment
Cell viability was assessed via MTT test as previously described [27]. The absorbance of the samples was measured using a microplate reader at 570 nm.
Cell Cycle Analysis
Cells seeded in six well plates were harvested when they are 70–80% confluent, and then washed twice with PBS. The cells were resuspended in 0.5 ml of PBS and fixed with 70% ethanol. Cells were resuspended in 0.2 mg/ml of propidium iodide (PI) containing 0.1 mg/ml RNase A, and incubated in the dark for 30 min at room temperature before analyzed on FACScan flow cytometer. The percentage of cells in different phases of the cell cycle was assayed using a ModFit 5.2 computer program.
Colony Formation Assay
Soft agar colony formation assay was used to assess the anchorage-independent growth ability of cells as previously described [27]. Agarose (0.6%) in DMEM was casted on six-well plates. Cells (1×103 cells/well) were mixed with 0.3% agarose in DMEM containing 10% FBS at 37°C and seeded over the agarose. Colonies larger than 50 µm were counted at 15 days after plating.
Propidium Iodide Staining
Briefly, cells (1×106 cells/mL) cultured in 24-well flat bottom plates were harvested, washed with cold phosphate-buffered saline and resuspended in PBS containing propidium iodide. After 20 minutes incubation, cells were examined with fluorescence microscope at emission 636 nm.
Ultrastructural Analysis
Cell monolayers were washed, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 15 min, gently scraped, and centrifuged at 1200 rpm. Cell pellets were further fixed by glutaraldehyde for 1 h. All specimens were post fixed in OsO4, alcohol dehydrated, and embedded in araldite, as previously described [30]. Thin sections were stained with uranyl acetate and lead citrate and analyzed with a Philips CM10 electron microscope.
Nude Mouse Xenograft Model
To examine the effects of Net gene overexpression on tumour growth in vivo, xenograft model of nude mice with pancreatic cancer was established. Balb/c nude mice were maintained under specific pathogen-free condition in Shanghai Experimental Animals Centre of Chinese Academy of Sciences. All protocols and procedures in animal study were approved by the Ethics Committee of Shanghai First People’s Hospital and following current China Guidelines. PL45 cells (1×107 suspended in 100 µl of PBS) were subcutaneously injected into the hind flank of 5–6 weeks old Balb/c nude mouse. As the tumour grew larger, it was resected, slashed into small even slices in aseptic condition, and replanted into the hind flanks of Balb/c nude mice. Tumours were implanted subcutaneously in mice to more than passage three. When the tumours were palpable, animals were randomly divided into three groups (8 in each group): (a) control group, animals were injected directly into the tumours with PBS every third day. (b) Ad5/F35-GFP group, animals were injected directly into the tumours with 1×108 pfu (plaque-forming units) Ad5/F35-GFP every third day; and (c) Ad5/F35-Net group, animals were injected directly into the tumours with 1×108 pfu adenovirus containing Net gene (Ad5/F35-Net) every third day. Body weight and volume of xenografts were measured in a blinded fashion using callipers every third day during the three weeks treatment period. Tumour volume (V) was estimated from tumour length (l) and width (w) using the formula V = lw2 π/6. Total mRNA was prepared from every group tumours and RT-PCR analyses were performed as described previously. Tunel assay and PCNA staining were used to evaluate cell proliferation and apoptosis according to the procedure previously described [28]. Positively stained cells were sorted as the percentage of total tumour cells in five high resolution fields.
Statistical Analysis
Results are expressed as the mean ± S.E.M. Each experiment was repeated at least three times. Statistical significance of difference between test groups was assessed by one-way ANOVA followed by Scheffe’s test (post hoc). The Kruskal-Wallis or Mann-Whitney U-tests was used to compare the difference of Net expression in age, sex, tumor size, tumor classification, localization and pathological stage. All statistical analyses were performed using SPSS 16.0 software. Statistical significance was defined at p<0.05.
Results
Down-regulation of Net in Pancreatic Carcinoma
36 cases of human pancreatic ductal adenocarcinoma tissues and matched adjacent normal tissue samples were examined for the expression of Net by immunohistochemistry. The neoplastic cells of low proportions (16.7%) of pancreatic carcinoma tissues expressed Net (6 cases in 36 cases), which was highly expressed on majority (88.9%) of paired normal adjacent tissues (32 cases in 36 cases). On the contrary, high expression of c-fos and Ras were detected in pancreatic carcinoma tissues compairing with low expression in peritumor tissues. While both Net and c-fos were mainly located in nucleus, Ras was located in cytoplasmic, the brown-yellowish staining indicate the positive cells (Fig. 1A). Semi-quantitative RT-PCR and Western blot were used to evaluate the changes of Net expression in both mRNA and protein levels in pancreatic carcinoma tissues. The results showed that Net expression was down regulated both at mRNA and protein levels in pancreatic carcinoma tissue in relative to that in peritumor tissues (Fig. 1B). Pancreatic carcinoma cell lines PL45, SW1990, PANC-1 and human pancreatic cell line hTERT-HPNE were employed and similar results were obtained, expression of Net mRNA and protein in pancreatic carcinoma cell lines (especially in PL45 cell line) were significantly lower than that in human pancreatic cell line (Fig. 1C).
10.1371/journal.pone.0057818.g001Figure 1 Net is down-regulated in pancreatic ductal adenocarcinoma.
(A) Immunohistochemical analysis of Net, c-fos and Ras expression in pancreatic ductal adenocarcinoma tissues (top row) and matched adjacent normal tissue samples (bottom row). a and b is H&E staining with low resolution (×100); c, d denote the expression of Net representative with high resolution (×200); e, f denote the expression of c-fos representative with high resolution (×200); g, h denote the expression of Ras representative with high resolution (×200). (B) Expression of Net was examined at mRNA and protein levels using RT-PCR and western blotting on human pancreatic ductal adenocarcinoma tissues (T) and matched adjacent normal tissue samples (N) (presentive 5 cases of 36). (C) Expression of Net was examined at mRNA and protein levels using RT-PCR and western blotting in pancreatic cancer cell lines (SW1990, PANC-1 and PL45) and human pancreatic cell hTERT-HPNE. PDAT, pancreatic ductal adenocarcinoma tissues; NAT, normal adjacent tissue.
The correlation of Net Immunohistochemistry score with a variety of clinicopathologic factors was evaluated with Kruskal-Wallis or Mann-Whitney U-tests in 36 patients with pancreatic ductal adenocarcinoma. Patients with weak or no expression of Net appeared to have high level of CA19-9 and node involvement (P<0.01). However, Net expression was not correlated with age, gender, localization of tumor, or differentiation. Although there was difference in tumour classification and clinical staging between patients with high expression of Net and patients with weak or no expression of Net, no statistical significance was observed. The few case number of patients may account for that.(As shown in Table 2).
10.1371/journal.pone.0057818.t002Table 2 Relationship between Net IHC score and clinicopathologic factors in 36 patients with pancreatic ductal adenocarcinoma.
Parameter n Net IHC score
<30 ≥30
P Valuea
Age 1.0
≤60 14 12 2
>60 22 18 4
Gender 0.655
Male 21 17 4
Female 15 13 2
Localization 1.0
Head 20 17 3
Other 16 13 3
CA19-9 (U/L) 0.014
≤100 8 4 4
>100 28 26 2
Size (cm) 0.662
≤3 19 15 4
>3 17 15 2
Differentiation 0.643
Well/moderate 25 20 5
Poor 11 10 1
Tumour classificationb
0.385
T1/T2 23 18 5
T3/T4 13 12 1
Nodal status 0.001
N0 13 7 6
N1 23 23 0
Pathological stage 0.304
I/II 26 20 6
III/IV 9 9 0
a Kruskal-Wallis test and Mann-Whitney U-test.
b Tumor classification was made according to International Union Against Cancer (2002).
Overexpression of Net Inhibits the Growth and Proliferation of Pancreatic Ductal Adenocarcinoma Cell PL45
PL45 cells that expressed low level of Net were transfected with adenovirus vector that encodes Net (Ad5/F35-Net) to make cells that expressed high level of Net. Cells were examined for growth and proliferation following transfection. Results indicated that the growth and proliferation ability of PL45 cell transfected with Ad5/F35-Net was significantly inhibited in relative to PL45 cells without transfection (control) or transfected with control vector Ad5/F35-GFP (P<0.01) (Fig. 2A, 2B); The number of colony formation was significantly reduced as well in cells transfected with Ad5/F35-Net (Fig. 2C). All those results suggested that Net play a role in inhibiting the proliferation of pancreatic ductal adenocarcinoma cell PL45. Moreover, cell cycle results revealed that most of PL45 cells transfected with Ad5/F35-Net were delayed in G0/G1 phase (59.96±8.54%) after 48 hours transfection in comparing with cells transfected with Ad5/F35-GFP (44.45±4.75%) or control PL45 cells (44.45±4.75%) respectively. The cells in S phase (26.57±5.64%) were significantly lower in cells transfected with Ad5/F35-Net than that in cells transfected with Ad5/F35-GFP (45.73±4.68%) or control cells (45.84±5.36%) (Fig. 2D) (P<0.01), which suggested that the overexpression of Net can delay pancreatic ductal adenocarcinoma cell PL45 at G0/G1 phase.
10.1371/journal.pone.0057818.g002Figure 2 Net inhibited growth and proliferation in pancreatic ductal adenocarcinoma cell PL45.
(A) Growth of PL45 cells transfected with or without Ad5/F35-Net was measured by MTT assay. (B)The growth curve of PL45 cells transfected with or without Ad5/F35-Net was obtained by counting cell numbers per well on each day. (C) Colony formation assay was performed using PL45 cells transfected with or without Ad5/F35-Net after 48 h. (D) Cell cycle of PL45 cells transfected with or without Ad5/F35-Net was evaluated. (E) Cell cycle related genes were examined at mRNA and protein levels 48 hours after Ad5/F35-Net transfection. *p<0.05, **p<0.01.
Net Inhibits Cell Cycle Progression through p21-Cyclin D1/CDK4 Pathway
To further investigate the mechanisms by which Net inhibits pancreatic ductal adenocarcinoma cell PL45 proliferation, several cell cycle associated genes (p21, p27, CDK2, CDK4, Cyclin D1, Cyclin E,c-Jun, and etc.) were evaluated at mRNA and protein levels 48 hours after Ad5/F35-Net infection. The results showed that overexpression of Net inhibited c-fos expression in both mRNA and protein levels. While the expression of p21 was up-regulated by overexpression of Net, the expression of Cyclin D1 and CDK4 were down-regulated following transfection of Net. No obvious changes were observed for the expression of p27, CDK2, Cyclin E and c-Jun before and after transfection (Fig. 2E).
Net Expression Induces the Apoptosis of Pancreatic Ductal Adenocarcinoma Cell PL45
The apoptosis of PL45 cell was evaluated by AnnexinV/PI staining method after 48 hours of Ad5/F35-Net transfection. The early period apoptosis potential of control cells, cells transfected with Ad5/F35-GFP or Ad5/F35-Net group was 5.81±1.6%, 5.90%±1.3 and 46.32±8.1% respectively. The differences between Ad5/F35-Net and control or Ad5/F35-GFP group were significant (P<0.01), while there was no significant difference between control and Ad5/F35-GFP group (P>0.05). The enhanced apoptotic ability of cell transfected with Ad5/F35-Net occurred in the early stage but not in the late stage, there were no statistical difference in the late stage of apoptosis among three groups (32.32±4.1%, 32.61%±4.4 and 34.64±4.7%, respectively. Fig. 3A, 3B). Ultrastructures of cells were observed by TEM (Transmission Electron Microscope) after transfection. We found that PL45 cell transfected with Ad5/F35-Net demonstrated concentrated phenomena of cytoplasm, density increasing, nuclear chromatin margination, karyopyknosis and karyorrhexis, apoptotic body formation, while no obvious changes was observed in neither control group or Ad5/F35-GFP group (Fig. 3C). Our results suggested that overexpression of Net induced the apoptosis of PL45 cell.
10.1371/journal.pone.0057818.g003Figure 3 Net induces the apoptosis in pancreatic ductal carcinoma cell PL45.
(A) Cell apoptosis was examined in PL45 cells after 48 hours of Ad5/F35-Net transfection using AnnexinV/PI method. (B) The rates of early and late apoptosis were evaluated after 48 hours of Ad5/F35-Net transfection. (C) Ultrastructures of cells were observed by transmission electron microscope. Concentrated phenomena of cytoplasm, karyopyknosis, karyorrhexis and apoptotic body formation were detected in PL45 cell transfected with Ad5/F35-Net, no obvious changes was observed in control group and Ad5/F35-GFP group. Black arrow indicated karyopyknosis andkaryorrhexis. *p<0.05, **p<0.01.
Overexpression of Net Inhibits Growth of Transplanted Pancreatic Carcinoma in Nude Mice
To examine the effect of Net on human pancreatic carcinoma growth in in vivo, xenograft model of human pancreatic carcinoma in nude mice was established. The average tumor size and weight were significantly reduced in nude mice injected with PL45 cells transfected with Ad5/F35-Net 21 days later compared with that in mice injected with control cells or cells transfected with Ad5/F35-GFP (P<0.05, Fig. 4A, 4B) indicating that the overexpression of Net inhibits the growth of pancreatic carcinoma in mice. RT-PCR and Immunohistochemistry assay showed that over-expression of Net resulted in decreased c-fos expression and increased P21 expression both in mRNA and protein levels in xenograft tissues (Fig. 4C, 4D). PCNA stain results demonstrated that PCNA labeling index was significant lower in xenograft tissues with cells transfected with Ad5/F35-Net (42.9%) than that with control cells (74.1%) or cells transfected with Ad5/F35-GFP (68.7%) (P<0.01, Fig. 4E). On the other hand, apoptotic cells detected by TUNEL assay showed that the apoptotic index of xenograft tissues with cells transfected with Ad5/F35-Net(18.2%) was significant higher than that with control cells (5.4%) or cells transfected with Ad5/F35-GFP group (4.9%) (P<0.01, Fig. 4E), which suggested that Net had the ability to prohibit pancreatic carcinoma xenograft growth in nude mice through inhibiting proliferation and inducing cell apoptosis.
10.1371/journal.pone.0057818.g004Figure 4 The effects of Net expression on tumor growth in vivo.
(A) Tumor size was measured every three days interval in each group. (B) Weight of dissectable xenograft tumors was measured in each group. (C) Expression of Net, c-fos and P21 in mRNA levels in xenograft tissues. (D) Representative H&E staining, histological staining of Net, c-fos and P21, PCNA staining and TUNEL assay in xenogrft tissues. (E) Index of PCNA and apoptosis were accounted in xenograft tumour tissues. *p<0.05, **p<0.01.
Discussion
Net, an important transcription regulator and downstream target of Ras-MAPKs pathway, has the ability to regulate protooncogene c-fos transcription by forming a ternary complex on the promoter of target gene and eventually affect cell proliferation and differentiation [21], [23]. Low expression of Net has been reported in some carcinoma cells such as cervical cancer and overexpression of Net could inhibit the growth of cervical carcinoma cell [31], but the mechanism is still unknown. It was not known whether other tumors (including pancreatic carcinoma) expressed Net and its fnctional significance. In this study, human pancreatic ductal adenocarcinoma tissues and pancreatic adenocarcinoma cell lines were examined for the expression of Net using immunohistochemistry, RT-PCR, and Western blot. Results showed that pancreatic ductal adenocarcinoma tissues and cell lines expressed relatively low level of Net and an inverse correlation was found between the expression of Net and c-fos or Ras in the tissues and cells, which was in agreement with our previous reports and implied that Net could inhibit the expression of c-fos [27]. The clinicopathologic factors analysis revealed low level of Net expression was correlated with high level of CA19-9 and node involvement. These results suggested that lack of Net expression might have biological and clinical importance on pancreatic ductal adenocarcinoma.
Genetically manipulating the expression of Net provides a useful tool to examine the impact of Net on cancer growth. Previous studies suggested that Net could inhibit the growth of pancreatic cancer cell BxPC-3 transfected with Net [27]. In present study, adenovirus vector Ad5/F35-Net was constructed and transfected into human pancreatic ductal adenocarcinoma cell PL45. We found that overexpression of Net could inhibit the growth and colony formation of pancreatic carcinoma and induced delayed G0/G1 phase. TEM examination showed that stable expression of Net promoted apoptosis of PL45. Ultrastructure damage was found in Net overexpression pancreatic carcinoma cells, which was characterized by forming apoptotic bodies. Overexpressing of Net induced apoptosis. We hence postulated that Net might inhibit the proliferation of pancreatic ductal adenocarcinoma cells and promote apoptosis, and eventually preventing the development of pancreatic cancer. To confirm our hypothesis, xenograft model of the human pancreatic carcinoma in nude mice were used to study the effects of Net expression on tumor development, results also indicated that Net overexpression inhibits the growth of xenograft pancreatic carcinoma with down-regulation of c-fos and up-regulation of P21 expression in vivo. In addition, we also noticed that decreased PCNA index and increased apoptotic index by overexpression of Net. These results suggested that Net has the inhibitory effects on pancreatic tumor cell growth and development in vitro and in vivo.
Given the fact that progression of cell cycle from G1 to S phase in mammalian cell is controlled by the cyclin A, cyclin D, and cyclin E, which bind to and activate different kinases in G1, such as CDK4, CDK6 and CDK2. Activation of cyclin D1/CDK4, cyclin D1/CDK6 and cyclin E/CDK2 complex is required for cell transition from G1 to S phase. Cyclin D1/CDK4 or cyclin D1/CDK6 complex are involved in early G1 phase and cyclin E/CDK2 complex is involved in mid-to-late G1 stage. Former studies showed that cyclin-CDK complexes were associated with cyclin kinase inhibitors (ckis), that bind and inactivate cyclin-CDK complexes [32],[33],[34]. P21 and p27 are proteins that bind CDK2- and CDK4-cyclin complexes, and p21 is a major inhibitor during the G1 phase of the cell cycle [33],[35],[36]. Net is activated through phosphorylation by mitogen activated protein kinases (MAPKs) [32], The phosphorylated Net in turn binds to the response element of c-fos gene promoter, leading to c-fos transcription. c-Fos protein dimerizes with c-Jun protein to form AP-1 complex, thereby regulating various target genes involved in cell proliferation and cell cycle progression [37], [38]. AP-1 complex has been shown to transactivate cyclin D1 gene and stimulate G1 to S progression [26], [39]. To understand the molecular mechanisms by which Net inhibits cell cycle progression, multiple genes associated with cell cycle such as Cyclin D, Cyclin E, CDK2, CDK4, P21, P27, c-fos and c-Jun were examined by RT-PCR and Western Blot, we found that P21 expression was increased in PL45 cells following transfection of Ad5/F35-Net, while Cyclin D1 and CDK4 expression was decreased, expression of CDK2 showed no obvious change. All these suggested that the effect of Net on delaying cell cycle might through the pathway of P21-Cyclin D1/CDK4. Since AP-1 is a heterodimer constituted by c-fos and c-Jun, c-Jun expression was also checked. However, no obvious change of c-Jun was found due to the expression of Net, suggesting that down-stream transcription regulation of Net was probably modulated by c-fos pathway but not c-Jun pathway.
In summary, the present results suggested that Net has the potential to inhibit the growth of human pancreatic ductal adenocarcinoma cell PL45 and induce cellular apoptosis in vitro and in vivo. The inhibitory mechanism is presumably by inhibiting the expression of c-fos, subsequently inactivating the transcription activity of AP-1, followed by activating of p21 to antagonize the effects of Cyclin D/CDK4 on cell cycle progression, and ultimately leading to cell death. On the contrary, intra or extracellular signals through Ras-MAPKs pathway induce phosphorylation of Net, which results in downregulating Net expression and disabling inhibitory ability of Net on c-fos transcription, thus accelerating cell cycle progression and promoting cell proliferation (the possible regulatory model as showed in Fig. 5). Further studies will be pursued to fully characterized the mechanism.
10.1371/journal.pone.0057818.g005Figure 5 The possible regulatory model of effect of Net on pancreatic ductal adenocarcinoma.
Net inhibits the growth of pancreatic ductal adenocarcinoma cell by inhibiting the expression of c-fos, subsequently inactivating the transcription activity of AP-1, followed by activation of p21 to antagonize the effects of Cyclin D/CDK4 on cell cycle progression, and ultimately leading to cell death. On the contrary, phosphorylation of Net is activated by intra or extracellular stimulation signals through Ras-MAPKs pathway, which results in downregulation of Net expression and lack of inhibitory ability on c-fos transcription, thus promoting cell proliferation.
We gratefully thank the staff members in the department of gastroenterology at Shanghai First People’s Hospital of Shanghai Jiaotong University for their suggestion and assistance.
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18 : 3085 –3097 .10340380 | 23469073 | PMC3585156 | CC BY | 2021-01-05 17:20:58 | yes | PLoS One. 2013 Feb 28; 8(2):e57818 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23468839PONE-D-12-2081510.1371/journal.pone.0053072Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionGeneticsHuman GeneticsGene TherapyGenomicsGenomic MedicineGene TherapyMolecular Cell BiologyGene ExpressionMedicineClinical GeneticsGene TherapyOncologyBasic Cancer ResearchTumor PhysiologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaCancer TreatmentAlpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy AFP Promoter Driven HCC Specific RNAi TherapyPeng Yuan-Fei
1
Shi Ying-Hong
1
Ding Zhen-Bin
1
Zhou Jian
1
Qiu Shuang-Jian
1
Hui Bo
1
Gu Cheng-Yu
1
Yang Hua
1
Liu Wei-Ren
1
Fan Jia
1
2
*
1
Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University; Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Shanghai, People's Republic of China
2
Institutes of Biomedical Sciences, Fudan University, Shanghai, People's Republic of China
Samant Rajeev Editor
University of Alabama at Birmingham, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Technical assistance: JZ SJQ. Conceived and designed the experiments: YFP YHS JF. Performed the experiments: YFP ZBD WRL CYG BH HY. Analyzed the data: YFP YHS JF. Contributed reagents/materials/analysis tools: JZ SJQ. Wrote the paper: YFP JF.
2013 28 2 2013 8 2 e5307214 7 2012 28 11 2012 © 2013 Peng et al2013Peng et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Backgroud
RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.
Methods
Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.
Results
The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.
Conclusions
An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.
This work was supported by the grants from National Natural Science Foundation of China (81030038, 81001060, 81272389), National Key Sci-Tech Project (2012ZX10002011-002), China Postdoctoral Science Foundation (20100470639, 201104240), and Shanghai Postdoctoral Science Foundation (11R21410300). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death [1]. The prognosis of HCC is poor for most of patients because HCC is often diagnosed at a late stage and current treatment options are rather limited. The inherent difficulty of treating this malignancy has prompted many to consider new therapeutic approach.
In recent years RNA interference (RNAi) has emerged as a potential treatment modality for cancer therapy. RNAi is a sequence-specific posttranscriptional gene silencing process. Since its discovery, RNAi has been rapidly developed into a powerful technique for both therapeutic and gene functional research. Several clinical trials published so far and numberous in vivo animal studies have demonstrated the success and feasibility of RNAi for the treatment of various diseases [2]–[5]. HCC is a disease of altered genes. In recent years, unprecedented advances in genome-wide analysis have resulted in important discoveries regarding HCC. The cancer-causing genes identified by the new genome-wide technologies have provided great opportunities for HCC therapy [6]. In addition, since RNAi can be easily delivered into the liver, HCC is amenable to RNAi targeting and it has been extensively used as a disease model for testing RNAi therapy [5], [7]. Therefore, RNAi therapy has tremendous potential as a treatment modality for HCC therapy. It is foreseeable that RNAi technique will be widely appliedapplicable for HCC treatment in the near future.
However, although RNAi is highly attractive as a therapeutic approach, several hurdles must be overcome before RNAi can be successfully introduced into the clinic. These include efficiency of cellular uptake, specific guidance to target tissue or cell, and sustained efficacy [3]–[5], [8]. The RNAi should be limited to target cells and nonspecific cytotoxicity should be avoided. It is especially important to the RNAi therapy for HCC because HCC frequently occurs in patients with liver cirrhosis and compromised hepatic function reserve. The currently most popular materials used for RNAi are chemically synthesized small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) driven by Pol III promoters (U6 or H1). However, either siRNA or Pol III promoter-driven shRNA can silence target gene in all cell types, and therefore may destroy not only target tumor cells but also non-tumor cells. Besides, the duration of siRNA or vector-based shRNA induced silencing was short-term and lasted less than a few weeks [5]. But RNAi therapy usually requires much longer duration of action (in some cases, years). In addition, the in vivo cellular uptake and intracellular delivery still remain a major challenge for therapeutic application of RNAi [9]. To obtain tissue-specific, long-term and high efficient RNAi, one possible solution is to employ tissue-specific promoter (to restrict therapeutic RNAi expression in target tissue) in combination with lentiviral vector (to obtain high transduction efficiency and stable gene silencing). One of the major characteristics of hepatocellular carcinoma is the transcriptional reactivation of alpha-fetoprotein (AFP) [10]. The AFP promoter is highly specific to HCC. It has been employed to drive therapeutic gene expression to develop HCC-specific gene therapy, although there have been few reports on its application in RNAi therapy [11]–[16]. The long-term high-efficiency gene silencing required for RNAi therapy can be achieved by using lentiviral vectors. Lentivirus-mediated RNAi is a powerful method for long-term inhibition of gene expression in vitro and in vivo
[17]. The small interfering RNA can be delivered through lentiviral vector in a form of shRNA driven by a Pol III promoter or a miRNA-like structure expressed from a Pol II promoter [17]. The shRNA or miRNA contained in lentiviral vectors can be stably integrated into the genome, which allows permanent, heritable gene silencing. The lentivector technology has been widely used in basic and translational research, and it has been applied in clinic and provides therapeutic benefit [17].
The purpose of this study was to develop an efficient HCC tissue-specific RNAi system and investigate the possibility of the system for HCC treatment. We constructed two different HCC tissue-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by an AFP promoter (AFP-miRNA system and AFP-Cre/LoxP-shRNA system). The effectiveness and tissue-specificity of the systems were examined in HCC and non-HCC cells in vitro and in vivo. The feasibility of the system as new agent against HCC was tested based on the finding in previous research that silencing Atg5 gene could enhance sorafenib lethality [18]. The HCC tissue-specific RNAi system targeting Atg5 gene was constructed and the efficacy of the system for sensitizing HCC cells to sorafenib treatment was examined in vitro and in vivo.
Materials and Methods
Cell Lines and Animals
Human HCC cell lines Hep3B, HepG2, PLC/PRF/5 (ATCC), HCCLM3 [19], [20], and MHCC97L [20], [21], human normal hepatic cell line L-02 (Cell Bank, China), human cervical carcinoma cell line HeLa and colon cancer cell line SW1116 (ATCC) were routinely maintained. Male BALB/c nu/nu mice (6 weeks old, Chinese Academy of Science) were bred in specific pathogen-free conditions. All mice were cared for and handled according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Experimental protocol was approved by Shanghai Medical Experimental Animal Care Committee.
AFP promoter assay
A SV40-hAFP promoter from pDrive-SV40-hAFP (InvivoGene) was chosen as the AFP promoter. The activity and tissue-specificity of the AFP promoter was assayed using GFP reporter in AFP-producing and non-AFP-producing cell lines. A recombinant lentivirus vector containing the AFP promoter and GFP reporter gene was constructed as follow. The AFP promoter was synthesized according to the instruction of the pDrive-SV40-hAFP with adding NheI and EcoRI restriction enzyme cutting sites (RECS) (See File S1). After amplification it was cloned into NheI-EcoRI sites of pLV-CMV-GFP lentiviral plasmid (MicroSCI, China) by replacing the CMV promoter. The recombinant lentiviral plasmid and the mother plasmid pLV-CMV-GFP were packaged by transient co-transfection of HEK 293 cells with helper-packaging plasmids (psPAX2 and pMD2.G, Trono Lab). The recombinant lentiviral vector was named as AFP-GFP while the mother vector was termed as CMV-GFP (served as positive control). To evaluate the activity of the AFP promoter, AFP-producing HCC cells were infected with either AFP-GFP (MOI = 5, 10, 20, 40 and 80) or CMV-GFP (MOI = 5). Three days later, the cells were observed under fluorescence microscope (Leica). The images were analyzed and the intensity of GFP fluorescence was quantitated by Image-Pro plus 6 software (MediaCybernetics). To evaluate the tissue-specificity of the AFP promoter, the HCCLM3, HepG2, Hep3B, L-02, HeLa, and SW1116 cells were all infected with AFP-GFP at MOI of 20. Three days later, the cells were observed and the GFP expression was quantified as mentioned above.
Construction of AFP-miRNA and evaluation of RNAi efficacy
The AFP-miRNA targeting Beclin 1 was constructed as follow. Four miRNAs targeting Beclin 1 with negative control were designed and constructed into pcDNA6.2-GW/EmGFP-miR (Invitrogen). The vectors were named as pcDNA-miR-1, pcDNA-miR-2, pcDNA-miR-3, pcDNA-miR-4 and pcDNA-miR-NC. Then the 900 bp CMV-miRNA-EGFP structure was amplified from the templates pcDNA-miR-1/2/3/4/NC by PCR using primers Gecp-1,2 (Table 1). Then the CMV-miRNA-EGFP fragments were cloned into the NheI-NotI sites of a lentiviral vector pSil01 (MicroSCi, China) and formed new vectors (CMV-miRNA-1/2/3/4/NC). The HCCLM3 cells were infected with the CMV-miRNA-1/2/3/4/NC at MOI of 20. Q-PCR and western blot analysis of Beclin 1 gene silencing were performed to determine the most effective miRNA (CMV-miRNA-3). Then a 740 bp fragment A was amplified from template AFP-GFP by PCR using primers Gecp-3,4 (Table 1). A 230 bp fragment B was amplified from CMV-miRNA-3 or CMV-miRNA-NC by PCR using primers Gecp-5,6 (Table 1). A fragment C was generated from the templates fragment A and fragment B by PCR. Then the fragment C was cloned into the NheI-AvrII sites of the AFP-GFP and formed new vectors AFP-miRNA (AFP-miRNA-3/NC). The CMV-miRNA-3 was used as positive control. The AFP-miRNA-3/NC and CMV-miRNA-3/NC were then packaged into lentiviral particles. To evaluate the effectiveness of AFP-miRNA, the AFP-producing HCC cell lines (HCCLM3, HepG2, Hep3B) were infected with either CMV-miRNA-3 (MOI = 20) or AFP-miRNA-3 (MOI = 5, 10, 20, 40, 80, 160 and 320). Three days later, the cells were observed under fluorescence microscope (Leica). Q-PCR and western blot analysis of Beclin 1 gene silencing were performed one week later.
10.1371/journal.pone.0053072.t001Table 1 Primers used for the construction of HCC-tissue specific RNAi systems.
Primer Sequence
Gecp-1
CTAGAGCTAGCGCCACCATGGTGAGCAAGGGCGAGG
Gecp-2
ATCCTTGCGGCCGCTAGATATCTCGAGTGCGGCC
Gecp-3
TAACACGCTAGCGCCACCA
Gecp-4
GACGGCCACGAAGTGCTTAGCTTACTTGTACAGCTCGTCCATGC
Gecp-5
GCTAAGCACTTCGTGGCCGTC
Gecp-6
TCGACGCCTAGGGCGGCCGCTAGATATCTCGAGTGCGGCCA
Gecp-7
TTTTATCGATACTAGTGGCCTGAAATAACCTCTGAAAG
Gecp-8
CCTCTTCTTCTTGGGCATGGTGGCGTGTTATTGGCAGTGGTGG
Gecp-9
CATGCCCAAGAAGAAGAGGAAGGTGTCCAATTTACTGACCGTAC
Gecp-10
ATTCGCTAGCTCTAGACTAATCGCCATCTTCCAGCAG
Gecp-11
GGGCTCGAGCCCGGGAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATT
Gecp-12
ATTACTTTCTACGTCACGTATTTTG
Gecp-13
TTTTAAAATGGACTATCATATGCTTACC
Gecp-14
AAGAATTCACGCGTGCATACCTGCGG
Gecp-15
CAAAATACGTGACGTAGAAAGTAATATAACTTCGTATAGCATACATTATAC
Gecp-16
AACTTCGTATAGCATACATTATACGAAGTTATGTTAACGTGGATAACCGTATTACCGCC
Gecp-17
CTTCGTATAATGTATGCTATACGAAGTTATGGATCCTTACTTGTACAGCTCGTCCATGC
Gecp-18
GGTAAGCATATGATAGTCCATTTTAAAAATAACTTCGTATAATGTATGCTATAC
The miRNA sequence: Beclin 1-F: TGCTGTAATGGAGCTGTGAGTTCCTGGTTTTGGCCACTGACTGACCAGGAACTCAGCTCCATTA; Beclin 1-R: CCTGTAATGGAGCTGAGTTCCTGGTCAGTCAGTGGCCAAAACCAGGAACTCACAGCTCCATTAC.
Construction of AFP-Cre/LoxP-shRNA system and evaluation of effectiveness and tissue-specificity
The AFP-Cre/LoxP-shRNA system consists of AFP-Cre and LoxP-shRNA lentiviral vectors. The AFP-Cre vector contains a Cre gene driven by the AFP promoter. The LoxP-shRNA vector has a modified U6 promoter followed by shRNA against target gene (Beclin 1). The modified U6 promoter harbors a LoxP-CMV-eGFP-LoxP inside. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter will drive the expression of Cre recombinase which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates the U6 promoter. The activated U6 promoter then drives the down-stream shRNA expression and silences the target gene. The CMV-eGFP inside the U6 promoter serves as an indicator (when the CMV-eGFP is cut by the Cre recombinase, the GFP fluorescence will diminish or disappear). The AFP-Cre and LoxP-shRNA vectors were constructed as follow: (1) AFP-Cre vector: The pCDH-CMV-MCS-EF1-Puro (SBI) was used to generate the AFP-Cre. The AFP promoter fragment was amplified from the template AFP-GFP by PCR using primer Gecp-7,8 (Table 1). The Cre (NLS-Cre) was amplified from the template pLV-CRE (MedSCI, China) using primer Gecp-9,10 (Table 1). The AFP-Cre structure fragment was generated from the template AFP promoter and Cre by PCR using primer Gecp-7,10 (Table 1). The AFP-Cre structure (See File S2) was then cloned into the SpeI-XbaI sites of pCDH-CMV-MCS-EF1-Puro by In-Fusion cloning technique (Clontech) and formed the AFP-Cre vector. (2) LoxP-shRNA vector: Fragment A and fragment B were cloned from template pSil02 vector (MicroSCI, China) by PCR using primers Gecp-11,12 and Gecp-13,14 (Table 1), respectively. A fragment C was generated by PCR from template pSil04 (MicroSCI, China) using primers Gecp-15,16,17,18 (Table 1). A fragment D (U-LoxP-CMV-eGFP-U6 structure, See File S2) was generated by PCR from templates fragments A, B, C using primers Gecp-11,14 (Table 1). The fragment D was then cloned into the XhoI-EcoRI sites of the pSil02 vector and formed the LoxP-shRNA vector. A previously validated shRNA targeting Beclin 1 (See File S3) was cloned into the AgeI-EcoRI sites of LoxP-shRNA vector. The AFP-Cre and LoxP-shRNA were then separately packaged into lentiviral particles. The effectiveness and tissue-specificity of the AFP-Cre/LoxP-shRNA system were examined in vitro and in vivo. (1)In vitro evaluation: The AFP-producing HCC cells (HCCLM3/HepG2/Hep3B) and non-AFP-producing cells (L-02/Hela/SW1116) were infected with the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) (AFP-Cre:LoxP-shRNA = 1, MOI = 1,5,10,20 for each). The cells were observed under fluorescence microscope every day. Q-PCR and western blot analysis of Beclin 1 gene silencing were performed one week later. (2)In vivo evaluation: Xenograft nude mouse model of HCC via orthotopic implantation and subcutaneous inoculation of HCCLM3 cells were established as previously described [19]. Intravenous injection: Twelve mice were randomly divided into two groups (n = 6 per group). One week after orthotopic implantation, the AFP-Cre/LoxP-shRNA system was intravenously injected via tail vein (LoxP-shRNA group: LoxP-shRNA 1.8×108 TU in 180 µl Opti-MEM; Cre+LoxP group: AFP-Cre 0.9×108 TU+LoxP-shRNA 0.9×108 TU in 180 µl Opti-MEM). The mice were sacrificed two weeks later. Intratumoral injection: Thirty-six mice were randomly divided into four groups (n = 6 per group). When the subcutaneous tumors reached a size of about 4 mm maximal diameter, the AFP-Cre/LoxP-shRNA system was intratumorally injected (OMEM group: 50 µl Opti-MEM; AFP-Cre group: 1×108 TU AFP-Cre in 50 µl Opti-MEM; LoxP-shRNA group: 1×108 TU LoxP-shRNA in 50 µl Opti-MEM; Cre+LoxP group 1: 0.25×108 TU AFP-Cre+0.25×108 TU LoxP-shRNA vectors in 50 µl Opti-MEM; Cre+LoxP group 2: 0.5×108 TU AFP-Cre+0.5×108 TU LoxP-shRNA vectors in 50 µl Opti-MEM). All mice were sacrificed two weeks later. All of the tumors were resected. The frozen sections of the tumors were stained with DAPI (Invitrogen) and observed under fluorescence microscope as mentioned below. Q-PCR and Western blot analysis of Beclin 1 silencing were performed.
The Beclin 1 shRNA sequence: Beclin 1-F: CCGGCCCGTGGAATGGAATGAGATTCTCGAGAATCTCATTCCATTCCACGGGTTTTTG;Beclin 1-R: AATTCAAAAACCCGTGGAATGGAATGAGATTCTCGAGAATCTCATTCCATTCCACGGG.
Quantitative real-time PCR analysis
The quantitative real-time PCR (Q-PCR) analysis of Atg5 and Beclin 1 expression was performed as previously described [22].
Western blot analysis
Western blot analysis was performed as previously described [22]. The following antibodies were used: anti-human antibodies against Atg5 (1∶500; Cell Signaling), Beclin 1 (1∶1000; Cell Signaling), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1∶5000; Millipore).
Cell viability assay
Cell viability was determined with WST-8 cytotoxicity assay using Cell Counting Kit-8 (Dojindo, Japan) as previously described [23].
Fluorescence microscopy of tissues
After the mice were killed, the tumors were resected immediately and embedded in Tissue-Tek OCT compound using liquid nitrogen and stored at −80°C. Then the tissue samples were sectioned at 40 µm thickness with a cryostat. The tissue sections were stained with DAPI (1∶1000 dilution, 15 min; Invitrogen). Then they were observed and analyzed using confocal fluorescence microscope (Carl Zeiss).
Construction of AFP-Cre/LoxP-shRNA targeting Atg5 and evaluation of its efficacy for sensitizing HCC cells to sorafenib treatment in vitro and in vivo
A previously validated Atg5 shRNA (See File S3) was cloned into the AFP-Cre/LoxP-shRNA system in a similar fashion as mentioned above and formed the AFP-Cre/LoxP-shRNA targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5). The AFP-producing HCC cells (MHCC97L and HepG2) were infected with AFP-Cre/LoxP-shRNA-Atg5 (MOI = 1, 5, 10 and 20). Q-PCR and western blot analysis of Atg5 gene silencing were performed one week later. The AFP-Cre/LoxP-shRNA-Atg5 -mediated sensitization of HCC cells to sorafenib treatment was evaluated in vitro and in vivo. (1)In vitro evaluation: The MHCC97-L and PLC/PRF/5 cells were infected with the AFP-Cre/LoxP-shRNA-Atg5 (AFP-Cre:LoxP-shRNA = 1, MOI = 20 for each). One week later, the virus-infected cells were exposed to sorafenib (20 µM, Bayer) for 24 h. Cells without infection and cells infected with AFP-Cre, LoxP-shRNA, or AFP-Cre/LoxP-shRNA-NC served as controls. Apoptosis was measured using Annexin V and PI flow cytometry as previously described [23]. (2)In vivo evaluation: Xenograft nude mouse model of HCC via subcutaneous inoculation of MHCC97-L cells (1×107) was established as previously described [24]. Twelve mice were randomly divided into two groups (n = 6 per group): (i) Sorafenib group (sorafenib treatment alone); (ii) Sorafenib+Cre/LoxP group (AFP-Cre/LoxP-shRNA-Atg5 treatment followed by sorafenib treatment). The AFP-Cre/LoxP-shRNA-Atg5 treatment alone was not included as pilot studies showed that the AFP-Cre/LoxP-shRNA-Atg5 treatment had no effect on the tumorigenesis of MHCC97L cells (see File S4). Tumor size was determined by caliper measurements every 3 days. When the subcutaneous tumors reached a size of about 4 mm maximal diameter, the mice in the Sorafenib+Cre/LoxP group were treated with the AFP-Cre/LoxP-shRNA-Atg5 by intratumorally injection (0.5×108 TU AFP-Cre+0.5×108 TU LoxP-shRNA-Atg5 in 50 µl Opti-MEM). One week after AFP-Cre/LoxP-shRNA-Atg5 treatment, all mice in the Sorafenib group and Sorafenib+Cre/LoxP group were treated daily with sorafenib (30 mg/kg) via intraperitoneal injection. The animals were monitored every day and sacrificed 6 weeks after tumor implantation. All of the tumors were resected and the tumor weights were measured.
The Atg5 shRNA sequence: Atg5-F: CCGGGCTAGCTGGCTGTCCATATTTCAAGAGAATATGGACAGCCAGCTAGCTTTTTTG;Atg5-R: AATTCAAAAAAGCTAGCTGGCTGTCCATATTCTCTTGAAATATGGACAGCCAGCTAGC.
Statistical Analysis
Statistical analysis was performed with SPSS 13.0 software (SPSS, Chicago, IL). Results were presented as mean ± standard deviation (SD). Comparisons of quantitative data were analyzed using Student's t test between two groups or by one-way ANOVA for multiple groups. P values<0.05 were considered statistically significant.
Results
AFP-promoter is active and specific in AFP-producing HCC cells and tissues
A recombinant lentiviral vector containing GFP reporter gene driven by AFP promoter was constructed (AFP-GFP) (Fig. 1A). The activity and tissue-specificity of the AFP promoter were assayed by infecting the AFP-producing and non-AFP-producing cells with the AFP-GFP vector. GFP reporter assay showed that the AFP-promoter could efficiently drive GFP expression in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (L-02, SW1116, and Hela) (Fig. 1B,D). The GFP expression was vector dose-dependent (Fig. 1 C,E). However, as compared with the CMV promoter which is known to be one of the strongest promoters in a wide range of cell lines, the transcriptional activity of the AFP promoter was remarkably lower (Fig. 1E).
10.1371/journal.pone.0053072.g001Figure 1 Activity and tissue-specificity of the AFP promoter.
(A) Construction of recombinant lentiviral vectors containing GFP reporter gene driven by AFP promoter or CMV promoter (AFP-GFP and CMV-GFP) for AFP promoter assay. (B,D) The AFP promoter is active and HCC tissue-specific. GFP was highly expressed in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (normal hepacyte L-02, cervical cancer cell Hela, and colon cancer cell SW1116). (C,E) The AFP promoter was efficient for transgenic expression but its activity was weaker than CMV-promoter, as exemplified by HCCLM3 cells infected with AFP-GFP (MOI = 5–80) and CMV-GFP (MOI = 5).
AFP-miRNA system can silence target gene but requires high titer
We first investigated the strategy of AFP-promoter driven miRNA for HCC-specific gene silencing. An AFP-miRNA lentiviral vector (AFP-miRNA) targeting Beclin 1 gene was constructed (Fig. 2A). A CMV promoter driven miRNA vector (CMV-miRNA) served as positive control (Fig. 2A). To assess the effectiveness of the AFP-miRNA, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) were infected with the AFP-miRNA at various MOI. The AFP-miRNA was efficient in infecting all cell lines in vitro (Fig. 2B). Q-PCR and western blot analysis of Beclin 1 gene silencing showed that the AFP-miRNA could knockdown Beclin 1 but required considerably high titer (Fig. 2C). A MOI of more than 80 was required to achieve efficient gene silencing (Fig. 2C). As compared with positive control CMV-miRNA, the efficacy of the AFP-miRNA was remarkably weaker (Fig. 2C). The CMV-miRNA infection at a MOI of 20 could achieve silencing level equal to that of the AFP-miRNA infection at a MOI of 320 in AFP-producing HCC cells (Fig. 2C). As high titer was needed for effective gene silencing, the cytotoxicity of the AFP-miRNA against target cells was determined. Cell viability assay showed that the AFP-miRNA infection at required titer (MOI>80) led to considerable cell death. With the increase of MOI, the cytotoxicity significantly enhanced (Fig. 2D).
10.1371/journal.pone.0053072.g002Figure 2 Effectiveness of AFP-miRNA system for HCC tissue-specific RNAi.
(A) Construction of recombinant lentiviral vectors contained miRNA targeting Beclin 1 gene driven by AFP promoter or CMV promoter (AFP-miRNA and CMV-miRNA). (B) The AFP-miRNA system could efficiently infected HCC cells (more than 99%), as exemplified by HCCLM3 cells. (C) Q-PCR and western blot analysis showed that the AFP-miRNA system could downregulate target gene (Beclin 1) of HCC cells but required high titer (MOI>80), as compared with positive control CMV-miRNA. (D) Cell viability assay showed that the high titer required for effective AFP-miRNA-mediated RNAi was cytotoxic to target HCC cells, as exemplified by HCCLM3 and HepG2 cells.
As the AFP-miRNA required a high titer which was cytotoxic to target cells, it was not desirable for target therapy because it might cause damage to the adjacent normal cells and tissues. Therefore, another system (AFP-Cre/LoxP-shRNA) was constructed (Fig. 3A).
10.1371/journal.pone.0053072.g003Figure 3 Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.
(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.
AFP-Cre/LoxP-shRNA system can efficiently silence target gene in a HCC-specific manner
The AFP-Cre/LoxP-shRNA system was successfully established (Fig. 3A). The CMV-eGFP indicator inside could well indicate whether the system worked as desired (Fig. 3B,C). The GFP fluorescence diminished after AFP-Cre and LoxP-shRNA co-infected AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) while it did not change as AFP-Cre and LoxP-shRNA infected the non-HCC cells (L-02, Hela, and SW1116) (Fig. 3C). To assess the effectiveness and tissue-specificity of the AFP-Cre/LoxP-shRNA system, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B), normal hepatocyte (L-02), and non-HCC cancer cells (Hela and SW1116) were infected with the AFP-Cre/LoxP-shRNA system at a MOI of 1,5,10 or 20. Q-PCR and western blot analysis showed that the system could efficiently silence Beclin 1 gene in only AFP-producing HCC cells (Fig. 3C,D). It could effectively knockdown target gene at a MOI of 10–20 instead of the high titer required for the AFP-miRNA system (Fig. 3D). The effectiveness of the system was further examined in vivo. Systemic administration of the AFP-Cre/LoxP-shRNA system was firstlytested. Nude mouse model of HCC via orthotopic implantation was established using AFP-producing HCC cells (HCCLM3) and the AFP-Cre/LoxP-shRNA-Beclin1 was intravenously injected (Fig. 4B). Q-PCR and western blot analysis indicated that there was no significant decrease of Beclin 1 gene after the AFP-Cre/LoxP-shRNA-Beclin1 was given (Fig. 4A). Intravenous injection of LoxP-shRNA with CMV-eGFP indicator showed no GFP expression in HCC tumor, which suggested that no vectors were delivered into the tumor cells (Fig. 4B). The ineffectiveness of systemic administration was thought to be attributed to the disadvantages of the nude mouse model and lentivirus-based vector. The effectiveness of in vivo application of the system was then examined by intratumorally administration. Nude mouse model of HCC via subcutaneous injection was established using HCCLM3 cells (Fig. 4D). The AFP-Cre/LoxP-shRNA-Beclin1 was intratumorally injected into the subcutaneous tumor. The GFP indicator analysis showed that the system could efficiently infect HCC cells (Fig. 4D). Q-PCR and western blot analysis showed that it could efficiently knockdown the Beclin 1 gene of the HCCLM3 tumor tissues in vivo (Fig. 4C).
10.1371/journal.pone.0053072.g004Figure 4 Efficacy of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi in vivo.
(A,B) Mouse model of HCC via orthotopic implantation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intravenous injection of the AFP-Cre/LoxP-shRNA-Beclin1 did not knockdown Beclin 1 gene. GFP indicator analysis showed no GFP expression after the LoxP-shRNA vector was intravenously given, suggesting that the system did not enter the tumor in the liver. (C,D) Mouse model of HCC via subcutaneous inoculation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Beclin1 could efficiently silence target gene (Beclin 1) in vivo. GFP indicator indicated that the AFP-Cre/LoxP-shRNA could efficiently infect and work in HCC tissue in vivo (GFP fluorescence diminished after intratumoral injection of the AFP-Cre/LoxP-shRNA, the LoxP-shRNA alone served as control). (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Beclin1; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone).
AFP-Cre/LoxP-shRNA-Atg5 mediated Atg5 silencing sensitizes HCC cells or tissues to sorafenib-mediated lethality
Our previous study showed that silencing of Atg5 gene could enhance sorafenib-mediated lethality [18]. We next used the Atg5 as target gene to validate the effectiveness of the AFP-Cre/LoxP-shRNA system and test the value of the system for HCC treatment. The AFP-Cre/LoxP-shRNA system targeting Atg5 (AFP-Cre/LoxP-shRNA-Atg5) was successfully constructed. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in AFP-producing cells (MHCC97L and PLC) (Fig. 5A). We then examined whether the AFP-Cre/LoxP-shRNA-Atg5 could sensitize HCC cells to sorafenib treatment. The efficacy of the AFP-Cre/LoxP-shRNA-Atg5 was firstly examined in vitro. Apoptosis assay showed that the apoptotic rate of cells (MHCC97L and PLC) treated with AFP-Cre/LoxP-shRNA-Atg5 plus sorafenibwas significantly higher than that of cells receiving sorefenib alone (all P<0.05) (Fig. 5B). To further evaluate whether the AFP-Cre/LoxP-shRNA system can be translated into a practical therapeutic modality, the AFP-Cre/LoxP-shRNA-Atg5 was next tested in vivo. A nude mouse model of HCC was established using MHCC97L cells and the AFP-Cre/LoxP-shRNA-Atg5 was intratumorally administrated. Q-PCR and western blot analysis showed that the AFP-Cre/LoxP-shRNA-Atg5 could efficiently downregulate Atg5 in vivo (Fig. 5C). The average tumor weight of mice receiving AFP-Cre/LoxP-shRNA-Atg5 plus sorafenib (1.04±0.33) was significantly lower than that of mice subjected to sorafenib alone (1.42±0.29) (P = 0.039) (Fig. 5D). These results indicated that the AFP-Cre/LoxP-shRNA-Atg5 could enhance sorafenib treatment.
10.1371/journal.pone.0053072.g005Figure 5 Application of AFP-Cre/LoxP-shRNA targeting Atg5 gene for enhancement of sorafenib treatment.
(A) Construction of the AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5). Q-PCR and western blot analysis showed that it could efficiently knockdown Atg5 gene. (B) Combination of AFP-Cre/LoxP-shRNA-Atg5 infection and sorafenib treatment induced a significantly increase in apoptosis of HCC cells (MHCC97L and PLC) as compared to sorafenib treatment alone. Apoptosis was quantified by annexin V/PI FCM analysis. (C) Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 efficiently silenced Atg5 gene in vivo. (D) The AFP-Cre/LoxP-shRNA-Atg5 significantly enhanced sorafenib-induced suppression of tumorigenicity of MHCC97L cells in vivo. The tumor weight of mice receiving intratumoral injection of the AFP-Cre/LoxP-shRNA-Atg5 combined with sorafenib was significantly lower than that of mice subjected to sorafenib alone. (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Atg5; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone). (*P<0.05, **P<0.01).
Discussion
In this study, an efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. In vitro and in vivo analysis showed that it could efficiently silence target gene in an HCC-specific manner. Application of the system targeting Atg5 was shown to be able to enhance sorafenib treatment in vitro and in vivo, suggesting its value for HCC therapy. Our system provides a usable tool for HCC tissue-specific RNAi therapy, which holds significant promise as new approach for the management of HCC.
In recent years, with tremendous advance in HCC research, increasing HCC-related genes have been discovered. Translating these findings into clinical therapy, especially target therapy, holds great promise for HCC treatment [1], [6], [7]. The RNAi is a powerful tool for gene silencing. It can exert target therapy while avoid off-target side-effects. Increasing evidence shows that RNAi has great potential to be developed into HCC therapy [7], [17]. However, some obstacles must be overcome before the successful therapeutic application. [4]. RNAi should be capable of sustained inhibition of target gene while exerting its effect within target cells or tissues. With regard to targeting cells or tissues of interest, there are two strategies, including envelope-mediated entry targeting strategy and tissue-specific promoter -mediated targeting strategy [17]. (1) Envelope-mediated entry targeting strategy. Combining viral particles with a foreign envelope glycoprotein can alter the host tropism of viral vector. Using the hepatic virus envelope glycoprotein to pseudotype the host tropism and generate HCC tissue-type viral vectors may obtain targeting RNAi to HCC tissues; (2) Tissue-specific promoter -mediated targeting strategy. Using a tissue-specific promoter as an internal promoter can target expression to the cells of interest. Unlike the pseudotyping vectors in envelope-mediated strategy, vectors with a tissue-specific promoter can basically enter and integrate in any cell types. But their expression is limited to a certain cell type by the internal promoter. Various tissue-specific promoters have been incorporated into lentiviral vectors, including the AFP promoter [17]. Given the more complex structure of the envelop-mediated target delivery and difficulty de novo design, we chose to use HCC specific promoter (AFP-promoter) in combination with lenti vector to construct the RNAi system required. The AFP promoter is a commonly used HCC tissue-specific promoter because of its high specificity. It has been used to drive therapeutic genes expression to achieve HCC-specific target therapy [11]–[16]. Lenti vector-mediated RNAi is a powerful method for persistent and specific gene silencing. RNAi can be triggered by microRNA (miRNA) or small interfering RNA (siRNA) [7]. Regarding lentiviral vectors, RNAi can be delivered as shRNA driven by a Pol III promoter (such as U6) or microRNA (miRNA) expressed from a Pol II promoter (such as CMV or tissue-specific promoter) [7], [17]. The lentivirus-delivered shRNA or miRNA can be stably integrated into the target cell genome, allowing permanent and heritable gene silencing [17]. In this study, we designed two systems, including miRNA-based AFP-miRNA and shRNA-based AFP-Cre/LoxP-shRNA. The miRNA can be directly driven by Pol II promoter. We firstly tested whether the AFP promoter (Pol II) could drive miRNA expression to obtain effective RNAi. Our data showed that the AFP-miRNA could decrease target gene but required a high titer that was lethal to target cells, which excluded the in vivo application as it posed threat to adjacent normal cells or tissues. The low efficacy of the AFP-miRNA was attributed to the weak activity of the AFP promoter. Although the AFP promoter was shown to able to drive GFP expression, the transcriptional activity appeared to be insufficient to mediate efficient miRNA expression. As the AFP-miRNA could not achieve satisfatory efficacy, we then attempted the shRNA strategy. The shRNA cannot be directly driven by the AFP promoter (Pol II) but it can be efficiently driven by Pol III promoter (such as U6). However, the Pol III promoters mediated RNAi cannot be controlled in a tissue-specific manner because the Pol III promoters are constitutively expressed in all cell types. To circumvent this problem, a switch structure (Cre/loxP system combined with a modified U6 promoter) was designed to obtain shRNA expression driven by AFP promoter. Our results strongly demonstrated that the novel system could efficiently mediate shRNA expression and specifically silence target gene of HCC cells or tissues. Further application of the system for HCC treatment showed that the system was valuable for HCC therapy. The system-mediated HCC-specific Atg5 silencing enhanced sorafenib treatment. Although the Atg5 gene was shown to be not an optimal target gene for HCC RNAi therapy as its efficacy was weak, the validation using Atg5 had demonstrated the value of the AFP-Cre/LoxP-shRNA system. With the application of new better target gene, a satisfactory effect of the system-mediated RNAi therapy will be achieved.
Although the AFP-Cre-LoxP-shRNA system is shown efficient for HCC tissue-specific RNAi, it still carries some inherent disadvantages. The main one is that intravenous application of the system appears to be ineffective. This is not consistent with our expectations. The systemic administration of the system was thought to be able to achieve satisfactory efficacy since numerous studies had demonstrated that RNAi agents (siRNAs) could be easily delivered into the liver and liver tumor was suitable for intravenous application [7]. However, our data showed that intravenous injection of the system was not effective. This may be due to several reasons. First, intake of the system is limited. Due to the limitations of the lentiviral vector (maximum concentration is 2×109 TU/ml) and the nude mouse model (maximum volume of intravenous injection is 180 µl), the maximum intake is only 3.6×108 TU. Second, there is a considerable loss of the system during intravenous administration. The lentivrial vector used for this study has a wide host tropsin. It can quickly transfect any type of cells, although it works in only AFP-producing HCC cells. After being intravenously injected, it may immediately infect blood, endothelial cells or any other cells it encountered and then is considerably consumed before it reaches the tumor in the liver. The commonly used hydrodynamic injection was not used in this study. Although it is an effective way of delivering therapeutic agents into the liver in HCC mouse models [25], [26], this delivery method is found to be associated with significant mortality in our pilot study, which excluded it from systemic application as human HCC therapy was the final objective. Significant efforts are still required to improve the current system. The vector manipulation may be a possible solution. Lentiviral vector is a system easy to be manipulated [17]. Limiting the lentiviral vector entry through pseudotyping with heterologous viral glycoproteins is a practical strategy. Using the hepatic virus envelope glycoprotein for pseudotyping the host tropism to generate HCC tissue-type lentivectors may obtained targeted delivery of RNAi to HCC tissues and efficiently overcome the biological barriers. The vector manipulation is under investigation and the promising result is expected to be presented in the future.
Taken together, in this study, an HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully developed. The AFP-Cre/LoxP-shRNA system can efficiently silence target gene in an HCC tissue-specific manner. The system mediated silencing of therapeutic gene is valuable as adjuvant therapy for HCC treatment. Our system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new therapeutic modality for the management of HCC.
Supporting Information
File S1 The sequence of AFP promoter.
(DOC)
Click here for additional data file.
File S2 The AFP-Cre structure and U-LoxP-CMV-eGFP-LoxP-U6 structure.
(DOC)
Click here for additional data file.
File S3 Determination of the effective shRNAs targeting Beclin 1 and Atg5.
(DOC)
Click here for additional data file.
File S4 Comparison of the effects of AFP-Cre/LoxP-shRNA-Atg5 among sorafenib alone, and Cre/PoxP with and without sorafenib treatment.
(DOC)
Click here for additional data file.
We thank Dafeng Xu and Jin Zhen for technical assistance. We also thank David Duchenne, Jenny Xu, Eddie K. Kwong and Susan Zhao for linguistic advice and editorial assistance.
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11 : 675 –682 .14724673 | 23468839 | PMC3585287 | CC BY | 2021-01-05 17:21:22 | yes | PLoS One. 2013 Feb 28; 8(2):e53072 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23468836PONE-D-12-2609110.1371/journal.pone.0052469Research ArticleBiologyBiochemistryEnzymesEnzyme ClassesDehydrogenasesModel OrganismsAnimal ModelsRatMolecular cell biologySignal transductionSignaling in cellular processesProtein kinase C signalingSignaling PathwaysMedicineDrugs and DevicesCardiovascular PharmacologyIsoflurane Preconditioning Confers Cardioprotection by Activation of ALDH2 Cardioprotection Mechanism of IsofluraneLang Xiao-E
1
*
Wang Xiong
1
Zhang Ke-Rang
2
Lv Ji-Yuan
1
Jin Jian-Hua
3
Li Qing-Shan
4
1
Department of Cardiology, The First Clinical Medical College of Shanxi Medical University, Taiyuan, Shanxi, China
2
Department of Psychiatry, The First Clinical Medical College of Shanxi Medical University, Taiyuan, Shanxi, China
3
Department of Nuclear Medicine, The First Clinical Medical College of Shanxi Medical University, Taiyuan, Shanxi, China
4
School of Pharmaceutical Science, Shanxi Medical University, Taiyuan, China
Singh Shree Ram Editor
National Cancer Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: X-EL. Performed the experiments: X-EL XW K-RZ J-HJ. Analyzed the data: X-EL XW J-YL. Contributed reagents/materials/analysis tools: X-EL Q-SL XW K-RZ J-YL J-HJ. Wrote the paper: X-EL.
2013 28 2 2013 8 2 e5246925 8 2012 13 11 2012 © 2013 Lang et al2013Lang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.
This study is supported by National Natural Science Foundation of China (81172938). The funder’s website is www.nsfc.gov.cn. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Acute myocardial infarction (AMI) is responsible for the death of millions of persons worldwide each year [1]. Murry et al. demonstrated that a succession of short periods of myocardial ischemia and reperfusion prior to the continuous maintenance of coronary reperfusion protects the myocardium against subsequent prolonged ischemic insults, which has been termed ‘ischemic preconditioning’ (IPC) [2]. This phenomenon is achieved by several pharmacological agents, including volatile anesthetics. Volatile anesthetics such as isoflurane have cardioprotective effects when administered before a period of myocardial ischemia and reperfusion, and this phenomenon is referred to as anesthetic preconditioning (APC) [3], [4]. APC is a cardioprotective strategy that increases resistance to ischemia and reperfusion (I/R) by eliciting innate protective mechanisms, and was described in various animal models [3]–[6], as well as in humans [7], [8]. APC has been shown to reduce infarct size, and attenuate contractile dysfunction and serum CK-MB concentration caused by myocardial ischemia. Cellular signaling during APC is complex, and in many aspects, comparable to that of IPC. The intracellular mechanisms involved in APC have not been completely identified. It has become clear that multiple cellular pathways participate in the establishment of a cellular phenotype that makes the heart more resistant to ischemic damage. Mechanisms reported to date involve inhibition of mitochondrial permeability transition pore (mPTP) opening [9], the activation of kinases such as protein kinase C (PKC) [10], [11], the generation of reactive oxygen species (ROS) [12], [13], and opening of adenosine triphosphate-sensitive potassium channels (KATP) [3], [14], [15]
.
Translocation of PKC isoforms from the cytosol to the membranes is known to be a key mediator in IPC. PKC epsilon (PKCε) activation is required and is sufficient to protect the heart from ischemia and reperfusion (I/R) injury [16], [17]. Recent evidence suggests that PKCε is targeted to the mitochondria and interacts with many mitochondrial proteins, including mitochondrial aldehyde dehydrogenase 2 (ALDH2) [18]. The mitochondrial isoform of ALDH2 plays a key role in the metabolism of acetaldehyde and other toxic aldehydes, whose phosphorylation and activation by PKCε is required to confer cardioprotection [19], [20]. Overexpression of the ALDH2 transgene alleviates I/R injury, post-I/R and ischemic ventricular dysfunction [21], [22]. Consistent with this, ALDH2 knockout exacerbated I/R injury [23]. These data support the essential role of ALDH2 against I/R injury in the heart. Nonetheless, the mechanism(s) behind ALDH2-induced protection against I/R injury may be diverse, involving bioactivation of nitroglycerin, and reducing the production of free radicals [24], and ultimately mitochondrial dysfunction [23], all hallmarks of I/R injury. As anesthetic-induced preconditioning can also be demonstrated in humans, a thorough understanding of the signal transduction involved might have an impact on the clinical applicability of cardioprotection by APC. However, the role of ALDH2 in isoflurane-induced APC has not been investigated. Thus, the current study tested the hypothesis that PKCε-mediated activation of mitochondrial ALDH2 plays a critical role in isoflurane preconditioning.
Materials and Methods
Animals
Male Sprague-Dawley (SD) rats, weighing 200–220 g, were used in this study. The animals were provided by Experimental Animal Center of Tsinghua University. They were placed in a quiet, temperature (23±3°C) and humidity (60±5%) controlled room with a 12∶12 hours light-dark cycle (light beginning at 8 a.m.). This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the China National Institutes of Health.
Ischemia/reperfusion Injury Experimental Protocol
The acute myocardial I/R injury model was performed as previously described [25]. Male SD rats were anesthetized with pentobarbital sodium (30 mg/kg, i.p.). After a tracheotomy had been performed, rats’ lungs were ventilated mechanically with positive pressure ventilation using 30–40% air/oxygen mixture to maintain arterial blood gas pH within a physiological range by adjusting the respiratory rate and tidal volume throughout the experiment. Myocardial infarction (MI) was created by ligation of the left anterior descending (LAD). The thorax was opened at the fourth or fifth left intercostal space. After left thoracotomy and pericardiotomy, MI was induced by LAD ligation 2–3 mm from the origin with a 6–0 silk suture. All animals (except for the rats in the sham groups) were subjected to 40 min of regional myocardial ischemia followed by 120 min of reperfusion. To confirm isoflurane-induced APC, a minimal alveolar concentration of isoflurane of 1.0 (2.1%) was started at the end of the stabilization period and administered for 30 min, followed by 30 min of washout before coronary occlusion.
Rats were randomly assigned to one of the following groups subjected to different protocols: Sham group, non-ischemic control group of sham-operated rats; non-ischemic control group of sham-operated rats with isoflurane; I/R group with isoflurane; I/R group without isoflurane (n = 5, respectively). To evaluate the role of ALDH2 in isoflurane-induced APC, the direct activator of ALDH2, Alda-44 (40 µM) [18], was given 5 min prior to ischemia in the groups without and with isoflurane (n = 8, respectively), and the ALDH2 inhibitor, cyanamide (5 mM) [20], was given without and with isoflurane (5 min prior to isoflurane) (n = 8, respectively). To verify that PKCε participates in the phosphorylation of ALDH2, the PKCε inhibitor, PKCε v1–2 (1 µM), was given 5 min prior to ischemia without and with isoflurane (n = 8, respectively, 5 min prior to isoflurane).
After taking blood samples, the heart was removed and perfused with Langendorff apparatus for 10 min to wash out the blood. The coronary artery was re-occluded and Evans Blue was infused into the aortic root to label the normally perfused zone with a deep blue colour. The heart was sectioned into transverse slices, which were incubated with 1% trimethyl tetrazolium chloride (TTC), and photographed by a digital camera. Since TTC stains viable tissue a deep red colour, non-stained tissue was presumed to be infarcted. Area at risk (negative for Evans Blue) and infarct area (negative for TTC) were quantified using Imageproplus software (Version 4. 1, Media Cybernetics, LP, USA) and infarct size was expressed as percentage of the area at risk (infarct area/AAR)×100 (%).
Levels of Lactate Dehydrogenase and Creatine Kinase-MB in Plasma
Serum CK-MB analysis is a widely used biomarker to detect cardiac injury. Proportionally greater serum CK-MB, relative to the total CK activity can evaluate acute myocardial injury [8]. Before removing the heart, 5 ml blood samples were taken. Serum was separated by centrifugation at 5000 g for 5 min on a tabletop centrifuge; the supernatant was stored in liquid nitrogen. The samples were thawed for analysis. Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assayed using commercial kits (Roche, Germany) by an automatic analyzer 7600 (Hitachi, Japan).
Western Blotting Analysis
Upon completion of the experimental period, the myocardium and cardiomyocytes were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin at 4°C for 15 min. The homogenate was incubated and centrifuged. The supernatant was collected and the protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit according to the manufacturer's protocol (Pierce). The detergent soluble supernatant was frozen with liquid N2, and stored at −70°C.
The supernatant was mixed with 5× loading buffer and heated for 5 min at 100°C. Soluble extracts (50 µg) were loaded in each lane and separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) filter membrane (0.45 µm, Gehealthcare). The membrane was blocked in Tris-buffered saline Tween-20 (TBST) with 5% non-fat milk and incubated overnight with the corresponding primary antibodies at 4°C. The membrane was then incubated for 1 h with secondary antibody (horseradish peroxidase-conjugated antirabbit IgG) diluted with TBST (1∶2000). The signals of detected proteins were visualized by an enhanced chemiluminescence reaction (ECL) system (Millipore, Billerica, MA, USA). The staining was quantified by scanning the films and the band density was determined with Image-Pro software.
Adenovirus Construction and Infection
Previous studies [18], [26] demonstrated that the constitutively active ALDH2 amino acid 487 must be Glu not Lys, and Thr185, Thr412 and Ser 279 must be constitutively phosphorylated. Accordingly, we obtained constitutively active mutant ALDH2 (CA-ALDH2) by nucleotide substitutions leading to the mutations Lys487Glu, Thr185Asp, Thr412Asp, and Ser279Asp introduced into the wild-type rat Aldh2 cDNA.
Short RNA hybrids (siRNAs) of 19 bp were formed by annealing two 21-mer oligoribonucleotides (Eurogentec, Belgium), each having two thymidines at their 3′ end. Rat-Aldh2 siRNA sequence was GCAACCAGATTCATTAATT
[27]. The sense and antisense oligonucleotides were incubated together (1.5 nmol each) in 75 µL of 50 mM Tris (pH 7.5) and 100 mM NaCl for 2 min at 94°C, 5 min at 78°C and 5 min at 65°C. Finally, the annealed siRNAs were cooled to 20°C, aliquoted, and stored at −80°C. The cooling transitions was carried out at a rate of 2°C/min.
Viral vectors that expressed RFP, si-ALDH2 and RFP, the constitutively active mutant of ALDH2 (CA-ALDH2), and CA-ALDH2 and RFP were generated using the AdEasy system (Stratagene) [23]. Cardiomyocytes were prepared from ventricles and cultured in 60-mm dishes at a density of 1×105 cells/cm2 in NCS-DMEM. After 24 h incubation at 37°C and 5% CO2, cell density reached approximately 70%. Cells were cultured overnight in 10% FBS-containing medium and infected with adenovirus for 6 h at a multiplicity of infection (MOI) of 20, then cultured in serum-free medium for an additional 24 h, before the addition of reagents.
Cell Culture and Hypoxia/reoxygenation Treatment
Hearts were obtained from one-day old neonatal Sprague–Dawley rats, retaining the ventricles only, and kept in cold PBS without Ca2+ and Mg2+ on ice. The ventricles were rapidly minced and dissociated with 0.1% trypsin enzyme solution. The cells released after the first digestion were discarded, whereas the cells from subsequent digestions were added to NCS-DMEM (DMEM supplemented with 20% NCS, 100 U/ml penicillin, and 100 µg/ml streptomycin). After stepwise trypsin dissociation (10 min, 4–5 times), the mixture was centrifuged (1500 r/m, 5 min). The cells were resuspended in NCS-DMEM and first transferred to tissue culture dishes for 1 h in a 37°C incubator to plate out the fibroblasts [28]. The suspended cells were then replated at a density of 1×104 cells/cm−2 and incubated under the same conditions as above. Bromodeoxyuridine (BrdU, 0.1 mM) was added to the medium for the first 2 days after plating to inhibit the growth of fibroblasts.
Simulated I/R was achieved by culturing the cells in 0.5% FBS DMEM in a hypoxia chamber, saturated with 5%CO2/95%N2 and supplemented with an anaerobic pouch (Mitsubishi Gas Chemical Company, Inc.) at 37°C for 24 h and following reoxygenation for 12 h using 0.5% FBS DMEM in the normal incubating condition [29]. Exposure to isoflurane was carried out by incubating the cells for 5 min in 0.5 mM isoflurane (approximately 1.0 minimum alveolar concentration) in 0.5% FBS DMEM. The isoflurane-containing medium was removed immediately before the onset of hypoxic conditions and the cells were washed with phosphate-buffered saline (PBS) [9]. Anesthetic concentrations were measured by gas chromatography (Gas chromatograph GC-8A; Shimadzu, Kyoto, Japan). Cells were grouped as follows: vector-infected with and without isoflurane, Adeasy-Si-ALDH2-treated with and without isoflurane; vector-infected with and without isoflurane, Adeasy-CA -ALDH2-treated with and without isoflurane.
Apoptosis Assay
To determine cardiomyocyte apoptosis in a quantitative manner, the in situ detection of apoptotic cardiomyocytes was performed using terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) with an in situ cell death detection kit, Fluorescein (Roche, Germany) according to the manufacturer’s protocol for cultured cells. Cells (105 cells/ml) from different treatment groups were cultured in a 6-well chamber slide and fixed in 4% paraformaldehyde followed by digestion with proteinase K (10 µg/ml) for 15 min at 37°C and permeabilization with 0.1% Triton X-100 for 5 min at 4°C. After washing twice with PBS, the cardiomyocytes were incubated with 50 µl TUNEL reaction mixture that contains TdT and fluorescein-dUTP for 1 h at 37°C. The percentage of TUNEL positive cells was determined by randomly chosen fields in each slide. In each group, at least 500 cells were counted. Sample evaluation was performed in a blinded manner, and samples from 3 independent experiments were scored per group.
Measurement of Caspase 3 Activity
The Caspase 3 Colorimetric assay kit (MBL, MA, USA) was used to measure the activity of caspase 3 according to the manufacturer’s protocol. Cells were grown in a 6 well plate. After the appropriate treatment, cells were resuspended in lysis buffer and centrifuged. The supernatant was diluted with 50 ul cell lysis buffer for each assay. Then, the reaction buffer containing DTT and DEVD-pNA substrate were added to each assay and incubated at 37°C for 2 hours. After the correct incubation time, each sample was transferred to each well in a 96 well plate, and read at 405 nm using a microplate reader. Cell lysates were also analyzed by Western blotting with an antibody (Cell Signaling, Beverly, MA, USA) which allowed detection of inactive procaspase 3 and activated cleaved caspase 3.
Statistical Analysis
Continuous values are expressed as mean ± standard error of the mean (SEM). Comparisons between multiple-group means were performed using one-way analysis of variance (one-way ANOVA) and comparisons between groups were performed using the least significant difference test (LSD-test). The number of animals/group and statistical significance for all data are listed in the figures and figure legends. P values <0.05 were considered to be statistically significant. All statistical analyses were performed using SPSS version 15.0.
Results
Isoflurane Preconditioning Attenuated the Release of LDH and CK-MB and Reduced Infarct Size in vivo I/R Injury
Regional myocardial ischemia for 40 min by LAD ligation followed by 120 min of reperfusion markedly increased the leakage of LDH (Figure 1A) and CK-MB (Figure 1B) compared to sham controls. Isoflurane-induced APC significantly reduced the I/R-induced increase in LDH and CK-MB release in rat heart.
10.1371/journal.pone.0052469.g001Figure 1 The influence of anesthetic-induced preconditioning with 1.0 MAC of isoflurane on leakage of LDH and CK-MB, and infarct area in rat hearts.
A, B. Serum LDH and CK-MB concentrations were analyzed. The increase in LDH and CK-MB concentrations was lower in rats pretreated with isoflurane than in I/R group. C. Isoflurane preconditioning significantly decreased infarct area compared with I/R group animals. Representative cross-sectional slices derived from a single heart with and without isoflurane. The infarct size normalized to the area at risk. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. sham group, and #P<0.05 vs. the I/R control group.
As shown in Figure 1C, regional myocardial ischemia for 40 min by LAD ligation followed by 120 min of reperfusion significantly increased myocardial infarct size compared with sham groups. Isoflurane preconditioning substantially decreased I/R-induced myocardial infarct size.
Phosphorylation of ALDH2 Participated in Cardioprotection Induced by Isoflurane Pretreatment
Representative gels for the different treatment groups are shown in Figure 2Aa. Figure 2Ab summarizes the quantitative data on the ratio of phosphoALDH2 to total ALDH2, and shows that pretreatment with isoflurane prior to ischemia increased the phosphorylation of ALDH2. The ALDH2 inhibitor, cyanamide, significantly inhibited isoflurane-induced activation of ALDH2. The direct activator of ALDH2, Alda-44, substantially increased the phosphorylation of ALDH2, but did not enhance the phosphorylation of ALDH2 by isoflurane.
10.1371/journal.pone.0052469.g002Figure 2 Phosphorylation of ALDH2 associated with isoflurane-induced cardioprotection.
A. Effects of isoflurane preconditioning with and without the ALDH2 inhibitor (cyanamide), and direct activator of ALDH2 (Alda-44) on phosphorylation of ALDH2. (a) Representative of western blot analysis of the phosphorylation of ALDH2 (phos-ALDH2 top lanes) and total ALDH2 (middle lanes). β-actin (lower lanes) was used to demonstrate equal protein loading. (b) Quantification of phos-ALDH2 to the total ALDH2 from 3 independent experiments. B, C. Serum LDH and CK-MB concentrations were analyzed. D. The effects of ALDH inhibitor (cyanamide), and direct activator of ALDH2 (Alda-44) on infarct area in rat hearts. (a) Representative cross-sectional slices derived from a single heart. (b) The infarct size normalized to the area at risk. Values are means ± S.E.M., n = 8 in each group. ##P<0.01 vs. the saline control group and *P<0.05, **P<0.01 vs. the corresponding control.
Consistent with ALDH2 phosphorylation, we observed that isoflurane and Alda-44 markedly attenuated I/R-induced leakage of LDH and CK-MB in plasma, as well as myocardial infarct size. However, the isoflurane-induced decrease in LDH and CK-MB release, and reduction of infarct size was significantly blocked by the ALDH2 inhibitor cyanamide (Figure 2B–D). These findings suggest that isoflurane-mediated cardioprotection is mainly mediated by activation of ALDH2.
Isoflurane Preconditioning Alleviated in vitro H/R Injury by Activation of ALDH2
To further confirm the critical role of ALDH2 in isoflurane-induced cardioprotection, we constructed an ALDH2 knockdown adenovirus and constitutively active ALDH2 mutant adenovirus. We used TUNEL, caspase 3 activity, and LDH release as quantitative assays to determine the functional significance of manipulating ALDH2 expression.
After 24 h of hypoxia followed by 12 h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry (LSC;) and caspase 3 activity in the vector control group. Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 3B–E). However, when ALDH2 was downregulated (Figure 3A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level (Figure 3B), more intense cleaved caspase-3 staining (Figure 3C), caspase 3 activity (Figure 3D) and LDH release (Figure 3E) were observed, which supports the hypothesis that phosphorylation of ALDH2 might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2 level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure 4A). Constitutively active ALDH2 significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase 3 activity and leakage of LDH (Figure 4 B–E), which were not further increased by isoflurane treatment.
10.1371/journal.pone.0052469.g003Figure 3 ALDH2 knockdown blocks isoflurane-induced protection against hypoxia/reoxygenation.
A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and ALDH2shRNA (Ad-Si-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. ALDH2 down-regulation inhibited attenuation of TUNEL positive staining level by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and full length caspase-3 (FL caspase-3) in (a), and 3 independent experiments were quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05, vs. the corresponding Ad-RFP group.
10.1371/journal.pone.0052469.g004Figure 4 ALDH2 activation induced protection against hypoxia/reoxygenation.
A. Schematic representation of adenoviruses encoding RFP (Ad-RFP) and CA-ALDH2 (Ad-CA-ALDH2-RFP, left panel); right panel, immunoblotting analysis of ALDH2 protein level. β-actin was used as a control. B. Expression of constitutively active mutant of ALDH2 attenuated TUNEL positive staining level, which was not reinforced by isoflurane. (a) Apoptotic cells were examined using TUNEL assay for DNA fragmentation. Cardiomyoctes were photographed by fluorescence microscopy after 24 hours of hypoxia followed by 12 hours of reoxygenation. TUNEL-positive nuclei are shown in green and transfection efficiency of adenoviruses in red. (b) Quantitative analysis (percentage of apoptotic cells versus total) is shown in histogram. C. Representative Western blots of cleaved caspase-3 and FL caspase-3 in (a), and 3 independent experiments are quantitated in (b). β-actin was used as a loading control. D. Cell lysates under each condition were quantitatively assayed for caspase-3 activity. E. LDH concentrations in cell culture media were analyzed. Values are means ± S.E.M., n = 5 in each group. *P<0.05, **P<0.01 vs. Control group, and #P<0.05 vs. the corresponding Ad-RFP group.
PKCε is Involved in Isoflurane-induced Phosphorylation of ALDH2 and Cardioprotection
PKCε translocation to mitochondria and then phosphorylation of ALDH2 is required to protect the heart from I/R injury. Here we demonstrate that pretreatment with isoflurane resulted in elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2. Isoflurane-induced phosphorylation of ALDH2 was inhibited by the PKCε inhibitor, PKCε V1–2. Because mitochondrial translocation of PKCε occurs rapidly, with a corresponding decline in cytosolic PKCε levels, and because the total cellular PKCε levels do not change (Figure 5A), our data suggest that isoflurane enables dynamic mitochondrial translocation of PKCε in response to I/R. Consistent with PKCε translocation to mitochondria, PKCε V1–2 had a detrimental effect on isoflurane-induced attenuation of LDH and CK-MB leakage (Figure 5B, 5C), and the decrease in myocardial infarct size (Figure 5D).
10.1371/journal.pone.0052469.g005Figure 5 PKCε translocation is involved in isoflurane preconditioning.
A PKCε translocation was associated with isoflurane-induced phosphorylation of ALDH2. (a) Representative of western blot analysis of phos-ALDH2, total ALDH2, β-actin, mitochondria PKCε (mito-PKCε) and total PKCε (from top lanes to bottom lanes). β-actin was used to demonstrate equal protein loading. (b) Quantification of the phos-ALDH2, normalized to the total ALDH2, and PKCε translocation to the mitochondria from 3 different experiments. B, C. Isoflurane-induced inhibition of LDH and CK-MB release by I/R was restored by PKCε v1–2. Serum LDH and CK-MB concentrations were analyzed. D. PKCε v1–2 inhibits the decrease in heart infarct size caused by isoflurane following I/R. Representative cross-sectional slices derived from a single heart. The infarct size normalized to the area at risk. Values are means ± S.E.M., n = 8 in each group. *P<0.05, **P<0.01 vs. the saline control group and #P<0.05, ##P<0.01 with PKCε v1–2 vs. the corresponding group without PKCε v1–2.
Discussion
In the present study, we observed that (1) isoflurane pretreatment reduced I/R injury in vivo and stimulated H/R insult in vitro associated with phosphorylation of ALDH2; (2) isoflurane-induced phosphorylation of ALDH2 and cardioprotection was mediated by PKCε translocation from the cytosol to mitochondria. Thus, phosphorylation of ALDH2 is critical for the cardioprotective effects of isoflurane preconditioning.
Volatile anesthetics have a long history in the clinical management of anesthesia. Consistent with our results, numerous studies have shown that volatile anesthetics can protect the myocardium when applied before a harmful ischemic event and at the beginning of reperfusion, and that the characteristics of this protection are similar to those observed during classic IPC. Studies have attempted to characterize the mechanisms involved. Cardioprotective mechanisms produced by APC were shown to involve activation of phosphoinositide 3-kinase [25], extracellular regulated kinases 1 and 2 (ERK1/2) [30], the 70-kDa ribosomal protein S6 kinase, endothelial ROS (eNOS) [31], mitochondrial KATP channels [3], [14], [15] and inhibition of glycogen synthase kinase 3-β [32], but the precise mechanism responsible for APC remains undefined. However, it is unlikely that stimulation of pro-survival signaling pathways occurs rapidly enough to prevent damage resulting from the initial injury during reperfusion. Recently, attention has focused on mitochondria as a target of cardioprotection by volatile anesthetics [11], [33], [34].
Mitochondria are essential for cell survival and play important roles in the complex signaling pathways leading to cardioprotection by volatile anesthetics, and in the production of adenosine triphosphate and the regulation of cell death [35]. The mechanisms by which isoflurane ultimately limits infarct size are not known. Apoptosis and inflammation have been implicated in cardiac I/R injury [36]–[39]. In agreement with our results, isoflurane-treated mice subjected to ischemia and 2 weeks of reperfusion showed reduced expression of proapoptotic genes, significantly decreased expression of cleaved caspase-3, and TUNEL staining [5].
ALDH2 is best known for its role in metabolizing the ethanol intermediate, acetaldehyde. These highly toxic, reactive aldehydes can create aldehydic adducts with proteins, causing protein dysfunction and tissue injury, and have been linked to various diseases, such as cancer and MI, in humans [40]. It has been reported that overexpression of the ALDH2 transgene may alleviate I/R injury, post-I/R and ischemic ventricular dysfunction [21], [41]. Consistent with this, I/R injury may be exacerbated by ALDH2 knockout [23], [41]. These data support our results that ALDH2 plays an essential role in isoflurane-induced cardioprotection against I/R injury. It was shown that overexpression of ALDH2 significantly attenuated acetaldehyde and ethanol-induced oxidative stress (ROS generation), activation of stress signal molecules and apoptosis in fetal human cardiac myocytes [21]. Here, we showed that isoflurane preconditioning increased the phosphorylation of ALDH2. These data also support our notion that isoflurane pretreatment attenuated I/R-induced apoptosis which is associated with phosphorylation and activation of ALDH2.
We observed that isoflurane pretreatment led to PKCε translocation to mitochondria. Although phosphorylation and translocation of PKC are thought to be pivotal steps in cardioprotection by IPC, and PKCε seems to play a critical role in the signaling cascade underlying preconditioning [11], [16], there are few data suggesting the involvement of PKC in APC [11], [42]. It has been shown that phosphorylation and translocation of PKCε depends on the concentration of the volatile anesthetic and that alternative pathways may exist at higher concentrations [30]. Recent studies reported that PKCε targeted the inner mitochondrial membrane and phosphorylated a number of intra-mitochondrial proteins [18], [43], [44]. Mitochondrial ALDH2 has been identified as a PKCε substrate, whose activity correlates with cardioprotection against I/R [18], [20]. Our results showed that isoflurane-induced ALDH2 activation was accompanied by translocation of PKCε from the cytosolic to the mitochondria fraction, which was inhibited by the PKCε inhibitor. It was shown that ERK1/2 blockade abolished PKCε activation, suggesting ERK pathway was involved in activation of PKCε, during desflurane-induced preconditioning [30]. Opening of mitochondrial adenosine triphosphate-sensitive potassium channels and generation of reactive oxygen species were upstream events of PKCε activation in isoflurane-induced preconditioning [45]. Activation of ALDH2 can attenuate ROS production [21], indicating that ALDH2 might be a critical mediator of isoflurane-induced protection. Further research needs to be carried out to identify the ALDH2 mechanism in mitochondria.
Although polymorphism in ALDH2 gene is an independent risk factor for myocardial infarction [46], [47], a recent study showed that inhibited ALDH2 activity during cardiac surgery got less I/R injury and better cardiac function [48]. The contradicted clinical results might need larger sample and stronger evidence to testify. However, from point of view of clinical application of APC in the future, in patients with lower Aldh2 activity who experience an ischemic event, the use of isoflurane may need to be reconsidered.
In summary, our results demonstrate that isoflurane preconditioning increased the phosphorylation of mitochondrial ALDH2 which was mediated by mitochondrial PKCε and is required for cardiac protection against I/R (Figure 6). This work suggests a possible mechanism by which isoflurane can access cytoprotective substrates located within the mitochondria to confer cardioprotection [49]. Our data provide an insight into the mitochondrial-dependent basis of isoflurane-induced, and PKCε and ALDH2-mediated protection against cardiac ischemia, in vivo and in vitro [19]. The current study extends our understanding of APC cardiac protection, which is relevant for extrapolation to the clinic.
10.1371/journal.pone.0052469.g006Figure 6 Hypothetical scheme demonstrate that the phosphorylation of ALDH2 through mitochondrial translocation of PKCε plays an important role in the cardioprotection of isoflurane preconditioning in myocardium I/R injury.
Ischemic preconditioning/postconditioning and pharmacological agents result in the activation of the RISK pathway, which lead to the phosphorylation and mitochondrial translocation of PKCε.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23468978PONE-D-12-3369710.1371/journal.pone.0057382Research ArticleBiologyBiochemistryProteinsTransmembrane ProteinsDevelopmental BiologyMorphogenesisCell MigrationGeneticsGene ExpressionMedicineGastroenterology and HepatologyOncologyBasic Cancer ResearchMetastasisCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaCancer Detection and DiagnosisThe Significance of Notch1 Compared with Notch3 in High Metastasis and Poor Overall Survival in Hepatocellular Carcinoma Notch1 and Notch3 in HepatomaZhou Liang Zhang Ning Song Wenjie You Nan Li Qingjun Sun Wei Zhang Yong Wang Desheng Dou Kefeng
*
Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shannxi, People’s Republic of China
Man Kwan Editor
The University of Hong Kong, Hong Kong
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: DW KD. Performed the experiments: LZ NZ. Analyzed the data: WS NY. Contributed reagents/materials/analysis tools: QL WS YZ. Wrote the paper: LZ.
2013 28 2 2013 8 2 e5738226 10 2012 21 1 2013 © 2013 Zhou et al2013Zhou et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The prognosis for patients with hepatocellular carcinoma (HCC) is poor, and the mechanisms underlying the development of HCC remain unclear. Notch1 and Notch3 may be involved in malignant transformation, although their roles remain unknown.
Materials and Methods
HCC tissues were stained with anti-Notch1 or -Notch3 antibody. The migration and invasion capacities of the cells were measured with transwell cell culture chambers. RT-PCR was used to measure the expression of Notch1 and Notch3 mRNA. Additionally, western blot analysis was used to assess the protein expression of Notch1, Notch3, CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA). RNA interference was used to down-regulate the expression of Notch1 and Notch3. Cell viability was assessed using MTT.
Results
Based on immunohistochemistry, high Notch1 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage. High Notch3 expression was only strongly correlated with metastasis, venous invasion and satellite lesions. Kaplan-Meier curves demonstrated that patients with high Notch1 or Notch3 expression were at a significantly increased risk for shortened survival time. In vitro, the down-regulation of Notch1 decreased the migration and invasion capacities of HCC cells by regulating CD44v6, E-cadherin, MMP-2, MMP-9, and uPA via the COX-2 and ERK1/2 pathways. Down-regulation of Notch3 only decreased the invasion capacity of HCC cells by regulating MMP-2 and MMP-9 via the ERK1/2 pathway.
Conclusions
Based on the migration and invasion of HCC, we hypothesize that targeting Notch1 may be more useful than Notch3 for designing novel preventive and therapeutic strategies for HCC in the near future.
This work was supported by grants from the National Natural Science Foundation of China (Grants No. 30872480) and the Major Program of the National Natural Science Foundation of China (Grants No. 81030010/H0318). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Currently, systemic chemotherapy is ineffective in hepatocellular carcinoma (HCC), as evidenced by low response rates and no demonstrated survival benefit. Additionally, liver transplantation is considered the only curative treatment option for HCC. However, its use has been restricted by factors such as the scarcity of donor organs and the risks associated with primary hepatic resection. Many patients undergo different therapies, yet the prognosis of HCC remains dismal, which is mainly attributed to the aggressive metastasis and recurrence of HCC [1]. However, the mechanisms underlying the development of HCC remain unclear.
The Notch pathway is important for cell fate determination, tissue patterning and morphogenesis, and cell differentiation, proliferation and death [2]. Most studies have focused on Notch1 and Notch3, which may be involved in malignant transformation. Notch1 has been shown to be up-regulated in prostate cancer, small cell lung cancer, pancreatic cancer and HCC and is involved in tumor cell invasion in pancreatic cancer, lingual squamous cell carcinoma, and breast cancer [3]–[8]. Additionally, high Notch1 expression has been reported to be related to poor overall survival rates in breast and colorectal cancer [9], [10]. Aberrant Notch3 expression has been reported in virtually all cases of T cell acute lymphoblastic leukemia (T-ALL), colorectal cancer, HCC, lung cancer, pancreatic cancer, and ovarian cancer [11]–[16]. However, the relationship among Notch1 or Notch3, clinicopathological manifestations and the survival rate in patients with HCC has not been explored. Furthermore, the potential mechanisms of Notch1 and Notch3 involvement in HCC are unclear.
In the present study, we investigated Notch1 and Notch3 expression in HCC tissues and, for the first time, explored the possible relationships between Notch1 and Notch3 expression and prognosis in HCC. We further explored the potential mechanism of Notch1 and Notch3 involvement in the migration and invasion of HCC in vitro.
Materials and Methods
Patients and Tissue Specimens
Tissue specimens from HCC and adjacent non-cancerous hepatic tissues (at least 1.5 cm away from the tumor) were collected from 86 patients who underwent surgical treatment for primary HCC in the Department of Hepatobiliary Surgery at Xijing Hospital (Xi’an, China) between 2004 and 2007. Specimens were collected from patients who had not received preoperative treatment. There were 54 male and 32 female patients, with a median age of 45.3 years (range, 30–80 years). This study was approved by the Ethics Committee of the Fourth Military Medical University and conformed to the ethical guidelines of the 2004 Declaration of Helsinki. Written informed consent was obtained from each patient or his or her legal guardians. Clinical parameters, such as gender, age, tumor location, tumor size, tumor grade, metastasis, satellite lesions, tumor number, AJCC TNM stage, and AFP, were collected. In patients diagnosed with metastasis, we also analyzed vascular invasion. Among the 24 cases diagnosed with metastasis, complications included venous invasion (n = 16), bile duct tumor thrombi (n = 9) and lymph node metastasis verified by pathological analysis (n = 4). Enrolled patients were followed for 5 years for survival calculations.
Cell Culture and Reagents
A human liver non-tumor cell line (HL-7702, obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences) and HCC cell lines (HepG2, and SMMC-7721, obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and MHCC97H, obtained from the Liver Cancer Institute of Fudan University) were cultivated in DMEM supplemented with 10% fetal calf serum (Sigma Chemical Co., St. Louis, MO). The cells (1 × 105 cells/well) were seeded into 6-well cell culture plates for 48 h until the next experiments. Primary antibodies against Notch1, Notch3, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, urokinase-type plasminogen activator (uPA), cyclooxygenase-2 (COX-2) and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD44v6, extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p-ERK1/2 were purchased from Abcam (Cambridge, UK). All secondary antibodies were obtained from Pierce (Rockford, IL, USA). An SP immunostaining kit purchased from ZYMED (ZSGB; Beijing, China) was used. Notch1 small interfering RNA (siRNA), Notch3 siRNA and an siRNA control were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). To inhibit endogenous COX-2 activity, 50 µmol/l NS-398 (Sigma-Aldrich) and 70 nmol/l SC58125 (Ann Arbor, MI) were used. To inhibit ERK1/2 activity, 10 µmol/l PD98059 (Calbiochem, San Diego, CA) and 1 µmol/l U0126 (Ellisville, Missouri, USA) were used. NS-398, SC-58125, PD98059, and U0126 were dissolved in DMSO (Sigma-Aldrich). All other chemicals and solutions were purchased from Sigma-Aldrich, unless otherwise indicated.
Immunohistochemistry and Evaluation of Staining
Immunohistochemistry involved the use of the avidin-biotin-peroxidase complex method on all tissues. All sections were deparaffinized in xylene and dehydrated through a graduated alcohol series before endogenous peroxidase activity was blocked with 0.5% H2O2 in methanol for 10 min. Nonspecific binding was blocked by incubating sections with 10% normal goat serum in PBS for 1 h at room temperature. Without washing, sections were incubated with anti-Notch1 or anti-Notch3 (1∶50) in PBS at 4°C overnight in a moist box. The sections were incubated with biotinylated IgG for 2 h at room temperature and detected with a streptavidin-peroxidase complex. The brown color indicative of peroxidase activity was obtained by incubating the section with 0.1% 3,3-diaminobenzidine in PBS with 0.03% H2O2 for 10 min at room temperature. The tissue specimens were scored independently by two pathologists blinded to the clinicopathology and outcome of the patients using an immunoreactivity score system described previously [10]. Based on the score, we divided all HCC specimens into two subgroups: the low expression group (score of 0–4) and the high expression group (score of 5–12 score).
Small Interfering RNA Transfection
According to the LipofectAMINE 2000 protocol (Carlsbad, CA, USA), HCC cells were transfected with Notch1 siRNA, Notch3 siRNA or control siRNA. The cells transfected with siRNA (1 × 105 cells/well) were seeded into 6-well cell culture plates and allowed to continue growing for 24 h before harvesting for further analysis.
Real-time Reverse Transcription PCR
Total RNA was extracted and reverse transcribed. The primers used in the PCR are as follows: Notch1, forward primer (5′-CACCCATGACCACTACCCAGTT-3′) and reverse primer (5′-CCTCGGACCAATCAGAGATGTT-3′); Notch3, forward primer (5′-AAGGACGTGGCCTCTGGT-3′) and reverse primer (5′-TCAGGCTCTCACCCTTGG-3′); GAPDH, forward primer (5′- AAATCCCATCACCATCTTCC-3′) and reverse primer (5′- TCACACCCATGACGAACA-3′). The primers were evaluated by running a virtual PCR, and the primer concentration was optimized to avoid primer dimer formation. Additionally, dissociation curves were evaluated to avoid nonspecific amplification. Real-time PCR amplifications were undertaken in the Mx4000 Multiplex QPCR System (Stratagene, La Jolla, CA) using 2×SYBR Green PCR Master Mix (Applied Biosystems). Data were analyzed according to the comparative C
t method and normalized by the GAPDH expression in each sample.
Protein Extraction and Western Blotting
The cells were lysed in lysis buffer [8] after incubation for 20 minutes at 4°C. The protein concentration was determined using the Bio-Rad assay system (Bio-Rad, Hercules, CA, USA). Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked with 5% nonfat dried milk or bovine serum albumin in 1×TBS buffer containing 0.1% Tween 20 and then incubated with appropriate primary antibodies. Horseradish peroxidase–conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody, and the protein bands were detected using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The quantification of western blots was performed using laser densitometry, and relative protein expression was normalized to GAPDH levels.
MTT Assay
The treated cells (1×104 cells/well) were seeded into 96-well cell culture plates and grown for up to 48 h. Cell viability was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Sigma Chemicals Co.) in accordance with the manufacturer’s protocol. Each experiment included six replications and was repeated three times. The data were summarized as means ± SDs.
Migration and Invasion Assays
Cell migration and invasion were analyzed using non-matrigel-coated or matrigel-coated transwell cell culture chambers (8 µm pore size) (Millipore, Billerica, MA, USA). Briefly, treated cells (5 × 104 cells/well) were serum starved for 24 h and plated in the upper insert of a 24-well chamber in serum-free medium. Medium containing 10% serum as a chemoattractant was added to the well, and the cells were incubated for 24 h. Cells on the upper side of the filters were mechanically removed by scrubbing with a cotton swab. The membrane was subsequently fixed with 4% formaldehyde for 10 min at room temperature and stained with 0.5% crystal violet for 10 min. Finally, invasive and migrated cells were counted at 200× magnification in 10 different fields of each filter.
Statistical Analysis
Statistical analysis was conducted using SPSS 15.0 software (Chicago, IL, USA). Each experiment was repeated at least three times. All data were summarized and presented as means ± SDs. The differences among means were analyzed statistically using a t-test. Associations between Notch1 and Notch3 expression and categorical variables were analyzed using χ2 tests and Fisher’s exact tests, as appropriate. Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazard model was used for univariate and multivariate analysis to explore the effect of clinicopathological factors and Notch1 and Notch3 expression on survival. P<0.05 was considered statistically significant.
Results
Notch1 and Notch3 Immunohistochemistry
Notch1 and Notch3 were mainly localized in the cytoplasm and cell membrane. Neither Notch1 nor Notch3 was significantly expressed in adjacent non-cancerous hepatic tissues, with only weak staining of Notch1 and Notch3 in the cell membrane and cytoplasm. As Fig. 1 (a–h) shows, the expression of Notch1 and Notch3 was different in HCC tissues. Notch1 staining was not detected in 9 samples of HCC. In contrast, weak positive staining of Notch1 was detected in 31 samples of HCC, moderate positive staining was detected in 17 samples, and strong positive staining was detected in 29 samples. Notch3 staining was not detected in 11 samples of HCC. In contrast, weak positive staining of Notch3 was detected in 30 samples of HCC, moderate positive staining was detected in 19 samples, and strong positive staining was detected in 26 samples.
10.1371/journal.pone.0057382.g001Figure 1 Expression of Notch1 and Notch3 in HCC tissues and Kaplan-Meier statistical analyses of postoperative survival curves according to Notch1 and Notch3 expression.
Notch1 expression in HCC tissues (a–d): (a) negative, (b) weakly positive, (b) moderately positive, (d) strongly positive. Notch3 expression in HCC tissues (e-h): (e) negative, (f) weakly positive, (g) moderately positive, (h) strongly positive. (i) Kaplan-Meier statistical analyses of postoperative survival curves according to Notch1 expression; (j) Kaplan-Meier statistical analyses of postoperative survival curves according to Notch3 expression.
Relationship between Notch1 and Notch3 Expression and Clinicopathological Characteristics
We divided the 86 patients into two subgroups: a high Notch1 or Notch3 expression group and a low Notch1 or Notch3 expression group. The relationship between Notch1 and Notch3 expression and clinicopathological factors is summarized in Table 1. The results showed that high Notch1 expression was strongly correlated with tumor size (P<0.001), tumor grade (P = 0.003), metastasis (P = 0.045), venous invasion (P = 0.014) and AJCC TNM stage (P<0.001). However, the high expression of Notch3 was only strongly correlated with metastasis (P = 0.002), venous invasion (P = 0.010) and satellite lesions (P = 0.033).
10.1371/journal.pone.0057382.t001Table 1 Association of Notch1 or Notch3 expression with clinicopathologic factors of the HCC patients.
Tumor characteristic n Notch1 p-value χ2
Notch3 p-value χ2
High(5–12 score) Low(0–4 score) High(5–12 score) Low(0–4 score)
All cases 86 46(53.5%) 40(46.5%) 45(52.3%) 41(47.7%)
Gender
Male 54 29(53.7%) 25(46.3%) 0.959 0.003 27(50.0%) 27(50.0%) 0.575 0.315
Female 32 17(53.1%) 15(46.9%) 18(56.3%) 14(43.8%)
Age (years)
≤50 48 26(54.2%) 22(45.8%) 0.887 0.020 27(56.3%) 21(43.8%) 0.413 0.671
>50 38 20(52.6%) 18(47.4%) 18(47.4%) 20(52.6%)
Tumor location
Left 40 18(45.0%) 22(55.0%) 0.141 2.166 19(47.5%) 21(52.5%) 0.403 0.698
Right 46 28(60.9%) 18(39.1%) 26(56.5%) 20(43.5%)
Tumor size (cm)
≤5 36 11(30.6%) 25(69.4%) <0.001*
13.090 23(63.9%) 13(36.1%) 0.068 3.319
>5 50 35(70.0%) 15(30.0%) 22(44.0%) 28(56.0%)
Tumor grade (differentiation)
Well 29 22(75.9%) 7(24.1%) 0.003*
8.804 14(48.3%) 15(51.7%) 0.592 0.288
Moderately or poorly 57 24(42.1%) 33(57.9%) 31(54.4%) 26(45.6%)
Metastasis
Yes 24 17(70.8%) 7(29.2%) 0.045*
4.026 19(79.2%) 5(20.8%) 0.002*
9.614
No 62 29(46.8%) 33(53.2%) 26(41.9%) 36(58.1%)
Venous invasion
+ 16 13(81.3%) 3(18.8%) 0.014*
6.090 13(81.3%) 3(18.7%) 0.010*
6.593
– 70 33(47.1%) 37(52.9%) 32(45.7%) 38(54.3%)
Satellite lesions
+ 24 13(54.2%) 11(45.8%) 0.937 0.006 17(70.8%) 7(29.2%) 0.033*
4.571
– 62 33(53.2%) 29(46.8%) 28(45.2%) 34(54.8%)
Tumor number
Solitary 65 32(49.2%) 33(50.8%) 0.164 1.940 35(53.8%) 30(46.2%) 0.619 0.247
Multiple 21 14(66.7%) 7(33.3%) 10(47.6%) 11(52.4%)
AJCC TNM stage
I and II 24 4(16.7%) 20(83.3%) <0.001*
18.143 13(54.2%) 11(45.8%) 0.832 0.045
III and IV 62 42(67.7%) 20(32.3%) 32(51.6%) 30(48.4%)
AFP (ng/ml)
≤400 18 7(38.9%) 11(61.1%) 0.163 1.950 9(50.0%) 9(50.0%) 0.824 0.049
>400 68 39(57.4%) 29(42.6%) 36(52.9%) 32(47.1%)
* Statistically significant difference.
Correlation between Notch1 and Notch3 Expression and Prognosis of HCC Patients
A Kaplan-Meier postoperative survival curve was used to evaluate the overall survival rate of HCC patients in relation to Notch1 and Notch3 expression. The log-rank test showed that the survival time was significantly different between the low and high Notch1 (P<0.001) and Notch3 (P<0.001) expression groups. Moreover, the low Notch1 and Notch3 expression groups had better survival (Fig. 1 i and j). But the survival time between the high Notch1 and Notch3 groups had not different (Fig. S1). The cumulative 5-year survival rates were 30.0% and 26.8% in the low Notch1 and Notch3 expression group, respectively, and only 15.2% and 8.9% in the high Notch1 and Notch3 expression groups, respectively.
For Notch1 expression, a univariate Cox regression analysis showed that tumor size, metastasis, venous invasion, tumor number, AJCC TNM stage, and Notch1 protein expression were significantly associated with overall survival (Table 2). Furthermore, to evaluate the potential of high Notch1 expression as an independent predictor for overall survival of HCC, multivariate Cox regression analyses were performed. Although other characteristics failed to demonstrate an independent prognostic role, tumor size, metastasis, venous invasion, tumor number, and Notch1 expression may play a role in the prediction of overall survival in HCC (Table 2). However, in the data for Notch3 expression, a univariate Cox regression analysis also showed that metastasis, venous invasion, AJCC TNM stage, and Notch3 protein expression were significantly associated with overall survival (Table 3). Multivariate Cox regression analyses showed that metastasis, venous invasion and Notch3 expression may play a role in the prediction of overall survival in HCC (Table 3).
10.1371/journal.pone.0057382.t002Table 2 Univariate and multivariate analysis for overall survival of 86 patients (analyze data of Notch1 expression).
Tumor characteristic Relative risk (95% CI) P-value
Univariate
Gender 0.664(0.407–1.083) 0.101
Age (years) 1.472(0.907–2.388) 0.118
Tumor location 0.852(0.525–1.383) 0.517
Tumor size 2.026(1.210–3.392) 0.007*
Tumor grade (differentiation) 0.712(0.427–1.188) 0.193
Metastasis 25.045(10.773–58.225) <0.001*
Venous invasion 23.858(10.333–55.088) <0.001*
Satellite lesions 1.587(0.938–2.684) 0.085
Tumor number 2.266(1.325–3.876) 0.003*
AJCC TNM stage 4.940(2.544–9.593) <0.001*
AFP (ng/ml) 1.231(0.672–2.258) 0.501
Notch1 2.353(1.434–3.859) 0.001*
Multivariate
Tumor size 3.296(1.245–8.725) 0.016*
Metastasis 18.415(5.968–56.825) <0.001*
Venous invasion 7.625(2.631–22.096) <0.001*
Tumor number 2.790(1.522–5.116) 0.001*
AJCC TNM stage 1.034(0.313–3.418) 0.956
Notch1 1.787(1.012–3.155) 0.045*
95% CI: 95% confidence interval.
* Statistically significant difference.
10.1371/journal.pone.0057382.t003Table 3 Univariate and multivariate analysis for overall survival of 86 patients (analyze data of Notch3 expression).
Tumor characteristic Relative risk (95% CI) P-value
Univariate
Gender 0.856(0.531–1.379) 0.523
Age (years) 1.062(0.664–1.696) 0.802
Tumor location 0.996(0.624–1.590) 0.988
Tumor size 1.105(0.682–1.789) 0.686
Tumor grade (differentiation) 0.674(0.414–1.100) 0.114
Metastasis 8.965(4.845–16.590) <0.001*
Venous invasion 18.410(8.600–39.407) <0.001*
Satellite lesions 1.395(0.830–2.345) 0.209
Tumor number 1.352(0.798–2.289) 0.262
AJCC TNM stage 2.328(1.311–4.136) 0.004*
AFP (ng/ml) 0.925(0.522–1.639) 0.790
Notch3 2.848(1.743–4.654) <0.001*
Multivariate
Metastasis 3.142(1.352–7.300) 0.008*
Venous invasion 8.774(3.143–24.492) <0.001*
AJCC TNM stage 1.787(0.969–3.296) 0.063
Notch3 3.114(1.853–5.233) <0.001*
95% CI: 95% confidence interval.
* Statistically significant difference.
siRNA can Efficiently Down-regulate Notch1 and Notch3 Expression
The mRNA and protein expression levels of Notch1 and Notch3 were significantly up-regulated in HCC cells compared with HL7702 cells. In particular, in parallel with the increase in metastatic potential in HCC cells (MHCC97H>SMMC7721>HepG2), the mRNA and protein expression levels of Notch1 and Notch3 were markedly up-regulated (Fig. S2a and S2b). The migration and invasion capacity was lowest in HepG2 cells and highest in MHCC97H cells. Therefore, we only used HepG2 and MHCC97H cells for the subsequent experiments. In HepG2 and MHCC97H cells, siRNA down-regulated the expression of Notch1 and Notch3 mRNA and protein levels (Fig.S2c–f). To further confirm that the inhibitory effects of siRNA on Notch1 and Notch3 expression were independent of apoptosis, we used an MTT assay to detect Notch1 and Notch3 siRNA-transfected cells. As the results of the MTT assay show, Notch1 and Notch3 siRNA had no effect on the cell growth or viability of HCC cells (Fig. S2 g).
Notch1 and Notch3 Play Different Roles in the Migration and Invasion of HCC Cells
Using transwell cell culture chambers, we measured the migration and invasion of Notch1 and Notch3 siRNA-transfected HepG2 and MHCC97H cells. HepG2 and MHCC97H cells transfected with control siRNA were used as controls. Only Notch1 down-regulation significantly decreased the numbers of migratory HCC cells (Fig. 2a–c). However, Notch1 or Notch3 down-regulation significantly decreased the numbers of invasive HCC cells (Fig. 2d–f). These data indicated that Notch1 down-regulation can reduce the migration and invasion of HCC cells. However, Notch3 down-regulation had no effect on migration and could only reduce invasion in HCC cells. Furthermore, to determine the potential mechanisms of Notch1 and Notch3 in the migration and invasion of HCC cells, we examined the effect of down-regulated Notch1 and Notch3 on metastasis-associated molecules, such as CD44v6, E-cadherin, MMP-2, MMP-9, and uPA. As Fig. 3a shows, Notch1 down-regulation decreases the protein expression of CD44v6, MMP-2, MMP-9, and uPA while increasing the protein expression of E-cadherin in HCC cells. However, Notch3 down-regulation only decreased the protein expression of MMP-2, MMP-9, and uPA.
10.1371/journal.pone.0057382.g002Figure 2 Down-regulation of Notch1 or Notch3 can decrease the migration and invasion of HepG2 and MHCC97H cells in vitro.
(a–c) Migrated HCC cells analyzed by transwell assays compared with siRNA controls. (d–f) Invaded HCC cells analyzed by transwell assays compared with siRNA controls. The data are presented as the mean ± SD, *P<0.05 compared with control siRNA-transfected HepG2 cells; #P<0.05 compared with control siRNA-transfected MHCC97H cells. NT: No transfection; Cs: control siRNA transfection; N1s: Notch1 siRNA transfection; N3s: Notch3 siRNA transfection.
10.1371/journal.pone.0057382.g003Figure 3 Effects of down-regulated Notch1 or Notch3 on the expression of CD44v6, E-cadherin, MMP-2, MMP-9, uPA, COX-2, ERK1/2, and p-ERK1/2 in HepG2 and MHCC97H cells.
(a) The protein expression of CD44v6, E-cadherin, MMP-2, MMP-9, and uPA was measured by western blot analysis in differently treated HCC cells. (b) The protein expression of COX-2, ERK1/2 and p-ERK1/2 were measured by western blot analysis in differently treated HCC cells. NT: No transfection; Cs: control siRNA transfection; N1s: Notch1 siRNA transfection; N3s: Notch3 siRNA transfection.
Notch1 and Notch3 Play Different Roles in Regulating COX-2 and the ERK1/2 Pathway
COX-2 can regulate the expression of CD44v6 and E-cadherin, while the ERK1/2 pathway can regulate the expression of MMP-2, MMP-9, and uPA in some cancers. In HCC cells, inhibitors of COX-2 can also effectively decrease the expression of CD44v6 and increase the expression of E-cadherin (Fig. S3). Inhibitors of ERK1/2 can effectively decrease the expression of MMP-2, MMP-9, and uPA (Fig. S4). But it was unknown that if the regulated roles of COX-2 or ERK1/2 were via regulating Notch1 or Notch3. We also examine the proteins expression of Notch1 or Notch3 in HepG2 and MHCC97H cells treated with inhibitors of COX-2 or ERK1/2. As Fig. S5 and Fig. S6 showed, the inhibitors of COX-2 or ERK1/2 can not affect the expression of Notch1 and Notch3 in protein level. Further, we explored the effect of down-regulated Notch1 or Notch3 on COX-2 and the ERK1/2 pathway. As Fig. 3b shows, down-regulated Notch1 decreased the expression of COX-2 and p-ERK1/2. However, down-regulated Notch3 only decreased the expression of p-ERK1/2.
Discussion
The Notch pathway interacts with several other signal transduction pathways, and its activation can lead to different outcomes ranging from the control of proliferation to apoptosis, differentiation, and cell fate decision [2]. The Notch pathway includes Notch ligands, receptors, negative and positive modifiers, and Notch target transcription factors. To date, four Notch receptors (Notch1-4) have been identified in mammals. But different Notch receptors play a paradoxical role, either as a tumor suppressor or oncogene. Notch1 and Notch3 are up-regulated in many types of tumors and are involved in the metastasis of tumor cells [3]–[8], [11]–[17]. It has also been reported that high levels of Notch1 and Notch3 expression are related to poor overall survival rates in cancer [9], [10], [18]–[20].But in many tumor, Notch2 may play the opposite roles. The expression of Notch2 was down-regulated in HCC, colorectal cancer, and breast cancer [8], [21], [22]. Low levels of Notch2 expression are related to poor overall survival rates and poor differentiation in cancer [21], [22]. Though Notch4 levels are up-regulated in tumor and involved in tumor [23], [24], Notch4 appears to have committed vascular functions. Notch4 (as a endothelial arterial markers) are expressed by vascular endothelial cells [25] and are involved in sprouting angiogenesis [26]. So it indicated Notch1 and Notch3 may play similar role in tumor cells. However, the research about Notch1 and Notch3 in HCC is limited, especially, the relationships between Notch1 and Notch3 and the prognosis of HCC patients is unknown.
In the present study, we examined the expression of Notch1 and Notch3 by immunohistochemistry in HCC samples as other researchs [27]–[30]. Though immunohistochemistry is a good tool to detect a specific protein expression, while it is not a good tool for quantify a specific protein expression. Western blotting may be good for quantify a specific protein expression. But Notch1 expression in tumor vasculature and is known to be involved in vascular endothelial cells [25]. If total tissue proteins (perhaps including vascular cells) were subjected to western blotting, tumor vascular endothelial cells, in addition to tumor cells, might be evaluated in western blotting. To confirm the results precisely, figures of immunohistochemistry will give us the definite information on distribution and intensity of Notch1 protein both within tumor itself and within tumor vasculature. By using immunohistochemistry, we showed that in tumor tissues, high levels of Notch1 expressions were correlated with tumor size, tumor grade, metastasis, venous invasion and TNM stage, whereas Notch3 expression was correlated with metastasis, venous invasion and satellite lesions. These clinical parameters are also indications of an advanced tumor. The results strongly suggested that Notch1 and Notch3 may play key roles in the advancement of HCC. Prognostic molecular biomarkers are invaluable for the clinician to evaluate patients and to aid in tumor control. The Kaplan-Meier analysis of the survival curves showed a significantly worse overall survival rate for patients whose tumors had high Notch1 (log-rank test, P<0.001) and Notch3 (log-rank test, P<0.001) levels, indicating that high Notch1 and Notch3 protein levels are markers of poor prognosis for patients with HCC. But overall survival rate between high Notch1 and Notch3 was not statistical different (log-rank test, P>0.005). Moreover, a multivariate analysis showed Notch1 and Notch3 expression to be indicators of worse outcomes independent of the known clinical prognostic indicators. These data suggest that high Notch1 or Notch3 expression was correlated with worse outcomes and might be independent prognostic factors for patients with HCC. Thus, expression of Notch1 or Notch3 could constitute a useful additive prognostic marker to the TNM staging system for patients who are more likely to have disease recurrence and are thus good candidates to receive aggressive adjuvant chemotherapeutic treatment. Our present findings suggest that not only Notch1 but also Notch3 can be good for determining the prognosis of HCC patients. However, there is no consensus on which protein plays the predominant role in HCC, thus limiting their clinical predicative value for the prognosis of HCC patients.
Metastasis is an important factor that affects the prognosis of HCC patients. Metastasis is responsible for cancer-associated mortality, yet it remains the most poorly understood component of cancer. For individual and small groups of cancer cells to break away from the primary tumor and initiate the metastatic process, these cells must acquire the ability to migrate and invade. These traits enable cells to degrade and move through the extracellular matrix of the surrounding tissue toward blood and lymphatic vessels, which in turn provide highways for their passage to distant secondary sites. Thus, to determine which one of Notch1 or Notch3 played the more predominant role in HCC, we focused on evaluating the roles of Notch1 and Notch3 in HCC migration and invasion, which are two important processes of metastasis in vitro.
Adhesion processes are involved at all levels of the migration cascade. Most of the adhesion receptor families reported so far, including integrins, cadherins, selectins, immunoglobulins, and proteoglycans, play a significant role in various stages of tumor progression and metastasis. In our experiments, we focused on two important adhesion molecules, CD44v6 and E-cadherin. The CD44 family comprises important cell adhesion molecules. One of its variants, CD44v6, regulates tumor progression and metastasis formation [31]. Previous reports have indicated that the over-expression of CD44v6 is correlated with the poor prognosis of human cancers [32], [33]. E-cadherin, a member of the cadherin family, is involved in homotypic, calcium-dependent cell-cell adhesion in epithelial tissues [34]. A great deal of previous research has shown that a reduction in E-cadherin is relevant for tumor migration, metastasis, and unfavorable prognosis [35], [36], [37]. The loss of E-cadherin expression and disassembly of E-cadherin adhesion plaques on the cell surface enable tumor cells to disengage from the primary mass and move through conduits of dissemination [38]. In the present study, it was interesting that Notch1 down-regulation could reduce the migration of HCC cells, whereas Notch3 down-regulation could not. A potential explanation could be that Notch1 down-regulation can decrease the protein expression of CD44v6 and increase the protein expression of E-cadherin. Conversely, down-regulated Notch3 had no effect on the protein expression of CD44v6 or E-cadherin.
The Notch signaling pathway is required to convert the hypoxic stimulus into changes in E-cadherin, for increased motility, and for the migration of cervical, colon, glioma, and ovarian cancer cells [39]. In contrast, Lim et al. demonstrated that the Notch1 intracellular domain (N1ICD) can increase the expression of E-cadherin, thereby resulting in a decrease in the invasion of Snail-dependent HCC cells [40]. In the present study, we found that down-regulated Notch1 can increase the expression of E-cadherin, which is involved in cancer invasion and migration. Our results are consistent with the results shown by Wang et al. [41]. The mechanism through which Notch1 mediates E-cadherin regulation in tumor cells is complex and depends on the tissue and cell type. In contrast, the relationship between Notch3 and E-cadherin is unknown. Moreover, the relationship between Notch1 or Notch3 and CD44v6 is unclear. To further explore the mechanism by which Notch1 but not Notch3 can regulate the expression of E-cadherin and CD44v6, we focused on one important pathway, COX-2, which is upstream of CD44v6 and E-cadherin [42], [43]. Tumor COX-2 plays important roles in regulating diverse cellular functions under physiologic and pathologic conditions [44], [45]. Elevated COX-2 expression is often associated with metastasis in cancer [42], [46]. COX-2 contributes to the modulation of E-cadherin and CD44v6 expressions which are involved in cancer metastasis [42], [43]. Knowledge about the relationship between the Notch signaling pathway and COX-2 is limited. One previous study showed that Notch1 can regulate COX-2 expression in gastric cancer through N1IC bound to a COX-2 promoter [47]. In contrast, the relationship between Notch3 and COX-2 is unknown. The results of the current experiments show that down-regulated Notch1 can decrease the expression of COX-2, whereas down-regulated Notch3 cannot. On other hand, inhibition of COX-2 can not affect the protein expression of Notch1 and Notch3. Thus, we speculated that the Notch signaling pathway played different roles in regulating the expression of COX-2. We also speculated that Notch3 cannot affect migration in HCC because Notch3 cannot regulate the expression of COX-2. However, many factors are involved in cancer migration. Our results may demonstrate one possible mechanism. If there are other mechanisms, further studies should be conducted. Our findings suggested that during the migration process of HCC cells, Notch1 is more important than Notch3.
We also examined whether Notch1 or Notch3 is more important in invasion, which is another process in metastasis. Tumor metastasis occurs by a series of steps, including cell invasion, and the degradation of basement membranes and the stromal extracellular matrix, ultimately leading to tumor cell metastasis. Many molecules are involved in tumor invasion, such as matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA). MMPs are a family of related enzymes that degrade the extracellular matrix (ECM). Additionally, the activation of these enzymes allows tumor cells to access the vasculature, invade target organs, and develop into tumor metastases [48]. Among the previously reported human MMPs, MMP-2 and MMP-9 play the most important role in tumor invasion and metastasis because of their specificity for type IV collagen, which is the principal component of the basement membrane [49], [50]. The plasminogen activator system is involved in multiple physiological and pathologic processes, including cell migration, angiogenesis, wound healing, embryogenesis, tumor growth, and metastasis. uPA binds to its receptor (uPAR), which facilitates the conversion of plasminogen to plasmin. Plasmin, either directly or indirectly through metalloproteinases (MMP), can degrade components of the extracellular matrix, contributing to cancer cell invasion and metastases [51]. In the present study, Notch1 and Notch3 showed no difference in regulating invasion by HCC cells. Not only down-regulated Notch1 but also down-regulated Notch3 can reduce the invasion of HCC cells. The potential mechanism may be that Notch1 and Notch3 can both regulate the expression of MMP-2, MMP-9, and uPA.
ERK1/2 belongs to the family of mitogen-activated protein kinases (MAPKs), which play a major role in signaling pathways concerning scattering/motility, invasion, proliferation and survival [52], [53]. ERK1/2 activation has also been reported to regulate the expression of a variety of important genes in some cellular responses, including metastasis-related genes, such as MMP-2/−9 and uPA [54], [55]. Because the ERK1/2 pathway plays important roles in many cellular processes, studies on the interaction of ERK1/2 activation with other cell signal transduction pathways, including the Notch signaling pathway, have received increased attention in recent years. Our findings suggest that down-regulated Notch1 and Notch3 can decrease the expression p-ERK1/2, whereas ERK1/2 inactivation can decrease the expression of MMP-2/−9 and uPA. On other hand, inhibition of ERK1/2 can not affect the protein expression of Notch1 and Notch3. This may be one mechanism through which Notch1 and Notch3 are involved in invasion by HCC.
During the study, we found another interesting result. Down-regulated Notch1 and Notch3 did not affect the cell growth or viability of HepG2 and MHCC97H cells. However, Li et al. showed that Notch1 down-regulation inhibits tumor growth in the human HCC cell lines HEP3B, SK-Hep-1 and SNU449 [56], whereas Qi et al. showed that Notch1 over-expression was able to inhibit the growth of SMMC7721 cells [57]. These results also indicated that Notch1 plays a complex role in tumor cells and depends on the tissue and cell type. However, a similar role for Notch3 is unknown. Many studies need to be performed. Thus, the Notch signaling pathway plays a critical role in maintaining the balance between cell proliferation and apoptosis. Moderate changes in the Notch signaling pathway may be caused by the cells’ self-regulation mechanisms, which can protect cells and keep them from being damaged. Non-spontaneous changes in the Notch signaling pathway may affect the results of the experiment and is a limitation of our study.
In summary, our findings strongly suggested that high levels of Notch1 and Notch3 expression were significantly correlated with HCC progression and unfavorable prognosis. Thus, Notch1 and Notch3 expression can be used as an adjunct to the TNM staging system to improve prognostication for individual patients. Further, we can conclude that Notch1 may interact with more signal transduction pathways related to HCC metastasis than Notch3 from the results above. Therefore, based on the migration and invasion of HCC, we hypothesize that targeting Notch1 in specific cell types may be more useful than Notch3. Additionally, in the near future, targeting the Notch pathway may be used for devising novel preventive and therapeutic strategies for HCC. Furthermore, more mechanisms of Notch1 and Notch3 involvement in HCC should be explored.
Supporting Information
Figure S1
Kaplan-Meier statistical analyses of postoperative survival curves according to Notch1 and Notch3 high expression.
(TIF)
Click here for additional data file.
Figure S2
siRNA can effectively inhibit the expression of Notch1 and Notch3 mRNA and protein levels in HCC cells. (a and b) RT-PCR and western blot analysis of the mRNA and protein expression level of Notch1 and Notch3 in different HCC cells. (c–f) RT-PCR and western blot analysis of the mRNA and protein expression of Notch1 and Notch3 in differently treated HepG2 and MHCC97H cells. (g) MTT analysis of the cell viability of differently treated HepG2 and MHCC97H cells. The expression of Notch1 and Notch3 was normalized to GAPDH (Notch1 or Notch3/GAPDH). The data are presented as the mean ± SD, *P<0.05 compared with control siRNA-transfected HepG2 cells; #P<0.05 compared with control siRNA-transfected MHCC97H cells. NT: No transfection; Cs: control siRNA transfection; N1s: Notch1 siRNA transfection; N3s: Notch3 siRNA transfection.
(TIF)
Click here for additional data file.
Figure S3
Effects of COX-2 inhibitors on the protein expression of CD44v6 and E-cadherin in HepG2 and MHCC97H cells. The protein expression of CD44v6 and E-cadherin was measured by western blot analysis. The HepG2 and MHCC97H cells were treated with 50 µmol/l NS-398 and 70 nmol/l SC58125 for 48 h. Cells were treated with DMSO as a control.
(TIF)
Click here for additional data file.
Figure S4
Effects of ERK1/2 pathway inhibitors on protein expression of MMP-2, MMP-9 and uPA in HepG2 and MHCC97H cells. Protein expressions of MMP-2, MMP-9 and uPA were measured by western blot analysis. The HepG2 and MHCC97H cells were treated with 10 µmol/l PD98059 and 1 µmol/l U0126 for 48 h. Cells were treated with DMSO as a control.
(TIF)
Click here for additional data file.
Figure S5
Effects of COX-2 inhibitors on the protein expression of Notch1 and Notch3 in HepG2 and MHCC97H cells. The protein expression of Notch1 and Notch3 was measured by western blot analysis. The HepG2 and MHCC97H cells were treated with 50 µmol/l NS-398 and 70 nmol/l SC58125 for 48 h. Cells were treated with DMSO as a control.
(TIF)
Click here for additional data file.
Figure S6
Effects of ERK1/2 pathway inhibitors on protein expression of Notch1 and Notch3 in HepG2 and MHCC97H cells. Protein expressions of Notch1 and Notch3 were measured by western blot analysis. The HepG2 and MHCC97H cells were treated with 10 µmol/l PD98059 and 1 µmol/l U0126 for 48 h. Cells were treated with DMSO as a control.
(TIF)
Click here for additional data file.
We are grateful to Fuqin Zhang who provided me the technical help.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23469244PONE-D-12-2817310.1371/journal.pone.0057816Research ArticleBiologyBiochemistryGeneticsEpigeneticsHistone ModificationChromatinGene ExpressionHistone ModificationModel OrganismsAnimal ModelsRatMolecular Cell BiologyGene ExpressionHistone ModificationNeuroscienceAnimal CognitionLearning and MemorySocial and Behavioral SciencesPsychologyCognitive PsychologyMemoryDetection of Histone Acetylation Levels in the Dorsal Hippocampus Reveals Early Tagging on Specific Residues of H2B and H4 Histones in Response to Learning Learning-Dependent Acetylation in the HippocampusBousiges Olivier
1
Neidl Romain
1
Majchrzak Monique
1
Muller Marc-Antoine
1
Barbelivien Alexandra
1
Pereira de Vasconcelos Anne
1
Schneider Anne
1
Loeffler Jean-Philippe
2
Cassel Jean-Christophe
1
Boutillier Anne-Laurence
1
*
1
Laboratoire de Neurosciences Cognitives et Adaptatives, UMR7364, Université de Strasbourg-CNRS, GDR CNRS 2905, Faculté de Psychologie, Strasbourg, France
2
Inserm, U692, Laboratoire de Signalisations Moléculaires et Neurodégénérescence, Université de Strasbourg, Faculté de Médecine, UMRS692, Strasbourg, France
Baudry Michel Editor
Western University of Health Sciences, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ALB JCC MM OB. Performed the experiments: OB RN MAM AS. Analyzed the data: ALB AB AP OB RN. Contributed reagents/materials/analysis tools: JPL. Wrote the paper: ALB JCC OB RN.
2013 4 3 2013 8 3 e5781614 9 2012 26 1 2013 © 2013 Bousiges et al2013Bousiges et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain - notably in the hippocampus - and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.
This work was supported by CNRS, INSERM, University of Strasbourg, ANR (ANR-12-MALZ-0002-01 to ALB) and granted by associations that the authors warmly thank: FRM-Alsace (to JCC and ALB), Ligue Européenne Contre la Maladie d’Alzheimer (LECMA project #10702 to ALB), Alsace Alzheimer 67 (to ALB and Dr. F. Blanc [FB], HUS, Strasbourg, France), as well as France Alzheimer Haut-Rhin (to JCC). OB received salary support from the LECMA, FORNASEP (to FB). RN and MAM were recipients of a doctoral fellowship from the French government, and RN received additional salary support from the LECMA. AS has been supported by the Fondation Unistra-don Pierre Fabre and ANR-12-MALZ-0002-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
As a result of dynamic interactions between environmental constraints and an organism’s genome, synaptic plasticity and formation of enduring memories require modulations of gene transcription (expression, repression) at critical periods following learning [1], [2], [3]. Such changes implicate in part chromatin structure modifications catalyzed by epigenetic mechanisms, among which histone acetylation appears to be one of the critical processes [4]. Among the 5 core histones, studies investigating global changes in histone acetylation levels in the hippocampus of rodents after learning have mainly focused on H3 and H4. A few examples are rodents subjected to either fear conditioning [5], [6], [7], subsequent extinction [8], [9], object recognition [10], [11], [12], or place learning in the Morris water maze [13] (for reviews [14], [15]). However, a series of indirect evidence suggests that H2B histone could be an additional target for regulations involved in memory formation and consolidation processes. Indeed, HDAC2 knock-out mice have recently been shown to display improved memory functions, and increased acetylation levels of H2B (among others) were measured in their hippocampus [16]. Genetic inhibition of protein phosphatase 1 (PP1) in the mouse brain, previously shown to produce animals with prolonged vividness of a spatial memory [17], also presented increased H2B acetylation in the hippocampus [11]. A recent paper described that depolarization of hippocampal slices maintained in vitro induced H2BK5K12K15K20 acetylation within minutes [18], suggesting that the tetra acetylation of H2B could mediate activity-dependent signaling in the hippocampus. Finally, our recent work showed that acetylations of H2B histones on its N-terminus were dynamically regulated during the consolidation of a spatial memory: tetra acetylated H2B was increased in the dorsal hippocampus of rats having learned the location of an escape platform hidden in a water maze for 3 days [13]. Acetylated H2B was enriched on gene promoters involved in memory and plasticity, such as the BDNF promoter IV, cFos, FosB and Zif268. Moreover, spatial training-induced H2B acetylation was strongly diminished in a rat model invalidating spatial memory consolidation by selective damage to cholinergic and glutamatergic hippocampal inputs [13]. Together, these data strongly suggest a particular involvement of H2B acetylation in the molecular processes involved in spatial memory formation. However, it is yet unknown whether these acetylation changes measured on H2B histone N-terminus specifically concern place learning or more generally hippocampus-dependent learning. Therefore, in this paper, we compared the acetylation status of H2B in different hippocampal-dependent learning tasks; one taxed spatial memory formation, the other contextual fear conditioning. Moreover, we compared acetylation levels in the dorsal hippocampus of learning animals (location of a hidden platform, fear to context association) to a series of control situations that did not require the formation of a memory for spatial cues (rats had to swim to a visible platform) or for context signification (rats were exposed to context or shock-only conditions, or taken from their home). Together with H2B tetra-ac, we also assessed H2B acetylation on lysine 5, which, according to Valor and colleagues [19], seems to be dynamically regulated in CBP deficient mice. Lastly, we measured the acetylation levels of two other histones: H3 and H4. To this end, we chose specific acetylation modifications: H3K9K14 and H4K12, previously reported to be associated with learning and memory. Histone H3 acetylation on lysine 14 was one of the first modifications described to be modulated by experience-dependent behavior: H3K14 was found hyperacetylated in the CA1 region of the hippocampus of rats after a contextual fear conditioning vs. naive rats [6]. H3K9 acetylation, together with that of K14, was recently shown over promoters of actively transcribed genes in mouse cells [20] and we previously observed H3K9K14 hyperacetylation in the hippocampus of rats undergoing a spatial memory task compared to naïve rats [13]. H4K12 acetylation modification was selected for its described role in fear conditioning in mice [7]. Moreover, authors showed that aged mice displayed a specific deregulation of histone H4K12 acetylation during learning and failed to initiate a hippocampal gene expression program associated with memory consolidation [7]. Restoration of physiological H4K12 acetylation with HDAC inhibitors reinstated the expression of learning-induced genes and led to the recovery of cognitive abilities [7]. We previously showed an increase of H4K12 acetylation in rats undergoing spatial training, associated with the cFos and Zif268 gene promoters [13].
Herein we report that H2B acetylation is increased in both learning situations (spatial or fear memory) as compared to the respective controls, suggesting that this modification is not specific to spatial learning but seems to be part of the molecular mechanisms involved in hippocampus-dependent memory formation. Our results also point to distinct regulations on specific histone sites, that seem to depend on which component of a behavioural test rats have to deal with (overall environmental context vs. specific goal therein) and which are detectable in whole dorsal hippocampus homogenates: while histone modifications detected on H2B N-terminus and H4K12 are induced in learning conditions, H3K9K14 seems more responsive to contextual and environmental changes. In addition, our results show that such changes are very precocious during the timing of learning, as they are detected early in the course of task acquisition.
Materials and Methods
Animals and Ethics Statement
Seventy nine 3-4 month-old Long-Evans male rats (Centre d’Elevage René Janvier, France) were used. They were individually housed in standard cages with food and water provided ad libitum, in a temperature- and humidity-controlled room (22±1°C and 55±5%, respectively) under a 12 h–12 h light-dark cycle (lights on at 8:00 a.m.). Experimental protocols and animal care were in compliance with the institutional guidelines (council directive 87/848, October 19, 1987, Ministère de l’agriculture et de la Forêt, Service Vétérinaire de la Santé et de la Protection Animale ) and international (directive 86–609, 24 November 1986, European Community) laws and policies (personal authorizations N° 67–167 for A.B., N° 67–289 for M.M N° 67–215 for JCC). All efforts were made to minimize suffering.
Morris Water Maze
The specifications of the water maze and the testing procedures have been described previously [13]. Briefly, after a four-trial session using a visible platform (VPf), two groups of rats which had to learn the location of a hidden platform (HPf) were given four successive acquisition trials per day for 1 day or 3 consecutive days. Control rats had to swim to a visible platform (VPf) emerging 1 cm above the water surface, and of which the location was changed from trial to trial on each day. One hour after the last acquisition trial, rats trained with the HPf for 1 or 3 days were tested for retention in a probe trial (for the control group, rats had to swim to a VPf ). Rats from the day1 group were immediately euthanized for biochemical studies. For biochemical studies (see below), a group of control rats taken from their home cage (HC) was also used.
Contextual Fear Conditioning
Rats were handled for 6 consecutive days (1 min/day/rat) before conditioning. Fear conditioning was performed in two identical Plexiglas chambers (25×27×18 cm) placed in ventilated light- and sound-attenuated boxes (57×38×38 cm, Campden Instruments LTD). The grid floor of each chamber consisted of parallel 0.3 cm diameter stainless-steel bars, 0.8 cm apart, connected to a shock generator (0.6 mA, 0.8 s, scrambled) controlled by a computerized interface (Med-PC, Med Associates, Inc., St Albans, VT, USA). Four conditions were used. Contextual fear conditioned rats received 3 foot shocks 180 s, 241 s and 362 s after the placement in the chamber (context-shock, CS). A first control group received 3 foot shocks delivered 1 s, 3s and 5s after their placement in the chamber (immediate-shock, IS). Another control group was left in the context, receiving no foot shock during the session (context group, CX). A last control group consisted of rats taken from their home cage without any exposure to shock or context (HC). Each training condition lasted 8 min. After training, all rats were returned to their home cage and left undisturbed until either euthanasia for biochemical studies (1 h delay) or behavioural testing for retention (24 h delay). To this end, automatic freezing measurements were carried out during an 8-min session, as described in detail by Marchand et al. [21].
Preparation of Tissues for Western Blot Analyses
All animals were killed by decapitation, their brains rapidly removed from the skull and transferred on an ice-cold glass plate. Freshly dissected dorsal hippocampi were immediately frozen in liquid nitrogen and kept at −80°C. Western blots were performed as described previously [13] with polyclonal antibodies against acetylated-H2B histone (H2B tetra-Ac, H2BK5) and acetylated-H3 histone (Upstate Biotechnology, New York, NY, USA), acetylated-H4 histone (Active motif Carlsbad, CA, USA), H3 and H4 histones (Abcam, Cambridge, UK), H2B histone (Euromedex, France). Secondary HRP-conjugated antibodies were from Jackson Immunoresearch (Suffolk, UK). Blots were revealed with BioFX® HRP chemiluminescent substrates SERI (SurModics, Eden Prairie, MN, USA) and exposed with Kodak BioMax light film (Sigma-Aldrich). Results were quantified using the ImageJ software. For each histone (either total or modified), we performed western blot analyses on increasing amounts of a total protein extract mix and determined the adequate amount within the linear range of detection to be assessed for quantitative western blots analyses.
Statistical Analysis
Behavioural studies
The analysis of spatial learning performance recorded during acquisition used a two-way ANOVA with repeated measures considering days (1–3) and platform condition (HPf vs. VPf). Probe trial performance was analyzed using a one-way ANOVA. An additional one sample t-test was performed to compare the time spent in the each quadrant to chance level (i.e., 15 s). When appropriate, post hoc comparisons used the Newman–Keuls multiple range statistic. Freezing was computed as the percentage of time spent at freezing over the 8-min test session. It was analyzed using an ANOVA with “Training condition” as the between-subject factor. The ANOVA was complemented by post hoc Newmann-Keuls tests when appropriate. In all cases, the threshold for rejecting the null hypothesis was set at α<0.05. Biochemical studies. Statistical analyses were performed using one-way ANOVA followed by Newman-Keuls multiple comparison tests. Data are expressed as the mean ± SEM. Differences at p<0.05 were considered significant.
Results
Histone Acetylation Profiles during Spatial Reference Memory Formation
We investigated whether histone acetylation was modulated at the beginning of a spatial memory training (1-day training) in rats having to search for a hidden platform (HPf) in the Morris Water Maze. Acetylation levels were compared to those measured in naive rats (HC) or rats that had swum to a visible platform (VPf). At this time point, rats had experienced the learning task, but did not present any behavioural evidence for a consolidated memory trace during a probe trial (figure 1A). In order to verify that our test conditions permitted learning with prolonged training, another group of rats was trained for 3 days. Acquisition (distance to the platform, either hidden or visible) and retention (time spent in the target quadrant, no platform, HPf group only) performances are shown in figure 1A. As expected, the retention results now clearly showed that after three acquisition days, the probe trial performance was significantly above chance in the target quadrant (quadrant effect 2 way-Anova F(3,12) = 11.84, p<0.001; time in target quadrant versus 15 sec: t(3) = 3.18, p<0.05), indicating efficient memory formation. Histone acetylation profiles of 3 major histones (H2B, H3 and H4) at specific lysine residues in the 1-day experimental group were established by western blot analyses in dorsal hippocampi of the 3 animal groups (HPf, VPf and HC; figure 1B). Representative western blots are shown on the left (duplicates) and quantification is shown on the right (n = 5). Global H2B histone acetylation (tetra Ac) was significantly increased in the HPf group as compared to VPf and HC control groups (1.50-fold, when compared to VPf, p<0.01; 1.56-fold, when compared to HC, p<0.05). Tetra-acetylated-H2B histone levels were not significantly different between VPf and HC groups. As tetra-acetylated-H2B, H2B acetylation on the single lysine 5 (H2BK5) was also significantly up-regulated in the HPf group compared to VPf and HC groups (1.38-fold, when compared to VPf, p<0.001; 1.3-fold, when compared to HC, p<0.05). H4K12 acetylation was also significantly increased in the HPf group compared to controls (1.41-fold, when compared to HC, p<0.05; 1.29-fold, when compared to VPf, p<0.05). Here again, no significant difference was found between HC and VPf groups, a result similar to that found for H2B acetylation. Finally, H3K9K14 histone acetylation levels were increased in HPf rats as compared to HC rats (1.74-fold, p<0.001). However, and this is a major difference with the other histone marks measured on H4 and H2B, there was no significant difference between HPf and VPf rats, the latter also showing H3 K9K14 histone acetylation levels that significantly exceeded those found in HC rats (1.79-fold, p<0.001).
10.1371/journal.pone.0057816.g001Figure 1 Short spatial memory training differentially modulates histone acetylation in the rat hippocampus.
(A) Performance of rats trained in the Morris water maze task during one or three consecutive days in the Morris Water Maze (left panel) and probe trial performance after 1 or 3 days of training (right panel). During training, rats had to search for the location of a platform hidden at a constant location (HPf); their controls swam to a visible platform (VPf) whose location was changed from trial to trial. Probe trial performances of the HPf groups are presented after 1- or 3 days of training (right panel) as the mean time (+ SEM) spent in the target quadrant. After 3 days of training, the rats trained with the hidden platform performed significantly above chance (i.e., 15 s), *p<0.05, an effect not observed after only 1 day of training. (B) Comparison of acetylated and total histone levels between home cage rats (HC, n = 5), rats trained to swim to a visible platform (VPf, n = 5) and rats trained to learn the location of a hidden platform (HPf, n = 5) in a single daily session (4 trials). Acetylation levels were measured by western blot performed on total extracts from dorsal hippocampus with specific antibodies (Tetra Ac: H2BK5K12K15K20, K5Ac: H2BK5, H4K12 and H3K9K14). Typical western blots are presented in duplicates on the left. Corresponding quantifications are shown on the right. Ratios of acetylated/total histone corresponding to the home cage rats (HC) were arbitrarily set at 100% and other values normalized accordingly. Newman-Keuls multiple comparisons test: ***p<0.001, **p<0.01, *p<0.05, for comparisons with the HC group or as indicated. Both H2B and H4 histones showed hyperacetylation in the group trained to find the hidden platform (HPf) compared to either control (VPf or HC), while H3 was hyperacetylated in the VPf and HPf groups, thus more reflecting task-related context processing.
In summary, these observations show that some acetylation modifications on H2B (K5 and tetra-acetylation) and H4 (K12) histones are consistently associated with early stages of spatial learning. Similarly, acetylation of H3K9K14 histones are also rapidly increased, but conversely to tetra-acetylated-H2B and H4K12 histone marks, it is also the case under all control situations when compared to HC, thus suggesting a role of this histone mark in task/context processing (swimming, stress, exploration…). Are these changes specific to a spatial learning situation? To address this question, we used a similar approach in rats that were subjected to a task that, being non spatial by nature, is also hippocampus-dependent, namely contextual fear conditioning (CFC).
Histone Acetylation Profiles during Contextual Fear Conditioning
CFC is one of the most widely used tests to study memory processes, and a few studies have reported histone modifications during the consolidation of conditioned fear. Indeed, H3 histone acetylation was consistently found up-regulated in the rat hippocampus after contextual fear conditioning [5], [6], [7]. H4 histone acetylation was reported unchanged in early studies [6], but was found to be increased in more recent ones [7], [22]. To the best of our knowledge, H2B has never been investigated in relation with this type of memory.
We thus analyzed histone acetylation of H2B, H4 and H3 in rats trained for contextual fear conditioning using 3 shocks at random time points within an 8-min training period (CS). As illustrated in Figure 2A, histone acetylation was compared to that found in context-only rats (CX) and in immediate-shock rats (IS). An additional group consisted of rats taken from their home cage (HC). As shown in figure 2B, only rats of the Context-Shock group exhibited conditioned freezing to the context after this delay. Freezing levels were very low in the two other groups. The ANOVA showed a significant effect of “Training condition” [F
(1,27) = 112,28 P<0.0001] and the post hoc comparisons indicated that freezing levels in the CS group significantly differed from those measured in the CX and IS groups (p<0.001 in each case), which did not differ significantly from each other.
10.1371/journal.pone.0057816.g002Figure 2 Impact of contextual fear conditioning on histone acetylation in the rat hippocampus.
(A) Experimental design. Three groups of rats (n = 16/group) were used. In one group, rats were kept in the context but received no shock (CX). Others received three immediate and consecutive shocks and were subsequently left in the context for 8 min (IS). In the last group, rats received three randomly-distributed shocks while being kept in the context as noted (CS). Animals (n = 10/group) were then either tested for freezing behavior after 24 h (probe) (B; n = 10/group) or euthanized after 1 h for tissue collection (dorsal hippocampus) and western blot analyses of acetylated histones (C; n = 6/group). (B) Freezing levels at 24 h. Notice that marked freezing was observed only in the Context-shock group (CS), demonstrating that rats of this group were the only ones to have associated the shock with the context and memorized this association. (C) Comparison of acetylated and total histone levels in the three groups relative to their counterparts taken from the home cage (HC, n = 6). Lysine acetylations measured were H2BK5 (K5Ac, plain histograms), H2BK5K12K15K20 (Tetra Ac, stripped histograms), H4K12 (K12Ac) and H3K9K14 (K9K14Ac). Typical western blots are shown in duplicates. Quantified results are represented as % induction of the Acetylated/total ratio for each histone. The ratio obtained in the HC condition was arbitrarily set at 100%. Newman-Keuls multiple comparisons test: ***p<0.001**p<0.01, *p<0.05, as compared to HC group. Global H2B and H4 histone acetylation levels were clearly increased in the group exhibiting fear towards the context (CS) as compared to the other situations, while H3 acetylation levels were increased in CS and both controls (CX and IS ) as compared to rats completely naive to the test situation (HC).
Histone acetylation levels were measured by immunoblotting in the dorsal hippocampus of rats trained in parallel and euthanized one hour after the training (figure 2C). When fear conditioned rats (CS) were compared to home-cage rats (HC), all histone marks measured on H2B, H3 and H4 displayed a significant increase in acetylation (H2BK5, 2.35 fold, p<0,001; tetra-Ac, 1.42-fold, p<0.05; H3K9K14, 1.52 fold, p<0.05; H4K12, 1.74 fold, p<0.01). Nevertheless, these marks were differentially responsive to the control situations. In is noteworthy that H3K9K14 histone acetylation was significantly increased in both the CX (1.3 fold, p<0.05) and the IS (1.42 fold, p<0.05) control groups as compared to the HC group. H2B N-terminus and H2BK5 acetylations showed a non significant trend to increase in response to IS (H2B tetra-Ac, 1.22 fold, p = 0.163; H2BK5, 1.34 fold, p = 0.052), while H4K12 acetylation remained unchanged in the CX or IS condition. Altogether, and as was also the case in the water maze test, these observations suggest that acetylation on H2B N-terminus and H4K12 are increased when shocks are paired with the context (i.e. when training subsequently results in established fear), whereas the increased H3K9K14 acetylation appears less specific to the establishment of such a context-shock association.
Discussion
We recently identified H2B tetra-acetylation as a major chromatin mark associated with plasticity/memory gene promoters in the hippocampus of rats which had learnt a spatial reference memory task over three consecutive days [13]. In the current report, we describe that this chromatin mark is consistently activated in response to learning engaging the hippocampus (spatial memory or contextual fear conditioning). We also report that the H4K12 acetylation pattern follows that of H2B N-terminus in the two behavioral tasks. Finally, we confirm that H3K9K14 acetylation seems more sensitive to manipulations of the rats’ environmental context in the Morris water maze and we extend this observation to contextual fear memory formation. Our results emphasize that the integration of memory-associated behaviors at the level of histone acetylation occurs on specific lysine residues, that can be detected at a global level in the dorsal hippocampus. In addition, our results suggest that such changes may reflect the type of information to be stored.
Acetylation of H2B and H4 Histones at Specific Sites is Induced in Tasks Requiring Memory Formation
A remarkable result presented herein is that the tetra-acetylated-H2B and H4K12ac histones were consistently found to be hyperacetylated in the hippocampus of rats subjected to a training resulting in memory formation, be it for the location of a platform hidden in the water maze or for the context-associated shocks in the fear conditioning paradigm. The acetylation status of these histone marks (H2BK5, H2BK5K12K15K20 and H4K12) could represent a molecular step towards memory formation.
The functions of H2B histone modifications are poorly documented. Nevertheless, the few available data suggest interesting features in relation with transcription and memory. At the level of gene transcription, it is noteworthy that H2BK5 was recently reported to be consistently found within the 5′ proximal region of high CpG content promoters (HCP) [23]. Hence, H2BK5Ac binding seems predictive for expression of HCP genes [23], which represent about 70% of the regulated genes expressed in most tissues [24]. These include memory/plasticity-related immediate-early genes (e.g., zif268,…), kinases (e.g. catalytic subunit of cAMP-dependent protein kinase,…), and neurotrophic factors (e.g. BDNF,…) [24]. In line with this, we previously showed that tetra-acetylated-H2B histones were enriched at specific plasticity/memory-related promoters (bdnf exon IV, cFos and zif268) in the hippocampus during consolidation of spatial memory, an event associated with higher gene expression levels [13]. At the global level, increased acetylated-H2B levels have been measured in hippocampi of transgenic mice models displaying enhanced long term potentiation (LTP) and improved memory functions (HDAC2 knock-out mice [16] and NIPP1 mice [11]). H2B tetra-acetylation at K5K12K15K20 can also be rapidly triggered by depolarization in hippocampal slices [18]. Altogether, these data suggest that H2B tetra-acetylation could represent an early subcellular step of memory formation, triggering the transcription of specific genes likely related to memory consolidation. Of note, H2B is itself the preferred histone target of CBP in the hippocampus [19], [25], [26], an acetyltransferase playing an important role in memory formation and consolidation [10], [19], [25], [26], [27]. We showed that CBP is up-regulated during spatial learning, while its proximal promoter was enriched in acetylated-H2B histone [13]. Thus, CBP-induced acetylation of H2B might be a means to activate specific plasticity/memory-related gene transcription programs. CBP-dependent transcription has also been described as an important mediator of environmental enrichment-induced adult neurogenesis, acetylated-H2B histone being associated with neurogenesis-related gene promoters [28]. Future studies using ChIP-sequencing will certainly help to identify and characterize acetylated H2B-regulated genetic programs in the hippocampus during memory formation. Remarkably, our previous immunohistochemistry studies performed on VPf and HPf after 3 days of training showed that acetylated H2B N-terminus levels were increased in all nuclei of hippocampal neurons (data not shown) - as was already the case for CBP [13] - rather than in a subset of the neuronal population [29]. This is in line with the fact that these changes are detectable by western blot analyses performed on total dorsal hippocampi extracts and further suggests that a global response to behavior takes place in the dorsal hippocampus. If, and also how this general modification will be subsequently integrated into only a subpopulation of neurons to sustain the memory trace remains to be established.
Acetylation of H4K12 has been more widely studied and its association with memory formation is documented, particularly after fear conditioning [7], [22], latent inhibition training [6] and spatial memory formation [13]. Acetylated H4K12 enrichment has been shown on different bdnf promoters in response to fear conditioning in the hippocampus [7], [30], [31] or in the frontal cortex [9], and our recent data show an enhancement of acetylated H4K12 on cFos and zif268 promoters in the hippocampus after spatial memory training [13]. A recent study remarkably showed that H4K12 acetylation was altered by aging in mice subjected to fear conditioning [7]. Histone H4 acetylation, including other lysine residues than K12, might also be involved in Alzheimer’s disease (AD) pathology as it is reduced in transgenic models of this disease [22], [32], [33], [34]. Furthermore, acetylated H4K12 associated genetic programs were recently identified by ChIP-sequencing in the hippocampus during fear learning [7]. Thus, the study presented here further emphasizes that this epigenetic mark is specific to memory formation as it is consistently induced in hippocampus-dependent learning paradigms, and not in the different control situations used herein and elsewhere.
H3 Histone Acetylation Might be a Marker of Contextual Changes Processing
Another striking result is that H3 was found to be significantly acetylated at K9 and K14 in the hippocampus of rats subjected to learning, but to almost the same extent than in rats exposed to other control situations as compared to naive home cage rats. H3K14 acetylation is known to be induced in the hippocampus of animals undergoing unpleasant shocks paired to a context [5], [6], [35]. However, in all these studies, tissue collected in learners was compared to tissue from naive controls or from animals exposed to unpaired shocks. Our results indicating that a « new » situation, even when not associated with fear learning, is able to modify this epigenetic mark, further suggest that certain acetyltransferases could be rapidly activated in the hippocampus of animals placed in a novel situation to acetylate K9 and/or K14 of H3 histone in the nucleus. This would result in the opening of the chromatin and favor some gene transcription. Contextual fear learning was actually reported to induce bdnf mRNA in the CA1 area of the hippocampus, bdnf exon IV being more specifically activated when the context was paired to shocks and bdnf exon I being activated in the context-only situation [36]. It is noteworthy that bdnf exon I transcripts in the hippocampus are very responsive to a HDAC inhibitor directly modulating histone acetylation levels [37]. Thus, it is likely that acute changes in usual situations, either mild, such as having to wander in a novel environment when having been taken out from the home cage, or strong, such as having to experience unpaired shocks or swimming towards a visible escape platform, impact H3K9K14 histone acetylation, whereby the chromatin structure can be modified and specific gene profiles regulated. Of note, K9 and K14 acetylation has been recently shown to co-occur at active enhancers, and it was found to trigger transcriptional activation in mouse cells [20]. Which genetic programs are indeed activated in the behavioral conditions remains to be established, but they should definitely depend on how stressful and/or novel environmental changes may be. An interesting study demonstrated that rats either trained in associative or in non associative fear learning displayed similar gene expression profiles in the hippocampus, whereas greater levels of gene regulation were seen in the amygdala in response to associative fear conditioning compared to the non associative control [38]. This study was performed 30 min after training, a time point chosen to optimally detect immediate-early gene induction. In light of our observation that the acetylated-H3K9K14 histone is increased in all conditions compared to home cage controls in the hippocampus, these results suggest that there is a step of hippocampal activation in response to conditioning, whether more specific associative learning-dependent responses have to be formed or not. It would be of prime interest to compare the dependency of these genes [38] to acetylated-H3K9K14 histone versus acetylated H2B N-terminus or H4K12.
Early Engagement of Histone Acetylation in Memory Processing
Little is known about biochemical studies of memory formation in the Morris Water Maze (MWM). Indeed, MWM is a complex protocol requiring several days of training and daily repetitions of several learning trials. Thus, acquisition/consolidation/recall signals are mixed all along the learning days. In our previous studies [13], we measured increased H2BK5K12K15K20 and H4K12 acetylation levels after the 3rd day of acquisition, a moment at which performance can still be improved and thus memory undergo further consolidation, suggesting a role of this modification in memory consolidation. However, the study presented here in the Morris Water Maze shows that specific acetylation modifications occurring on H2BK5K12K15K20, H2BK5 and H4K12 are already elevated in the hippocampus after a single day of training, when no evidence for consolidation can be measured yet in a probe trial and learning experience has just started, suggesting that these modifications accompany or might even be a substrate of the earliest stages of task integration/memory formation. This does not necessarily mean that the processes brought to light in the current study are associated with short term memory processes, as early molecular events could serve to implement the transcriptional response for long-term memory processes over repetition of the task. A hypothesis could be that iterative training allows a gradual increase of acetylation marks over days. Repetition of the training could also impact persistence of the acetylation marks over time, thereby maintaining specific memory−/plasticity- gene transcription throughout the memorisation/consolidation process. It is noteworthy that levels of acetylation on H2B measured in this study at day 1 seem comparable to those measured in the study by Bousiges et al. [13] at day 3, suggesting that repetitive training would in fact not support accumulation of molecular events over the three days, but rather reflect behavior-induced molecular events after a given training session. However, measurements of acetylation levels by western blot are technically limited to assess subtle changes at the global level. Therefore, this kind of study should be conducted at the promoter level by chromatin immunoprecipitation on specific loci. In addition, whether or not acetylated chromatin is present on the same genes at early and later time points (day 1 and day 3) is not known. It must be considered that other epigenetic changes, such as histone phosphorylation [35] or histone methylation [39] could take place at later time points (between day 1 and days 3) and act in concert with acetylation modifications. Lastly, our global approach might have missed more discrete changes occurring in different hippocampal sub-structures (e.g. CA1, dentate gyrus….).
Taken together, our water maze and fear conditioning data support the idea that specific acetylation modifications might be engaged in the hippocampus at early stages of task training (water maze and fear conditioning) and maintained during further training over the process of memory formation in tasks based on cumulative learning (water maze). In addition, our findings indicate that H3K9K14 might be the more sensitive to changes in the environmental context than to the mnemonic dimension of the task itself, whereas H2BK5K12K15K20/H4K12 seem more sensitive to the formation of a memory for the platform location or for the meaning of the context. These outcomes support the hypothesis of a language within the chromatin [40] in response to behavior/environment and might therefore contribute to identify co-activator recruitment (e.g. CBP-dependent acetylation of H2B in the hippocampus, [19], [26]) to specific plasticity/memory-related promoters. Such knowledge will help to better define therapeutic options, especially in the perspective of treating cognitive alterations by a pharmacological action on acetylation or deacetylation of specific lysine residues on histones in order to directly stimulate appropriate transcriptional programs [41], [42], [43].
The authors are grateful to O Bildstein, D Egesi, and G Edomwonyi (UMR 7364) for their assistance in animal care, and to MJ Ruivo (U692), as well as B Cosquer, K Herbeaux and MM Klein (UMR 7364) for their technical assistance.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23469181PONE-D-12-1898310.1371/journal.pone.0057020Research ArticleBiologyDevelopmental BiologyCell DifferentiationStem CellsMolecular Cell BiologyCellular TypesStem CellsStem Cell LinesMedicineOncologyCancers and NeoplasmsLung and Intrathoracic TumorsNon-Small Cell Lung CancerSmall Cell Lung CancerSquamous Cell Lung CarcinomaBasic Cancer ResearchSurgeryCardiothoracic SurgeryIdentification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines Stem Cell-Like Properties in Primary Lung CancerWang Ping
1
2
3
8
9
Gao Quanli
1
3
9
Suo Zhenhe
4
8
Munthe Else
3
5
Solberg Steinar
6
Ma Liwei
7
Wang Mengyu
2
5
Westerdaal Nomdo Anton Christiaan
3
Kvalheim Gunnar
2
8
Gaudernack Gustav
1
3
8
*
1
Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway
2
Department of Cellular Therapy, Oslo University Hospital, Radiumhospitalet, Oslo, Norway
3
Cancer Stem Cell Innovation Centre, Oslo, Norway
4
Department of Pathology, Oslo University Hospital, Radiumhospitalet, Oslo, Norway
5
Department of Tumor Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway
6
Department of Thoracic Surgery, Oslo University Hospital, Rikshospitalet, Oslo, Norway
7
Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet, Oslo, Norway
8
Faculty of Medicine, University of Oslo, Oslo, Norway
9
Department of Hematology, Henan Tumor Hospital, Zhengzhou, People's Republic of China
Tang Dean G. Editor
The University of Texas M.D Anderson Cancer Center, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: PW QG GG LM. Performed the experiments: PW QG EM MW NW. Analyzed the data: PW QG ZS EM NW GG. Contributed reagents/materials/analysis tools: PW QG ZS EM SS LM GK GG. Wrote the paper: PW GK GG.
2013 4 3 2013 8 3 e5702026 6 2012 21 1 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population.
This work was supported by a grant from the Norwegian research council (SFI-CAST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The “cancer stem cell” (CSC) theory implies a hierarchical organization within the tumor in which CSCs represents the apex of the hierarchy. Similar to normal stem cells, CSCs have the capacity to undergo self-renewal as well as asymmetric cell division. These key features enable CSCs to initiate and maintain tumors. In addition to the classical term, i.e. CSC, various terms have been used in the recent scientific literature to describe essential characteristics of CSCs such as self-renewal and tumor initiating/maintaining property. Among others are such terms as tumor initiating cell (TIC), cancer initiating cell (CIC) and tumor propagating cell (TPC). In this report we will use the CSC term to characterize cells that were able to initiate and sustain tumor formation in animals and long-term liquid culture.
Current studies in the field of cancer stem cell research have provided increasing evidence for the existence and identification of CSCs using several specific biomarkers, such as CD44, CD133 and CD90. These markers have been widely accepted for isolation of CSCs in human haematological malignancies [1], [2] as well as in solid tumors [3]–[11]. Furthermore, CSCs have been found to be more resistant to conventional chemotherapy and radiotherapy than the major population of more differentiated cancer cells, indicating that the CSCs may remain in residual tumors after treatment and contribute to cancer recurrence and spreading. Therefore, new treatments targeting CSCs may potentially prevent tumor recurrence and prolong survival of patients. The epithelial-mesenchymal transition (EMT) plays an important role in embryonic development [12]. It causes epithelial cells to lose their epithelial behavior, changing their morphology and cellular properties to resemble mesenchymal cells [13]. EMT has been suggested to contribute to the invasive and metastatic growth of many types of cancers [14]–[17]. Recent study showed that stem cell-like cells from epithelial cancers having a mesenchymal phenotype express markers associated with EMT [15].
Lung cancer is the leading cause of cancer-related mortality worldwide, and has a poor prognosis with 5-year survival rates of approximately 15%. According to the histological heterogeneity, lung carcinomas are categorized into four major subtypes: small cell lung cancer (SCLC), squamous cell carcinoma (SCC), large cell carcinoma (LCC) and adenocarcinoma (AC). In this study, following the “Cancer Stem Cell” hypothesis, we focused on the identification and characterization of CSCs in above mentioned subtypes of lung cancer.
The cell surface marker CD133 has previously been identified as a reliable marker for CSCs in some of lung cancer subtypes [18]. However, the reliability of this marker as a CSC marker for lung cancer has recently been disputed [19]. Therefore, we focused on another marker, i.e. CD44, which has been suggested to characterize CSCs in breast, prostate, head and neck, colorectal, pancreatic and gastric cancers [3], [4], [9]–[11], [20].
In this study, we took advantage of the primary lung cancer tissues removed during resection and focused on establishing of PLCCLs from freshly isolated tumors. We assumed that PLCCL may provide a more representative and appropriate source of cancer cells that can be used for identification of cells or cell populations with stem cell-like properties. We first successfully established a panel of the primary lung cancer cell lines from freshly obtained specimens of the major subtypes of lung cancer. Based on detailed phenotypic and functional analysis of representative cell lines from SCLC, LCC and SCC, we provide evidence indicating that CD44high population may harbour CSCs in these cancer subtypes. In addition, co-expression of CD90 further narrowed down population of CSCs in SCLC and LCC. The picture for AC, however, remains less clear, and at the present we have no convincing evidence that either of the markers, i.e. CD44 and CD90, is characteristic for CSCs in this particular cancer subtype.
Materials and Methods
Collection of tumor specimens and establishment of primary lung cancer cell lines
This study was approved by the Regional Ethical Committee and the Institutional Review Board of Oslo University Hospital and performed according to the guidelines of the Helsinki Convention. Upon signed informed consent, human lung cancer tissues were obtained from 20 patients with primary lung cancer (age range 55–81 years) undergoing lobectomy or pneumonectomy at Oslo University Hospital from July 2007 to October 2009. Histological diagnosis was determined based on microscopic features of carcinoma cells (
Table 1
).
10.1371/journal.pone.0057020.t001Table 1 Summary of the primary cultures included in this study.
Code No Surgery date Patient sex/age(years)#
PLCCL Tumor type Diagnosis pTMN
1 03.07.2007 M/70 LC003 AC T1N0MXG2
2 18.07.2007 M/61 LC005 AC T2N0MXG1
3*
25.07.2007 F/63 LC007 AC T2N0MXG4
4 04.02.2008 M/73 LC013 AC T3N1MXG3
5 06.02.2008 F/69 LC014 AC T1N0MXG3
6 12.03.2008 F/72 LC016 AC T2NxM1G2
7 29.09.2009 M/81 LC024 AC T1N0MXG2
8 12.03 2008 M/80 LC017 SCC T2NXMXG2
9 12.03.2008 M/71 LC018 SCC T2N0MXG4
10 16.06.2008 M/56 LC019 SCC T3N2MXG2
11*
21.07.2008 M/68 LC021 SCC T2N2MXG2
12 23.07.2008 M/67 LC022 SCC T2N0MXG2
13 21.08.2008 M55 LC023 SCC T1N2MXG2
14*
24.07.2007 M/62 LC006 LCC T2N1MXG4
15*
09.07.2007 F/71 LC004 SCLC T1N0MXG2
# M, male; F, female.
* Primary cell lines highlighted in blue were used for further characterization.
Freshly obtained tumour tissue (within 1–2 hours after surgical removal) was washed in RPMI-1640 medium (Invitrogen, USA) containing penicillin-streptomycin (PS, penicillin 100 U/ml and streptomycin 100 μg/ml; Lonza, Belgium). Blood vessels and connective tissue were carefully removed and the cancerous part was then minced into small pieces less then 1 mm3 using scalpel, followed by extensive washing in RPMI-1640 medium and centrifugation at 300 g for 5 min. Finally, the cells were re-suspended in RPMI-1640 medium containing collagenase II (Invitrogen, USA) at the concentration of 200 U/ml and digested for 2–4 hours at 37°C in a humidified incubator. The enzymatic digestion was stopped when most of the cells were in the single cells suspension. Following washing in RPMI-1640 and 3x centrifugation at 300 g for 5 minutes, cells were transferred into standard tissue culture coated flasks (Corning Life Sciences, USA) and cultured in the Defined Keratinocyte-Serum Free Medium (DK-SFM) supplemented with L-glutamine (Invitrogen, USA), EGF 20 ng/ml, basic-FGF 10 ng/ml (PeproTech Inc., USA), 2% B27 (Invitrogen, USA), PS and amphotericin B (0.25 μg/ml; Invitrogen, USA). The cultures of all PLCCLs were maintained at 37°C in a humidified incubator with 5% CO2. Culture medium was changed every 2–3 days. Cells were passaged after detachment with TrypLE™Express (Invitrogen, USA), when the cells reached 80–90% confluence. All the studies were performed with the initial five passages of established PLCCLs.
Isolation of nucleic acid and DNA fingerprinting assay
To establish a genetic fingerprint for each of the new cell lines, DNA fingerprinting assay was performed on the representative PLCCLs (LC004, LC006, LC007 and LC021) as well as on a re-established cell line from xenograft of LC021. Genomic DNA was isolated from Dulbecco's phosphate-buffered saline (DPBS, Invitrogen) washed cell pellets. Total genomic DNA was obtained using NucleoSpin Tissue kit (Macherey-Nagel, Germany) according to manufacturer's protocol. The identity of the DNA profiles was determined by STR profiling using Powerplex 16 System (Promega, Madison, WI). This kit amplifies 15 STR loci and amelogenin for gender identification: Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. The PCR products were size determined in a MegaBace 1000 (Applied Biosystems, USA) using the software Fragment Profiler (GE Healthcare). These fingerprints were then compared to DNA profiles in a fingerprint database of previously established lung cancer cell lines to ensure their uniqueness.
Immunohistochemistry
The primary cultured lung cancer cell lines were embedded into paraffin blocks by the following method: cells from the liquid culture were detached and washed with DPBS and subsequently centrifuged at 300 g for 5 minutes. The supernatant was carefully removed before 3 drops of plasma and 2 drops of thrombin were added and mixed by tube rotation. Buffered formalin (4%) was added after the mixture was coagulated. The coagulated mass was then placed in linen paper before being embedded in paraffin blocks by standard procedure. Some sections were stained with Hematoxylin and Eosin (H&E) to assess cellular morphology, while other sections were used for immunohistochemistry (IHC) analysis. Four micrometer thick sections were dewaxed and hydrated in graded ethanol. To unmask the epitopes, sections were then microwaved in different buffers optimised for different antibodies. Low pH 10 mM citrate buffer (pH = 6.0) was used for antibodies P53, Ber-EP4 and CD44. To inhibit the endogenous peroxidase, the sections were incubated with 3% hydrogen peroxide (DakoCytomation, USA) for 5 minutes at room temperature (RT). Sections were then incubated with primary mouse anti-human P53, CD44 and Ber-EP4 (DAKO, 1∶1000, 1∶100 and 1∶300 dilutions, respectively) and CD133 (Miltenyi biotec, clone AC133/1 1∶40 dilution) antibodies for 30 minutes at RT followed by incubation with corresponding secondary antibodies for 30 minutes at RT and stained with 3,3′-diaminobenzidine tetrahydrochloride (Dako EnVision™+System Peroxidase (DAB) (K4007, DakoCytomation, USA)) before they were counterstained with hematoxylin, dehydrated and mounted in Diatex. All sections were rinsed thoroughly with TBS-Tween washing buffer (Dako Cytomation, USA) after each incubation step. Sections from paraffin blocks containing human seminoma and breast cancer specimen were used as positive controls for antibodies P53, Ber-EP4 and CD44, respectively; Sections from the paraffin block containing colon cancer cell line CaCo-2 were used as positive control for CD133 antibody. Isotype control antibodies at the same concentration were used as negative controls. All controls were performed and the antibody reactivity was verified before experimental applications of antibodies. Each stained section was reviewed independently by two pathologists.
Animal experiments
All animal experiments were approved by and performed according to the guidelines set by the Animal Research Ethics Board at University of Oslo. Female NOD/SCID mice (NODSC-M-F/M-M Homozygous NOD/mrkBOMTac-Prkdcscid; Taconic, Denmark) were purchased. After being kept in quarantine one week for monitoring in the sterile environment of the animal facility in the Oslo University Hospital, Rikshospitalet, 4–5 weeks old NOD/SCID mice were used in all experiments. To assess the tumorigenicity of established PLCCLs, 3×106 cells of each cell line suspended in DK-SFM were inoculated subcutaneously into the right flank of NOD/SCID mice (3 mice in each group) at a maximum volume of 100 µl. The mice were inspected twice a week and tumor size was measured with callipers. Mice were sacrificed by cervical dislocation when the size of the tumor reached a diameter of 15 mm. Xenografts were then removed and used for histological analysis and establishing of liquid cell cultures. Serial xeno-transplantations were performed with the LCC LC006 cell line by implanting pieces of the primary xenograft subcutaneously into secondary recipient NOD/SCID mice. Xenografts derived from each passage were taken out and fixed in 4% buffered formalin for histological analysis.
Flow cytometry analysis and Fluorescence-Activated Cell Sorting (FACS)
Flow cytometry analysis was performed on PLCCLs at logarithmic growth phase. Cells were first detached with TrypLE™Express and washed with DPBS. Re-suspended cells were counted and transferred into 75 mm polystyrene round-bottom test tubes (BD Falcon, USA) at a cell concentration 1×106 cells/ml, and subsequently stained with antibodies at dilutions determined by previous titration. Human immunoglobulin staining buffer (DPBS+0.1% human serum albumin (Octapharma, Stockholm, Sweden)) and 5 µL of 10 mg/ml gamma globulin (Gammagard, Baxter, UK) was added to the cells to block the FcR and minimize unspecific binding of antibodies. Fluorochrome coupled monoclonal antibodies (mAbs) were added to the test tubes at saturating concentrations and the cells were incubated for 20 minutes on ice avoiding light exposure. A screening for markers was performed with a panel of mAbs (see detailed data in Table 2) on four PLCCLs representing each lung cancer subtype. Cells stained with isotype-matched mAbs (BD Pharmingen, USA) served as negative controls. Different populations of cells were sorted based on the expressions of CD44 alone, or in combination with CD90 using a FACSAria II cell sorter (Becton Dickinson, Franklin Lakes, NJ). Flow data were acquired on the FACSAria II or LSR II cytometer (Becton Dickinson, Franklin Lakes, NJ). The results were analyzed using the BD FACSDiva software (Version 6.1.3). The viability of sorted cells was routinely checked using Trypan blue (Invitrogen, USA) staining and usually was >70%.
10.1371/journal.pone.0057020.t002Table 2 Antibodies used in flow cytometry analysis.
Antigen Conjugate Clone Source Volume
CD15 APC HI89 BD 20 µl
CD24 PE ML5 BD 20 µl
CD29 PE MAR4 BD 20 µl
CD31 PE WM59 BD 20µl
CD34 PE, APC 563 BD 20 µl
CD44 FITC G44-26 (C26) BD 20 µl
CD45 FITC, PerCP HI30,2D1 BD 20 µl
CD49b PE 12F1 BD 20 µl
CD49f PE-Cy5 GoH3 BD 20 µl
CD90 APC 5E10 BD 5 µl
CD142 PE HTF-1 BD 20 µl
CD117 PE, PerCP-Cy5.5 YB5. B8,104D2 BD 5 µl, 20 µl
CD166 PE 3A6 BD 20 µl
CD184 PE 12G5 BD 20 µl
CD326 APC EBA-1 BD 5 µl
CD133/1 APC AC133 Miltenyi Biotec 10 µl
CD133/2 PE 293C3 Miltenyi Biotec 10 µl
FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridium chlorophyll protein; APC, allophycocyanin; Cy, cyanine dye.
Cell proliferation assay
FACS sorted CD44high and CD44low/− cells at density of 150 or 500 cells per well were plated in triplicate in 200 µl DK-SFM with supplements in standard coated 96-well plates. Wells containing medium only were used as a background negative control. The cells were allowed to adhere at 37°C in a 5% CO2 humidified incubator for one day before they were used in the proliferation assay. Cell proliferation was measured during the following 8 days using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the protocol provided by the manufacturer. Absorbance at 490 nm was measured on a Wallac Victor2 plate reader (Perkin Elmer, Waltham, MA). The growth curves were drawn according to the mean value of absorbance, related to the background.
2 dimensional (2D) single cell colony forming and heterogeneity assay
CD44high and CD44low/− cells from the SCLC cell line LC004 and the LCC cell line LC006 were sorted and seeded at a single cell per well into standard coated 96 well plates containing 200 μl DK-SFM supplemented with EGF 20 ng/ml, basic-FGF 10 ng/ml, and 2% B27. Single cell plating was validated by phase-contrast microscope (Nikon, Germany). Cultures were kept at 37°C in a 5% CO2 humidified incubator. After 2 weeks the number of positive wells containing colonies was counted and morphological characteristics of the colonies were evaluated. To study self-renewal and differentiation of single cells, the colonies of different types i.e. holoclones, meroclones and paraclones, were subjected to serial passages by picking and re-seeding them first into 24-well, then 6-well plates (Corning Life Science, USA), and eventually into the flasks (Corning Life Science, USA) where they were further expanded. Colony growth in 2D single cell colony forming assay and the long-term liquid cultures derived from these colonies were observed using phase-contrast microscope (Nikon, Germany).
2D colony formation efficiency assay
FACS sorted CD44high, CD44low/− and CD44highCD90+ and CD44highCD90− cells from the cell lines SCLC LC004 and LCC LC006 were seeded in triplicates at a density of 200 cells per well into standard coated 6 well plate (Corning Life Science, USA) containing 5 ml DK-SFM with supplements per well. The sorted cells were kept in culture at 37°C in 5% CO2 humidified incubator for ∼10 days. When the generated colonies were visible and contained >100 cells, the supernatant was aspirated and the wells were rinsed twice with DPBS, fixed in 4% buffered formalin for 15 min at RT followed by staining with crystal violet for another 15 min at RT. After rinsing away the dye, the colony number was counted and colony formation efficiency (CFE), i.e. the number of colonies per plated cell, was calculated and compared between different sub-populations.
Spheroid formation assay
FACS sorted cells were seeded at density of 100 cells per well into ultra low attachment (ULA) 96 well plate (Corning Incorporated) containing 200 μl DK-SFM with supplements and cultured at 37°C in a 5% CO2 humidified incubator until cells started forming spheroids. The medium was changed every 2 days by carefully removing 50 μl of the upper layer of medium and replacing it with equal volume of fresh medium. The wells were inspected every 2 days using phase-contrast microscope. When the spheroids were big enough (most of the cell spheroids were >200 μm), they were counted and photographed (Nikon, Germany).
RNA extraction and real-time PCR analysis
Total RNA was extracted from 105 FACS sorted cells and subsequently reverse transcribed using Taqman Cell-to-CT kit (Applied Biosystems, USA) according to the protocol provided by the manufacturer. Real-time PCR was performed using Taq-Man Gene Expression Assay system (Applied Biosystems, USA) on 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) according to the manufacturer's instructions and the data were analyzed by the SDS 2.3 Software (Applied Biosystems, USA). Each sample, including no template controls, was performed in duplicate. PCR reaction without template served as the negative control. Thermal cycling conditions were 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 15 s at 95°C, followed by 1 min at 60°C. The expression levels were determined for the following genes Nanog (assay ID 8S02387400_gl), Oct4 (assay ID Hs03005111_g1), Sox2 (assay ID Hs01053049_sl), E-Cadherin (assay ID Hs01023895_m1), N-Cadherin (assay ID Hs00169953_m1), Vimentin (assay ID Hs00958111_ml), high mobility group AT-hook 2 (HMGA2) (assay ID HS00171569_ml) and phosphoglycerate kinase 1 (PGK1) (assay ID Hs99999906_m1). The expression of target genes was related to the expression of PGK1 and normalized to the unsorted control cells using the ddCT method.
Radiation sensitivity assay
FACS sorted cells were seeded at a density of 500 cells per well in duplicates in standard coated 96 well plates (Corning Incorporated) containing 200 μl DK-SFM with supplements. After 24 hours of conventional incubation, cells in the monolayer cultures were irradiated at RT with doses of 1Gy, 2Gy and 4Gy, respectively using a Siemens Stabilipan X-ray unit, operated at 200 kV, 20 mA, with 0.5 mm copper filtration (Siemens, Germany). The cells were then cultured in regular culture conditions for 5 additional days with medium change every 2 days. The effect of irradiation on different cell populations was tested by the CellTiter 96® AQueous One Solution Cell Proliferation Assay as described above. The effect of irradiation on proliferation is given as the inhibition ratio and was calculated according to the formula: inhibition ratio = ((non-irradiated control well absorbance – irradiated well absorbance)/non-irradiated control well absorbance) ×100%.
Results
1. Primary lung cancer cell lines
To provide a basis for the present study, a novel, robust method for culture of lung cancer cells from freshly resected lung carcinomas was established (see material and methods). Fifteen primary lung cancer cell lines were successfully established from 20 lung cancer tissues removed. These included 7 ACs, 6 SCCs, 1 LCC and 1 SCLC (
Table 1
). The success-rate of establishing primary cultures was 75%. The majority of failures occurred in the first part of the study, before optimal concentrations of the growth factors had been determined. The DK-SFM did support the selective growth of typical epithelial cells, clearly inhibiting the growth of co-existing fibroblasts. This was in contrast to cultures containing serum used in the beginning, where rapid overgrowth by fibroblasts occurred (data not shown). Monolayer cells with typical epithelial morphology were obtained at very high purity in each of the established PLCCLs (
Figure 1
). The primary cell lines maintained under these conditions were passaged for multiple generations with the longest passage so far over 30 generations, without any sign of growth decline.
10.1371/journal.pone.0057020.g001Figure 1 Typical epithelial cells were obtained at high purity under the serum-free condition.
A. The SCLC cell line LC004; B. The LCC cell line LC006; C. The AC cell line LC007; D. The SCC cell line LC021. All the representative images were from primary cultured lung cancer cell lines at the second passage. Photomicrograph magnification, ×200.
To ensure that the established cell lines were of unique origin and not contaminated by previously established cell lines [21], DNA profiles using a set of highly polymorphic microsatellite markers were generated from a subset of the cell lines representing the four different types of lung cancer. The results demonstrated that the four cell lines were unique and unrelated (Table Sl).
2. Phenotype of PLCCLs and parental cancer tissues have similar/are comparable for expression of P53, Ber-EP4 and CD44
We focused our work on four PLCCLs representing the four lung cancer subtypes: the SCLC cell line LC004, the LCC cell line LC006, the AC cell line LC007 and the SCC cell line LC021. From the selected representative PLCCLs, we prepared paraffin blocks, and sections from these cell lines were stained with H&E. Morphological evaluation of the PLCCLs confirmed their epithelial origin with signs of malignant transformation. Moreover, the primary cell lines derived from each of the four lung cancer subtypes were morphologically heterogeneous both at the cellular and nuclear level (
Figure 2A
).
10.1371/journal.pone.0057020.g002Figure 2 Comparison of morphological and phenotypic features between the PLCCLs and corresponding tumor tissues.
A. H&E staining of the 4 representative primary lung cancer cell lines for different lung cancer subtypes: the SCLC cell line LC004; the LCC cell line LC006; the AC cell line LC007 and the SCC cell line LC021. The cells derived from the four subtypes of lung cancer showed heterogeneity in cellular and nuclear morphology. Cells of each primary cell line had epithelial morphology. Photomicrograph magnification, ×200. B. Analysis the expression of P53, Ber-EP4 and CD44 in the 4 primary cell lines and their corresponding archival patients' tumor tissues. All the primary cell lines investigated showed diffuse positive staining for P53 except the original SCC tissue of cell line LC021. Epithelial membrane antigen Ber-EP4 was widely positive in all the original lung cancer tissues and diffuse positive in all the primary cell lines. CD44 showed diffuse positive staining with different intensity in the primary cell lines and their corresponding archival patients' cancer tissues except weakly positive in the original tumor tissue of the AC cell line LC007. Sections from paraffin blocks containing human seminoma and breast cancer specimen were used as positive controls for antibodies P53, Ber-EP4 and CD44, respectively. Photomicrograph magnification, ×200.
Tumor-suppressor protein P53 mutations is a hallmark of cancer and is generally up-regulated [22]. We next compared expression levels of P53 in serial sections from the archival patients' tumor tissues and the newly established PLCCLs using IHC. All four PLCCLs showed diffuse positive staining for P53. The same type of staining was observed in the original patient's cancer tissue with the exception of the SCC parental tissue, which was negative for P53 (Figure 2B). Interestingly, the cell line derived from the SCC (LC021) expressed P53. The expression levels of the Ber-EP4 and CD44 were investigated in the same way. The archival patients' tumor tissues and their corresponding PLCCLs showed broadly positive staining for the pan-epithelial marker Ber-EP4. Diffuse positive staining with different intensity in the archival patients' tumor tissues and their corresponding cell lines was seen with CD44 staining, except for the AC parental tumor tissue, which was weakly positive over most of the section studied (Figure 2B, Table S2). Expression of CD133 in the parental tumor tissues was also investigated by IHC staining. CD133 displayed a more variable pattern, with all the parental tumors being negative, except the SCLC (LC004) where expression in isolated areas was observed (data not shown). All PLCCLs derived from the corresponding parental cancer tissues were uniformly negative for CD133 expression by IHC staining.
Taken together the summarized experiments suggest that the PLCCLs in early passages (up to 5) are representative of the parental tumor tissue in all four cancer subtypes.
3. Primary cultures of lung cancer cells are as tumorigenic as xenotransplants
Malignant potential of the PLCCLs was investigated in an experimental animal model. A tumorigenesis assay was performed on 5 to 6-week old female NOD/SCID mice. Seven of the PLCCLs were tested in early passages and all were able to generate xenografts within 3 weeks after injection. The four cell lines SCLC LC004-P4, LCC LC006-P2, SCC LC021-P2 and AC LC007-P3, were chosen for further studies, propagating subcutaneous bulk tumors in 2/2, 3/3, 2/2 and 2/2 animals, respectively. Moreover, we were able to re-established PLCCLs in vitro from the xenografts taken from these animals (
Figure S1
). In addition, DNA fingerprinting was useful to track individual cell lines from the xenografts of LC021 (
Table S1
and
Figure S1
).
Based on these results we conclude the PLCCLs with the tumorigenesis capacity in immunodeficient mice can easily and reproducibly be generated from all four subtypes of lung cancer and propagated in in vitro liquid culture.
Secondary xeno-transplantation was performed by implanting subcutaneously pieces of the primary LCC xenograft (LC006) into secondary recipient. Primary recipient transplanted with LC006 cells revealed lung metastasis in addition to subcutaneous tumor. However, in the following serial xeno-transplantations, tumors were formed only subcutaneously. When morphological features of the PLCCL and the serial xenografts and parental tissues were compared, they displayed high similarity supporting the observation that PLCCLs and xenotransplants represent the original tumor.
4. Expression of CSC associated cell surface markers is dynamic in the long-term culture of PLCCLs
To identify subpopulations of putative lung cancer stem cells in the primary cancer cell lines, we investigated the expression of a broad panel of markers previously described as differentially expressed in a variety of human cancer stem cells. Data from six representative PLCCLs analyzed at different passages are given in
Table S3
. The expression profile revealed a variable pattern with some common features. In two cell lines (SCLC LC004 and SCC LC021) tested repeatedly the phenotype shifted during long term in vitro culture (Table S3). Some markers (CD29, CD49b and CD49f) were uniformly expressed at high levels in all passages and all cell lines studied, while others, such as CD44, CD166 and CD142, showed a higher expression in later compared to earlier passages. The opposite was observed for CD90 and CD326 expression, where the proportion of positive cells diminished with increasing passage number. Interestingly, distinct minor subpopulations expressing CD117, CD184 and CD15 were present in each of the cell lines tested. CD24 and CD326 showed variable expression level in different PLCCLs. The expression of CD133 was low, as tested by two different antibodies, and varied between 0–2.4% confirming the results obtained by IHC.
Thus, the expression level and the frequency of CD44 and/or CD90 positive sub-populations was dynamic, revealing differential expression of the antigens at different time points. These distinct changes that occurred during long-term culture indicate either possible selection of certain sub-populations in long-term culture or response to in vitro culture conditions.
All in all, our results clearly show that multiple cell surface antigens described to be differentially expressed in human CSCs, were also expressed on established PLCCLs, independent of the cancer subtype.
5. CD44high cells from primary SCLC and LCC primary cell lines harbour cells with CSC characteristics
CD44 has been reported as a candidate CSC marker in many types of cancer [3], [4], [9]–[11]. All our PLCCLs expressed CD44 (Table S2). The flow cytometry analysis revealed the existence of distinct sub-populations with differential expression of CD44: a small sub-population of CD44high cells could be identified in both the SCLC cell line LC004 and the LCC cell line LC006, representing 6.63% (Figure 3A) or 2.79% of total cells (Figure 3B), respectively. Similar results were obtained for cell lines AC LC007 and SCC LC021 (Percentage of CD44high cells was 6.69% and 14.95%, respectively) (Figure 3C, D). The main cell population was either negative or weakly positive for CD44. To investigate possible functional differences between these subpopulations, CD44high and CD44low/− cells of SCLC LC004 and LCC LC006 were FACS-sorted and tested in several functional assays. The results from the colony formation efficiency assay suggest that CD44high cells from the SCLC LC004 and LCC LC006 cell lines have a significantly higher potential to form colonies compared to CD44low/− cells (data not shown and Figure 4A). The fact that CD44high cells formed bigger colonies suggests that CD44high cells from LC006 cell line proliferate more vigorously and for longer time than cells with low expression or negative for CD44.
10.1371/journal.pone.0057020.g003Figure 3 Identification of CD44high cells in the PLCCLs by flow cytometry analysis.
A small sub-population of CD44high cells could be identified in the SCLC cell line LC004 (6.63%) (A); the LCC cell line LC006 (2.79%) (B); the AC cell line LC007 (6.69%) (C) and the SCC cell line LC021 (14.95%) (D), respectively. Left panel: isotype control Ab; right panel: CD44 Ab. Data shown are from representative experiments (n>3).
10.1371/journal.pone.0057020.g004Figure 4 Comparison of colony forming potential of different cell sub-populations from PLCCLs by colony formation efficiency assays.
A. Comparison of FACS sorted CD44high and CD44low/− cells from the LCC cell line LC006. Cells were seeded at 200 cells per well and grown in standard 6 well plates for ∼10 days. Upper wells: CD44high cells. Lower wells: CD44low/− cells. B. Comparison of FACS sorted CD44highCD90+ and CD44highCD90− cells from the SCLC cell line LC004 (upper plate) and the LCC cell line LC006 (lower plate). Cells were seeded at 200 cells per well and grown in standard 6 well plates for ∼10 days. In each 6-well plate: upper wells: CD44highCD90+ cells; lower wells: CD44highCD90− cells. X denotes empty wells.
To investigate the proliferative potential of single cancer cells from PLCCLs, we next performed a single cell colony formation assay. Colony formation was observed 7∼10 days following plating. Only the CD44high cells from the two cell lines SCLC LC004 and LCC LC006 were capable of forming a full range of colonies: holoclones, which consisted of tightly packed small cells, paraclones representing loosely packed colonies and meroclones with characteristics between holoclone and paraclone. CD44low/− cells did not form holoclones, but were able to form a few meroclones and paraclones (Figure S2 and Table S4). To further study self-renewal and differentiation potential of cells isolated from the different colony types, the single cell derived colonies were picked, expanded and maintained in common cell culture flasks for long-term serial passages. Only the cells from holoclones, which were derived from the sorted CD44high cells, had potential to maintain in vitro long-term cultures. When re-seeded into the colony assays, these cells formed all three types of colonies. Paraclones and meroclones both from CD44high and CD44low/− sorted cells stopped proliferating after one passage (data not shown).
These results demonstrate that CD44high cells from SCLC and LCC cell lines contain cells with self-renewal and long-term growth potential. These stem cell-like properties were found only in CD44high expressing cells.
We next compared the spheroid formation potential of CD44high and CD44low/− cells under serum-free conditions. Sorted cells were seeded at the concentration of 100 cells per well in ULA 96-well plate. Development of spheroids was evaluated 2 weeks after plating. Seven of ten wells with CD44high cells from the SCLC cell line LC004 and 6/10 wells with CD44high cells from the LCC cell line LC006 were identified with spheroid formation. Wells containing CD44low/− cells from both cell lines did not reveal any formation of spheroids, and the cells eventually died (
Figure S3
).
Finally, growth curves of cell line LCC LC006 were determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. At the highest cell concentration tested (500 cells/well) no significant difference in the proliferation of CD44high and CD44low/− cells was observed (
Figure S4
). However, when cells were seeded at a lower density (150 cells/well) CD44high cells demonstrated higher proliferative potential compared to CD44low/− cells (
Figure S4B
). Taken together, these results indicate that CD44high population harbour cells with stem cell-like properties.
6. The CD44highCD90+ phenotype identifies sub-population of cells with stem cell-like properties in the SCLC and LCC
In order to narrow down the phenotype of the CSC population, the expression of additional putative stem cell markers were investigated. The initial analysis of PLCCLs by flow cytometry suggested that both CD44high and CD44low/− populations could be further sub-divided based on CD90 expression, namely CD44highCD90+, CD44highCD90−, CD44low/−CD90+, and CD44low/−CD90− sub-populations (
Figure 5
). Since CD44low/− population did not seem to contain cells with stem cell properties, CD44low/−CD90+, and CD44low/−CD90− cells were sorted as one CD44low/− population irrespective of CD90 expression. We then repeated the experiments described above, now testing 3 different sub-populations. In the 2D single cell colony forming and heterogeneity assay, only the CD44highCD90+ cells from the SCLC LC004 and LCC LC006 cell lines were able to form all three types of colonies (
Figure 4B
). Importantly, the cells derived from holoclones of sorted CD44highCD90+ cells maintained long-term in vitro growth and formed all three types of colonies when re-plated. CD44highCD90− cells from the SCLC cell line LC004 gave rise to a single holoclone. This clone, however, did not sustain cell growth for more than one generation (Table S5). Repeatedly, CD44low/− cells from SCLC and LCC cell lines, were able to form only a few meroclones and paraclones, which were not able to sustain a long-term growth in liquid culture and stop proliferating after one passage. These data indicate that CD44high cells from SCLC and LCC cell lines that co-express CD90 have stem cell-like characteristics and sustain cell growth in liquid culture.
10.1371/journal.pone.0057020.g005Figure 5 Flow cytometry analysis of CD44 and CD90 expression in the PLCCLs.
Staining by anti-CD44 FITC and anti-CD90 APC. All the cell lines showed heterogeneous staining and could be divided into 4 sub-populations: CD44highCD90+, CD44highCD90−, CD44low/−CD90+ and CD44low/−CD90−. For the SCLC cell line LC004 (A), the frequency of CD44highCD90+ cell was 16.6%, and the CD44highCD90− cells was 8.2%. For the LCC cells line LC006 (B), the frequency of CD44highCD90+ cells was 1.1%, the CD44highCD90− cells was 2.5%. For the AC cell line LC007 (C), the frequency of CD44highCD90+ cells was 9.4%, and the CD44highCD90− cells was 23.4%. For the SCC cell line LC021 (D), the frequency of CD44highCD90+ cells was 2.3%, the CD44highCD90− cells was 1.1%.
Above mentioned cell populations were also tested in the spheroid forming assay. Cell spheroids were detected in 8/10 wells with sorted CD44highCD90+ cells from SCLC LC004 (Figure S5A) and in 8/10 from LCC LC006 (Figure S5B) cell line. In contrast, only 1/10 wells with CD44highCD90− cells of both cell lines formed spheroids. As expected, no spheroids were formed in the wells with CD44low/− cells irrespective of CD90 expression supporting the data from the 2D colony forming assay. These results clearly indicate that the spheroid forming cells are mainly harboured within the CD90+ subpopulation of the CD44high cells.
We also tested the spheroid-forming capacity of the CD44highCD90+, CD44highCD90−and CD44low/− cell populations in the SCC cell line LC021. Here 10/10 wells of CD44highCD90+ cells formed spheroids (Figure S6A), but contrary to the results of the SCLC cell line LC004 and LCC cell line LC006, 9/10 wells with CD44highCD90− cells also formed spheroids (Figure S6B). No spheroids were formed in the wells with CD44low/− cells (Figure S6C). Taken together, these results indicate that cells with stem cell-like characteristics are enriched in the CD44high population also in the SCC cell line LC021. CD90, however, may not enrich further for the cells with stem cell-like properties.
A CD44highCD90+ population was also identified in the AC cell line LC007. However, neither CD44highCD90+, CD44highCD90− nor CD44low/− cells formed spheroids. Since this cell line did not form spheroids under the defined culture conditions, we cannot conclude whether either of the markers, i.e. CD44 and CD90, is a stem cells markers in the tested AC cell line.
7. The CD44highCD90+ sub-population exhibits stem cell and EMT associated gene expression profile in LC004 and LC021 primary cell lines
The observations that some sub-populations sorted based on expression of CD44 and CD90 had stem cell-like properties, prompted us to investigate whether the expression of stem cell related genes, such as Nanog, Oct4 and Sox2, was different in these sub-populations of the cell lines LC004, LC006 and LC021. Detectable expression levels of these genes were found in all the sorted sub-populations. Interestingly, the expression of Nanog, Oct4 and Sox2 were higher in the CD44high population compared to the CD44low/− population in all the examined cell lines (
Figure 6
). Furthermore, the expression level of Nanog and Oct4 genes was higher in the CD44highCD90+ population compared to the CD44highCD90− population in the cell lines LC004 and LC006 (
Figure 6A, B
). However, no differences in gene expression levels except Nanog were observed between CD44highCD90+ and CD44highCD90− populations in the LC021 cell line (
Figure 6C
).
10.1371/journal.pone.0057020.g006Figure 6 Analysis of the expression of stem cell and EMT related genes in different sub-populations from the PLCCLs.
Detectable expression levels of the genes were found in all sorted sub-populations from the cell lines LC004 (A), LC006 (B) and LC021 (C). PCR reaction without template served as a negative control. The relative expression of target genes was related to the expression of PGK1 and normalized to the unsorted control cells. X axis shows the target genes, Y axis shows the relative expression level (RQ). The error bars reflect the variation within the triplicates, P<0.05. (B). The FACS sorted cell sub-populations derived from the cell line LC006 were cultured in the serum-free culture system for 1–2 weeks and revealed different cell morphology for different sorted sub-populations: (a) CD44highCD90+ cells; (b) CD44highCD90− cells; (c) CD44low/−CD90+ cells, and (d) CD44low/−CD90− cells. Photomicrograph magnification, ×200.
CSCs are proposed to play an important role in cancer invasion and metastasis seeding. Increasing evidence shows that epithelial-mesenchymal transition may impose stem cell characteristics on tumour cells, and accordingly, stem cell-like cells from carcinomas have a mesenchymal phenotype and express markers associated with EMT [15]. Culture of the different sorted cell populations derived from the LC006 cell line in our serum-free culture system shows that the sorted cell populations display different cell morphologies (Figure 6B). For example, the CD44highCD90+ and CD44highCD90− cells have a mesenchymal morphology, while the CD44low/−CD90+ and CD44low/−CD90− cells grow as cobblestones, a hallmark of epithelial cells. Therefore, we examined the expression profiles of EMT associated genes N-Cadherin, E-Cadherin, Vimentin, and HMGA2 in the sorted sub-populations from the cell lines LC004, LC006 and LC021. Interestingly, higher expression of the mesenchymal marker genes N-Cadherin, Vimentin and HMGA2, accompanied by a decreased expression for the epithelial marker gene E-Cadherin was found in the CD44high population compared to the CD44low/− population in all three cell lines (Figure 6A, B, C). The expression level of N-Cadherin and Vimentin were higher in the CD44highCD90+ population compared to the CD44highCD90− population from the cell lines LC004 and LC021 (Figure 6A, C), however, this was not the case for the same populations derived from the cell line LC006 (Figure 6B). The expression level of HMGA2 was higher in the CD44highCD90+ population compared to the CD44highCD90− population in the cell line LC004 (Figure 6A), no difference was observed between the subpopulations CD44highCD90+ and CD44highCD90− of the cell lines LC006 and LC021 (Figure 6B, C).
Altogether, our results of stem cell and EMT associated gene expression profile indicate a higher stem cell-like potential for the CD44high population for all the examined cell lines. Co-expression of CD90 further enriched putative lung CSCs in the cell lines LC004 and LC006.
8. CD44highCD90+ cells from LCC primary cell line display the highest resistance to radiotherapy
The resistance to irradiation of CD44high and CD44low/− cells from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. Five days post irradiation with different doses, CellTiter 96® AQueous One Solution Cell Proliferation Assay was performed to compare the difference in cell proliferation of irradiated monolayer cultures from different cell sub-populations. The inhibition ratio of X-ray to the cells was calculated. The CD44high cells were more resistant to irradiation at each dose tested (
Figure 7A
). To investigate whether resistance to irradiation was associated with the CD44highCD90+ phenotype, the same assay was applied to determine the relative resistance to irradiation of the four cell populations derived from the same cell line. Among the four sub-populations, CD44highCD90+ cells displayed the highest resistance to irradiation at each dose tested (
Figure 7B
). This result further supports the notion that CD44highCD90+ population is enriched for putative lung CSCs.
10.1371/journal.pone.0057020.g007Figure 7 Relative irradiation resistance of different sorted cell populations from the cell line LC006. A.
The relative resistance to irradiation of the FACS sorted CD44high and CD44low/− cells derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. The inhibition ratios were compared among the different cell populations after irradiating the monolayer cultures at 1, 2, 3 and 4Gy. The CD44high cells displayed the higher resistance to X-ray radiation at each dose tested. P<0.01. B. relative resistance to irradiation of the four different FACS sorted cell populations derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. Four populations: CD44highCD90+, CD44highCD90−, CD44low/−CD90+ and CD44low/−CD90− cells were sorted into 96-well plates at 500 cells per well in 10 replicates. The inhibition ratios were compared among the four sub-populations after irradiation of the monolayer layer of the cultures at 1, 2 and 4Gy. The CD44highCD90+ cells displayed the highest resistance to irradiation at 2 and 4G. P<0.01.
9. The primary cell lines from long-term cultures changed their morphological features and the phenotype with time
In order to study the potential changes of established primary cell lines during long-term culture in vitro, we monitored the morphology and phenotype in the cell lines LC004 and LC021 at different passages. Although these primary lung cancer cell lines can successfully sustain long-term in vitro culture (the longest passage so far is over 30 generations of LC006) without growth decline, we observed certain changes in cell morphology. Cells from the first several passages demonstrated predominantly epithelial-like morphology, and then, the cells showed the change to mesenchymal features. Thereafter, shift towards more stressed epithelial-like morphology was observed following prolonged in vitro liquid culture.
We further employed flow cytometry to monitor phenotypic changes of the LC004 and LC021 cells at different passages, focusing on the CD44 and CD90 expression. In early passages (within passage 5), the cultured primary lung cancer cells maintained a sub-population of CD44highCD90+ cells. In later passages, the expression level of the CD44high cells markedly increased while the expression level of CD90 decreased. The majority of the cells thus moved to the CD44highCD90− region (Figure S7 A, B).
Concomitant with the changes in phenotype, cells from early passages demonstrated better colony forming potential compared to cells from later passages, corresponding to the decrease in the CD44highCD90+ subset from the prolonged culture (Figure S7 A, B).
The changes of morphology and phenotype during passaging of the cell lines may be caused by selection of certain sub-populations with distinct properties in an in vitro culture environment.
Discussion
Tumor growth is hypothesized to depend on CSCs within a tumor, which shares the similar self-renewal function and capacity to generate differentiated cells with their normal stem cell counterparts. These cells can be identified by tumor stem cell associated antigens and other markers. Identification of reliable markers for CSC/TIC, which can be detected by robust tests such as flow cytometry using monoclonal antibodies, may open new opportunities for early diagnosis and staging, as well as the identification of novel targets leading to improvement of treatment for cancer in the clinic. By the use of different surface markers, CSCs/TICs have been identified in a variety of human cancers including acute myeloid leukemia, brain tumor, breast cancer, colon cancer, pancreatic cancer and prostate cancer [1]–[11].
In 2005, bronchioalveolar stem cells (BASCs) localized at the bronchioalveolar duct junction were identified as the tumor initiating cells for adenocarcinoma in a mouse model [23]. Recent attempts to identify lung CSCs in humans have suggested that CD133 [18] and also CD44 independently [24] could define a cell population with CSC characteristics. Later, several lines of evidence indicated that the ability of CD133 expression to discriminate lung CSCs may have been overestimated [19]. This is in line with our own observations showing that CD133 expression on lung cancer cell lines may be gained upon culture of sorted CD133 negative subpopulations and lost upon culture of CD133 positive subpopulation (Ping et al. unpublished). In the present study we accordingly focused on differential expression of CD44 in 4 representative primary cancer cell lines established from freshly resected tumors.
In order not to be restricted by the low number of cells available from surgical specimens and to be able to perform consecutive and repeated experiments on cancer cells derived from the same primary sample, we choose to study primary cell lines, which may also more accurately represent the situation in the patient compared to cell lines that have been established decades ago. In the present study, we successfully established a panel of novel cell lines from the primary cultures directly from different subtypes of freshly resected primary lung tumors. We used early passages of the generated cell lines to isolate phenotypically distinct sub-populations with stem cell features from these cell lines.
CD44 has been identified as a CSC marker for many cancer types, including breast cancer [4], prostate cancer [11], colorectal cancer [3] and lung cancer [24]. Flow cytometry and immunohistochemistry analyses showed that all the primary cultured lung cancer cell lines expressed CD44, but with marked differences in intensities and frequencies. A series of experiments aimed to evaluate whether lung CSCs could be enriched by the expression of CD44, demonstrated that CD44high cells always showed a stronger proliferative potential than CD44low/− cells, and only CD44high cells in the LC004 and LC006 cell lines were enriched in cells that could form holoclones, meroclones and paraclones reported to be associated with cancer stem cells [25]. The CD44high cells could form cell spheroids, whereas CD44low/− cells lacked this potential. Moreover, the CD44high sub-population showed a more mesenchymal morphology than the CD44low/− population. Together these results indicated that CD44high expression was associated with several stem cell characteristics in cell lines both from primary SCLC and LCC. These results confirm and extend the finding reported recently [24], which were mainly based on studies of established non-small cell lung cancer (NSCLC) cell lines. We did not investigate the potential co-expression of CD133 on the CD44high subpopulation. Such a co-expression might have solved the apparent controversy regarding the role of these two markers on lung cancer stem cells. The main reason for not doing so was the absence of CD133 expression in the surgical samples and the corresponding low or absent expression in the early passages of the cell lines derived from these samples.
CD90 is a marker expressed in mesenchymal stem cell [26] and liver cancer stem cell [27]. We found that it is also expressed in the primary lung cancer cell lines. By further co-staining with CD44 and CD90, all four representative primary cultured lung cancer cell lines could be divided into 4 heterogeneous sub-populations: CD44highCD90+, CD44low/−CD90+, CD44highCD90− and CD44low/−CD90− cell populations. In the SCLC cell line LC004 and the LCC cell line LC006, CD44highCD90+ cells had much stronger colony and spheroid forming potential. The self-renewal and differentiation analysis, demonstrated that only CD44highCD90+ cells were capable of forming holo-, mero- and paraclones. All the holoclones from CD44highCD90+ cells could be serially passaged and were capable of giving rise to all the same set of clones, while the only holoclone developed from CD44highCD90− cells was only able to sustain one generation. These results indicate that CD44highCD90+ cells may be able to undergo asymmetric divisions, giving rise to mature cells with a limited life span [28]. Fitting with the other findings, CD44highCD90+ cells in the LCC cell line LC006 also showed the highest level of resistance to irradiation. For the lung squamous cancer cell line LC021, CD44high expression alone may be a stem cell marker, since no further enrichment for colony formation or clonal growth was observed when CD90 was included. For the AC cell line LC007, only marginal differences in colony formation was observed in the four different sub-populations (data not shown). We are therefore unable to make any firm conclusion about the role of CD44 or CD90 as CSC marker in AC. Taken together, these result therefore indicate that CSCs in the four main subtypes may differ considerably in their phenotype.
Increasing evidence shows a direct link between the EMT and CSCs. Both EMT and CSC are implicated in the generation of invasive cells and formation of distant metastases. Furthermore, CSCs have been found to express EMT associated genes in addition to stemness associated genes [15]. Our observation that CD44high cells exhibited an increased expression of EMT associated genes indicates that CD44high cells may play a key role in progression of SCLC and LCC. Interestingly, CD44+CD90+ cells isolated from human hepatocellular carcinomas demonstrated a more aggressive phenotype than single positive, here CD90+/CD44− cells, forming more metastatic lung lesions in immunodeficient mice [27], indicating an important role of CD44 also in this type of cancer. The high mobility group A2 gene (HMGA2) is abundantly expressed in embryonic stem cells [29]. Aberrant re-expression of HMGA2 is correlated with tumor aggressiveness in a variety of human cancers [30], [31]. Furthermore, there is an increased expression of HMGA2 in cancer cells with stem cell characteristics, and the expression increase upon EMT [32]–[33]. In our study, the expression of HMGA2 was increased in the CD44high population compared to the CD44low/− population in three cell lines investigated, consistent with the increase of other EMT associated genes and stem cell related genes in this population.
In CSC research, established cancer cell lines are widely used to isolate and identify CSCs. Although it is relatively easy to use established cancer cell lines that have been grown for a long time in vitro, the CSCs identified in such cell lines may not truly reflect all biological features of primary CSC due to culture adaptation and genetic alterations taking place during long-term culture under hyperoxic culture conditions. The robust method for generating such primary cell lines from surgical specimens of lung cancer and identification of lung CSCs in these cell lines may provide a valuable and unique tool for further studies. With more reliable biomarkers available, these cell lines can also be used for screening of panels of different drugs targeting CSC related pathways.
Supporting Information
Figure S1
The flow cytometry analysis of xeno-cell line LC021. Xeno-cell line LC021 displayed a hierarchical organization similar to that found in primary cultures, CD44highCD90+ cells were found in xeno-cell line LC021. Anti-Tra-1-85 Ab was used to gate human cells; Anti-H2Kd Ab was used to gate mouse cells.
(TIF)
Click here for additional data file.
Figure S2
Clonal heterogeneity assay of sorted cell populations from the cell lines LC004 and LC006. Sorted cells were cultured under serum-free condition for 2 weeks after seeding of 1 single cell per well in 96-well standard plate. Two weeks later, heterogeneous colonies of distinct morphologies were identified in CD44high cell populations from the SCLC cell line LC004 (A) and the LCC cell line LC006 (B). Left column: Typical holoclones which are generally more round and tightly packed composed of homogenous cells. Middle column: meroclones which are colonies made of cells of intermediate morphology and cell numbers. Right column: paraclones which are irregular in shape and contain fewer and more elongated or flattened cells. Representative colony images were acquired at 100×.
(TIF)
Click here for additional data file.
Figure S3
Potential of sorted CD44high and CD44low/− cells to form spheroids under serum-free culture condition. Cells were seeded at 100 cells per well in ULA 96-well under serum-free condition. CD44high cells of the SCLC cell line LC004 (A) and the LCC cell line LC006 (C) developed cell spheroids 7–10 days after plating, while CD44low/− cells from both cell line LC004 (B) and LC006 (D) formed no spheroids. The assay was repeated twice with similar results. The photomicrograph shows representative sections of the wells. Photomicrograph magnification 200×.
(TIF)
Click here for additional data file.
Figure S4
Proliferation of CD44high and CD44low/− cells from the PLCCL LC006 at different cell concentrations and different time points. A. Proliferation of sorted CD44high (blue line) and CD44low/− (purple line) cells seeded at 500 cells per well in 96 well plate. B. Proliferation of sorted CD44high and CD44low/− cell populations seeded at 150 cells per well under identical culture conditions. Data represent the mean value of at least three wells.
(TIF)
Click here for additional data file.
Figure S5
Potential of sorted sub-populations from LC004 and LC006 PLCCLs to form spheroids under serum-free culture condition. Four populations were sorted based on expression of CD44 and CD90 surface markers. Results are shown only for the CD44highCD90+ sub-population that had the potential to form spheroids. Sorted cells were seeded at 100 cells per well in ULA 96-well under serum-free condition. Cell spheroids were formed only in wells with CD44highCD90+ cells from LC004 (A) and from LC006 (B). Photomicrograph magnification 200×.
(TIF)
Click here for additional data file.
Figure S6
Comparison of cell spheroid formation of different sub-populations from the LC021 under serum-free condition. CD44highCD90+, CD44highCD90− and CD44low/− cell populations were sorted from the SCC cell line LC021. Spheroids were formed by CD44highCD90+ cells (A) and by CD44highCD90− cells (B). None of spheroids was formed by CD44low/− cells (C). Photomicrograph magnification 200×.
(TIF)
Click here for additional data file.
Figure S7
Morphological and phenotypic changes of PLCCLs LC004 and LC021 upon long-term
in vitro
culture. A. Monitoring of the morphology and phenotype of the cell line LC004 at different passages upon long-term in vitro culture. a. In early passages, the cultured cells predominantly demonstrated mesenchymal morphology, while a shift towards a more stressed epithelial-like morphology was observed following prolonged in vitro culture in serum free medium. Photomicrograph magnification 200×. b. Monitoring of the phenotypical changes of the LC004 cells at different passages, based on the expression level of CD44 and CD90 by flow cytometry. c. The charts showing the changes of phenotype (upper left and right), colony forming efficiency (lower left) and propagation (lower right) of the LC004 cells upon long term in vitro culture. B. Monitoring of the morphology and phenotype of the cell line LC021 at different passages upon long-term culture in vitro. a. Monitoring of the phenotypical changes of the LC004 and LC021 cells at different passages based on expression of CD44 and CD90 by flow cytometry. b. The charts show the changes of phenotype (upper left and right), colony forming efficiency (lower left) and propergation (lower right) of the LC021 cells upon long term in vitro culture.
(TIF)
Click here for additional data file.
Table S1
DNA fingerprinting data on PLCCLs.
(DOC)
Click here for additional data file.
Table S2
Summary of immunoshistochemical analysis of P53, Ber-EP4, and CD44 of PLCCLs and their corresponding parental tumor tissue.
(DOC)
Click here for additional data file.
Table S3
Expression of a broad panel of cancer stem cell associated markers analyzed in six representative cell lines at different passages.
(DOC)
Click here for additional data file.
Table S4
Single cell 2D colony forming and heterogeneity assay.
(DOC)
Click here for additional data file.
Table S5
Single cell 2D colony forming and heterogeneity assay.
(DOC)
Click here for additional data file.
We thank Ingjerd Solvoll from the Pulmonary Biobank project at Radiumhospitalet for her assistance in collection of tumor samples and clinical data; Dr. Lars Jørgensen from the Thoracic Surgery Department at Rikshospitalet for the assistance in obtaining tumor samples; Anna Berit Wennerstrøm and Menaka Sathermugathevan from SFI-CAST for their assistance with the long-term cultures; Karen-Marie Heintz from Tumorbiology at Radiumhospitalet for her assistance in performing DNA fingerprinting assay. This work was supported by the Cancer Stem Cell Innovation Centre (SFI-CAST).
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23472088PONE-D-12-3601710.1371/journal.pone.0057525Research ArticleBiologyImmunologyImmune ResponseMicrobiologyVirologyAnimal Models of InfectionAntiviralsMechanisms of Resistance and SusceptibilityViral VaccinesMedicineGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BChanges in Innate and Permissive Immune Responses after HBV Transgenic Mouse Vaccination and lLong-Term-siRNA Treatment Disrupt Immune Tolerance Status to HBVRen Guang-Li
1
2
*
Huang Guang-Yu
3
Zheng Hong
4
Fang Ying
5
Ma Heng-Hao
1
Xu Man-Chun
1
Zhang Hong-Bin
1
Zhang Wei-Yun
1
Zhao Ya-Gang
6
Sun Da-Yong
6
Hu Wen-Kui
1
7
Liu Jian
1
7
1
Department of Pediatrics, General Hospital of GuangZhou Military Command of PLA, GuangZhou, China
2
The Biochemistry Institute of the Technology University of South China and the HuaBo Biopharmceutics Institute of GuangZhou, GuangZhou, China
3
Department of Infectious Disease, Southwest Hospital, Third Military Medical University, ChongQing, China
4
Department of Oral Orthopaedic, Dental Hospital of Fourth Military Medical University, Xi’an, China
5
Department of Stomatology, Medical College of GuangZhou, GuangZhou, China
6
Department of Gastroenterology General Hospital of GuangZhou Military Command of PLA, GuangZhou, China
7
Department of Gerontology and the Deanery, General Hospital of GuangZhou Military Command of PLA, GuangZhou, China
Vartanian Jean-Pierre Editor
Institut Pasteur, France
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: RGL LJ. Performed the experiments: RGL HGY ZH ZHB ZWY. Analyzed the data: FY MHH XMC ZHB. Contributed reagents/materials/analysis tools: MHH ZYG SDY. Wrote the paper: RGL HWK.
2013 5 3 2013 8 3 e5752515 11 2012 22 1 2013 © 2013 Ren et al2013Ren et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Currently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators.
Methodology/Principal Findings
To explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine.
Results
Functional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group.
Conclusions/Significance
For the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.
This research was funded by China postdoctoral Science Foundation [20100470917; 201003353]; Natural Science Foundation of Guangzhou province [8451001002000762]; and Guangdong province & GuangZhou Science and Technology Program Funds [12C19732961131678, 20120314]. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Approximately 400 million people have persistent hepatitis B virus (HBV) infection in the world, and most of them will eventually develop chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) [1]. In addition, the risk of dying from HBV-related diseases is approximately 15–25% each year, and about one million deaths occur annually due to end-stage cirrhosis and HCC. Although the HBV vaccine has a strong protective effect, about 10% of the population does not to it. Each year newly infected patients are highly represented by specific populations, such as immunocompromised adults and infants; however, the available treatments are limited [2]. Unfortunately, there are no licensed therapies capable of thoroughly eradicating HBV from the body.
RNA interference (RNAi) is considered an evolutionarily conserved mechanism to protect the genome against invasion by mobile genetic elements, such as transposons and viruses. The effective molecules are small interfering RNAs (siRNAs) and microRNAs (miRNAs) [3]. It is now known to be an elegant and valuable tool against viral infection and cancer. The effectiveness of synthetic siRNAs, vector-generated siRNA, and short hairpin RNA (shRNA) for the inhibition of HBV replication and antigen production has recently been shown [4]–[7]. Previously, we developed several HBV-specific siRNA vectors using rational design tools for testing HBV RNAi-based therapeutics for evaluating the transient and long-term effects in both HepG2.2.15 cells and transgenic mice. Using Polymerase II (Pol II)-driven siRNAs, we [4]–[5] and others [8] have shown that Pol II can drive efficient silencing without off-target effects compared to Pol III-driven siRNAs. Thus, it appears that siRNAs offer a promising method for antiviral therapy.
It is not yet known whether these siRNAs can thoroughly eradicate chronic HBV infection. We know that HBV is an enveloped DNA virus that replicates through an RNA intermediate. HBV relies on a retroviral replication strategy [3], [9]–[10], and the eradication of HBV infection is difficult because stable, long enduring, a covalently closed-circular DNA (cccDNA) becomes established in hepatocyte nuclei and subsequently integrated into the host genome. Thus, if we want to eliminate HBV from the body, the cccDNA must be persistently depleted. Considering these problems, will siRNAs be capable of curing chronic HBV infection? Despite that RNAi is able to split RNA levels, HBV-specific siRNAs do not affect pre-existing HBV cccDNA. Similar to nucleoside analogues, long-term stable siRNA is required to exhaust HBV cccDNA. Furthermore, previous studies demonstrated that siRNA was capable of reducing the formation of HBV cccDNA, but has no effect on established cccDNA [11]. Thus, a combination of RNAi with multiple treatments might achieve HBV clearance.
Recent studies indicated that HBV could counteract the antiviral response of the local innate immune system of the liver by antagonizing the toll-like receptor (TLR)-mediated induction of proinflammatory cytokines [12]–[13]. Furthermore, the natural killer and T-cell responses were attenuated in patients during the early stages of HBV infection [14]–[15]. In response to this, we propose that the body’s immunity could be modified by long-term HBV-specific siRNAs/miRNAs treatment. Improving the innate immune system would likely help control HBV infection [12], [14] and may offer another opportunity for using the HBV vaccine. Here, we compared the effects of long-term anti-HBV RNAi pSilencer5.1/HBV in HBV transgenic mice with those of two recently developed siRNA vectors driven by H1 and cytomegalovirus, which also showed high liver transduction efficiency [4]–[5]. Different concentrations of pSilencer/HBV were also investigated to determine a dosing effect in the transgenic mice. Changes in the innate immune system were evaluated by detecting levels of antiviral cytokines and specific antigen presentation markers of dendritic cells (DCs). We also examined whether repeated injections of recombinant hepatitis B vaccine into transgenic mice with transfected siRNA would initiate production of the HBV surface antigen (HBsAg) antibody (HBsAg-Ab). The immunopotentiators, CpG oligonucleotides, which are recognized by TLR9 and lead to strong immunostimulatory effects [16], were injected with HBV vaccine to further determine whether the immune system would be reinforced.
Here, we confirm that appropriate siRNA concentrations driven by pSilencer4.1-cytomegalovirus can regulate the immune system of HBV transgenic mice. Furthermore, long-term siRNA-treated transgenic mice produced detectable HBsAg-Ab after three injections of the HBV vaccine. Lastly, the CpG oligonucleotide immune stimulators will aid HBsAg-Ab production when the innate immune system has been strengthened in the transgenic mice. Overall, our studies demonstrate for the first time that HBV-specific siRNAs with immunopotentiators of vaccine can strengthen the immune response and break immune tolerance against HBV of the HBV transgenic mice.
Materials and Methods
Ethics Statement
Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health were adhered in our study on mice. Our animal protocol was approved by the Institutional Animal Care and Use Committee of the Third and Fourth Military Medical University of People Liberation Army.
siRNA Vector Preparation
The HBV siRNA expression vectors pSilencer3.1/HBV and pSilencer4.1/HBV have been described in our previous research. The pSilencer5.1/HBV was constructed as described previously [4]–[5]. Briefly, DNA duplexes encoding shRNAs were cloned downstream of the modified cytomegalovirus promoter (an RNA Pol II promoter) and human H1 promoter (an RNA Pol III promoter) for the pSilencer 4.1 and pSilencer 3.1 vectors, respectively (Ambion, Inc, Austin, TX, USA). For the target sites, we chose the sequences for the C open reading frame region 282 and of the S open reading frame region 366 located in the wild type HBV genome (GenBank accession no. U95551, ayw subtype). The siRNA sites were chosen based on the conserved region of HBV, which was obtained by aligning and analyzing the major infectious HBV subtypes. The target sites were 5′-AATGACTCTAGCTACCTGGGT-3′ (C2) and 5′-AACCTGCATGACTACTGCTCA-3′ (S2), and the scrambled siRNA plasmid (negative-control plasmid [p-NC]; Ambion) were set as the control. The pSilencer 5.1 vector (Ambion) conferred retroviral-mediated gene transfer, which is a well-characterized and effective tool for the delivery of DNA sequences both in vivo and in vitro.
Mice, Vector, and Vaccine Administration
The SCXK-Balb/c mice (purchased from Fourth Military Medical University, Xi’an, China) and GK-Balb/c-HBV1.3 HBV transgenic mice, which contain 1.3-times over-length of the ayw subtype of the HBV genome, were obtained from the Key Liver Army Laboratory (458 Hospital, Guangzhou, China) at 8–10 weeks of age and weighing between 20 and 23 g. The characterization of the mice has been described in detail previously [5], [17]. All mice were housed in a specific pathogen-free environment and cared for according to guidelines of Laboratory Animals of the National Institutes of Health.
Three types of pSilencer/HBV driven by different promoters as well as a plasmid negative control vector were injected into HBV transgenic mice via hydrodynamic tail vein injection. Mice received 5 mg/kg plasmid diluted in 0.08 ml/g phosphate-buffered saline (PBS). Two different concentrations (3 and 10 mg/kg) of pSilencer/HBV were used to investigate a dose-dependent relationship in the body. The effects of siRNAs on transgenic mice were observed at 5 days (d), 19d, 1 month (M), 3 M, 6 M, 12 M, and 14 M after injection. At 3 and 6 months, the different vector group transgenic mice were repeatedly injected with the corresponding siRNA vector to determine differences in metabolism-dynamic siRNAs. To evaluate the response of the siRNA-treated transgenic mice to HBV vaccine (recombinant protein vaccine expressed by yeast, HBsAg+Al(OH)3), the mice received three HBV-vaccine (20 µg/ml/mice) and immunopotentiators CpG injections at 4 (0), 5 (1) and 10 M (6) after the administration of siRNA vectors. The normal Balb/c mice and the PBS-treated transgenic mice were set as controls.
Detection of siRNA Expression
To evaluate the expression of specific siRNAs in the liver tissue, northern blots were carried out. Total RNA was extracted and isolated total RNA was digested with DNAse I, as described in our previous study [4]–[5]. For siRNA northern blot analysis, 30 µg of total liver RNA from siRNA-treated mice was separated on a 15% polyacrylamide-urea gel and transferred onto nylon membranes (Amersham International, Amersham, UK). Subsequently, the blots were hybridized to 32P-labeled oligonucleotides (19 nt) corresponding to the antisense strand of the HBV-C2 siRNA, HBV-S2 siRNA, miRNA-122, or 5S rRNA. Equal RNA loading was assessed by ethidium bromide staining. ImageQuant software (GE Healthcare, Uppsala, Sweden) was used to quantify small RNA signals. All primers were synthesized by the Invitrogen Corporation (Carlsbad, CA, USA), and all experiments were performed in quadruplicate.
Long-term Silencing Effect of siRNA
Serum and liver samples were collected at different time points, and DNA and RNA were extracted to measure the HBV viral load and RNA. Quantitative analysis of HBsAg in mouse sera was determined using the AXSYM system kit (Abbott Diagnostic Division, Wiesbaden, Germany). Real-time, fluorescent, quantitative polymerase chain reaction (RT-qPCR) was performed to quantify HBV viral genomic DNA using HBV fluorescence RT-qPCR Diagnostic Kits (DaanGene, Guangzhou, China), as described previously. And northern blot analysis for HBV RNA was performed as described in our previous research [4]–[5]. Briefly RT-qPCR was carried out using the TaqMan PCR master mix, the HBV forward primer (5′- CCGTCTGTGCCTTCTCATCTG-3′) and reverse primer (5′-AGTCCAAGAGTCCTCTTATGTAAGACCTT- 3′) targeting positions 1551 and 1646, respectively, and the Taqman probe (FAM-5′- GTGTGCACTTCGCTTCACCTCTGCACGTC-3′-TAMRA, 1551–1646). All reactions were performed in triplicate in 96-well optical reaction plates on an ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA, USA) and analyzed with GeneAmp7700 SDS software. The reagents were denatured for 2 min at 95°C, followed by 50 cycles of 45 s at 95°C and 60 s at 55°C. The northern blot analysis for HBV RNA was carried out. Briefly, 30 µg total liver RNA was separated by electrophoresis on a 1.5% agarose formaldehyde gel and transferred to a nylon membrane. The blot was then hybridized with a biotin-labeled HBV or glyceraldehyde 3-phosphate dehydrogenase probe, which was prepared with a Biotin Random Prime DNA Labeling Kit (Pierce, Rockford, IL, USA).
Detection of Cytokines
To evaluate the vaccine response of the long-term siRNA-treated transgenic mice, the cytokines tumor necrosis factor (TNF)-α (R&D Systems, Wiesbaden, Germany), interferon (IFN)-β, nuclear factor-kappa b (NF-κb), extracellular signal-regulated kinase (ERK) (Cell Signaling Technology Inc., Beverly, Massachusetts, USA), interleukin (IL)-2, IL-6, IL-12, and IL-15 (R&D Systems, Minneapolis, MN, USA; ShanHai, China Co. Ltd.) in mouse sera were detected using enzyme-linked immunosorbent assays (ELISA). Levels of TLR3, TLR8, and TLR9 in liver tissues were evaluated by western blot. The extraction of nuclear, cytosolic, and total proteins was carried out as described previously. From each sample, 10 µg total protein was loaded on a sodium dodecyl sulfate polyacrylamide gel and electrophoresed. For western blot analysis, gels were transferred to nitrocellulose membranes and incubated with anti-TLR3 (Chemicon International, Temecula, CA, USA), anti-TLR8 (LifeSpan Biosciences, Seattle, WA, USA), and anti-TLR9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies overnight at 4°C. The corresponding secondary antibody conjugated with peroxidase (Chemicon) was added for an additional hour at room temperature. Antibody–antigen complexes were visualized by the chemiluminescent SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) detection system on radiographic film (CL-XPosure Film, Pierce). The housekeeping genes glyceraldehyde 3-phosphate dehydrogenase and β-actin were used as internal controls. ELISA was carried out according to the manufacturer’s instructions.
Isolation, Culture, and Functional Assessment of Liver DCs
At the different time points indicated above, liver samples from siRNA-treated, siRNA and vaccinated, vaccinated single and PBS control transgenic mice were collected. The detailed procedure for generating monocyte-derived DCs has been described previously [18]. Briefly, liver vessels were flushed with HBSS containing type IV collagenase (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA), DNAse (50 ng/ml; Roche Diagnostics, Indianapolis, IN, USA), and 3% endotoxin-free fetal calf serum (Invitrogen Corporation) to remove contaminating circulating blood cells. Liver tissue was then morselized and digested in the collagenase solution at 37°C for 30 min. The digestion was quenched with cold HBSS containing DNAse (25 ng/ml) passed through a 100-µm filter. The cell suspension was centrifuged twice at 250×g for 10 min to remove fat and debris. The cell pellet was reconstituted with HBSS and DNAse, and the parenchymal hepatocytes were removed with a low speed spin (30 µg for 1 min). Mononuclear cells from the remaining neural stem/precursor cells were isolated by Ficoll PaquePlus density centrifugation. To detect the function of the DCs, the culture supernatants were collected, and the levels of IL-6, IL-12, and TNF-α were evaluated by ELISA. The CD80+ and CD86+ DCs were sorted by flow cytometry, as described previously [19]. mAbs specific for CD80, CD86, and CD11b were purchased from BD Bioscience (Bilerica, MA, USA) and were used as allophycocyanin, fluorescein isothiocyanate, or phycoerythrin conjugates. Cell enumeration and acquisition were performed using FACSAria and FACSDiva software (BD Bioscience, San Jose, CA, USA).
Administration of CpG Oligonucleotides
CpG oligonucleotides are synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts of CpG motifs, which are recognized by TLR9 and cause robust immunostimulatory effects [16]. Type B CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides, and strongly activate B cells but stimulate weak IFN-a secretion. B cell activation is the key element for producing specific antibodies. Thus, the CpG dinucleotides (ODNs 1826, InvivoGene, San Diego, CA, USA) and HBV vaccine were co-injected into siRNA-treated transgenic mice three times. The bases are 5′-TCCATGACGTTCCTGACGTT-3′ -(20 mer), which were phosphorothioate. Briefly, sterile endotoxin-free water was added to obtain a 500 µM stock solution (315 µl sterile endotoxin-free water to 1 mg vial of ODN 1826), and a 5 mg/kg dose of CpG ODNs was used for injection. The ODN 1826 Control contains GpC dinucleotides instead of CpGs and was used as a negative control.
Quantitation of HBsAg-Ab, CD4+ and CD8+ T Cells
To determine the response of the adaptive immune system, HBsAg-Ab quantification was carried out using MEIA technology at 3 (7 months after siRNA), 5 (9 months after siRNA), 7 (11 months after siRNA) and 9 (13 months after siRNA) months following the first vaccination (Abbott Diagnostic Division, Santa Clara, CA, USA). The sorting of CD4+ and CD8+ T cells in serum and liver samples by flow cytometry was accomplished as previously described [19]. mAbs specific for CD3, CD4, and CD8 were purchased from BD Biosciences and were used as allophycocyanin, fluorescein isothiocyanate, or phycoerythrin conjugates. Cell enumeration and acquisition was performed using FACSAria and FACSDiva software.
Statistical Analysis
For all experiments, one-way analyses of variance were performed to determine the effects of different siRNA treatments at different time points. When the analysis of variance indicated a significant difference among the groups, statistical differences between individual groups were evaluated with the Student-Newman-Keuls test. All experiments were repeated at least four times. All data are expressed as means ± standard deviation. A value of p<0.05 was considered statistically significant. Statistics were performed using the SPSS statistics base 17 (SPSS Inc. Chicago, IL, USA).
Results
Production of siRNAs by Vectors
Recent research had shown that a retroviral vector could produce a high level RNAi effect and enable stable long-term in vivo research [20]. Therefore the pSilencer5.1-H1 retroviral vectors were constructed as pSilcencer5.1/C2 and pSilencer5.1/S2, which were compared with the previous constructed pSilencer4.1/HBV and pSilencer3.1/HBV siRNA vectors (Figure S1). Two different siRNA target sequences were chosen based on our previous research [4]–[5] according to their efficient HBV silencing effect, the rational design, and their conservation among the major HBV genotypes. The sequence homology of the wild type ayw HBV genome was blasted and compared to the ayw HBV of the HBV transgenic mice, which showed 99.97% similarity. Importantly, the target sites of the open reading frame region had identical sequences. Thus, the specificity of the siRNAs were considered reliable.
Comparison of the Transduction Efficiency of the Different siRNA-vectors
Northern blot showed that the expression of siRNAs was stable in the liver of three different vector groups at 3 and 12 months post-transfection (Figure 1a). Moreover siRNAs expressions in the pSilencer4.1-S2/C2 groups were significantly lower than the other two vectors (p<0.05; Figure 1a, b). In addition, the siRNAs showed a slight decline at 3 and 6 months, but recovered after repeated injection in the pSilencer3.1-S2/C2 and pSilencer4.1-S2/C2 groups. So the repeated injection of specific siRNAs vectors can sustain a stable concentration of siRNAs in mice liver. However, in the pSilencer5.1-S2/C2 groups, the siRNAs were unchanged at 3 and 6 months (Figure 1c, d). The expression of miRNA-122 and 5S rRNA were set as internal controls.
10.1371/journal.pone.0057525.g001Figure 1 Specific expression of siRNA in the liver of transgenic mice.
The HBV-C2 siRNA and HBV-S2 siRNA produced by different vectors were detected by northern blot at 3 and 12 months post-transfection (a). The gray scale analysis chart showed the relative siRNA levels of different vectors at 12 months post-transfection (b). The negative-control plasmid (p-NC) which has a mismatched sequence served as control groups in vitro. The siRNAs were quantitated by qRT-PCR after both single and repeated injection with vectors (c). The HBV-specific siRNAs in the liver were measured by qRT-PCR in different groups, such as p-4.1-C2, p-4.1-C2 repeated injection, and pSilencer5.1-C2 (p-5.1-C2) (d). Error bars represent standard error (SE) of the mean. Rep., repeated injection; p-, pSilencer-; qRT-PCR, quantitive reverse transcription PCR. (*p<0.05).
Long-term Efficacy of siRNAs for HBV Suppression
MEIA quantitative analysis of HBsAg showed that gene expression silencing peaked on day 19 after transfection in the specific siRNA groups, and that functional siRNAs caused a marked decrease in viral markers through 14 months in transgenic mice (Figure 2a). Moreover, the 5 mg/kg dose was more inhibitory than the 3 mg/kg dose. The greater 10 mg/kg siRNA dose did not result in a greater silencing effect (Figure 2b). Additionally, the pSilencer5.1/C2 showed the strongest anti-HBV effect, reducing the serum HBV marker by an average of 95%, while the pSilencer3.1/C2 and pSilencer4.1/C2 groups were equivalent in HBV suppression, resulting in an average of 90% and 89% reduction in serum proteins, respectively. The RT-qPCR results revealed that each vector significantly inhibited HBV DNA replication for 14 months (Figure 2c), with the pSilencer5.1/C2 vectors yielding the greatest inhibition (310-fold reduction in serum HBV DNA copy number compared to the 201- and 190-fold reduction by the pSilencer4.1/C2 and pSilencer3.1/C2 vectors, respectively). Consistent with the serum HBV DNA copy results, northern blots showed that treatment with pSilencer5.1/C2 led to the greatest decrease in HBV mRNA levels (Figure 2d). Compared to the PBS-treated control, pSilencer5.1/C2 resulted in an average 90% reduction in the steady state levels of the 3.5, 2.4/2.1, and 0.7 kb RNA transcripts at 12 months post-transfection when normalized to the expression of endogenous glyceraldehyde 3-phosphate dehydrogenase mRNA. A significant reduction in HBV mRNA level was also measured in the pSilencer3.1/C2 and pSilencer4.1/C2-treated mice; however, this was lower than in pSilencer5.1/C2-treated mice with an average 80% and 85% reduction in the HBV transcripts, respectively (Figure 2d). The highest inhibition effect of pSilencer5.1/C2 on HBV among three different vectors may due to the highest siRNAs expression in the mice liver. The scrambled (p-NC) siRNAs did not affect gene expression and the result was similar to that of PBS-treated group. Consistent with the siRNAs expressions in liver of different vectors targeting S and C gene, the silence effect of vectors pSilencer/S2 and pSilencer/C2 were almost the same (Figure 2b, d). Here, the figures were representatively chosen with the vector pSilencer/C2.
10.1371/journal.pone.0057525.g002Figure 2 The silencing effect of specific siRNAs on HBV in transgenic mice.
The HBsAg in mouse sera were quantitated by microparticle enzyme immunoassay (MEIA) at different time points post-tansfection (a). The silencing effects of siRNA on the HBsAg were compared among the different siRNA vector doses at 12- and 14-month post-ransfecion (b). The sample rate/cutoff rate (S/CO) values represent the relative amounts of HBsAg according to the AXSYM system. The long-term silencing effect of siRNA on the HBV-DNA in mouse sera was measured by real time fluorescent quantitative polymerase chain reaction (RT-qPCR) (c). Northern blot and reverse transcription PCR (RT-PCR) showed that HBV transcripts (3.5, 2.4/2.1 and 0.7 kb RNA) from the liver tissues of transgenic mice at 12-month post-transfection were substantially reduced by specific siRNAs (d). Housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin were used as internal controls. n = 6–8 mice per group. PBS, phosphate-buffered saline; p-NC, plasmid negative control; Rep., repeated injection; p-, pSilencer-. (*p<0.05).
Innate Immune System Strengthening after Long-term Treatment with HBV-specific siRNAs
Previous studies [12] including ours [21] have indicated that the TLR family is critical for the expression of cytokine genes at baseline and the up-regulation of certain cytokines by stimulatory signals. Compared to the PBS-treated control group, the antiviral cytokines were significantly elevated in the HBV-specific siRNA groups, particularly for the pSilencer4.1/C2 group (Figure 3). The siRNAs appeared to be capable of reversing the declined innate immunity mediated by HBV chronic infection. Figure 3a demonstrates the TNF-α, IFN-β, IL-2, IL-6, IL-12 and IL-15 levels, which are primarily induced by the TLR family and were markedly upregulated (p<0.05) compared to PBS-treated transgenic mice and almost the same as normal Balb/c mice. Furthermore, the transcription factors NF-κb and ERK were activated after long-term treatment with siRNAs, which would result in the production of proinflammatory cytokines (Figure 3b). Innate immune system changes in the siRNA-treated transgenic mice peaked at 10 months, then declined slightly over the next 4 months, which may be due to the aging process in the mice. The levels of cytokines were almost the same between pSilencer/S2 and pSilencer/C2. In addition, Figure 3c shows that the expression of TLR3, TLR8, and TLR9 were all increased in the pSilencer4.1/C2 groups compared to the controls (p-NC and PBS injected groups) (p<0.05). Figure 3d gray quantification analysis was shown as well. As well as PBS, the p-NC had no effect on the immune response of transgenic mice. Additionally, the expression of the TLR family was lower in the pSilencer5.1/C2 and pSilencer3.1/C2 groups compared to pSilencer4.1/C2 group (p<0.05; Figure 3e). It seems that the pSilencer5.1/C2 vector with highest inhibition on HBV could not induce a better immune response than pSilencer4.1/C2 did. The results obtained with pSilencer4.1/C2 were mainly presented here after initial comparisons the superiority of the vector over the others.
10.1371/journal.pone.0057525.g003Figure 3 Effect of specific siRNAs on the innate immune system of HBV transgenic mice.
The changes in cytokines and TLRs of HBV transgenic mice were measured by enzyme linked immunosorbent assay (ELISA) and western blot after 6, 10, 12, and 14 months treated with pSilencer4.1-C2 (p-4.1-C2) (a, b, c). The cytokines TNF-α, IFN-β, IL-2, IL-6, IL-12, and IL-15 were determined in the serum of transgenic mice by ELISA (a). And the transcription factors NF-κb and ERK were semi-quantitatively assessed in the serum of transgenic mice by ELISA (b). Western blots (c) showed the differences in TLR3, TLR8, and TLR9 between control and p-4.1-C2-treated groups. And the quantification of the results were displayed as well (d). The p-NC and PBS injected groups were set as controls. The expression of TLRs were higher in the p-4.1-C2 group to the other two groups at 12-month post-transfection (e). Error bars represent the standard error (SE) of septuple specimens. Results are representative of at least four separate experiments. n = 6–8 mice per group. p-NC, negative-control plasmid; PBS, phosphate-buffered saline; Mon, month. (*p<0.05).
Modification of DC Function and the Innate Immune System in siRNA-treated Mice after Repeated HBV Vaccine Injections
Figure S2a shows that serum levels of the cytokines IL-6, IL-12, and TNF-α in the siRNA-treated mice were higher prior to vaccine administration. Additionally, DCs were isolated at 3, 5, 7, and 9 months after the first injection, and the DCs were cultured for 6 days. The supernatant levels of IL-6, IL-12, and TNF-α were measured by ELISA on day 6. Consistent with the level of cytokines in the serum, the expression of the antiviral cytokines in the vaccine plus siRNAs treatment group were significantly elevated compared to the PBS- and siRNA-injected groups (Figure S2b). Furthermore, flow cytometry demonstrated that the CD80+ and CD86+ DCs from the liver of siRNA-treated transgenic mice markedly increased after vaccination (Figure 4a). Cultured DCs were also harvested on day 6, and the CD80+ and CD86+ DCs were sorted by flow cytometry. Figure 4b indicated that both the CD80+ and CD86+ DCs from the HBV vaccinated siRNA-treated mice were markedly upregulated after 6 days in culture. The changes of CD80+ and CD86+ DCs in transgenic mice vaccinated single control have no difference with that of PBS and p-NC control groups. To evaluate the effect of different vectors on the immune response, we assessed cytokines and DC functionality among the different vector groups. Despite that the pSilencer5.1/C2 could induce the highest silencing effect among the three types of vectors, it mediated less influence on the innate immune system than the pSilencer4.1/C2 (p<0.05; Figure 4c).
10.1371/journal.pone.0057525.g004Figure 4 Accommodation effect of the HBV vaccine on the innate immune system in siRNA-treated HBV transgenic mice.
The isolated and cultured dendritic cells (DCs) were stained with CD11b, CD86 and CD80 specific abs. CD11b gated liver DCs were examined for CD86 and CD80 expression respectively at different time points in siRNAs (pSilencer 4.1-C2) and siRNAs with vaccination group by flow cytometry (a). And cultured DCs were sorted to identify the changes of CD80 and CD86 on the sixth day (b). The transgenic mice vaccinated single group, negative-control plasmid (p-NC) and PBS injected mice are set as controls. Moreover, the changes in the CD80+ and CD86+ DCs between pSilencer 4.1-C2 and pSilencer 5.1-C2 treated groups were counted to evaluate the different effects of the two vector-driven siRNAs on the innate immune system (c). Results are representative of at least four separate experiments. Error bars represent the standard error (SE) of mean. (*: p<0.05, a: p>0.05).
The Permissive Immune Response of Transgenic Mice to the HBV Vaccine
The MEIA quantitation indicated that the titer of HBsAg-Ab was detectable at 3 months after two vaccine injections, while the titers were lower in the siRNA-treated group compared to normal Balb/c mice 5 months after HBV vaccination (p<0.05). Gratifyingly, the HBsAg-Ab titers in the siRNA-treated mice were almost the same as the normal vaccination Balb/c mice at 7 and 9 months after receiving three vaccinations (Figure 5a). We also assessed CD4+ and CD8+ T-cell counts in both the serum and liver samples by flow cytometry. Data from the flow cytometry analysis showed that the absolute value of both CD4+ and CD8+ T cells were markedly increased after vaccination in the siRNA-treated mice (Figure 5b). Additionally, the pSilencer5.1/C2 and pSilencer3.1/C2 groups could not produce the levels of HBsAg-Ab (Figure 5c), CD4+ (Figure 5d), and CD8+ T cells as the pSilencer4.1/C2 group. Conversely, the pSilencer4.1/C2 group was considered the best for these parameters compared to the other two siRNA vectors. The Figure S3 showed the therapeutic protocol of the vaccine and CpG ODNs.
10.1371/journal.pone.0057525.g005Figure 5 Immune tolerance rescue in the HBV transgenic mice after siRNA treated and vaccine.
The Abbott MEIA quantitation of HBsAg-Ab in mouse serum was carried out among the pSilencer4.1-C2 treated transgenic mice, PBS injected transgenic mice and normal Balb/c mice after HBV vaccination (a). The mononuclear cells were stained with CD3, CD4 and CD8 specific abs. CD3 gated cells were examined for CD4 and CD8 expression. The graph indicates the effect of pSilencer4.1-C2 and vaccination on the changes of CD4+ and CD8+ T cells in the liver samples of transgenic mice (b). The CD4+ T cells count (c) and HBsAg-Ab titers quantitation (d) were measured by flow cytometry and MEIA respectively, and figures showed their difference expressions among three kinds of siRNA vector treated transgenic mice after HBV vaccination. The symbol ‘a’ in the brackets indicate no differences between two groups. n = 6–8 mice per group. Balb/c mice, normal mice; PBS, phosphate-buffered saline; vac., vaccination. (*: p<0.05, a: p>0.05).
Immunologic Enhancement of CpG ODNs
As a TLR-9 agonist, CpG ODNs further strengthened the adaptive immune response to the HBV vaccine after the innate immunity had been reinforced by siRNAs. Fortunately, the level of HBsAg-Ab in the siRNA-treated mice was significantly higher in the vaccine and CpG ODNs co-injected group compared to the vaccine groups alone (Figure 6a). Flow cytometry showed that CD4+ and CD8+ T cells in the co-injection group were almost the same as those in normal Balb/c mice (Figure 6b). Moreover the HBsAg expression and HBV DNA copy were negative in the co-injection siRNA-treated transgenic mice. The TLR9-agonists could further increase the induction of HBsAg-Ab and the response of T cells.
10.1371/journal.pone.0057525.g006Figure 6 CpG ODNs enhance adaptive immunity after HBV vaccination.
HBsAg-Ab quantification was carried out using microparticle enzyme immunoassay (MEIA) at 5-, 7-, 9-month following the first vaccination. Compared to controls, the CpG ODNs raised the level of HBsAg-Ab in the serum of transgenic mice after three injections of HBV vaccine (a). The mononuclear cells were stained with CD3, CD4 and CD8 specific abs. CD3 gated cells were examined for CD4 and CD8 expression. The percentage of CD4+ and CD8+ T cells sorted by flow cytometry in the livers of CpG ODN vaccine co-injection group were identical to Balb/c mice with vaccine only (b). The symbol ‘a’ above the brackets indicate no differences between two groups. n = 8 mice per group. Balb/c mice, normal mice; Vac., vaccination; TM, transgenic mice; siRNAs, pSilencer4.1-C2. (*: p<0.05, a: p>0.05).
Discussion
Chronic HBV carriers is a big population in the world, for example there are more than 130 million people in China, which have high tendency to develop chronic hepatitis, cirrhosis, or HCC. But physicians have no appropriate strategies for them to clear HBV from the body. For instance, the hydrodynamic method to transfer antiviral sequences will not be applicable in people. Alternative gene transfer methods have been insufficiently robust for clinical applications. Use of integrating vectors, e.g., lentiviral or AAV-derived, could be suboptimal in the setting of chronic HBV with DNA integrants. Therefore, it should be appropriate to examine whether inhibition and clearance of HBV by combination of multiple treatments will be successful for chronic HBV infection. The pre-formed HBV cccDNA is extremely stable in the nucleus, which is required for HBV transcription, it is likely resilient to some types of antiviral attack, such as lamivudine. Thus, failure to completely eradicate HBV cccDNA by a particular therapy would fail to conquer HBV chronic infection. This was proven by a recent report, which showed that, although the U6S shRNA sequence significantly reduces HBV transcripts and inhibits HBV replicative intermediates and extracellular DNA in chronically infected cells, it does not affect HBV cccDNA levels [11]. Now that siRNAs are not unique sufficient to conquer the disease, it is extremely necessary to explore multiple treatments in combination with RNAi to thoroughly eliminate HBV.
In this study, we showed that the pSilencer5.1/C2 could more efficiently inhibit the expression and replication of the HBV subtype ayw in transgenic mice than the pSilencer3.1/C2 and pSilencer4.1/C2 vectors. This may be due to the ability of the pSilencer5.1 vector to stably integrate into the host genome. The pSilencer4.1 driven by Pol II promoters was better at effectively suppressing the expression and replication of the HBV for long periods in vivo than the pSilencer3.1 driven by the Pol III promoter, which corroborated our previous research [5]. Secondly, the inhibitory effects of siRNA on HBV gene expression and replication were sequence specific and dose-dependent to a certain extent, as increasing the siRNA vector dosage to 10 mg/kg did not obtain a more efficacious silencing effect, which might be due to the saturation of RISC binding [22]. Thirdly, we demonstrated an obvious strengthening of the innate immune system in the HBV transgenic mice after long-term treatment with HBV-specific siRNAs. Once the TLR family was upregulated, the inflammatory cascade, including transcription factors and antiviral cytokines, was propagated. Certainly, these processes will help mediate both the clearance of the viral infection and the functional recovery of the antigen presenting cells. Additionally, the siRNA-treated mice first received the HBV vaccine 4 months after siRNA administration, and the adaptive immune system parameters were significantly increased after three vaccinations, especially in the vaccine and CpG ODNs co-injected group, indicating that the type B CpG ODNs are effective adaptive immune stimulators for enhanced vaccine responses. Lastly, we found that the pSilencer4.1/C2 was the best for stimulating the immune system after long-term treatment instead of the pSilencer5.1/C2, which showed the strongest silencing effect on HBV expression and replication. This may be due to altered expression of siRNAs by the pSilencer5.1 vector, which would compete for RISC binding with endogenous miRNAs. In general, our research is the first to evaluate immune system effects after long-term treatment with siRNAs coupled to HBV vaccination. This work offers important insight for potential treatment paradigms for chronic HBV infection patients. The combination of the power of RNAi with the vaccine-mediated immunity has the potential to completely eradicate HBV infection.
At present, much research has shown that specific HBV siRNAs are powerful silencers of HBV in vitro and in vivo [4]–[7], [11], [23], but few have evaluated the long-term silencing effects [7] and the influence of siRNAs on the immune system in vivo. To evaluate the long-term stability of this new therapeutic method, it is important for the test systems to be consistent. Here, we selected HBV transgenic mice (HBV ayw) that continuously produce viral proteins, RNA, and DNA. Furthermore, the siRNAs used were expressed by viral vectors and plasmids, which were considered to be more stable for in vivo transfer. Importantly, our previous reports and those by others have proved the stability of these vectors [4]–[5], [7], [21]. To explore more efficient vectors, three types of vectors were used in the current study, and they all mediated efficient HBV silencing. Furthermore, the detection of specific siRNAs by northern blot demonstrated that the vector-based siRNAs were stably expressed in the mouse liver. Previous studies have demonstrated that the Pol II promoter can direct efficient shRNA synthesis and strong RNAi effects, and RNA Pol II was primarily responsible for mRNA transcription in vivo [24]. This was supported by this research and our previous report [4]–[5], [21], which indicated that the pSilencer4.1 vector driven by the Pol II promoter was a more potent suppressor than the pSilencer3.1 vector driven by Pol III in vivo. However, in this study, the retroviral vector pSilencer5.1 driven by Pol III showed a higher inhibition compared to pSilencer4.1 driven by Pol II promoter, which may be because the retroviral vector could integrate into the liver cell genome. Additionally, we and others have shown that 21-nucleotide siRNAs driven by Pol III, but not Pol II, could activate the IFN response pathway resulting in a non-specific silencing effect, which has been named an off-target effect [25]. In the current study, the immune system of the transgenic mice did include IFN up-regulation after long-term siRNA treatment, which may initially appear to contradict previous studies. However, both our previous study and those conducted by others indicated that the siRNA-mediated IFN response occurred during the early stage in vitro. While we did not measure increases in IFN at the early time points in vivo, in our opinion, the IFN up-regulation in vivo was caused by the clearance of HBV by siRNAs and the immune system reconstruction. As we know natural killer and T-cell responses are attenuated in patients at early stages after HBV infection, and HBV can counteract the antiviral responses of the liver’s innate immune system by antagonizing proinflammatory cytokines, including IFN, IL-2 and others [12], [14]–[15].
Nonetheless, it is very difficult to clear chronic HBV infections, particularly in patients with immune tolerance. Rebuilding the immune system presents another challenge for conquering the disease. Recent reports indicate that HBV could suppress the TLR-induced antiviral activity of liver cells [12]. To date, at least 13 members of TLRs (TLR1-TLR13) have been identified in mammals and all have been found in humans. Each TLR member recognizes distinct components of microbial pathogens and regulates the innate immune to limit invading microbes [26]. It has been shown that TLR-activated murine nonparenchymal liver cells can suppress HBV replication [27]. HBV can almost completely abrogate TLR-induced antiviral activity, which has been correlated with the suppression of IFN-β production and subsequent gene induction as well as suppressed activation of NF-κb, and ERK 1/2 [28]. Thus, we deduced that HBV clearance could modify the immune system. Our data demonstrates that the expression of TLR3, TLR8, and TLR9 were clearly upregulated in the mouse liver after receiving long-term siRNA treatment. Accordingly, the levels of cytokines, such as TNF-α, IFN-β, IL-2, IL-6, IL-12 and IL-15, which are associated with antiviral effects and T-cell activation and proliferation, were markedly increased in the transgenic mouse liver after long-term siRNA exposure. Based on these results, it appears that the clearance of HBV is useful for rebuilding the innate immune, but this will take a long period of time. This study also supports another hypothesis that postulates that HBV has developed strategies to suppress the initial antiviral response. Indeed, our results suggest that the global CD8+ T-cell population in HBV patients may be skewed toward IFN-γ/TNF-α production and are impaired in their ability to produce IL-2 and proliferate due to the chronic HBV infection.
DCs play a vital role in the initiation of innate and adaptive immune responses, and their role as immune mediators in cancer and infection have been studied extensively. More recently, several lines of evidence have emerged supporting the importance of DCs in the induction and maintenance of tolerance [29]. Furthermore, cross-presentation by human DCs requires activation via TLRs, which is in contrast to mouse CD80+/CD86+ DCs. The magnitude of antigen presentation in human DCs assays is determined by measuring the number of IFN-γ producing cells elicited by the DCs. Thus, factors other than the efficiency of generation of MHC I-peptide complexes may have influenced the outcome [30]. Additionally, DCs can sense the extracellular environment and modulate cellular responses, especially the TLR-dependent inflammatory response. Accordingly, we observed that the DCs’ functionality in the HBV transgenic mice rose after long-term treatment with siRNAs as well as mice treated with siRNAs and vaccination. On the other hand, the adaptive immune responses were improved by the up-regulation of CD4+ and CD8+ T cells and detectable levels of HBsAg-Ab. Furthermore, differences in the CD4+/CD8+ ratio between the serum and liver samples may be due to the antiviral immunity and clearance of HBV. Moreover, the adaptive immunity in the siRNA-treated transgenic mice was further enhanced by co-injection of vaccine with CpG ODNs. As the TLR-induced innate immunity of the transgenic mice was strengthened by long-term treatment of siRNAs, the agonist type B CpG ODNs bound to TLR-9 and strongly activated B cells [16]. Thus, modulation of the HBV vaccine response with immunopotentiators may be useful for increasing the rate of response. In a word, the up-regulation of TLRs, markers for antigen presentation of DCs and T lymphocytes by the long-term siRNAs treatment and HBV vaccination would be contribute to production of antiviral cytokines. The pSilencer5.1/C2 with the greatest silencing ability could not induce the remodification of the immune system as well as the pSilencer4.1/C2. The present data showed that appropriate vectors and a specific siRNA concentrations could modulate the immune response. Importantly, excessive exogenous siRNAs will compete with endogenous miRNAs for the RISC. Emerging evidence suggests that the host endogenous miRNAs and host RNA-silencing machinery are considered to be a fundamental layer of host immune response control. Furthermore, specific siRNAs and miRNAs may contribute to the adaptive immune response, especially in the effector phases, including the differentiation into functional T-cell lineages and the activation of antigen-presentation cells through pattern-recognition pathways [31].
Taken together, our results show that the vector-delivered HBV-specific siRNAs offer a powerful therapy for chronic HBV infection in transgenic mice. After long-term treatment with siRNAs, the innate and adaptive immune responses were both strengthened, and it appeared that the functional siRNAs could break the immune tolerance status against HBV after HBV clearance. Looking forward, HBV-specific siRNAs coupled to the immune-modified HBV vaccine could represent a sharp double-edged sword for ameliorating chronic HBV infection. The data in the current study provides a foundational theory for how immune-modified HBV vaccination may efficacious in the persistent HBV infection patient after the initial HBV clearance with siRNAs. For clinical application of siRNA treatment, many problems must be solved, such as siRNA delivery, safety evaluation for introduction of exogenous DNA to humans, and the immune response to the viral vector, among others. Further exploration will be required to assess these issues; however, our study provides important evidence supporting the treatment of chronic HBV infections by combination of RNAi with CpG-enhanced vaccination. Further study will be required to evaluate the relationship of the siRNAs with the immune system.
Supporting Information
Figure S1
Design and construction of siRNA vectors. The two effective targets of the HBV C and S open reading frames are indicated by arrows (a). The synthesized complementary 55-mer siRNA template oligonucleotides targeting HBV-C2 were inserted into the pSilencer5.1-H1 retro vector (b). The siRNA template oligonucleotides were confirmed by sequencing. The siRNA template oligonucleotides encode a hairpin siRNA, which will be cut by Dicer for a 21nt siRNAs (c).
(TIF)
Click here for additional data file.
Figure S2
Detection cytokines. Mouse serum cytokine levels for IL-6, IL-12, and TNF-α were quantified by ELISA at 3, 5, 7, and 9 months after the first injection (a). Moreover, levels for antiviral cytokines IL-6, IL-12, and TNF-α in the culture supernatants of DCs obtained from liver of mouse at 3, 5, 7, and 9 months after the first injection were both measured by ELISA on culture day 6 (b). n = 6–8 mice per group. PBS, phosphate-buffered saline; vac., vaccination.
(TIF)
Click here for additional data file.
Figure S3
The therapeutic protocol of the vaccine and CpG ODNs. The therapeutic protocol of the vaccine and CpG ODNs. The schematic represent the therapeutic protocol of the HBV vaccine and CpG ODNs. n = 8 mice per group. Balb/c mice, normal mice; TM, transgenic mice.
(TIF)
Click here for additional data file.
We thank the staff at the State Key Laboratory of Trauma and Tissue Repair, the Biochemistry Institute of the Technology University of South China and the Department of Medical Laboratory of the General Hospital of Guangzhou Military Command. We appreciate Prof. Lin Xu for the helpful discussion and technical assistance in this study.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23554869PONE-D-12-1904910.1371/journal.pone.0057801Research ArticleBiologyBiochemistryNeurochemistryNeurochemicalsNitric OxideMarine BiologyMicrobiologyImmunityInflammationModel OrganismsAnimal ModelsMouseImmunologyImmunityInflammationImmune CellsImmune ResponseImmunomodulationNeuroscienceNeurochemistryNeurochemicalsNitric OxidePerthamide C Inhibits eNOS and iNOS Expression and Has Immunomodulating Activity In Vivo Perthamide C Immunomodulating ActivityBucci Mariarosaria
1
Cantalupo Anna
1
Vellecco Valentina
1
Panza Elisabetta
1
Monti Maria Chiara
2
Zampella Angela
3
Ianaro Angela
1
*
Cirino Giuseppe
1
1
Department of Experimental Pharmacology, University of Naples Federico II, Naples, Italy
2
Department of Biomedical and Pharmaceutical Sciences, University of Salerno, Fisciano (SA), Italy
3
Department of Natural Products Chemistry University of Naples Federico II, Naples, Italy
Bonini Marcelo G. Editor
University of Illinois at Chicago, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: MRB AZ AI. Performed the experiments: AC VV EP MCM. Analyzed the data: MRB AZ AI. Contributed reagents/materials/analysis tools: AZ GC. Wrote the paper: AI GC.
2013 12 3 2013 8 3 e578013 7 2012 29 1 2013 © 2013 Bucci et al2013Bucci et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H.
In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.
The research was supported by the Italian Ministry of Scientific Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Within marine invertebrates, sponges have developed a highly complex immune system associated to the capacity to produce efficiently secondary metabolites as a defence mechanism. Moreover, sponges have provided a number of compounds exhibiting anti-inflammatory activity [1]. The cellular targets of these drugs are often different from those of non-steroidal anti-inflammatory drugs (mainly cyclooxygenase-1 or -2 inhibition) and in some cases relate to the inhibition of inflammatory gene transcription.
In our studies on bioactive compounds from sponges collected at Solomon Islands, [2] we found a single specimen of the sponge Theonella swinhoei, (Order Lithistida, Class Demospongia), as an extraordinary source of new metabolites. Analysis of the polar extracts afforded anti-inflammatory peptides such as the large library of perthamide C derivatives [3]–[6] and solomonamides A–B, [7] and anti-inflammatory sulfated sterols, solomonsterols A and B [8], potent agonists of the human nuclear receptor and xenobiotic sensor, pregnane-X-receptor (PXR) and new leads in the treatment of immune-driven inflammatory bowel diseases [9].
From a structural point of view, perthamide C has a peculiar unprecedented primary structure that comprises a 25-membered macrocycle with 6 out of the 8 residues constituted by unusual amino acids: γ-methylproline, N
δ-carbamoyl-β-OSO3Asparagine, o-tyrosine, d-ABU, O-methylthreonine, and the β-amino acid AHMHA (3-amino-2-hydroxy-6-methylheptanoic acid). Acute inflammatory response is characterized by an increase in vascular permeability and cellular infiltration leading to oedema formation as a result of extravasation of fluid and proteins, and accumulation of leukocytes at the inflammatory site. Following these changes, many other mechanisms are activated, contributing to the amplification of the inflammatory response and tissue damage, leading to the development of a more complex ‘scenario’ e.g. the chronic inflammatory reaction. Recently we have shown that perthamide C, among the different derivatives screened, significantly inhibited the inflammatory reaction in vivo [3]. Since perthamide C given systemically resulted active at very low dose e.g 0.3 mg/kg ip we have investigated in details the mechanism of this powerful anti-inflammatory activity.
Materials and Methods
Induction of edema in mouse paw
Male Swiss (CD-1; Harlan, Italy) weighing >30 g were divided into groups (n = 6 each group) and lightly anaesthetized with isoflurane. Each group of animals received subplantar injection of 50 µl of λ-carrageenan 1% (w/v) or 50 µl of saline in the left hind paw. Paw volume was measured by using an hydropletismometer specially modified for small volumes (Ugo Basile, Comerio, Italy) immediately before the subplantar injection and 2, 4, 6, 24, 48, 72 and 96 h thereafter. The same operator always performed the double-blind assessment of paw volume. The increase in paw volume was calculated as the difference between the paw volume measured at each time point and the basal paw edema. Each group of animals received 100 µl of perthamide C (0.3 mg/kg), or vehicle (polyethilenglycole, PEG) given i.p immediately before the injection of λ-carrageenan and 24 h thereafter. In a separate set of experiments dexamethasone (4 mg/kg) or vehicle (saline) were administered i.p.1 h prior λ-carrageenan injection and 24 h thereafter.
All procedures were performed according to Italian ministerial authorization (DL 116/92) and European regulations on the protection of animals used for experimental and other scientific purposes. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Italian Ministry of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Naples Federico II (Protocol Number: 2009/0047185). The paw edema was performed under enflurane anesthesia, and all efforts were made to minimize suffering.
Myeloperoxidase (MPO) measurement in injected paws
Mice from different groups were euthanized with CO2 at different time points from λ-carrageenan or vehicle injection. Paws were cut, weighed and homogenized in 1 ml of hexadecyltrimethylammonium bromide (HTAB) buffer on ice by using a Polytron homogenizer (two cycles of 10 sec at maximum speed). After centrifugation at 10,000 rpm for 2 min, supernatant fractions were assayed for MPO activity as an estimate of cellular migration according to the method described in Posadas et al., 2004. Briefly, samples (20 µl) were mixed with phosphate buffer (180 µl) containing 1 mM O-dianisidine dihydrochloride and 0.001% hydrogen peroxide in a microtiter plate. Absorbance was measured at 450 nm, performing three readings at 30-s intervals. Calculation of MPO units was evaluated considering that 1 U MPO = 1 µmol H2O2 split and 1 µmol H2O2 gives a change in absorbance of 1.13×10−2 (change in absorbance = nm min).
Western blot analysis
Paws were harvested from different groups of mice at different time points after λ-carrageenan or vehicle injection, and homogenized in modified RIPA buffer (Tris HCl 50 mM, pH 7.4, triton 1%, Na-deoxycholate 0.25%, NaCl 150 mM, EDTA 1 mM, phenylmethanesulphonylfluoride 1 mM, aprotinin 10 µg/ml, leupeptin 20 mM, NaF 50 mM) using a polytron homogenizer (two cycles of 10 sec at maximum speed) on ice. After centrifugation at 12,000 rpm for 15 min, protein concentration was determined by Bradford assay using BSA as standard (Bio-Rad Laboratories, Milan, Italy). 40 µg of the denatured proteins were separated on 10% SDS/PAGE and transferred to a PVDF membrane. Membranes were blocked in PBS-tween 20 (0.1%, v/v) containing 3% non fat dry milk for 1 hour at room temperature, and then incubated with anti-COX1 (1∶1000), anti COX-2 (1∶1000), anti-eNOS (1∶1000) or anti-iNOS (1∶500) overnight at 4°C. The filters were washed with PBS-tween 20 (0.1%, v/v) extensively for 30 min, before incubation, for 2 hours at 4°C, with the secondary antibody (1∶5000) conjugated with horseradish peroxidase antimouse IgG. The membranes were then washed and immunoreactive bands were visualized using an Enhanced Chemiluminescence Substrate (ECL; Amersham Pharmacia Biotech, San Diego, CA, USA).
Preparation of cell suspension
Cell suspension was obtained as previously described [10]. Briefly, peripheral lymph nodes (PLN) (axillary, brachial, inguinal, cervical and mesenteric) were harvested from different groups of animals and at different time points from λ-carrageenan injection. Cells were pooled and prepared as single cell suspensions, by passing through a Nitex sieve (Cadisch Precision Meshes, London,UK) using a syringe plunger, and washed in sterile RPMI-1640 (Invitrogen Life Technologies, Paisley, UK). Monocytes were obtained by separation with Histopaque 10771 (Sigma, Milan, Italy). The cell suspensions obtained were used for the evaluation of the proliferative response to Concanavalin A (Con A).
Proliferation assay
The MTT colorimetric assay, based on the ability of viable cells to reduce the yellow MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) salt to the blue MTT formazan product, was used to assess lymphocytes proliferation in vitro. Preliminary experiments determined that lymphocytes number and absorbance (570 nm) formed a linear relationship thereby providing a reliable assay for measurement of cell proliferation. PLN lymphocytes obtained from mice of each study group, were dispensed into 96-well plates at a density of 2×106 cells per well in RPMI media containing streptomycin/penicillin (1%), sodium pyruvate (0.1%), 2-mercaptoethanol (0.1%) and foetal calf serum (10%) in a final volume of 150 µl and kept in an incubator at 37°C and in an atmosphere of, 5% CO2+95% air., To induce T cells proliferative responses cells were pulsed with Con A (5 µg/ml) for 72 h. In the last 4 h of incubation, 25 µl MTT reagent was added to each well. This step was followed by the addition of detergent reagent (100 ml per well) for 2 h to enable complete solubilisation of formazan product (plates kept in the dark). The absorbance of the samples was then measured using a microplate reader at 570 nm. Each treatment/assay was based on n = 6 replicate samples.
In a separate set of experiment we tested the biological activity of perthamide C on lymphocytes obtained from mice of the näive group. In this set of experiments, cells were pulsed for 72 h with Con A (5 µg/ml) in presence or absence of different concentrations (3-10-30 µM) of perthamide C.
Sample preparation for mass spectrometry-based detection of perthamide C and H in mouse plasma
Plasma samples were collected from mice of each group at different time points after perthamide C administration. The clean-up procedure of plasma was performed by solid-phase extraction as previously described [11]. The Waters Sep-Pak Vac tC18 cartridges (50 mg) were initially washed with 2 ml ACN and then pre-conditioned with 5 ml of H2O containing 0.05%TFA and 2% HCOOH. 100 µl of ice plasma were put at 37 °C for 15 min and then diluted at 2 ml with H2O. Each sample was then applied onto the cartridge, the cartridge was washed with 2 ml of H2O containing 0.05% TFA and 2% HCOOH and the analytes were eluted using 100% ACN containing 0.05% TFA and 2% HCOOH. The eluted substances perthamides C and H were dried and the residues were dissolved in 100 µl of 100% ACN containing 0.05% TFA and 2% HCOOH. This procedure has been repeated 3 times for each of mice plasma at 0, 1, 4, 16 and 24 h, after each perthamide C administration.
Finally, in order to measure the recovery rate of extraction, the same procedure has also been carried out on blank samples spiked with both perthamides C and H and at different concentrations for 3 times; the recovery rate of the procedure is around 90%.
RP-HPLC-MS/MS method for detection of perthamide C (1) and H (2) in mouse plasma
25 µl of the extracts were injected into the HPLC–MS system LTQ XL ThermoScientific equipped with Accelera 600 Pump and Accelera AutoSampler system. The mixture was separated on a Jupiter 5 µ C18 column from Phenomenex (150×2.00 mm) using a mobile phase consisted on H2O-0.05% TFA-2% HCOOH (eluent A) and ACN-0.05% TFA-2% HCOOH (eluent B). The eluents were linearly changed from 10% B to 70% B within 14 min; the columns flow rate was set at 200 µl/min. The mass-spectrometer was set to operate with an ESI ionization source in positive and negative mode. The capillary temperature was kept at 320 °C, the sheath gas flow rate and the auxiliary flow rate were set at 10 and 5, respectively. Instrument optimization was performed by direct infusion and manual tuning. Detection of perthamides C and H was performed using the selected reaction-monitoring mode (SRM). The transition of the deprotonated perthamide C molecule to its corresponding product ions was recorded at m/z 1066.5→1023.5 (-CONH) and m/z 1068.5→1025.5 (-CONH) and the transition of the protonated perthamide H molecule to its corresponding product ions was recorded at m/z 988.5→971.5 (-NH3). The collision energy used was set at 35.
As standard for calculating the relative percentage of these metabolites, several runs of samples containing perthamide C alone, perthamide H alone or the same concentration of perthamides C and H upon clean up procedure were also performed.
Sponge material and isolation of perthamides C and H
Theonella swinhoei (order Lithistida, family Theonellidae) was collected on the barrier reef of Vangunu Island, Solomon Islands, in July 2004. Authorization to collection and exportation of sponge samples was released by Fisheries Department of Solomon Islands Government to IRD (Institut de Recherche pour le Dèveloppement, Polynesian Research Center on Island Biodiversity, BP529, 98713 Papeete, Tahiti, French Polynesia) in the frame of a research project entitled: ‘Coral Reef Initiative in The South Pacific’ (CRISP) ‘Biodiversité et substances marines actives’, volet Molécules actives, soutenu par l′Agence Française pour le Développement’, authorization IRD–AFDCZZ3012-02U. The sponge lyophilized material was kindly provided by Dr. Cecile Debitus, IRD. The sponge is not on the endangered and protected species list (CITES list, www.cites.org) and no specific permits were required for the described field studies. Taxonomic identification was performed by Dr John Hooper of Queensland Museum, Brisbane, Australia, where specimens are deposited under the accession number G3122662. Identity of perthamide C and H was established by comparison of their RM and mass data with those previously reported [6].
Evaluation of ulcerogenic effect of perthamide C
Mice were randomly divided in three groups (n = 3 for each group) and deprived of food, but allowed to drink water at libitum, for 16 hours. Administration of perthamide C (0.3 mg/kg i.p.) indomethacin (20 mg/kg per os, 100 µl), or vehicle (PEG) were then performed. Indomethacin was used as positive control. Three hours after the drugs administration, mice were euthanized by an overdose of anesthetic and stomachs were harvested and washed in saline. A longitudinal incision along the greater curvature was performed and the stomachs were inverted over the index finger. Lesions were blindly evaluated macroscopically with an arbitrary score as follows:
0 = healthy; 1 = hyperemic
2 = lesions without blood
3 lesions with blood
Drugs and Reagents
Bradford reagent was obtained from Bio-Rad (Bio-Rad laboratories, Segrate, Milan, Italy). The antibodies against COX-2, eNOS and iNOS were purchased from BD Transduction laboratories (U.S.A.). The antibody against COX-1 was from Santa Cruz Biotechnology, Inc (Milan, Italy). All other reagents and compound used were obtained from Sigma-Aldrich (Milan, Italy).
Statistical analysis
Results were expressed as mean±s.e.m. The level of statistical significance was determined by one way ANOVA followed by Dunnett's test for multiple comparisons or t-test analysis where appropriate, using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Differences were considered statistically significant when p was less than 0.05. Each sample was processed at least in triplicate.
Results
Perthamide C inhibits MPO activity in carragenan-induced mouse paw edema
We have previously shown, that systemic administration of perthamide C significantly reduced both the early (0–6 h) and the late phase (24–96 h) of λ-carrageenan-induced paw edema displaying a dose-dependent (0.1, 0.3 and 1 mg/kg, i.p.) anti-inflammatory activity that reached almost 60% of oedema inhibition at the dose of 0.3 mg/kg [6].
To clarify the mechanism/s of action of perthamide C we firstly evaluated the MPO activity, as an index of neutrophil infiltration in response to λ-carrageenan injection. As shown in Figure1 A and B, λ-carrageenan injection induced a significative increase in neutrophil infiltration into the mouse paw. Administration of perthamide C reduced MPO activity as compared to vehicle group and this effect resulted statistically significant at 6 and 24 h after λ-carrageenan injection, when MPO activity reaches its highest levels (Figure1 A and B) [12].
10.1371/journal.pone.0057801.g001Figure 1 Perthamide C reduces MPO activity in both phases of oedema.
Paws were harvested at different time points 2-4-6 h (A) and 24-48-72-96 h (B) from each group of animals and processed in order to measure MPO activity. Values are expressed as mean±s.e.m. (n = 4–6 paws for each time point). *P<0.05 **P<0.01, ***P<0.001 vs S (saline), ° P<0.05, °° P<0.01 vs vehicle.
Perthamide C reduces the expression of NOS but not COX isoforms
Next we evaluated if perthamide C administration was able to modify the expression levels of the constitutive enzymes eNOS and COX-1 as well as of their respective inducible isoforms, iNOS and COX-2. Thus, we performed western blot analysis on paw homogenates obtained from each groups of animals. As shown in figure 2 (panels A, B, C), during the early phase of edema both eNOS and COX-1 were expressed in total extracts obtained from saline- and λ-carrageenan-injected paw homogenates (Figure 2A and B). As expected, their inducible isoforms, iNOS and COX-2, were not detectable in this timeframe (data not shown). Systemic administration of perthamide C significantly reduced eNOS expression after 2 h and 4 h from λ-carrageenan injection (Figure 2A and B), while COX-1 expression was not modified by perthamide C treatment all throughout the early phase of edema (Figure 2A and C).
10.1371/journal.pone.0057801.g002Figure 2 Western blot analysis of eNOS and COX-1 in the early phase of the paw edema.
Expression of eNOS and COX-1 (A) were evaluated in total tissue extracts from paw homogenates obtained from mice treated with perthamide C, vehicle or saline at 2, 4 and 6 h following λ-carragenan injection (n = 4 paws for each treatment and time point). In panel B and C are shown the relative densitometric analysis °P<0.05, °°P<0.01 vs vehicle. Image is representative of three separate experiments.
We observed a different ‘scenario’ in the late phase of oedema following administration of perthamide C. In fact, as shown in figure 3 (panels A, B), systemic administration of perthamide C significantly reduced the expression of both NOS isoforms while it did not inhibit either COX-1 or COX-2 (Figure 4A and B).
10.1371/journal.pone.0057801.g003Figure 3 Western blot analysis of eNOS and iNOS in the late phase of the paw edema.
Expression of eNOS (A) and iNOS (B) were evaluated in paws homogenates obtained from mice treated with perthamide C, vehicle or saline at 24, 48, 72, 96 h following λ-carragenan injection. (n = 4 paws for each treatment and time point)**P<0.01vs saline, ° P<0.05, °° P<0.01, °°° P<0.001 vs vehicle. Image is representative of three separate experiments.
10.1371/journal.pone.0057801.g004Figure 4 Western blot analysis of COX-1 and COX-2 in the late phase of the paw edema.
Expression of COX-1 (A) and COX-2 (B) were evaluated in paws homogenates obtained from mice treated with perthamide C, vehicle or saline at 24, 48, 72, 96 h following λ-carrageenan injection. (n = 4 paws for each treatment and time point) *P<0.05 **P<0.01 vs saline. Image is representative of three separate experiments.
Perthamide C inhibits Con-A induced cellular proliferation
Injection of λ-carrageenan in the hind paw causes activation and proliferation of T cells in the draining lymph nodes. Proliferation assays showed that lymphocytes harvested from control mice (mice receiving only λ-carrageenan) responded to Con A stimulation as compared to cells obtained from vehicle group (Figure 5). Treatment of mice in vivo with perthamide C (0.3 mg/Kg/i.p.) significantly suppressed the proliferative response to Con A of lymphocytes at all time points considered (4-48-96 hrs) as compared to the control group (Figure 5). Dexamethasone (4 mg/kg) was used as reference drug (Figure 5).
10.1371/journal.pone.0057801.g005Figure 5 Proliferation assays on lymphocytes.
Lymphocytes were obtained from PLN of control (vehicle) or perthamide C treated mice 4 h, 8 h or 96 h after λ-carrageenan edema induction. Values are means±SEM of three separate experiments with n = 5–6 mice. ***P<0.001 vs Naive+Con A; °°°P<0.001 vs CTL+Con A.
In a separate set of experiment we tested the biological activity of perthamide C in vitro on lymphocytes obtained from näive mice. Cells were pulsed for 72 h with Con A (5 µg/ml) in presence or absence of different concentrations (3-10-30 µM) of perthamide C (data not shown). Surprisingly, in contrast with the ex-vivo experiments, perthamide C added to the cells in vitro, did not modify Con A-induced cellular proliferation at any of the concentrations tested. Thus we hypothesized that the molecule we were using in vitro was probably subjected, in vivo, to a metabolic activation, most likely at the sulfate group on the N
δ-carbamoyl-β-OSO3Asparagine unit, thus acting as a pro-drug. To prove this hypothesis, we decided to test the effect of the desulfated perthamide C derivative, perthamide H, recently isolated by our research group from a deep re-investigation of the polar extracts of Theonella swinhoei
[6]. As we hypothesized, the elimination of the sulfate group in perthamide C led to the active form of the molecule that was able to inhibit also in vitro and in a concentration-dependent manner, the Con A-induced proliferation of lymphocytes from näive mice (Figure 6).
10.1371/journal.pone.0057801.g006Figure 6 Proliferation assays on lymphocytes harvested from näive mice.
Perthamide H significantly inhibited, in a dose-dependent manner, Con A-induced proliferation of lymphocytes. Values are means±SEM of three separate experiments with n = 5–6 mice. ***P<0.001 vs basal (Bas);°°°P<0.001 vs Con A.
Perthamide C is converted in vivo in its desulfated derivative, perthamide H
In order to measure the relative levels of perthamide C and H in mice plasma upon different time from each perthamide C administration, we resorted to a selective quantification of these metabolites by RP-HPLC-MSMS analysis. Plasma samples of three different mice collected at 1, 4 and 16 and 24 h from each perthamide C administration have been pre-cleaned by solid-phase extraction and the fractions eluted at 100% ACN have been submitted to RP-HPLC-MSMS runs operating in selected reaction monitoring (SRM) mode. Perthamide C shows two chromatographic peaks respectively at 12.25 min (MH− at 1066.5) and at 22.50 min (MH+ at 1068.5); this behaviour was probably due to the charge state of N
δ-carbamoyl-β-OSO3Asparagine residue (Figure 7A and B). Perthamide H retention time is 12.33 min and its MH+ is 988.50 as expected (Figure 7A and B). The area of these three peaks has been integrated in each run and the results has been reported as relative percentage of the two metabolites (Figure 7C). At a starting point, 100% of perthamide C has been arbitrary set.
10.1371/journal.pone.0057801.g007Figure 7 Chemical structure and LCMS runs of perthamide C and H.
Panel A shows the chemical structure of perthamide C and H. Panel B shows one of the LCMS runs for detecting perthamides C and H in mice plasma samples using the selected reaction-monitoring mode (SRM). The peak at rt of 12.25 min corresponds to deprotonated perthamide C, the peak at rt of 12.33 min corresponds to protonated perthamide H, the peak at rt of 22.62 min corresponds to protonated perthamide C. Panel C shows the % of pertamides C and H detected by LCMS in mice plasma samples after 0, 1, 4, 16 and 24 h from each perthamide C administration.
It's noteworthy that, upon 1 h from each perthamide C administration, around 35% of this pro-drug has been desulfated giving rise to its active derivative perthamide H whereas, after 4, 16 and 24 h from each administration, decreasing levels of perthamide C and perthamide H have been detected.
Perthamide C does not exert ulcerogenic action
In order to verify if perthamide C treatment could induce gastric lesions, a standard animal model for ulcerogenic activity was performed. As shown in Figure 8, perthamide C, at the dose that exerts anti-inflammatory action, did not induce any damage to gastric mucosa. Indeed, the score obtained from perthamide C treatment group (Figure 8B) was comparable to one obtained from vehicle group (Figure 8A). Conversely, administration of NSAID drug indomethacin, used as positive control, induced a massive ulcerogenic action (Figure 8C), as highlighted by the high score reached.
10.1371/journal.pone.0057801.g008Figure 8 Perthamide C does not possess ulcerogenic action.
Blindly arbitrary score was assessed as follows: 0 = healthy; 1 = hyperemic; 2 = lesions without blood; 3 lesions with blood; n = 3 for each group indomethacin was used as a control ulcerogenic drug. Pictures are representative of three different animals for each group of treatment.
Discussion
In the present study we have investigated, in details, the molecular mechanism of the anti-inflammatory activity exhibited by perthamide C [3]. We have used to address this issue the λ-carrageenan mouse paw edema since due to its, sub-chronic nature it has allowed us to dissect the early event triggered within few hours from those that are deployed later within 3–4 days. In fact, the λ-carrageenan induced mouse paw edema as opposite to rats, produces a biphasic edema. In the first phase, which developed up to 6 h, edema is of low intensity, while in the second phase, after 24 h, edema is more pronounced and peaks at 72 h after λ-carrageenan injection [10], [12], [13]. Another important feature of this model is that, as the inflammation progresses, an increasing number of T cells infiltrate the draining lymph nodes of the λ-carrageenan-injected paws [10] thereby this model is also reminiscent of a typical type III immune reaction. In this reaction regional lymph nodes are the source of effector T cells that are postulated to extravasate at the site of antigen challenge and in turn, recruit and activate a variety of nonspecific inflammatory cells [14]. Perthamide C significantly inhibited the myeloperoxidase activity, a marker of neutrophil infiltration in tissue, in the early phase of the inflammatory reaction. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) while COX-1, iNOS and COX-2 expression were unaffected. Thus, perthamide C inhibitory effect in vivo is fast on onset and its action is possibly linked to modulation of eNOS-derived NO. This feature well fit with the finding that, in this model, the increase in vascular permeability in the early phase is dependent upon eNOS-derived NO [15]. Most likely the modulation of perthamide C operated on eNOS is linked to its ability to bind hsp90 [16]. Indeed, hsp90 is instrumental for eNOS activation as elegantly demonstrated in the paper by García-Cardeña et al. [17].
Perthamide C reduced up to 96 h the expression of both NOS isoforms i.e. eNOS and iNOS without affecting COXs expression i.e. COX-1 and COX-2. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation that is typical of the second phase of this animal model. Indeed, it is widely accepted that NO plays an important role in immunomodulation and neutrophil trafficking [18], [19]. In order to be consistent, we approached this issue by studying the proliferation of PLN lymphocytes harvested from mice receiving in vivo perthamide C. Lymphocytes challenged in vitro with Con A displayed a reduced proliferation demonstrating that the effect of perthamide C in vivo involves also these cellular population. This result suggested a potential immuno-suppressor activity for perthamide C.
In order to further investigate this effect a study was carried out in vitro on lymphocytes recovered from naïve mice. Surprisingly, in contrast with the ex-vivo experiments, perthamide C added in vitro to lymphocytes harvested from naïve mice, did not modify Con A-induced cellular proliferation at any of the concentrations tested. Therefore, we hypothesized that perthamide C was most likely subjected, in vivo, to a metabolic activation, probably at the sulfate group on the N
δ-carbamoyl-β-OSO3Asparagine unit, thus acting as a pro-drug. Our assumption was confirmed by the selective quantification of both metabolites by RP-HPLC-MSMS analysis carried out on plasma samples collected at different time points following each perthamide C administration in vivo to mice. This hypothesis was confirmed by the finding that desulfated perthamide C, namely perthamide H, did inhibit, in a concentration-dependent manner, the Con A-induced proliferation of lymphocytes harvested from naïve mice as opposite to perthamide C. The finding that perthamide C induces a state of immune-suppression acting in vivo only after metabolic activation represents an important outcome of our data since it give us a first insight into a structure/activity relationship.
The marine environment has proven to be a very rich source of extremely potent compounds that have demonstrated significant activities in anti-tumor, anti-inflammatory, analgesia, immuno-modulation, allergy and anti-viral assays. There are now significant numbers of very interesting molecules that have come from marine sources, or have been synthesized as a result of knowledge gained from a prototypical compound, that are either in or approaching Phase III clinical trials in cancer, analgesia and allergy. In conclusion we have defined an immunomodulatory activity of perthamide C in vivo and clarified that the action is due to its metabolite Perthamide H. Therefore this cyclic peptide could represent a new leading structure to develop therapeutics.
We thank Professor J.L. Wallace for his valuable and constructive suggestions for the evaluation of the ulcerogenic effect of perthamide C.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23554911PONE-D-12-3337010.1371/journal.pone.0058647Research ArticleBiologyAnatomy and PhysiologyPhysiological ProcessesHomeostasisImmune PhysiologyBiochemistryProteinsImmune System ProteinsImmunologyImmune ResponseMolecular Cell BiologyCellular TypesEpithelial CellsMedicineAnatomy and PhysiologyImmune PhysiologyImmune CellsPhysiological ProcessesHomeostasisGastroenterology and HepatologySurgeryUp-Regulation of Intestinal Epithelial Cell Derived IL-7 Expression by Keratinocyte Growth Factor through STAT1/IRF-1, IRF-2 Pathway IL-7 Expression of Intestinal Epithelial CellsCai Yu-Jiao
1
Wang Wen-Sheng
1
Yang Yang
1
Sun Li-Hua
1
Teitelbaum Daniel H.
2
Yang Hua
1
2
*
1
Department of General Surgery, Xinqiao Hospital, Third Military Medical University, Chongqing, China
2
Department of Surgery, The University of Michigan Medical School, Ann Arbor, Michigan, United States of America
Singh Shree Ram Editor
National Cancer Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: HY. Performed the experiments: YJC WSW. Analyzed the data: YJC YY. Contributed reagents/materials/analysis tools: LHS. Wrote the paper: YJC DHT.
2013 12 3 2013 8 3 e5864724 10 2012 5 2 2013 © 2013 Cai et al2013Cai et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine.
Methods
Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA).
Results
KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively.
Conclusion
KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.
This study was supported by the National Natural Science Foundation of China (No. 30973113 to H.Y.; No. 81020108023 to H.Y.; No. 81000830 to Y.J.C.), and the Chongqing Science and Technology Commission International Key Collaboration Project (CSTC 201110008 to H.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Intestinal epithelial cells (IECs) function as active participants in local immune regulation via secreting a variety of cytokines. Among these, interleukin-7 (IL-7) is particularly important in terms of its pleiotropic function in the intestinal immune system [1]. In the intestine, IL-7 is produced by IECs, and in turn IL-7 receptors (IL-7R) have been detected on intraepithelial lymphocytes (IELs) [2]. Studies have demonstrated that IEC-derived IL-7 stimulates the proliferation of lamina propria lymphocytes and IELs [3], [4] and also enhances cytokine release from these lymphocytes in humans [5]. In addition, IL-7 is essential for early developmental processes such as the differentiation of pre-T cells into mature thymocytes. This latter function cannot be performed by any other known cytokines [6]. In the absence of IL-7, homeostatic proliferation of naive T-cells is almost completely abolished, and the lifespan of naive T cells is greatly reduced [7]. In vivo, our group found administration of IL-7 has been demonstrated to enhance IEL functional capacity and population [8]. Geiselhart et al. [9] reported that IL-7 administration altered the peripheral T cell CD4-to-CD8 ratio and resulted in an increase in peripheral T cell numbers and altered function. Watanabe et al. [4] observed that exogenous IL-7 administered to mice resulted in a stimulation of lamina propria lymphocytes. All these data suggest that IL-7 may be essential for ongoing maintenance of IEL function and growth.
Keratinocyte growth factor (KGF) is produced exclusively by mesenchymal cells and IELs, and acts on epithelial cells [10], [11], through its receptors FGFR, indicating that the intestine can both synthesize and respond to KGF [10], [12], [13]. KGF has been reported to play a critical role in intestinal epithelial growth and maintenance. An interest finding shows, after bone marrow transplantation (BMT), KGF could lead to increased IL-7 production [14], and the protective effects of pre-BMT were improved by KGF administration on thymopoiesis [14]. Our previous study reported KGF could up-regulate IL-7 expression through the KGF-KGFR pathway both in an intestinal ischaemia/reperfusion (I/R) mouse model and in LoVo cells [15]. However, the mechanism by which pathway involved in this regulation of IL-7 expression is still unclear.
STATs are a family of latent cytoplasmic proteins that are involved in transmitting extracellular signals to the nucleus. KGF-stimulated increase in GM-CSF levels in lung tissue, which was associated with STAT5 phosphorylation in alveolar macrophages, was consistent with epithelium-driven paracrine activation of macrophage signaling through the KGF receptor/GM-CSF/GM-CSF receptor/ JAK-STAT axis [16]. Epidermal growth factor (EGF) is another important growth factor contributing to normal homeostasis and healing of the ocular surface [17], [18]. EGF has been reported to mediate its effect on target cells through the JAK-STAT pathway [19]-[20]. We sought to determine whether KGF, similar to EGF, activates this pathway in mediating effects on intestinal epithelial cells.
Interferon regulatory factors (IRFs) are a large family of transcription factors, in which IRF-1 and IRF-2 were first identified as activator and repressor, respectively [21]. The regulation of CIITA pIV by IFN-γ in B cells depends on the binding of signal transducer and activator of transcription (STAT)1 to IFN regulatory factor (IRF)-1 and IRF-2 to an interferon regulatory factor element (IRF-E) [22]. It has also been found that the STAT1 activation of IRF-1 plays an important role of STAT1 in promoter IV activation [23], [24]. Another study showed the transcriptional regulation via an IRF-E was important for IL-7 production in human IECs [1], which is consistent with the previous report on murine keratinocytes[25]. Of note, it was found that not only IRF-1 but also IRF-2, could up-regulate IL-7 production [1].
In this study, we demonstrated for the first time that the KGF signaling pathway was involved in the regulation of IL-7 expression in LoVo cells, and hypothesized that up-regulation of intestinal epithelial cell derived IL-7 expression by KGF through STAT1, IRF-1/IRF-2 pathway. This study would gain a better understanding of the functions of this cytokine on local immune regulation.
Materials and Methods
Ethics Statement
The study has been approved by the ethics committee of Xinqiao Hospital, Third Military Medical University. Animals were handled according to the guideline for the care and use of laboratory animals.
Cell culture
Human intestinal epithelial LoVo Cells (ATCC CCL-229) were used in our experiments. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 50 units/ml penicillin, and 50 mg/ml streptomycin, refreshed every 48 h, and subcultured serially when 80% confluent. Cells were seeded at identical cell densities and were typically used 12–15 days after reaching confluence.
Cell treatment
Cells were grown on 6-well plates and incubated with Recombinant human KGF (rHuKGF) (150 ng/ml) for 0, 30 min, 1, 2, 3, 6, 48 h, respectively. Then cells were fixed for staining experiments and nuclear extracts or total protein extraction of cells was used for Western blotting detection.
For STAT signaling pathway analysis, inhibitors including rapamycin (RPM) and AG490 were used. The cells were randomly allocated into three groups. Two of the groups were treated with either RPM (50 ng/ml, catalogue no. 37094; Sigma), or AG490 (50 µmol/l, catalogue no. S1509; Sigma) and one group of cells was left untreated as a control. The samples were harvested 24 h after the onset of stimulation for further study.
IRF1 and IRF2 expression were silenced by using interfering RNA and the plasmids 663, 664 and 665 (Shanghai SunBio Medical Biotechnology Co., Ltd) were transfected into LoVo cells for silencing IRF1 and plasmids 691, 692 and 693 (Shanghai SunBio Medical Biotechnology Co., Ltd) for IRF2, respectively, with lipofectamine 2000 (Invitrogen) following the manufacturer's instructions as previously.
Flow cytometric analysis
LoVe cells were cultured as described above. The amount of BrdU incorporated into the cells was measured following the procedures according to the manual in the kit (Flow Cytometry BrdU Testing Kit, GENMED). Briefly, 1×106 cells were pulse-labeled for 30 min with 10 mM BrdU, washed in ice-cold PBS, and pelleted. Cell pellets were resuspended in PBS and cells fixed in ice-cold ethanol. Incorporation of BrdU was measured with a fluorescein isothiocyanate (FITC, green)-conjugated anti-BrdU antibody and propidium iodide (PI, red). Flow-cytometric analysis was done with BD VERSE (Becton Dickinson) and accompanying BD FACSuite software, with forward and side scatter gates set to exclude nonviable cells.
Enzyme linked immunosorbent assay(ELISA)
ELISA analysis was used to evaluate the expression of secreted IL-7. For quantification of IL-7 in the supernatant of cultured LoVo cells, conditioned culture media were collected and centrifuged at 1200 rpm for 5 min to remove particulates; cleared supernatant was collected, concentrated, and stored at −80 °C until use. A human IL-7 ELISA Quantikine™ HS (High Sensitivity) from R&D Systems was used for detection of IL-7. The protocol was performed according to the manufacturer's instruction. The absorbance for IL-7 was assayed and the concentrations of each were determined by interpolation against a standard curve.
Animals
Male, 6-8week-old, specific pathogen-free, C57Bl/6 mice were purchased from Laboratory Animal Center, Third Military Medical University, Chongqing, P.R. China, maintained in temperature, humidity, and light-controlled conditions. Mice were divided into two groups: KGF group and control. Recombinant human KGF (rHuKGF) administration to mice was given daily by intraperitoneal injection (5 mg/kg/ day) for five days. In all experiments, six animals were analyzed per group and three times of experiments were repeated. There was no significant difference in survival between treatment and control groups.
Histological score
Segments of jejunum were harvested, were fixed with 4% paraformaldehyde and used for histological analysis. Tissues were then dehydrated with ethanol and embedded in paraffin. Sections were cut and stained with hematoxylin-eosin(H&E). Histological changes were assessed by a pathologist in a blinded fashion. Especially, the villus height and depth of crypt were measured using a calibrated micrometer. Each measurement of villus height and crypt depth consisted of the mean of 7 different fields.
Epithelial Cells Proliferation Assay
Crypt cell proliferation rate was calculated by the ratio of the number of crypt cells incorporating PCNA to the total number of crypt cells. Samples fixed by 4% paraformaldehyde were cut into 8 m-thick sections, treated with 0.5% hydrogen peroxide in methanol solution, blocked for 45 min, and then incubated with an anti-PCNA(catalogue no. 10205-2-AP; Proteinteck) or purified rabbit IgG (10 mg/ml; negative control) overnight at 4°C. The sections were incubated with biotinylated goat antirabbit IgG for 60 min and reacted with streptavidin-enzyme conjugates (Vector Laboratories Inc), and then the peroxidase activities were developed by diaminobenzidin. The total number of proliferating cells per crypt was defined as a mean of proliferating cells in 10 crypts (Original magnification ×400).
Mucosal wet weight, RNA and protein measurements
At the time of death, 10 cm of jejunum was excised and this segmental of intestine was weighed and was used for the measurement of intestinal RNA and protein content. Intestinal mucosal RNA was determined by spectrophotometry using a modified Schmidt-Tannhauser method as described by Munro and Fleck [26]. Protein determination was performed by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). RNA is expressed in µg/cm segment of intestine and protein results are expressed in mg/cm segment of intestine.
RNA Isolation, Reverse Transcriptase, and Polymerase Chain Reaction(PCR)
Total RNA specimens were isolated by using the Trizol reagents (catalogue no. 15596026; Invitrogen). Total RNA was reverse transcribed into cDNA using SuperScript II H-reverse transcriptase (catalogue no. 18064071; Invitrogen). PCR amplification primers of IL-7 were as follows: Up 5-TCTAATggTCAgCATCgATCA-3 and Down 5-gTggAgATCAAAATCACCAgT-3; Taqman probe was 6FAM-CCgCCgCCCgTCCACACCCgCCph, as described previously [27]. Amplification standard curves of target genes and of the reference gene β-actin were established, as previously described [27]. A PCR reaction mixture (30 µL) containing 1 mM dNTP (Life Technologies), 0.3 µM of each oligonucleotide primer, 1 µM Taqman probe, 1 U AmpliTaqGold DNA polymerase (Roche, Branchburg, NJ), and 100 ng of sample cDNA in PCR buffer was amplified on an ABI Prism 7700 sequence detector (Applied BioSystems). Cycling conditions included initial denaturation at 94 °C for 10 minutes, 30 seconds at 94 °C, 30 seconds at 60 °C, and 45 seconds at 72 °C for 45 cycles. All assays were performed in triplicate. The quantities of IL-7 gene expression and of the reference gene β-actin were determined by using standard curves. The mRNA copy numbers of these targets were calculated for each sample from the standard curve by measuring the threshold cycle value. The target amount was then divided by the reference gene amount to obtain a normalized target value and presented as relative rates compared with the expression of the reference gene β-actin.
Western blot assay
The nuclear extracts and total proteins were prepared from treated LoVo cells, as described previously [28]. Protein concentrations were measured, and equal amounts of nuclear extracts or total proteins were fractionated on 10% SDS polyacrylamide gel and transferred to 0.2-µm nitrocellulose membrane. Nitrocellulose blots were blocked by incubation in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% milk for 1 h. Blots were incubated with appropriate antibodies against IL-7(catalogue no. ab-9628; Abcam Inc.), IRF-1 (catalogue no. sc-13041; Santa Cruz Biotechnology), IRF-2 (catalogue no. sc-13042; Santa Cruz Biotechnology), STAT1 (catalogue no. 10144-2-AP; Proteinteck), or P-Tyr-STAT1 (Tyr701) (catalogue no. 7649; Cell Signaling Technology). After washed, the membrane was incubated with HRP-conjugated secondary antibodies (Cell Signaling) and then visualized with enhanced chemiluminescence (Cell Signaling). β-tubulin (Sigma, Dorset, UK) was used as an internal control.
Immunofluorescence staining
Cells and sections were fixed for staining experiments. Cells were incubated with the following primary antibodies: anti-IRF-1 rabbit polyclonal antibody (catalogue no. sc-13041; Santa Cruz Biotechnology) and anti-IRF-2 rabbit polyclonal antibodies (catalogue no. sc-13042; Santa Cruz Biotechnology) and sections were incubated with anti-IL-7 rabbit polyclonal antibody (catalogue no. bs-1811R; Beijing Boisynthesis Biotechnology Co.,Ltd.) overnight at 4C. Then the cells and sections were stained with FITC-conjugated goat anti-rabbit IgG. Nuclear staining for total cell counting was performed by 5 min addition of 1 mg/ml of DAPI (40,60-diamidino-2-phenylindole) and the fluorescence signals were analyzed by recording and merging single-stained images, using confocal laser microscope (Leica TCS SP2). Images were processed using Adobe Photoshop (Adobe Systems, San Jose, Calif., USA) and was analyzed by Leica's software system.
Immunohistochemistry staining
Samples fixed by 4% paraformaldehyde were incubated with either an anti-IRF-1 (catalogue no. sc-13041; Santa Cruz Biotechnology) antibody, an anti-IRF-2 (catalogue no. sc-13042; Santa Cruz Biotechnology) antibody or purified rabbit IgG (10 mg/ml; negative control). After the samples were counterstained with hematoxylin, the localization of IRF-1, IRF-2 was examined by light microscopy (Original magnification ×400).
Statistical analysis
Data are expressed as means ± standard deviation (SD). Statistics were performed using SPSS 13.0 software. Results were analyzed using analysis of variance (ANOVA). Statistical significance was defined as P<0.05.
Results
KGF administration leads to EC proliferation both in vivo and in vitro
Proliferation in a cell culture model
To investigate if the LoVo cells were proliferated after KGF treatment, we analyzed BrdU-incorporation expression by using flow cytometry. Results showed LoVo cells treated with KGF at different concentrations (0, 80 and 150 ng/ml) for 48 h displayed BrdU+ population increased to 26.9% and 31.1% with treatment of KGF (80 and 150 ng/ml, respectively) compared with control (12.0%), suggesting that cell viability was induced by KGF (Figure 1).
10.1371/journal.pone.0058647.g001Figure 1 Detection of cell viability by flow cytometric analysis of BrdU-incorporation.
Incorporation of BrdU was measured with a fluorescein isothiocyanate (FITC, green)-conjugated anti-BrdU antibody and propidium iodid (PI, red), shows a typical example of flow cytometric analysis of LoVo cells proliferation after KGF treatment, where a BrdU+ population is clearly visible. Control (A), KGF treated groups with different concentrations including 80 ng/ml (B) and 150 ng/ml (C).
Intestinal Morphology
To investigate the effect of KGF on mice intestinal mucosa, histopathological evaluation was used. There was a significant increase in both villus height and crypt depth in the group after KGF treatment. KGF treatment led to an increase in jejunal villus height 387±49 µm), as compared with control (243±42 µm) (P<0.05). The crypt depth was also greater in the KGF group (116±21 µm) than in control (53±17 µm) (P<0.05), respectively (Figure 2).
10.1371/journal.pone.0058647.g002Figure 2 Alterations in villus height and crypt depth in mice after KGF treatment.
* P<0.05 vs. control group, n = 6 per group.
PCNA-positive cells were all distributed in the crypt of Lieberkuhn of the small intestine. KGF also significantly increased the number of PCNA positive cells to (57.2±5.4%) when compared with control (23.7±3.9%) (P<0.05) (Figure 3). There was no significant difference in the positions of positive cells between groups, and all positive cells remained in the crypts.
10.1371/journal.pone.0058647.g003Figure 3 Alterations in PCNA expression in small intestine of KGF treated mice by immunohistochemistry.
PCNA expression was significantly increased in KGF group (A), as compared to the control group (B). PCNA expression is expressed as means ± SD (C), *P<0.05 vs control group. Original magnification: ×400; n = 6 per group. Scale bar = 25 µm
Indexes of jejunum
Mucosal wet weight of the jejunum (mg/10 cm) was significantly increased in KGF group compared with the control. KGF administration significantly increased RNA content (39.7±6.4 µg/cm) when compared with control (16.2±4.5 µg/cm) (P<0.05). The changes of jejunum mucosal protein contents were similar to changes of RNA content. There was significant difference of protein contents between the KGF group (2.65±0.19 mg/cm) and the control group (1.78±0.26 mg/cm) (P<0.05) (Table 1).
10.1371/journal.pone.0058647.t001Table 1 Intestinal wet weight and contents of jejunal protein and RNA.
Control KGF group
Protein (mg/cm) 1.78±0.26 2.65±0.19*
RNA (µg/cm) 16.2±4.5 39.7±6.4*
Intestinal wet weight (mg/10 cm) 387.8±8.4 576.4±11.7*
*
P<0.05 vs Control group.
KGF administration results in an increased expression of EC -derived IL-7 both in vivo and in vitro
To investigate the role of KGF in the regulation of IL-7, both in vitro and in vivo models were used. KGF administration at different concentrations (20, 40, 80, 100 and 150 ng/ml) for 48 h in the LoVo cells resulted in an increased IL-7 expression detected by Western blot assay, showing a dose-dependent manner [15], which was also confirmed by ELISA. We found that IL-7 levels in cell culture supernatant rose from 4.43±0.47, 5.52±0.41, 6.47±0.45, 8.72±0.53 pg/mL in the KGF (20, 40, 80,150 ng/ml) treated group to 2.33±0.28 pg/mL in the control. Furthermore, IL-7 expression in the intestinal mucosa was dramatically increased in protein nearly 4-folds compared with control in a health mouse model [15]. Moreover, KGF up-regulated IL-7 in a mouse model of intestinal I/R, which was confirmed by the results from immunofluorescence staining [15].
STAT1 pathway is involved in the regulation of IL-7 after KGF treatment
KGF treatment leads to increased levels of P-Tyr-STAT1
To gain direct evidence for the activity of the STAT1 signaling pathway induced by KGF in LoVo cells, the STAT1 activity was evaluated by Western blot analysis (Figure 4). Compared with control cells, KGF (150 ng/ml) treatments of different time point (30 min, 1, 2, 6 and 24 h) resulted in significantly increased levels of P-Tyr-STAT1 (P<0.05) (Figure 4), but not STAT1 proteins at all time points including 30 min, 1 h, 2 h, 6 h and 24 h.
10.1371/journal.pone.0058647.g004Figure 4 Changes of P-Tyr-STAT1 and STAT1 expression after KGF treatment in LoVo cells.
Increased expression of P-Tyr-STAT1, but not STAT1, were confirmed by western blot in LoVo cells with KGF (150 ng/ml) treatment. Tubulin was used as internal control.
To inhibit STAT1 expression causes a significant down-regulation of IL-7 expression in LoVo cells
Both RAPA and AG490 are inhibitors of STAT1. The effects of RPM or AG490 on STAT1 and IL-7 protein expression were determined by Western blot analysis (Figure 5A). The results showed that the protein levels of P-Tyr-STAT1 and IL-7 were significantly decreased by the treatment with RPM or AG490 partially counteracted the effects of KGF, while STAT1 proteins did have not significantly decreased when compared with the control (P<0.05) (Figure 5A). Similarly, we analyzed the IL-7 mRNA expression by using quantitative real-time PCR. The results were shown in Figure 5B, which were similarly to IL-7 protein expression.
10.1371/journal.pone.0058647.g005Figure 5 Changes of P-Tyr-STAT1, STAT1 and IL-7 expression after STAT1 blockade following KGF treatment, by western blot in LoVo cells (A).
Tubulin was used as internal control. Suppressions of P-Tyr-STAT1 and IL-7 expression, but not STAT1, were observed with STAT1 inhibitors including AG490 (50 µmol/l) and RPM (50 ng/ml) following KGF (150 ng/ml) treatment. Changes of IL-7 mRNA expression after STAT1 blockade following KGF treatment were detected by quantitative real-time PCR (B), * indicates significant difference between RPM (or AG490) group and control, ** indicates significant difference between RPM (or AG490)+KGF group and control+KGF group, P<0.05.
IRF-1 and IRF-2 are involved in the up-regulation of IL-7
This study showed the evidence for the activity of the STAT1 signaling pathway induced by KGF, and the previous report found that the transcriptional regulation via an IRF-E including IRF-1 and IRF-2, was important for IL-7 production in human IECs [1], which suggest IRF-1 and IRF-2 are involved in the regulation of IL-7.
KGF treatment results in an increased expression of IRF-1 and IRF-2 both in vivo and in vitro
To further investigate the pathway involved in this regulation of IL-7 expression, LoVo cells were treated with KGF (150 ng/ml) for 0 h, 2 h, 3 h and 6 h, and IRF-1, IRF-2 expressions of the nuclear extracts and total proteins were detected by Western blot. Results showed a significantly increased IRF-1 and IRF-2 expression in 6 h both in nuclear extracts and total proteins respectively; P<0.05 compared with controls) (Figure 6A, 6B). These results were confirmed with another finding, which showed that LoVo cells were treated with KGF (150 ng/ml), for 0 h, 1 h, 3 h and 6 h, and immunofluorence staining was performed to detect the expressions of IRF-1, IRF-2 in the nucleus. Results showed the fluorescence band of IRF-1 and IRF-2 in nuclear, which were most obvious at 6 h than other time points in LoVo cells (Figure 7A, 7B). All these results suggest that KGF treatment caused increased expressions of IRF-1 and IRF-2 with a time dependent manner (Figure 6A, B, 7A, B).
10.1371/journal.pone.0058647.g006Figure 6 KGF administration resulted in an increased IRF-1 and IRF-2 expression of the nuclear extracts and total proteins in vitro.
Dose-dependent increased expression both of IRF-1 (A) and IRF-2 (B) were confirmed by western blot in LoVo cells with KGF treatment. Tubulin and H1 were used as internal control.
10.1371/journal.pone.0058647.g007Figure 7 Increased expression of IRF-1 and IRF-2 were confirmed by immunofluorenscence staining with KGF treatment in vitro.
Increased expression of IRF-1 and IRF-2 in the nucleus were observed after 6 h with KGF treatment.
Immunohistochemistry was done to detect the IRF-1 and IRF-2 expression 5 days after KGF administration in a mouse model. Results showed that KGF administration also increased the number of positive cells which express IRF-1 and IRF-2 preferentially exhibited nuclear patterns, indicating that these IRF proteins function as transcriptional regulators in IECs in vivo (Figure 8). Furthermore, the number of the IRF-2-positive cells was much more than IRF-1 -positive cells (Figure 8). These findings were consistent with our present report in vitro. Recombinant KGF acts on the intestinal epithelial cells leading to the up-regulation of IRF-1 and IRF-2 expressions and subsequent IL-7 expression.
10.1371/journal.pone.0058647.g008Figure 8 Alterations in IRF-1 and IRF-2 expression in small intestine of KGF treated mice by immunohistochemistry.
IRF-1 expression in control group (A) and in KGF group (B), IRF-2 expression in control group (C) and in KGF group (D). Original magnification: ×400; n = 6 per group. Scale bar = 25 µm.
Changes of IL-7 expression after IRF-1 and IRF-2 expression were silenced
To further confirm the pathway of KGF through IRF-1 and IRF-2 to regulate IL-7 expression, IRF-1 and IRF-2 expression were silenced by using interfering RNA, and then the effect of KGF on the IL-7 expression was investigated in the LoVo cells. The IL-7 protein and mRNA expression was determined by Western blot analysis and quantitative real-time PCR. The plasmids 663, 664 and 665 used for IRF-1, plasmids 691, 692, 693 for IRF-2 were transfected into LoVo cells and the IRF-1 and IRF-2 expression of the nuclear extracts and total proteins were detected by Weston blot, respectively. Results showed the IRF-1 expression, both in the nuclear extracts and total proteins, were dramatically reduced, when treated with 665 plasmid, compared to controls (p<0.05), while the same condition was found in IRF-2 expression (treated with 693 plasmid) compared to controls, p<0.05) (Figure 9A, 9B). Following IRF-1 silencing by plasmids 665 and IRF-2 silencing by plasmids 693, LoVo cells were treated with 150 ng/ml KGF for 48 h, respectively and significant reduction of IL-7 expression were noted. IL-7 protein expression significantly reduced by treated with 665 plasmid for IRF-1(p<0.05) and by treated with 693 plasmid for IRF-2 (p<0.05), compared to control, respectively (Figure 9C, 9D), which were also found in IL-7 mRNA expression detected by quantitative real-time PCR. These results showed that transfection of plasmid 665 and plasmid 693 could result in obvious suppression of IRF-1 and IRF-2 expression respectively, so that decreased IL-7 expression was observed in LoVo cells following KGF treatment. However, transfection of control plasmid had no influence on the mRNA and protein expression of IL-7. These findings further confirm that KGF can regulate IRF-1 and IRF-2 expressions and subsequent IL-7 expression in IECs.
10.1371/journal.pone.0058647.g009Figure 9 IL-7 is up-regulated by KGF through IRF-1/IRF-2 pathway.
Tubulin and H1 were used as internal control. (A) Reduced the nuclear extracts and total proteins of IRF-1 was confirmed by western blot in LoVo cells following IRF-1 RNA interference. Plasmids 663, 664 and 665 were transfected into LoVo cells and IRF-1 expression was detected. Plasmid 665 can definitely inhibit IRF-1 expression. (B) Reduced the nuclear extracts and total proteins of IRF-2 was confirmed by western blot in LoVo cells following IRF-2 RNA interference. Plasmids 691, 692 and 693 were transfected into LoVo cells and IRF-2 expression was detected. Plasmid 693 can definitely inhibit IRF-2 expression. (C) Reduced expression of IL-7 was confirmed by western blot and quantitative real-time PCR in LoVo cells following IRF-1 RNA interference. Decreased expression of IL-7 was observed in LoVo cells following KGF treatment in response to RNA interference of IRF-1 by plasmid 665 in both mRNA and protein levels. *P<0.05 vs. control group. (D) Reduced expression of IL-7 was confirmed by western blot and quantitative real-time PCR in LoVo cells following IRF-2 RNA interference. Decreased expression of IL-7 was observed in LoVo cells following KGF treatment in response to RNA interference of IRF-2 by plasmid 693 in both mRNA and protein levels. *P<0.05 vs. control group.
Discussion
In this study, we found that KGF administration resulted in EC proliferation both in vivo and in vitro study. KGF treatment led to increased levels of P-Tyr-STAT1, and RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. KGF also up-regulated IRF-1 and IRF-2 in vivo and in vitro studies, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by using interfering RNA in LoVo cells, respectively. All these results suggest that KGF could up-regulate the IL-7 expression through the STAT1/ IRF-1, IRF-2 signaling pathway.
It is believed that KGF plays a critical role in intestinal epithelial growth and maintenance [29]. Our present study showed that KGF administration led to proliferation in LoVo cells, and also found that there was a significant increase in villus height, crypt depth and the number of PCNA positive cells in mice after KGF treatment. In addition, KGF significantly increased the intestinal mucosal wet weight, RNA and protein contents. These results suggested the important role of KGF in the intestinal epithelial growth, which was confirmed by the study of Farrell et al
[30], who found that wet weights of the intestinal segments were increased by the KGF treatment and morphometric measurement showed that both crypt depth and villus height were also increased in mice.
Recent studies have demonstrated that the interactions between intestinal EC and mucosal lymphocytes are crucial in regulating maintenance intestinal function and immune response [4], [31]. KGF can expand thymic epithelium cells (TECs) and intestinal epithelial cells (ECs) [15], [32] and has been reported to increase IL-7 production in treated mice [15], [32], and also potently augments thymopoiesis and protects from thymic and intestinal damage [15], [32] by signaling via FGFR2IIIb [15], [29], [33]-[35]. Meanwhile, it is believed that IL-7 has effects on developing and mature lymphocytes, and is essential for the ongoing maintenance of the IEL growth and function. In our previous study, we found that IL-7 and KGFR were both expressed in the intestinal epithelial cells (IECs), and KGF could up regulate the IL-7 expression both in vivo and in vitro
[15]. Through ELISA assay, we also found that KGF significantly increased IL-7 protein expression. When the KGFR was blocked, the above findings were absent [15]. These results suggest that KGF could up-regulate the IL-7 expression through interacting with KGFR pathway in IECs. However, the further mechanism which is involved in transmitting extracellular signals to the nucleus remains unknown.
KGF is a multifunctional cytokine, and it would not be surprising if this growth factor initiates different signals to modulate effects on different epithelial cells. A report of signaling pathways used by KGF in corneal epithelial cells show that the p42 and p44 MAPKs are activated by KGF in human corneal epithelial cells [36]. In this study, our studies were directed toward identifying the signaling molecules activated by KGF to mediate effects on intestinal epithelial cell functions. We found that KGF activated STAT1 in human intestinal epithelial cells, which was the first report of the STAT1 signaling pathways involved by KGF in intestinal epithelial cells. We found KGF increased P-Tyr-STAT1 but not STAT1 in LoVo cells, and P-Tyr-STAT1expression was decreased by blocking agents used, including RPM and AG490. RPM is a streptomyces derivative that is critical for the regulation of cell growth, cell proliferation, cell motility and cell survival [37]. More importantly, other data directly support the idea that RPM inhibits the activity of STAT1 [38]. Furthermore, AG490 is an inhibitor of the JAK-2, JAK-3/STAT signaling pathway and potently inhibits cytokine-independent cell growth in vitro
[39]. In the present study, treatment with RPM and AG490 inhibited the activity of STAT1 and the IL-7 mRNA and protein expression in KGF-stimulated LoVo cells, which suggested KGF regulated IL-7 though the STAT1 signaling pathway. The binding of KGF to its receptor results in the activation of receptor-associated phosphorylation of STAT1, and phosphorylated STAT1 forms homodimers, which migrate to cell nucleus and activate transcription.
Interferon regulatory factor-1 and -2 (IRF-1 and -2) are two structurally related members of the IRF family of transcription factors, which are both involved in signal transducing. IFN-γ regulated CIITA pIV by activating transcription (STAT1), which binds to IFN regulatory factor IRF-1 and IRF-2 to an IFN regulatory factor-element (IRF-E) [22]. The role of IRFs in the regulation of IL-7 expression has been explored previously [1], and this work sheds new light on the role of IRF-1 and IRF-2 in the transcriptional regulation of IL-7 by KGF in intestine in vivo and in vitro. We found a significantly increased IRF-1 and IRF-2 expression in the total proteins and in the nucleus, which were detected by Western blot and immunofluorence assay, respectively. We also found KGF significantly increased the number of positive cells which express IRF-1 and IRF-2 in the nucleus by immunohistochemistry staining in the mice intestine. In addition, decreased IL-7 mRNA and protein expressions were observed in LoVo cells by obvious suppression of IRF-1 and IRF-2 expression respectively, even following KGF treatment. The studies reported suggest that STAT1 and IRF transcription factors, including IRF-1 and IRF-2 contribute to the transcriptional regulation of IL-7 by KGF.
In this study, we found KGF up-regulated IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which was the first report of regulation of IL-7 by KGF in intestinal epithelial cells. All of these data may suggest the indirect data to support that KGF may play an important role in mucosal immune responses by regulating IL-7 to help to regulate IEL. This is important because these data would shed new light on the potential role of KGF in therapies aiming to enhance the ability of the immune system in intestine.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23516624PONE-D-12-1482310.1371/journal.pone.0059296Research ArticleBiologyBiochemistryBiochemistry SimulationsBioinorganic ChemistryChemical BiologyEnzymesImmunochemistryHistologyImmunologyImmunopathologyModel OrganismsAnimal ModelsRatSynthetic BiologyChemistryMedicinal ChemistryOrganic ChemistryAcute Toxicity and Gastroprotection Studies with a Newly Synthesized Steroid Gastroprotective Effect of AMDCPA. Ketuly Kamal
1
A. Hadi A. Hamid
1
Golbabapour Shahram
2
3
Hajrezaie Maryam
2
3
Hassandarvish Pouya
2
Ali Hapipah Mohd
1
Majid Nazia Abdul
3
Abdulla Mahmood A.
2
*
1
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
2
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
3
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
Tache Yvette Editor
University of California, Los Angeles, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: MAA KAK AHAH HMA NAM. Performed the experiments: KAK PH MH SG. Analyzed the data: KAK PH MH SG. Contributed reagents/materials/analysis tools: MAA KAK AHAH HMA NAM. Wrote the paper: KAK MAA SG.
2013 13 3 2013 8 3 e5929623 5 2012 14 2 2013 © 2013 A2013AThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Synthetic steroids, such as 9α-bromobeclomethasonedipropionate, have shown gastroprotective activity. For example, the potent glucocorticoid steroid, beclomethasone dipropionate, has been used for treatment of bowel ulcerations. The purpose of the present study was to evaluate the effect of a synthetic steroid, (20S)-22-acetoxymethyl-6β-methoxy-3α,5-dihydro-3′H-cyclopropa[3α,5]-5α-pregnane (AMDCP), on ethanol-induced gastric mucosa injuries in rats.
Methodology/Principal Finding
Rats were divided into 8 groups. The negative control and ethanol control groups were administered Tween 20 (10%v/v) orally. The reference control group, 20 mg/kg omeprazole (10% Tween 20, 5 mL/kg), was administrated orally. The experimental groups received 1, 5, 10, 15 or 20 mg/kg of the AMDCP compound (10% Tween 20, 5 mL/kg). After 60 min, Tween 20 and absolute ethanol was given orally (5 mL/kg) to the negative control group and to the rest of the groups, and the rats were sacrificed an hour later. The acidity of gastric content, gastric wall mucus and areas of mucosal lesions were assessed. In addition, histology and immunohistochemistry of the gastric wall were assessed. Prostaglandin E2 (PGE2) and malondialdehyde (MDA) content were also measured. The ethanol control group exhibited severe mucosal lesion compared with the experimental groups with fewer mucosal lesions along with a reduction of edema and leukocyte infiltration. Immunohistochemical staining of Hsp70 and Bax proteins showed over-expression and under-expression, respectively, in the experimental groups. The experimental groups also exhibited high levels of PGE2 as well as a reduced amount of MDA. AMDCP decreased the acidity and lipid peroxidation and increased the levels of antioxidant enzymes.
Conclusion/Significance
The current investigation evaluated the gastroprotective effects of AMDCP on ethanol-induced gastric mucosal lesions in rats. This study also suggests that AMDCP might be useful as a gastroprotective agent.
This study was funded by the University of Malaya HIR-MOHE [HIR-MOHE (F000009-21001)] and UMRG grants (RG035/10 BIO and RG0373/11 HTM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Peptic ulcers are a common disorder that may occur throughout the entire gastrointestinal tract but mainly occur in the stomach and the proximal duodenum [1]. The gastric mucosal membrane is continuously exposed to potentially harmful agents, such as HCl, pepsin, bile acids, food seasonings, bacterial products and drugs. These agents are involved in the pathogenesis of gastric injury by promoting an increase in the secretion of gastric acid and pepsin and a decrease in gastric blood flow, suppressing the output of endogenous prostaglandins, inhibiting cellular proliferation and growth of the gastric mucosa and altering gastric motility [2]. The basic pathophysiology of gastric ulcers and mucosal lesions results from an imbalance of multiple endogenous aggressive factor(s), such as hydrochloric acid, pepsin, refluxed bile, leukotrienes and reactive oxygen species (ROS) and protective factors, which include a functional mucus-bicarbonate barrier, surface active phospholipids, prostaglandins (PG), mucosal blood flow, cell renewal and migration, non-enzymatic and enzymatic antioxidants and some growth factors [3], [4]. Heat shock proteins, generated by gastric epithelial cells such as Hsp70 are critical macromolecule chaperones [5] which associate in maintaining of physiology of gastric tissue in response to stress, such as oxidative stress through protecting protein against denaturation [6], [7]. Under stress imbalance between anti-apoptotic protein, such as Bcl2 family, and pre-apoptotic protein (Bax) cause gastric ulcer [8]. Several studies evaluated the expression of Hsp70 and Bax to evaluate gastric mucosa protection and damage respectively [9]–[11]. In spite of the multi-faceted pathogenesis of peptic ulcers and mucosal lesions, gastric acid secretion is still recognized as a central component of this disease. Therefore, the main therapeutic goal is to control acid secretion using antacids, H2 receptor blockers (ranitidine and famotidine) or proton pump inhibitors (omeprazole and lansoprazole) [12]. However, current gastric ulcer therapies show limited efficacy against gastric mucosal lesions/ulceration and are often associated with several side effects [4]. In this study, the synthetic steroid acetoxymethyl-6β-methoxy-3α,5-dihydro-3′H-cyclopropa[3α,5]-5α-pregnane (AMDCP) was tested to evaluate its ability to prevent mucosal lesions. Other synthetic steroids, such as 9α-bromobeclomethasone dipropionate, have shown gastroprotective properties at larger dosages [13] than AMDCP. The potent glucocorticoid steroid, beclomethasone dipropionate, has been used for the treatment of bowel ulcerations [14]. However, there is no data in the literature on the gastroprotective activities associated with AMDCP. Hence, the current study was undertaken to evaluate the gastroprotective effects of AMDCP on ethanol-induced gastric mucosal lesions in rats and the effect of ethanol and AMDCP treatment on Hsp70 and Bax proteins in immunohistochemical staining. In addition, the antioxidant status of gastric tissue homogenate was assessed by determining the levels of malondialdehyde (MDA) and Prostaglandin E2 (PGE2).
Materials and Methods
Omeprazole
In this study, omeprazole (obtained from the University of Malaya Medical Centre (UMMC) Pharmacy) was used as a control for anti-ulcer medicine. The medicine was dissolved in (10% Tween 20, 5 mL/kg) and administered orally to the rats in a single dose of 20 mg/kg body weight (5 mL/kg) according to the recommendations of Mahmood et al.
[15].
Synthesis of (20S)-22-Acetoxymethyl-6β-methoxy-3α,5-dihydro-3′H-cyclopropa[3α,5]-5α-pregnane (AMDCP)
(20S)-22-Hydroxymethyl-6β-methoxy-3α,5-dihydro-3′H-cyclopropa[3α,5]-5α-pregnane [16] (250 mg) was dissolved in pyridine (2 mL) and acetic anhydride (2 mL). The solution was heated at 80°C for 1 h. The solvent was evaporated to isolate the crude product (280 mg), which was then purified by recrystallization from methanol (140 mg) to yield AMDCP (m.p. 122–123°C, mass spectrum: M+ 388). Anal. calcd for C25H40O3, C 77.27, H 10.38% (found: C 77.06, H 10.44%) for the X-ray structure [17]. AMDCP was administered orally to rats at doses of 1, 5, 10, 15 or 20 mg/kg body weight (5 mL/kg body weight).
Acute toxicity test and experimental animals
Healthy male and female ICR mice (6–7 weeks old) were obtained from the Animal House, Faculty of Medicine, University of Malaya, Kuala Lumpur (Ethic No. PM/27/07/2010/MAA (R)). The mice weighed between 25 and 30 g. The animals were fed a standard rat pellet diet and tap water. The acute toxicity study was used to determine a safe dose for AMDCP. Thirty-six mice (18 males and 18 females) were randomly assigned into 3 groups: vehicle (10% Tween 20, 5 mL/kg), low-dose (100 mg/kg) and high-dose (2000 mg/kg) of AMDCP (10% Tween 20, 5 mL/kg) according to OECD [18]. Prior to dosing, the animals were fasted overnight (i.e., receiving water but not food). Food was withheld for an additional 3 to 4 h after dosing. The animals were observed continuously for 30 min, they were then monitored frequently (2, 4, 8, 24 and 48 h) for the onset of any clinical or toxicological symptoms. Mortality, if any, was observed over a period of 2 weeks. The animals were sacrificed on the 15th day. Histological, hematological and serum biochemical parameters were determined according to the OECD [18]. The study was approved by the Ethics Committee for Animal Experimentation, Faculty of Medicine, University of Malaya, Malaysia. During the experiments, all animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health.
Experimental animals for gastric mucosal lesions
Healthy adult Sprague Dawley male rats were obtained from the Experimental Animal House, Faculty of Medicine. The rats were divided randomly into 8 groups of 6 rats each. Each rat that weighed between 180 and 220 g was placed individually in a separate cage (1 rat per cage) with wide-mesh wire bottoms to prevent coprophagia during the experiment. The animals were maintained on a standard pellet diet and tap water. The study was approved by the Ethics Committee for Animal Experimentation, Faculty of Medicine, University of Malaya, Malaysia.
Gastric mucosal lesion-induction by ethanol
The rats were fasted for 24 h, but they had access to drinking water up to 2 h prior to the experiment. The negative control group (group 1) was orally administered 10% Tween 20 (5 mL/kg). The ethanol control group (group 2) was orally administered 10% Tween 20 (5 mL/kg). The reference control group (group 3) received a single oral dose of 20 mg/kg omeprazole (10% Tween 20, 5 mL/kg). AMDCP at doses of 1, 5, 10, 15 and 20 mg/kg (10% Tween 20, 5 mL/kg) was orally administered to the experimental groups (groups 4–8). One hour after treatment, 10% Tween 20 (5 mL/kg) was orally administered to the negative control group, and absolute ethanol was orally administered to the rest of the groups to induce gastric mucosal lesions (5 mL/kg). The rats were euthanized 1 h later with an overdose of xylazine and ketamine anesthesia, and their stomachs were immediately excised.
Measurement of gastric juice acid content (pH)
Samples of gastric contents were analyzed for hydrogen ion concentration by pH metric titration with 0.1 N NaOH solutions using a digital pH meter.
Determination of gastric wall mucus (GWM)
The gastric wall mucus was evaluated according to the modified procedure of Corne et al.
[19]. Glandular stomach segments were separated from the lumen, weighed and transferred immediately to 10 mL of a 0.1% w/v Alcian blue solution (in a 0.16 M sucrose solution buffered with 0.5 mL of sodium acetate, pH 5). The tissue was stained for 2 h in Alcian blue. Excess dye was removed by 2 consecutive rinses with 10 mL of 0.25 M sucrose. The dye that complexed with the gastric wall mucus was extracted using 10 mL of 0.5 M magnesium chloride. This mixture was intermittently shaken for 1 min at 30 min intervals over 2 h. Four milliliters of the blue extract was then vigorously shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged at relative centrifugal force (RCF) = 2361× g for 10 min. The absorbance of the aqueous layer was recorded at 580 nm. The quantity of Alcian blue that was extracted from the wet glandular tissue was then calculated.
Macroscopic gastric lesions evaluation
The presence of elongated bands of red hemorrhagic lesions parallel to the long axis of the stomach is a symptom of mucosal lesions of the gastric mucosa. The gastric mucosa of each rat was then examined for damage. The length and width of the lesion (mm) were measured using a planimeter (10×10 mm2 = lesion area) under a dissecting microscope (1.8×). The ulcerated area was measured by counting the number of small squares (2 mm ×2 mm) covering the length and width of each mucosal lesion band. The sum of the areas of the all lesions for each stomach was used to calculate the mucosal lesion area (LA), in which sum of the small squares ×4×1.8 = LA (mm2), according to the previously used protocol [20]. The inhibition percentage (I%) was calculated using the following formula according to the method of Abdulla et al.
[21].
Histological evaluation of gastric lesions
Specimens from the gastric walls of each rat were fixed in 10% buffered formalin and processed in a paraffin tissue processing machine. Sections of the stomach were sliced at a thickness of 5 µm and stained with hematoxylin and eosin for histological evaluation [13], [21].
Immunohistochemistry
Tissue section slides were heated at 60°C for approximately 25 min in a hot air oven (Venticell, MMM, Einrichtungen, Germany). The tissue sections were de-paraffinized in xylene and rehydrated using an alcohol gradient. The antigen retrieval process was performed in 10 mM sodium citrate buffer. Immunohistochemical staining was conducted according to the manufacturer's protocol (Dako Cytomation, USA). Briefly, endogenous peroxidase was blocked in a peroxidase blocking solution (0.03% hydrogen peroxide containing sodium azide) for 5 min. Tissue sections were washed gently with washing buffer and subsequently incubated with Hsp70 (1∶500) or Bax (1∶200) biotinylated primary antibodies for 15 min. The sections were rinsed gently with washing buffer and placed in a buffer bath. The slides were then placed in a humidified chamber with a sufficient amount of streptavidin – HRP (streptavidin conjugated to horseradish peroxidase in phosphate-buffered saline (PBS) containing an anti-microbial agent). The slides were incubated for 15 min. Subsequently, the tissue sections were rinsed gently in washing buffer and placed in a buffer bath. A diaminobenzidine-substrate-chromogen was added to the tissue sections and incubated for 5 min, followed by washing and counterstaining with hematoxylin for 5 sec. The sections were then dipped in weak ammonia (0.037 M/L) 10 times, rinsed with distilled water and cover slipped. Positive immunohistochemical staining was observed as brown stains under a light microscope.
Biological activity of gastric homogenate
Sample preparations
The gastric tissue homogenate from each rat was prepared for PGE2 and MDA assays. The entire experiment was performed at 4°C. Gastric tissue was cut into 3 small pieces (approximately 200 mg for each), and the exact weight of each piece was recorded [22]. The tissues were homogenized in a teflon homogenizer (Polytron, Heidolph RZR 1, Germany) using the appropriate buffer. The amount of buffer used was dependent on the weight of the tissue used. After centrifugation at 4,500 rpm for 15 min at 4°C, the supernatant was used for the PGE2 and MDA assays.
Measurement of membrane lipid peroxidation (MDA)
The rate of lipoperoxidation in the gastric mucosal membrane was determined by measuring the level of MDA using the Thiobarbituric Acid Reactive Substances test. The tissues were washed with phosphate-buffered saline to minimize the interference of hemoglobin and to remove blood adhered to the gastric mucosa. The stomachs were homogenized with 10% of the tissue using potassium phosphate buffer. Then, 250 μL of homogenate were incubated at 37°C for 1 h, 400 μL of 35% perchloric acid was added, and the mixture was centrifuged at 4,500 rpm for 20 min at 4°C. The supernatant was removed, mixed with 400 μL of 0.6% thiobarbituric acid and incubated at 95–100°C for 1 h. After the homogenate was cooled, the absorbance was measured at 532 nm. A standard curve was generated using 1,1,3,3-tetramethoxypropane. The results were expressed as nM of MDA/mg of protein. The concentration of the protein was measured using the method described by Bradford [23], which is based on the interaction of Coomassie Blue G250 dye with proteins. The amount of total protein in each tissue sample was measured after the lesions were induced by ethanol treatment. The interaction between the high molecular weight proteins and the dye causes a shift in the dye to its anionic form, which exhibits a strong absorbance at 595 nm. Solutions of albumin standard, distilled water, buffer (Borate 50 mM, Tris 25 mM, HEPES 100 mM and Phosphate 100 mM) and each sample were added to the wells. For sample preparation, 2 μL of sample and 38 μL of buffer were added to each well. Then, 200 μL of Bradford's solution (diluted 5×) was added to each well. After a 5 min incubation, absorbance at the wavelength of 595 nm was recorded, according to the Bradford method [23].
Measurement of PGE2 formation using enzyme immunoassays
The gastric mucosa was weighed, minced with scissors, and homogenized at 4°C in PBS buffer. Homogenates were centrifuged at 1400 rpm for 10 min. The supernatants were analyzed by PGE2 assay using a PGE2 Monoclonal Enzyme Immunoassay Kit (Sigma-Aldrich, Malaysia).
Measurement of protein concentration
Protein concentrations (mg/mL tissue) were determined using the Biuret reaction, as described by Gornall et al.
[24].
Statistical analysis
All values are reported as the means ± S.E.M. The statistical significance of the differences between groups was assessed using a one-way ANOVA. A p-value of p<0.05 was considered to be significant.
Results
Acute toxicity study
An acute toxicity study did not show any sign of toxicity in any groups within 14 days. There were no histological signs of hepatic or renal toxicity. Moreover, the blood biochemistry analysis appeared normal.
Effect of AMDCP on mucosal lesion area
The experimental groups showed significant prevention of gastric lesion formation as well as a significant increase in the percent inhibition of gastric mucosal lesions (Figure 1).
10.1371/journal.pone.0059296.g001Figure 1 Effect of AMDCP on gastric mucosal lesions and inhibition percentage in rats.
Inhibition of gastric lesions (%) is indicated in brackets above the columns. Groups 2 and 3 represent the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
Macroscopic evaluation of gastric lesions
The gastroprotective activity of AMDCP in the ethanol-induced gastric lesion model is shown in Figure 2. The results showed that rats in the reference control group and in the experimental groups showed a pronounced reduction in the formation of gastric mucosal lesions compared with the ethanol control group (Figure 2). Ethanol produced extensive visible red hemorrhagic lesions of the gastric mucosa. AMDCP dramatically suppressed the formation of mucosal lesions and produced a notable flattening of the gastric mucosal folds in rats pre-treated with 20 mg/kg of AMDCP (Figure 2). The remarkable inhibition of gastric mucosal lesions in rats pre-treated with 10 mg/kg of AMDCP (group 7) was comparable with the reference control group (omeprazole, 20 mg/kg) (Figure 2).
10.1371/journal.pone.0059296.g002Figure 2 Macroscopic appearance of the gastric mucosa in rats.
The negative control group showed no injuries to the gastric mucosa (A). Severe injuries were observed in the gastric mucosa of the ethanol control group. Ethanol treatment produced extensive visible hemorrhagic lesion of the gastric mucosa (B). The reference control group, treated with omeprazole (20 mg/kg), showed milder injuries to the gastric mucosa compared to the injuries observed in the ethanol control group (C). Group 4 (1 mg/kg AMDCP) showed moderate injuries to the gastric mucosa (D). Group 5 (5 mg/kg AMDCP), mild injuries were observed in the gastric mucosa. AMDCP reduced the formation of gastric lesions induced by ethanol (E). Groups 6, 7, and 8 (10, 15, and 20 mg/kg AMDCP, respectively) showed no injuries to the gastric mucosa. Instead, flattening of the gastric mucosa was observed (F, G and H).
Gastric mucosal wall evaluation
Treatment with ethanol caused a significant decrease in the mucus content of the gastric wall in the ethanol control group (Figure 3). The depleted gastric mucus was significantly replenished in the experimental groups. It was also found that the experimental groups significantly increased the amount of gastric mucus (Figure 3).
10.1371/journal.pone.0059296.g003Figure 3 Effect of AMDCP on ethanol-induced changes in the Alcian blue binding capacity of gastric mucosa, gastric mucosal lesions area and inhibition percentage in the gastric mucosa of rats.
Groups 1, 2 and 3 represent the negative control group, the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
pH of gastric content
The acidity of the gastric content in the experimental groups was decreased significantly compared with that of the ethanol control group (p<0.05) (Figure 4).
10.1371/journal.pone.0059296.g004Figure 4 Effect of AMDCP on the pH of gastric content.
Groups 1, 2, and 3 represent the negative control group, the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
Histological evaluation of gastric lesions
Histological evaluation of the ethanol-induced gastric lesions in the ethanol control group showed extensive damage to the gastric mucosa, necrotic lesions penetrating deeply into the mucosa, extensive edema and leukocyte infiltration of the submucosal layer (Figure 5). Rats in the experimental groups had better protection of the gastric mucosa compared with the controls as observed by a reduction of mucosal lesions, submucosal edema and leukocyte infiltration (Figure 5). AMDCP has been shown to exert protective effects in a dose-dependent manner.
10.1371/journal.pone.0059296.g005Figure 5 Histological study of ethanol-induced gastric mucosal damage in rats.
In the negative control group no injuries to the gastric mucosa were observed (A). The ethanol control group showed severe disruption to the surface epithelium (black arrow), gastric lesions penetrating deeply into the mucosa, and extensive edema of the submucosal layer (yellow arrow), and leukocyte infiltration (blue arrow) was present (B). The reference control group showed mild disruption of the surface epithelium mucosa. There was edema and leukocyte infiltration of the submucosal layer (C). Group 4 (1 mg/kg AMDCP) exhibited moderate disruption of the surface epithelium. There was edema with leukocyte infiltration of the submucosal layer (D). Group 5 (5 mg/kg AMDCP) had mild disruption of the surface epithelium. There was no edema or leukocyte infiltration of the submucosal layer (E). Groups 6, 7 and 8 (10, 15 and 20 mg/kg AMDCP, respectively) did not show any disruption to the surface epithelium. There was no edema or leukocyte infiltration of the submucosal layer (F, G and H) (H & E stain, 10x).
Immunohistochemistry
The immunohistochemical results of the experimental groups demonstrated that the pre-treatment caused over-expression of Hsp70 protein. The expression of Hsp70 protein in the experimental groups was up-regulated compared to Hsp70 expression in the control groups (Figure 6). Immunohistochemical staining of Bax protein demonstrated that the experimental groups showed decreased expression of the Bax protein. Expression of Bax protein in the experimental groups was down-regulated compared to the ethanol control group (Figure 6).
10.1371/journal.pone.0059296.g006Figure 6 Immunohistochemical analysis of Hsp70 and Bax proteins expression in the stomachs of rats with ethanol-induced gastric mucosal lesions.
Immunohistochemical staining of the Hsp70 proteins (First row) and Bax proteins (second row); the negative control group (A and D), the ethanol control group (B and E) and the treated group with 20 mg/kg AMDCP (C and F). Arrows indicate the proteins in situ (10x).
PGE2, MDA levels and protein concentration of the gastric tissue homogenate
In gastric tissue homogenates, PGE2 activity in the ethanol control group was significantly lower than that in the negative control group (Figure 7). Administration of AMDCP before ethanol treatment significantly increased the level of PGE2 compared to that of the ethanol control group. Administration of ethanol significantly increased the level of MDA in gastric homogenate in the ethanol control group compared to the negative control group. Administration of AMDCP decreased the MDA level in gastric tissues compared to the ethanol control group (Figure 8). Protein concentration in gastric homogenates was significantly decreased in the ethanol control group compared with the negative control group. Administration of AMDCP significantly increased the protein content of gastric homogenate compared with the ethanol control group (Figure 9).
10.1371/journal.pone.0059296.g007Figure 7 Effects of AMDCP on PGE2 in the gastric mucosal homogenates from rats.
Groups 1, 2 and 3 represent the negative control group, the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
10.1371/journal.pone.0059296.g008Figure 8 Effect of AMDCP on the level of MDA in the gastric mucosal homogenates from rats.
Groups 1, 2 and 3 represent the negative control group, the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
10.1371/journal.pone.0059296.g009Figure 9 Effect of AMDCP on protein concentration in the gastric mucosal homogenates from rats.
Groups 1, 2 and 3 represent the negative control group, the ethanol control group and the reference control group, respectively. The experimental groups are presented as groups 4–8. All values are expressed as the means ± standard error of the mean. Mean difference is significant at the p<0.05 level (one-way between groups ANOVA with post-hoc analysis). * significant when compared with the group 2. # significant when compared with the group 3.
Discussion
Imbalance between the protective and aggressive mechanisms of the mucosa, which may be triggered by several endogenous factors and aggressive exogenous factors, is the main cause of peptic ulcers [25]. Ethanol treatment produces necrotic lesions by direct necrotizing action, which in turn reduces defensive factors, such as the secretion of bicarbonate and the production of mucus [26]. Ethanol can reach the mucosa by disrupting the mucus-bicarbonate barrier, causing cell rupture in the walls of blood vessels. These effects are most likely due to biological actions, such as lipid peroxidation, formation of free radicals, intracellular oxidative stress, changes in permeability and depolarization of the mitochondrial membrane prior to cell death [27]. In addition, ethanol treatment produces linear hemorrhagic lesions, extensive submucosal edema, mucosal friability, inflammatory cell infiltration, and epithelial cell loss in the stomach. These symptoms are typical characteristics of alcohol injury [28].
In the present study, AMDCP did not show any signs of toxicity or mortality in any of the acute toxicity tests performed. Behavioral changes, such as irritation, restlessness, respiratory distress, abnormal locomotion and catalapsy, over a period of 14 days were not observed. The observed decrease in acidity and increase in the gastric wall mucus in response to AMDCP is consistent with the results reported previously by Al-Attar [29]. Similarly, Hajrezaie et al.
[10] reported a reduction in gastric acidity in treated animals. Omeprazole exhibits both an anti-secretory and a protective effect [30]. Omeprazole, a proton pump inhibitor (PPI), offered some protection to the gastric mucosa and has been widely used as an acid inhibitor in the treatment of disorders related to gastric acid secretion [31]. In addition to its anti-secretory effect and effectiveness in acid-dependent ulcer models, omeprazole is also effective in acid independent models, such as in the ethanol-ulcer model. In the ethanol-ulcer model, omeprazole exhibits mucosal protection at doses that do not inhibit secretion [32], [33]. Similarly, H2 blocking drugs can also induce gastroprotection at non-anti-secretory doses [34].
Absolute alcohol extensively damaged the gastric mucosa, leading to increased neutrophil infiltration into the gastric mucosa. Activation and infiltration of neutrophils appear to be involved in the initial processes of lesion formation. In the present study, histopathology results also revealed the protection of gastric mucosa and inhibition of leukocyte infiltration into the gastric wall of rats pre-treated with AMDCP. Previous studies demonstrated that the reduction of neutrophil infiltration into gastric lesion promotes the prevention of gastric mucosal lesions in rats [10], [21], [35]. Wasman et al.
[36] showed that oral administration of the Polygonum minus aqueous leaf extract prior to ethanol administration significantly decreased neutrophil infiltration into the gastric mucosa. In the present study, we observed flattening of the mucosal folds, which suggests AMDCP exerts a gastroprotective effect. Relaxation of circular muscles may protect the gastric mucosa by flattening the folds. Flattening of the mucosal folds increases the mucosal area exposed to necrotizing agents and reduces the volume of the gastric irritants on the rugal crest [21], [36]. It was shown that enhanced gastric motility may contribute to the development of gastric mucosal lesions [6]. Ethanol produces a marked contraction of the circular muscles of the rat fundic strip. Such a contraction can lead to mucosal compression at the site of the greatest mechanical stress, the crests of mucosal folds, leading to necrosis and lesion [20]. Gastric tissue homogenates from experimental groups showed significantly decreased levels of MDA and elevated levels of PGE2 in response to oxidative stress due to absolute ethanol administration. MDA is the final product of lipid peroxidation and is used to determine the level of lipid peroxidation in tissues [37]. PGE2 plays an important role in the regulation of gastric mucus secretion [38]. PGE2 has exhibited protective effects in various models of gastric lesion [39], [40]. PGE2, the most abundant gastrointestinal prostaglandin, regulates functions of the gut, including motility and secretion. PGE2 has also been shown to protect the stomach by the activating EP receptors [41] and by modulating gastrointestinal mucosal integrity [5]. The results of the present study suggest that the gastroprotective effect of AMDCP is partially mediated by PGE2. A direct measurement of the PGE2 mucosal level confirmed that its biosynthesis was significantly enhanced by AMDCP. It has been shown that prostaglandins influence virtually every component of the mucosal defense [38]; stimulating mucus and bicarbonate secretion, maintaining mucosal blood flow, enhancing the resistance of epithelial cells to injury induced by cytotoxins and inhibiting leukocyte recruitment [42].
Hsp70 proteins defend cells from oxidative stress or heat shock. Ethanol-generated ROS normally inhibits the expression of Hsp70 and increases the expression of Bax. Hsp70 prevents partially denatured proteins from aggregating and allows them to refold. The over-expression of Hsp70 observed in this study suggests that AMDCP protected the gastric tissues by up-regulating Hsp70.
Conclusion
In conclusion, the acute toxicity study demonstrated that rats treated with AMDCP (2000 mg/kg) manifested no abnormal signs. This synthetic steroid could significantly protect the gastric mucosa against ethanol-induced injury. This protection was ascertained grossly by a significant increase in the gastric wall mucus in comparison with the ethanol control group. Additionally, a reduction of mucosal lesions in the gastric wall and reduction or inhibition of edema and leukocyte infiltration in the submucosal layers was shown histologically. Immunohistochemical staining for the Hsp70 and Bax proteins showed over-expression of the Hsp70 protein and down-regulation of the Bax protein in rats pre-treated with the synthetic steroid. Assays for the levels of PGE2 and MDA in gastric tissue homogenates revealed that AMDCP significantly increased the amount of PGE2 and decreased the level of lipid peroxidation (MDA) in the experimental groups compared to the ethanol control group. This study provides evidence that AMDCP possesses a gastroprotective effect, which is partially due to the preservation of gastric mucus secretion, increased production of Hsp70 protein, and the presence of antioxidant enzymes.
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23516466PONE-D-12-2180410.1371/journal.pone.0058361Research ArticleBiologyGeneticsCancer GeneticsMolecular Cell BiologyGene ExpressionMedicineOncologyBasic Cancer ResearchMetastasisTumor PhysiologyCancer TreatmentCytokine TherapyCancers and NeoplasmsLung and Intrathoracic TumorsNon-Small Cell Lung CancerOncology AgentsSurgerySurgical OncologyDifferential Expression of the RANKL/RANK/OPG System Is Associated with Bone Metastasis in Human Non-Small Cell Lung Cancer RANKL/RANK/OPG Expression in Human NSCLCPeng Xianbo Guo Wei
*
Ren Tingting Lou Zhiyuan Lu Xinchang Zhang Shuai Lu Qunshan Sun Yifeng
Musculoskeletal Tumor Center, Peking University People's Hospital, Beijing, People's Republic of China
Samant Rajeev Editor
University of Alabama at Birmingham, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: WG XP. Performed the experiments: XP TR XL. Analyzed the data: XP WG ZL. Contributed reagents/materials/analysis tools: SZ QL YS. Wrote the paper: XP WG.
2013 13 3 2013 8 3 e5836118 7 2012 6 2 2013 © 2013 Peng et al2013Peng et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Human non-small cell lung cancer (NSCLC) patients exhibit a high propensity to develop skeletal metastasis, resulting in excessive osteolytic activity. The RANKL/RANK/OPG system, which plays a pivotal role in bone remodeling by regulating osteoclast formation and activity, is of potential interest in this context.
Materials and Methods
Reverse transcriptase polymerase chain reaction, western blotting, and immunohistochemical analysis were used to examine the expression of RANKL, RANK, and OPG in human NSCLC cell lines with different metastatic potentials, as well as in 52 primary NSCLC samples and 75 NSCLC bone metastasis samples. In primary NSCLC patients, the expression of these proteins was correlated with clinicopathological parameters. Recombinant human RANKL and transfected RANKL cDNA were added to the PAa cell line to evaluate the promoter action of RANKL during the process of metastasis in vitro and in vivo.
Results
Up-regulated RANKL, RANK, and OPG expression and increased RANKL:OPG ratio were detected in NSCLC cell lines and in tumor tissues with bone metastasis, and were correlated with higher metastatic potential. The metastatic potential of NSCLC in vitro and in vivo, including migration and invasion ability, was significantly enhanced by recombinant human RANKL and the transfection of RANKL cDNA, and was impaired after OPG was added. The increased expression of RANKL and OPG correlated with tumor stage, lymph node metastasis, and distant metastasis.
Conclusions
Differential expression of RANKL, RANK, and OPG is associated with the metastatic potential of human NSCLC to skeleton, raising the possibility that the RANKL/RANK/OPG system could be a therapeutic target for the treatment of metastatic NSCLC patients.
This work was funded by the National Natural Science Foundation of China (No. 30973020 and 81001193). Http://www.nsfc.gov.cn/Portal0/default152.htm. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Non-small cell lung cancer (NSCLC) is the most commonly diagnosed malignancy and the main cause of cancer-related deaths in Asian and Western populations, with over 150,000 people expected to die annually from this disease[1]. The skeleton represents the most common site of tumor metastasis. Approximately 9% to 30% of patients with lung cancer develop bone metastases, which lead to significant morbidity from spinal cord compression, pathologic fractures, and intractable pain[2], [3]. Despite the high rate of metastasis, the underlying molecular mechanisms that regulate the ability of lung cancer cells to proliferate and invade remain poorly understood, and successful treatment modalities remain elusive.
To develop effective treatments for bone metastasis, it is necessary to clarify the molecular mechanisms underlying tumor-induced changes in the bone microenvironment. Normally, there is a closely coordinated balance in which osteoclast-mediated bone resorption and osteoblast-mediated bone formation counteract and contribute to the constant remodeling of bone structure[4]. When lung cancer metastasizes to bone, disruption of this balance can lead to increased bone resorption, resulting in excessive osteolytic activity and consequent skeletal disease[5].
Recent reports have brought to light a new receptor-ligand system belonging to the tumor necrosis factor (TNF) superfamily: the receptor activator of nuclear factor (NF)-kB (RANK), its ligand (RANKL), and the protein osteoprotegrin (OPG)[6], [7]. RANKL, a membrane-bound protein expressed primarily on the surface of osteoblasts and bone marrow stromal cells, binds to RANK on the surface of osteoclast precursors, stimulating their differentiation into mature osteoclasts[8]–[10]. OPG, a decoy receptor of RANKL that is also produced by osteoblast/stromal cells, can prevent bone destruction by blocking the binding between RANKL and RANK, thereby inhibiting osteoclast differentiation and activation[6], [11]. Dysregulation of the RANKL/RANK/OPG system has been detected in several tumors, such as breast cancer[12], [13], prostate cancer[14], malignant bone tumors (e.g., multiple myeloma, giant cell tumors of bone, and chondroblastoma)[15]–[17], squamous cell carcinoma[18], and Hodgkin disease[19]. Previous studies have shown that RANKL is necessary for the development of osteolytic lesions in bone[20]. In addition, by blocking the RANKL-RANK interaction, osteolytic lesions have been successfully inhibited in several types of cancer, including multiple myeloma and prostate cancer[21]–[23].
In lung cancer, the RANKL/RANK/OPG system has been detected in both the presence and absence of bone metastases. Elevated serum levels of soluble RANKL and OPG have been reported in lung cancer patients with bone metastases[24]. Recently, it was reported that RANKL also triggers the migration of human tumor cells that express RANK[25]. Furthermore, RANK-Fc, a chimeric protein that inhibits the RANK-RANKL interaction, has been proven to resist osteoclastogenesis[26]. Accordingly, we hypothesize that the expression of RANKL/RANK/OPG may correlate with NSCLC progression. In this study, expression of RANKL, RANK, and OPG were examined in various human lung cancer cell lines with different metastatic potentials. Recombinant human RANKL and transfected RANKL cDNA were added to an NSCLC cell line to evaluate the promoter action of RANKL in the process of metastasis. Immunohistochemical analysis was carried out to assess the expression of RANKL, RANK, and OPG in primary NSCLC tumors, as well as in bone metastatic tissues of NSCLC. Expression of these proteins was correlated with clinicopathological parameters of primary NSCLC.
Materials and Methods
Patients
The present study investigated surgical biopsy samples of 127 patients with NSCLC, including 52 cases of newly diagnosed NSCLC and 75 cases of bone metastasis of NSCLC. All patients were treated at the Peking University People's Hospital and received no therapy at the time of sample collection. Detailed clinical data regarding these patients is provided in Table 1. The NSCLC patients were staged according to the American Thoracic Society TNM classification, and graded histologically as either well, moderate, or poorly differentiated. The study was approved by the Institutional Review Boards of Peking University People's Hospital. The ethics committees approved this procedure. Informed consent for the experimental use of surgical specimens was obtained from all patients in written form according to the hospital's ethical guidelines.
10.1371/journal.pone.0058361.t001Table 1 Correlations between RANKL, RANK, and OPG expression and clinicopathological parameters in NSCLC patients.
NO. of positive(%)
Parameters Patients(no.) RANKL RANK OPG
Gender
Male 36 18(50%) 21(58.3%) 28(77.8%)
Female 16 10(62.5%) 11(68.8%) 13(81.3%)
P
0.404 0.476 0.777
Age
<59.3 28 14(50%) 18(64.3%) 20(83.3%)
>59.3 24 14(58.3%) 14(58.3%) 21(87.5%)
P
0.548 0.66 0.157
Smoking history
No 20 11(55%) 13(65%) 15(75%)
Yes 32 17(53.1%) 19(59.4%) 26(81.3%)
P
0.895 0.685 0.591
Histotype
adenocarcinoma 30 16(53.3%) 20(66.7%) 25(83.3%)
squamous carcinoma 22 12(54.5%) 12(54.5%) 16(72.7%)
P
0.931 0.375 0.355
Pathological tumor stage
T1/2 38 15(39.5%) 15(39.5%) 12(31.6%)
T3 14 11(78.6%) 10(71.4%) 9(64.3%)
P
0.012* 0.041* 0.033*
Lymph node metastasis
Negative 31 13(41.9%) 19(61.3%) 13(41.9%)
Positive 21 15(71.4%) 13(61.9%) 16(76.2%)
P
0.036* 0.964 0.015*
Distant metastasis
Negative 43 21(48.8%) 23(53.5%) 15(34.9%)
Positive 9 8(88.9%) 7(77.8%) 7(77.8%)
P
0.028* 0.18 0.018*
Histological grade
Poorly-differentiated 11 10(90.9%) 7(63.6%) 8(72.7%)
Well differentiated 41 18(43.9%) 25(60.9%) 33(80.5%)
P
0.005* 0.255 0.576
Cell culture and reagents
Human lung cancer cell lines PG-BE1, PG-LH7, and PAa were purchased from the Institute of Pathology, Peking University Health Science Center (Beijing, China). Cells were cultured in RPMI 1640 (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Recombinant human protein RANKL and OPG were purchased from Prospec Biotechnology Inc. (Prospec, Ness-Ziona, Israel, Cat No: CYT-334 and CYT-633). Antibodies raised against RANK, RANKL, and OPG were purchased from Abcam Inc. (Abcam, MA, USA, Cat No: ab12008, ab9957 and ab73400). β-actin antibody was acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA, Cat No: sc-130065).
Animals
Six week-old male severe combined immunodeficient (SCID) mice weighing 20–25 g were used for this study. The animal study was approved by the Institutional Review Boards of Peking University People's Hospital. Animals were obtained from the Model Animal Research Center of Peking University Health Science Center, housed under pathogen-free conditions in accordance with the NIH guidelines using an animal protocol approved by the Animal Care and Use Committee at the college.
RT-PCR and quantitative real-time PCR
Total RNA was extracted from PG-BE1, PG-LH7, and PAa cells by a single-step method using TRIzol reagent (Invitrogen, La Jolla, CA, USA) according to the manufacturer's instructions, and the RNA was subsequently reverse transcribed into complementary DNA. The specific primers used for DNA amplification are summarized in Table 2. The amplified PCR product was fractionated through 1.5% agarose gel electrophoresis, photographed under ultraviolet light, and analyzed by densitometry. Quantitative real-time PCR was performed in an ABI Prism7300 Sequence Detection System (Applied Biosystems, Beverly, MA, USA) using a GoTaq qPCR Master Mix A6001 kit (Promega, Madison, WI, USA). The thermal profile was 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 58°C for 30 s. The mRNA expression of RANKL/RANK/OPG was analyzed using the 2−(ΔΔCt) method (PAa cell line as a calibrator) based on Ct values for both target and reference genes. The quantity of each transcript was normalized against a known quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. Each experiment was performed in triplicate. Results of real-time PCR analysis are given as mean±the standard error of the mean (SEM). Thermal dissociation plots were examined for biphasic melting curves, indicative of whether primer-dimers or other nonspecific products could be contributing to the amplification signal.
10.1371/journal.pone.0058361.t002Table 2 Primers used in RT-PCR.
Gene Sequence (sense and antisense) Product size(bP)
RANKL
5′- GCCAGTGGGAGATGTTAG -3′
486
5′- TTAGCTGCAAGTTTTCCC -3′
RANK
5′- CAGGGATCGATCGGTACAGT -3′
592
5′- GTTTGAGACCAGGCTGGGTA -3′
OPG
5′- GAACCCCAGAGCGAAATACA -3′
602
5′- TATTCGCCACAAACTGAGCA -3′
MMP-9
5′- TCCCTGGAGACCTGAGAACC -3′
307
5′- GGCAAGTCTTCCGAGTAGTTT -3′
GADPH
5′- ACCACAGTCCATGCCATCAC -3′
450
5′- TCCACCACCCTGTTGCTGTA -3′
β-actin
5′- CTCCATCCTGGCCTCGCTGT -3′
268
5′- GCTGTCACCTTCACCGTTCC -3′
Western blot analysis
Western blot analysis was performed as previously described[27]. Briefly, proteins were separated on a 10% denaturing polyacrylamide gel and transferred to a PVDF membrane. Individual immunoblots were performed with primary antibodies raised against RANK (1∶500), RANKL (1∶1000), OPG (1∶500), and β-actin (1∶1000). Membranes were blocked in TBS-T containing 5% nonfat dry milk, and then incubated overnight with primary antibody. Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, CA, USA) for 1 h. ECL reagent (GE Healthcare, NJ, USA) was used for protein detection. Each experiment was performed in triplicate.
Immunohistochemistry
Paraffin sections were reacted with rabbit polyclonal anti-RANKL antibodies (1∶500 dilution), mouse polyclonal anti-RANK antibodies (1∶200 dilution) or rabbit anti-OPG antibodies (1∶100 dilution), as described previously[27]. Sections stained with non-immune rabbit or mouse serum (1∶200 dilution) in phosphate-buffered saline (PBS) instead of primary antibody served as negative controls. To evaluate RANKL, RANK, and OPG staining, cancer cells exhibiting positive staining on cell membranes and in cytoplasm were counted in at least 10 representative fields (400× magnification) and the mean percentage of positive cancer cells was calculated. Cases in which the proportion of positive cancer cells was ≥50% were defined as positive, and those containing <50% positive cancer cells were defined as negative. Immunostaining was assessed by two independent pathologists blinded to clinical characteristics and outcomes.
Computer-assisted image analysis of immunohistochemistry
The immunostaining of all antibodies was quantitatively analyzed by using a computer-assisted image analysis system. Briefly, images of stained sections were captured with a Leica digital camera and processed using Image Pro Plus analysis software (version 6.0; Media Cybernetics, Rockville, MD, USA). The threshold was set by determining the positive staining of control sections and was used to automatically analyze all recorded images of all samples that were stained in the same session under identical conditions. The area of immunostained regions was calculated automatically by the software in each microscopic field. Pixel counts of the immunoreaction product were calculated automatically and were given as total density of the integrated immunostaining over a given area of the sections. This reflects the relative amount of proteins detected by the antibodies on the tumor sections. The ratio of RANKL/OPG was calculated from integrated immunostaining densities of RANKL and OPG in each group.
RANKL cDNA transfection
Full-length human RANKL cDNA(RefSeq: NM_033012.2) was inserted into the eukaryotic vector pCMV6-XL5 (purchased from OriGene Technologies, Inc. Cat No: SC305532). PAa cells were transfected with the recombinant plasmid or vector alone (mock transfectants) using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA, USA) as described by the manufacturer, and cultured in RPMI1640 supplemented with 10% FBS and 500 µg/ml G418 (AMRESCO, OH, USA). RANKL stable transfectants(named PAa-RANKL) and mock transfectants(named PAa-mock) were established and maintained in RPMI1640 supplemented with 10% FBS and 250 µg/ml G418.
In vitro migration and invasion assays
Migration and invasion assays were performed by seeding 3×105 cells in 200 µl RPMI1640 on top of Transwell cell culture inserts containing a polyethylene terephthalate membrane pre-coated with or lacking Matrigel (24-well inserts, 8.0 µm pore size; Coster, Corning Inc., Corning, NY, USA). The lower chamber was filled with 0.8 ml RPMI1640, with or without added human recombinant RANKL and with or without human recombinant OPG. After incubation for 24 h, the non-migrating cells were scraped off and the membranes were fixed and stained using the Diff-Quik stain kit (Sysmex Co., Hyogo, Japan). Cells that had migrated through the membranes were quantified by determination of the cell number in three randomly chosen visual fields at 200× magnification.
Tibial implantation of lung cancer cells
A murine intratibial injection model of bone metastasis was used to create osteolytic lesions in this study.[28]–[30] The lung cancer cells (5×105 cells) were suspended in 10µl of 1% PBS and mixed with 10µl of matrigel (BD biosciences, San Jose, CA) for each tibial injection. 20µl of the cells and matrigel mixture was injected into the proximal tibia of 6-weeks old SCID mice as published previously[29], [31]. Briefly, the mice were anesthetized using isoflurane (1.5–2%) and oxygen. The overlying skin was prepped in sterile fashion with 70% ethanol and betadine. A 3-mm longitudinal incision was made over the patellar ligament with a number 12 scalpel blade, and then a 2-mm longitudinal incision was made along the medial border of the patellar ligament to the tibial plateau. A 26 1/2 gauge needle was introduced through the proximal tibial plateau and 20µl of lung cancer cells and matrigel mixture was injected into the medullary cavity. The wound was closed with a single 5–0 Vicryl suture (Ethicon Inc.).
Animal study groups
In this study, fifty male SCID mice underwent tibial implantation with lung cancer cells and were equally divided into five study groups. Group I (PAa) animals received intratibial injection of PAa cells alone. Group II (PAa-Mock) tibias received PC-3 cells that were transfected with an empty vector to control for transfection. Group III (PAa-RANKL) animals received PAa cells that were transfected with a vector over expressing RANKL cDNA. Tibias in Group IV (PAa-RANKL+OPG) were implanted with PAa-RANKL cells and animals were subsequently treated with OPG. OPG was used in dose of 10 mg/kg dissolved in a 100µl of phosphate buffer saline(PBS) and was injected subcutaneously three times a week starting on the day of tibial implantation of cancer cells and continued for a total of 8 weeks. Group V(PAa-RANKL+PBS) tibias were implanted with PAa-RANKL cells and the mice were treated with 100µl PBS, which was also injected subcutaneously three times a week for 8 weeks.
Radiotracer preparation
Fluoride ion was produced using O-18 water and proton bombardment using a RDS cyclotron (CTI). 18F-fluoride ion was produced at specific activities of approximately 1000 Ci/mmol and 18F-FDG was synthesized at specific activities of approximately 5000 mCi/mmol as published previously[30].
Micro PET/CT imaging protocol
Animals in the imaging subgroups underwent positron emission tomography(PET) and micro CT scans at 8 weeks at the author's institution according to a previously published protocol[30]. Briefly, mice were anesthetized with isoflurane (1.5–2%) and oxygen in induction chambers. The mice were then directly injected with approximately 250 μCi of 18F-FDG via tail vein using a 27 gauge needle threaded to a polyethylene catheter. The animals were administered maintenance anesthesia with 2% isoflurane in the isolation bed system during the period of radiotracer uptake. Bladders were manually expressed 5-min prior to imaging and animals were positioned in a portable multimodality bed system consisting of a lucite chamber with anesthesia ports and raised platform. Whole-body scans were performed with a 10-minute acquisition time using a MicroPET® FOCUS 220 system (CTI Concorde Microsystems LLC). Immediately afterwards, a non-contrast enhanced micro CT study using microCAT® II (ImTek Inc.) imaging system was used to scan animals with a 10-minute acquisition time. PET scan images were reconstructed using filter-back projection. MicroPET and microCAT® images were then merged for analysis for use with AMIDE® software.
Quantitative analysis of micro PET/CT data
PET and CT scan data was analyzed and quantified by AMIDE® (A Medical Image Data Examiner) version 0.7.154 as published previously[30]. 18F-FDG uptake correlates with the cellular glucose metabolism and was used in micro PET imaging for the detection and longitudinal monitoring of tumor burden. Briefly, regions of interest (ROIs) were drawn using a ROI tool over bilateral tibial plateaus that were three-dimensionally reconstructed to confine all discernible signal uptakes. Using ROI boxes of the same size, data analysis tools were used to calculate maximum and mean signal intensity in both tumor implanted tibias and the contralateral uninjected tibias. The contralateral tibia was used as an internal control for each animal. To quantify the tumor size, 3D isocontour ROI was drawn in the tumor tibia using the maximum FDG signal intensity in the contralateral tibia as the threshold, and FDG signal volume (mm3) was then calculated in the tumor implanted tibias. Micro CT images were used to identify and quantify osteolytic lesions.
Tumor burden measurements
Animals were sacrificed after micro PET/CT scan at 8 weeks, and their tumors in hind limb were harvested for soft-tissue measurement. The soft tissue tumor burden was calculated using the formula as published previously[29], [32].
Statistical analysis
Data from image analysis of sections are expressed as mean±the standard error of the mean (SEM) of each group. Statistical analyses were performed using t-test and analysis of variance (ANOVA), with p-values<0.05 considered statistically significant. Pearson's chi-squared test was used to determine the correlation between RANKL/RANK/OPG expression and clinicopathological parameters. All data were analyzed using SPSS 15.0 software (SPSS Inc., Chicago, Illinois, USA).
Results
Expression of RANKL, RANK, and OPG in human lung cancer cell lines
First, we determined the metastatic potential of PG-BE1, PG-LH7, and PAa cells. PG-BE1 cells more strongly penetrated the filter than the other cell lines, and the number of migrated PAa cells was minimal (p<0.05; Figure 1A,B). Moreover, the level of matrix metalloproteinase-9 (MMP9) mRNA observed through RT-PCR analysis was similar to the level observed with Transwell inserts (p<0.05; Figure 1C). All of above indicated that these three cell lines had different metastatic potentials. Next, the expression of RANKL, RANK, and OPG was evaluated in all three cell lines at transcriptional and protein levels using quantitative real-time PCR and western blotting. All three NSCLC cell lines exhibited different levels of RANKL, RANK, and OPG expression. Of the three cell lines, PG-BE1 cells (which had the highest metastatic potential) demonstrated the strongest expression of RANKL, RANK, and OPG (p<0.05 vs. other cell lines; Figures 2 and 3). PAa cells, which were the least invasive, exhibited only minimal RANKL, RANK, and OPG staining. Immunocytochemistry confirmed the RANKL, RANK and OPG expression observed with quantitative real-time PCR and western blotting. In addition, a significantly higher RANKL: OPG density ratio was observed in the cells with higher metastatic potential (p<0.05; Figures 2D and 3D).
10.1371/journal.pone.0058361.g001Figure 1 The metastatic potential of PG-BE1, PG-LH7, and PAa cells.
The metastatic potential of three NSCLC cell lines was first determined using an in vitro migration assay (A,B). Differential MMP9 expression in three NSCLC cell lines was further analyzed by RT-PCR (C). Results are expressed as the mean±the standard error of the mean (SEM) of three separate experiments. **p<0.05 for PG-BE1 versus PG-LH7. *p<0.05 for PG-LH7 versus PAa.
10.1371/journal.pone.0058361.g002Figure 2 RANKL, RANK, and OPG mRNA expression in three NSCLC cell lines.
RT-PCR was performed to detect RANKL, RANK, and OPG mRNA levels in PG-BE1, PG-LH7, and PAa cells(A). Quantitative real-time PCR revealed the relative expression of RANKL, RANK, and OPG mRNA in three NSCLC cell lines using the 2−(ΔΔCt) method(PAa cell line as a calibrator). GAPDH and β-actin were used as the internal reference(B,C). Next, the ratio of RANKL: OPG mRNA expression in three NSCLC cell lines was calculated based on Ct values for both target and reference gene(D). Bars represent the mean±the standard error of the mean (SEM) of three different experiments. **p<0.05 for PG-BE1 versus PG-LH7. *p<0.05 for PG-LH7 versus PAa.
10.1371/journal.pone.0058361.g003Figure 3 RANKL, RANK, and OPG protein expression in three NSCLC cell lines.
Western blot analysis was performed to detect RANKL, RANK, and OPG protein levels in PG-BE1, PG-LH7, and PAa cells. Band intensities were normalized to β-actin (A–C). Next, the ratio of RANKL: OPG protein expression was calculated in three NSCLC cell lines (D). Bars represent the mean±the standard error of the mean (SEM) of three different experiments. **p<0.05 for PG-BE1 versus PG-LH7. *p<0.05 for PG-LH7 versus PAa.
Recombinant RANKL stimulated PAa migration and invasion In vitro
With the lowest metastatic potential and the least RANKL expression, the low number of migrated PAa cells was increased by three-fold in the presence of recombinant human RANKL (p<0.05). This effect was blocked by the addition of recombinant human OPG in a dose-dependent manner (Figure 4B). Stimulated invasion also increased by two-fold when recombinant RANKL was added to PAa cells. Similarly, OPG produced a dose-dependent reduction in PAa cell invasion. These results indicated that the RANKL/RANK/OPG system was functional in lung cancer cells.
10.1371/journal.pone.0058361.g004Figure 4 Recombinant RANKL and RANKL cDNA stimulated PAa migration.
Western blot analysis demonstrated higher RANKL protein expression in PAa-RANKL cells compared with PAa and PAa-Mock cells (A). Recombinant RANKL stimulated PAa cell migration, and the effect of RANKL administration was blocked by adding OPG to the culture medium in a dose-dependent manner. *p<0.05 for 300 ng/ml recombinant RANKL versus the control and 200 ng/ml OPG-treated samples (B). Increased migration of PAa-RANKL cells in vitro was demonstrated, and could be blocked by adding OPG to the culture medium. *p<0.05 for PAa-RANKL versus PAa, PAa-Mock and 200 ng/ml OPG-treated samples (C). Results are reported as the mean±the standard error of the mean (SEM) of triplicate assays.
RANKL cDNA transfection stimulated PAa migration and invasion in vitro and vivo
To further elucidate the biological functions of the RANKL/RANK/OPG system in NSCLC, we used the transfection technique to specifically regulate RANKL gene expression in PAa cells. PAa cells were transfected with plasmid DNA encoding the full-length RANKL gene. After G418 screening for several weeks, the stable transfectant cell line PAa-RANKL was established. RANKL expression was significantly up-regulated in this cell line compared with the original PAa line and the mock transfectant with pCMV6-XL5 vector (PAa-Mock) (p<0.05; Figure 4A).
Next, we investigated the effect of up-regulation of RANKL expression on the metastatic behavior of tumor cells by transwell assay. The number of migrated cells was over two-fold higher in PAa-RANKL cells than in PAa and PAa-Mock cells; the number of invaded cells was also two-fold higher. Both of these effects were blocked by adding OPG to the culture medium in a dose-dependent manner (Figure 4C).
To further investigate the role of RANKL in NSCLC in vivo, we established xenograft mice model by intratibial injection of PAa cells or PAa-RANKL cells into the SCID mice. After 8 weeks, we observed that the volume of tumor derived from PAa-RANKL cells was significantly larger than that derived from PAa cells(Table 3). Micro PET analysis also revealed significant difference of bone lesion size between Group(PAa) and Group(PAa-RANKL)(Table 3; Figure 5). In addition, with OPG subcutaneously injected three times a week, both tumor volume and 18F-FDG bone lesion size of Group(PAa-RANKL+OPG) were significantly smaller than Group(PAa-RANKL) and Group(PAa-RANKL+PBS) (Table 3; Figure 5).
10.1371/journal.pone.0058361.g005Figure 5 Plain radiographs and 18F-FDG micro PET/CT at 8-weeks in the study groups.
A—Plain radiograph; B—micro CT(transverse view); C—micro PET/CT overlay(transverse view). Plain radiograph and micro PET/CT demonstrated increased bone destruction(white arrows) and 18F-FDG uptake in group(PAa-RANKL) and group (PAa-RANKL+PBS) at 8-weeks following intratibial injection of tumor cells, whereas the increase was inhibited in group(PAa-RANKL+OPG).
10.1371/journal.pone.0058361.t003Table 3 Tumor volumes and 18F-FDG micro PET lesion sizes at 8-weeks.
Group Tumor volume
18F-FDG lesion size
I (PAa) 22.2±6.6 32.3±5.5
II (PAa-Mock) 25.3±7.1 29.6±6.3
III (PAa-RANKL) 95.5±20.3*
120.1±23.2*
IV (PAa-RANKL+OPG) 39.6±13.7**
55.8±12.7**
V(PAa-RANKL+PBS) 92.1±27.8 116.4±25.5
*
P<0.05 versus Groups I and II.
**
P<0.05 versus Groups III and V.
Taken together, these data above demonstrated that the RANKL/RANK/OPG system plays a crucial role in bone metastases of NSCLC in vivo. These findings are consistent with the in vitro studies and clinicopathologic data.
Protein expression of RANKL, RANK, and OPG in primary NSCLC lesions and metastases
Next, we used immunostaining to examine RANKL, RANK, and OPG protein levels in human NSCLC tissue samples. Of 75 NSCLC metastases to bone included in the current study, 60 cases (80%) and 54 cases (72%) exhibited expression of RANKL and RANK, respectively, while 62 cases (82.7%) exhibited OPG expression (Table 4). RANKL and RANK staining were mainly observed in the cell membrane and cytoplasm of cancer cells. Non-neoplastic bone tissues showed weak and focal RANKL, RANK, and OPG staining, and the staining intensity of all three proteins was stronger at the cancer cell/bone interfaces than in the center of the cancer cell nests (Figure 6A). By comparison, in 52 primary cancers only 53.8% and 59.6% of cases exhibited RANKL and RANK expression, respectively, and 63.5% of cases demonstrated OPG expression (p<0.05; Table 4). Significantly stronger immunostaining for all three proteins was observed in bone metastases than in tumor cells at the primary site. This finding was confirmed by quantitative analysis of the immunostaining density of the proteins in each group.
10.1371/journal.pone.0058361.g006Figure 6 Protein staining and RANKL: OPG ratio in NSCLC primary lesions and bone metastases.
Immunocytochemistry for RANKL, RANK, and OPG was performed in tissue sections from primary NSCLC lesions and bone metastases originating from NSCLC, and the staining intensities were evaluated (A). Next, the ratio of RANKL: OPG immunostaining density was calculated in primary NSCLC lesions and bone metastases originating from NSCLC (B). Results are expressed as the mean±the standard error of the mean (SEM) of three separate experiments. *p<0.05.
10.1371/journal.pone.0058361.t004Table 4 RANKL, RANK, and OPG expression in primary and metastatic NSCLC.
NO. of positive(%)
Group Patients(no.) RANKL RANK OPG
Primary 52 28(53.8%) 31(59.6%) 33(63.5%)
Metastasis 75 60(80%) 57(76%) 62(82.7%)
χ2
9.872 4.597 6.009
P
0.002* 0.032* 0.014*
Next, to more accurately assess the immunostaining results, we calculated RANKL: OPG ratios by measuring the optical density of tissues from primary NSCLC and NSCLC bone metastases. The analysis revealed significantly higher RANKL: OPG ratios in bone metastases compared with primary NSCLC tissues (Figure 6B).
Correlation of RANKL, RANK, and OPG expression with clinicopathological parameters of lung cancer
Various clinicopathological features of primary NSCLC patients were compared based on the expression levels of RANKL, RANK, and OPG. As shown in Table 1, RANKL, RANK, and OPG expression were not associated with age, biological sex, smoking history, or histotype. However, clear correlations were established between RANKL and OPG expression and tumor stage, lymph node metastasis, and distant metastasis. Therefore, higher RANKL (78.6%, 71.4%, and 88.9%) and OPG expression (64.3%, 76.2%, and 77.8%) were observed in more advanced metastatic tumors (p<0.05; Table 1). Nearly three-fourths (71.4%) of stage T3/4 tumor samples stained positive for RANK, compared with 39.5% of stage T1/2 tumor samples (p<0.05; Table 1). There was no significant association between RANK expression and lymph node metastasis or distant metastasis. Finally, patients with poorly differentiated histological grade exhibited much higher RANKL expression than those with well differentiated histological grade (90.9% vs. 43.9%, p<0.05); no significant association was observed regarding RANK or OPG.
Discussion
Bone metastases that originate from lung cancer and other malignancies are associated with severe skeletal complications, and lung cancer metastasis to bone remains a significant source of morbidity, with few successful treatment options. Most NSCLC metastases to bone are typically characterized as osteolytic according to radiographic appearance[20], [33], [34]. In normal bone, there is a dynamic balance between osteoclast-regulated bone resorption and osteoblast-regulated bone formation[4]. In the process of bone metastasis, during which that balance is broken, it is vital for tumor cells to arrest in bone marrow, attach to bone surfaces, destroy bone structure, and colonize in bone[35].
Tumor cells within the bone can secrete a variety of factors that stimulate bone cell function, often resulting in osteolysis. Since the 1990s, the RANKL/RANK/OPG system has been regarded as a key mediator of osteoclastogenesis and bone resorption that likely contribute to the underlying pathogenesis of tumor cell metastasis to bone[6], [36]. RANKL is the focus of these events, and previous studies have also reported dysregulation of the RANKL/RANK/OPG system in a number of cancers; the levels of these components appear to be associated with various tumor characteristics[37], [38]. Multiple myeloma, a bone malignancy with purely lytic lesions, has been shown to exhibit high levels of RANKL and low levels of OPG[15]. Similar results have been reported in breast cancer with osteolytic bone metastasis[39]. Studies in several prostate cancer mouse models have shown that RANKL inhibition with OPG-Fc or RANK-Fc can attenuate RANKL-mediated pathologic bone loss and the progression of prostate-originated tumors in bone[38], [40]. These findings strongly suggested the importance of the RANKL/RANK/OPG system in NSCLC bone metastasis, and encouraged us to examine the relationship between the expression levels of three components in the system and the various clinical features of NSCLC and to evaluate the potential of the RANKL/RANK/OPG system as a therapeutic target in the context of bone metastasis of NSCLC origin.
We initially proposed that most NSCLC would contain carcinoma cells with various degrees of RANKL/RANK/OPG expression at the protein and mRNA levels, and that NSCLC characterized by high RANKL expression would have high metastatic potential. To show this, RANKL/RANK/OPG expression was detected in PG-BE1, PG-LH7, and PAa cell lines, which have differing metastatic potentials. PG-BE1, the cell line with the highest metastatic potential, exhibited the strongest expression of RANKL, RANK, and OPG, which revealed a striking relationship between RANKL-RANK interaction and clinical metastasis of NSCLC. Previous studies have shown different levels of RANKL, RANK, and OPG expression in serum from NSCLC patients[24], while the immunohistochemical characterization of these three components has been limited. In this study, immunohistochemical staining demonstrated that the expression of these three system components was significantly higher in bone metastases than in primary lesions. Statistical analyses regarding clinicopathological features showed that RANKL and OPG expression were associated with tumor stage, lymph node metastasis, and distant metastasis, while RANK level correlated with tumor stage only. This finding suggested that the movement of NSCLC cells from primary sites to secondary sites (i.e., regional lymph nodes or distant organs) might depend on the level of RANKL expression. RANK, the receptor for RANKL, is constitutively expressed in a broad range of tissues. Therefore, when high-RANKL tumor cells invade regional tissue or metastasize to lymph nodes, they are liable to be subjected to high concentrations of RANK.
In normal bone, osteoclast activation and inactivation are tightly controlled by a balance between RANKL and OPG, which act as a positive and negative regulator, respectively. Tumor cells may shift osteoclast activation by producing RANKL and/or OPG directly, or through the production of other factors that indirectly stimulate osteoblast/stromal cells to produce RANKL or OPG. This shift may then create a favorable local environment in bone for the seeding of tumor cells and the development of metastases[10]. Previous studies in various tumor types have implicated an altered RANKL: OPG ratio in bone metastasis with severe osteolysis[41]. In the present work, RANKL: OPG ratio was also calculated in NSCLC cell lines and different tumor tissues. The RANKL: OPG ratio was increased at both transcript and protein levels in the most metastatic cell line. Moreover, NSCLC tissues that metastasized to bone exhibited higher RANKL: OPG ratios compared with primary lesions. These findings indicate that there is a predominance of RANKL production toward OPG during the process of metastasis. In other words, RANKL produced by cancer cells may play a pivotal role in bone metastasis, while the level of OPG is increased to counterbalance the high RANKL concentration produced by tumor cells. In this case, OPG acts as a decoy receptor of RANKL, and may be considered a protector of bone environment[6]. However, in bone metastasis with severe osteolysis, OPG production still cannot compensate for the high levels of RANKL released by tumor cells. Such an imbalance of RANKL/OPG has been envisaged for myeloma cells, which modify the human bone marrow environment and induce osteoclastogenesis[41], [42]. This result strengthens our interest in studying RANKL and OPG together by exploring the RANKL: OPG ratio.
The migration and invasion of a particular tumor cell are believed to be associated with its metastatic potential, which could be triggered by chemokine binding to chemokine receptor on the cell surface[43], [44]. According to the literature, RANKL triggers the migration of RANK-expressing cancer cells[45], [46]. We demonstrated that PAa cell lines could express RANK, and that recombinant RANKL protein stimulated the migration and invasion of PAa cells in vitro. Similar results were observed after PAa cells were transfected with RANKL cDNA, which indicated that RANKL expressed in cancer cells can accelerate their migration and invasion in vitro. The effect of RANKL in vitro was inhibited by adding OPG to the culture medium in a dose-dependent manner.
To demonstrate that RANKL and OPG contribute to NSCLC development in vivo, we employed xenograft mice model and found that RANKL overexpression promoted bone destruction and tumor growth of NSCLC cells. The promotion of RANKL was significantly inhibited by OPG subcutaneously injection regularly. In light of the discussion above concerning the compensatory role of OPG with respect to RANKL, it is implied that blocking the RANKL-RANK interaction with OPG might lead to a novel tool in the prevention and treatment of human metastatic NSCLC.
In summary, differential levels of RANKL, RANK, and OPG expression in NSCLC were found to correlate with metastatic potential in vitro and in vivo, suggesting that the movement of NSCLC cells from primary sites to metastatic nodes might depend on RANKL level. Disruption of the RANKL-RANK interaction by antagonists of RANKL, such as OPG, may lead to the design of novel therapeutic tools with which to treat NSCLC patients. Additional studies are warranted to examine the mechanism of action of these proteins in the progression of metastasis, as well as possible crosstalk with other signaling pathways.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23516546PONE-D-12-3711610.1371/journal.pone.0058738Research ArticleMedicineSurgeryTransplant SurgeryArtificial Liver Support System Combined with Liver Transplantation in the Treatment of Patients with Acute-on-Chronic Liver Failure ALSS Combined with LT in Patients with ACLFXu Xiao
1
Liu Xiaoli
2
Ling Qi
1
Wei Qiang
1
Liu Zhikun
1
Xu Xiaowei
2
Zhou Lin
1
Zhang Min
1
Wu Jian
1
Huang Jianrong
2
Sheng Jifang
2
Zheng Shusen
1
*
Li Lanjuan
2
*
1
Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
2
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
Man Kwan Editor
The University of Hong Kong, Hong Kong
* E-mail: [email protected] (LL); [email protected] (SZ)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: XX SZ LL. Performed the experiments: XL QL QW. Analyzed the data: ZL XX LZ. Contributed reagents/materials/analysis tools: MZ JW JH JS. Wrote the paper: XX.
2013 14 3 2013 8 3 e587387 11 2012 5 2 2013 © 2013 Xu et al2013Xu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The search for a strategy to provide temporary liver support and salvage the patients with acute-on-chronic liver failure (ACLF) remains an important issue. This study was designed to evaluate the experience in artificial liver support system (ALSS) combined with liver transplantation (LT) in the treatment of ACLF.
Methodology/Principal Findings
One hundred and seventy one patients with HBV related ACLF undergoing LT between January 2001 and December 2009 were included. Of the 171 patients, 115 received 247 sessions of plasma exchange-centered ALSS treatment prior to LT (ALSS-LT group) and the other 56 received emergency LT (LT group). The MELD score were 31±6 and 30±7 in ALSS-LT group and LT group. ALSS treatment resulted in improvement of liver function and better tolerance to LT. The average level of serum total bilirubin before LT was lower than that before the first time of ALSS treatment. The median waiting time for a donor liver was 12 days (2–226 days) from the first run of ALSS treatment to LT. Compared to LT group, the beneficial influences of ALSS on intraoperative blood loss and endotracheal intubation time were also observed in ALSS-LT group. The 1-year and 5-year survival rates in the ALSS-LT group and LT group were 79.2% and 83%, 69.7% and 78.6%.
Conclusions/Significance
Plasma exchange-centered ALSS is beneficial in salvaging patients with ACLF when a donor liver is not available. The consequential LT is the fundamental treatment modality to rescue these patients and lead to a similar survival rate as those patients receiving emergency transplantation.
Financial support to the project: this work was supported by the National Science and Technology Major Project (2012ZX10002004), the National High Technology Research and Development Program of China (863 Program 2012AA020204) and the National Basic Research Program of China (973 program 2009CB522404). The funders had no role in study design, data collection and analysis, decision to publish, and preparation of the manuscript.
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Introduction
Liver failure remains a disease associated with high mortality. In China, because of the high prevalence of hepatitis B, hepatitis B virus-related acute-on-chronic liver failure (ACLF) is a common cause of liver failure, which is much different from the western countries where drugs, alcohol, and hepatitis C are the major causes. According to Asian Pacific Association for the Study of the Liver, ACLF has been recently defined as ‘acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease [1].
Liver transplantation (LT) is the best treatment for salvaging patients with ACLF. However, LT is not always possible because of donor shortage. Enormous attempts at providing temporary liver support have been made, with an aim to increase survival rate or improve the condition of the patient until a donor is available. In the past decades, a variety of artificial liver support system (ALSS), including plasma exchange (PE), hemoperfusion, PE plus continuous hemodiafiltration, MARS and fractionated plasma separation, adsorption and dialysis system, have been employed in the management of liver failure [2].
Our previous study has demonstrated that ALSS can efficiently decrease the mortality of patients with severe hepatitis of early and middle stages [3]. However, for patients with ACLF, since hepatocytes undergo massive denaturation, necrosis and dysfunction, it is difficult to completely reverse the clinical course of the disease with routine medical management and even combined with ALSS. Under this circumstance, LT is usually indispensable. In the present study, we described our experience of PE-centered ALSS combined with LT in the rescue of patients with ACLF.
Methods
2.1 Patients
This study was approved by the First Affiliated Hospital, Zhejiang University School of Medicine and the current regulation of the Chinese Government, and the Declaration of Helsinki were strictly followed for each organ donation and transplant performed in our center. Written informed consents from each donor and recipient were obtained. No donor livers were harvested from executed prisoners. All the data was analyzed anonymously.
From January 2001 to December 2009, 796 patients underwent LT at our hospital. United Network for Organ Sharing status was used as the organ allocation system before December 2002, and Model for End-Stage Liver Disease (MELD) score was applied after January 2003. Their primary diseases included hepatic malignancies (n = 310) and benign end-stage liver diseases (n = 486). Malignancies, retransplantation, and combined transplantation were excluded. Among the 486 cases of benign end-stage liver diseases, 171 cases of ACLF with a known history of chronic hepatitis B or cirrhosis were finally enrolled in this retrospective study. Of these 171 cases, 115 received 247 sessions of ALSS treatment before LT (ALSS-LT group), whereas the other 56 cases fortunately gained appropriate donor livers and received emergency LT within 72 h after their referral to our centre, without any prior ALSS treatment (LT group). The baseline characteristics including age, gender, serum total bilirubin, MELD scores, and major complications of the above two groups were collected at the time of admission and summarized in Table 1. MELD score was calculated as 9.57×loge (Cr mg/dl)+3.78×loge (TB mg/dl)+11.20×loge (international normalized ratio)+6.43 [4]. Lamivudine combined with low-dose intramuscular hepatitis B immunoglobulin therapy was applied in all patients [5]. Immunosuppressive regimen was triple therapy incorporating tacrolimus or cyclosporin A, mycophenolate and steroid [6].
10.1371/journal.pone.0058738.t001Table 1 The baseline patient characteristics.
ALSS-LT group (n = 115) LT group (n = 56)
P value
Age (years) 46±10 45±10 NS
Gender (male/female) 100/15 45/11 NS
TB ( µmol/L) 557±195 537±201 NS
MELD score 31±6 30±7 NS
Infections 33 15 NS
Encephalopathy 54 24 NS
Hepatorenal syndrome 29 12 NS
Abbreviations: ALSS, artificial liver support system; LT, liver transplantation; TB, total bilirubin; MELD, model for End-Stage Liver Disease; HBV, hepatitis B virus.
2.2 ALSS treatment
Blood access was established through a double-lumen catheter via the patient's jugular or femoral vein. The methods of ALSS included PE, plasma perfusion, continuous hemodiafiltration and MARS. PE was performed with plasma separator Plasmacure PS-06 (Kuraray Co., Tokyo, Japan). The total volume of exchanged plasma was about 3300 ml, and the exchange rate of plasma was 22–25 ml/min. Continuous hemodiafiltration was performed with Diafilter D-30NR (Minntech Co., Minneapolis, MN). Plasma perfusion utilizing Adsorba 300C contained 300 g cellulose coated charcoal (Gambro Dialysatoren GmbH Co., KG, Hechingen, Germany). The MARS system (MARS monitor, Teraklin AG, Rostock, Germany) was used, and its albumin circuit, containing 600 mL 20% human albumin, was driven at 150 mL/min. Dexamethasone (5 mg) and prophylactic antibiotics were routinely given. Totally 20–60 mg heparin and 10–30 mg protamine sulphate were given in one run of ALSS treatment.
The detailed methods of PE-centered ALSS were performed based on individuals' conditions. For example, patients with coagulopathy were indicated for PE; when the patient had hepatic encephalopathy, we used PE plus plasma perfusion or continuous hemodiafiltration. For patients complicated with hepatorenal syndrome or imbalance of water or electrolytes, we applied PE plus continuous hemodiafiltration or MARS. In ALSS-LT group, 247 sessions of ALSS were applied to 115 patients, with PE 162 times, PE plus plasma perfusion 52 times, PE plus continuous hemodiafiltration 18 times and MARS 15 times.
2.3 Data Collection
Patient demographic, surgical, and postoperative data were collected by chart review and from surgical records. Serum parameters of serum total bilirubin, alanine aminotransferase, aspartate aminotransferase, total bile acid, creatinine, prothrombin time and electrolytes were closely monitored before and after every session of ALSS treatment or during the perioperative period.
2.4 Statistical analysis
The values were expressed as mean±SD. The data were statistically analyzed by SPSS 10.0 software package (SPSS Inc, Chicago, IL). The laboratory data were compared by Wilcoxon's rank-sum test or Mann-Whitney U test. Chi-square test was used to compare categorical variables. Survival analysis was estimated using Kaplan-Meier method. A P value less than 0.05 was considered statistically significant.
Results
3.1 Efficacy of ALSS
Before ALSS treatment, 115 patients in ALSS-LT group were in poor general condition, complicated by cachexia, fatigue, loss of appetite, abdominal distention, jaundice, hepatorenal syndrome or hepatic encephalopathy. After ALSS treatment, general conditions and clinical symptoms including spirit, sleeping, appetite, and hepatic encephalopathy were improved. The changes of main laboratory parameters in 4 subgroups of ALSS are listed in Table 2. The levels of serum total bilirubin declined markedly by almost 50% on average in all subgroups. PE plus continuous hemodiafiltration and MARS obviously decreased serum creatinine level with the removal rates of 45±9% and 28±4%, respectively. Prothrombin time decreased significantly in PE involved ALSS subgroups (P<0.05).
10.1371/journal.pone.0058738.t002Table 2 Changes of key laboratory parameters pre- and post-artificial liver support system (ALSS) treatment in different subgroups.
Parameters PE (n = 162) PE+continuous hemodiafiltration (n = 52) PE+Plasma perfusion (n = 18) MARS (n = 15)
TB
Pre-treatment ( µmol/L) 575±174 530±165 558±183 511±137
Post-treatment ( µmol/L) 260±96 253±82 261±95 331±115
Removal rate (%) 55±6 52±5 54±5 34±4
P value <0.05 <0.05 <0.05 <0.05
Cr
Pre-treatment ( µmol/L) 80±19 301±102 78±15 198±43
Post-treatment ( µmol/L) 76±15 169±52 74±13 145±29
Removal rate (%) 4±5 45±9 4±4 28±4
P value >0.05 <0.05 >0.05 <0.05
PT
Pre-treatment (s) 32.4±10.3 31.6±8.0 31.2±7.7 28.4±8.4
Post-treatment (s) 20.7±3.6 22.5±6.3 23.6±5.6 30.1±8.8
P value <0.05 <0.05 <0.05 >0.05
Removal rate was calculated as: (pre-treatment concentration—post-treatment concentration)/pre-treatment concentration.
Abbreviations: PE, plasma exchange; TB, total bilirubin; Cr, creatinine; PT, prothrombin time.
3.2 Impact of ALSS on patients' transplantability
One hundred and twenty five patients of ALSS-LT group were successfully bridged to LT after attaining proper donor organs. The average level of TB before LT was significantly lower than that before the first session of ALSS treatment (476±169 µmol/L vs. 557±195 µmol/L, P
<0.05). The average levels of MELD pre-ALSS and pre-LT were 31±6 and 29±9, but no significant difference was found between them (Table 3). The median waiting time for a donor liver was 12 days (range from 2 days to 226 days) from the first run of ALSS treatment to LT.
10.1371/journal.pone.0058738.t003Table 3 Liver function and model for End-Stage Liver Disease (MELD) score before artificial liver support system (ALSS) and before liver transplantation (LT).
TB ( µmol/L) ALT (U/L) AST(U/L) TBA µmol/L) MELD score
Pre-first ALSS 557±195 127±113 182±152 246±98 31±6
Pre-LT 476±169 105±80 165±207 144±108 29±9
P value <0.05 >0.05 >0.05 <0.05 >0.05
Abbreviations: TB, total bilirubin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBA, total bile acid;
3.3 Impact of ALSS on intraoperative blood loss and ICU staying
Compared to those in LT group, there was less blood loss during the operations and shorter endotracheal intubation time for the patients in ALSS-LT group (3941.8±1997.4 ml vs. 5058.3±2193.6 ml, P<0.05; 3.3±2.6 days vs. 4.5±3.6 days, P<0.05). A similar trend was observed in the average ICU staying time but the reduction was not statistically significant (9.8±4.5 days vs. 10.5±4.7 days, P>0.05). (Table 4).
10.1371/journal.pone.0058738.t004Table 4 Intraoperative blood loss, intubation and ICU staying time in the artificial liver support system (ALSS)-liver transplantation (LT) group and LT group.
Blood loss (ml) Intubation time (days) ICU staying time (days)
ALSS-LT group 3941.8±1997.4 3.3±2.6 9.8±4.5
LT group 5058.3±2193.6 4.5±3.6 10.5±4.7
P value <0.05 <0.05 >0.05
3.4 Impact of ALSS combined with LT on patients' survival
In ALSS-LT group, the survival rates of 1-year and 5-year were 79.2% and 69.7%, respectively. Compared with that in ALSS-LT group, the survival of LT group did not show significant difference, with the 1-year and 5-year survival rate of 83% and 78.6%, respectively. No significant differences in survival were found between two groups (Figure 1). However, in another cohort of ACLF patients (n = 158) receiving only conventional medical therapy (no ALSS and no LT), the 1- and 3-month mortality was 79.7% and 90.5%, respectively.
10.1371/journal.pone.0058738.g001Figure 1 Comparison of patient cumulative survival between ALSS-LT group and LT group.
(P = 0.406) (ALSS, artificial liver support system; LT, liver transplantation).
Discussion
The past two decades witnessed the progress of LT [7], [8]. As the only efficient procedure to treat ACLF, LT has been applied with a perioperative mortality rate of less 3% and 1-year survival rate of exceeding 80% for recipients in some major transplant centers in China [9]. At our center, ACLF related to HBV infection has been one of the main indications of LT. The shortage of donor livers, however, will undoubtedly make the patients with critical condition lose the opportunity for LT. For those who undergo LT in a deteriorating status with cachexia and disturbance of internal environment, the outcome would be unsatisfactory. The increasing discrepancy between the number of potential candidates for LT and the number of donor livers available suggests that some therapeutic alternatives for temporary liver support to patients with ACLF should be necessary [10], [11].
Recent studies have shown that extracorporeal liver support systems could temporarily support patients' liver function, improve their preoperative condition, and enhance their tolerance to surgery, thus extending the waiting time for a donor liver as a bridge to LT [12]–[16].
As a promising liver assist system, ALSS can perform partial functions of liver, with important therapeutic potentials in various patients with hepatitis, liver cirrhosis, and acute liver failure [17], [18]. ALSS treatment seemed to reduce the mortality in patients with ACLF [19]. As an important part of ALSS, PE has been long applied in fulminant hepatic failure with aim of removing overabundant toxic substances and correcting the severe coagulopathy [20]. For removal of the hepatic encephalopathic substances such as aromatic amino acids, ammonia and middle molecules, plasma perfusion or continuous hemodiafiltration are often used with PE simultaneously [21], [22]. MARS is actually combining hemodialysis/filtration and plasma perfusion, and serves to remove albumin-bound toxins and water-soluble toxins [23]. To patients complicated with hepatorenal syndrome, PE plus continuous hemodiafiltration and MARS also helped to improve renal function. In this study, PE-centered ALSS including PE alone, PE plus continuous hemodiafiltration, PE plus plasma perfusion and MARS were applied based on individuals' conditions.
Our early study has found that non-biological artificial liver techniques can efficiently decrease the mortality of patients with severe hepatitis of early and middle stages [3]. Our results supported the favorable effects of PE-centered ALSS. Before ALSS treatment, all the patients developed pre-terminal or terminal clinical manifestations, such as hepatic encephalopathy, hepatorenal syndrome, disturbance of water and electrolytes, and other severe complications. The initial serum bilirubin level of 115 patients with ACLF in the ALSS-LT group was as high as 557±195 µmol/L and still in an increasing tendency. After treatment of ALSS, liver and renal function and coagulopathy improved evidently. Neurological improvements were found in patients with encephalopathy following repeated sessions of ALSS treatment. Disorders of the internal environment prior to LT were also corrected to a certain extent, thus facilitating improvement of patients' general condition. The result of this study suggests that for patients with ACLF who are in the waiting list for LT, ALSS should be considered as an important part of preoperative management. When a donor liver is not available, salvaging ALSS should be carried out timely to support liver function and win precious waiting time till a donor liver is available. In the present study, patients after each ALSS treatment showed marked improvement in liver function and stabilized general condition, sustaining patients' lives with a median time of nearly 2 weeks and the longest time of 226 days.
The beneficial influences of ALSS on intraoperative blood loss and ICU staying were also observed. Furthermore, it was demonstrated from our results that the combined treatment of ALSS and LT achieved the same 1-year survival rate as emergency LT which was applied to those critical patients in 72 hours. Although a prospective, randomized, and controlled trial is needed to confirm the beneficial effects of ALSS, our study has undoubtedly demonstrated the efficacy and safety of ALSS in supporting liver function and extending the waiting time for donor livers. In this regard, ALSS for ACLF patients will broaden the indications of LT, and more patients in the waiting list of LT will achieve a new life.
For ACLF patients, however, what ALSS may provide is still a transient liver function support [3], [24]. The biochemical manifestation of liver failure may relapse and approach or even exceed the level before the previous ALSS treatment [25], [26]. Therefore, for the patients who developed massive necrosis of hepatocytes and lost ability of liver regeneration, several times of ALSS and sequential timely LT were needed. Although further prospective and randomized studies should be performed, we believe that ALSS is beneficial in salvaging patients with ACLF when a donor liver is not available and LT is the fundamental treatment modality to rescue these patients.
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Pan Afr Med JPan Afr Med JPAMJThe Pan African Medical Journal1937-8688The African Field Epidemiology Network PAMJ-14-2810.11604/pamj.2013.14.28.1347ResearchDetection of ESBL among ampc producing enterobacteriaceae using inhibitor-based method Bakthavatchalu Sasirekha 1&Shakthivel Uma 1Mishra Tannu 11 Department of Microbiology, Centre for Post Graduate Studies, Jain University, Bangalore, Karnataka- 560 011, India& Corresponding author: Sasirekha Bakthavatchalu, Department of Microbiology, Centre for Post Graduate Studies, Jain University, Bangalore, Karnataka- 560 011, India20 1 2013 2013 14 2810 11 2011 03 1 2013 © Sasirekha Bakthavatchalu et al.2013The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction
The occurrence of multiple β-lactamases among bacteria only limits the therapeutic options but also poses a challenge. A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in bacterial isolates further different phenotypic methods are compared to detect ESBL and AmpC.
Methods
A total of 259 clinical isolates of Enterobacteriaceae were isolated and screened for ESBL production by (i) CLSI double-disk diffusion method (ii) cefepime- clavulanic acid method (iii) boronic disk potentiation method. AmpC production was detected using cefoxitin alone and in combination with boronic acid and confirmation was done by three dimensional disk methods. Isolates were also subjected to detailed antibiotic susceptibility test.
Results
Among 259 isolates, 20.46% were coproducers of ESBL and AmpC, 26.45% were ESBL and 5.40% were AmpC. All of the 53 AmpC and ESBL coproducers were accurately detected by boronic acid disk potentiation method.
Conclusion
The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method that accurately detects the isolates that harbor both AmpCs and ESBLs.
boronic acidESBLampc beta-lactamasesenterobacteriaceaecefepime
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Introduction
The rapid global dissemination of Enterobacteriaceae harboring plasmid-borne extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases represents a significant clinical threat [1, 2]. ESBLs producing organism confer resistance to penicillin, cephalosporins, and monobactams. They cannot hydrolyze cephamycins and are inhibited by clavulanic acid (CA) [3]. Like ESBLs, plasmid-mediated AmpC β-lactamases have a broad substrate profile that includes penicillin, cephalosporins, and monobactams. In contrast to ESBLs, they hydrolyze cephamycins and are not inhibited by commercially available β-lactamase inhibitors [4, 5]. Inappropriate use of cephalosporins in clinical practice led to the emergence of bacteria producing multiple β-lactamases. This leads to therapeutic failure when β-lactam drugs or β-lactam/inhibitor combination are used [6].
The ESBL confirmation methods have been established by Clinical Laboratory Standards Institute (CLSI) and are used worldwide [7]. Currently there are no CLSI recommended guidelines to detect AmpC β-lactamases. Several methods of phenotypic detection of AmpC β-lactamases are described; however, these methods are labor intensive and subjective, lack sensitivity and/or specificity and cannot be adopted on a routine basis. PCR gives satisfactory results, but it is costlier and time consuming, and equipment availability is limited to few laboratories [8–15].
The CLSI recommended phenotypic confirmatory test would fail to detect ESBL in the presence of AmpC, as the latter enzyme is resistant to clavulanic acid [10]. Clavulanic acid induces high level expression of chromosomal AmpC β-lactamases, masking the synergy arising from the inhibition of an ESBL. Thus, the coexistence of both ESBL and AmpC β-lactamases in the same strain may result in false-negative tests for the detection of ESBLs [16].
Boronic acid (BA) derivatives were reported as reversible inhibitors of AmpC enzymes [17, 18]. Several studies have validated the use of boronic acid to detect AmpC β-lactamases among Gram-negative bacteria [16, 19–21]. Rapid and accurate detection of ESBLs and AmpC β-lactamases is important to guide proper antimicrobial therapy and for appropriate infection control measures. Therefore the present study was aimed to evaluate the usage of boronic acid in a phenotypic confirmatory test to detect ESBL among AmpC β-lactamases producing isolates.
Methods
Bacterial isolates
A total of 259 consecutive nonrepetitive clinical isolates of Enterobacteriaceae were isolated from various clinical samples such as urine (n= 103), pus (n=83), sputum (n= 60), blood (n= 9) over a period of six months from January 2010 to June 2010. Samples were processed and isolates were identified on the basis of conventional microbiological procedures [22].
Antimicrobial susceptibility testing
Antibiotic susceptibility was determined by Kirby- Bauer disk diffusion method and the results were interpreted according to the guidelines of the Clinical Laboratory Standard Institute [23]. The antibiotics used were ampicillin (10µg), ticarcillin(75µg), piperacillin(100µg), amoxycillin/ clavulanic acid (20/10µg), ticarcillin/clavulanic acid (75/10µg), piperacillin- tazobactum (100/10µg), aztreonam (30µg), cephotaxime (30µg), ceftazidime(30µg), ceftriaxone (30µg), cefepime (30µg), impenem (10µg), gentamicin (10µg), amikacin (30µg), tetracycline (30µg) and ciprofloxacin (5µg), chloramphenicol (30µg). E. coli ATCC 25922 was used as a quality control strain.
All the 259 isolates were screened for ESBL production by (i) CLSI double-disk diffusion method [23] (ii) cefepime- clavulanic acid method (iii) boronic disk potentiation method. AmpC production was detected using cefoxitin alone and in combination with boronic acid and confirmation was done by three dimensional disk method. Briefly, 5µl of the freshly prepared clavulanic acid (2g/l of PBS at pH 6) was added to cefotaxime (30µg; CTX+CA) and cefepime (30µg; CPM+CA) disks. Then 5 µl of 3- amino phenyl boronic acid (Sigma Aldrich, India) stock solution (60g/l of DMSO) was added to cefotaxime disc with(CTX+CA+BA) and without clavulanic acid(CTX+BA) and also to cefoxitin disc(FOX+BA). The discs were placed onto Mueller hinton agar plates containing lawn culture of 0.5 McFarland unit of test organism. The plates were incubated at 37° C for 18-24 hrs. The results were interpreted as follows:A ≥ 5 mm increase in the zone diameter of the cefotaxime alone (CTX) and in combination with clavulanic acid (CTX+CA) or boronic acid (CTX+BA) was indicative of ESBL or AmpC production
A ≥ 5 mm increase in the zone diameter of CTX+BA and CTX+CA versus CTX+CA+BA was indicative of combined ESBL and AmpC production
A ≥ 5 mm increase in the zone diameter of the CPM alone and in combination with clavulanic acid (CPM+CA) was indicative of ESBL production
A ≥ 5 mm increase in the zone diameter of the Cefoxitin (FOX) alone and in combination with boronic acid (FOX+BA) was considered positive for AmpC production
All 259 isolates were subjected to a modified three dimensional extract test to confirm AmpC production [23].
Results
Of the total 259 Enterobacteriaceae isolates, 115 were Escherichia coli (44.4%), 59 (22.77%) were Klebsiella pneumonia, 41 (15.83%) were Proteus mirabilis, 29(11%) were Enterobacter cloacae, and 15 Citrobacter spp. Among 259 clinical isolates of Enterobacteriaceae, 68 (26.25%) and 14(5.4%) were pure ESBL and AmpC producers respectively; 53 (20.46%) isolates were combined ESBL and AmpC producers; and 124 (47.87%) of the isolates did not harbor any type of enzyme (Table 1). In our study the prevalence of ESBL and AmpC co- producing isolates was 20.46%, which is relatively low (27.5% and 33.7%) compared to the previous report [25, 26]. This variation may be due to different pattern of antibiotic use and differences in the study group.
Table 1 Extended-spectrum beta-lactamases and AmpC producing Enterobacteriaceae
Organisms Pure ESBL (%) Pure AmpC ESBL+ AmpC Negative Total
E coli
30 (26) 3 (2.60) 18 (15.65) 64 (55.65) 115
K pneumoniae
19 (32.20) 4 (6.77) 21 (35.59) 15 (25.42) 59
E cloacae
5 (12.19) 3 (7.31) 6 (14.63) 27 (65.85) 41
P mirabilis
11(37.93) 2(6.89) 5 (17.24) 11(37.93) 29
Citrobacter spp 3 (20) 2 (13.33) 3 (20) 7 (46.66) 15
Total
68 (26.25)
14 (5.40)
53 (20.46)
124 (47.87)
259
ESBL: Extended-spectrum beta-lactamases
CLSI double-disk diffusion method detected all ESBL producers (100%) but in combined ESBL and AmpC failed to detect 16 (30.18%), ESBL producers. The average increases in the zone diameters of the CTX discs in the presence of either CA and BA was 14.1 mm and 13.2 mm respectively were higher than those for the CLSI confirmatory test 11.3 mm and 10.9 mm, respectively. The rate of detection of ESBLs by the CLSI confirmatory test for clinical isolates that produce both ESBLs and AmpC (20.46%) was lower than that for clinical isolates that produce ESBLs but not AmpC (26.45%). If CLSI double-disk diffusion method was used alone, 6% of ESBL producing organisms would have been missed. The average increases in the zone diameters of the CTX disc in the presence of both CA and BA was 10.7 mm and 8.3 mm, which is higher than that of CLSI confirmatory test 7.1 mm and 5.1 mm, respectively. CLSI double-disk diffusion method was able to detect only 105 of 121 ESBL producing isolates but it detected all ESBL negative isolates correctly. Sturenburg et al
[27] reported that the cefepime-clavulanic acid (CPM-CA) method could reliably detect ESBL in the presence of AmpC, in our study CPM+CA potentiated disc detected all ESBL producers whether alone or in combination with AmpC correctly (Table 2).
Table 2 Comparison of phenotypic method with boronic acid disk potentiation method for extended-spectrum beta-lactamases detection
Phenotypes CLSI double-disk diffusion method CTX+BA for AmpC CTX +CA+BA for ESBL + AmpC CPM + CA for ESBL
Positive Negative
Pure ESBL (n= 68) 68 0 68 68 0
Pure AmpC (n= 14) 0 14 14 0 14
ESBL + AmpC (n= 53) 37 43 53 53 0
Negative (n=124) 0 0 0 0 124
Total (n= 259) 105 57 135 121 138
CLSI: Clinical Laboratory Standards Institute; CTX+CA+BA: Cefotaxime disc with clavulanic acid; CTX+BA: Cefotaxime disc without clavulanic acid; ESBL: Extended-spectrum beta-lactamases; CPM-CA: Cefepime-clavulanic acid
Discussion
The occurrence of multiple β-lactamases among bacteria only limits the therapeutic options but also poses a challenge for microbiology laboratories to identify them [6]. The detection of the co-production of ESBL and AmpC is essential for enhanced infection control and effective anti-microbial therapy. There is no CLSI described guidelines for the detection of multiple β-lactamases. There is a paucity of data from Indian laboratories on the coexistence of multiple beta lactamases in individual isolates. Possible approaches to overcome this difficulty of ESBL detection in the presence of AmpC include the use of tazobactam or sulbactam, which are much less likely to induce AmpC β-lactamases or preferable use of inhibitors to ESBL detection tests [24].
All AmpC enzymes can hydrolyze cephamycins except ACC-1, which makes this drug better screening agents for AmpC production [28]. In the present study cefoxitin resistance was seen in 86/259 (33.20%) isolates. All the 67 (100%) AmpC producing isolates (100%) showed resistance to cefoxitin disc, but only 62/67 (93%) showed ≥ 5mm zone diameter with FOX+BA discs. None of the cefoxitin sensitive isolates showed AmpC production. The isolate which does not harbor AmpC, zone sizes of disks containing FOX and FOX+BA were the same. Modified three dimensional extract method detected 61 isolates (91%) as AmpC producers. All the negatives were identified correctly (Table 3). FOX resistance in isolates that did not show any enhancement with the addition of BA, resistance may be due other mechanisms like porin channel alterations in these isolates. Our study correlated with that of Song et al. [20] who showed 97.7% sensitive for AmpC detection by FOX-BA method, where our study showed 91% sensitivity. Pure AmpC β-lactamases were detected only in 5.40% of the isolates. This prevalence was lower than compared to the reports from other parts of the world [12, 29]. Two Indian studies reported 8 and 43% prevalence of AmpC β-lactamases [15, 30]. In all these AmpC producers, we were not able to distinguish between the chromosomal derepressed and plasmid mediated enzymes as this requires genotypic confirmatory tests.
Table 3 Occurrence of cefoxitin resistance and efficacy of FOX-BA disk test for detection of AmpC among Enterobacteriaceae
Phenotypes FOX (Cefoxitin disk resistance) FOX+BA disc for AmpC
R (%) S (%) ≥5mm enhancement FOX disc resistant, no zone enhancement FOX disc senstitive, no zone enhancement
Pure ESBL (n= 68) 19 49 0 19 49
Pure AmpC (n= 14) 14 0 12 2 0
ESBL+AmpC (n= 53) 53 0 50 3 0
Negative (n=124) 0 124 0 0 124
Total (n= 259) 86 (33) 173 (67.79) 62(23.93) 24(9.26) 173(66.79)
ESBL: Extended-spectrum beta-lactamases
In our study ESBL and AmpC co producing isolates were predominantly from K. pneumonia (35.59%) followed by E. coli (15.65%). Isolates producing both ESBL and AmpC showed greater resistance to most of the antibiotics. Greater resistance to β-lactam and non β-lactam antibiotics was evident in isolates coproducing both ESBL and AmpC producers than in pure ESBL/AmpC isolates. Combination of β-lactam/ β-lactam inhibitor showed greater activity in both groups, this is likely to be due to the heavy selection pressure from overuse of these antibiotics and seem to be losing the battle [31]. Piperacillin/ tazobactum showed less resistance as compared to ticarcillin/ clavulanic acid and amoxycillin/ clavulanic acid. Among aminoglycosides, amikacin showed grater activity against all the isolates irrespective of their resistance status (Table 4). Sensitivity to imipenem was observed to be 100%, which is in concordance with the studies conducted by other workers. Sensitivity to imipenem, which again advocates the usage of carbapenem antibiotics as the therapeutic alternative to β-lactam antibiotics as indicated in many studies [32, 33].
Table 4 Comparison of antimicrobial resistance patterns of isolates harboring both extended-spectrum beta-lactamases and AmpC
Antimicrobials Resistant pattern of ESBL and AmpC producer (n =53) % Resistant
Ampicilin 97.95
Ticarcillin 95.91
Piperacillin 81.63
Amoxycillin/clavulanic acid 42.85
Ticarcillin/clavulanic acid 73.46
Piperacillin- tazobactum 36.73
Aztreonam 83.67
Cephotaxime 85.71
Ceftazidime 81.63
Ceftriaxone 83.67
Cefepime 63.26
Impenem 0
Gentamicin 69.38
Amikacin 73.46
Tetracycline 65.30
Ciprofloxacin 53.06
Chloramphenicol 48.97
ESBL: extended-spectrum beta-lactamases
Conclusion
A mixed type of drug resistance mechanisms seem to operate in the isolates tested. The results of the study indicate that the current CLSI recommended methods to confirm ESBL enzymes by conducting clavulanate synergy tests with ceftazidime and cefotaxime may be insufficient for ESBL detection in clinical isolates of Enterobacteriaceae since these organisms often produce multiple β-lactamses. Inhibitor based method using boronic acid disc test, practical and efficient method that uses current CLSI methodology to detect co- producing ESBL and AmpC β-lactamase is a suitable alternative to test ESBL.
Acknowledgments
We gratefully acknowledge Mrs. Geetha Bhat, Consultant Microbiologist, Sri Bhagawan Mahaveer Jain hospital for providing clinical specimens
Competing interests
The authors declare that they have no competing interests
Authors’ contributions
Sasirekha Bakthavatchalu, conceived the study, analysed data, and drafted manuscript. Uma Shakthivel and Tannu Mishra were involved in sample collection literature search, analysis and processing of samples.
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J Diabetes Metab DisordJ Diabetes Metab DisordJournal of Diabetes and Metabolic Disorders2251-6581BioMed Central 2251-6581-11-710.1186/2251-6581-11-7Research ArticleMobile phone text messaging and Telephone follow-up in type 2 diabetic patients for 3 months: a comparative study Zolfaghari Mirta [email protected] Seyedeh A [email protected] Hamid [email protected] Nursing & Midwifery Care Research Center, Tehran University of Medical Science, Tohid Sq. East Nosrat Street, Tehran, Iran2 School of Nursing and Midwifery, Tehran University of Medical Science, Tehran, Iran3 Department of Biostatistics, Tehran University of Medical Sciences, Tehran, Iran2012 24 8 2012 11 7 7 18 7 2012 18 7 2012 Copyright ©2012 Zolfaghari et al.; licensee BioMed Central Ltd.2012Zolfaghari et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
To investigate and to compare the effectiveness of a nurse short message service (SMS) by cellular phone and telephone follow-up by nurse on Glycosylated hemoglobin (HbA1c) levels in people with type 2 diabetes.
Methods
Semi experimental study consisted of 77 patients with type 2 diabetes that randomly assigned to two groups: telephone follow-up (n = 39) and short message service (n = 38). Telephone interventions were applied by researcher for 3 months. SMS group that received message daily for 12 weeks. Data gathering instrument include data sheet to record HbA1c and questionnaire that consisted of demographic characteristics. Data gathering was performed at two points: initial the study and after 12 weeks. Data analyzed using descriptive and inferential statistics methods with SPSS version 11.5.
Results
Demographic variables were compared and all of them were homogenous. Results of this study showed that both interventions had significant mean changes in HbA1c; for the telephone group (p = 0.001), with a mean change of −0.93% and for the SMS group (p = 0.001), with a mean change of −1.01%.
Conclusion
Finding of this research showed that intervention using SMS via cellular phone and nurse-led-telephone follow up improved HbA1c for three months in type 2 diabetic patients and it can consider as alternative methods for diabetes control.
Glycosylated hemoglobinCellular phoneShort Message ServiceType 2 diabetes mellitusTelephone follow-up
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Introduction
The prevalence of diabetes has been alarmingly increasing [1]. Each year, 7 million people are diagnosed with the disease, and every 10s, a person dies from diabetes-related causes [1]. Diabetes is a chronic disease requiring lifelong medical and nursing intervention and lifestyle adjustment [2]. The National Survey of Risk Factors for Non-Communicable of Iran, which was conducted in 2005, demonstrated that the prevalence of diabetes mellitus in Iranian citizens aged 25–64 years was 7.7% (2 million individuals) [3], among whom half are undiagnosed [4]. An additional 16.8%, or 4.4 million, of Iranian adults have impaired fasting glucose [4]. If current trends continue, based on the World Health Organization forecast for Iran, there will be 5.2 million Iranians with diabetes mellitus in 2025[3].This high prevalence of diabetes in working aged adults is an ominous sign for this developing nation [4]. As the relatively young Iranian population ages in the future, and urbanization continues or accelerates, the prevalence of diabetes will likely escalate [4].
The Diabetes Control and Complications Trial showed that for every 1% reduction in the HbA1c levels, there was a 40% to 50% reduction in risk for microvascular and neuropathic complications [5,6]. The American Diabetes Association (ADA) has recommended that all persons with diabetes should attempt to achieve near normalization of blood glucose levels [7].
Algorithms for diabetes care exist but may be complex and difficult for physicians to follow, given the patient load, diversity of patient seen, lack of information systems, and time constraints [8]. In addition, economic and technical barriers to providing diabetes care in the community health system are recognized [9].
Since control of diabetes has been shown to decrease mortality and prevent long-term complications, it is critical that healthcare systems develop innovative ways to improve diabetes management, and provide timely care to patients [1]. Several research studies have shown that various telemedicine approaches can have a positive impact on patient blood glucose control and that over the long term; these approaches can result in reduction or elimination of the complication related to diabetes mellitus [10]. Telemedicine has, in particular, caught the attention of patients and caregivers [1]. For patients, it has the advantage of providing a quick, efficient way to communicate with their providers [1]. Especially, for those patients living in rural areas, it is potentially invaluable to have access to their caregiver from the comfort of their homes, thus sparing them the time and cost of traveling [11]. Although the magnitude of the impact of telemedical support on diabetes care remains debatable [11]. Telemedicine cannot replace patient visit and direct interaction with providers, but it can supplement between-visit care and improve ‘quality of care’ [1]. Patient suffering from chronic diseases in general and diabetes in particular, have benefited from support systems and telephone education with improved clinical outcomes [11], a telephone care program has been shown to be a viable strategy for bringing diabetes management services into patients’ homes, improving their glycemic control and provide timely care to patients [8]. To maintain normal range of blood glucose and prevent diabetic complications, patients ought to contact more frequently with their health care providers, but this will in turn increase health care expenditure [12]. And the telephone intervention was more time- consuming than an Internet-based blood glucose monitoring system [13].
Recently mobile phones as a new delivery system can provide medical recommendations and prescriptions at the appropriate time and to accommodate for patients’ behavioral changes and to normalize of blood glucose levels [14]. Mobile phones are an integral part of everyday life, although mobile phone technology is a relatively new and innovative methodology [15]. This method is becoming an important way of encouraging better nurse-patient communication and will undoubtedly increase in application over coming years [15]. Because of widespread usage and ubiquitous availability of mobile phones these devices may be can maximize the efficiency [16] Using short message service enables users to send and receive text messages to and from mobile phones up to 160 characters [16]. SMS allows rapid reception and reply at low cost. It is an interactive service, and is simple, fast and confidential [17]. Although it has been used for patient reminders, psychological support, medical appointments, to report critical medical events or laboratory results and even for surveys in other countries [17], but no research has been done to test the direct effects of Short Message Service on controlling (HbA1c) in people with type 2 diabetes mellitus and in healthcare delivery in Iran as far as we are aware.
The present study evaluated and compared whether an intervention using the short message service (SMS) of cellular phone by a nurse and nurse-led telephone follow up could improve HbA1c levels in patients with type-2 diabetes mellitus.
Methods
Participants
This study is quasi experimental research. Participants were recruited from the Iranian Diabetes Association. We studied this intervention during a three month period starting in May 2008. Diabetes was diagnosed according to the American Diabetes Association (ADA) criteria. The age range was 18–65 years. Patients had to have telephone access in their homes and have their own personal mobile phone, or have access to one belonging to a relative. Although selection criteria required that participants should be diabetic patients that only use Oral anti-diabetic medications, should be able to read and write, have power vision sufficient, no problem in hearing and vocalization and no history of psychiatric diseases. Patients were excluded if they had a clinical history of an important illness such as renal insufficiency with a creatinine level >1.5 mg/dl, hepatic insufficiency, were mentally ill or had less than 7% of HbA1c.
Seventy nine patients met the above criteria and agreed to participate. They were randomized by random permuted block design using a random number table and assigned to one of two groups: SMS group (N = 39) or Telephone group (N = 40). Only 77 subjects completed the entire study, 38 SMS and 39 Telephone. Two subjects were lost before completing the post-test in the Telephone group: one decided to opt out of the programme before completing the post-test and one expired during intervention. One subject was lost before completing the post-test in the SMS group because the change of therapeutic regimen from oral anti-diabetic agents to insulin.
Ethics committee approval was required. For ethical considerations, the research protocol was approved by the Medical Research Ethics Committee of the Tehran University of Medical Sciences. Written consent was obtained from those patients who agreed to participate in the study. Anonymity and confidentiality were guaranteed to participants.
Intervention
The goal of the intervention was to maintain blood glucose levels within a normal range. Participants attended in three days diabetes self-care education in Iranian Diabetes Association. Before the intervention, each patient was instructed, for 10 minutes by researcher, about how to use their own cell phones and to check their ability to read Short Messages and match the time for telephone follow-up. The researcher provided the intervention for 12 weeks. Patients in the SMS group received about 4 messages weekly consisted of diet, exercise, diabetic medication taking and frequent self-monitoring blood glucose levels. Participants in SMS group could receive our messages at any place where access was possible by cellular phone. The researcher sent optimal recommendations back to each patient, 4 times by short message service of cellular phone weekly. For example, recommendations included: ‘Do you know, the best bread for you is pebble bread’; ‘Please eat vegetables and salad in every meal’; ‘Please for prevention of high glycemic fluctuations’, eat your meals in six times instead of three times’; ‘Please consume your drugs on prescribed times’ ; ‘Do you know, eating in regular times, helps you to control your diabetes better’; ‘Lack of exercise may be the cause of the aggravated glucose level’; ‘Try to exercise 3 times daily and at least 15 minutes every time’; ‘Do at least 30 minutes of physical exercise or walking’; ‘Please check the amount that you eat’; ‘Take your recommended diabetic medication’; ‘If you consume Glybenclamide, please eat it, 30 minutes before your meal’ and so on. The 12 weeks of intervention consisted of continuous education and reinforcement of diet, exercise, medication taking, as well as frequent self-monitoring of blood glucose levels.
The intervention for Telephone group was provided via telephone for 12 weeks. The 12 weeks of intervention consisted of counseling on the nature of the disease, risk factors, importance of maintaining blood glucose levels within a near-normal range, continuous education and reinforcement of diet, exercise, medications taking, hypoglycemia management, illness management how to record daily blood glucose and frequent self-monitoring of blood glucose levels. The researcher contacted the telephone group at least twice a week for the first month and then weekly for the second and third month. The total frequency of telephone counseling averaged 16 times per subject. The duration of each counseling session was an average of 20 minutes. The researcher was asking questions such as: “did you take your recommended diabetic medication?” “When did you consume your prescribed tablet?” Do you know how you’re consuming medications, act in your body? “How many times did you do physical exercise or walking during last days?” “When is the best time to do exercise?” “Did you feel better after doing exercise?” Do you know that doing exercise is as important as diabetic medication?“ How many days did you follow your recommended diet over the past days?” Did you eat salad and vegetable before every meal?” Before initiation of recommendation, researcher asked every patient about problems they were facing during last days and patients could ask their questions and could solve their problems. Sometimes they feel stress, so the researcher educate some ways of decreasing stress such as: deep breathing, distraction methods, taking a bath, go to the country, concentrate to good points of their life, be more with their family members or closed or lovely friends, try to laugh more and so on.
Procedure
Before the intervention, demographic characteristics and HbA1c value were collected as pre-test data at the Iranian Diabetes Society. The intervention was provided to the telephone group with counseling appointments scheduled when convenient to the subject. The HbA1c was measured again three months later. Patients’ blood was drawn in veins for HbA1c measurement. HbA1c was measured in the metabolism and endocrinology laboratory of the Tehran University-affiliated medical center. HbA1c was determined by a high-performance liquid chromatography technique. HbA1c level was measured after 12 weeks as posttest data.
Data analysis
The data were analyzed using the SPSS (Version 11.5) program. Chi square test, Paired t-test, independent t-test and Fisher’s exact test were used to test for the homogeneity of demographic and clinical characteristics between the SMS and Telephone groups. The paired t test was used for comparison of differences between pretest and posttest values in the group. The unpaired t test was used for comparing the differences between the SMS and Telephone groups.
Results
The characteristics of the SMS and Telephone groups are shown in Table 1. The mean age of the SMS group was 51.07 years and that of Telephone group was 53.71 years. The mean BMI of the SMS group was 29.008 kg/m2 and that of Telephone group was 27.334 kg/m2. There was no significant difference in age, gender, BMI, duration of diabetes, treatment method and blood glucose levels between the two groups.
Table 1 Baseline demographic and clinical data of the SMS and intervention groups
Characteristics SMS group (n = 38) Telephone group (n = 39) t / χ2 p
Age (years) 51.70 ± 9.90 53.71 ± 9.04 1.221 0.226
Sex:Male/.Female 18/20 18/21 0.011 0.915
Body mass index (kg.m2) 29.008 ± 6.65 27.334 ± 3.53 1.374 0.175
Diabetes duration (months) 95.57 ± 72.96 74.55 ± 61.93 1.365 0.176
Glycosylated haemoglobin (%) 8.97 ± 1.65 9.44 ± 1.72 1.219 0.227
Data are Means ± SD (%).
At the pre-test, no significant differences were found in HbA1c between the groups in Table 1. HbA1c did not differ significantly with two groups (SMS group vs. Telephone group) (p = 0.227). There was a significant percentage change in HbA1c for the SMS group (p = 0.000), with a mean change of −1.01 (8.97% pre-test to 7.96% three months), and also there was a significant percentage change in HbA1c for the Telephone group (p = 0.000), with a mean change of −0.93 (9.44% pre-test to 8.51% three months).
Glycosylated hemoglobin (HbA1c) decreased 0.93% points at three months compared with baseline in the Telephone group and 1.01% points at three months compared with baseline in the SMS group (Table 2). There was no significant difference were found between two intervention (p = 0.186).
Table 2 Effect of the intervention on Glycosylated haemoglobin (%) levels
Variable HbA1C (%) Baseline 3 months Difference (Post-test) – (Pre-test) t p
SMS group 8.97 ± 1.65 7.96 ± 1.75 −1.01 ± 0.01 4.254 0.000
Telephone group 9.44 ± 1.72 8.51 ± 1.85 −0.93 ± 0.13 4.150 0.000
Discussion
Glycosylated haemoglobin reflects mean blood glucose levels over the previous six weeks. HbA1c has become a standard assessment of glycaemia and a standard part of diabetes management [7]. Therefore, large studies of this relationship, such as the Diabetes Control and Complications Trial Research Group (1993) and the United Kingdom Prospective Diabetes Study Group (UKPDS, 1998), used HbA1c as the primary index of glycaemia [5,6].
This study evaluates and compares whether an intervention using the short message service (SMS) of cellular phone by a nurse and nurse led telephone follow-up would improve HbA1c levels in patients with type-2 diabetes mellitus for three months.
In this study, HbA1c levels decreased 1.01% points in SMS group and 0.93% points in Telephone group after 12 weeks compared with baseline. Previous studies showed the following results: Kwon et al. (2004) reported that the 12-weeks follow-up examination HbA1c levels in diabetic patients by web-based management system using short message service (SMS) caused to mean decrease of 0.9% points in HbA1c [12], People with diabetes in a nurse short-message service by cellular phone intervention group had mean decrease of 1.15% points in HbA1c levels during the 3-months study period and 1.05% points at six months [14]. A short message service by cellular phone study in type 2 diabetic patients resulted in a decrease of HbA1c of 1.31% points at nine months and 1.32% points at twelve months [18]. An SMS intervention study by a nurse showed that the HbA1c levels decreased 1.1 percentage points after 12 weeks [2]. At the end of Internet diabetic patient management study using a Short Messaging Service A1C levels were significantly (0.72%) decreased in the intervention group [19]. The study of evaluating the impact of nurse’s education by short message service of cellular phone and wire internet revealed a significant percentage change in a baseline HbA1c ≥7.0% for the intervention group with a mean change of −2.15(9.35% pretest to 7.20% post-test) at 3-months follow-up [13]. An intervention using SMS of personal cellular phone and internet showed great efficacy in HbA1c control in obese type 2 diabetes. The intervention group showed a marked decrease in HbA1c levels after 12 months of follow-up versus the baseline levels (a mean percentage change of −1.22 at 3 months, -1.09 at 6 months, -1.47 at 9 months and −1.49 at 12 months [20]. Kim, Oh and Lee (2005) reported that a nurse-coordinated intervention by telephone decreased HbA1c levels 1.2 percentage points after 12 weeks [8]. In a prior randomized trial, the effect of nurse telephone calls on HbA1c levels was evaluated. After 12 weeks, patients in the telephone intervention group had a mean decrease of 1.2% in HbA1c levels [21]. These results confirm that the use of various telemedicine approaches can have a positive impact on patients’ HbA1c control.
Therefore, mean end-point HbA1c levels in the intervention groups of the 2 studies were essentially the same. However, in the telephone intervention group, nurse spent more time and money with patients than the SMS intervention by nurse. Overall, the findings suggested that the SMS intervention as improved HbA1c level as telephone group. Patients with diabetes wanted more frequent contact with their health care providers for managing the disease. By using telemedicine management systems such as short message service via cellular phones and telephone follow-up, patients can contact their nurses frequently. The patients in the Telephone group have more frequent contact with the nurse than those in the short message service of cellular phone group. In addition, the patients in the Telephone group received advice according their most recent data, confirming their current state. These factors may have stimulated and motivated the patients to control glucose levels enthusiastically.
These results have important clinical implications because the service for diabetic patients provided via mobile phone is now increasing, whereas the efficacy of the short message service for glucose control has not been evaluated extensively. Also, this study results show evidence that short message services are as effective as telephone calls guidance in managing diabetes. One major advantage of short message service is that the researcher could send short messages without location limitations. An intervention that involves the use of SMS and personal cellular phone can also be applicable to other chronic diseases such as hypertension, hyperlipidemia, obesity, and metabolic syndrome.
This study adds that a nurse educational intervention programme using the telephone calls and an SMS by cellular phone improved levels of glycosylated haemoglobin levels for three months in patients with type2 diabetes. The SMS by cellular phone can be used as a means of providing education for patients with diabetes.
The trial demonstrated the usability of the system. The effectiveness of the system relied on regular contact. The contact frequency provided an indication of the level of acceptance and the extent to which use of the system had become a habit. Therefore, nurse’s education using SMS of cellular phone not only allowed for the patients to maintain steady levels of HbA1c, but also improved the degree of their HbA1c control as well. These results confirm that the use of various telemedicine approaches by the nurse have a positive impact on patient’s HbA1c control.
Although this study demonstrated that an SMS of cellular phone intervention by a nurse could maintain and reduce .HbA1c during a short-term study period of 12 weeks, the long-term effectiveness remains to be determined.
In this study, Iranian patients with type 2 diabetes who received short massages and followed by telephone had improved HbA1c levels. This study suggests both telephone follow-up intervention and using SMS of personal cellular phone improved HbA1c levels remarkably during three months in type 2 diabetic patients. Regard to convenience of SMS can be used as an appropriate alternative method for following up in diabetic patients.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MZ had substantial contributions to conception and design of the study, analysis of the data and drafting the manuscript. SAM had substantial contributions to acquisition of data and analysis. HH involved in the data analysis and the interpretation of results. All authors read and approved the final manuscript.
Acknowledgement
This study was supported by Vice-chancellor for Research of Tehran University of Medical Sciences [TUMS] (grant no: 7091). We thank all the project partners for their support. We acknowledge the help of all participants in this study.
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J Diabetes Metab Disord
J Diabetes Metab Disord
Journal of Diabetes and Metabolic Disorders
2251-6581 BioMed Central London
23497656
29
10.1186/2251-6581-11-29
Research Article
The association between depression, socio-economic factors and dietary intake in mothers having primary school children living in Rey, South of Tehran, Iran
Payab Moloud [email protected] 1 Motlagh Ahmad-reza Dorosty [email protected] 1 Eshraghian Mohammadreza [email protected] 1 Rostami Reza [email protected] 2 Siassi Fereydoun [email protected] 1 Abbasi Behnood [email protected] 3 Ahmadi Mehrnaz [email protected] 4 Karimi Tina [email protected] 4 Mahjouri Mohammad Yoosef [email protected] 5 Seifirad Soroush [email protected] 5 1 grid.411705.60000000101660922School of Public Health & Institute of Public Health Researches, Tehran University of Medical Sciences, Tehran, Iran
2 grid.46072.370000000406127950Institute for Psychology and Educational Sciences, University of Tehran, Tehran, Iran
3 grid.411600.2Faculty of Nutrition & Food Technology, Shahid Beheshti University of Medical Science and Health Service, Tehran, Iran
4 grid.411463.50000000107062472Islamic Azad University, Sciences and Research Branch, Tehran, Iran
5 grid.411705.60000000101660922Endocrinology and Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran
22 12 2012
22 12 2012
2012
11 2925 10 2012 25 10 2012 © Payab et al.; licensee BioMed Central Ltd. 2012This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
According to the WHO report released in 2000, about 121 million people worldwide suffer from depression. The present study aimed to explore factors influencing depression in mothers from Rey, South of Tehran, Iran; who had elementary school children.
Methods
The cross-sectional survey was conducted in spring 2010. Four hundred thirty mothers, who had elementary school children, were selected through a two stage cluster sampling. Beck Depression Inventory (BDI) was used to assess depression in the mothers and a 24-hour food recall was used to collect information regarding their dietary intake. General information regarding economic condition and socio-economic status were also gathered using a questionnaire. The data was analyzed using chi-square, one-way analysis of variance and simple regression tests.
Results
In our study, 51.4% of the mothers suffered from depression. There was an inverse correlation between the educational level of the mothers and the heads of household, their occupational status, their marital status, their socio-economic condition and depression. Conversely, any increase in the family size worsened the depression. The daily intake of different macronutrients, except for fat, was lower in individuals of depressed group.
Conclusion
The present study emphasized the fact that more attention should be paid to the educational level and economic condition of the family in order to reduce maternal depression. Family size also plays an important role in this regard.
Keywords
DepressionDietMothersSocio-economic factorsissue-copyright-statement© The Author(s) 2012
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Introduction
The major depressive disorder (MDD), also known as unipolar depression, is one of the most common mood disorders experienced during life. The condition is characterized by any changes in appetite, weight, sleeping pattern and routine activities, lack of energy, guilt feeling, difficulty in decision making, lack of motivation and pleasure [1, 2].
According to the 2000 WHO report, 121 million people worldwide suffer from depression and MDD is considered as the fourth cause of burden and the most cause of disability worldwide. The report also predicts that MDD will become the second leading cause of burden disease on 2020. It is also mentioned that 15% of patients with MDD eventually commit suicide [3].
In Iran, studies have shown that depression with a prevalence of 3.8% is on the top of the mental disorders and 21% of population suffers from this disease [4]. Hadavi et al. [5] reported that the mild, moderate and major depression prevalence in women referred to Rafsanjan’s health centers respectively was 18%, 19.1% and 3.4%. Also Ahmadi and Yousefi [6] found that in Bakhtiari nomads the overall prevalence of depression is 29.6%. In another study, Kavyani et al. [7] demonstrated that in Tehran, the prevalence of depression in women is 12.6% and in men is 8.47%.
In general, studies have shown that nutritional status and nutrient intake is associated with depression and may be poor diet is a main cause of depression [8].
The prevalence of depression is higher in women; mainly often married women aged 18–24 years who have a child suffering from depression [9]. Considering the fact women constitute half of the population in each society, being depressed reduces their efficacy whether they are working outside or are a housekeepers. Regarding this issue, it is clear that depression imposes significant costs on society, therefore studying the association between depression levels and factors, such as nutrients intake in women, could be necessary. To the best of our knowledge, there is no similar study on the association between depression, socio-economic factors and dietary intake in Iran.
Potentially, some of studied factors of this study are preventable, and thus dispelling them could decrease depression rates in the society.
Methods
Study population
The cross-sectional study was conducted in spring 2010 on 430 mothers having primary school children living in Rey. In order to obtain the desired number of samples, a pilot study was conducted in Rey elementary schools. The number of regional schools in Rey was identified with the help of “Rey Education Center”. After contacting their administrators, forty three schools were randomly selected among 102 schools. Subsequently, ten students selected from each school and their mothers were invited to participate in the study. Random selection of 430 mothers was commenced with sorting, based on the economic situation, the list of all primary schools (102 public and private schools) in Rey. Thus, the total cumulative frequency of the primary school students was calculated (36039) from the sorted list. Dividing the total cumulative frequency by the number of clusters (43), the distance between clusters (838) was obtained. Selecting a random number from 1–838 and determining their place in the total cumulative frequency, first school was selected. In the second step, in each selected school, in accordance with the chosen grade, about 10 mothers were selected.
Measurements
Demographic data
A self report general information questionnaire was used to record the demographic characteristics such as age, marital status, socio-economic characteristics, and education level of all participants.
In order to determine the socio-economic status, participant were asked to specify if household appliances such as furniture, handmade carpet, refrigerator, washing machine, dish washing machine, microwave, personal computer, car, and property are amongst their household appliances. Having 1–3, 4–6, and 7–9 of these nine household appliances, participants were categorized as low, middle, and high socioeconomic status respectively.
Body weight and height
Standing height was measured while the subjects had no shoes and their soles were stuck to the wall using a SECA height meter with 0.1cm accuracy. Weight was measured on using a SECA digital scale with 0.1 kg accuracy, again with subjects having no shoes and minimal clothing. Then Body Mass Index (BMI) was calculated as weight (kg)/height2 (m2). Weight classification was made based on WHO standard guidelines. In this regard, those with BMI of above 30 were considered as obese, fewer than 18.5 as extremely underweight, between 18.5 and 25 as normal, and between 25 and 30 as overweight [10].
Depression assessment
The Persian version of Beck Depression Inventory (BDI) was used to measure depressive symptoms [11]. Individuals gaining higher scores on the 21-question psychological test are more depressed [12]. Thereafter based on their BDI scores, subjects were divided into 6 groups: normal, slightly depressed, required consultation with a psychiatrist, relatively depressed, severely depressed, and excessively depressed (Table 1). Then these groups were combined into three groups: normal, mild to moderate depressed and severely depressed. Many groups were studied the relationship between credibility and reliability of BDI [13]. For instance, internal consistency of BDI 0.73-0.92 with a mean of 0.86 was reported, and the coefficient alpha for psychiatric populations and non-psychiatric populations were measured 0.86 and 0.81 respectively. Beck test-retest reliability with regard to the distance between the two tests and studied population was measured 0.48-0.86 [1, 2]. Furthermore, a study on 116 participants in Iran revealed the correlation coefficient of 0.23-0.68 for BDI.Table 1
BDI scores
Depression status Scores
Normal 1-10
Slightly depressed 11-16
Require consultation with a psychiatrist 17-20
Relatively depressed 21-30
Severely depressed 31-40
Excessively depressed >40
Dietary assessment methods
The 24 hours Food Recall is a suitable method to evaluate the food intake of different population. The test involves asking subjects to recall and describe all the food and drinks they have consumed in the past 24 hours [14]. In our study the participants completed a 24-hours dietary recall form twice (In the middle of the week, and three days later).
Statistical analyses
Data were entered and analyzed using Statistical Package for the Social Sciences (SPSS Inc. Chicago, IL, USA). The relationship between qualitative variables with depression levels were assessed using Chi-Square test. The mean and standard deviation were calculated for quantitative variables. Also simple (linear) regression was used to examine the relationship between depression levels and the studied variables. The variables that were significantly associated with depression, were entered into stepwise multiple regression model.
Food intake was converted into nutrients and its grams amount was calculated and encoded. Then, these values were entered in Dorosty Food Processor for Windows software (DFPW- 2.1), and the amounts of energy, carbohydrate, protein and fat intake were calculated.
Result
Demographic data
Totally 430 participants were evaluated. The mean age and BMI of the subjects were 34.8 (SD= 5.3) years and 28 (SD= 5) respectively. Also 96.7% of the participants were married and 3.3% were widow. Occupational status of the head of household and socioeconomic, and BMI status respectively in two, fifteen, and one subjects were not recorded. All demographic characteristics are showed in Table 2. The prevalence of depression was 51.4% whereas 46.5% of them suffered from mild to moderate depression, and severe form of the condition was only reported in 4.9%. Depression rate was higher among the older subjects (P= 0.001); it was significantly associated with family size and the number of children (p< 0.001).Table 2
Demographic data (n= 430)
Characteristics %
Age*
34.8± 5.3
Marital status
Married 96.7
Widow 3.3
Occupational status
Housekeepers 95.8
Employed 4.2
Education level
Illiterate/Elementary school 16.8
Secondary school/ High school 74.2
University 9
Occupational status of the head of household (n= 415)
Unemployed 3.7
Worker 21.9
Government employee 30
Self-employed 37.2
Retired 3.7
Education level of the head of household
Illiterate/Elementary school 16.1
Secondary school/ High school 65.1
University 18.8
Residential house ownership status
Personal house 55.6
Rent or mortgage 29.1
Living with parents or other relatives 15.3
Family size
Less than 4 persons 30.7
More than 4 persons 69.3
Socioeconomic status (n= 428)
low 32.2
Middle 56.8
High 11
*Data for the age is mean±SD.
Educational level and occupational status of the mothers and the heads of household, being married and socioeconomic status were inversely associated with depression (Table 3). Based on simple regression fitting, with increasing educational levels of mothers and the heads of household the score of depression were decreased. From among occupations of heads of household, being self-employed was associated with lower rates of depression among the studied population. Also living with of the relatives rather than a personal house, and being widowed was associated with high levels of depression (Table 4).Table 3
Correlation between levels of depression and qualitative variables
Variables Depression levels Total
X2 test / P-Value
Non Mild & Moderate Major
Marital status
Married 207 (49.8) 192 (46.2) 17 (4.1) 416 (100) <0.001
Widow 2 (14.3) 8 (57.1) 4 (28.6) 14 (100)
Total 209 (48.6) 200 (46.5) 21(4.9)
Occupational status
Housekeepers 199 (48.3) 195 (47.3) 18 (4.4) 412 (100) 0.031
Employed 10 (55.6) 5 (27.8) 3 (16.7) 18 (100)
Education level
Illiterate/Elementary school 26 (35.6) 41 (56.2) 6 (8.2) 73 (100) 0.025
Secondary school/ High school 158 (49.5) 146 (45.8) 15 (4.7) 319 (100)
University 25 (65.8) 13 (34.2) 0 (0.0) 38 (100)
Occupational status of the head of household (n= 415)
Unemployed 2 (12.5) 11 (68.8) 3 (18.8) 16 (100) 0.005
Worker 45 (47.9) 45 (47.9) 4 (4.3) 94 (100)
Government employee 74 (57.4) 54 (41.9) 1 (0.8) 129 (100)
Self-employed 76 (47.5) 75 (46.9) 9 (5.6) 160 (100)
Retired 3 (56.3) 7 (43.8) 0 (0.0) 16 (100)
Education level of the head of household
Illiterate/Elementary school 22 (32.8) 39 (58.2) 6 (9.0) 67 (100) 0.013
Secondary school/ High school 143 (52.8) 118 (43.5) 10 (3.7) 271 (100)
University 42 (53.8) 35 (44.9) 1 (1.3) 78 (100)
Residential house ownership status
Personal house 119 (49.8) 111 (46.4) 9 (3.8) 239 (100) 0.161
Rent or mortgage 66 (52.8) 51 (40.8) 8 (6.4) 125 (100)
Living with parents or other relatives 24 (36.4) 38 (57.6) 4 (6.1) 66 (100)
Family size
Less than 4 persons 76 (57.6) 51 (38.6) 5 (3.8) 132 (100) 0.046
More than 4 persons 133 (44.6) 149 (50.0) 16 (5.4) 298 (100)
Socioeconomic status (n= 428)
low 52 (37.7) 74 (53.6) 12 (8.7) 138 (100) 0.003
Middle 127 (52.3) 109 (44.9) 7 (2.9) 243 (100)
High 30 (63.8) 15 (31.9) 2 (4.3) 47 (100)
Table 4
Regression fitting between depression and qualitative independent variables
Variable β±SE P-value
Marital status
Constant value 12.34±0.45 <0.001
Being married (Basic group) - -
Being widowed 8.73±2.48 0.003
Occupational status
Constant value 12.54±0.46 <0.001
Housekeepers (Basic group) - -
Employment 1.96±2.23 <0.719
Education level
Constant value 14.93±1.07 <0.001
Illiterate (Basic group) - -
Diploma −2.32±1.19 <0.001
University −6.59±1.83 <0.001
Occupational status of the head of household (n= 415)
Constant value 19.87±1.61 <0.001
Unemployed (Basic group) - -
Worker −9.19±1.86 0.082
Government employee −7.3±1.8 <0.001
Self-employed −9.37±1.77 <0.001
Retired −6.5±2.77 0.084
Education level of the head of household
Constant value 16.53±1.0 <0.001
Illiterate (Basic group) - -
diploma −4.7±1.15 <0.001
University −5.22±1.44 <0.001
Residential house ownership status
Constant value 11.91±0.6 <0.001
Personal house (Basic group) - -
Rental house 0.88±0.01 0.007
Relatives home 2.97±1.28 0.011
The mean height, weight and BMI had no significant impact on depression. (Tables 5, 6) The mean daily energy, carbohydrate, and protein intake however had a significant inverse association with depression; such a line was not found for daily fat intake. Moreover, percentage of energy intake from carbohydrates, protein, and fat were not associated with depression (Table 7). Based on stepwise multiple regression low socio-economic status, being widowed (In comparison with being married), and having more child were the main factors contributing to depression.Table 5
Correlation between Depression and studied variables
Variable p-value Pearson correlations
Age
0.014 0.118
Family size
> 0.002 0.147
Number of children
> 0.001 0.181
Socio- economic level
> 0.001 −0.179
Weight
0.565 0.028
Height
0.224 −0.059
BMI
0.277 0.053
Education level of mother
0.001 −0.161
Education level of the head of household
0.004 −0.142
Occupational status of the head of mother
0.022 0.017
Occupational status of the head of household
0.002 −0.151
Residential house ownership status
0.011 0.077
Marital Status
> 0.001 0.182
Table 6
Regression fitting between depression and quantitative independent variables
Variable β±SE P-value
Age
9.19±2.98 0.002
0.1±0.08 0.244
Family size
6.5±1.91 0.001
1.53±0.46 0.001
Number of children
9.44±1.03 > 0.001
1.61±0.47 0.001
Socio- economic Level
17.79± 1.15 > 0.001
−1.21±0.25 > 0.001
Weight
11.42±2.445 >0.001
0.02±0.03 0.617
Height
17.47±12.26 0.155
−0.03±0.08 0.692
BMI
11.02±2.56 > 0.001
0.06±0.09 0.524
*Constant.
**Coefficient.
Table 7
Mothers daily energy and macronutrient intake based on depression levels
Variables Normal Mild and moderate depression Major depression Total ANOVA
n= 209 n= 200 n= 21 n= 430 P-Value
Energy (kcal)
2246.7±746.8 2110.6±753.3 1698.4±682.9 2158.1±754.5 0.019
Carbohydrate (gr)
330.3±120.6 311.4±119.3 248.5±97.8 317.8±120.1 0.033
Protein (gr)
64.9±23.7 58.7±19.0 44.5±18.0 61.1±21.8 0.001
Fat (gr)
74.0±29.3 70.0±34.7 58.5±31.3 71.4±32.2 0.172
Percentage of energy intake from carbohydrates
58.47±6.8 58.66±8.31 59.05±6.03 58.59±7.51 0.478
Percentage of energy intake from protein
11.7±2.54 11.49±2.66 10.48±1.51 11.55±2.57 0.11
percentage of energy intake from fat
29.83±6.92 29.85±8.49 30.46±6.26 29.87±7.66 0.476
Data are set as the mean±SD.
Discussion
The present study reported a high prevalence of depression among Iranian mothers (51.4%). Previous studies have estimated the prevalence of depression in women and men to be 34% and 30% respectively [15]. Kavyani et al. [7] also demonstrated that the prevalence of depression in 20–65 years old women and men living in Tehran was 12.6% and 8.47%. Moreover the prevalence of mild, moderate and major depression in women referred to Rafsanjan’s health centers was 18%, 19.1% and 3.4% respectively [5]. Compared to the other published studies, it seems that depression prevalence in our study is higher than the other studied populations.
Previous studies have highlighted the difference noted in the prevalence of depression in several countries. For example, Ballenger et al. [16, 17] reported the prevalence of depression in the United States to have increased from 6% in early 1960 to 28% in early 1990. Furthermore, the results of another study showed that the prevalence of depression among premenopausal and postmenopausal women in Malatya, Turkey was 41.8% [18].
In accordance with several previous studies, our findings indicated a significant positive relationship between depression and the mean age of the participants [5, 15, 19]. This comes while several studies have shown the contrary [20, 21]. On the contrary to the Kerman study, our study revealed a significant positive association between family size and depression [20].
Our findings also showed a significant negative relationship between depression and socio-economic condition. This is consistent with the results of several previous researches [22]. Controversial reports have been stated regarding correlation between income and depression [5, 18, 21, 23]. It seems that inappropriate economic and social conditions can lead to or exacerbate psychological problems in the family members. Furthermore, we found a significant correlation between depression and the occupational status of the head of household; in other words depression was more prevalent among mothers whose husbands were unemployed. Occupational status of the mothers similarly, affected their mood. The condition was more common among housekeepers. Hadavi et al. [5] reported that the prevalence of depression was higher in housekeepers and those married to workers, farmers and self-employed men. Our study however reports a higher rate of depression among women whose husbands were government employee.
In line with many published studies, our findings revealed an inverse correlation between depression and educational levels of the mothers and the heads of household [15, 18, 19, 21]. However, some studies have shown the contrary [19]. It seems that higher educational level can help people adjust better with the environment and have a better performance in dealing with problems and it can improve their mental health.
In addition, our study results revealed a significant correlation between depression and mother’s marital status, so that women who had lost their husbands were more depressed. This association was also reported in several previous published studies [15, 18, 21, 24]. A higher rate of depression among women who have lost their husbands is reported in our study. It was predictable since they are more responsible for their children, additionally lack of support from spouse in the various fields can impose high psychological stress on these women.
The results of present research indicated that the mean daily energy intake significantly and inversely affected depression levels. Moreover, those who took more carbohydrates and protein were significantly less likely to be depressed. Results of Park et al. study [8] contradicts our results in favor of carbohydrate consumption relation with depression while the results of protein consumption in their study were in accordance with our findings.
One of the major limitations of this study is the adopted cross-sectional design. Thus, it was not possible to show the causal relationship between the variables. Moreover, other possible causes of depression were not examined. Further researches are needed to study these variables.
Considering the fact that any change in appetite is a symptom of depression, it is likely the changes in macronutrient intake would lead to depression. It could be concluded that to prevent and reduce the rise of depression, more attention should be paid to education, employment and economic status.
Competing interests
The authors have no financial competing interests. However the data may disclose prevalence of depression in mothers having primary school children living in Rey, South of Tehran, Iran.
Authors’ contribution
MP participated in the study design, data acquisition, statistical analysis, and interpretation. AD participated in the study design and interpretation. ME participated in the statistical analysis. RR acted as psychology advisor in collection and interpretation of data. FS participated in the study design. BA participated in the data acquisition. MA participated in the data acquisition. TK participated in the data acquisition. MM drafted the manuscript and given final approval of the version to be published. SS drafted the manuscript and submitted the paper, made critical review, and study interpretation, also given final approval of the version to be published. All authors read and approved the final manuscript.
Change history
5/14/2013
An Erratum to this paper has been published: 10.1186/2251-6581-12-21
Acknowledgment
The authors wish to thank all participants of this study for their cooperation. This study was financially supported by Tehran University of Medical Sciences (TUMS).
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Nanoscale Res LettNanoscale Res LettNanoscale Research Letters1931-75731556-276XSpringer 1556-276X-8-1002343291910.1186/1556-276X-8-100Nano ExpressSingle-wall carbon nanohorns inhibited activation of microglia induced by lipopolysaccharide through blocking of Sirt3 Li Lihong [email protected] Jinqian [email protected] Yang [email protected] Qiang [email protected] Li [email protected] Yanlong [email protected] Tao [email protected] Xingye [email protected] Guoan [email protected] Yongmei [email protected] Zujin [email protected] Ming [email protected] Guodong [email protected] Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, 710038, Xi’an, China2 Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, 100015, Beijing, China3 Department of General Surgery, the Second People’s Hospital of Guangdong Province, 510515, Guangzhou, China4 International Mongolian Medical Hospital of Inner Mongolia, 010065, Hohhot, China5 Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering, Peking University, 100871, Beijing, China6 Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, 100191, Beijing, China2013 22 2 2013 8 1 100 100 29 11 2012 20 1 2013 Copyright ©2013 Li et al; licensee Springer.2013Li et al; licensee Springer.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate in cytotoxic levels within organs of various animal models and cell types, which emerge as a wide range of promising biomedical imaging. Septic encephalopathy (SE) is an early sign of sepsis and associated with an increased rate of morbidity and mortality. Microglia activation plays an important role in neuroinflammation, which contributes to neuronal damage. Inhibition of microglia activation may have therapeutic benefits, which can alleviate the progression of neurodegeneration. Therefore, we investigated the functional changes of mice microglia cell lines pre-treated with or without lipopolysaccharide (LPS) induced by SWNHs. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of microglia cell lines in mice (N9 and BV2) pre-treated with or without LPS had been performed. Our results indicate that the particle diameter of SWNHs in water is between 342 to 712 nm. The images in scanning electron microscope showed that SWNHs on polystyrene surface are individual particles. LPS induced activation of mice microglia, promoted its growth and proliferation, and inhibited its apoptosis. SWNHs inhibited proliferation, delayed mitotic entry, and promoted apoptosis of mice microglia cells. The effects followed gradually increasing cultured time and concentrations of SWNHs, especially in cells pre-treated with LPS. SWNHs induced a significantly increase in G1 phase and inhibition of S phase of mice microglia cells in a dose-manner dependent of SWNHs, especially in cells pre-treated with LPS. The transmission electron microscope images showed that individual spherical SWNH particles smaller than 100 nm in diameters were localized inside lysosomes of mice microglia cells. SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in cells pre-treated with LPS. SWNHs inhibited expression of Sirt3 and energy metabolism related with Sirt3 in mice microglia cells in a dose-dependent manner, especially in cells pre-treated with LPS. The role of SWNHs on mice microglia was implicating Sirt3 and energy metabolism associated with it.
Single-walled carbon nanohornsSeptic encephalopathyCell proliferationApoptosisLipopolysaccharideSepsisSirt3
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Background
Sepsis-induced encephalopathy is caused by systemic inflammation in the absence of direct brain infection and clinically characterized by slowing of mental processes, impaired attention, disorientation, delirium, or coma. Importantly, septic encephalopathy (SE) is an early sign of sepsis and associated with an increased rate of morbidity and mortality. The pathogenesis of SE is unlikely to be directly induced by a pathogenic toxin, because similar encephalopathy can develop as a result of a number of systemic inflammatory response syndromes that lack an infectious etiology (e.g., acute pancreatitis and burns). Clinical and experimental data suggested that a number of factors including the local generation of pro-inflammatory cytokines and impaired cerebral microcirculation. The imbalance of neurotransmitters or the negative impacts of peripheral organ failure contribute to the development of SE [1-3].
Microglia, innate immune cells of the CNS, become activated in response to injury and appear to have important role in the defense against invading microbes and in wound repair [4]. Microglia, the resident immune cells of the central nervous system, are normally quiescent but become activated after infection or injury [5]. Because activated microglia can promote both damage and protection [5], their numbers require strict regulation, in part by ‘activation-induced cell death’ (AICD). In view of the key participation of microglia in neurological disorders [6], the knowledge of the molecular mechanism about AICD is important. However, under certain pathophysiological circumstances, microglia may also contribute to neuronal toxicity. For example, factors released from activated microglia can amplify inflammatory processes that contribute to neurodegeneration [7]. To harness and modulate the activity of microglia, it would be useful to be able to target biologically active compounds specifically to these powerful cells.
Since Iijima’s laboratory first synthesized single-walled carbon nanohorns (SWNHs) in 1999 [8], most of researchers have drawn their attention to theoretical and applicative fields relating to the material. With its tip-closed single-wall nanoscale cavum structure and advantages of high purity, uniform size, and ease of dispersion in solvents, SWNHs have been considered as a promising carrier for drug delivery system [9-14]. Nevertheless, interaction between unmodified SWNHs and cells has not been reported, although effects of modified SWNHs on HeLa and murine macrophage RAW 264.7 cells were shown recently [15,16]. More researches were focused on biological effects of fullerene, graphene, and carbon nanotubes (CNTs) modified with various bioactive groups on multiple type cells [17-38]; they revealed that carbon nanoparticles could be internalized in cells and react with subcellular organelles, such as endosome, mitochondria, lysosome, and nucleus [24-28,30]. Besides, an endocytic and a passive diffusion pathway for multi- and single-walled CNTs transmembrane process [27,28], and an oxidative stress pathway for cellular apoptosis induced by carbon nanoparticles, were proposed [39,40].
It is very important how SWNHs material reacts with the cells for evaluating its biological functions. Moreover, researches on the interactions between SWNHs and the cell lines will be helpful for examining the difference of cytotoxic effects of the material on the cells. So far, the role and functional mechanism of SWNHs material itself in the microglia cells are still unclear. Herein, to address this question, direct mechanisms of raw SWNHs on the growth, proliferation, and apoptosis of mice microglia cell lines were studied. The remarkable behavior of SWNHs in N9 and BV2 cells will be revelatory for further study on the interactive mechanisms in mice microglia cells with SWNHs and their possible applications in clinical treatment of SE or other neurodegenerative diseases associated with microglia.
Methods
Synthesis of single-wall carbon nanohorns by arc discharge in air
For the synthesis of SWNHs, we used pure graphite rods (Ø8 mm) as the electrodes. Direct current arc discharge was carried out in a water-cooled stainless steel chamber. The discharge between two electrodes was ignited in buffer gas with a pressure of 400 Torr and the current was held at 120 A. As the anode was consumed, the rods were kept at a constant distance from each other of about 1 mm by rotating the cathode. When the discharge ended, the soot generated was collected under ambient condition. In the arc discharge process, graphitic particles dropped to the bottom of the chamber, so we only collected the soot deposited on the inner and upper wall of the reaction chamber. Morphology analysis of the samples was carried out on JEOL JSM-7401 (JEOL Ltd., Tokyo, Japan) scanning electron microscope (SEM). The SEM was operated at 100 and 10 kV, respectively. Raman spectra were recorded from 1,000 to 2,000 cm−1 with a Jobin Yvon HR-800 spectrometer (Horiba Instruments, Tokyo, Japan) with an excitation wavelength of 633 nm. Thermogravimetric analysis was performed on a Q50TGA thermogravimetric analyzer (Thermal Analysis Inc., New Castle, DE, USA) from room temperature to 1,173 K at a rate of 10 K/min under an air flow of 30 ml/min [41].
Preparation of SWNHs-coated dishes
Purified SWNHs were synthesized by the arc discharge method [41]. C, H, N analysis was carried out on Vario EL III Element Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany). Other elemental contents were determined on a S4-Explorer X-ray fluorescence spectrometer (Bruker Corporation, Billerica, MA, USA) with 1 kW power and wavelength dispersion mode. The SWNHs had a purity of >95 wt.% and contained <5 wt.% amorphous carbon as the dominant impurity. To prepare the homogeneous SWNHs coating, a diluted solution of SWNHs in ultrapure water (produced from Milli-Q system, Millipro, Billerica, MA, USA) was dispersed. The aliquot (10 μg/ml) of the dispersed SWNHs was immediately spotted onto a 60-mm non-treated polystyrene dish (normal PS), which has a low adhesive surface for suspension culture in order to decrease the influence of the base material layer. The dishes were dried at 60°C for 3 h and sterilized by UV irradiation (DM-5; Daishin Co., Ltd., Osaka, Japan) for 16 h.
The following abbreviations have been used in this paper for the SWNHs-coated dishes: SWNHs-coated dishes, SWNHs10 (0.21 μg/cm2), SWNHs20 (0.42 μg/cm2), SWCNHs30 (0.64 μg/cm2), and SWNHs40 (0.85 μg/cm2).
SWNHs40 PS dishes with a bottom area of about 1 cm2 were prepared for SEM measurements and contact angle determinations. Uncoated PS dishes were used as control. After pre-treated by spraying gold on the films of samples, SEM measurements were carried out using a SIRION field emission scanning electronic microscope (FEI Corporation Ltd., Hillsboro, OR, USA) with accelerating voltage of 10.0 kV.
Contact angles of water droplets (volumes 2 to 5 μl) on SWNHs/PS and uncoated PS surfaces were determined on Dataphysics OCA20 Contact Angle Measuring System (Dataphysics, Filderatadt, Germany) at 293 K.
Surface roughness and topography
The surface area and mesopore size of SWNHs were determined by ASAP 2010 V3.02 E surface area analyzer (Micromeritics Instrument Corp., Norcross, GA, USA) with BET method. The sample was pre-treated at 298.15 K under vacuum for half an hour. Adsorptive gas is N2 and saturation pressure is about 765 mm Hg. Temperature of analysis bath liquid N2 is 77.41 K. for 5 s. Particle density of SWNHs was determined on AccuPyc 1330 Pycnometer at 291.3 K. The particle density was estimated from the high-pressure He buoyancy effect. This effect was measured gravimetrically up to 30 Mpa by an electronic micro-balance and pressure transducers. The particle size of 10 μg/ml SWNHs aqueous suspension was determined on Zetasizer V 2.0 (Malvern Instrument Ltd., Worcestershire, UK) at 298.3 K.
A film with 0.83 μg/cm2 SWNHs/Ps was prepared for SEM and contact angle determination. The culture dish was cut, and the area of every film is about 1 cm2. For comparison, polystyrene films of same area without SWNHs were also prepared. SEM measurements were carried out on XL30 S-FEG scanning electronic microscopy (FEI Corporation Ltd) with accelerating voltage of 10.0KV. The samples were treated by spraying gold on films.
Cell culture
Mice microglia cell lines N9 and BV2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, CA, USA) and 1% penicillin-streptomycin-neomycin (PSN) antibiotic mixture (Invitrogen) at 37°C in a humidified 5% CO2/95% air environment for 5 days. Lipopolysaccharide (LPS) from Escherichia coli serotype O111:B4 (Sigma-Aldrich, St. Louis, MO, USA) were used in this study. The cells were treated with 100 ng/ml LPS.
Cells were seeded onto 60-mm SWNHs-coated dishes and then were cultured in DMEM with FBS and PSN at 37°C in a humidified 5% CO2/95% air environment for 48 h treated with or without LPS at the same time. All results from BV-2 were similar to those from N9.
Cell synchronization, BrdU labeling, and mitotic index
The cells were synchronized by double thymidine block. Briefly, cells were plated at 40% confluency and arrested with 2 mM thymidine. The cells were incubated in DMEM with FBS and PSN at 37°C in a humidified 5% CO2/95% air environment for 48 h, and after which were incubated with DNA-lipid mixture for 3 h, then the cells were washed twice and incubated in fresh medium for additional 5 h. Subsequently, cells were cultured in medium containing 2 mM thymidine and 2 μg/ml puromycin for the second arrest and drug selection. After 16 h incubation, the cells were released into the cell cycle by incubation in fresh medium at SWNHs-coated dishes for 48 h treated with or without LPS at the same time. Cells were collected or fixed at indicated time points and subjected to specific analyses.
BrdU labeling was used to evaluate DNA synthesis. After release from the second thymidine arrest at indicated time points, cells were cultured for 48 h in 12-well plate coated with SWNHs, then the cells were pulse labeled with BrdU (50 μM) for 30 min. After three washes of phosphate buffered solution (PBS), cells were fixed with 1 ml of Carnoy’s fixative (three parts methanol 1:1 part glacial acetic acid) at −20°C for 20 min, and followed by three washes of PBS. Subsequently, DNA was denatured by incubation of 2M HCl at 37°C for 60 min, followed by three washes in borate buffer (0.1 M borate buffer, pH 8.5). After incubation with the blocking buffer, cells were stained with anti-BrdU antibody (1:100; BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C. After three washes of PBS, the cells were incubated with Texas Red-conjugated anti-mouse goat IgG for 30 min at real-time. After washes, the cells were mounted and BrdU positive cells were manually scored under immunofluorescence microscope.
Mitotic events were scored by time-lapse video microscopy and DNA staining. The cells were synchronized as described above and then cultured in SWNHs-coated for 48 h treated with or without LPS at the same time. Real-time images were captured every 10 min with Openlab software (PerkinElmer Inc., Waltham, MA, USA). Mitotic events of control, cells were scored by their morphological change (from flat to round-up). For each experiment, at least 800 cells were videotaped, tracked, and analyzed. Alternatively, nocodazole (100 ng/ml) was added into the medium and after release, the cells were collected, fixed, and stained with DNA dye (Hoechst 33258; Invitrogen, Carlsbad, CA, USA). Mitotic cells were scored by nuclear morphology and DNA condensation.
Cell cycle analysis
The cells cultured in SWNHs-coated for 48 h treated with or without LPS at the same time were dissociated with trypsin, washed, and resuspended in PBS as a single-cell suspension after cultured 48 h. The cells were fixed in 70% ethanol overnight, stained with propidium iodide (25 μg/ml) (Sigma), and incubated for 30 min at 37°C with RNase A (20 μg/ml). The cells group treated with PBS was used as the controls. The cells were assessed by flow cytometer (Becton Dickinson, San Jose, CA, USA) and the results were analyzed with Modifit software. The DNA content of the cells was then evaluated by fluorescence-activated cell sorting with a FACSCalibur (BD Immunocytometry Systems).
Cell growth and proliferation assay
Cell growth in SWNHs-coated dishes for 48 h treated with or without LPS at the same time was determined by the colorimetric tetrazolium derived sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay (Roche Applied Science, Mannheim, Germany), and DNA synthesis of the cells was assessed by the BrdU (bromodeoxyuridine) incorporation assay (Roche Applied Science). For the cell growth and proliferation assay, at 48 h after culture, the cells of each group were re-seeded in SWNHs-coated 96-well plates at a density of 0.3 to 1 × 104 cells per well. After 48 h, XTT and incorporated BrdU were measured colorimetrically using a microtiter plate reader (Bio-Rad Corp., Hercules, CA, USA) at a wavelength of 450 nm [42].
Cell viability assay
Cell viability was determined using a CCK-8 cell viability assay kit (DOJINDO Laboratories, Japan). All cells (5 × 103 cells/well) were pre-treated with various methods as indicated and then incubated 16 h in a 96-well plate. A 10 μL of cell viability assay kit solution was added to each well of the plate. After incubation for 1 h at 37°C in the dark, absorbances were measured at 450 nm using a multi-well plate reader [43].
Determination of apoptosis
Apoptotic cells treated with SWNHs were identified by fluorescence-activated cell sorting (FACS) using Annexin V-Fluos (Biolegend, San Diego, CA, USA) following the protocol of the manufacturer.
TEM
Cells were seeded onto 60-mm SWNHs-coated and control dishes and then cultured in DMEM at 37°C in a humidified 5% CO2/95% air environment for 48 h, then collected and fixed with 3% glutaraldehyde. For transmission electron microscope (TEM), ultrathin cells slices of 100 nm thickness were cut using an ultramicrotome and mounted on grids. The slices were contrasted with aqueous solution of uranyl acetate and lead citrate and examined on JEM-1400 Transmission Electron Microscope (JEOL Ltd, Japan) with accelerating voltage of 80 kV.
Cellular oxygen consumption assay
Steady state cell respiration in cells was measured in nonbuffered DMEM containing 5.5 mM glucose for cells with XF24 analyzer (Seahorse Bioscience, North Billerica, MA, USA) according to the manual.
ATP production assay
Steady state cellular ATP levels were measured by using ATP bioluminescence assay kit CLS II in accordance with the protocol (Roche).
NAD assay
Nicotinamide adenine dinucleotide (NAD) assay was performed as previously described [44-46]. Cells were extracted in 0.5 N HClO4, neutralized with 3 M KOH/125 mM gly-gly buffer (pH 7.4), and centrifuged at 10,000×g for 5 min. Supernatants were mixed with a reaction medium containing 0.1 mM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), 0.9 mM phenazine methosulfate, 13 units/ml alcohol dehydrogenase, 100 mM nicotinamide, and 5.7% ethanol in 61 mM gly-gly buffer (pH 7.4). The A560 nm was determined immediately and after 10 min, and results were calibrated with NAD standards.
Western blot analysis
Western blots were prepared as described [45]. Neuron cultures were lysed and collected in radioimmunoprecipitation assay buffer (cell signaling) with 1 mM PMSF on ice for 30 min. Cell lysates were centrifuged at 14,000×g for 10 min, and cell extracts were mixed with a 1:4 volume of SDS-PAGE loading buffer (10% β-mercaptoethanol, 10% glycerol, 4% SDS, 0.01% bromophenol blue, and 62.5 mM Tris–HCl, pH 6.8) and heated to 65°C for 15 min. Five samples were loaded on a 10% resolving SDS-polyacrylamide gel and transferred to polyvinyldifluoridine membranes. Membranes were incubated overnight at 4°C with rabbit polyclonal anti-Sirt3 (1:500; Abcam, Pak Shek Kok, New Territories, Hong Kong), rabbit polyclonal anti-acetyl-lysine (1:1,000; Biomol, Enzo Life Sciences, Inc., Farmingdale, NY, USA), P53 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:1,000 dilution), β-actin (Santa Cruz, 1:1,000), caspase 3, 7 (Cell Signaling Technology Inc., Danvers, MA, USA; 1:1,000), and then reacted with anti-rabbit or anti-goat secondary antibodies (1:10,000; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected with luminol reagent (GE, Munich, Germany).
Statistics
Continuous normally distributed variables were represented graphically as mean ± standard deviation (SD). For statistical comparison of quantitative data between groups, analysis of variance (ANOVA) or t test was performed. To determine differences between groups not normally distributed, medians were compared using Kruskal-Wallis ANOVA. The χ2 test was used when necessary for qualitative data. The degree of association between variables was assessed using Spearman’s non-parametric correlation. All statistical analyses were carried out using SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Probabilities of 0.05 or less were considered to be statistically significant.
Results and discussion
Characterization of SWNHs
The result of elemental composition determination of the SWNHs material used in this work is shown in Additional file 1: Table S1. The result showed that the material contained 95.3% of carbon. The content of each of the transition metals was less than 0.1%. The total metal content was about 0.25%. Due to catalyst-free preparation method of the material, its metal impurities are from the graphite raw material.
The adsorptive isotherm plot and BJH pore size distribution of SWNHs material are shown in Additional file 1: Figures S1 and S2. The result showed that BET surface area was 631.55m2/g, higher than that reported previously [47]. Single point total pore volume of pores (diameter less than 308.7 nm at P/P0 0.994) was 1.57 cm3/g. The particle density was 1.0077 g/cm3 (RSD 0.91%). It implies the existence of many closed pores in SWNHs (see Additional file 1).
The measurement of SWNHs particle size distribution (Additional file 1: Figure S3) showed that it ranged from 342 to 712 nm in aqueous suspension. An individual SWNHs particle is a dahlia-like spherical aggregate of nanohorns with a diameter of 80 to 100 nm. Thus, our result showed that the particles were secondary aggregations of primary spherical SWNHs aggregates in aqueous suspension.
SEM and contact angle measurements of SWNHs-coated dishes
SEM images (Additional file 1: Figure S4) showed that SWNHs were individual spherical particles with diameters of 60 to 100 nm on the PS surface. The comparison with the diameter of SWNHs aggregates in aqueous suspension was shown in above section. It meant that the structure of secondary SWNHs aggregates were disintegrated into individual primary aggregates of SWNHs on PS surface after evaporation of water in the suspension. It is conjectured that the disintegration is due to the stronger stacking interactions between the benzene ring on the surface of PS and SWNHs than that between SWNHs aggregates.
Because SWNHs particles were unstable coated on PS surface, partial SWNHs particles on PS surface diffused to water droplet and suspended by buoyancy of water. Then a new SWNHs/PS surface with less SWNHs particles than original SWNHs/PS surface was formed, as a result, the hydrophobicity of the surface was lowered and it resulted in decrease of the contact angle (Additional file 1: Figure S5).
SWNHs inhibited mitotic entry of N9 cells, especially in pre-treated with LPS
To assure how the SWNHs affect cellular mitosis, we incorporated BrdU into the control. We found that the accumulation of mitotic N9 cells pre-treated with or without LPS were significantly delayed by SWNHs at every time point followed with the increasing concentrations of SWNHs (P < 0.01). The accumulation of mitotic N9 cells pre-treated with LPS (Figure 1B) was much more than that without LPS (Figure 1A).
Figure 1 SWNHs inhibited mitotic entry of N9 cells, especially in pre-treated with LPS. To assure how the SWNHs affect cellular mitosis, we incorporated BrdU into the control. We found that the accumulation of mitotic N9 cells pre-treated with or without LPS was significantly delayed by SWNHs at every time point followed with the increasing concentrations of SWNHs (P < 0.01), and the accumulation of mitotic N9 cells pre-treated with LPS (B) were much more than that without LPS (A). The mitotic entry of N9 cells pre-treated with LPS (D) was more than N9 cells (C). SWNHs inhibited mitotic entry of N9 cells pre-treated with or without LPS significantly at every time point followed with the increasing concentrations of SWNHs (P < 0.01). The mitotic entry inhibited by SWNHs in N9 cells pre-treated with LPS (D) was more significant than N9 cells (C). All data are represented as mean ± SEM.
The mitotic entry of N9 cells pre-treated with LPS (Figure 1D) was more than N9 cells (Figure 1C). SWNHs inhibited mitotic entry of N9 cells pre-treated with or without LPS significantly at every time point followed with the increasing concentrations of SWNHs (P <0.01). The mitotic entry inhibited by SWNHs in N9 cells pre-treated with LPS (Figure 1D) was more significant than N9 cells (Figure 1C).
SWNHs inhibited growth and proliferation of N9 cells, especially in pre-treated with LPS
By XTT assays, we investigated the effect of SWNHs on cell growth and found that the growth of N9 cells pre-treated with LPS (Figure 2B) was much more significant than that in N9 cells (Figure 2A). The growth of cells was significantly inhibited by SWNHs at each time point in a dose-dependent manner (P < 0.001), especially in cells pre-treated with LPS (Figure 2B).
Figure 2 SWNHs inhibited growth and proliferation of N9 cells, especially in pre-treated with LPS. By XTT assays, we investigated the effect of SWNHs on cell growth and found that the growth of N9 cells pre-treated with LPS (B) was much more significantly than N9 cells (A). The growth of cells were significantly inhibited by SWNHs at each time point in a dose-dependent manner (P < 0.001), especially in cells pre-treated with LPS (B). Cell viability was evaluated by CCK-8 assay. The result showed that the proliferation of N9 cells pre-treated with LPS (D) was much more significantly than N9 cells (C). The proliferation of N9 cells treated with SWNHs was significantly inhibited at each time point in a time and dose-dependent manner (C and D). The effect induced by SWNHs on N9 cells pre-treated with LPS (D) was far more than that cells pre-treated without LPS (B). All data are represented as mean ± SEM.
Cell viability was evaluated by CCK-8 assay. The result showed that the proliferation of N9 cells pre-treated with LPS (Figure 2D) was much more significant than that in N9 cells (Figure 2C). The proliferation of N9 cells treated with SWNHs was significantly inhibited at each time point in a time- and dose-dependent manner (Figure 2C,D). The effect induced by SWNHs on N9 cells pre-treated with LPS (Figure 2D) was far more significant than that cells pre-treated without LPS (Figure 2B).
SWNHs affected cell cycle of N9 cells, especially in pre-treated with LPS
The cell cycle of N9 cells was affected by SWNHs in a dose-dependent manner, especially in cells pre-treated with LPS (Figure 3B). Followed with the increasing concentrations of SWNHs, the ratio of the G1 phase increased and S phase decreased significantly in N9 cells pre-treated with LPS (P < 0.01). The ratio of G2 phase decreased in N9 cells and it decreased until SWNHs30 and increased abruptly at the concentration of SWNHs40 in N9 cells pre-treated with LPS (P > 0.05). The effect induced by SWNHs on N9 cells pre-treated with LPS was more significant than on N9 cells (Figure 3A).
Figure 3 SWNHs affected cell cycle of N9 cells, especially in pre-treated with LPS. Cell cycle of N9 cells was affected by SWNHs in a dose-dependent manner, especially in cells pre-treated with LPS (B). Followed with the increasing concentrations of SWNHs, the ratio of the G1 phase increased and S phase decreased significantly in N9 cells pre-treated with LPS (P < 0.01), the ratio of G2 phase decreased in N9 cells and it decreased until SWNHs30 and increased abruptly at the concentration of SWNHs40 in N9 cells pre-treated with LPS (P > 0.05). The effect induced by SWNHs on N9 cells pre-treated with LPS was more significant than on N9 cells. All data are represented as mean ± SEM.
SWNHs promoted cell apoptosis of N9 cells, especially in pre-treated with LPS
After the cells had been cultured onto SWNHs-coated dishes for 48 h, the effect of SWNHs on cell apoptosis distribution was determined by flow cytometry. Apoptosis of N9 cells (Figure 4A) and N9 cells pre-treated with LPS (Figure 4B) was promoted with the increasing concentrations of SWNHs (P < 0.001). The effect on apoptosis induced by SWNHs on N9 cells pre-treated with LPS was more significant than N9 cells.
Figure 4 SWNHs promoted cell apoptosis of N9 cells, especially in pre-treated with LPS. After the cells had been cultured onto SWNHs-coated dishes for 48 h, the effect of SWNHs on cell apoptosis distribution was determined by flow cytometry. Apoptosis of N9 cells (A) and N9 cells pre-treated with LPS (B) was promoted with the increasing concentrations of SWNHs (P < 0.001). The effect on apoptosis induced by SWNHs on N9 cells pre-treated with LPS was more significant than N9 cells. All data are represented as mean ± SEM.
The growth N9 cells affected by SWNHs, especially in pre-treated with LPS
The 3 × 105 liver cells were seeded onto 60-mm SWNHs-coated dishes, and then all cells were countered after cultured for 48 h. The number of N9 cells pre-treated with LPS (Figure 5B) was more significant than N9 cells (Figure 5A). Followed with the increasing concentrations of SWNHs, the number of N9 cells was decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (Figure 5B) (P < 0.01).
Figure 5 The growth N9 cells affected by SWNHs, especially in pre-treated with LPS. The 3 × 105 liver cells were seeded onto 60-mm SWNHs-coated dishes, and then all cells were countered after cultured for 48 h. The number of N9 cells pre-treated with LPS was more significant than N9 cells (A). Followed with the increasing concentrations of SWNHs, the number of N9 cells decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (B) (P < 0.01). All data are represented as mean ± SEM.
TEM images of N9 cells treated with SWNHs
N9 cells were treated with SWNHs untreated with LPS as control (Figure 6A). The size of N9 cells pre-treated with LPS (Figure 6B) and their nucleus were larger than that in control. The apoptotic bodies were observed in cytoplasm. The size of lysosome and mitochondria in N9 cells pre-treated with LPS (Figure 6B) were larger than that in control (Figure 6A). A lot of secretory vesicles were observed outside of cells treated with SWNHs.
Figure 6 TEM images of N9 cells treated with SWNHs. (A) N9 cells treated with SWNHs untreated with LPS as control (×15,000 magnification). Scale bar represents 1 μm. (B) N9 cells cultured onto SWNHs-coated dishes (0.85 μg/cm2) for 48 h pre-treated with LPS (×15,000 magnification). Scale bar represents 1 μm. The size of N9 cells pre-treated with LPS and their nucleus were larger than that of control. The apoptotic bodies were observed in cytoplasm. The size of lysosome and mitochondria in N9 cells pre-treated with LPS (B) were larger than that of control (A). A lot of secretory vesicles could be observed outside of cells treated with SWNHs. All data are represented as mean ± SEM.
The mitochondrial functions of N9 cells affected by SWNHs, especially in pre-treated with LPS
Intact cellular basal oxygen consumption rates (OCR) of N9 cells pre-treated with or without LPS induced by SWNHs were measured by Seahorse XF24 analyzer. The OCR of N9 cells pre-treated with LPS (Figure 7B) was more significant than that in N9 cells (Figure 7A). Followed with the increased concentrations of SWNHs, the OCR of N9 cells decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (Figure 7B) (P < 0.01).
Figure 7 The mitochondrial functions of N9 cells affected by SWNHs, especially in pre-treated with LPS. Intact cellular basal OCR of N9 cells pre-treated with or without LPS induced by SWNHs measured by Seahorse XF24 analyzer. The OCR of N9 cells pre-treated with LPS (B) was more significant than N9 cells (A). Followed with the increasing concentrations of SWNHs, the OCR of N9 cells decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (B) (P < 0.01). Steady state cellular ATP levels of N9 cells pre-treated with or without LPS induced by SWNHs were measured too. The steady state cellular alkaline phosphatase (APT) level of N9 cells pre-treated with LPS (D) was more significant than N9 cells (C). Followed with the increasing concentrations of SWNHs, the steady state cellular ATP level of N9 cells decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (D) (P < 0.01). All data are represented as mean ± SEM.
Steady state cellular ATP levels of N9 cells pre-treated with or without LPS induced by SWNHs were measured too. The steady state cellular APT level of N9 cells pre-treated with LPS (Figure 7D) was more significant than that in N9 cells (Figure 7C). Followed with the increased concentrations of SWNHs, the steady state cellular ATP level of N9 cells was decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (Figure 7D) (P < 0.01).
The NAD levels of N9 cells affected by SWNHs, especially in pre-treated with LPS
NAD levels were measured in N9 cells pre-treated with or without LPS induced by SWNHs. NAD level of N9 cells pre-treated with LPS (Figure 8B) were more significant than in N9 cells (Figure 8A). Followed with the increased concentrations of SWNHs, the NAD level of N9 cells pre-treated with (Figure 8B) or without LPS (Figure 8A) was decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (Figure 8D) (P < 0.01).
Figure 8 NAD levels of N9 cells affected by SWNHs, especially in pre-treated with LPS. NAD levels were measured in N9 cells pre-treated with or without LPS induced by SWNHs. NAD level of N9 cells pre-treated with LPS (B) were more significant than in N9 cells (A). Followed with the increasing concentrations of SWNHs, NAD level of N9 cells decreased significantly in a dose-dependent manner, especially in pre-treated with LPS (B) (P < 0.01). All data are represented as mean ± SEM.
Key factors involved in apoptosis in vivo
The expression levels of Sirt3 in N9 cells pre-treated with LPS (Figure 9B) was much more than that in the control of N9 cells (Figure 9A). The increased expression levels of Sirt3 decreased followed with the increasing concentrations of SWNHs, which is especially significant in pre-treated with LPS (Figure 9B). The expression levels of activation cleavage of P53, caspase-3, and caspase-7 correlated with apoptosis increased followed with the increasing concentrations of SWNHs, especially in pre-treated with LPS (Figure 9B).
Figure 9 Key factors involved in apoptosis in vivo. The expression levels of Sirt3 in N9 cells pre-treated with LPS (B) was much more than control of N9 cells (A). The increased expression levels of Sirt3 decreased followed with the increasing concentrations of SWNHs, which is especially significant in pre-treated with LPS (B). The expression levels of activation cleavage of P53, caspase-3, and caspase-7 correlated to apoptosis increased followed with the increasing concentrations of SWNHs, which is especially significant in pre-treated with LPS (B).
Sepsis and its complications are the leading causes of mortality in intensive care units accounting for 10% to 50% of deaths. Up to 71% of septic patients develop potentially irreversible acute cerebral dysfunction. Sepsis-induced SE is the leading cause of death in septic patients. On one side, the brain is especially susceptible to damage during sepsis and on the other side the brain dysfunction may actively contribute to the pathogenesis of SE. The existence of reciprocal interactions between the immune and central nervous systems (CNS) makes the brain be one of the most vulnerable organs during sepsis. Furthermore, brain dysfunction can influence the function of the autonomic nervous system and neuroendocrine system, which accelerates the occurrence of SE [1-3]. Microglia is the resident immune cell in the brain tissue and is among the first to respond to brain injury. Microglia are rapidly activated and migrate to the affected sites of neuronal damage where they secrete both cytotoxic and cytotrophic immune mediators [48]. Microglial activation plays an important role in neuroinflammation and SE, which contributes to neuronal damage. Inhibition of microglial activation may have therapeutic benefits that can alleviate the progression of neurodegeneration and SE [7].
Our results indicated that LPS induced activation of microglia, promoted its growth and proliferation, and inhibited its apoptosis. The status was converted by SWNHs. Our result showed that in aqueous suspension, the particles were secondary aggregations of primary spherical SWNHs aggregates. In the present study, we prepared SWNHs-coated dishes with homogeneous thin or thick films by coating non-modified SWNHs on the surface of a commercially available non-treated polystyrene dish (normal PS). The main advantages of our research with SWNHs-coated dishes were as follows: (1) coating with non-modified SWNHs and without binder, (2) coating with gradual concentrations of SWNHs, (3) PS surface as individual particles of SWNHs and high transparency, and (4) no influence from the base substrate (normal PS as a base material has a proper adhesiveness for cells). Therefore, we can evaluate the natural properties of SWNHs films for cell responses. Thin films were promising materials because they have individual particles of SWNHs, which are known to largely influence cell functions.
The contact angle of water droplet on PS surface was 44.9° which was less than SWNHs/PS, 74.5°. The phenomena indicated higher surface hydrophobicity of SWNHs/PS than PS film. After a few minutes, contact angle of water droplet on SWNHs/PS surface decreased to 64.7° (Additional file 1: Figure S5). Because SWNHs particles were unstable covered on PS surface, SWNHs particles were suspended by buoyancy force of water. The image of SEM showed that distances between neighbor SWNHs particles were about 500 nm which was far less than the diameter of water droplet. Such a surface phenomena similar to lotus leaf effect can be observed (Additional file 1: Figure S4).
We found that LPS induced activation of microglia, promoted its growth and proliferation, and inhibited its apoptosis. SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in activation microglia cells induced by LPS. The results of Ding et al. showed that at high dosages, carbon nanoparticles can seriously impact the cellular functions in maintenance, growth, and differentiation [49]. These different cellular behaviors cited above can be partially ascribed to the differences of properties for different carbon nanomaterials-surface area, pore structure, particle size, length, diameter and curvature, and partially ascribed to different cell types. Besides, the status of modification of carbon nanomaterials - modified with different functional groups or compounds, or not modified at all - will affect their biological functions on cells [50,51].
Apoptosis is an active process of cell death that both involves physiological and pathogenic processes. We observed the distended nuclei and scant cytoplasm, cell shrinkage, membrane blebbing, chromatin condensation, and apoptotic body in the cytoplasm of mice microglia, especially in cells pre-treated with SWNHs. The features of these phenomena were typical during the apoptotic process [52-54].
Our results showed that the roles of SWNHs on mice microglia cells were related to energy metabolism. Sirt3 was the only sirtuin implicated in extension of life span in human [55]. It has been shown Sirt3 involved with mitochondrial energy metabolism and biogenesis [56] and preservation of ATP biosynthetic capacity in the heart [57]. Sirt3 was shown to regulate the activity of acetyl-CoA synthetase 2 (AceCS2), an important mitochondrial enzyme involved in generating acetyl-CoA for the tricarboxylic acid (TCA) cycle. In these studies, Sirt3 knockout resulted in a marked decrease of basal ATP level in vivo[58]. Recent studies in cardiomyocytes demonstrated the protective role of Sirt3 from oxidative stress and hypertrophy [59,60]. Accordingly, the evidences above suggest that Sirt3 also has a pivotal role in protecting neurons from injury due to conditions that promote bioenergetic failure, such as excitotoxicity. Mitochondrial localization of Sirt3 plays a role in various mitochondrial functions, such as maintaining basal ATP level and regulating apoptosis. Sirt3 has been shown to regulate energy homeostasis [57].
Continuous supply of energy is crucial for the neuron survival due to the requirement for large amounts of energy for high metabolic processes coupled with an inability to store energy [61,62]. Therefore, the neurons are highly susceptible to insults that lead to energy depletion, such as oxidative stress, excitotoxicity, and DNA damage [63,64]. As a critical factor in energy metabolism for cell survival, NAD has drawn considerable interest. NAD is an essential molecule playing a pivotal role in energy metabolism, cellular redox reaction, and mitochondrial function. Recent studies have revealed that it is important for maintaining intracellular NAD in promoting cell survival in various types of diseases, including axonal degeneration, multiple sclerosis, cerebral ischemia, and cardiac hypertrophy [59,65-70]. Loss of NAD decreases the ability of NAD-dependent cell survival factors to carry out energy-dependent processes, leading to cell death. Our results coincide with those; the roles of SWNHs on mice microglia cells related to energy metabolism were associated with Sirt3.
Mitochondrial enzymes play central roles in anabolic growth, and acetylation may provide a key layer of regulation over mitochondrial metabolic pathways. As a major mitochondrial deacetylase, Sirt3 regulates the activity of enzymes to coordinate global shifts in cellular metabolism. Sirt3 promotes the function of the TCA cycle and the electron transport chain and reduces oxidative stress. Loss of Sirt3 triggers oxidative damage and metabolic reprogramming to support proliferation. Thus, Sirt3 is an intriguing example of how nutrient-sensitive, posttranslational regulation may provide integrated regulation of metabolic pathways to promote metabolic homeostasis in response to diverse nutrient signals. The expression levels of Sirt3 in mice microglia cells was increased as induced by LPS (Figure 9B). However, increased expression levels of Sirt3 were decreased followed with the increasing concentrations of SWNHs, which is especially significant in pre-treated with LPS (Figure 9B). The roles of SWNHS on mice microglia was implicating Sirt3 and energy metabolism associated with it.
P53 and SIRT3 regulated the apoptosis of various mammalian cells. Caspase-3 and caspase-7 are the key factors among cysteine proteases which are critical for apoptosis of eukaryotic cells. Our results showed that the expression levels of SIRT3 were decreased, and those of caspase-3 and caspase-7, as well as that of activation cleavage of P53 increased for mice microglia cells (Figure 9) in a dose-dependent manner with the increasing concentrations of SWNHs, which is especially significant in pre-treated with LPS (Figure 9B). These results also indicate that SWNHs promoted cell apoptosis. The phenomenon was associated with Sirt3 and energy metabolism was related to Sirt3.
SWNHs may be as a novel opportunity or method for the research on treatment of septic encephalopathy by inhibiting activation of microglia through blocking of Sirt3.
Conclusions
SWNHs inhibited mitotic entry, growth and proliferation of mice microglia cells, and promoted its apoptosis, especially in cells pre-treated with LPS. SWNHs inhibited expression of Sirt3 and energy metabolism related with Sirt3 in mice microglia cells in a dose-dependent manner, especially in cells pre-treated with LPS.
Abbreviations
AceCS2: Acetyl-CoA synthetase 2; AICD: Activation-induced cell death; BrdU: Bromodeoxyuridine; CNS: Central nervous system; CNTs: Carbon nanotubes; DMEM: Dulbecco’s modified Eagle’s medium; FACS: Fluorescence-activated cell sorting; FBS: Fetal bovine serum; LPS: Lipopolysaccharide; NAD: Nicotinamide adenine dinucleotide; PSN: Penicillin–streptomycin-neomycin; SD: Standard deviation; SE: Septic encephalopathy; SEM: Scanning electron microscope; SWNHs: Single-wall carbon nanohorns; TCA: Tricarboxylic acid
Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
LL, JZ, YY, QW, YC, ZS, MZ, and GG have carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. LL, JZ, LG, YY, TC, XZ, GX, and GG participated in the design of the study and performed the statistical analysis. JZ and GG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1
Supporting Informations. This file contain descriptions of the elemental contents of SWNHs material, adsorptive isotherm plot and BJH pore size distribution of SWNHs material, particle density of SWNHs, particle sizes distribution of SWNHs in aqueous suspension and the films of SWNHs/PS observed by SEM, and contact angle of water droplet on the surfaces of PS and PS coated with SWNHs(SWNHs/PS) films.
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Acknowledgments
This work was supported by granted from the National Natural Science Foundation of China (nos. 30600524, 81071990, 81172383 and 81201758), Science and Technology Planning Project of Guangdong Province (nos. 2012A030400055, 2010B080701088, 2011B080701096, and 2011B031800184), Science and Technology Application infrastructure projects of Guangzhou (nos. 2011J410010 and 2011J4300066). The study sponsors had no involvement in the study. We thank Ms. Kening Xu and Ms. Yuan Wang in the State Key Laboratory of Natural and Biomimetic Drugs, Peking University (PKU); Ms. Ling Sun, Ms. Fei Zhang, and Ms. Li Zhang in Analytical Instrumentation Center, PKU; Ms. Qin Xie in Center for Nanochemistry, PKU; Ms. Shenglan Wang in Electronic Microscope Laboratory, Pathology Department, PKU; Mr. Dongwu Chang in Department of Thermal Engineering, Tsinghua University; and Mr. Xinan Yang in Institute of Physics, Chinese Academy of Sciences for their kind help to perform physicochemical data determination and microscope measurement. We also thank Dr. Bingjiu Xu in School of Pharmaceutical Sciences, Capital Medical University for his kind proposals to the research.
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Front PsycholFront PsycholFront. Psychol.Frontiers in Psychology1664-1078Frontiers Media S.A. 10.3389/fpsyg.2013.00073PsychologyOriginal ResearchRecursive Fury: Conspiracist Ideation in the Blogosphere in Response to Research on Conspiracist Ideation Lewandowsky Stephan 1*Cook John 12Oberauer Klaus 13Marriott Michael 41School of Psychology, University of Western AustraliaCrawley, WA, Australia2Global Change Institute, The University of QueenslandSt. Lucia, QLD, Australia3School of Psychology, University of ZurichZurich, Switzerland4Climate Realities ResearchMelbourne, VIC, AustraliaEdited by: Viren Swami, University of Westminster, UK
Reviewed by: Elaine McKewon, University of Technology Sydney, Australia; Viren Swami, University of Westminster, UK
*Correspondence: Stephan Lewandowsky, School of Psychology, University of Western Australia, Crawley, WA 6009, Australia. e-mail: [email protected] article was submitted to Frontiers in Personality Science and Individual Differences, a specialty of Frontiers in Psychology.
18 3 2013 2013 4 7305 11 2012 02 2 2013 Copyright © 2013 Lewandowsky, Cook, Oberauer and Marriott.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Conspiracist ideation has been repeatedly implicated in the rejection of scientific propositions, although empirical evidence to date has been sparse. A recent study involving visitors to climate blogs found that conspiracist ideation was associated with the rejection of climate science and the rejection of other scientific propositions such as the link between lung cancer and smoking, and between HIV and AIDS (Lewandowsky et al., in press; LOG12 from here on). This article analyses the response of the climate blogosphere to the publication of LOG12. We identify and trace the hypotheses that emerged in response to LOG12 and that questioned the validity of the paper’s conclusions. Using established criteria to identify conspiracist ideation, we show that many of the hypotheses exhibited conspiratorial content and counterfactual thinking. For example, whereas hypotheses were initially narrowly focused on LOG12, some ultimately grew in scope to include actors beyond the authors of LOG12, such as university executives, a media organization, and the Australian government. The overall pattern of the blogosphere’s response to LOG12 illustrates the possible role of conspiracist ideation in the rejection of science, although alternative scholarly interpretations may be advanced in the future.
science denialconspiracy theoriesconspiracist ideationInternet blogsclimate change
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Introduction
Conspiratorial thinking, also known as conspiracist ideation, has been repeatedly implicated in the rejection of scientific propositions (Diethelm and McKee, 2009; Kalichman, 2009; Goertzel, 2010; McKee and Diethelm, 2010). Conspiracist ideation generally refers to the propensity to explain a significant political or social event as a secret plot by powerful individuals or organizations (Sunstein and Vermeule, 2009). When conspiracist ideation is involved in the rejection of science, ideations tend to invoke alternative explanations for the nature or source of the scientific evidence. For example, among people who reject the link between HIV and AIDS, common ideations involve the beliefs that AIDS was created by the U.S. Government to control the African American population or that people who take medicines for HIV are guinea pigs for the government (Bogart and Thorburn, 2005; Kalichman, 2009). Among African Americans, 16 and 44% of respondents, respectively, have been found to endorse those two beliefs (Bogart and Thorburn, 2005). Given that such conspiracist ideation has been associated with sexual risk-taking behaviors (Bogart et al., 2011), the prominence of conspiracist ideation among people living with HIV should give rise to concern. AIDS denial also invokes ideations of censorship to explain why dissenting scientists who question the link between HIV and AIDS fail to insert their ideas into the peer-reviewed literature (Kalichman, 2009)1.
The belief that censorship, rather than evidence-based peer-review, underlies a consensus in the scientific literature also suffuses other arenas of science denial, such as in climate science (e.g., Solomon, 2008; McKewon, 2012a) and medical research other than HIV/AIDS. For example, the tobacco industry referred to research on the health effects of smoking in internal documents as “a vertically integrated, highly concentrated, oligopolistic cartel” (Abt, 1983, p. 127), which in combination with “public monopolies” … “manufactures alleged evidence, suggestive inferences linking smoking to various diseases, and publicity and dissemination and advertising of these so-called findings” (Abt, 1983, p. 126).
Because peer-review tends to eliminate ideas that are not supported by evidence (e.g., questioning the link between HIV and AIDS lost intellectual respectability decades ago; Nattrass, 2010, 2011), much of science denial involves the internet. The internet provides a platform for individuals who reject a scientific consensus to affirm “each other’s feelings of persecution by a corrupt elite” (McKee and Diethelm, 2010, pp. 1310–1311). Internet sites such as blogs dedicated to a specific issue have therefore become hubs for science denial and they arguably play a major role in the creation and dissemination of conspiracist ideation.
The role of conspiracist ideation, and its communication through the blogosphere are also prominent in the denial of the benefits of vaccinations. Content analyses have shown that YouTube videos critical of HPV vaccinations (Briones et al., 2012) and anti-vaccination blogs (Zimmerman et al., 2005) are suffused with conspiratorial content. Common conspiracist themes include alleged government cover-ups of vaccine information or suggestions that a vaccine solely exists to maximize the profit of pharmaceutical companies (Kata, 2010; Briones et al., 2012). The anti-vaccine movement has had demonstrably serious adverse public-health impacts (Poland and Jacobson, 2012). For example, nations that discontinued or reduced use of the pertussis (whooping cough) vaccine under public pressure now experience an incidence of the often fatal disease that is 10–100 times greater than countries that have continued vaccinations (Gangarosa et al., 1998). Lewandowsky et al. (2012a) provide a review of the societal and cognitive processes that underlie the spread of misinformation provided by groups such as the anti-vaccination movement.
The rejection of climate science has been particularly infused with notions of a conspiracy among scientists. Accusations of conspiracies within the Intergovernmental Panel on Climate Change (IPCC) were aired in the opinion pages of the Wall Street Journal (WSJ) as early as 1996 (Lahsen, 1999; Oreskes and Conway, 2010), in a piece that alleged a “… disturbing corruption of the peer-review process.” The charges focused on Chapter 8, a key component of the 1995 IPCC report that was concerned with the attribution of global warming to human activities. The WSJ piece was authored by an individual who had no part in the IPCC process, and subsequent scholarly work traced the origin of the charge of conspiracy and corruption to a document produced by the Global Climate Coalition, a lobby group representing 60 companies and trade associations, primarily from the energy sector (Lahsen, 1999). In her analysis of this controversy, Lahsen identified clear conspiracist themes and concluded that conspiracy theories are “… rhetorical means by which to cast suspicion on scientific and political opponents” (p. 133).
Accordingly, the titles of recent popular books critical of mainstream climate science are replete with hints of a conspiracy, with terms such as “hoax” (Bell, 2011; Inhofe, 2012), “corruption” (Montford, 2010), “scam” (Sussman, 2010), “fraud” (Solomon, 2008), or “junk science” (Isaac, 2012) being quite common. Some books have appealed directly to an alleged “conspiracy” (Inhofe, 2012), whereas others invoked a conspiracy obliquely by referring to global warming as an “assertion” by the United Nations (Alexander, 2009). Similarly McKewon (2012a) identified broad conspiracist themes in a narrative analysis of press coverage in response to one particular climate-“skeptic” book in Australia.
Conspiracist ideation is arguably also exhibited on climate blogs, for example when expressing the belief that climate scientists “colluded with government officials to ignore the law” (Condon, 2009), or that “… the alarmists who oversee the collection and reporting of the data simply erase the actual readings and substitute their own desired readings in their place” (Taylor, 2012). The role of the blogosphere in climate denial cannot be ignored: one blogger triggered several Congressional investigations into a Nature paper on paleoclimatology in the 2000’s, and the blogosphere continues to reverberate with alleged scandals involving climate scientists. Analyses of the blogosphere, and how it contributes to conspiracist ideation and science denial are therefore of considerable importance.
We are aware of only two reports that have quantitatively linked conspiracist ideation to the rejection of scientific propositions: Smith and Leiserowitz (2012) found that among people who reject the findings from climate science, up to 40% of affective imagery invoked conspiracy theories. That is, when asked to provide the first word, thought, or image that came to mind in the climate context, statements such as “the biggest scam in the world to date” would be classified as conspiracist. Similarly, a recent survey of visitors to climate blogs found general conspiracist ideation to be linked to the rejection not only of climate science but also of the link between HIV and AIDS and between lung cancer and smoking (Lewandowsky et al., in press). The present article examines the denialist blogosphere’s response to the study of Lewandowsky et al.(in press; hereafter; LOG12), which we therefore present in some detail.
Conspiracist ideation and rejection of science among climate blog visitors
Lewandowsky et al. (in press) placed links to their study on a number of climate blogs with a pro-science orientation but a diverse audience of readers, including a notable proportion of climate “skeptics.” The survey queried people’s belief in the free-market (which previous research had identified as an important predictor of the rejection of climate science; Heath and Gifford, 2006), their acceptance of climate science, their acceptance of other scientific propositions such as the link between HIV and AIDS, and most important in the present context, conspiracist ideation. The main results of Lewandowsky et al. (in press) are shown in Figure 1.
Figure 1 Latent variable model reported by Lewandowsky et al. (in press) that predicts acceptance of climate science and acceptance of other scientific propositions on the basis of free-market ideology, the perception that earlier environmental problems have been solved, and conspiracist ideation. All regression weights and correlations are significant and standardized. Weights and correlations that are not shown were set to zero (e.g., correlation between the residuals of climate science and other sciences). Manifest variables for each latent variable are omitted for clarity. Adapted from Lewandowsky et al. (in press). Reprinted with permission.
The figure shows the structural-equation model that captured the relationship between latent variables (the large circles in the figure). Each latent variable was measured by several items (manifest variables; not shown). For example, people’s endorsement of the free-market was measured by items such as “an economic system based on free-markets unrestrained by government interference automatically works best to meet human needs” and (reverse-coded) “free and unregulated markets pose important threats to sustainable development” (Heath and Gifford, 2006). In replication of much previous research (e.g., Heath and Gifford, 2006; Kahan et al., 2012), endorsement of free-market ideology emerged as a strong predictor of the rejection of climate science. Free-market ideology was also found to predict the rejection of other scientific propositions.
Of greater interest in the present context is the association between conspiracist ideation and the rejection of climate science and other scientific propositions, although the strength of this association was considerably less than that of free-market ideology. The conspiracy test items were adapted from previous research (e.g., Swami et al., 2009) and consisted of various conspiracies that respondents could endorse or reject, such as “a powerful and secretive group known as the New World Order are planning to eventually rule the world through an autonomous world government which would replace sovereign governments” and “The Apollo moon landings never happened and were staged in a Hollywood film studio,” and so on2.
When the article by Lewandowsky et al. (in press) became available for download in July-August 2012, the climate denialist blogosphere responded with considerable intensity along several prongs: complaints were made to the first author’s university alleging academic misconduct; several freedom-of-information requests were submitted to the first author’s university for emails and documents relating to LOG12; multiple re-analyses of the LOG12 data were posted on blogs which purported to show that the effects reported by LOG12 did not exist; and a number of hypotheses were disseminated on the internet with arguably conspiracist content. This response is not altogether surprising in light of research which has shown that threats – in particular to people’s sense of control – can trigger targeted small-scale conspiracy theories (Whitson and Galinsky, 2008), especially those involving a specific opponent (Sullivan et al., 2010).
The remainder of this article reports a content analysis of the hypotheses generated by the blogosphere to counter LOG12. The extent and vehemence of contrarian activity provided a particularly informative testbed for an analysis of how conspiracist ideation contributes to the rejection of science among web denizens. Unlike previous analyses of web content, the present project was conducted in “real time” as the response to LOG12 unfolded, thus permitting a fine-grained temporal analysis of the emerging global conversation. Moreover, the tight focus of the response on a single paper permitted the content analysis to be quite encompassing while still remaining manageable in size.
Materials and Methods
Sampling of content
Internet activity related to LOG12 was sampled using Google search and PsychInfo. Results were limited to English-speaking sites and text.
The first phase of the search placed LOG12 into a scholarly and public context. All peer-reviewed publications on conspiracist ideation published in 2012 were obtained from PsychInfo on 18 October, 2012. Papers were located using the search terms “conspiracy” or derivatives (e.g., “conspiracist” or “conspiratorial”). For each paper obtained in this search (N = 21), we recorded the total number of Google hits, limited to the first 10 months of 2012, using the author’s last name and the article’s title (or first phrase of title for titles exceeding a single phrase) as search string. Each of those hits was then examined to establish whether it contained any recursive hypotheses, defined as any potentially conspiracist ideation that pertained to the article itself or its author, such as “Dr Smith is a government agent,” or unsubstantiated and potentially conspiracist allegations pertaining to the article’s methodology, intended purpose, or analysis (e.g., “there were no human subjects”).
The second phase of the search traced the response to LOG12 in the blogosphere. An on-going web search in real time was conducted by two of the authors (John Cook and Michael Marriott) during the period August-October 2012. This daily search used Google Alerts to detect newly published material matching the search term “Stephan Lewandowsky.” If new blog posts were discovered that featured links to other relevant blog posts not yet recorded, these were also included in the analysis. To ensure that the collection of hypotheses pertaining to LOG12 was exhaustive, Google was searched for links to the originating blog posts (i.e., first instances of a recursive theory), thereby detecting any further references to the original hypothesis or any derivatives.
Although the second phase of the search encompassed the entire (English-speaking) web, it became apparent early on that the response of the blogosphere was focused around a number of principal sites. To formally identify those sites, we began by analyzing the 30 most-frequently read “skeptic” websites, as identified by Alexa rankings. Alexa is a private company, owned by Amazon, that collects data on web browsing behavior and publishes web traffic reports for the higher trafficked sites. This enables comparison of the relative traffic of websites covering similar topics.
Each of those 30 sites was then searched by Google for instances of the name of the first author of LOG12 that fell within the period 28 August-18 October 2012. Sites that returned more than 10 hits were considered a principal site, and they are shown in Table 1.
Table 1 Principal web sites involved in the blogosphere’s response to the publication of LOG12.
Website Google hitsa Blog Postsb
wattsupwiththat.com 747 11
joannenova.com.au 82 8
junkscience.comd 40 3
climateaudit.org* 36 11
bishophill.squarespace.com 33 4
australianclimatemadness.comc 30 7
climatedepot.com*d 20 17
rankexploits.com/musings 18 6
warwickhughes.com 16 0
noconsensus.wordpress.com 13 2
Sites identified with an asterisk were among the 5 sites contacted by LOG12 with an invitation to participate in the study.
aTotal number of hits on each site to the name of the first author of LOG12 that fell within the period 28 August-18 October, 2012.
bTotal number of blog posts featuring recursive theories about LOG12 posted within the period 28 August-18 October, 2012.
cThis blog is not among the top-30 “skeptic” sites but was a principal player in the response to LOG12 because its proprietor launched several freedom-of-information requests relating to LOG12.
dThese blogs reposted content from other blogs but published no original content of their own.
Blog posts that published recursive theories were excerpted (see Online Supplementary Material for all recorded instances) with each excerpt representing a mention of the recursive theory (see Table 3; Figure 2). Unless prevented by the website, all Google hits from the second phase were archived using www.webcitation.org.
Figure 2 Timeline of principal recursive theories developed by the blogosphere in response to LOG12. Density of shading reflects the number of mentions of each particular theory on a particular date.
Conspiracist classification criteria
We derived six criteria from the existing literature to permit classification of hypotheses pertaining to LOG12 as potentially conspiracist (see Table 3). Our criteria were exclusively psychological and hence did not hinge on the validity of the various hypotheses. This approach follows philosophical precedents that have examined the epistemology of conspiratorial theorizing irrespective of its truth value (e.g., Keeley, 1999; Sunstein and Vermeule, 2009). The approach also avoids the need to discuss or rebut the substance of any of the hypotheses.
First, the presumed intentions behind any conspiracy are invariably nefarious (Keeley, 1999): conspiracist ideation never involves groups of people whose intent is to do good, as for example when planning a surprise birthday party. Instead, conspiracist ideation relies on the presumed deceptive intentions of the people or institutions responsible for the “official” account that is being questioned (Wood et al., 2012). There is evidence that climate denial is infused with this assumption of nefarious intent, for example when climate science and research on the harmful effects of DDT are interpreted as a globalist and environmentalist agenda designed to impoverish the West and push civilization back into the stone age (Delingpole, 2011). When presenting the results, we refer to this criterion by the acronym NI, for nefarious intention (see Table 3).
A corollary of the first criterion is the pervasive self-perception and self-presentation among conspiracy theorists as the victims of organized persecution. The theorist typically considers herself, at least tacitly, to be the brave antagonist of the nefarious intentions of the conspiracy; that is, the victim is also a potential hero. The theme of the victimization of conspiracy theorists or their allies features prominently in science denial, for example when isolated scientists who oppose the scientific consensus that HIV causes AIDS are presented as persecuted heroes and are likened to Galileo (Kalichman, 2009; Wagner-Egger et al., 2011). We refer to this persecution-victimization criterion as PV for short.
Third, during its questioning of an official account, conspiracist ideation is characterized by “… an almost nihilistic degree of skepticism” (Keeley, 1999, p. 126); and the conspiracy theorist refuses to believe anything that does not fit into the conspiracy theory. Thus, nothing is at it seems, and all evidence points to hidden agendas or some other meaning that only the conspiracy theorist is aware of. Accordingly, low trust (Goertzel, 1994) and paranoid ideation (Darwin et al., 2011) feature prominently among personality and attitudinal variables known to be associated with conspiracist ideation. The short label for this criterion is NS (for nihilistic skepticism).
Fourth, to the conspiracy theorist, nothing happens by accident (e.g., Barkun, 2003). Thus, small random events are woven into a conspiracy narrative and reinterpreted as indisputable evidence for the theory. For example, the conspiracy theory that blames the events of 9/11 on the Bush administration relies on “evidence” (e.g., intact windows at the Pentagon; Swami et al., 2009) that are at least equally consistent with randomness. We refer to this criterion, that nothing is an accident as NoA for short.
Fifth, the underlying lack of trust and exaggerated suspicion contribute to a cognitive pattern whereby specific hypotheses may be abandoned when they become unsustainable, but those corrections do not impinge on the overall abstraction that “something must be wrong” and the “official” account must be based on deception (Wood et al., 2012). In the case of LOG12, the “official” account is the paper’s conclusions that conspiracist ideation contributes to science denial; and it is this conclusion that must be wrong. At that higher level of abstraction, it does not matter if any particular hypothesis is right or wrong or incoherent with earlier ones because “… the specifics of a conspiracy theory do not matter as much as the fact that it is a conspiracy theory at all” (Wood et al., 2012, p. 771). Thus, the specific claims and assumptions being invoked by conspiracist ideation may well be fluctuating, but they are all revolving around the fixed belief that the official version is wrong. In consequence, it may not even matter if hypotheses are mutually contradictory, and the simultaneous belief in mutually exclusive theories – e.g., that Princess Diana was murdered but also faked her own death – has been identified as an aspect of conspiracist ideation (Wood et al., 2012). We label this criterion MbW, for “must be wrong.”
Finally, contrary evidence is often interpreted as evidence for a conspiracy. This ideation relies on the notion that, the stronger the evidence against a conspiracy, the more the conspirators must want people to believe their version of events (Keeley, 1999; Bale, 2007; Sunstein and Vermeule, 2009). This self-sealing reasoning necessarily widens the circle of presumed conspirators because the accumulation of contrary evidence merely identifies a growing number of people or institutions that are part of the conspiracy. Concerning climate denial, a case in point is the response to events surrounding the illegal hacking of personal emails by climate scientists, mainly at the University of East Anglia, in 2009. Selected content of those emails was used to support the theory that climate scientists conspired to conceal evidence against climate change or manipulated the data (see, e.g., Montford, 2010; Sussman, 2010). After the scientists in question were exonerated by 9 investigations in 2 countries, including various parliamentary and government committees in the U.S. and U. K., those exonerations were re-branded as a “whitewash” (see, e.g., U.S. Representative Rohrabacher’s speech in Congress on 8 December, 2011), thereby broadening the presumed involvement of people and institutions in the alleged conspiracy. We refer to this “self-sealing” criterion by the short label SS.
Results
Recursive hypotheses
Table 2 summarizes the impact of LOG12 as revealed by Google hits and, for comparison, the impact of the other 21 peer-reviewed papers published in 2012 on conspiracist ideation. The table shows that LOG12 represents an outlier compared to other papers on the same topic, especially when considering that LOG12 only received public attention in late August 2012. Thus, less than 2 months elapsed between its release and the data summarized in Table 2, which represent a snapshot during October 2012. It is particularly notable that unlike any of the other papers, LOG12 engendered at least 10 recursive hypotheses during that 2-month period. This count subsumes all hypotheses advanced against LOG12, irrespective of whether they addressed presumed flaws in the methodology or accused the authors of deception, incompetence, or outright conspiracies.
Table 2 Summary of impact of peer-reviewed psychological articles on conspiracist ideation published in 2012.
Citation Google hitsa Recursive hypotheses
LOG12 443 (2) 10
Grebe and Nattrass (2012) 13 (9) 0
Briones et al. (2012) 11 (9) 0
Hamdy and Gomaa (2012) 12 (5) 0
Nattrass (2012) 13 (3) 0
Hoyt et al. (2012) 11 (1) 0
Vu et al. (2012) 10 (3) 0
de Zavala and Cichocka (2012) 8 (6) 0
Clark (2012) 7 (1) 0
Aupers (2012) 5 (2) 0
Baleta (2012) 6 (1) 0
Tun et al. (2012) 5 (2) 0
Moritz et al. (2012) 4 (1) 0
Swami et al. (2012) 3 (3) 0
Barbieri and Klausen (2012) 3 (2) 0
Collins and Chamberlain (2012) 3 (1) 0
Cook (2012) 3 (1) 0
Schneider-Zioga (2012) 3 (1) 0
Drinkwater et al. (2012) 2 (1) 0
Gholizadeh and Hook (2012) 2 (1) 0
Liebich (2012) 2 (1) 0
Krychman (2012) 1 (1) 0
aTotal number of hits, with hits in Google Scholar in parentheses.
The hypotheses are classified into distinct clusters in Table 3. The table also identifies the criteria, using the short labels introduced earlier, that support the classification of each hypothesis as conspiracist. We do not comment on the validity of any hypothesis other than those that can be unambiguously classified as false (namely, hypotheses 2, 6, 7, and 8).
Table 3 Summary of recursive – and at least partially conspiracist – hypotheses advanced in response to LOG12 during August–October 2012.
ID Date Originatora Summary of hypothesis Criteriab
1 29 Aug JN Survey responses “scammed” by warmists NI, PV, MbW, SS
2 29 Aug JN “Skeptic” blogs not contacted NI NS PV
3 3 Sep ROM Presentation of intermediate data NI, NS, MbW, UCT
4 4 Sep GC “Skeptic” blogs contacted after delay NI, NS, MbW, NoA, UCT
5 5 Sep SMcI Different versions of the survey NI, MbW, UCT
6 6 Sep SMcI Control data suppressed NI, NoA
7 10 Sep SMcI Duplicate responses from same IP number retained NS, MbW
8 14 Sep SMcI Blocking access to authors’ websites NI, PV, NoA
9 Various Various Miscellaneous hypotheses See text
10 12 Sep AW Global activism and government censorship NI, PV, SS
aAttribution is based on where and by whom a hypothesis was first proposed in public. JN, Jo “Nova” of joannenova.com.au; ROM, Anonymous commenter with pseudonym ROM at www.bishop-hill.net; GC, Geoff Chambers (commenter at www.shapingtomorrowsworld.org); SMcI, Steve McIntyre of www.climateaudit.com; AW, Anthony Watts of wattsupwiththat.com.
bNI, nefarious intent; NS, nihilistic skepticism; PV, persecuted victim; MbW, must be wrong; NoA, no accident; SS, self-sealing; UCT, unreflexive counterfactual thinking.
Creation of those hypotheses was propelled mainly by the sites shown in Table 1, with a further 10 domain names making lesser contributions to the hypothesis-generation process. The ID numbers in Table 3 are cross-referenced in the section headings of our analysis below.
Survey responses “scammed” (1)
Whenever people express their opinions it cannot be ruled out that they are “faking” their responses by providing answers that are intended to please (or deceive) the experimenter. This possibility may be exacerbated with internet surveys that are completed outside a controlled laboratory environment. In a politically charged context, such as climate change, the further risk arises that groups of respondents may “scam” the survey by “faking” responses to deliver a “desired” outcome. This risk was instantly perceived by the blogosphere, and almost immediately (on 29 August, 2012) the concern was expressed that: “The survey was so transparently designed to link climate skeptics with ‘conspiracy nutters’ it would hardly be surprising if a percentage of alarmists readers of those blogs understood what was required, and dutifully performed3.”
The notion of “scamming” took center-stage in the blogosphere’s response to LOG12, although not all comments went so far as to suggest “… there are no ‘Human Subjects4.’ ” On numerous blogs, it appeared to be taken for granted that the data was “faked” or “scammed.” In one blog post that repeated the words “scam” or “scammed” 21 times (the post ran to approximately 5,100 words), the author asserted that some respondents of the survey “… were almost certainly warmists caricaturing skeptics5.”
The persistence of this hypothesis is illustrated in Figure 2. During exploration of this hypothesis, initial focus by the blogosphere rested on responses to the LOG12 survey items that targeted conspiracist ideation, with the assertion that the few people who endorsed all (or all but one) conspiracy theories (N = 3) might not represent authentic responses (see text footnote 5).
This assertion transmuted into several additional “scamming” hypotheses: on 8 September, a blogger claimed to have identified a “second strategy of fake responses” involving the participants (N ≃ 120) who disagreed with one of the survey items, namely that “fossil fuels increases atmospheric temperature to some measurable degree” (see text footnote 5). In support, the blogger argued that those responses represented an extremist position belonging to so-called “skydragons” (“Skydragons” deny the thermal properties of greenhouse gases that were discovered in the mid nineteenth century.) Based on “nothing more than an impression” (see text footnote 5), the blogger estimated the actual proportion of skydragons as being no higher than 20% among “skeptics” in general. Because the observed proportion of “skydragons” was around 50% of the total number of “skeptics” in the LOG12 sample (≃120 out of ≃250), this was taken to imply that “as much as 75% of the skydragon-style responses are fake.”
On 23 September, the same blogger identified a further 48 participants who registered zealous support for free-market ideology. This zealous support was taken to imply that those responses, too, represented scammed data as they “showed significantly greater incidence of super-zealous pro-free-market sentiment” than an alternative survey conducted on a “skeptic” blog after the controversy over LOG12 erupted6. The blogger concluded “that these super-zealots are fake responses by warmists acting out their caricature of skeptics7.”
The pursuit of the scamming hypothesis without clear a priori statement of what response pattern would represent a “faked” response, and the continual shifting of the criteria for what constitutes “scamming,” reveals either an inconsistent and purely ad hoc approach to data analysis or hints at an agenda-driven effort to invalidate the LOG12 data8. Several of our earlier criteria for conspiracist ideation point toward the latter possibility. For example, the blogosphere’s response appeared driven by the need to resist the “official” explanation of an event (i.e., the LOG12 results in this instance; criterion MbW) and propose a sinister hidden alternative (i.e., “scamming” in this instance; NI). The scamming theory was also explicitly motivated by the presumption that the LOG12 survey was intentionally designed to make “skeptics” look like “nutters”; this meshes with criteria NI and PV. Finally, without a priori specification of what constitutes faked responses, the scamming hypothesis is in principle unfalsifiable: there exists no response pattern that could not be considered “fake” by an innovative theorist. This self-sealing attribute of the hypothesis (criterion SS) may explain its longevity (Figure 2).
“Skeptic” blogs not contacted (2)
Initial attention of the blogosphere also focused on the method reported by LOG12, which stated: “Links were posted on 8 blogs (with a pro-science science stance but with a diverse audience); a further 5 “skeptic” (or “skeptic”-leaning) blogs were approached but none posted the link.” Speculation immediately focused on the identity of the 5 “skeptic” bloggers. Within short order, 25 “skeptical” bloggers had come publicly forward9 to state that they had not been approached by the researchers. Of those 25 public declarations, 5 were by individuals who were invited to post links to the study by LOG12 in 2010. Two of these bloggers had engaged in correspondence with the research assistant for further clarification.
This apparent failure to locate the “skeptic” bloggers led to allegations of research misconduct by LOG12 in blog posts and comments. Those suspicions were sometimes asserted with considerably confidence; “Lew made up the “5 skeptical blogs” bit. That much we know10.” One blog comment airing the suspicion that “skeptic” bloggers had not been contacted also provided the email address to which allegations of research misconduct could be directed at the host institution of LOG12’s first author. This comment was posted by an individual (SMcI; see Table 3) who had been contacted twice by the researchers’ assistant.
The names of the “skeptic” bloggers became publicly available on 10 September, 2012, on a blog post by the first author of LOG12; http://www.shapingtomorrowsworld.org/lewandowskyGof4.html. Although this information invalidated the hypothesis, the blogosphere’s suspicion about LOG12 seemed undiminished (cf. criteria MbW, NS) and attention shifted to various other hypotheses. Two aspects of the process underlying this hypothesis shift are noteworthy.
First, the hypothesis that bloggers were not contacted was abandoned gradually. For example, one blogger opined that “…even if he [first author of LOG12] offered skeptical blogs to participate in his survey, it’s pretty obvious and he must have known that most of them and probably all of them would refuse to give room to a survey organized by an alarmist whose results were likely to be distorted in a way to try to harm skeptics11,12.” This hypothesis imputes a pervasive stance of suspicion among “skeptic” bloggers (criterion NS) because they are presumed to assume that any survey would be intended to “harm skeptics.” This statement also illustrates the self-perception as a victim of persecution (PV).
Similarly, it was pointed out that “He [first author of LOG12] himself emailed or was named in emails to alarmist anti-skeptic bloggers, while he used an unknown assistant to email skeptical blogs13.” This “inconsistent delivery” sub-hypothesis lasted for 48 h (11–13 September) and meshes well with criteria MbW, NoA, and NI.
Notwithstanding the abandoning of the initial “no-contact” hypothesis, the allegation that the survey was designed to be biased by excluding “skeptics” remained in the public domain. That is, the hypothesis that LOG12 sought to exclude “skeptics” from their survey persisted in people’s inferences, even though the original basis for that inference was no longer maintained. Over a week after the “skeptic” bloggers had been revealed, one blogger argued (on 18 September): “None of the skeptic blogs approached publish it (maybe because it’s so painfully obvious to them what he’s attempting to achieve and don’t want a bar of it)14”; criteria PV and NI.
It is notable that concerns about the representativeness of the LOG12 sample were rarely mentioned outside the context of the hypotheses just reviewed. Only two blog comments (shown in the Supplementary Material) noted that because “skeptic” blogs did not post links to the survey, the LOG12 sample may have been skewed toward people who endorse the science, without also accompanying that critique with a hypothesis of nefarious intentions or malfeasance on the part of LOG12.
Once hypothesis-shifting was complete, several new hypotheses emerged in short order to counter the conclusions of LOG12. Several of those new hypotheses were based on what we call unreflexive counterfactual thinking; that is, the hypothesis was built on a non-existent, counterfactual state of the world, even though knowledge about the true state of the world was demonstrably available at the time. Table 3 indicates which of the remaining hypotheses involved this reliance on counterfactuals (marked by UCT in the final column). We argue later that this unreflexive counterfactual thinking is indicative either of the absence of a collective memory for earlier events, or of the lack of a cognitive control mechanism that requires an hypothesis to be compatible with all the available evidence (which is a hallmark of scientific cognition but is known to be compromised in conspiracist ideation; Wood et al., 2012). Unreflexive counterfactual thinking may therefore represent a distinct aspect of conspiracist ideation that has received little scientific attention to date.
Presentation of intermediate data (3)
The first author of LOG12 presented a talk at Monash University on 23 September, 2010. The slides for that talk were posted on the web on 27 September, 2010 and contain a single brief reference (10 words; “conspiracy factor without climate item predicts rejection of climate science”) to the LOG12 data, based on the responses received by that date (virtually the entire sample).
Because this date fell within 3 days of the second (unsuccessful) approach to a “skeptic” blogger to post the link to the survey (the first one had been made 2 weeks earlier, at which time other “skeptic” bloggers were also contacted), the suggestion arose that “…even if he [first author of LOG12] didn’t send out final emails inviting his primary sources (skeptic blogs) to participate until September 20th. It almost seems as if he [first author of LOG12] had decided on the number and nature of responses before the final data could possibly have been received15.” This hypothesis implies that the data would have been different at a later point. Given that none of the “skeptic” blogs posted a link, and therefore could not have affected the result at any point in time, this hypothesis rests on a counterfactual assumption about the world.
A more extreme variant of this hypothesis proposed that “…the results of the survey were already a foregone pre-ordained result of which the survey was only to give it the appearance of legitimacy” (see text footnote 10). This hypothesis identifies the survey as a “cover-up” for pre-ordained results that, presumably, were fabricated by LOG12: it thus goes a step beyond the hypothesis that a subset of the responses were “scammed.”
These comments arguably reveal an intense degree of suspicion (criterion NS), an assumption of nefarious intent by the LOG12 authors (NI), and the belief that something must be wrong (MbW).
“Skeptic” blogs contacted after delay (4)
The “skeptic” blogs were contacted at least a week after the links to the study had already been posted on the 8 other blogs that agreed to participate in the study. This delay was greeted with suspicion by the blogosphere, with one blogger arguing “Inviting Morano on September 23 when the survey had been been initiated at least as early as August suggests less than reputable behavior on the part of the lead researcher16.”
This hypothesis never matured to the point of clarifying how this delay could have had any bearing on the outcome of the study given that none of the “skeptic” blogs posted the link. The hypothesis therefore represents another instance of unreflexive counterfactual thinking, in addition to suspicion and the attribution of nefarious intent (NI, NS, MbW). We also suggest that this hypothesis meshes well with the criterion that “nothing is an accident” (NoA) because it imputes significance and intentionality into an event (i.e., a delayed email) that could equally have been accidental.
Different versions of the survey (5)
Because question order was counterbalanced between different versions of the LOG12 survey, links to the various versions were quasi-randomly assigned to participating blogs. The existence of different versions of the survey gave rise to several hypotheses, for example that “…the most troubling new revelation appears to be that some climate skeptic blogs got different questionnaires [sic] than their counterpart AGW advocate blogs. …this negates the study on the basis of inconsistent sampling17.”
This hypothesis rests on counterfactual thinking: even if survey versions had differed on some variable other than question order, given that none of the “skeptic” blogs posted the link and hence did not contribute responses, any claim regarding the published data based on those differences among versions rests on a counterfactual state of the world. Arguably, this hypothesis also rests on the presumption of nefarious intent and the belief that something must be wrong (NI, MbW).
On 7 September, the first author of LOG12 published a blog post explaining the reason for the different versions of the survey18. Within a day, instances of this theory ceased.
Control data suppressed (6)
Data collection for LOG12 also involved an attempt to recruit a “control” sample via an emailed invitation to participate in the survey among the first author’s campus community. Because this invitation returned only a small number of respondents (N < 80), only the sample of blog denizens was reported in LOG1219.
When the survey invitation was discovered by a blogger, questions emerged about those data: “What was the results of UWA staff who actually took the survey. Surely this would have made an interesting comparison group with the bloggers who are the target of the Moon landing paper. It would have been a logical comparison. Was it done and discarded? If so, why? If it wasn’t, why wasn’t it done20?” Reflecting the pervasive belief that something must be wrong (NI, MbW), those questions metamorphosed into the suggestion that the data reported by LOG12 were “cherry-picked21.”
Duplicate responses from same IP number retained (7)
Following standard internet research protocols (e.g., Gosling et al., 2004), LOG12 filtered the data such that whenever more than one response was submitted from the same IP address, all those responses were eliminated from consideration. This was stated in the LOG12 Method section available for download in August 2012 as “…duplicate responses from any IP number were eliminated.”
Some members of the blogosphere interpreted this statement to mean that LOG12 “…accepted multiple responses from the same IP address as long as there was a slight variation in any answer22.” Although this statement was initially qualified by noting that it was “only an interpretation,” this parenthesized qualifier was dropped from subsequent re-posts of the allegation by other bloggers. The re-posts thus presented the unqualified claim that multiple responses from the same IP address could be included in the LOG12 data. The spread of this hypothesis despite being based on “only an interpretation” reveals considerable suspicion (NS) and also arguably the belief that something had to be wrong (MbW).
This theory lasted 2 days and was mentioned on a News Limited blog in Australia, albeit without the qualifier that it rested only on an interpretation23.
Blocking access to authors’ websites (8)
On 14 September, the websites of the first two present authors (Stephan Lewandowsky24; John Cook25) were temporarily inaccessible (for at least 9 h) from parts of the world, most likely owing to Internet blockages between certain regions and the website server.
This gave rise to the claim by a blogger that both sites had specifically targeted his IP number to prevent access: “I tried both sites via Hide My Ass and got through. So Lewandowsky (and Cook) are definitely blocking my IP address. It seems pretty unethical for a publicly funded university website to slag me and simultaneously block my IP address from accessing their site or responding.” This claim was subsequently qualified by the same individual by removing the “unethical” charge (original version archived)26. This hypothesis is illustrative of the conspiracist tendency to assign intentionality to random events: against the background of a presumed nefarious intent (NI), nothing is an accident (NoA), and the conspiracy theorist is a victim (PV).
The claim of IP blocking then escalated into a more intricate alleged plot by LOG12 to paint their critics as paranoid. One commenter warned “Watch, they may unblock you just so they can say you are paranoid, hyper-sensitive, were never really blocked27.” A different commenter similarly interpreted the IP blocking as a deliberate attack: “Yep, if your argument is that X is paranoid, bombard him with attacks that are deniable and leave no traces, then the moment he squeals say ‘Told you so”’ (see text footnote 27). Another commenter applauded the alleged cunning strategy to goad bloggers into paranoid behavior: “If it’s true they are selectively blocking, I have to begrudgingly respect the skill with which they are playing this audience: there is no way for anyone to complain without matching the stereotypical conspiracist of the study!28”
This reasoning is reminiscent in its complexity of other conspiracist ideation, for example surrounding the events of 9/11: “After 9/11, one complex of conspiracy theories involved American Airlines Flight 77, which hijackers crashed into the Pentagon. Even those conspiracists who were persuaded that the Flight 77 conspiracy theories were wrong folded that view into a larger conspiracy theory. The problem with the theory that no plane hit the Pentagon, they said, is that the theory was too transparently false, disproved by multiple witnesses and much physical evidence. Thus the theory must have been a straw man initially planted by the government, in order to discredit other conspiracy theories and theorists by association” (Sunstein and Vermeule, 2009, p. 223, emphasis added).
The blogosphere’s apparent concern over being “baited” into “acting paranoid” is consonant with the excessive level of suspicion identified earlier as a criterion (NS) of conspiracist ideation and it reveals the pervasive self-perception of climate deniers as victims (PV). The hypothesis also exemplifies the conspiracist tendency to detect meaning and intentionality behind accidental events (NoA).
The IP blocking hypothesis persisted for a day. The originator of the claim updated his comment (without however acknowledging the removal of the “unethical” charge), stating that “… it is possible that the blocking was caused by internet blocks enroute to Australia, with Hide My Ass access occurring because it used a different route. Seems not only possible, but likely29.”
Miscellaneous hypotheses (9)
Two miscellaneous hypotheses deserve mention as they provide insight into the recursive and self-reinforcing nature of conspiracist ideation.
A regular contributor to the blog of the second author of the present paper (see text footnote 25) posted a public critique of LOG1230. While this post was welcomed and reposted by critics of LOG12, one commenter treated it with suspicion, arguing that: “In fact it looks more that your critisism [sic] of Lewandowsky article title was a false flag operation meant to confuse/distract scrutiny of SkS [see text footnote 25] dubious involvement in this unreliable survey. It failed. You have not shot yourself in the foot but somewhere else, more fatal31.” This reasoning is reminiscent of the “decoy theory” just described in the context of 9/11 and illustrates the self-sealing nature of conspiracist reasoning (SS).
A further hypothesis supposed that the real purpose of LOG12 was to provoke conspiracist ideation from climate deniers: “Here’s a conspiracy theory for you: this is the subject of the study, not the survey. The reactions of the skeptic community to a controlled publication with obvious flaws, presented as caustically as possible and with red herrings presented for them to grasp at. There’s some evidence for this theory in internal mails at SkepticalScience, where John Cook can be heard talking enthusiastically about his discussions with Stephan about gaming blogs32.” This theory inconsistently assumes (a) that LOG12 does not contain valid results, although (b) for this theory to be true, the conclusions of LOG12 (a positive correlation between climate denial and conspiracist ideation) must be true because otherwise no such expectation about the “skeptic” response can be formulated. Notwithstanding its inconsistency, the existence of the present article is consonant with this theory.
Beyond recursion: global activism and government censorship (10)
Thus far, we considered only strictly recursive theories – that is, hypotheses that were spawned by LOG12 and pertained to the methodology and results of LOG12. We conclude with an analysis of theories that were spawned by LOG12 but expanded beyond being recursive.
The expansion commenced when one blogger suggested: “That’s quite a little activist organization they have running out of the University of Western Australia. I wonder if UWA officials realize the extent that UWA has become a base for this global climate activism operation and if they condone it33?”
Another blogger further promoted this theory, linking to the above post and commenting “SkepticalScience [the blog of the present second author, J.C.] seems to becoming the ringleader for conspiratorial activities by the green climate bloggers34.” Notably, this blogger explicitly referred to conspiratorial activities by, presumably, the authors of LOG12 and their associates.
A commenter sought to clarify the extent of this presumed conspiratorial activity, claiming that: “It’s mostly a 3-man show: Lew [Lewandowsky], Cook and UWA maths professor Kevin Judd, who is the real strategist behind all this35.” Kevin Judd’s apparent leadership role in this conspiracy was reinforced in a subsequent comment: “As local I can confirm that the Maths Prof Kevin Judd is the mastermind behind UWA AGW. He is apparently a brilliant mathematician, chess and go player, and computerwizz. He is a typical reclusive mad scientist. There is no doubt he is behind all UWA” (see text footnote 36).
A more extended variant of this hypothesis cited the research funding for the first author of LOG12 available on his webpage: “Here Lewandowsky proudly details his $4.4 million in grants. Which includes $762,000 specifically related to Climate Research funding in the last year or two, and none of that includes the $6 million the Federal Government provided him and a few colleagues to found and run ‘The Conversation’ which provides a substantial forum for his ‘Climate Change position36.”’ “The Conversation” refers to an online newspaper37 that is primarily written by academics and is funded by a consortium of major Australian universities and other scientific organizations. This hypothesis thus widens the scope of the presumed activism by LOG12 authors to include a national online media initiative.
The expanding scope of the presumed conspiracy exhibited considerable longevity, as evidenced by a blogpost several months later that was triggered by a radio interview with the first author of LOG12 on the Australian Broadcasting Corporation’s (ABC) science show: “The government, via the Australian Research Council [ARC] is involved in suppressing dissent. …Lewandowsky has received over $2 million worth of ARC funding to support his efforts to equate climate change skepticism with mental disorder. ‘Punitive psychology’ as it is called, was widely used in the Soviet Union to incarcerate dissidents in mental institutions. In modern Australia the walls of the prison are not brick or stone, but walls of censorship, confining the dissident to a limbo where no-one will report what they say for fear of being judged mentally deficient themselves. …But the problem is obviously more widespread and involves the University of Western Australia, where Lewandowsky holds his chair, the ARC, the ABC, and possibly even the government38.”
Common to all these hypotheses is the presumption of widespread nefarious intent among the authors of LOG12 and colleagues (NI) and a potentially self-sealing propensity to broaden the scope of the presumed malfeasance (SS): extending the presumed malfeasance to include the Australian government may amplify a self-perception of being victimized (PV).
Freedom-of-information release
On 10 October, 2012, the host institution of the first author of LOG12 released a tranche of emails and documents that had been requested by a “skeptic” climate blogger under Freedom-of-information (FOI) legislation. One set of emails involved all correspondence between the researchers and the blogs that were contacted to host the survey, including those that by an initial hypothesis – number 2 in Table 3 – were presumed not to exist. The remaining documents and emails pertained to the institutional ethics approval for the study reported by LOG12. Because the FOI release occurred about a month after the last hypothesis spontaneously emerged in response to LOG12, it is considered separately from the other hypotheses summarized in Table 3.
Although the released correspondence confirmed the chronology that underpinned an earlier hypothesis, relating to the dates at which “skeptic” bloggers were contacted (hypothesis 4 in Table 3), this confirmatory evidence was ignored and no further mention of this hypothesis was made. Instead, the blogosphere focused on the ethics approvals underlying the study.
The existence of ethics approval was met by a broadening of the scope of presumed malfeasance, from the authors of LOG12 to the ethics committee and its chair at the first author’s institution. To illustrate, one blogger claimed that the “…approval originally obtained was for a fundamentally different project, and the nature of the amendment and its rapid approval raises a number of questions for the university… How was it possible that the EC [ethics committee] could have reviewed such substantive changes and come to a decision within 24 hours?39”
The broadening of the scope of purported malfeasance to include additional people or institutions in light of disconfirmatory evidence is a principal attribute of conspiracist ideation (Keeley, 1999). The self-sealing response to the freedom-of-information release therefore illustrates several of our classification criteria (viz., NI, NS, MbW, and in particular SS). The alternative hypothesis, namely that the existence of ethics approvals in conformance with applicable procedures might confirm that there were no ethical problems with the LOG12 study was not considered by the blogosphere.
Discussion
Potential limitations
Our analysis was concerned with the blogosphere’s response to a single 4,000-word article. One might therefore question the generality of our results. In response, we note that at least one other scientific report in the climate arena engendered a sustained critique that subsequent scholarly analysis identified as conspiracist (Lahsen, 1999). Likewise, conspiratorial themes have been found to be prominent in media coverage of climate-related issues (McKewon, 2012a), and accounts by climate scientists of the strategies of climate denial are replete with accounts of conspiratorial accusations against individual papers (e.g., Mann, 2012). We therefore suggest that the present analysis illuminated not just an isolated incident but the broader propensity of climate denial to involve a measure of conspiracist ideation; a suggestion that is consonant with the slant of recent popular books espousing denial (e.g., Solomon, 2008; Alexander, 2009; Montford, 2010; Sussman, 2010; Bell, 2011; Inhofe, 2012; Isaac, 2012).
A second criticism might cite the fact that we have considered the “blogosphere” as if it were a single entity, analyzed within the context of psychological processes and constructs that typically characterize individuals rather than groups. Our response is twofold: first, at the level of purely descriptive discourse analysis, our work fits within established precedent involving the examination of communications from heterogeneous entities such as the U.S. Government (Kuypers et al., 1994) or the Soviet Union (Kuypers et al., 2001). Second, at a psychological level, numerous psychological constructs – such as cognitive dissonance, social dominance orientation, or authoritarianism – have been extended to apply not only to individuals but also to groups or societies (e.g., Moghaddam, in press). We therefore argue that our extension of individual-level work on conspiracist ideation to the level of amorphous groups fits within precedent in two areas of scholarly enquiry.
A further criticism might hold that although we may have presented some evidence for the presence of conspiracist ideation, the evidence falls far short of “real” conspiracy theories involving events such as 9/11 or the moon landing. In response, we note that the hypotheses leveled against LOG12 do not differ qualitatively – that is, in terms of magnitude or scope – from others that have been identified as conspiracist in the context of another paper in the climate arena (Lahsen, 1999) or that have been observed in response to experimental manipulations (Whitson and Galinsky, 2008). We suggest that conspiracist ideation, like most other psychological constructs (e.g., extraversion), represents a continuum that finds expression to varying extents in theories of varying scope.
In a related vein, critics might propose an alternative explanation for the behavior of the blogosphere based on a dissonance effect. Science denial commonly involves “skeptics”’ self-perception of being the only rational consumers of information in a sea of corrupt or self-serving scientists (Kalichman, 2009; Wagner-Egger et al., 2011). Given that the data of LOG12 arguably challenged that perception, the resultant dissonance – rather than some underlying general predisposition – may have triggered the observed conspiracist response. This alternative explanation meshes with previous observations that conspiracist ideation can arise in response to threats in a random sample of participants (e.g., Whitson and Galinsky, 2008); however, it meshes less well with the conspiratorial undercurrent that suffused public climate denial even before the LOG12 data became public (e.g., Solomon, 2008; Alexander, 2009; Montford, 2010; Sussman, 2010; Bell, 2011; Inhofe, 2012; Isaac, 2012). In any case, this hypothesis is not in opposition to ours: we would expect that a person’s disposition to engage in conspiratorial thinking is more likely to become manifest when triggered by factors such as cognitive dissonance.
Critics might furthermore argue that our analysis of the response to LOG12 was over-extensive, and that some of the hypotheses advanced by the blogosphere in fact constituted legitimate criticism. This criticism is rendered less potent by the fact that our analysis was conducted at a psychological level, without regard to the truth value of any of the hypotheses other than those that could be unambiguously classified as false (i.e., hypotheses 2, 6, 7, and 8 in Table 3). We remain neutral with respect to the question whether the remaining hypotheses presented valid criticisms. The issue of validity of those hypotheses – or indeed the validity of the conclusions of LOG12 – is orthogonal to the psychological question at issue here, viz. whether the response to LOG12 constituted conspiracist ideation.
Our decision not to address the validity of any of the hypotheses also helps allay one important remaining issue: two of the present authors also contributed to LOG12, and the present analysis may therefore be biased by a potential conflict of interest. This possibility cannot be ruled out, although a balanced evaluation would note that the present article arguably goes against the interests of those two authors, because it placed several criticisms of LOG12 into the peer-reviewed literature that previously had been limited to internet blogs. Given the well-known resistance of information to subsequent correction (e.g., Lewandowsky et al., 2005, 2012a), the present article could therefore equally be taken to run counter to the interests of the LOG12 authors. In addition, because data collection (via internet search) was conducted by two authors who were not involved in analysis or report of LOG12, the resulting “raw” data – available in the Online Supplementary Material – cannot reflect a conflict of interest involving the LOG12 authors. The availability of these raw data enables other scholars to bring an alternative viewpoint to bear during any re-analyses.
Theoretical and pragmatic implications
Implications for understanding conspiracist ideation
Our principal thesis is that some of the responses to LOG12 voiced in the blogosphere satisfy attributes of conspiracist ideation by the criteria defined at the outset. Two attributes deserve to be highlighted: first, most of the hypotheses can be unified under the immutable belief that “there must be something wrong” (MbW in Table 3) and that the authors of LOG12 engaged in intentional malfeasance (NI, NS). Those underlying beliefs infused conspiracist elements even into those hypotheses that would be expected to arise during routine scholarly critique. For example, the “scamming” hypothesis evolved continuously without being guided by clear a priori assumptions about what would constitute a “scammed” response profile, thereby ultimately rendering this hypothesis self-sealing and unfalsifiable (criterion SS). It is this psychological attribute that points toward a conspiracist component rather than conventional scholarly critique.
Second, self-sealing reasoning also became apparent in the broadening of the scope of presumed malfeasance on several occasions. When ethics approvals became public in response to an FOI request, the presumption of malfeasance was broadened from the authors of LOG12 to include university executives and the university’s ethics committee. Similarly, the response of the blogosphere evolved from an initial tight focus on LOG12 into an increasingly broader scope. Ultimately, the LOG12 authors were associated with global activism, a $6 million media initiative, and government censorship of dissent, thereby arguably connecting the response to LOG12 to the grand over-arching theory that “climate change is a hoax.” Notably, even that grand “hoax” theory is occasionally thought to be subordinate to an even grander theory: one of the bloggers involved in the response to LOG12 (cf. Table 1) considers climate change to be only the second biggest scam in history. The top-ranking scam is seen to be modern currency, dismissed as “government money” because it is not linked to the gold standard40.
The observed broadening of scope meshes well with previous research that has identified stable personality characteristics that predict the propensity for conspiracist ideation (cf. Goertzel, 1994; Swami et al., 2009; Douglas and Sutton, 2011). It is therefore not altogether surprising that suspicions about a single scholarly paper can rapidly mature into more encompassing hypotheses. We suggest that some of the variables that predict conspiracist ideation – viz. low trust (Goertzel, 1994) and paranoid ideation (Darwin et al., 2011) – were observable in the response to LOG12. Those variables are revealed by statements such as: “Given the lack of evidence that he [first author of LOG12] tried to contact skeptic blogs, and his bizarre excuse for not reporting the blogs he tried to contact when describing his methodology, some people suspect he didn’t try very hard to contact skeptic blogs. But that suspicion is not a conspiracy theory” (emphasis added)41.
Whereas suspicion on its own is insufficient to identify conspiracist ideation, it arguably constitutes one of its core attributes. For example, the suspicion that LOG12 did not contact “skeptic” bloggers tacitly invokes several major presumptions, namely (a) that the authors of LOG12 were willing to engage in research misconduct; (b) that they would invent a claim about a non-event and publish it in the Section “Materials and Methods” when there was no incentive or reason to do so; and (c) that they should have somehow provided “evidence” beyond writing an accurate Method section. The ease with which those presumptions about misconduct and malfeasance were made and accepted provides a fertile environment for the subsequent unfolding of conspiracist ideation (cf. Keeley, 1999; Wood et al., 2012).
Our research also points to at least two issues that merit further investigation. The first issue arises from the well-established fact that the rejection of climate science is strongly associated with right-wing political leanings and the embrace of a “fundamentalist” laissez-faire vision of the free-market (e.g., Heath and Gifford, 2006; Dunlap and McCright, 2008; Feygina et al., 2010; Kahan, 2010; Hamilton, 2011; Kahan et al., 2011; McCright and Dunlap, 2011a,b). There is a parallel literature that has linked conspiracist ideation, at least in some cases, with right-wing political leanings: for example Swami (2012) found endorsement of an anti-Semitic conspiracy theory in Malaysia to be associated with right-wing authoritarianism. Similar associations with authoritarianism have been reported by Abalakina-Paap et al. (1999) and Swami et al. (2012) in samples of Western participants. One might therefore be tempted to consider conspiracist ideation another manifestation of the “paranoid style” in American politics – mainly focused on the political Right – that was famously highlighted by Hofstadter (1966). On this view, the involvement of conspiracist ideation in climate denial would be expected as a likely by-product of the strong ideological drivers underpinning rejection of climate science. There are several indications that acceptance of this view would be premature: LOG12 found no association between conspiracist ideation and free-market ideology in their structural-equation model (see Figure 1), and in a similar study involving a representative sample, Lewandowsky et al. (2013) found conspiracist ideation to be negatively associated with free-market ideology and conservatism. (Related results were reported by Swami et al., 2009.) The relationship between conspiracist ideation and political worldviews thus remains to be pinned down.
Second, we uncovered a potentially novel aspect of conspiracist reasoning when some of the later hypotheses were found to involve a residual impact of earlier, discarded hypotheses. For example, whereas critics initially argued that the results of LOG12 were invalid because “skeptic” bloggers were not contacted (hypothesis 2 in Table 3), upon release of evidence to the contrary, the same conclusion of invalidity was reached by other means; either because of a preliminary report of the data during a colloquium (hypothesis 3); or because of the presumedly faulty timing of the correspondence (hypothesis 4); or because “skeptic” bloggers were emailed different versions of the survey (hypothesis 5). All of those hypotheses rely on counterfactual thinking because no “skeptic” blogger posted links to the survey, and therefore neither the dates of correspondence nor the version of the survey (nor any other event involving those bloggers) could have affected the data as reported in LOG12.
Although there appears to be ample evidence to classify the response to LOG12 at least in part as conspiracist, one must guard against overextending this conclusion: there are other streams of science denial that are detectable in the response to LOG12. For example, the repeated re-analysis of data, involving the elimination of “inconvenient” subsets of data points based on fairly fluid criteria, has a long-standing history in other contentious arenas. Michaels (2008) reviews the extensive history of epidemiological data that were subject to industry-sponsored re-analysis because of their regulatory implications, such as reports of the association between tobacco and lung cancer, or the link between bladder cancer and chemicals used in dye production. Re-analyses by industry bodies often fail to detect previously-published links between, say, tobacco use and cancer or heart disease (e.g., Cataldo et al., 2010; Proctor, 2011). A common technique underlying those re-analyses involves the selective removal of data points based on ad hoc criteria (Michaels, 2008); this technique is also detectable in the various re-“analyses” of the LOG12 data to buttress hypothesis 1 from Table 3.
Implications for understanding science denial
The discovery by John Tyndall that CO2 is a greenhouse gas dates back over 150 years. Recognition of the possibility that industrial CO2 emissions may alter the planet’s climate dates back more than a century, and during the last two decades the scientific evidence for the fact that humans are interfering with the climate has become overwhelming. The vast majority of domain experts agree that the climate is changing and that human CO2 emissions are causing this change (Oreskes, 2004; Doran and Zimmerman, 2009; Anderegg et al., 2010).
Given this broad agreement on the fundamentals of climate science, what cognitive mechanism would underlie people’s dissent from the consensus? We suggest that if a person rejects an overwhelming scientific consensus, such as the one for climate science, then that person needs to deny that the consensus emerged as the result of researchers converging independently on the same evidence-based view. Rejection of the scientific consensus thus calls for an alternative explanation of the very existence of that consensus. The ideation of a secretive conspiracy among researchers can serve as such an explanation (Diethelm and McKee, 2009; McKee and Diethelm, 2010; Smith and Leiserowitz, 2012). Moreover, the ideation of a conspiracy may also serve as a “fantasy theme” that permits groups to develop and share a symbolic reality. Such fantasy themes (e.g., the denier as “Galileo” who opposes a corrupt iron-fisted establishment) operate as bonding agents that build group cohesion by creating a shared social reality. Fantasy themes are known to play a major role in climate denial (McKewon, 2012a,b).
Accordingly, there is growing evidence of the involvement of conspiracist ideation in climate science denial (McKewon, 2012a; Smith and Leiserowitz, 2012; Lewandowsky et al., in press) as well as the denial of other scientific propositions (Diethelm and McKee, 2009; Goertzel, 2010; McKee and Diethelm, 2010). The prevalence of conspiracist ideation has notable implications for science communicators.
Implications for science communication
A defining attribute of conspiracist ideation is its resistance to contrary evidence (e.g., Keeley, 1999; Bale, 2007; Sunstein and Vermeule, 2009). This attribute is particularly troubling for science communicators, because providing additional scientific information may only serve to reinforce the rejection of the evidence, rather than foster its acceptance. A number of such “backfire” effects have been identified, and they are beginning to be reasonably well understood (Lewandowsky et al., 2012a). Although suggestions exist about how to rebut conspiracist ideations – e.g., by indirect means, such as affirmation of the competence and character of proponents of conspiracy theories, or affirmation of their other beliefs (e.g., Sunstein and Vermeule, 2009) – we argue against direct engagement for two principal reasons.
First, much of science denial takes place in an epistemically closed system that is immune to falsifying evidence and counterarguments (Kalichman, 2009; Boudry and Braeckman, 2012). We therefore consider it highly unlikely that outreach efforts to those groups could be met with success. Second, and more important, despite the amount of attention and scrutiny directed toward LOG12 over several months, the publication of recursive hypotheses was limited to posts on only 24 websites, with only 13 blogs featuring more than one post (see Table 1). This indicates that the recursive theories, while intensely promoted by certain bloggers and commenters, were largely contained to the “echo chamber” of climate denial. Although LOG12 received considerable media coverage when it first appeared, the response by the blogosphere was ignored by the mainstream media. This confinement of recursive hypotheses to a small “echo chamber” reflects the wider phenomenon of radical climate denial, whose ability to generate the appearance of a widely held opinion on the internet is disproportionate to the smaller number of people who actually hold those views (e.g., Leviston et al., 2012). This discrepancy is greatest for the small group of people who deny that the climate is changing (around 6% of respondents; Leviston et al., 2012). Members of this small group believe that their denial is shared by roughly half the population. Thus, although an understanding of science denial is essential given the importance of climate change and the demonstrable role of the blogosphere in delaying mitigative action, it is arguably best met by underscoring the breadth of consensus among scientists (Ding et al., 2011; Lewandowsky et al., 2012b) rather than by direct engagement.
Author Note
Preparation of this paper was facilitated by a Discovery Outstanding Researcher Award from the Australian Research Council to the first author. This project was funded by the School of Psychology at the University of Western Australia under the auspices of an Adjunct Professorship awarded to the third author. We thank Alexandra Freund for comments on an earlier version of the manuscript. Address correspondence to the first author at the School of Psychology, University of Western Australia, Crawley, WA 6009, Australia. Electronic mail may be sent to [email protected]. Personal web page: http://www.cogsciwa.com.
Supplementary Material
The Supplementary Material for this article can be found online at http://www.frontiersin.org/Personality_Science_and_Individual_Differences/10.3389/fpsyg.2013.00073/abstract
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
1In current scholarly usage the term “denial” is often reserved to describe an active public denial of scientific facts by various means, such as the use of rhetoric to create the appearance of debate where there is none (Diethelm and McKee, 2009; McKee and Diethelm, 2010). The term “rejection of science,” by contrast, has been used in research aimed at identifying the factors that predispose people to be susceptible to organized denial (e.g., Lewandowsky et al., in press). In the present article, we frequently use the term “denial” because the object of our study is on the active and public dissemination of information.
2The study also queried whether several long-standing environmental issues, such as acid rain, continue to present a problem. Figure 1 shows that the perception that previous environmental problems have been solved was negatively associated with climate science but was unrelated to other sciences; this effect is of little interest in the present context.
3http://joannenova.com.au/2012/08/lewandowsky-shows-skeptics-are-nutters-by-asking-alarmists-to-fill-out-survey/
4http://www.shapingtomorrowsworld.org/ccc1.html#198
5http://climateaudit.org/2012/09/08/lewandowsky-scam/
6http://wattsupwiththat.com/2012/09/08/replication-of-lewandowsky-survey/
7http://climateaudit.org/2012/09/23/more-deception-in-the-lewandowsky-data/
8The criteria for this hypothesis may also have shifted in response to a blogpost by two of the authors of LOG12 which demonstrated the resilience of their main findings to the removal of outliers on the measure of greatest interest, the endorsement of the various conspiracy theories, on 12 September, 2012 (http://www.shapingtomorrowsworld.org/lewandowskyScammers1.html). This analysis is reproduced in the Online Supplementary Material for LOG12.
9http://www.webcitation.org/6APs1GdzO
10http://www.bishop-hill.net/blog/2012/8/31/lewandowskys-data.html?currentPage = 2#comments
11http://motls.blogspot.com.au/2012/09/stephan-lewandowskys-incredible-blog.html
12This statement was made on the same day that the bloggers’ names were released and it is impossible to ascertain whether it predated or postdated the release.
13http://joannenova.com.au/2012/09/lewandowsky-science-by-taunts-and-smears/
14http://www.australianclimatemadness.com/2012/09/lew-a-few-final-thoughts/
15http://www.shapingtomorrowsworld.org/ccc2.html#225
16http://rankexploits.com/musings/2012/the-five-blogs/
17http://wattsupwiththat.com/2012/09/05/stephan-lewandowskys-slow-motion-social-science-train-wreck/
18http://www.shapingtomorrowsworld.org/lewandowskyVersionGate.html
19The authors subsequently obtained a control sample via a professional survey firm in the U.S: This representative sample of 1,000 respondents replicated the results involving conspiracist ideation reported by LOG12 (Lewandowsky et al., 2013).
20http://climateaudit.org/2012/09/12/lewandowskys-unreported-results/
21http://joannenova.com.au/2012/09/lewandowsky-gets-1-7m-of-taxpayer-funds-to-demonize-people-who-disagree-with-him/
22http://climateaudit.org/2012/09/10/the-third-skeptic/#comment-350166
23http://blogs.news.com.au/heraldsun/andrewbolt/index.php/heraldsun/comments/lewandowsky_was_warned_his_survey_was_no_good/
24www.shapingtomorrowsworld.org
25www.skepticalscience.com
26http://www.webcitation.org/6AhCviEOE
27http://climateaudit.org/2012/09/14/the-sks-link-to-the-lewandowsky-survey/#comment-352577
28http://climateaudit.org/2012/09/14/the-sks-link-to-the-lewandowsky-survey/#comment-352753
29http://climateaudit.org/2012/09/14/the-sks-link-to-the-lewandowsky-survey/#comment-352542
30http://www.skepticalscience.com/AGU-Fall-Meeting-sessions-social-media-misinformation-uncertainty.html#84306
31http://climateaudit.org/2012/09/12/lewandowsky-study-useless-unless-authors-demonstrate-data-integrity/#comment-351497
32http://www.shapingtomorrowsworld.org/news.php?p=2&t=118&n=161#751
33http://wattsupwiththat.com/2012/09/12/the-cook-lewandowsky-social-internet-link/
34http://judithcurry.com/2012/09/15/bs-detectors/
35http://wattsupwiththat.com/2012/09/12/the-cook-lewandowsky-social-internet-link/#comment-1076866
36http://watchingthedeniers.wordpress.com/2012/09/13/watts-explains-why-lewandowsky-paper-on-conspiracy-theories-is-wrong-its-a-conspiracy-between-john-cook-and-the-prof/#comment-14459
37http://theconversation.edu.au/who_we_are
38http://www.ambitgambit.com/2012/11/24/paedophilia-climate-science-and-the-abc/
39http://www.australianclimatemadness.com/2012/10/lewandowsky-foi-substantial-last-minute-changes-to-project-waved-through-by-uwa-ethics-committee/
40http://joannenova.com.au/2012/03/we-are-all-austrians-now/
41http://rankexploits.com/musings/2012/conspiracy-theory-get-lewindowsky-a-dictionary-stat/
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23526983PONE-D-12-2847810.1371/journal.pone.0058428Research ArticleBiologyAnatomy and PhysiologyEndocrine SystemEndocrine PhysiologyInsulin-like Growth FactorImmunologyImmunityImmune DeficiencyImmunoregulationImmunologic SubspecialtiesTumor ImmunologyAntigen Processing and RecognitionMajor Histocompatibility ComplexMedicineClinical ImmunologyImmunologic SubspecialtiesTumor ImmunologyOncologyBasic Cancer ResearchImmune EvasionCancer TreatmentImmunotherapyCancers and NeoplasmsNeurological TumorsGlioblastoma MultiformeRescue of MHC-1 Antigen Processing Machinery by Down-Regulation in Expression of IGF-1 in Human Glioblastoma Cells Modulation of Antigen Processing by IGF-1Pan Yuexin
1
2
Trojan Jerzy
3
Guo Yajun
4
Anthony Donald D.
1
2
*
1
Division of General Medical Sciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America
2
Department of Medicine, University Hospitals of Cleveland, Cleveland, Ohio, United States of America
3
INSERM U542 and U602, Paul-Brousse Hospital, Paris XI University, Villejuif, France
4
International Joint Cancer Institute, Second Military Medical University, Shanghai, China
Castresana Javier S. Editor
University of Navarra, Spain
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: DDA YP. Performed the experiments: YP JT. Analyzed the data: YP DDA YG JT. Contributed reagents/materials/analysis tools: DDA YP YG. Wrote the paper: DDA YP.
2013 20 3 2013 8 3 e5842817 9 2012 5 2 2013 © 2013 Pan et al2013Pan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Escape from immune recognition has been hypothesized to be a factor in carcinogenesis. It may be mediated for many cancers through down-regulation in the MHC class 1 antigen processing and presentation pathway. TAP-1, TAP-2, tightly linked to LMP-2 and LMP-7 are multiple components of the endogenous, antigen presentation pathway machinery. We addressed the question of alterations in this pathway in human Glioblastoma (HGB) and of its relationship to modulation in expression of IGF-1 that is highly expressed in this cancer. Deficiencies in expression of TAP-1 were demonstrated by RT-PCR and/or by immuno-flow cytometry in the HGB cell line T98G obtained from ATCC, and in 3 of 4 human cell lines established from patients with Glioblastoma Multiforme. Deficiencies in expression of TAP-2 were observed in 3 of 4, deficiencies in expression of LMP-2 in 4 of 4 and deficiencies in LMP-7 in 3 of 4 HGB cell lines examined by RT-PCR and Western blot. Following down-regulation of IGF-1 by transfection with the pAnti IGF-1 vector that expresses IGF-1 RNA in antisense orientation, or by the exogenous addition of IGF-1 receptor monoclonal antibody to cell culture media, the deficiencies in components of the MHC-1 antigen presentation pathway were up-regulated and/or rescued in all HGB cell lines tested. Moreover, this up-regulation in expression was aborted by addition of 100 ng/ml of IGF-1 to the culture media. Unlike in the case of IFN-γ, the restoration of TAP-1 and LMP-2 by down-regulation of IGF-1 in Glioblastoma cells was not correlated to the tyrosine phosphorylation of STAT 1. In summary, the simultaneous reversion in expression of the multiple constituents of MHC-1 antigen processing path and up-regulation in expression of MHC-1 occurring with down-regulation in IGF-1 may have a role in reinforcement of immunity against tumor antigen(s) in some animal cancers and in humans with Glioblastoma Multiforme.
The work was supported by American Cancer Society grant CN-8 A, a grant from the Ohio Board of Regents and by gifts from the following benefactors: Marcia & Robert Williams, Regina & David Letterman, the Paul Newman Foundation, Mrs. Helen Wright, Mr. & Mrs. Edward Yoman, Mrs. Elizabeth Becker, Kimberly Birn & family, Mrs. Verna Warpula, Herbert Braverman, John D. Proctor, Harold & Clare Minoff, James & Rosemary Koehler, Wm. Gustaferro & family, Edward & Mary Lasko, and other unnamed donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Body
Introduction
Major histocompatibility complex (MHC) genes in humans are referred to as human leukocyte antigen (HLA) genes. The HLA locus spans approximately four megabases on chromosome 6P21.3. Its gene products are predominately associated with the immune system. HLA-1 and II molecules are membrane-bound glyco-proteins, which have key roles in the presentation of antigens to T-lymphocytes [1], [2]. HLA-1 molecules are ubiquitously expressed in accordance with their essential functions in mediating immune responses against endogenously derived virus and tumor cell antigens [3]. Endogenous antigen peptides are generally produced in the cytosol by large multicatalytic proteolytic molecules named proteasomes (LMPs). LMP-2, LMP-7 and LMP-10 subunits of the proteasomes are inducible by interferon-gama (IFN-γ) [4], [5]. The 8–9 amino acids antigen peptides produced by this reaction are then translocated to the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP-1 and TAP-2) [6], [7]. Assembly with HLA class 1 heavy chain and the β2-microglobulin light chain occurs here [8]. The HLA class 1 peptide complex is then transported to the cell surface to be presented to cytotoxic T lymphocytes (CTL). This antigen-processing machinery and HLA-1 restricted antigen-presentation pathway is believed to have a role in the activation of CTL mediated immunogenicity [9]. Importantly, this machinery and the MHC-1 restricted antigen presentation pathway are down-regulated in many different cancer tissues and cancer cell lines [10]–[14]. This has led to the hypothesis that the defective pathway may have a significant role in loss of immuno-surveillance and possibly in causation of cancer.
We previously showed, in several different animal cancer models (rat C6 glioma [15], murine teratocarcinoma [16], transgenic spontaneous hepatoma [17], commentary rat/LFCI2A-hepatocarcinoma [18]), and, in human glioblastoma cell lines [19], an up-regulation in expression of MHC class 1 following down-regulation in cellular IGF-1 by transfection with the pAnti IGF-1 (an IGF-1 antisense RNA expression vector) [19]–[21]. We show in this paper, the association between down–regulation in expression of IGF-1 and enhancement in the cell surface expression of HLA class 1 molecules in human Glioblastoma cells and Glioblastoma cell lines. Along with this, we show a concomitant increase in mRNA expression for TAP-1, TAP-2, LMP-2 and LMP-7 components of the endogenous antigen presentation pathway. Increase in the TAP-1 peptide was demonstrated, and, increase and/or rescue in the expression of TAP-2, LMP-2 and LMP-7 peptides were demonstrated when down-regulation of IGF-1 by IGF-1 antisense RNA or when blockade of the IGF-1 receptor (IGF-1R) by its monoclonal antibody occurred. We conclude that loss and/or down-regulation in expression of the endogenous antigen processing pathway machinery in human Glioblastoma (HGB) and HGB cell lines can be modulated and rescued by down-regulation of IGF-1 expression in HGB cells.
Materials and Methods
Ethical Considerations
Human experiments were done in accordance with the Declaration of Helsinki (1964). The experiments were conducted with the understanding and informed consent of the human subjects. The Institutional Review Board of University Hospitals of Cleveland has approved these experiments.
Cell Lines and Cell Culture
The human Glioblastoma cell line T98G was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Primary human Glioblastoma cell lines were obtained from 16 patients with diagnosis of human Glioblastoma multiforme as determined by histo-pathology in accordance with the World Health Organization system of classification. Primary human Glioblastoma cell cultures were derived according to techniques previously described [22]. Cells were grown in supplemented Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/l Glucose, 4 mM L-Glutamine (Biowhittaker) to which was added 1×non-essential amino acids solution (MEM) (GIBCO/BRL, Carlsbad, CA), 1 mM sodium pyruvate (GIBCO/BRL), 5 μg/ml human transferrin (GIBCO/BRL), 3.46 ng/ml sodium selenite (GIBCO/BRL) and 500 ng/ml insulin (SIGMA, St. Louis, MO). The complete medium was adjusted to a final concentration of 10% fetal bovine serum (FBS) (Biowhittaker, Walkersville, MD). Cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
pAnti IGF-1 Plasmid
The E. coli bacterial strain DH5α bearing the IGF-1 antisense RNA vector, pAnti-IGF-1, was kindly provided by Dr. J. Ilan (Case Western Reserve University, Cleveland, Ohio, USA). This expression vector includes the Epstein-Barr virus origin of replication and the gene encoding nuclear antigen 1, which together drive extra-chromosomal replication. The construct shown in
Fig 1A
is as previously described [22]. The vector replicates episomally within human cells providing a copy number up to 100 per cell [23]. Vector is isolated and purified using a Plasmid Maxi Kit (QIAGEN, Valencia, CA) according to manufacturer's instructions. A control plasmid, pAnti IGF-2 has the same construction map accept the 1 kb of IGF-2 cDNA, in antisense orientation, replaced the IGF-1 antisense sequence. This was also previously described by us [16].
10.1371/journal.pone.0058428.g001Figure 1 Transfection of HGB cells with the vector pAnti IGF-1.
A, Physical map of pAnti IGF-1. MT-1: metallothionein - 1 promotor; IGF-1 3′-5′: human IGF-1 DNA sequence in antisense orientation; SV40 poly A: SV40 poly A termination sequence; Ori: origin of replication; Hyg R: hygromycin resistance gene; Amp R: Ampicillin resistance gene; EBNA-1: Epstein Barr Virus (EBV) encoded nuclear antigen 1; EBV ori-P: EBV origin of replication. B, Expression of IGF-1 cDNA in transfected HGB cells. Primer pairs for RT-PCR used to detect and amplify antisense IGF-1 cDNA are as designated in methods. This set of primers gives rise to a 424 bp cDNA band containing exons 1, 2, 3 and 5 of the IGF-1 molecule in antisense orientation. Lane 1 shows the molecular weight markers of ØX174 DNA cut by HaeIII. Lanes 2, 3 and 4 demonstrate the 424 bp band, and a 327 bp β-actin band (internal control). Lane 5 shows a negative clone, which did not express IGF-I antisense cDNA. Lane 6 depicts a negative control in which all constituents of the reaction were present but not the DNA template. C, Detection of IGF-1 antisense RNA transcripts in HGB cells by Northern blot analysis. 30ug of total RNA from non-Transfected and transfected clones of HGB cells were applied to 1.0% formaldehyde agarose gel. 32P-labeled IGF-I cDNA was used as probe. Lanes 1, 3, 4, 5, 6, 8, 9 and 10 demonstrate the dominant 1 kb IGF-1 antisense RNA band from each of 8 separately transfected clones. Lanes 2, 7 and 11 represent RNA of non-transfected clones.
Fig 1
C represents result for one set of the experiments;
Fig 1
D represents the semi-quantitative densitometry analysis for C as determined by the NIH image J program.
Transfection of Human Glioblastoma Cells
The ATCC human glioblastoma (HGB) cell line T98G and cell lines established in our laboratory from primary cultures of HGB patients were transfected with pAnti IGF-1 as previously described using the lipofectin reagent kit available from GIBCO/BRL [22]. Isolated foci (clones) of transfected cells were obtained using cloning cylinders. Separated clones were transferred to six-well plates, expanded and transferred to 60 mm or larger culture dishes as appropriate. The populations of cells were then further expanded under growth conditions that included the selective pressure of hygromycin B. Cell passages used to reach the point at which transfection could be done were 1 – 2 [22]. To complete transfection to point of a stably transfected cell line required an additional 3 – 4 cell passages.
Immunofluorescent Flow Cytometry
Immunofluorescent staining for HLA Class 1 and B-7.1 antigens was performed by modification of standard techniques at 4°C in a 96 well micro-titer plate [22]. HGB cells were first trypsinized and washed×3 with phosphate buffered saline (PBS) as previously described. Cells (2.5 – 5.0×105) in 150 μl PBS were loaded into each well of a 96 well plate and centrifuged 800 g×5 min at 4°C. Supernatants were aspirated and primary antibody was added at a dilution of 1∶50 at 4°C for 30 min. Cells were then washed×3 with 150 μl of PBS as previously described [22]. Secondary antibody binding was performed using fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG (Kirkegaard & Perry Lab, KPL, Gaithersburg, MA) at a dilution of 1∶200 in absence of light. Surface antigen B-7.1 was detected, using mouse anti-human B-7.1 monoclonal antibody conjugated with R-phycoerythrin (R-PE) (Ancell, Bayport, MN), by direct staining technique in the absence of light. To detect intracellular IGF-1 and TAP-1, trypsinized and PBS washed cells were pretreated with 2% paraformaldehyde at 25°C ×5 minutes and permeabilized with 0.1% Triton x-100 at 25°C for 1 min. Cells were treated with primary antibody, mouse anti-human IGF-1 (Upstate Biotechnology, Waltham, MA) or mouse anti-human TAP-1 antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany), and, then stained with secondary antibody (FITC conjugated goat anti-mouse IgG) and analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) on the day of staining. Histogram analysis was performed using LYSYS II software.
For immuno-cytochemistry and flow cytometry studies, non -transfected or transfected cells were incubated×18–20 hrs in supplemented DMEM (with no added FBS) in presence or absence of 50 uM ZnSO4. Incubations were done to complete induction and expression of IGF-1 antisense RNA and the down-regulation of IGF-1 prior to cell staining procedures.
PCR and RT-PCR
Cell lysates were prepared for polymerase chain reaction (PCR) by addition of cells in 100 ul aliquots of PCR buffer (50 mM KCL, 10 mM Tri-HCL pH 8.3, 2.5 mM MgCL2) containing the 0.45% non-ionic detergent NP40, 0.45% Tween-20 and 100 µg/ml proteinase K, to each well of a 48 well-plate. Following incubation×1 hr at 55°C, temperature was increased to 95°C ×10 min to inactivate proteinase K. Detection of IGF-1 antisense cDNA by PCR was carried out in a Thermal Cycler 480 (Perkin Elmer) programmed for 30 cycles of 60/45/90 sec at 94/56/72°C respectively, using Taq polymerase. Reverse transcription PCR (RT-PCR), for detection of IGF-1 antisense RNA and TAP-1, TAP-2, LMP-2, and, LMP-7 mRNA, was performed using Trizol reagent (GIBCO/BRL) in the presence of RNase inhibitor (RNasin). Briefly, HGB cells cultured in 100 mm dishes were washed with PBS×1, PBS was aspirated and 2 ml of Trizol reagent was added to each dish. The mixtures were then incubated at room temperature×5 min. 0.4 ml of chloroform was added and the homogenates were formed with gentle shaking. After incubation×2 min at room temperature, mixtures were centrifuged at 1,500 rpm×7min at 4°C to separate phases. The upper aqueous phase was transferred to a fresh tube, mixed with an equal volume of isopropyl alcohol and then incubated at room temperature×10 min. Total RNA precipitates were obtained by centrifugation at 12,000 g×15 min at 4°C in a microfuge. Pellets were washed×1 in 75% ethanol, air-dried and re-suspended in DEPC (diethylpyrocarbonate)-treated water. DNase digestion was then done in 50 ul aliquots of total RNA as previously described [22]. After incubation at 37°C ×I hr, 100ul of Trizol reagent+20 ul of chloroform were added and reactions were continued at 25°C ×5 min. The mixtures were then centrifuged at 12,000 g×15 min at 4°C in a micro-centrifuge. The upper phases were separated and precipitated with an equal volume of isopropyl alcohol. The pellets were washed with 75% alcohol and air-dried×5 min. Reverse transcription was done as follows: 16ul (2 uM) of the DNAse treated total RNA was combined with 5ul (1.5 uM) random primer (GIBCO/BRL), 10 ul of 5×RT-buffer and 6.5ul of DEPC-treated water; and, the mixture was then incubated at 70°C ×5 min. After chilling on ice, 5ul of M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (200 u/ul), 5ul of 0.1 M DDT, 1ul of 4×dNTP and 1.5ul of 40u/ul RNasin were added. The reaction was then incubated at 42°C ×50 min, and terminated at 99°C ×5 min. PCR was carried out combining 2.5 ul of the cDNA solution, 2.5 ul of 10×PCR buffer, 0.75ul of 50 mM MgCL2, 1.25 ul of dNTP mix (4 mM each), 1.0 ul of the forward and reverse primer pair (50 ng), 0.5 µl of the β-actin primer pair (50 ng), 0.25 ul of Taq polymerase (0.5u) and 15.5 ul of water. PCR was done in a Perkin Elmer thermal cycler 480 as previously described. The sequences of primer pairs used for amplification of cDNA are shown in
Table 1
.
10.1371/journal.pone.0058428.t001Table 1 Primer pairs used for RT-PCR.
Product 5′ Sequence 3′ Function Length of cDNA
ANTISENSE IGF1
GAAGATGCACACCATGTCCT
Forward 424 bp
TCACTCTTCACTCCTCAGGAG
Reverse
TAP-1
GGGCTGTAAGCAGTGGGAACC
Forward 197 bp
CAAGGCCCTCCAAGTGTAAGGG
Reverse
TAP-2
CACGGCTGAGCTCGGATACCAC
Forward 428 bp
CGACTCAGCATCAGCATCTGC
Reverse
LMP-2
GGGATAGAACTGGAGGAACC
Forward 312 bp
AGATGACACCCCCGCTTGAG
Reverse
LMP-7
GAACACTTATGCCTACGGGGTC
Forward 174 bp
TTTCTACTTTCACCCAACCATC
Reverse
Western Immunobloting
HGB cells grown to 90% confluence were incubated in fresh serum-free, supplemented DMEM containing 50 µM ZnSO4×18–20 hrs. Cells were washed ×1with cold 1×DPBS, then solubilized in 0.5 ml of cold lysis buffer cocktail containing protease inhibitors (complete mini) (Roche, Indianapolis, IN) at 4°C. Cell lysates were sonicated×10–15 sec to shear DNA and reduce viscosity of samples. Protein concentration was determined for samples, using the DC protein assay kit (Bio-Rad laboratories, Hercules, CA). 50 µg of each sample was heated to 95–100°C ×5 min, cooled in ice and centrifuged at 12,000 g×5 min. Supernatants were maintained at room temperature, then loaded on SDS-PAGE gel and electrophoresis was carried out at 125 V for 1 hr. The separated proteins were electro-transferred to PVDF membrane (Immobilon™– P membrane) (Millipore, Bedford, MA) at 30 V overnight or at 120 V for 1 hr at 4°C. Non-specific protein binding was blocked by incubation in TBS-T (20 mM Tris/HCL, PH 7.6, 137 mM NaCL, 0.1% Tween 20) containing 5% non-fat milk for 1 hr at room temperature. Immuno-detection was performed using primary mouse anti-human TAP-1 monoclonal antibody (a gift from Dr. Robert Tampe, Philipps-University of Marburg, Germany) and secondary rabbit anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, Beverly, MA). LumiGLO chemiluminesence was used as the detection system. Blots were stripped with Western-Re-Probe (Gene Technology, Inc, St. Louis, MO) and probed with primary mouse anti-human actin (Ab-1) monoclonal antibody (IgM) and secondary goat anti-mouse IgM antibody conjugated with HRP (Oncogene Research Products, San Diego, CA).
Quantification and Statistical Analysis
Quantification by densitometry for images from RT-PCR was performed with an Epson Scanner densitometer. The relative optical densities were measured by sampling 5×5 pixels. The density for each sample band was determined relative to the corresponding band of actin, with subtraction of the background density for a similar-sized area in a control zone of the image section. The quantity of each protein band in the Western blot was determined using the NIH IJ (image J) densitometry program. Statistical analysis was performed using SDA WINKS Software (Cedar Hill, Texas). All values are expressed as means (+/-SEM) for similar sized samples from two - three independent experiments. The One-way ANOVA/t-test analysis of variance was performed to assess significance. P Values<0.05 were considered statistically significant.
Results
Molecular Characterization and Down-regulation of IGF-I in pAnti-IGF-I Transfected HGB Cell Lines
Human Glioblastoma (HGB) cell lines, obtained from primary cultures of six patients with histo-pathologically diagnosed Glioblastoma, each demonstrated glial fibrillary acidic protein (GFAP) and IGF-1 positivities by indirect immuno-cytochemical staining technique using mouse anti-human IGF-1 monoclonal antibody. IGF-1 positive cells were characterized by yellow-brown staining in peri-nuclear cytoplasm. The HGB cell line T98G, obtained from ATCC, demonstrated similar staining. In contrast, pAnti-IGF-1 transfected HGB cells stained negatively [22].
Fig 1
A shows a map of the 10.8 kb pAnti-IGF-1 vector. This plasmid expresses 1 kb of IGF-1 RNA in antisense orientation. The suppression of endogenous cellular IGF-1 RNA transcripts by IGF-1 antisense RNA in tumor cells was previously described [16], [24]. Data in Fig1 B show that IGF-I cDNA product from pAnti-IGF-1 vector in transfected cells was present in antisense orientation. In 3 of 4 separate clones tested using a primer pair that bridges IGF-I cDNA from exons 1 to 5, the PCR product was a 424 bp fragment characteristic of the IGF-I cDNA, which contains exons 1, 2, 3 and 5 but not exon 4 (lanes 2, 3, 4). This band was not appreciable in the RNA obtained from non-transfected cells (lane 5). The number of copies of vector in transfected cells was estimated by restriction enzyme analysis and Southern blot. The 10.8 kb DNA fragment characteristic of extra-chromosomal pAnti-IGF-I vector averaged 4 –10 copies per Human Glioblastoma transfected cell (data not shown). The expression of the 1 kb IGF-I antisense RNA in separately transfected cell clones is demonstrated by Northern blot in
Fig 1
C (lanes I, 3, 4, 5, 6, 8, 9, 10). The degree of expression varies greatly among different transfected cell clones. Lanes 2, 7 and 11 demonstrate that there was no hybridization of the IGF-I cDNA probe to RNA of non-transfected cell clones.
Fig 1
D represents a bar graph of quantitated intensities from bands shown in
Fig 1
C. Analysis by flow cytometry showing down-regulation of IGF-I in pAnti-IGF-I transfected, compared to non-transfected and mock transfected HG-2 cells is depicted in
Fig 2
A. Among the different HGB cell lines tested, IGF-I levels, as determined by specific fluorescence, were decreased variably by 28 to 93% in transfected (TX) relative to corresponding non-transfected (NT) clones (
Fig 2
B).
10.1371/journal.pone.0058428.g002Figure 2 Down-regulation in expression of IGF-1 in pAnti IGF-1 transfected HGB cell lines. A,
Demonstration of intracellular IGF-1 levels in the HG-2 cell line by Flowcytometry. Isotype control (non-transfected Cells+mouse IgG FITC); Non-transfected (non-transfected cells+mouse anti-human IGF-1 mAb+goat antimouse IgG FITC); Transfected (transfected cells+mouse antihuman IGF-1 mAb+goat antimouse IgG FITC); Mock transfected (cells transfected with vector minus antisense IGF-1 cDNA+mouse antihuman IGF-1 mAb+goat antimouse IgG FITC). B, Bar graph comparison of IGF-1 expression in transfected and corresponding parental, non-tranfected HGB Cell Lines. Cell lines were established from discarded tumor tissue of Glioblastoma patients. The experiment design was as depicted in legend A of this Fig. % IGF-1 content = %Fs (specific fluorescence) = [Target (Fluorescence mean value) – Control (Fluorescence mean value)]/Target (Fluorescence mean value)×100%. NT = non-transfected, TX = pAnti-IGF-1 transfected. The experiments of
Fig 2B
were done×3. The paired t-test was used to determine P values. The statistical procedures were performed on the average difference for each cell line before (NT) and after (TX) transfection. The calculated t and associated p-values are given. Grouped comparisons between TX and NT cell lines for IGF-1 from summarized data by two-way ANOVA were statistically significant at p<0.001 (5 cases) or p <0.05 (3 cases).
Major Histocompatibility Complex (HLA-1) and Co-stimulatory B-7.1 in pAnti-IGF-1 Transfected HGB Cell Lines
Expression of HLA -1 in transfected compared to non-transfected clones of 6 separate HGB cell lines and the ATCC cell line T98G were analyzed by immunofluorescent flow cytometry. Similar comparisons were obtained for the expression of the B-7.1 co-stimulatory molecule in 4 of the 6 HGB cell lines. Each of the pAnti-IGF-1 transfected HGB cell lines tested was down-regulated in IGF-1 content. IGF-1 was also down-regulated by 90% in the transfected T98G cell line (data not shown). The results for up-regulation of HLA-1 and B-7.1 are summarized in
Table 2
. Parental T98G cells and 6 HGB cell lines expressed low levels of HLA-1 molecules on cell surfaces. Following transfection with pAnti-IGF-1, transfectants of the T98G cells and each of the 6 HGB cell lines tested showed a greater than 45% increase, with a range of increase up to seven fold in expression of HLA-1 (p< 0.05). Analysis by Flow cytometry demonstrating relatively significant increase in expression of HLA-1 in transfected, when compared to non-transfected and mock transfected, T98G cells is depicted in
Fig 3
A and B (P<0.05). Also depicted in
Figure 3
is transfection with the vector pAnti-IGF-2. These data showed no significant differences from the non-transfected or mock transfected controls (
Fig 3
C). Two HGB cell lines, HG-2 and HG-3, showed a 16 fold and 5 fold increase, respectively, in expression of the B-7.1 (
Table 2
). However, the increments of change in 2 of the 4 HGB cell lines tested (HG-4 and HG-25) were not sufficient to give statistically significant results.
10.1371/journal.pone.0058428.g003Figure 3 Comparison of HLA-1 cell surface antigens in pAnti IGF-1 transfected, mock transfected, pAnti IGF-2 transfected and non-transfected T98G cells.
A, Histogram of fluorescence intensity from isotype control (non-transfected cells+mouse IgG FITC); NT (non-transfected cells+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); Mock TX (cells transfected with vector devoid of anti IGF-1 cDNA+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); pAnti IGF-2 TX (cell transfected with vector expressing IGF-2 RNA in antisense orientation+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC); and, pAnti IGF-1 TX (cells transfected with vector pAnti IGF-1+mouse anti-human HLA-1 mAb+goat anti-mouse IgG FITC). B, Specific fluorescence showing differential expression of HLA-1 from pAnti IGF-1 transfected cells when compared to parental non-transfected cells and other controls (P <0.05). C, Group ANOVA comparison was done by boxplot with Newman-Keuls graphical representation for comparison in expression of HLA-1 in the experimental groups. The data in Fig 3 B was repeated×3.
10.1371/journal.pone.0058428.t002Table 2 Comparison in expression of HLA-1 and B-7.1 for pAnti IGF-1 transfected and non-transfected HGB cell lines by flow cytometry.
Cell line HLA-1%Fs B-7.1%Fs
HG-1
Control 22.1 ND
TX 32.1
% change +45.0
SD 1.13
P value P = 0.019
HG-2
Control 21.7 3.9
TX 63.3 66.6
% change +191.7 +1607.0
SD 2.65 10.15
P value P = 0.001 P = 0.008
HG-3
Control 12.5 7.5
TX 36.3 45.1
% change +190.4 +501.3
SD 4.41 1.65
P value P = 0.007 P = 0.001
HG-4
Control 17.8 44.9
TX 35.6 45.8
% change +100.0 +2.0
SD 1.58 1.56
P value P = 0.025 P = 0.766
HG-9
Control 6.3 ND
TX 54.4
% change +763.5
SD 4.13
P Value P = 0.001
HG-25
Control 11.9 27.6
TX 50.9 33.0
% change +327.7 +19.6
SD 4.13 1.50
P value P = 0.001 P = 0.07
T98G
Control 20.8 ND
TX 35.9
% change +72.6
SD 3.14
P value P = 0.001
Fs: Specific fluorescence. Fs defined as total mean fluorescence of sample (Ft) minus that of the background fluorescence (Fb) divided by Ft, i.e. %Fs = (Ft-Fb)/Ft×100.
Control: cells not transfected with vector pAnti IGF-1.
TX: cells transfected with vector pAnti IGF-1.
SD: standard deviation.
P value: Grouped comparison of TX vs. NT (control) by two-way ANOVA at p<0.05 is statistically significant.
Enhanced Expression of TAP-1, TAP-2, LMP-2 and LMP-7 mRNA
Analysis by semi-quantitative RT-PCR demonstrated a relative decrease or absence in transcription of antigen processing products in 4 different parental, non-transfected, compared to corresponding transfected HGB cell lines. The data are shown in
Fig 4
A and B Lanes 1, 2, 3, 4 compared to lanes 5, 6, 7, 8 for TAP-1 and TAP-2 gene expression respectively, and, in
Fig 4
C and D lanes 1, 2, 3, 4 compared to lanes 5, 6, 7, 8 for LMP-7 and LMP-2 gene expression respectively. Following transfection, increases in the expression of TAP-1 mRNA, LMP-2 and LMP-7 mRNA for HG-3 were relatively less than in the cases of other transfected cell lines, accept in the case of TAP-2 mRNA (see lanes 6 of respective photographs). In two of the non-transfected cell lines, HG-5 and HG-9, TAP-2 mRNA expression was modest (lanes 3 and 4 of
Fig 4
B). However, its expression in transfected HG-5 and HG-9 cells in comparison to the corresponding non-transfected cells was substantially increased (lanes 7 and 8 of
Fig 4
B). Using the Epson perfection photo scanner, the optical intensities for Fig A, B, C and D are shown by the bar graphs of Fig E, F, G, H, respectively. The panels of each bar graph represent intensity in expression of the component molecule for each cell line as depicted. The intensity was calculated against intensity of the internal control, β-actin. Also subtracted was background in each of the respective sites. According to these measurements as shown in Figs E, F, G and H, following transfection the expression in TAP-1 was increased in HG-2, HG-5 and HG-9 (p<0.05); expression in TAP-2 was increased in HG-2 (p<0.001) and HG-3 (p<0.05); expression in LMP-7 was enhanced in HG-2 (p<0.001), HG-5 and HG-9 (p<0.05); and, expression in LMP-2 was up-regulated in HG-2 (p<0.001), HG-5 and HG-9 (p<0,05). These increments of increase are significant.
10.1371/journal.pone.0058428.g004Figure 4 Comparison in expression of TAP and LMP transcripts in pAnti IGF-1 transfected (TX) and non-transfected (NT) HGB cell lines.
RNA was prepared from cell lines established from four different patients. Following incubation of cells in the presence of 50 µM ZnSO4 for 18–20 hrs, agarose gel eletrophoresis and ethidium bromide staining were used to characterize products of RT-PCR for A, TAP-1; B, TAP-2; C, LMP-7; and D, LMP-2. Primer pairs for β-actin were included in each experiment as internal control. Expected sizes of RT-PCR products were: TAP-1, 197 bp; TAP-2, 428 bp; LMP-7, 174 bp; LMP-2, 312 bp; β-actin, 289 bp except in experiments concerning expression of LMP-2 in which the primer pair for the 618 bp fragment was used. Molecular weight markers are multiples of 50 bp from 50 to 800. Please note that Fig 4 A – D represent the results for one set of three similar RT-PCR experiments; E – H represent the results for the three sets of data analyzed together using the Epson Scan program for densitometry. Each TX cell line was compared to the respective NT cell line. Significance ** p<0.05, *** p<0.001.
Up-regulation of TAP-1 and LMP-7 Peptides, and, Rescued Expression of TAP-2, LMP-2 Peptides and the B-7.1 Molecule
Down-regulation of IGF-1 or the IGF-1/IGF-1R signaling pathway by transfection with the pAnti- IGF-1 vector or by the exogenous addition of IGF-1R monoclonal antibody (mAb) to culture medium of HGB cell lines resulted in enhanced and/or rescued expression in antigen processing machinery components by Western Immuno-blotting analysis.
Fig 5
A demonstrates two transfectants, one from T98G and one from HG-2. Both showed a 70 kd band characteristic of the TAP-1 peptide. The corresponding non-transfected (NT) T98G and HG-2 cell lines did not show this band. Data are consistent with results from the RT-PCR experiments of
Fig 4
.
Fig 5
B demonstrates that three cell lines (T98G, HG-2 and HG-9) cultured in medium plus 10 ug/ml of IGF-1R monoclonal antibody were up-regulated in expression of TAP-1 and LMP-7, and that rescue in the expression of TAP-2 and LMP-2 occurred when compared to these cell lines cultured in medium minus the IGF-1R mAb. In
Fig 5
B row 5, the up-regulated expression of the co-stimulatory 60 kd B-7.1 molecule is also shown in these cell lines following blockade of the IGF-1 receptor by its monoclonal antibody.
Fig 5
C demonstrates the densities of the protein bands in Western blot of
Fig 5
B, and in an additional set of data from repeated experiments, as determined by the NIH image J program. It was shown that each of the components of the antigen processing machinery tested in these three HGB cell lines were significantly up-regulated following addition of the IGF-1R mAb to culture media (P<0.05).
10.1371/journal.pone.0058428.g005Figure 5 Comparison in expression of TAP, LMP and B-7.1 peptides in parental and pAnti IGF-1 transfected and/or IGF-1R monoclonal antibody (mAb) treated HGB cell lines.
A, Regulation of TAP-1 peptide in T98G and HG-2 TX cells was determined by Western Blot. Cell lysate was prepared from cells of TX and corresponding NT cell lines pretreated as described in Fig 4 and then subjected to SDS-PAGE and electronically blotted to nitrocellulose membrane. TAP-1 peptide on the membrane was probed by anti-human TAP-1 monoclonal Ab+anti-mouse IgG HRP-linked Ab; and, signal was detected by LumiGLO reagent. Lanes 1, 2 are NT cells from the T98G and HG-2 cell lines respectively. Lane 3, 4 are TX cells from clone b and c of the T98G cell line; Lane 5, 6 are TX cells from clone 5 and 11 of the HG-2 cell line. Re-probed membrane with anti-actin Ab demonstrates presence of the 42 kd actin. Up-regulation in expression of the 70 kd TAP-1 in the two pAnti IGF-1 transfected cell clones were demonstrated in this experiment. B, Up-regulation in TAP-1 and LMP-7 peptides, and, rescue in expression of TAP-2 and LMP-2 peptides following the exogenous addition of 10ug/ml IGF-1R mAb into cell culture medium for 48 hours were demonstrated by Western Blot. The lysates of wild type and IGF-1R antibody treated cells were prepared as described in A. Lanes 1, 2 and 3 represent T98G, HG-2 and HG-9 cell lines cultured in medium with no IGF-1R mAb added, while lanes 4, 5 and 6 are the corresponding respective cell lines cultured in medium with addition of exogenous IGF-1R mAb (10ug/ml), respectively. Rows 1, 2, 3 and 4 represent TAP-1, TAP-2, LMP-2 and LMP-7, respectively. Row 5 demonstrates the rescue in expression of the B-7.1 peptide in IGF-1R mAb treated T98G, HG-2 and HG-9 cell lines when compared to the wild types of the corresponding non-treated cells. The 42 kd band of actin is also shown as an internal control for the quantity of samples loaded. B represents the results from one set of the experiments, that were repeated×2; C, The densitometry analysis for the two sets of similar experiments was obtained by the NIH image J program. The differences in results between the groups with and without IGF-1R mAb in culture media were significant at p<0.05 or p<0.001.
Specificity for Enhanced Expression of the TAP-1 peptide in pAnti-IGF-1 Transfected HGB Cell Lines
Quantitative assessment of intracellular TAP-1 peptide in HGB cell lines was demonstrated by immunofluorescent flow cytometry using mouse anti-human TAP-1 mAb as primary antibody (kindly provided by Dr. Robert Tampe, Philipp-University, Marburg, Germany). Enhancement of intracellular TAP-1 level was demonstrated in 8 pAnti-IGF-1 transfected T98G, 2 pAnti-IGF-1 transfected HG-2 and 1 pAnti-IGF-1 transfected HG-3 clones when compared to the respective non-transfected cell clones. The variation in degree of enhancement in transfected compared to corresponding non-transfected cell lines is shown in
Fig 6
A. The increase in each cell line is statistically significant at the p<0.05.
Fig 6
B and C demonstrate the relative specificity for enhancement of TAP-1 in pAnti IGF-1 transfected cells. For this experiment, the established ATCC T98G cell line was used. The data show a five fold increase in TAP-1 in pAnti IGF-1 transfected, IGF-1 down-regulated cells (8 clones), when compared to non-transfected cells (4 clones); a greater than 3 – 4 fold increase when compared to pEMT (vector containing no IGF-1 antisense sequence) transfected cells (4 clones), or to pAnti IGF-2 transfected cells (4 clones) (P<0.001). The degree of significance is shown in the
Fig 6
C box plot as determined by one-way ANOVA analysis. The results of the analysis showed that the group 4, T98G cells transfected with pAnti IGF-1, is significantly different from the other treatments (P<0.001); the differences between groups 1 (NT), 2 (EMP), and 3 (pAnti IGF-2) are not significant (P = 0.46).
10.1371/journal.pone.0058428.g006Figure 6 Variability and specificity in expression of TAP-1 in HGB cell lines. A,
Variability of TAP-1 levels in pAnti IGF-1 transfected and non-transfected HGB cell lines. Different clones of transfected (TX) and corresponding non-transfected (NT) HGB cell lines, eight from T98G (circles), two from HG-2 (rectangles) and one from HG-3 (triangle), were examined by flow cytometry for the expression of TAP-1. Expression of TAP-1 in TX and NT cell clones were determined using mouse anti-human TAP-1 monoclonal antibody (courtesy of Dr.Robert Tampe) as described in methods. The up-regulated expression of TAP-1 in TX compared to NT clones was significant at the P< 0.05. B, Relative specificity in up-regulation of TAP-1 in pAnti IGF-1 transfected HGB cells. T98G cells were transfected with the vector containing antisense IGF-1 cDNA (TX pAnti IGF-1), with vector containing antisense IGF-2 cDNA (TX pAnti IGF-2), or, with vector containing no antisense sequence (TX pEMT). Transfected cells were comparatively examined by flow cytometry relative to non-transfected T98G cells (NT) for content of TAP-1. The antibody used and flow cytometry analysis were as described in A of this Fig and methods. This experiment was repeated×3. C, TAP-1 expression in comparison of treatments was tested by t-test/ANOVA using Winks SDA software. Boxplot illustrates the comparison in specific fluorescence value among the experimental groups, P<0.001. Represented below the boxplot is the graphical description of Newman-Keuls multiple comparisons.
Enhanced Expression of TAP-1 Peptide in pAnti IGF-1 Transfected HGB Cells was Down-regulated by Exogenous Addition of IGF-1
To detect whether the enhanced expression of TAP-1 peptide can be suppressed by IGF-1, the pAnti-IGF-1 transfected T98G and HG-2 cells were first cultured in serum- free medium in the presence of ZnSO4 (50uM) ×8 hours (Zn2+ binds to metal responsive element of metallothionein-1 promoter in the pAnti IGF-1 vector. This activates transcription of down-stream IGF-1 antisense cDNA). The cell culture was then incubated with addition of IGF-1 (100 ng/ml) overnight. The cell lysates of IGF-1 treated and transfected HGB cells were subjected to analysis by Western blot. The down-regulation in expression of TAP-1, after addition of IGF-1, in tested TX cell lines is shown in
Fig 7
A. The data showed that exogenous IGF-1 can reverse the up-regulation of TAP-1 that occurs with antisense IGF-1 transfection. The kinetics of decrease in expression of TAP-1 in pAnti IGF-1 transfected T98G cells by addition of IGF-1 (100 ng/ml) is shown in
Fig 7
B, C. In this case, the expression of TAP-1 was abolished after 60 minutes of treatment with IGF-1. The data therefore also demonstrate that IGF-1 may be involved in regulation of the TAP-1 gene expression.
10.1371/journal.pone.0058428.g007Figure 7 Effect of exogenous IGF-1 on expression of TAP-1, pStat1(Tyr701) and pStat3(Tyr705). A,
Expression of TAP-1 peptide in T98G cells was down-regulated by addition of IGF-1 to culture medium. TX cells were incubated in absence of serum and in the presence of 50 μM ZnSO4 and IGF-1 (100 ng/ml) over-night. Lanes 1 and 2 are NT cells from the T98G and HG-2 cell lines respectively. Lane 3 and lane 4 are T98G TX (clone b) and HG-2 TX (clone 5), respectively, into which IGF-1 was added overnight. Lanes 5 and 6 are as in lanes 3 and 4 except without added IGF-1. B. The expression of TAP-1 was examined as a function of time following addition of IGF-1 (100 ng/ml) in TX T98G cells in absence of serum and in the presence of Zn2+. Cells were treated with exogenous IGF-1 for the time periods as depicted. The cell lysates were prepared and subjected to Western Blot analysis as described in methods. The primary antibodies to TAP-1, pStat1 (Tyr701), pStat3 (Tyr705) and Stat3 were also obtained as described. The 70 kd TAP-1, 91 kd pStat1, and 86 kd pStat3 and 86 kd Stat3 peptides produced during the time course were identified by monoclonal antibody used and molecular weights. The data represent one of three sets of similar experimental results. C, Data from
Fig 7
B and similar results from two repeat experiments were used for this bar graph. The densitometry quantification was done using the NIH image J program as described in methods.
Down-regulation in Expression of TAP-1 in pAnti IGF-1 Transfected HGB Cells by Exogenous Addition of IGF-1 Occurs with Inhibition of Phosphorylated STAT3(Tyr 705) and in Absence of Phosphorylated STAT1(Tyr 701)
In order to determine whether Stat signaling is involved in the IGF-1 regulated expression of the TAP-1 gene, the pAnti IGF-1 transfected T98G and/or HG-2 cells were treated with addition of IGF-1 (100 ng/ml) in absence of serum and with ZnSO4 (50uM) added to culture medium. During different time intervals of treatment, cells were collected and cell lysates were obtained as described in methods and analyzed by Western blot using the antibodies (Cell Signaling Technology Inc.) specific for phosphorylated Stat1(Tyr 701) or phosphorylated Stat3(Tyr 705). The expression of pStat1 in T98G NT (data not shown) and T98G TX cells was not detected; and it was also not detected after the exogenous addition of IGF-1 (
Fig 7
B, C). In contrast, pStat3 was expressed in both T98G NT (data not shown) and T98G TX cells; and it was transiently decreased following addition of IGF-1 to T98G TX cells for a limited time interval (
Fig 7
B, C). At greater than 120 min, its expression returned to a high level. The same result was obtained from experiments using HG-2 TX cells (data not shown). These data, therefore, demonstrate that expression of pStat1 and pStat3 was differentially regulated in Glioblastoma cancer cells in response to IGF-1 growth factor signaling, and, that pStat1 and pStat3 may have different roles in regulating expression of the TAP-1 gene.
Discussion and Summary
Previous data showed that down-regulation in IGF-1 leads to an increase in cell surface MHC-1 [16]–[21], [25], [26]. This led to the hypothesis that decrease in intracellular IGF-1 would enhance components of the endogenous antigen processing and presentation pathway. The decrease in expression of endogenously processed tumor cell antigens in cancer tissues and cancer cell lines is a well-reported phenomenon [10]–[12], [27], [28]. It was previously demonstrated that the reverse of this process could be obtained by down-regulation in the expression of IL-10 [29]. We show in this paper an association between down–regulation in expression of IGF-1 and enhancement in the cell surface expression of HLA class 1 molecules. Along with this, there is a concomitant increase in the TAP-1, TAP-2, LMP-2 and LMP-7 components of the endogenous antigen presentation pathway.
Our previously reported work demonstrated enhancement in survival of animals that is consistent with immunity in four different animal model systems in which the intracellular expression of IGF-1 was down-regulated by transfection with vector expressing an IGF-1 antisense RNA or by transfection with vector expressing an IGF-1 RNA that can form a triple-helix oligonucleotide sequence with IGF-1 DNA [15]–[21]. The established role for the IGF-1 molecule in early differentiation of tissues as well as in cell proliferation, and, the possible need for the developing embryo to modulate the immune reaction toward protein antigens specifically expressed from the male parental genes provide a teleological rationale for a dual role in its regulation of development and immunity [30]–[32]. This together with the well reported anti- apoptotic effect of IGF-1 on programmed cell death provides insight into fundamental mechanisms for a causal role of this molecule in the progression toward frankly invasive cancer. One explanation for the results obtained, would concern whether the impact of IGF-1 is directed to selection of a specific subset of cells rather than to the modulation of IGF-1 in a given glial cell fraction. The care to select cloned cell fractions as starting material would seem to rule against this possibility. In addition, the reversal of the up-regulation in antigen processing machinery by addition of IGF-1 to culture medium would also tend to rule against this possibility. Furthermore, the morphological characteristics of transfected cells do not differ from the parental non-transfected cells. In addition, and importantly, similar results were obtained using the ATCC T98G stably cloned cell line of Glioblastoma as a standard control. Thus, we conclude that the decrease in the antigen processing machinery is affected by cell content of IGF-1.
Although a one base deletion of the TAP-1 promoter sequence of a melanoma cell line has been reported [33], other reports document that the decreased expression of TAP-1, 2 and LMP-2, 7 in cancer cell lines can be modulated or rescued by induction with IFN-γ [34]–[36]. Here we report that the deficiencies can be rescued by transfection of HGB cells with a vector expressing antisense IGF-1 RNA and/or by exogenous addition of antibody to IGF-1 receptor. We show further that the rescue can be aborted by addition of IGF-1 to culture medium (
Fig 7
).
The appropriate regulation of transport associated proteins and large multicatalytic proteasomes is critical for initiation and continuation of the cellular immune response through the endogenous antigen process pathway [28]. The TAP-1 and LMP-2, constituents of this processing pathway are interferon-gamma (IFN-γ) inducible genes. TAP-1 and LMP-2 were reported to be up-regulated or rescued by induction of IFN-γ via tyrosine phosphorylation of Stat 1 (signaling transducer and activator of transcription -1) [37]–[40]. We report in this paper that the restoration of TAP-1 and LMP-2 by down-regulation of IGF-1 in Glioblastoma cells was not related to the tyrosine phosphorylation of STAT 1, i.e. there was no pSTAT 1 detected in either wild type Glioblastoma cells or in cells transfected with the pAnti-IGF-1 vector that expresses IGF-1 antisense RNA and in which expression of TAP-1 and LMP-2 were restored by induction of the MT-1 promoter of pAnti IGF-1 in the absence of serum. There was also no pSTAT 1 detected in the pAnti IGF-1 transfected cells when IGF-1 was exogenously added to the serum-free culture medium (
Fig 7
). That is, the down-regulation of TAP-1 and LMP-2 in cells and/or the restoration of TAP1 by down-regulation of IGF-1 in these tumor cells are pSTAT 1 independent. Thus, we assume that the mechanism for the restoration of TAP-1 and LMP-2 by down-regulating IGF-1 is different, or partially different, from the one in which restoration of expression of genes by induction of IFN-γ occur in cancer cells. In the experiments described here, we found that the restoration of TAP-1 and LMP-2 may be related to the phosphorylation of STAT 3. Firstly, pSTAT 3 was detected in pAnti IGF-1 transfected, IGF-1 down-regulated T98G cells. Secondly, the exogenous addition of IGF-1 led to suppression in expression of pSTAT 3 for up to 6 hours; this suppression is paralleled by suppression in expression of TAP-1 (
Fig 7
B, C). These data show that STAT 3 may have a tumor-suppressive function in Glioblastoma cells, such as in the T98G cell line. This is similar to the findings of the unexpected Stat 3 function linked to PTEN gene activities in brain cancer cells by Bonni A, et al. [41]. Moreover, STAT 3 is an important transcription factor, and, activated STAT 3 can also bind to GAS element-like sequence or ISRE element to activate a promoter of downstream genes [42], [43]. Accordingly, we postulate, that STAT 3 may have a role in the restoration of TAP-1 and LMP-2 in IGF-1 down-regulated Glioblastoma cells. Further investigation is needed to elucidate the mechanisms involved in the modulation of antigen processing machinery by down-regulation of IGF-1 in cancer cells.
Fig 7
B also shows the effect of IGF-1 on expression of pAkt. PAkt was not detectable in pAnti IGF-1 transfected T98G cells cultured in serum-free medium plus 50 uM Zn2+. It was, however, detected 30–60 min after addition of IGF-1. This suggests that IGF-1 stimulates the PI3K/Akt signaling pathway, and is consistent with the work of others [44]. It is very likely that inhibition of IGF-1 leads to down-regulation in expression of the MHC-1 antigen processing [45].
It is clear, however, that IGF-1, in addition to its other functions, can modulate the endogenous antigen processing machinery and possibly the immune response as well as having its known role in modulating the process of apoptosis. Furthermore, in those cancers that over-express this molecule, strategies for treatment that modulate IGF-1 have promise for leading to efficacious immuno-gene therapy.
We are indebted to Dr. Joseph Ilan, Lei Yan, Russell Catanese and Drs. Frauka Rinesland and Adisek Wongkajornsilp for their contributions to this work. We also thank Dr. Robert Tampe, former director of the Maxplanck Institute, Philipps-University, Marburg, Germany for his kind gift of the TAP-1 monoclonal antibody.
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J NanobiotechnologyJ NanobiotechnologyJournal of Nanobiotechnology1477-3155BioMed Central 1477-3155-10-462324471110.1186/1477-3155-10-46ResearchSynthesis, characterization and in vitro studies of doxorubicin-loaded magnetic nanoparticles grafted to smart copolymers on A549 lung cancer cell line Akbarzadeh Abolfazl [email protected] Mohammad [email protected] Sang Woo [email protected] Maryam [email protected] Younes [email protected] Hamid Tayefi [email protected] Soodabeh [email protected] Department of Medical Nanotechnology, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran2 Department of Endodontics,Dental School, Tabriz University of Medical Sciences, Tabriz, Iran3 School of Mechanical Engineering, WCU Nanoresearch Center, Yeungnam University, Gyeongsan 712-749, South Korea2012 18 12 2012 10 46 46 26 9 2012 6 12 2012 Copyright ©2012 Akbarzadeh et al; licensee BioMed Central Ltd.2012Akbarzadeh et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The aim of present study was to develop the novel methods for chemical and physical modification of superparamagnetic iron oxide nanoparticles (SPIONs) with polymers via covalent bonding entrapment. These modified SPIONs were used for encapsulation of anticancer drug doxorubicin.
Method
At first approach silane–grafted magnetic nanoparticles was prepared and used as a template for polymerization of the N-isopropylacrylamide (NIPAAm) and methacrylic acid (MAA) via radical polymerization. This temperature/pH-sensitive copolymer was used for preparation of DOX–loaded magnetic nanocomposites. At second approach Vinyltriethoxysilane-grafted magnetic nanoparticles were used as a template to polymerize PNIPAAm-MAA in 1, 4 dioxan and methylene-bis-acrylamide (BIS) was used as a cross-linking agent. Chemical composition and magnetic properties of Dox–loaded magnetic hydrogel nanocomposites were analyzed by FT-IR, XRD, and VSM.
Results
The results demonstrate the feasibility of drug encapsulation of the magnetic nanoparticles with NIPAAm–MAA copolymer via covalent bonding. The key factors for the successful prepardtion of magnetic nanocomposites were the structure of copolymer (linear or cross-linked), concentration of copolymer and concentration of drug. The influence of pH and temperature on the release profile of doxorubicin was examined. The in vitro cytotoxicity test (MTT assay) of both magnetic DOx–loaded nanoparticles was examined. The in vitro tests showed that these systems are no toxicity and are biocompatible.
Conclusion
IC50 of DOx–loaded Fe3O4 nanoparticles on A549 lung cancer cell line showed that systems could be useful in treatment of lung cancer.
Superparamagnetic iron oxide nanoparticles (SPIONs)Drug loading efficiencyRadical polymerizationN-Isopropylacrylamide-methyl metacrylc acid (NIPAAm-MAA)
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Background
Functionalization of nanomaterials with chemical or biological molecules exhibits novel properties for various likely applications. The distinctive physico-chemical properties of these materials when utilized in conjunction with the remarkable biomolecular recognition capabilities could lead to miniature biological, optical and electronics devices [1,2].
However, an essential issue for in vivo application is its biocompatibility. Central focus to tackling this problem is surface modification of nanomaterials to prevent the spontaneous aggregation and elucidating the interface between nanomaterials and biosystem. Among inorganic nanomaterials, iron oxide nanoparticles (IOPs) have a high potential for the use in a lot of in vitro and in vivo applications. Based on their unique mesoscopic physical, chemical, thermal, and mechanical properties, IOPs offer a high potential for several biomedical applications such as: [3,4].
(1) cellular therapy, cell labelling, and targeting as a tool for cell-biology research (2) tissue repair (3) drug delivery (4) magnetic resonance imaging (MRI); (5) hyperthermia; (6) magnetofection; etc. For these applications surfaces modification of the nanoparticles by creating a few atomic layer of organic (e.g. polymers) or inorganic (e.g. gold) material or oxide surfaces (e.g. silica or alumina) could be an excellent job for the further functionalization with various bioactive molecules. MNPs may soon play a significant role in meeting the healthcare requirements of tomorrow.
A significant challenge associated with the application of these MNP systems is their behavior in-vivo. The efficacy of many of these systems is often compromised due to recognition and clearance by the reticuloendothelial system (RES) prior to reaching target tissue, as well as by an inability of to overcome biological barriers, such as the vascular endothelium or the blood brain barrier. The fate of these MNP upon intravenous administration is highly dependent on their size, morphology, charge and surface chemistry. These physicochemical properties of nanoparticles directly affect their subsequent pharmacokinetics and biodistribution. To increase the effectiveness of MNPs, several techniques, including: reducing size and grafting non-fouling polymers have been employed to improve their “stealthiness” and increase their blood circulation time to maximize the likelihood of reaching targeted tissues [5,6].
The major disadvantage of most chemotherapeutic approaches to cancer treatment is that most of them are non-specific. Therapeutic (generally cytotoxic) drugs are administered intravenously leading to general systemic distribution (Figure 1). The non-specific nature of this technique results in the well-known side effects of chemotherapy as the cytotoxic drug attacks normal, healthy cells in addition to its primary target and tumor cells [7,8]. Magnetic nanoparticles (MNPs) can be used to overcome this great disadvantage. Nanoparticle can be used to treat tumors in three different ways: (i) specific antibodies can be conjugated to the MNPs to selectively bind to related receptors and inhibit tumor growth; (ii) targeted MNPs can be used for hyperthermia for tumor therapy; (iii) drugs can be loaded onto the MNPs for targeted therapy [9-11]. The targeted delivery of anti-tumor agents adsorbed on the surface of MNPs is a promising alternative to conventional chemotherapy. The particles loaded with the drug are concentrated at the target site with the aid of an external magnet. The drugs are then released on the desired area [12]. Magnetic particles smaller than 4 μm are eliminated by cells of the RES, mainly in the liver (60–90%) and spleen (3–10%). Particles larger than 200 nm are usually filtered to the spleen, whose cut-off point extends up to 250 nm. Particles up to 100 nm are mainly phagocytosed through liver cells. In general, the larger the particles are the shorter their plasma half-life-period [13].
Figure 1 Applications of superparamagnetic iron oxide nanoparticles (SPION)10.
Functionalization of MNPs with amino group, silica, polymer, various surfactants or other organic compounds is usually provided in order to achieve better physicochemical properties. Moreover, the core/shell structures of MNPs have the advantages of good dispersion, high stability against oxidation and appreciable amount of drug can be loaded to the polymer shell. Furthermore, lots of functional groups from polymers on the surface can be used for further functionalization to get various properties [14]. It is favored that MNPs retain sufficient hydrophilicity with coating, do not exceed 100 nm in size to avoid rapid clearance by reticuloendothelial system (RES) [15]. It was found the surface functionalization plays also the key role in nanoparticle toxicity [16].
It was found the surface functionalization plays also the key role in nanoparticle-toxicity.In this research we intend to investigate the in vitro characteristics of our nanoparticles for drug delivery applications [17]. Of these temperature-sensitive polymer-grafted MNPs, poly-(N-isopropylacrylamide) (PNIPAAm)-grafted MNPs are of particular interest because of their stimuli (temperature) responsiveness and enhanced drug-loading ability. These characteristics are due to their large inner volume, amphiphilicity, capacity for manipulation of permeability, and response to an external temperature stimulus with an on-off mechanis [18-20]. However, one potential problem with using PNIPAAm as a polymer coat is that its lower critical solution temperature (LCST), the temperature at which a phase transition occurs, is below body temperature (32°C). To increase the LCST of PNIPAAm above body temperature, it has been co-polymerized with different monomers (Figure 2) [21,22].
Figure 2 Structure of the PNIPAAm-MAA copolymer.
To manufacture the PNIPAAm-MAA-grafted Magnetic nanoparticles, two synthetic steps were used [23]. First, magnetic nanoparticles were covalently bound with a silane coupling agent, vinyltriethoxysilane (VTES), to produce a template site for a radical polymerization. NIPAAm and MAA were then polymerized on the silicon layer around the magnetic nanoparticles via methylene-bis-acrylamide and ammonium persulfate as a cross-linking agent and an initiator, respectively. The resultant particles were characterized by X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM). The in-vitro cytotoxicity test for the PNIPAAm-MAA-grafted magnetic nanoparticles was analyzed. The drug release behavior of doxorubicin (an anticancer drug model) from the nanoparticles at various pH and at different temperatures below and at the lower critical solution temperature (LCST) was also analyzed. Being able to monitor the location of the drug-loaded nanoparticles after administration proved to be a considerable advantage in cases such as cancer therapy, in which the drug has serious side effects on healthy tissues [24,25].
Materials and methods
Materials
Ferric chloride hexahydrate (FeCl3.6H2O), Ferrous chloride tetrahydrate (FeCl2. 4H2O) and ammonium hydroxide (25 wt.%) were purchased from Fluka (Buchs, Switzerland). 1,4 dioxan, Ammonium persulfate, AIBN(2 Azo Bis Iso Butyro Nitrile), MAA, NIPAAm, and DMSO , methylene-bis-acrylamide (BIS), VTES, acetic acid, ethanol were purchased from Sigma-Aldrich (St. Louis, Missouri) . Doxorubicin hydrochlorid was purchased from Sigma-Aldrich. XRD, Rigaku D/MAX-2400 X-ray diffractometer with Ni-filtered Cu Kα radiation, scanning electron microscopy (SEM) measurements were conducted using a VEGA/TESCAN. The drug loading capacity and release behavior were determined using a UV–vis 2550 spectrometer (Shimadzu). The infrared spectra of copolymers were recorded on a Perkin Elmer 983 IR spectrometer (Perkin Elmer, USA) at room temperature. The magnetic property was measured on VSM/AGFM (Meghnatis Daghigh Kavir Co Iran) vibrating sample magnetometer at room temperature. The drug loading capacity and release behavior were determined using a UV–vis 2550 spectrometer (Shimadzu). The organic phase was evaporated by rotary (Rotary Evaporators, Heidolph Instruments, Hei-VAP Series).
Preparation of superparamagnetic magnetite nanoparticles
Superparamagnetic magnetite nanoparticles (MNPs) were prepared via improved chemical co-precipitation method [26]. According to this method, 3.17 g of FeCl2 · 4H2O (0.016 mol) and 7.68 g of FeCl3 · 6H2O (0.008 mol) were dissolved in 50 ml of deionized water, such that Fe2+/Fe3+ = 1/2. The mixed solution was stirred under N2 at 85°C for 1 h (Figure 3). Then, 40 ml of NH3 · H2O was injected into the mixture rapidly, stirred under N2 for another 1 h and then cooled to room temperature. The precipitated particles were washed several times with hot water and separated by magnetic decantation. Finally, magnetic MNPs were dried under vacuum at 65°C.
Figure 3 Magnetite-hexane suspension attached to a magnet.
Synthesis of Silane–grafted magnetic nanoparticles for loading of doxorubicin
Synthesis of VTES-grafted magnetic nanoparticles
VTES-modified magnetite nanoparticles were synthesised by the reaction between VTES and the hydroxyl groups on the surface of magnetite. Nearly, 2 g of Fe3O4 nanoparticles were dispersed in 100 ml of ethanol by sonication for about 1 h, then 24 ml of NH3.H2O was added and sonicated to homogenize for 12 min. Under continuous mechanical stirring, 10 ml of VTES was added to the reaction mixture. The reaction was allowed to proceed at 60°C for 6 h under continuous stirring. The resultant products were obtained by magnetic separation with permanent magnet and were thoroughly washed with ethanol and deionized water until neutral, then were dried at room temperature under vacuum for 24 h.
Copolymerization of PNIPAAm-MAA on the surface of VTES-grafted magnetic nanoparticles
The graft polymerization was conducted under various reaction conditions. VTES-grafted magnetic nanoparticles were used as a template to polymerize PNIPAAm-MAA in a 1, 4 dioxan. BIS was used as cross-linking agent. In brief, 0.06 g of VTES-grafted magnetic nanoparticles, 0.3 g of NIPAAm, 0.026 g of MAA and 0.027 g of BIS were sonicated in 200 ml cold water for 50 minutes. Then, 0.16 g of ammoniumpersulfate was added to the solution, and the reaction was carried out at room temperature under N2 gas for 10 hours. The product was purified several times with deionized water by using a magnet to collect only PNIPAAm-MAA-grafted magnetic nanoparticles. PNIPAAm-grafted magnetic nanoparticles were also formulated using the same synthesis process as with PNIPAAm-MAA-grafted magnetic nanoparticles, but without addition of MAA monomers (Figure 4) [27].
Figure 4 (A) Chemical modification of Fe3O4 surface by grafting polymerization without cross-linking (B) In presence of cross-linking.
Drug-loading of the PNIPAAm-MAA-grafted magnetic nanoparticles
For drug-loading doxorubicin was used as a model drug. In brief, 2 mg of freeze-dried PNIPAAm-MAA-grafted magnetic nanoparticles and 2 mg of doxorubicin were dispersed in 30 ml phosphate buffer solution (PBS). The solution was stirred at 4°C for 2 days. The doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles were separated from the solution using an external magnet. The solution was then analyzed using an ultraviolet-visible (UV-vis) spectrofluorometer (Shimadzu) to determine the amount of unencapsulated doxorubicin (λex 470 nm and λem 585 nm). This value was then compared to the total amount of added doxorubicin to determine the doxorubicin-loading efficiency of the nanoparticles [28].
In vitro drug release
To study drug release, four different sets of experiments were performed. They include two different temperatures (40 and 37°C) and two different pHs (5.8 and 7.4). In each drug release experiment, 3.0 mg of the drug carrier bonded with smart polymer was sealed in a 30 ml of Na2HPO4– NaH2PO4 buffer solution with pH of 5.8 or 7.4. The test tube with the closer was placed in a water bath maintained at 40°C up the lower critical solution temperature (>LCST), 37°C (>LCST). The release medium (~3 ml) was withdrawn at predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 24, 36, 48, 70, 90, 110, 120, 170, 180 and 250 h) and after the experiment the samples were analyzed using a UV–vis spectrometer (Shimadzu) to determine the amount of doxorubicin released (λex 470 nm and λem 585 nm for doxorubicin measurement) [29-31]. The amount of doxorubicin entrapped efficiency within nanoparticles was calculated by the difference between the total amount used to prepare nanoparticles and the amount of doxorubicin present in the aqueous phase. Loading efficiency was calculated according to the following formula: [32].
(1) Loadingefficiency%=amountofloaddruginmgamountofaddeddruginmg×100%
Cell culture
In-vitro cytotoxicity and Cell culture study
A549 lung cancer cell line (kindly dedicated from pharmaceutical nanotechnology research center, Tabriz University of Medical Sciences, Tabriz, Iran) were cultured in RPMI1640 (Gibco, In-vitro gen, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, UK), 2 mg/ml sodium bicarbonate, 0.05 mg/ml penicillin G (Serva co, Germany), 0.08 mg/ml streptomycin (Merck co, Germany) and incubated in 37°C with humidified air containing 5% CO2. After culturing sufficient amount of cells, cytotoxic effect of PNIPAAm-MAA-grafted magnetic nanoparticles was studied by 24, 48 and 72 h MTT assays (Carmichael et al., 1987). Briefly, 1000 cell/well were cultivated in a 96 well plate (Figure 5). After 24 h incubation in 37°C with humidified atmosphere containing 5% CO2, cells were treated with serial concentrations of the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles (0 mg/ml to 0.57 mg/ml) for 24, 48 and 72 h in the quadruplicate manner as cells which received 0 mg/ml extract + 200 μl culture medium containing 10% DMSO served as control. After incubation, the medium of all wells of plate were exchanged with fresh medium and cells were leaved for 24 h in incubator. Then, medium of all wells were removed carefully and 50 μl of 2 mg/ml MTT (Sigma co, Germany) dissolved in PBS was added to each well and plate was covered with aluminum foil and incubated for 4.5 h. After removing of wells’ content, 200 μl pure DMSO was added to wells. Then, 25 μl Sorensen’s glycine buffer was added and immediately absorbance of each well was read in 570 nm using ELx800 Microplate Absorbance Reader (Bio-Tek Instruments) with reference wavelength of 630 nm [33].
Figure 5 XRD patterns of (a) pure Fe3O4 nanoparticles.
Cell treatment
After determination of IC50, 1 × 106 cells were treated with serial concentrations ofthe doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles (0.028, 0.057, 0.114, 0.142,0.171 and 0.199 mg/ml). For control cells, the same volume of 10% DMSO without the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles was added to flask of control cells. Then, culture flasks were incubated in 37°C containing 5% CO2 with humidified atmosphere incubator for 24 h exposure duration.
Characterization
The IR spectra were recorded by a Fourier transform infrared spectrophotometer (FT-IR, Nicolet NEXUS 670, USA), and the sample and KBr were pressed to form a tablet. The magnetization curves of samples were measured with a vibrating sample magnetometry (VSM, Meghnatis Daghigh Kavir Co Iran) at room temperature. Powder X-ray diffraction (XRD, Rigaku D/MAX-2400 X-ray diffractometer with Ni-filtered Cu Kα radiation) was used to investigate the crystal structure of the magnetic nanoparticles. The infrared spectra of copolymers were recorded on a Perkin Elmer 983 IR spectrometer (Perkin Elmer, USA) at room temperature. The size and shape of the nanoparticles were determined by scaning electron microscope (SEM, VEGA/TESCAN), the sample was dispersed in ethanol and a small drop was spread onto a 400 mesh copper grid.
Results
Synthesis of poly (NIPAAm-MAA) grafted Fe3O4 nanoparticles
The processes for synthesis of poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles and the loading of doxorubicin onto them are shown in Figure 4. The Fe3O4 nanoparticles were prepared by a chemical co-precipitation of Fe2+ and Fe3+ ions under alkaline condition. The concentration ratio of Fe2+ /Fe3+ was selected to be 1:1.8 rather than the stoichiometric ratio of 1:2, because Fe2+ is prone to be oxidized and become Fe3+ in solution. The Fe3O4 nanoparticles prepared by the co-precipitation method have a number of hydroxyl groups on the surface from contacting with the aqueous phase. VTES-modified Fe3O4 nanoparticles were achieved by the reaction between VTES and the hydroxyl groups on the surface of magnetite. Two reactions were involved in the process. First, the VTES was hydrolyzed to be highly reactive silanols species in the solution phase under alkaline condition. Then, their condensation with surface free -OH groups of magnetite to render stable Fe–O–Si bonds takes place. Oligomerization of the silanols in solution also occurs as a competing reaction with their covalent binding to the surface. Surface-grafted polymerization by NIPAAm and MAA also involves two reactions, which take place simultaneously. On the surface of VTES-modified Fe3O4 nanoparticles, the graft polymerization occurs, while the random polymerization takes place in the solution. In order to decrease the random polymerization, the following strategies were adopted. On the one hand, after AIBN was dissolved in the modified nanoparticles suspended solution, the solution was placed overnight to make the nanoparticles absorb AIBN onto the surface furthest. On the other side, an optimal concentration of initiator was selected. In the other work BIS was used as cross-linking agent and the monomers were added dropwise in the reaction. The unreacted oligomers would be separated by magnetic decantation after reaction.
Characterization of Fe3O4 and poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles
XRD patterns
Figure 6 shows the XRD patterns of pure Fe3O4. It is apparent that the diffraction pattern of our Fe3O4 nanoparticles is close to the standard pattern for crystalline magnetite. The characteristic diffraction peaks marked, respectively, by their indices (2 2 0), (311), (4 0 0), (4 2 2), (511), and (4 4 0) could be well indexed to the inverse cubic spinel structure of Fe3O4 (JCPDS card no. 85–1436), were also observed from poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles. This reveals that modified and grafted polymerized, on the surface of Fe3O4 nanoparticles, did not lead to their crystal phase change. The average crystallite size D was about 15 nm, obtained from Sherrer equation D = Kλ/ (βcos θ), where K is constant, λ is X-ray wavelength, and β is the peak width of half-maximum.
Figure 6 The SEM micrographs of (a) pure Fe3O4 nanoparticles (b) Fe3O4 nanoparticles grafted by poly-(NIPAAm-MMA) (c) Hydrodynamic sizes of PNIPAAm-MAA-grafted MNPs.
Size, morphology, and core-shell structure of nanoparticles
The SEM micrographs of pure Fe3O4 nanoparticles (Figure 6 (a)) and Fe3O4 nanoparticles grafted by poly (NIPAAm-MAA) (Figure 6 (b)) are shown. Observing the photograph (a), nanoparticles were aggregated seriously, which was due to the nanosize of the Fe3O4, and they were about 20–75 nm, according to the result of XRD. After graft polymerization, the size of particles was changed to be 60–100 nm, and the dispersion of particles was improved greatly (Figure 6 (b)), which can be explained by the electrostatic repulsion force and steric hindrance between the polymer chains on the surface of Fe3O4 nanoparticles.
FT-IR spectroscopy of nanoparticles
To evaluate the effect of graft polymerization, the homopolymers and unreacted monomers were extracted in ethanol to be separated from the grafted nanoparticles. FT-IR spectroscopy was used to show the structure of Fe3O4 (Figure 7 (a)), VTES-modified Fe3O4 (Figure 7 (b)) and poly (NIPAAm-MAA)-grafted Fe3O4 (Figure 7(c)). From the IR spectra presented in Figure 8, the absorption peaks at 568 cm-1 belonged to the stretching vibration mode of Fe–O bonds in Fe3O4. Comparing with the IR spectrum (a), the IR spectrum (b) of VTES-modified Fe3O4 possessed absorption peaks presented at 1603 and 1278 cm-1 should be attached to the stretching vibrations of C = C and the bending vibration of Si–C bonds, peak at 1411 cm-1 due to the bending vibration of = CH2 group, additional peaks centered at 1116, 1041, 962 and 759 cm-1 were most probably due to the symmetric and asymmetric stretching vibration of framework and terminal Si–O– groups. All of these revealed the existence of VTES. It indicated that the reactive groups had been introduced onto the surface of magnetite. The absorption peaks of C = C and = CH2 groups disappeared, and additional peaks at 1724, 1486, 1447 and 1387 cm-1 due to the stretching vibrations of C = O, the bending vibration of –CH2–, –CH– and –CH3 absorption peaks at 1147, 906 and 847 cm-1 belonged to the stretching vibration of the alkyl groups fromNIPAAm. However, the identification of peak attributable to the stretching vibrations of C–N (normally at about 1100 cm-1) was problematic due to overlapping other peaks, but the element analysis method demonstrated the presence of N element of the NIPAAm in poly (NIPAAm-MAA)-grafted Fe3O4 nanoparticles. Overall, these FT-IR spectra provided supportive evidence that the –CH = CH2 group initiated polymerization of NIPAAm and MAA polymer chains were successfully grafted onto the Fe3O4 nanoparticles surface.
Figure 7 FT-IR spectra of (a) pure Fe3O4 nanoparticles, (b) Fe3O4 nanoparticles modified by VTES, (c) poly(NIPAAm-MMA)-grafted Fe3O4 nanoparticles.
Figure 8 The magnetic behavior of magnetic nanoparticles. (1.Fe3O4, 2. VTES-Fe3O4, 3. VTES-Fe3O4-PNIPAAm-MAA).
Magnetism test
The magnetic properties of the magnetic nanoparticles were analyzed by VSM at room temperature. Figure 8 shows the hysteresis loops of the samples. The saturation magnetization was found to be 34.5 and 17.6 emu/g for VTES-modified Fe3O4 and poly(NIPAAm-MAA)-grafted Fe3O4, respectively, less than the pure Fe3O4 nanoparticles (70.9 emu/g). With the large saturation magnetization, the poly (NIPAAm-MAA)-grafted Fe3O4 could be separated from the reaction medium rapidly and easily in a magnetic field. In addition, there was no hysteresis in the magnetization with both remanence and coercivity being zero, suggesting that these magnetic nanoparticles were superparamagnetic. When the external magnetic field was removed, the magnetic nanoparticles could be well dispersed by gentle shaking. These magnetic properties were critical in the applications of the biomedical and bioengineering fields.
In vitro release experiment
The release behavior of the nanoparticles was studied for ~200 hours in PBS (0.1 M, pH 7.4, 5.8) at 37°C, and 40°C. The percentage of cumulative release of doxorubicin at 40°C was significantly higher than at 37°C (Figure 9). The pH-responsive release profiles from the hybrid nanoparticles are shown in Figure 10 (pH 5.8, and 7.4). The release rate decreased with the increase of pH values. The pKa value of the amino group in doxorubicin is about 8.2. Thus the electrostatic interaction existed at neutral surrounding and disappeared at acid surrounding. The pH value of the tumor was 5.0–6.0, which was lower than the pH value of the normal tissue, so the doxorubicin on hybrid nanoparticles could be released at the tumor.
Figure 9 Release profiles of doxorubicin from the hybrid nanoparticles at different pH /temperature values.
Figure 10 IC50 of (a) the doxorubicin-loaded linear PNIPAAm-MAA-grafted magnetic nanoparticles (b) the doxorubicin-loaded cross linker PNIPAAm-MAA-grafted magnetic nanoparticles (c) Pure doxorubicin on A549 tumor cell line after 24, 48 and 72 h treatment.
In-vitro cytotoxicity study of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line
MTT assay is an important method to evaluate the in-vitro cytotoxicity of biomaterials. In MTT assay, the absorbance is in a significant linear relationship with cell numbers. The corresponding optical images of cells are shown in Figure 10. In the current work, MTT assay showed that doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles has time-dependent but not dose-dependent cytotoxicity on the A549 lung cancer cell line(IC50 = 0.16 to 0.20 mg/ml). Also, MTT assay showed that pure doxorubicin has dose-dependent but not time-dependent cytotoxicity on the A549 lung cancer cell line(IC50 = 0.15 to 0.16 mg/ml). Therefore, there is need for further study of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line in the future. However, results of current work demonstrated that IC50 of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles and pure doxorubicin are about 0.16, 0.20 mg/ml and 0.15 mg/ml respectively, in A549 lung cancer cell line.
Discussion
In this work we have characterized in vitro behavior of Poly NIPAAm-MAA-grafted magnetic nanoparticles for targeted and controlled drug delivery applications. The XRD data only showed peaks attributable to magnetite (Fe3O4) and discovered that grafted polymerized, on the surface of Fe3O4 nanoparticles, did not lead to their crystal phase transform. FT-IR spectroscopy was used to show the structure of Fe3O4, VTES-modified Fe3O4 and poly (NIPAAm-MAA)-grafted Fe3O4. The saturation magnetization was found to be 34 and 17 emu/g for VTES-modified Fe3O4 and poly(NIPAAm-MAA)-grafted Fe3O4, respectively, less than the pure Fe3O4 nanoparticles (70.9 emu/g) by VSM. This difference suggests that a large amount of silane and polymers grafted on the surface of Fe3O4 nanoparticles. The size and morphology of the synthesized nanoparticles were analyzed by SEM. This method was carried out to study the core shell structure, morphology, and size of the nanoparticles. A close examination of the SEM image (Figure 6) reveals the presence of magnetic nanoparticles (~10 nm diameter) at the center with a PNIPAAm-MAA coating surrounding them. The size of the magnetic core was similar to earlier reported values of magnetic nanoparticles synthesized by similar methods [34]. In comparison with PNIPAAm-grafted magnetic nanoparticles [35], there was clearly less agglomeration of magnetic nanoparticles in the core. This might be a result of the higher mixing capability due to utilization of a mechanical stirrer and the electrostatic charge repulsion from the carboxylic group of MAA in the PNIPAAm-MAA coating, which would further reduce the magnetic dipole interactions and promote stability [36]. We believe that grafting magnetic nanoparticles with a biocompatible copolymer is necessary when high concentrations of magnetic nanoparticles are used. The drug release study indicates that the Poly NIPAAm-MMA is a temperature-sensitive polymer, whereby at its lower critical solution temperature (LCST) the nanoparticles go through the phase change to fall down and release more drugs. After 250 hours, 55% of the bonded doxorubicin was released at 40°C, whereas at 37°C ~40% was released. The release profile of the doxorubicin over the first 40 minutes is also shown in Figure 9. After 40 minutes the percentages of growing release of doxorubicin were only 0.05% at 37°C, whereas at 40°C it was 2.5%. The system is shown to release its payload over a short burst release period with changes in temperature. Since the measurement time was very short while the drug release fixed time interval was significantly large, the influence of the returned medium on drug release during the measurement time is expected to be insignificant [37]. The doxorubicin release profiles from our nanoparticles established that our nanoparticles xwere responsive to temperature with a significantly higher release at 40°C than at 37°C. The in-vitro cytotoxicity test showed that the doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles had no cytotoxicity and were biocompatible, which means there is potential for biomedical application [38]. Also IC50 of doxorubicin-loaded PNIPAAm-MAA-grafted magnetic nanoparticles on A549 lung cancer cell line showed that they are time-dependent.
Conclusions
SPIONs were synthesised via co-precipitation method and then Fe3O4 nanoparticles were grafted by Vinyltriethoxysilicane, and created reactive groups onto the nanoparticles’ surface therefore, NIPAAm and MAA were bonded onto the surface of modified-Fe3O4 nanoparticles by surface initiated radical polymerization with presence and without presence cross linker. The results indicate that the copolymer chains had been effectively encapsulated Fe3O4 nanoparticles and effectively grafted onto the surface of Fe3O4 nanoparticles. The functionalized particles remained dispersive and superparamagnetic. These particles were employed in encapsulation of doxorubicin under mild conditions and could significantly used in the drug delivery. The resultant particles were characterized by vibrating sample magnetometry (VSM), Fourier transform infrared spectroscopy (FT-IR), Scanning electron microscopy (SEM), and X-ray powder diffraction (XRD). The in vitro cytotoxicity study demonstrated that the grafted-Fe3O4 nanoparticles had no cytotoxicity and were biocompatible. This study suggests that supercritical fluid technology is a promising technique to produce drug-copolymer magnetic composite nanoparticles for the design of drug controlled release systems. Current work demonstrated that doxorubicin-loaded with modified-Fe3O4 nanoparticles has potent anti-growth effect on A549 and time-dependently inhibits cell growth in this cell line. As a result, these nanoparticles can be normal potent chemotherapeutic agent for lung cancer patients and constituents of these nanoparticles can be suitable candidate for drug development [39-42].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SD conceived of the study and participated in its design and coordination. AA participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgments
The authors thank Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University for all supports provided. This work is funded by 2012 Yeungnam University Research Grant.
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PLoS GenetPLoS GenetplosplosgenPLoS Genetics1553-73901553-7404Public Library of Science San Francisco, USA 23555281PGENETICS-D-12-0181610.1371/journal.pgen.1003361Research ArticleBiologyBiochemistryNeurochemistryEvolutionary BiologyComparative GenomicsEvolutionary GeneticsEvolutionary ImmunologyGeneticsGene FunctionAstakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans AST2 Links Melatonin to Circadian RhythmWatthanasurorot Apiruck
1
Saelee Netnapa
1
¤
Phongdara Amornrat
2
Roytrakul Sittiruk
3
Jiravanichpaisal Pikul
1
4
Söderhäll Kenneth
1
Söderhäll Irene
1
*
1 Department of Comparative Physiology, Uppsala University, Uppsala, Sweden2 Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Songkhla, Thailand3 Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, Pathumthani, Thailand4 Aquatic Molecular Genetics and Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), NSTDA, Pathumthani, ThailandBeltz Barbara S. EditorWellesley College, United States of America* E-mail: [email protected] authors have declared that no competing interests exist.
Conceived and designed the experiments: AW NS PJ SR KS IS. Performed the experiments: AW NS SR. Analyzed the data: AW NS AP PJ KS IS. Contributed reagents/materials/analysis tools: PJ SR AP. Wrote the paper: AW KS IS.
¤ Current address: Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Songkhla, Thailand
3 2013 21 3 2013 9 3 e100336118 7 2012 5 1 2013 © 2013 Watthanasurorot et al2013Watthanasurorot et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.
Author Summary
Most living organisms are able to sense the time and in particular time of day by their internal clocks. So-called clock proteins control these internal clockworks. BMAL1 and CLK are two important clock proteins, which together form a complex that serves as a transcription factor and controls the production of diurnal proteins. These diurnal proteins, in turn, inhibit the formation of clock proteins so that the concentration of the different proteins in the cell oscillates back and forth throughout the day. External factors may affect the balance of clock proteins, and one such important factor is light. Melatonin is a darkness hormone produced in the brain of most animals during the night, and here we show that melatonin controls the formation of a protein named AST2 in crayfish. AST2 belongs to a group of proteins found in many arthropods, such as spiders, scorpions, crustaceans, and some insects, whose function has been unknown until now. Now we demonstrate that AST2 is induced by melatonin at night and then functions in the internal biological clock by preventing BMAL1 and CLK to form a complex. In this way, AST2 acts as a link between melatonin and the internal biological clock.
This work was supported by the Swedish Research Council VR (http://www.vr.se) (319-2010-650, 621-2009-5715 to KS and 621-2011-4797 to IS) and by the Swedish Science Research Council FORMAS to KS (223-2011-606). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The physiology and behavior of most organisms are regulated according to daily environmental changes in a circadian manner. Circadian rhythms are often monitored by assaying behavioral and/or molecular fluctuations [1]. Clock genes and hormones are core regulators or pacemakers in circadian regulation, which reside in the suprachiasmatic nucleus (SCN) in mammals and the brain visual center in insects [2], [3]. In Drosophila, the proteins encoded by clock genes form two distinct heterodimers: Period (PER) and Timeless (TIM) form one heterodimer, and Clock (CLK) and Cycle (CYC) form the other [4]. In mammals, the homologues of the CLK and PER proteins have the same name, while the homologues of TIM and CYC are called cryptochrome (CRY) and brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) respectively [5]. The PER-TIM heterodimer functions as an autoregulatory negative feedback loop that causes a decrease in transcriptional activation by CLK and CYC [6]. Conversely, the CLK-CYC heterodimer directly interacts with the upstream E-boxes (CACGTG) of the PER and TIM genes to activate their transcription [7]. These clock genes are detected in several neural and non-neural tissues, suggesting that these feedback loops are not restricted to neurons [8]. RACK1 was recently identified as an inhibitor of mammalian CLK-BMAL1 activity by recruiting PKCα during the negative feedback phase of the cycle [9].
As described above, the SCN serves as the “master clock” in the mammalian brain [10]. The SCN is reset on a circadian basis by light input from the retina during the day and by melatonin secretion from the pineal gland at night [11], [12]. Several animal studies have documented that melatonin is mainly synthesized and released during darkness, while the melatonin level is low in the presence of light [13]. This hormone is formed not only in the pineal gland but also in the photoreceptive structures of both vertebrates and invertebrates [14]. The melatonin synthesis pattern is known to reflect both daily rhythms and changes in photoperiod, demonstrating that melatonin has major roles in regulating both circadian and seasonal rhythms [15]. Several circadian rhythms, such as the rest/activity cycle, core body temperature, neuronal electrical activity and locomotor activity, are driven by melatonin [16], [17], [18], [19]. However, melatonin does not directly induce immediate changes in clock gene mRNA expression in the rat SCN, suggesting that the phase-shifting effect of melatonin on the SCN molecular loop implicates a post-translational rather than a transcriptional effect [15]. Little is known about the molecular mechanism by which melatonin drives the clock gene feedback loops in the SCN. In addition, the nocturnal increase in the circadian secretion of the cytokine IL-2 and its immunobiological activity are under melatonin control during the dark period [20]. There is a link between the rest/activity cycle and the immune system via melatonin-cytokine interactions [21].
Neuronal tissues that contain circadian clocks are present in the brain of the crayfish Cherax destructor and Procambarus clarkii, the latter of which is highly related to Pacifastacus leniusculus. These species have photoreceptor cells not only within their eyes, but also in extraretinal parts of the brain. In Cherax this area is located in the anterior median brain soma cluster 6 [22], and the axons from these cells terminate in an area within the protocerebral bridge [23]. It is clear that these brain photoreceptors are sufficient to maintain the circadian locomotor rhythm in these animals [23]. These areas have also recently been shown to be immunoreactive for proteins encoded by the clock genes CLK, PER and TIM [24]. Several studies imply a conserved role of melatonin as an important transducer of circadian information in invertebrates and vertebrates, and melatonin production has been demonstrated in the eyestalks of several crustacean species [25], [26]. The presence of MT2 melatonin receptors have not been conclusively shown in crayfish, but indirect evidences for such a receptor were presented by Mendoza-Vargas et al [27] since the application of the MT2 receptor selective agonist 8-M-PDOT or antagonist DH97 had a significant effect on how melatonin affected the retinal photoreceptors [27].
In mammals, the secretion of prokineticin 2 (PROK2) by the SCN is implicated in the regulation of the sleep/wake cycle and locomotor activity and is known to suppress the function of melatonin [28]. The transcription of PROK2 is activated by the CLK-BMAL1 heterodimer and light [29], [30].
Crayfish astakines are invertebrate homologues of the prokineticins (PROK), and there are two forms in the crayfish P. leniusculus: astakine 1 (AST1, AAX14635) and astakine 2 (AST2, EF568370) [31]. These astakines have been identified in several invertebrates, and similar to PROK2, the expression profiles of crayfish AST1 and AST2 follow a daily rhythm [32]. In contrast to AST1, the rhythmic expression of AST2 is at its maximum level in the dark phase [32]. Although the patterns of melatonin and AST2 release are circadian-regulated in crayfish [32], [33], nothing is known about their role in regulating rhythmic oscillations. In this study, we provide evidence that melatonin regulates the circadian rhythm and that this regulation is mediated by AST2 during the dark phase. Our results imply that AST2 acts as novel negative feedback regulator of CLK-BMAL1 activity.
Results
Melatonin induces the expression of AST2
In a recent report, we showed that AST2 mRNA is expressed in the hematopoietic tissue (HPT) of P. leniusculus at higher levels at night than during the day, the time when melatonin is also at a high level [32], [33]. Thus, we hypothesized that there may be a physiological link between these two molecules. We examined the expression of AST1 and AST2 in HPT cells incubated with melatonin in vitro (Figure 1A–1B) and in HPT, hemocytes and brain injected with melatonin in vivo (Figure 1C–1H). Melatonin incubation or injection clearly affected AST2 expression (Figure 1B, 1D, 1F and 1H), whereas AST1 expression was unaffected by this treatment (Figure 1A, 1C, 1E and 1G). These results were also confirmed at the level of translation using an ELISA assay (Figure 2A–2C), in which melatonin treatment resulted in higher levels of the AST2 protein. When cell lysates from the brain and HPT were analyzed by western blot at 9:00 and 20:00, respectively, the level of AST2 was clearly higher at night than during the day (Figure 2D).
10.1371/journal.pgen.1003361.g001Figure 1 AST2 expression is induced by melatonin treatment in vitro and in vivo.
A) Relative expression of AST1 mRNA estimated by qPCR, after incubation with melatonin in cultured HPT cells in vitro at daytime. Black bars = melatonin (3 µM), white bars = control. B) Relative expression of AST2 mRNA estimated by qPCR, after incubation with melatonin in cultured HPT cells in vitro at daytime. Black bars = melatonin (3 µM), white bars = control. C) Relative expression of AST1 mRNA in hemocytes estimated by qPCR, after injection of melatonin in live crayfish. Black bars = melatonin (4.3 nmol/g), white bars = control. D) Relative expression of AST2 mRNA in hemocytes estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. E) Relative expression of AST1 mRNA in HPT estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. F) Relative expression of AST2 mRNA in HPT estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. G) Relative expression of AST1 mRNA in the brain estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. H) Relative expression of AST2 mRNA in the brain estimated by qPCR, after injection of melatonin in live crayfish at daytime. Black bars = melatonin (4.3 nmol/g), white bars = control. Expression of the 40S ribosomal protein was used as an internal control. Error bars indicate standard deviation (SD) from three replicates and the experiment has been repeated three times with similar results. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test.
10.1371/journal.pgen.1003361.g002Figure 2 Melatonin induces higher AST2 protein levels in vitro and in vivo.
A) Relative levels of AST1 or AST2 protein in cultured HPT cells in vitro as estimated by ELISA, after incubation with melatonin. Black bars = melatonin (3 µM), white bars = control. The level of β-actin was used as an internal control. B) Relative levels of AST1 in the HPT, brain and hemocytes in live crayfish as estimated by ELISA, after injection of melatonin. Black bars = melatonin (4.3 nmol/g), white bars = control. The level of β-actin was used as an internal control. C) Relative levels of AST2 in the HPT, brain and hemocytes in live crayfish as estimated by ELISA, after injection of melatonin. Black bars = melatonin (4.3 nmol/g), white bars = control. The level of β-actin was used as an internal control. The asterisks indicate significant differences (*P<0.05); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. D) Western blot analysis of AST2 in the brain and HPT at 9:00 and 20:00, using an antibody against AST2. The expression level of actin was used as an internal control. Light and dark periods are indicated on the top of the blot.
AST2 binds to PlRACK1
In contrast to AST1, which is a secreted protein, AST2 is mainly an intracellular protein (Figure S1). To identify any protein that interacts with AST2 in HPT cells, recombinant AST2 (GST-AST2) was used as a bait to bind to proteins in HPT and hemocyte lysates by far overlay or pull-down assays (Figure 3A–3B). After excluding the background proteins found in the control, a protein with high similarity to Penaeus monodon RACK1 was identified as an AST2-binding protein by mass spectra analysis. The Mascot search resulted in significant hits where P. monodon RACK1 had the highest significant protein score 781 (Table S1). This protein was the significant hit detected in both the far overlay and the GST pull-down assay.
10.1371/journal.pgen.1003361.g003Figure 3 AST2 interacts with PlRACK1.
A) PlRACK1 was identified as an AST2 binding protein using a far western assay with recombinant GST-AST2 or GST as a control. A HPT protein extract was subjected to SDS-PAGE, electroblotted to a PVDF membrane and overlayed with either GST (left) or GST-AST2 (right) alone and binding was detected using a GST antibody. B) The binding between recombinant AST2 with PlRACK1 was confirmed by a GST pull-down assay using GST-AST2 as the binding protein and proteins in a HPT cell lysate as bait. GST was used as a control in both assays. C) GST pull-down of His-Trx-AST2 by GST-PlRACK1. The bound proteins were analyzed by 12.5% SDS-PAGE. Bands corresponding to GST and GST-PlRACK1 were detected with an anti-GST antibody (lanes 1–4). The eluted material was also examined for the presence of AST2 with an anti-His antibody (lanes 5–8). Lanes 9 and 10 contain purified His-Trx-AST2 and HisTrx, respectively.
By designing degenerate primers from the obtained amino acid sequences, a complete RACK1 cDNA was identified by RACE technique from P. leniusculus (PlRACK1). The open reading frame of PlRACK1 contains 957 bp (JQ686053), encoding 318 amino acids with a calculated molecular mass of 35.7 kDa (predicted MW using ProtParam program), and its deduced amino acid sequence was found to consist of seven conserved WD40 repeat binding sites (Figure S2). PlRACK1 mRNA was highly expressed in different crayfish tissues (Figure S3). To confirm the interaction between PlRACK1 and AST2, recombinant GST-PlRACK1 was produced and purified with a predicted molecular mass of 62 kDa (Figure S5A). The GST-PlRACK1 was used in an in vitro binding assay with a His-Trx-AST2 fusion protein (predicted mass of 33 kDa). The His-Trx and GST proteins were also expressed and purified to serve as control proteins (Figure S5B: the predicted mass of His-Trx and GST proteins are 20 and 26 kDa, respectively). As shown in Figure 3C, western blot analysis revealed that the His-Trx-AST2 protein was pulled down by GST- PlRACK1, but not by GST alone. These data indicate that there is an interaction between PlRACK1 and AST2.
PlBMAL1 forms a complex with PlRACK1
The finding that PlRACK1 is a binding partner of the melatonin-induced protein AST2 led us to examine whether PlRACK1 associates with BMAL1 to thereby act as a regulator of circadian rhythm, as has recently been reported in mice [9]. BMAL1 cDNA was amplified from P. leniusculus (PlBMAL1, JQ670886) with an open reading frame of 1,995 bp that encodes a 664 amino acid protein with a predicted molecular mass of 73.64 kDa. One close sequence to PlBMAL1 was that of BMAL1 from Drosophila melanogaster (identity = 55%). Domain homology analysis using SMART revealed that the deduced amino acid sequence was highly conserved in its HLH (helix-loop-helix) DNA-binding domain and the protein dimerization PAS and PAC regions (Figure S4A). The expression of PlBMAL1 was highest in hemocytes, followed by testis, nerve, intestine and brain (Figure S4B). An interaction between PlBMAL1 and PlRACK1 was demonstrated using a GST pull-down experiment with recombinant GST-PlRACK1 (predicted mass of 62 kDa) and His-Trx-PlBMAL1 (predicted mass of 94 kDa as shown in Figure S6A–S6B). This in vitro GST pull-down assay showed an apparent binding between these two recombinant proteins (Figure 4A). The far western blotting using PlRACK1 without GST tag (predicted mass of 36 kDa) as a prey and GST- PlBMAL1 (predicted mass of 100 kDa as shown in Figure S6C) as bait, also confirmed this interaction (Figure 4B). The results show that the GST-PlBMAL1 was detected at the place where the PlRACK1 was located, whereas the GST control protein could not be detected. Binding of PlRACK1 to AST2 as well as to PlBMAL1 was detected using far western after SDS-PAGE at reducing condition. The crystal structure of human RACK1, with high homology to PlRACK1, is known as a sevenfold ß-propeller structure [34]. A comparison of the structure of human RACK1 with yeast RACK1 (that lacks most of the cysteines present in animal RACK1's) show a high similarity in the overall structure, indicating that the cysteines are of minor importance for the main structure characteristics of this group of proteins, which may explain retained binding properties in the far western assay.
10.1371/journal.pgen.1003361.g004Figure 4
PlRACK1 can bind to PlBMAL1.
A) In vitro GST pull-down assay of His-Trx-PlBMAL1 by GST-PlRACK1. The elution fractions of the GST-PlRACK1 pull-down assay were examined by western blot analysis using anti-GST and anti-His antibodies. Lanes 1 and 5: elution fraction of GST-PlRACK1 pull-down of HisTrx-PlBMAL1; Lanes 2 and 6: GST-PlRACK1 pull-down of His-Trx; Lanes 3 and 7: GST pull-down of HisTrx-PlBMAL1; Lanes 4 and 8; GST pull-down of His-Trx. B) The protein-protein interaction of PlRACK1 and PlBMAL1 was analyzed by far western blotting. C) Binding of AST2 and PlBMAL1 to GST-PlRACK1. GST-PlRACK1 (0, 5, 50 or 500 ng) was bound to Glutathione Sepharose beads and incubated with 500 ng His-Trx-PlBMAL1 and 500 ng His-Trx-AST2. Bound proteins were eluted and immunoblotted for PlBMAL1 and AST2 using an anti-His antibody and for PlRACK1 with an anti-GST antibody.
Interaction of AST2 with PlRACK1-PlBMAL1 in vitro
To investigate if and how AST2 regulates an oscillatory mechanism, we examined whether AST2 interacts with the PlBMAL1-PlRACK1 complex. This interaction is mediated by AST2 binding to PlRACK1, since we could show that AST2 is not able to bind to BMAL1 alone (Figure S7). The results from GST pull-down experiments showed that recombinant His-Trx-AST2 strongly associated with the recombinant GST-PlRACK1/His-Trx-PlBMAL1 complex, and the binding of His-Trx-PlBMAL1 and His-Trx-AST2 was dependent on the concentration of GST-PlRACK1 (Figure 4C). These results suggest that AST2 plays an essential role as a stabilizer of the PlBMAL1-PlRACK1 complex, thereby suppressing the CLK-BMAL1 interaction during the dark period.
AST2 associates to form an AST2-PlRACK1-PlBMAL1 complex during the dark period in vivo
The amount of AST2 protein varies in a circadian manner and is highest during early night in the brain and HPT, while PlBMAL1 and PlRACK1 protein levels do not change significantly according to daily rhythms (Figure 5A–5B). Because our in vitro experiments indicated an interaction between AST2 and PlRACK1 and between PlRACK1 and PlBMAL1, we confirmed these two interactions with in vivo experiments. To study the in vivo interactions between these three proteins during the light and dark periods, we performed immunoprecipitation experiments of brain and HPT extracts. A high-molecular-weight complex was present at night and disappeared during the day, as shown in Figure 5C and 5D. This complex was abundant in the brain; it was also found at very low levels in a few HPT samples (Figure 5C). Reducing SDS-PAGE revealed that AST2, PlBMAL1 and PlRACK1 were components of this approximately 200 kDa complex (Figure 5C) (the native mass of these three proteins are 14, 74 and 36 kDa, respectively). During the light period, this high molecular weight complex could not be detected; instead, a smaller complex of AST2-PlRACK1 was identified (Figure 5D).
10.1371/journal.pgen.1003361.g005Figure 5
PlRACK1, PlBMAL1, and AST2 form an approximately 200-kDa protein complex during the dark period.
A) Relative amounts of AST2, PlRACK1 and PlBMAL1 proteins in brain extracts as determined by ELISA at 3 h after light turned off or on. B) Relative amounts of AST2, PlRACK1 and PlBMAL1 proteins in HPT extracts as determined by ELISA at 3 h after light turned off or on. The asterisks indicate significant differences (*P<0.05); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. C) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed that a PlRACK1-AST2 complex was present in the brain (B) during the day. D) Immunoprecipitation (IP) of brain extract using antibodies against RACK1, BMAL1 and AST2 revealed the presence of a high-molecular-weight complex (approximately 200 kDa) composed of all three proteins in the brain (B) at night. C–D) The immunoprecipitated complex was analyzed by western blotting (WB) using another antibody. “B” and “HPT” represent brain and hematopoietic tissue, respectively. The antibodies used for immunoprecipitation (IP) and detection (WB) is indicated at the top and bottom of the blots, respectively, and +DTT and −DTT represent reducing and non-reducing conditions, respectively. Molecular masses are indicated at the left.
The CLK protein is detected as a component of the 400-kDa complex
To determine whether CLK (JQ670885) forms a complex with BMAL1, crayfish brain lysates were prepared, immunoprecipitated with antibodies against BMAL1 and probed for CLK. The CLK protein with a molecular mass of 95 kDa clearly coimmunoprecipitated with endogenous PlBMAL1 (Figure 6A), indicating that CLK is one protein component of this large approximately 400 kDa complex. Confirmation of complex formation was accomplished by switching the antibodies used for immunoprecipitation and for western blot detection, respectively (Figure 6A). We then monitored the CLK protein levels in the brain during the day and night by western blotting. As shown in Figure 6B, the CLK protein level oscillated in a circadian rhythm, and its expression peaked in the light period (Figure 6F). Furthermore, the approximately 400 kDa CLK-PlBMAL1 complex was barely detectable at night and reached maximal levels in the morning (Figure 6B and 6C). More interestingly, the lowest level of the CLK-PlBMAL1 complex in brain lysates coincided with the time at which the AST2-PlRACK1-PlBMAL1 complex was at its highest level (Figure 6B and 6D). Thus, these data indicate that AST2 is an important regulator of circadian rhythm by interfering with formation of the CLK-PlBMAL1 complex.
10.1371/journal.pgen.1003361.g006Figure 6 A 400-kDa CLK-PlBMAL1 complex is present in the light in the crayfish brain.
A) Immunoprecipitation (IP) of endogenous CLOCK (CLK) and PlBMAL1 in the brain (B) and HPT. Total proteins were extracted from the brain and HPT and were immunoprecipitated (IP) with the antibodies (Ab) indicated on the top of the blots, followed by western blot (WB) detection with antibodies against CLK or BMAL1 as shown at the bottoms of the blots. Reducing and non-reducing conditions of the samples are indicated by +DTT or −DTT, respectively. B) The levels of the CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The AST2-PlRACK1 heterodimer and CLK were detected by western blotting using antibodies against AST2 and CLK, respectively. Time points were taken at 03:00, 06:00, 12:00, 18:00, and 21:00 (n = 4). An actin protein was used as an internal control. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). C) Relative amounts of CLK-PlBMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. D) Relative amounts of AST2-PlRACK1-PlBMAL1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. E) Relative amounts of AST2-PlRACK1 protein complex in brain extracts at different time points (n = 3) as determined by western blotting. F) Relative amounts of CLK protein in brain extracts at different time points (n = 3) as determined by western blotting. Average protein level in Graphs C–F was quantitated using Quantity One. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test. Results are representative of three independent experiments. Error bars indicate SD from three replicates and the experiment has been repeated three times with similar results. G) Melatonin injection inhibited the formation of the CLK-PlBMAL1 complex; this inhibition is mediated by the AST2-PlRACK1-PlBMAL1 complex. The levels of the CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 protein complexes were analyzed by SDS-PAGE under non-reducing conditions (sample without DTT) followed by western blotting using an antibody against BMAL1. The level of β-actin was used as an internal control. H) Relative levels of CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 complexes in vivo, in the brain of crayfish after injection of melatonin and then brain extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). I) Relative levels of CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 complexes in vivo, in the HPT of crayfish after injection of melatonin and then HPT extracts were analyzed by western blotting using an antibody against BMAL1. Grey bars = melatonin (4.3 nmol/g), white bars = control injection (PBS). The level of β-actin was used as an internal control. Asterisks indicate significant differences (*P<0.05, **P<0.01). Quantity One analysis was used to quantify the intensity of protein bands. Graphs (H and I) represent the quantification of each complex formation, using Quantity one. Results are representative of three independent experiments. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).
The effect of melatonin injection during the light period
Because we detected a clear stimulatory effect of melatonin on the expression of AST2 (Figure 2C), we decided to further confirm this putative role of AST2 by determining the presence, in vivo, of the approximately 400 kDa CLK-PlBMAL1 and approximately 200 kDa AST2-PlRACK1-PlBMAL1 complexes, respectively, after melatonin injection during the light period. Six hours after melatonin injection, the approximately 200 kDa complex was detected in the crayfish brains, but not in the HPT (Figure 6G–6I). Conversely, the amount of the approximately 400 kDa complex containing CLK-PlBMAL1 was reduced post-melatonin injection (Figure 6G and 6H). These results indicate that either secretion of natural melatonin at night or melatonin treatment causes an increase in the AST2 protein level and thereby increases the amount of the approximately 200 kDa complex.
AST2 is required for delayed CLK-BMAL1 formation
To further test whether AST2 is crucial for the interference of CLK-BMAL1 heterodimer formation, AST2 mRNA expression was partially knocked down by RNAi, to ca 50% of the control. As shown in Figure 7A and 7B, AST2 expression could be partly decreased in the brain by injection of dsRNA. A reduction of the approximately 200 kDa complex occurred during the night as a result of this AST2 deficiency when compared to injection of the dsGFP control (Figure 7C and 7E). In contrast to the control group, the formation of the ≈400 kDa complex in AST2-silenced animals occurred at 21:00, as shown in Figure 7C and 7D. Thus, this experiment further confirms that AST2 is required to interfere with the formation of the CLK-PlBMAL1 heterodimer, and that high AST2 levels at night cause lower amounts of CLK-PlBMAL1 heterodimer formation during the dark period (Figure 7C–7G).
10.1371/journal.pgen.1003361.g007Figure 7 AST2 is required to regulate the CLK-PlBMAL1 heterodimer formation.
A) The relative expression levels of AST2 in the brain at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) in the brain by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). B) The relative expression levels of AST2 in hemocytes at 03:00, 06:00, 12:00, and 21:00 (n = 9) after partial knock down of AST2 (grey) by dsRNA injection and detection by qPCR; dsGFP injection was used as a control (white). Graph A and B represent AST2 mRNA levels, using qPCR. Results are representative of three independent experiments. Error bars indicate SD of nine replicates. The asterisks indicate significant differences (*P<0.05, **P<0.01); Student's paired t-test. C) The effect of dsAST2 on complex formation in the brain in vivo was examined by western blotting of brain extracts and detection as follows: for the CLK-PlBMAL1 complex an anti-BMAL1 antibody, for the AST2- PlRACK1-PlBMAL1 complex an anti-BMAL1 antibody, and for the AST2-PlRACK1 an anti- AST2 antibody was used. The horizontal band at the top of the histogram indicates the light condition (white = light, black = dark). D) Relative amounts of CLK-PlBMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. E) Relative amounts of AST2-PlRACK1-PlBMAL1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against BMAL1. Grey bars = dsAST2, white bars = dsGFP. F) Relative amounts of AST2-PlRACK1 protein complex in the brain in vivo was determined at different time points (n = 9) by western blotting of brain extracts, using an antibody against RACK1. Grey bars = dsAST2, white bars = dsGFP. G) Relative amounts of AST2 protein in the brain in vivo was determined at different time points (n = 3) as determined by western blotting of brain extracts, using an antibody against AST2. Grey bars = dsAST2, white bars = dsGFP. Quantity One analysis was used to quantify the intensity of protein bands from three independent experiments and results are presented in graphs D to G. Statistical significance: *P<0.05, **P<0.01 using Student's paired t-test (error bars indicate SD from nine replicates).
AST2 gene knockdown induces CLK/BMAL1 E-box binding in the dark
Since knockdown of AST2 resulted in more CLK-PlBMAL1 complex formation during the dark period, we decided to test whether AST2 also had an effect on CLK-PlBMAL1 E-box binding. We assayed the CLK-PlBMAL1 E-box binding by a DNA-protein binding assay. As expected, we could demonstrate CLK-PlBMAL1 specific E-box binding during the night in AST2 silenced animals, whereas the control group (dsGFP) did not show any E-box binding in the dark (Figure 8A–8B). A super shift of this E-box binding was induced after incubation with anti-CLK antibodies (Figure 8A), and controls using mutated or unlabelled E-box oligonucleotides did not show any binding (Figure 8B and Figure S8A–S8B). In contrast, melatonin injection during the day (which leads to enhanced levels of AST2, Figure 2A) reduced the DNA binding activity of CLK-PlBMAL1 compared to other groups (Figure S8B). The complex formation of CLK-PlBMAL1 or AST2-PlRACK1-PlBMAL1 was followed by western blotting after knockdown of AST2 expression by dsRNA or increasing AST2 by addition of melatonin (Figure 8C–8E). These results reveal that AST2 is an important regulator of the E-box binding activity of the CLK-PlBMAL1 complex.
10.1371/journal.pgen.1003361.g008Figure 8 AST2 knockdown enhanced the DNA binding activity of CLK/PlBMAL1.
A) Crayfish received dsGFP or dsAST2 treatments, then crude brain lysates were harvested during day and night, and were used to study E-box binding with fluorophore labeled oligonucleotides in an EMSA assay. As a control the brain lysates were incubated with an antibody against CLK, to show a supershift. The E-box binding activity of CLK-PlBMAL1 is indicated on the right side of the gel. B) The effect of AST2 knock down on CLK-PlBMAL1 E-box binding was analyzed by EMSA. A mutant E-box was used as control. C) Western blot showing the effect of AST2 knock down or melatonin treatment on the protein levels of CLK/PlBMAL1, AST2-PlRACK1-PlBMAL1 complexes (upper panel), and AST2 level (middle panel). Actin was used as an internal control (lower panel). The horizontal band on top of this histogram indicates day (white) or night (black). D) Relative amounts of CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 protein complexes in brain extracts isolated during the day (n = 9) as determined by western blotting, using an antibody against BMAL1. The AST2 protein was also detected by western blotting, using an antibody against AST2. White bars = dsAST2, black bars = melatonin, grey bars = dsGFP. E) Relative amounts of CLK-PlBMAL1 and AST2-PlRACK1-PlBMAL1 protein complexes in brain extracts isolated during the night (n = 9) as determined by western blotting, using an antibody against BMAL1. The AST2 protein was also detected by western blotting, using an antibody against AST2. White bars = dsAST2, and grey bars = dsGFP. In three independent experiments, proteins were visualized by western blotting and quantitated using the Quantity One software. These results are shown in graph D and E. Error bars indicate SD from nine replicates. The asterisks indicate significant differences (*P<0.05, **P<0.01); one-way ANOVA with Duncan's new multiple-range test and the Tukey test.
Discussion
Various proteins are regulated by the circadian rhythm, and there are a growing number of examples of circadian regulation of stem cell activities, such as the proliferation and recruitment of hematopoietic progenitor cells [33], [35]. We have recently shown that the hematopoietic cytokine AST1 is regulated by light and that this regulation has an impact on hemocyte synthesis [26]. AST1 contains a prokineticin domain (pfam06607) and shares similarities with vertebrate PROK2, which is regulated by light at the transcriptional level by core clock genes [22]. Apart from functioning in circadian clocks, vertebrate PROKs have documented roles in the regulation of intestinal muscle contractility, neurogenesis, pain perception, food uptake, and appetite regulation [36], [37]. In similarity with invertebrate astakines the vertebrate PROKs also play roles in hematopoiesis and proinflammatory immune responses. Therefore, the structural and functional similarities between astakines and the vertebrate PROKs point to an ancient similar role for these proteins [38].
The oscillation in PROK2 transcription in mice occurs in the SCN, and it acts as an output molecule, signaling the rhythm to other cells and tissues and thereby changing circadian behavior [39]. Signal transduction from vertebrate PROK's are mediated by two closely related G-protein coupled receptors, and the AVIT motif has been shown to be crucial to this receptor binding and the activity of the PROK's [40]. An analysis of the gene structure for human PROK2 reveals that exon 2–4 share similarities to crayfish AST2, while the first exon encodes the signal peptide and the AVIT motif [41], [42]. Thus, all invertebrate astakines found so far, differ from the vertebrates PROKs in their N-terminal and lack of the AVIT-motif [25] a fact that indicates different receptor binding properties for this invertebrate group of PROK domain containing proteins. Similar to PROK2, AST1 is a secreted molecule with circadian expression, and these data indicate a conserved role for this domain throughout evolution, from arthropods to humans [25], [26]. In this study, we report a more direct role for another crayfish prokineticin, namely AST2, as an intracellular regulator of the heterodimeric transcription factors CLK and PlBMAL1. The expression of AST2 does not exhibit a light-dependent pattern that is similar to that of AST1 or PROK2 in mice; instead, we detected high levels of AST2 expression in the HPT and brain during the dark period when melatonin levels are high. We also showed that melatonin administration clearly increased the expression of AST2 at both the mRNA and protein levels.
It is well established that the neurohormone melatonin has an important function in the SCN in regulating the feedback loop of the core clock genes and also functions as an output signal that has an effect on peripheral cells [43]. Melatonin is mainly produced within the eyestalks of crayfish, and its production is elevated during the dark period [27]. Melatonin is known to influence numerous physiological processes in crayfish [18], [33], [44], although the molecular mechanism by which melatonin exerts its effect has not been fully clarified. However, AST2 is an intracellular protein that is present in several crustaceans and insect species [25]. Here, for the first time, we identify a direct role for AST2 in the regulation of the circadian clock and show that the protein acts as a link between melatonin and the heterodimeric transcriptional activator CLK-PlBMAL1. Intracellular PlRACK1 was identified as an AST2-binding protein, and in the brain, the binding of PlBMAL1 to this complex mediated a decrease in the formation of the CLK-PlBMAL1 heterodimer. This process may be very important in all animals that have AST2 molecules i.e., spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, Blattodea but not Diptera and Coleoptera. If any vertebrate PROK is performing the same function still remains to be demonstrated.
Recently, RACK1 and protein kinase Cα (PKCα) were shown to interact with BMAL1 in a circadian manner, resulting in suppressed CLK-BMAL1 activity [9]. This result is consistent with our observation of the PlRACK1-PlBMAL1 interaction in the crayfish brain. At night, we detected an approximately 200 kDa complex in the crayfish brain and found that the components of this complex consisted of AST2, PlRACK1 and PlBMAL1. Conversely, this complex disappeared during the light period due to lower levels of AST2 during the day. Thus, our results show that AST2 acts as a regulator of the CLK-PlBMAL1 complex by binding to PlRACK1-PlBMAL1 in an oscillatory manner that is induced by changes in the level of melatonin.
Interestingly, enhancement of the approximately 400 kDa complex consisting of PlBMAL1 and CLK was detected when the level of the approximately 200 kDa complex was decreased either during the light period or by knockdown of AST2 expression. A similar high-molecular-weight complex of PlBMAL1 and CLK (approximately 400 kDa) was also recently detected in mice [9]. The formation of the approximately 400 kDa crayfish complex is dependent on the expression of CLK. The expression of mammalian CLK is not rhythmic [45], [46], while zebrafish CLK shows robust rhythmic expression, as was the case for crayfish CLK in our study [47], [48], [49]. As with zebrafish and Drosophila, the CLK of crayfish is directly regulated by light [50], [51]. Unlike those in mammals, the protein products of PER and TIM (or CRY in mammals) in Drosophila act to inhibit the transcriptional activity of the CLK-CYC complex (CLK-BMAL1) by interacting with CLK or a CLK-containing complex during the night but not during most of the day [52]. In our experiments, the AST2-PlRACK1-PlBMAL1 complex (approximately 200 kDa) competes for binding to PlBMAL1 to interfere with the formation of the CLK-PlBMAL1 heterodimer during the dark period. Further it is clear from our DNA mobility shift assay that AST2 can inhibit the transcriptional activation activity of CLK-PlBMAL1. This 200 kDa AST2-containing complex directly interacts with different proteins and is clearly induced by melatonin. Therefore, our findings reveal an ancient evolutionary role for a prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops, as indicated in the model (Figure 9).
10.1371/journal.pgen.1003361.g009Figure 9 Molecular model of the circadian regulation by CLK, PlBMAL1, PlRACK1, and AST2.
The protein level of CLK is enhanced during the light period, and a CLK-PlBMAL1 complex is formed that acts as a transcriptional activator. During the dark period melatonin secretion causes an upregulation of AST2 and this high AST2 level results in the formation of a complex between PlBMAL1 and PlRACK1. Then AST2 binds to PlRACK 1 in the PlBMAL-PlRACK1 complex and forms the AST2-PlRACK1-PlBMAL1 complex and this interferes with the formation and activity of the CLK-PlBMAL1 heterodimer.
Materials and Methods
Crayfish
Healthy intermolt freshwater crayfish (P. leniusculus) were obtained from Lake Hjälmaren, Sweden and were maintained in aerated tap water at 10°C.
Cloning and sequence analysis of full-length P. leniusculus RACK1 (PlRACK1) and PlBMAL1 cDNAs
Total RNA was isolated from crayfish brains using TRIzol LS (Invitrogen) followed by RNase-free DNase I (Ambion) treatment. cDNA synthesis was accomplished with an oligo (dT) primer and ThermoScript RT-PCR (Invitrogen) according to the manufacturer's protocol. A partial sequence of PlRACK1 was amplified using primers that were designed based on the shrimp (P. monodon) PmRACK1 sequence (GenBank accession no. EF569136), whereas PlBMAL1 primers were based on the cDNA sequences from several organisms (listed in Table S2). To obtain the full-length cDNAs of the PlRACK1 and PlBMAL1 genes, 5′ and 3′ rapid amplification of cDNA ends (RACE) technology was performed using the SMARTer RACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. The complete PlRACK1 cDNA sequence was amplified using gene-specific primers for PlRACK1 (Table S2) and the SMART Universal Primer A. The PCR products were then cloned into the TOPO TA cloning vector and sequenced. The deduced amino acid sequence of the PlRACK1 gene was searched against the GenBank database with the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Sequence alignment of PlRACK1 was generated by ClustalW, and the protein domains were analyzed with the SMART program (http://smart.embl-heidelberg.de/).
Tissue distribution analysis
Total RNAs were isolated from different crayfish tissues using TRIzol reagent (Invitrogen) followed by RNase-free DNase I (Ambion) treatment. For reverse transcription, 1 µg of total RNA was reverse transcribed to produce cDNA using ThermoScrip RT-PCR (Invitrogen). To amplify and visualize the cDNAs of PlRACK1 and PlBMAL1, two sets of primers were used: the forward and reverse primers listed in Table S2. The PCR cycles were as follows: 1 cycle (95°C for 2 min); 28 cycles (95°C, 30 s; 55°C, 30 s; 72°C, 40 s) followed by an extension step (72°C for 5 min). The transcription of the 40S ribosomal protein was used as an internal control. All PCR products were analyzed on 1% GelRed-stained agarose gels.
Crayfish HPT cell culture and maintenance
The hematopoietic tissue was dissected, washed with CPBS (crayfish phosphate buffered saline (pH 6.8), 10 mM Na2HPO4, 10 mM KH2PO4, 150 mM NaCl, 10 µM CaCl2, and 10 µM MnCl2,) and incubated in 600 µl of 0.1% collagenase (types I and IV) (Sigma) in CPBS at room temperature for 45 min to separate the HPT cells. The separated cells were washed twice with CPBS by centrifugation at 800×g for 5 min at room temperature. The cell pellet was re-suspended in modified L-15 medium, and cells were then seeded at a density of 2.5×106 cells/150 µl in 96-well plates. The HPT cells were supplemented with partially purified plasma as a source of AST1 after 1 h of attachment at room temperature. The culture plates were incubated at 16°C, and 1/3 of the medium was changed at 48 h intervals.
Melatonin treatment
For the in vivo experiments, 100 µl of a melatonin solution (Sigma) was dissolved in ethanol and diluted in crayfish saline (to a final ethanol concentration of 1%). This solution was injected into the base of a walking leg such that the amount of injected melatonin was 4.3 nmol/g fresh weight. Control crayfish received 100 µl of saline with 1% ethanol. The hemolymph, brain, and HPT of these crayfish were collected at 0, 1, 2, 3, and 6 h post-injection for the extraction of total RNA, whereas samples for total protein extraction were dissected at 3 h post-injection. All injections were performed during the day.
For the in vitro experiments, HPT cell cultures were prepared and incubated at 16°C for 12 h. The medium was then replaced with 150 µl of L-15 medium containing 5 µl of the melatonin solution at final concentration of 0.43 nmol/well (3 µM) (the control group was supplemented with 5 µl of saline with 1% ethanol) and 5 µl of crude AST1, followed by incubation for 0, 0.25, 1, 3, 6, and 12 h at 20°C. Thereafter, the cells were harvested at each time point for extraction of total RNA and protein. The transcription and translation levels of AST1 and AST2 in vitro and in vivo were detected by quantitative RT-PCR and ELISA, respectively.
Sampling times
The light in the aquarium room was turned on and off at 4:00 and 20:00, respectively. Brains were dissected at 06:00, 12:00, 16:00, 21:00, and 03:00 from three crayfish at each sampling time, which were kept in normal light/dark conditions. The experiments were repeated at least twice. Complex formation and CLK expression were determined by western blotting using antibodies against BMAL1 and CLK, respectively. An anti-actin antibody (Santa Cruz Biotechnology) was used as an internal control.
Generation of dsRNA
A T7 promoter sequence was incorporated into gene-specific primers for AST2 and GFP (italic letters) at their 5′ ends (AST2 RNAi-F, 5′- TAA TAC GAC TCA CTA TAG GGT CCA CGC CTC TGA GTC TTT T-3′; AST2 RNAi-R, 5′- TAA TAC GAC TCA CTA TAG GGA TGC CCA GAG TGT TGT CCT C-3′ and GFP 63+, 5′- TAA TAC GAC TCA CTA TAG GGC GAC GTA AAC GGC CAC AAG T-3′; GFP 719-, 5′- TAA TAC GAC TCA CTA TAG GGT TCT TGT ACA GCT CGT CCA TG-3′). These primers were used to amplify PCR products as templates for dsRNA synthesis. A GFP transcript was amplified with the pd2EGFP-1 vector (Clontech) as a template and was used as a control. The amplified products were then purified using a GenElute Gel extraction kit (Sigma) followed by in vitro transcription using a MegaScript kit (Ambion), and the dsRNA was purified with TRIzol LS (Invitrogen).
dsRNA in vivo
Small intermolt crayfish (15±2 g of fresh weight) were divided into two groups with three crayfish in each group (n = 3). The first and second groups were injected with 300 µg of GFP control dsRNA and 300 µg of dsAST2, respectively, at the base of the fourth walking leg. Twenty-four hours after the first dsRNA injection, four drops of crayfish hemolymph were bled for total RNA isolation to test the efficiency of the RNAi. Then, dsRNA was injected a second time into both groups as described above. Twenty-four hours after the second dsRNA injection, the brains were dissected at 06:00, 12:00, 21:00, and 03:00 to determine complex formation by western blotting.
Plasmid constructs
PlRACK1 was amplified using the synthesized forward primer RACK1_F1 and reverse primer RACK1_R1 (Table S2) by PCR. The conditions for the PCR reactions were essentially identical to those described in the manufacturer's protocol for the vector. All constructs were sequence-verified. The PCR product was cloned into the pGEX-4T-1 bacterial expression vector at the BamHI and SalI cleavage sites.
PlBMAL1 was amplified from crayfish testis cDNA by PCR using 1 unit of Paq5000 DNA polymerase (Stratagene) and a pair of specific primers listed in Table S2. EcoRI and XhoI sites were engineered into the primers to facilitate subsequent cloning. PlBMAL1 was further subcloned into the pGEX-4T-1 and pET-32a vectors. The recombinant plasmids containing the PlRACK1 and PlBMAL1 genes were confirmed by DNA sequencing.
Bacterial protein expression and purification
The full-length cDNA encoding PlRACK1was cloned into the pGEX-4T-1 bacterial expression vector to express a glutathione S-transferase (GST)-fused PlRACK1 protein in the Escherichia coli strain BL21(DE3). Protein expression was induced by adding IPTG to a final concentration of 1 mM at 25°C for 6 h. The cells were harvested by centrifugation followed by suspension in lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl and 2% Triton X-100) and lysis by sonication. Inclusion body pellets were solubilized in denaturing buffer (50 mM Tris (pH 8.0), 8 M guanidine (GdnHCl) and 10 mM DTT) followed by centrifugation (13,000 rpm for 15 min). The protein was refolded in refolding buffer (55 mM Tris-HCl, 0.44 M L-arginine, 21 mM NaCl, 0.88 mM KCl and 10 mM DTT). The refolded GST-PlRACK1protein was dialyzed in phosphate-buffered saline (PBS, pH 7.4) and applied to Glutathione Sepharose 4B resin (Amersham Biosciences, Inc.) for 30 min at room temperature. The beads were washed with ice-cold PBS followed by incubation in reducing buffer (50 mM Tris-HCl and 20 mM reduced glutathione, pH 8.0) for 30 min at room temperature. After centrifugation at 1,000×g for 5 min, the supernatant was collected and analyzed by 12.5% SDS-PAGE. The protein concentration was measured, and the proteins were stored at −80°C. The full-length sequence of PlBMAL1 was ligated into EcoRI/XhoI-linearized pGEX-4T-1 and pET-32a vectors to create a plasmid that expresses GST- and His-Trx fusion proteins, respectively. The recombinant GST-PlBMAL1 protein was expressed and purified in a similar manner as the GST-PlRACK1 protein. The His-Trx-PlBMAL1 recombinant protein was purified using a Ni-NTA column. His-Trx-AST2 was expressed and purified as described in earlier publications [25]. Briefly, His-Trx tagged AST2 was expressed in E. coli BL21 (DE3) and induced by the addition of 0.02 mM IPTG. The cells were resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl and 2% Triton X-100) and purified using HisPur Cobalt Resin (Thermo Scientific). The protein products of the His-Trx tag expression vector pET-32a and the expression vector pGEX-4T-1, which are used for the incorporation of N-terminal thioredoxin and GST tags, respectively, were expressed in E. coli BL21(DE3) cells. The thioredoxin and GST proteins were purified with HisPur Cobalt or Glutathione Sepharose resins, respectively, as control proteins. All samples were analyzed by 12% SDS-PAGE.
In vitro GST pull-down assay
To identify putative AST2-binding proteins, total protein was extracted from crayfish brains in buffer containing 50 mM Tris-HCl (pH 7.2), 100 mM NaCl, 10% glycerol, 1 mM PMSF, 10 µM MG132 and Complete Mini Protease Inhibitor tablets according to the manufacturer's instructions (Roche Diagnostics). Cell debris was removed by centrifugation at 10,000×g for 10 min. Recombinant GST–AST2 was expressed in E. coli and purified using glutathione Sepharose 4B resin according to standard protocols. GST–AST2 or GST (3–4 µg) was incubated with 800 µg of crude brain total protein extract at 4°C for 1 h unless otherwise specified. Glutathione beads were recovered by brief centrifugation and washed three times with 1 ml of washing buffer (50 mM Tris-HCl (pH 7.2), 100 mM NaCl, 10% glycerol and 0.1% Tween 20). GST was used as a control protein in this experiment. Pull-down mixtures were separated by SDS–PAGE, and after excluding the background proteins found in the control, the remaining band of about 30 kDa was sequenced by mass spectrometry after trypsin cleavage. Identification of the protein was based on raw MS/MS data, and ions search using the Mascot program (http://www.matrixscience.com).
The interaction between GST-PlRACK1and HisTrx-AST2 was examined by incubating purified GST-PlRACK1or the GST control with Glutathione Sepharose 4B resin (50 µl a 50% bed slurry) at room temperature for 1 h. The beads were washed three times with PBS, purified HisTrx-AST2 or the His-Trx control (5 µg) was added, and incubated for another 2 h at room temperature in PBS buffer. After washing with PBS, the precipitated proteins were eluted with SDS-sample buffer.
To determine the formation of a PlRACK1-PlBMAL1 complex, 5 µg purified His-Trx-PlBMAL1 protein was incubated with 5 µg purified GST-PlRACK1 protein bound to Glutathione Sepharose 4B beads. The beads were then washed five times, and the remaining proteins were eluted with SDS-sample buffer.
To explore whether AST2 can bind to PlRACK1 together with PlBMAL1, purified His-Trx-PlBMAL1 (500 ng) and His-Trx-AST2 (500 ng) were incubated with Glutathione Sepharose 4B-bound GST-PlRACK1 (0, 5, 50, 500 ng) in PBS buffer at 25°C for 2 h. The protein-bead complexes were washed, and the bound proteins were eluted with SDS-sample buffer. All protein samples were resolved by SDS-PAGE and were analyzed by western blot.
Blot overlay assays
In addition to an in vitro GST pull-down assay, a far western overlay assay was used to identify any putative AST2 binding protein. The protein was extracted from crayfish HPT and then subjected to 12% SDS-PAGE, transferred to PDVF membranes, and blocked with 10% skim milk in TBST buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.05% Tween 20) at room temperature. After three washes with TBST, the membranes were incubated with 25 mM GST-AST2 or GST alone in 10 ml TBST buffer at room temperature. The blots were washed twice and incubated for 1 h with 1∶2,000 dilution of anti GST antibody and then washed twice, incubated for 1 h with 1∶1,000 dilution of anti mouse antibody for 1 h, and washed three times before detection with an ECL western blotting reagent kit. The control reaction was incubated with GST instead of GST-AST2.
Purified GST-PlRACK1 protein was cleaved by incubation with thrombin (GE Healthcare, 1 µl thrombin per 100 µg recombinant protein) at 22°C overnight to remove the GST fusion tag. Free PlRACK1was eluted with PBS from the Glutathione Sepharose 4B beads. PlRACK1 was run on 12.5% SDS-PAGE, transferred to PDVF membranes and incubated at 4°C overnight with purified GST-PlBMAL1 or GST alone (7 µg) in 10 ml TBST buffer. The blot was washed three times (10 min each) in TBST containing 0.05% Tween 20. Protein interactions were detected by western blotting using an anti-GST antibody. Blot overlay assays were also used to confirm the binding of AST2. Preparation of total crayfish brain homogenates and gel overlays with recombinant GST-AST2 (far western blots) were performed as described above.
Protein extraction
Whole brains were dissected, and HPT cells were isolated, rinsed three times with PBS and prepared for western analysis, immunoprecipitation and ELISA. Cell extracts for western blotting and immunoprecipitation were prepared by suspending the PBS-washed cells in NP-40 lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, and 1.0% NP-40) with protease inhibitor cocktail tablets (Roche). Following incubation on ice for 30 min, non-extractable material was removed by centrifugation at 13,000 rpm for 10 min at 4°C. Cleared supernatants were used for immunoprecipitation, whereas whole homogenized cells were mixed with 1X SDS loading buffer with and without DTT, followed by western analysis. Cell extracts analyzed directly by ELISA were homogenized in bicarbonate/carbonate coating buffer (pH 9.6).
Western blot analysis
Protein samples were separated by 12.5% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, America) in electroblotting buffer (25 mM Tris, 190 mM glycine, 20% methanol) for 120 min. After rinsing with TBST buffer, the membranes were immersed in blocking buffer (5% skim milk powder in TBST) at 4°C, overnight, followed by incubation with primary antibody for 1 h (anti-GST or anti-His diluted in 5% skim milk in TBST). Subsequently, the membranes were incubated in HRP-conjugated rabbit anti-mouse IgG (GE Healthcare) for 1 h and visualized by the enhanced chemiluminescence detection system (Amersham Biosciences). The X-ray images from western blotting were analyzed according to the Quantity One user guide. Briefly, the western blotting films were scanned and saved as digital images as a quantity-one file (or .1sc) by GS-800 calibrated imaging densitometer. These quantity-one files were used for protein analysis. Protein bands in all scanned images (.1sc file) were quantified and the values of the band intensities were calculated by using the Volume and Match tools (Quantity One software version 4.6.0; Bio-Rad Laboratories). These values were used to perform statistical analysis as described in the “Statistical analysis” part below.
Immunoprecipitation
Immunoprecipitations were performed by first incubating cell lysates from brain or HPT with the indicated primary antibodies overnight at 4°C. Immune complexes were then precipitated by incubating reactions for an additional 2 h at 4°C with either Sepharose-conjugated protein A or agarose-conjugated protein G. The immunoprecipitates were washed four times with 1% Triton X-100 lysis buffer and were resuspended in SDS-PAGE sample buffer for western blotting.
Transcription analysis
The transcript levels of AST1 and AST2 were detected by quantitative RT-PCR using QuantiTect SYBR green PCR kit (QIAGEN). The relative expression was normalized to the expression of the mRNA encoding the crayfish ribosomal protein gene (R40s) for each sample. The primers used are shown in Table S2. The qPCR reactions contained 5 µl of 1∶10 diluted cDNA template, 1xQuantiTect SYBR Green PCR master mix (QIAGEN) and 5 µM forward and reverse primers in a 25 µl reaction volume. The following amplification profile was used: 95°C for 15 min, followed by 45 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s. All qPCR reactions were performed in triplicates. The hemocytes, HPTs and brains from a least three crayfish were used for each time point. The statistical comparisons were performed as shown below (Statistical analysis).
ELISA
Cell extracts, containing protein at 20 µg/ml were coated in wells and incubated for 2 h at room temperature, followed by incubation with blocking solution (1% BSA in PBS). Following three careful washes with PBS, the different proteins were detected with the corresponding primary antibody (1∶1000) followed by HRP-conjugated secondary antibodies (1∶3000). After the addition of 100 µl tetramethylbenzidine (TMB) substrate (Sigma) and incubation for 20 min, the reaction was terminated by 100 µl of stop solution (0.5 M sulfuric acid) and the absorbance of the resulting color was measured at 450 nm. All samples were performed in triplicates and each experiment was repeated three times.
Antibodies
Polyclonal antibodies against recombinant crayfish AST1 and AST2 were raised in rabbit as have been described earlier [31]. A goat polyclonal antibody to human BMAL1 (N-5: SC-8550) and goat polyclonal antibody to mouse CLK (S-19: SC-6927) were obtained from Santa Cruz Biotechnology. A goat polyclonal antibody to RACK1 (GNB2L1: Orb22484) was obtained from Biorbyt. A goat polyclonal antibody against actin (C-11: SC-1615) was used as internal control. The specificity of each antibody was tested by western blots, and all were found to be specific for each protein.
Electrophoretic mobility shift assay (EMSA)
Mobility shift assays were performed according to reference [53]. Briefly, a synthetic fluorophore labeled oligonucleotide probe including an E-box element (
CACGTG
) was used as a substrate in this assay. To generate an E-box containing double-stranded DNA fragment, the two fluorophore labeled E-box oligonucleotides described below were annealed. A 30 µL EMSA reaction mixture contained ∼100 mM KCl, 50 ng of crude crayfish brain lysates, 1 µg poly (dI-dC), and 10 fmol of labeled probe. For control 10 fmol of unlabeled competitor oligonucleotide or mutant E-box oligonucleotide were incubated as control. After incubation for 1 hour on ice, antibodies against CLK protein was added and incubated another 20 minutes on ice. Protein-DNA complexes were resolved by 5% polyacrylamide gel electrophoresis. Specific DNA-protein and antibody-supershifted complexes were observed as retarded bands in the gel. E-box (5′-TTT AGT GAA AAG CCG CCG CTC ACG TGG CGA ACT GCG TGA CTT G-3′ and 5′-TTT CAA GTC ACG CAG TTC GCC ACG TGA GCG GCG GCT TTT CAC T-3′) and E-box mutant (5′-TTT AGT GAA AAG CCG CCG CTC AGC TGG CGA ACT GCG TGA CTT G-3′ and 5′-TTT CAA GTC ACG CAG TTC GCC AGC TGA GCG GCG GCT TTT CAC T-3′) sequences were used in the gel shift analysis (the E-box, and E-box mutant sequence is underlined).
Statistical analysis
All statistical comparisons were examined by one-way analysis of variance, followed by Duncan's new multiple-range test and the Tukey test. When two treatments were compared, a Student's paired t-test was used. Differences were considered statistically significant at P<0.05 and/or P<0.01. The results are expressed as the mean ± standard deviation (SD).
Supporting Information
Figure S1 A) The specific binding of AST1 and AST2 antibodies was detected by western blotting of a hemocyte lysate. B) AST2 protein was detected only inside the cell, whereas the AST1 could be detected in both plasma and cytoplasm of the hemocytes.
(TIF)
Click here for additional data file.
Figure S2 Amino acid sequence alignment of PlRACK1 with other RACK1 sequences from other species: mouse-RACK1 (Mus musculus, BAE40059.1), human-RACK1 (Homo sapiens, BAD96208.1), Zebrafish (Danio rerio, NP571519.1), shrimp-RACK1 (P. monodon, ABU49887.1), crayfish-RACK1 (P. leniusculus) and fruit fly-RACK1 (D. melanogaster, NP477269.1). The sequences of PlRACK1 were subjected to conserved domain analysis at NCBI to predict the presence of the WD40 repeats. Positions of the seven WD repeats (WD1–7) are indicated with arrows.
(TIF)
Click here for additional data file.
Figure S3
PlRACK1 is expressed in all tested tissues in P. leniusculus. The experimental tissues examined included muscle (M), hemocytes (HC), heart (HE), gill (G), hepatopancreas (HP), hematopoietic tissue (HPT), abdominal nerve (N), eyestalk (ES), intestine (IN), testis (TT), and brain (B). A 40S ribosomal gene was used as internal control.
(TIF)
Click here for additional data file.
Figure S4 A) Schematic representation of the alignment in BMAL1 proteins. Full-length amino acid sequences and conserved domains of BMAL1 from three organisms were aligned. Overall % identity is shown. The black boxes represent the conserved domains and % identities of these domains. B) RT-PCR analysis of PlBMAL1 in different crayfish tissues, consisting of hemocytes (HC), hepatopancreas (HP), testis (TT), heart (HE), intestine (IN), gill (G), abdominal nerve (N), muscle (M), eyestalk (ES), brain (B), and hematopoietic tissue (HPT). The 40S ribosomal gene was used as the internal control in all tissues.
(TIF)
Click here for additional data file.
Figure S5 The expression and purification of GST-PlRACK1, His-Trx-AST2, and tag proteins. A) The GST-PlRACK1 was analyzed by 12.5% SDS-PAGE. Lane M is protein molecular weight markers; lane 1 and 2 show the expression of the GST-PlRACK1 before and after induction; lane 3 is the soluble fraction and lane 4 is the insoluble pellet of the cell lysate; lane 5 is the purified- GST-PlRACK1 protein from the column after dissolution of the inclusion bodies and refolding. B) The expressed proteins: GST, thioredoxin and His-Trx-AST2 were analyzed by 12.5% SDS-PAGE. Lane M is protein molecular weight markers; lane 1 and 2 show the expression of proteins before and after induction; lane 3 shows the purified proteins.
(TIF)
Click here for additional data file.
Figure S6 The expression of recombinant PlBMAL1 in E.coli. A) Recombinant His-Trx-PlBMAL1 was analyzed by 10% SDS-PAGE, and B) western blot. Lane M, protein molecular weight markers; lane 1 and 2 show expression of recombinant protein before and after induction; lane 3 and 4 show the protein from supernatant and insoluble pellet of the cell lysate respectively; lane 5 shows the purified His-Trx-PlBMAL1. C) Recombinant GST-PlBMAL1 was analyzed by 10% SDS-PAGE. Lane M, protein molecular weight markers; lane 1–3 show the expression of the GST-PlBMAL1 at 0 h, 4 h and overnight respectively; lane 4–5 show the soluble fraction and insoluble pellet of the cell lysate; lane 6 shows the purified-GST-PlBMAL1 protein.
(TIF)
Click here for additional data file.
Figure S7 AST2 can't bind to BMAL1 directly. In vitro GST pull-down assay of His-Trx-PlBMAL1 by GST-AST2, the eluted fractions were examined by western blotting using anti-GST or anti-His antibodies. The recombinant GST was used as internal control.
(TIF)
Click here for additional data file.
Figure S8 A) Crayfish were treated with dsGFP or dsAST2, and then crude brain lysates were harvested during day and night, and used in EMSA assay. The E-box binding activity of CLK-PlBMAL1 is indicated on the right side of the gel. A competitive binding with unlabeled E-box was used as a control. The horizontal band on top of the gel indicates day (white) or night (black). B) The crayfish were injected with melatonin or crayfish saline as control, during daytime. At 3 h post injection, the DNA binding activities of brain lysates were analyzed by EMSA (lane 1 and 2). A supershift EMSA using preincubation of the lysate with anti-CLK Ab (lane 3 and 4) or competitive binding with unlabeled E-box (lane 5 and 6) were also performed. The CLK-PlBMAL1 specific mobility shift is indicated on the right side of the gel. C) The presence of CLK-PlBMAL1 proteins in the specific shift bands were confirmed by cutting the bands and homogenizing in SDS-PAGE buffer followed by detection by western blot using antibodies against CLK and BMAL1 respectively.
(TIF)
Click here for additional data file.
Table S1 Mascot search result for protein detected as AST2-binding in a GST pull-down and far overlay assay. Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores >61 indicate identity or extensive homology (p<0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. A protein score >70 is considered as significant (p<0.05).
(DOCX)
Click here for additional data file.
Table S2 Sequences of the primers used in this study.
(DOC)
Click here for additional data file.
==== Refs
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==== Front
Part Fibre ToxicolPart Fibre ToxicolParticle and Fibre Toxicology1743-8977BioMed Central 1743-8977-10-42340620410.1186/1743-8977-10-4ResearchIntragastric exposure to titanium dioxide nanoparticles induced nephrotoxicity in mice, assessed by physiological and gene expression modifications Gui Suxin [email protected] Xuezi [email protected] Lei [email protected] Yuguan [email protected] Xiaoyang [email protected] Lei [email protected] Qingqing [email protected] Zhe [email protected] Jie [email protected] Renping [email protected] Ling [email protected] Fashui [email protected] Meng [email protected] Medical College of Soochow University, Suzhou, 215123, China2 Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, 210009, China3 Jiangsu key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, 210009, China2013 13 2 2013 10 4 4 26 11 2012 3 2 2013 Copyright ©2013 Gui et al.; licensee BioMed Central Ltd.2013Gui et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) induced nephrotoxicity in animals. However, the nephrotoxic multiple molecular mechanisms are not clearly understood.
Methods
Mice were exposed to 2.5, 5 and 10 mg/kg TiO2 NPs by intragastric administration for 90 consecutive days, and their growth, element distribution, and oxidative stress in kidney as well as kidney gene expression profile were investigated using whole-genome microarray analysis technique.
Results
Our findings suggest that TiO2 NPs resulted in significant reduction of renal glomerulus number, apoptosis, infiltration of inflammatory cells, tissue necrosis or disorganization of renal tubules, coupled with decreased body weight, increased kidney indices, unbalance of element distribution, production of reactive oxygen species and peroxidation of lipid, protein and DNA in mouse kidney tissue. Furthermore, microarray analysis showed significant alterations in the expression of 1, 246 genes in the 10 mg/kg TiO2 NPs-exposed kidney. Of the genes altered, 1006 genes were associated with immune/inflammatory responses, apoptosis, biological processes, oxidative stress, ion transport, metabolic processes, the cell cycle, signal transduction, cell component, transcription, translation and cell differentiation, respectively. Specifically, the vital up-regulation of Bcl6, Cfi and Cfd caused immune/ inflammatory responses, the significant alterations of Axud1, Cyp4a12a, Cyp4a12b, Cyp4a14, and Cyp2d9 expression resulted in severe oxidative stress, and great suppression of Birc5, Crap2, and Tfrc expression led to renal cell apoptosis.
Conclusions
Axud1, Bcl6, Cf1, Cfd, Cyp4a12a, Cyp4a12b, Cyp2d9, Birc5, Crap2, and Tfrc may be potential biomarkers of kidney toxicity caused by TiO2 NPs exposure.
Titanium dioxide nanoparticlesNephrotoxicityOxidative stressGene-expressed profileMice
==== Body
Background
The dynamically development of the nanotechnolo-gy industry has led to the wide-scale production and application of nanomaterials. Among the various nanomaterials, customarily titanium dioxide nanoparticles (TiO2 NPs), owing to their high surface area to particle mass ratio and high reactivity, have been used as nontoxic, chemical inert and biocompatible pigment products or photocatalysts in cosmetics, pharmaceuticals and paint industries [1-7]. However, their attractive properties are the source of reservations. The potential human toxicity and environmental impact of TiO2 NPs have attracted considerable attention with their increased use in industrial applications.
Recently, published data indicated that the toxicity of TiO2 NPs. Liu et al. had found that TiO2 NPs were absorbed and accumulated in the liver, lungs, brain, lymph nodes, and red blood cells [8]. Park et al. observed that TiO2 NPs induced apoptosis and micronuclei formation in Syrian hamster embryo fibroblasts and increased the production of nitric oxide and hydrogen peroxide in human bronchial epithelial cells [9]. Furthermore, an in vitro study showed that high concentration of TiO2 NPs caused renal proximal cell death [10]. TiO2 NPs were also supposed to impair nephric functions and cause nephric inflammation, which through reactive oxygen species (ROS) accumulation to reveal its toxicity [11]. Our previous study also demonstrated that exposure to TiO2 NPs induced nephric inflammation and nephric cell necrosis [12]. We hypothesize that TiO2 NPs -induced kidney damages in mice may have special biomarkers of toxicity.
Newly, a large body of in vivo animal model studies have shown the toxicologic characteristics which cause striking changes of gene expression of some nanomaterials in kidney. For instance, curcumin treatment can alter the gene expression profile of kidney in mice on endotoxin-induced renal inflammation [13]. In addition, nanocopper can result in widespread renal proximal tubule necrosis and dramatically gene expression alterations in rat kidney [14]. Furthermore, a recent report found that proteins were differentially expressed in mouse kidney by exposure to TiO2 NPs [15]. However, the synergistic molecular mechanisms of multiple genes activated by TiO2 NP-induced renal toxicity in animals and humans remain unclear. In this study, mice were exposed to 2.5, 5 and 10 mg/kg body weight (bw) TiO2 NPs for 90 consecutive days, and their growth, element distribution, and oxidative stress as well as kidney gene expression profile were investigated. Our findings suggested that exposure to TiO2 NPs resulted in histopathological changes, apoptosis, oxidative stress, and impairment of element balance in kidney with increased TiO2 NPs doses. Furthermore, microarray analysis showed marked alterations in the expression of 1006 genes were associated with immune/inflammatory responses, apoptosis, biological processes, oxidative stress, metabolic processes, the cell cycle, transport, signal transduction, cell component, transcription, translation, and cell differentiation in the 10 mg/kg TiO2 NPs-exposed kidney. Therefore, the application of TiO2 NPs should be carried out cautiously.
Results
TiO2 NPs characteristic
XRD measurements show that TiO2 NPs exhibit the anatase structure (Figure 1), and the average grain size calculated from the broadening of the (101) XRD peak of anatase was roughly 5.5 nm using the Scherrer’s equation. TEM demonstrated that the average size of the particles of powder (Figure 2a) and nanoparticles which suspended in HPMC solvent after 12 h and 24 h incubation ranged from 5—6 nm, respectively (Figure 2b and c), which is consistent with the XRD results. The value of the sample surface area was generally smaller than the one estimated from the particle size, and it would seem that the aggregation of the particles may cause such a decline (Table 1). To investigate the dispersion and the stability of the suspensions of TiO2 NPs, we detected the aggregated size and the zeta potential of TiO2 NPs in HPMC. After the 12 h and 24 h incubation, the mean hydrodynamic diameter of TiO2 NPs in HPMC solvent ranged between 208 and 330 nm (mostly being 294 nm), as measured by DLS (Figure 3a and b), which indicates that the majority of TiO2 NPs were clustered and aggregated in solution. In addition, the zeta potential was 7.57 mV and 9.28 mV, respectively, and the particle characteristics for the TiO2 NPs used in this study are summarized in Table 1. The leakage of Ti4+ ions from 12 h, 24 h and 48 h incubation of TiO2 NPs in HPMC solvent after centrifugation was measured by ICP-MS. However, Ti4+ contents were not detected in filtrate, which are lower than the detection limit of 0.074 ng/mL (not listed). Therefore, these results suggested that the Ti4+ ions leakage from TiO2 NPs is limited in HPMC incubation.
Figure 1 The (101) X-ray diffraction peak of anatase TiO2 NPs. The average grain size was about 5 nm by calculation of Scherrer’s equation.
Figure 2 Transmission electron microscope image of anatase TiO2 NPs particles. (a) TiO2 NPs powder; (b) TiO2 NPs suspended in HPMC solvent after incubation for 12 h; (c) TiO2 NPs suspended in HPMC solvent after incubation for 24 h. TEM images showed that the sizes of the TiO2 NPs powder or suspended in HPMC solvent for 12 h, 24 h were distributed from 5 to 6 nm, respectively.
Figure 3 Hydrodynamic diameter distribution of TiO2 NPs in HPMC solvent using DLS characterization. (a) Incubation for 12 h; (b) Incubation for 24 h.
Table 1 Characteristics of TiO2 NPs
Sample Crystllite size (nm) Phase Surface area (m2/g) Composition Zeta potential
TiO2 NPs 5.5 Anatase 174.8 Ti, O 7.57(a), 9.28(b)
(a) Zeta potential after the 12 h incubation in 0.05% w/v HPMC solvent; (b) Zeta potential after the 24 h incubation in 0.05% w/v HPMC solvent.
Body weight, coefficient of kidney and titanium accumulation
Titanium accumulation, bw, and kidney indices of mice are listed in Table 2. As shown, an increased TiO2 NPs dose led to a gradual decrease in bw, whereas kidney indices and titanium content were significantly increased (P < 0.05), indicating growth inhibition and kidney damage in mice. These findings were confirmed by subsequent renal histological and ultrastructural observations and oxidative stress assays.
Table 2 Body weight, coefficient of kidney and titanium accumulation in mice kidney by intragastric administration of TiO2 NPs for 90 consecutive days
Index TiO2 NPs (mg/kg bw)
0 2.5 5 10
Net increase of body weight (g) 22.55 ± 1.13a 17.59 ± 0.88b 14.22 ± 0.71c 12.05 ± 0.61d
Relative weight of kidney (mg/g) 10.07 ± 0.50a 11.58 ± 0.58b 13.31 ± 0.67c 15.69 ± 0.78d
Ti content (ng/g tissue) Not detected 105 ± 5a 193 ± 10b 366 ± 18bc
Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM(N = 10).
Mineral element contents
The contents of mineral elements in kidney provide insight into how mineral elements in the kidneys of mice responded to treatment with TiO2 NPs. The mineral elements in the kidney, such as Ca, Na, K, Mg, Zn, Cu and Fe, were determined and listed in Table 3. It can be seen that with increased doses, TiO2 NPs exposure led to marked increased Ca, K, Mg, Zn, and Cu contents, whereas Na, and Fe contents decreased (P < 0.05, Table 3).
Table 3 Accumulation of metal elements in mouse kidney by intragastric administration of TiO2 NPs for 90 consecutive days
TiO2 NPs (mg/kg bw) Metal element contents (μg/g tissue)
Ca Na K Mg Zn Cu Fe
0 1054 ± 53a 3540 ± 177a 2383 ± 119a 138 ± 7a 9.88 ± 0.49a 1.986 ± 0.10a 33.26 ± 1.66a
2.5 1259 ± 63b 3083 ± 154b 3039 ± 152b 159 ± 8b 18.72 ± 0.94b 5.69 ± 0.28b 17.16 ± 0.86b
5 1486 ± 74c 2772 ± 139c 3866 ± 193c 215 ± 11c 31.89 ± 1.59c 10.27 ± 0.51c 9.81 ± 0.49c
10 1823 ± 91cd 2511 ± 125d 4839 ± 242d 300 ± 15d 47.88 ± 2.39d 18.48 ± 0.92d 2.326 ± 0.12d
Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM(N = 5).
Histopathological evaluation of kidney
Figure 4 presents the histopathological changes of kidneys in mice treated by TiO2 NPs exposure for 90 consecutive days. Unexposed kidney did not suggest any histological changes (Figure 4a), while those exposed to increased TiO2 NPs concentrations exhibited severe pathological changes, including significant reduction of renal glomerulus number, apoptosis or vacuolization, infiltration of inflammatory cells, cell abscission on vessel wall as well as tissue necrosis or disorganization of the renal tubules (Figure 4b, c and d), respectively. In addition, we also observed significant black agglomerates in the 10 mg/kg bw TiO2 NPs exposed kidney (Figure4d). Confocal Raman microscopy further showed a characteristic TiO2 NPs peak in the black agglomerate (148 cm-1), which further confirmed the aggregation of TiO2 NPs in kidney (see the spectrum B in the Raman insets in Figure 4d). The results also suggested that exposure to TiO2 NPs deposited in the kidney and resulted in mouse renal injury.
Figure 4 Histopathological observation of kidney caused by intragastric administration of TiO2 NPs for 90 consecutive days. (a) Control, (b) 2.5 mg/kg TiO2 NPs, (c) 5 mg/kg TiO2 NPs, (d) 10 mg/kg TiO2 NPs. Yellow arrows indicate apoptosis or vacuolization, green arrows indicate cell abscission, fatty degeneration or cell necrosis, green virtual circle indicates infiltration of inflammatory cells, blue arrows indicate tissue necrosis or disorganization of renal tubules. Yellow virtual circle indicates TiO2 NPs aggregation. Arrow A spot is a representative cell that not engulfed the TiO2 NPs, while arrow B spot denotes a representative cell that loaded with TiO2 NPs. The right panels show the corresponding Raman spectra identifying the specific peaks at about 148 cm-1.
Nephric ultrastructure evaluation
Changes to the nephric ultrastructure in mouse kidney are presented in Figure 5. As shown, the untreated mouse renal cells (control) contained round nucleus with homogeneous chromatin (Figure 5a), whereas with increased TiO2 NPs doses, the ultrastructure of renal cell from the TiO2 NPs-treated groups indicated a classical morphology characteristic of apoptosis, including mitochondria swelling, nuclear shrinkage, and chromatin marginalization in the renal cell (Figure 5b, c, and d). In addition, black deposits were also observed in the 10 mg/kg TiO2 NPs -exposed nephric cell via TEM (Figure 5d), Raman signals of TiO2 NPs was also exhibited via confocal Raman microscopy (Figure 5d).
Figure 5 Ultrastructure of kidney in male mice caused by intragastric administration of TiO2 NPs for 90 consecutive days. (a) Control, (b) 2.5 mg/kg TiO2 NPs, (c) 5 mg/kg TiO2 NPs, (d) 10 mg/kg TiO2 NPs. Yellow arrows indicate nucleus shrinkage, chromatin marginalization, green arrows indicate mitochondria swelling, and red arrows show presence of TiO2 NPs. Arrow A spot is a representative cell that not engulfed the TiO2 NPs, while arrow B spot denotes a representative cell that loaded with TiO2 NPs. The right panels show the corresponding Raman spectra identifying the specific peaks at about 148 cm-1.
Oxidative stress
Alterations in ROS levels such as ( O2– O2- and H2O2) in the kidney can be regarded as markers of adaptive response of kidney to oxidative damage. As shown in Table 4, the levels of both O2– O2- and H2O2 in mouse kidney following exposure to TiO2 NPs significantly increased compared with control values (P <0.05, Table 4). To prove the effects of TiO2 NPs on ROS generation, the levels of lipid peroxidation (MDA), protein peroxidation (carbonyl) and DNA peroxidation (8-OHdG) in mouse kidney were evaluated and presented in Table 4. The great increases of MDA, carbonyl and 8-OHdG in the TiO2 NPs -exposed kidney were also observed with increased TiO2 NPs doses (P < 0.05), suggesting that ROS accumulation led to lipid, protein, and DNA peroxidation in the kidney.
Table 4 Oxidative stress in mouse kidney after intragastric administration of TiO2 NPs for 90 consecutive days
Oxidative stress TiO2 NPs (mg/kg BW)
0 2.5 5 10
O2.- (nmol/mg prot. min) 18 ± 0.9a 25 ± 1.23b 38 ± 1.91c 43 ± 2.15d
H2O2 (nmol/mg prot. min) 31 ± 1.55a 46 ± 2.28b 77 ± 3.845c 92 ± 4.6d
MDA (μmol/ mg prot) 1.02 ± 0.05a 1.97 ± 0.10b 3.05 ± 0.15c 4.88 ± 0.24d
Carbonyl (μmol/mg prot) 0.51 ± 0.03a 1.13 ± 0.06b 1.89 ± 0.09c 2.79 ± 0.14d
8-OHdG (mg/g tissue) 0.48 ± 0.02a 2.16 ± 0.11b 3.58 ± 0.18c 5.87 ± 0.29d
Different letters indicate significant differences between groups (p < 0.05). Values represent means ± SEM (N = 5).
Change in the gene expression profile
Treatment with high dose of 10 mg/kg bw of TiO2 NPs resulted in the most severe kidney damages, and these tissues were used to detect gene expression profiles to further explore the mechanisms of kidney damages induced by TiO2 NPs. Whole-genome expression profiling using mRNAs from pulmonary tissues of vehicle control groups and those treated with 10 mg/kg bw of TiO2 NPs exposed groups for 90 consecutive days were analyzed with the Illumina Bead Chip. Compared to the vehicle control group, 1, 246 genes of total genes (45, 000 genes) were found to be differentially expressed in the 10 mg/kg TiO2 NPs group (Additional file 1: Table S1), including 610 genes up-regulated and 636 down-regulated. Using the ontology-driven clustering algorithm included with the PANTHER Gene Expression Analysis Software (http://www.pantherdb.org/) as a tool for biological themes analysis, indicating that the 1, 006 genes among 1, 246 genes were associated with immune/inflammatory responses, apoptosis, biological processes, oxidative stress, metabolic processes, the cell cycle, ion transport, signal transduction, cell component, transcription, translation, and cell differentiation, another 240 genes function are unknown (Figure 6), respectively.
Figure 6 Functional categorization of 1246 genes. Genes were functionally classified based on the ontology-driven clustering approach of PANTHER.
RT-PCR
To verify the accuracy of the microarray analysis, twenty-eight genes that demonstrated significantly different expression patterns were further evaluated by qRT-PCR due to their association with immune/inflammatory responses, apoptosis, oxidative stress, cell cycle, signal transduction and biological process. These 14 genes including Psmb5, Ngfrap1, Cycs, Tnfrsf12, Birc5, Fn1, Cd55, Cfi, Bub1b, Egr1, Nid1, Odc1, Cd34, and Apaf1 were up-regulated, whereas 14 genes including Bcl2l1, Ccl19, Ccl21a, Bmp6, Cd74, Cfd, Cxcl12, C3, Bcl6, Cygb, Klf1, Txnip, and Serpinalb were down-regulated (Table 5). The qRT-PCR analysis of all 28 genes displayed expression patterns comparable with the microarray data (i.e., either up- or down-regulation; Additional file 1: Table S1).
Table 5 RT-PCR validation of selected genes from microarray data
Function Gene ΔΔCt Fold Microarray
Apoptosis Apaf1 0.516327 0.699149555 0.5535156
Ngfrap1 0.15624 0.897360758 0.6713378
Cycs 0.34444 0.787613639 0.7045653
Tnfrsf12a 0.04954 0.966244365 0.655954
Bcl2l1 -1.131909 2.191485298 1.663064
Birc5 3.213009 0.107841995 0.10103
Fn1 0.470814 0.721557364 0.4940369
Ccl19 -2.490308 5.618978967 4.313047
Ccl21a -1.826821 3.547545038 2.850085
Immune/Inflammatory response Bmp6 -1.291201 2.447317025 40.56928
Cd55 0.54583 0.684997202 0.3824578
Cd74 -1.327117 2.509007877 1.832029
Cfd -1.861094 3.632830363 2.601335
Cfi 1.259912 0.417569429 0.3720603
Cxcl12 -1.913449 3.76708608 2.043431
C3 -1.580409 2.990546189 1.886813
Cd34 0.282201 0.822335491 0.4588617
Bcl6 -2.069258 4.196707749 3.073497
Oxidative stress Cygb -1.439087 2.711492162 1.646112
Gpx7 0.585512 0.666412792 0.3389097
Psmb5 0.057543 0.960899198 0.6914788
Cell cycle Klf1 -1.646997 3.131810673 2.100916
Bub1b 3.216187 0.1076047 0.2573011
Txnip -1.615053 3.063228525 1.648611
Signal transduction Egr1 0.348792 0.785241322 0.5866718
Nid1 0.918823 0.528940372 0.3838886
Biological process Odc1 1.576015 0.335407069 0.3957967
Serpinalb -2.035634 4.100028676 3.299695
Discussion
NPs were shown to attain the systemic circulation after ingestion, inhalation or intravenous injection. They can distribute to several organs like kidney, liver, spleen, heart, brain, and ovary [16-20]. The kidney has been known to eliminate harmful substances from the body, thus NPs assimilate in the systemic circulation can be filtered by renal clearance [21,22]. In this study, we found that intragastric administration of 2.5, 5, and 10 mg/kg bw of TiO2 NPs for 90 consecutive days induced bw reduction, increased kidney indices, TiO2 NPs deposition (Table 2), renal inflammation, tissue necrosis or disorganization of renal tubules (Figure 4), and renal apoptosis (Figure 5) in mouse kidney tissues coupled with element unbalance (Table 3), and severe oxidative stress, significant production of O2.– O2.-and H2O2, and peroxidation of lipids, proteins, and DNA (Table 4). The renal damages and oxidative stress following exposure to TiO2 NPs may be involved in impaired immune function and antioxidant capacity in mice and, thus, may be associated with changed gene expression in renal tissue. Large-scale gene expression analysis provides an approach to obtain a global view of the genomic changes and to gain insights into the detailed mechanisms behind the pathogenesis of various diseases [23]. To elucidate the molecular mechanisms of kidney damages and identify specific biomarkers induced by TiO2 NPs exposure, RNA microarray analysis of mouse kidney was performed to establish a global gene expression profile and identify toxicity-response genes in mice induced by exposure to 10 mg/kg bw of TiO2 NPs for 90 consecutive days. Our analysis indicated that the expression levels of 1, 246 genes were significantly changed and 1, 006 of these genes were involved in immune-inflammatory responses, oxidative stress, apoptosis, metabolism, the cell cycle, signal transduction, and ion transport etc. The main results are discussed below.
As we known, the development of kidney immune/nflammatory responses is result from the interaction between multifactor, multigene, multi-cell, multi-stage and inherent kidney cells, such as infiltration of inflammatory cells (Figure 4). The pathogenesis is involved in expression alterations of immune/inflammation-related genes. In this study, 36 genes linked to immune/inflammatory responses were significantly altered by exposure to 10 mg/kg TiO2 NPs (Figure 6). Of these genes altered, 29 genes were up-regulated and 7 genes were down-regulated. Ye et al. investigated that BCL-6 may regulate specific T-cell-mediated responses and can control germinal centre formation as a transcriptional switch. Modification of expression of BCL-6 in lymphoma results in the unnormal B cell proliferation and a deregulation of germinal centre formation [24], while B cell is an immune cell, so the up-regulated of the differentiation of B cell triggers the immune responses in the kidney. In our data, Bcl6 gene was greatly increased with a DiffScore of 67.89 in the kidney (Additional file 1: Table S1), suggesting that TiO2 NPs disordered the process of B cell differentiation, thus interfering with immune responses in mice. The inflammatory kidney disease membranoproliferative glomerulonephritis type II (MPGN2) is following the presence of complement C3. At the same time, complement factor I (cfi) can modulate the activation of C3 through the alternative pathway. And the breakdown of activated C3 is regulated by factor I, the deficiency of factor I causes uncontrolled C3 activation [25]. Our results showed that c3 gene up-regulated with a Diffscore of 30.23 and cfi gene down-regulated with a DiffScore of -54.62 following exposure to TiO2 NPs (Additional file 1: Table S1). The renal inflammation following exposure to TiO2 NPs was closely associated with overexpression of c3 gene and decreased expression of cfi gene in the kidney. While, our result also showed that complement factor D (Cfd) gene was observably up-regulated with a DiffScore of 52.09. Cfd is expressed in the kidney and plays a central role in the activation of the alternative pathway as a serine protease [26]. So the significant increased expression of Cfd gene demonstrated that TiO2 NPs exposure affected renal biochemical functions in the kidney [12]. CXCL12 (stromal cell-derived factor-1) is not only a unique homeostatic chemokine but also a potent small proinflammatory chemoattractant cytokines that binds primarily to CXC receptor 4 (CXCR4; CD184). As an inflammatory chemokine, CXCL12 has been immunodetected not only in normal tissues but also in many different inflammatory diseases [27]. In the present study, CXCL12 gene was up-regulated with a DiffScore of 28.02 after TiO2 NPs treatment, which was associated with infiltration of inflammatory cells in the kidney.
The current study suggested that TiO2 NPs exposure increased ROS significant production and led to peroxidation of lipids, proteins, and DNA in mouse renal tissue (Table 4), and caused renal cell apoptosis (Figure 5), which may be associated with alterations of oxidative stress-related or apoptosis-related gene expression. The overproduction of ROS has been shown to be closely associated with the induction of apoptotic and necrotic cell death in cell cultures [28]. This breaks down the balance of the oxidative/antioxidative system in the kidney, resulting in lipid peroxidation, which increased the permeability of mitochondrial membrane [11]. In our previous studies, TiO2 NPs were also shown to mediate apoptosis in the liver, spleen, brain, lung, and ovary in mice through the induction of ROS [21,29-35]. Meena et al. also showed that TiO2 NPs can induce oxidative stress which causes cell apoptosis in the kidney [36]. However, the apoptotic mechanism following TiO2 NPs -induced nephrotoxicity remains unclear. In the present study, our findings indicated that about 49 genes involved in oxidative stress and about 35 genes involved in apoptosis were dramatically altered in the 10 mg/kg TiO2 NPs exposed kidney, in which 49 were up-regulated and 35 were down-regulated (Figure 6). For example, Cyp4a12a, Cyp4a12b, Axud1, Ccl19, and Ccl21a genes were greatly up-regulated with DiffScores of 38.54, 123.6, 60.66, 83.27, and 28.86, respectively; while Cyp24a1, Akrlc18, Birc5, and E2F1 genes were significantly down-regulated with DiffScores of -33.79, -56.24, -101.23, and -66 (Additional file 1: Table S1), respectively. As we know, the cytochrome P450 (CYP) is a gene superfamily of enzymes encodes many isoforms and reveals a variety of catalytic activity, regulatory mechanisms and substrates [37]. Cyp4a12a, and Cyp4a12b are members of Cyp4 family of cytochrome P450 proteins and can hydroxylated arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE) effectively. Furthermore, Cyp4a12a and Cyp4a12b also effectively transformed eicosapentaenoic acid (EPA) into 19/20-OH- and 17, 18-epoxy-EPA, which are the predominant 20-HETE synthases in mouse kidney [38]. The up-regulation of Cyp4a12a and Cyp4a12b genes following exposure to TiO2 NPs illustrated that these abnormal expression may cause the disorder of oxidation-reduction process involved in 20-HETE production. The catabolic enzyme product of Cyp24a1 regulates the levels of hormonal 1, 25-dihydroxyvitamin D(3) (1, 25(OH)2D3) intracellular. The regulation of expression of this enzyme is crucial to the biological activity of 1, 25(OH)2D3[39]. Therefore, down-regulated of Cyp24a1 gene following exposure to TiO2 NPs suggested may disrupt the metabolism of 1, 25(OH)2D3 in the kidney. Aldo–keto reductases (AKRs) are members of a large enzymes family that catalyze NADPH- and NADH-dependent oxidoreduction of a wide variety of substrates, including 20α-Hydroxysteroid dehydrogenase (20α-HSD) simple carbohydrates and steroid hormones [40,41]. It is well-known that the AKR1C18 (20α-HSD) is a member of the AKR superfamily that catalyze the inactivation of progesterone, which stereoselective converts progesterone to its inactive metabolite 20α-hydroxy-4- pregnen-3-one (20α-HP) [40-43]. Down-regulation of Akrlc18 gene by TiO2 NPs exposure implied that TiO2 NPs may induce the activation of progesterone, which affects renal physiological processes. Axud1 (cysteine-serine-rich nuclear protein-1) also known as Csrnp-1 is an immediate early gene which strongly caused as a response to IL-2 in mouse T cells [44]. Overexpression of Axud1 conducts to apoptosis through the activation of the JNK pathway and inhibits mitosis [44]. In the study, significant increase of Axud1 gene expression caused by TiO2 NPs promoted renal cell apoptosis. Birc5 is a member of the inhibitor of apoptosis (IAP) gene family that encodes negative regulatory proteins which block apoptotic cell death. What’s more, the functions of Birc5 (survivin) are to enhance proliferation and survival of cells in the kidney [45]. Whereas, Birc5 gene down-regulation following exposure to TiO2 NPs may result in decreased survival of cells, and renal cells apoptosis in the kidney (Figure 5). It was previously reported that the stimulation of DCs with CCR7 ligands CCL19 and CCL21 inhibits well-known apoptotic hallmarks of serum-deprived DCs, including increased membrane blebs and membrane phosphatidylserine exposure, nuclear changes, and loss of mitochondria membrane potential [46]. In this study, we observed significant nucleus shrinkage, chromatin marginalization and mitochondria swelling in renal cell following exposure to TiO2 NPs (Figure 5). Ccl19 and Ccl21a upregulation, however, may serve as a protective role for kidney following TiO2 NPs-induced apoptosis. In addition, E2F1 is suggested to induce apoptosis and activation of p53-responsive target genes which coincides with an ability of E2F1 to induce accumulation of p53 protein. By affecting the accumulation of p53, E2F1 serves as a specific signal for the induction of apoptosis [47]. Decreased expression of E2F1 gene may also be associated with a protective role for kidney following TiO2 NPs–induced nephrotoxicity.
The equilibrium of various elements is essential for immune integrity in the kidney and plays an important role in renal physiology. Our data indicated that TiO2 NPs exposure led to significant increases in Ca, K, Mg, Zn, and Cu concentrations, but decreased Na, and Fe concentrations in the kidney (Table 3). The changes of these elements can provide useful information on physiology and pathology of kidney. To further clarify the molecular mechanisms of mineral element unbalance, we analyzed microarray data and found significant alterations of related-gene in the kidney. Sri gene overexpresses sorcin in K562 cells by gene transfection, which results in marked decrease of the level of cytosolic calcium and increased the ability of cell to resistance to apoptosis [48]. Intracellular Ca2+ homeostasis plays an important role in sustaining the biological functions of the cell and Ca2+overload may trigger apoptosis [49]. In contrast, our results showed that Sri gene was down-regulated with a Diffscore of -16.77 by TiO2 NPs exposure (Additional file 1: Table S1), which resulted in a significant Ca2+ overload in the kidney, thus leading to renal cell apoptosis. Slc10a6, also known as Soat, encodes protein of SOAT [50]. The transport conducted by SOAT is highly sodium dependence, indicating a symport transport with Na+ of the substrate [51]. In our data, Slc10a6 overexpressed with a Diffscore of 25.48, therefore, the increased Na+ concentration may be closely associated with Slc10a6 up-regulation in the kidney following exposure to TiO2 NPs. Establishing and maintaining high K+ and low Na+ in the cytoplasm are required for normal resting membrane potentials and various cellular activities. Therefore, the imbalance of Na+ and K+ caused by TiO2 NPs disturbed the ion homeostasis and cause a series of physiological disorders in the kidney. We also found that Cp gene was up-regulated with a Diffscore of 17.04, and Tfrc gene was dramatically down-regulated with a diffscore of -67.61 in the kidney (Additional file 1: Table S1). Ceruloplasmin (Cp), a copper-containing ferroxidase, is essential for body iron homeostasis as selective iron overburden takes place in aceruloplasminemia. Copper is an essential metal cofactor for numerous cuproenzymes which can catalyze some important biochemical reactions [52]. And the sticking point in the molecular mechanisms associated with copper-iron hypothesis is the cuproenzyme Cp [53]. Therefore, the increased Cu concentration and decreased Fe concentration may correlate with the up-regulation of Cp gene expression. In addition, Cu is a heavy metal, its overload following exposure to TiO2 NPs would lead to Cu poisoning in the kidney. Iron-restricted erythropoiesis is a common clinical condition in patients with chronic kidney disease. Iron status can be monitored by different parameters such as ferritin, transferrin saturation etc. Transferrin receptors (TfRc) are the principal pathway by which various organ cells to obtain iron for physiological requirements [54]. The number of TfRc on the cell surface displays the requirement of iron, so the synthesis of transferrin receptor is closely related to the iron requirements [55]. Decreased Fe level caused by TiO2 NPs may be also closely correlated to significant reduction of Tfrc gene expression, whereas Fe deficit would aggravate renal anemia and decrease immune capacity in the TiO2 NPs-exposed mice.
Conclusions
The present study suggested that long-period exposure to TiO2 NPs resulted in severe kidney pathological changes and apoptosis, coupled with unbalance of mineral elements and severe oxidative stress. Furthermore, the nephrotoxicity following exposure to TiO2 NPs may be closely related to significant alterations in the expression of genes involved in immune/inflammatory responses, apoptosis, biological processes, oxidative stress, metabolic processes, the cell cycle, ion transport, signal transduction, cell component, transcription, translation, and cell differentiation. Axud1, Bcl6, Cf1, Cfd, Cyp4a12a, Cyp4a12b, Cyp2d9, Birc5, Crap2, and Tfrc genes may be potential biomarkers of kidney toxicity caused by TiO2 NPs exposure. Therefore, the application of TiO2 NPs in food, toothpastes, cosmetics and medicine should be carried out cautiously.
Methods
Preparation and characterization of TiO2 NPs
Nanoparticles anatase TiO2 was prepared via controlled hydrolysis of titanium tetrabutoxide. The details of the synthesis TiO2 NPs were previously described [34,56]. Briefly, colloidal titanium dioxide was prepared via a controlled hydrolysis of titanium tetrabutoxide. In a typical experiment, 1 mL of Ti (OC4H9)4 was dissolved in 20 mL of anhydrous isopropanol, and was added dropwise to 50 mL of double-distilled water that was adjusted to pH 1.5 with nitric acid under vigorous stirring at room temperature. The temperature of the solution was then raised to 60°C, and maintained for 6 h to promote better crystallization of TiO2 nanoparticles. Using a rotary evaporator, the resulting translucent colloidal suspension was evaporated yielding a nano-crystalline powder. The obtained powder was washed three times with isopropanol, and then dried at 50°C until the evaporation of the solvent was complete. A 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4M was used as a suspending agent [57]. TiO2 powder was dispersed onto the surface of 0.5% w/v HPMC solution, and then the suspending solutions containing TiO2 particles were treated ultrasonically for 15–20 min and mechanically vibrated for 2 min or 3 min.
The particle sizes of both the powder and nanoparticles suspended in 0.5% w/v HPMC solution after incubation for 12 h and 24 h (5 mg/L) were determined using a TecnaiG220 transmission electron microscope (TEM) (FEI Co., USA) operating at 100 kV, respectively. In brief, particles were deposited in suspension onto carbon film TEM grids, and allowed to dry in air. The mean particle size was determined by measuring > 100 randomly sampled individual particles. X-ray-diffraction (XRD) patterns of TiO2 NPs were obtained at room temperature with a charge-coupled device (CCD) diffractometer (Mercury 3 Versatile CCD Detector; Rigaku Corporation, Tokyo, Japan) using Ni-filtered Cu Kα radiation. The surface area of each sample was determined by Brunauer–Emmett–Teller (BET) adsorption measurements on a Micromeritics ASAP 2020M + C instrument (Micromeritics Co., USA). The average aggregate or agglomerate size of the TiO2 NPs after incubation in 0.5% w/v HPMC solution for 12 h and 24 h (5 mg/L) was measured by dynamic light scattering (DLS) using a Zeta PALS + BI-90 Plus (Brookhaven Instruments Corp., USA) at a wavelength of 659 nm. The scattering angle was fixed at 90°. The Ti4+ ions leakage from TiO2 NPs at time 0 and/or after 12, 24, 48 h of incubation in 0.5% w/v HPMC was measured by Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co., Finland) after sample was centrifugated at 1,719 × g for 10 min and filtrated with a 0.001 μm membrane filter.
Animals and treatment
One hundred and twenty male CD-1 (Imprinting Control Region) mice aged 5 weeks with an average bw of 23 ± 2 g were purchased from the Animal Center of Soochow University (Jiangsu, China). All mice were housed in stainless steel cages in a ventilated animal facility with a temperature maintained at 24 ± 2°C and relative humidity of 60 ± 10% under a 12-h light/dark cycle. Distilled water and sterilized food were available ad libitum. Prior to dosing, the mice were acclimated to the environment for 5 days. All animals were handled in accordance with the guidelines and protocols approved by the Care and Use of Animals Committee of Soochow University (Jiangsu, China). All procedures used in the animal experiments conformed to the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals [58].
An HPMC concentration of 0.5% was used as a suspending agent. TiO2 NPs powder was dispersed onto the surface of 0.5% w/v HPMC, and then the suspending solutions containing TiO2 NPs were treated ultrasonically for 30 min and mechanically vibrated for 5 min. For the experiment, the mice were randomly divided into four groups (N = 30 each), including a control group (treated with 0.5% w/v HPMC) and three experimental groups (treated with 2.5, 5, and 10 mg/kg bw TiO2 NPs, respectively). About the dose selection in this study, we consulted the report of World Health Organization in 1969. According to the report, LD50 of TiO2 for rats is larger than 12,000 mg/kg bw after oral administration. In addition, the quantity of TiO2 nanoparticles does not exceed 1% by weight of the food according to the Federal Regulations of US Government. The mice were weighed and then the TiO2 NPs suspensions were administered by intragastric administration every day for 90 days. All symptoms and deaths were carefully recorded daily. After the 90-day period, all mice were weighed, anesthetized with ether, and then sacrificed. Blood samples were collected from the eye vein by rapidly removing the eyeball and serum was collected by centrifuging the blood samples at 1, 200 × g for 10 min. The kidneys were quickly removed and placed on ice, and the kidneys were dissected and frozen at -80°C.
Coefficient of kidney
After weighing the body and kidneys, the coefficients of kidney mass to bw were calculated as the ratio of kidney (wet weight, mg) to bw (g).
Elemental content analysis
The frozen kidneys tissues were thawed and ~ 0.1 g samples were weighed, digested, and analyzed for titanium, sodium, magnesium, potassium, calcium, zinc, and iron content content. Briefly, prior to elemental analysis, the kidney tissues were digested overnight with nitric acid (ultrapure grade). After adding 0.5 mL of H2O2, the mixed solutions were incubated at 160°C in high-pressure reaction containers in an oven until the samples were completely digested. Then, the solutions were incubated at 120°C to remove any remaining nitric acid until the solutions were colorless and clear. Finally, the remaining solutions were diluted to 3 mL with 2% nitric acid. Inductively coupled plasma-mass spectrometry (Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA) was used to determine the titanium, sodium, magnesium, potassium, calcium, zinc, and iron concentration. Indium (20 ng/mL) was chosen as an internal standard element. Data are expressed as nanograms per gram fresh tissue.
Histopathological evaluation of kidney
For pathological studies, all histopathological examinations were performed using standard laboratory procedures. The kidneys were embedded in paraffin blocks, then sliced (5-μm thickness), and placed on glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments using light microscopy (U-III Multi-point Sensor System; Nikon, Tokyo, Japan).
Observation of kidney ultastructure
The kidneys were fixed in fresh 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4°C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2—7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate, then the specimens were dehydrated in a graded series of ethanol (75, 85, 95, and 100%) and embedded in Epon 812 resin. Ultrathin sections were made, contrasted with uranyl acetate and lead citrate, and observed by TEM (model H600; Hitachi, Ltd., Tokyo, Japan). Kidney apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation).
Confocal Raman microscopy of kidney sections
Raman analysis of renal glass or TEM slides was performed using backscattering geometry in a confocal configuration at room temperature in a HR-800 Raman microscope system equipped with a 632.817 nm HeNe laser (JY Co., France). It has been previously reported that when the size of TiO2 NPs reached to 6 nm, the Raman spectral peak was 148.7 cm-1[59]. Laser power and resolution were approximately 20 mW and 0.3 cm-1, respectively, while the integration time was adjusted to 1 s. The slides were scanned under the confocal Raman microscope.
Oxidative stress assay
Superoxide ion (O2·–) in the kidney tissues was measured by monitoring the reduction of 3′-{1-[(phenylamino) carbonyl]-3, 4-tetrazolium}–bis (4-methoxy- 6-nitro) benzenesulfonic acid hydrate (XTT) in the presence of O2·–, as described by Oliveira et al. [60]. The detection of H2O2 in the kidney tissues was carried out by the xylenol orange assay [61].
Lipid peroxidation of kidneys was determined as the concentration of malondialdehyde (MDA) generated by the thiobarbituric acid (TBA) reaction as described by Buege and Aust [62]. Protein oxidation of kidneys was investigated according to the method of Fagan et al. by determining the carbonyl content [63]. DNA of kidneys was extracted using DNeasy Tissue Mini Kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) as described by the manufacturer. Formation of 8-OHdG was determined using the 8-OHdG ELISA kit (Japan Institute for the Control of Aging, Haruoka, Japan). This kit provides a competitive immunoassay for quantitative measurement of the oxidative DNA adduct 8-OHdG. It was carefully performed according to manufacturer’s instructions, and using a microplate varishaker-incubator, an automated microplate multi-reagent washer, and a computerized microplate reader.
Microarray assay
Gene expression profiles of the kidney tissues isolated from 5 mice in the control and TiO2 NPs-treated groups were compared by microarray analysis using Illumina BeadChip technology (Illumina Inc., USA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no. 1755, Ambion, Inc., Austin, TX, USA) according to the manufacturer’s protocol, and stored at -80°C. RNA amplification is the standard method for preparing RNA samples for array analysis [64]. Total RNA was then submitted to Biostar Genechip, Inc. (Shanghai, China) to analyze RNA quality using a bioanalyzer and complementary RNA (cRNA) was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized for 18 hrs at 55°C on Illumina HumanHT-12 v3.0 BeadChips, containing 45,200 probes (Illumina, Inc., San Diego, CA, USA), according to the manufacturer’sprotocolandsubsequently scanned with the Illumina BeadArray Reader 500. This program identifies differentially expressed genes and establishes the biological significance based on the Gene Ontology Consortium database (http://www.geneontology.org/GO.doc.html). Data analyses were performed with GenomeStudio software version 2009 (Illumina Inc., San Diego, CA, USA), by comparing all values obtained at each time point against the 0 hrs values. Data was normalized with the quantile normalization algorithm, and genes were considered as detected if the detection p-value was lower than 0.05. Statistical significance was calculated with the Illumina DiffScore, a proprietary algorithm that uses the bead standard deviation to build an error model. Only genes with a DiffScore ≤ -13 and ≥13, corresponding to a p-value of 0.05, were considered as statistical significant [65,66].
Quantitative real-time PCR (qRT-PCR)
The levels of mRNA expression of Apaf1, Bcl2l1, Bcl6, Bmp6, Birc5, Bub1b, C3, Ccl19, Ccl21a, Cd74, Odc1, Cd34, Cd55, Cfd, Cfi, Cxcl12, Cygb, Cycs, Egr1, Fn1 Klf1, Ngfrap1, Nid1, Psmb5, Serpinalb, Tnfrsf12, and Txnip in the mouse kidney were determined using real-time quantitative RT polymerase chain reaction (RT-PCR) [67-69]. Synthesized complimentary DNA was generated by qRT-PCR with primers designed with Primer Express Software (Applied Biosystems, Foster City, CA, USA) according to the software guidelines, and PCR primer sequences are listed Table 6. The quantitative real-time PCR was performed by SYBR Green method using the special primers (as shown in Table 6) by the Applied Biosystems 7700 instrument (Applied Biosystems, USA).
Table 6 Real time PCR primer pairs
Gene name Description Primer sequence Primer size (bp)
Refer-actin Mactin-F 5′-GAGACCTTCAACACCCCAGC-3′
Mactin-R 5′-ATGTCACGCACGATTTCCC-3′ 263
Apaf1 mApaf1-F 5'-TAGCGGCTCATCTGTTCTGTAG-3'
mApaf1-R 5'-CCACTTGAAGACAAAAGACCAA-3' 87
Bcl2l1 mBcl2l1-F 5'- ATTTCCCATCCCGCTGTG-3'
mBcl2l1-R 5'-GGCTAAAAGCACCTCACTCAAT-3' 82
Bcl6 mBcl6-F 5'- TTTCAATGATGGACGGGTGT-3'
mBcl6-R 5'- ACGCAGAATGTGGGAGGAGT-3' 118
Birc5 mBirc5-F 5'-TCTAAGCCACGCATCCCA-3'
mBirc5-R 5'-CAATAGAGCAAAGCCACAAAAC-3' 150
Bmp6 mBmp6-F 5'-ATTAAATATCCCTGGGTTGAAAGAC-3'
mBmp6-R 5'-CTGGGAATGGAACCTGAAAGAG-3' 117
Bub1b mBub1b-F 5'-AATGGGTGGGGCTTTTGA-3'
mBub1b-R 5'-CCTGGCTGCTTGTCTTGC-3' 117
C3 mC3-F 5'-GGAGAAAAGCCCAACACCAG-3'
mC3-R 5'-GACAACCATAAACCACCATAGATTC-3' 148
Ccl19 mCcl19-F 5'-CCTCCTGATGCTCTGTCCCA-3'
mCcl19-R 5'-CGGTACCAAGCGGCTTTATT-3' 145
Ccl21a mCcl21a-F 5'-CACGGTCCAACTCACAGGC-3'
mCcl21a-R 5'-TTGAAGCAGGGCAAGGGT-3' 102
Cd34 mCd34-F 5'-CTCAGTCCCCTGGCAGATTC-3'
mCd34-R 5'-GGACCCCTGTTCTCCCCTTA-3' 147
Cd55 mCd55-F 5'-AAATCCAGGAGACCAACCAAC-3'
mCd55-R 5'-CTGTAGATGTTCTTATTGGATGACG-3' 113
Cd74 Cd74-F 5'-ACGGCAAATGAAGTCAGAACA-3'
Cd74-R 5'-AAGACTACTAATGGGTCAGAAATGG-3' 97
Cfd mCfd-F 5'-AGCAACCGCAGGGACACTT-3'
mCfd-R 5'-TTTGCCATTGCCACAGACG-3' 108
Cfi Cfi-F 5'-CCCGAGTTCCCAGGTGTTTA-3'
Cfi-R 5'-GAAGGAGGTCATAGCTTCAGACA-3' 112
Cxcl12 mCxcl12-F 5'-CCAGTCAGCCTGAGCTACCG-3'
mCxcl12-R 5'-TTCTTCAGCCGTGCAACAA-3' 128
Cycs mCycs-F 5'-CAACTCCGACTACAGCCACG-3'
mCycs-R 5'-GACACCACTATCACTCATTTCCCT-3' 134
Cygb mCygb-F 5'-GCTCAGTGCCCTGCATTCC-3'
mCygb-R 5'-CCGTGGAGACCAGGTAGATGAC-3' 120
Egr1 mEgr1-F 5'-TTACCTACTGAGTAGGCTGCAGTT-3'
mEgr1-R 5'-GCAATAGAGCGCATTCAATGT-3' 141
Fn1 mFn1-F 5'-TGAAGCAACGTGCTATGACGA-3'
mFn1-R 5'-GTTCAGCAGCCCCAGGTCTAC-3' 149
Klf1 mKlf1-F 5'-ACCACCAGATAAATCAACTCAAATG-3'
mKlf1-R 5'-ATAGTAACGACAACAATCCTAGCAGA-3' 146
Ngfrap1 mNgfrap1-F 5'-GCCTTTAATGACCCGTTTGTG-3'
mNgfrap1-R 5'-TCCATGCTAATGGGCAACACT-3' 147
Nid1 mNid1-F 5'-ACCTCCTTTTCTTCTACTTTCACTG-3'
mNid1-R 5'-TCCAATTATTTAAGTAAAGACTCCCT-3' 122
Odc1 mOdc1-F 5'-TGCTGAGCAAGCGTTTGTAG-3'
mOdc1-R 5'-ATTCCCTGATGCCCAGTTATT-3' 107
Psmb5 mPsmb5-F 5'-GCTTCTGGGAGCGGTTGTT-3'
mPsmb5-R 5'-CATGTTAGCGAGCAGTTTGGA-3' 101
Serpina1b mSerpina1b-F 5'-TGAGTCCACTGGGCATCAC-3'
mSerpina1b-R 5'-GCTTCTGTTCCTGTCTCATCG-3' 136
Tnfrsf12a mTnfrsf12a-F 5'-CCAAGGACTGGGCTTAGAGTT-3'
mTnfrsf12a-R 5'-CCTTAGTATGGGTCGCTTTGTG-3' 114
Txnip mTxnip-F 5'-CCTGGGTGACATTCTACATTGA-3'
mTxnip-R 5'-TAAGGCTTAGTGAGCTTCCGAG-3' 141
PCR primers used in the gene expression analysis.
Statistical analysis
All results are expressed as means ± standard error of the mean(SEM). The significant differences were examined by unpaired Student's t-test using SPSS 19 software (USA). A p-value <0.05 was considered as statistically significant.
Competing interests
The authors declare that they have no competing financial interests.
Authors’ contributions
Conceived and designed the experiments: FH, MT, SG, XS, LZ, YZ, and XZ. Performed the experiments: FH, SG, XS, LZ, YZ, and XZ. Analyzed the data: FH, SG, XS, LZ, YZ, XZ, LS, QS, ZC, JC, RH, LW. Contributed reagents/materials/analysis tools: LS, QS, ZC, JC, RH, LW. Wrote the paper: FH, MT, SG, XS, LZ, YZ, and XZ. All authors read and approved the final manuscript.
Supplementary Material
Additional file 1 Table S1
Genes which related to apoptosis, oxidative stress, immune/inflammatory, biological process, translation, cell differentiation, cell cycle, transcription, transport, metabolic process, cell component and signal transduction altered significantly by intragastric administration with TiO2 NPs for consecutive 3.
Click here for file
Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant No. 81273036, 30901218, 81172697), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Major State Basic Research Development Program of China (973 Program) (grant No. 2006CB705602), National Important Project on Scientific Research of China (grant No. 2011CB933404) and the National Ideas Foundation of Student of Soochow University (grant No.111028534).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23555716PONE-D-12-3844810.1371/journal.pone.0059580Research ArticleBiologyModel OrganismsAnimal ModelsMouseRatNeuroscienceNeurophysiologyCentral Nervous SystemSynapsesBehavioral NeuroscienceLearning and MemoryMolecular NeuroscienceNeurobiology of Disease and RegenerationNeuropsychologyNeurotransmittersInhibition of Spontaneous Recovery of Fear by mGluR5 after Prolonged Extinction Training mGluR5 Inhibits Spontaneous Recovery of FearMao Sheng-Chun
1
Chang Chih-Hua
1
Wu Chia-Chen
1
Orejanera Maria Juliana
2
Manzoni Olivier J.
2
*
Gean Po-Wu
1
*
1
Institute of Basic Medical Sciences and Department of Pharmacology, National Cheng-Kung University, Tainan, Taiwan
2
INSERM U901, Marseille, France
Baudry Michel Editor
Western University of Health Sciences, United States of America
* E-mail: [email protected] (P-WG); [email protected] (OJM)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: PWG OJM. Performed the experiments: SCM CHC CCW MJO. Analyzed the data: SCM CHC CCW MJO. Contributed reagents/materials/analysis tools: SCM CHC CCW MJO. Wrote the paper: PWG OJM.
2013 21 3 2013 8 3 e595806 12 2012 15 2 2013 © 2013 Mao et al2013Mao et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Fear behavior is vital for survival and involves learning contingent associations of non-threatening cues with aversive stimuli. In contrast, excessive levels of fear can be maladaptive and lead to anxiety disorders. Generally, extensive sessions of extinction training correlates with reduced spontaneous recovery. The molecular mechanisms underlying the long-term inhibition of fear recovery following repeated extinction training are not fully understood. Here we show that in rats, prolonged extinction training causes greater reduction in both fear-potentiated startle and spontaneous recovery. This effect was specifically blocked by metabotropic glutamate receptor 5 (mGluR5), but not by mGluR1 antagonists and by a protein synthesis inhibitor. Similar inhibition of memory recovery following prolonged extinction training was also observed in mice. In agreement with the instrumental role of mGluR5 in the prolonged inhibition of fear recovery, we found that FMR1−/− mice which exhibit enhanced mGluR5-mediated signaling exhibit lower spontaneous recovery of fear after extinction training than wild-type littermates. At the molecular level, we discovered that prolonged extinction training reversed the fear conditioning-induced increase in surface expression of GluR1, AMPA/NMDA ratio, postsynaptic density-95 (PSD-95) and synapse-associated protein-97 (SAP97). Accordingly, delivery of Tat-GluR23Y, a synthetic peptide that blocks AMPA receptor endocytosis, inhibited prolonged extinction training-induced inhibition of fear recovery. Together, our results demonstrate that prolonged extinction training results in the mGluR5-dependent long-term inhibition of fear recovery. This effect may involve the degradation of original memory and may explain the beneficial effects of prolonged exposure therapy for the treatment of phobias.
This study was supported by grants NSC99-2923-B-006-001-MY3 from the National Science Council, NHRI-EX101-10117NI from the National Health Research Institute and Aim for the Top University Project of the National Cheng-Kung University of Taiwan. INSERM (O.J.M.), ANR-Blanc France-Taiwan RescueMemo (O.J.M. and P.W.G.), FRAXA research foundation (O.J.M.), a NARSAD 2010 Independent Investigator Grant given by the Brain & Behavior Research Foundation (O.J.M.), and Fondation pour la Recherche Médicale (M.J.O.) also supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Fear behavior involves the contingent associations of non-threatening cues with aversive stimuli. Although necessary to survival, excessive levels of fear can be maladaptive and lead to anxiety disorders.
A commonly used protocol referred to extinction training is to repeatedly present non-threatening cues (conditioned stimulus, CS) to the subject without pairing with aversive stimuli (unconditioned stimulus, US) which results in a gradual decrease in conditioned response (CR) [1], [2]. This extinction process represents an explicit model of behavioral therapy and is an effective treatment for anxiety disorders including phobias and post-traumatic stress disorder [3]. Unfortunately, extinction is a new inhibitory learning that inhibits expression of the original association rather than its erasure [4]–[7] and reduction of fear through behavioral therapy is often followed by a return of fear. This idea is supported by a variety of experimental maneuvers that cause fear return including changing the test context (renewal) [8], presenting unsigned US (reinstatement) [9], or simply allowing time to pass (spontaneous recovery) [10].
Previous studies have shown that long-term potentiation (LTP) of synapses from auditory thalamus and cortex to the lateral amygdala (LA) is a key molecular event leading to the encoding of fear memory [11], [12]. Fear conditioning drives the synaptic insertion of AMPA receptors in the amygdala [13]. Indeed, by labeling surface receptors with biotin or using membrane fractionation approaches, we have reported that fear conditioning resulted in an increase in surface expression of GluR1 subunit of AMPA receptors in the amygdala [14]. More recently, we also found that 3 sessions of 10 presentations of light-alone trials applied 24 h after training reduced fear-potentiated startle without influencing the conditioning-induced increase in surface GluR1 [15]. Consistent with previous reports, the extinguished rats exhibited reinstatement and spontaneous recovery of fear.
Extinction of fear often takes more trials than acquisition and once initiated further CS presentations are more effective with spaced than with massed CS presentations [16]. To investigate the mechanisms underlying extinction, we apply two to eight sessions of 15 presentations of light-alone trials to test their effects on fear-potentiated startle, spontaneous recovery and conditioning-induced increase in surface GluR1.
Results
Recovery of Fear after Extinction Training Depends on the Number of CS-alone Trials
We have previously shown how rats that had received 30 CS-alone trials 24 h after training exhibited spontaneous recovery and reinstatement of fear [15]. Here we tested whether increasing the number of extinction trials could prevent spontaneous recovery and thus result in a more enduring reduction in fear responses. To that end, we randomly divided rats into 8 groups after 10 light-shock pairings. Groups 1–7 were given extinction training consisting of 2–8 sessions of 15 presentations of light-alone trials respectively and the percentage of fear-potentiated startle was measured 24 h after trials (Day 3, Test 1). The 8th group was exposed to the context at equivalent time without receiving light-alone trials (context control group). Figure 1A shows that light-alone trial resulted in a reduction in startle potentiation. Startle potentiation were 201.2±19.2% (n = 7) in context controls, 74.4±11.4% (CS30, n = 7), 60.1±10.0% (CS45, n = 7), 53.0±10.4% (CS60, n = 7), 47.2±10.9% (CS75, n = 7), 41.6±6.7% (CS90, n = 7), 38.0±11.8% (CS105, n = 7) and 43.4±10.4% (CS120, n = 7) in extinction animals. The ANOVA for startle scores showed a significant effect for group (F(7,48) = 24.61, p<0.001). In addition, less startle reflex occurred in the CS90, CS105 and CS120 groups than in the CS30 group (p<0.05), indicating that the effect depended on the number of CS-alone trials.
10.1371/journal.pone.0059580.g001Figure 1 Recovery of fear after extinction training depends on the number of CS-alone trials.
(A) Plot of percent startle potentiation in context control and extinction rats. Rats received 10 light-shock pairings and were randomly assigned to 2 to 8 sessions of extinction training groups. Rats in 2 or 8 sessions of extinction groups received 2 or 8 sessions of 15 presentations of light-alone trials without footshock and memory retention was assessed 24 h later (Test 1). Context control rats were returned to the startle box at the equivalent time without receiving light-alone trials. All groups were also tested on day 8 (Test 2) and Day 15 (Test 3). ***p<0.001, **p<0.01, *p<0.05 vs. context controls. (B) Inhibition of spontaneous recovery by prolonged extinction training is blocked by mGluR5 antagonist. MTEP was administered intraperitoneally (2.5, 5 or 10 mg/kg) 60 min before light-alone trials or was infused into the amygdala (10 µg/per side) abilaterally 30 min before CS-alone trials. ***p<0.001, *p<0.05 vs. saline. (C) Distribution of cannula tips in the amygdala from rats infused with MTEP (10 µg/per side) in experiments B. (D) MTEP was without effect on the 30 CS-alone trials-induced extinction memory. (E) Distribution of cannula tips in the amygdala from rats infused with MTEP (10 µg/per side) in experiments D. (F) CPCCOEt (5 or 10 mg/kg) injected intraperitoneally 60 min before light-alone trials failed to affect 90 CS-induced extinction of fear memory.
To determine whether the fear response could spontaneously recover some time after extinction training, all groups were also tested on day 8 (Test 2) and day 15 (Test 3) and their fear-potentiated startles were compared with their respective Test 1. As shown in figure 1A, fear responses were markedly reduced in animals receiving 90, 105 and 120 CS-alone trials. One-way ANOVA revealed a significant effect of groups among Test 2 (F(7,48) = 10.69, p<0.001) and Test 3 (F(7,48) = 9.97, p<0.001). Looking at each group individually we first found that the CS30, CS45, CS60 and CS75 groups showed increased startle potentiation (spontaneous recovery) in Test 3 relative to Test 1. On the other hand, CS90, CS105 and CS120 groups did not. Such absence of spontaneous recovery did not reflect a passive loss of fear memory because context control rats retained a stable memory for 14 days.
These results suggest that spontaneous recovery of fear after extinction can be retarded if sufficient CS-alone trials are delivered. Since little spontaneous recovery occurred after 90, 105 or 120 CS trials, 90 CS trials were used in the following experiments to elucidate the underlying mechanisms.
mGluR5 Drive the Inhibition of Spontaneous Fear Recovery after Prolonged Extinction Training
A series of experiments were conducted to identify the receptor that mediated prolonged extinction training. Activation of group 1 metabotropic glutamate receptors (mGluRs) induce a distinct form of long-term depression (LTD) attributable to the endocytosis and decreased surface expression of postsynaptic AMPARs [17]–[19]. To test the possible involvement of group 1 mGluRs, we examined the effect of 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), a non-competitive antagonist of mGluR5 [20], [21] on prolonged extinction training. Rats were conditioned with 10 light-shock pairings and 24 h later received 6 sessions of 15 presentations of light-alone trials. MTEP was administered intraperitoneally (2.5, 5 or 10 mg/kg) 60 min before light-alone trials. These doses of MTEP have been shown to impair auditory fear conditioning [22]. Fear-potentiated startles were measured at 2, 7 and 14 days after conditioning. The results showed a significant effect for group (F(3,24) = 17.85, p<0.001), and Newman-Keuls post hoc tests revealed that the level of startle potentiation in the 5 and 10 mg/kg rats was significantly higher than that of the 2.5 mg/kg rats (p<0.001) indicating a dose-dependent effect. There were no differences between saline and 2.5 mg/kg MTEP for Test 2 and Test 3 (P>0.1). Furthermore, the levels of startle potentiation in the 5 and 10 mg/kg rats were equivalent to the levels observed in rats conditioned without light (p>0.05) (Fig. 1B). These results suggest that systemic application of MTEP before CS-alone trials attenuated inhibition of spontaneous recovery of fear memory. Similar to systemic administration, direct infusion of MTEP (10 µg/side, n = 7) into the amygdala 30 min before light-alone trials attenuated inhibition of fear recovery, demonstrating the key role of amygdala mGluR5 in this effect.
To examine whether mGluR5 was involved in the effect of 30 CS-alone trials, we repeated the experiments except that 90 CS-alone trials were replaced by 30 CS-alone trials. Comparison among 30 CS-alone (no drug treatment) and two MTEP groups revealed no significant differences on Test 1 (F(2,18) = 0.105, n = 7 in each group, p>0.5), Test 2 (F(2,18) = 0.129, p>0.5) and Test 3 (F(2,18) = 0.181, p>0.5) in these 3 groups. Thus, in contrast to 90 CS-alone trials, mGluR5 is not involved in 30 CS-alone extinction memory.
To exclude a contribution of mGluR1, we evaluated the effect of mGluR1 receptor selective antagonists [7-(hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt) [23] on extinction. CPCCOEt (5 or 10 mg/kg, i.p.), at the doses reported to induce central effects [24], had no effect on the 90 CS-alone-induced extinction (Fig. 1F). Together results suggest that mGluR5 but not mGluR1 receptor is involved in the extensive extinction training-induced extinction of fear memory.
mGluR5-dependent Inhibition of Fear Recovery Requires Protein Synthesis
Induction of LTD by group 1 metabotropic glutamate receptor required rapid dendritic protein synthesis [25], [26]. Thus, we determined the effect of a potent protein synthesis inhibitor on extinction. Anisomycin was administered intraperitoneally (100 mg/kg) 60 min before light-alone trials. One-way ANOVA of startle amplitude for vehicle, anisomycin and context control rats indicated a main effect of group (F(2,18) = 41.18, p<0.001) with anisomycin rats showing significantly higher startle amplitude than that of vehicle-treated rats (p<0.001) and equivalent startle amplitude to context control rats (p>0.05) (Fig. 2A). Similarly, intra-amygdala application of anisomycin (62.5 µg/side, n = 7) 30 min before light-alone trials blocked extinction (F(2,18) = 29.69, p<0.001).
10.1371/journal.pone.0059580.g002Figure 2 Protein synthesis is required for 90 CS-alone trials-induced inhibition of spontaneous recovery.
(A) Rats were conditioned with 10 light-shock pairings and 24 h later received 6 sessions of 15 presentations of light-alone trials. Anisomycin was administered intraperitoneally (100 mg/kg, n = 7) 60 min before light-alone trials or was infused into the amygdala (62.5 µg/side, n = 7) 30 min before light-alone trials. ***p<0.001 vs. vehicle. (B) Distribution of cannula tips in the amygdala from rats infused with Anisomycin (62.5 µg/side, n = 7).
Contextual Fear Conditioning and Prolonged Extinction in Wild Type and FMR1−/− Mice
To extend our previous observation in rats, we reproduced our main results in mice. Mice were placed in a context for 3 min and received a footshock every 60 sec for 5 times. After training, mice were randomly assigned to saline, 2.5 mg or 5 mg MTEP groups. As shown in figure 3A, all experimental groups acquired equivalent amount of conditioned freezing. On day 2, mice were placed in the same context for 10 min without receiving footshock. The procedure was repeated 3 times with interval of 30 min (Extinction training). Contextual freezing responses were measured on day 3 and day 8. Figure 3A shows that all groups exhibited a similar decrement of freezing. The effect of drug was not significant at the last min of extinction training on day 2 (F(2, 27) = 0.021, p>0.5). However, on day 3 and day 8, mice of pre-extinction injection of MTEP 5 mg/kg showed higher level of freezing compared with saline and MTEP 2.5 mg/kg groups (Day 3: F(2, 27) = 4.45, p<0.05; Day 8: F(2, 27) = 4.90, p<0.05). In addition, intraperitoneal injection of CPCCOEt (10 mg/kg) to mice before extinction training did not affect contextual freezing in both day 3 (T(18) = 0.35, p>0.5 vs. vehicle) and day 8 (T(18) = 0.54, p>0.5 vs. vehicle) (Fig. 3B). These results suggest that mGluR5 also is involved in the inhibition of spontaneous fear recovery after prolonged extinction training in mice.
10.1371/journal.pone.0059580.g003Figure 3 Effects of mGluR5 antagonist and protein synthesis inhibitor on prolonged extinction training-induced inhibition of spontaneous recovery in mice.
(A) On day 1, mice were placed in a context for 3 min and received a footshock every 60 sec for 5 times. On day 2, mice received intraperitoneal injection of saline, 2.5 mg/kg or 5 mg/kg of MTEP and 60 min later they were placed in the same context for 10 min without receiving footshock (extinction training). The extinction training was repeated 3 times with an interval of 30 min. Contextual freezing responses were measured as an index for memory retention on day 3 and day 8. *p<0.05 vs. saline or MTEP 2.5 mg. (B) CPCCOEt (10 mg/kg) injected intraperitoneally 60 min before extinction training failed to affect 90 CS-induced extinction of fear memory. (C) Same experimental procedure as (A) except anisomycin (62.5 µg/side, n = 10) or vehicle (n = 10) was infused into the hippocampus bilaterally 30 min before extinction training. *p<0.05 vs. vehicle. (D) Distribution of cannula tips in the hippocampus from mice infused with Anisomycin (62.5 µg/side, n = 10) or vehicle (n = 10).
We also determined the effect of protein synthesis inhibitor on extinction in mice. Since contextual fear memory is thought to depend upon the hippocampus [27], anisomycin was injected bilaterally into the hippocampus. Figure 3C shows that mice that had received intra-hippocampal injection of anisomycin (62.5 µg/per side, n = 10) before extinction training exhibited higher levels of freezing on both day 3 (T(18) = 2.98, p<0.01 vs. vehicle) and day 8 (T(18) = 2.67, p<0.05 vs. vehicle). These results suggest that protein synthesis is required for the inhibition of fear memory recovery.
Genetic deletion of fragile X mental retardation protein (FMRP) is generally linked to the enhancement of mGluR5 signaling and dendritic protein synthesis-dependent mGluR long-term depression (mGluR-LTD) is upregulated in FMR1−/− mice [26], [28]. To confirm our hypothesis of a key role of mGluR5 in fear memory recovery, we examined whether extinction training was altered in FMR1−/− mice. Acquisition and extinction of contextual fear conditioning in FMR1−/− mice (n = 8) and WT littermates (n = 7) are shown on figure 4. At the end of the acquisition session both genotypes exhibited similar levels of freezing behavior. Two-way repeated measures ANOVA (time x genotype) revealed a non-significant main effect of genotype [F(1, 13) = 0.348, p>0.05] and a significant main effect of time [F(7,91) = 41.577, p<0.001] (Fig. 4A). On day 2, mice underwent three 10 min extinction sessions in the conditioning context. FMR1−/− mice showed decreased freezing levels from minute one to ten indicating both impaired fear conditioning and increased rate of extinction [F(1,13) = 21.277, p<0.001]. Lower freezing levels were maintained in FMR1−/− mice compared to WT animals in subsequent extinction sessions, two [F(1,13) = 33.394, p<0.001] and three [F(1,14) = 8.242, p<0.05] (Fig. 4B, C, D), showing that inhibitory learning was increased in FMR1−/− mice. This effect was maintained in time as revealed by lower freezing levels on day 3 [F(1,14) = 6.251, p<0.05] and 8 [F(1,14) = 16.052, p<0.01] after conditioning (Fig. 4E). These results are compatible with our working hypothesis that mGluR5 are instrumental to fear recovery.
10.1371/journal.pone.0059580.g004Figure 4 Contextual fear conditioning and extinction in FMR1−/− mice and WT littermates.
(A) Acquisition of contextual fear conditioning. Mice were placed in a context for 3 min and received a foot shock every 60 sec for 5 times. Mice significantly increase freezing responses when compared to baseline (BL) measures before US presentation **p<0.01; ***p<0.001 vs. baseline (pairwise comparison test). (B) Extinction training. Mice underwent 3 extinction sessions with and interval of 30 min on day 2. *p<0.05; **p<0.01; ***p<0.001 (genotype effect). (C) Retention test. Freezing behavior was measured on day 3 and day 8 to evaluate long-term extinction memory *p<0.05; **p<0.01 (genotype effect). Data are expressed as mean ±s.e.m.
Reversal of Conditioning-induced GluR1 Expression by 90 CS-alone Trials
We have previously shown that fear conditioning elicited an increase in surface expression of GluR1 subunit of AMPA receptors in the amygdala which was unaffected by 30 CS-alone trials [15]. Thus, we evaluated whether 90 CS-alone trials produced differential action on the conditioning-induced GluR1 expression. Rats were conditioned and 24 h later received 2–6 sessions of light-alone trials. Twenty-four hours later, LA and Basolateral Amygdala (BLA) tissues were dissected out and surface receptors were labeled with biotin. Biotinylated receptors were precipitated and surface GluR1 was determined by immunoblotting. Figure 5A shows that conditioning-induced increase in GluR1 was absent after 6 sessions of light-alone trials but was not affected by 2–4 sessions of trials (F(5,30) = 7.36, p<0.001). Similarly, 90 CS-alone trials reversed conditioning-induced increase in GluR2 (F(5,30) = 5.91, p<0.001) (Fig. 5A, right).
10.1371/journal.pone.0059580.g005Figure 5 Reversal of conditioning-induced increases in surface expression of GluR1 and GluR2 is mediated by mGluR5.
(A) Representative blots and mean ± SEM of GluR1 (left) and GluR2 (right) immunoreactivities from rats that had been conditioned with 10 light-shock pairings and 24 h later received 2–6 sessions of 15 presentations of light-alone trials. Lateral (LA) and basolateral (BLA) amygdala tissues were dissected out, and surface GluR1 and GluR2 levels were determined by biotin labeling. **p<0.01, *p<0.05 vs. context control. (B) GluR1 and GluR2 surface levels were normalized to total protein in the left, and GluR1 and GluR2 intracellular levels were normalized to total protein in the right. Conditioning-induced increase in GluR1 and GluR2 surface levels and decrease in GluR1 and GluR2 intracellular levels were reversed after 90 CS-alone trials (CS). The effect of 90 CS-alone trials was blocked in a dose dependent manner by MTEP (2.5–10 mg/kg, i.p.) or by intra-amygdala infusion of MTEP (10 µg/side). ***p<0.001 vs. unpaired and naive, ### p<0.001, #p<0.05 vs. saline. (A) Representative immunoblots shown in B.
A protein cross-linking assay [29] was used to compare the distribution of GluR1 and GluR2 after 90 CS-alone trials. Briefly, LA and BLA tissues were removed and cross-linked with BS3. BS3 selectively cross-links cell surface (S) proteins, forming high-molecular-weight aggregates. Intracellular (I) proteins are not modified and thus retain their normal molecular weight. A measure of total receptor subunit protein is obtained by summing S+I [30]. Analyses of GluR1 and GluR2 from naïve, unpaired, paired and 90 CS-alone trial rats are shown in figure 5B. The levels of surface expression of GluR1 and GluR2 relative to total (S/S+I) were significantly higher (Fig. 5B, left) (GluR1: F(2,15) = 33.97, p<0.001; GluR2: F(2,15) = 77.13, p<0.001) whereas intracellular GluR1 and GluR2 relative to total (I/S+I) were significantly lower in paired (Fig. 5B, right) than in naïve and unpaired rats. This conditioning-induced increase in surface expression of GluR1 and GluR2 was reversed by 90 CS-alone trials (p<0.001 vs. paired). Furthermore, the effect of 90 CS-alone trials could be blocked by MTEP in a dose-dependent manner (GluR1: F(4,25) = 13.04, p<0.001; GluR2: F(2,15) = 20.09, p<0.001).
To assess whether the decrease in surface receptors by prolonged extinction training resulted in changes of excitatory synaptic transmission, we measured the relative contribution of AMPA receptors and NMDA receptors to the EPSCs [31]. Amygdala slices were made from naïve, unpaired, paired, 30 CS-alone trials, 90 CS-alone trials and 90 CS-alone trials plus MTEP pretreatment rats 24 h after CS-alone trials. As shown in figure 6A, the AMPA/NMDA ratios were 1.59±0.10 (n = 6) and 1.57±0.30 (n = 6) in slices from the naïve and unpaired rats. The ratio was significantly higher in the paired rats (4.14±0.11, n = 6, F(2,15) = 19.2, p<0.001) suggesting that fear conditioning persistently increased AMPA-mediated synaptic transmission. There was no difference in the AMPA/NMDA ratio between paired and 30 CS-alone trials rats (4.08±0.46, n = 6, p>0.5), suggesting that 30 CS-alone trials did not affect conditioning-induced increase in AMPA/NMDA ratio. However, in the 90 CS-alone trials-treated rats, AMPA/NMDA ratio (1.67±0.19, n = 6) was significantly lower than those of paired and 30 CS-alone trials rats (F(2,15) = 12.25, p<0.001). There was no difference in AMPA/NMDA ratio between naïve, unpaired and the 90 CS rats (F(2,15) = 0.6, p>0.5). These results suggest that 30 CS-alone trials failed to affect conditioning-induced increase in AMPA/NMDA ratio. Only by 90 CS-alone trials did extinction training reverse the increase. Moreover, the effect of 90 CS-alone trials was blocked by MTEP pretreatment (3.80±0.34, n = 6, p<0.001 vs. 90 CS-alone trials).
10.1371/journal.pone.0059580.g006Figure 6 Effects of 90 CS-alone trials on conditioning-induced increases in AMPA/NMDA ratio and the expression of PSD-95 and SAP97.
(A) Plot of AMPA/NMDA ratios in naïve, unpaired, paired, 30 CS, 90 CS and 90 CS+MTEP rats. ***p<0.001 vs. paired and 30 CS, ###p<0.001 vs. 90 CS. Scale: 50 ms, 100 pA. (B) Representative blots and mean ± SEM of PSD-95 immunoreactivity from rats that have been conditioned and 24 h later received 2–6 sessions of 15 presentations of light-alone trials. Lateral (LA) and basolateral (BLA) amygdala tissues were dissected out, and PSD-95 levels were determined by Western blot analysis. ***p<0.001, **p<0.01 vs. context control. (C and D) Representative blots and mean ± SEM of PSD-95 and SAP97 immunoreactivities from rats that received 90 CS-alone trials. LA and BLA tissues were dissected out at various time points after extinction training as indicated, and PSD-95 and SAP97 levels were determined by Western blot analysis. *p<0.05, **p<0.01, ***p<0.001 vs. context control.
Postsynaptic density-95 (PSD-95) is a scaffolding protein of the postsynaptic density, which interacts with GluR indirectly through stargazin and regulates GluR trafficking [32], [33]. We examined whether PSD-95 expression was altered after 90 CS-alone trials. Rats were conditioned and 24 h later received 2–6 sessions of CS-alone trials. Twenty-four hours later, LA and BLA tissues were dissected out and PSD-95 was determined by immunoblotting. As shown in figure 6B, conditioning-induced increase in PSD-95 was absent after 6 sessions of light-alone trials but was not affected by 2–4 sessions of trials. In the next experiment, rats were conditioned and 24 h later received 90 CS-alone trials. LA and BLA tissues were dissected out at 5, 10, 30, 60, 120, 240 and 360 min after 90 CS-alone trials. As shown in figure 6C, conditioning-induced increase in PSD-95 was reduced within 30 min after 90 CS-alone trials and abolished by 240 min (F(7,40) = 11.98, p<0.001).
A PDZ-domain-containing protein SAP97 binds to GluR1 and traffics GluR1 into spines [34]. In the lateral amygdala, the coupling of A-kinase anchoring proteins (AKAPs) and protein kinase A (PKA) to GluR1 through SAP97 is essential for fear memory formation [35]. We examined whether SAP97 expression was altered after 90 CS trials. As shown in figure 6D, conditioning-induced increase in SAP97 was reduced at 120 min and abolished 240 min after 90 CS-alone trials (F(7,40) = 6.89, p<0.001). In contrast to what was observed with 90 CS trials, 30 CS-alone trials failed to affect the level of PSD-95 (Fig. 6B).
Effects of Disruption of AMPA Receptor Endocytosis on 90 CS Extinction
A synthetic peptide containing a short C-terminal sequence of GluR2 (869YKEGYNVYG877, GluR23Y) has been shown to block LTD in the hippocampus and the nucleus accumbens [36], [37], and the extinction of learned fear [38], [39]. When fused to the cell membrane transduction domain of the HIV-1 Tat protein (Tat-GluR23Y) creating a cell membrane permeable peptide, Tat-GluR23Y impairs extinction of fear memory. We performed behavioral assessment to determine whether Tat-GluR23Y influenced extinction. Rats received 10 light-shock pairings, followed next day by 90 CS-alone trials and percentage of fear-potentiated startle was measured 24 h after extinction training (Day 3, Test 1). Rats were given Tat-GluR23Y (869YKEGYNVYG877, 15 pmol in 0.8 µl saline per side, n = 7) or the control peptide Tat-GluR23A (869AKEGANVAG877, n = 6) bilateral to the amygdale 30 min before 90 CS alone trials. One rat with incorrect Tat-GluR23Y infusion placement was excluded from the analysis. Figure 7 shows that light-alone trial resulted in a reduction of startle potentiation in Tat-GluR23A-treated rats and fear-potentiated startle was significantly less than that in the Tat-GluR23Y rats (t(12) = 5.218, p<0.001). Rats were also tested at 6 days (Day 8, Test 2) and 13 days (Day 15, Test 3) after the extinction training and Tat-GluR23Y -treated rats showed higher level of startle potentiation than those of Tat-GluR23A-treated rats.
10.1371/journal.pone.0059580.g007Figure 7 Tat-GluR23Y blocks the effect of 90 CS-alone trials on extinction.
(A) Rats received Tat-GluR23Y (15 pmol in 0.8 ml saline per side) or Tat- GluR23A (15 pmol in 0.8 ml saline per side) bilaterally into the amygdala 30 min before extinction training. Retention of memory was assessed 1 (Test 1), 6 (Day 8, Test 2) and 13 days (Day 15, Test 3) after extinction training. **p<0.01, ***p<0.001 vs. GluR23Y. (B) Distribution of cannula tips in the amygdala from rats infused with Tat-GluR23Y (△) or Tat- GluR23A (▴).
Discussion
The role of amygdala glutamate receptors in fear learning, fear-potentiated startle, and extinction has been proposed before [40]. Although mGluR5 in the lateral amygdala is implicated in the induction of long-term potentiation and the formation of fear memory [41], [42], its role in fear extinction in the limbic circuitry is not well established. A previous study has shown that intra-LA injection of CPCCOEt impaired extinction suggesting a contribution of mGluR1 [43]. In mGluR5 knock-out mice, acquisition of fear conditioning is partially impaired whereas the extinction of both contextual and auditory fear was completely abolished [44], [45]. Here we showed that prolonged extinction training resulted in an inability to retrieve memory at later times consistent with either a persistent or permanent inhibition of fear. An mGluR5 antagonist blocked the long-term inhibition whereas an mGluR1 antagonist was without effect. Furthermore we found that six- but not two-sessions of extinction training reversed conditioning-induced increase in surface expression of GluR1 and AMPA/NMDA ratio in an mGluR5-dependent manner. Finally, a GluR2-derivative peptide that blocked regulated AMPAR endocytosis reversed the inhibition of spontaneous recovery after prolonged extinction training. We interpret these data to suggest that prolonged extinction training results in an mGluR5-dependent long-term inhibition of fear recovery that may involve the degradation of the original memory. However, conditioned fear is usually measured by either fear-potentiated startle or freezing response. We used fear-potentiated startle paradigm and did not measure within-session extinction and extinction recall. Therefore, we could not differentiate between whether prolonged extinction makes it more likely that test sessions will produce more efficient and long lasting within-test-session extinction as opposed to prolonged extinction reducing spontaneous recovery directly.
Extensive extinction training induces greater reduction of conditioned response and reduced spontaneous recovery, implying that repeated extinction triggers the permanent depression of memory recovery [16], [46]. Once sufficient numbers of CS have been presented and extinction successfully induced, additional spaced presentations are more effective [16]. We used 6 sessions of 15 presentations of light-alone trials spaced by 10 min between each session and found that this protocol produced a greater reduction of fear-potentiated startle and less spontaneous recovery than that of 2 sessions. Loss of spontaneous recovery was not seen after application of the mGlu5 receptor antagonist MTEP.
Fragile X syndrome (FXS) is a monogenic developmental disorder associated with a complex neuropsychiatric phenotype [47]. FXS is caused by transcriptional silencing of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP)–an RNA-binding protein that regulates translation of its interacting mRNAs [28]. It has been proposed that exaggerated mGlu5-mediated signaling in the absence of FMRP plays a causal role in FXS. In FMRP-knockout animals, there is a marked increase of LTD in the hippocampus but no alterations in LTP [48]. The enhanced LTD in hippocampal neurons of knockout mice is a consequence of increased activity mediated by type 1 metabotropic glutamate receptors [49], [50]. In the present study, we first demonstrated that mGluR5 and new protein synthesis are involved in prolonged extinction training-induced inhibition of memory recovery in mice as seen in rats. Then using FMR1−/− mice, we find reduced spontaneous recovery of fear memory after extinction training compared with wild type mice. These results further support the involvement of mGluR5 in the inhibition of spontaneous recovery of fear memory after prolonged extinction training.
It seems likely that our prolonged CS-alone extinction protocol that results in a long-term inhibition of spontaneous recovery acts through one of two mechanisms. First, it could be the protocol enhances consolidation of extinction memory. Alternatively, the loss of spontaneous recovery could be the consequence of unlearning. Notably, conditioning-induced increase in GluR1 was reversed after the prolonged CS-alone trials. Consistent with the latter hypothesis, inhibition of AMPA receptor endocytosis in the amygdala restored spontaneous recovery suggesting that extinction might be mediated by degradation of the original memory. It is conceivable that prolonged extinction training results in a large amount of glutamate release, which acts on perisynaptic mGluR5 leading to formation of inositol 1,4,5-trisphosphate (IP3) and diacyl glycerol. IP3 induced Ca++ release from internal stores may lead to the activation of calcineurin, whereupon calcineurin can cause the dephosphorylation of GluR1 Ser845 likely through the inactivation of phosphatase 1A inhibitor that may initiate endocytosis of GluR1 and GluR2 from the surface of neurons [51], [52]. Generally the signal transduction coupling mGluR5 to AMPAR endocytosis depends on the experimental conditions and the brain areas studied [53]. At cerebellar parallel fiber to Purkinje cell (PF-PC) synapses, mGluR5 activation is coupled to PLC activation, diacylglycerol production and protein kinase C (PKC) activation resulting in phosphorylation of GluR2 at Ser880 [54], [55] which reduces its affinity for the AMPAR scaffold GRIP leading to internalization of AMPAR. At hippocampal CA1 synapses, mGluR5 activation is coupled to protein synthesis [26] via PI3K-Akt mammalian target of the rapamycin (mTOR) [56] or ERK-MAPK [57] signaling pathways.
People suffering from phobias show impaired extinction of aversively conditioned responses [58], [59] and increased amygdala activity when exposed to traumatic stimuli [60], [61]. Therefore, methods of preventing the return of fear after exposure therapy may lead to more effective therapeutic interventions. In the present study, we have demonstrated that extended extinction leads to persistent attenuation of fear recovery via an mGluR5-dependent mechanism. These results may open a new avenue for the treatment of anxiety disorders.
Materials and Methods
Animals
All procedures were approved by the Institutional Animal Care and Use Committee of the College of Medicine, National Cheng-Kung University or Animals and animals were treated in compliance with the European Communities Council Directive (86/609/EEC). Animals were housed in cages of four rats or mice each in a temperature (24°C)-controlled animal colony; pelleted rat chow and water were available ad libitum. They were maintained on a 12∶12 light–dark cycle with lights on at 0700 h. All behavioral procedures took place during the animal light cycle. Male fmr1−/− mice on a C57BL/6J genetic background [62] aged 10 to 11 weeks (P70 - 80) (fmr1−/− ) were used, with wild-type littermates used as control group. Mice were genotyped by tail PCR as described by [62].
Surgery
Male Sprague-Dawley rats (175–200 g), anesthetized with sodium pentobarbital (50 mg/kg, i.p.), were mounted on a stereotaxic apparatus and a cannula made of 22 gauge stainless steel tubing was implanted into the lateral (LA) or basolateral (BLA) amygdala [anteroposterior, –2.8 mm; mediolateral, ±4.5 mm; dorsoventral, –7.0 mm]. A 28 gauge dummy cannula was inserted into each cannula to prevent clogging. The rats were monitored and handled daily and were given 7 days to recover. MTEP (dissolved in saline. 10 µg/side for intra-amygdalar injection, and 2.5, 5 or 10 mg/kg for intraperitoneal injection) and CPCCOEt (dissolved in saline containing 45% 2-hydroxypropyl-β- cyclodextrin w/v. 5 mg/kg or 10 mg/kg for intraperitoneal injection) were purchased from Tocris Cookson Ltd (Northpoint, UK). Anisomycin (3 drops of Tween 80 in 2.5 ml of 7.5% DMSO in artificial CSF, and adjusted to pH 7.4 with NaOH. 62.5 µg/side for intra-amygdalar injection, and 100 mg/kg for intraperitoneal injection) was obtained from Sigma (St. Louis, Missouri). A TAT-conjugated peptide (GluR23Y, YKEGYNVYG) designed to impair AMPA receptor endocytosis was dissolved in 0.9% NaCl and infused into the LA or BLA (15 pmol/side) bilaterally 30 min before extinction training. The control peptide had the sequence AKEGANVAG (GluR23A). Dose was chosen with reference to Brebner et al. (2005) [35]. Drug was administered bilaterally to the amygdala in a volume of 0.5–0.8 µl at a rate of 0.1 µl/min. The infusion cannulas were left in place for 2 min before being withdrawn.
Behavioral Apparatus and Procedures
Rats were trained and tested in a stabilimeter device. Behavioral experiments of fear conditioning and extinction training were performed in standard operant chamber (San Diego Instrument, San Diego). The acoustic startle stimulus was a 50 ms white-noise at the intensity of 95 dB. The visual CS was a 3.7 s light produced by an 8 W fluorescent bulb attached to the back of stabilimeter. The US was a 0.65 mA footshock with duration of 0.5 s. Rats were placed in the training/testing chamber for 10 min and returned to their home cages on three consecutive days to habituate them to the test chamber and to minimize the effect of contextual conditioning. Following two days, Rats were handled in the same chamber before fear conditioning for pre-exposure. During pre-exposure, baseline startle was measured on each of 2 d by presenting 30 startle stimuli at a 10 sec interstimulus interval (ISI). Rats having equivalent baseline mean startle amplitudes were then divided into separated matched groups. On the day of fear conditioning, the animal was brought to the room, allowed to habituate, and placed in the chamber as before. The CS–US pairing began after a 3 min acclimation period in the chamber.
Training
The rats were placed in the startle boxes and received 10 light-footshock pairings at an ITI of 2 min. Unpaired controls received the same number of light and footshock presentation, but in a pseudorandom fashion in which the US could occur at anytime except at 3.2 sec following the CS.
Extinction
The rats were returned to the stabilimeter 24 h after the training and given 2, 3, 4, 5, 6, 7 or 8 sessions of 15 presentations of the 3.7-s light with neither shock or a startle-elicited noise burst (light-alone trials). Each session was separated by 10 min with an ITI of 1 min.
Test
The rats were tested for fear-potentiated startle 1(Test 1), 6(Test 2) or 13(Test 3) days after Extinction training. This involved 10 startle-eliciting noise bursts presented alone (noise-alone trial) and 10 noise bursts presented 3.2 s after onset of the 3.7-s light (light-noise trials). The two trial types were presented in a balanced mixed order (ITI, 30 s). The percentage of fear-potentiated startle was computed as: [(startle amplitude on CS-noise minus noise-alone trials)/(noise-alone trials)]×100.
Fear conditioning for mice occurred in 30×24×21 cm operant chamber (Med Associates, St. Albons, VT). The chamber was cleaned with 75% ethanol before each mouse was trained or tested for contextual fear conditioning. On the first day, mice were placed in a context for 3 min and received a footshock every 60 sec for 5 times. After training, mice were randomly assigned to saline, MTEP 2.5 mg or MTEP 5 mg groups in which they received intraperitoneal injection of saline, MTEP 2.5 mg/kg or MTEP 5 mg/kg respectively one hour before extinction training. On day 2, mice were placed in the same context for 10 min without receiving footshock. The procedure was repeated 2 times with an interval of 30 min (Extinction training). Contextual freezing responses were measured on day 3 and day 8. The behavior of mice was recorded by video camera mounted above the conditioning chamber. Freezing was defined as the absence of any movement except for respiration and measured automatically using FreezeScan software. Freezing data are presented as percent time spent freezing.
Whole-cell Patch-clamping Recordings for AMPA/NMDA Ratio
Whole-cell patch-clamp recordings from LA projection neurons were performed at ∼32°C in a superfusing chamber. Neurons were visualized with infrared video microscope using a 40× water immersion objective on an upright microscope. EPSCs were evoked at 0.03 Hz by extracellular stimulation of fibers emerging from the internal capsule which originate in the medial geniculate nucleus of the thalamus and project monosynaptically to the LA using a bipolar electrode. Patch electrodes were pulled from thick wall glass capillary (0.75 mm I.D., 1.5 mm O.D.) to a tip resistance of 3–5 MΩ. The composition of the internal solution was (in mM): Cs-gluconate 115, NaCl 5, EGTA 1, CaCl2, 0.3, MgCl2 2, Na-ATP 5, Na-GTP 0.4, HEPES 10. The final pH of the internal solution was adjusted to 7.3 by adding 1 M KOH; the final osmolarity was adjusted to 280 mOsm by adding sucrose. Recordings were low-pass-filtered at 2.5–20 kHz and digitized at 5–50 kHz. The signal was monitored and recorded with an Axopatch 200B amplifier. On-line analysis and control of experimental acquisition is accomplished via a 586 (Intel)-based PC clone and a Digidata 1200 computer interface. AMPAR-mediated EPSC was evoked when the neurons were voltage-clamped at −70 mV whereas NMDAR-mediated EPSC was determined as current amplitude at 50 ms after peak EPSC amplitude at a holding potential of +40 mV. Bicuculline (10 µM) was present in the perfusion solution. To avoid bias, data were collected from one cell per animal.
Surface Biotinylation of AMPA Receptor GluR1/R2 Subunits
Brain slices containing only LA and BLA were placed on ice and washed twice with ice-cold ACSF. The slices were then incubated with ACSF containing 0.5 mg/ml Sulfo -NHS- LC- Biotin (Pierce Chemical Co., Rockford, IL) for 1 h on ice. Next, the slices were rinsed in ACSF and then sonicated briefly in homogenizing buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, and 4 µg/ml aprotinin). After sonication, the samples were centrifuged at 14000 rpm for 30 min at 4°C and the supernatant was obtained. Protein concentration in the soluble fraction was then measured using a Bradford assay, with bovine serum albumin as the standard. Biotinylated protein (400 µg) from the supernatant was precipitated with 50 µl of 50% Neutravidin agarose (Pierce Chemical Co.) for 16 h at 4°C and washed 4 times with homogenizing buffer. Bound protein was re-suspended in 4 µl of SDS sample buffer and boiled. Biotinylated protein was resolved in 8.5% SDS-polyacrylamide gels, blotted electrophoretically to PVDF membrane, and blocked overnight in TBS buffer containing 5% non-fatty milk. Surface GluR1, GluR2 receptors and pan-cadherin (surface protein control) were detected by a biotinylation assay, followed by Western blot analysis that used either a GluR1 (1∶4000, Millipore), GluR2 (1∶5000, Millipore) or pan-cadherin (1∶2500; Sigma) antibodies, followed by HRP-conjugated secondary antibody for 1 hr. Other membranes were incubated overnight with PSD-95 (1∶10,000, Millipore), SAP97 (1∶5,000, Stressgen, Victoria, BC, Canada) and Actin (1∶10,000, Millipore). An enhanced chemiluminescence kit was used for detection. Western blots were developed in the linear range used for densitometry. GluR1 and GluR2 levels in the conditioned animals were expressed as a percentage of those in naïve controls without receiving light-shock pairings.
Synaptoneurosome Preparation
Brain slices containing only LA and BLA were homogenized in 70 µl of ice-cold lysis buffer in an Eppendorf tube. The buffer consisted of 118.5 mM NaC1, 4.7 mM KC1, 1.18 mM MgSO4, 2.5 mM CaCl2, 1.l8 mM KH2PO4, 24.9 mM NaHCO3, 10 mM dextrose, 10 µg/ml adenosine deaminase, pH adjusted to 7.4 by bubbling with 95% O2+5% CO2. Proteinase inhibitors (0.0l mg/ml leupeptin, 0.005 mg/ml pepstatin A, 0.l mg/ml aprotinin and 5 mM Benzamide) were included in the buffer to minimize proteolysis. The homogenate was diluted with 350 µl of additional ice-cold lysis buffer. This mixture was loaded into a l-m1 tuberculin syringe attached to a 13-mm diameter Millipore syringe filter holder. The diluted filtrate was forced over three layers of nylon (Tetko, 100 µm pores) pre-wetted with 150 µl of lysis buffer, and collected in a 1.5 m1 Eppendorf tube. The nylon-pre-filtered mixture was loaded into another 1 ml tuberculin syringe and forced through a pre-wetted 5 µm Millipore nitrocellulose filter. The homogenate was kept ice cold at all times to minimize proteolysis. The filtered particulate was then spun at l000×g for l5 min at 4°C. The supernatant was removed, and the pellet (synaptoneurosome) was re-suspended in 80 µl of lysis buffer for Western blot analysis.
Surface Receptor Cross-linking with bis(sulfosuccinimidyl)suberate
Surface and intracellular GluR1 and GluR2 levels were determined with a protein cross-linking assay. After conditioning or extinction training, rats were decapitated, brains were removed rapidly. Slices were added to Eppendorf tubes containing ice-cold artificial CSF spiked with 2 mM bis(sulfosuccinimidyl)suberate (BS3; Pierce Biotechnology, Rockford, IL). Incubation with gentle agitation proceeded for 15 min at 4°C. Crosslinking was terminated by quenching the reaction with 100 mM glycine (10 min at 4°C). The slices were pelleted by brief centrifugation, and the supernatant was discarded. Pellets were resuspended in ice-cold lysis buffer containing protease and phosphatase inhibitors [1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, pH 7.5, 0.3 M sucrose, 5 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, and 4 µg/ml aprotinin, 1 µM microcystin-LF, 1 µM okadaic acid, 1×protease inhibitor mixture (EMD Biosciences, San Diego, CA), and 0.1% Nonidet P-40 (v/v)] and homogenized rapidly by sonicating for 10 s. A brief centrifugation was performed, and the supernatant fraction was used for further studies. Total protein concentration of the supernatant was determined by the Lowry method. Samples were aliquoted and stored at –80°C for future analysis.
Histology
To identify cannula placements, animals received an overdose of pentobarbital (100 mg/kg) at the end of behavioral experiments. The brains were removed from the skull and fixed in buffered 4% paraformaldehyde for 48 h. Brains were sectioned with a microslicer (DTK-1000, Dosaka) and 40-μm-thick sections were stained for Nissl bodies.
Data Analysis
All values in the text were mean±SEM. Differences among the groups were evaluated with one-way ANOVA followed by the Newman-Keuls post hoc tests. Single-factor ANOVA and Newman–Keuls post hoc comparisons were used to analyze the differences in AMPA/NMDA ratio among naïve, paired, unpaired and extinction groups. Unpaired t test, was used to analyze differences of startle reflex between drug-treated and vehicle control groups. The level of significance was p<0.05.
The authors thank Dr. Min-Der Lai for critical comments on the manuscript and Dr. Henry Martin for expert editing of the manuscript.
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21 : 549 –555 .16257225 | 23555716 | PMC3605338 | CC BY | 2021-01-05 17:24:00 | yes | PLoS One. 2013 Mar 21; 8(3):e59580 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23533599PONE-D-13-0172710.1371/journal.pone.0059021Research ArticleBiologyBiochemistryDrug DiscoveryBiotechnologyDrug DiscoveryComputational BiologyMolecular GeneticsGene ExpressionMolecular Cell BiologyGene ExpressionMedicineDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryOncologyBasic Cancer ResearchOphthalmologyRetinal DisordersThe Nexus between VEGF and NFκB Orchestrates a Hypoxia-Independent Neovasculogenesis Hypoxia-Independent Retinal NeovasculogenesisDeNiro Michael
1
2
*
Al-Mohanna Falah H.
2
Alsmadi Osama
3
Al-Mohanna Futwan A.
4
5
1
Research Department, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia [Affiliate of the Wilmer Eye Institute/Johns Hopkins Medicine, Baltimore, Maryland, United States of America]
2
Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
3
Dasman Genome Centre, Dasman Diabetes Institute, Kuwait City, Kuwait
4
Department of Cell Biology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
5
Medical College, Al-Faisal University, Riyadh, Saudi Arabia
St-Pierre Yves Editor
INRS, Canada
* E-mail: [email protected]; [email protected] Interests: The authors have declared that no competing interests exist.
Conceived the project and obtained the required software for data analysis: MD FAAM. Conceived and designed the experiments: MD FAAM. Performed the experiments: MD FAAM FHAM OA. Analyzed the data: MD FAAM. Contributed reagents/materials/analysis tools: MD FAAM FHAM OA. Wrote the paper: MD FAAM.
2013 22 3 2013 8 3 e590219 1 2013 9 2 2013 © 2013 DeNiro et al2013DeNiro et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.
This study was supported by King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia (http://www.kkesh.med.sa/kkeshweb/en/), and King Faisal Specialist Hospital and Research Centre (http://www.kfshrc.edu.sa/wps/portal/En). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Angiogenesis is the formation of new blood vessels and capillary beds from existing vessels, which plays a fundamental role in physiological and pathological processes. In physiological conditions, angiogenesis occurs primarily in embryonic development, during tissue and wound repair, and in response to ovulation. However, pathological angiogenesis, or the abnormal rapid proliferation of blood vessels, is implicated in various diseases, including cancer, psoriasis, diabetic retinopathy [DR], and rheumatoid arthritis. Vascular endothelial growth factor [VEGF] is one of the most potent stimuli for new blood vessel growth, and therefore it has emerged as one of the most important growth factors controlling angiogenesis. Under pathological conditions, ischemia/hypoxia develops within the neovascular retina, which in turn increases VEGF levels in part through stabilization of VEGF mRNA [1]. This ischemic effect is mediated primarily by hypoxia inducible factor-1 [HIF-1], which is often considered as the master regulator of angiogenesis under ischemia/hypoxia. Retinal ischemia often precedes the onset of such NV, and the ischemic retina has been identified as a potential source of diffusible angiogenic factors. Retinal neovascularization [NV] is a major cause of the blindness that is associated with ischemic retinal disorders such as DR, retinopathy of prematurity [ROP], and retinal vein occlusion. Despite the prevalence of DR and ROP, an effective treatment for retinal NV remains elusive. Retinal NV is induced by complex interactions among multiple cytokines and adhesion molecules. Several potential inhibitors of retinal NV, including soluble VEGF receptor and antagonists of both αv-integrin and growth hormone have been identified with the use of a highly reproducible model of ischemia-induced retinal NV. Although VEGF is one of the central angiogenic factors induced in the neovascular retina, other growth factors may play crucial roles in the development and progression of retinal NV, many of which are hypoxia-independent. Various therapeutic modalities to inhibit VEGF have shown efficacy in the treatment of ischemia/hypoxia-driven retinal NV [2], [3], [4]. However, hoard evidence indicates that nonischemic microenvironment may also induce retinal NV [5], [6]. Furthermore, it has been demonstrated that in the streptozotocin [STZ]-induced type 1 diabetic rat model [7], [8]; the retinas exhibit most of the pathological features of DR seen in humans, including blood vessel dilation, blood retinal barrier [BRB] breakdown, microaneurysm formation, and intraretinal microvascular abnormalities, which makes this model widely used in studies of the early stages of DR, especially in those examining vascular hyperpermeability in the retina. [9], [10], [11]. In addition, streptozotocin [STZ]-induced diabetes failed to cause any significant increase in either HIF-1α or hypoxia. Interestingly, however, there was even a tendency for hypoxia levels to be decreased [tissue more highly oxygenated] [12].
Nuclear factor kappa-B [NFκB] is a heterodimeric complex of Rel family of proteins that is physically confined to the cytoplasm in unstimulated cells through the binding to inhibitor of κB [IκB] proteins [13]. It has been suggested that VEGF’s activation of NFκB signaling pathway is largely dependent on the cellular context. Both activation [14], [15] and inhibition [16] of NFκB in response to VEGF have been reported. Furthermore, it has been indicated that expression of pro-angiogenic factors, such as; SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression, are mediated by NFκB activation and may contribute to the pathogenesis of intraocular NV in individuals with DR or retinal vein occlusion. YC-1; 3-(5′-Hydroxymethyl-2′-furyl)-1-benzylindazole, has been identified as an sGC, and was shown to increase the intracellular cGMP concentration in platelets [17]. It was further demonstrated that YC-1 may activate the sGC/cGMP/PKG pathway to induce Ras and PI3K/Akt activation, which in turn initiates IKKα/β and NF-κB activation [18]. In addition, it has been established that cyclic GMP regulates NFκB in T Lymphocytes, neuronal cells, cardiomyocytes, endothelial cells, and hepatocytes [19]. These observations suggest that NFκB may represent a suitable target for therapeutic intervention in retinal NV. With the animal model of oxygen-induced retinopathy [OIR], it has been previously shown that nuclear factor KB [NFκB] activation may be important to induce retinal NV. In that model, exposure of neonatal animals to hyperoxic conditions results in extensive obliteration of retinal capillaries. When the animals are returned to room air, the inner retina presumably becomes relatively hypoxic, which results in the activation of NFκB, production of many cytokines, adhesion molecules, and retinal NV. Here, we investigate the molecular nexus between VEGF and NFκB in relation to retinal neovasculogenesis in the absence of the hypoxic microenvironment, and examine the effects of YC-1 on retinal neovasculogenesis in human retinal microvascular endothelial cells [hRMVECs].
Materials and Methods
Ethics Statement
All experiments were conducted in compliance with the laws and the regulations of the Kingdom of Saudi Arabia. In addition, all protocols were approved by the Institutional Review Board. This research study was approved by: The King Khaled Eye Specialist Hospital’s Human Ethics Committee & Institutional Review Board [HEC/IRB], Riyadh, Saudi Arabia. The permit number/approval ID is “RP 0630-P”.
Reagents
YC-1 [Fig. 1A] was purchased from A.G. Scientific [San Diego, CA] and dissolved in sterile DMSO. SN50 [a cell-permeable synthetic peptide, known to inhibit the nuclear translocation of NFκB, and its negative control mutant peptide SN50M were obtained from Calbiochem [San Diego, CA]. TSA™ Kit #4 with Alexa Fluor® 568 tyramide was purchased from Molecular Probes, Inc [Eugene, OR]. Recombinant human VEGF165 was purchased from Chemicon [Temecula, CA]. Mouse monoclonal antibody that recognizes the active subunit [12H11] of RELA [NFκB/p65] [MAB3026] was obtained from Millipore [Billerica, MA, USA]. Anti-phospho-IkBα antibody was purchased from Cell Signaling Technology [Beverly, MA, USA]. Anti-IκBα monoclonal antibody [clone 6A920] was obtained from Caymen [Ann Arbor, MI]. Rabbit polyclonal anti-human SDF-1 [CXCL12] antibody was obtained from Novus Biologicals [Littleton, CO]. Rabbit polyclonal anti-CXCR4 antibody was obtained from Abcam [Cambridge, UK]. Rabbit monoclonal FAK Antibody [EP695Y] was purchased from Novus Biologicals [Littleton, CO]. Monoclonal Mouse IgG1 anti human αVβ3 [Clone 23C6] was obtained from R&D Systems [Minneapolis, MN]. Anti-human integrin α5β1 monoclonal antibody [clone JBS5] was obtained from Bioscience Research Reagents [formerly Chemicon] [Temecula, CA]. Anti-EPO monoclonal antibody was purchased from R&D Systems [Minneapolis, MN]. Polyclonal rabbit anti-Endothelin-1 [ET-1] antibody was purchased from Abbiotec [San Diego, CA]. Monoclonal mouse anti-MMP-9 antibody was purchased from Neuromab [Davis, CA]. Rabbit anti-human β-actin monoclonal antibody was purchased from OriGene Technologies [Rockville, MD]. Rabbit anti-human IgG was purchased from Abcam [Cambridge, MA] and was used as an isotype control antibody for Western Blot and immunohistochemistry studies.
10.1371/journal.pone.0059021.g001Figure 1 YC-1 Exhibits Anti-Proliferative and Anti-Chemotactic Activities Under a Hypoxia-Independent Microenvironment in hRMVECs.
A. Chemical structure of YC-1. B. Cell Proliferation Assay. YC-1 Inhibited VEGF-stimulated proliferation of hRMVECs that were grown for 48 hours. Treatments with SN50 or YC-1 for 48 hours suppressed VEGF-induced ECs growth. Whereas treatments with DMSO or SN50M had no impact on cell proliferation. Evaluation of DNA contents reflected the proliferative vitality rates in different groups. Values are presented as mean ± SEM obtained from triplicate experiments [*P<0.05; **P<0.01; ***P<0.001]. C. YC-1 Inhibits hRMVECs Chemotaxis. VEGF165 caused a significant increase in hRMVECs chemotaxis [***P<0.001] as compared to cells that were cultured in medium only. Treatment with YC-1 inhibits VEGF-induced cell migration. The inhibitory effects of SN50 as compared to SN50M exhibited the specificity of NFκB-mediated increase in chemotaxis induced by VEGF. The lack of VEGF presence in the control medium has significantly suppressed the hRMVECs migratory ability. Means ± SEM obtained from 6 wells/treatment of 4 independent experiments [*P<0.05; **P<0.01; ***P<0.001 vs. DMSO-treated cells].
Tissue Culture
Human retinal microvascular endothelial cells [hRMVECs], attachment factor, complete growth medium were purchased from Cell Systems [Kirkland, WA]. Cells were cultured in 75-cm2 tissue culture flasks coated with attachment factor maintained in CS-C medium containing 10% FBS and CS-C growth factor at 1X in humidified conditions [5% CO2] at 37°C.
Assessments of Cell Proliferation
hRMVECs [40,000 cells/well] were grown in a 96-well plate and cultured in 150 ml of CS-C medium supplemented with 10% FBS, placed in a CO2 incubator at 37°C and allowed to adhere overnight. Fresh medium was added and the cells were cultured for 48 hours at 37°C in CS-C medium and under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated [100 µM], or VEGF-stimulated/YC-1-treated [100 µM]. In each of these experiments, the cells were lysed in 0.1% sodium dodecyl sulfate [SDS]; DNA content was measured by means of Hoechst-33258 dye and a fluorometer [model TKO-100; Hoefer Scientific Instruments, San Francisco, CA]. It has been shown that total cellular DNA content measured in this manner correlates closely with actual cell number, as determined by hemocytometer counting of trypsinized retinal ECs.
Chemoinvasion Assay
The invasiveness of hRMVECs was examined in vitro using a QCM™ 24-Transwell fluorimetric cell migration assay with polycarbonate filter inserts with 8.0-µm-sized pores. Briefly, the lower side of the filter was coated with gelatin [10 µl, 1 mg/ml], and the upper side was coated with Matrigel [10 µl, 3 mg/ml]. HRMVECs were grown to confluence in CS-C medium. To ensure precise results, hRMVECs cells were starved for 24 hours prior to the assay serum-free CS medium. HRMVECs cells [1×105 cells] were suspended in 200 µl of serum-free CS-C medium with 0.1% bovine serum albumin and loaded into the upper chamber of a transwell. YC-1 [100 µM], or DMSO [0.2%], or SN50 [20 µM], or SN50M [20 µM] were added 1 hour before the assay and remained in the culture throughout the experiment. VEGF was diluted to 30 ng/ml in 0.6 ml of M199/0.1% bovine serum albumin and added to the lower wells of the chamber. Other lower wells were left with medium only. The chambers were incubated for 24 hour at 37°C in an atmosphere of 95% air and 5% CO2. The migrated cells were fixed with cold 70% methanol for 15 minutes and stained with the CyQuant Cell Stain Solution [Chemicon, CA]. The dye mixture was transferred to a 96-well microtiter plate suitable for measurement. Cell migration was identified by fluorescence plate reader using 480/520 nm filter.
In vitro Angiogenesis Assay
The assay was conducted according to the manufacturer’s instruction [Chemicon, Temecula, CA] with modifications. Matrigel [10 mg/mL] was added to a 48-well plate and allowed to polymerize for 1 hour at 37°C. HRMVECs [6×104] were seeded onto the surface of the matrigel and cultured in CS-C medium, under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated [100 µM], or VEGF-stimulated/YC-1-treated [100 µM]. DMSO, YC-1, SN50, SN50M, were added 30 minutes prior to the incubation. Tube-like structure formation was examined 24 hours after treatment. The enclosed networks of complete tubes from four randomly chosen fields/well at a magnification of X10 objective were photographed using inverted bright field microscopy [Zeiss Axiovert 135, Thornwood, NY]. Cells were labeled by adding 50 ml/well of Calcein AM [8 mg/ml]. Images were acquired using fluorescence microscopy [Zeiss Axiovert] and a digital camera [AxioCam, NY]. A mean of the total tube length at the four different fields was determined by Axiovision® 3.1 software and measured according to the branching points between two ECs. The images were printed at a constant magnification, and the length of the tubes formed was measured using the Axiovision imaging software [Zeiss], followed by calculation of the total relative length of the tube-like structures formed as percentage to the control. The inhibition percentage was calculated using the following formula: IR = [1–(tubes YC-1/tubes control)]×100%.
Measurements of hRMVECs Lumen Formation
The assay was conducted according to the manufacturer’s instruction [Chemicon, Temecula, CA] with modifications. Matrigel [10 mg/ml] was added to a 48-well plate and allowed to polymerize for 1 h at 37°C. HRMVECs [6×104] were suspended in 3D collagen gels and cultured in CS-C medium for 24 hours. The effects of exogenous addition of VEGF, DMSO, SN50M, SN50, and YC-1 on EC lumen formation over the time course of 24 hours were evaluated. Immunofluorescence still photography was performed using a fluorescence microscopy [Zeiss Axiovert] and a digital camera [AxioCam, NY]. After image acquisition, the values of immunofluorescence staining were analyzed and quantified using Metamorph™ imaging analysis software version 6.0 [Universal Imaging, Sunnyvale, CA]. EC Lumen areas per high power field were determined by tracing EC lumens using Metamorph software from acquired images.
Evaluation of NFκB/p65 Transcription Factor Activity [ELISA]
ELISA assay was done after culturing the cells for 18 hours at 37°C in CS-C medium, under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated, or VEGF-stimulated/YC-1-treated [100 µM]. DMSO, YC-1, SN50, SN50M, were added 30 minutes prior to the incubation. Activation of the transcription factor NFκB was measured using a DNA-binding assay [Trans-AM™ NFκB/p65 Transcription Factor Assay Kit, Active Motif, Carlsbad, CA] according to manufacturer’s instructions. This is an ELISA-based method designed to specifically detect and quantify NFκB/p65 subunit activation, with high sensitivity and reproducibility. Nuclear protein extract was obtained using Nuclear Extract Kit [Active Motif] according to manufacturer’s instruction. Subsequently, a specific double stranded DNA sequence containing the NFκB response element was immobilized onto the bottom of the wells of the plate. Fifty [50 µg] of nuclear proteins were prepared, added to the wells, and incubated overnight at 4°C. NFκB binding specifically to the NFκB response element was detected by addition of specific primary antibody directly against NFκB/p65. A secondary antibody conjugated to HRP was added and incubated for 1 h at room temperature to provide a sensitive colorimetric readout at 450 nm.
Subcellular Fractionation: Cytoplasmic and Nuclear Fractions
Adherent cells were scraped into ice-cold PBS harvested by centrifugation and washed once with ice-cold PBS. Cell-pellets were then lysed in hypotonic lysis buffer [5 mM HEPES, 1 mM MgCl2, 0.2 mM EDTA, 0.5 M NaCl, 25% glycerol, pH 7.0]. After incubation on ice for 10 minutes, lysates were centrifuged [13,000 g, for 5 minutes, at 4°C] to remove nuclei and cell debris. The cleared lysates were then removed to fresh tubes, frozen and stored at –20°C for subsequent estimation of protein concentration and use in Western blotting. The nuclei pellets were resuspended in hypertonic extraction buffer [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, pH 7.9] for 1–2 hours at 4°C under agitation. After centrifugation [13,000 g for 10 minutes at 4°C], supernatants containing the nuclear protein were removed to fresh tubes and stored at –70°C. Protein concentrations were assessed by reaction with Bradford reagent [0.1% Coomassie blue G, 5% methanol, orthophosphoric acid].
Western Blot
To determine the effects of YC-1 on VEGF-dependent IκBα phosphorylaion and degradation, and p65 translocation; hRMVECs were seeded overnight in 6-well plates [2×106/ml]. Subsequently, hRMVECs were cultured for 8 hours at 37°C in CS-C medium, under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated, or VEGF-stimulated/YC-1-treated [100 µM]. DMSO, YC-1, SN50, SN50M, were added 30 minutes prior to the incubation. Cytoplasmic and nuclear extracts were prepared as previously described in the “Subcellular Fractionation: Cytoplasmic and Nuclear Fractions” section. Nuclear fraction was collected to examine the effects of YC-1 on nuclear translocation of p65. The protein concentration was determined using the Bradford assay [Bio-Rad] with BSA as a standard. Equal amount of cytoplasmic protein [30 mg] was resolved on 10% SDS-PAGE gel. Separated proteins were transferred to a nitrocellulose membrane [Roche, USA]. To avoid unspecific binding, the membranes were incubated in TBS containing 5% skim milk and 0.1% Tween 20 for 1 h at room temperature, and then immunoblotted with specific antibodies against; IκBα [1∶1000], phosphorylated IκBα [1∶1000], and β-actin [1∶1000]. To determine the effect of YC-1 on p65 translocation, 50 µg of nuclear protein were resolved on 10% SDS-PAGE, transferred and blotted with specific p65 antibody [1∶1000]. Negative control experiments consisted of omission of the primary antibody and utilizing a rabbit anti-human IgG [isotype control antibody] as a replacement. All the membranes were incubated overnight at 4°C. The membranes were washed, exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and finally the blots were detected by enhanced chemiluminescence reagent [Amersham Pharmacia Biotechnology, Little Chalfont] and analyzed using Quantity One software [BioRad, USA].
In another set of experiments; we investigated the influence of YC-1 on the protein expression levels of other molecules [SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9]. Briefly, hRMVECs were seeded overnight in 6-well plates [2×106/ml]. Subsequently, hRMVECs were treated with YC-1 [100 µM], or DMSO [0.2% v/v], SN50 [20 µM], or SN50M [20 µM] and then stimulated with 30 ng/mL VEGF for 48 hours at 37°C. Reactions were terminated by addition of lysis buffer [Cell Signaling, Beverly, MA]. Protein content of the cell lysates was determined according to the Bradford method [Bio-Rad, Hercules, CA]. Aliquots [40 µg] of whole-cell lysates were separated on 7.5% SDS-PAGE, and electro-transferred onto polyvinylidene membranes [Amersham Pharmacia Biotech, Little Chalfont]. After blocking with 5% nonfat dry milk in TBS-T, the blots were incubated overnight with anti-[SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, MMP-9, and β-actin] antibodies. Negative control experiments consisted of omission of the primary antibody and utilizing a rabbit anti-human IgG [isotype control antibody] as a replacement. Then blots were washed 3×10 minutes washes in PBS/tween and subsequently incubated with peroxidase-conjugated anti-mouse IgG secondary antibody at 1∶3000. The signals were obtained by enhanced chemiluminescence [Amersham Biosciences], and visualized by exposure to X-ray film. Upon completion of chemiluminescence, equal lane loading was checked by Ponceau S Solution [Sigma, St. Louis, MO]. X-ray films were scanned with a computer-assisted densitometer [model G-710; Bio-Rad] to quantify band optical density [Quantity One software; Bio-Rad].
Immunocytochemistry: Immunolocalization of NFκB/p65
The effect of YC-1 on the nuclear translocation of p65 was examined by immunocytochemistry. Briefly, hRMVECs [105 cells/well] were grown on 8-well chamber slides and cultured for 8 hours at 37°C in CS-C medium and under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated, or VEGF-stimulated/YC-1-treated [100 µM]. DMSO, YC-1, SN50, SN50M, were added 30 minutes prior to the incubation. The cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.2% Triton™ X-100 in PBS. The cells were incubated for 2 hours with anti-NFκB/p65 antibody. Negative control experiments consisted of omission of the primary antibody and utilizing a rabbit anti-human IgG [isotype control antibody] as a replacement. Cells were then incubated with HRP-conjugate working solution, followed by the addition of the Tyramid solution [TSA Kit#4] at 1∶100 dilutions [Molecular Probes, Carlsbad, CA]. Digitized images were acquired utilizing AxioVision software [Zeiss Axiovert 135 and AxioCam]. Intensity values of immunofluorescence staining of NFκB/p65 in cells was analyzed and quantified using Metamorph™ imaging analysis software version 6.0 [Universal Imaging, Sunnyvale, CA]. The staining intensity in our series ranged from a weak blush to moderate or strong. The amount of cells staining with the antibody was further categorized as focal [<10%], patchy [10%–50%], and diffuse [>50%]. For semiquantitative analysis, focal and/or weak staining was considered equivocal staining, and patchy or diffuse staining was subcategorized as either moderate or strong.
ELISA: Measurements of the Nuclear Translocation of NFkB/p65
Cells [106 cells/well] were cultured for 8 hours at 37°C in CS-C medium and under one of the following conditions; VEGF-stimulated [30 ng/ml], VEGF-stimulated/DMSO-treated [0.2%], VEGF-stimulated/SN50M-treated [20 µM], VEGF-stimulated/SN50-treated, or VEGF-stimulated/YC-1-treated [100 µM]. DMSO, YC-1, SN50, SN50M, were added 30 minutes prior to the incubation. The relative increase of NFκB/p65 translocation into the nucleus was measured using an ELISA according to the manufacturer’s protocol [IMGENEX, San Diego, CA]. In brief, the cells were centrifuged at 400 g for 1 minute and washed with cold PBS. The cells were lyzed by 400 µl of hypotonic buffer and 30 µl of 10% NP-40 was added. The mixture was centrifuged at 18000 g for 30 s. The supernatant was used as cytoplasmic extract. To the pellet was added 220 µl of nuclear extraction buffer and centrifuged at 18000 g for 1 minute. The supernatant was used as nuclear extract. The anti-p65 antibody coated plate captured nuclear or cytoplasmic free p65 of samples [0.5–1 mg/ml of protein] and the amount of bound p65 was detected by adding a secondary antibody followed by alkaline phosphatase-conjugated secondary antibody. The absorbance value for each well was determined at 405 nm by a microplate reader [Bio-Rad]. The relative ratio of nuclear to cytoplasmic p65 was calculated from the absorbance value of nucleus divided by that of cytoplasm.
Quantitative RT-PCR by Molecular Beacon Assays
The mRNA levels for all genes [NFκB/p65, SDF-1, CXCR4, FAK, αV, α5, β3, β1, EPO, ET-1, and MMP-9] were quantified by Real time RT-PCR using specific primers. Gene-specific molecular beacons and primers were designed to encompass the genes of interest, with beacon’s annealing site to overlap with the exon-exon junctions for additional specificity [Beacon Designer 6.0, Premier Biosoft International, Palo Alto, CA, USA]. Threshold cycle [Ct] values for the different samples were utilized for the calculation of gene expression fold change using the formula 2 to the minus power of delta delta ct. Fold changes in the [NFκB/p65, SDF-1, CXCR4, FAK, αV, α5, β3, β1, EPO, ET-1, MMP-9] genes relative to the β-actin endogenous control gene were determined by the following equation: fold change = 2–Δ [ΔC
T
], where change in threshold cycle [ΔC
T] = C
T [gene of interest] – C
T [β-actin] and Δ [ΔC
T] = ΔC
T [treated] – ΔC
T [untreated].
Statistical Analysis
Data are given as means ± S.E.M. All experiments were repeated at least three times independently. Statistical analysis between two groups was performed using Student’s t-test. One-way ANOVA, combined with Tukey’s multiple-comparison test, was used to evaluate the statistical significance of differences between three or more groups. Statistical significance was defined as *P<0.05; **P<0.01; ***P<0.001.
Results
YC-1 Specifically Inhibits VEGF-stimulated Proliferation of hRMVECs
To determine whether YC-1 [Fig. 1A] could suppress VEGF-induced cell proliferation, hRMVECs were stimulated with VEGF [30 ng/ml] for 48 hours and DNA content was evaluated. VEGF165 induced a significant increase in hRMVECs proliferation, by 70.8% ±0.1% [***P<0.001], as compared to cells that were cultured in the absence of VEGF [medium only] [Fig. 1B]. However, the proliferation rate was reduced to 80% ±0.02 [***P<0.001] in the presence of 100 uM YC-1, as compared to DMSO-treated cells. Treatment with SN50 [20 uM] for 48 hours suppressed VEGF-induced ECs growth by 86.2% ±0.3 [***P<0.001] as compared to mutated control peptide, SN50M [20 µM]. The IC50 value for the antiproliferative effects of YC-1 in the presence of VEGF was 55.30±0.1 µM. Dye-exclusion assay revealed that there were no significant differences observed in cell viabilities between YC-1-treated cells, as compared to DMSO-treated controls [data not shown].
YC-1 Inhibits hRMVECs Chemotaxis
VEGF165 caused a significant increase in hRMVECs chemotaxis [***P<0.001] [Fig. 1C]. This increase was inhibited by 4.65 folds in the presence of 100 uM YC-1 [***P<0.001], as compared to DMSO-treated cells. To determine whether NFκB induced the ECs activation and chemotaxis; NFκB peptide inhibitor, SN50 [20 µM] was added. Our results indicated that SN50 caused the inhibition of VEGF-induced chemotaxis by 7.50 folds, as compared to the mutated control peptide, SN50M [20 µM] treated cells. Under such conditions, no inhibition was observed indicating the specificity of NFκB-mediated increase in chemotaxis induced by VEGF. Cells that were incubated in medium only, exhibited an 815 folds decrease in their migratory ability, as compared to the VEGF-treated cells [***P<0.001].
Anti-angiogenic Effects of YC-1 on VEGF-induced Tube Formation
HRMVECs cultured on the surface of three-dimensional type I collagen gel have a cobblestone-like appearance when cultured in the absence of VEGF [Fig. 2A]. Stimulation of hRMVECs with VEGF165 [30 ng/ml] induced the formation of three-dimensional capillary-like tubular structures within 24 hours [Fig. 2B]. Furthermore, VEGF stimulation increased the elongation of these structures, and augmented the numbers of their tube multicentric junctions. Treatment of hRMVECs with SN50M or DMSO didn’t have any influence on the growth or the integrity of these tube-like structures [Fig. 2C and 2D]. The angiogenic ability of hRMVECs to spontaneously form branching and thick anastomosing capillaries in vitro was severely abrogated by either SN50 [20 uM] [Fig. 2E] or YC-1 [100 uM] [Fig. 2F], as compared to their respective controls; SN50M- or DMSO-treated cells, respectively. Tubular morphogenesis, an indicator of NV, was significantly abrogated at 24 hours after SN50- or YC-1-treatments. SN50 and YC-1 blocked VEGF-promoted angiogenesis, as evidenced by the significant shortening of the capillary tubules and the presence of isolated cell clumps with few sprouting capillaries. The mean tube lengths were 101±2 um and 160±21 um in the presence of SN50 and YC-1, respectively. This represented a significant decrease of 81% ±0.03 and 77% ±0.02 in tube length in the presence of SN50 and YC-1, respectively [***P<0.001], as compared to their respective controls; SN50M- and DMSO-treated cells, respectively [Fig. 3A]. SN50 and YC-1 treatments significantly [**P<0.01] decreased the number of lumen formed in these ECs by 76% ±0.03 and 70% ±0.1, respectively [Fig. 3B], as compared to their respective controls; SN50M- and DMSO-treated cells, respectively.
10.1371/journal.pone.0059021.g002Figure 2 YC-1 Displays Anti-Angiogenic Properties via the Inhibition of VEGF-Stimulated Tube Formation of hRMVECs.
Representative micrographs exhibit the presence of cobblestone-like appearance when cells are grown in the absence of VEGF [A]. In the control groups [B, C, and D]; exposure of hRMVECs to VEGF165 [30 ng/ml] within 24 hours caused a rapid alignement of cells with one another to form tube-like structures. VEGF stimulation caused an increase in the elongation of the tube-like structures and the numbers of their tube multicentric junctions. Treatment with SN50 [E] or YC-1 [F] or caused a severe abrogation to the tube formation. Scale bars 75 mm.
10.1371/journal.pone.0059021.g003Figure 3 Assessments of the Inhibitory Effects of YC-1 on hRMVECs.
A. Quantitative Assessments of the Inhibitory Effects of YC-1 on Tube Formation. The capillary tubules were significantly shortened when treated with YC-1 or SN50, as compared to their respective controls; DMSO or SN50M, respectively. Data represent means ± SEM from three independent experiments with 3 replications in each experiment [*P<0.05; **P<0.01; ***P<0.001, as compared with the DMSO-treated controls by Student’s two-tailed t-test]. B. Quantitative Assessments of the Inhibitory Effects of YC-1 on Lumen Formation. The EC lumenal area were quantitatively measured over 24 hours by tracing EC lumenal areas using the Metamorph software program in order to systemically analyze EC lumen formation. The effects of addition of VEGF, DMSO, SN50M, SN50, and YC-1 on EC lumen formation over the time course of 24 hours, with the bar graph highlighting EC lumens per high power field [HPF] at 24 hour time point. SN50 and YC-1 treatments significantly [***P<0.001] decreased the number of lumen formed in hRMVECs as compared to their respective controls, SN50M and DMSO, respectively. Graph showing the mean number of EC lumens per high power field [HPF] ± S.D. [n = 3]. ***P<0.001; **P<0.01, as compared with controls.
YC-1 Inhibits VEGF-induced NFκB Activation
In order to determine that VEGF effects on hRMVECs were mediated via the increase in the level of NFκB binding activity, we measured the NFκB/p65 activity by ELISA in hRMVECs, as compared to cells cultured in medium only [Fig. 4A]. VEGF induced a significant [***P<0.001] [98.3% ±0.01] upregulation in NFκB/p65 binding activity, as compared to cells that were incubated in medium only [no VEGF]. Treatment of cells with SN50 or YC-1 resulted in a significant [***P<0.001] attenuation of the VEGF-induced activation of NFκB binding activity. The extent of NFκB/p65 inhibition with SN50 or YC-1 was found to be 85% ±0.01, and 67% ±0.4, respectively, as compared to their respective controls; SN50M- and DMSO-treated cells, respectively [Fig. 4A]. Taken together, these data demonstrate that VEGF-stimulated effects are mediated via the activation of NFκB pathway, and YC-1 significantly inhibits such activity.
10.1371/journal.pone.0059021.g004Figure 4 YC-1 Impairs VEGF-Induced NFκB Activation by Inhibiting IκBα Phosphorylation in VEGF-Treated Cells.
A. The Influence of YC-1 on VEGF-Induced NFκB Transcriptional Activity.The graph illustrates the suppression of NFκB Activation by YC-1 in hRMVECs. ELISA assay was done after 18 hours of incubation with YC-1. Columns represent the means derived from three individual experiments. [*P<0.05; **P<0.01; ***P<0.001, as compared with controls. B. Inhibition of IκBα Phosphorylation and Accumulation of IκBα by YC-1. Cells were incubated in the absence or presence of 100 µl YC-1 for 8 hours. Cell extracts were then subjected to Western blotting using IκBα and p- IκBα antibodies. The blots exhibit the inhibitory influence of SN50 and YC-1 on the expression of the phosphorylated form of IκBα. C. The Effects of YC-1 on Intranuclear Expression of NFκB/p65. Cells were treated with one of the following conditions; DMSO, SN50M, SN50, or YC-1 in the presence or absence of VEGF for 8 hours. Nuclear extracts were prepared and assayed for NFκB/p65 by Western-blot as described in materials and methods. Both SN50 and YC-1 specifically inhibited the intranuclear expression of NFκB/p65 in cell preparations.
Inhibition of IκBα Phosphorylation and the Accumulation of IκBα in YC-1-treated Cells
In order to determine; 1) whether VEGF influence on hRMVECs was mediated via NFκB pathway; and 2) whether the inhibition of NFκB activation by YC-1 was due to decreased degradation of IκBα, we examined IκBα degradation in response to VEGF stimulation. Because the degradation of IκBα normally requires the inhibitor to be phosphorylated, it was of interest to examine the extent of IκBα phosphorylation in SN50- and YC-1-treated cells, as compared to their respective controls; SN50M- and DMSO-treated cells. Western blotting for IκBα was done as an index of total inhibitor expression levels. Our data demonstrate that VEGF treatment promoted NFκB/p65 activation via upregulating the phosphorylation status of IκBα, which peaked at 8 hours following exposure to VEGF165; in addition, it increased its intrinsic hydrolysis activity [Fig. 4B]. Furthermore, our data reveal that blockade of IκBα phosphorylation with the specific NFκB inhibitor, SN50 [20 µM], significantly [***P<0.001] attenuated VEGF-stimulated IκBα phosphorylation, as compared to its respective control SN50M [Fig. 4B]. Likewise, the effects of VEGF on IkBα phosphorylation and degradation was significantly attenuated [***P<0.001] in the presence of YC-1 [100 µM] as compared to DMSO-treated cells [Fig. 4B]. Taken together, SN50 and YC-1 induced significant downregulations in the expression level of the phosphorylated form of IκBα [p- IκBα], while it induced significant upregulations in the levels of total IκBα expression levels, as compared to their respective controls; SN50M- and DMSO-treated cells, respectively [Fig. 4B]. These results are indicative that YC-1 induced the downregulation of the NFκB activity via; 1) blocking the degradation of IκBα; and 2) promoting the dephosphorylation and the accumulation of IκBα.
YC-1 Impairs VEGF-induced Nuclear Translocation of NFκB/p65 Subunit
The effects of YC-1 on NFκB signaling were further explored by examining the nuclear translocation of the NFκB/p65 subunit in controls versus treated hRMVECs preparations. Our Western blot studies have indicated that SN50- and YC-1-treatments inhibited the nuclear translocation of NFκB/p65 protein, as compared to their respective controls; SN50M- and DMSO-treated cells [Fig. 4C].
In a different set of studies, our immunocytochemistry data have revealed that cells that were cultured in medium only exhibited the lack of p65 cytoplasmic and/or nuclear staining [Fig. 5A]. Whereas the cells that were stimulated with VEGF exhibited a significant increase [***P<0.001] in the signal intensity levels of dissociated nuclear p65, which was increased by 83.5 folds following VEGF treatment [Fig. 5B], as compared to cells that were cultured in medium only, which indicate that VEGF activates the canonical NFκB pathway. Cells that were stimulated with VEGF only, and/or treated with SN50M or DMSO; exhibited high levels of NFκB/p65 immunoreactivity, which was preferentially localized in the nuclei of the cells. In addition, there was a positive strong staining signal of NFκB/p65 deposited over the cytoplasms of the cells of these groups [Fig. 5B and 5C]. No NFκB/p65 staining was observed in experiments in which the primary antibody was omitted [data not shown]. Treatment of cells with SN50 or YC-1 had significantly abolished the VEGF-induced nuclear shuttling mechanism of p65 subunit, and ultimately blocked 88% and 82% of the VEGF-induced increase in NFκB/p65 levels, respectively, as compared their respective controls; SN50M-treated cells or DMSO-treated cells, respectively [Fig. 5C and 5D versus Fig. 5E and 5F]. Hence in the SN50 or YC-1-treated groups; the nuclear expression was virtually eliminated, yet few cells displayed the presence of cytoplasmic localization but then with equivocal “moderate or weak” staining intensity, in addition, a few stained regions were still detected in the nuclei. We demonstrate that VEGF-induced nuclear translocation of NFκB/p65 was severely abrogated in the presence of YC-1. This is in parallel with our Western blot data, which indicated a significant downregulation in the nuclear p65 levels in the SN50- and YC-1-treated groups. Taken together, these data indicate that YC-1 impairs VEGF-induced NFκB/p65 nuclear translocation.
10.1371/journal.pone.0059021.g005Figure 5 YC-1 Impairs VEGF-Induced Nuclear Translocation of NFκB. A–F. Immunofluorescence Analysis.
hRMVECs were incubated for 8 hours under various conditions and were subsequently fixed for immunocytochemistry. The staining intensity and the subcellular localization of NFκB/p65 were determined by immunofluorescence microscopy using anti-NFκB/p65 antibody. Photomicrographs, which exhibit intense nuclear and/or cytoplasm staining was considered a positive signal. Cells that were stimulated with VEGF alone (B), or incubated with either VEGF/SN50M (C) or VEGF/DMSO (D); exhibited high levels of NFκB/p65 immunoreactivity, which was preferentially localized in the nuclei of the cells. In addition, there was a positive intense staining signal of NFκB/p65 deposited over the cytoplasms and nuclei of the cells of these groups. Treatment of cells with SN50 [20 uM] or YC-1 [100 uM] resulted in a significant and almost complete inhibition of NFκB/p65 nuclear translocation. Images are representatives of three independent experiments. Scale bars, 200 mm.
In another series of experiments, cells were treated with either; YC-1 [20 µM], or DMSO [0.2%], or SN50 [20 µM], or SN50M [20 µM] in the presence or absence of VEGF [30 ng/ml], and later cells were analyzed by two different types of ELISA assays. The first ELISA assay detected the nuclear translocation of p65 [Fig. 6A], while the second assay was utilized to further quantify the nuclear/cytoplasmic ratio of p65 [Fig. 6B]. To conduct both assay, nuclear protein extracts were prepared from hRMVECs after exposure to VEGF165 [30 ng/ml]. Our results indicated that VEGF induced a significant [***P<0.001] increase 94.6% ±0.04 in NFκB/p65 nuclear translocation, as compared to cells cultured in medium only. Furthermore, treatment of cells with SN50 or YC-1 significantly [***P<0.001] reduced the nuclear NFκB/p65 translocation by 83.5% ±0.2 and 77% ±0.03, as compared to their respective controls; SN50M-and DMSO-treated cells, respectively.
10.1371/journal.pone.0059021.g006Figure 6 YC-1 Induced Specific Alterations in the Cytoplasmic-Nuclear Shuttling of NFκB/p65. A. ELISA Assay Analysis: YC-1 Inhibits Nuclear Translocation of NFκB/p65.
The relative nuclear translocation of NFκB/p65 in hRMVECs was measured by ELISA. ELISA was performed as described in Materials and methods. NFκB/p65 translocation to the nucleus was inhibited in YC-1- and SN50-treated cells. Data are expressed as Mean ± SD of triplicate cultures from at least three independent experiments. B. The Relative Ratio of Nuclear to Cytoplasmic p65 in the Cells Treated with YC-1. Data represent the mean ± SD of three [3] independent experiments. Statistical analysis was performed [*P<0.05; **P<0.01; ***P<0.001, as compared with the DMSO-treated cells by Student’s two-tailed t-test].
Our second ELISA assay has indicated that treatment of cells with SN50 or YC-1 resulted in a significant [***P<0.001] reduction in the p65 nuclear/cytoplasmic ratio, as compared to their respective controls; SN50M-treated and DMSO-treated cells, respectively [Fig. 6B]. These data are indicative that YC-1 inhibited nuclear translocation of NFκB/p65 subunit followed suppression of NFκB/p65 activity.
YC-1 Downregulates the Pro-angiogenic Gene Expression Profile in VEGF-stimulated hRMVECs
We have utilized quantitative real time RT-PCR to elucidate the molecular mechanisms involved in the regulation of VEGF-induced NFκB-dependent retinal neovasculogenesis. The mRNA expression levels of; NFκB/p65, SDF-1, CXCR4, FAK, αV, α5, β3, β1, EPO, ET-1, and MMP-9 were evaluated. The data were normalized to β-actin mRNA expression level. Our results demonstrate that there were significant upregulations in the message levels of the above mentioned pro-angiogenic genes in the VEGF-stimulated cells, as compared to cells that were cultured in medium only [Fig. 7A–F] and [Fig. 8A–E]. Treatment of hRMVECs preparations with YC-1 [100 µl] significantly downregulated the mRNA expression levels of the above mentioned genes, as compared with DMSO-treated cells. However, their expression level remained slightly higher than that of the cells that were cultured in medium only. The effects of sham treatment [DMSO] on the gene expression patterns paralleled those seen in the VEGF-stimulated cells. The mRNA expression levels of; NFκB/p65, SDF-1, CXCR4, FAK, αV, α5, β3, β1, EPO, ET-1, and MMP-9 were evaluated by using the primers that were summarized in [Fig. 9A].
10.1371/journal.pone.0059021.g007Figure 7 YC-1 Inhibits the Expression of NFκB/p65, SDF-1, CXCR4, αV, α5, and β1 in VEGF Stimulated Cells. A–F. Real Time RT-PCR Analysis.
The mRNA levels for NFκB/p65, SDF-1, CXCR4, FAK, αV, α5, β3, β1, EPO, ET-1, MMP-9, was quantified by Real time RT-PCR. The mRNA levels of these genes were upregulated in the cells that were cultured in the presence of VEGF [30 ng/ml] and either treated with DMSO [0.2%], or SN50M [20 µM]. Whereas the cells that were cultured in medium only exhibited significant low mRNA levels. Treatment of VEGF-stimulated cells with SN50 [20 µM] or YC-1 [100 µM] resulted in a significant downregulation of the mRNA expression of the genes listed above, as compared to respective controls; SN50M and DMSO-treated cells, respectively. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [***P<0.001 and **P<0.01, as compared to respective controls]. Data are representative of 3 independent experiments.
10.1371/journal.pone.0059021.g008Figure 8 YC-1 Inhibits the Expression of Various Pro-Angiogenic Molecules in VEGF-Stimulated Cells.
A–E. Real Time RT-PCR Analysis. The mRNA levels for β3, FAK, EPO, ET-1, and MMP-9, was quantified by Real time RT-PCR. The expression level was upregulated in the cells that were cultured in the presence of VEGF and either treated with DMSO or SN50M. Treatment with SN50 or YC-1 resulted in a significant downregulation of the mRNA expression, as compared to their respective controls; SN50M and DMSO-treated cells, respectively. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [***P<0.001 and **P<0.01, as compared to respective controls]. Data are representative of 3 independent experiments.
10.1371/journal.pone.0059021.g009Figure 9 YC-1 Inhibits the Protein Expression of Various Pro-Angiogenic Molecules in VEGF-Stimulated Cells.
A. Sequence for the Primer Sets Used for the Quantitative Real-Time PCR Analysis.
B. Western Blot Analysis. Protein expression levels were significantly elevated in the cells that were treated with SN50M or DMSO in the presence of VEGF [30 ng/ml]. In VEGF-stimulated/SN50-treated [20 µM], and VEGF stimulated/YC-1 treated [100 µM] cells, the protein expression levels were significantly decreased, as compared with SN50M and DMSO-treated cells. Statistical significance was determined by ANOVA [**p<0.01]. Data are representative of 3 independent experiments.
YC-1 inhibits the pro-angiogenic proteins expression in VEGF-stimulated hRMVECs
Western blot analysis demonstrated that cell exposure to VEGF induced a significant [***P<0.001] upregulation in the expression levels of SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9, as compared to cells that were incubated in medium only [Fig. 9B]. Treatment with YC-1 [100 µM] significantly inhibited the expression of levels [***P<0.001] of these proteins as compared to DMSO-treated cells [Fig. 9B]. Since YC-1 treatment did not inhibit β-actin, this indicates that YC-1 influence on the expression of the above proteins was specific.
Discussion
Hypoxia-induced expression of VEGF is a crucial mechanism that triggers an angiogenic response under physiological and pathological conditions. VEGF has been proposed to play an important role in the pathogenesis of diabetic vascular complications. Therefore, with the progress of DR; retinal ischemia and subsequent hypoxia may become a major determinant of VEGF. It has been suggested that there is a significant elevation of VEGF levels in ocular fluids obtained from patients with DR [20]. Conversely, neutralizing anti-VEGF antibodies in experimental animals have been implicated in the inhibition of VEGF signaling, the suppression of retinal NV [21], and the reversal of high glucose–induced vascular hyperpermeability [22]. Our current study demonstrates that VEGF treatment in hRMVECs promotes NFκB activation via; 1) upregulating the phosphorylation status of IκBα and increasing its intrinsic hydrolysis activity; 2) promoting the nuclear accumulation of p65; and 3) increasing the NFκB activity. Whereas YC-1 treatment induced the downregulation of the NFκB activation by preventing IκBα degradation, and hence inhibiting the nuclear translocation of NFκB/p65 subunit.
Previous studies have indicated that NFκB can regulate VEGF transcription [23]. Analyses of the VEGF promoter have not identified consensus and functional κB sites [24], and therefore, NFκB may regulate VEGF indirectly through other transcription factors. Our data suggest a pivotal role of NFκB activation in the development of diabetic microvascular angiopathy under a hypoxia-independent mechanism. The current study reveals that treatment of hRMVECs with VEGF enhances NFκB binding activity and invokes the expression of several pro-angiogenic factors [SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9] via NFκB-dependent mechanism. This upregulation was attenuated by the NFκB inhibitor SN50 and by YC-1 suggesting a common effecter target for the peptide SN50 and the sGC activator, YC-1. Our observations exhibits that YC-1 exerted an inhibitory effect on several essential steps of retinal neovasculogenesis, including cell invasion and migration, through NFκB signaling pathway. These observations are in parallel with previous studies, which indicated that YC-1 abolished constitutive nuclear translocation and activation of NF-kappaB/p65 in PC-3 cells [25]. Furthermore, it has been demonstrated that in STZ-induced diabetes, there was a significant increase in HIF-2α in the retinas of the diabetic rats, which was independent of hypoxia [12]. Our observations underscore the complexity and diversity of such neovascular angiogenic response, specifically in the absence of hypoxia.
During this investigation we report for the first time the molecular nexus between VEGF and NFκB in relation to retinal neovasculogenesis in the absence of the hypoxic microenvironment, and investigated the possible pathological role, which may play in instigating retinal NV. Furthermore, we have now analyzed the effects of YC-1 on VEGF-induced stimulation of NFκB, which mediated a significant upregulation in the expression of various pro-angiogenic molecules and augmented retinal neovasculogenesis in hRMVECs. The use of YC-1, with its pleiotropic effects [26], [27] may be necessary to offset such compensatory angiogenic responses and maximize therapeutic outcomes. Although our data may suggest the potential therapeutic use of YC-1 in ocular diseases, it is imperative that a suitable in vivo model is utilized to demonstrate the full potential of sGC agonists in retinal vasculopathy.
The authors owe a considerable debt of sincere gratitude to Mr. Gabriel DeNiro and Ms. Adara DeNiro, who were abundantly helpful and offered invaluable technical assistance in the quantification of the immunohistochemical staining, processing and analysis [Metamorph Analysis], as well as editing the references [Endnote]. We owe special thanks to Mr. Melvin Velasco for his design expertise throughout the various stages of this project. We thank Mr. Fadi Alkayal, Dasman Diabetes Institute, Kuwait, for his technical assistance in conducting minor segments of the real-time PCR experiments.
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Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 10.1155/2013/140509Review ArticleChemistry and Biology of Essential Oils of Genus Boswellia
Hussain Hidayat
1
2
*Al-Harrasi Ahmed
1
Al-Rawahi Ahmed
1
Hussain Javid
1
1Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, Birkat Al-Mouz, 616 Nizwa, Oman2Department of Chemistry, University of Paderborn, Warburger Straße 100, 33098 Paderborn, Germany*Hidayat Hussain: [email protected] Editor: Ruixin Zhang
2013 6 3 2013 2013 1405092 12 2012 28 1 2013 Copyright © 2013 Hidayat Hussain et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The properties of Boswellia plants have been exploited for millennia in the traditional medicines of Africa, China, and especially in the Indian Ayurveda. In Western countries, the advent of synthetic drugs has obscured the pharmaceutical use of Boswellia, until it was reported that an ethanolic extract exerts anti-inflammatory and antiarthritic effects. Frankincense was commonly used for medicinal purposes. This paper aims to provide an overview of current knowledge of the volatile constituents of frankincense, with explicit consideration concerning the diverse Boswellia species. Altogether, more than 340 volatiles in Boswellia have been reported in the literature. In particular, a broad diversity has been found in the qualitative and quantitative composition of the volatiles with respect to different varieties of Boswellia. A detailed discussion of the various biological activities of Boswellia frankincense is also presented.
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1. Introduction
The genus Boswellia has approximately 20 species occurring in the dry regions spanning from west Africa to Arabia and south to the northeast region of Tanzania. In addition, its species have been found in India and Madagascar. The genus is centered in northeast Africa, where approximately 75% of the species are endemic to the area. Members of this genus are trees or shrubs that are described as having outer barks that peel in parchment flakes, a greenish inner bark, watery aromatic resins, and wood with a milky latex [1].
Frankincense, or olibanum, is the oleogum resin that is harvested from several different trees belonging to the genus Boswellia. The word frankincense is derived from the ancient French name “frankincense,” meaning “pure incense.” Frankincense is also known in Arabic as “luban,” which means “white” or “cream;” in Greek as “libanos;” in Ethiopia as “etan” [2–8]. Olibanum (frankincense) has been used as incense since ancient times [9, 10]. In recent years, it has been important in the preparation of cosmetics and perfumes. In addition, olibanum has anti-inflammatory, sedative, anti-hyperlipidemic, and antibacterial activities in Unani (Islamic) and Chinese traditional medicines [9–15].
For at least 5000 years, olibanum has been an important trade material for the civilizations located in North Africa and the Arabian Peninsula. It was a precious commercial material even before Christian times because of the interest in this incense material of the old kings and queens, such as the Queen of Saba in 700 B.C. With the dawn of Christianity, it was mentioned in the Bible as one of the presents in which the three wise men had brought to Jesus on the night he was born [16]. The wide use of this resin in religious ceremonies as incense material is still important in the Roman Catholic, Episcopal, and eastern Orthodox churches. Therefore, both the production and export of olibanum is an economic priority for countries like Somalia, Ethiopia, Oman, South Arabia, and India. Olibanum has been utilized as an important fixative in perfumes, soaps, creams, lotions, and detergents in the leading products of the perfume and cosmetic industry, as it has an oriental note in its scent. The interest of pharmaceutical companies created a third market for olibanum. Since ancient times, it has been used in folk medicine for its antiseptic, antiarthritic, and anti-inflammatory effects. For this reason, olibanum has gained increasing attention from scientists in the last 20 years to better define its medical effects and identify the constituents that are responsible for these effects [16].
2. Volatile Constituents of Boswellia
Investigations of the resin and essences or extracts of Boswellia, with regard to the specific volatile constituents, were reported in a series of studies [16–35] (Table 1). The chemical characterization procedures enabled a total of 340 volatiles to be identified. Due to the resin nature of frankincense, it is not surprising that the major part of the constituent volatiles belonged to the terpene and sesquiterpene families or their terpenoid derivatives (Table 1). The common compounds included α- and β-pinene, limonene, myrcene, linalool, and several others. Additionally, a series of purely hydrocarbon-terpene compounds was found. More than 30 compounds from the sesquiterpene and diterpene fractions have been identified, for example, α-cubebene, γ-cadinene, β-bourbonene, and α-phellandrene dimer, which was first reported by Basar [16]. Several oxygenated isoprenoid derivatives have also been identified, such as carbonyl derivatives (e.g., carvone, fenchone) and alcohol-containing terpene and sesquiterpene derivatives (e.g., trans-pinocarveol, cis-verbenol, and cembrenol), as well as ester-containing compounds (e.g., α-terpinyl acetate and bornyl acetate). In addition to these isoprenoid-derived compounds, a series of straight-chain alkyl-esters, such as octyl acetate and alcohols (decanol, hexanol), have been detected. Analyzing B. serrata by hydrodistillation, Singh et al. [22], Verghese et al. [23], and Camarda et al. [24] each found α-thujene to be the dominant substance. However, the specific values were in the range 22.7–61.4%. Although, Basar [16] claimed that myrcene was the prevalent volatile (38%), followed by α-thujene (12%). α-Pinene was found in all samples, with the relative amounts in the range of 3.3–11.2%. In the hydrodistillate of B. carter and B. sacra, the values of the detected substances differed to a major extent. Basar [16] claimed that octyl acetate (39.9%) was the main constituent, followed by 1-octanol (11.9%). Al-Harrasi and Al-saidi [25] found limonene (33.5%) and (E)-β-ocimene (32.2%) to be the predominant compounds, whereas Marongiu et al. [26] claimed that octanol acetate was the main volatile (45.2%), followed by phyllocladene (13.2%) and incensole acetate (13%). Mikhaeil et al. [20] identified duva-3,9,13-trien-1,5a-diol-1-acetate as the main volatile (21.4%), followed by octyl acetate (13.4%). Like Al-Harrasi, Camarda et al. [24] also reported limonene to be the dominant substance, albeit at half the abundance found by Al-Harrasi (18.2%). Furthermore, Camarda identified α-pinene as the second most abundant substance (15.1%). However, this finding is in contrast to other studies, in which the α-pinene contents were reported in the range of 0.7–15.1%. Baser et al. [27], Basar [16], and Camarda et al. [24] all claimed that limonene was the main constituent of B. rivae, with values in the range of 14.8–28.0%. Again, another dominant substance seemed to be α-pinene, with relative values of 5.3–16.7%. In agreement with this, α-pinene, together with α-thujene, was found as the dominant substance in B. neglecta, whereas the most abundant volatile in B. papyrifera was octyl acetate at 63.5% [24] and 56% [36]. Furthermore, Basar [16] found that trans-verbenol was the main volatile (15.5%) in B. pirottae and α-pinene (38%) in B. frereana. Summarizing the data from the analyses of the hydrodistillation extracts, α-pinene, limonene, octyl acetate, a-thujene, and (E)-β-ocimene can be regarded as those compounds that have been most frequently reported to be the dominant volatile constituents of the frankincense distillate. Nevertheless, these results do not allow for chemosensory interpretation of their odor contribution to the characteristic frankincense smell. In accordance with the results of the hydrodistillation, Hamm et al. [18] found α-thujene (with a value of 11.7%) to be the dominant volatile in a B. serrata extract obtained by SPME. In Basar's study [16], thujene was also mentioned, but no quantitative data was provided. For the SPME analyses, Basar reported only qualitative data and no quantitative data. Hamm reported α-pinene, limonene, and β-caryophyllene to be the main constituents in B. carteri and B. sacra. By comparison, the α-phellandrene dimer was the dominant volatile (20.2%) in B. frereana, followed by α-pinene (12.4%). Octyl acetate was found in the greatest abundance (64.6%) in B. papyrifera, followed by octanol (13.9%) [1].
Al-Saidi et al. [34] reported the volatile chemical profile and physicochemical characteristics of four commercial grades of botanically certified oleogum resins of B. sacra, known as Hoojri, Najdi, Shathari, and Shaabi, were studied. The striking feature of Al-Saidi et al. [34] study was the presence of high amounts of α-pinene in all four oils, which can be considered as a chemotaxonomical marker that confirms the botanical and geographical source of the resins. Even though many Boswellia species produce frankincense, the major sources of commercial frankincense are B. serrata (India), B. sacra (Oman), and B. carteri (Somalia). The reports on the oil composition of B. serrata revealed a wide variation of major constituents, but none of the studies reported α-pinene as the most abundant component. Similarly, reports on the oil composition of B. carteri from Somalia also showed variation within the major constituents, suggesting a possible existence of different chemotypes. The Al-Saidi et al. [34] study reveals that the composition pattern of three of the Omani luban oils, that is, the Hoojri, Shathari, and Shaabi oils, was different from the above cited studies in terms of the major constituent α-pinene. The Najdi oil, however, showed some similarity with the Somalian B. carteri sample studied by Abdel Wahab et al. [37], with α-pinene and limonene as the major constituents. The variation observed might be expected, based on several factors such as climatic changes, harvest conditions, and geographical source.
Since the entire history of Boswellia nomenclature is fraught with misidentification, Woolley et al. [35] showed that the history of inaccurate frankincense taxonomy also applies to the widespread error of identifying B. carterii as being synonymous with B. sacra. Woolley et al. [35] studied the essential oil of B. carterii and B. sacra and showed that B. carterii can always be identified by the key markers viridiflorol, cembrenol, dimethyl ethermorcinol, and most importantly incensole. B. sacra was distinguished by higher quantities of α-pinene and delta-3-carene, while B. carterii possessed higher quantities of α-thujene, myrcene, limonene, trans-β-caryophyllene, germacrene D, and incensole. Woolley et al. [35] hypothesize that the differences in enantiomeric pair ratios of monoterpenes in Arabian B. sacra and African B. carterii resins were due to the differences in the abundance of genetically expressed chiral-specific enzymes for monoterpene biosynthesis. The most likely cause of genetic shift and speciation of B. sacra trees in Arabia and B. carterii trees in East Africa was the geological isolation created by the Red Sea Rift Valley that has separated these two tectonic plate land masses. Genetic mapping of these species might provide conclusive data to support Woolley et al. observations. Woolley et al. concluded that B. sacra and B. carterii are different species based on enantiomeric pair ratios and optical rotation.
Although α-pinene was the major compound and found in high concentrations in all grades of Omani frankincense (B. sacra) [34], this compound cannot be considered as a chemotaxonomic marker for B. sacra because of its frequent occurrence in other species of Boswellia (Table 2). Frankincense is a natural oleo-gum resin whose ingredients may depend on many factors, such as location, climate, time of harvest, and other environmental conditions. An indication of this variance could be clearly seen when comparing the different results of the samples of the same species (Table 2). A remarkable diversity of the predominant compounds in similar Boswellia species reported by different authors is clearly seen in Table 2. For example myrcene and α-thujene were individually reported to be major compounds for B. serrata. Mikhaeil et al. [20] reported duva-3,9,13-triene-1a-ol-5,8-oxide-1-acetate to be a major compound in the essential oil of B. carteri whereas Basar [16] found octyl acetate to be a major compound for the same species. Limonene, α-pinene, and octanol were reported individually to be major constituents for B. rivae. The incompatibility between the results mentioned in different literature could still be logical due to the influence of several factors mentioned earlier such as the climate, harvest conditions, and geographical source. However, such contradicting results make it difficult to rely on the chemical profile of the oil as a chemotaxonomic marker to distinguish between the different commercial varieties of frankincense.
3. Biological Activities of Essential Oils
3.1. Antioxidant Activity
Awadh Ali et al. [29] evaluated essential oils of Boswellia species for antioxidant activity. The essential oils were able to reduce the stable free radical DPPH with IC50 values of 121.4 μg/mL, 211.2 μg/mL, and 175.2 μg/mL for B. socotrana, B. elongate, and B. ameero, respectively. The positive factor for B. socotrana essential oils was the higher concentration of oxygenated monoterpenes [38, 39], but there are no data on the antioxidant activity of the oxygenated monoterpene (E)-2,3-epoxycarene, which is the main constituent in this oil. Boswellia essential oils showed lower free radical scavenging activity in comparison to other reported essential oils rich in oxygenated monoterpenes, such as Melissa officinalis and M. piperita with IC50 = 7.58 and 2.53 μg/mL, respectively [40, 41].
Recently, Mothana et al. [31] also evaluated the antioxidant activity of essential oils of Boswellia, demonstrating only weak antioxidant abilities in the reduction of DPPH. The three essential oils of B. dioscorides, B. elongate, and B. socotrana exhibited weak radical scavenging effects (22%, 21%, and 28%, resp.) at a concentration of 1 mg/mL. In comparison, ascorbic acid had a 96% antioxidant effect. This observation was certainly associated with the low content of phenolic components, such as thymol and carvacrol, in the three investigated oils [39].
3.2. Acetylcholinesterase Inhibition
Awadh Ali et al. [29] also evaluated the AChE inhibition of essential oils of Boswellia species. At a concentration of 200 μg/mL, essential oils of B. socotrana (59.3% inhibition) exhibited higher AChE inhibition than the essential oils of B. elongata and B. ameero (29.6 and 41.5% inhibition, resp.). The AChE inhibitory activity of B. socotrana oil may be due to the presence of (E)-2,3-epoxycarene and p-menth-1(7)-en-2-one, which belong to a group of monoterpenoid skeletons reported to have AChE inhibitory activity [42, 43]. Pulegone, a monoterpene with a p-menthane skeleton in Mentha spp, showed AChE inhibition with an IC50 of 890 μM [42].
3.3. Antimicrobial Activity
Camarda et al. [24] investigated the antimicrobial efficacy of B. carteri against Escherichia coli, Pseudomonas aeruginosa, and three strains of Staphylococcus aureus. Inhibitory activity was found against all pathogens, with the highest sensitivity noted for P. aeruginosa at concentrations as low as 6.6 μg/mL. Conversely, the essential oil of B. carterii was investigated for inhibitory activity against a Methicillin-resistant Staphylococcus aureus (MRSA) strain using a disc diffusion assay and found to have no inhibitory activity. In addition, Van et al. [32] reported that different fractions of essential oils of B. carteri, B. neglecta, B. sacra, B. thurifera, and B. frereana showed moderate to poor activity against a reference S. aureus strain (ATCC 12600).
Mothana et al. [31] evaluated the antimicrobial activity of B. dioscorides, B. elongate, and B. socotrana oils against two Gram-positive bacteria, two Gram-negative bacteria, and one fungal strain. The results indicated that the oils had varying degrees of growth inhibition against the bacterial strains. However, no activity was registered against the fungus Candida albicans. The Gram-positive strains showed more susceptibility to the tested essential oils than the Gram-negative ones. The essential oil of B. socotrana demonstrated the strongest activity with the lowest MIC values (1.87 mg/mL) obtained against Staphylococcus aureus and Bacillus subtilis.
Another study by Camarda et al. [24] demonstrated that the essential oils of four Boswellia species exhibited significant antifungal activity against both Candida albicans and Candida tropicalis. Camarda et al. [24] and Shao et al. [43] reported that limonene present in the essential oils was the component responsible for the antifungal activity. Hence, the absence of limonene in the essential oils explored by Mothana et al. [31] explained the lack of antifungal activity.
The MIC values of the tested essential oils were relatively lower than those of the positive controls (3.5–7.0 lg/mL). However, as crude oils, the overall antimicrobial activity screening results were still indicative of the potential of these herbal drugs to be effective treatments for bacterial infections. Moreover, oxygenated monoterpenes, such as camphor, borneol, linalool, and α-terpineol, were reported to be responsible for the antimicrobial activity of several essential oils [44, 45]. Consequently, the high antibacterial efficacy of B. socotrana could be attributed to the high percentage of oxygenated monoterpenes, such as camphor, α-fenchol, terpinen-4-ol, and borneol. Moreover, the predominance of 2-hydroxy-5-methoxy-acetophenone (16.3%) could have contributed to the strong activity [31].
Furthermore, B. rivae resin essential oil was tested for its antifungal activity against Candida albicans ATCC 10231. In previous reports, B. rivae essential oils showed the lowest MIC value of 2.6 μg/mL (0.3% v/v) against the same strain of C. albicans [24] among the oils tested. As hyphal formation is a morphogenetic process that contributes to the virulence of C. albicans [46], Schillaci et al. [47] opted to test the oil anti-germ tube formation activity. In this case, B. rivae oil demonstrated a particularly good activity as an inhibitor of germ tube formation with an IC50 value of 0.12 μg/mL (0.014% v/v). Thus, such low IC50 values for B. rivae were a good indication that this oil also had demonstrable antibiofilm activity. In fact, the authors observed the prevention of adhesion and biofilm formation at a sub-MIC concentration of 0.88 μg/mL (0.1% v/v). Moreover, the oil was significantly active at a concentration of 44.1 μg/mL (5% v/v) against a preformed 24 h old C. albicans biofilm [47]. The chief chemical component of B. rivae oleogum resin oil was limonene (28%), a monoterpene hydrocarbon with demonstrated antifungal activity [48]. However, it is difficult to attribute the anti-biofilm activity to one single component, and further studies are needed to understand the role of the components of such oil in the biological activity.
Al-Saidi et al. [34] reported antibacterial activity of oleo-gum resins of B. sacra, known as Hoojri, Najdi, Shathari, and Shaabi against. All the four oils were effective against both Gram-positive and Gram-negative bacteria. The clinical isolates of Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Klebsiella pneumoniae, and Enterobacter aerogenes were sensitive to all the oils, while those of Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris were resistant to the Shathari, Najdi, and Hoojri oils, respectively. The resistance of some of the Gram-negative bacteria to some of the oils could be due to the more hydrophilic outer membrane containing lipopolysaccharide. Small hydrophilic molecules are able to pass the outer membrane through porin channels, while the outer membrane acts as a penetration barrier for macromolecules and hydrophobic compounds. But the outer membrane is not completely impermeable to hydrophobic molecules; some of them can slowly pass through the porins. Hence, passing through the outer membrane contributes to the bactericidal activity of a compound. This could be the possible explanation for the sensitivity of some Gram-negative bacteria to the different luban oils [34].
Recently Abdoul-latif et al. [49] reported the antibacterial activity of essential oils of B. sacra and B. papyrifera. The best zone of inhibition of essential oil of B. sacra for bacteria was obtained for Enterococcus faecalis (37 mm), Shigella dysenteria (37 mm), Salmonella enterica (35 mm), Bacillus cereus (34 mm), and Listeria innocua (34 mm). Similarly best zone of inhibition of essential oil of B. papyrifera for bacteria was obtained for Salmonella enterica (40 mm), Bacillus cereus (39 mm), Enterococcus faecalis (39 mm), Shigella dysenteria (31 mm), and Staphylococcus camorum (30 mm). Interestingly essential oils of B. sacra and B. papyrifera present an antimicrobial activity stronger than the tetracycline.
3.4. Anticancer Activity
Frankincense oil-induced cell viability was investigated for essential oils of B. carterii in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells [50]. The results showed that within a range of concentrations, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activated genes that were responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis. Therefore, frankincense oil appeared to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that could be activated by frankincense oil to induce bladder cancer cell death [50].
Recently, Suhail et al. [51] showed that B. sacra essential oil suppressed important malignant features of tumor cells, such as invasion and multicellular tumor spheroid growth. Tumor cell plasticity enables highly malignant tumor cells to express endothelial cell-specific markers and form vessel-like network structures on basement membranes. The in vitro Matrigel-based tumor invasion model has been shown to correlate with in vivo metastatic potential [52]. This in vitro model has been used to study mechanisms of cancer aggressive behavior, metastasis, and poor prognosis [53], and it has been used as a tool to screen therapeutic agents for antimetastatic activity [54, 55]. MDA-MB-231 cells grown on Matrigel are more resistant to essential oil-suppressed cell viability than cells grown on tissue culture plates. This difference may result from the protective effects of the Matrigel basement membrane matrix enriched with various growth factors. In addition, cancer cells can form multicellular spheroid aggregates, which afford protection for cancer cells against some chemotherapeutic agents [56].
Multicellular tumor spheroids in culture have been used as an in vitro model for screening and testing anticancer drugs [57]. Similar to results from cytotoxicity and apoptosis, B. sacra essential oil obtained at 100°C in hydrodistillation is more potent than essential oil obtained at 78°C in disruption of cellular networks on Matrigel and spheroids. More importantly, observations obtained in the above-described experimental models were consistent with clinical responses in human cancer cases. These results suggested that B. sacra essential oil might represent an effective therapeutic agent for treating invasive breast cancer.
Aberrant activations of Akt and ERK1/2 MAPK signaling molecules have been identified in various cancers including breast cancer, and activations of Akt and ERK1/2 have been suggested as independent cancer prognostic markers. The Akt pathway has been found to be activated in early stages of breast cancer development [58], and activation of Akt signaling protects breast cancer cells from tamoxifen-induced apoptosis in vitro and confers poor prognosis in cancer patients [59, 60]. Activation of ERK1/2 has also been shown to be associated with the development of tamoxifen resistance and patient survival [61, 62]. Both Akt and ERK1/2 have been proposed as molecular targets for treating breast cancer, particularly in antiestrogen-resistant states [63, 64]. Targeting Akt signaling by inhibiting mTRO signaling has been shown to restore cancer responses to chemotherapy drugs [65, 66], and inhibition of both epidermal growth factor receptor (EGFR)/HER2 and MAPK signaling has been shown to result in growth inhibition and apoptosis of EGFR-expressing breast cancer cells [67]. Studies have shown that boswellic acids and AKBA activate the PI3 K/Akt pathway in human colon cancer HT29 cells [68]. Suhail et al. [51] demonstrated that B. sacra essential oil suppressed Akt and ERK1/2 activation in human breast cancer cell lines, except MDA-MB-231. The differences observed between boswellic acids and B. sacra essential oil may result from different tested tumor cell types or components other than boswellic acids present in the essential oil [51].
3.5. Antibiofilm Activity
Schillaci et al. [47] evaluated anti-biofilm activity of the commercially available essential oils from B. papyrifera against the preformed 24 h old biofilms of two bacterial strains: Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213. Interestingly, the anti-biofilm activity exhibited at the lowest concentrations of 13.6 μg/mL (1.5% v/v) and 6.8 μg/mL (0.75% v/v) was below that of the MIC concentration of 22.6 μg/mL (2.6% v/v), as determined against planktonic forms of both bacterial strains. These data were curious, considering that staphylococcal biofilms are usually resistant to conventional antibiotics at concentrations up to 1000 times the MIC.
4. Conclusion
None of the volatiles identified in the different studies have, according to our knowledge, ever been attributed to the specific smell of frankincense. It is interesting to note, however, that an olibanum-like odour has been reported elsewhere for a substance found in orange oil residue [21] which was identified as cis-iso-cascarilla acid. Nevertheless, this compound has not been reported as a constituent of frankincense in the literature discussed in this paper.
Furthermore, it remains unanswered whether there are substances with frankincense-specific odour qualities, or whether the characteristic smell of frankincense is due to a specific blend of dorants, as often observed in other food or plant aromas. Further research is therefore necessary to elucidate the specific contributors to the aroma profile of frankincense and frankincense pyrolysate. The essential oils of Boswellia plants showed different activities which is summarized in Table 3.
Table 1 Essential oil of Boswellia spp.
Number Compound
1 5,5-Dimethyl-1-vinylbicyclo-[2.1.1]-hexane
2 Anethol
3 Benzyl tiglate
4
trans-α-Bergamotene
5 Bornyl acetate
6
β-Bourbonene
7 Cadinene
8
γ-Cadinene
9 Camphene
10 Camphor
11
m-Camphorene
12
p-Camphorene
13 Carene-3
14 (E)-β-Caryophyllene
15 Cembrene A
16 Cembrenol
17 1,8 Cineol
18 Citronellol
19
α-Copaene
20
β-Copaene
21
p-Cymene
22
m-Cymene
23 Elemol
24 Elemicine
25
epi-Cubenol
26 Estragol
27 Eudesmol
28 10-epi-γ-Eudesmol
29 Fenchone
30 Geraniol
31 Germacrene D
32 Humulene epoxide
33 Isoincensole
34 Isomenthone
35 Kessane
36 Limonene
37 Linalool
38 Linalyl acetate
39 Menthone
40 Methylchavicol
41 Methylisoeugenol
42 Methyleugenol
43
γ-Muurolene
44 Myrcene
45 Neocembrene A
46 Nerolidol
47
cis-β-ocimene
48
(Z)-Ocimene
49
(E)-β
-Ocimene
50 Perillene
51
α-Phellandrene
52
β-Phellandrene
53
α-Pinene
54
β-Pinene
55
trans-Pinocarveol
56 Sabinene
57
cis-Sabinol
58 Terpinin-4-ol
59 Terpinen-4-ol
60 Terpinolene
61
α-Terpineol
62
α-Terpinene
63
α-Terpinene
64
γ-Terpinene
65 Terpinyl acetate
66 Terpinyl isobutyrate
67 Tetrahydrolinalool
68
α-Thujene
69
α-Thujone
70
β-Thujone
71 Tricyclene
72 Undecenol
73
trans-Verbenol
74
β-Ylangene
75 Zingiberene
76 Abieta-8,12-diene
77
α-Amorphene
78
alloaromadendrene
79 Benzyl benzoate
80 Beyerene
81 Bisabolene
82 Isopentyl-2-methylbutanoate
83
cis-Calamenene
84
α-Cadinene
85
τ-Cadinol
86 2-Carene
87 Campholenealdehyde
88 Caryophyllene oxide
89
cis-Carveol
90 (+) trans-Carveol
91 Carvone
92
α-Cedrene
93 Cedrol
94 Cembra-1,3,7,11-tetraene
95 Cembra-3,7,11,15-tetraene
96 Cembrene
97 Cembrene C
98 Citronellyl acetate
99
α-Cubebene
100
β-Cubebene
101
o-Cymene
102 Chrysanthenone
103 1,4-Cyclohexadiene
104
p-Cymen-8-ol
105 Decanol
106 Decyl acetate
107 2,6-Dimethoxytoluene
108 3,5-Dimethoxytoluene
109 Duva-3,9,13-trien-1,5α-diol
110 Duva-4,8,13-trien-1a,3α-diol
111 Duva-3,9,13-trien-1,5α-diol-1-acetate
112 Duva-3,9,13-triene-1α-ol-5,8-oxide-1-acetate
113
β-Elemene
114 Farnesyl acetate
115 Geranyl acetate
116
α-Gurjunene
117 Hedycariol
118 1,3,6-Trimethylencycloheptane
119 1-Hexanol
120 Hexyl acetate
121 Hexyl hexanoate
122
α-Humulene
123 Incensole
124 Incensole acetate
125
Isodurene
126 Isocembrene
127 Isophyllocladene (kaur-15-ene)
128 Kaurene
129 Ledol
130 Maaliane
131
p-Mentha-1,5-dien-8-ol
132
o-Methyl anisole
133
α-Muurolene
134
α-Muurolol
135 Myrtenal
136 Naphthalene
137
Naphthalene 1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl
138 Neryl acetate
139
cis-Nerolidol
140 (S)-trans-Nerolidol
141 (E)-Nerolidol
142 1-Octanol
143
n-Octanol
144 Octanol acetate
145 Octyl acetate
146 Octyl formate
147
allo-Ocimene
148 Phenanthrene-7-ethenyl-9,10,10a-dodeca-hydro-1-1-4a-
7-tetramethyl
149
α-Phellandrene epoxide
150 Phyllocladene
151
α-Pinene-epoxide
152 1-β-Pinene
153 2-β-Pinene
154 Isopinocampheol
155 Piperitone
156
Pyrimidine
157 Sabinyl acetate
158 Sandaracopimara-8(14)-15-diene
159 Sclarene
160
α-Selinene
161
β-Selinene
162
δ-Selinene
163
trans-Terpine
164 4-Terpineol
165 Terpinolene
166 Isoterpinolene
167 2,4(10)-Thujadiene
168 Thujopsene
169 Thunbergol
170 Isomyl-valerate
171 Verticilla-4(20),7,11-triene
172 Verbenone
173
cis-Verbenol
174 Verticiol
175 Viridiflorol
176 Benzene, 1methoxy-2-methyl
177
endo-Borneol
178
γ-Campholene aldehyde
179
α-Campholene aldehyde
180 Cara-2,4-diene
181 Carvacrol
182 Carvotanacetone
183
trans-Dihydrocarvone
184 Cumin alcohol
185
m-Cymene-8-ol
186
p-Cymene-9-ol
187
p-Cymenene
188 Dodecanol
189 Eucalyptol
190 Eucarvone
191 Isopropyl benzaldehyde
192 Isopropyl benzalcohol
193
cis-1,2-Limonene epoxide
194 8,9-Limonene epoxide II
195 8,9-Limonene-epoxide I
196
trans-1,2-Limonene epoxide
197
cis-Linalool oxide
198
trans-Linalool oxide
199
p-Mentha-1,5-diene-7-ol
200
p-Mentha-1,8-diene-4-ol
201
cis-p-Menth-2-en-1-ol
202
cis-p-Mentha-1(7),8-diene-2-ol
203
cis-p-Mentha-2,8-diene-1-ol
204
trans-p-Menth-2-en-1-ol
205
trans-p-Mentha-1(7),8-diene-2-ol
206
trans-p-Mentha-2,8-diene-1-ol
207 2,4(8)-p-Menthadiene
208
p-Mentha-6,8-dien-2-one
209
p-Methylanisole
210 Myrtenol
211 Nerol
212
trans-Ocimene
213 (E)-β-Ocimene epoxide
214
α-Phellandrene-dimer
215
α-Phellandrene-8-ol
216
α-Pinene oxide
217 Pinocamphone
218 Pinocarvone
219 Piperitenone
220 Isopiperitenone
221
trans-Piperitol
222
α-Terpineol
223 Sabina ketone
224
cis-Sabinene hydrate
225
trans-Sabinene hydrate
226
trans-Sabinol
227 2,5-Dimethylstyrene
228
cis-1,2-Epoxy-terpin-4-ol
229 Thuj-3-en-10-al
230 Thujanol
231 Thunbergene
232 Thymol
233 Umbellulone
234 Verticellol
235 5,5-Dimethyl-1-vinylbicyclo-[2.1.1]-hexane
236
p-Anisaldehyde
237 Aromadendrene
238 Benzyl tigilate
239
p-Camphorene
240 Isocaryophyllene
241 Cumaldehyde
242 Cyclosativene
243
γ-Eudesmol
244 Guaioxide
245 5-Guaiene-11-ol
246 Isogermacrene D
247 4-Methylene-1-(1-methylethyl)-bicyclo[3.1.0]hex-2-ene
248 2-Methyl-5-(1-methylethyl)-1,3-cyclohexadiene monoepoxide
249
n-Pentadecan
250 Perilla alcohol
251 Perillol
252 Thujol
253
m-Thymol
254
α-Ylangene
255
γ-Campholene aldehyde
256
n-Decanoic acid
257
β-Eudesmene
258
β-Cyclogeranylacetate
259
n-Hexanoic acid
260 Hexylcaprylate
261 Incensyl acetate
262 Incensole oxide
263 Incensole oxide acetate
264 Lauric acid
265
p-Methylacetophenone
266
p-Methyleugenol
267
β-Myrcene
268
n-Nonanoic acid
269
n-Octanoic acid
270 3,4-Dimethoxystyrene
271
α-Cadinol
272 1,Hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene
273 1,5,5,8-Tetramethyl-12-oxabicyclo-[9.1.0]-dodeca-3,7-diene
274 1-Methyl-4-(1-methylethenyl)-1,2-cyclohexanediol
275
trans-p-Mentha-2,8-dienol
276 1,2,3,4,6,8a-hexahydro-1-isopropyl-4,7-dimethyl-
naphthalene
277 2-Isopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,8a-ctahydronaphthalene
278 3,5-Dimethoxytoluene
279 (Z)-α-Hydroxymanool
280 Hydroxy-manool
281 Methyl linoleate
282 1-Acetyl-4-isopropenylcyclopentene
283 2,4-Dimethylacetophenone
284
α-Amyrenone
285
β-Amyrenone
286 10-Hydroxy-4-cadinen-3-one
287 2-Hydroxy-1,4-cineole
288 Cryptone
289 Eucarvone
290 Isopropylidencyclohexane
291 1,2,4-Trihydroxy-p-menthane
292 Δ4-p-Menthen-2-one
293 5-Hydroxy-p-menth-6-en-2-one
294 Myrtenoic acid
295 Nopinone
296 3,6,6,-Trimethyl-norpinan-2-one
297
o-Methylacetophenone
298 Perillaaldehyde
299 Phellandra
300 Pinocamphone/isopinocamphone
301 Thujone
302 24-Noroleana-3,12-diene
303 24-Noroleana-3,9(11),12-triene
304 24-Norursa-3,12-diene
305 24-Norursa-3,9(11),12-triene
306 24-Norursa-3.12-dien-11-one
307
α-Amyrine
308
epi-α-Amyrine
309
β-Amyrine
310 Lupeol
311 Terpinenyl acetate
312 1,5-Isopropyl-2-methylbicyclo[3.1.0]hex-3-en-2-ol
313
α-Campholenal
314 (3E,5E)-2,6-Dimethyl-1,3,5,7-octatetraene
315 (E)-2,3-Epoxycarene
316 3,4-Dimethylstyrene
317 1-(2,4-Dimethylphenyl)ethanol
318 4-Methylbenzoic acid
319
p-Menth-1(7)-en-2-one
320 Caryophyllene
321 Methylcycloundecanecarboxylate
322 Nonanoic acid
323 Hexadecanoic acid
324 1,4-Cineol
325 Sabinene hydrate
326 Methyl-trans-2-cis-4-decadienoate
327 2-Hydroxy-5-methoxy-acetophenone
328 (E)-β-Farnesene
329 2-Dodecenoic acid methyl ester
330 Calacorene
331
n-Dodecanoic acid
332
α-Guaiol
333 Caryophylla-3(15),7(14)-dien-6-ol
334 Cadalene
335 Eudesma-4(15),7-dien-1β-ol
336
n-Heptadecane
337
n-Tetradecanoic acid
338
n-Octadecane
339 Galaxolide
340 Manool
Table 2 Percentages of major compounds in the essential oils of reported Boswellia species.
Boswellia specie Method of obtaining resin Predominant compound(s) Percentage (%) Literature
B. serrata
Obatined from Willy Benecke GmbH (Hamburg, Germany) Myrcene 38 [16]
B. serrata
NA
α-Thujene 22.7–47.4 [22]
B. serrata
NA
α-Thujene 29.3 [24]
B. serrata
NA
α-Thujene 61.36 [23]
B. carteri
Purchased from the local market of herbs and spices in Egypt Duva-3,9,13-triene-1a-ol-5,8-oxide-1-acetate 21.4 [20]
B. sacra
Botanically certified oleogum resin
E-β-Ocimene 32.3 [25]
B. carteri/sacra
NM Octanol acetate 45.2 [26]
B. carteri
Authentic sample from Ethiopia certified for its authenticity from the Agricultural Department of the Ethiopian government Octyl acetate 39.3 [16]
B. rivae
NA Limonene 28.0 [24]
B. rivae
Authentic sample from Ethiopia
α-Pinene 16.7 [16]
B. rivae
NA
α-Pinene 13.3 [24]
B. rivae
NA Octanol 17.8 [24]
B. neglecta
NA
α-Pinene 16.7 [27]
B. neglecta
Authentic sample from Ethiopia
α-Pinene 21.3 [16]
B. papyrifera
NA Octyl acetate 63.5 [24]
B. papyrifera
NA Octyl acetate 56.0 [36]
B. pirottae
NA
Trans-Verbenol 15.5 [27]
B. pirottae
NA Terpinen-4-ol 14.6 [27]
B. frereana
Willy Benecke GmbH (Hamburg, Germany)
α-Pinene 38.0 [16]
NA: not available.
Table 3 Biological activities of essential oils of Boswellia genus.
Plants Biological activities of essential oils of Boswellia planta
Antioxidant AchEI inhibition Antimycrobial Anticancer Antibiofilm
B. socotrana
IC50 121.4 μg/mL, 59.3% Moderate activity NK NK
B. elongata
IC50 211.2 μg/mL 29.6 Moderate activity NK NK
B. ameero
IC50 175.2 μg/mL 41.5 NK Good activity NK
B. carteri
NKa
NK Moderate activity NK
B. neglecta
NK NK Moderate activity NK
B. sacra
NK NK Good activity Good activity NK
B. thurifera
NK NK Moderate activity NK
B. frereana
NK NK Moderate activity NK
B. dioscorides
NK NK Moderate activity NK
B. rivae
NK NK Good activity NK
B. papyrifera
NK NK Good activity Good activity
aNK: not known.
==== Refs
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23556005PONE-D-12-3345210.1371/journal.pone.0060827Research ArticleBiologyAnatomy and PhysiologyEndocrine SystemDiabetic EndocrinologyImmunologyImmune SystemCytokinesImmunityInflammationModel OrganismsAnimal ModelsMouseMedicineAnatomy and PhysiologyEndocrine SystemDiabetic EndocrinologyCardiovascularCardiomyopathiesEndocrinologyDiabetic EndocrinologyMyocardial Remodeling in Diabetic Cardiomyopathy Associated with Cardiac Mast Cell Activation Cardiac Mast Cells Mediate Diabetic CardiomyopathyHuang Zhi Gang
1
Jin Qun
2
Fan Min
1
Cong Xiao Liang
1
Han Shu Fang
2
Gao Hai
3
*
Shan Yi
4
*
1
Department of Cardiology, Chang Zheng Hospital, Second Military Medical University, Shanghai, China
2
Department of Cardiology, The General Hospital of Jinan Military Region, Jinan, China
3
The Third People's Hospital of Haiyang, Haiyang, Shandong, China
4
Department of Emergency Medicine, Chang Zheng Hospital, Second Military Medical University, Shanghai, China
Madeddu Paolo Editor
Bristol Heart Institute, University of Bristol, United Kingdom
* E-mail: [email protected] (HG); [email protected] (YS)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: ZH QJ YS. Performed the experiments: MF XC SH. Analyzed the data: ZH QJ HG YS. Contributed reagents/materials/analysis tools: HG YS. Wrote the paper: ZH QJ.
2013 29 3 2013 8 3 e6082729 10 2012 3 3 2013 © 2013 Huang et al2013Huang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Diabetic cardiomyopathy is a specific disease process distinct from coronary artery disease and hypertension. The disease features cardiac remodeling stimulated by hyperglycemia of the left ventricle wall and disrupts contractile functions. Cardiac mast cells may be activated by metabolic byproducts resulted from hyperglycermia and then participate in the remodeling process by releasing a multitude of cytokines and bioactive enzymes. Nedocromil, a pharmacologic stabilizer of mast cells, has been shown to normalize cytokine levels and attenuate cardiac remodeling. In this study, we describe the activation of cardiac mast cells by inducing diabetes in normal mice using streptozotocin (STZ). Next, we treated the diabetic mice with nedocromil for 12 weeks and then examined their hearts for signs of cardiac remodeling and quantified contractile function. We observed significantly impaired heart function in diabetic mice, as well as increased cardiac mast cell density and elevated mast cell secretions that correlated with gene expression and aberrant cytokine levels associated with cardiac remodeling. Nedocromil treatment halted contractile dysfunction in diabetic mice and reduced cardiac mast cell density, which correlated with reduced bioactive enzyme secretions, reduced expression of extracellular matrix remodeling factors and collagen synthesis, and normalized cytokine levels. However, the results showed nedocromil treatments did not return diabetic mice to a normal state. We concluded that manipulation of cardiac mast cell function is sufficient to attenuate cardiomyopathy stimulated by diabetes, but other cellular pathways also contribute to the disease process.
These authors have no support or funding to report.
==== Body
Introduction
Diabetic cardiomyopathy is defined as a primary disease process distinct from coronary artery disease and hypertension [1]–[3]. Hyperglycemia contributes to this condition as it induces metabolic disturbances that cause oxidative damage and deregulated cytokine signaling that result in cellular injury, impairment of cell-cell coupling, and apoptosis of myocardial cells. These events, in turn, activate collagen deposition and remodeling of the extracellular matrix [4]. The cumulative result is the stiffening of cardiac tissues that impair normal contractile functions. Therefore, structural abnormalities in the left ventricle (LV), such as interstitial and perivascular fibrosis, are common hallmarks attributed to diabetic cardiomyopathy [4].
Mast cells are recognized as active participants in allergic and anaphylactic reactions [5], [6]. Recent studies showed that mast cells also mediate a wide range of non-allergic reactions including autoimmunity [7], inflammation [8], and infection [9]. Mast cells are tissue-specific and respond to different stimulants in different tissues [10]. The cells function by producing secretory granules that release an assortment of bioactive molecules including cytokines, chemokines, and proteases, such as chymase and tryptase, into the surrounding tissues. In the context of diabetic cardiomyopathy, metabolic byproducts associated with diabetes, such as ROS and oxidized lipoproteins, may trigger mast cell activation [11], but the exact mechanism is unclear because mast cells are sensitive to many environmental stressors and those specific to diabetes have not been identified. However, there are evidence that mast cell activation may contribute, in part, to alterations in cardiac tissue [11]–[13].
There is indirect evidence that cardiac mast cell activation may contribute to cardiac remodeling. Mast cell degranulation has been observed in the human heart [14]. Increased mast cell density has been implicated in human cardiomyopathy [15], and LV fibrosis in hypertensive rat hearts [16]. The biomolecules released by cardiac mast cells may contribute to cardiovascular disease [11], [16]. In particular, chymase has been shown to promote cardiac remodeling by increasing angiotensin II (Ang II) independently from the renin-angiotensin system (RAS) and by altering collagen metabolism [17]–[19]. Furthermore, mast cell density has been associated with MMP activation [20] as chymase was shown to cleave pro-MMP-9 and pro-MMP-2 [21], which may contribute to collagen degradation and remodeling of the cardiac extracellular matrix. Pharmacological inhibition of degranulation using nedocromil (Ned) had not only reduced cardiac remodeling, but also normalized the expression of cytokines such as interferon-γ and tumor necrosis factor-α (TNF-α), as well as anti-inflammatory cytokine interleukin (IL)-4 and IL-10 [16], [22]–[24]. Furthermore, mast cell-deficient rats were protected against adverse cardiac remodeling and showed lower matrix metalloproteinase (MMP) activity, reduced TNF-α activation, and fewer collagen deposits [25].
Based on these previous studies, we propose to use an experimental diabetic cardiomyopathy model to observe if hyperglycemia is associated with cardiac mast cell activation and if mast cell activation correlated with collagen deposition and cardiac remodeling in the heart. We modulate cardiac mast cell activity by administering the mast cell-stabilizing agent nedocromil (Ned) to diabetic mice and then monitor cardiac function, cardiac mast cell activity, expression levels of proteins associated with cardiac remodeling, and the extent of collagen deposition as indirect measurements of the contribution of cardiac mast cell degranulation in diabetic cardiomyopathy.
Materials and Methods
Ethic statement
The study protocol was approved by the medical ethics committee of Shanghai Changzheng Hospital, conforms to the Principles of Laboratory Animal Care (National Society for Medical Research), and was conducted according to National Institutes of Health guidelines.
Animals and treatments
We used previously described methods to induce diabetes in our mouse model [16], [26]. Briefly, 8–12 weeks old C57/BL6 male mice between 23–25 g received intraperitoneal (i.p.) injections of 50 mg/kg streptozotocin (STZ, Sigma, St. Louis, MO), dissolved in 100 mM citrate buffer pH 4.5, for five consecutive days. At 72 h after the final STZ injection, whole blood samples were obtained from the mice using mandibular puncture blood sampling. Blood glucose levels were measured with an Ascensia Counter Glucometer (Bayer health care, NY). Hyperglycemic mice with blood glucose above 15 mmol/L were considered diabetic and were used for our experiments.
Diabetic mice (13-week-old) were randomly divided into three groups: 1) untreated group; 2) nedocromil group, with nedocromil released at the rate of 30 mg/kg per day from a subcutaneous (s.c.) pellet implantation [26]; and vehicle group, with an inactive pellet implanted. Normal mice (non-diabetic) and normal mice that received nedocromil (30 mg/kg per day) were also included in this study for comparison. All sample groups included 15 mice (n = 15).
Cardiac function assay
To assess cardiac function, mouse hearts were isolated and perfused using the Langendorff system [27] according to published methods [26], [28], [29]. The mice were injected (i.p.) with heparin (10,000 U/kg, Sigma) before the operation. Twenty minutes after the injection, the mice were killed using cervical dislocation. The chest was opened to isolate the heart, which was preserved in ice-cold saline. The perfusion tube was inserted into the aortic root and the heart was perfused with Krebs solution. The solution was maintained at 37°C in a thermostatic water bath with a gas mixture composed of 95% O2 and 5% CO2 continuously bubbled into the solution. The physiological pressure transducer was sutured to the apical area with 4-0 thread.
Cardiac function indicators were recorded using Powerlab multi-channel physiological recorder. The recorded indicators include maximal contractile rate (+dF/dt), maximal relaxation rate (-dF/dt), heart rate (bpm), and heart work (g.bpm). Heart work was calculated by multiplying the force (g) by the heart rate and normalized to heart weight. After the recording, the left ventricle was placed on ice and then cut transversely into three equal slices perpendicular to the long axis. The slices were either processed immediately for molecular characterization or flash-frozen in liquid nitrogen and stored at -80°C for future analysis.
Histological characterization
Histological analyses were performed as previously described [30]. Briefly, a slice of the left ventricle was fixed in neutral formaldehyde, embedded in paraffin, and sectioned onto glass slides. The sections were prepared and stained with modified Masson's trichrome to detect myocardial fibers and interstitial fibrosis and estimate collagen deposition. Collagen around blood vessels was not included in the analyses. The collagen volume fraction (CVF) was estimated from five random visual fields of the myocardial interstitium using NIH ImageJ imaging software (version 1.60) and was calculated as the ratio of collagen area/total area. The data shown represent the averaged CVF from five fields.
Immunohistochemical analysis was performed to determine the number of mast cells and mast cell activation based on chymase expression [16], [17]. Briefly, formalin-fixed, paraffin-embedded, 3-μm mouse heart sections were deparaffined in xylol and rehydrated in a graded ethanol series. Antigen retrieval was performed by microwave heating for 20 min in 1 mM EDTA buffer (pH 8.0). The sections were incubated in nonimmune serum for 30 min and then incubated overnight at 4°C in chymase primary monoclonal antibody (dilution 1∶100; Abcam, Cambridge, UK). After washing in TBST, the immunolabeled sections were incubated with HRP-conjugated secondary antibody (dilution 1∶200; Abcam, Cambridge, UK) for 20 min at room temperature, then visualized with 3,3′-diaminobenzidin and counterstained with hematoxylin. The areas of positive and negative staining were calculated using Image-Pro Plus 5.1 software (Media Cybernetics, Silver Spring, MD, USA).
RT-PCR
Real time-polymerase chain reaction (RT-PCR) was used to measure mRNA expression levels of type I and III collagen, and to confirm chymase mRNA expression. Total cellular RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA (0.2 µg) was reverse-transcribed using High-Capacity cDNA synthesis kit (Applied Biosystems, California, USA). RT-PCR was performed using the QuantiFast Sybr Green PCR kit (Qiagen, USA). The samples were amplified using Light Cycler 480 real-time PCR machine (Roche Diagnostics, USA). GAPDH expression was used for normalization. mRNA levels were expressed as fold-change relative to untreated normal or untreated diabetic mice. Primers specific for the target genes, the primer's annealing temperature and amplicon length are listed in Table 1.
10.1371/journal.pone.0060827.t001Table 1 Primer sequences, annealing temperatures, and amplicon lengths for RT-PCR.
Target gene Upstream sequence Downstream sequence Length (bp) Temp. (oC)
Type I collagen
5′-TGCCGTGACCTCAAGATGTG-3′
5′-CACAAGCGTGCTGTAGGTGA-3′
462 60
Type III collagen
5′-AGATCATGTCTTCACTCAAGTC-3′
5′-TTTACATTGCCATTGGCCTAG-3′
480 64
Chymase
5′-GAGGCCTGTAAAATCTATAGAC-3′
5′-TGTGTATCTTTGAGAGCCTCAA-3′
351 58
GAPDH
5′-ACGGCAAATTCAACGGCACAGTCA-3′
5′-TGGGGGCATCGGCAGAAGG-3′
231 61
Western blot
Western blots were used to detect the protein expression levels of type I and III collagen, MMP-2, MMP-9, tryptase, histamine, and chymase in cardiac tissues. Protein extraction was performed using a protein extraction kit (Pierce, Rockford, IL, USA) and the concentration was determined using a protein assay kit (Pierce, Rockford, IL, USA). The extracts were resolved on SDS-PAGE gels by electrophoresis and transferred onto nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ). The membranes were block with 5% nonfat milk in PBS-T. The primary antibodies used were: rabbit anti-mouse collagen I and collagen III antibodies (dilution 1∶5000); rabbit anti-mouse MMP2 and MMP9 antibodies (dilution 1∶1000); rabbit anti-mouse tryptase (dilution 1∶100), rabbit anti-mouse histamine (1∶1000), and goat anti-mouse chymase antibody (dilution 1∶200). Goat anti-rabbit HRP conjugated IgG (dilution 1∶2000), and donkey anti-Goat HRP conjugated IgG (dilution 1∶5000) were used as secondary antibodies to resolve the signal. All antibodies were purchased from Abcam (Cambridge, UK).
ELISA
Commercial ELISA kits (R&D System, Minneapolis, MN, USA) were purchased to determine the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6, IL-10, and angiotensin (Ang) II in cardiac tissue. The assays were performed according to manufacturer's instructions using extracted proteins as described above.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). ANOVA with Scheffe's-F test was used to determine statistical significance between treatment groups, when appropriate. A p-value of <0.05 was considered significant.
Results
STZ treatment induced hyperglycemia and weight loss in normal mice
Hyperglycemia has long been implicated in the development of cardiac diseases including cardiac remodeling [1]–[4]. Therefore, we first induced hyperglycemia in normal mice to observe diabetes development and diabetic cardiomyopathy in a controlled manner. We treated C57/BL6 male mice with STZ for five days as previously described, and then monitored blood glucose levels over the following weeks to confirm hyperglycemia.
As shown in Figure 1A, the mice used in the diabetic sample groups displayed a blood glucose level of >15 mmol/L after the final STZ dose, while that of normal mice remained below 10 mmol/L. The diabetic mice then received a continuous dose of nedocromil or vehicle for the next 12 weeks. The glucose levels of the diabetic mice fluctuated between 17 mmol/L and 22 mmol/L during the subsequent weeks. Nedocromil and vehicle treatments did not significantly affect baseline readings in normal or diabetic mice.
10.1371/journal.pone.0060827.g001Figure 1 The blood glucose level and body weight of normal and STZ-induced diabetic mice were monitored for 12 weeks after nedocromil (Ned) treatment, as described in Methods.
(A) The blood glucose levels of the different treatment groups at the indicated time points. (B) The body weights of the different treatment groups at the indicated time points. (n = 15 per group).
Figure 1B showed the average body weight of normal and diabetic mice for all treatment protocols. All diabetic mice displayed very similar body weights after the final STZ treatment. However, the body weights between the normal and diabetic mice began to diverge on week 4 as all diabetic mice began to lose weight, while all normal mice began to gain weight. Figure 1B also showed that nedocromil did not affect the baseline body weight of either the normal or the diabetic group.
Nedocromil significantly improved cardiac function in diabetic mice
Cardiac function was evaluated based on four parameters: maximal cardiac contractility, maximal cardiac relaxation, heart rate, and heart work. Heart work was calculated by multiplying the contraction force by the heart rate and normalized to the heart weight. Figure 2A showed that heart work is significantly lower for hearts in untreated diabetic animals when compared with normal hearts. This observation is likely the result of the lowered magnitude of contractile forces (Figures 2C, 2D), rather than decreased number of heart beats because the heart rate for all mice did not differ significantly (Figure 2B).
10.1371/journal.pone.0060827.g002Figure 2 Cardiac function of isolated and perfused hearts from normal and STZ-induced diabetic mice after nedocromil (Ned) treatments.
Contractile function of the heart was determined using a multi-channel physiological recorder. Changes in heart work (A), heart rate (B), maximal rate of contraction (C), and relaxation (D) are presented. Quantitative data are shown as means ± SD (n = 15 per group). *P<0.05 vs. untreated normal group; #P<0.05 vs. untreated diabetic group.
Nedocromil-treated diabetic mice showed significantly improved heart function compared with controls (Figure 2A). The contractility and relaxation forces showed similar improvements (Figures 2C, 2D). However, the cardiac function of nedocromil-treated diabetic mice remained significantly impaired when compared with normal mice (Figures 2A, 2C, 2D). The data indicated that nedocromil can significantly improve cardiac function in mice with diabetic cardiomyopathy, but the treatment cannot restore normal function.
Mast cell density and activity increased in the hearts of diabetic mice
Previous studies have shown that cardiac mast cells are activated in injured cardiac tissue to mediate cardiac remodeling [15], [16]. Therefore, we decided to probe the cellular and molecular characteristics of cardiac tissues from diabetic mice to assess mast cell activity.
We performed immunohistochemical studies on cardiac tissues from normal and diabetic mice that underwent nedocromil treatment. We examined mast cell activation by assessing chymase level because the enzyme is overexpressed and released during the degranulation process [17].
The number of chymase-positive cells was significantly higher in the cardiac tissues of diabetic mice than in normal mice. Nedocromil significantly reduced the number of chymase-positive cells in samples from diabetic mice, and had little effect on samples from normal mice (Figure 3A, Figure S1). The calculated mast cell density and chymase mRNA expression level reflected a similar trend (Figure 3B, 3C). Overall, the data confirmed mast cell activation in diabetic mice. Nedocromil-treated diabetic mice showed a significant reduction in mast cell number and chymase expression compared with controls, but remained statistically above those of normal mice (Figure 3B, 3C).
10.1371/journal.pone.0060827.g003Figure 3 Characterization of cardiac mast cell activation in normal or STZ-induced diabetic mice after nedocromil (Ned) treatments.
(A) Quantification of chymase-positive mast cells. Chymase-positive cells from immunohistochemically stained cardiac tissues were counted in 100 random fields at 400X magnification. (B) Mast cell density in sample tissues was determined by dividing the number of chymase-positive cells by the total number of cells in the visual fields. (C) mRNA expression levels of chymase in sample tissues were normalized to GAPDH expression and depicted as fold-change relative to the untreated normal group. (D) Representative results of protein expression analysis for chymase, tryptase, and histamine in sample tissues using western blot. GAPDH expression is shown as loading control. Quantitative data are displayed as means ± SD (n = 15 per group). *P<0.05 vs. untreated normal group; #P<0.05 vs. untreated diabetic group.
We further confirmed cardiac mast cell activation by evaluating the protein levels of additional mast cell-associated enzymes, which are also induced upon mast cell activation [11], [16]. The western blot in Figure 3D showed an increase in chymase, tryptase, and histamine that corresponded with diabetes and mast cell activation. The addition of nedocromil induced a modest decrease in the chymase level of diabetic and normal mice compared with vehicle-treated control, but no significant reduction of tryptase or histamine was observed in diabetic or normal mice (Figure 3D). This observation suggests that the modulatory effect of nedocromil is limited and degranulation was not completely blocked.
Nedocromil treatment decreases factors associated with cardiac remodeling
We have observed nedocromil treatment improved overall cardiac function in diabetic mice (Figure 2). So, we examined the expression profile of factors associated with cardiac remodeling to determine whether the functional improvements correlated with decreased remodeling. We used the expression of type I and type III collagen to monitor collagen deposition. We also measured MMP-2 and MMP-9 protein expression, which were reported to modulate the extracellular matrix by degrading collagen and were associated with cardiac remodeling [26], [31].
Trace amount of collagen deposits were observed in the hearts from normal mice, while those from diabetic mice showed significant areas of collagen deposition (Figures 4A, 4B) that correlated with molecular characterization (Figures 4A, 4B). MMP-2 and MMP-9 were also overexpressed, which suggested a high rate of collagen turnover in diabetic mice that may indicate active cardiac remodeling. Nedocromil treatment significantly reduced collagen deposits and MMP-2 and -9 expression levels in diabetic mice when compared with controls, but they remained greater than in normal mice (Figures 4A, 4B). Nedocromil may have stabilized collagen turnover and halted cardiac remodeling associated with diabetic cardiomyopathy.
10.1371/journal.pone.0060827.g004Figure 4 Collagen deposits in the left ventricle (LV) of normal and STZ-induced diabetic mice after nedocromil (Ned) treatments.
(A) Representative images of LV sections stained with Masson's trichrome at 100X magnification. Blue color indicates collagen deposits. (B) Quantification of LV collagen volume fraction (CVF). CVF is represented as the ratio of collagen area/total area. (C) The mRNA expression levels of type I and type III collagen are normalized to GAPDH expression and depicted as fold-change relative to the untreated normal group. (D) Representative results of protein expression analysis for collagen I, collagen III, MMP-2, and MMP-9 in sample tissues using western blot. GAPDH expression is shown as loading control and used for normalization. (E) Quantification of type I and type III collagen, MMP-2, and MMP-9 protein levels normalized to GAPDH. Quantitative data are displayed as means ± SD (n = 15 per group). *P<0.05 vs. untreated normal group; #P<0.05 vs. untreated diabetic group.
Nedocromil treatment modulated cytokines associated with cardiac remodeling in diabetic mice
Cardiac mast cell activation was reported to modulate cytokines that contribute to cardiac remodeling [16]. Therefore, we evaluated the extent to which mast cell activation modulate various cytokine levels in the heart. We used commercial kits to quantify several pro-inflammatory cytokines—specifically tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and also anti-inflammatory cytokine interleukin (IL)-4 and IL-10, which was reported to block inflammation and attenuate left ventricle remodeling [32]. We also evaluated angiotensin II, which is a cytokine-like factor known to mediate cardiac remodeling and is produced primarily by chymase in the heart, rather than by the RAS [18], [19].
Figures 5A and 5B showed diabetic mice had significantly higher levels of TNF-α and IFN-γ than normal mice. Nedocromil treatment was able to significantly decrease TNF-α and IFN-γ in diabetic mice relative to control. The levels of IL-4 and IL-10 were significantly reduced in diabetic mice compared with normal mice (Figures 5C, 5D). This pattern is consistent with the known anti-inflammatory properties of IL-4 and IL-10 [22]–[24]. Nedocromil treatment reversed this trend and the expression of both cytokines increased in diabetic mice to normalized levels (Figures 5C, 5D). Ang II was induced in diabetic mice, and nedocromil treatment was able to significantly reduce Ang II levels in diabetic mice relative to vehicle-treated control (Figure 5E). The results suggested nedocromil has indirect modulatory effects on cytokine levels by stabilizing mast cell activity.
10.1371/journal.pone.0060827.g005Figure 5 ELISA assay for cytokine expression in cardiac tissues of normal and STZ-induced diabetic mice treated with nedocromil (Ned) or controls.
The graphs show the quantitative levels of (A) TNF-α, (B) IFN-γ, (C) IL-4, (D) IL-10, and (E) Angiotensin II (Ang II) of the indicated sample groups. Quantitative data are displayed as means ± SD (n = 15 per group). *P<0.05 vs. untreated normal group; #P<0.05 vs. untreated diabetic group.
Discussion
Hyperglycemia has long been implicated in cardiac diseases by producing metabolic stress and triggering cardiac remodeling [1]–[4]. Mast cells have been suggested to regulate cardiac fibrosis by Panizo et al. [33], and later studies bolstered the claim by showing cardiac mast cell activity was stimulated in injured cardiac tissue to mediate cardiac remodeling [15], [16]. The cumulative evidence suggests cardiac mast cell activation is correlated with cardiomyopathy, but the role of hyperglycemia in cardiac mast cell activation remains unclear.
We have presented data that showed cardiac mast cell activation and cardiac remodeling occurs following hyperglycemia. The hearts of STZ-induced diabetic mice showed significantly impaired contractile function that resulted from increased collagen deposits and initiation of extracellular matrix degradation. We also examined chymase and tryptase levels in the heart because the enzymes are known mast cell products and have been linked to increased collagen production and to induce remodeling of the extracellular matrix by cleaving pro-forms of MMP-2 and MMP-9 [17], [19], [21], [34], [35]. We confirmed that mast cell numbers and protease production were significantly increased in hyperglycemic mice, which is consistent with a previous study [36]. Taken together, we speculate that mast cell activation and protease secretion may be responsible for cardiac remodeling by increasing collagen turnover, which in turn, destabilizes the cardiac extracellular matrix in diabetic mice. When this phenomenon is combined with the mechanical forces in a beating heart, it may be sufficient to induce cardiac remodeling and disrupt cardiac functions.
Many of these features were reversed upon mast cell stabilization using nedocromil. We showed the drug had protective effects against cardiac dysfunction in diabetic mice by reducing mast cell density and chymase secretion, and confirmed the expression of remodeling factors had decreased using biochemical assays.
We also observed aberrant cytokine levels in diabetic mice, which may contribute to cardiac remodeling. Nedocromil treatment was able to normalize them in diabetic mice. Cardiac mast cell activation has been reported to modulate cytokines that contribute to cardiac remodeling [16], [18], [19], and our results support this idea in the context of hyperglycemic mice.
Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and angiotensin Ang (II) have long been associated with cardiac remodeling. They were all significantly increased in diabetic mice. TNF-α is known to increase collagen production by stimulating the angiotensin II type 1 receptor [37], while knockout experiments showed TNF-α is required for cardiac remodeling under hypertensive conditions [38]. IFN-γ expression was also increased in our diabetic mice. IFN-γ is a marker not only of mast cell activation, but is also used to identify T-cell activation [39]. This is interesting because it suggests other factors may also modulate IFN-γ expression and extracellular matrix remodeling [40]. In fact, this is likely the case because nedocromil treatment was able to improve cardiac function in our model, but not restore normalcy. Ang II production is primarily mediated by chymase in the heart [18], [19], which further support mast cell activation and chymase secretion in diabetic mouse hearts. Nedocromil treatment reduced the expression levels of both chymase and Ang II, suggesting the drug may reduce Ang II levels indirectly via chymase inhibition.
The role of IL-6 in cardiac remodeling is unclear because its expression has been shown to vary greatly at different stages of cardiac remodeling in hypertensive rats [41], [42]. Its role is further complicated by the modulatory effects exerted by anti-inflammatory cytokine IL-10 [22]–[24], which was significantly decreased in diabetic mice and increased after nedocromil treatment.
Our data also showed nedocromil had no effect on blood glucose levels in diabetic mice, indicating mast cell activation is likely correlated with blood glucose levels and not by possible nonspecific effects from nedocromil. We also confirmed the effects of nedocromil had no significant effects in normal mice. However, the drug did not completely inhibit the degranulation process, as shown by the stable tryptase and histamine levels. We speculate that heightened levels of tryptase and histamine in diabetic mice may contribute to cardiac remodeling by an unknown mechanism, which may explain why nedocromil treatment did not completely abrogate the molecular changes observed in diabetic hearts.
We have shown a correlation between mast cell activation and cardiac remodeling in diabetic mice. We concluded that regulating mast cell activity was sufficient to significantly improve heart function in diabetic mice, and reduce signs of collage deposition and extracellular matrix destablization. Nedocromil treatment alone normalized the levels of several cytokines that are known contributors to cardiac remodeling. Our results propose that the manipulation of cardiac mast cells can attenuate cardiomyopathy by modulating aberrant cytokine levels. However, this mechanism is insufficient to fully restore normal cardiac functions and suggests other cellular pathways may contribute to the disease process. In addition, the molecular mechanisms that link hyperglycemia and cardiac mast cell activation remain unclear and warrant further study.
Supporting Information
Figure S1
Immunohistochemical staining for chymase in cardiac tissues (400X magnification).
(TIF)
Click here for additional data file.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23555924PONE-D-12-3550810.1371/journal.pone.0060197Research ArticleBiologyBiochemistryHormonesDevelopmental BiologyCell DifferentiationModel OrganismsAnimal ModelsRatMolecular Cell BiologyCellular TypesLeydig CellsMedicineAnatomy and PhysiologyReproductive SystemTGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells Effect of TGF-β1 on Leydig CellsLiu Man-Li
1
2
Wang Huan
3
Wang Zong-Ren
1
Zhang Yu-Fen
1
Chen Yan-Qiu
1
Zhu Fang-Hong
1
Zhang Yuan-Qiang
2
Ma Jing
1
*
Li Zhen
2
*
1
Department of Traditional Chinese Medicine, Xijing Hospital, the Fourth Military Medical University, Xi'an, People's Republic of China
2
Department of Human Anatomy and Histology and Embryology, the Fourth Military Medical University, Xi'an, People's Republic of China
3
Department of Dermatology, Tangdu Hospital, the Fourth Military Medical University, Xi'an, People's Republic of China
Yue Junming Editor
The University of Tennessee Health Science Center, United States of America
* E-mail: [email protected] (JM); [email protected] (ZL)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: MLL ZL JM. Performed the experiments: MLL HW YFZ YQC. Analyzed the data: MLL ZL. Contributed reagents/materials/analysis tools: ZL JM YQZ ZRW FHZ. Wrote the paper: MLL ZL.
2013 29 3 2013 8 3 e6019712 11 2012 22 2 2013 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E2) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.
Methodology/Principal Findings
Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E2 as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E2 secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E2 and TGF-β1, and E2 treatment in turn restored the inhibition of TGF-β1 on GJIC.
Conclusions
Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E2 secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.
This work was supported by Natural Science Foundation of China (NSFC) (3087326, 2008). The web site is http://isis.nsfc.gov.cn/portal/proj_search.asp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Leydig cells situated in the testicular interstitium are the main sites of testosterone production [1]. It has become increasingly clear that in the adult testis besides androgens, other steroid hormones commonly synthesized by Leydig cells and the germ cells, estrogens [2], also play an important role in the development, growth and differentiation of the male reproductive system and maintenance of spermatogenesis [3]. In particular, the demonstration that spermatogenesis is impaired in mice lacking aromatase or the estrogen receptor α (ERα) has shed new light on the role for estrogen in male reproduction [4]. Concomitantly, the discovery of mutations in both the human ERα and aromatase genes [5], [6] has reinforced the idea that estrogen plays a key role in the human male reproductive system.
Leydig cell estradiol (E2) is converted from testosterone (T), catalyzed by the microsomal enzymatic complex termed cytochrome P450 aromatase (P450arom), encoded by the single-copy Cyp19 gene. Despite the presence of aromatase in germ cells in several species, including the mouse, rat, brown bear, bank vole, rooster and man [7], it is worth noting that Leydig cells in the adult testis have also been identified as the major sites of expression of this enzyme [8], which has been shown to be controlled by various factors, such as LH, cyclic cAMP and testosterone, together with other paracrine factors produced by germ cells, such as TNF-α and TGF-β1 [9]. Aromatase transcription occurs via the alternative use of nine distinct tissue-specific promoters located in the first exon of the Cyp19 gene [10], and promoter PII is the principal promoter active in rat Leydig cells [11]. This promoter contains several cAMP response element (CRE)-like motifs that mediate the effects of the cAMP transduction pathway that potentiates aromatase gene expression and activity. In Leydig cells, both nuclear receptor steroidogenic factor-1 (SF-1) [12] and liver receptor homologue-1 (LRH-1) are able to activate aromatase transcription by binding to the aromatase promoter PII [13]–[14]. Therefore, transcriptional regulation of Cyp19 is a major mechanism controlling the activity of aromatase which affects E2 synthesis. Several lines of evidence suggest a crucial role for TGF-β1 in regulation of Leydig cell function. For instance, TGF-β1 has been shown to inhibit testosterone secretion [15], to suppress proliferation of Leydig cells [16] and to be involved in the morphological differentiation of immature Leydig cells into the adult form [17]. TGF-β1 has also been found to regulate aromatase expression in a tissue-specific manner. It increases aromatase mRNA levels and activity in osteoblast-like cells, THP-1 cells and the leukaemic cell line FLG29.1 [18]–[19]. However, in germ cells [20], granulosa cells [21] and trophoblast cells [22], TGF-β1 suppresses aromatase gene expression. So far, its role in regulating the expression of Cyp19 and aromatase activity in Leydig cells is not clear.
Gap junctional intercellular communication (GJIC) directs the exchange of small molecules, including ions, second messengers, and other metabolic precursors less than 1 kDa, between adjacent cells, and this function is mainly mediated by proteins called connexins (Cx) [23]. GJIC between testicular cells is also essential for the initiation and maintenance of spermatogenesis [24]; it is also involved in several cellular processes including control of cell proliferation and differentiation [25]. To date, Cx43 is the only Cx detected in Leydig cells of different species [24], and it forms the gap junctions between them. The regulation of Cx43 expression by estrogen has been reported in human myometrium, rat cardiomyocytes and rat prostate [26]–[28]. The rat Cx43 promoter contains several sequences resembling half the palindromic estrogen response elements (half-EREs), which are functional when co-transfected with estrogen receptor cDNA into HeLa cells [29]. In addition, it has been demonstrated that estrogens in osteocyte and mouse embryonic stem cells, also bound to membrane ERα, induce activation of phosphatidylinositol 3-kinase (PI3K)/Akt, and that one of the tyrosine kinases, PI3K/Akt, participates in the up-regulation of Cx43 [30]–[31]. However, the effect of E2 on GJIC in Leydig cells and their only gap-junctional connexin Cx43 has not been studied.
To determine whether TGF-β1 regulates E2 synthesis in adult rat Leydig cells and to assess the role of E2 and TGF-β1 in GJIC between Leydig cells, in the present study we have examined the effects of TGF-β1 on Leydig cell E2 synthesis and aromatase activity. The probable function of E2 and TGF-β1 in Cx43-based GJIC between rat Leydig cells was also investigated.
Materials and Methods
Ethics Statement
The Ethics Committee for Animal Experiments of the Fourth Military Medical University approved all animal work (Permit number: 08014) and the experimental protocols strictly complied with the institutional guidelines and the criteria outlined in the “Guide for Care and Use of Laboratory Animals”. All surgery was performed under sodium pentobarbital anesthesia.
Isolation and culture of rat Leydig cells
Male SD rats were sacrificed at 3 months of age and the testes were decapsulated under aseptic conditions. Leydig cells were isolated by enzymatic digestion and purified on a continuous Percoll gradient as described previously [32]. Briefly, the testes were incubated for 20 min in culture medium containing 0.25 mg/ml collagenase (Type II, Sigma) in a shaking water bath at 34°C. Separated cells were filtered through two layers of nylon mesh (100 µm pore), centrifuged at 250 g and re-suspended in 55% isotonic Percoll. Following density gradient centrifugation at 20,000 g for 60 min at 4°C, Leydig cell fractions with densities between 1.070–1.088 g/ml were collected from the Percoll gradient. The cells were washed twice with Hanks' buffered saline solution and then cultured in a 37°C, 5% CO2 humid incubator in DMEM-F12 for further studies. The purity of Leydig cells was assessed by histochemical staining of 3β-HSD and viability was determined by trypan blue exclusion. The purity was 90–95% and viability was 95–98%.
Radioimmunoassay of E2 and T
Culture medium with TGF-β1 (final concentrations of 1, 2, 5, 10 ng/ml according to previous studies [17], [33]–[35]) was collected and the production of E2 and T was measured by employing a commercially available radioimmunoassay kit (Beijing North Institute of Biological Technology, China), in accordance with the manufacturer's instructions. All measurements were repeated three times independently, and data were presented as mean ± standard deviation (SD).
Aromatase activity determination
The catalytic activity of aromatase in Leydig cells was assayed by the formation of tritiated water from [1β-3H]-androstenedione as described previously [36]. Briefly, TGF-β1-treated Leydig cells were incubated with 20 nM 1β-3H-androstenedione (New England Nuclear Research Products, Boston, MA, USA) in serum-free medium for 6 h. Incubations were conducted in an identical fashion in the absence of cells to establish background values. Then the medium (600 µl) was extracted with 1,500 µl of ice-cold chloroform and centrifuged at 12,000 g at 4°C for 1 min. The aqueous phase was transferred to a vial containing 400 µl dextran-coated charcoal to remove the residual labelled and unlabelled steroids. The mixture was vortexed for 10 s and centrifuged at 15,000 g at 4°C for 15 min. The supernatant was decanted, mixed with scintillation fluid and counted in a beta-spectrometer; thus, tritiated water formed during the aromatization of [1-3H]-androstenedione to estrogen was determined by measuring the radioactivity in the supernatant. Aromatase activity was expressed as rate of incorporation of tritium into water per mg protein per h for Leydig cells. Each experiment was conducted in triplicate and was repeated at least two times to ensure that the results were quantitatively reproducible.
Western blotting
Western blotting was performed by following a routine protocol. Briefly, Leydig cells were washed with PBS and scraped into 200 µl sodium dodecyl sulfate (SDS) electrophoresis sample buffer (10 mM Tris, pH 6.8, 15% w/v glycerol, 3% w/v SDS, 0.01% w/v bromophenol blue and 5% v/v 2-mercaptoethanol). Then cell lysates were sonicated (60 Hz, 10 s for 3 times) and heated for 10 min at 95°C. Protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The blotting membranes were blocked with 5% w/v non-fat milk and probed with rabbit anti-Cyp19 (1∶400 dilution, Sigma-Aldrich), mouse anti-Cx43 (1∶200 dilution, Santa Cruz Biotechnology) or rabbit anti-β-actin (1∶400 dilutions, Sigma-Aldrich). After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1∶3,000 dilution, Sigma-Aldrich) for 1 h at room temperature. The bound antibodies were visualized by an electric control loading system (Amersham Biosciences). Semi-quantitative densitometric analysis of Western blotting was performed by Image J software (NIH, Bethesda, USA).
Real-time RT-PCR
Total RNA was extracted from rat Leydig cells (2×106) using the RNeasy mini kit (Qiagen, Hiddens, Germany) according to the manufacturer's instructions. Routine Dnase (Ambion, Austin, TX, USA) treatment was performed before reverse transcription. First strand cDNA was synthesized from 1 µg RNA with the Omni RT kit (Qiagen). Real-time PCR was set up with the Corbett Rotor-gene TM 6000 (Corbett, Sydney, Australia) by using SYBR Green (Sigma-Aldrich). Primer sequences were as follows: Cyp19, 5′-ggtaaattcattgggcttgg-3′ and 5′- cctgtcgtggtcttggtca-3′; Gapdh, 5′-cgaccacctttgtcaagctca-3′ and 5′-aggggtctacatggcaactg -3′. The Gapdh from the same exacts were used as internal control. The amount of Cyp19 was normalized to the Gapdh value. Data were calculated from the mean of three experiments.
Immunofluorescence microscopy analysis
Leydig cells were incubated with the aromatase inhibitor Letrozole (final concentration 10 ng/ml) or different concentrations of TGF-β1 for 20 h and then fixed with 4% formalin for 30 min at room temperature, rinsed with PBS and permeabilized with 0.1% v/v Triton. Cells were incubated with mouse anti-Cx43 (diluted 1∶200) in PBS containing 1% w/v bovine serum albumin (BSA) overnight at 4°C. After three rinses, cells were incubated at room temperature for 1 h with goat anti-mouse TRITC-conjugated antibodies (1∶100). Finally, cells were mounted in Vecta shield medium with DAPI (Biovalley, Marne-La-Vallée Cedex 3, France) to label nuclei. For negative controls, cover slips were processed without the primary antibody, and no signals were detected. Immunofluorescence images were captured using a Nikon E800 microscope with a Spot-2 camera (Tokyo, Japan).
Analysis of GJIC
Leydig cells were seeded in a 6-well culture plate overnight. At 70% cell confluence, a GJIC inhibitor Carbenoxolone (Sigma-Aldrich, St. Louis, MO, USA; final concentration 40 µM), aromatase inhibitor Letrozole (Sigma-Aldrich, St. Louis; final concentration 10 ng/ml) or human recombinant TGF-β1 (R&D Systems, Lille, France; final concentration 5 ng/ml) was added. Alternatively, E2 (Sigma-Aldrich, St. Louis, MO, USA; final concentration 5 µM) was added 4 h ahead of TGF-β1 exposure. After 20 h of incubation, GJIC was measured by fluorescence recovery after photo-bleaching (FRAP). This method was performed as described previously [37]. Briefly, cells were washed twice with PBS, and then were incubated in culture medium without phenol red, containing 5, 6-carboxyfluorescein diacetate (Research Organics, Ohio, USA) at a final concentration of 50 µg/ml and incubated for 20 min in a 37°C 5% CO2 humid incubator. Individual cells were then bleached by strong laser pulses (488 nm 100% and 50 iterations) with a Zeiss confocal microscope LSM 510 (Service Commun de Microscopie, IFR Biomédicale des Saint-Pères, Paris, France). Confocal images were taken every 2 min during a 15 min period after calcein photo-bleaching. Fluorescence recovery was analyzed using LSM software before bleaching, immediately afterwards, and 8 min afterwards. The percentage fluorescence recovery in bleached cells was determined by averaging all cells (n>50) for each experiment.
Statistical Analysis
All data are expressed as the mean ± SD of three or more independent experiments carried out with different cell preparations. Statistical analysis was performed by using the one-way ANOVA parametric test and significance was accepted at p<0.05.
Results
Effects of TGF-β1 on E2 and T production in Leydig cells
To investigate whether TGF-β1 could influence E2 and T production, various concentrations of TGF-β1 (1–10ng/ml) were added to isolated adult Leydig cells for 20 h incubation period (Fig. 1). Compared with the control group, TGF-β1 produced a dose-dependent inhibitory effect on the E2 and T secretion of Leydig cells. With increasing concentration TGF-β1 significantly suppressed the basal E2 secretion (Fig. 1A) from initial 64 pg/ml to 1 pg/ml with **p<0.01, as well as the basal T secretion (Fig. 1B) from initial 22 ng/ml to 2 ng/ml with **p<0.01.
10.1371/journal.pone.0060197.g001Figure 1 Effects of various concentrations of TGF-β1 on E2 and T production by purified rat Leydig cells.
TGF-β1 induced a dose-dependent inhibition of E2 as well as T synthesis. Each column represents mean ± S.D of three independent experiments. Significant differences between groups were analyzed with one-way ANOVA. *p<0.05; **p<0.01 compared with control cells.
Effects of TGF-β1 on aromatase activity and Cyp19 expression in Leydig cells
To explore the effect of TGF-β1 on aromatase activity, we next carried out a tritiated water-release assay to address the activity of P450arom. Incubations of Leydig cells were performed in the absence or presence of TGF-β1 for a 20 h period. Although there was no difference between control group and the low dose group (1 ng/ml), when the concentration of TGF-β1 was 2 ng/ml, the activity of aromatase was decreased by 29% in Leydig cells (Fig. 2A). To delineate the mechanism of TGF-β1 inhibition with aromatase activity in Leydig cells, the effect of TGF-β1 on Cyp19 expressions at the protein and mRNA levels was evaluated. TGF-β1 inhibited Cyp19 protein expression in a dose-dependent manner (Fig. 2B, C) and the mRNA levels changed in parallel to its protein expression (Fig. 2C). We observed that at 1 ng/ml, TGF-β1 significantly decreased Cyp19 protein and mRNA levels in Leydig cells from values in the solvent controls and that this inhibitory effect was most significant when the concentration of TGF-β1 was 10 ng/ml.
10.1371/journal.pone.0060197.g002Figure 2 Dose-related effects of TGF-β1 on aromatase activity and Cyp19 expression in purified rat Leydig cells.
(A) Inhibition of aromatase activity by pretreatment of Leydig cells with TGF-β1. Cells were serum starved for 24 h and stimulated with TGF-β1 at concentrations between 1 and 10 ng/ml for 20 h. Aromatase activity was evaluated by the tritiated water release assay. The results are expressed as mean ± S.D of three independent experiments (*p<0.05; **p<0.01). Significant differences between groups were analyzed with one-way ANOVA. (B) Representative Western blot of Cyp19 immunoreactivity in Leydig cells, treated for 20 h with TGF-β1 at concentrations between 1 and 10 ng/ml. The β-actin expression is shown as the loading control. (C) The relative amounts of Cyp19 mRNA (real-time quantitative PCR analysis) and protein (Western blotting analysis) levels in Leydig cells treated with TGF-β1. Data shown are means ± S.D of three independent experiments (*p<0.05; ## p<0.01; **p<0.01). Significant differences between groups were analyzed with one-way ANOVA.
Effect of E2 and TGF-β1 on Cx43 expression
Cx43 is the major gap junction protein expressed in Leydig cells and has been shown to form functional gap junctions. To determine whether the reduction of E2 induced by Letrozole or TGF-β1 could affect Cx43 expression in Leydig cells, the distribution of Cx43 was observed by confocal immunofluorescence microscopy. As shown in Fig. 3A, Cx43 in untreated Leydig cells was predominantly in the form of defined spots uniformly distributed at sites of cell-cell contact and in the cytoplasm. However, a clear decrease in staining intensity of Cx43 in Leydig cells was observed in the presence of the aromatase inhibitor Letrozole. After TGF-β1 exposure for 20 h at the concentration of 1 ng/ml, Cx43 was almost the same as that of control in the pattern of location but distinctly decreased in immunoreactivity in Leydig cells and at the cell borders. The decrease obtained by immunofluorescence staining was confirmed by Western blot analysis. This revealed that the aromatase inhibitor Letrozole and TGF-β1 down-regulated total Cx43 immunoreactivity in Leydig cells (Fig. 3B). This down-regulation was significant when cells were treated with TGF-β1 at concentrations of 2 ng/ml and higher (Fig. 3C).
10.1371/journal.pone.0060197.g003Figure 3 Effects of Letrozole and various concentrations of TGF-β1 on Cx43 expression in purified rat Leydig cells.
(A) Subcellular distribution of Cx43 in Leydig cells treated with Letrozole or TGF-β1 observed by confocal microscopy. In untreated cells, Cx43 immunoreactivity is detected both at the cell membrane and in cytoplasm. After treatment with Letrozole or TGF-β1 a distinct decrease of Cx43 immunoreactivity in Leydig cells and at the cell borders is detected. (B) Representative western blot of Cx43 immunoreactivity in Leydig cells, treated for 24 h with Letrozole or TGF-β1 at different concentrations. The β-actin expression is shown as the loading control. (C) The relative amounts of Cx43 protein as shown in (B), demonstrating a significant down-regulation of Cx43 immunoreactivity by TGF-β1 at concentrations of 5 and 10 ng/ml (*p<0.05; **p<0.01). Significant differences between groups were analyzed with one-way ANOVA.
Effect of E2 and TGF-β1 on GJIC between Leydig cells
Since Cx43 is the major functional protein forming gap junction channels in Leydig cells and plays critical role in regulating gap junction communication, we investigated if GJIC of Leydig cells is affected by E2 and TGF-β1. The results showed that Carbenoxolone, an established GJIC inhibitor, reduced the dye transfer rate by almost 90% (Fig. 4A, B). This result serves as a positive control for the reliability of the gap-FRAP technique to measure changes in GJIC [38]. Compared with PBS-treated cells, the transfer rate of the fluorescent dye 5, 6-carboxylfluorescein diacetate was significantly lower in both Letrozole- and TGF-β1-treated Leydig cells (Fig. 4A, B). However, the TGF-β1-induced down-regulation of GJIC was attenuated when the cells were treated with E2 before the addition of TGF-β1 (Fig. 4).
10.1371/journal.pone.0060197.g004Figure 4 FRAP analysis of GJIC in Leydig cells.
(A) Effects of various treatments on dye transfer in Leydig cells. Left panel: Image of the target cell before bleaching (white collar). Middle panel: Image of the target cell after bleaching. Right panel: Image of the target cell after 8 min of fluorescence recovery. Recovery of fluorescence in the target cell was caused by influx of dye from adjacent cells. NC: In normal control group, cells were treated with phosphate-buffered saline; Carbenoxolone: cells were treated with 40 µM Carbenoxolone for 20 h; Letrozole: cells were treated with Letrozole of 10 ng/ml for 20 h; TGF-β1: cells were treated with 5 ng/ml TGF-β1 for 20 h; E2: cells were treated with 5 µM E2 for 4 h and 5 g/ml TGF-β1 for 20 h. (B) Histograms representing the percentage of GJIC in each condition (*p<0.05; **p<0.01). Significant differences between groups were analyzed with one-way ANOVA.
Discussion
The results from the present study showed that TGF-β1 down-regulated the level of E2 secretion, the activity of P450arom and the expression of Cyp19 in adult rat Leydig cells. Such reduction of E2 could explain the dysfunction of Cx43-mediated GJIC between Leydig cells treated with TGF-β1, since E2 restored the TGF-β1 inhibition of GJIC. These unique effects of TGF-β1 imply that it may have novel functions in the testis, moderating the intercellular communication between Leydig cells.
The main source of estrogen in the testis is conversion of androgen catalyzed by P450arom. Leydig cells have been demonstrated to be a major site of aromatase expression in the adult testis [8]. In this study we demonstrated that TGF-β1 treatment decreased E2 and T production, as well as aromatase activity in Leydig cells, in a dose-dependent manner, and in parallel decreased expression of Cyp19 mRNA and protein was also observed. Interestingly, TGF-β1 reduced E2 synthesis significantly at the concentration of 1 ng/ml but had no effect on P450arom activity at the same concentration. This observation suggests that the production of E2 may well be the sum of decreased T and reduced aromatase expression. These findings concur with those obtained in germ cells, where TGF-β1 significantly inhibits aromatase activity and P450arom transcripts [39]. In addition, it has been demonstrated that TGF-β1 inhibits E2 production in cultured human trophoblast cells [40], human fetal hepatocytes [41], and skin fibroblasts [42]. Although the mechanism by which TGF-β1 exerts its inhibitory effect on aromatase gene expression in Leydig cells remains to be established, one possible explanation is transcriptional regulation of Cyp19. Expression of P450arom in rat testicular cells is controlled primarily by promoter PII, proximal to the translation start site [43]. It has been proved that TGF-β1 inhibits the promoter activity via the Smad2 signalling pathway in human trophoblast cells [44]. Moreover, the orphan receptor steroidogenic factor-1 (SF-1) binding site has been identified as the response element in the proximal promoter PII of the rat aromatase gene [45], and TGF-β1 has been proved to inhibit SF-1 expression both at the transcriptional and translational levels in the mouse adrenocortical cell Y-1 [46] and human adrenocortical cell H295R [47].
It has been proved that stimulation of testosterone production by hCG is associated with a decrease in Cx43 mRNA levels in Leydig cells both in vitro and in vivo [48]. Given that Leydig cells are the main site of conversion of androgens into estrogens in the testis, and that GJ-protein expression is regulated in part by steroid hormones in steroid-sensitive organs, it is essential to investigate whether there is a correlation between E2 synthesized by Leydig cells and Cx43-mediated intercellular communication. In the present study, we showed that administration of Letrozole or TGF-β1 to rat Leydig cells decreased Cx43 expression and down regulated GJIC. Considering that both Letrozole and TGF-β1 inhibit E2 production by Leydig cells, it is possible that E2 operates as a local regulator in the fine-tuning of the gap junction between Leydig cells. Recent studies demonstrate that the expression of Cx43 is greatly reduced by ovariectomy and is restored by treatment with estrogen [49]. Also, GJ coupling and the amount of Cx43 protein are reduced after anti-estrogen treatment in bovine myocytes from the circular layer of myometrium [50]. One possible explanation for these results is the regulation of transcripts encoding Cx43 by estrogen. Activated ERs are transcription factors that bind to estrogen response elements (EREs) in the regulatory region of target genes. Two related estrogen receptors ERα and ERβ have been demonstrated to be expressed in rat Leydig cells [51]; and a series of half- palindromic EREs is present in the promoter of the rat connexin43 gene [45], [52], [53]. Transcription of Cx43 can be induced by estrogen via an ER-dependent pathway during preimplantation [54]. Therefore, estrogen may increase Cx43 through a genomic pathway mediated by the nuclear receptors in rat Leydig cells. Although TGF-β1 distinctively down-regulated gap junctional communication between Leydig cells, prior addition of estradiol to the culture medium attenuated this inhibition to an extent. Up-regulation of cell-to-cell communication and Cx43 expression by estrogen has been observed in the human myometrium [26], similarly, in rat cardiomyocytes, estrogen has been shown to increase GJIC via ER-mediated signalling at a pharmacological concentration [55]–[56]. Since Cx43 is a gap junctional protein expressed in Leydig cells, we speculate that E2 modulates GJIC in Leydig cells through ER/Cx43 pathway.
In conclusion, the present study demonstrates that TGF-β1 has a significant inhibitory effect on estrogen production by rat Leydig cells possibly via down-regulation of aromatase gene expression and activity. This could decrease Cx43 expression, leading to Cx43-mediated gap junction between Leydig cells. Future studies are required to clarify further the mechanisms of regulation of E2 in the gap junctions between Leydig cells.
We are indebted to Dr. TG Cooper and Dr. CH Yeung for their careful assistance during the preparation of the manuscript.
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Med Oral Patol Oral Cir BucalMed Oral Patol Oral Cir BucalMedicina Oral S.L.Medicina Oral, Patología Oral y Cirugía Bucal1698-44471698-6946Medicina Oral S.L. 232292481806810.4317/medoral.18068Research-ArticleClinical and Experimental dentistryerbB expression changes in ethanol and 7, 12- dimethylbenz
(a) anthracene-induced oral carcinogenesis Jacinto-Alemán Luis F. 1García-Carrancá Alejandro 2Leyba-Huerta Elba R. 3Zenteno-Galindo Edgar 4Jiménez-Farfán María D. 1Hernández-Guerrero Juan C. 11 Immunology Laboratory, Postgraduate and Research Division, Dental School, National Autonomous University of Mexico, Mexico, D.F., Mexico2 Biomedic Cancer research, Biomedic Reseach Institute, National Autonomous University of Mexico & National Cancer Institute, Health Ministry. México, D.F., Mexico3 Oral Pathology Laboratory, Postgraduate and Research Division, Dental School, National Autonomous University of México, México, D.F., Mexico4 Biochemestry department, Medicine School, National Autonomous University of México, México, D.F., Mexico Laboratorio de Inmunología. DEPeI
Facultad de Odontología
Universidad Nacional Autónoma de México
Circuito Institutos s/n Ciudad Universitaria
C.P. 04510, México, D.F., Mexico
, E-mail: [email protected] 3 2013 10 12 2012 18 2 e325 e331 7 6 2012 8 11 2011 Copyright: © 2013 Medicina Oral S.L.2013This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Objetive: The aim of this study was to determine erbB expression in normal mucosa, oral dysplasia, and invasive carcinomas developed in the hamster’s buccal pouch chemical carcinogenesis model.
Study design: Fifty Syrian golden hamsters were equally divided in five groups (A-E); two controls and three experimental group exposed to alcohol, DMBA, or both for 14 weeks. Number of tumors per cheek, volume, histological condition, erbB expression were determined and results were analyzed by the Mann–Whitney U and Dunn’s test.
Results: Control groups and those exposed to alcohol (A, B and C respectively) only presented clinical and histological normal mucosa; while those exposed to DMBA or DMBA plus alcohol (D and E groups) developed dysplasia and invasive carcinomas. erbB2, erbB3, and erbB4 increased their expression in alcohol-exposed mucosa, dysplasia, and invasive carcinomas. We observed a similar expression level for erbB2 in dysplasia and carcinomas; while, erbB3 and erbB4 were similar only in carcinomas.
Conclusion: The DMBA and alcohol can be considered as carcinogen and promoter for oral carcinogenesis. The erbB expression is different according to their histological condition, suggesting differential participation of the erbB family in oral carcinogenesis induced by alcohol and DMBA.
Key words:erbB, 7,12- dimethylbenz(a)anthracene, oral squamous cell carcinoma.
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Introduction
Worldwide, approximately 274,300 new cases and 127,500 deaths have been attributed to oral cancer during 2002 (1). Oral squamous cell carcinoma (OSCC) is the most frequent neoplasm in the mouth, and many OSCC are preceded by leukoplakia and erythroplakia, which could present epithelial dysplasia (ED), an indicative of malignancy development (2). 7,12-dimethylbenz(a)anthracene (DMBA), a polycyclic aromatic hydrocarbon has been found in high concentration (40-100 ng per cigarette) in the tar fraction of cigarette smoke (3). In mammalian cells, DMBA is bioactivated to the diolepoxide metabolite, which subsequently promotes adducts to adenine and guanine residues in DNA (4). Another important compound is the ethanol, however, the ethanol carcinogenic effect is controversial; some reports consider it as co-carcinogen and/or tumor promoter (5,6). Nevertheless, ethanol consumption has been associated to a 2- to 3-fold increased risk for cancer in the oral cavity, pharynx, larynx, and esophagus (7).
The cellular and molecular analysis, had improve our understanding of OSCC. The analysis of the tyrosine kinase erbB family receptors (erbB1/EGFR, erbB2/Neu/HER2, erbB3/HER3, and erbB4/HER4) had indicated their participation in embryogenesis, proliferation, differentiation, and malignant transformation of breast, renal, colon, and oral cancer. These receptors are activated upon ligand-induced receptor dimerization and, consequently, numerous dimmers are formed, promoting downstream transduction pathway activation, such as MAPK, PI3K, and STAT (8). erbB1 and erbB2 over-expression plays a significant role in driving cancer cells through the cell cycle checkpoints in G1-S transition. erbB2, erbB3, and erbB4 signaling has been associated with apoptosis evasion, invasion, metastases to lymph nodes and angiogenesis (9,10). Specific determination of erbB expression to each tumor could improve our understanding of their biological behavior. Our hypothesis is that erbB receptor expression changes is according to histological condition, for that reason, the aim of this study was to determine the relationship of erbB expression in normal mucosa, dysplasia, and invasive carcinomas developed in the hamster buccal pouch chemical carcinogenesis model.
Material and Methods
-Population, clinical and histological analysis
Fifty male Syrian golden hamsters (Mesocricetus auratus), 5-week-old, were employed following the guidelines of the Ethics committee of the Postgraduate and Research Division, Dental School, National Autonomous University of México. All animals were housed at four animals per cage, with 12 h light:dark cycles. At 8-week-old animals were divided randomly in five groups; ten animals for each group (A to E groups). The weight of each animal was measured weekly. Animals from group A remained untreated while those in group B received mineral oil (Sigma-Aldrich, M8410, St. Louis, MO) on the right cheek pouch applied three times per week during fourteen weeks at 10 a.m., using a camel’s hair brush No. 4. The animals from groups C and E were left to consume ethanol solution at 15% (Mallinckrodt Baker, V568, Xalostoc, Mexico) ad libitum, consumption was measured two times per week. Groups D and E received DMBA (Sigma-Aldrich, D3254) at 0.5%, dissolved in mineral oil, as above. The amount of carcinogens delivered to each animal was quite uniform using the ‘‘wiped-brush’’ method (11). All animals were simultaneously sedated and killed by a chloroform overdose (Sigma-Aldrich, C2432). The right cheek pouch was dissected and washed in Hank’s balanced salt solution (Gibco-BRL, Cat. 0-125 Pailey, Scotland, UK). Tumor per cheek pouch and two diameters from each tumor were registered. Tumor volume was calculated through diameter measures and the equation V = a x b2 x 0.52; where a is the largest diameter and b is the largest diameter perpendicular to a (12). The cheek pouch was divided in two halves; a portion was fixed in 4% paraformaldehyde solution for 24 h for its histological and immunohistochemical analysis. Another portion was immediately frozen in liquid nitrogen and stored at -80ºC until their western blot analysis.
The fixed samples were processed for paraffin embedding, 4 µm serial sections, and hematoxylin and eosin staining for histopathological analysis was made. The histological classification in normal mucosa, epithelial dysplasia (ED; mild, moderate and severe), and OSCC (well differentiated, WD; moderately differentiated, MD and poorly differentiated, PD) was performed by an oral pathologist.
-Western blot (WB) analysis
Frozen samples were homogenized and lysed in 300 µl of lysis buffer (225 mM saccharose, 10 mM Tris, 0.3 mM ethylene glycol tetraacetic acid, 1% Triton X-100, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium carbonate; and a mixture of proteinase inhibitors, including 1 mM phenymethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 5 mM benzamidine, 10 µg/ml phenantrholine; BD, Bioscience Pharmigen). Tissue lysate was cleared by centrifugation at 10,000 rpm at 4ºC for 10 minutes. Protein content was quantified using Lowry’s protein assay (13). Protein (25 µg) was electrophoresed in a 10% gradient SDS-PAGE for 120 minutes at 150 mV. The resolved protein was transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membrane was blocked with PBS-1% Triton with 5% non-fat dry milk for 2 h for posterior overnight primary antibodies incubation (erbB1, sc-03, rabbit polyclonal, 1:1000; erbB2, sc-7301, mouse monoclonal, 1:1000; erbB3, sc-285, rabbit polyclonal, 1:1500, and erbB4, sc-283, rabbit polyclonal, 1:1000, all antibodies from Santa Cruz Biotechnology, Santa Cruz, CA) in heat sealed plastic bags at 4ºC. Horseradish peroxidase-labeled (goat anti-mouse sc-2005, mouse anti-rabbit, sc-2357, mouse anti-goat, sc-2354, Santa Cruz Biotechnology) secondary antibodies were used for 1 h at room temperature incubation. The immune complex was visualized using the ECL Western blot detection system (Amersham Pharmacia Biotech, Arlington Heights, IL) according to manufacturer’s instruction. Membranes were exposed one minute to Kodak Biomax light film (Eastman Kodak, Co., Rochester, NY). ß-actin and GAPDH (Sigma A1978, mouse monoclonal, IgG1, 1:2000 and sc-20357 goat polyclonal, 1:1500, Santa Cruz Biotechnology, respectively) detection was used as charge control. The film was scanned for densitometric analysis using Scion Image software (Scion, Frederick, MD). Each representative sample according to histological condition was analyzed in triplicate.
-Immunohistochemistry (IHC)
The IHC analysis for erbB1, erbB2, erbB3, and erbB4 was performed according to reported (14). Eight slides for each sample were deparaffinized and rehydrated in xylene and alcohol washes. Antigenic retrieval in 10 mM citrate buffer; endogenous peroxidase blockade in 3% hydrogen peroxide, unspecific blockade with serum-free protein block (DakoCytomation, Dako, Carpinteria, CA) and posterior immersion in 0.2% Triton X100 were performed. Four slides were incubated overnight with anti-erbB primary antibodies at 4°C in 1:100 dilution. Biotinylated link universal secondary antibody and streptavidin-HRP incubation for 30 minute each were performed (Dako, Carpinteria, CA). Posterior 3,3’diaminobenzidine revealing (Dakocytomation, Dako, Carpinteria, CA) and Hill’s hematoxylin nuclear counterstaining were done, for posterior dehydration in alcohol-xylene serial washes and mounting with hydrophobic resin (14). For negative control (the rest four slides), the primary antibody was substituted by phosphate buffer saline (PBS) solution. From each slide, a digital photomicrograph of 2000 × 1500 pixels was obtained at 1000X magnification with a digital camera (Olympus C-3040 Tokyo, Japan). Cellular expression zone was determined semiquantitatively as follows: zone (membrane, cytoplasm, and nucleus) and percentage of positive cells (0 = 0% cells, 1 = 1 to 29% cells, 2 = 30 to 69% cells, and 3 = >70 % cells).
-Statistical analysis
All data are presented as means ± SE. Mean values were analyzed by ANOVA with a post hoc Mann–Whitney U and Dunn’s test to compare differences between groups and similar histological conditions. Statistical significance was at P < 0.05, SPSS v.13.0. Software package (SPSS Inc., Chicago IL) was used for statistical analysis.
Results
-Clinical and histological analysis
The final population was 47 hamsters; in groups D and E two and one animal were lost by territorial disputes, respectively. The mean weight of all specimens was of 99.2 ± 6.6 g, none important variation was observed. The alcohol consumption for C and E groups was 20.4 ± 2ml and 18.9 ± 2.3ml, respectively. In clinical examination, only D and E groups developed tumors. The number of tumors per cheek in D group was of 3.2 ± 1.7 with a mean volume of 15.4 ± 9.8 mm3; animals of group E showed 2 ± 0.7 tumors with a mean volume of 84.6 ± 44.9 mm3.
The histological analyses indicate that all specimens of A, B, and C groups showed normal mucosa. Group D showed four speci-mens diagnosed as severe ED and 4 WD OSCC. The E group presented two specimens classified as severe ED, 5 WD OSCC, one MD OSCC and one PD OSCC.
-erbB expression
The WB analysis indicates that erbB1 did not present changes in expression in normal mucosa of groups A, B, and C. The severe ED and WD OSCC developed in D and E groups revealed noticeable variation between similar histological condition and comparing to control normal mucosa. erbB2 showed a significant increase (P = 0.04) in alcohol-exposed mucosa compare to control normal mucosa. In severe ED showed similar expression patterns, while WD OSCC of E group showed significant increase. erbB3 presented significant changes in alcohol-exposed mucosa (P = 0.01), severe ED and WD OSCC compare to control; since, only severe ED showed significant difference between similar histological condition. erbB4 showed variation in severe ED and WD OSCC compare to control; since, comparing similar histological condition only severe ED showed significant variation in their expression ( Table 1), (Fig. 1).
Table 1 erbB expression analysis according their histological condition.
Figure 1 Expression of erbB family members in oral carcinogenesis.
a) Western Blot analysis according to histological condition. erbB1 showed similar expression for normal mucosa (1 to 3) and different expression for severe ED and carcinomas. erbB2 increase their expression in alcohol exposed mucosa, severe ED and carcinomas. 1) Normal mucosa group A, 2) Normal mucosa group B, 3) Normal mucosa group C, 4) Severe ED group D, 5) Severe ED group E, 6) WD OSCC group D, 7) WD OSCC group E, 8) MD OSCC group E and 9) PD OSCC group E.
b) IHC analysis. a) erbB1 expression in normal mucosa with predominant membrane expression pattern b) erbB1 in WD OSCC of group D with cytoplasm expression pattern, c) erbB2 expression in normal mucosa of group A showed cytoplasm expression pattern without presence in corneous stratum, d) erbB2 expression in WD OSCC of group D with cytoplasm expression pattern even in cells next to keratinization area, e) erbB3 expression in normal mucosa, f) erbB3 expression in alcohol exposed mucosa, it shows an important expression increase, g) and h) showed similar expression pattern of erbB4 in WD carcinomas of D and E groups. Objective 100X. Scale bar = 8 µm
-Cellular expression zone
To determine the cellular expression zone IHC analysis was performed. We observed that erbB1 in normal mucosa (A to C groups) was expressed predominantly in membrane, however, severe ED and all OSCC showed differential expression patterns compare to normal mucosa and between similar histological conditions. erbB2 presented cytoplasm expression in all normal mucosa and increase their membrane expression in severe ED and WD carcinoma, with a similar expression pattern between similar histological condition. erbB3 and erbB4 presented variation in alcohol-exposed mucosa, severe ED and WD carcinomas compared to control normal mucosa, and differential expression between similar histological condition ( Table 2), (Fig. 1).
Table 2 erbB expression according to their cellular zone.
Discussion
Alcohol and tobacco consumption are very common in some areas of the world, this behavior increases the relative risk to develop larynx, pharynx, esophagus, and oral cancer (3-6,15). We confirm above, when observed that alcohol, DMBA and both exposure induced clinical, histological, and molecular alterations related to oral carcinogenesis. DMBA is an organ-specific carcinogen, which can mediate neoplasm transformation by inducing DNA damage, generating excess reactive oxygen species, mediate the chronic inflammatory process and tumor develop (16). Our results from D and E groups are according with that above, however, the tumors developed in animals receiving DMBA and alcohol (group E) were bigger than those from animals receiving only DMBA (D group); this would be associated to alcohol’s promoter effect (17). Nevertheless, the promoter effect of alcohol is controversial, some reports attribute this to its metabolic product, acetaldehyde, because that interferes with DNA synthesis and repair, induces point mutation, sister chromatin exchanges, and chromosomal aberration (5,18). Another possible promoter effect derived from alcohol is the increase in mucosa’s permeability to toxic and carcinogenic compounds, it has been reported that diluted ethanol (15%) may be more effective than higher concentration of ethanol, because, this concentration allows molecules and compounds to cross the membrane (19). Both possible effects could induced malignant transformation, however to confirm the malignant phenotype a histological analysis is required. The histological analysis of A, B and C groups showed normal mucosa, D group developed severe ED and WD carcinoma, whereas group E also presented ED, however, invasive carcinoma was the predominant histological condition. This could be compared to patients that consume both compounds, in whom the relative risk to develop oral carcinomas is higher with ethanol and tobacco consumption (15). The golden Syria Hamster chemical oral carcinogenesis is a model that closely resembles a human oral tumor, both histologically and morphologically. Lipid peroxidation, epigenetic control, angiogenesis and apoptosis had been analyzed; however, the implication of an important receptor family, as erbB, during sequential pathogenesis of this model had not been reported (16,20-22).
The role of erbB receptors in carcinogenesis has been established in breast, colon, lung cancer, and head and neck carcinoma. Their expression and function analysis is an important research issue because the possible approaches could be applied in specific therapies (10). Our results indicate that erbB1 showed similar level and pattern expression in normal mucosa; and variations in its level and zone expression in severe dysplasia and invasive carcinomas developed in D and E groups. The above suggest that erbB1 expression is necessary in histological normal condition, but when an early or severe malignant transformation is present the level and cellular zone expression can modify. An interesting feature was the change in expression zone of membrane to cytoplasm in dysplasia and carcinomas as compared to normal mucosa. Lin et al. have reported that erbB can translocate from membrane to nucleus promoting transcription, and enhancing malignant transformation (23). It is possible that this effect is carrying out, nevertheless, to determinate what are the particular mechanism involved, transcription or proteomic analysis is necessary.
Some reports suggest that only erbB1 and erbB2 are sufficient to induce cell transformation, however, others indicate that erbB3 and erbB4 could participate (24-26). Our results suggest an increase in erbB2, erbB3, and erbB4 expression in alcohol exposed mucosa, severe ED and invasive OSCC compared to control normal mucosa. It has been reported that perturbation in epithelial permeability barrier leads to increasing mRNA levels of IL-1a, IL-1b, TNFa, and GM-CSF, as well as in the inflammatory re-sponse. The increase of erbB receptors could be a response mechanism that promotes epithelial maintenance, repair, differentiation, and enhancement of survival in exposed cells, however, if a carcinogen appears the malignant transformation could be more possible (19,27).
In our experimental model erbB2 showed level expression increase in alcohol exposed mucosa, severe ED and carcinomas, with similar membrane cellular expression only for severe ED and well differentiated OSCC. This result agrees with the report by Fong et al., which indicated that erbB2 expression in normal mucosa was almost undetectable, very low in ED, and significantly higher in OSCC, suggesting the participation of this receptor in early transformation (26). Respect to erbB3 a similar level expression pattern to erbB2 was observed in alcohol exposed mucosa, severe ED and carcinomas; however, in their cellular zone expression a heterogeneous pattern was observed. Xia et al. have reported that principally erbB2 and erbB3 expression to be associated to malignant phenotype, suggesting that these receptors may help predict the outcome of patients with OSCC (25). However, our results in this animal model suggest that erbB4 and erbB2 are the predominant receptors in well differentiated OSCC. These changes could be derivate from reduction of erbB1 expression, heterogeneous erbB3 expression and molecular structure of erbB4. The role of erbB4 in neoplasm biology is controversial, because some reports suggest a protector action for this receptor and other consider it as an indicator of aggressiveness and metastasis (24,25,28-30). Today, the golden Syrian hamster model is an important tool in the oral cancer research; nevertheless, the compare to human cancer is necessary, in our opinion this is the principal limitation of that experimental model. Until one does not demonstrate similarities between both carcinogenic processes, the applications will be reduced.
The understanding of cellular and molecular carcinogenesis, could give to us the possibility of restrain the advance of malignant transformation using specific inhibitors, monoclonal antibodies or chemopreventive compounds directed to erbB2, erbB3 or erbB4. The above means a promising advance in oral oncology because currently many young people consume ethanol, tobacco or both and the oral cancer risk is increasing. In this study we observe the carcinogenic potential of DMBA, the promoter effect of ethanol and the particular behavior of erbB receptors, suggesting a homogenous pattern for erbB2 and erbB4 in well differentiated carcinomas. The underlying revelation of the mechanism involved is necessary to lead to the application of new therapeutic strategies derived from this knowledge.
This work was supported by grants from PAPIIT IN217912, Universidad Nacional Autónoma de México and CONACYT-167474.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23565150PONE-D-12-3371510.1371/journal.pone.0059378Research ArticleBiologyModel OrganismsAnimal ModelsMouseChemistryMaterials ScienceMaterial by AttributeNanomaterialsNanotechnologyNanomaterialsMedicineObstetrics and GynecologyFemale SubfertilityUrologyInfertilitySocial and Behavioral SciencesSociologyDemographyFertility RateNanosized TiO2-Induced Reproductive System Dysfunction and Its Mechanism in Female Mice Nano TiO2-Induced Ovary ToxicityZhao Xiaoyang Ze Yuguan Gao Guodong Sang Xuezi Li Bing Gui Suxin Sheng Lei Sun Qingqing Cheng Jie Cheng Zhe Hu Renping Wang Ling Hong Fashui
*
Medical College, Soochow University, Suzhou, People’s Republic of China
Forloni Gianluigi Editor
"Mario Negri" Institute for Pharmacological Research, Italy
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist. All authors have read the manuscript, agreed that the work is ready for submission to PLOS ONE, and accepted responsibility for the manuscript's contents.
Conceived and designed the experiments: FH XZ YZ GG XS. Performed the experiments: FH XZ YZ GG XS. Analyzed the data: FH XZ YZ GG XS BL SG LS QS JC ZC RH LW. Contributed reagents/materials/analysis tools: BL SG LS QS JC ZC RH LW. Wrote the paper: FH XZ.
2013 2 4 2013 8 4 e5937830 10 2012 13 2 2013 © 2013 Zhao et al2013Zhao et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Recent studies have demonstrated nanosized titanium dioxide (nano-TiO2)-induced fertility reduction and ovary injury in animals. To better understand how nano-TiO2 act in mice, female mice were exposed to 2.5, 5, and 10 mg/kg nano-TiO2 by intragastric administration for 90 consecutive days; the ovary injuries, fertility, hormone levels, and inflammation-related or follicular atresia-related cytokine expression were investigated. The results showed that nano-TiO2 was deposited in the ovary, resulting in significant reduction of body weight, relative weight of ovary and fertility, alterations of hematological and serum parameters and sex hormone levels, atretic follicle increases, inflammation, and necrosis. Furthermore, nano-TiO2 exposure resulted in marked increases of insulin-like growth factor-binding protein 2, epidermal growth factor, tumor necrosis factor-α, tissue plasminogen activator, interleukin-1β, interleukin -6, Fas, and FasL expression, and significant decreases of insulin-like growth factor-1, luteinizing hormone receptor, inhibin α, and growth differentiation factor 9 expression in mouse ovary. These findings implied that fertility reduction and ovary injury of mice following exposure to nano-TiO2 may be associated with alteration of inflammation-related or follicular atresia-related cytokine expressions, and humans should take great caution when handling nano-TiO2.
This work was supported by the National Natural Science Foundation of China (grant No. 81273036, 30901218), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, National New Ideas Foundation of Student of China (grant No.11028534). The authors acknowledged in the manuscript all financial support for the work and confirmed that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The manufacture and application of various synthetic nanoparticles are expanding at a rapid rate, and therefore increased environmental and occupational exposures of these materials seem inevitable. Nanosized titanium dioxide (nano-TiO2) presents various uses in industry, in commerce such as cosmetics, sunscreen, toothpaste, food additives and paints, and even in the treatment of contaminated environments [1]–[4]. However, toxicological studies suggested that nano-TiO2 had adverse effects on human health and environmental species. The bio-safety of nano-TiO2 is still an argumentative issue.
Toxicological properties of nano-TiO2 have been studied in kidney [5]–[8], and brain [9]–[14] of animals. Especially, recent studies demonstrated that exposure to nano-TiO2 resulted in toxicity in reproductive system. For example, nano-TiO2 can reduce sperm density and motility, increase sperm abnormality and germ cell apoptosis of male mice in vivo
[15], inhibit follicle development and oocyte maturation of rat in vitro
[16], affect development, reproduction, and locomotion behavior of Caenorhabditis elegans [17], induce genotoxicity and cytotoxicity in Chinese hamster ovary cells in vitro
[18], and impair zebrafish reproduction [19]. Nano-TiO2 was also administered subcutaneously to pregnant mice and transferred to the offspring and impaired the genital and cranial nerve systems of the male mice offspring [20]. However, the mechanisms of nano-TiO2–induced toxicity in reproductive system have yet to be understood. The present study was designed to investigate histopathological changes of ovary, fertility, and sex hormone levels in female mice following exposure to 2.5, 5, and 10 mg/kg body weight TiO2 NPs 90 consecutive days. Furthermore, the inflammation-related or follicular atresia-related cytokine expressions were also examined by real time RT-PCR and ELISA after female mice exposure to nano-TiO2. Our results showed that nano-TiO2 can induce ovarian dysfunction measured by histological assessment, mating rate, pregnancy rate, number of newborns, and sex hormone levels. Nano-TiO2 also significantly altered the inflammation-related or follicular atresia-related cytokine expressions in the mouse ovary.
Materials and Methods
Preparation and Characterization of Nano-TiO2
Nanoparticulate anatase TiO2 of the synthesis and characterization of nano-TiO2 was described in our previous reports [13], [21]. In a typical experiment, 1 mL of Ti (OC4H9)4 was dissolved in 20 ml of anhydrous isopropanol, and was added dropwise to 50 mL of double-distilled water that was adjusted to pH 1.5 with nitric acid under vigorous stirring at room temperature. The temperature of the solution was then raised to 60°C, and maintained for 6 hours to promote better crystallization of nanoparticulate TiO2 particles. Using a rotary evaporator, the resulting translucent colloidal suspension was evaporated yielding a nanocrystalline powder. The obtained powder was washed three times with isopropanol, and then dried at 50°C until the evaporation of the solvent was complete. A 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4M was used as a suspending agent. TiO2 powder was dispersed onto the surface of 0.5% w/v HPMC solution, and then the suspending solutions containing TiO2 particles were treated ultrasonically for 15–20 min and mechanically vibrated for 2 min or 3 min.
The particle sizes of both the powder and nanoparticle suspended in 0.5% w/v HPMC solution after incubation for 24 h (5 mg/mL) were determined using a TecnaiG220 transmission electron microscope (TEM) (FEI Co., USA) operating at 100 kV, respectively. In brief, particles were deposited in suspension onto carbon film TEM grids, and allowed to dry in air. Mean particle size was determined by measuring more than 100 individual particles that were randomly sampled. X-ray-diffraction (XRD) patterns were obtained at room temperature with a MERCURY CCD diffractometer (MERCURY CCD Co., Japan) using Ni-filtered Cu Kα radiation. The surface area of each sample was determined by Brunauer–Emmett–Teller (BET) adsorption measurements on a Micromeritics ASAP 2020M+ C instrument (Micromeritics Co., USA). The average aggregate or agglomerate size of the TiO2 NPs after incubation in 0.5% w/v HPMC solution for 24 h (5 mg/mL) was measured by dynamic light scattering (DLS) using a Zeta PALS+BI-90 Plus (Brookhaven Instruments Corp., USA) at a wavelength of 659 nm. The scattering angle was fixed at 90°. The average particle sizes of powdered nano-TiO2 that suspended in 0.5% w/v hydroxypropylmethylcellulose (HPMC) K4M solvent after 24 h incubation ranged from 5 to 6 nm. The surface area of the sample was 174.8 m2/g. The mean hydrodynamic diameter of TiO2 NP in HPMC solvent ranged from 208 to 330 nm (mainly 294 nm), and the zeta potential after 24 h incubation was 9.28 mV, respectively [13].
Ethics Statement
All experiments were conducted during the light phase, and were approved by the Animal Experimental Committee of the Soochow University(Grant 2111270) and in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals(NIH Guidelines).
Animals and Treatment
CD-1 (ICR) female mice were used in this study. 400 CD-1 (ICR) female mice and 40 CD-1 (ICR) male mice (18±2 g) were purchased from the Animal Center of Soochow University (China). All mice were housed in stainless steel cages in a ventilated animal room. Room temperature of the housing facility was maintained at 24±2°C with a relative humidity of 60±10% and a 12-h light/dark cycle in Animal Center of Soochow University (China). Distilled water and sterilized food were available for mice ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 days.
A 0.5% HPMC was used as a suspending agent. Nano-TiO2 powder was dispersed onto the surface of 0.5%, w/v HPMC, and then the suspending solutions containing TiO2 NPs were treated by ultrasonic for 30 min and mechanically vibrated for 5 min. About the dose selection in this study, we consulted the report of World Health Organization in 1969. According to the report, LD50 of TiO2 for rats is larger than 12,000 mg/kg body weight (BW) after oral administration. In addition, the quantity of TiO2 nanoparticles does not exceed 1% by weight of the food according to the Federal Regulations of US Government. In the present study, we selected 2.5, 5, and 10 mg/kg BW TiO2 NPs exposed to mice by intragastric administration every day. They were equal to about 0.15–0.7 g TiO2 NPs of 60–70 kg body weight for humans with such exposure, which were relatively safe doses. For the experiment, the female mice were randomly divided into four groups (each group N = 100), including a control group (treated with 0.5% w/v HPMC) and three experimental groups (2.5, 5, and 10 mg/kg body weight nano-TiO2 of fresh solutions). The mice were weighed, and the fresh nano-TiO2 suspensions within 30 min were administered to the mice by intragastric administration every day for 90 days. Any symptom or mortality was observed and recorded carefully everyday during the 90 days.
After the 90-day period, all mice were weighed and then sacrificed after being anesthetized with ether. Blood samples were collected from the eye vein by rapidly removing the eyeball. Serum was collected by centrifuging blood at 2500 rpm for 10 min. The ovaries of all animals were quickly removed and placed on ice and then dissected and frozen at −80°C except for 40 ovaries for histopathological examination, respectively.
Mating of Animals
To evaluate the effect of nano-TiO2 on the fertility and growth of newborns, we treated three groups of treated female mice (10 in each mating group) for 90 days. After last day of nano-TiO2 administration, 10 male and 10 control or treated female mice from each group were put in a common cage for mating. The number of newborns from each pregnant mouse were counted and weighed.
Relative Weight of Ovary
After weighing the body and ovary, the relative weight of ovary was calculated as the ratio of ovary (wet weight, mg) to body weight (g).
Titanium Content Analysis
The ovaries were removed from the freezer (−80°C) and thawed. Approximately 0.1 g of the ovary was weighed, digested, and analyzed for titanium content. Inductively coupled plasma-mass spectrometry ([ICP-MS] Thermo Elemental X7; Thermo Electron Co., USA) was used to analyze titanium concentration in the samples.
Hematological Parameters Determination
Blood samples were collected in tubes containing EDTA as anticoagulant. Red blood cells (RBC), lymphocytes (LYMPH), reticulocytes (Ret), white blood cells (WBC), haemoglobin (HGB) were measured using a hematology autoanalyzer (Cell-DYN 3700).
Serum Parameters Analysis
Biochemical parameters were evaluated by serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), uric acid (UA), blood urea nitrogen (BUN), creatinine (Cr). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A, Hitachi, Japan).
Sex Hormone Assays
Sex hormones were evaluated with serum levels of estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), testosterone (T), and sex hormone binding globulin (SHBG) using commercial kits (Bühlmann Laboratories, Switzerland). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A; Hitachi Co., Japan).
Histopathological Examination of Ovary
For pathologic studies, all histopathologic examinations were performed using standard laboratory procedures. The ovaries were embedded in paraffin blocks, then sliced (5 µm thickness) and placed onto glass slides. After hematoxylin–eosin (HE) staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (Nikon U-III Multi-point Sensor System, Japan).
Confocal Raman Microscopy in Ovarian Sections
Raman analysis was performed using backscattering geometry in a confocal configuration at room temperature in a HR-800 Raman microscope system equipped with a 632.817 nm HeNe laser (JY Co., France). Laser power and resolution were approximately 20 mW and 0.3 cm−1, respectively, while the integration time was adjusted to 1 s. Ovaries were embedded in paraffin blocks, then sliced into 5 µm in thickness and placed onto glass slides. The slides were dewaxed, hydrated, and then scanned under the confocal Raman microscope.
Expression Assay of Cytokines
The levels of mRNA expressions of insulin-like growth factor-1 (IGF-1), insulin-like growth factor-binding protein 2 (IGFBP-2), epidermal growth factor (EGF), tumor necrosis factor (TNF-α), tissue plasminogen activator (tPA), luteinizing hormone receptor(LHR), inhibin-α (INH-α), interleukin-1β (IL-1β), IL–6, Fas, Fas ligand (FasL), and growth differentiation factor 9 (GDF-9) in mouse ovary tissue were determined using real-time quantitative RT polymerase chain reaction (RT-PCR) [22]–[24]. Synthesized cDNA was used for the real-time PCR by employing primers that were designed using Primer Express Software according to the software guidelines, and PCR primer sequences are available upon request. To determine levels of protein expressions of IGF- 1, IGFBP-2, EGF, TNF-α, tPA, LHR, INHA, IL-1, IL –6, Fas, FasL, and GDF-9 in the mouse ovary tissue, ELISA was performed using commercial kits that are selective for each respective protein (R&D Systems, USA). Manufacturer’s instruction was followed. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of IGF-1, IGFBP-2, EGF, TNF-α, tPA, LHR, INHA, IL-1β, IL–6, Fas, FasL, and GDF-9 were calculated from a standard curve for each sample.
Statistical Analysis
All results are expressed as means ± standard error (SE). The significant differences were examined by unpaired Student's t-test using SPSS 19 software (USA). A p-value <0.05 was considered as statistically significant.
Results
Body Weight, Relative Weight of Ovary and Titanium Accumulation
The body weight, relative weight of ovary, titanium accumulation in the mouse ovary caused by exposure to nano-TiO2 for 90 consecutive days are exhibited in Figs. 1, 2, respectively. It can be seen that with increased nano-TiO2 doses, the body weight (Fig. 1 a) and relative weight of ovary (Fig. 1 b) were significantly decreased (P<0.05 or P<0.01), while titanium contents in the ovary were significantly increased (Fig. 2, P<0.01).
10.1371/journal.pone.0059378.g001Figure 1 Changes of body weight and relative weight of ovary of mice caused by intragastric administration of nano-TiO2 for 90 consecutive days.
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 10).
10.1371/journal.pone.0059378.g002Figure 2 Titanium accumulation in mouse ovary caused by intragastric administration of nano-TiO2 for 90 consecutive days.
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
Hematological and Biochemical Parameters
Results of hematological detection indicate that WBC, LYMPH, NEUT, RBC, and HGB in the nano-TiO2-treated female mice were significantly reduced with increased exposure doses (P<<0.05 or p<0.01, Table 1). It can be also seen from Table 1 that nano-TiO2 exposure significantly increased the activities of ALT, AST, ALP, LDH, and the levels of Cr, and reduced UA and BUN in sera (P<<0.05 or p<0.01, Table 1), respectively.
10.1371/journal.pone.0059378.t001Table 1 Hematological and biochemical parameters in female mice by intragastric administration of nano-TiO2 for 90 consecutive days.
Index Nano-TiO2 (mg/kg BW)
0 2.5 5 10
WBC (109/L)
7.88±0.39a 5.78±0.29b 4.08±0.20c 2.76±0.14d
LYMPH
(109/L)
6.05±0.30a 3.79±0.19b 2.29±0.11c 1.68±0.08d
NEUT (109/L)
1.62±0.08a 1.18±0.06b 0.81±0.04c 0.52±0.03d
RBC (1012/L)
9.45±0.47a 8.57±0.43b 7.59±0.38c 6.89±0.34d
HGB (g/L)
146.12±7.30a 131.55±6.60b 127.11±6.40b 116.55±5.9c
ALT (U/L)
19.29±0.96a 22.53±1.13b 26.38±1.32c 35.82±1.79d
AST (U/L)
77.27±3.86a 86.25±4.31b 97.56±4.88c 115.39±5.77d
ALP (U/L)
92.81±4.64a 108.66±5.43b 118.61±5.93c 136.86±6.84d
LDH (U/L)
659.66±32.98a 715.38±35.77b 796.71±39.84c 896.79±44.84d
UA(µmol/L)
228.76±11.44a 157.39±7.87b 105.26±5.26c 89.87±0.49d
Cr (µmol/L)
8.45±0.422a 9.89±0.49b 12.23±0.61c 14.68±0.73d
BUN (mmol/L)
9.56±0.48a 8.02±0.40b 6.88±0.34c 6.05±0.30d
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
Reproduction
With increased nano-TiO2 exposed doses, decreased mating rate, pregnancy rate, number of newborns and weight of neonates of mice were significantly observed in Table 2 (P<0.05). In addition, nano-TiO2 led to reduction of survival of young mice at 28th day after birth (P<0.05).
10.1371/journal.pone.0059378.t002Table 2 Effects of nano-TiO2 on conception of female mice, number of newborns, and weight of neonates after intragastric administration of nano-TiO2 for 90 consecutive days.
Index Nano-TiO2 (mg/kg BW)
0 2.5 5 10
Mating rate (%)
100±5a 85±4.25b 75±3.75c 65±3.25d
Pregnancy rate (%)
100±5a 81±4.05b 72±3.6c 58±2.90d
Number of newborns
14±0.7a 10±0.5b 8±0.4c 6±0.3d
Weight of neonates female mice in 1st
after birth (g)
1.54±0.077a 1.52±0.076a 1.45±0.073b 1.40±0.07b
Weight of neonates male mice in 1st
after birth (g)
1.57±0.079a 1.54±0.077a 1.47±0.074b 1.41±0.07b
Survival rate of young mice in 28th day
after birth (%)
98±4.90a 89±4.45b 81±4.05c 72±3.60d
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
Sex Hormone Levels
As shown in Table 3, serum E2 levels were gradually increased (P<0.05), contrary, P4, LH, FSH, and T were significantly decreased in female mice (P<0.05) with increased nano-TiO2 exposed doses. However, no significant differences between serum PRL and SHBG levels in the nano-TiO2 exposed female mice and those of control were observed (P>0.05).
10.1371/journal.pone.0059378.t003Table 3 Effects of nano-TiO2 on sex hormone levels in sera of female mice.
Hormone level Nano-TiO2 NPs (mg/kg BW)
0 2.5 5 10
E2 (pmol/L)
83.66±4.18a 91.09±4.554b 101.98±5.10c 111.88±5.59d
P4 (nmol/L)
34.99±1.75a 30.11±1.506b 26.49±1.32c 23.42±1.17d
LH (IU/L)
0.12±0.006a 0.061±0.003b 0.038±0.002c 0.021±0.001d
FSH (IU/L)
0.48±0.024a 0.42±0.021b 0.37±0.018c 0.28±0.014d
PRL (µg/L)
0.60±0.030a 0.64±0.032a 0.67±0.033a 0.73±0.036a
T (ng/dL)
71.13±3.56a 61.55±3.08b 55.01±2.75c 49.02±2.45d
SHBG (nmol/L)
0.43±0.021a 0.42±0.021a 0.42±0.021a 0.42±0.021a
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
Histopathological Evaluation
Figure 3 presents histological changes in ovary. Normal development of primary follicle and secondary follicle from the control ovary was observed (Fig. 3 a, b). In the nano-TiO2-treated groups, however, a large of atretic follicles, severe inflammatory cell infiltration, and necrosis were observed (Fig. 3 d–e), respectively. In addition, we also observed significant black agglomerates in the ovary samples exposed to 10 mg/kg of nano-TiO2 (Fig. 3e). Confocal Raman microscopy further showed a characteristic nano-TiO2 peak in the black agglomerate (148 cm−1), which further confirmed the deposition of nano-TiO2 in the ovary (see spectrum B in the Raman insets in Fig. 3f). The results also suggest that exposure to nano-TiO2 dose-dependently deposited in the ovary, thus severely resulted in the ovarian injuries.
10.1371/journal.pone.0059378.g003Figure 3 Histopathological observation of ovary of mice caused by intragastric administration of nano-TiO2 for 90 consecutive days.
(a) control groups (unexposed mice) present normal development of primary follicle and secondary follicle; (b) 2.5 mg/kg nano-TiO2-exposed group: green cycle suggest inflammatory cell infiltration, yellow arrows indicate atretic follicle, red arrows present apoptosis or tissue necrosis; (c) 5 mg/kg nano-TiO2-exposed group: green cycle suggest severe inflammatory cell infiltration, yellow cycles present nano-TiO2 deposition, yellow arrows indicate atretic follicle, red arrows present apoptosis or tissue necrosis in ovary; (d) 10 mg/kg nano-TiO2 -exposed group: green cycle suggest severe inflammatory cell infiltration, yellow arrows indicate atretic follicle, red arrows present tissue necrosis, yellow cycle may show aggregation of nano-TiO2 in ovary. Arrow A spot is a representative cell that not engulfed the nano-TiO2, while arrow B spot denotes a representative cell that loaded with nano-TiO2. The right panels show the corresponding Raman spectra identifying the specific peaks at about 148 cm−1.
Cytokines Expression
To confirm molecular mechanisms of nano-TiO2 on the ovary injury, the expression of the inflammation-related genes or follicular atresia-related genes and their proteins in the ovary were examined (Tables 4, 5). It can be observed that exposure to nano-TiO2 resulted in significant increases of IGFBP-2, EGF, TNF-α, tPA, IL-1β, IL –6, Fas, and FasL expression, while obviously decreased IGF-1, LHR, INH-α, and GDF-9 expression in the ovary compared with the control (P<0.05 or 0.01), which are consistent with the trends of fertility reduction and ovary injury.
10.1371/journal.pone.0059378.t004Table 4 Effect of nano-TiO2 on the levels of cytokine gene mRNA expression in mouse ovary.
Ratio ofgene/actin Nano-TiO2 (mg/kg BW)
0 2.5 5 10
IGF-1/actin
1.35±0.068a 0.92±0.046b 0.58±0.029c 0.32±0.016d
IGFBP-2/actin
0.41±0.021a 0.69±0.035b 0.99±0.050c 1.37±0.069d
EGF/actin
0.71±0.036a 1.03±0.052b 1.38±0.069c 1.75±0.088d
TNF-α/actin
0.27±0.014a 0.43±0.022b 0.69±0.035c 0.97±0.049d
tPA/actin
0.07±0.004a 0.28±0.014b 0.42±0.021c 0.56±0.028d
LHR/actin
0.46±0.023a 0.25±0.013b 0.12±0.006c 0.05±0.003d
INH-α/actin
0.95±0.048a 0.61±0.031b 0.38±0.019c 0.12±0.006d
IL-1β/actin
0.22±0.011a 0.39±0.020b 0.68±0.034c 1.05±0.053d
IL-6/actin
0.09±0.005a 0.25±0.013b 0.48±0.024c 0.76±0.038d
Fas/actin
0.55±0.028a 0.78±0.039b 1.06±0.053c 1.67±0.084d
FasL/actin
0.33±0.017a 0.54±0.027b 0.86±0.043c 1.13±0.057d
GDF-9/actin
1.07±0.054a 0.72±0.036b 0.46±0.023c 0.29±0.015d
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
10.1371/journal.pone.0059378.t005Table 5 Effects of nano-TiO2 on the levels of cytokine protein expression in mouse ovary.
Protein expression (ng/g tissue) Nano-TiO2 (mg/kg BW)
0 2.5 5 10
IGF-1
117.62±5.88a 92.29±4.61b 74.19±3.71c 61.24±3.06d
IGFBP-2
34.38±1.72a 40.41±2.02b 47.30±2.36c 61.61±3.08d
EGF
41.22±2.06a 65.38±3.27b 82.85±4.14c 102.42±5.12d
TNF-α
20.00±1.00a 31.03±1.75b 50.14±2.51c 71.49±3.57d
tPA
11.32±0.56a 18.50±0.93b 26.44±1.32c 34.22±1.71d
LHR
39.53±1.98a 29.73±1.49b 21.76±1.09c 15.21±0.76d
INH-α
82.90±4.14a 66.77±3.34b 52.18±2.61c 38.85±1.94d
IL-1β
22.98±1.15a 30.52±1.53b 38.20±1.91c 47.38±2.37d
IL-6
10.99±0.55a 19.19±0.96b 31.59±1.58c 42.04±2.10d
Fas
43.37±2.17a 64.47±3.22b 89.99±4.50c 125.98±6.30d
FasL
31.45±1.57a 42.24±2.11b 54.05±2.70c 67.11±3.35d
GDF-9
85.37±4.27a 66.70±3.33b 47.85±2.39c 31.19±1.56d
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SE (N = 5).
Discussion
To confirm effects of reproductive system of female mice caused by 90 consecutive days exposure to low dose of nano-TiO2, the present study was designed to investigate the changes of ovarian morphology, fertility, hormone levels and expression of relevant genes and their proteins in mouse ovary.
Our findings indicated that the oral nano-TiO2 with 2.5, 5, and 10 mg/kg BW doses for 90 consecutive days led to atretic follicle increases, severe inflammatory response and necrosis in the ovary (Fig. 3). Increased atretic follicles were closely associated with premature ovarian failure following nano-TiO2–induced toxicity. Furthermore, the present study also suggested that exposure to nano-TiO2 reduced mating rate, pregnancy rate, number of newborns and growth of neonates (Table 2) and altered sex hormone levels, including a significant increase of E2 concentration and great decreases of P4, LH, FSH, and T concentrations in the sera (Table 3). Decreased mating capacity of female mice following exposure to nano-TiO2 may be associated with imbalance of sex hormone levels. Follicular atresia is not only the break-down of the ovarian follicles, but also is hormonally controlled apoptosis.Therefore, FSH and LH reduction by exposure to nano-TiO2 resulted in the follicular atresia in mouse ovary. Theoretically, increased effect on E2 levels may be due to the activation of cytochrome P450 aromatase, which converts T into E2 [25]. Elevated E2 and decreased T caused by nano-TiO2 may be related to activate cytochrome P450 aromatase, promoting transformation from T to E2, but it needs to study in future. Taken together, increased atretic follicles and decreased fertility were due to reduction of FSH, P4, LH, and T levels in the nano-TiO2 treated female mice. In addition, T has wide ranging roles in ovarian function, including granulosa cells, theca cells, oocytes, and interstitial cells, because T enhances IGF-I and IGF-I receptor mRNAs in primates [26]. Activins, inhibins, GDF-9, and TGF-β of growth and differentiation factors can influence follicular development [27]. Therefore, the ovarian injuries and changes of sex hormone levels in female mice may be due to nano-TiO2 alter the expression of relevent genes and their proteins in the ovary. To identify the mechanisms of multiple cytokines working together caused by nano-TiO2, mRNA and protein expression of IGFBP-2, EGF, TNF-α, tPA, LHR, INH-α, IL-1β, IL–6, Fas, FasL and GDF-9 from ovary were examined. The assays indicated that the levels of these cytokines were significantly altered (Tables 4, 5). The main results are discussed below.
In mammals, most of ovarian follicles undergo atresia during development, and only a few differentiate to mature finally. Apoptosis occurs in ovarian follicular granulosa cell of majority of animals during follicular atesia, which is the cause of atretic initiation and progression [28]. Therefore apoptosis is suggested to regulated by various factors and atretogenic factors [29]. Intrafollicular IGF-1 plays a critical role in the enhanced response (estradiol production) of the future dominant follicle to the small rise in FSH that initiates the follicular wave. The binding of IGF to its receptors is strongly modulated by a family of six high-affinity IGF-binding proteins (IGFBPs). IGFBP-2 inhibits IGF effect on gonadotropin-induced follicular growth and differentiation. EGF is involved in regulation of ovarian cell proliferation and differentiation. So, EGF, IGF-1 and gonadotropins are determined to be survival factors, but IGFBPs are atretogenic factors [30]. Luo and Zhu demonstrated that FSH concentration, and IGF-1 expression were decreased, contrary IGFBP-2 and EGF expression were increased in process of induced follicular atresia of female rat [31]. Our data indicated that the levels of IGFBP-2 and EGF expression were significantly elevated, whereas IGF-1 expression was greatly inhibited in the nano-TiO2–treated ovary (Tables 4, 5), suggesting that follicular atresia caused by nano-TiO2 (Fig. 3) may be involved in increased IGFBP-2 and EGF, and decreased IGF-1 in the ovary.
Granulosa cells produce estrogen which can synergistically promote FSH-induced self-production of LHR and aromatase activity in cells. In contrast to estrogen, androgen is capable of inducing follicular atresia. Inhibin has also been demonstrated to be an atretic factor. To form bioactive dimers linked by disulfate bonds, one α subunit combines with one of the two β subunits will form two types of inhibin, and combination of two types of β subunits will form three types of actin. Inhibin and actin are involved in coordination between gonadotropins or other factors in regulation of follicular selection, development and atesia [32]. tPA is responsible for the cumulus cell expansion, dispersion and oocyte maturation. Yan et al indicated that tPA expression was significantly increased, but LHR and inhibin subunits were not expressed in the follicle undergoing atresia in rats [33]. Our findings also showed that nano-TiO2 greatly promoted tPA expression, but inhibited LHR and INH-α expression in the ovary (Tables 4, 5), which may lead to follicular atresia in female mice (Fig. 3).
It had been suggested that Fas can be not only found in murine oocytes obtained from atretic follicles [34], but also in granulosa cells of follicles undergoing atresia in the ovary [35]. FasL was also demonstrated to express in the granulosa cells and antral atretic follicles from rat [35], and in granulosa cells of atretic follicles from mouse ovary [36]. Fas and FasL expression in the ovary raise the possibility of Fas-FasL interaction as a mediator of apoptosis during follicular atresia [37]. In the present study, the significant increases of Fas and FasL expression in the ovary are observed following nano-TiO2 induced toxicity (Tables 4, 5), which may conduct to atretic follicle formation of female mice (Fig. 3).
As we know, TNF-α induces apoptosis in several cellular models, and is produced locally in the rat, ovine ovarian granulosa cells and oocytes, and may act as a paracrine regulatory factor [38]. IL- 1β, IL-6 and androgens produced locally in the ovary are also demonstrated to induce follicular atresia [39]. GDF-9 is a growth factor secreted by oocytes in growing ovarian follicles, which is essential for normal follicular development [27]. GDF-9-deficient female mice are infertile because of an early block in folliculogenesis at the type 3b primary follicle stage [40]. Increased levels of TNF-α, IL-1β and IL–6 expressions are also demonstrated to be closely associated with inflammation generation in human and animals. The previous studies suggested that expressions of TNF-α, IL-1β and IL–6 were significantly elevated in the nano-TiO2 exposed lung [41]–[44], liver [45]–[49], kidney [5], [8], [50], and spleen [5], [19], [51]–[53] of animals. The present findings showed that nano-TiO2 exposure markedly promoted expression of TNF-α, IL-1β and IL –6, but significantly inhibited GDF-9 expression in mouse ovary (Tables 4, 5), which resulted in inflammation and follicular atresia in mouse ovary (Fig. 3). A scheme that links the nano-TiO2 and the changes of IGFBP-2, IGF-1, EGF, tPA, LHR, INH-α, Fas, FasL, GDF-9, TNF-α, IL-1β, and IL –6 is depicted in Figure 4.
10.1371/journal.pone.0059378.g004Figure 4 A schematic showing possible mechanisms of nano-TiO2 induced follicular atresia in mouse ovary.
Conclusion
In the present study, we demonstrate that the exposure to nano-TiO2 could result in the fertility reduction, ovarian inflammation and follicaular atresia in a dose-dependent manner, which were closely related to reduction of immunity, biochemical dysfunction, imbalance of sex hormones, and changes of IGFBP-2, IGF-1, EGF, tPA, LHR, INH-α, Fas, FasL TNF-α, IL-1β, IL –6, and GDF-9 expressions in the ovary. Therefore, our findings suggested the need for great caution to handle the nanomaterials for workers and consumers.
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100(4) : 894 –902 . | 23565150 | PMC3615008 | CC BY | 2021-01-05 17:24:34 | yes | PLoS One. 2013 Apr 2; 8(4):e59378 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23573234PONE-D-12-3828210.1371/journal.pone.0060092Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionGeneticsGene ExpressionModel OrganismsAnimal ModelsMouseMolecular Cell BiologyGene ExpressionNeuroscienceLearning and MemoryToxicologyNeurotoxicologyMedicineGene-Expression Changes in Cerium Chloride-Induced Injury of Mouse Hippocampus Brain Injury Following Exposure to CeCl3Cheng Zhe
1
Zhao Haiquan
1
2
Ze Yuguan
1
Su Junju
1
Li Bing
1
Sheng Lei
1
Zhu Liyuan
1
Guan Ning
1
Gui Suxin
1
Sang Xuezi
1
Zhao Xiaoyang
1
Sun Qingqing
1
Wang Ling
1
Cheng Jie
1
Hu Renping
1
Hong Fashui
1
*
1
Medical College of Soochow University, Suzhou, P. R. China
2
College of Life Sciences, Anhui Agriculture University, Hefei, P. R. China
Pant Aditya Bhushan Editor
Indian Institute of Toxicology Research, India
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: FH ZC HZ YZ JS. Performed the experiments: FH ZC HZ YZ JS. Analyzed the data: FS ZC HZ YZ JS BL JC LZ NG RH SG XS XZ LS QS LW. Contributed reagents/materials/analysis tools: BL JC LZ NG RH SG XS XZ LS QS LW. Wrote the paper: FH ZC HZ YZ JS.
2013 3 4 2013 8 4 e6009229 11 2012 19 2 2013 © 2013 Cheng et al2013Cheng et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Cerium is widely used in many aspects of modern society, including agriculture, industry and medicine. It has been demonstrated to enter the ecological environment, is then transferred to humans through food chains, and causes toxic actions in several organs including the brain of animals. However, the neurotoxic molecular mechanisms are not clearly understood. In this study, mice were exposed to 0.5, 1, and 2 mg/kg BW cerium chloride (CeCl3) for 90 consecutive days, and their learning and memory ability as well as hippocampal gene expression profile were investigated. Our findings suggested that exposure to CeCl3 led to hippocampal lesions, apoptosis, oxidative stress and impairment of spatial recognition memory. Furthermore, microarray data showed marked alterations in the expression of 154 genes involved in learning and memory, immunity and inflammation, signal transduction, apoptosis and response to stress in the 2 mg/kg CeCl3 exposed hippocampi. Specifically, the significant up-regulation of Axud1, Cdc37, and Ube2v1 caused severe apoptosis, and great suppression of Adcy8, Fos, and Slc5a7 expression led to impairment of mouse cognitive ability. Therefore, Axud1, Cdc37, Ube2v1, Adcy8, Fos, and Slc5a7 may be potential biomarkers of hippocampal toxicity caused by CeCl3 exposure.
This work was supported by the National Natural Science Foundation of China (grant No. 81273036, 30901218), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the National Bringing New Ideas Foundation of Student of Soochow University (grant No. 201210285036). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Due to their diverse physical, chemical, and biological effects, lanthanides (Ln) have been increasingly used in many aspects of modern society, including agriculture (fertilizers and animal breeding), industry (color TV, photographic cameras, semiconductors, movie films) [1], [2] and in medicine as anticancer, anti-inflammatory, and antiviral agents [3]. Recently, numerous studies have also suggested that CeCl3 and cerium oxide nanoparticles have potential positive effects on fibroblast and osteoblast proliferation and differentiation [4], cutaneous wound healing [5], and downregulation of tumor growth and invasion [6]. Due to the widespread application of Ln,they inevitably enter the ecological environment and are then transferred to humans through food chains [7]. It was reported that the average intake of Ln was about 1.75–2.25 mg/day in China; and may be 5–10 times higher in the Ln ore district [8]. Therefore, the toxicological effects of Ln in humans are a concern.
Cerium, is a typical rare earth element, and is one of the most frequently used elements in Ln. It was found that high doses of Ce3+ induced liver and kidney lesions and induced enzyme metabolic disorders in animals [9], [10]. Li et al. found increases in creatinine, ketone bodies, succinate, lactate, and various amino acids in the serum of rats acutely exposed to Ce3+ for 48 h, as well as a reduction in serum dextrose [11]. These authors suggested that Ce3+ at high doses damaged a specific region of the liver. A recent study showed that exposure to Ce3+ damaged the spleen [12], [13], lung [14]), and liver [15]–[17] of mice. In addition, La3+, Ce3+ and Nd3+ were demonstrated to result in brain injuries in mice [18]–[20]. In the early 1990s, it was reported that the mean memory and intelligence quotient (IQ) value in children aged 6 to 9 years in rare-earth polluted areas were significantly lower than those in non-polluted areas [21]. Feng et al. suggested that La3+ could affect learning ability, which may be ascribed to disturbance in the homeostasis of trace elements, enzymes and neurotransmitter systems in the rat brain [22]. Other studies showed that short-term exposure to La3+- and Ce3+ significantly decreased total antioxidant level and the activities of Na+, K+-ATPase, and increased the activities of acetylcholinesterase in rat brain [23. 24]. Our previous study suggested that cerium was significantly accumulated in the mouse hippocampus, and in turn caused hippocampal apoptosis and impairment of spatial recognition memory in mice exposed to CeCl3 for 60 consecutive days [20]. These data indicated that Ln affects the central nervous system (CNS), especially cognitive ability. Although the above-mentioned studies clarified the neurotoxic effects of Ln, further studies are needed to elucidate the synergistic molecular mechanisms of multiple genes activated by Ln-induced nuerotoxicity in animals and humans. We speculate that CeCl3-induced CNS damages in mice may have special biomarkers of nuerotoxicity.
Microarray technology has been used as a screening tool for the identification of molecular mechanisms involved in toxicity [25]. Large-scale gene expression analysis provides a logical approach for researchers to identify those genes and their products which are involved in conferring resistance or susceptibility to toxic substances. In the present study, we aimed to investigate hippocampal dysfunction caused by CeCl3 exposure and alterations in the gene expression profile in mouse hippocampus using microarray analysis. The data on gene expression profiling showed significant changes in genes involved in learning and memory, immunity and inflammation, signal transduction, apoptosis, and response to stress in the CeCl3 exposed hippocampi. Our findings may provide a reference for future mechanistic studies on the effects of Ln on brain tissues in animals or humans.
Materials and Methods
CeCl3 was purchased from Shanghai Chem. Co. (China) and was of analytical grade (99.99%).
Ethics Statement
All experiments were conducted during the light phase, were approved by the Animal Experimental Committee of Soochow University (Grant 2111270) and were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Guidelines).
Animals and Treatment
Male CD-1 (ICR) mice (18±2 g, 4 weeks old) were purchased from the Animal Center of Soochow University (Suzhou, China), and were housed in polypropylene cages with corncob bedding in a controlled-environment animal room (temperature, 24±1°C; relative humidity, 60±10%; photoperiod, 12 h light/dark cycle).
Three hundred animals were randomly divided into four groups (75 mice/group): control group (treated with purified water) and three experimental groups (treated with 0.5, 1, and 2 mg/kg body weight [BW] CeCl3, respectively). With regard to the dose selection in this study, we identified the average intake of Ln (1.75–2.25 mg/day) in humans [8]. The mice were weighed and the CeCl3 solutions were administered intragastrically every day for 90 days. Symptoms and mortality were carefully observed and recorded every day for 90 days (death not observed).
Behavioral Experiment
Following 90 days of CeCl3 administration, the acquisition of spatial recognition memory in mice (N = 20) was determined using the Y-maze test. In order to avoid any stress-related interference with the learning procedure, mice were not handled by the experimenter, but were allowed to voluntarily enter the maze. To assess spatial recognition memory, the Y-maze test consisted of two trials separated by an intertrial interval (ITI). The Y-maze was made of green–blue painted timber and consisted of three arms with an angle of 120° between each two arms. Each arm was 8 cm × 30 cm × 15 cm (width × length × height). The three identical arms were randomly designated: Start arm, in which the mouse started to explore (always open), Novel arm, which was blocked during the 1st trial, but open during the 2nd trial, and the Other arm (always open).
The maze was placed in a sound attenuated room with low illumination. The floor of the maze was covered with sawdust, which was mixed after each individual trial in order to eliminate olfactory stimuli. Visual cues were placed on the walls of the maze, and the observer was always in the same position at least 3 m from the maze.
The Y-maze test consisted of two trials separated by an ITI to assess spatial recognition memory. The first trial was of 10-min duration and allowed the mouse to explore only two arms (Start arm and Other arm) of the maze, with the third arm (Novel arm) being blocked. After a 1 h ITI, the second trial (retention) was conducted, during which all three arms were accessible and novelty vs. familiarity was analyzed by comparing behavior in all three arms. For the second trial, the mouse was placed back into the maze in the same starting arm, with free access to all three arms for 5 min. By using a ceiling-mounted CCD camera, all trials were recorded on a VCR. Video recordings were later analyzed and the number of entries and time spent in each arm were analyzed. Data were also expressed as percentage of total time and distance spent in arms every 30 s and during the total 5 min period [26]. In the second trial, we also assessed which of the arms was entered first as another way of recognizing the Novel arm–discrimination memory. Because retention in the Y-maze test does not last longer than a few hours, this task can be assessed three times in the same animal [27]. All mice were therefore tested in the Y-maze three times using a 1 h ITI. There was no arm difference in animals treated with CeCl3, but when compared to the control, animals in this group spent less time in the Novel arm indicating that animals in this group failed to recognize the Novel arm after the 1 h ITI.
To measure spatial recognition memory, the number of entries and time spent in each arm of the maze by each mouse was recorded and novelty versus familiarity was analyzed by comparing behavior in all three arms. The number of arms visited was taken as an indicator of locomotor and exploratory activity.
Preparation of Hippocampus
After the behavioral experiments, all animals were first weighed and then sacrificed after being anesthetized by ether. The brains were quickly removed and placed in ice-cold, and the hippocampi were dissected and frozen at −80°C.
After weighing the body and brains, the coefficient of brain mass to BW was calculated as the ratio of brain (wet weight, mg) to BW (g).
Histopathological Examination of the Hippocampus
For pathologic studies, all histopathologic examinations were performed using standard laboratory procedures. The hippocampi were embedded in paraffin blocks, then sliced (5 µm thickness) and placed onto glass slides. After hematoxylin–eosin staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (Nikon U-III Multi-point Sensor System, Japan).
Observation of Hippocampus Ultrastructure
Hippocampi were fixed in a fresh solution of 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde and 2% formaldehyde followed by a 2 h fixation period at 4°C with 1% osmium tetroxide in 50 mM sodium cacodylate (pH 7.2–7.4). Staining was performed overnight with 0.5% aqueous uranyl acetate. The specimens were dehydrated in a graded series of ethanol (75, 85, 95, and 100%), and embedded in Epon 812. Ultrathin sections were obtained, contrasted with uranyl acetate and lead citrate, and observed with a HITACHI H600 Transmission Electron Microscope (TEM) (HITACHI Co., Japan). Hippocampal apoptosis was determined based on the changes in nuclear morphology (e.g., chromatin condensation and fragmentation).
Analysis of Hippocampal Cerium Content
The frozen hippocampal tissues were thawed and ∼ 0.1 g samples were weighed, digested, and analyzed for cerium content. Briefly, prior to elemental analysis, the lung tissues were digested overnight with nitric acid (ultrapure grade). After adding 0.5 mL of H2O2, the mixed solutions were incubated at 160°C in high-pressure reaction containers in an oven until the samples were completely digested. The solutions were then incubated at 120°C to remove any remaining nitric acid until the solutions were colorless and clear. Finally, the remaining solutions were diluted to 3 mL with 2% nitric acid. Inductively coupled plasma-mass spectrometry (Thermo Elemental X7; Thermo Electron Co., Waltham, MA, USA) was used to determine the cerium concentration in the samples. Indium (20 ng/mL) was chosen as an internal standard element. The detection limitation of cerium was 0.074 ng/mL and data are expressed as ng/g of fresh tissue.
Oxidative Stress Assay
Reactive oxygen species (ROS) (O2
− and H2O2) production and levels of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxy deoxyguanosine (8-OHdG) in the hippocampal tissues were assayed using commercial enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions.
Microarray and Data Assay
Gene expression profiles in hippocampal tissue isolated from 5 mice in the control and CeCl3-treated groups were compared by microarray analysis using Illumina BeadChip purchased from Illumina, Inc. (San Diego, CA, USA). Total RNA was isolated using the Ambion Illumina RNA Amplification Kit (cat no. 1755) according to the manufacturer’s protocol, and stored at −80°C. RNA amplification is the standard method for preparing RNA samples for array analysis [28]. Total RNA was then submitted to the Biostar Genechip Inc. (Shanghai, China) where RNA quality was analyzed using a BioAnalyzer, and cRNA was generated and labeled using the one-cycle target labeling method. cRNA from each mouse was hybridized for 18 h at 55°C on Illumina Human HT-12 v 3.0 BeadChips, containing 45, 200 probes (Illumina, Inc., San Diego, CA, USA), according to the manufacturer’s protocol and subsequently scanned with the Illumina BeadArray Reader 500. This program identifies differentially expressed genes and establishes the biological significance based on the Gene Ontology Consortium database (http://www.geneontology.org/GO.doc.html, GSE44906). Data analyses were performed with GenomeStudio software version 2009 (Illumina Inc., San Diego, CA, USA), by comparing all values obtained at each time point against the 0 h values. Data were normalized with the quantile normalization algorithm, and genes were considered as detected if the detection p-value was lower than 0.05. Statistical significance was calculated using the Illumina DiffScore, a proprietary algorithm that uses the bead standard deviation to build an error model. Only genes with a DiffScore ≤-13 and ≥13, corresponding to a p-value of 0.05, were considered statistically significant(You et al., 2010; Grober Oli et al., 2011).
Quantitative Real-time PCR (qRT-PCR)
Expression levels of Adcy8, Axud1, Cdc37, Fos, Slc5a7, and Ube2v1 mRNA in mouse hippocampal tissues were determined using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) [29]. Synthesized complimentary DNA was generated by qRT-PCR with primers designed with Primer Express Software (Applied Biosystems, Foster City, CA, USA) according to the software guidelines, and PCR primer sequences are listed in Table 1.
10.1371/journal.pone.0060092.t001Table 1 Real time PCR primer pairs. PCR primers used in the gene expression analysis.
Gene name Description Primer sequence Primer size (bp)
Refer-actin
mactin-F
5′-GAGACCTTCAACACCCCAGC-3′
mactin-R
5′-ATGTCACGCACGATTTCCC-3′
263
Slc5a7
mSlc5a7-F
5′-TTCCAGATTCAGGCAGTAGACG-3′
mSlc5a7-R
5′-GGGAGGGAAACTCCTATCTTGT-3′
125
Fos
mFos-F
5′-TGTACTGTAGTCCTTCAGCGTCA-3′
mFos1-R
5′-TGTCAGAACATTCAGACCACCTC-3′
82
Adcy8
mAdcy8-F
5′-GGAGAAACAGACTTCCTGGGTACAA-3′
mAdcy8-R
5′-CATTGTGCTCCCTTAACTCCTTCATTTC-3′
175
Axud1
mAxud1-F
5′-CGCCCTTCATTAGCTGATGTT-3′
mAxud1-R
5′-CAGAGCCTGCGTTTCTTGG-3′
117
Ube2v1
mUbe2v1-F
5′-GATAGAGTGTGGGCCTAAGTACC-3′
mUbe2v1-R
5′-TGAGCAGTTCGAATGGAGTG-3′
120
Cdc37
mCdc37-F
5′-CTGTCAGACAACGTCCACCTG-3′
mCac37-R
5′-CGAAGCACTTCTGGAGTTCCT-3′
88
ELISA Assay
To determine Adcy8, Axud1, Cdc37, Fos, Slc5a7, and Ube2v1 levels in mouse hippocampal tissues, Enzyme Linked Immunosorbent Assay (ELISA) was performed using commercial kits that were selective for each respective protein (R&D Systems, USA), following the manufacturer’s instructions. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of Adcy8, Axud1, Cdc37, Fos, Slc5a7, and Ube2v1 were calculated from a standard curve for each sample.
Statistical Analysis
All results are expressed as means ± standard error of the mean(SEM). The significant differences were examined by unpaired Student’s t-test using SPSS 19 software (USA). A p-value <0.05 was considered as statistically significant.
Results
Spatial Recognition Memory
The effects of CeCl3 on the spatial recognition memory of mice are presented in Fig. 1. From this figure it can be seen that the percentage duration in the novel arm in control mice was significantly higher than that in the start and other arms throughout the experiment, while the percentage duration in the novel arm in 0.5, 1 and 2 mg/kg CeCl3 -treated mice was lower than that of control mice (P<0.05), respectively. These results suggest that exposure to low-dose CeCl3 for a long period impaired the spatial recognition memory of mice. This might be related to brain injury, which was confirmed by morphological examination.
10.1371/journal.pone.0060092.g001Figure 1 Effect of CeCl3 on spatial recognition memory in mice in the Y-maze test following intragastric administration of CeCl3 for 90 consecutive days.
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 20).
Locomotor Activity
The effect of CeCl3 on arm visits is presented in Fig. 2. The results indicated that CeCl3 dose-dependently decreased the number of arm entries compared to the control. Measurement of total number of arm entries during the second trial revealed a significant difference between the three arms in each group after the 1 h ITI.
10.1371/journal.pone.0060092.g002Figure 2 Effects of acute CeCl3 locomotor activity of mice in the Y-maze test following intragastric administration of CeCl3 for 90 consecutive days.
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 20).
BW, Relative Brain Weight, and Cerium Accumulation
BW, relative brain weight and cerium accumulation in mice are listed in Table 2. As shown, an increase in CeCl3 dose led to a gradual decrease in BW, and relative brain weight, whereas cerium content was significantly increased (p<0.05), indicating growth inhibition and brain damage in mice. These findings were confirmed by subsequent hippocampus histological and ultrastructural observations and oxidative stress assays.
10.1371/journal.pone.0060092.t002Table 2 Body weight, relative weight of brain and cerium accumulation in mouse hippocampus after intragastric administration of CeCl3 for 90 consecutive days.
Index CeCl3 (mg/kg BW)
0 0.5 1 2
Net increase of body weight (g)
23±1.15a 19±0.95b 14±0.70c 10±0.50d
Relative weight of brain (mg/g)
16.08±0.80a 14.59±0.73a 11.37±0.57b 8.98±0.45c
Cerium content
(ng/g tissue)
Not detected 150±7.50a 288±14.40b 609±30.45c
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 10).
Histopathological Evaluation
The histopathological changes in mouse hippocampus are presented in Fig. 3. It can be seen that exposure to CeCl3 resulted in abnormal pathological changes in the hippocampi when compared with the control group, including overproliferation of all glial cells, tissue necrosis and bleeding, indicating that the CNS was injured by exposure to CeCl3.
10.1371/journal.pone.0060092.g003Figure 3 Histopathology of hippocampi in mice caused by intragastric administration of CeCl3 for 90 consecutive days (N = 5).
(a) Control group; (b) 0.5 mg/kg CeCl3 group indicates significant proliferation of all glial cells; (c) 1 mg/kg CeCl3 group indicates significant proliferation of all glial cells and tissue necrosis; (d) 2 mg/kg CeCl3 group indicates significant proliferation of all glial cells, tissue necrosis and bleeding.
Hippocampal Cell Ultrastructure
The changes in hippocampal cell ultrastructure in mice are shown in Fig. 4. It was observed that the neurons in the control group contained elliptical nuclei with homogeneous chromatin. However, the hippocampal cell ultrastructure in the CeCl3-treated mice showed typical apoptosis, including irregularity of nuclear membrane, shrinkage of the nucleus, chromatin marginalization, mitochondrial swelling and ectatic endoplasmic reticulum. These results suggested that exposure to low doses of CeCl3 caused hippocampal apoptosis, which might affect spatial recognition memory in mice.
10.1371/journal.pone.0060092.g004Figure 4 Ultrastructure of hippocampal cells in mice caused by intragastric administration of CeCl3 for 90 consecutive days (N = 5).
(a) Control group indicates nucleus with homogeneous chromatin; (b) 0.5 mg/kg CeCl3 group indicates irregularity of nuclear membrane, shrinkage of nucleus, chromatin marginalization, and mitochondrial swelling; (c) 1 mg/kg CeCl3 group indicates irregularity of nuclear membrane, severe shrinkage of nucleus, chromatin marginalization, and mitochondrial swelling; (c) 2 mg/kg CeCl3 group indicates significant irregularity of nuclear membrane, severe shrinkage of nucleus, chromatin marginalization, mitochondrial swelling, and ectatic endoplasmic reticulum.
Analysis of Oxidative Stress
To further confirm hippocampal apoptosis, we detected ROS generation rate, and levels of lipids, proteins, and DNA peroxidation. The effects of CeCl3 on the production of O2
− and H2O2 in mouse hippocampal tissues are shown in Table 3. With increased CeCl3 dose, the rate of ROS generation in the CeCl3-exposed groups was significantly elevated (p<0.05), suggesting that exposure to CeCl3 accelerated ROS production in the hippocampal tissues. As shown in Table 3, levels of MDA, PC, and 8-OHdG in the hippocampal tissues from the CeCl3-exposed groups were markedly elevated (p<0.05), suggesting that CeCl3–induced ROS accumulation led to lipid, protein, and DNA peroxidation.
10.1371/journal.pone.0060092.t003Table 3 Oxidative stress in mouse hippocampus after intragastric administration of CeCl3 for 90 consecutive days.
Oxidative stress CeCl3 (mg/kg BW)
0 0.5 1 2
O2− (nmol/mg
prot. min)
31.05±1.55 40.86±2.04 52.89±2.64 67.50±3.38
H2O2 (nmol/mg prot. min)
58.05±2.90 82.64±4.13 106.60±5.33 148.50±7.42
MDA (µmol/mg prot)
1.46±0.07 2.15±0.11 3.90±0.19 6.95±0.35
Carbonyl (µmol/
mg prot)
0.73±0.04 1.32±0.07 2.50±0.12 4.10±0.21
8-OHdG (mg/g tissue)
0.57±0.03 3.05±0.15 5.74±0.29 9.61±0.48
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 5).
Gene Expression Profile
Treatment with 2 mg/kg BW of CeCl3 resulted in the most severe hippocampal damage and these tissues were used to determine gene expression profiles to further explore the mechanisms of hippocampal damage induced by CeCl3. The results showed that 244 genes were obviously altered in brain tissue following exposure to CeCl3 compared with the control group. Of the genes altered, 194 genes were up-regulated and 50 genes were down-regulated. Alterations in the known gene expression profile induced by exposure to CeCl3 for 90 consecutive days are shown in Table S1. The gene expression profile of the hippocampal tissues from the CeCl3-treated mice was classified using the ontology-driven clustering algorithm included with the PANTHER Gene Expression Analysis Software (www.pantherdb.org/), which suggested that 154 genes in the gene expression profile were divided into 10 cluster categories including learning and memory, transcription, signal transduction, cell structure and cytoskeleton, growth and development, metabolism, immunity and inflammation, apoptosis, response to stress and translation (Fig. 5), and the function of 90 genes was unknown.
10.1371/journal.pone.0060092.g005Figure 5 Gene expression changes in mouse hippocampus following intragastric administration of 2 mg/kg BW CeCl3 for 90 consecutive days.
Selection of over-represented biological categories that include differentially expressed genes. Rows represent gene categories and the percentage of the genes included in each category to the total number of differentially expressed genes, and the number of genes is indicated on the right side of each bar.
qRT-PCR
To verify the accuracy of the microarray analysis, six genes that demonstrated significantly different expression patterns were further evaluated by qRT-PCR due to their association with learning and memory, apoptosis and oxidative stress. These 3 genes including Axud1, Cdc37, and Ube2v1 were up-regulated, whereas 3 genes including Adcy8, Fos, and Slc5a7 were down-regulated (Table 4). The qRT-PCR analysis of all 6 genes displayed expression patterns comparable with the microarray data (i.e., either up- or down-regulation; Table S1).
10.1371/journal.pone.0060092.t004Table 4 RT-PCR validation of selected genes from Microarray Data.
Function Gene △△Ct Fold Microarray data (Fold)
learning and memory
Slc5a7
0.71±0.04 0.61±0.03a 0.52±0.03a
Fos
1.82±0.09 0.28±0.01a 0.43±0.02b
Adcy8
0.40±0.02 0.76±0.04a 0.54±0.03b
Apoptosis
Axud1
−3.40±0.17 10.59±0.53a 14.13±0.71b
Ube2v1
−1.77±0.09 3.40±0.17a 2.59±0.13b
Cdc37
−1.33±0.07 2.51±0.13a 26.25±1.31b
Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 5).
ELISA
In order to further confirm expression of Adcy8, Axud1, Cdc37, Fos, Slc5a7, and Ube2v1 in hippocampal tissues, their protein levels were measured by ELISA. The expression levels of Axud1, Cdc37, and Ube2v1 proteins in hippocampus were gradually elevated following exposure to 2 mg/kg CeCl3, whereas the levels of Adcy8, Fos, and Slc5a7 in CeCl3-exposed mice were lower than those in unexposed mice (Fig. 6).
10.1371/journal.pone.0060092.g006Figure 6 Effect of CeCl3 on the levels of protein expression in mouse
hippocampus following intragastric administration of CeCl3 for 90 consecutive days. Different letters indicate significant differences between groups (p<0.05). Values represent means ± SEM (N = 5).
Discussion
The results of this study indicated that exposure to low dose CeCl3 for 90 days resulted in decreases in spatial recognition memory (Fig. 1). According to Dellu et al., the two-trial Y-maze task is a specific and sensitive test of spatial recognition memory in rodents [27]. Our data supported this view by showing that there were always significant arm effects on percentage measures of total duration of visits and number of visits during the retention test. Following exposure to increased doses of CeCl3, the time spent in the unfamiliar novel arm, and or the frequency with which mice entered this arm, were not statistically different from the familiar start and other arms after the 1 h ITI. However, in unexposed mice, the time spent in the unfamiliar novel arm, and or the frequency with which mice entered these arms, were higher than those for the familiar start and other arms (Fig. 1). This suggested that mice were highly sensitive to their spatial and contextual environment. Moreover, our data were consistent with previous findings in CD1 mice which demonstrated a very high level of novelty exploration [27]. In the retention test, mice had to make a choice between the novel arm (unfamiliar) and the other arm (familiar) when they were released from the start arm in the Y-maze. Mice exposed to CeCl3 showed a lower score in discrimination memory than unexposed mice. Our findings demonstrated that CeCl3 may impair spatial recognition memory in mice in the Y-maze. Locomotor activity is a function of the level of excitability of the central nervous system [30]. We found that CeCl3 reduced locomotor activity in mice. Furthermore, in the CeCl3-exposed mice, the reduction in learning and memory was coupled with a reduction in brain indices, cerium accumulation (Table 2), severe hippocampal lesions (Figs. 2, 3) as well as oxidative stress (Table 3). These injuries may be associated with alterations in gene expression in the hippocampus. To identify the molecular mechanisms of multiple genes working together following exposure to CeCl3, microarray assays of hippocampal RNA were performed to establish a global gene expression profile. These assays suggested (Table S1) that the expression levels of 154 known genes were obviously altered, and these genes were involved in learning and memory, immune/inflammatory responses, apoptosis, response to stress, and signal transduction. The main results are discussed below.
Overproliferation of all glial cells is a universal event in many types of CNS damage. Glial cells are required for the development, formation and repair of neurons, and the axonal regeneration process is guided by these cells. In the present study, overproliferation of all glial cells caused by exposure to CeCl3 indicated that inflammatory/immune responses occurred in hippocampal tissue in addition to hippocampal injury. Secretoglobin, family 1A, member 1 (SCGB1A1) is the most studied member of the SCGB gene superfamily, its encoded product is a multifunctional protein with anti-inflammatory properties and a manifestation of anti-allergic, anti-chemotactic and anti-tumorigenic activity [31], [32]. Our data suggested that SCGB1A1 was significantly up-regulated with a Diffscore of 74.04 (Table S1), which may be associated with inflammatory/immune responses in the Ce3+-treated hippocampus. Adenosine a2B receptor (ADORA2B) is a member of the adenosine receptor group of G-protein-coupled receptors. ADORA2B plays a role in the relaxation of smooth muscle in the vasculature and intestines, inhibits monocyte and macrophage function and stimulates mast cell mediator release. Studies have shown that genetic deficiency of ADORA2B increased the death rate of mice suffering from cecal ligation and puncture-induced sepsis, and the increased mortality of ADORA2B knockout mice may be associated with increased levels of inflammatory cytokines, chemokines, augmented NF-κB and p38 activation in the spleen, heart, and plasma [33]. In the study by Frick et al., the endogenous protective molecule, ADORA2B, was expressed on intestinal epithelial cells [34]. In the current study, ADORA2B was down-regulated with a Diffscore of −16.41 (Table S1), which may promote inflammatory responses in the hippocampus following exposure to CeCl3. FCRLS belongs to a family which includes an intriguing group of cell surface receptors with preferential B cell expression. It has been shown to positively or negatively modulate the antibody-mediated responses of lymphocytes and inflammatory cells [35] and has important pathophysiologic roles in autoimmunity, allergy and inflammation [36]. Fc receptor-like s, scavenger receptor (FCRLS) deficient mice have profoundly altered humoral immune responses, immediate hypersensitivity, cytotoxic inflammatory responses and immune complex mediated inflammation [37], [38]. CeCl3 exposure led to significant down-regulation of FCRLS with a Diffscore of −68.11 (Table S1), which may also be related to inflammatory/immune responses in the Ce3+-treated hippocampus. In addition, guanine nucleotide binding protein, alpha q polypeptide (GNAQ), neurexin III (NRXN3) and rho/rac guanine nucleotide exchange factor (GEF) 2 (LBCL1) levels involved in the development, maturation, repair and regeneration of neurons were also significantly increased with Diffscores of 39.97, 29.42 and 28.26, respectively (Table S1). It is known that GNAQ transduces signals from the α1-adrenergic receptor to stimulate inositol-1,4,5-trisphosphate and diacylglycerol generation via phospholipase C, and then activates additional downstream effectors, to regulate a diverse range of biological functions. Mueller et al. indicated that as a strategy, increased GNAQ could improve neurite growth and sprouting after nerve damage [39]. NRXN3 is a cell adhesion molecule which helps to specify and stabilize synapses and provide receptors for neuroligins, neurexophilins, dystroglycans and a-latrotoxins [40]. It has been linked to many addictions such as cocaine, alcohol and morphine, suggesting that NRXN3 may play a crucial role in the synaptic plasticity of neurons in the basal ganglia, where they regulate reward-related learning [41]. LBCL1 and its negative regulator dynlt1, dynein light chain tctex-type 1d (Tctex-1) determine the genesis of neurons from precursors in the embryonic murine cortex [42]. In addition, Tctex-1 is selectively enriched in almost all cycling progenitors, young neuronal progeny, immature progenitors and migrating neuroblasts, but not in mature granular cells and astrocytes [43]. Increased LBCL1 may inhibit the differentiation, maturity and migration of neurons in mammalian brain. FBJ osteosarcoma oncogene (Fos) is a proto-oncogene. Its product dimerizes with members of the Jun family to form the transcription factor AP-1, which regulates a series of genes in response to many stimuli [44]. It has been proposed that Fos has a critical role in signal transduction, cell proliferation and differentiation [45]. Reports have demonstrated that the level of Fos expression was decreased in the LC (locus coeruleus) from rats treated acutely or chronically with morphine [46], and Fos knockout mice were growth-retarded, developed osteopetrosis with deficiencies in bone remodelling and tooth eruption, and have altered hematopoiesis [47]. In the present study, FOS gene was significantly down-regulated with a Diffscore of −19.89 (Table S1). Therefore, up-regulation of GNAQ, NRXN3 and LBCL1, and down-regulation of FOS by exposure to CeCl3 may result in inhibition of the development, maturation, repair and regeneration of neurons, and subsequently decreased spatial recognition memory in mice.
In the present study, our findings suggested that long-term exposure to low dose CeCl3 promoted ROS production (such as O2
− and H2O2) and led to peroxidation of lipids, proteins, and DNA (Table 3), which led to hippocampal apoptosis (Fig. 3). ROS are one of the triggers for intrinsic apoptosis. The overproduction of ROS has been shown to be closely associated with the induction of apoptotic and necrotic cell death in cell cultures [48]. This breaks down the balance of the oxidative/antioxidative system in the brain, resulting in lipid peroxidation, which increased the permeability of mitochondrial membrane [49]. In our previous studies, Ln3+ was also shown to mediate apoptosis in the spleen, liver in mice through the induction of ROS [13], [17]. However, the apoptotic mechanism in Ln3+-induced neurotoxicity remains unclear. In the present study, our data indicated that the expression of genes related to apoptosis, such as apoptosis antagonizing transcription factor (TRB), ubiquitin-conjugating enzyme e2 variant 1 (UBE2V1), csrnp1, cysteine-serine-rich nuclear protein 1 (AXUD1) and cell division cycle 37 homolog (CDC37), were significantly increased with Diffscores of 28.24, 41.69, 72.68 and 28.98 (Table S1), respectively. It was demonstrated that the protein encoded by TRB is involved in cell cycle control, gene transcription, and apoptotic signaling in neural tissues, therefore, TRB in cortical neurons exhibits anti-apoptotic activity, protecting cells from neuronal damage [50]. UBE2V1 is known to encode an ubiquitin conjugating enzyme variant. Syed identified UBE2V1 as a potential proto-oncogene, showing that high-level expression of UBE2V1 in cultured human cells caused a significant increase in NF-κB activity as well as the expression of Bcl-2, which is a target anti-apoptotic protein [51]. The up-regulation of UBE2V1 also conferred prolonged cell survival and protected cells against apoptosis induced by diverse stressors. AXUD1 is a member of a novel gene family encoding nuclear proteins which contain cysteine- and serine-rich domains, and is often considered to be related to reduced apoptosis [52]. CDC37 is an essential component of the mitogen-activated kinase protein (MAPK) signaling pathway. The protein encoded by CDC37 is thought to specifically strengthen the interaction between Hsp90 and kinase clients [53], which are involved in tumorigenesis, and interruption of Hsp90-managed pathways has a broad effect on cell growth and susceptibility to apoptosis [54]. It was demonstrated that CDC37 is required for G1 cell cycle progression, plays a critical role in v-Src oncogenesis and is required to maintain multiple protein kinase pathways implicated in apoptosis induction [55]. Therefore, brain apoptosis following exposure to CeCl3 may lead to upregulation of TRB, UBE2V1, AXUD1 and CDC37 expressions. Specifically, increased TRB, UBE2V1, and AXUD1 levels perhaps promoted anti-apoptotic activities, protecting cells from neuronal damage following Ce3+-induced neurotoxicity.
Previous studies have suggested that the reduction in learning and memory in rats or mice caused by La3+ and Ce3+ were related to significant increases in acetylcholine (Ach), glutamic acid (Glu), and nitric oxide (NO), and marked decreases in neurotransmitters such as norepinephrine (NE), 5-hydroxy tryptophan and its metabolite 5-hydroxy indole acetic acid, as well as dopamine and its metabolite 3, 4-dihydroxyphenylacetic acid [18], [22]. In addition, Ce3+ exposure resulted in a significant reduction in Ca2+ concentration in mouse brain [18]. Ln3+-induced neurotransmitter and Ca2+ changes were thought to be related to alterations in gene expressions in the brain [18]. In the present study, our data showed decreases in Slc5a7 and ADCY8 expression with Diffscores of −14.98, and −16.62, and an increase in DCAMKL1 expression with a Diffscore of 23.33 in the CeCl3-treated hippocampus (Table S1), respectively. Solute carrier family 5 (choline transporter), member (Slc5a7) is expressed in cholinergic neurons and is efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis [56]. Ferguson et al. demonstrated that Slc5a7 is an essential and regulated presynaptic component of cholinergic signaling and Slc5a7 warrants consideration as a candidate gene for disorders characterized by cholinergic hypofunction [57]. In the CNS, the cholinergic system plays an important role in learning and memory ability, and brain cholinergic dysfunction results in dementia with symptoms such as memory loss and disorientation in cerebrovascular or Alzheimer’s disease [58]. Adenylate cyclase 8 (ADCY8 or AC8) is a pure Ca2+ sensor, catalyzing the transformation of ATP to cAMP, with an important role in neuronal plasticity. The deletion of ADCY8 can exacerbate neuroapoptosis in mice exposed to ethanol [59]. The AC8 knockout mice exhibited mossy fiber long-term potentiation (LTP) defects comparable with wild type mice, and short-term plasticity was also disrupted [60]. All these reports provided us with credible evidence that the decrease in Ca following Ce3+ exposure was related to down-regulation of ADCY8 expression, which in turn, exacerbated neuroapoptosis and disrupted mossy fiber LTP. Decreased Slc5a7 and Adcy8 expression due to CeCl3 exposure may be related to overproduction of Ach, Ca reduction, and subsequent mental retardation. The doublecortin-like kinase 1 (DCAMKL1) gene encodes a member of the protein kinase superfamily and the doublecortin family. The protein encoded by DCAMKL1 has microtubule-polymerizing activity and is involved in several different cellular processes, including neuronal migration, retrograde transport, neuronal apoptosis and neurogenesis. The DCAMKL1 gene can be up-regulated by brain-derived neurotrophic factor and is associated with memory and general cognitive abilities. Transgenic mice with over-expression of brain CaMK Related Peptide (CARP) were shown to have decreased hippocampal CA3/CA1 network excitability. Schenk et al proposed that the DCAMKL1 gene product affects glutamatergic neuronal transmission in response to neurological stimuli [61, 62]. Therefore, the decrease in learning and memory ability, and Glu overaccumulation induced by Ln3+ may be associated with increased DCAMKL1 expression.
Conclusion
The present study suggests that long-term exposure to CeCl3 at 0.5, 1, and 2 mg/kg dose resulted in hippocampal damage, oxidative stress, and a decrease in spatial recognition memory. Furthermore, hippocampal dysfunction following exposure to CeCl3 may be closely related to significant changes in the expression of genes involved in learning and memory, apoptosis, response to stress, immunity and inflammation, and signal transduction. Considering the average intake of Ln (1.75–2.25 mg/day) in humans, the application of cerium in crop production, animal breeding, and medicine should be carried out cautiously.
Supporting Information
Table S1 Genes of known function were significantly altered after intragastric administration of 2 mg/kg BW CeCl3 for 90 consecutive days.
(DOC)
Click here for additional data file.
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23577194PONE-D-12-3133910.1371/journal.pone.0061089Research ArticleBiologyMicrobiologyMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesOncogenic SignalingMedicineGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis CInfectious diseasesViral diseasesHepatitisHepatitis COncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaHepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells HCV Core Protein Regulates miR-345 and p21Shiu Tzu-Yue
1
2
Huang Shih-Ming
3
Shih Yu-Lueng
2
Chu Heng-Cheng
2
Chang Wei-Kuo
2
Hsieh Tsai-Yuan
1
2
*
1
Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, R.O.C.
2
Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, R.O.C.
3
Department of and Graduate Institute of Biochemistry, National Defense Medical Center, Taipei, Taiwan, R.O.C.
Yu Ming-Lung Editor
Kaohsiung Medical University Hospital, Kaohsiung Medical University, Taiwan
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Final approval of the version to be published: SMH TYH. Conceived and designed the experiments: TYS TYH. Performed the experiments: TYS SMH. Analyzed the data: YLS HCC WKC. Wrote the paper: TYS.
2013 8 4 2013 8 4 e6108911 10 2012 5 3 2013 © 2013 Shiu et al2013Shiu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21Waf1/Cip1 expression in human hepatoma cells.
Methods
Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay.
Results
HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting microRNA-345 in Huh7 cells.
Conclusion and Significance
HCV core protein enhances the expression of microRNA-345 which then down-regulates p21Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21Waf1/Cip1 gene expression through miR-345 targeting.
This study was supported in part by Grants from the Foundation for Medical Research of Tri-Service General Hospital and National Defense Medical Center (TSGH-C98-47, TSGH-C101-066 and DOD-100-I-27). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
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Introduction
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [1]. The viral genome encodes a single polyprotein precursor of approximately 3000 amino acids, which is cleaved by cellular and viral proteases into three structural proteins (Core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) [2]. The HCV core protein, a component of the viral capsid, is initially split from the viral polyprotein between amino acid residues 191 and 192 by a signal peptidase within the endoplasmic reticulum (ER) lumen. Subsequently, this full-length core protein (amino acids 1–191, 191 a.a.) is further cleaved into a mature form (amino acids 1–173, 173 a.a.) by a signal peptide peptidase (SPP) [3], [4]. Further processing of the core protein in the C-terminal region produces a more truncated form, which can only be produced from limited HCV genotypes [5]. The full-length core protein localizes in the cytoplasm, whereas truncated core proteins (amino acids 1–173 and 1–151) localize in the nucleus [3], [5], [6]. Some cytoplasmic and nuclear proteins have been reported to interact with HCV core protein [7]–[10]. Recently, Moriishi et al. have reported that HCV core protein binds to the proteasome activator PA28γ in the nucleus, which induces liver steatosis and hepatocarcinogenesis through a PA28γ-dependent pathway in core transgenic mice [11], [12]. These studies suggest that HCV core as a regulatory protein may be involved in hepatocarcinogenesis during chronic HCV infection.
MicroRNAs are small, endogenous non-coding RNA molecules that regulate the expression of at least one-third of human genes by inhibiting mRNA translation or inducing its degradation depending on the degree of complementarity [13], [14]. Studies have shown that cellular microRNAs play an important role in various physiological and pathological processes including human cancers [15], [16]. It has been reported that some cellular microRNAs are misregulated in HCV-related HCC [17], [18]. Recently, HCV-specific effects on the modulation of cellular microRNAs have been shown in full-length HCV genome-expressing HepG2 cells [19]. These studies suggest that HCV proteins-modulated microRNAs may function as oncogenes or as tumor suppressor genes.
The misregulation of p21Waf1/Cip1 gene expression is frequently observed in human cancers [20]. Hepatocarcinogenesis requires continuous cellular stresses such as viral replication, oxidative stress, inflammation, and continued cell death after regeneration, to receive DNA damage in hepatocytes [1], [21]. The up-regulation of p21Waf1/Cip1 gene expression by cellular stresses may prevent hepatocytes from transformation by inducing a sufficient G1 span to trigger apoptosis or repair DNA damage [20]. Although p21Waf1/Cip1 deficiency may not be sufficient to hepatocarcinogenesis, p21Waf1/Cip1 gene misregulation may be involved in multistep hepatocarcinogenesis. Moreover, p21Waf1/Cip1 may be a target for cancer therapy [20]. Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [22]. Curcumin exerts its effect on anticancer, at least in part, by triggering apoptosis. Curcumin may induce apoptosis through p21Waf1/Cip1-dependent pathway [23]. Recently, curcumin has been reported to induce apoptosis in human hepatoma cell lines [24].
The HCV core protein is considered to be involved in hepatocarcinogenesis [11], [12], [25]. HCV has been reported to regulate cellular microRNAs [19]. Moreover, the core protein can regulate p21Waf1/Cip1 expression [26]–[29]. In this study, we analyzed the expression profiles of cellular microRNAs in core-overexpressing human hepatoma cells compared to cells nonexpressing core. The HCV core protein was able to up-regulate microRNA-345 (miR-345) expression in human hepatoma cells. The HCV core-induced miR-345 suppressed endogenous p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region (3′UTR) in HepG2 cells and curcumin-stimulated Huh7 cells. In addition, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting miR-345 in Huh7 cells.
Materials and Methods
Antibodies, MicroRNA mimics, Mutant microRNA mimic, Small interfering RNA, MicroRNA inhibitor, and Chemicals
Anti-HA epitope antibody (catalog no. 11583816001) was purchased from Roche Applied Science, anti-p21Waf1/Cip1 antibody (catalog no. sc-6246) was from Santa Cruz Biotechnology, and anti-β-actin antibody (catalog no. A1978) was from Sigma-Aldrich. Anti-HCV core (catalog no. ab2740) and anti-HCV NS5B (catalog no. ab35586) antibodies were purchased from Abcam. Human microRNA-345 (hsa-miR-345, MIMAT0000772) and microRNA-93 (hsa-miR-93, MIMAT0000093) mimics were purchased from Thermo Scientific. Mutant microRNA-345 mimic was synthesized by Thermo Scientific. The sequence was 5′- GCUCUGACCUAGUCCAGGGCUC-3′. The four mutated sites are underlined. Human p21Waf1/Cip1 small interfering RNA (siRNA, catalog no. sc-29427) was purchased from Santa Cruz Biotechnology. Human miR-345 inhibitor (MH12733) was purchased from Applied Biosystems. Curcumin (catalog no. C7727) and dimethyl sulfoxide (DMSO, catalog no. D2650) were purchased from Sigma-Aldrich. Curcumin was dissolved in DMSO to 100 mM and stored at −20°C until use.
Plasmid Construction
Plasmid pT-REx-HA-Core was generated by cloning the N-terminus HA-tagged HCV core coding sequence (genotype 1b strain) [30] into pT-REx-DEST30 vector (Invitrogen) according to the manufacturer's instructions. HA-Core was amplified by PCR using pcDNA3-HA-Core191 (amino acids 1–191) as a template. Primers used for cloning HA-Core191 were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGCGGAAGCTGGGATGG-3′. Primers used for cloning HA-Core173 (amino acids 1–173) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAAGAGCAACCGGGCAGAT-3′. Primers used for cloning HA-Core153 (amino acids 1–153) were forward 5′-ACCATGTATCCATATGATGT-3′ and reverse 5′-TCAATGTGCCAGGGCTCTGG-3′. A control vector, pT-REx-Mock, was created by deleting HCV core coding sequence from the above construct using BsrGI restriction enzyme. Wild-type human p21Waf1/Cip1 3′UTR was amplified by PCR from human genomic DNA and cloned into pGL3-Control vector (Promega) immediately downstream of luciferase reporter gene but upstream of poly (A) signal using XbaI restriction enzyme to generate pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) vectors. Primers used for cloning human p21Waf1/Cip1 3′UTR were forward 5′-CCCTCTAGATCCGCCCACAGGAAGCCTGC-3′ and reverse 5′-CCCTCTAGAAAAGTCACTAAGAATCATTT-3′. The XbaI site is underlined. The mutant p21Waf1/Cip1 3′UTR in the seed sequence of hsa-miR-345 was generated by PCR and ligation of two pieces of DNA fragments, and then cloned into pGL3-Control vector to yield a pGL3-Control-p21 3′UTR Mutant vector. Primer pairs used for cloning mutated p21Waf1/Cip1 3′UTR were forward 5′-CCCTCTAGATCCGCCCACAGGAA-3′ and reverse 5′-CCTTGTTCCGCTGCTAATCA-3′, and were forward 5′-TCAGAGACATTTTAAGATGGTGGC-3′ and reverse 5′-CCCTCTAGAAAAGTCACTAAGAA-3′. The XbaI site is underlined.
Full-length HCV Replicon
A genotype 1b strain of full-length HCV replicon (HCV-N) [31] was kindly provided by Dr. Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C.. A control replicon HCV-A357T, which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N (Fig. 1D, upper panel) using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer's instructions.
10.1371/journal.pone.0061089.g001Figure 1 HCV core protein up-regulates miR-345 and miR-93 expression in human hepatoma cells.
(A) Huh7 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1–191, 1–173 and 1–153, respectively. At 48 hours after transfection, the expression of HCV core protein was analyzed by immunoprecipitation. (B) Huh7 cells were transiently transfected with empty vector and two HCV core gene-expressing vectors, pT-REx-HA-Core191, and pT-REx-HA-Core173, for core protein with amino acids 1–191 and 1–173, respectively. At 48 hours after transfection, cellular microRNA profiling was analyzed by TaqMan low density array. Three microRNAs, miR-21, miR-345 and miR93, of thirty-one microRNAs were indicated. (C) Huh7 and HepG2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1–191, 1–173 and 1–153, respectively. At 48 hours after transfection, relative expression of miR-345 or miR-93 was determined by TaqMan real-time qPCR in Huh7 cells (left upper panel) and HepG2 cells (right upper panel). The expression of HCV core protein was analyzed by Western blotting (left lower and right lower panels). (D) The genotype 1b strain of full-length HCV replicon (HCV-N) and control replicon HCV-A357T which expresses only the initial five amino acids of the core protein due to introduction of a termination codon, was created by the change of one nucleotide at HCV nt 357 in pHCV-N (upper panel). HCV core and NS5B gene expression in full-length HCV replicon-expressing cells was analyzed by immunoprecipitation followed by Western blotting and Western blotting only respectively (left lower panel). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR in full-length HCV replicon-expressing Huh7 cells (right lower panel). Data was shown as the means ± S.D. from triplicate experiments. *P<0.05, **P<0.001.
In Vitro Transcription of HCV RNA
pHCV-N DNA was linearized with XbaI, purified by phenol/chloroform extraction and ethanol precipitation [31], and then the linearized DNA was used as template to transcribe full-length HCV RNA using Riboprobe in vitro Transcription Systems (Promega, catalog no. P1440) following the manufacturer's protocol. The integrity of RNA transcripts was determined by agarose gel electrophoresis and ethidium bromide staining.
Cell Culture and Transfection
Human hepatoma cell lines, Huh7 and HepG2 cells, were cultured at 37°C in a humidified incubator containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone). Plasmid DNA was transfected into cells using GenJet In Vitro DNA Transfection Reagent Ver. II (SignaGen Laboratories) following the manufacturer's protocol. To establish the full-length HCV replicon-expressing system in Huh7 cells, HCV RNA was transfected into cells using TransIT-mRNA Transfection Kit (Mirus Bio LLC) according to the manufacturer's instructions. The siRNA, microRNA mimic and inhibitor were transfected into cells using GenMute siRNA & DNA Transfection Reagent (SignaGen Laboratories) following the manufacturer's protocol.
RNA Extraction
Total RNA was extracted using miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. The trace quantities of genomic DNA were further removed using Turbo DNA-free Kit (Applied Biosystems) following the manufacturer's instructions.
TaqMan Low Density Array for MicroRNA
TaqMan low density array was performed using Applied Biosystems 7900HT Fast Real-Time PCR System according to the manufacturer's protocol. Briefly, cDNA templates were synthesized using TaqMan MicroRNA Megaplex RT Kit (Applied Biosystems) following the manufacturer's instructions. A total reaction mixture containing RT products and TaqMan Universal PCR Master Mix (Applied Biosystems) was added to each line of TaqMan low density array card after gentle vortex mixing. Thermal cycler conditions were as follows: 50°C for 2 minutes, 94.5°C for 10 minutes, and then 40 cycles of 97°C for 30 seconds and 59.7°C for 1 minute. Cycle threshold was automatically given by SDS software v2.2 (Applied Biosystems) using automatic baseline settings and a threshold of 0.2. MammU6 was used as an internal control to normalize the amount of individual microRNA in each sample. Significant difference of relative microRNA expression was determined using the 2−ΔΔCt method [32].
TaqMan Real-time qPCR for Single MicroRNA Assay
cDNA template for single microRNA was synthesized using TaqMan MicroRNA RT Kit (Applied Biosystems) according to the manufacturer's protocol. Expression of specific microRNA was determined by real-time qPCR using TaqMan MicroRNA qPCR Kit (Applied Biosystems) following the manufacturer's instructions. MammU6 was used as an internal control to normalize the amount of specific microRNA in each sample. Significant difference of relative microRNA expression was determined as described above.
Real-time qPCR
cDNA template was synthesized using ImProm-II Reverse Transcription System (Promega) according to the manufacturer's protocol. Expression of human p21Waf1/Cip1 mRNA was determined by real-time qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer's instructions. The β-actin mRNA was used as an internal control to normalize the amount of human p21Waf1/Cip1 mRNA in each sample. Primer sequences for human p21Waf1/Cip1 and β-actin mRNAs were described previously [33]. Significant difference of relative gene expression was determined as described above.
Luciferase Reporter Assay
Cells were transfected with luciferase reporter vector in combination with microRNA mimic. All transfections included pRL-TK vector (Promega) for normalization. Luciferase activity was analyzed by Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions. Renilla luminescence, expressed from pRL-TK vector, was used as an internal control to normalize the luciferase activity.
Immunoprecipitation
Cell lysates were extracted using RIPA buffer (Sigma-Aldrich) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific). The supernatants were incubated for 2 hours at 4°C with anti-HA epitope or anti-HCV core antibody, and then overnight at 4°C with Protein A-agarose beads (Roche Applied Science, catalog no. 11719408001). Following four washes with RIPA buffer, proteins were eluted for 10 minutes at 100°C with 2× Laemmli sample buffer (Sigma-Aldrich), electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes (Millipore).
Western Blotting
Cell lysates were extracted as described above. Cell lysates were electrophoresed on SDS-PAGE and electroblotted onto PVDF membranes. Anti-HA epitope, anti-HCV core, anti-HCV NS5B, anti-p21Waf1/Cip1, and anti-β-actin antibodies were used in Western blotting according to the manufacturer's protocol. β-actin was used as an internal control. Relative protein expression was quantified by BioSpectrum Imaging System (UVP).
DNA Fragmentation Analysis
Genomic DNA from cells was extracted using Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. DNA ladder was determined by agarose gel electrophoresis and ethidium bromide staining.
Annexin V-FITC Apoptosis Assay
Apoptotic cells were detected and quantified by fluorescence microscopy and FACS Calibur (Becton Dickinson) respectively, using Annexin V-FITC Apoptosis Detection Kit (BioVision) following the manufacturer's protocol.
Cell Cycle Analysis
Cells were subjected to serum starvation for 24 hours to arrest cell growth, and then cultured in fresh serum-containing medium for 24 hours for cell cycle re-entry. Cells were harvested after trypsinization in serum-containing medium, centrifuged and suspended in phosphate buffered saline (PBS). The absolute ethanol was added drop-wise and cells were maintained overnight at −20°C to complete fixation. Cells were centrifuged, resuspended in PBS plus RNase A and propidium iodide (Sigma-Aldrich), and incubated at 37°C for 30 minutes. Fluorescence was measured and analyzed by FACS Calibur.
Cell Proliferation Assay (MTT assay)
Cell proliferation was determined using CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer's instructions. To analyze cell proliferation, Huh7 cells were seeded in 96-well culture plates at 3000 cells per well. After 24 hours, the medium was replaced with fresh medium supplemented with increased amount of curcumin. At 24 hours after treatment, Dye Solution was added to each well and cells were incubated at 37°C for 2 hours. Solubilization/Stop Solution was then added to each well and the absorbance was measured at 570 nm using an ELISA reader. Relative cell number was calculated by normalizing the absorbance to untreated cells. Relative cell viability was compared to untreated cells.
Statistical Analysis
Data was shown as the means ± SD from triplicate experiments. The two-sided Student's t-test was used for comparisons between experimental groups. P<0.05 was considered statistically significant.
Results
MicroRNA-345 and microRNA-93 are overexpressed in HCV core-overexpressing human hepatoma cells
It has been reported that the full-length HCV core protein (amino acids 1–191) is further cleaved into truncated forms (amino acids 1–173 and 1–153) [3], [5]. Although the truncated core protein localizes in the nucleus [3], [5], [6], only a small quantity of the core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [25], [34], [35]. In this study, three core gene-expressing vectors for core protein with amino acids 1–191, 1–173 and 1–153, respectively, were transfected into Huh7 cells. Immunoprecipitation followed by Western blotting was used to determine HCV core expression. The results showed that three forms of HCV core protein were overexpressed in Huh7 cells (Fig. 1A). Interestingly, a small quantity of a product with lower molecule weight was observed when the full-length core gene was overexpressed in Huh7 cells (Fig. 1A, lane 2). This product may suggest a truncated core protein trimmed at the C-terminal region due to the intracellular processing of the full-length core protein. To investigate the effect of the full-length and mature core proteins on the modulation of cellular microRNAs, two core gene-expressing vectors for core protein with amino acids 1–191 and 1–173, respectively, were transfected into Huh7 cells, and then the expression profiles of cellular microRNAs was determined by TaqMan low density array at 48 hours after transfection. The result showed that thirty-one microRNAs exhibited a greater than 2-fold up- or down-regulation in full-length core (191 a.a.) or mature core (173 a.a.)-overexpressing Huh7 cells compared to cells nonexpressing core (Fig. 1B). The HCV core protein can regulate p21Waf1/Cip1 expression [26]–[29]. Recently, Wu et al. have reported that human p21Waf1/Cip1 gene expression can be inhibited by twenty-eight microRNAs in HEK 293 cells [33]. In this study, we indicated that two p21Waf1/Cip1-targeting microRNAs, microRNA-345 (miR-345) and microRNA-93 (miR-93), were up-regulated in core-overexpressing Huh7 cells (Fig. 1B). Array data were further confirmed by TaqMan real-time qPCR for miR-345 and miR-93 in Huh7 and HepG2 cells. Three core gene-expressing vectors for core protein with amino acids 1–191, 1–173 and 1–153, respectively, were transfected into cells, and then the relative expression of miR-345 and miR-93 was determined at 48 hours after transfection. The results showed that miR-345 and miR-93 were overexpressed with more than 3.5- and 2-fold changes, respectively, in mature core (173 a.a.) and more truncated core (153 a.a.)-overexpressing Huh7 cells but not in full-length core (191 a.a.)-overexpressing cells compared to cells nonexpressing core (Fig. 1C, left upper panel). Similar results also indicated in mature core and more truncated core-overexpressing HepG2 cells but not in full-length core-overexpressing cells (Fig. 1C, right upper panel).
To further verify the expressions of cellular microRNAs in full-length HCV replicon-expressing system, the full-length HCV replicon was transfected into Huh7 cells. At 96 hours after transfection, immunoprecipitation followed by Western blotting was used to determine HCV core expression, and Western blotting only for NS5B expression (Fig. 1D, left lower panel, lane 2). The relative expressions of miR-345 and miR-93 were determined by TaqMan real-time qPCR. The results showed that miR-345 induction was not significantly affected in full-length HCV replicon-expressing system in Huh7 cells compared to control cells and untreated cells (Fig. 1D, right lower panel, third bar pair). Indeed, there is no detectable or little, if any, amount of truncated form of HCV core protein expression in full-length HCV replicon-expressing system in Huh7 cells (Fig. 1D, left lower panel, lane 2). Furthermore, miR-93 overexpression in full-length HCV replicon-expressing cells but not in full-length core-overexpressing cells suggested that other HCV proteins might up-regulate miR-93 expression. Together, these results demonstrate that truncated HCV core proteins (amino acids 1–173 and 1–153) up-regulate cellular miR-345 and miR-93 expression in human hepatoma cells.
MicroRNA-345 down-regulates p21Waf1/Cip1 gene expression in human hepatoma cells
It has been reported that human p21Waf1/Cip1 gene expression can be inhibited by miR-345 and miR-93 in HEK 293 cells [33]. Because the different cell types might generate different results, the effects of miR-345 and miR-93 on human p21Waf1/Cip1 gene expression were examined in human hepatoma cells. Two luciferase reporter vectors which bear the sense and antisense 3′UTRs from human p21Waf1/Cip1 gene, respectively, were used in luciferase reporter assay (Fig. 2A, upper panel). The relative luciferase activity was determined in Huh7 and HepG2 cells at 24 hours after transfection. The results showed that the treatment with miR-345 mimic led to a significant reduction of luciferase activity in p21 3′UTR Sense construct-transfected Huh7 cells, but treatment with miR-93 mimic had no significant inhibition in luciferase activity (Fig. 2A, left lower panel, second bar cluster). Moreover, treatment with a mixture of miR-345 and miR-93 mimics also had no double reduction of luciferase activity (Fig. 2A, left lower panel, second bar cluster). As expected, miR-345 and miR-93 mimics had no effect in control vector-transfected and p21 3′UTR Antisense construct-transfected Huh7 cells (Fig. 2A, left lower panel, first and third bar clusters). Similar results also indicated in HepG2 cells (Fig. 2A, right lower panel). We further examined human p21Waf1/Cip1 gene expression at protein level when miR-93 mimic was transfected into HepG2 cells. As the results in luciferase reporter assay (Fig. 2A, left lower and right lower panels), p21Waf1/Cip1 expression at protein level was not suppressed by miR-93 in HepG2 cells (Fig. S1). These results demonstrated that human p21Waf1/Cip1 gene may be not a target of miR-93 in human hepatoma cells. We further focused on miR-345 to identify the seed sequence in human p21Waf1/Cip1 3′UTR by using computational tools, miRanda (http://www.microrna.org/microrna/home.do) and TargetScan (http://www.targetscan.org/) (Fig. 2B, left panel). To further verify the seed sequence of miR-345, two luciferase reporter vectors which bear the wild-type and mutant (mutated in the seed sequence of miR-345) 3′UTRs from human p21Waf1/Cip1 gene, respectively, were used in luciferase reporter assay (Fig. 2B, left panel, mutated sites are underlined). The results showed that wild-type miR-345 mimic can significantly inhibit the luciferase activity in wild-type p21 3′UTR construct-transfected Huh7 cells but not in mutant p21 3′UTR construct-transfected cells (Fig. 2B, right panel, second and third bar pairs). However, an additional experiment with mutant miR-345 mimic (Fig. 2B, left panel, mutated sites are underlined) and mutant p21 3′UTR with restoring complementarity was able to show a significant inhibition of luciferase activity (Fig. 2B, right panel, fourth bar pair). These results showed that miR-345 may down-regulate human p21Waf1/Cip1 gene expression through targeting its 3′UTR in human hepatoma cells. To further verify the down-regulation of endogenous p21Waf1/Cip1 gene expression by miR-345, p21Waf1/Cip1 mRNA and protein levels were examined in HepG2 cells at 24 hours after transfection with miR-345 mimic. As expected, p21Waf1/Cip1 gene expression at mRNA and protein levels was suppressed with the increased amount of miR-345 mimic in HepG2 cells (Fig. 2C, left and right panels). Together, these results demonstrate that miR-345 down-regulates p21Waf1/Cip1 gene expression through targeting its 3′UTR in human hepatoma cells.
10.1371/journal.pone.0061089.g002Figure 2 MicroRNA-345 down-regulates p21Waf1/Cip1 gene expression through targeting its 3′UTR but not microRNA-93 in human hepatoma cells.
(A) Two luciferase reporter vectors, pGL3-Control-p21 3′UTR Sense (S) and pGL3-Control-p21 3′UTR Antisense (AS) which bear the sense and antisense 3′UTRs from human p21Waf1/Cip1 gene, respectively, were constructed (upper panel). Huh7 cells and HepG2 cells were transfected with p21 3′UTR sense or antisense luciferase reporter vector in combination with miR-345 mimic, miR-93 mimic or a mixture of miR-345 and miR-93 mimics. At 24 hours after transfection, Huh7 cells (left lower panel) and HepG2 cells (right lower panel) were collected for luciferase reporter assay. (B) Base-pairing between mature hsa-miR-345 and target site in human p21Waf1/Cip1 3′UTR is shown (left panel). One luciferase reporter vector, pGL3-Control-p21 3′UTR Mutant (mutated in the seed sequence of miR-345), was constructed. Mutant miR-345 mimic was synthesized. The mutated site is underlined (left panel). Huh7 cells were transfected with wild-type p21 3′UTR sense or mutant p21 3′UTR luciferase reporter vector in combination with wild-type or mutant miR-345 mimic. At 24 hours after transfection, Huh7 cells were collected for luciferase reporter assay. The experiment with mutant miR-345 mimic and mutant p21 3′UTR with restoring complementarity was performed (right panel). (C) HepG2 cells were transiently transfected with the increased amount of miR-345 mimic for 24 hours. The p21Waf1/Cip1 gene expression was analyzed by real-time qPCR (left panel) and Western blotting (right panel). β-actin served as an internal control. Data was shown as the means ± S.D. from triplicate experiments. *P<0.05.
MicroRNA-345 inhibits curcumin-induced apoptosis through down-regulation of p21Waf1/Cip1 gene expression in Huh7 cells
Curcumin, a potential anticancer agent, has been used in preclinical in vitro and in vivo models of HCC [22]. Recently, curcumin has been reported to induce apoptosis in Huh7 cells [24]. In this study, we showed that p21Waf1/Cip1 gene expression at protein level was enhanced with increased amount of curcumin in Huh7 cells (Fig. 3A, upper panel). To investigate the functional relevance of p21Waf1/Cip1 up-regulation and curcumin treatment in Huh7 cells, annexin V-FITC apoptosis assay, MTT assay, and cell cycle analysis were performed. The results showed that curcumin induced apoptosis and inhibited cell viability in Huh7 cells in a dose-dependent manner (Fig. 3A, middle and left lower panels). Furthermore, the result also showed that S-phase entry of cell cycle was slightly inhibited in Huh7 cells in response to high doses of curcumin (Fig. 3A, right lower panel). To further verify that the up-regulation of p21Waf1/Cip1 gene expression involved in curcumin-induced apoptosis in Huh7 cells, curcumin-stimulated cells were transfected with increased amount of p21Waf1/Cip1 siRNA, and then apoptosis was analyzed by using DNA fragmentation analysis at 24 hours after transfection. The result showed that apoptosis was inhibited as presented in DNA ladder disappeared when p21Waf1/Cip1 gene expression at protein level was suppressed in curcumin-stimulated Huh7 cells (Fig. 3B), indicating that curcumin induced apoptosis through up-regulation of p21Waf1/Cip1 gene expression in Huh7 cells. Similar results were also showed in Figure 3C. Curcumin had no effect on the modulation of cellular miR-345 expression in Huh7 cells (Fig. S2). As expected, p21Waf1/Cip1 gene expression at protein level was suppressed by miR-345 mimic in curcumin-stimulated Huh7 cells in a dose-dependent manner (Fig. 3C, upper panel). Curcumin-induced apoptosis was inhibited with increased amount of miR-345 mimic (Fig. 3C, left middle panel). Moreover, the introduction of miR-345 mimic enhanced cell viability in curcumin-stimulated Huh7 cells (Fig. 3C, right middle panel). The number of apoptotic cells (early and late apoptotic cells) had a maximum reduction of about 55% when curcumin-stimulated cells were transfected with 50 nM miR-345 mimic (Fig. 3C, left lower and right lower panels). In HepG2 cells, we also showed that curcumin induced apoptosis, but curcumin had no effect on the modulation of p21Waf1/Cip1 gene expression. The results indicated that curcumin was able to induce apoptosis through a p21Waf1/Cip1-independent pathway in HepG2 cells (Fig. S4). Together, these results demonstrate that miR-345 inhibits curcumin-induced apoptosis through down-regulation of p21Waf1/Cip1 gene expression in Huh7 cells.
10.1371/journal.pone.0061089.g003Figure 3 MicroRNA-345 inhibits curcumin-mediated apoptosis through down-regulation of p21Waf1/Cip1 gene expression in Huh7 cells.
(A) Huh7 cells were treated with different doses (6.25, 12.5, 25 and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The p21Waf1/Cip1 gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (middle panel). Cell proliferation was analyzed by MTT assay (left lower panel). Cell cycle distribution was examined by cell cycle analysis (right lower panel). (B) Huh7 cells were transfected with the increased amount of p21Waf1/Cip1 siRNA in response to curcumin stimulation (50 µM) for 24 hours. Apoptosis was analyzed by DNA fragmentation analysis. The p21Waf1/Cip1 gene expression at protein level was examined by Western blotting. (C) Huh7 cells were transfected with the increased amount of miR-345 mimic in response to curcumin stimulation (50 µM) for 24 hours. The p21Waf1/Cip1 gene expression at protein level was examined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was analyzed by fluorescence microscopy (left middle panel) and FACS Calibur (left lower and right lower panels) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Cell proliferation was analyzed by MTT assay (right middle panel). Data was shown as the means ± S.D. from triplicate experiments. *P<0.05, **P<0.001.
HCV core-induced microRNA-345 inhibits p21Waf1/Cip1 gene expression in HepG2 cells and curcumin-stimulated Huh7 cells
It has been reported that the mature form (amino acids 1–173) of HCV core protein in the nucleus suppresses p21Waf1/Cip1 gene expression in HepG2 cells [27]. To determine that HCV core-induced miR-345 down-regulated endogenous p21Waf1/Cip1 gene expression, core expressing HepG2 cells were transfected with or without miR-345 inhibitor, and then p21Waf1/Cip1 gene expression at protein level was determined at 24 hours after transfection. As expected, the mature form (amino acids 1–173) of the core protein suppressed p21Waf1/Cip1 gene expression (Fig. 4A, lane 3). However, the suppression of p21Waf1/Cip1 gene expression was attenuated in the presence of increased amount of miR-345 inhibitor (Fig. 4A, lane 3, lane 6 and lane 7). The full-length core protein (amino acids 1–191) slightly suppressed p21Waf1/Cip1 gene expression (Fig. 4A, lane 2), this result may be due to the expression of a small quantity of the truncated core protein (Fig. 1A, lane 2). We have shown that only little amount of p21Waf1/Cip1 protein can be detected in Huh7 cells (Fig. 3A, upper panel, lane 1). Therefore, curcumin was used to induce endogenous p21Waf1/Cip1 gene expression to investigate the inhibition of p21Waf1/Cip1 gene expression by HCV-core-induced miR-345 in Huh7 cells. Similar results were also showed in Figure 4B. As expected, the mature form (amino acids 1–173) of the core protein suppressed p21Waf1/Cip1 gene expression in curcumin-stimulated Huh7 cells (Fig. 4B, upper panel, lane 3). Similarly, the suppression of p21Waf1/Cip1 gene expression was attenuated when mature core-expressing cells were transfected with miR-345 inhibitor (Fig. 4B, upper panel, lane 3, lane 6 and lane 7). Additionally, the suppression of curcumin-induced apoptosis by mature core protein (amino acids 1–173) was significantly attenuated in the presence of increased amount of miR-345 inhibitor (Fig. 4B, middle and lower panels). Together, these results demonstrate that HCV core-induced miR-345 inhibits endogenous p21Waf1/Cip1 gene expression in HepG2 cells and curcumin-stimulated Huh7 cells.
10.1371/journal.pone.0061089.g004Figure 4 HCV core-induced microRNA-345 inhibits p21Waf1/Cip1 gene expression in HepG2 cells and curcumin-stimulated Huh7 cells.
(A) HCV core (amino acid 1–191 or 1–173)-expressing HepG2 cells were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, p21Waf1/Cip1 gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control. (B) HCV core (amino acid 1–191 or 1–173)-expressing Huh7 cells in response to curcumin stimulation were transfected with miR-345 inhibitor (5 nM or 10 nM). At 24 hours after transfection, p21Waf1/Cip1 gene expression at protein level was analyzed by Western blotting. β-actin served as an internal control (upper panel). Apoptosis was analyzed by fluorescence microscopy (middle panel) and FACS Calibur (lower panel) using Annexin V-FITC apoptosis assay. Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow. Data was shown as the means ± S.D. from triplicate experiments. *P<0.05, **P<0.001.
Discussion
In this study, we determine the effect of a mature HCV core protein on miR-345 induction in human hepatoma cells. Moreover, HCV core-induced miR-345 suppresses p21Waf1/Cip1 gene expression in HepG2 cells, and inhibits curcumin-mediated apoptosis through down-regulation of p21Waf1/Cip1 gene expression in Huh7 cells.
HCV core protein has an effect on cellular microRNA regulation. HCV core protein is initially separated from HCV polyprotein by a signal peptidase. The full-length core protein (amino acids 1–191) localizes in the cytoplasm, which mainly functions as a component of the viral capsid. HCV core protein is considered to play a crucial role in hepatocarcinogenesis [11], [12], [25]. Many studies have reported that HCV core protein can interact with cytoplasmic and nuclear proteins [7]–[10]. Chen et al. has shown that HCV core protein interacts with Dicer, an RNase enzyme that generates mature microRNAs, in the cytoplasm, and then inhibits the function of Dicer [36]. This inhibitive effect may contribute to HCV replication [37]. The mature form (amino acids 1–173) of HCV core protein cleaved by SPP, which lacks the 174–191 peptide for attachment to ER membrane, enables subcellular distribution of the core protein [3]–[6]. Unlike full-length core protein (amino acids 1–191), mature form (amino acids 1–173) has been reported to mainly localize to nucleus [3], [5], [6]. Some studies have shown that a small quantity of HCV core protein localizes to the nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [25], [34], [35]. In this study, we observed a small quantity of a product with lower molecule weight when full-length HCV core gene was expressed in cultured Huh7 cells. These studies suggest that the nuclear localization or truncated form of the core protein may play an important role in chronic HCV infection. Moriishi et al. has reported that mature (amino acids 1–173) and more truncated (amino acids 1–151) forms of HCV core protein can bind to PA28γ in the nucleus and hence induces liver steatosis and HCC development through a PA28γ-dependent pathway in core gene-transgenic mice [11], [12], suggesting that the nuclear localization of the truncated core proteins may function as a transcriptional factor or regulator. In this study, up-regulation of miR-345 expression by mature form (amino acids 1–173) of the core protein may be also associated with its nuclear localization. Indeed, we also demonstrated that more truncated core protein (amino acids 1–153) which deletes more residues of hydrophobic C-terminal region up-regulated miR-345 expression, indicating that the up-regulation of miR-345 expression may be associated with the nuclear localization of HCV core protein. MiR-345 is down-regulated in full-length HCV genome-expressing cells as reported by Braconi et al.
[19]. Our result was not consistent with the observation from Braconi's group, since the different expressing system and different hepatoma cells were used in the experiments. There is no detectable or little, if any, amount of truncated form of HCV core protein expression in our full-length HCV replicon-expressing system. However, truncated form of HCV core protein has been demonstrated to localize in nuclei of hepatocytes in chronically HCV-infected patients and core transgenic mice [25], [34], [35].
The up-regulation of miR-345 expression by HCV core protein may be associated with the demethylation of its promoter. Tang et al. has demonstrated that miR-345 with a CpG island in the promoter is a methylation-sensitive microRNA and is highly induced by demethylating agent in human colorectal cancer cell lines [38]. Recently, miR-21 has been identified to indirectly down-regulate DNA methyltransferase 1 (DNMT1) expression by directly targeting human RASGRP1 gene, a known critical regulator of the upstream Ras-MAPK pathway of DNMT1 [39]. In this study, we also determined the up-regulation of miR-21 expression in HCV core-overexpressing Huh7 and HepG2 cells (Fig. S3). The up-regulation of miR-21 expression by mature form (amino acids 1–173) of the core protein may contribute to miR-345 promoter hypomethylation. Up-regulation of miR-21 expression has been reported in HCV infectious clone-infected Huh7.5 cells and patients with chronic HCV infection [40]. In our study, the mature form (amino acids 1–173) of the core protein can enhance miR-21 expression, however, the full-length core protein had no effect on miR-21 induction. This finding suggested that the mature form (amino acids 1–173) of the core protein was relevant to the regulation of cellular microRNAs in chronic HCV infection. In fact, we also initially observed the modulation of most of cellular microRNAs by mature form (amino acids 1–173) of the core protein (Fig. 1B). Further confirmation will be needed.
Up-regulation of miR-345 expression may involve in cancer development. Some studies have described the up-regulation of miR-345 expression in human cancers including oral squamous cell carcinomas and malignant mesothelioma [41], [42]. A recent study also reveals that some tumor-related microRNAs including miR-345 are up-regulated from 28 published tumor studies by analyzing microRNA expression microarray datasets, indicating that miR-345 may be an oncomiR in human cancers including HCC [43]. MiR-345 has been reported to down-regulate BAG3 gene expression, an anti-apoptosis gene, as a tumor suppressor microRNA in human colorectal cancer [38]. However, in our study, miR-345 down-regulates p21Waf1/Cip1 gene expression as an oncomiR in human hepatoma cells. These two studies suggest that the same microRNA may function as an oncogene or as a tumor suppressor gene depending on cell types and microenvironment. Our study shows that the up-regulation of miR-345 expression may be related to hepatocarcinogenesis during chronic HCV infection.
In conclusion, our study demonstrates that mature or more truncated HCV core protein (amino acids 1–173 or 1–153) can up-regulate the expression of miR-345 which then suppresses p21Waf1/Cip1 gene expression in human hepatoma cells. We also show that mature HCV core-induced miR-345 can suppress endogenous p21Waf1/Cip1 gene expression in HepG2 and curcumin-stimulated Huh7 cells. Furthermore, we demonstrate that mature HCV core-induced miR-345 involves in anti-apoptosis through down-regulation of p21Waf1/Cip1 gene expression in curcumin-stimulated Huh7 cells. In conclusion, it is the first time that HCV core protein has ever been demonstrated to inhibit human p21Waf1/Cip1 gene expression through miR-345 targeting.
Supporting Information
Figure S1
MicroRNA-93 cannot down-regulate endogenous
p21Waf1/Cip1
gene expression in human hepatoma cells. HepG2 cells were transiently transfected with the increased amount of miR-93 mimic for 24 hours. The p21Waf1/Cip1 gene expression at protein level was determined by Western blotting. β-actin served as an internal control.
(TIF)
Click here for additional data file.
Figure S2
Curcumin has no effect on endogenous miR-345 expression. Huh7 cells were treated with curcumin in dose- (left panel) and time- (right panel) dependent manners. The relative expression of miR-345 was determined by TaqMan real-time qPCR. Data was shown as the means ± S.D. from triplicate experiments. *P<0.05.
(TIF)
Click here for additional data file.
Figure S3
HCV core protein up-regulates miR-21 expression in human hepatoma cells. Huh7 and Hepg2 cells were transiently transfected with empty vector (labeled with Mock) and three HCV core gene-expressing vectors, pT-REx-HA-Core191 (labeled with HA-Core191), pT-REx-HA-Core173 (labeled with HA-Core173), and pT-REx-HA-Core153 (labeled with HA-Core153), for core protein with amino acids 1–191, 1–173 and 1–153, respectively. At 48 hours after transfection, relative expression of miR-21 was determined by TaqMan real-time qPCR in Huh7 cells (left panel) and HepG2 cells (right panel). Data was shown as the means ± S.D. from triplicate experiments. *P<0.05, **P<0.001.
(TIF)
Click here for additional data file.
Figure S4
Curcumin induces apoptosis through p21Waf1/Cip1-independent pathway in HepG2 cells. HepG2 cells were treated with different doses (6.25, 12.5, 25, and 50 µM) of curcumin for 24 hours. DMSO served as control (labeled with 0 µM). The p21Waf1/Cip1 gene expression at protein level was determined by Western blotting (upper panel). β-actin served as an internal control. Apoptosis was determined by annexin V-FITC apoptosis assay (lower panel). Original magnifications ×200. Cells from early apoptotic stage were stained with annexin V-FITC, and appeared green. Cells from late apoptotic stage were stained with both annexin V-FITC and PI, and merged to be yellow.
(TIF)
Click here for additional data file.
We thank King-Song Jeng and Michael M.C. Lai at Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, R.O.C. for reading the manuscript and providing helpful suggestions and the full-length HCV replicon of a Japanese genotype 1b strain (pHCV-N).
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23593420PONE-D-12-2146110.1371/journal.pone.0061167Research ArticleBiologyBiochemistryNucleic acidsRNARNA interferenceBiotechnologyPlant BiotechnologyMarker-Assisted SelectionMedicineDermatologySkin NeoplasmsMalignant Skin NeoplasmsMelanomasEpidemiologyBiomarker EpidemiologyOncologyCancer TreatmentAntibody TherapyCancers and NeoplasmsHematologic Cancers and Related DisordersLeukemiasInhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells ROR1 Inhibition in MelanomaHojjat-Farsangi Mohammad
1
2
*
Ghaemimanesh Fatemeh
3
Daneshmanesh Amir Hossein
1
Bayat Ali-Ahmad
3
Mahmoudian Jafar
3
Jeddi-Tehrani Mahmood
3
Rabbani Hodjatallah
3
Mellstedt Hakan
1
1
Department of Oncology-Pathology, Immune and Gene therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institute, Stockholm, Sweden
2
Department of Immunology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran
3
Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
Rich Benjamin Edward Editor
Dana-Farber Cancer Institute, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Supervised the experiments: MHF HM. Reviewed the manuscript: MHF AAB AHD JM MJT HM. Designed the ROR1 peptides and provided the mAbs for this study: HR. Conceived and designed the experiments: MHF. Performed the experiments: FG AAB AHD JM. Wrote the paper: MHF HM.
2013 8 4 2013 8 4 e6116716 7 2012 7 3 2013 © 2013 Hojjat-Farsangi et al2013Hojjat-Farsangi et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.
This study was supported by grants from the CLL Global Research Foundation, VINNOVA, the Swedish Research Council, the Cancer and Allergy Foundation, the Swedish Cancer Society, the Cancer Society in Stockholm, the King Gustaf Vth Jubilee Fund, the Karolinska Institute Foundations, the Stockholm County Council and Avicenna Research Institute, ACECR, Tehran, Iran. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Melanoma is a skin cancer arising from melanocytes located in the epidermis. The incidence of melanoma is rapidly increasing. The frequency of melanoma is only 4% of all dermatological cancers but responsible for 80% of the mortality in skin cancer. Early detection and treatment may improve prognosis [1]. A series of melanoma-associated antigens (MAGE) has been identified on melanoma cells [2]–[4]. Large efforts have been done to use different MAGEs for immunotherapy of melanoma patients, but most clinical trials have failed [5].
Receptor tyrosine kinases (RTKs) are important structures involved in cell signaling, differentiation and proliferation of normal and malignant cells [6]. RTKs and their signaling pathways may contribute to the dysregulation of malignant cells, as self-sufficiency for growth factors, evasion from apoptosis, unlimited cell replication and metastasis [7]. The receptor tyrosine-kinase-like orphan receptor 1 (ROR1) is a member of the RTK families [8]–[11] and a highly conserved receptor with no clearly identified ligand/s [12]. Wnt5a has however been suggested as a candidate ligand for ROR1 [9], [13]–[14].
ROR1 is a transmembrane protein consisting of 937 amino acid residues with an extra and intracellular part. The extracellular part consists of 3 regions, including the Ig-like, cysteine rich (CRD) and kringle (KNG) domains. The CRD and KNG domains might be ligand binding sites [13], [15]. The intracellular part contains a tyrosine kinase domain that might be triggered to phosphorylation by other cytoplasmic signaling proteins [16]. ROR1 is expressed during the development of the nervous system and regulates survival and maintenance of neural progenitor cells in the brain [14]. It is also expressed in other organs during embryogenesis and of importance for the morphogenesis of several organs [12].
The role of ROR1 in various malignancies is not well understood. No mutations have been noted [17]. ROR1 is however considered to be a survival factor for various malignancies including chronic lymphocytic leukemia (CLL) [18], breast cancer [13] and lung adenocarcinoma [15]. ROR1 might be a promising antigen to be targeted. Anti-ROR1 monoclonal antibodies (mAbs) and ROR1 specific siRNAs have been shown to induce apoptosis and necrosis of malignant cells [16], [19]–[20].
In the current study, we analysed the expression and phosphorylation of ROR1 in a series of malignant melanoma cell lines using RT-PCR, immunocytofluorescence (IF), flow cytometry and western blot. The cytotoxic effects of anti-ROR1 mAbs were evaluated in the absence or presence of complement (complement dependent cytotoxicity) (CDC) or immune effector cells (antibody dependent cell-mediated cytotoxicity) (ADCC) and ROR1 siRNA was used for gene silencing.
Materials and Methods
Cell lines and controls
The melanoma cell lines ESTDAB049, 075, 081, 094 and 112 were obtained from the European Searchable Tumor Cell Line Data Base (ESTDAB project, contract no. QLRI-CT-2001- 01325) [21]. The DFW melanoma cell line was derived from a metastatic lesion from a patient at Radiumhemmet, Karolinska Hospital University Solna, Stockholm, Sweden [22]. A375 (melanoma cell line) and T47D (human ductal breast epithelial tumor cell line) were obtained from American Type Culture Collection (ATCC). After thawing, cells were grown in RPMI-1640 (Gibco, Life Technologies, Karlsruhe, Germany) containing 10% FCS (Gibco), 2% glutamine (Biochrom KG, Berlin, Germany) and 100 ug/ml penicillin/streptomycin (Biochrom KG) (complete medium) at 37°C in a humidified incubator with 5% CO2.
Production of anti-ROR1 monoclonal antibodies
Mouse monoclonal antibodies against ROR1 were generated against the extracellular part of ROR1 as previously described [20]. Out of more than 20 clones, four clones including 1A8, 1E9, 5F1 and 3H9 (all of the IgG1 isotype) were selected. The characterization and specificity of the anti-ROR1 mAbs (Avicenna Research Center, Tehran, Iran) were checked by ELISA and after transfection of the HEK293 cell line with the extracellular domain of ROR1 in western blot as previously described [17].
RNA preparation, cDNA synthesis and RT-PCR
Total RNA was purified from cells, using pure link RNA mini-kits (Ambion, Inc., Austin, Texas, USA). One ug of high quality RNA was reversely transcribed using a first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. PCR amplification was performed as previously described [23], using 150 ng of cDNA for PCR amplification. ROR1 specific primers, 5′-CTGCTGCCCAAGAAACAGAG-3′ (position 455–474) as the sense and 5′-CATAGTGAAGGCAGCTGTGATCT-3′ (position 977–999) as antisense primers, with a PCR product of 545 bp (reference: g.b. M97675) were used for ROR1 amplification [17]. Beta-actin was used as a control for cDNA quality and integrity (Sense primer: ATTAAGGAGAAGCTGTGCTACGTC, Anti-sense primer: ATGATGGAGTTGAAGGTAGTTTCG) [17].
Immunocytofluorescence (IF)
Cells were grown on coverslips (Marienfeld GmbH & Co, Lauda-Königshofen, Germany) placed in 35-mm dishes in an incubator with humidified air and 5% CO2 at 37°C. After 24 h of incubation, medium was removed. Cells were dried at room temperature (RT) and fixed with cold neutral buffered formalin for 5 min. The slides were washed with tris-buffered saline (pH = 7.4), containing 0.1% Bovine Serum Albumin (TBS-BSA) and blocked with 5% sheep serum diluted in TBS-BSA for 30 min. Slides were then incubated with 5 ug/ml of ROR1 mAbs as well as with a non-relevant mAb (mouse IgG1 isotype) (eBioscience, Inc., San Diego, California, USA) for 1 h at RT. Following three washes, slides were incubated with FITC- conjugated sheep anti-mouse IgG (1∶100) (Avicenna Research Center) for 1 h. After three washes in TBS-BSA, the nuclei were counterstained with 1 ug/ml of 4′, 6-Diamidino-2-Phenylindole Dihydrochloride (DAPI) (Sigma-Aldrich Corp., Saint Louis, MO) for 5 min. Finally, cells were mounted in PBS-glycerol 50% and examined with a fluorescent microscope (Zeiss Axioplan2, Oberkochen, Germany).
Flow cytometry analysis
Surface staining of cells was performed as previously described [24]. Briefly, 106 cells were washed in PBS and suspended in 100 ul of FACS buffer (PBS, 0.1% sodium azide, and 0.5% BSA). Five ug/ml of the respective anti-ROR1 mAbs or one ug/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) was added to the cells and incubated at 4°C for 1 h. Cells were washed with FACS buffer and FITC conjugated sheep anti-mouse Ig or FITC conjugated rabbit anti-goat Ig (Dako, Glostrup, Denmark) (1∶100) were added and incubated at 4°C for 1 h. Finally, cells were washed with FACS buffer and fixed with 1% paraformaldehyde in PBS. A FACSCalibur flow cytometer (BD Bioscience, Mountain View, CA, USA) was used to analyse ROR1 expressing cells. 5×104 events were counted. Cells were analyzed using the FlowJo software program (Tree Star Inc. Ashland OR, USA).
Western blot analysis
10×106 cells were lysed in 200 ul of lysis buffer [0.1% SDS, 1% Triton X-100, 50 mM Tris- HC1, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor (Roche, Stockholm, Sweden)] and incubated on ice for 30 min with 5 min interval and vortexed for 10 sec. Bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, IL, USA) was used to measure the protein concentration according to the manufacturer's instructions. Ten ug of the cell lysate or immunoprecipitated ROR1 (using goat anti-ROR1 antibody) was run on 10% Bis-Tris SDS-PAGE gel at 120V/90mA for 2 h (IP-WB). After electrophoresis, proteins were transferred to PVDF membrane (Millipore Corporation, Bedford, MA, USA) and blocked overnight at 4°C with 5% nonfat dry milk (skim milk) or BSA in TBS containing 0.1% Tween 20 (TBS-T). Filters were incubated with anti-phospho-tyrosine (0.5 ug/ml) (PY99, Santa Cruz Biotechnology, Inc., CA, USA), anti-phospho-serine mAbs (clone 4A4) (0.5 ug/ml) (Millipore Corporation) or goat anti-ROR1 polyclonal antibody (R&D system) (0.2 ug/ml) for 1 h at RT. After washing with TBS-T, filters were incubated with peroxidase-conjugated rabbit anti-goat or rabbit anti-mouse immunoglobulin (Dako) for 1 h at RT followed by washings and developed using the advanced ECL chemiluminescence detection system (GE Healthcare, Uppsala, Sweden).
Cleaved PARP as well as caspase-8 and 9 and MCL-1 expression were analyzed using cell lysates from the apoptosis experiments (see below). Briefly, 10 ug of the protein lysate was run in western blot. Filters were incubated with rabbit anti-PARP, cleaved caspase-8 (p 43/41 and p18), cleaved caspase-9 (p37) and MCL-1 antibodies (Cell Signaling Technology, Danvers, MA, USA) respectively overnight at 4°C, and subsequently with a peroxidase-conjugated goat anti-rabbit antibody (Dako). Finally, blots were developed with a chemiluminescence detection system [20].
Annexin-V/PI apoptosis assay
5×104 cells/well were cultured in 6 replicates in 24 well plates. After 24 h, medium was replaced and cells were incubated with 5 ug/ml of the ROR1 mAbs in 1 ml of complete medium. Cells treated with a non-relevant isotype control mAb (mouse IgG1 isotype) (eBioscience) or 1 uM staurosporine (Sigma-Aldrich) were used as controls, respectively. After 24 h of incubation at 37°C in humidified air with 5% CO2, cells were collected (24 well plates were incubated on ice for 10 min and then cells were suspended by pipetting), washed twice with PBS and resuspended in 150 ul of binding buffer. Five ul of FITC-conjugated Annexin-V and PI (propidium Iodide) (BD Biosciences) was added to the cells, vortexed and incubated at RT in the dark for 10 min. Apoptosis was measured by flow cytometry (FACSCalibur, BD Biosciences). Cells were analyzed using the FlowJo software program.
XTT cytotoxicity assay
XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) cytotoxicity assay was used as previously described [25]. Briefly, 104 cells were incubated in 200 ul complete medium in 6 replicates using 96 well plates. After 24 h, medium was replaced and anti-ROR1 mAbs were added to the cells (5 ug/ml) as well as the non-relevant isotype control mAb (mouse IgG1 isotype). The T47D cell line treated with mAbs was used as a negative control [16]. Cells were incubated for 24 h with mAbs and 20 ul of XTT (5 mg/ml) (Sigma-Aldrich) in PBS was added after activation of XTT with PMS (N-methyl dibenzopyrazine methyl sulfate) (Sigma-Aldrich). Cells were incubated for further 2 h at RT. Optical density (OD) was measured at 450 nm. Cytotoxicity was calculated as follows: % cytotoxicity = (Test OD - background OD)/(positive control OD - background OD)×100. The positive control OD was defined as the value of untreated cells at time zero and background OD as the value of medium alone.
CDC and ADCC assays
The effect of anti-ROR1 mAbs in CDC was evaluated as previously described [26]. Briefly, 5×104 target cells were plated in V-bottomed microtiter plates (Nunc, Roskilde, Denmark) in 100 ul complete medium. Cells were incubated with 5 ug/ml of each of the anti-ROR1 mAb as well as with 5 ug/ml of the non-relevant isotype control mAb (mouse IgG1 isotype) for 30 min at RT followed by washings with RPMI-1640. Twenty percent fresh normal human serum (NHS) in 100 ul complete medium was added and cells incubated at 37°C in a humidified air with 5% CO2 for 2 h. Finally, cells were collected, washed twice with PBS and resuspended in 150 ul of Annexin-V/PI binding buffer. Five ul of PI was added to the cells, vortexed and incubated at RT in the dark for 10 min. The frequency of PI stained cells was measured by flow cytometry.
ADCC assay was performed as previously described [20]. Briefly, cells were labeled with 2.8 MBq sodium Cr51 (PerkinElmer Inc. Wellesley, MA, USA) for 3 h at 37°C. After 3 washes with RPMI-1640, 104 cells in 100 ul medium were added to each round-bottomed microtiter well (Nunc) and natural killer (NK) cells enriched from healthy donors buffy coat [26] were added to yield target: effector cell ratios of 1∶25 and 1∶50 to a final volume of 200 ul containing 5 ug/ml of the anti-ROR1 mAbs or the non-relevant isotype control mAb (mouse IgG1 isotype). Each experiment was run in six wells. After 4 h at 37°C, the reaction was stopped by centrifugation. Cr51 release was measured by a gamma counter (Beckman Gamma 5500, Beckman Coulter, Fullerton, CA). The percentage of target cell lysis was calculated based on the following formula: % specific lysis = (experiment cpm- spontaneous cpm)/(maximum cpm-spontaneous cpm)×100. Maximum Cr51 release was determined by adding 1% of Triton X-100 to the target cells and spontaneous release was measured in the absence of antibodies and effector cells.
ROR1 siRNA transfection
Downregulation of endogenous ROR1 mRNA was performed as previously described [19]. The siRNA sequences used to target 5′-ATGAACCAATGAATAACATC-3′ ROR1 mRNA with antisense 5′-GAUGUUAUUCAUUGGUUCAdTdT-3′ and sense 5′-UGAACCAAUGAAUAACAUCdTdT-3′ sequences. Control siRNA (MISSION siRNA Universal Negative Control; Sigma-Aldrich) was used as a negative control. Cells were harvested after 6, 12, 24 and 36 h of incubation for mRNA preparation, the Annexin-V/PI apoptosis assay and for western blot.
Data analysis
Statistical analyses were performed using student's t-test and Mann–Whitney U test as appropriate. Analyses were conducted using the SPSS statistical package (SPSS, Chicago, IL). P-values less than 0.05 were considered to be significant.
Results
ESTDAB (European Searchable Tumor Cell Line Database) contains more than 100 melanoma cell lines with defined HLA class I and II genotypes in the ESTDAB Melanoma Cell Bank (Tubingen, Germany). These cell lines also have been characterized for glycan composition in relation to clinical tumor progression [21], [27]–[29]. In the current study, we randomly selected five cell lines (049, 075, 081, 094 and 112) from this collection and two other melanoma cell lines (A375 and DFW).
ROR1 expression and phosphorylation
The expression of ROR1 mRNA was determined by RT-PCR. ROR1 was expressed in all melanoma cells at the mRNA level but not in the T47D cell line. The protein expression of ROR1 was assessed by IF. All melanoma cell lines expressed the ROR1 molecule (anti-ROR1 mAb clone 3H9). Representative results for the ESTDAB112 cells are shown in Figure 1A. In IP-WB (immunoprecipitation followed by western blot), a 130 kDa band representing fully glycosylated ROR1 was detected using a goat anti-ROR1 polyclonal antibody for immunoprecipitation. This band could also be shown to be phosphorylated using anti-phospho-tyrosine and phospho-serine mAbs. No expression of ROR1 was seen in the T47D cell line [16] (Fig. 1B).
10.1371/journal.pone.0061167.g001Figure 1 Protein expression of the receptor tyrosine kinase ROR1 in melanoma cell lines.
Representative experiment (IF) showing the expression of ROR1 on the ESTDAB112 cell line using the anti-ROR1 (clone 3H9) mAb (40×). Nuclei were counterstained with DAPI (blue). A non-relevant isotype control mAb (mouse IgG1 isotype) was used as a negative control (A). Western blot analysis of ROR1 protein expression and phosphorylation in melanoma cells detected by a goat anti-ROR1 antibody, anti-p-tyrosine (PY99) and anti-p-serine (clone 4A4) mAbs (B). ROR1 protein was shown to be phosphorylated in all cell lines using immunoprecipitation of ROR1. A 130 kDa band corresponding to the fully glycosylated/phosphorylated ROR1 was observed. The T47D cell line was used as a ROR1 negative control [16].
ROR1 surface expression and intensity was also analysed by flow cytometry. All cell lines expressed ROR1 as detected by the four anti-ROR1 mAbs and a polyclonal goat anti-ROR1 antibody (Table 1). However it should be noted that a proportion (about 50%) of the melanoma cells did not express ROR1.
10.1371/journal.pone.0061167.t001Table 1 Frequency of ROR1 positive melanoma cells.
ROR1 mAb ESTDAB
049 075 081 094 112 DFW A375 T47D
1A8
60 (17.1) 52.5 (16) 60 (60.3) 59.1 (54.5) 52.3 (19.7) 59.4 (52.1) 57.9 (36.7) 5 (3)
1E9
64.3 (27) 53.6 (14) 63.5 (38.6) 75.2 (113) 58.6 (23) 65 (74) 44 (12.5) 11 (4)
5F1
68.9 (29.3) 59.3 (22.3) 62.5 (40.4) 70.9 (46.6) 56.8 (15.8) 68.3 (47.5) 71.3 (86.4) 9 (5)
3H9
78.1 (99.6) 58.4 (22.2) 70 (74.2) 70.4 (46.7) 63.7 (24.1) 62.4 (85.7) 58.7 (56.9) 5 (3)
Frequency (%) of ROR1 positive cell lines and Geometric Mean Fluorescence Intensity, stained by 4 anti-ROR1 mAbs in flow cytometry.
Induction of apoptosis by anti-ROR1 mAbs
Induction of apoptosis by the anti-ROR1 mAbs in the absence of complement or effector cells was analysed after 24 h incubation (Fig. 2). The frequency of apoptotic/necrotic cells (lower right, upper left and right quadrants) induced by a non-relevant isotype control mAb (mouse IgG1 isotype) was deducted from the frequency of cells treated with the anti-ROR1 mAbs. Anti-ROR1 mAbs alone induced apoptosis of melanoma cells varying between 4% and 54% as determined by Annexin-V/PI staining (Fig. 2A) and the XTT assay (Fig. 2B). Representative experiments are shown in Figure 2C. Different anti-ROR1 mAbs had various effects on the individual cell lines. The anti-ROR1 mAb clones 5F1, 3H9 and 1A8 were the most effective using ESTDAB049, ESTDAB112, DFW and A375 cell lines, while anti-ROR1 mAb clone 1E9 was the most effective using ESTDAB075 cells. ESTDAB081 and 094 cell lines were resistant to the direct cytotoxic effects of the anti-ROR1 mAbs. The frequency of ROR1 positive cells did not differ significantly comparing the various melanoma cell lines. No effect on apoptosis of the non-relevant isotype control mAb (mouse IgG1 isotype) was observed. The anti-ROR1 mAbs did not induce apoptosis of the ROR1 negative cell line T47D.
10.1371/journal.pone.0061167.g002Figure 2 Induction of apoptosis in melanoma cells using anti-ROR1 mAbs.
Frequency (%) of apoptotic/necrotic cells in Annexin-V+/PI+ (A) and XTT cytotoxicity assay (B) induced by anti-ROR1 mAbs in the absence of complement or immune effector cells [anti-ROR1 mAb clones 1A8 (□), 1E9 () and 5F1 ()3H9 (▪)]. Dot plot diagrams of apoptosis induced by anti-ROR1 mAbs (clones 1A8 and 3H9) in melanoma cells and ROR1 negative cell line T47D (Annexin-V/PI) (C). Western blot for cleaved PARP, caspase 8, 9 and MCL-1 expression in apoptotic ESTDAB049 and ESTDAB112 cells induced by the anti-ROR1 mAb clone 5F1 (D). (−) cells treated with a non-relevant isotype control mAb (mouse IgG1 isotype). (+) cells treated with the anti-ROR1 mAb clone 5F1. (S) cells treated with staurosporine.
Western blot analyses for cleaved PARP and caspase-8/9 as well as downregulation of MCL-1 were done using protein lysates prepared from the 24 h apoptosis experiments. The effects of the anti-ROR1 mAb clone 5F1 were tested on the ESTDAB049, ESTDAB112 and A375 melanoma cells. The anti-ROR1 mAbs induced cleavage of PARP, caspase 8 and caspase 9 as well as down-regulation of MCL-1 (Fig. 2D).
Staurosporine was used as a positive control for apoptosis induction. More than 70% of cells had gone into apoptosis after 24 h incubation (data not shown). Staurosporine also induced a significant PARP, caspase 8 and 9 cleavage as well as MCL-1 downregulation (Fig. 2D).
Cytotoxic effects of anti-ROR1 mAbs in CDC
The effect of anti-ROR1 mAbs in CDC was evaluated after 2 h incubation of cells in the presence of human complement. Anti-ROR1 mAbs could activate complement to lyse melanoma cells to a varying degree. The CDC activity of the various mAbs against the different cell lines are shown in Figure 3A–C. The anti-ROR1 mAb clone 1E9 had the lowest CDC activity against the A375 cell line. Lysis of all mAbs in the presence of complement using the 7 melanoma cell lines (except for anti-ROR1 mAb clone 1E9 on A375 cell line) was statistically significant compared to no complement (p<0.01). No CDC activity was seen using the ROR1 negative T47D cell line (Fig. 3C). Comparison of 4 mAbs in CDC showed a significantly better effect of mAb 5F1 compared to 3H9 using ESTDA049 (p = 0.01), of 3H9 on the ESTDAB075 cell line compared to the other 3 mAbs (p = 0.01-0.001), of mAb 3H9 compared to 5F1 on ESTDAB094 (p = 0.01) and of mAbs 1A8, 5F1 and 3H9 compared to 1E9 mAb on the A375 cell line (p = 0.01-0.0001).
10.1371/journal.pone.0061167.g003Figure 3 Anti-ROR1 mAbs in complement dependent cytotoxicity (CDC).
Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V+/PI+) induced by 4 anti-ROR1 mAbs with (▪) or without (□) human complement using various ESTDAB (A, B), DFW and A375 melanoma cell lines (C). The T47D cell line did not express ROR1. *P = 0.01; **P = 0.001. P-values refer to comparison with and without complement for the respective mAbs. NR mAb: non-relevant isotype control mAb (mouse IgG1 isotype), C: Complement.
Antibody dependent cell-mediated cytotoxicity (ADCC)
Purified NK cells were used as effector cells. Various effects of the anti-ROR1 mAbs were noted (Fig. 4). The lowest and highest cytotoxic activity was observed for the anti-ROR1 mAb clone 5F1 on DFW (Fig. 4A) and ESTDAB081 (Fig. 4B). The effects of the anti-ROR1 mAbs on melanoma cell lines was statistically significant compared to the non-relevant isotype control mAb (mouse IgG1 isotype) as well as in comparison to the ROR1 negative cell line T47D (P = 0.05-0.0001).
10.1371/journal.pone.0061167.g004Figure 4 Cytotoxic effects of anti-ROR1 mAbs in the presence of NK cells (ADCC).
Frequency (%) (mean+SEM) of apoptotic/necrotic cells (Annexin-V+/PI+) induced by 4 anti-ROR1 mAbs and a non-relevant isotype control mAb (mouse IgG1 isotype) in the presence of NK cells at different target: effector ratios. Target cells: ESTDAB049 (□), 075 (), DFW (▪), A375 () (A) and ESTDAB081 (□), 094 (), 112 (▪) melanoma cells and T47D () as a ROR1 negative cell line (B). ADCC of the melanoma cells induced by the anti-ROR1 mAbs compared to the non-relevant isotype control mAb (mouse IgG1 isotype) as wells as to the T47D cell line was statistically significant (P = 0.05-0.0001).
Silencing of ROR1 in melanoma cells by siRNA
A ROR1 specific siRNA was used to suppress ROR1 expression in those melanoma cell lines that were most sensitive to the direct apoptotic effect of the anti-ROR1 mAbs (ESTDAB049, 075, 112, DFW and A375) as well as the ESTDAB081 melanoma cell line which was resistant to apoptosis by anti-ROR1 mAbs. Transfection of melanoma cells induced a marked decrease in the ROR1 gene-expression (RT-PCR) (Fig. 5A). ROR1 was also downregulated at the protein level (Fig. 5B). Silencing of ROR1 induced apoptosis of the ROR1 positive melanoma cell lines but not of the T47D ROR1 negative cell line (Fig. 6). Morphological changes of the cells typical for apoptosis were noted by light microscopy as well as loss of adherence to tissue culture plates (data not shown).
10.1371/journal.pone.0061167.g005Figure 5 Transfection of melanoma cells (n = 6) using ROR1 suppressing siRNA.
Downregulation of ROR1 mRNA (RT-PCR) (A). Downregulation of the ROR1 protein (130 kDa) expression (B). (−) untreated cells, (C) control siRNA treated cells, (+) ROR1 siRNA treated cells.
10.1371/journal.pone.0061167.g006Figure 6 Apoptosis of melanoma cells treated with siROR1.
Dot plot (frequency) of apoptotic/necrotic melanoma cells (Annexin-V+/PI+) treated with siRNA, control siRNA and untreated. Within each quadrant the frequency of apoptotic cells is shown. Results are presented for the ESTDAB049, ESTDAB075, A375, ESTDAB112 (sensitive to apoptosis by anti-ROR1 mAbs) and ESTDAB081 (resistant to apoptosis by anti-ROR1 mAbs), The cell lines T74D cell line was used as a ROR1 negative control.
Discussion
ROR1 is not only overexpressed in hematologic malignancies, but also in solid tumors [13], [15], [30]–[32]. ROR1 knockdown prevented growth of primary leukemic cells as well as of breast cancer cells in vitro and in vivo [13], [16], [19]. ROR1 was constitutively phosphorylated in CLL and cell lines of different origins [16], [18]. Current evidence suggests that ROR1 might play a role as a survival factor for various malignancies and to be an interesting target for therapy [13], [15], [17], [20].
In the present study we could show that melanoma cell lines expressed a 130 kDa ROR1 protein, corresponding to the fully glycosylated isoform [33]. A proportion of melanoma cell lines did however not express ROR1 on the surface or at least not detectable by our anti-ROR1 mAbs. This subpopulation might represent melanoma cells with a low proliferative activity as ROR1 has been shown to be expressed in less mature cells with a high rate of cell division [15]. ROR1 was phosphorylated at serine and tyrosine residues. Transfection of melanoma cells using ROR1 siRNA, downregulated ROR1, which was proceeded by apoptosis. Specific anti-ROR1 mAbs induced apoptosis of the melanoma cells.
Functional characteristics of cellular proteins are related to post-translational modifications, as glycosylation, forming a unique functional glycan in the tissue [34]–[35]. Aberrant glycosylation has been demonstrated for various proteins in melanoma cells with functional consequences [27]. The ESTDAB series of melanoma cell lines showed different glycan patterns which related to clinical characteristics of the patients from which the cell lines were derived [21], [27]–[29].
Depending on the glycosylation pattern of an antigen, a targeting mAb may induce various effects upon binding [36]. The frequency of ROR1 positive cells did not differ markedly between the various melanoma cell lines, but a significant variation in the cytotoxic effects of the different anti-ROR1 mAbs was noted. The ESTDAB081 cell line was resistant to the apoptotic effect of the anti-ROR1 mAbs, but not to siRNA. These variations might mirror post-translation modifications of ROR1, as well as the epitopes recognized by the mAbs in addition to other factors of importance for drug resistance [36]–[37]. Gene silencing of ROR1 induced apoptosis also in mAb resistant cells indicating that some ROR1 mAbs may not mediate a proper apoptotic signal.
The induction of direct apoptosis of melanoma cells is in agreement with recent findings demonstrating the same phenomenon in primary CLL leukemic cells [18], [20]. Targeting ROR1 in CLL with anti-ROR1 mAbs induced rapid dephosphorylation of ROR1 preceding apoptosis [18], [20]. Mechanisms of action for induction of apoptosis by anti-ROR1 antibodies are not well understood but pathways as AKT/CREB may be involved [18], [20], [31], [38]. The A375 melanoma cell line has been shown to express activated BRAF and mediate a strong BRAF/MEK/ERK signal [39]. Whether ROR1 activation might be associated with the BRAF/MEK/ERK signaling pathway or if blocking of ROR1 may mediate cell death through this pathway is not known.
Phosphorylation of serine and tyrosine residues is important for regulating protein activities including RTKs [40]–[42]. ROR1 as well as ErbB2 are both members of the type I RTK subclass, contributing to the malignant transformation of various human cancers. High expression of HER1/2, VEGFR2/KRD and estrogen receptors and their tyrosine phosphorylation in breast cancer correlated with a poor prognosis [40], [43]–[44]. Our findings, showing phosphorylation of ROR1 at tyrosine and serine residues in melanoma cell lines is of interest. We have recently shown that ROR1 is highly phosphorylated in progressive compared to non-progressive CLL [18]. Furthermore, mouse ROR1 is phosphorylated at the serine position 652 located in the activation segment of ROR1 both in the human and mouse ROR1 protein and may be an important site to be triggered by serine/threonine kinases [45]. It is not clear if phosphorylation of ROR1 at tyrosine [46]–[48] or serine [49]–[50] residues is due to autophosphorylation or not. Tyrosine and serine phosphorylation might be triggered by other kinases [51].
Expression of ROR1 has previously been shown in 3 melanoma cell lines including SK-MEL 2, 5 and 28 and ROR1 was phosphorylated at tyrosine and serine residues [16]. SiRNA transfection prevented cell growth only in a low numbers of cells, probably due to a low expression and phosphorylation of ROR1. However, a high degree of growth inhibition was observed in the ROR1 high expressing non-melanoma cell lines NCI-H1993 and HS746T [16]. These two cell lines had an abnormality in the Met oncogene inducing activation of Met. ROR1 might have been phosphorylated by Met as a result of transphosphorylation but a low degree of autophosphorylation could also be seen [16]. To our knowledge, there is no report showing aberrant expression of the Met oncogene in those melanoma cell lines we used. Our data may support the suggestion that the Met RTK is not the only RTK to phosphorylate ROR1 [13], [15]–[16], [31]. Other modifications might contribute to the functional properties of ROR1 [52]–[53]. Furthermore, cells expressing endogenously upregulated ROR1 might be differently activated compared to transfected cells [54]–[55].
In summary, we described for the first time the expression of ROR1 at the mRNA and protein levels in melanoma cells and could show that targeting melanoma cells by anti-ROR1 mAbs and ROR1 suppressing RNA induced apoptosis of the cells. Further studies on the biology of ROR1 in melanoma are warranted as well as to develop anti-ROR1 targeted therapy.
The secretarial help from Ms Leila Relander is highly appreciated. We thank Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran, for producing and providing us with the anti-ROR1 mAbs.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23585825PONE-D-12-3228810.1371/journal.pone.0055695Research ArticleBiologyBiochemistryProteinsDefense ProteinsMolecular cell biologyCellular TypesEpithelial CellsNucleic acidsRNARNA interferenceCell DeathMedicineClinical ImmunologyImmunityAging and ImmunityInnate ImmunityPulmonologyEnvironmental and Occupational Lung DiseasesRegulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2 Regulation of Smoke-Induced Autophagy by Nrf2Zhu Lingxiang
1
Barret Erika C.
1
Xu Yuxue
1
2
Liu Zuguo
2
Manoharan Aditya
1
Chen Yin
1
*
1
Department of Pharmacology and Toxicology, University of Arizona, Tucson, Arizona, United States of America
2
School of Medicine, Xiamen University, Xiamen, China
Chu Hong Wei Editor
National Jewish Health, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YC. Performed the experiments: LZ ECB YX AM. Analyzed the data: YC LZ. Contributed reagents/materials/analysis tools: ZL. Wrote the paper: YC.
2013 9 4 2013 8 4 e5569517 10 2012 2 1 2013 © 2013 Zhu et al2013Zhu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome.
The study was supported by an Arizona Biomedical Research Commission grant, a National Institutes of Health grant RO1AI061695, and a Flight Attendant Medical Institute's (FAMRI) Clinical Innovator Award (CIA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Autophagy is a regulated catabolic process, by which cellular components are sequestered in the vesicular system and later delivered to lysosomes for degradation and recycling of biogenic components [1]. Autophagy has been demonstrated to play a critical role in maintenance of cellular homeostasis and the adaption to environmental stress such as oxidative stress, starvation, hypoxia and infection [1]–[4]. The final outcome of autophagy could be either cell death or survival, and its morphological and biomedical features are distinct from other cell death pathway (e.g. apoptosis). A set of autophagy-related genes (ATG) have been identified to be responsible for the regulation of each step of autophagic pathway [5].
Most of the ATG proteins are highly conserved in mammals. Class III phosphoinositide 3-kinase (PI3K) and ATG6 initiate the formation of autophagosomes. Furthermore, two ubiquitination-like conjugation systems are required for autophagosome formation. One of these systems mediates the conjugation of ATG12 to ATG5 [6], and the other mediates a covalent linkage between LC3B (Atg8) and phosphatidylethanolamine (PE) [7]. An ATG12-ATG5 conjugate is present on the outer side of the isolation membrane and is required for elongation of the isolation membrane [8]. A PE-conjugated form of LC3B localizes on the isolation membrane and the autophagosome membrane [9], [10]. The unconjugated (designated as LC3B-I) and conjugated forms (designated as LC3B-II) of LC3B can be easily separated by SDS-PAGE and detected by antibody staining on the western blot [11]. The intracellular LC3B-II can usually be recognized by its unique “punctate dot” structure [9], [11]when the cells are transfected with GFP-LC3B and challenged with autophagy-inducers. Because the level of LC3B-II is generally correlated with the number of autophagosomes [9], the currently established standard [12] uses the amount of LC3B-II (through either in vitro western blot or in vivo punctate formation) as the hallmark and surrogate for autophagic activity.
Despite the overwhelming studies on autophagy in diseases, very few have been carried on non-malignant airway diseases. Recently, several studies have demonstrated that cigarette smoke (CS) or cigarette smoke extract (CSE) induces autophagy in lung cells [13]–[15], and this autophagic process appears to play a critical role in the pathogenesis of emphysema [13], [16]. In these studies, reactive oxygen species (ROS) has been suggested to mediate CSE-induced autophagy, but the detailed mechanism is not entirely clear. Interestingly, it is well established that anti-oxidant systems such as Nrf2 protect the animal from CS-induced lung injury and airway inflammation [17], and the deregulation of Nrf2 contributes to the pathogenesis of emphysema [18]–[20]. Thus, we speculate that Nrf2 may also have the ability to regulate autophagy in CS/CSE exposure model.
Materials and Methods
1. Chemicals, antibodies, and plasmids
3-Methyladenine (3-MA), E64D, Pepstatin A were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against LC3B, p62 and HO-1 were from MBL International (Woburn, MA). Nrf2, Keap1 and Actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p-mTOR (Ser 2448) and p-S6K (Thr 389) were purchased from Cell signaling technology (Danvers, MA). The plasmids of Nrf2 and GFP-LC3B were kind gifts from Dr. Jingbo Pi (The Hamner Institutes, RTP, NC) and Dr. Tamotsu Yoshimori (Osaka University, Osaka, Japan), respectively. P62 plasmid and two ARE-reporters (pARE_Gst-luc contains ARE sequence from mouse glutathione S-transferase Ya subunit promoter [21]; pARE_NQO1-luc contains ARE sequence from human NQO1 promoter [22]) were kind gifts from Dr. Donna Zhang (University of Arizona, AZ).
2. Cell culture
BEAS-2B was cultivated on regular tissue culture dish in a humidified atmosphere of 5% CO2/balanced air at 37°C as described before [23]. Before experiments, the cells were cultivated in growth factor free medium for 16 hrs.
3. CSE preparation and treatment
CSE preparation was based on the protocol described previously with some modifications [15]. Briefly, Kentucky 1R3F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, Kentucky) were smoked using a peristaltic pump (VWR International). Cigarette smoke from three cigarettes was bubbled through 30 ml of F12 medium. This solution was regarded as 100% strength CSE. pH was adjusted to 7.4 and used within 20 min after preparation. For the treatment, CSE was diluted directly into the culture medium for the indicated percentage.
4. Transient transfection and dual luciferase reporter assay
For the dual-luciferase reporter gene assay, BEAS-2B cells were transfected with the pARE_Gst-luc or pARE_NQO1-luc along with the Renilla luciferase expression plasmid pGL4.74 (hRluc/TK) (Promega). At 24 hrs post-transfection, the transfected cells were treated with CSE for 24 hrs or immersed in the starve media for 6 hrs. Then, the cells were lysed with passive lysis buffer (Promega) and both firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay system purchased from Promega. Firefly luciferase activity was normalized to Renilla luciferase activity. The experiment was carried out in triplicate and expressed as the mean ± the standard deviation (SD).
5. Live imaging of autophagosome
BEAS-2B cells were transfected with GFP-LC3B. At 24 hrs post-transfection, the cells were treated with or without CSE for 24 hrs. Fluorescence Images were acquired by using of a confocal microscope (LSM 510 meta, Carl Zeiss, Thornwood, NY). Six random areas (20X) were selected for the quantification on each slide, and triplicate samples were included for each experimental group. Cells containing “punctate dot” were counted and expressed as percentage of the total cells.
6. Western blot
Total cellular proteins were collected and western blot analysis were performed based on the methods described previously [24]. The sources of antibodies have been described in the “Chemicals, antibodies, and plasmids”. Equal protein load was confirmed using the staining of anti – actin antibody.
7. Real-time PCR
Real-time PCR was performed as described previously [25]. cDNA was prepared from 3 µg of total RNA with Moloney murine leukemia virus (MoMLV)–reverse transcriptase (Promega, Inc.) by oligo-dT primers for 90 min at 42°C in a 20-µl reaction solution, and was then further diluted to 100 µl with water for the following procedures. Two microliters of diluted cDNA were analyzed using 2x SYBR Green PCR Master Mix by an ABI 5700 or ABI Prism 7900HT Sequence Detection System (Applied Biosystems Inc., Foster City, CA), following the manufacturer's protocol. Primers (Table 1) were used at 0.2 µM. The PCR reaction was performed in 96-well optical reaction plates, and each well contained a 50-µl reaction mixture. The SYBR green dye was measured at 530 nm during the extension phase. The relative mRNA amount in each sample was calculated based on the ΔΔCt method using housekeeping gene GAPDH. The purity of amplified product was determined from a single peak of a dissociation curve. Efficiency curves were performed for each gene of interest relative to the housekeeping gene, based on the manufacturer's instructions. Results were calculated as fold induction over control, as described previously [25].
10.1371/journal.pone.0055695.t001Table 1 Realtime Primers.
Gene Primers
LC3B forward
AGAGCAGCATCCAACCAAAAT
reverse
TGAGCTGTAAGCGCCTTCTAA
GAPDH forward
CAATGACCCCTTCATTGACC
reverse
GACAAGCTTCCCGTTCTCAG
8. Small interference RNA (siRNA) and transient transfection
Two different siRNAs were used for the knockdown of each gene. siRNA targeting Nrf2 (#1: GTAAGAAGCCAGATGTTAA) [26], #2: AAGAGTATGAGCTGGAAAAAC
[27]), Keap1 (#1: GGGCGTGGCTGTCCTCAAT) [26], #2: TGAACGGTGCTGTCATGTA
[28]) NQO1 (#1: GGACATCACAGGTAAACTG, #2: GAACCTCAACTGACATATA
[29]) and P62 (#1: GCATTGAAGTTGATATCGA) [30]) were synthesized by Ambion (Austin, TX). The second set of P62 was the pooled siRNA purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Since the results were identical, only the data from #1 were presented. In addition, non-targeted control siRNA (siRNA #1 from Ambion) and individual randomized sequence for Nrf2, Keap1, NQO1 and P62 were also tested as described in the previous publication [31]. Because of identical results, only siRNA #1 was used as control for all the siRNA knockdown studies. siRNA was transfected into cells using lipofectamineTM 2000 (Invitrogen, Carlsbad, CA) based on manufacturer's instruction. Successful knockdown of the target was confirmed by realtime RT-PCR and western blot.
9. Measurement of Intracellular Reactive Oxygen Species by Live Cell Imaging
Reactive oxygen species (ROS) production was determined using chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR) according to the manufacturer's instruction. Briefly, cells were rinsed twice with warm phenol red-free medium and then loaded for 30 min with CM-H2DCFDA (3 μM) in medium without phenol red. The cells were rinsed three times for removal of the extracellular dye, and the medium was replaced with phenol red-free medium. Then the cells were loaded into a live cell imaging incubator (Carl Zeiss, Thornwood, NY) at 37°C in a 5% CO2 atmosphere. Live cell images were continuously recorded before and after CSE treatment by confocal microscopy (LSM 510 meta, Carl Zeiss).
10. Statistical analysis
Experimental groups were compared using a two-sided Student's t test, with significance level set as P<0.05. When data were not distributed normally, significance was assessed with the Wilcoxon matched-pairs signed-ranks test, and P<0.05 was considered to be significant. Matlab 6.0 with statistics toolbox (MathWorks, Inc., Natick, MA) was used for analyses of the data.
Results
1. CSE induced LC3B-I and II as well as autophagosomes, but CSE-induced autophagy was different from that induced by starvation
Starvation-induced autophagy has been extensively studied. Recently, CSE has also been shown to induce autophagy in airway cells [13]–[15]. The hallmark of autophagy is the elevation of LC3B-II [12]. We compared these two stimuli in the induction of autophagy in airway epithelial cells. Indeed, both starvation and CSE dramatically increased cellular autophagosomes as compared with non-treated control (Fig. 1A). Significant increase of LC3B-II and decrease of LC3B-I, thus the increase of LC3B-II: LC3B-I ratio, was observed in the starved cells at both 2 hrs and 6 hrs (Fig. 1B). This conversion (from LC3B-I to LC3B-II) was significantly blocked by 3-MA (type III PI3K kinase inhibitor) treatment, suggesting that a classical autophagy dependent mechanism is responsible for this process. Starvation for 24 hrs caused significant cell death and the reduction of overall LC3B in these cells (data not shown). Thus, we did not include this time point. Different from starvation, CSE increased both LC3B-I and LC3B-II time- and dose-dependently (Fig. 1C). At lower dose (4%), CSE treatment slightly induced both LCB-I and II level; but at the higher dose (10%), LCB-I and II were elevated at 6 hrs and markedly increased at 24 hrs. CSE also increased the level of Nrf2 (Fig. 1C), a master regulator of cellular antioxidant response. Nrf2 appeared to be elevated early (6 hrs) and later slightly subsided (24 hrs). Consistent with Nrf2 increase, the activities of two different ARE reporters (pARE_Gst-luc or pARE_NQO1-luc) were significantly increased in CSE treated as compared with non-treated controls (Fig. 1D). The highest dose (i.e. 20%) appeared to cause outright toxic effects that shut down all the processes. Thus, we used 10% CSE for the following studies. In contrast, starvation had no effect on Nrf2 (Fig. 1B) or ARE promoter activities (Fig. 1D).
10.1371/journal.pone.0055695.g001Figure 1 CSE-enhanced autophagy is different from the starvation-induced autophagy in airway epithelial cells.
All western blot images were the representative image from at least three independent analyses, and Actin was use as a loading control. A) Increase of cellular autophagosome by live imaging. Cells stably transfected with GFP-LC3B were treated either with 10% CSE for 24 hrs or were immersed in HBSS buffer (starvation or S) for 6 hrs. Control (C) was sham (medium)-treated sample. Live images were recorded. “punctate dots” of GFP-LC3B were the indication of autophagosomes. B) Starvation induced autophagy but not Nrf2. Cells were immersed in HBSS (S) without or with 3-Methyladenine (3-MA). Total protein was collected at the indicated time point and subject to western blot analysis. C) CSE induced both autophagy and Nrf2. Cells were treated with different dose (as indicated in the figure) for either 6 hrs or 24 hrs. Total protein was collected and subject to western analysis. D) CSE induced ARE-reporter activation. Two different ARE-reporters were transfected into cells. 24 hrs later, the cells were treated with 10% CSE for 24 hrs or immersed in HBSS (S) for 6 hrs. Cell lysates were prepared and subject to dual luciferase assay. Relative luciferase unit (RLU) was calculated as described in the Materials and Methods. Fold induction = RLU of treated cells/RLU of control cells. *: p<0.05, n = 5. NS: not significant.
2. CSE regulated LC3B-I and II at transcriptional and post-translational levels
Since LC3B is the critical player of autophagy and its dynamics in CSE treated cells were clearly different from the classical autophagy inducer-starvation, we examined the potential molecular mechanism underlying CSE-induced LC3B-I and II. As shown in Fig. 2A, CSE increased LC3B-I as early as 1 hrs; but only starting at 6 hrs, LC3B-II was elevated. As comparison, Nrf2 induction by CSE peaked at 6 hrs and decreased at 24 hrs. By realtime PCR, we found that LC3B mRNA was moderately (but significantly) increased (Fig. 2B). Since LC3B is known to be degraded in the lysosomal compartment after the fusion between autophagosome and lysosome, we asked the question whether or not the elevation of LC3B protein by CSE was perhaps caused by the blockade of this process. LC3B-I was not changed (or slightly increased) either by the blockade of lysosomal protease activity via the treatment of E64D + pepstatin A (two common lysosomal inhibitors), or by the inhibition of autophagosome formation using 3-MA (Fig. 2C). But, LC3B-II level was significantly increased by E64D + pepstatin A and was significantly reduced by 3-MA (Fig. 2C). This observation corroborates the well-established paradigm that LC3B-II level is indeed the surrogate of autophagic activity [12]. In contrast, CSE-induced LC3B-I, which is not a common phenomenon by other autophagy stimuli, was not dependent on autophagy; thus it appears to be a CS-specific phenomenon. We also did a pulse-chase study to further understand the kinetic change of LC3B. The cells were treated with/without CSE for 24 hrs, then CSE was removed and the level of LC3B was determined after 6 hrs. CSE significantly elevated both LC3B-I and II at 24 hrs (5th lane, Fig. 2D). CSE removal for 6 hrs significantly reduced LC3B-I and II almost to the baseline (6th lane, Fig. 2D), suggesting that this process is reversible. The decrease of LC3B-I (but not LC3B-II) after CSE removal was restored by 3-MA treatment (7th lane, Fig. 2D), suggesting that autophagy was the main mechanism to clear up the increased LC3B protein. Enhanced autophagy flux even after CSE removal was demonstrated by the significantly elevated LC3B-II in CSE+ E+P treated cells (8th Lane, Fig. 2D) as compared with E+P treated only cells (4th Lane, Fig. 2D). Therefore, CSE regulates LC3B protein at both transcriptional and post-translational levels.
10.1371/journal.pone.0055695.g002Figure 2 CSE induced LC3B through at multiple levels.
All western blot images were the representative image from at least three independent analyses, and Actin was use as a loading control. A) Time course of CSE induced LC3B and Nrf2. Cells were treated with 10% CSE. At each indicated time point, total protein was collected and subject to western blots analysis. B) CSE increased LC3B steady-state RNA. Cells were treated with 10% CSE. At each indicated time point, total RNA was collected and subject to Real-time PCR analysis. *: p<0.05, n = 5. C) CSE regulates LC3B-I and II at posttranslational levels. Cells were treated with 10% CSE for 24 hrs with either 3-MA (3MA) or lysozymal protease inhibitors-E64D and Pepstatin A (E+P). D) Pulse-chase study. Cells were treated with or without 10% CSE for 24 hrs. Then, CSE was removed and thoroughly washed. The cells were left alone (C) or treated with various inhibitors: cycloheximide (Cy), 3-MA (3MA) or E64D + Pepstatin A (E+P) for additional 6 hrs. Total protein was collected and subject to western blots analysis.
3. Nrf2 negatively regulated LC3B and autophagy, but independent of mTOR
Since CSE elevated both LC3B and Nrf2, we decided to examine the role of Nrf2 in the regulation of LC3B. We artificially increased Nrf2 level by overexpressing Nrf2. In these cells, LC3B-I and LC3B-II were significantly decreased (4th lane, Fig. 3A) as compared to the mock-transfected cells (2th lane, Fig. 3A). Likewise, increased Nrf2 expression by siRNA knockdown of Keap1 (the cellular inhibitor of Nrf2) also repressed both LC3B-I and LC3B-II levels (6th lane, Fig. 3B). Note: CSE appeared to significantly repress Keap1 expression (Fig. 3B). Conversely, Nrf2 knockdown further increased LC3B-I and II level by CSE (4th lane, Fig. 3B), but not at the non-treatment condition (3th lane, Fig. 3B). In addition, when both Nrf2 and Keap1 were knocked down, the levels of LC3B-I and II were restored to the similar level (8th lane, Fig. 3B) as if Nrf2 was knocked down (4th lane, Fig. 3B). Because LC3B-II is the hallmark of autophagy, we examined the impact of Nrf2 on autophagy. As shown in Fig. 3C, overexpression of Nrf2 (3C–e) or siRNA knockdown of Keap1 (3C–f) significantly repressed CSE induced autophagosomes. Fig. 3D shows the statistics of Fig. 3C from triplicates. Taken together, Nrf2 activation appears to repress CSE-induced LC3B-I and II as well as autophagosomes.
10.1371/journal.pone.0055695.g003Figure 3 Nrf2 negatively regulate LC3B I and II.
All western blot images were the representative image from at least three independent analyses, and Actin was use as a loading control. A) Effect of Nrf2 overexpression. Cells were transfected with Nrf2 plasmid (Nrf2) or empty vector (PCDNA3, or PC). 24 hrs later, cells were treated without (C) or with (CSE) 10% CSE for 24 hrs. B) Effect of siRNA knockdown of siNrf2 and/or Keap1. Cells were transfected with siRNA control (siC), siRNA against Nrf2 (siNrf2), siRNA against Keap1 (siKeap1), and siNrf2+siKeap1 (siN+K). 24 hrs later, cells were treated without (C) or with (CSE) 10% CSE for 24 hrs. C) Negative regulation of autophagosome by Nrf2. Cells stably transfected with GFP-LC3B were transfected with Nrf2 plasmid (Nrf2) or siRNA against Keap1 (siKeap1). Control was the mock-transfected cells. These cells were treated without (C) or with (CSE) 10% CSE for 24 hrs. Live images were recorded. “punctate dots” of GFP-LC3B were the indication of autophagosomes. D) Quantification of autophagosome (GFP-LC3B+ dots) was described in Materials and Methods. *, $: p<0.05, n = 3.
Since mTOR is the master inhibitor of autophagy, we tested whether or not the repression of CSE-induced LC3B and autophagosome by Nrf2 was due to the activation of mTOR. CSE significantly repressed activated (phosphorylated) mTOR (Fig. 3A–B). The repression of mTOR activity was further corroborated by the decreased phosphorylated S6K (p-S6K) (Fig. 3A–B), a substrate of mTOR. However, neither Nrf2 overexpression nor Keap1 knockdown could restore the activations of both mTOR and S6K (Fig. 3A–B). Thus, mTOR is not the target of Nrf2 activation.
4. Increase of cellular GSH repressed CSE-induced LC3B and autophagosome but not mTOR
Nrf2 is a master regulator of antioxidant response partly through increasing the cellular reduced glutathione (GSH) level via activating several key GSH synthesizing enzymes [17]. Thus, we tested if the enhancement of GSH synthesis could recapitulate the effect of Nrf2 activation. Indeed, when supplementing the cells with N-acetylcysteine (NAC, a GSH precursor) or GSH itself, LC3B I and II were significantly repressed (Fig. 4B). In addition, these supplements repressed cellular ROS generation (Fig. 4A) and CSE-induced autophagosomes (Fig. 4C–D). Similar to Nrf2 activation, NAC or GSH was not able to restore the repressions of mTOR and S6K by CSE (Fig. 4B). Taken together, Nrf2 appears to repress CSE-induced LC3B level and autophagosomes through increasing the cellular antioxidants (e.g. GSH), thereby inhibiting CSE-induced oxidative stress. But, it has no effect on mTOR activity.
10.1371/journal.pone.0055695.g004Figure 4 Antioxidant supplement repressed CSE-induced LC3B and autophagosome.
A) Antioxidants neutralized CSE-induced cellular ROS. Cells were treated with 10% CSE or plus NAC (CSE+NAC) or GSH (CSE+GSH). Images were recorded 30 min after dye loading. Green fluorescence indicates generation of reactive oxygen species that oxidized the dye to emit fluorescence. B) Antioxidants downregulated CSE-induced LC3B. C) Antioxidants downregulated CSE-induced autophagosome. D) Quantification of autophagosome (GFP-LC3B+ dots) was described in Materials and Methods. *, $: p<0.05, n = 3.
5. P62 is another Nrf2-responsive gene and responsible for repressing CSE-induced LC3B and autophagosomes
To further understand the molecular mechanism underlying Nrf2-mediated LC3B repression, we searched for downstream effectors of Nrf2. HO-1, an Nrf2 downstream gene, has been shown to modulate CSE-induced LC3B [15]. We extended this finding by examining two additional well-known Nrf2 dependent genes- P62 [32] and NQO1 [33]. They are both elevated by CSE (2nd lane, Fig. 5A–B). As shown in the previous study, single knockdown of Keap1 repressed LC3B-I and II (6th lane, Fig. 3B) while double knockdown of Keap1 and Nrf2 could restore CSE-induced LC3B-I and II (8th lane, Fig. 3B). Thus, we used the same strategy to determine whether or not either of these two proteins is the downstream effector of Nrf2. As shown in Fig. 5A, double knockdown of Keap1 and NQO1 couldn't restore the CSE-induced LC3B-I and II (8th lane, Fig. 5A), and single knockdown of NQO1 also had no effect (4th lane, Fig. 5A). In contrast, when both Keap1 and P62 were knocked down, CSE-induced LC3B-I and II were restored (8th lane, Fig. 5B). To confirm this observation, we overexpressed P62 and it indeed repressed the elevations of LC3B-I and II induced by the increasing doses of CSE (6th-8th lanes, Fig. 5C). Consistently, P62 overexpression also repressed CSE-induced autophagy (Fig. 5D–E). Thus, P62 appears to be the downstream effector of Nrf2 that mediates the repression of CSE-induced LC3B and autophagosomes.
10.1371/journal.pone.0055695.g005Figure 5 P62, but not NQO1, replicated Nrf2 phenotype.
All western blot images were the representative image from at least three independent analyses, and Actin was use as a loading control. A) NQO1 knockdown couldn't rescue Keap1 knockdown. Cells were transfected with siRNA control (siC), siRNA against NQO1 (siN), siRNA against Keap1 (siK), and their combinations as indicated. 24 hrs later, cells were treated without or with 10% CSE for 24 hrs. Total proteins were collected for western blot analysis. B) P62 knockdown couldn't rescue Keap1 knockdown. Cells were transfected with siRNA control (siC), siRNA against P62 (siP), siRNA against Keap1 (siK), and their combinations as indicated. 24 hrs later, cells were treated without or with 10% CSE for 24 hrs. Total proteins were collected for western blot analysis. C) Effect of P62 overexpression. Cells were transfected with P62 plasmid (P62) or empty vector (PCDNA3, or PC). 24 hrs later, cells were treated without (C) or with (CSE) at increasing doses (8%, 10%, 12%) for 24 hrs. D) overexpression of P62 represses LC3B. Cells stably transfected with GFP-LC3B were transfected with P62 plasmid (P62) or empty vector (PCDNA3, or PC). These cells were treated without (C) or with (CSE) 10% CSE for 24 hrs. Live images were recorded. “punctate dots” of GFP-LC3B were the indication of autophagosomes. E) Quantification of autophagosome (GFP-LC3B+ dots) was described in Materials and Methods. *, $: p<0.05, n = 3.
Discussion
CSE-induced autophagy appears to be different from the autophagy induced by conventional stimuli such as starvation. The conventional stimuli mostly increase the conversion from LC3B-I to LC3B-II, while the overall amount of LC3B appears to be constant. In contrast, CSE significantly increase the total amount of LC3B. The induction of LC3B-I appears to be much earlier than the increase of autophagy (as shown by the increase of LC3B-II). However, it is unclear whether CSE accelerates the autophagy biogenesis, which leads to more LC3B-I to LC3B-II conversion; or large amount of LC3B-I induced by CSE drives more LC3B-II conversion through a concentration-dependent passive mechanism. To investigate these possibilities, we blocked autophagy biogenesis using class III PI3K inhibitor 3-MA. There was no change in LC3B-I and II levels in untreated cells, but 3-MA significantly decreased LC3B-II in CSE-treated cells without affecting LC3B-I. In addition, when lysosomal protease activity was blocked, LC3B-II level was increased. These suggest that CSE did increase the autophagy biogenesis. It is surprising that, even without CSE treatment, LC3B was still recycled through lysozyme but is 3-MA insensitive. This process is perhaps mediated through other non-canonical autophagic pathways. In our pulse/chase study, we further demonstrate that CSE-induced LC3B is reversible. In the CSE-treated cells, autophagosome/lysosome-mediated post-translational degradation pathway plays an important role in maintaining LC3B level. The excessive LC3B-I was cleared within 6 hrs by autophagy. The increase of LC3B-I protein (also perhaps mRNA) by CS (or CSE) exposure appears not to be restricted in the lung epithelial cells [13], [15]. Similar observations have been made in other cell types such as macrophage [14], fibroblast [14], keratinocyte [34], endothelial cells [35], the follicle-associated epithelium and M-cells in peyer patches [36]. Thus, CS (or CSE) exposure is capable of modulating autophagy in various tissues of the body, and autophagy may play critical role in the pathogenesis of different CS-related illnesses.
In addition to its traditional pro-survival role in the cellular response to stress conditions. excessive autophagy can also lead to cell death (type II) [37]. In the above-mentioned CS (or CSE) induced models, excessive autophagy appears to be largely associated with cellular damage and cell death. Chen ZH et, al. have elegantly demonstrated that LC3B, through interacting with Fas and Cav, regulates extrinsic apoptosis in CS-induced emphysema model [16]. However, CS (or CSE)-induced autophagy in immune related cells such as macrophage [14] and M cells [36] may have other functions. Indeed, autophagy has been well studied for its broad function in innate and adaptive immunity, including microbicidal activity [38], modulation of pattern recognition receptors [39], [40] and antigen presentation [41], [42]. Interestingly, microbials can also develop counter measures to hijack autophagic machinery for their own survival. For example, Staphylococcus aureus co-localizes with LC3B and its replication is dependent of autophagy [43]. In addition, Picornavirus (e.g. polio virus and rhinovirus) utilizes the surface of autophagosome as their replicating factory [44], [45]. Thus, increased autophagosomes by CS (or CSE) may facilitate the replication and growth of certain viruses or bacteria. To support this speculation, markedly enhanced rhinovirus replication has been demonstrated in the experimental rhinoviral infections on COPD patients [46] or on the epithelial cells derived from the airways of COPD patients [47]. Thus, further studies may be required to completely elucidate the interactions between virus and autophagosome in the context of virus-induced COPD exacerbation [48]–[50].
Another major distinction between CSE- and starvation-induced autophagy is the involvement of ROS and Nrf2 in the former. Nrf2 has been well established as the master regulator of cellular antioxidant pathway mainly via the activation of a battery of antioxidant gene expression [51]. The importance of Nrf2 in various lung diseases (e.g. acute lung injury, acute respiratory distress syndrome, asthma, COPD, lung cancer, infection etc.) has been extensively reported (reviewed in [51]). Although CSE induces both Nrf2 activation and autophagy, the interaction between these two pathways hasn't been clearly defined. The present study is the first report regarding their interactions in the CS-related models. Previous reports suggest that the lack of autophagy causes the accumulation of P62, which leads to persistent activation of Nrf2 via Keap1 (the cellular inhibitor of Nrf2) sequestration [22], [52]. Thus, Autophagy appears to act upstream to positively regulate Nrf2 activation. In the present study, we have found that the peak Nrf2 activation (6 hrs) occurred early than the peak of autophagy marker-LC3B-II level (24 hrs). The activation of Nrf2 either by overexpression or by Keap1 knockdown repressed LC3B-I and II and also decreased autophagosome number. Thus, our study indicates that Nrf2 acts upstream to negatively regulate CS-induced autophagy. Even more intriguingly, we found that P62 was the downstream effector of Nrf2 that repressed autophagy. Both studies are not entirely conflicting though, because a recent report suggests an Nrf2->p62->Nrf2 positive loop that regulates Nrf2 mediated anti-oxidant gene expression [32]. In their model of non-canonical Nrf2 activation, p62 is transcriptionally activated by Nrf2 via ARE site on its promoter; then the accumulated p62 sequesters Keap1 and subjects it to autophagic degradation, which leads to persistent Nrf2 activation [32]. However, this model has been established entirely in the non-pulmonary cell types such as HEK293, mouse embryonic fibroblasts, kidney cells and hepatocytes [22], [52]. Whether or not it also exists in pulmonary cells require further investigation. Another possibility is that CS-induced autophagy may be different from or be regulated differentially by the canonical autophagy. Previously, Kim HP et al. has demonstrated the protective role of HO-1 in repressing CSE-induced autophagy [15]. This observation supports our finding about the protective role of Nrf2 in CSE-induced autophagy because HO-1 is a classical Nrf2-dependent gene [51]. The present study identifies a second Nrf2-dependent gene product-P62 as another autophagy repressor. Thus, Nrf2 system is likely to play an indispensible role in regulating autophagy.
Recently, Fujita, K et, al. indicates that Nrf2-P62 is required for TLR4 mediated aggresome-like induced structure (ALIS) formation [53]. This ALIS has autophagosome-like features such as LC3 positive and being degradable through lysosomal compartment. In their study, Nrf2-P62 appears to be a positive regulator of ALIS formation. However, classical autophagy inhibitors (3-MA and wortmannin) and siRNA knockdown of essential ATG (ATG5, ATG7) failed to prevent this process [53]. Thus, the formation of ALIS is different from the classical autophagy. Nonetheless, this study has reflects the complicated nature of Nrf2-P62 system in controlling a variety of LC3 associated cellular structures that warrant further study.
In summary, we have, for the first time, demonstrated that Nrf2 system negatively regulates CSE-induced LC3B expression and autophagosomes partly through the elevation of P62. The elucidation of this important interaction between Nrf2 systems and autophagy will advance our understanding of the pathogenic effect of CS-associated chronic airway diseases and reveal novel drug targets for the development of effective treatments.
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108 : 1427 –1432 .21220332 | 23585825 | PMC3621864 | CC BY | 2021-01-05 17:25:08 | yes | PLoS One. 2013 Apr 9; 8(4):e55695 |
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10.1371/journal.pone.0059556
Research Article
Biology
Genetics
Population Genetics
Genetic Polymorphism
Population Biology
Population Genetics
Genetic Polymorphism
Mathematics
Statistics
Biostatistics
Medicine
Clinical Research Design
Meta-Analyses
Oncology
Cancer Risk Factors
Genetic Causes of Cancer
Cancers and Neoplasms
Lung and Intrathoracic Tumors
Genetics and Genomics
Oncology
Mathematics
Association of CYP2A6*4 with Susceptibility of Lung Cancer: A Meta-Analysis
A Meta-Analysis for Lung Cancer
Wang Lishan 1 2
1 Bio-X Institutes and Affiliated Changning Mental Health Center, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, P.R. China
2 FengHe (ShangHai) Information Technology Co., Ltd, Minhang District, Shanghai, China
Miao Xiao-Ping Editor
MOE Key Laboratory of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, China
Competing Interests: LW is employed by a commercial company (FengHe (ShangHai) Information Technology Co., Ltd). This does not alter the author's adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: LW. Wrote the paper: LW.
2013
9 4 2013
8 4 e5955610 12 2012
15 2 2013
© 2013 Lishan Wang
2013
Lishan Wang
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Objectives
To assess the association between the variant of Cytochrome P450 2A6 whole gene deletion (CYP2A6*4) polymorphism and risk of lung cancer.
Methods
Two investigators independently searched the PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for CYP2A6*4 and lung cancer were calculated in a fixed-effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate.
Results
This meta-analysis included seven eligible studies, which included 2524 lung cancer cases and 2258 controls (cancer–free). Overall, CYP2A6*4 was associated with the risk of lung cancer (allele*4 vs. allele non-*4, pooled OR = 0.826, 95% CI = 0.725−0.941, P-value = 0.004). When stratifying for population, significant association was observed in Asian (additive model, pooled OR = 0.794, 95% CI = 0.694−0.909, P-value = 0.001; dominant model, pooled OR = 0.827, 95% CI = 0.709−0.965, P-value = 0.016; recessive model (pooled OR = 0.444, 95% CI = 0.293−0.675, P-value <0.0001). In the overall analysis, a comparably significant decrease in the frequency of *4/*4 genotype was detected between cases and controls in Asian while no *4/*4 genotype was detected in Caucasian in collected data.
Conclusion
This meta-analysis suggests that the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer in Asian. The whole gene deletion of CYP2A6 may decrease the risk of lung cancer in Asian samples.
The authors have no support or funding to report.
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pmcIntroduction
Lung cancer is the most common cancer in the world, representing approximately 12% of all new cancer cases, with over 1 million deaths annually, which is the leading cause of cancer death [1]. It is well-known that smoking is a major cause of lung cancer. It is reported that environmental tobacco smoke increases the risk of lung cancer in nonsmokers by approximately 20–50% [2].
Tobacco carcinogens are the most significant factors for smoking induced cancers. To exert their effects, most tobacco carcinogens require metabolic activation, which is generally carried out by cytochrome P450 (CYP) enzymes. Short-lived electrophile agents produced in metabolic activation process react with DNA, thus causing DNA damage and inducing tumors. Such process, mostly, only happen in tissues where it is generated. Therefore, tissue-specific metabolic activation is of vital importance for tissue susceptibility to carcinogen-induced tumors. Among tobacco's potent carcinogens, tobacco-specific nitrosamines (TSNA) and other nitrosamines are activated by CYP2A enzymes, nicotine is also metabolized by CYP2A enzymes [3]–[4].
There are three CYP2A genes in humans (CYP2A6, CYP2A7 and CYP2A13) and one pseudogene (CYP2A18)[5], but there is no catalytic activity shown for CYP2A7 so far. CYP2A6 expression is mainly found in the liver, but its protein or mRNA is also expressed in other tissues such as nasal epithelium, trachea, lung and esophagus [6]–[7]. There are 31 numbered CYP2A6 allelic variants identified to date, however, not all have been functionally characterized. The different alleles are described at the Human CYP Allele Nomenclature Committee Homepage (www.cypalleles.ki.se/cyp2a6.htm). CYP2A6*4 presents a gene deletion that accounts for the majority of poor metabolizer individuals (PM) in Asian populations, and various alleles have been described [8]–[9]. Currently, three deletion variants are known for CYP2A6*4. CYP2A6*4A lacks the 3′-UTR of the CYP2A7 gene and the whole CYP2A6 gene is deleted and an unequal crossover junction is located in the 3′-UTR. CYP2A6*4B has a normal CYP2A7 gene, while the whole CYP2A6 gene is deleted. In CYP2A6*4D, an unequal crossover junction is located at the end of the CYP2A7 gene in either exon 8 or 9 and the whole CYP2A6 gene is deleted. Formerly, a CYP2A6*4C allele is recognized, but subsequent observations reveal that this allele is the same as the CYP2A6*4A allele. Because all these variants result in a whole gene deletion of CYP2A6, most studies do not discriminate between the variants and the term ‘CYP2A6*4’ is designated to all deletions [8]–[9].
During this decade, a number of studies have assessed the association between CYP2A6*4 polymorphism and risk of lung cancer in different populations; however, the results are inconsistent and inconclusive [[10]–[11]]. Different methodologies have been used, however, in particular, most of the studies use a small sample size and it is therefore not surprising that there has been a lack of replication in the various studies. As meta-analysis is an effective way to increase the statistical power by pooling all the available data together and analyzing with a large dataset, in which all the published case-control studies are processed to confirm whether the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer.
Materials and Methods
Literature search
The PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI) for all articles were searched with the following search terms: (‘CYP2A6’ OR ‘Cytochrome P450 2A6’) AND (‘lung cancer’). The date of the last search was Sep 20, 2012. Publication date and publication language were not restricted in our search. Reference lists were examined manually to further identify potentially relevant studies. Unpublished reports were not considered. If more than one article was published by the same author using the same case series, we selected the study where the most individuals were investigated.
Inclusion and exclusion criteria
Abstracts of all citations and retrieved studies were reviewed. Studies meeting the following criteria were included: (1) Using a case – control design; (2) Detecting the relationship between variant CYP2A6*4 and lung cancer; (3) Providing available genotype data of CYP2A6*4; (4) Control group is cancer-free. Studies were excluded if one of the following factors existed: (1) the design is based on family or sibling pairs or case-only; (2) the genotype frequency of CYP2A6*4 is not reported; or (3) there is insufficient information for extraction of data.
Data extraction
All data were extracted independently by two reviewers (XXX and XXX) according to the inclusion criteria listed above. Disagreements were resolved by discussion between the two reviewers. The following characteristics were collected from each study: first author, year of publication, country of sample, ethnicity, number of cases and controls, main background of samples, matching criteria, and genotyping methods (Table 1).
10.1371/journal.pone.0059556.t001 Table 1 Characteristics of studies included in the meta-analysis.
Author Year Country Population No.(cases /controls) Gender Smoking status Matchingcriteria Genotypingmethods Ref.
Miyamoto 1999 Japan Asian 246/201 Mixed Mixed Age, sex, race PCR-RFLP [15]
Loriot 2001 France Caucasian 244/250 Male All smokers Age, sex, race PCR-RFLP [16]
Tan 2001 China Asian 151/326 Mixed Mixed Age, sex, race PCR-RFLP [17]
Fujieda 2004 Japan Asian 1094/611 Male All smokers Age, sex, race PCR-RFLP [18]
Gu 2005 China Asian 180/224 Mixed Mixed Age, sex, race PCR-RFLP [19]
Tamaki 2011 Japan Asian 192/203 Mixed Mixed Age, sex, race PCR-RFLP [20]
Wassenaar 2011 Canada Caucasian 417/443 Mixed All smokers Age, sex, race PCR-RFLP [21]
PCR-RFLP: Polymerase chain reaction-restriction fragment length polymorphism.
Statistical analysis
The statistical analysis was conducted using STATA 11.0 (Stata Corp LP, College Station, TX, United States); P-value <0.05 was considered statistically significant. Hardy Weinberg Equilibrium (HWE) in the controls was tested by the chi-square test for goodness of fit, and a P –value <0.05 was considered as significant disequilibrium. Pooled ORs were calculated for allele frequency comparison (*4 vs. non-*4), dominant model (*4/*4+*4/non-*4 vs. non-*4/non-*4), and recessive model ((*4/*4 vs.*4/non-*4 +non-*4/non-*4)), respectively. The significance of pooled ORs was determined by Z-test and P-value <0.05 was considered statistically significant.
The OR and 95% CI were estimated for each study in a random-effects model or a fixed-effects model. Heterogeneity among studies was examined with the χ2 -based Q testing and I2 statistics [12]. P-value <0.1 was considered significant for the χ2-based Q testing and I2 was interpreted as the proportion of total variation contributed by between-study variation. If there was a significant heterogeneity (P-value <0.1), a random-effects model (the DerSimonian and Laird method) was selected to pool the data. If not, a fixed-effects model (the Mantel-Haenszel method) was selected to pool the data. Heterogeneity was also quantified using the I2 metric (I2<25%, no heterogeneity; I2 = 25–50%, moderate heterogeneity; I2>50%, large or extreme heterogeneity) [25]. Publication bias was examined with funnel plots and with the Egger's tests [13]–[14]. If there is evidence of publication bias, the funnel plot is noticeably asymmetric. For the Egger's tests, the significance level was set at 0.05.
Results
Study Characteristics
A total of 52 papers were retrieved after the first search. After our selection, 7 case-control studies including 2524 lung cancer cases and 2258 controls fulfilled the inclusion criteria [10]–[11]. The qualities of the studies were considered acceptable for our meta-analysis. HWE were calculated for all seven publications and found that only Tamaki's study was inconsistent with Hardy-Weinberg disequilibrium (P-value = 0.042). The flow chart of selection of studies and reasons for exclusion is presented in Figure 1. Studies had been carried out in Japan, France, China and Canada. Five studies [10], [15]–[16] used Asian samples while two studies [17] , [11] used Caucasian samples. Characteristics of studies included in the meta-analysis are presented in Tables 1 and 2.
10.1371/journal.pone.0059556.g001 Figure 1 Flow chart of selection of studies and specific reasons for exclusion from the meta-analysis.
10.1371/journal.pone.0059556.t002 Table 2 Genotype frequencies of CYP2A6*4 in studies included in the meta-analysis.
Author Year Case genotypea Control genotypeb HWEc Ref.
*4/*4 *4/non-*4 non-*4/non-*4 *4/*4 *4/non-*4 non-*4/non-*4
Miyamoto 1999 5 48 193 9 60 132 0.3507 [15]
Loriot 2001 0 24 220 0 19 231 1.0000 [16]
Tan 2001 1 38 112 5 46 275 0.0735 [17]
Fujieda 2004 25 301 768 28 186 397 0.3082 [18]
Gu 2005 0 23 157 1 30 193 1.0000 [19]
Tamaki 2011 7 63 122 19 66 118 0.042 [20]
Wassenaar 2011 0 6 411 0 0 443 1.0000 [21]
a Absolute number of patients; b Absolute number of controls; c HWE: Hardy-Weinberg equilibrium, which was evaluated using the goodness-of-fit chi-square test. P < 0.05 was considered representative of a departure from HWE.
Evaluation of CYP2A6*4 and association with lung cancer
There were seven case-control studies [10]–[11] which had been performed to study the CYP2A6*4 polymorphism and lung cancer risk. Results of the meta-analysis are shown in Table 3. Results showed that there was a significant association between CYP2A6*4 polymorphism and lung cancer risk (additive model, pooled OR = 0.826, 95% CI = 0.725–0.941, P-value = 0.004). But no significant association was observed under dominant model (pooled OR = 0.867, 95% CI = 0.747–1.006, P-value = 0.06).
10.1371/journal.pone.0059556.t003 Table 3 Pooled odds ratio for CYP2A6*4 in meta-analyses.
Population Genetic Model Pooled OR(95% CI) P-valuea P-valueb(Publication bias) P-valuec(heterogeneity) I2
All Additive 0.826(0.725–0.941) 0.004 0.177 0.002 71.4%
Dominant 0.867(0.747–1.006) 0.06 0.211 0.001 72.9%
Asian Additive 0.794(0.694–0.909) 0.001 0.622 0.005 73.0%
0.737(0.640–0.848)d <0.0001 0.800 0.370 4.5%
Dominant 0.827(0.709–0.965) 0.016 0.791 0.002 75.7%
0.751(0.637–0.884)e 0.001 0.804 0.324 13.7%
Recessive 0.444(0.293–0.675) <0.0001 0.486 0.990 0.0%
Caucasian Additive 1.640(0.919–2.927) 0.094 – 0.105 62.0%
Dominant 1.674(0.927–3.024) 0.088 – 0.106 61.8%
a Random-effects model was used when the p-value for heterogeneity test<0.10, otherwise the fixed-effect model was used. b Egger's test to evaluate publication bias. P –value <0.05 is considered statistically significant. c P-value <0.1 is considered statistically significant for Q statistics. d and e The results of Asian samples after exclusion of Tan's study. OR: Odds ratio; CI: confidence interval.
When studies were divided according to the population, the results indicated that significant associations were observed in Asian samples under all models (additive model, pooled OR = 0.794, 95% CI = 0.694–0.909, P-value = 0.001; dominant model, pooled OR = 0.827, 95% CI = 0.709–0.965, P-value = 0.016; recessive model (pooled OR = 0.444, 95% CI = 0.293–0.675, P-value <0.0001). Reversely, no significant associations were observed in Caucasian samples under any model (allele, pooled OR = 1.640, 95% CI = 0.919–2.927, P-value = 0.094; dominant model, pooled OR = 1.674, 95% CI = 0.927–3.024, P-value = 0.088; recessive model was not available as no *4/*4 genotype was observed in Caucasian samples). Results of the meta-analysis are shown in Table 3 and Figure 2.
10.1371/journal.pone.0059556.g002 Figure 2 Forest plots of all studies and studies with Asian samples under different genetic models.
a. All studies (Additive model). b. Studies with Asian samples (Additive model). c. Studies with Asian samples (Recessive model). d. Studies with Asian samples (Dominant model).
Sensitivity analysis
The influence of a single study on the overall meta-analysis estimate was investigated by omitting one study at a time, and the omission of any study made no significant difference, indicating that our results were statistically reliable.
Evaluation of heterogeneity and publication bias
For all studies, statistically significant heterogeneity was observed (P-values by χ2-based Q testing <0.1 and I2 >50%). Then subgroup analysis was carried out. Studies were divided according to the population. For Caucasian, no statistically significant heterogeneity was observed under either additive model (*4 vs. non-*4, P-value = 0.105) or dominant model (*4/*4+*4/non-*4 vs. non-*4/non-*4, P-value = 0.106). For Asian, no statistically significant heterogeneity was observed under recessive model (*4/*4 vs. *4/non-*4 + non-*4/non-*4, P-value = 0.990, I2 = 0), but significant heterogeneity was still observed under both additive model (*4 vs. non-*4, P-value = 0.005) and dominant model (*4/*4+*4/non-*4 vs. non-*4/non-*4, P-value = 0.002). However, when Tan's study was excluded, no statistically significant heterogeneity was observed anymore under either additive model (*4 vs. non-*4, P-value = 0.370, I2 = 4.5%) or dominant model (*4/*4+*4/non-*4 vs. non-*4/non-*4, P-value = 0.324, I2 = 13.7%). Funnel plot and Egger's test were performed to assess the publication bias of the literature. No publication bias was observed (all P-value of Egger's test >0.05) and symmetrical funnel plots were obtained. Results of heterogeneity and publication bias are shown in Table 2.
Discussion
As previous research reported, allele frequency of CYP2A6*4 differed significantly between Asian and non-Asian. CYP2A6*4 is more prevalent among Japanese individuals, with an allele frequency of approximately 0.200[18],[19]–[20]. The frequency is also relatively high among Koreans and Thais (0.110 and 0.140, respectively)[20],[21]. Among Brazilians, French individuals and Canadians, the frequency is 0.010 or lower [22],[23],[24]. The data from this meta-analysis showed a significant decrease of genotype frequency of *4/*4 for the CYP2A6*4 polymorphism in patients with lung cancer than controls in Asian, which suggest that genotype *4/*4 of CYP2A6*4 may decrease the risk of lung cancer in Asian. Therefore, significant results were only discovered in Asian, but not non-Asian population, which may be caused by low frequency of CYP2A6*4 polymorphism. In addition, it is reported that the plasma concentration of cotinine, a major metabolite of nicotine, is considerably higher in carriers of wild-type alleles of CYP2A6 than that in carriers of null or reduced-function alleles of CYP2A6, raising the possibility that cotinine plays an important role in the development of lung cancer [25]. It is also reported that lung tumorigenesis can be promoted by anti-apoptotic effects of cotinine through activation of PI3K/Akt pathway, which is mediated by CYP2A6 [26]. These previous findings support our results and give us possible explanation to the mechanism.
The degree of heterogeneity is one of the major concerns in a sound meta-analysis because non-homogeneous data are liable to result in misleading results. In the present study, the Q testing and I2 statistics were carried out to test the significance of heterogeneity. For all studies, there existed significant heterogeneity. So subgroup analysis was made according to the ethnicity of samples. No significant heterogeneity was observed in Caucasian under any model or in Asian under recessive model. But significant heterogeneity existed in Asian under the other two models, and Tan's study was found to be responsible for heterogeneity. After removing this study, no significant heterogeneity was observed (both P-value of Q testing>0.1, shown in Table 2). Moreover, we performed a sensitivity analysis by removing one study each time and rerunning the model to determine the effect on each overall estimate. The estimates changed little, which implied that our results were statistically reliable.
However, there are still some limitations in this meta-analysis. (1) In seven studies included for our analysis, only two of them are Caucasian samples; (2) The samples from 4 countries and controls are not uniform; (3) CYP2A6*4 is related with smoking, but the smoking status of samples is not uniform in our study. Thus, results should be interpreted with caution; (4) Number of studies and number of subjects in the studies included in the meta-analysis are still small; and (5) Meta-analysis is a retrospective research that is subject to some methodological limitations. In order to minimize the bias, we used explicit methods for study selection, data extraction and data analysis. Nevertheless, our results should be interpreted with caution.
This meta-analysis suggests that the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer in Asian and the whole gene deletion of CYP2A6 may decrease the risk of lung cancer. The pooled ORs in this study suggest that *4/*4 genotype has a modest but definite genetic effect in Asian. Larger and well-designed studies based on different ethnic groups are needed to confirm our results.
Supporting Information
Checklist S1 PRISMA Checklist.
(DOC)
Click here for additional data file.
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ISRN CardiolISRN CardiolISRN.CARDIOLOGYISRN Cardiology2090-55802090-5599Hindawi Publishing Corporation 10.1155/2013/248476Clinical StudyIntracoronary Adenosine versus Intravenous Adenosine during Primary PCI for ST-Elevation Myocardial Infarction: Which One Offers Better Outcomes in terms of Microvascular Obstruction? Doolub Gemina
1
*Dall'Armellina Erica
2
1Cardiology Department, John Radcliffe Hospital, Oxford OX3 9DU, UK2Oxford Centre for Clinical Magnetic Resonance, John Radcliffe Hospital, Oxford, UK*Gemina Doolub: [email protected] Editors: Y. Hayabuchi and T. Ishimitsu
2013 27 3 2013 2013 2484764 2 2013 7 3 2013 Copyright © 2013 G. Doolub and E. Dall'Armellina.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aims. Previous studies have suggested that intravenous administration of adenosine improves myocardial reperfusion and reduces infarct size in ST-elevation myocardial infarction (STEMI) patients. Intracoronary administration of adenosine has shown conflicting results. Methods. In this retrospective, single-centre, blinded clinical study, we assessed whether selective intracoronary administration of adenosine distal to the occlusion site immediately before initial balloon inflation reduces microvascular obstruction (MVO) as assessed with cardiac magnetic resonance imaging (MRI). Using contrast-enhanced sequences, microvascular obstruction (MVO) was calculated. We found 81 patients presenting with STEMI within 12 h from symptom onset who were eligible for the study. In 80/81 (100%) patients receiving the study drug, MRI was performed on Day 1 after primary angioplasty. Results. The prevalence of MVO was reduced in the patients treated with intracoronary adenosine, (45%) compared to 85% of patients who were administered intravenous adenosine (P = 0.0043). We found that the size of MVO in patients receiving intracoronary adenosine was significantly reduced compared to 0.91 g in the intravenous-treated group (P = 0.027). There was no statistically significant difference in TIMI flow and clinical outcomes after primary PCI. Conclusion. We found significant evidence that selective high-dose intracoronary administration of adenosine distal to the occlusion site of the culprit lesion in STEMI patients results in a decrease in microvascular obstruction.
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1. Introduction
The concept of “no reflow” refers to a state of myocardial tissue hypoperfusion in the presence of a nonoccluded epicardial coronary artery. The underlying cause of no reflow is microvascular obstruction, which may be produced by various mechanisms [1, 2].
“Reperfusion no reflow” [2] occurs after primary percutaneous coronary intervention (PCI) for reperfusion of an infarct artery in the setting of acute myocardial infarction (AMI) and may be asymptomatic or may present clinically with continued chest pain and ST-segment elevation [3].
Reperfusion no reflow is an independent predictor of adverse clinical outcome after AMI, regardless of infarct size. Despite successful recanalization, perfusion of the ischaemic myocardium is either unrestored or incompletely restored in up to 30% of patients as a result of microvascular obstruction (MVO) [4], which is associated with higher incidence of left ventricular (LV) dysfunction, complications, and death.
Multiple therapies [5] for no reflow have been tested in animals and to a lesser degree in humans.
Beneficial effects of intracoronary diltiazem, verapamil, epinephrine, nitroprusside, and adenosine have been reported [5–8].
While large randomized trials [4, 5, 9] have suggested that intravenous administration of adenosine during intervention for STEMI may reduce infarct size, especially at higher doses, the effect of intracoronary adenosine in reducing microvascular obstruction is still relatively controversial [9, 10].
In a trial of 54 patients who underwent primary percutaneous transluminal coronary angioplasty, intracoronary adenosine significantly decreased the rate of noreflow [9]; however, in a later trial of 112 patients undergoing primary PCI with stenting [10, 11], adenosine use was not associated with an improvement in the degree of microvascular obstruction or in a decrease in infarct size at four months [12, 13].
2. Study Aims
The aim of this study was to investigate the effect of selective intracoronary administration of adenosine immediately before initial balloon inflation on microvascular obstruction in an unselected population of patients with STEMI.
3. Materials and Methods
This study was a single-centre, retrospective, randomized, blinded clinical study with blinded evaluation of endpoints. All consecutive patients presenting with a suspected acute STEMI to a large tertiary referral centre for interventional cardiology in the UK were eligible for the study. Data was collected using our trust local interventional cardiology database (Datacam), electronic discharge summaries, and medical notes. The study was designed and carried out in the Oxford Centre for Clinical Magnetic Resonance Research Centre (OCMR), located at the John Radcliffe Hospital, Oxford.
Inclusion criteria were as follows:symptoms of chest pain suggestive of myocardial ischaemia for at least 20 min,
a time from onset of symptoms of <12 h,
an ECG showing ST-segment elevation of >0.1 mV in two or more limb leads or >0.2 mV in two or more contiguous praecordial leads,
presumed new left bundle-branch block,
raised troponin (cut-off taken to be troponin >0.04 in Oxford Radcliffe Hospitals NHS Trust),
patients undergoing primary angioplasty with lesions amenable to stenting,
patients consenting to MRI within 24 hours of primary angioplasty,
patients aged >18.
Exclusion criteria were as follows:patients receiving intravenous adenosine during the procedure,
contraindication to heparin, low-molecular-weight heparin, or clopidogrel,
anticipated difficult vascular access,
cardiogenic shock,
inability to give informed consent,
High-grade atrioventricular block
severe asthma,
treatment with theophylline, glibenclamide, or dipyridamole,
prior coronary artery surgery,
participation in any investigational drug or device study within the past 30 days,
contraindications to MRI, for example, an incompatible pacemaker in situ,
Patients with non-STEMI,
Patients with ST-elevation MI but absent coronary artery disease angiographically.
4. Treatment
After coronary angiography, eligible patients received a bolus injection of intracoronary adenosine (900 micrograms in 5 mL of 0.9% sodium chloride solution). Control patients received an intravenous bolus injection of adenosine (900 micrograms in 20 mL sodium chloride) during the procedure.
After crossing the obstruction of the infarct-related coronary artery with a long guide wire, an over-the-wire balloon (Maverick) was positioned at the level of the obstruction. The guide wire was removed and a small quantity of diluted contrast medium was carefully injected through the central lumen of the balloon catheter to confirm positioning of the catheter tip downstream of the obstruction and to assess patency of the distal vessel. Consequently, the study drug solution was injected by hand through the central lumen of the balloon catheter into the distal vascular bed over 60 seconds. The guide wire was then reinserted through the balloon catheter and advanced to a distal position, and the balloon was inflated. After deflation of the balloon, the procedure was continued per operator preference. As the study was carried out after 2006, we had to ensure that we excluded cases of thrombus aspiration or the use of any other additional device except coronary balloons and stents as per protocol.
All patients received aspirin (300 mg), low-molecular-weight heparin (in the form of dalteparin ACS treatment dose as per patient's weight), and clopidogrel (600 mg) after confirmation of ST-segment elevation on the first ECG. Before commencing primary PCI, patients received a bolus of the glycoprotein IIb/IIIa inhibitor abciximab (0.25 mg/kg), followed by a 12 h infusion. The standard treatment in all patients after primary PCI included aspirin, clopidogrel, β-blockers, lipid-lowering agents, and angiotensin-converting enzyme inhibitors.
5. Endpoints
The primary endpoint was microvascular obstruction (MVO), which was defined as the very dark region within a bright area of myocardial infarction scar tissue (Figure 2). MVO on MRI at Day 1 was expressed as a percentage of the infarcted area.
Secondary endpoints were TIMI flow grade, major adverse cardiac events at 30 days, left ventricular (LV) function as assessed using MRI at Day 1, and evolution of cardiac markers in the first 24 hours (Figures 3 and 4).
6. Cardiac Magnetic Resonance Imaging
Cardiac MRI studies were performed on Day 1 of acute STEMI. Both qualitative and semiquantitative methods were used to assess the extent of early MVO on MRI (Table 2).
On each MRI slice, a region of interest (ROI) was drawn in the centre of the left ventricular (LV) cavity, avoiding papillary muscles.
The three contours (epicardium, endocardium, and blood pool) were copied (registered) to the other images, adjusting for breathing motion.
The myocardium was divided into the relevant segments, using a reference point at the inferior/anterior LV-RV junction. The remaining segments were automatically assigned. The start of the upslope of the time-intensity segment was identified for each slice. The time-intensity curve for each myocardial segment was viewed (Figures 5 and 6).
7. Angiographic Analysis
Coronary angiograms obtained before and after primary PCI were reviewed by two experienced interventional cardiologists and a report created on the Datacam software. On the initial angiogram and on the angiogram after stenting, TIMI flow grade was assessed. Site of infarct, door-to-balloon times, medications used during the procedure, and intraprocedural adverse events were also recorded.
8. Biomarkers Reflecting Infarct Size
Serum troponin I measurements were collected in all patients during hospitalization. Blood was collected on admission and at 12 hours after revascularization.
9. Followup
Followup data at 30 days after primary PCI was collected from hospital records and discharge summaries.
10. Acute Adverse Events
These were recorded onto Datacam by the interventional cardiologist performing the procedure.
11. Statistical Analysis
Continuous normally distributed variables are presented as mean values and standard deviations. For skewed distributed variables, median values with interquartile range (IQR) are shown. Comparisons between the groups were done by means of an analysis of variance (ANOVA) using randomized treatment and stratum as factors in the model. Subgroup analyses were performed for MVO by age, gender, time to PCI, diabetes, baseline TIMI, final TIMI, and infarct location. All tests were two-sided and assessed at the 5% significance level. In view of the exploratory nature of this study, no correction was applied for multiple testing. Statistical analysis was performed using the SPSS and Excel software, version 2010 for Mac.
12. Results
This study comprised 81 patients who underwent primary PCI for STEMI at the John Radcliffe Hospital, Oxford Radcliffe Hospitals NHS Trust. One patient was excluded from the study, as he could not tolerate MRI scanning due to severe claustrophobia. Of the remaining 80 patients, half (n = 40) were given intracoronary adenosine during primary PCI, whereas the remainder (n = 40) received intravenous adenosine. Patients randomised to intracoronary adenosine had a higher heart rate on admission compared to patients who had intracoronary adenosine. Patients who received intracoronary adenosine more often had a medical history of diabetes and previous PCI, whereas patients who received intravenous adenosine more often had a history of hypertension and hypercholesterolaemia. TIMI flow 0 or 1 was present in 36 of the 40 patients (90%) having intracoronary adenosine and in 32 of the 40 patients (80%) receiving intravenous adenosine.
All other procedural characteristics did not significantly differ between the two treatment groups—including the stent type and size used, as well as the use of Abciximab (Reopro) infusion during the procedure (Table 1). All patients included in this study had drug-eluting stents (DESs) used during primary PCI.
13. Magnetic Resonance Imaging Results
Of the 80 patients included in the study, all underwent MRI during the first 24 hours of admission for primary PCI. Infarct volume was 19.6 g (17.9, 29.9) in the intracoronary-treated patients versus 19.9 g (17.8, 32.4) in the intravenous group of patients (P = 0.35) or—expressed as a percentage of the left ventricular mass—19.2% (8.6, 29.7) versus 16.8% (10.9, 22.7) (P = 0.11).
Microvascular obstruction (MVO) was observed in 18 of the 40 intracoronary-treated patients (45%) when compared with 34 of the 40 (85%) intravenous treated patients (P = 0.0043). The extent of early MVO was 0.35 g (0, 0.53) in the patients receiving intracoronary adenosine and 0.91 g (0.19–1.25) in the intravenous treated group (P = 0.027). Expressed as a percentage of the infarct area, this resulted in an MVO of 2.04% (0, 3.75) in the intracoronary group, compared to 7.83% (2.85, 12.13) in the intravenous group (P = 0.0014) (Figure 1).
This treatment effect was present in both strata, irrespective of time of symptom onset. Other MRI analyses were comparable between the two treatment groups, with the exception of a higher ejection fraction in the coronary-treated patients.
Additional analyses, adjusted for baseline TIMI, diabetes, time to PCI, and infarct location, were performed for the main endpoints of interest. Subgroup analyses for microvascular obstruction (MVO) revealed a statistically significant interaction between treatment and baseline TIMI flow grade (0/1 versus 2/3) (P = 0.042), whereby a statistically significant treatment benefit of intracoronary adenosine was found for patients with TIMI 2/3 at baseline (P = 0.037). A similar beneficial effect of treatment was found in the group of patients with TIMI flow grade 0/1 at baseline, but this difference was not statistically significant (P = 0.068).
14. Angiographic Results
After primary PCI, TIMI flow grade 3 was present in 40 of the 40 patients (100%) receiving intracoronary adenosine and in 36 of the 40 patients (90.0%) receiving intravenous adenosine (P = 0.077; Table 3).
15. Biomarkers
Calculation of the area under the curve of cardiac markers as an estimation of infarct size was not performed if either the first or last measurement was missing. Other missing values were imputed by linear interpolation. The area under the curve for troponin I could only be calculated for 78 of the 80 patients and showed no significant differences between treatment groups (Table 4), irrespective of time from symptom onset. One major limitation in the measurement of troponin was the fact that the assay used by the Oxford Hospitals Trust did not enable measurements of troponin, which meant that any troponin value exceeding this range was consistently reported as being >50.
16. Complications during Primary PCI
An increase of chest pain during or immediately after selective administration of adenosine 900 micrograms distally to the occlusion site was not observed at all. Occlusion of a significant side branch, no reflow phenomenon, bradycardia, ventricular tachycardia, and ventricular fibrillation were not seen in either of the two treatment groups. Dissection was numerically more frequent in the intravenous group of patients compared with patients receiving intracoronary adenosine, the difference, however, not reaching statistical significance (2/40 (5%) versus 0/40 (1.9%), P = 0.87). The incidence of second- and third-degree atrioventricular block was also numerically higher in the intravenous group of patients (4/40 (10%) and 2/40 (5%)) than in intracoronary-treated patients (2/40 (5%) 0/40 (0%)). These conduction blocks disappeared within 4 minutes, without clinical sequelae (Table 5).
17. Clinical Outcomes
None of the patients died during initial hospitalization: one patient from the control group developed heart block 9 hours after stenting of the right coronary artery (RCA), which required urgent pacing.
Between discharge and 30-day followup, an additional patient randomized to intravenous adenosine developed a recurrent myocardial infarction at 28 days, for which he underwent repeat coronary angioplasty.
At 30 days, none of the patients had died in each treatment group.
18. Discussion
This retrospective, blinded, case-controlled clinical study demonstrated that selective intracoronary administration of adenosine distally to the occlusion site and immediately before initial balloon inflation improved reperfusion in patients with STEMI. There were significant differences in MVO and TIMI flow as assessed by angiography and MRI. However, there were no significant difference in enzymatic infarct size and clinical outcome at 30 days after primary PCI between patients given intracoronary adenosine, as compared to the control group.
In the present study, we attempted to avoid some of the methodological limitations of earlier trials [11–13]. First, we used MRI to assess the primary endpoint, microvascular obstruction, because it is less influenced by differences in the extent of ischaemic myocardium at baseline. Second, all study personnel remained blinded to treatment assignment until completion of all analyses. Third, the study drug was consistently administered subselectively to the ischaemic myocardium, prohibiting inadvertent spillover into other coronary branches. Fourth, we used a consistently high dose of adenosine, 900 micrograms, proven to be safe and well tolerated. We also directly compared the effects of intracoronary versus intravenous adenosine, which has not been done in previous studies. Finally, in each case the intracoronary adenosine was consistently administered before initial balloon inflation.
One may argue that the presence of a beneficial effect of intracoronary adenosine is due to differences in baseline clinical characteristics, favouring intracoronary-treated patients. However, there were no significant differences in baseline characteristics between the two treatment groups with the exception of a slightly higher heart rate on admission in patients who received intracoronary adenosine and a higher incidence of diabetes mellitus and previous PCI in the same group of patients. On the contrary, such differences should logically have favoured the intravenous group, thus inducing smaller amounts of MVO—which was clearly not the case in this study.
19. Limitations of the Study
First, the primary endpoint was MVO and we cannot comment on clinical endpoints, for which the study was underpowered. Second, 28% of patients had limited spontaneous reperfusion before intracoronary administration of the study drug and in a few more mere passage of the guide wire may have mediated some degree of reperfusion. Hence, in these patients, adenosine was not truly given before reperfusion. However, for MVO, we identified a large interaction between treatment and baseline TIMI, whereby a statistically significant treatment benefit of intracoronary adenosine was found for patients with TIMI 2/3 flow at baseline and a nonstatistically significant benefit was found for the TIMI 0/1 group. At this stage, it is not very clear to us how this finding can be explained. Third, the trial included a significant proportion of patients with small and/or completed infarcts. Fourth, infarct size cannot be reliably assessed when biomarkers are drawn every 8 h as in this study. And finally, intracoronary adenosine boluses may be too short acting for any beneficial effect and we cannot exclude the efficacy of prolonged intracoronary administration.
20. Conclusions and Implications
We found convincing evidence that selective intracoronary administration of high-dose adenosine as adjunctive therapy to primary PCI reduces MVO in patients with STEMI. Future attempts at improving myocardial reperfusion, preventing reperfusion injury, and salvaging ischaemic myocardium should focus on finding the optimal dose of intracoronary adenosine that should be used in order to create a maximum beneficial effect.
Key Messages
Microvascular obstruction (MVO) is now recognised as a key factor in incomplete perfusion.
Our study shows evidence that intracoronary adenosine significantly reduced the extent of MVO after STEMI, compared to intravenous adenosine.
Conflict of Interests
The authors can confirm that they have no direct financial relation with the commercial identities mentioned in the paper that might lead to a conflict of interests.
Acknowledgments
The authors wish to thank the staff of the Oxford Centre for Magnetic Resonance (OCMR) at the John Radcliffe Hospital, for their valuable support and help.
Abbreviations
ACS:Acute coronary syndrome
Cx:Circumflex artery
LAD:Left anterior descending artery
MRI:Magnetic imaging resonance
MVO:Microvascular obstruction
PCI:Percutaneous coronary intervention
RCA:Right coronary artery
STEMI:ST-elevation myocardial infarction
TIMI:Thrombolysis in myocardial infarction.
Figure 1 Microvascular obstruction: three-chamber MRI demonstrates near full-thickness transmural infarct (arrow) in the basal anteroseptum. There is a black area (curved arrow) within the bright scar consistent with microvascular obstruction [14].
Figure 2 Delineation and calculation of MVO by drawing the relevant contours: white area demonstrates the area of infarct, while the black area within shows the extent of microvascular obstruction (MVO).
Figure 3 Left ventricle before contrast.
Figure 4 Left ventricle after contrast.
Figure 5 Myocardial time-intensity curve.
Figure 6 Box plot showing microvascular obstruction (%) (primary endpoint). Box plot shows median and interquartile range. Q1, Q3 = first and third quartile, IQR = Q3 − Q1. “+” sign indicates mean value.
Table 1 Baseline patient characteristics (% in brackets).
Characteristic Intracoronary adenosine (n = 40) Intravenous adenosine (n = 40)
Age (years) 61.0 59.8
Male gender 30 (75) 32 (80)
Heart rate (b.p.m.) 96 87
Systolic blood pressure (mm Hg) 156 162
Diastolic blood pressure (mm Hg) 98 104
Current smoker 26 (65) 30 (75)
History
Myocardial infarction 6 (15) 4 (10)
Diabetes mellitus 6 (15) 2 (5)
PCI 4 (10) 2 (5)
Family history 12 (30) 10 (25)
Hypertension 18 (45) 22 (55)
Hypercholesterolaemia 12 (30) 22 (55)
Door-to-balloon time (median, IQR), min 16.1 15.8
Angiographic
Infarct-related vessel
RCA 20 (50) 22 (55)
LAD 18 (45) 16 (40)
Cx 2 (5) 2 (5)
TIMI flow before PCI
0 32 (80) 20 (50)
1 4 (10) 12 (30)
2 4 (10) 8 (20)
3 0 (0) 0 (0)
0/1 36 (90) 32 (80)
2/3 4 (10) 8 (20)
Stent diameter, mm 3.0 3.0
Stent length, mm 19.5 21.7
GPIIb/IIIa inhibitor during PCI 40 (100) 40 (100)
Table 2 Magnetic resonance imaging results at Day 1.
Intracoronary adenosine (n = 40) Intravenous adenosine (n = 40)
P value
Myocardial evaluation (median, IQR)
Microvascular obstruction present 18/40 (45%) 34/40 (85%) 0.0043
Microvascular obstruction (g) 0.35 (0, 0.53) 0.91 (0.19, 1.25) 0.027
Microvascular obstruction (%) 2.04 (0, 3.75) 7.83 (2.85, 12.13) 0.0014
Infarct volume (g) 19.60 (17.9, 29.9)
19.90 (17.8, 32.4) 0.35
Infarct volume (% LV mass) 19.20 (8.6, 29.7) 16.80 (10.9, 22.7) 0.11
LV function (mean, SD)
SV (mL) 23.81 (11.10) 24.42 (14.31) 0.44
EF (%) 31.54 (11.49) 29.40 (10.15) 0.27
LV mass (g) 70.40 (24.8) 99.89 (22.3) 0.35
Table 3 TIMI flow grade following primary PCI.
Adenosine Control
P value
TIMI flow grade
0 0 0
1 0 4
2 0 0
3 40 36 0.077
0/1 0 4 0.068
2/3 40 36 0.037
Table 4 Infarct based on cardiac markers (median (interquartile range)).
Intracoronary adenosine Intravenous adenosine
P value
Troponin I maximum (microg/L) 50 (32.4; 50) (n = 19) 50 (23.5; 50) (n = 20) 0.31
Troponin AUC (microg/L × h) 400 (296.8; 400) (n = 19) 400 (196.1; 400) (n = 20) 0.28
Table 5 Complications during primary percutaneous coronary intervention [n (%)].
Complication during/immediately after PCI Intracoronary adenosine (n = 40) Intravenous adenosine (n = 40)
P value
Increase of chest pain during study drug administration 0 (0%) 0 (0%) 0.91
Occlusion of side branch ≥ 2 mm 0 (0%) 0 (0%) 0.76
Distal embolization 0 (0%) 0 (0%) 0.76
No reflow 0 (0%) 0 (0%) 1.53
Dissection 0 (0%) 2 (5%) 0.27
Second-degree AV block 2 (5%) 4 (10%) 0.63
Third-degree AV block 0 (0%) 2 (5%) 0.75
VT (ventricular tachycardia) 0 (0%) 0 (0%) 0.57
VF (ventricular fibrillation) 0 (0%) 0 (0%) 0.98
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BMC OphthalmolBMC OphthalmolBMC Ophthalmology1471-2415BioMed Central 1471-2415-13-112357031010.1186/1471-2415-13-11Research ArticleCental macular thickness in patients with type 2 diabetes mellitus without clinical retinopathy Demir Mehmet [email protected] Ersin [email protected] Burcu [email protected] Erhan [email protected] Efe [email protected] Sisli Etfal Training and Research Hospital, Eye Clinic, Karayolları Mah. Abdi ipekci bulvarı. N0:32 Avrupa tem konutları 28. Blok. Daire:14. 34250 GOP, Sisli, Istanbul 34400, Turkey2013 9 4 2013 13 11 11 2 10 2012 15 3 2013 Copyright © 2013 Demir et al.; licensee BioMed Central Ltd.2013Demir et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
An increase in macular thickness due to fluid accumulation in the macula in patients with diabetes mellitus. Optical coherence tomography (OCT) has been shown to be highly reproducible in measuring macular thickness in normal individuals and diabetic patients. OCT can detect subtle changes of macular thickness. The aim of this study is to compare central macular thickness (CMT) of diabetic patients with type 2 diabetes without clinical retinopathy and normal controls, in order to assess possible increased macular thickness associated with diabetes mellitus.
Methods
Optical coherence tomography (OCT) measurements were performed in 124 eyes of 62 subjects with diabetes mellitus without clinically retinopathy (study group: 39 female, 23 male, mean age: 55.06 ± 9.77 years) and in 120 eyes of 60 healthy subjects (control group: 35 female, 25 male, mean age: 55.78 ± 10.34 years). Blood biochemistry parameters were analyzed in all cases. The data for central macular thickness (at 1 mm) and the levels of the fasting plasma glucose and glycosylated hemoglobin (HbA1c) were compared in both groups.
Results
The mean central macular thickness was 232.12 ±24.41 μm in the study group and 227.19 ± 29.94 μm in the control group.
The mean HbA1c level was 8.92 ± 2.58% in the study group and 5.07 ± 0.70% in the control group (p=0.001). No statistically significant relationship was found between CMT, HbA1c, and fasting plasma glucose level in either group (p=0.05).
Conclusions
Central macular thickness was not significantly thicker in patients with type 2 diabetes without clinical retinopathy than in healthy subjects.
Diabetes mellitusCentral macular thicknessGlycosylated hemoglobinFasting plasma glucose level
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Background
Diabetic retinopathy is the leading cause of blindness in working aged adults in westernized countries. Diabetic macular edema (DME) has been reported at rates of 10% and occurs more frequently in type 2 diabetes mellitus than in type 1. Diabetic patients also have multiple risk factors for retinopathy, such as hyperglycemia and hypertension [1]. Their visual acuity is often dependent the central foveal involvement, perifoveal capillary blood flow velocity, severity of perifoveal capillary occlusion, and retinal thickness at the central fovea [2,3]. The clinical findings of diabetic retinopathy are microaneurysms, soft exudates, accumulation of hard exudates, and neovascularisation.
Macular edema can develop at any stage of diabetic retinopathy. In the past, macular edema was diagnosed with slit-lamp view. Fundus fluorescein angiography provides guidance for treatment of macular edema. Optical coherence tomography (OCT) has been used for detection of macular edema secondary to different pathologies, such as diabetes mellitus, central or branched retinal vein occlusion, uveitis, and age related macular degeneration [4]–[11].
Methods
The central macular thickness (CMT) was measured in both groups by OCT (Optovue Inc. Co., RTVue 100 model, Fremont, CA). The CMT was measured after providing pupil dilation with tropicamide drops 2 times, 10 minutes before measurement (Tropicamide 1%, Alcon Lab. Inc, USA). Three measurements were taken from each patient after pupillary dilatation. Blood biochemical tests for glycosylated hemoglobin (HbA1c) and fasting plasma glucose levels were run on all patients. All cases underwent ophthalmological examinations including best corrected visual acuity (BCVA), anterior and posterior segment examinations under slit-lamp, intraocular pressure (IOP) (applanation tonometer model AT 900; Haag-Streit, Switzerland), and central macular thickness measured by OCT Visual acuity was measured with an Early Treatment Diabetic Retinopathy Study chart at 4 meters. Each subject gave written informed consent to participate in the study. Ethic Committee approval was obtained from local committee.
Participiants
The study group included 62 patients (124 eyes; 39 female, 23 male, mean age: 55.06 ± 9.77 years) who had type 2 diabetes mellitus without clinical retinopathy and a control group of 60 patients (120 eyes; 35 female, 25 male, mean age: 55.78 ± 10.34 years) (Table 1). Inclusion criteria for the study group included: no visible findings of diabetic retinopathy (hard-soft exudates, microaneurysms) on retina at slit-lamp fundus examination with a +78 D lens, type 2 diabetes mellitus, no other problems (such as hypertension, uveitis), and no history of ophthalmologic trauma, intravitreal injection, high refractive errors (spherical equivalent; between: +1.00 D to −1.00 D) or use of drugs(s) for retinal problems. Inclusion criteria for the control group patients included: no ophthalmologic or systemic problems, no history of intraocular surgery or treatment of the retina, and no high refractive errors (spherical equivalent: between −1.0 D to +1.0 D). Exclusion criteria for both groups were visible retinopathy or uveitis, hypertension, or previous ophthalmologic surgery. In the study group, the duration of diabetes mellitus ranged from 0 – 20 years and the average was 7.19 ± 4.87 years. Five patients were newly diagnosed, 19 patients had been diagnosed for 1–5 years, 23 patients had been diagnosed for 6–10 years, 9 patients had been diagnosed for 11–15 years, and 6 patients had been diagnosed for more than 15 years. In the study group; five patients were newly diagnosed, 49 patients were undergoing insulin treatment, and 8 patients were taking oral antidiabetic drugs (Table 2). Both groups were compared based on mean age, central macular thickness, fasting plasma glucose, and HbA1c levels.
Table 1 Demographic characteristics, values for central macular thickness (CMT), and biochemical analysis in patients with type 2 diabetes without clinical retinopathy
Parameters Study group (n=62) Control group (n=60) p
BCVA 0.00 (log MAR) 0.00 (logMAR) NS
IOP mmHg 17,8 ±2.3 mmHg 18.1 ±2.1 mmHg NS
Age(year) 55.06±9.77 55.78±10.34 NS
Male/Female Gender 23/39 25/35 NS
CMTμm(±SD) 232.12±24.41 227.19±29.94 NS
HbA1c ( mean±SD) 8.92±2.58 5.07±0.70 0.001
Fasting blood glucose Average ±SD 202.14±104.78 (median:178 ) 92.17±7.75 (median:92) 0.001
BCVA: Best corrected visual acuity, IOP: Intraocular pressure, CMT: Central macular thickness, μm:micrometer, SD: standard deviation, logMAR: logarithm of the minimum angle of resolution, HbA1c: glycosylated hemoglobin, n: number of patients, logMAR: logarithm of the minimum angle of resolution, NS: Non significant; Study group: Patients with type 2 diabetes without clinical retinopathy; Control group: healthy controls.
Table 2 Duration and treatment of diabetes mellitus in patients with type 2 diabetes without clinical retinopathy
Duration of DM n (=62) %
New diagnosis 5 8.1
1-5 years 19 30.6
6-10 years 23 37.1
11-15 years 9 14.5
>15 years 6 9.7
Insulin treatment 49 79
OAD (oral anti-diabetic drug) 8 12.9
DM: Diabetes mellitus, n: number of patients.
Statistical analysis
The NCSS (Number Cruncher Statistical System) 2007 and the PASS 2008 Statistical Software (Utah, USA) programs were used to evaluate the results of the study.
Descriptive statistical methods (mean, standard deviation) and Student’s t- test were used together to compare the data from the two groups and the parameters that showed normal distribution. The Mann Whitney U test was used to compare parameters of the two groups that did not show normal distribution. A Chi-square test was used to compare the quality of the data. Pearson correlation analyses were conducted to evaluate the relationship between the parameters showing normal distribution and Spearman’s rho correlation analyses have been used to evaluate correlation between the parameters not showing normal distribution. A value of p<0.05 was considered significant.
Results
Best corrected vision (BCVA) was 0.00 (log MAR) in both groups. No significant differences were found for the mean age, IOP, or gender distribution (Table 1).
The mean HbA1c level was 8.92 ± 2.58% in the study group, and 5.07 ± 0.70% in the control group. The mean level of HbA1c was statistically higher in the study group than in the control group (Table 1, p=0.001). Fasting plasma glucose level was statistically higher in the study group than in the control group (Table 1, p=0.01). The duration of diabetes mellitus was 7.19 ± 4.8 (range: 0–20) years. The mean of CMT was 232.12 ± 24.41 μm in the study group and 227.19 ± 29.94 μm in the control group (Table 1). The CMT was thicker in the study group than in the control group but this difference was not statistically significant.
No relationship was found between CMT and fasting plasma glucose level in the study (p=0.483) and control (p=0.399) groups. No relationship was found between CMT and HbA1c level in the study (p=0.550), and control (p=0.997; Table 3).
Table 3 Relationship between central macular thickness (CMT), glycosylated hemoglobin (HbA1c), and fasting blood glucose levels in patients with type 2 diabetes without clinical retinopathy
Parameters Study group Study group Control group Control group
r p r p
CMT-HbA1c −0.077 NS 0.001 NS
CMT-Fasting glucose level −0.091 NS 0.011 NS
CMT: Central macular thickness, HbA1c: glycosylated hemoglobin, p; statistic value, r: relation between two variables.
NS: Non significant; Study group: Patients with type 2 diabetes without clinical retinopathy; Control group: healthy controls.
Discussion
We found no studies in the literature which reviewed CMT, fasting plasma glucose level, and level of HbA1c less than HbA1c 8%.
Several previous studies [12]–[17] determined that optical coherence tomography can help in the evaluation of macular edema in diabetic or non-diabetic patients, and also help in the follow-up of the patients during treatment to establish quantitative or qualitative responses to therapy.
We reviewed the relationship between central macular thickness, HbA1c, and fasting plasma glucose levels in patients with type 2 diabetes without clinical diabetic retinopathy. Optical Coherence Tomography (OCT) was used for objective measurement and monitoring of central macular thickness. Browning and Hee, et al. [18,19] described that a change in the OCT measurements greater than 10% of the baseline thickness is likely to represent a true change in macular thickness. Glycosylated hemoglobin is a parameter that can be used to follow up hyperglycemia over the long term. Moon, at al [20] suggested that a high baseline HbA1c and a large reduction in HbA1c were risk factors for increase in macular thickness. Yeung, et al [21], showed that HbA1c level positively correlated with macular thickness in patients with type1 and 2 diabetes of10 or more years’ duration without diabetic macular edema. Chou, Moreira at al [22]. showed that a HbA1c level of 8% or above was associated with an increase in macular thickness in diabetic patients with diabetic retinopathy. Yeung, http://at al. [21]–[23] concluded that meticulous diabetes control may slow the progression of early diabetic retinopathy and may play an important role in preventing macular dysfunction. In type 1 and 2 diabetes patients, strict follow-up of plasma glucose level could reduce the progression and development of diabetic retinopathy.
The purpose of this study was to examine central macular thickness in patients with type 2 diabetes mellitus without retinopathy. This study showed the following four results: 1) The mean central macular thickness is thicker in diabetic patients without diabetic retinopathy than in healthy subjects, but this difference was not statistically significant; 2) No positive relationship was found between fasting plasma glucose level and the central macular thickness in patients with diabetes mellitus without retinopathy; 3) Central macular thickness was not increased by mild or high levels of HbA1c (8.92 ± 2.59%); and 4) Central macular thickness was not affected by the duration of diabetes mellitus in patients with diabetes type 2 without retinopathy. There are limitations to our study. One of these is the small sample size in both groups and another is that no patients had diabetes mellitus for longer than 20 years.
Conclusion
Our opinion is that the truly effective parameter on macular thickness is vascular permeability in patients with diabetes mellitus.
In this study, glycosylated HbA1c and fasting plasma glucose levels were significantly higher in diabetic patients without retinopathy than in the control group, although there was no difference in central macular thickness between the two groups.
Competing interests
The authors have no finacial competing interests.
Authors’ contributions
All authors conceived of and designed the experimental protocol. MD and EO contributed to the study design and did critical revision of the manuscript for important intellectual content. MD, EO and BD participated in the eye examinations. EO and EC collected the data. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2415/13/11/prepub
Acknowledgements
Thanks to Celeste Krauss (and her team) who provided writing in good medical English CEO of http://www.Mededit.net
Thanks to Bendisah Karaer for helping to transfer of participiants to room of Optic Coherence Tomography.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23593473PONE-D-12-3617910.1371/journal.pone.0061363Research ArticleBiologyBiochemistryLipidsFatty AcidsProteinsRecombinant ProteinsGeneticsGene ExpressionGene FunctionMolecular Cell BiologyGene ExpressionPlant ScienceAgronomyPlant BreedingPlantsFlowering PlantsPlant GeneticsPlant GenomicsPlant Growth and DevelopmentOverexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli
Peanut Diacylglycerol Acyltransferase 2 ExpressionPeng Zhenying
1
2
Li Lan
3
Yang Lianqun
1
2
Zhang Bin
1
2
Chen Gao
1
2
Bi Yuping
1
2
3
*
1
High-Tech Research Center, Shandong Academy of Agricultural Science, Jinan, China
2
Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China
3
College of Life Science, Shandong Normal University, Jinan, China
Bennett Malcolm Editor
University of Nottingham, United Kingdom
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YB. Analyzed the data: ZP. Wrote the paper: ZP. Vector construction, transformation, bacterial culture and growth rate observations: LL. Fatty acid tests: LY. Morphological observations: BZ. Western blot analysis: GC.
2013 11 4 2013 8 4 e6136322 11 2012 7 3 2013 © 2013 Peng et al2013Peng et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.
This work was supported by the National Natural Science Foundation of China (30871541), International Science & Technology Cooperation Program of China (2012DFA30450), and the Science and Technology Development Project of Jinan (201004044). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme of the Kennedy pathway for synthesizing triacylglycerols (TAGs) in eukaryotes. DGAT genes have been identified in a wide range of organisms [1]–[4], but TAGs are especially important for energy storage in oil-producing plants, especially peanuts, soybeans and rape. At least four different types of DGAT have been identified in plants. DGAT1 and DGAT2 are transmembrane domain proteins with essential roles in TAG biosynthesis in plants and other eukaryotes [3]. Only one soluble DGAT (DGAT3) has been identified in peanut cotyledons; however, BLAST analyses have identified several potential orthologs in EST collections from Arabidopsis, rice, and other plant species [5]. The fourth type, phospholipid:diacylglycerol acyltransferase (PDAT), catalyzes the acyl-CoA-independent formation of TAG in yeast and plants [6]. DGAT1 makes the major contribution to seed oil accumulation [1], [4], [7], whereas DGAT2 and PDAT both affect the specific accumulation of unusual fatty acids (FAs) in seed oil [2], [8]–[11]. These enzymes have unique expression patterns in a variety of plant tissues [7] and may have other roles besides seed oil accumulation, such as FA mobilization [12] and leaf senescence [13]. The process by which such enzymes are regulated in developing seeds and other tissues remains poorly understood.
Much research has focused on DGATs because of their important roles in TAG synthesis. Overexpression of such genes can greatly increase the oil content of transgenic organisms. For example, overexpression of the Arabidopsis DGAT1 gene in tobacco and yeast greatly enhanced the TAG content of the transformed lines [14]–[15]. Interestingly, Ricinus communis DGAT2 (RcDGAT2) has a strong preference for hydroxyl FAs containing diacylglycerol (DAG) substrates, the levels of which increased from 17% to nearly 30% when RcDGAT2 was expressed in Arabidopsis
[10]. In Ricinus seeds, RcDGAT2 expression was 18-fold higher than in leaves, whereas RcDGAT1 expression differed little between seeds and leaves. Hence, RcDGAT2 probably plays a more important role in castor bean seed TAG biosynthesis than RcDGAT1 [2]. In addition, OeDGAT1 from the olive tree Olea europaea is responsible for most TAG deposition in seeds, while OeDGAT2 may be a key mediator of higher oil yields in ripening mesocarps [16].
Recombinant proteins can be used as alternatives to endogenous ones to study their structures and functions or to make high-titer antibodies that recognize them. Because most DGATs are integral membrane proteins, they are difficult to express and purify in heterologous expression systems [17], [18]; thus far, only limited success has been achieved in this area [18]–[20]. Weselake et al. expressed the N-terminal 116 amino acid residues of Brassica napus (oilseed rape) DGAT1 as a His-tagged protein in Escherichia coli
[16]. The resulting recombinant BnDGAT1(1–116)His6 interacted with long chain acyl-CoA and displayed enhanced affinity for erucoyl (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-CoA [18]. Subsequently, the amino terminal 95 residues of mouse DGAT1 were expressed in E. coli with similar results [19]. Encouragingly, full-length DGAT1 expression from the tung tree (Vernicia fordii) in E. coli has been achieved [20]. In this case, the recombinant protein was mostly targeted to the membranes, and there were insoluble fractions with extensive degradation from the carboxyl end as well as association with other proteins, lipids, and membranes.
Arachis hypogaea (peanut, Fabaceae) is one of the most economically-important oil-producing crops, so the fact that peanut DGATs have not been extensively studied is surprising. Saha et al. identified a soluble DGAT3 from immature peanut cotyledons and expressed its full length in E. coli, where the recombinant protein had high levels of DGAT activity but no wax ester synthase activity [5]; this is the only published report on peanut DGATs thus far. Here, we identified two isozymes of DGAT2 in peanut and expressed both of them as full-length recombinant proteins in E. coli. This is the first time that a full-length recombinant DGAT2 protein from peanut has been successfully expressed in E. coli, and the first evaluation of its effects on the growth and FA content of the transformed E. coli strains studied.
Materials and Methods
Cloning of the full-length peanut DGAT2 cDNA
Total RNA (5 µg) from peanut cultivar ‘Luhua 14’ pods obtained 25 days after flowering (DAF) was reverse-transcribed into first-strand cDNAs using a cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) in a 20 µL reaction volume. Examination of the conserved domains of soybean GmDGAT2 and RcDGAT2 nucleotide sequences enabled us to design a pair of primers (AhD2-S: 5′ TCTTACACCAGCAACAAGGAAA 3′ and AhD2-A: 5′ GACCAAAGCAGAAAACAGGAAC 3′) (Sangon Co., Shanghai, China) that successfully amplified a 197-bp fragment of the gene. The 20 µL PCR mixture contained 1 µL cDNA, 1 µL of each primer (10 µM), 2 µL PCR buffer (10×), 2 µL dNTPs (2.5 mM each), and 1 unit of Pyrococcus furiosus (Pfu) DNA polymerase (Invitrogen). The reaction was denatured at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C; then 10 min at 72°C. PCR was performed in a PCR Thermal Cycler Dice-TP600 (Takara, Otsu, Japan). The AhDGAT2 fragment was purified using a MinElute™ Gel Extraction Kit (Qiagen, Hilden, Germany), cloned into a pMD18-T vector (Takara), and sequenced.
The full-length AhDGAT2 from ‘Luhua 14’ was cloned using a SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). Total RNA (1 µg) from the 25-DAF peanut pods was used for cDNA synthesis following the manufacturer's protocol. Rapid amplification of cDNA ends (RACE) primers were based on the sequence of the AhDGAT2 fragment described above as follows: AhD2-3O (5′ TCTTACACCAGCAACAAGGAAA 3′) and AhD2-3I (5′ CCCTCTTGGATAATGGCTACAGTTG 3′), and AhD2-5O (5′ ACTGTAGCCATTATCCAAGAGGG 3′) and AhD2-5I (5′ TTTCTTTGTTGCTGGTGTAA 3′). PCRs were performed according to the manufacturer's protocol. The fragments were sequenced and assembled into a full-length sequence.
Based on the full-length sequence of the AhDGAT2 gene, its full-length open reading frame (ORF) was amplified with gene-specific primers (AhD2-FS: 5′ TCAACAGCCACCGAATCCA 3′ and AhD2-FA: 5′ TAAAACAAGGAAGGGTGCCA 3′). The 20 µL PCR volume comprised 1 µL cDNA, 1 µL of each primer (10 µM), 2 µL PCR buffer (10×), 4 µL dNTPs (2.5 mM each), and 1 unit of Pfu DNA polymerase. The reaction was denatured at 94°C for 5 min; followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min 20 s at 72°C; then 10 min at 72°C. The full length fragment (AhDGAT2 ORF) was purified from an agarose gel and cloned into a pMD18-T vector for sequencing.
Translations of the full-length ORF sequences were analyzed for structural motifs. Transmembrane helices were predicted using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), conserved domains were found using the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) at the National Center for Biotechnology Information (NCBI), and putative functional motifs were identified using PROSCAN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_proscan.html). We also predicted the two- and three-dimensional structures of the genes using phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).
Phylogenetic analyses
To better understand the evolutionary origins of the AhDGAT2s, their protein sequences were aligned with those of other DGAT2 genes obtained from NCBI. Homologous sequences in GenBank were identified by a protein BLAST with E-value>6e-149. A multiple sequence alignment using hydrophilic and residue-specific penalties was conducted in DNAMAN 6.0 software (Lynnon Biosoft, Quebec, Canada), which was also used to reconstruct a phylogenetic tree using the observed
divergency distance method and default parameters. Two sequences from monocots, Zea mays and Oryza sativa, were used as outgroups. Statistical support for the tree was gauged using 500 bootstrap replicates.
Construction of recombinant AhDGAT2a and AhDGAT2b expression vectors and transformations
AhDGAT2a and AhDGAT2b ORFs were amplified using DGAT2a-S2 (5′ CGCGGATCCATGGAAGATCGAGGGAACGT 3′), DGAT2b-S2 (5′ CGCGGATCCATGAAGTCCGAGGGAACGT 3′) and DGAT2-A2 (5′ CCGCTCGAGTCAGACAATTCTCAACTCAAGG 3′) primers, after which the PCR products were restriction digested with BamHI and XhoI then ligated into the similarly-digested expression vector pGEX-4T-1 (Biovector Science Lab, Beijing, China) for expression as GST-tagged fusion proteins. The constructs were transformed into the E. coli Rosetta (DE3) strain (Transgen, Beijing, China) following the manufacturer's directions.
We evaluated the time course of expression of the fusion proteins by inducing cells transformed with the empty vector or with a GST-tagged fusion protein with 1.0 mM IPTG at 25°C or 37°C. Cells were collected after 0, 2, 4, and 6 h by centrifuging (5,000×g, 10 min), and the pellets were sonicated for 10 min in homogenization buffer (3–4 mL/g wet cells) containing 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 10 mM β-mercaptoethanol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 1∶100–1∶500 dilution of protease inhibitor cocktail (#P8340, Sigma–Aldrich, St. Louis, MO, USA). Cell debris, inclusion bodies, and protein aggregates in the homogenate were removed by two consecutive centrifugations (2,000×g, 10 min, followed by 10,000×g, 10 min). Samples were treated with 0.5 M NaOH and protein concentrations were determined with the Bradford method (Protein Assay Dye Reagent Concentrate, Bio-Rad, Hercules, CA, USA). The supernatant and the pellet were evaluated by SDS-PAGE (10%) and visualized by staining with Coomassie brilliant blue. Furthermore, the expression locations of the recombinant proteins were determined by purifying cytoplasmic and inclusion body fractions using a B-PER GST Fusion Protein Purification Kit (Thermo Scientific, Rockford, IL, USA). Protein expression was verified by a western blot analysis using an anti-GST tag monoclonal antibody (EarthOx, San Francisco, CA, USA).
Growth and morphology of AhDGAT2a and AhDGAT2b transformed E. coli strains
To examine how transformation with DGAT2 affected bacterial growth rate, wild-type (WT) Rosetta (DE3) E. coli strains and strains transformed with either empty vector, AhDGAT2a–GST, or AhDGAT2b–GST were cultured in Luria broth (LB) medium at 37°C at an initial density of OD600 = 0.05. IPTG (1.0 mM final concentration) was added after 2.0 h for protein induction. OD600 measurements were conducted every hour for 8 h using an Evolution 300 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Each treatment was examined in triplicate.
We also evaluated the morphological effects of transformation on these four cell types. At 0, 2, 4, and 6 h after IPTG induction, E. coli cells were smeared onto glass slides and heat fixed, after which a drop of crystal-violet Gram staining solution was added. Cells were stained for ∼1 min, washed three times in distilled water, air-dried, then visualized under a light microscope (Zeiss, Jena, Germany) at 1000× magnification under an oil immersion objective. Over 100 cells were observed at each time point in each of three replicates.
Fatty acid analysis
WT and transformed strains were cultured in liquid LB for 2 h, after which the fusion proteins were induced with 1.0 mM IPTG for 6 h. Isochronous WT cultures and un-induced transformed strains were used as controls. After induction, thalli were collected and freeze-dried for FA analysis. The whole procedure was repeated for a total of two replicates.
Thalli were accurately quantified and soaked in 2.0 mL 2% sulfuric acid in dry methanol for 16 h at room temperature followed by 80 min at 90°C to convert the FAs into FA methyl esters (FAMEs). Supelco™ 37 Component FAME Mix (Sigma–Aldrich, St. Louis, MO, USA) was added as an internal standard. After addition of 2.0 mL of distilled water and 3.0 mL of hexane, the FAMEs were extracted for analysis with a 6890N gas chromatograph (Agilent, Santa Clara, CA, USA). Lipids were measured with a programmed temperature method. An initial column temperature of 140°C was maintained for 5 min, then increased to 240°C at a rate of 4°C/min and held for 10 min. Injection and detector temperatures were 240°C and 260°C, respectively. Two microliters of the sample were injected into the column. FAMEs were identified by comparison of their retention times with those of known standards. The results were analyzed using Chrom Perfect® LSi system software (Fife, Scotland) with the FAME Mix peak area used as the reference.
Fatty acid content was computed as the absolute content (mg/g) using the gas chromatograph area counts for the different FAMEs. The FAME quantity in a sample was used to calculate the oil content using the following equation:
Where Ms is the weight of the internal standard added to a sample, Ai is the area count of an individual FAME, As is the area count of the corresponding FAME in the internal standard, and M is the weight of the E. coli extract used.
Statistical analyses
Differences in cell volumes, growth rates, and FA content among the four strains when induced and uninduced were statistically analyzed by one-way analysis of variance (ANOVA) using SPSS 16.0 (IBM, Chicago, IL, USA).
Results
Comparison of AhDGAT2a and AhDGAT2b proteins
By employing a homologous gene sequence method with RACE, we identified two isozymes of AhDGAT2 from the peanut cultivar ‘Luhua 14’ and designated them AhDGAT2a and AhDGAT2b (GenBank accession numbers JF897614 and JF897615, respectively). We identified 14 nucleotide differences between them (Figure S1), but the predicted amino acid sequences exhibited only three differences in the N-terminal region (Figure 1). A phylogenetic analysis of the amino acid sequences of the AhDGAT2s and other known DGAT2s (Figure 2) demonstrated that the two AhDGAT2s were closely related to one another and to the DGAT2 from another Fabaceae species (Medicago truncatula), to which they exhibited high similarity (91.02%). They were also quite similar to EaDGAT2 from Euonymus alatus (Celastraceae; 88.12%).
10.1371/journal.pone.0061363.g001Figure 1 Comparison of the amino acid sequences of AhDGAT2a and AhDGAT2b.
The three amino acid differences are shaded in gray. Two black underlined regions (amino acids 40–62 and 67–82) highlight the two predicted transmembrane domains. The red underline shows the conserved LPLAT_MGAT-like domain (amino acids 104–321).
10.1371/journal.pone.0061363.g002Figure 2 Phylogenetic tree showing relationships among the DGAT2 protein sequences from various plant species.
The tree was generated using DNAMAN software. Branch lengths indicate evolutionary distance. The GenBank protein ID numbers for the DGAT2s are as follows: MtDGAT2, Medicago truncatula, ACJ84867.1; AtDGAT2, Arabidopsis thaliana, NP_566952.1; EaDGAT2, Euonymus alatus, ADF57328.1; HaDGAT2, Helianthus annuus, ABU50328.1; OeDGAT2, Olea europaea, ADG22608.1; RcDGAT2, Ricinus communis, XP_002528531.1; VfDGAT2, Vernicia fordii, ABC94473.1; ZmDGAT2, Zea mays, NP_001150174.1; OsDGAT2, Oryza sativa Japonica Group, NP_001057530.1. The two peanut DGAT2s are underlined.
The two AhDGAT2s had the same predicted internal structures. Using TMHMM, we identified two potential transmembrane helices, at amino acid positions 40–62 and 67–82 (Figure 1), suggesting that these proteins are located in the membrane system. This program also predicted the presence of a small N-terminal domain and a large C-terminal domain on the cytoplasmic side of the membrane. The Conserved Domain search inferred that both AhDGAT2s possessed an LPLAT_MGAT-like domain at their C-terminus (amino acids 104–321). This domain is a putative acyl-acceptor binding pocket and suggests that AhDGAT2 has acyltransferase activity. PROSCAN identified six putative functional motifs, including N-glycosylation, cAMP- and cGMP-dependent protein kinase phosphorylation, protein kinase C phosphorylation, casein kinase II phosphorylation, N-myristoylation sites, and an amidation site (Table 1). Two putative functional motifs, namely the N-glycosylation (NVTA versus NVTV) and the first N-myristoylation sites, in AhDGAT2a and AhDGAT2b differed because of a mutation at the ninth amino acid position. N-glycosylation is a form of co-translational and post-translational modification. Recently, N-glycosylation of a protein was found to affect its catalytic activity, thermostability, folding, subcellular localization, and secretion, as well as having an impact on pathogen interactions [21]-[24]. N-myristoylation plays a vital role in membrane targeting and signal transduction in plant responses to environmental stress [25], [26]; AhDGAT2a contained four N-myristoylation sites, whereas AhDGAT2b contained only three. Moreover, the three-dimensional structure of AhDGAT2b contained three beta strands (amino acid positions 22–23, 211–214, and 265–267) that were absent in AhDGAT2a (Figure 3). We speculated that these motif and structural differences could lead to differences in function. Hence, we constructed AhDGAT2a and AhDGAT2b plasmids to enable us to investigate their individual functions in E. coli.
10.1371/journal.pone.0061363.g003Figure 3 Predicted two and three-dimensional structures of AhDGAT2a and AhDGAT2b.
Structures were predicted using phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). (A, B) Predicted two-dimensional structures of AhDGAT2a and AhDGAT2b, respectively. (C, D) Predicted three-dimensional structures of AhDGAT2a and AhDGAT2b. The rainbow coloration indicates the progression from N (red) → C (indigo) terminus.
10.1371/journal.pone.0061363.t001Table 1 Putative functional motifs in peanut AhDGAT2s predicted by PROSCAN.
Functional site AhDGAT2a AhDGAT2b
Position Amino acid Position Amino acid
N-glycosylation 6–9 NVTA 6–9 NVTV
cAMP- and cGMP-dependent protein kinase phosphorylation 90–93 RKLS 90–93 RKLS
Protein kinase C phosphorylation 29–31 SSK 29–31 SSK
118–120 SNR 118–120 SNR
178–180 TKK 178–180 TKK
Casein kinase II phosphorylation 184–187 SLLD 184–187 SLLD
298–301 TTEE 298–301 TTEE
312–315 SLQD 312–315 SLQD
N-myristoylation 5–10 GNVTAA – –
173–178 GLTPAT 173–178 GLTPAT
198–203 GVQETF 198–203 GVQETF
208–213 GTETAY 208–213 GTETAY
Amidation 88–91 FGRK 88–91 FGRK
AhDGAT2a and AhDGAT2b fusion proteins are located in both soluble and insoluble fractions of E. coli cells
The AhDGAT2a and AhDGAT2b fragments were inserted into pGEX-4T-1 expression vector, transformed into E. coli, and the recombinant AhDGAT2–GST proteins, containing a GST-tag at the N-terminus, were induced with IPTG at 25°C or 37°C. Recombinant AhDGAT2 expression at 25°C was somewhat lower than that at 37°C (data not shown), so we selected 37°C as the optimal induction temperature. Cells induced at 37°C were collected after 0, 2, 4, and 6 h and the recombinant proteins extracted for SDS–PAGE analysis (Figure 4). Recombinant AhDGAT2 expression increased over time and was highest at 6 h, so this time point was used for subsequent experiments. No obvious differences were detected between the AhDGAT2a and AhDGAT2b expression levels.
10.1371/journal.pone.0061363.g004Figure 4 SDS–PAGE analysis of the time course for expression of AhDGAT2a and AhDGAT2b recombinant fusion proteins in E. coli cell extracts.
Recombinant proteins transformed into E. coli were induced with IPTG and their expression levels evaluated after 0, 2, 4, and 6 h. Molecular weight standards are shown on the left. The relative mobilities of GST (26.97 kDa), AhDGAT2a–GST (64.5 kDa), and AhDGAT2b–GST fusion proteins (64.5 kDa) are indicated on the right.
Following IPTG induction of the AhDGAT2 proteins, their production was scaled up to facilitate purification from the cytoplasmic fraction and inclusion bodies. Fusion proteins were purified and loaded onto SDS–PAGE for analysis (Figure 5). The fusion proteins were present in both the soluble (cytoplasm) and insoluble (inclusion bodies) fractions, with the largest quantities present in the inclusion bodies. The expression patterns of AhDGAT2a and AhDGAT2b did not differ noticeably.
10.1371/journal.pone.0061363.g005Figure 5 Expression of AhDGAT2–GST fusion proteins after induction with 1 mM IPTG at 37°C for 6 h.
M: Protein molecular weight marker. (A) Lanes 1, 3: AhDGAT2a–GST and AhDGAT2b–GST extracted from the cytoplasmic fraction; Lanes 2, 4: AhDGAT2a–GST and AhDGAT2b–GST extracted from inclusion bodies. (B) Western blot analysis of the AhDGAT2–GST fusion proteins using anti-GST tag monoclonal antibody. Lane 1: Wild-type E. coli Rosetta (DE3) strain; Lane 2, GST expression from the empty-vector transformed strain; Lanes 3, 5: AhDGAT2a–GST and AhDGAT2b–GST from the cytoplasmic fraction; Lanes 4, 6: AhDGAT2a–GST and AhDGAT2b–GST from inclusion bodies.
Overexpression of AhDGAT2s affects the growth rate and morphology of transformed E. coli
Expression of GST or AhDGAT2–GSTs depressed the growth rate of the E. coli Rosetta (DE3) strain relative to the WT strain (Figure 6). This result was not unexpected, because carrying a foreign vector or expressing a foreign gene is likely to retard bacterial growth rates. After IPTG was added to the medium, the reduction in growth rate became more obvious. After 5 h in culture, the growth rate of the WT E. coli reached a plateau, whereas the growth rates of the three transformed lines increased for up to 7 h and showed no apparent differences among one another.
10.1371/journal.pone.0061363.g006Figure 6 Growth curves of the wild type and transformed E. coli strains in liquid culture.
The optical density (OD600) of the bacterial cultures is shown on the y axis. Bacteria were incubated aerobically at 37°C with shaking at 170 rpm. Vertical bars represent the standard deviation of three replicates.
We also studied the morphology of the E. coli strains before and after IPTG induction. Overexpression of both AhDGAT2a–GST and AhDGAT2b–GST substantially affected the morphology of the transformed E. coli cell lines (Figure 7; Table 2). Before IPTG induction, the WT and transformed E. coli lines showed no obvious differences in size or shape (Figure 7A,C,E,G). Unlike the WT line, which was unaffected by IPTG (Figure 7A–B), the empty-vector transformed line formed long filamentous structures instead of normal short rods within 2 h of IPTG induction. The filament lengths increased over time, and after 6 h of IPTG induction, the cell volume had significantly increased by about 1.5 times that of the un-induced control (Figure 7D; Table 2, P<0.05). The same phenomenon was observed in E. coli cells harboring AhDGAT2a–GST or AhDGAT2b–GST. The AhDGAT2a–GST and AhDGAT2b–GST vector-transformed cells increased markedly in size after IPTG addition, by ∼2.4–2.5 times (P<0.01) (Table 2; Figure 7F,H). Differences between AhDGAT2a–GST and AhDGAT2b–GST vector-transformed cells were less obvious. Of note, the length but not the width of the transformed E. coli cells increased (Table 2).
10.1371/journal.pone.0061363.g007Figure 7 Morphology of wild type and transformed E. coli before and 6 h after the addition of IPTG into the growth medium.
Cells were sampled and Gram stained before induction (A, C, E, G) and 6 h after induction (B, D, F, H). (A, B) wild-type E. coli cells; (C, D) empty-vector transformed cells; (E, F) AhDGAT2a–GST vector transformed cells; (G, H) AhDGAT2b–GST vector transformed cells.
10.1371/journal.pone.0061363.t002Table 2 Cell sizes (mean±SE) of the recombinant Escherichia coli strains.
WT strain Empty vector AhDGAT2a–GST AhDGAT2b–GST
Uninduced cells width ( µm) 4.10±0.12 4.08±0.14 4.08±0.13 4.07±0.11
length ( µm) 27.198±4.90 25.913±3.42 27.841±4.95 27.626±3.99
volume ( µm3) 358.90±61.57 341.95±42.91 367.38±62.19 364.55±50.14
Induced cells (6 h) width ( µm) 4.11±0.13 4.12±0.14 4.08±0.13 4.11±0.12
length ( µm) 27.412±4.23 41.975±7.09 a 66.175±5.16 b 67.032±10.37 b
volume ( µm3) 361.73±53.15 553.90±89.08 a 873.23±64.77 b 884.54±130.2 b
a: Significantly different from WT at the 0.05 level.
b: Significantly different from WT at the 0.01 level.
AhDGAT2 overexpression significantly affected the FA content of E. coli cells
The FA content of the IPTG-induced WT and transformed strains and their corresponding controls were analyzed by gas chromatography. Twenty-two types of FA were detected in the WT and transgenic E. coli strains (Table S1), with five main types accounting for 82.10–88.68% of the total FA content. C16:0 was the most abundant FA, comprising 45.52–61.5% of the total FA content. The other common FAs were C18:3n3 (12.42–19.88% of the total FA abundance), C14:0 (8.83–10.14%), C18:2n6t (6.46–9.01%), and C12:0 (4.91–5.94%). The contents of the remaining 17 FAs were below (0–8.34%).
In most cases when cell strains were un-induced, the empty-vector transformed E. coli strain showed no significant differences in FA content compared with the WT (Figure 8). Remarkably, transformation with AhDGAT2a–GST and AhDGAT2b–GST significantly increased the total FA content of the strains by 15.2–19.0% compared with the WT (P<0.01; Figure 8A) and also significantly affected some individual FA levels. The quantities of C14:0, C16:0, C16:1, and C18:1n9c showed significant increases compared with either the WT or empty-vector transformed strains (Figure 8C,E,F,G; P<0.01 or 0.05). The quantities of C12:0, and C18:3n3 showed significant increases only under IPTG induction compared with either the WT or empty-vector transformed strains (Figure 8B, I; P<0.01 or 0.05). The levels of C15:0 decreased significantly relative to the WT in both uninduced (P<0.01) and induced (P<0.05) AhDGAT2a–GST and AhDAGT2b–GST strains (Figure 8D), while the levels of C21:0 decreased significantly in the uninduced strains (P<0.01) and increased significantly in the induced AhDGAT2a strain (P<0.05; Figure 8J). However, the C18:2n6t content remained unchanged, except for a decrease in the uninduced AhDGAT2a–GST strain (P<0.05; Figure 8H).
10.1371/journal.pone.0061363.g008Figure 8 Fatty-acid content of wild-type E. coli strains and strains transformed with empty vector, AhDGAT2a–GST, and AhDAGT2b–GST.
Open columns represent the values in un-induced strains and filled columns the values in IPTG-induced cultures after 6 h. Vertical bars represent the standard deviations of three replicates. a,c,e, significant at the 0.05 level; b,d,f, significant at the 0.01 level. a and b, compared with WT; c and d, compared with the empty-vector strain; e and f, compared with AhDGAT2a-GST vector strain. P values are given in Table S1.
The effect of IPTG induction on the FA content of the different E. coli lines was also examined (Figure 8). The quantities of the individual FAs differed dramatically between the induced cultures and the WT. The C12:0, C14:0, C18:3n3, and C21:0 FA contents increased significantly (Figure 8B,C,I,J). In the AhDGAT2a–GST transformants, the respective increases were 10.06%, 3.63%, 40.38%, and 311.54%, and in the AhDGAT2b–GST transformants, the increases were 6.74%, 6.37%, 52.77%, and 240.91%, respectively (Table S1). In contrast, the C16:0, C16:1, and C18:1n9c contents decreased substantially after IPTG induction (Figure 8E,F,G). In the AhDGAT2a–GST transformants, the decreases were 9.45%, 88.09%, and 16.67%, respectively, while the AhDGAT2b–GST transformants decreased by 5.34%, 89.08%, and 35.56%.
The transformants carrying AhDGAT2a and AhDGAT2b also differed in their contents of some FAs, indicating possible functional differences between the two genes. When uninduced, the levels of C12:0 (P<0.05), C18:2n6t (P<0.01), CLA-c9t11 (P<0.01) (Figure 8B,H,K), and C22:ln9 (P<0.05) (Figure 8L) were significantly higher in the AhDGAT2b strain than in the AhDGAT2a strain. When induced, the AhDGAT2b strain contained significantly more C18:3n3 and C15:0 (P<0.01) (Figure 8I,D) and significantly less C21:0 and C22:ln9 (P<0.05) (Figure 8J,L).
Discussion
Overexpression of AhDGAT2 increases the FA content of E. coli cells
Increasing energy costs and environmental concerns have compelled the production of sustainable renewable fuels and chemicals. In recent years, biofuels have received significant attention and investment [27]–[35]. Prokaryotic expression systems, particularly E. coli systems, remain an effective way of producing large quantities of a variety of fusion proteins. For example, Lu et al. introduced four distinct genetic changes into the E. coli genome to achieve a high level of FA production (2.5 g/L) [27]. Zhang et al. studied the effects of overexpression of acyl-ACP thioesterase genes from four different plant species using E. coli that lacked FA production, and found that the transformed E. coli strains synthesized approximately 0.2 g/L of free FAs [35]. Jeon et al. cloned and overexpressed five genes (acetyl-CoA carboxylase A, acetyl-CoA carboxylase B, acetyl-CoA carboxylase C, malonyl-CoA-[acyl-carrier-protein] transacylase, and acyl-ACP thioesterase) in E. coli MG1655 and found that the FA (C16) levels from the recombinant strains were 1.23–2.41 times higher than those from the wild type [34]. Furthermore, Liu et al. engineered an E. coli cell line that produced 4.5 g/L/day FAs [33]. In addition, when a soluble DGAT3 from peanut was introduced into E. coli, the transformants showed high levels of DGAT activity and formation of labeled TAG [5].
In this study, we overexpressed two types of AhDGAT2 in E. coli Rosetta (DE3) and showed that the FA content of the transformants was significantly higher than in the WT or empty-vector transformed strains (increases from 15.18–18.94%; Table S1). The 6 h induction time used in our experiment was shorter than in most previous reports (5–48 h) [27], [34], [35]. Some reports have stated that the FA content and composition of certain E. coli strains (e.g. ML103 (pXZCO16, pXZ18, and pXZJ18)) changes over time [35]. We do not know whether the FA content of the AhDGAT2-transformed E. coli strains would increase with longer induction times, but our study clearly demonstrated the potential of AhDGAT2 for efficient FA production in E. coli.
Overexpression of AhDGAT2 in E. coli changed its morphology
Bacteria have evolved sophisticated systems to maintain their morphologies. However, in certain environments, rod-shaped bacteria can become more filamentous [36]. Numerous bacteria alter their shapes in response to the types and concentrations of internal and external compounds. For example, the E. coli DH5α strain forms long filamentous cells upon caffeine exposure [37], while over-production of penicillin-binding protein 2 causes morphological changes and lysis in E. coli
[38]. Nutritional stress most frequently induces filamentation, which can increase the total surface area of a bacterium without increasing its width; hence its surface-to-volume ratio does not change [39].
In this study, the transformed E. coli strains changed their general morphology from short rods to filamentous structures (Figure 7), a change similar to bacteria encountering nutritional stress [39]. These changes occurred gradually over time (data not shown) and were not caused by IPTG addition alone, because IPTG induction over 6 h caused no such morphological changes in the WT strain (Figure 7A–B). Furthermore, when fresh medium with or without IPTG was added to the 6-h induced cultures, the cells neither increased nor decreased in length when IPTG was included in the fresh medium, but they gradually shortened over several hours when IPTG was absent from the fresh medium (data not shown), suggesting that nutritional stress did not cause the changes in morphology. Perhaps the rapid accumulation of over-expressed proteins or FAs altered the cell shape. To some extent, cell size was related to the size of exogenous proteins produced. For example, GST transformants that produced ∼27 kDa proteins had cell sizes about 1.5 times those of their uninduced counterparts (Table 2). In contrast, AhDGAT2a–GST and AhDGAT2b–GST transformants (expressing 64 kDa AhDGAT2–GST fusion proteins) increased their sizes by about 2.4–2.5 times that of their uninduced counterparts (Figure 7E–F, G–H). Apparently, the larger the size of the exogenous protein, the larger the transformed cell will become.
IPTG induction and FA content in E. coli
Zhang et al. examined the effect of IPTG concentration on free FA accumulation and found that total free FA accumulation responded in a dosage-dependent way up to 500 µM of IPTG [35]. Below 500 µM, the cultures accumulated similar quantities of free FAs [35]; above this value, the percentages of the C14 and C16:1 straight chain FAs increased markedly, whereas the percentages of C16 and C18 fell dramatically [35]. In our study, IPTG affected FA accumulation in E. coli. The cellular content of the individual FAs differed dramatically between the un-induced and induced cultures (Figure 8). The C12:0, C14:0, C18:3n3, and C21:0 cell contents increased significantly, whereas the C16:0, C16:1, and C18:1n9c contents decreased significantly. Furthermore, the transformants with AhDGAT2a and AhDGAT2b showed significant differences in the production of some FAs, suggesting that these two isozymes have slightly different functions in peanut plants. We did not examine whether different IPTG concentrations affected cellular FA levels; however, our finding that IPTG induction altered FA content was consistent with that of Zhang et al. [35].
Conclusions
In this study, we cloned and successfully expressed the peanut DGAT2 genes in E. coli. The integral membrane proteins accumulated in the cells at a high level after IPTG induction, and the levels of several FAs were significantly higher in transformed cells, offering the possibility that these high-energy molecules might someday be generated for energy. In addition, we established an efficient way to express an integral membrane protein in E. coli that future studies can follow. We also identified the function of the peanut DGAT2 enzyme in E. coli.
Furthermore, the identification of these genes will help spur the creation of transgenic peanut germplasms with high oil content or other special characteristics. The DGAT antibody will be important for identifying DGAT protein expression levels in transgenic plants. Finally, the differences in enzyme activity in vitro will assist in identifying important motif sites or single nucleotide polymorphisms that can be used in molecular marker-assisted breeding.
Supporting Information
Figure S1
Comparison of the nucleotide sequences of
AhDGAT2a
and
AhDGAT2b
. The 14 nucleotide differences are shaded in gray. The red underlines show the initiation and termination codons.
(TIF)
Click here for additional data file.
Table S1
Original data and data analysis.
(XLS)
Click here for additional data file.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23593363PONE-D-13-0432310.1371/journal.pone.0060975Research ArticleBiologyBiochemistryImmunologyMolecular Cell BiologySignal TransductionSignaling in Cellular ProcessesRas SignalingSignaling in Selected DisciplinesOncogenic SignalingCell DeathMedicineOncologyCancer TreatmentChemotherapy and Drug TreatmentZoledronic Acid Restores Doxorubicin Chemosensitivity and Immunogenic Cell Death in Multidrug-Resistant Human Cancer Cells Zoledronic Acid Reverses Chemo-ImmuneresistanceRiganti Chiara
1
2
*
Castella Barbara
2
Kopecka Joanna
1
Campia Ivana
1
Coscia Marta
2
3
Pescarmona Gianpiero
1
2
Bosia Amalia
1
2
Ghigo Dario
1
2
Massaia Massimo
2
3
1
Department of Oncology, University of Torino, Torino, Italy
2
Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, Italy
3
Division of Hematology, University of Torino, Torino, Italy
Unutmaz Derya Editor
New York University, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: CR AB MM. Performed the experiments: CR BC JK IC. Analyzed the data: CR MC GP MM. Contributed reagents/materials/analysis tools: MC. Wrote the paper: CR BC DG AB MM.
2013 12 4 2013 8 4 e6097524 1 2013 1 3 2013 © 2013 Riganti et al2013Riganti et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Durable tumor cell eradication by chemotherapy is challenged by the development of multidrug-resistance (MDR) and the failure to induce immunogenic cell death. The aim of this work was to investigate whether MDR and immunogenic cell death share a common biochemical pathway eventually amenable to therapeutic intervention. We found that mevalonate pathway activity, Ras and RhoA protein isoprenylation, Ras- and RhoA-downstream signalling pathway activities, Hypoxia Inducible Factor-1alpha activation were significantly higher in MDR+ compared with MDR− human cancer cells, leading to increased P-glycoprotein expression, and protection from doxorubicin-induced cytotoxicity and immunogenic cell death. Zoledronic acid, a potent aminobisphosphonate targeting the mevalonate pathway, interrupted Ras- and RhoA-dependent downstream signalling pathways, abrogated the Hypoxia Inducible Factor-1alpha-driven P-glycoprotein expression, and restored doxorubicin-induced cytotoxicity and immunogenic cell death in MDR+ cells. Immunogenic cell death recovery was documented by the ability of dendritic cells to phagocytise MDR+ cells treated with zoledronic acid plus doxorubicin, and to recruit anti-tumor cytotoxic CD8+ T lymphocytes. These data indicate that MDR+ cells have an hyper-active mevalonate pathway which is targetable with zoledronic acid to antagonize their ability to withstand chemotherapy-induced cytotoxicity and escape immunogenic cell death.
Italian Association for Cancer Research (AIRC, www.airc.it; MFAG 11475 to Chiara Riganti, IG 13119 to Massimo Massaia); Italian Ministry of University and Research (www.miur.it; PRIN 2010–2011 to Massimo Massaia, FIRB 2012 to Chiara Riganti); Fondazione Internazionale Ricerche Medicina Sperimentale (www.cerms.it, to Amalia Bosia, Massimo Massaia); Regione Piemonte (Ricerca Sanitaria Finalizzata 2009 to Chiara Riganti, Amalia Bosia; Progetto Immonc to Massimo Massaia). Joanna Kopecka is the recipient of a “Mario and Valeria Rindi” fellowship from Italian Foundation for Cancer Research (FIRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The mevalonate (Mev) pathway is a highly conserved metabolic cascade which produces sterols, such as cholesterol, and isoprenoids, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). The latter are necessary for the isoprenylation and activity of small G-proteins such as Ras and Rho which control cell proliferation, cytoskeleton remodelling and angiogenesis [1].
Over-expression of Mev pathway enzymes has been correlated with a poor clinical outcome in several human cancers [2], [3]. Intracellular cholesterol levels may modulate resistance to a variety of anticancer drugs, within a functional phenotype termed multidrug-resistance (MDR), which can be constitutive or induced after exposure to repeated courses of non-eradicating chemotherapy [4]. The plasma membranes of MDR+ tumor cells are particularly rich in cholesterol which facilitates the activity of P-glycoprotein (Pgp) [5], an integral membrane transporter extruding chemotherapy drugs such as anthracyclines, taxanes, Vinca alkaloids, epipodophyllotoxins, topotecan, and mitomycin C [4]. Another hallmark of MDR+ tumor cells is the increased isoprenylation and activity of G-proteins which are also dependent on the rate of the Mev pathway activity [6], [7].
Accumulating evidence indicates that successful and durable tumor cell eradication is dependent on the ability of chemotherapy drugs to kill tumor cells in a way which is detectable by the immune system. The term immunogenic cell death (ICD) has been coined to describe the ability of certain drugs, such as doxorubicin (Dox), to kill tumor cells and concurrently induce antitumor immune responses triggered by dying tumor cells [8]. Molecular key events in Dox-induced ICD are the extracellular release of the high-mobility group 1 box (HMGB1) protein and the cell surface translocation of calreticulin (CRT) from the endoplasmic reticulum, where it exerts calcium-sensor and chaperone functions. These events trigger tumor cell phagocytosis by dendritic cells (DCs) and the subsequent DC-mediated recruitment of other immune subpopulations with antitumor activity [9], [10]. Interestingly, MDR+ cells are often refractory to ICD [11] indicating that tumor cells can afford multiple strategies to survive and proliferate in the chemotherapy-treated host.
Zoledronic acid (ZA) is an aminobisphosphonate widely used in clinics to prevent bone resorption and treat bone disease in solid tumors, including breast, prostate, lung cancer and multiple myeloma. ZA is a specific inhibitor of FPP synthase in the Mev pathway and exerts pleiotropic effects in tumor and non-tumor cells, such as osteoclasts, macrophages, endothelial cells and immune cells [12], [13]. These effects are due to the intracellular deprivation of isoprenylated proteins and/or the accumulation of isopentenyl pyrophosphate which is exploited to activate Vγ9Vδ2 T cells, a unique subset of unconventional T cells with regulatory and effector functions against microbes, stressed cells and tumor cells [14]–[16].
Previous data have shown that ZA enhances the anti-proliferative effect of Dox in drug-sensitive tumor cells [17], [18] and clinical studies have been initiated in breast cancer patients to take advantage of this synergy [19]. However, it is currently unknown whether ZA has any impact on MDR and/or ICD in tumor cells. The aim of this study was two-fold: 1) to investigate the activity of the Mev pathway and Ras/RhoA-downstream signalling pathways in MDR− and MDR+ tumor cells; 2) to evaluate the relationship, if any, between ZA-induced Mev pathway inhibition, Dox-induced cytotoxicity and ICD susceptibility. We found that an hyper-active Mev pathway is responsible for both chemo- and immune-resistance; thanks to the inhibition of Mev-pathway dependent signals, ZA restored Dox-induced cytotoxicity and ICD in MDR+ cells.
Materials and Methods
Chemicals
Fetal bovine serum (FBS) and culture medium were from Invitrogen Life Technologies (Carlsbad, CA). Plastic ware for cell cultures was from Falcon (Becton Dickinson, Franklin Lakes, NJ). ZA was a gift from Novartis (Basel, Switzerland). The specific inhibitors of farnesyl transferase FTI-277, geranylgeranyl transferase GGTI-286 and of RhoA kinase Y27632 were purchased from Calbiochem (San Diego, CA). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Hercules, CA). The protein content of cell monolayers and lysates was assessed with the BCA kit from Sigma Chemical Co. (St. Louis, MO). Unless otherwise specified, all the other reagents were purchased from Sigma Chemical Co.
Cell lines
Human colon cancer HT29, lung cancer A549, and breast cancer MCF7 are Dox-sensitive, MDR− tumor cell lines (ATCC, Rockville, MD). Dox-resistant counterparts (HT29-dx, A549-dx and MCF7-dx) were generated by culturing parental cells in the presence of increasing concentrations of Dox for up to 20 passages [11], [20], [21]. For the present work, HT29-dx cells were grown in medium containing 250 nmol/L Dox, A549-dx cells in medium containing 100 nmol/L Dox, MCF7-dx cells in medium containing 0.5 nmol/L, and represented models of acquired MDR. The human hepatoma HepG2 cell line (ATCC) and the HP06 and HMM cancer cells were used as prototypic models of cells with a constitutive MDR phenotype and were previously described ([7], [11], [21]; in all these works HMM cells were named MM98 cells). Primary HP06 cells (gift of Prof. Anna Sapino, Department of Biomedical Sciences and Oncology, University of Torino, Italy) were derived from the peritoneal metastasis of a female patient with an invasive breast cancer, while primary HMM cells (Malignant Mesothelioma Biologic Bank, Azienda Ospedaliera Nazionale, Alessandria, Italy) were derived from the pleural effusion of a patient with histologically confirmed malignant mesothelioma, after written informed consent from the patients. The use of HP06 cells was approved by the Bioethics Committee (“Comitato of Bioetica d'Ateneo”) of the University of Torino, Italy; the use of HMM cells was approved by the Bioethics Committee (“Comitato Etico Interaziendale”) of the “Azienda Ospedaliera Nazionale SS. Antonio e Biagio e Cesare Arrigo” of Alessandria, Italy. Primary cells were used at passages 2–4. All the cultures were supplemented with 10% FBS, 1% penicillin-streptomycin and 1% L-glutamine, and maintained in a humidified atmosphere at 37°C and 5% CO2.
Intracellular Dox accumulation
Intracellular Dox contents were detected with a fluorimetric-based assay as reported [20] and expressed as nmol Dox/mg cell proteins according to a previously prepared calibration curve.
Real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted and reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). RT-PCR was carried out with IQ™ SYBR Green Supermix (Bio-Rad). The same cDNA preparation was used for the quantitation of mdr1 gene, which encodes for human Pgp, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chosen as a housekeeping gene. The sequences of mdr1 primers were: 5′-TGCTGGAGCGGTTCTACG-3′, 5′-ATAGGCAATGTTCTCAGCAATG-3′. The sequences of GAPDH primers were: 5′-GAAGGTGAAGGTCGGAGT-3′, 5′-CATGGTGGAATCATATTGGAA-3′. The relative quantitation of each sample was performed by comparing the mdr1 PCR product with the GAPDH PCR product with the Bio-Rad Software Gene Expression Quantitation.
De novo synthesis of cholesterol and isoprenoids
Cells were labeled with 1 µCi/mL 3H-acetate (3600 mCi/mmol; Amersham Bioscience, Piscataway, NJ) and the synthesis of the radiolabeled cholesterol, FPP and GGPP was measured as described [22]. Results were expressed as pmol/mg cell proteins, according to the relative calibration curves.
Ras and RhoA isoprenylation and activity
To detect the isoprenylated membrane-associated Ras or RhoA proteins and the non-isoprenylated cytosolic forms, cells were lysed in MLB buffer (125 mmol/L Tris-HCl, 750 mmol/L NaCl, 1% v/v NP40, 10% v/v glycerol, 50 mmol/L MgCl2, 5 mmol/L EDTA, 25 mmol/L NaF, 1 mmol/L NaVO4, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride; pH 7.5) and centrifuged at 13 000× g for 10 min at 4°C. An aliquot of supernatant was taken out for the Western blot of total Ras and RhoA, while the remaining part was centrifuged at 100 000× g for 1 h at 4°C; both the supernatant (cytosolic extracts) and the pellet (membrane fractions) were solubilized in Laemli buffer (125 mmol/L Tris, 4% w/v SDS, 20% v/v glycerol and 1% w/v β-mercaptoethanol) and subjected to Western blotting, using an anti-Ras (Millipore, Billerica, MA) and an anti-RhoA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibody. To evaluate Ras and RhoA activity, the GTP-bound fraction, taken as an index of active G-proteins [23] was measured, using a pull-down assay (with the Raf-1-GST fusion protein, agarose beads-conjugates, Millipore) and an ELISA assay (with the G-LISA™ RhoA Activation Assay Biochem Kit, Cytoskeleton Inc, Denver, CO), respectively, as described previously [22].
RhoA kinase activity
RhoA kinase activity was evaluated with the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano, Japan) following the manufacturer's instructions [22].
Western blot analysis
Cells were lysed in MLB buffer, sonicated and centrifuged at 13 000× g for 10 min at 4°C. 10 µg cell lysates were subjected to Western blotting and probed with the following antibodies: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2 (Millipore); anti-ERK 1/2 (Millipore); anti-Hypoxia Inducible Factor-1α (HIF-1α; BD Bioscience, San Jose, CA); anti-Pgp (Santa Cruz Biotechnology Inc.); anti-GAPDH (Santa Cruz Biotechnology Inc.), followed by the secondary peroxidase-conjugated antibodies (Bio-Rad). Proteins were detected by enhanced chemiluminescence (PerkinElmer, Waltham, MA).
To assess HIF-1α phosphorylation, the whole cell lysate was immunoprecipitated with a polyclonal anti-HIF-1α antibody (Santa Cruz Biotechnology Inc.), then probed for 1 h with a biotin-conjugated anti-phosphoserine antibody (Sigma Chemical Co.) followed by polymeric streptavidin-horseradish peroxidase-conjugates (Sigma Chemical Co.).
HIF-1α transcriptional activity
Nuclear proteins were extracted using the Nuclear Extract Kit (Active Motif, Rixensart, Belgium). The activity of HIF-1 on 10 µg nuclear extracts was assessed with the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). For each set of experiments, blank (with double-distilled water), negative control (with mutated oligonucleotide) and competition assay (using 20 pmol of the wild type oligonucleotide with nuclear extracts of HMM cells grown at 3% O2 for 24 h) were included. In hypoxic conditions, the HIF-1 activity was 257.53±3.77 mU/mg prot; in the competition assay, the corresponding HIF-1 activity was reduced to 33.14±1.39 mU/mg prot (n = 5). The data were expressed as mU absorbance/mg cell proteins.
Chromatin Immunoprecipitation (ChIP) experiments to measure the binding of HIF-1α on the “Hypoxia Responsive Element” of the mdr1 promoter were performed as reported elsewhere [24].
Cytotoxicity assay
Lactate dehydrogenase (LDH) activity was measured in the extracellular medium and in the cell lysate as described [20]. To measure the extracellular release of HMGB1, 20 µL of the cells culture medium were boiled, resolved by SDS-PAGE and probe with an anti-HMGB1 antibody (Sigma Chemical Co.). Blots were pre-stained with Red Ponceau to check the equal loading of proteins. The ATP release was measured on 100 µL of the cell culture medium with the ATP Bioluminescent Assay Kit (FL-AA, Sigma Aldrich Co.), using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek, Winooski, VT). The results were expressed as nmol ATP/ml, according to the previously set titration curve.
Analysis of cell surface CRT
Cells were incubated for 45 min (4°C) with an anti-CRT antibody (Affinity Bioreagents, Rockford, IL), as reported in [11] and analyzed using a FACS-Calibur system (BD Biosciences). For each analysis 100,000 events were collected. The percentage of CRT-fluorescent viable cells (propidium iodide-negative) was calculated with Cell Quest software (BD Biosciences). Control experiments included incubating cells with non-immune isotypic antibodies followed by the appropriate secondary antibody. Flow cytometry results were confirmed by biotinylation assays [25], using the Cell Surface Protein isolation kit from Thermo Fisher Scientific Inc. (Rockford, IL). The entry of biotin in cells was excluded by checking the absence of cytosolic proteins (GAPDH, actin) in biotinylated extracts (not shown).
Dendritic cell (DCs) generation and in vitro phagocytosis assay
DCs were generated from peripheral blood samples obtained from healthy donors kindly provided by the local Blood Bank (Fondazione Strumia, Torino, Italy), as previously reported [11]. Cells were harvested on day 6 and confirmed as immature DCs by morphology and immunophenotype.
MDR− HT29 and MDR+ HT29-dx and HMM cells were green-stained with PKH2-FITC (Sigma Chemical Co.), washed twice and incubated with at a ratio of 1∶1 for 18 h at 37°C. Co-cultures were then stained for 20 min at 4°C with APC-conjugated HLA-DR antibody (Miltenyi Biotec, Tetrow, Germany) to mark DCs. Two-color flow cytometry was performed with a FACScan cell sorter and CellQuest software (Becton Dickinson). At least 10,000 events were accumulated specifically backgating on DC morphology (region 1: FSC versus SSC). Tumor cell phagocytosis was assessed as the percentage of double-stained (FITC plus APC) cells. Tumor cells do not express significant amounts of HLA-DR, and they are excluded from region 1 by their morphology. In each set of experiments, a phagocytosis assay was performed by co-incubating DCs and tumor cells at 4°C, instead of 37°C, and the percentage of double-stained cells obtained after the incubation at 4°C was subtracted from values observed at 37°C. The phagocytosis rate was expressed as a phagocytic index, calculated as previously reported [9].
In fluorescence microscopy-based assays, PKH2-FITC green-stained tumor cells were incubated for 6 h or 24 h at 37°C with 1×105 DCs at a 1∶1 ratio. Co-cultures were cytospun at 1500× g for 5 min onto glass slips, fixed with 4% w/v paraformaldehyde, washed and stained with a mouse polyclonal anti-MHCII antibody (Thermo Fisher Scientific Inc.). After washing, samples were incubated with an Alexa fluor 350-conjuganted goat anti-mouse IgG antibody (Invitrogen) for 1 h at room temperature, washed, mounted with Gel Mount Aqueous Mounting and examined under a fluorescence microscope, as described above.
DC-mediated CD8+ T-cell stimulation
After tumor cell phagocytosis, DCs were washed and co-cultured with autologous T cells, isolated from CD14-cells by immunomagnetic sorting with the Pan T Cell Isolation Kit (Miltenyi Biotec). DC and T cells were co-cultured for 10 days at a ratio of 1∶5 in complete medium supplemented with IL-2 (10 U/mL). On day 10, CD107 expression on CD8+ T cells was determined by flow cytometry to determine the activation of tumor-specific cytotoxic T cells [26]. At least 100,000 events in the lymphocyte gate were acquired and analyzed by two-color flow cytometry. Tumor cell death induced by CD8+ T cells was also quantified by CFSE-labeling, measuring the percentage of HT29-dx cells double-positive for CFSE and propidium iodide as previously reported [14], [15].
Statistical analysis
All data in the text and figures are provided as means ± SD. The results were analyzed by a one-way analysis of variance (ANOVA) with P<0.05 as the significance cut-off. The r coefficient was calculated with Fig.P software (Fig.P Software Inc., Hamilton, Canada).
Results
Correlation between mdr1 expression and Mev pathway activity
Intracellular Dox retention, mdr1 mRNA levels, and cholesterol synthesis were measured as markers of Pgp activity, Pgp expression, and Mev pathway activity in HT29, A549, MCF7 cells (MDR− cells), HT29-dx, A549-dx, MCF7-dx cells (acquired MDR+ cells), and HepG2 cells, HP06, HMM cells (constitutive MDR+ cells), respectively. Both acquired and constitutive MDR+ cells retained significantly less Dox (Figure 1A) and showed higher mdr1 mRNA levels (Figure 1B) than MDR− cells. Cholesterol synthesis was also significantly higher in MDR+ than in MDR− cells (Figure 1C). The differences between acquired and constitutive MDR+ cells were not statistically significant, even though the latter tended to retain less Dox, to express more mdr1 mRNA levels, and to generate more cholesterol than the former. A very significant correlation was observed between the rate of cholesterol synthesis and mdr1 mRNA levels (Figure 1D).
10.1371/journal.pone.0060975.g001Figure 1 Correlation between intracellular doxorubicin retention, mdr1 expression and Mev pathway activity in MDR− and MDR+ tumor cells.
A. Intracellular doxorubicin (Dox) concentrations in MDR− cells (HT29, A549 and MCF7), in the corresponding acquired MDR+ counterparts (HT29-dx cells, A549-dx cells, MCF7-dx), and constitutive MDR+ cells (HepG2, HP06, HMM). Significantly lower concentrations were detected in cells with acquired MDR vs MDR− cells (HT29-dx vs HT29: *p<0.002; A549-dx vs A549: *p<0.001; MCF7-dx vs MCF7: *p<0.001), and in cells with constitutive MDR vs MDR− cells (mean value of intracellular doxorubicin in HepG2/HP06/HMM vs mean value in HT29/A549/MCF7: °p<0.001). B. mdr1 mRNA expression. Significant higher mdr1 levels were observed in cells with acquired MDR vs MDR− cells (HT29-dx vs HT29: *p<0.002; A549-dx vs A549: *p<0.002; MCF7-dx vs MCF7: *p<0.001), and in cells with constitutive MDR vs MDR− cells (mean value of mdr1 levels in HepG2/HP06/HMM vs mean value in HT29/A549/MCF7: °p<0.001). C. Rate of cholesterol synthesis. Significant higher activity was measured in cells with acquired MDR vs MDR− cells (HT29-dx vs HT29: *p<0.002; A549-dx vs A549: *p<0.002; MCF7-dx vs MCF7: *p<0.002), and in cells with constitutive MDR vs MDR− cells (mean value of cholesterol synthesis in HepG2/HP06/HMM vs mean value in HepG2/HP06/HMM: °p<0.001). D. Direct correlation between the rate of cholesterol synthesis and the expression levels of mdr1 in individual cell lines (r2 = 0.95). For panels A, B, and C bars represent the mean ± SD of 3 independent experiments.
ZA inhibits Mev-dependent signalling pathways in MDR+ cells
ZA was used to investigate the effect of Mev pathway inhibition in HT29, HT29-dx and HMM cells which were selected as prototypic models of MDR−, acquired MDR+ and constitutive MDR+ tumor cells respectively.
ZA induced a dose- (Figure 2A, left panel) and time-dependent (Figure 2A, right panel) decrease of cholesterol synthesis with a significant inhibition at 1 µmol/L which was more evident in MDR+ HT29-dx and HMM cells than MDR− HT29 cells (Figure 2A). One µmol/L is also similar to the serum concentration observed in patients receiving ZA at clinically approved doses [27], [28] and therefore this concentration was used throughout the study.
10.1371/journal.pone.0060975.g002Figure 2 Effects of ZA on cholesterol and isoprenoid synthesis, Ras/RhoA isoprenylation, and ERK1/2 and RhoA kinase activity in MDR− and MDR+ cancer cells.
MDR− HT29, and MDR+ HT29-dx and HMM cells were cultured without (CTRL) or with zoledronic acid (ZA). For panels B–E, ZA (1 µmol/L) was used for 48 h, FTI-277 (10 µmol/L, FTI), GGTI-286 (10 µmol/L, GGTI), Y27632 (10 µmol/L, Y276) for 24 h. A. Left panel: dose-dependent inhibition of cholesterol synthesis in cells treated with 0.01–10 µmol/L ZA for 24 h. Inhibition was statistically significant in HT29 (*p<0.001), HT29-dx (°p<0.01) d HMM cells (◊p<0.005) vs baseline values (0). Right panel: time-dependent inhibition of cholesterol synthesis in cells treated with 1 µmol/L ZA for 24–72 h. Inhibition was statistically significant in HT29 (*p<0.001), HT29-dx (°p<0.0001) and HMM cells (◊p<0.001) vs baseline values (0). For both panels: HT29-dx/HMM vs HT29: *p<0.001. B. MDR+ cells synthesized higher amounts of FPP (left panel) and GGPP (right panel) than MDR− cells (*p<0.005). ZA significantly lowered FPP synthesis vs untreated (CTRL) cells (HT29: *p<0.001; HT29-dx: °p<0.002; HMM: ◊p<0.001) and GGPP synthesis vs untreated (CTRL) cells (HT29:*p<0.02; HT29-dx: °p<0.001; HMM:◊p<0.005). C. MDR+ cells displayed an unbalanced distribution between isoprenylated membrane-bound (M) and non isoprenylated cytosolic (C) Ras (left panel) and RhoA (right panel) compared with MDR− cells. ZA treatment increased the amount of cytosolic Ras and RhoA. T: amount of Ras and RhoA in whole cell lysates. D. ZA decreased Ras activity, measured as Ras-GTP amount, and phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2 amount. GAPDH data are shown to confirm equivalent protein loading. E. MDR+ cells had significantly higher amounts of RhoA-GTP (open bars) and RhoA kinase (hatched bars) than MDR− cells (*p<0.005); ZA decreased both RhoA-GTP and RhoA kinase vs untreated (CTRL) cells (HT29-dx: °p<0.02; HMM:◊p<0.02). The results shown in panels C and D are representative of 3 experiments. In panels A, B, and E the results represent the mean ± SD of 3 experiments.
According to the hyper-active Mev pathway, MDR+ HT29-dx and HMM cells showed higher FPP (Figure 2B, left panel) and GGPP (Figure 2B, right panel) synthesis than MDR− HT29 cells. Both MDR− and MDR+ cells had detectable amounts of isoprenylated, membrane-bound Ras and non-isoprenylated, cytosolic Ras, although the former was largely predominant in MDR+ cells as a consequence of the increased FPP supply favoring Ras isoprenylation (Figure 2C, left panel). Membrane-bound and cytosolic RhoA also showed a similar pattern in MDR+ HT29-dx and HMM vs MDR− HT29 cells as a consequence of the increased GGPP production and RhoA isoprenylation (Figure 2C, right panel).
The excess of membrane-bound Ras and RhoA resulted in increased activity of the corresponding downstream signalling pathways: intracellular levels of GTP-bound Ras (Figure 2D), a marker of Ras activation, and ERK1/2 phosphorylation (Figure 2D), as well as the amounts of GTP-bound RhoA (Figure 2E) and the activity of RhoA kinase (Figure 2E) were higher in MDR+ HT29-dx and HMM cells than MDR− HT29 cells.
ZA treatment abrogated the differences between MDR− and MDR+ cells. By inhibiting FPP and GGPP synthesis (Figure 2B), ZA decreased the amounts of membrane-bound Ras and RhoA (Figure 2C), intracellular Ras-GTP and RhoA-GTP contents, and the activity of their downstream signalling pathways (Figure 2D–E). These effects were more evident in MDR+ HT29-dx and HMM cells than MDR− HT29 cells according to their Mev pathway activity.
ZA decreases Pgp expression by reducing HIF-1α activation via Ras/ERK1/2- and RhoA/Rho-A kinase downregulation
HIF-1α, a master regulator of several genes including mdr1
[29], can become constitutively activated even under normoxic conditions upon serin phosphorylation by RhoA kinase [30] and MAP kinases [31]. Phosphorylated (pHIF-1α) and non-phosphorylated HIF-1α were undetectable by Western blot in MDR− HT29 cells, whereas both pHIF-1α and HIF-1α were expressed in MDR+ HT29-dx and HMM cells (Figure 3A). HIF-1α was transcriptionally active in MDR+ cells, as shown by the significantly higher amounts of nuclear HIF-1 bound to its specific DNA target sequence (Figure 3B). ZA had no effect on MDR− cells, whereas it reduced the amount of pHIF-1α and lowered total HIF-1α levels and activity in MDR+ cells (Figure 3A–B).
10.1371/journal.pone.0060975.g003Figure 3 ZA-induced inhibition of HIF-1α activity and Pgp expression in MDR+ cancer cells.
A. Detection of phosphorylated (pHIF-1α) and total HIF-1α in MDR− HT29, and MDR+ HT29-dx and HMM cells after 48-hour incubation without (CTRL) or with 1 µmol/L ZA (ZA). B. HIF-1 activity was higher (*p<0.001) in MDR+ HT29-dx and HMM cells than HT29 cells. After ZA treatment (as reported in A), a significant decrease of HIF-1 activity was observed in HT29-dx (°p<0.001) and HMM cells (◊p<0.001). C. Chromatin immunoprecipitation of HIF-1α on mdr1 promoter in MDR− and MDR+ cells, treated as reported in a. pro mdr1: PCR product from immunoprecipitated samples. Input: PCR product from non immunoprecipitated samples (genomic DNA). no Ab: samples incubated in the absence of anti-HIF-1α antibody. “-”: blank. D. Western blotting detection of Pgp in cells treated as described in A. E. Intracellular doxorubicin was measured spectrofluorimetrically: significantly lower concentrations were detected in HT29-dx and HMM vs HT29 cells (*p<0.002), significantly higher concentrations in ZA-treated cells vs untreated (CTRL) counterparts (HT29-dx: °p<0.02; HMM: ◊p<0.02). The results shown in panels A, C and D are representative of 3 experiments. For panels B and E the bars represent the mean ± SD of 3 independent experiments.
HIF-1α was constitutively bound to the Hypoxia Response Element of the mdr1 promoter in MDR+ cells HT29-dx and HMM cells, but not in MDR− HT29 cells (Figure 3C). This explains why the expression of the Pgp protein was only detectable in MDR+ cells (Figure 3D) and why these cells showed significantly lower Dox retention than MDR− cells (Figure 3E).
ZA treatment effectively abrogated HIF-1α-binding to the mdr1 promoter (Figure 3C) and Pgp expression (Figure 3D), and significantly increased intracellular Dox levels in MDR+ HT29-dx and HMM cells which became comparable to those observed in MDR− HT29 cells (Figure 3E). Intracellular Dox retention in MDR+ cells was significantly increased when ZA was administered before Dox, but neither vice versa, nor when the two drugs were used together (Figure S1).
To provide further evidence that the ZA-induced Pgp down-regulation in MDR+ cells was dependent on pHIF-1α suppression via ERK1/2 and RhoA kinase inhibition, side-by-side experiments were performed in the presence of specific inhibitors of ERK1/2 kinases (PD98059), RhoA kinase (Y27632) and HIF-1 (YC-1). In MDR+ HMM cells these inhibitors showed the same effects of ZA in terms of pHIF-1α levels (Figure 4A), HIF-1 transcriptional activity (Figure 4B), Pgp expression (Figure 4C), and Dox retention (Figure 4D).
10.1371/journal.pone.0060975.g004Figure 4 Effects of ZA and inhibitors of ERK1/2, RhoA kinase, HIF-1α on MDR+ cells.
A. Phospho(Ser)-HIF-1α (pHIF-1α) and total HIF-1α expression in HMM cells left untreated (CTRL) or treated for 24 h at 10 µmol/L with the ERK1/2 kinase inhibitor PD98059 (PD), RhoA kinase inhibitor Y27632 (Y27), HIF-1α inhibitor YC-1 (YC), and 1 µmol/L ZA for 48 h. GAPDH data are shown to confirm equivalent per lane protein loading. B. HIF-1 activity in HMM cells left untreated (CTRL) or treated as reported in panel A. All differences between treated vs untreated cells are statistically significant (* p<0.01). C. Pgp expression in HMM cells of untreated (CTRL) and treated as reported in panel A. D. Intracellular doxorubicin concentrations in HMM cells in cells incubated as reported above in medium alone (CTRL), followed by 1 µmol/L Dox for a further 24 h. Differences between treated vs untreated cells are statistically significant (*p<0.01). Results shown in panels A and C are representative data from one of 2 experiments. For panels B and D, the results represent the mean ± SD of 3 independent experiments.
Altogether, these data confirm that targeting the Mev pathway in MDR+ cells abrogates Pgp expression and promotes Dox retention by inhibiting the Ras/ERK1/2/HIF-1α/mdr1 and the RhoA/RhoA kinase/HIF-1α/mdr1 pathways.
ZA restores Dox-induced cytotoxicity and ICD in MDR+ cells
We then investigated whether the increased ZA-induced Dox retention was sufficient to induce cytotoxicity and ICD in MDR+ cells.
The release of intracellular LDH was used to assess Dox-induced cytotoxicity (Figure 5A). Dox alone sufficed to induce a significant cytotoxicity in MDR− HT29 cells, while ZA alone had no activity and did not increase Dox cytotoxicity when used in combination. On the contrary, MDR+ HT29-dx and HMM cells were resistant to Dox alone, but they became more vulnerable when Dox was used after ZA treatment (Figure 5A).
10.1371/journal.pone.0060975.g005Figure 5 ZA restores doxorubicin-induced cytotoxicity and ICD in MDR+ tumor cells.
MDR− HT29 and MDR+ HT29-dx, and HMM cells were incubated for 48 h without (CTRL) or with 1 µmol/L ZA, for 24 h with 1 µmol/L Dox, for 48 h with 1 µmol/L ZA, followed by 1 µmol/L Dox for additional 24 h (ZA+Dox). A. LDH release. Dox alone and ZA+Dox induced a significant cytotoxicity in HT29 cells (*p<0.005). ZA+Dox induced a significant increase of cytotoxicity in HT29-dx (°p<0.05) and HMM cells (◊p<0.02). B. Western blot analysis of extracellular HMGB1. Dox alone and ZA+Dox in HT29 cells, ZA+Dox in HT29-dx and HMM cells induced the release of HMGB1 in the cell culture medium. Red Ponceau staining was used to check the equal loading of proteins. C. Extracellular release of ATP. Dox and ZA+Dox induced a significant increase of extracellular ATP in HT29 cells (*p<0.01). ZA+Dox elicited a significant release of ATP in HT29-dx (°p<0.002) and HMM cells (◊p<0.001). D. Cell surface CRT exposure. Dox and ZA+Dox induced a significant CRT exposure in HT29 cells (*p<0.001). ZA+Dox induced a significant CRT exposure in HT29-dx (°p<0.001) and HMM cells (◊p<0.005). For panels A, C and D bars represent the mean ± SD of 3 independent experiments. For panel B the results are representative data from one of 2 experiments.
Hallmarks of ICD are the extracellular release of HMGB1 and ATP, and the translocation of CRT from the cytoplasm to the cell surface [10], [32]. Neither MDR− nor MDR+ cells showed detectable amounts of HMGB1 in the supernatants under baseline conditions or after ZA treatment (Figure 5B). Likewise, the release of ATP (Figure 5C) and the amount of cell surface CRT (Figure 5D) were low and not significantly different under baseline conditions or after ZA treatment in both MDR− and MDR+ cells.
Dox alone was sufficient to induce the release of HMGB1 and ATP, and CRT translocation in MDR− HT29 cells and these effects were not further enhanced by ZA treatment (Figure 5B–D). On the contrary, while MDR+ HT29-dx and HMM cells were refractory to Dox alone, they released HMGB1 (Figure 5B) and ATP (Figure 5C), and showed CRT translocation (Figure 5D) after ZA+Dox treatment. CRT translocation was confirmed with a biotinylation assay in both MDR− and MDR+ cells (Figure S2).
These results indicate that the increased Dox retention induced by ZA treatment is sufficient to induce tumor cell cytotoxicity and to promote ICD of MDR+ cells.
ZA enhances the phagocytosis of MDR+ tumor cells by DCs and induces the generation of tumor-specific CD8+ T cells
Next, we investigated whether ICD triggered by ZA+Dox could be sensed by the immune system via DCs [10], [32]. As expected, both untreated or ZA-treated tumor cells were poorly phagocytosed by DCs, with MDR+ cells being more resistant than MDR− cells (Figure 6A). Dox alone was sufficient to induce the phagocytosis of MDR− HT29 cells which was further enhanced by ZA treatment. By contrast, Dox alone was unable to induce MDR+ HT29-dx and HMM cell phagocytosis. However, when Dox was used in combination with ZA, MDR+ cells became recognizable by DCs and significant phagocytosis was detected in MDR+ cells (Fig. 6
Figure 6A). Multiple cell-to-cell contact sites were established after 6 hours incubation of DCs with ZA+Dox-treated MDR+ HT29-dx cells, and clear evidence of internalization was documented after 24 hours (Figure 6B).
10.1371/journal.pone.0060975.g006Figure 6 ZA increases the internalization of MDR+ cells by autologous DCs and the subsequent activation of cytotoxic CD8+ T cells.
A. DC-mediated internalization of HT29, HT29-dx, and HMM cells after incubation with ZA and/or Dox. Tumor cells were incubated for 48 h without (CTRL) or with 1 µmol/L ZA, for 24 h with 1 µmol/L Dox, for 48 h with 1 µmol/L ZA, followed by 1 µmol/L Dox for additional 24 h (ZA+Dox). Dox alone and ZA+Dox significantly increased internalization of HT29 cells (*p<0.02). ZA+Dox increased internalization of HT29-dx (°p<0.005) and HMM (◊p<0.005) cells. Results represent the mean ± SD of 3 independent experiments. B. Fluorescence microscopy analysis of HT29-dx internalization after 6 and 24 h incubation with DCs. HT29-dx cells were incubated with ZA+Dox as reported in A. Micrographs are from one representative of 3 experiments. C. Cytotoxic activation of CD8+ T cells after 10 days incubation of purified T cells with autologous DCs pulsed with HT29-dx tumor cells, treated as reported in a. Cytofluorometric analysis of cell surface CD107 expression was used as a marker of specific TCR-induced CD8+ T-cell degranulation. Results are from one representative of 4 experiments. D. Pooled data of CD107 expression on CD8+ T cells after incubation with autologous DC as reported above. Bars represent the mean ± SEM of 4 experiments.
Next, we investigated whether the phagocytosis of ZA+Dox-treated MDR+HT29-dx cells increased the immunostimulatory capacity of DCs. The highest CD86 expression, a marker of DC maturation, was observed in DCs exposed to ZA+Dox-treated MDR+ HT29-dx cells (Figure S3) and this resulted in a better capacity to activate autologous cytotoxic CD8+ T cells. Cytofluorometric analysis of cell surface CD107 expression on CD8+ cells was used as a surrogate marker of antigen-specific cytotoxic T-cell activation (Figure 6C–D). An increase of CD8+ CD107+ cells was observed after a 10-day incubation of T cells with autologous DCs loaded with MDR+ HT29-dx cells treated with ZA+Dox. The flow cytometry of a representative experiment is shown in Figure 6C, while pooled data from 4 experiments are given in Figure 6D. T cells incubated with DC exposed to ZA+Dox-treated MDR+ HT29-dx cells also showed increased cytotoxic activity against untreated MDR+ HT29-dx cells (Figure S4).
Discussion
Constitutive and/or acquired MDR is a major obstacle to successful anti-tumor chemotherapy and the development of safe and effective MDR-reversing agents remains an unmet clinical need. In this study, we investigated whether the Mev pathway is involved in MDR and is eventually targetable for therapeutic intervention.
We initially analyzed the Mev pathway activity in a panel of MDR−, acquired MDR+ and constitutive MDR+ cancer cells. The latter uniformly showed significantly higher Mev pathway activity than MDR− cells, probably reflecting an increased transcription of 3-hydroxy-3-methylglutaryl-CoA reductase, the pace-maker enzyme in the Mev pathway. Indeed, 3-hydroxy-3-methylglutaryl-CoA reductase is under the transcriptional control of the sterol regulated element binding protein-2, which is regulated by intracellular sterol levels, and by transcription factors such as HIF-1α [33], which can be constitutively activated in MDR+ tumor cells [34] (see also below).
Increased intracellular cholesterol levels can contribute to MDR by facilitating the localization and functional activation of Pgp in the plasma membrane [5], [21], [35]. Although the existence of a correlation between cholesterol levels and Pgp expression has previously been postulated [36], [37], there is yet to be a molecular or metabolic linkage. Here we show the existence of a correlation between the rate of cholesterol synthesis and mdr1 levels, indicating that the Mev pathway activity can directly regulate Pgp expression.
To further document this relationship, we targeted the Mev pathway with ZA, a selective FPPS inhibitor. ZA induced a significant decrease of cholesterol, FPP and GGPP synthesis which was more pronounced in MDR+ than in MDR− cells, according to the higher baseline Mev pathway activity in the former. Notably, ZA produced these effects at 1 µmol/L which is a clinically compatible concentration [13], [17], [28]. After intravenous administration, ZA avidly impregnates the bone mineralized component, where it reaches long-lasting millimolar concentrations, but levels around 1 µmol/L are also detectable for several hours in the peripheral blood [17], [38].
Intermediate metabolites of the Mev pathway are the isoprenoids FPP and GGPP, which are necessary for the isoprenylation and activity of the small GTPases Ras and RhoA. We enquired whether Ras and/or Rho-dependent signals were involved in the linkage between the Mev pathway, mdr1 expression and Pgp activity. Isoprenylated active Ras and RhoA GTPases were detectable in both MDR− and MDR+ cells, but much more abundantly expressed in the latter. By limiting isoprenoid supply, ZA decreased Ras and RhoA isoprenylation, increased the ratio between inactive cytosolic GTPase and active membrane-associated GTPase, reduced the GTP-binding capacity and impaired Ras and RhoA interactions with their downstream effectors ERKs and RhoA kinases. MDR+ cells were apparently more susceptible to ZA effects, most likely because they had a higher baseline rate of Mev pathway activity and are therefore more susceptible to the shortage of isoprenoid supply generated by ZA-induced Mev pathway inhibition. Under this perspective, a high Mev pathway activity should be considered not only as a marker of tumor aggressiveness and poor prognosis [2], [3], but also as a potential therapeutic target implying a “collateral sensitivity” of MDR tumors [39].
A striking difference between MDR− and MDR+ cells was the amount of intracellular HIF-1α, one of the downstream targets of ERK1/2 and RhoA kinase [30], [31]. The α subunit of HIF undergoes proteasomal degradation under normoxic conditions unless stabilized by serine phosphorylation. Under basal conditions, HIF-1α and Pgp, which is upregulated by HIF-1α [29], were undetectable in MDR− cells, whereas they were highly expressed in MDR+ cells with a higher Mev pathway activity. ZA significantly decreased both pHIF-1α and non-phosphorylated HIF-1α expression, abrogated HIF-1α-driven Pgp expression, and increased intracellular Dox retention in MDR+ cells to similar levels of MDR− cells.
Altogether, these data indicate that the Mev-pathway dependent Ras/ERK1-2 and RhoA/RhoA kinase axes promote the phosphorylation and nuclear translocation of HIF-1α in MDR+ cells where it induces the transcriptional activation of the mdr1 gene leading to Pgp expression and Dox extrusion. This hypothesis was further validated by the demonstration in HMM MDR+ cells that both ZA and the ERK1/2, RhoA kinase and HIF-1α inhibitors decreased Pgp expression and increased intracellular Dox concentrations. To the best of our knowledge, this is the first report demonstrating that the Mev pathway can regulate HIF-1α transcriptional activity. These data also provide a biochemical explanation to the recent observation that ERK1/2 increases the expression of membrane efflux pumps and Dox resistance in malignant mesothelioma [40].
MDR reversion occurs when Dox reaches a critical intracellular concentration sufficient to elicit direct cytotoxicity. Indeed, ZA significantly increased Dox-induced cytotoxicity in MDR+ HT29-dx and HMM cells indicating that the intracellular drug concentration had exceeded the critical threshold. Previous studies have reported a synergistic activity of ZA and Dox in breast cancer cell lines [18], [41]. This synergy was observed only when tumor cells were treated with Dox for 24 hours before ZA exposure. In our experiments, the highest intracellular accumulation of Dox was achieved in MDR+ cells if ZA treatment preceded Dox exposure. Even though this discrepancy might well be due to the different cell type and experimental conditions, it is reasonable from the biochemical standpoint that the synergy between ZA and Dox is enhanced if exposure to the latter is preceded by Mev pathway inhibition to trigger the cascade of events ultimately leading to Pgp down-regulation. These data suggest that targeting the Mev pathway may be exploited as a priming strategy to restore chemosensitivity in MDR+ cells.
In addition to killing cancer cells directly, some classes of chemotherapeutic drugs are endowed with unexpected immune activating properties [10]. These drugs trigger “danger signals” and “danger associated molecular patterns” from dying tumor cells which stimulate innate and adaptive immune responses [42]. Dox is a prototypic drug that induces ICD, whose hallmarks are the release of HMGB1 protein and ATP in the supernatant, and the translocation of CRT from the endoplasmic reticulum to the plasma membrane [32]. HMGB1 and CRT are sensed by dendritic cells (DCs) as an “eat me” signal, promoting tumor cell phagocytosis and cytotoxic T-cell activation [9], [10], [32].
Interestingly, it has been proposed that tumor cells that are resistant to direct chemotherapy-induced cytotoxicity are also have a tendency to escape ICD. A possible mechanism is the inadequate intracellular Dox retention in MDR+ cells which does not exceed the critical threshold to elicit CRT translocation [11]. Moreover, we, and others, have suggested that Pgp may have an immunosuppressive role by interfering with CRT functional activity [25], [43].
This work confirms the close relationship between MDR and ICD. As expected, direct cytotoxicity and ICD occurred jointly in Dox-treated MDR− HT29 cells that showed the release of extracellular LDH, an index of direct cell damage and necrosis, concurrently with the release of extracellular HMGB1 and ATP, and CRT translocation. These events culminated in the phagocytosis of Dox-treated MDR− HT29 cells by DCs and the induction of antigen-specific cytotoxic CD8+ cells. This immunogenic sequence was only minimally increased by concurrent ZA treatment. By contrast, the insufficient Dox retention in MDR+ HT29-dx and HMM cells did not induce any significant extracellular LDH and ATP release or any CRT translocation. This allowed MDR+ cells to escape DC recognition and phagocytosis and antigen-specific CD8+ T cells were not generated by DC exposed to Dox-treated MDR+ HT29-dx cells. Of note, susceptibility to ICD was restored when MDR+ cells were treated with ZA and Dox, probably because intracellular Dox retention was increased as a consequence of Pgp down-modulation. Under these conditions, MDR+ cells were sensed and effectively phagocytised by DC which increased the expression of activation markers such as CD86 and induced the generation of cytotoxic CD8+ T lymphocytes. Although we cannot exclude that ZA enhances the cytotoxicity of DOX in MDR+ cells with additional mechanisms, it is noteworthy that it is able to reinstate an ICD in previously refractory cells. Compared to other MDR-reversing agents [44], [45], ZA showed the unique property to combine the ability to improve chemotherapy-induced cytotoxicity with the capacity to promote chemotherapy-induced immunogenic cell death. This “multi-target” activity reinstates a complete chemosensitivity in MDR+ cells.
Conclusions
In conclusion, we propose that the hyper-activity of the Mev pathway is a metabolic signature of MDR+ cells and a common denominator linking the resistance to chemotherapy, due to the increased drug efflux and to the resistance to the pro-immunogenic effects of chemotherapy (Figure 7A). However, this hyper-activity might also be regarded as a potential “Achille's” heel of MDR+ cells because targeting the Mev pathway with appropriate inhibitors, such as ZA, can interrupt the pathways that sustain both chemo-and immune-resistance (Figure 7B). Overall, these results pave the way to the development of novel chemo-immunotherapy approaches based on the combination of selected chemotherapy drugs with Mev pathway targeting-agents.
10.1371/journal.pone.0060975.g007Figure 7 Schematic drawing of mechanisms operated by ZA to reverse chemoresistance and immune-resistance.
A. The accelerated Mev pathway in MDR+ cells leads to the constitutive activation of Ras/ERK1-2 and RhoA/RhoA kinase downstream signalling pathways which culminates into HIF-1α activation and plasma membrane Pgp expression. The higher amounts of plasma membrane-associated cholesterol in MDR+ cells also contribute to the functional Pgp activation. The higher efficiency to extrude Dox protects MDR+ cells from cytotoxicity and ICD epitomized by CRT exposure on the cell surface. B. By inhibiting the Mev pathway, ZA downregulates the Ras/ERK1-2 and RhoA/RhoA kinase signalling pathways, and decreases the HIF-1α-induced transcription of Pgp. As a result, Dox accumulates inside MDR+ cells at sufficient amounts to induce cytotoxicity and promote CRT exposure, turning the phenotype of these cells from a chemoimmunoresistant phenotype into a chemoimmunosensitive phenotype.
Supporting Information
Figure S1
Effects of different combinations of ZA and doxorubicin on the intracellular doxorubicin accumulation. HT29, HT29-dx and HMM cells were treated with 1 µmol/L Dox alone or Dox in combination with 1 µmol/L ZA under different conditions: 1) Dox and ZA were co-incubated for 48 h (ZA & Dox); 2) Dox was added first for 24 h, ZA was added for a further 48 h (Dox+ZA); 3) ZA was added first for 48 h, Dox was added for a further 24 h (ZA+Doz). Intracellular Dox concentrations were significantly higher in HT29 than in HT29-dx and HMM cells (*p<0.001). In MDR+ cells, intracellular Dox concentrations were significantly higher than untreated cells only when ZA preceded Dox (HT29-dx: °p<0.05; HMM: ◊p<0.02). Bars represent the mean ± SD of 3 independent experiments.
(TIF)
Click here for additional data file.
Figure S2
Biotinylation assays to detect surface calreticulin. HT29, HT29-dx, and HMM cells after incubation with ZA and/or Dox. Tumor cells were incubated for 48 h without (CTRL) or with 1 µmol/L ZA, for 24 h with 1 µmol/L Dox, for 48 h with 1 µmol/L ZA, followed by 1 µmol/L Dox for a further 24 h (ZA+Dox). Surface CRT was measured in biotinylated extracts; total CRT was measured in the whole cell lysates from the same cells by Western blotting. The results are representative data from one of 2 experiments.
(TIF)
Click here for additional data file.
Figure S3
CD86 expression on DC surface after internalization of MDR+ HT29-dx tumor cells. The CD86 expression was evaluated on DC surface in the immature status and after internalization of ZA and/or Dox-treated HT29-dx cells. The internalization of ZA+Dox treated-HT29dx cells induced an up-regulation of CD86 expression, in line with the concept that the exposure of DCs to Dox-treated tumor cells stimulates their maturation. The results represent the mean ± SD of 6 independent experiments.
(TIF)
Click here for additional data file.
Figure S4
Cytotoxic activity of T cells against MDR+ HT29-dx tumor cells. The cytotoxic activity exerted by T cells against MDR+ HT29-dx tumor cells was evaluated after incubation with autologous DCs pulsed with medium alone (CTRL), ZA, Dox and ZA+Dox-treated HT29-dx cells. Cytotoxicity was evaluated by CFSE staining of tumor target cells identifying the CFSE-labeled dead cells by propidium iodide. The highest cytotoxic activity was observed after T-cell stimulation by DCs loaded with HT29-dx cells exposed to ZA plus Dox. The results are from one experiment.
(TIF)
Click here for additional data file.
We are grateful to Mr. Costanzo Costamagna for technical assistance and to Mr. Andrew Martin Garvey for editorial assistance.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23614030PONE-D-13-0163610.1371/journal.pone.0062178Research ArticleBiologyBiochemistryMetabolismOxygen MetabolismBiotechnologyEnvironmental BiotechnologyBiodegradationApplied MicrobiologyMicrobiologyBacterial PathogensGram PositiveApplied MicrobiologyIndustrial MicrobiologyMedical MicrobiologyChemistryMetabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1 Metabolism of 2-Chloro-4-NitroanilineKhan Fazlurrahman Pal Deepika Vikram Surendra Cameotra Swaranjit Singh
*
Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Chandigarh, India
Johnson Stephen J. Editor
University of Kansas, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: SSC FK. Performed the experiments: FK DP SV. Analyzed the data: FK SSC. Wrote the paper: SSC FK.
2013 17 4 2013 9 7 2014 8 4 e6217810 1 2013 18 3 2013 © 2013 Khan, et al2013Khan, et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.
The authors have no support or funding to report.
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Introduction
2-Chloro-4-nitroaniline (C6H5ClN2O2, 2-C-4-NA) is a nitroaromatic compound, used as an intermediate in the synthesis of dyes, pharmaceuticals, corrosion inhibitors and in the manufacture of niclosamide, a molluscicide [1]–[3]. 2-C-4-NA is also reported as a photolysis product of niclosamide [4]. Espinosa-Aquirre et al. [5] reported the metabolism of niclosamide, which is used as an anti-helminthic drug, results in the formation of 2-C-4-NA and 5-chlorosalicylic acid metabolites by hydrolytic cleavage of amide bond. As a result of its extensive production and application it may get released into the environments through various waste streams and is considered to be an increasing threat into the environments and various life forms [6]. Hence the fate of 2-C-4-NA in the environments is of great concern. 2-C-4-NA causes severe cellular damage as studied in rat [7]. It is identified as a bacterial mutagenic compound as tested in Salmonella typhimurium, however, the potency is low compared to niclosamide [5]. The environmental fate of 2-C-4-NA can be determined either by non-biological (volatization, hydrolysis, photolysis, thermal decomposition) or by biological means. Volatization of 2-C-4-NA from the soil surface and water surface are not considered to be an important fate process owing to its Henry's Law constant of 9.5×10−9 atm-cu m mole−1
[8]. Similarly, 2-C-4-NA does not contain any functional group by which it would hydrolyze in the environment [9]. However, 2-C-4-NA contain chromophores that can absorb light at wavelength of >290 nm and may be susceptible to photolysis [9]. Thus, for the remediation of the heavy contamination in the environment, the microbial transformation and degradation could be used as the most effective, eco-friendly and technically challenging approach for the decontamination of soil, sediment and water bodies etc. 2-C-4-NA is considered to be non-biodegradable in the aquatic environment as well as in the industrial sewage treatment plants [10]. Similarly, Canton et al. [11] also classified 2-C-4-NA as a blacklist substance due to its poor biodegradability. However, based on the previous reports on the biodegradation of structural analogues of 2-C-4-NA such as 4-nitroaniline, 2-, 3-, 4-chloroaniline and 3, 4-dichloroaniline, possibilities for the biodegradation of 2-C-4-NA by microorganisms could also be presumed [12]–[18]. In this communication, we report metabolic characterization of 2-C-4-NA by Rhodococcus sp. strain MB-P1 which was previously characterized for the degradation of atrazine. The strain MB-P1 was capable of metabolizing 2-C-4-NA as the sole carbon, nitrogen, and energy source. The catabolic pathway for degradation of 2-C-4-NA by strain MB-P1 is initiated via oxidative hydroxylation resulting in the formation of 4-A-3-CP. Subsequent degradation occurs by dioxygenase mediated transformations as indicated by detection of ‘6-chlorohydroxyquinol’ (6-CHQ) as the terminal aromatic intermediate. This study has significant implications in terms of understanding the mechanism of aerobic degradation of 2-C-4-NA, related aromatic amines as well as determining their environmental fate.
Materials and Methods
Chemicals, strain, and growth medium
Analytical grade of 2-chloro-4-nitroaniline (2-C-4-NA) and standard 4-amino-3-chlorophenol (4-A-3-CP) were purchased from Sigma-Aldrich (St, Louis, MO, USA). Rhodococcus sp. strain MB-P1 was isolated from the contaminated soil sample and characterized for atrazine degradation [19]. Minimal salt medium (MSM) used in the present study was prepared as described earlier [19] with slight modification i. e. absence of nitrogen source [(NH4)2SO4]. Stock solution (10 mM) of 2-C-4-NA prepared in HPLC grade methanol was added to an empty Erlenmeyer flask to obtain the working concentrations. Further, the residual methanol in the flask was evaporated under a stream of air to leave the dry crystal of 2-C-4-NA in the bottom of the flask. Appropriate volume of MSM was added to the flask to attain desired working culture. Nutrient agar at one-quarter strength (1/4-NA) and nutrient broth (1/4-NB) were used as a rich media for bacterial growth and culture maintenance.
Metabolic activity of strain MB-P1 on 2-C-4-NA
Metabolic activity of strain MB-P1 on 2-C-4-NA was determined by growth studies carried out in carbon-free MSM supplemented with varying concentrations of 2-C-4-NA ranging from 50 to 500 µM. The positive metabolic activity was determined by time dependent bacterial growth measure in terms of increase in optical density of the culture medium monitored at 600 nm using Lambda EZ 201 UV-visible spectrophotometer (Perkin-Elmer Inc, USA). Bacterial growth was also monitored by measuring the total protein of the cultures grown on 2-C-4-NA with Pierce BCA protein assay kit (Thermo Scientific, USA). Release of nitrite ions (NO2
−), chloride ions (Cl−), and ammonia (NH3) in the growth medium and gradual decrease in concentration of 2-C-4-NA were monitored as the alternative methods for determination of metabolic activity of strain MB-P1 on 2-C-4-NA. Subsequent characterization was carried out to determine the kinetics of 2-C-4-NA degradation by strain MB-P1. Appropriate biotic and abiotic controls were included wherever necessary. Procedures used for growth studies and resting cell studies are as follows:
Growth studies
Growth studies were performed in 50 ml of carbon-free MSM supplemented with 2-C-4-NA (200 µM) by inoculating 1% (v/v) of overnight culture of MB-P1 cells grown in 1/4-NB. Cultures were incubated on a rotary shaker at 200 rpm at 30°C. Samples were withdrawn at every 8 h to monitor bacterial cell growth, release of NO2
−, Cl−, NH3, and degradation of the growth substrate. Cell growth was monitored spectrophotometrically as described above. Bacterial growth was also monitored by the indirect approach of measuring total protein concentration in the culture. Culture fluid samples (2.0 ml) were centrifuged at 8,000×g for 10 min to obtain cell-free culture supernatants that were subsequently analyzed for NO2
−, Cl−, and NH3 release, depletion of growth substrate and identification of degradation intermediates using methods described later. Non-inoculated flask and flask inoculated with heat killed cells of strain MB-P1 were used as abiotic control and negative control respectively.
Resting cell studies
Resting cell studies were performed with minor modification of the method described earlier [20]. Briefly, a seed culture (6%, v/v) of strain MB-P1 grown in 1/4-NB inoculated into 1.6 L of 1/4-NB supplemented with 200 µM of 2-C-4-NA and incubated at 30°C with aeration for 24 h (OD600, 1.3–1.4). The induced cells were harvested by centrifugation at 8,000×g at room temperature for 10 minutes, washed twice with phosphate buffer (20 mM, pH 7.2) and suspended in 100 ml of carbon-free MSM. This suspension was divided into four aliquots of 25 ml each; one aliquot was heat killed by incubating on boiling water for 30 minutes which was later used as negative control. The other two aliquots were supplemented with 200 µM of 2-C-4-NA and 4-A-3-CP separately. Each flask was then incubated at 30°C with shaking at 200 rpm. Similarly, another control for the above experiment was also made by suspending un-induced cells of strain MB-P1 in MSM supplemented with 200 µM of 2-C-4-NA and 4-A-3-CP separately. Samples (2.0 ml) were withdrawn from both control and experimental flasks at regular intervals of 2 h and subjected to NO2
−, Cl−, and NH3 release analysis, followed by high-performance liquid chromatography (HPLC) analysis and also gas chromatography-mass spectroscopy (GC-MS) analyses (methods described later).
Ring cleavage inhibition studies
There is a well known study to check the inhibition of ring cleavage catalyzed by ferrous ions dependent ring cleavage dioxygenase by using an iron chelator i. e. 2, 2-dipyridyl [21]. The ring cleavage experiment was performed in the same way as the resting cell study. The harvested 2-C-4-NA-induced resting cells were suspended in 25 ml carbon-free MSM supplemented with 200 µM of 2-C-4-NA and 1.0 mM 2, 2-dipyridyl. Similarly, another flask was also taken as a control containing 25 ml cell suspensions of 2-C-4-NA-induced resting cells in MSM supplemented with 200 µM of 2-C-4-NA only. Each flask was then incubated at 30°C with shaking at 200 rpm. Culture supernatant (2.0 ml) was collected at different time intervals and analyzed by HPLC.
Enzyme assays with cell-free lysates
2-C-4-NA-induced cells of strain MB-P1 were harvested by centrifugation and were washed twice with phosphate buffer (20 mM, pH 7.2) and finally re-suspended in lysis buffer (50 mM phosphate buffer, pH 7.2). Cell lysis was carried out by two passages through a French pressure cell (20,000 lb/in2). The lysed cell suspensions were centrifuged at 12,000 rpm for 30 min at 4°C and supernatant was carefully separated to obtain cell-free enzyme extract, which was subsequently used for the enzyme assays. Quantitation of protein content within cell-free extracts were performed routinely with Pierce BCA protein assay kit (Thermo Scientific, USA). The cell-free extract was tested for activity of monooxygenase since this enzyme is known to be involved in the first step of 2-C-4-NA degradation as indicated by the identification of the pathway metabolites. The monooxygenase enzyme assay was carried out as described below:
Enzyme assays for monooxygenase
Flavin-dependent monooxygenase enzyme activity was assayed to characterize the putative first step of 2-C-4-NA degradation by strain MB-P1. Cell-free protein extracts (5 mg ml−1 of protein) prepared from 2-C-4-NA-induced cells were added to phosphate buffer (20 mM, pH 7.0), 100 µM NADPH, 200 µM FMN in a total reaction volume of 10 ml. Reactions were initiated by the addition of 70 µM 2-C-4-NA and incubated at 28°C. Samples were withdrawn at regular time intervals and monooxygenase enzyme activity was determined by measuring the time dependent disappearance of 2-C-4-NA and NO2
− release. The 2-C-4-NA depletion was measured by HPLC analysis. Release of NO2
− ions were estimated with the colorimetric method described later.
Enzyme assay for aniline dioxygenase
The aniline dioxygenase enzyme activity by strain MB-P1 was measured with an oxygen electrode (YSI, Ohio, USA), according to the method described by Liu et al. [22]. MB-P1 cells grown in 500 ml 1/4-NB were harvested by centrifugation at 8,000×g at 4°C for 10 minutes. Pellets were washed twice with phosphate buffer (20 mM, pH 7.2) and suspended in 100 ml of MSM containing 2-C-4-NA (100 µM). In order to obtain the un-induced cells, the similar cell pellets were suspended in 100 ml of 1/4-NB. Further, these cells were incubated at 30°C under shaking at 200 rpm to obtain 2-C-4-NA-induced or non-induced cells respectively. After 24 h of incubation, cells were harvested, cell pellets were washed twice with phosphate buffer (20 mM, pH 7.2) and re-suspended in the same buffer. This suspension was used for the assay of aniline dioxygenase by measuring oxygen uptake at 30°C.
Analytical methods
The release of nitrite ions (NO2
−) was monitored with a colorimetric method using N-(1-naphthyl) ethylene-diamine-dihydrochloride and sulfanilic acid reagent as described earlier [23]. Ammonia (NH3) concentrations were also monitored with a colorimetric method using ‘Ammonia Estimation Kit’ (Sigma Aldrich, USA) according to the manufacturers' recommendation. Similarly, the release of chloride ions (Cl−) was also monitored colorimetrically with colorimetric method that uses mercuric thiocyanate [24]. Standard plots generated with known concentrations of NaCl, (NH4)2SO4 and NaNO2 were used to determine the concentrations of Cl−, NH4
+, and NO2
− ions released into the culture medium.
For the quantitative measurement of 2-C-4-NA degradation by strain MB-P1 and identification of metabolic products, 500 µl of cell-free aqueous culture supernatants from growth and resting cell studies were filtered with 0.2 µm filters (Millipore Inc. USA). The filtered culture supernatants were analyzed with minor modification of the quantitative HPLC method described earlier [25]. Briefly, the separation of 2-C-4-NA and their metabolic products was carried out on a C-18 reverse phase column at 30°C on UV-detector equipped Waters HPLC system (Waters, USA). The mobile phase used was a mixture of acetonitrile:water (30∶70, v/v) under an isocratic condition with the flow rate of 1.0 ml min−1. The peaks of analytes were detected with UV detector over a wavelength scan of 220–290 nm. Alternatively for the analysis of metabolites by GC-MS, cell-free aqueous culture supernatant samples were mixed with equal volume of ethyl acetate and performed liquid-liquid extraction by layer separation sequentially at neutral and acidic pH. Extracted organic phase was pooled, dried under nitrogen flow using RotaVapor II (BUCHI, Switzerland) and re-suspended in the appropriate volumes of ethyl acetate. The derivatization of metabolic products in the sample was performed by silylation as previously described [26]. The derivatized samples were analyzed by GC-MS using QP2010S (Shimadzu Scientific Instruments, USA) with temperature program and run parameter used for GC analyses as reported earlier [27]. Briefly, temperature program was: 1 min at 50°C, followed by ramping up to 150°C at a constant temperature increment rate of 20°C min−1, a slower ramping to 200°C at a rate of 5°C min−1, and then further ramped to 320°C at a rate of 20°C min−1, which was held for 5 min. Positive molecular ion mass spectra were scanned in mass/charge (m/z) range of 50–500.
Results
Growth study and degradation of 2-C-4-NA by strain MB-P1
Initial screening for the metabolic activity on 2-C-4-NA was carried out by using lab isolates (previously isolated from contaminated soil). A total 79 bacterial isolates were selected and screened for 2-C-4-NA degradation by inoculating in carbon-free MSM supplemented with 2-C-4-NA (100 µM). Thereafter 2-C-4-NA degrading abilities were examined by analyzing the growth of culture and release of NO2
−, Cl− ions, and NH3 in the medium. Five isolates showed apparent increase in bacterial cell mass and also the release of NO2
−, Cl− ions, and NH3. Among these isolates, strain MB-P1 was found to be an efficient degrader of 2-C-4-NA. The strain MB-P1 was incubated in carbon-free MSM supplemented with the different concentrations of 2-C-4-NA ranging from 50 to 500 µM. Noticeably complete inhibition of growth was observed when 2-C-4-NA was used at concentrations higher than 300 µM. However, 200 µM served as optimal concentration for growth of strain MB-P1 (data not shown). The growth study of strain MB-P1 carried out in carbon-free MSM supplemented with 2-C-4-NA (200 µM) showed an evident lag phase of 16 h (Figure 1). However, over time of subsequent log phase, the bacterial culture grew exponentially as observed with the increase of total protein concentration up to the value of 9.1 µg ml−1 (Figure 1). During the growth study, no metabolic products were detected from the sample analyzed by HPLC. However, NH3, Cl− ions, and NO2
− ions released in the culture media were quantified colorimetrically. The released NH3 and NO2
− ions were quantified colorimetrically to be 30 µM and 198.5 µM respectively at the end of the log phase of the growth (Figure 1). The accumulation of lesser amount of NH3 in the media clearly suggested that NH3 was used as a preferred nitrogen source for its growth. On the other hand, Cl− ions continued to accumulate in culture medium and reached a maximum concentration of 198.8 µM. The quantitative measurement of 2-C-4-NA by HPLC from growth medium showed its decrease from initial concentration of 200 µM to non-detectable amounts after∼90 h of incubation (Figure 1). The growth yield of strain MB-P1 on 2-C-4-NA was found to be 0.50 g of cells/g of 2-C-4-NA. Similarly, the rate of 2-C-4-NA degradation by strain MB-P1 during the growth studies was calculated to be 3.2 µmol mg protein−1 minute−1. The strain MB-P1 also utilized 4-A-3-CP as the sole carbon, nitrogen, and energy source with slight accumulation of NH3 (28 µM) in the culture media (Data not shown). The growth yield of strain MB-P1 on 4-A-3-CP was 0.62 g of cells/g of 4-A-3-CP and the rate of 4-A-3-CP degradation by strain MB-P1 was 3.0 µmol mg protein−1 minute−1. The growth yield of strain MB-P1 on 4-A-3-CP as well as the rate of 4-A-3-CP degradation was found almost similar as determined for 2-C-4-NA.
10.1371/journal.pone.0062178.g001Figure 1 Growth characterization of Rhodococcus sp. strain MB-P1 on 2-C-4-NA as the sole carbon, nitrogen, and energy source.
(•), 2-C-4-NA; (▴), NO2
−; (Δ), NH4
−; (○), Cl−; (▪), total protein. Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.
Elucidation of catabolic pathway for 2-C-4-NA degradation by strain MB-P1
Resting cell studies
During the growth study we could not identify any putative metabolites of 2-C-4-NA metabolism. Therefore, samples from resting cell studies and enzyme assays were used for the identification of intermediates. The aqueous sample collected at different time intervals from the resting cell studies was analyzed by HPLC. The resting cell study showed that the induced cells eliminate the lag phase for the depletion of 2-C-4-NA with transient accumulation of 4-A-3-CP metabolite, which subsequently depleted over the time of incubation (Figure 2A). However, when the same 2-C-4-NA-induced cells were also incubated with 4-A-3-CP, there was a shorter induction period for the disappearance of 4-A-3-CP in the first phase which underwent faster depletion over the time of incubation, suggesting that the enzyme responsible for the degradation of 4-A-3-CP is inducible in nature (Figure 2B). Noticeably, lesser amount of 6-CHQ metabolite was also identified, which disappeared over time of incubation (Figure 2). The above two metabolites identified in HPLC showed retention time (Rt) at 4.86 and 3.28 min respectively (Figure 3A). The Rt value corresponding to the 4.86 min matched with the authentic standard of 4-A-3-CP. However, the Rt value corresponding to the 3.28 min could not be characterized due to the non-availability of standard. Further, the confirmation and identification of unknown metabolites from the samples obtained from the resting cell study were subjected to mass fragmentation analysis by GC-MS. In GC-MS analysis two metabolic peaks appeared at Rt values of 7.62 and 9.52 min respectively (Figure 3B). The mass fragmentation pattern for metabolite with a Rt value of 7.62 min showed a conjugate ion at m/z value of 215 (representing silylated species: M+), 200 (M-CH3), 143, 73 [Si (CH3)3] (Figure 3C). The mass spectrum of this metabolite matched the known standard of 4-amino-3-chlorophenol. This metabolite would have probably resulted from direct denitrification of the aromatic nucleus without deamination. The second metabolite with a Rt value of 9.52 min showed a conjugate ion at m/z value of 378, 376 (M+), 366, 361 (M-CH3), 288, 275, 273, 179, 73 [Si (CH3)3] (Figure 3C). The fragmentation pattern and mass spectra of this metabolite were consistent and similar to that of 6-chlorohydroxyquinol as described earlier [27]. 6-chlorohydroxyquinol is reported as the terminal intermediate in the degradation of 2, 4, 6-trichlorophenol and 2, 6-dichlorophenol degradation by Azotobacter sp. strain GP1, Streptomyces rochei 303, Ralstonia eutropha JMP 134 and Cupriavidus necator JMP134 [28]–[32]. The formation of 6-chlorohydroxyquinol metabolite would be as a result of dioxygenation with the deamination of amino group from the benzene ring. Based on the above, HPLC and GC-MS analysis, we conclude that 4-A-3-CP and 6-CHQ are the major identified metabolites during aerobic degradation of 2-C-4-NA by strain MB-P1.
10.1371/journal.pone.0062178.g002Figure 2 Degradation kinetics of 2-C-4-NA and 4-A-3-CP during resting cell studies performed by 2-C-4-NA-induced cells of strain MB-P1.
(A) 2-C-4-NA degradation kinetics. (•), 2-C-4-NA; (▪), 4-A-3-CP; (Δ), 6-CHQ; (○), 2-C-4-NA in abiotic control. (B) 4-A-3-CP degradation kinetics. (•), 4-A-3-CP; (Δ), 6-CHQ. Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.
10.1371/journal.pone.0062178.g003Figure 3 Representative HPLC and GC-MS chromtograms along with mass spectra of metabolites identified during the degradation of 2-C-4-NA by resting cells of Rhodococcus sp. strain MB-P1.
(A) HPLC chromatograms of metabolites identified at different time intervals. Peak at Rt value of 8.5 min corresponds to 2-C-4-NA; Peak at Rt value of 4.86 min corresponds to 4-A-3-CP; Peak at Rt value of 3.28 min corresponds to 6-CHQ. (B) GC-MS chromatogram of the authentic standards and the identified metabolites. Peak at Rt value of 6.41 min corresponds to 2-C-4-NA; Peak at Rt value of 7.62 min corresponds to 4-A-3-CP; Peak at Rt value of 9.52 min corresponds to 6-CHQ. (C) Mass spectra of metabolites (Left side; 4-A-3-CP and right side; 6-CHQ) of 2-C-4-NA as analyzed by GC-MS.
Enzyme assays
To validate the formation of metabolites and illustrate the degradation pathway of 2-C-4-NA in strain MB-P1, the enzyme assays were carried out. The formation of 4-A-3-CP as the first degradation metabolite and the release of NO2
− ions from 2-C-4-NA suggested oxygenase mediated enzymatic reaction in the degradation pathway. Cell lysates prepared from 2-C-4-NA grown cells of strain MB-P1 showed positive activity for flavin-dependent monooxygenase enzyme and catalyzed the elimination of NO2
− ions and formation of 4-A-3-CP. The specific activity for this enzymatic reaction was 1.3±0.25 nmol min−1 mg of protein−1.
Oxygen uptake
There was rapid oxygen consumption by 2-C-4-NA, 4-nitroaniline and 4-chloroaniline by 2-C-4-NA-grown cells (Table 1). The oxygen uptake of the above results showed that the pathway enzymes involved in the degradation of 2-C-4-NA are induced. However, the lesser amount of oxygen consumption by 2-C-4-NA by 1/4-NB grown cells revealed the first enzyme is constitutive in nature. Whereas, there was a negligible amount of oxygen uptake by aniline and 4-A-3-CP in 1/4-NB grown cells indicating inducible nature of the aniline dioxygenase. The above results are in close agreement with the results as reported earlier [22], [33], [34]. The NH3 released were also quantitatively measured and showed stoichiometry to the amount of substrates added (data not shown). Liu et al. [22] reported the formation of catechol product from aniline occurs via the action of aniline dioxygenase. Thus, based on the above results it is concluded that the second enzyme responsible for the degradation of 2-C-4-NA is a type of dioxygenase.
10.1371/journal.pone.0062178.t001Table 1 Determination of aniline dioxygenase activity in strain MB-P1 measured by oxygen uptake by 2-C-4NA-grown versus 1/4-NB-grown cellsa.
Substrates O2 uptake (nmol O2/min/mg of protein) by cells grown in:
2-C-4-NA 1/4-NB
2-Chloro-4-nitroaniline 90.17±0.5 38.43±0.8
4-Amino-3-chlorophenol 59.70±1.5 0.15±0.2
4-Nitroaniline 83.13±0.2 42.41±1.0
4-Chloroaniline 69.10±0.8 23.35±0.1
Aniline 53.3±1.3 0.13±0.3
a The reaction was carried in 1.85 ml volume air-saturated phosphate buffer (20 mM, pH 7.2) containing substrates (40 µM), and cells (0.25 mg of protein). Data represents means of at least three separate experiments.
Ring cleavage inhibition studies
The HPLC analysis of the samples collected from the ring cleavage inhibition studies by 2, 2-dipyridyl on 2-C-4-NA showed the accumulation of 6-CHQ at the Rt value of 3. 28 minute with the liberation of only NO2
− ions and NH3 in the culture media (Figure 4A). The culture media became deep red in color, which also indicates accumulation of hydroxyquinol as reported previously [27], [35]. Noticeably, a slight accumulation of 6-CHQ in the control (without added 2, 2-dipyridyl) was observed which got disappeared within a short time of incubation with the liberation of NO2
−, Cl− ions, and NH3 (Figure 4B). The above results clearly suggested that 6-CHQ is the terminal intermediate for the degradation of 2-C-4-NA by strain MB-P1. It is well known that the reaction mechanism of 2, 2-dipyridyl used as an inhibitor for the ring cleavage require ferrous ions for their enzymatic activities [21]. The formation of hydroxyquinol as a terminal ring cleavage substrate has been exclusively reported in the degradation pathway of 4-nitrophenol by gram-positive bacteria [35]–[37]. Similarly, the formation of 6-chlorohydroxyquinol has also been reported as a ring cleavage substrate in the degradation pathway of 4-chlorophenol [21]. Based on the identified metabolite from the resting cell study, oxygen uptake, enzyme assays and ring cleavage inhibition study, the pathway for the degradation of 2-C-4-NA has been proposed as shown in Figure 5.
10.1371/journal.pone.0062178.g004Figure 4 Ring cleavage inhibition studies using 2, 2-dipyridyl.
(A) Treated with 2, 2-dipyridyl. (B) Untreated with 2, 2-dipyridyl. 2-C-4-NA (•); 4-A-3-CP (▴); and 6-CHQ (○).Values are presented as arithmetic means of data obtained from experiments carried out in triplicates; error bars represent standard deviation.
10.1371/journal.pone.0062178.g005Figure 5 Proposed metabolic pathway for aerobic degradation of 2-C-4-NA by Rhodococcus sp. strain MB-P1.
According to the results obtained presented here, 4-A-3-CP and 6-CHQ are identified as major intermediates during degradation of 2-C-4-NA.
Discussion
Biodegradation has been recognized as an economical and environment friendly approach for decontamination of sites polluted with toxic anthropogenic chemicals, released into environment as a result of industrial, agricultural, military and household activities [38], [39]. Conversely, its application has remained elusive and inefficient in case of highly recalcitrant compounds including chloro and nitro containing anilines [14], [40], [41]. To overcome this limitation, it is essential to isolate and characterize microorganism(s) with potential to metabolize these compounds. To date no aerobic degradation and pathway characterization has been reported for the microbial degradation of 2-C-4-NA. However, based on the earlier reports on the degradation of structural analogues of 2-C-4-NA such as anilines substituted with chloro and nitro groups, it is presumed that the degradation of 2-C-4-NA could also be possible in aerobic condition. Although, the enrichment of cultures could be used as an efficient approach for isolating microorganism(s) with desired degradative capabilities; however, determining metabolic diversity of naturally occurring degradative isolates could also be another approach. Results presented in few recent studies indicate some degradative strains to be metabolically versatile that they can degrade compounds analogous to their original enrichment substrate. The genus of Rhodococcus constitutes a diverse group of bacteria which exhibit a metabolic versatile activity on chloro and nitro substituted aromatic compounds [42]. The catabolic versatility of Rhodococcus is due to the presence of large genome and plasmid which harbors large number of metabolic genes, resulting in expansion of the multiple metabolic pathways [43]. Ghosh and co-workers reported Rhodococcus imtechiensis strain RKJ300 with the ability to degrade 4-nitrophenol, 2, 4-dinitrophenol as well as their chloro substituted analogue 2-chloro-4-nitrophenol [42]. Based on the above rationale, the screening for the degradation of 2-C-4-NA was performed by using previous lab isolates known for the degradation of nitroaromatic compounds. The 2-C-4-NA degrading abilities were examined by analyzing the increase in growth and also by measuring the release of NO2
−, Cl−, and NH3 in the medium. Results from primary screening, five strains belonging to the genus Rhodococcus showed the apparent increase of bacterial cell mass as well as the release of NO2
−, Cl− ions, and NH3. All these five isolates have been previously characterized for the degradation of chloro and nitro substituted aromatic compounds. Interestingly, the strain MB-P1 showed the faster degradation of 2-C-4-NA, hence it was selected for the detailed study. During the growth study, the rapid disappearance of 2-C-4-NA was accompanied by the release of NO2
− ions and NH3 followed by the release of Cl− ions in the media. The amounts of chloride and nitrite ions accumulated in growth media were stoichiometric; however, very limited amounts of NH3 was released. There was no accumulation of intermediates of 2-C-4-NA degradation in the growth study. Based on the growth study it could be argued that 2-C-4-NA was completely mineralized and NH3 is the preferred nitrogen source for the growth of strain MB-P1. The above results are in close agreement with the results of 5-nitroanthranilic acid degradation by Bradyrhizobium sp. strain JS329 [44]. Although, the growth study did not show any accumulation of metabolites, however, the resting cell studies showed the formation of 4-A-3-CP as the first intermediate, which subsequently disappeared with the formation of 6-CHQ as a second metabolite. Results from elucidation of the catabolic pathway in 2-C-4-NA degradation by strain MB-P1 clearly demonstrated identification of 4-A-3-CP and 6-CHQ as major intermediates. There are two ways for the removal of nitro group from the chloronitrobenzenes or chloronitrophenols out of which one is oxidative and another one is reductive reaction mechanism. In the oxidative reaction the nitro group is removed in the form of NO2
− ions as a results of hydroxylation, while a partial reduction of nitro group into hydroxylamine or amino group takes place in reductive reaction [42], [45]–[49]. Results showing transformation of 2-C-4-NA to 4-A-3-CP for initiation of 2-C-4-NA degradation by strain MB-P1 suggest the involvement of oxygenation reaction by a putative monooxygenase. The monooxygenase enzyme assays from the crude cell extracts confirm the involvement of flavin-dependent monooxygenase. The flavin-dependent monooxygenase reaction is very common for aerobic microbial degradation of nitro or chloro containing aromatic compounds such as 2-chloro-4-nitrophenol, 4-nitrophenol, 2,4-dichlorophenol and 4-chlorophenol by aerobic microorganisms [42], [50]–[51]. In the proposed pathway, conversion of 4-A-3-CP into 6-CHQ is expected to be catalyzed by an enzyme capable of removing amino group via oxidative reaction. Mostly the removal of amino group from the benzene ring occurs by the action of catechol dioxygenase [44]. Such catalytic reaction mechanisms are widely distributed among the microorganisms capable of degrading anilines and chloroanilines [22], [33], [40], [52]–[55].
These microorganisms utilize aniline as the sole carbon, nitrogen, and energy source and degradation occurs via the formation of catechol metabolite with the action of catechol dioxygenase [22], [56]–[60]. Schukat et al. [61] reported the co-metabolic degradation of chloroanilines by Rhodococcus sp. An117 via the formation of catechol as terminal intermediate. Similarly, Zeyer et al. [62] reported utilization of chloroaniline by Moraxella sp. strain G occurs by ortho-pathway. Thus, the enzyme which is involved in the second step degradation of 2-C-4-NA i. e. conversion of 4-A-3-CP to 6-CHQ should be almost similar with those responsible for aniline and chloroaniline catabolism. The identified metabolite 6-CHQ from the resting cell study constitute the ring cleavage substrate during the 2-C-4-NA degradation by Rhodococcus sp. strain MB-P1 as supported by the accumulation of 6-CHQ during the ring cleavage inhibition study. The above result is in close agreement with results reported for 4-chlorophenol degradation by Arthrobacter chlorophenolicus A6 [27]. Based on such reports as well as the results obtained during the present study, we propose the possible involvement of a similar bacterial monooxygenase in the first step of 2-C-4-NA degradation followed by aniline dioxygenase mediated reaction as the second step of degradation. Subsequent degradation of terminal intermediate 6-CHQ presumably proceeds via conventional 2, 4, 6-trichlorophenol degradation pathway.
Conclusion
In conclusion, we report metabolism of 2-C-4-NA by Rhodococcus sp. strain MB-P1 which was previously characterized for atrazine degradation. This is one of the first aerobic bacteria capable of degrading 2-C-4-NA as the sole carbon, nitrogen, and energy source. Strain MB-P1 degrades 2-C-4-NA via the formation of 4-A-3-CP and 6-CHQ as the novel intermediates. The first step of 2-C-4-NA degradation occurs by flavin-dependent monooxygenase mediated reaction with the formation of 4-A-3-CP which gets subsequently transformed to 6-CHQ via dioxygenation reaction. The lack of induction period and accumulation of 4-A-3-CP metabolite during the induction study of 2-C-4-NA degradation by strain MB-P1 confirmed the constitutive activity of first enzyme i. e. flavin-dependent monooxygenase. The above results were also supported by the results of oxygen uptake. However, the second enzyme responsible for the conversion of 4-A-3-CP to 6-CHQ is inducible in nature as confirmed from the oxygen uptake and induction studies. Strain MB-P1 could be used as a model system for studies focusing on this important transformation reaction for the degradation of chloro and nitro group containing anilines. Strain MB-P1 could also be used as an important model system for studies on biochemical and molecular evolution of microbial degradation of 2-C-4-NA. From application point of view, strain MB-P1 could be potentially used for bioremediation of ecological niches contaminated with 2-C-4-NA. The molecular components involved in the pathway of 2-C-4-NA degradation by strain MB-P1 are yet to be characterized.
FK, DP and SV acknowledge their research fellowship awards from CSIR, India. This is the IMTECH communication number 094/2012.
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Oncol LettOncol LettOLOncology Letters1792-10741792-1082D.A. Spandidos 10.3892/ol.2013.1188ol-05-04-1290ArticlesRNA interference-mediated USP22 gene silencing promotes human brain glioma apoptosis and induces cell cycle arrest LI ZHAO HUI 1YU YIN 1DU CHAO 1FU HONG 1WANG JIAN 2TIAN YU 11 Department of Neurosurgery, China-Japan Union Hospital of Jilin University, Changchun 130033;2 Department of Neurosurgery, Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000,
P.R. ChinaCorrespondence to: Dr Yu Tian, Department of Neurosurgery, China-Japan Union Hospital of Jilin University, 126 Xiantai Road, Changchun 130033, P.R. China, E-mail: [email protected] 2013 12 2 2013 12 2 2013 5 4 1290 1294 29 10 2012 04 2 2013 Copyright © 2013, Spandidos Publications2013This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.Ubiquitin-specific protease 22 (USP22) is a novel tumor stem cell marker that plays a key role in tumorigenesis and cell cycle progression. However, the effect of silencing the USP22 gene on human brain glioma cell growth is not well understood. In the present study, high gene expression of USP22 was identified in human brain glioma cells. In addition, RNA interference technology was used to silence USP22 gene expression in human brain glioma cells. Silencing the USP22 gene was found to effectively inhibit proliferation of human brain glioma cells, resulting in cell apoptosis and cell cycle arrest at the G2/M phase. USP22 silencing was also found to lead to reduced expression of cell cycle proteins, including CDK1, CDK2 and CyclinB1. In summary, in this study the USP22 gene was demonstrated to play a key regulatory role in the growth of human brain glioma cells by affecting progression of apoptosis and the cell cycle.
RNA interferenceUSP22 genehuman brain gliomacell apoptosiscell cycle
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Introduction
Originating in the nerve epithelial tissue, brain glioma is one of the most common malignant tumors in the central nervous system accounting for >70% of primary malignant brain tumors (1–3). The high incidence, poor prognosis and low efficacy of therapeutics associated with brain glioma has attracted considerable attention from researchers in the central nervous system disease field; however, progress remains challenging (4). At present, brain glioma is largely treated with surgery, radiotherapy and chemotherapy; however, the curative effect and prognosis are far from optimistic, and there is no significant improvement in treatment efficacy of brain glioma (5–7). In addition, current knowledge on the molecular mechanism of the generation and development of brain glioma remains extremely limited. Analysis of the roles played by specific molecules in the brain glioma generation and development process is important for the identification of effective treatment approaches and may provide effective molecular targets for future molecular therapies.
A previous study identified an 11-gene Polycomb/cancer stem cell signature that may predict the probability of treatment failure in cancer patients (8). Ubiquitin-specific protease 22 (USP22) is a novel putative cancer stem cell marker involved in the 11-gene Polycomb/cancer stem cell signature (9). USP22 belongs to a large family of proteins with ubiquitin hydrolase activity and has been identified to be an important subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex, which is required for activator-driven transcription and cell cycle progression. Within hSAGA, USP22 removes ubiquitin from histone H2B, thus regulating the transcription of downstream genes associated with epigenetic alteration and cancer progression.
A number of previous studies have reported high expression of USP22 in specific malignant tumors, affecting the progression and prognosis of malignant tumors, including esophageal carcinoma and gastric, colorectal and breast cancer (10–13). To date, the expression of USP22 in human brain glioma cells and its role in cell growth has not been determined. In the present study, high expression of USP22 in human brain glioma cells was identified. RNA interference was used to silence the USP22 gene, leading to apoptosis of human brain glioma cells and induction of cell cycle arrest. In addition, USP22 has been reported to be required for the correct function of MYC, which is widely hypothesized to play a significant role in the regulation of the tumor cell cycle and tumor invasion (14). USP22 has also been demonstrated to inhibit transcription of the p21 gene by deubiquitinating the transcriptional regulator, FBP1, leading to cell proliferation and tumorigenesis (15).
The aim of the present study is to determine the in vitro effect of USP22 gene silencing by RNA interference on human brain glioma cell apoptosis and the cell cycle and to elucidate its molecular mechanism.
Materials and methods
Cell culture
Human brain glioma U87 and U251 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Carlsbad, CA, USA) containing 10% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin in an incubator containing 5% CO2 and 95% O2 at 37°C. Experimental analyses were performed when cells reached logarithmic growth phase.
The study was approved by the Ethics Committee of the China-Japan Union Hospital of Jilin University, Changchun, China.
Small interfering RNA (siRNA) transfection
Human brain glioma cells were inoculated in 6-well culture plates containing DMEM in the absence of antibiotics at a density of 5×105 cell/ml. Transfection was performed when the cells reached >60% confluence. siRNA and Lipofectamine 2000 were added according to the manufacturer's instructions, as described previously (Invitrogen, Carlsbad, CA, USA) (16). The USP22 siRNA sequence was as follows: sense 5′-CACGGACAGUCUCAACAAUTT-3′ and anti-sense 5′-AUUGUUGAGACUGUCCGUGTT-3′. Non-specific control siRNA was used as the control group. The efficiency of siRNA interference was determined by RT-PCR and western blot analysis.
RNA extraction and RT-PCR determination
Total RNA was extracted from each experimental group and RNA concentration was calculated according to the manufacturer's instructions (RNAisoTM Plus; Takara Bio, Inc., Shiga, Japan). RT-PCR was performed using a RT-PCR kit obtained from Takara Bio, Inc. and performed in accordance with the manufacturer's instructions. USP22 and β-actin primers were synthesized by Invitrogen Life Technologies (Carlsbad, CA, USA). The primers for amplification were as follows: USP22, 5′-GGACAACTGGAAGCAGAACC-3′ (forward) and 5′-TGAAACAGCCGAAGAAGACA-3′ (reverse); β-actin, 5′-CTGGGACGACATGGAGAAAA-3′ (forward) and 5′-AAGGAAGGCTGGAAGAGTGC-3′ (reverse). Reaction conditions were as follows: 94°C for 2 min followed by 34 cycles of degradation at 94°C for 30 sec, annealing at 61°C for 30 sec and extension at 72°C for 30 sec. PCR products were subjected to electrophoresis on a 1.0% agarose gel, then scanned and analyzed with a gel imaging system.
Western blot analysis
Cells of all experimental groups were collected using a cell scraper and 2 ml lysis solution (50 mM Tris-HCl, 137 mM NaCl, 10% glycerin, 100 mM sodium vana-date, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% NP-40 and 5 mM cocktail containing protease inhibitors; pH 7.4) was added to extract proteins. Protein concentation was determined by the BCA method rationing and separated by SDS-PAGE. Next, proteins were transferred to PVDF membranes using the semi-dry method and blocked overnight using 5% skimmed milk powder at 4°C. Membranes were washed with TBST and incubated for 1 h at 37°C with primary antibodies against target proteins, followed by an additional TBST wash. Membranes were incubated with appropriate secondary antibodies for 1 h at 37°C and washed with TBST. Antibody reactions were developed by color reaction for 5 min using autoradiography and Quantity One (Bio-Rad, Hercules, CA, USA) was used to perform optical density analysis and quantification.
Cell viability
Cell viability was determined using the MTT method. Cells (2×104 cell/well) were cultured in 96-well culture plates 1 day prior to siRNA transfection. Following adherence to the culture plates, cells were transfected with USP22 and control siRNA on day 2. MTT solution (5 mg/ml; 20 μl) was added to each well and cells were cultured in a CO2 incubator for 4 h. Following this, the culture solution was removed and 150 μl DMSO was added to each well and agitated at room temperature for 10 min. OD values of each well were measured using a microplate reader. Analysis for each experimental group was performed in six double wells. Average values were calculated and the growth curves were constructed.
Test of apoptosis with flow cytometry
Cells from all experimental groups were digested in 0.25% trypsin and resuspended in PBS to prepare single cell suspensions. Cell density was adjusted to 1×106 cell/ml. Next, 5 μl Annexin V-FITC and 5 ml PI were added and cells were incubated for 30 min at 4°C prior to analysis by flow cytometry.
Cell cycle analysis
Cells were collected using the trypsin method and washed 3 times with PBS. Following this, cells were fixed at 4°C using 75% cold ethanol overnight. Ethanol was removed and cells were washed 3 times with PBS. Cell density was adjusted to 1×106 cells/ml at a final volume of 100 μl DNAStain comprehensive dye liquor (500 ml; with final concentrations of 50 mg/l RNase, 100 mg/l PI and 1 mg/l Triton X-100) for storage at room temperature in the dark for 15 min prior to analysis by flow cytometry.
Statistical analysis
SPSS 16.0 statistical software was used for statistical analysis. Values are presented as mean ± SD. Statistical analysis was performed using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
USP22 gene expression in human brain glioma cells
USP22 gene expression was analyzed by RT-PCR. USP22 mRNA was highly expressed in U87 and U251 cells of human brain glioma and USP22 mRNA expression was higher in U87 cells. In addition, western blot analysis of USP22 protein expression identified that protein levels in U87 and U251 cells were consistent with those of USP22 mRNA. The results indicate that USP22 mRNA and protein are expressed at high levels in U87 and U251 cells of human brain glioma (Fig. 1).
RNA interference-mediated USP22 gene silencing in human brain glioma cells
RNA interference was performed by transfecting U87 and U251 cells with control and USP22 siRNA. Following transfection, RT-PCR and western blot analysis were performed to determine USP22 mRNA and protein expression levels. USP22 siRNA transfection (58 nM) for 24 h was observed to significantly reduce expression of USP22 mRNA and protein in U87 and U251 cells (Fig. 2A–C). These results indicate that USP22 siRNA transfection for 24 h effectively silenced USP22 gene and protein expression.
RNA interference-mediated USP22 gene silencing and growth inhibition of human brain glioma cells
To investigate whether RNA interference-mediated USP22 gene silencing affects the growth of human brain glioma cells, the MTT method was used to analyze the viability of U87 and U251 cells at 12, 24, 48, 72 and 96 h following transfection with USP22 siRNA. The results indicate that the viability of U87 and U251 cells was significantly reduced compared with the control and control siRNA groups. These observations indicate that USP22 gene silencing by RNA interference inhibits growth of human brain glioma cells (Fig. 3A and B).
RNA interference-mediated USP22 gene silencing induced apoptosis of human brain glioma cells
To investigate the molecular mechanism by which USP22 silencing inhibits human brain glioma cell growth, flow cytometry was used to determine the rate of cell apoptosis and western blot analysis was performed to analyze expression levels of apoptosis-related protein changes. Following RNA interference-mediated USP22 gene silencing (24 h), the apoptosis rate of U87 and U251 cells was found to increase significantly (Fig. 4A and B). In addition, protein expression levels of procaspase-9, -8 and -3 were markedly reduced (Fig. 4C and D). The results indicate that inhibition of human brain glioma cell growth by USP22 gene silencing may be relevant to cell apoptosis.
USP22 gene silencing resulted in cell cycle arrest of human brain glioma cells in the G2/M phase
To determine the molecular mechanism by which USP22 gene silencing leads to inhibition of human brain glioma cell growth, the cell cycle was analyzed using flow cytometry. The percentage of USP22 siRNA-transfected U87 and U251 cells entering the G2/M phase was significantly higher than that of the control siRNA group (Fig. 5A and B), indicating that the majority of the cells were arrested in the G2/M phase. In addition, expression of cell cycle proteins, including CDK1, CDK2, CyclinB1 and CyclinD1, were analyzed by western blot analysis. CDK1, CDK2 and CyclinB1 expression in USP22 siRNA-transfected U87 and U251 cells was markedly reduced. By contrast, the expression of CyclinD1 protein was unchanged (Fig. 6A and B). The results indicate that inhibition of human brain glioma cell growth by USP22 gene silencing may induce G2/M phase cell cycle arrest via the downregulation of CDK1, CDK2 and CyclinB1 protein expression.
Discussion
USP22 was recently identified as a novel human deubiquitinating enzyme. High expression levels of USP22 are used to predict the time of recurrence, distant metastasis and treatment failure and correlate with poor prognosis in a number of types of tumor (11,17,18). Previous studies have reported that USP22 silencing inhibits the proliferation of various tumor cells (19–21). However, little is known with regards to the effect of USP22 gene silencing in human brain glioma cells. Therefore, the present study aimed to determine the role of USP22 in human brain glioma and its molecular mechanism. RT-PCR and western blot analysis were performed, revealing high USP22 gene and protein expression in human brain glioma cells, and indicating that USP22 gene expression may represent a novel biological marker and treatment target for brain glioma.
To establish the effect of the USP22 gene on human brain glioma, small RNA interfering technology was utilized to silence USP22 gene expression in human brain glioma cells. Following this, the biological status of the brain glioma cells was analyzed. Following USP22 gene silencing, the replication rate of brain glioma cells was markedly reduced, indicating that the USP22 gene is involved in the regulation of human brain glioma cell proliferation. Previous studies have demonstrated that the USP22 gene promotes the proliferation of esophageal carcinoma (10) and lung (22) breast (13), colorectal (21) and bladder cancer cells (20). Consistent with these observations, in the present study, USP22 was demonstrated to play an important role in the regulation of human brain glioma cell proliferation.
However, the mechanism by which USPCC gene silencing leads to inhibition of human brain glioma cell proliferation remains unknown. Flow cytometry analysis revealed that human brain glioma cell apoptosis occurred following USP22 gene silencing. Previous studies have reported that USP22 gene silencing induced apoptosis of bladder (20) and colorectal cancer cells (21). Consistent with these results, in the current study, USP22 inhibition of human brain glioma cell proliferation was hypothesized to be markedly associated with apoptosis. In addition, protein expression of procaspase-9, -8 and -3 was found to be significantly reduced following USP22 gene silencing. The activation of members of the caspase family is an important prerequisite for apoptosis (23). The rate of cell apoptosis was also found to increase, indicating that USP22 gene silencing may induce this process in brain glioma cells. Analysis by flow cytometry revealed that USP22 gene silencing arrested human brain glioma cells in the G2/M phase. In addition, expression of cell cycle regulatory proteins, including CDK1, CDK2 and CyclinB1, was markedly reduced while no change in the expression of CyclinD1 was identified. These observations indicate that USP22 gene silencing leads to the reduction in the number of human brain glioma undergoing cell division and subsequent inhibition of cell replication.
In the present study, high expression of the USP22 gene in human brain glioma cells was identified for the first time and USP22 gene silencing was found to inhibit human brain glioma cell replication via induction of apoptosis and cell cycle arrest. These observations indicate that the USP22 gene may represent a novel molecular targeted approach for future treatment of brain glioma.
The authors thank Professor Yuzhuo Pan for technical assistance. The present study was supported by grants from the National Science Foundation of China (no. 30672159) and New Century Excellent Talents in Chinese Universities (NCET-06-0306).
Figure 1 USP22 gene and protein expression in human brain glioma cells. (A) USP22 mRNA expression via RT-PCR analysis. (B) USP22 protein expression via western blot analysis. (C) Histogram of USP22 gene expression levels. Each independent experiment was repeated 3 times. USP22, ubiquitin-specific protease 22.
Figure 2 RNA interference to silence USP22 gene expression in human brain glioma cells. U87 and U251 cells were transfected with USP22 siRNA for 24 h (A) RT-PCR and (B) western blot analysis to determine interference efficiency following transfection. USP22, ubiquitin-specific protease 22.
Figure 3 USP22 gene silencing inhibits human brain glioma cell growth. MTT was performed to analyze viability of (A) U87 and (B) U251 cells following transfection with USP22 siRNA. Each independent experiment was repeated 3 times. USP22, ubiquitin-specific protease 22.
Figure 4 USP22 gene silencing led to human brain glioma cell apoptosis. (A) Flow cytometry to analyze cell apoptosis rate. (B) Quantification of cell apoptosis rate (%) in U87 and U251 cells. (C) Western blot analysis of procaspase-9, -8 and -3 protein expression. (D) Quantification of procaspase-9, -8 and -3 protein expression (%). Each independent experiment was performed 3 times. P<0.05, vs. control. USP22, ubiquitin-specific protease 22.
Figure 5 USP22 gene silencing arrested human brain glioma cells in the G2/M phase. (A) Flow cytometry to analyze cycle changes in U87 and U251 cells. (B) Quantification of cell cycle changes (%). Each independent experiment was performed 3 times.*P<0.05, vs. control. USP22, ubiquitin-specific protease 22.
Figure 6 USP22 gene silencing downregulated the expression of CDK1, CDK2 and CyclinB1 protein. (A) Western blot analysis of protein expression of CDK1, CDK2, CyclinB1 and CyclinD1 proteins. (B) Quantification of protein expression of CDK1, CDK2, CyclinB1 and CyclinD1 (%). *P<0.05, vs. control. Each independent experiment was performed 3 times. USP22, ubiquitin-specific protease 22.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23613781PONE-D-13-0252610.1371/journal.pone.0061052Research ArticleBiologyModel OrganismsAnimal ModelsRatMolecular Cell BiologySignal TransductionSignaling in Cellular ProcessesApoptotic SignalingNeuroscienceDevelopmental NeuroscienceNeurogenesisNeurochemistryNeurochemicalsBehavioral NeuroscienceCellular NeuroscienceMolecular NeuroscienceNeurobiology of Disease and RegenerationMedicineMental HealthPsychiatryMood DisordersVeterinary ScienceAnimal ManagementAnimal BehaviorSuppression of Neuroinflammatory and Apoptotic Signaling Cascade by Curcumin Alone and in Combination with Piperine in Rat Model of Olfactory Bulbectomy Induced Depression Olfactory Bulbectomy Induced DepressionRinwa Puneet
1
Kumar Anil
1
*
Garg Sukant
2
1
Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh, India
2
Department of Pathology, Dr. Harvansh Singh Judge Institute of Dental Sciences and Hospital, Panjab University, Chandigarh, India
Scavone Cristoforo Editor
Universidade de São Paulo, Brazil
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Performed the experiments: PR. Analyzed the data: PR. Contributed reagents/materials/analysis tools: SG. Wrote the paper: AK PR.
2013 17 4 2013 8 4 e6105212 1 2013 5 3 2013 © 2013 Rinwa et al2013Rinwa et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Objectives
Bilateral destruction of the olfactory bulbs is known to cause behavioral changes analogous to symptoms of depression. Curcumin, a traditional Indian spice is currently being investigated in different psychiatric problems including depression. Dietary phytochemicals are currently used as an adjuvant therapy to accelerate their therapeutic efficacy. Therefore, the present study is an attempt to elucidate the neuroprotective mechanism of curcumin and its co-administration with piperine against olfactory bulbectomy induced depression in rats.
Methods
Rats undergone olfactory bulbs ablations were analyzed after post-surgical rehabilitation period of 2 weeks. Animals were then treated with different doses of curcumin (100, 200 and 400 mg/kg; p.o.), piperine (20 mg/kg; p.o.) and their combination daily for another 2 weeks. Imipramine (10 mg/kg; i.p.) served as a standard control. Various behavioral tests like forced swim test (FST), open field behaviour and sucrose preference test (SPT) were performed, followed by estimation of biochemical, mitochondrial, molecular and histopathological parameters in rat brain.
Results
Ablation of olfactory bulbs caused depression-like symptoms as evidenced by increased immobility time in FST, hyperactivity in open field arena, and anhedonic like response in SPT along with alterations in mitochondrial enzyme complexes, increased serum corticosterone levels and oxidative damage. These deficits were integrated with increased inflammatory cytokines (TNF-α) and apoptotic factor (caspase-3) levels along with a marked reduction in neurogenesis factor (BDNF) in the brain of olfactory bulbectomized (OBX) rats. Curcumin treatment significantly and dose-dependently restored all these behavioral, biochemical, mitochondrial, molecular and histopathological alterations associated with OBX induced depression. Further, co-administration of piperine with curcumin significantly potentiated their neuroprotective effects as compared to their effects alone.
Conclusions
The present study highlights that curcumin along with piperine exhibits neuroprotection against olfactory bulbectomy induced depression possibly by modulating oxidative-nitrosative stress induced neuroinflammation and apoptosis.
Authors gratefully acknowledge the financial support of the Indian Council of Medical Research (ICMR), New Delhi Funding channel no. 45/54/2010) for carrying out this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Depression is one of the severe psychiatric disorders and has been estimated as the second biggest contributor of the global load of neurological diseases and disability for the year 2020 [1]. It is characterized by low mood, loss of interest in daily activities and marked reduction in pleasure activities. Although satisfactory progress has been made in understanding the pathophysiology of the disease but still there is need of a more valid animal model that mimics the manifestations of clinical depression and related problems.
Olfactory bulbectomy (OBX) has been widely used as an experimental model of depression [2]. Bilateral destruction of the olfactory bulbs caused complex alterations in behavioral, biochemical and cellular cascades, many of which are comparable to those seen in patients with major depression [2]. Bulb ablation results in anhedonia like state in sucrose preference test [3], increased hyperactivity in a novel environment [4] and increased immobility time [2]. Furthermore, OBX has also been reported to alter neurogenesis in several regions of brain, which is one of the putative pathogenic mechanisms to explain depression [5]. Studies have proved that damage to the hippocampal neurons can be reversed by chronic antidepressant treatments [6]. Since OBX-induced depressive symptoms respond to chronic, but not acute antidepressant treatment, thus OBX is considered as one of the best available models to evaluate antidepressant activity [2]. Interestingly, these OBX induced changes are independent of anosmia [7]. Since the olfactory bulb projects into different regions of the brain (cortex, amygdala and hippocampus), thus ablation of these bulbs results in neurodegeneration in the projection areas [2], which possibly explains OBX-induced behavioral changes. Although a lot of work is being carried out in this field, yet the therapeutic responses of newer antidepressant drugs are still not fully understood and hence often produce undesirable side effects. The approach towards development of safe and powerful antidepressant agents from traditional herbs may be a good novel therapeutic strategy for the treatment of depression.
Dietary and medicinal phyto-antioxidants these days are used as combination therapy with each other in order to limit their side effects and to increase their effectiveness. Curcumin, a polyphenolic compound derived from dietary spice turmeric, possesses diverse pharmacological effects including antioxidant [8], anti-inflammatory [9], and neuroprotective activities [10]. Curcumin have previously been reported to possess antidepressant-like effects in different experimental models [11], [12]. Studies have shown that antioxidant activities of curcumin are comparable to those of vitamin C and E [13]. Manganese complexes of curcumin are proved to have great capacity to protect brain lipids against peroxidation [14]. Studies from our laboratory also suggest that curcumin restored mitochondrial dysfunction and various mitochondrial enzyme complex activities [15]. Studies have documented a significant attenuated effect of curcumin on pro-inflammatory cytokines (TNF-a) [16]. Curcumin has also been known to enhance the level of brain derived neurotrophic factor (BDNF) [17]. Earlier, curcumin is also reported to significantly reduce stress induced increase in serum corticosterone levels in rats [18].
Piperine is a major alkaloidal constituent of black pepper. It is a powerful inhibitor of hepatic and intestinal glucuronidation, and increases the bioavailability of many drugs including curcumin [19], [20]. Since curcumin has a poor absorption rate and undergoes rapid metabolism which severely curtail its bioavailability, thus piperine has been tried as drug strategy with curcumin in the present investigation.
Based on this background, the present study has been designed with the aim to elucidate the neuroprotective mechanism of curcumin and its interaction with piperine against OBX induced behavioral, biochemical, mitochondrial, molecular and histopathological alterations.
Materials and Methods
Ethics Statement
The experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of Panjab University (IAEC/282/UIPS/39 dated 30/8/12) and conducted according to the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines of Government of India on the use and care of experimental animals.
Experimental Animals
Adult male Wistar rats (200–240 g) procured from Central Animal House, Panjab University, Chandigarh were used. Male rats were chosen to avoid the influence of female estrogen hormone on depression-like behavior. Animals were housed under standard (25±2°C, 60–70% humidity) laboratory conditions, maintained on a 12 hour natural day–night cycle, with free access to food and water. Animals were acclimatized to laboratory conditions before the experimental tests.
Surgical Procedure (Olfactory Bulb Ablation)
Bilateral olfactory bulb ablation was performed as modified method described by different investigators [21]. Briefly, animals were anaesthetized with ketamine (75 mg/kg, i.p) and xylazine (5 mg/kg i.p) combination. All the surgical equipments were sterilized before use. The animals were fixed in a stereotactic frame (Stoelting Co., USA) and skull was exposed by a midline incision and burr holes (2 mm in diameter) were drilled 8 mm anterior to bregma and 2 mm on either side of the midline at a point corresponding to the posterior margin of the orbit of the eye. Both olfactory bulbs were removed by suction and holes were then filled with haemostatic sponge (AbGel, Absorbable gelatin sponge USP, Srikrishna Laboratories, India) and then scalp was stitched with absorbable sutures (Ethicon 4–0, Absorbable surgical sutures USP (Catgut), Johnson and Johnson, India). Sham-operated rats were treated in the same way, including piercing of the duramater but their bulbs were left intact. To prevent post surgical infection, the animals were received Sulprim injectionR (each ml containing 200 and 40 mg of sulphadiazine and trimethoprim respectively), intramuscularly (0.2 ml/300 g) once a day for 3 days of post-surgery. The OBX/Sham animals were housed singly in cages. The animals were then housed for two weeks (14 days) and given extra care to avoid aggressive behaviour, which might have developed otherwise. Drug treatments were started after a 14 days surgical rehabilitation period. Pictogram of the entire protocol is represented in Table 1.
10.1371/journal.pone.0061052.t001Table 1 Experimental protocol design for olfactory bulbectomy experimental model.
Surgery and treatment schedule Behavioral tests
0th day 0th–1st day 2nd–14th day 15th–28th day 29th day 30th day 31st day
Surgery Recovery from surgery (Continuous care) Rehabilitation period (Daily observation and handling) Drug/vehicle treatment Sucrose preference test Open field exploration test Forced swim test
Drugs and Treatment Schedule
Curcumin and imipramine were purchased from Sigma Chemicals (St. Louis, MO, USA). Piperine was purchased from CDH, India. ELISA kit for TNF-α and caspase-3 were purchased from R&D Systems, USA, while Chemikine™ Brain Derived Neurotrophic Factor (BDNF) kit was procured from Millipore (USA). All other chemicals used for biochemical and mitochondrial estimations were of analytical grade. The animals were randomly divided into nine experimental groups with twelve animals in each. Entire study was conducted in multiple phases. First and second group was named as sham and OBX (ablation of olfactory bulbs) control group respectively. Curcumin (100, 200 and 400 mg/kg, p.o.) were treated as group 3–5 respectively. Piperine (20 mg/kg; p.o.) served as group 6. Co-administration of curcumin (100 and 200 mg/kg; p.o.) with piperine (20 mg/kg; p.o.) was categorized as group 7 and 8 respectively. Imipramine (10 mg/kg; i.p.) served as group 9. Curcumin and piperine were prepared in peanut oil and imipramine was dissolved in distilled water. Drugs were administered orally on the basis of body weight (5 ml/kg) and drug solutions were made freshly at the beginning of each day of the study protocol. Drugs were then administered once daily for a period of two weeks.
Behavioral Assessments
Sucrose preference test
Rats were tested for sucrose consumption as described earlier [22]. Animals were housed individually throughout the test duration and presented two bottles simultaneously in the home cage, one containing a 1% w/v sucrose solution, and other containing standard drinking water during the 48 h training session. To prevent the preference to position, the location of the two bottles was varied during this period. After an 18 h period of food and water deprivation, an 8 h test session was conducted. The amount of liquid remaining in each bottle was measured at the end of the testing period. The sucrose preference score was expressed as percent of total liquid intake. Sucrose preference (SP) was calculated according to the following equation:. Where, SI = sucrose intake in grams and WI = water intake in grams.
Open field exploration
Open field behavior of rats was recorded in a circular arena of diameter 80 cm, surrounded by a 30 cm high wooden wall [23]. The arena painted white, was divided in to 25 small sections. Each rat was carefully placed in the centre of circular arena and allowed to explore the open field for 5 min. During this period, the ambulatory activity, in terms of the number of sections crossed, and the frequency of rearing was recorded along with defecation and licking episodes and values expressed as counts per 5 min.
Immobility period
Forced swim test was performed as described [24]. One day prior to the test, a rat was placed for conditioning in a clear plastic tank (45 cm×35 cm×60 cm) containing 30 cm of water (24±0.5°C) for 15 min (pretest session). Twenty-four hours later (test session); the total immobility period within a 5-min session was recorded as immobility scores (in sec). A rat was judged to be immobile when its hind legs were no longer moving and the rat was hunched forward (a floating position). The immobility time was recorded manually by an observer who was blind to the drug treatment.
Biochemical Estimations
Immediately after the last behavioral test, animals were randomized into different sets; one set was used for the biochemical assays (n = 5/group). For biochemical analysis, animals were sacrificed by decapitation. Whole brain of each animal was put on ice and weighed. A 10% (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000×g for 15 min and aliquots of supernatants were separated and used for biochemical estimation.
Lipid peroxidation
The extent of lipid peroxidation was determined quantitatively by performing the method as described by Wills [25]. The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA). The values were calculated using the molar extinction co-efficient of chromophore (1.56×10 M−1 cm−1).
Nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric acid) [26]. Equal volumes of the supernatant and the Greiss reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA). The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve.
Reduced glutathione
Reduced glutathione in the brain was estimated according to the method of Ellman et al. [27]. Homogenate (1 ml) was precipitated with 1.0 ml of 4% sulfosalicylic acid and the samples were immediately centrifuged at 1200×g for 15 min at 4°C. The assay mixture contained 0.1 ml of supernatant, 2.7 ml of phosphate buffer of pH 8.0 and 0.2 ml of 0.01 M dithiobisnitrobenzoic acid (DTNB). The yellow color developed was read immediately at 412 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The results were expressed as micromoles of reduced glutathione per milligram of protein.
Catalase
Catalase activity was determined by the method of Luck [28], wherein the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2, phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and the change in absorbance was recorded at 240 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The results were expressed as micromoles of H2O2 decomposed per milligram of protein/min.
Protein
The protein content was estimated by biuret method [29] using bovine serum albumin as a standard.
Mitochondrial Enzyme Complex Estimations
Second set of animals (n = 5/group) were used for mitochondrial enzyme complex activities as described by Berman and Hastings [30]. The whole brain was homogenized in isolated buffer. Homogenates were centrifuged at 13,000 g for 5 min at 4°C. Pellets were re-suspended in isolation buffer with ethylene glycol tetraacetic acid (EGTA) and spun again at 13,000 g at 4°C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 g at 4°C for 10 min. Pellets containing pure mitochondria were re-suspended in isolation buffer without EGTA.
Complex-I (NADH dehydrogenase activity)
Complex-I was measured spectrophotometrically by method of King and Howard [31]. The method involves catalytic oxidation of NADH to NAD+ with subsequent reduction in cytochrome c. The reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl glycine buffer and 10.5 mM cytochrome c. The reaction was initiated by addition of requisite amount of solubilised mitochondrial sample and followed absorbance change at 550 nm for 2 min.
Complex-II (Succinate dehydrogenase activity)
Complex-II was measured spectrophotometrically according to King [32]. The method involves oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of mitochondrial sample and absorbance change was followed at 420 nm for 2 min.
Complex-III (MTT activity)
The MTT assay is based on the reduction of (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-H-tetrazolium bromide (MTT) by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the mitochondrial respiratory chain in isolated mitochondria by the method of Liu et al. [33]. Briefly, 100 µl mitochondrial samples were incubated with 10 µl MTT for 3 h at 37°C. The blue formazan crystals were solubilised with dimethylsulphoxide and measured by an ELISA reader at 580 nm filter.
Complex IV (cytochrome c oxidase)
Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa [34]. The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample and the changes in absorbance were recorded at 550 nm for 2 min.
Serum Corticosterone Estimations
Preparation of serum
Blood was collected (1.0 ml) between 8.00–9.00 AM through retro orbital bleeding in the test tube and allowed to clot at room temperature. The tubes were then centrifuged at 2000 rpm for 10 min. The straw colored serum was separated and stored frozen at −20°C.
Corticosterone assessment
For extraction of corticosterone the method of Silber et al. [35] was modified as described. 0.1–0.2 ml of serum was treated with 0.2 ml of freshly prepared chloroform: methanol mixture (2∶1, v/v), followed by 3 ml of chloroform instead of dichloromethane used in the procedure of Silber and its group [35]. The step of treatment of petroleum ether was omitted. The samples were vortexed for 30 sec and centrifuged at 2000 rpm for 10 min. The chloroform layer was carefully removed with the help of syringe with a long 16 gauge needle attached to it and was transferred to a fresh tube. The chloroform extract was then treated with 0.1 N NaOH by vortexing rapidly and NaOH layer was rapidly removed. The sample was then treated with 3 ml of 30 N H2SO4 by vortexing vigorously. After phase separation, chloroform layer on top was removed using a syringe as described above and discarded. The tubes containing H2SO4 were kept in dark for 30–60 min and thereafter fluorescence measurements carried out in fluorescence spectrophotometer (make Hitachi, model F-2500) with excitation and emission wavelength set at 472 and 523.2 nm respectively. The standard curve depicting the fluorescence yield versus corticosterone concentration was used for result analysis.
Molecular Estimations
BDNF and TNF-α ELISA
The quantifications of BDNF and TNF-α were done with the help and instructions provided by Chemikine and R&D Systems immunoassay kits respectively. All samples were assayed in duplicate and absorbance was read on an ELISA plate reader (iMark™ Microplate absorbance reader, BIO-RAD) and the concentration of each sample was calculated by plotting the absorbance values on standard curve with known concentrations generated by the assay.
Caspase-3 colorimetric assay
Caspase-3, also known as CPP-32 is an intracellular cysteine protease that exists as a pro-enzyme, becoming activated during the cascade of events associated with apoptosis. The tissue lysates/homogenates can then be tested for protease activity by the addition of a caspase specific peptide that is conjugated to the color reporter molecule p-nitroaniline (pNA). The cleavage of the peptide by the caspase releases the chromophore pNA, which can be quantitated spectrophotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate/homogenate is directly proportional to the color reaction. The enzymatic reaction for caspase activity was carried out using R&D systems caspase-3 colorimetric kit.
Histopathology of Brain Tissue
Tissue sections preparation
Remaining animals were deeply anaesthetized and perfused transcardially via the ascending aorta with cold phosphate buffered saline (0.1 M, pH 7.4) followed by fixative solution containing 4% (w/v) paraformaldehyde in 0.1 M PBS solution (pH 7.4). The whole brain was dissected out and fixed overnight at 4°C in the same buffer containing 4% (w/v) paraformaldehyde. The brain was then washed with 0.1 M PBS (pH 7.4) for 1 h, dehydrated in alcohol, and then embedded in paraffin wax. Serial coronal sections (5 µm thickness) of whole brain were then obtained.
Hematoxylin and Eosin (H&E) staining
The paraffin sections of whole brain (thickness 5 µm) were dewaxed and rehydrated with alcohol for hematoxylin-eosin (H&E) staining. The neurons in CA1 region of hippocampus and frontal cortex were examined under electron microscopy and photomicrographs were prepared.
Statistical Analysis
Data are expressed as mean ± S.E.M. The data was analyzed by One-way ANOVA followed by Tukey's test. p<0.05 was considered as statistically significant. All statistical procedures were carried out using sigma stat Graph Pad Prism (Graph Pad Software, version 5, San Diego, CA).
Results
Effects of Curcumin, Piperine and their Combination on Sucrose Preference Test
Removal of olfactory bulbs caused significant reduction in sucrose consumption as compared to sham group. Curcumin (200, 400 mg/kg) significantly and dose dependently attenuated the reduction in sucrose consumption as compared to control (OBX) rats. Further, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) significantly potentiated the sucrose consumption as compared to their effects alone. The efficacy of the combination was comparable to that of imipramine (10 mg/kg) [F (11, 131) = 37.48 (p<0.01)] (Fig. 1).
10.1371/journal.pone.0061052.g001Figure 1 Effects of curcumin, piperine and their combination on sucrose preference test (SPT).
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effects of Curcumin, Piperine and their Combination in Open Field Performance Task
OBX rats exhibited a characteristic hyperactivity in the open field as depicted by increase in ambulation, rearing, defecation (number of fecal pellets) and reduction in grooming/licking episodes which was significant as compared to sham group. Curcumin (200, 400 mg/kg) treatment significantly reduced ambulation, rearing, defecation and improved grooming/licking episodes as compared to OBX group. Further, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) potentiated their protective effects on open field performance task which were significant as compared to their effects alone. The efficacy of the combination was comparable to that of imipramine (10 mg/kg). However, piperine (20 mg/kg) alone did not produce any significant effect on ambulation [F(11, 131) = 123.54 (p<0.01)], rearing [F(11, 131) = 152.14 (p<0.05)], grooming [F(11, 131) = 52.24 (p<0.05)] and defecation [F(11, 131) = 73.22 (p<0.05)] parameters as compared to OBX control (Table 2).
10.1371/journal.pone.0061052.t002Table 2 Effect of curcumin, piperine and their combination on open field performance task.
Treatment(mg/kg) Ambulation Rearing Grooming/lickingepisodes Number of fecal pellets
Sham
102.6±5.95 28.2±2.49 8.4±1.26 0.5±0.18
OBX control
173.5±7.50a
43.8±2.18a
1.8±0.37a
4.8±0.51a
OBX+ C1
162.8±3.26 38.4±1.56 2.6±0.56 4.1±0.20
OBX+ C2
139.2±3.96b
34.6±1.41b
4.8±0.45b,c
2.6±0.28b,c
OBX+ C3
115.4±3.56c,d
31.8±1.07b,c
6.8±0.21c,d
1.2±0.20c,d
OBX+ P
169.6±4.34 44.2±3.56 1.8±0.56 4.4±0.64
OBX+ C1+P
134.6±4.57c,e
35.4±3.24b,e
4.6±0.87c,e
2.3±0.14c,e
OBX+ C2+P
118.4±5.56d,e
31.1±3.13c,e
6.4±0.93d,e
1.0±0.42d,e
OBX+I
110.4±5.11b
29.6±0.93b
7.3±1.17b
0.8±0.22b
Values are expressed as mean ± SEM. For statistical significance,
a P<0.05 as compared to sham group;
b P<0.05 as compared to OBX control;
c P<0.05 as compared to OBX+C1;
d P<0.05 as compared to OBX+C2;
e P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effects of Curcumin, Piperine and their Co-administration on Immobility Period
There was a significantly increase in the immobility period of OBX group as compared to sham treatment. Curcumin (200, 400 mg/kg) treatment significantly shortened immobility time as compared to OBX control. Curcumin (100 mg/kg) did not produce any significant effect on immobility period as compared to OBX group. Further, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) potentiated their protective effects (shortened immobility period) which were significant as compared to their effects alone. In addition, the efficacy of the combination was comparable to that of imipramine (10 mg/kg) F(11, 131) = 50.04 (p<0.01)] (Fig. 2).
10.1371/journal.pone.0061052.g002Figure 2 Effect of curcumin, piperine and their co-administration on immobility period.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effect of curcumin, piperine and their co-administration on lipid peroxidation (MDA), reduced glutathione (GSH), nitrite concentration and catalase enzymes level.
Olfactory bulbs ablation caused significant oxidative damage as evidenced by rise in MDA and nitrite levels, depletion of reduced GSH and catalase levels as compared to sham group. Chronic treatment with curcumin (200, 400 mg/kg) significantly attenuated oxidative damage (reduced MDA, nitrite levels, restoration of reduced GSH and catalase levels) as compared to OBX control. Further, co-administration of curcumin (100, 200 mg/kg) with piperine (20 mg/kg) significantly potentiated their antioxidant like effect which was significant as compared to their effect alone. The efficacy of the combination was similar to that of imipramine (10 mg/kg). However, piperine (20 mg/kg) alone did not produce any significant effect on LPO [F(11, 54) = 62.35 (p<0.01)], GSH [F(11, 54) = 49.12 (p<0.05)], Nitrite [F(11, 54) = 82.30 (p<0.01)] and Catalase [F(11, 54) = 40.21 (p<0.05)] activity as compared to control (Table 3).
10.1371/journal.pone.0061052.t003Table 3 Effect of curcumin, piperine and their co-administration on lipid peroxidation (MDA), reduced glutathione (GSH), nitrite concentration and catalase enzymes levels.
Treatment(mg/kg) LPO (mol of MDA/mgpr)(% of sham) GSH (µmol of GSH/mgpr)(% of sham) Nitrite (µg/ml)(% of sham) Catalase (µmol of H2O2 hydrolysed/min/mgpr)(% of sham)
Sham
0.161±0.009 (100) 0.086±0.005 (100) 312±16.54 (100) 0.703±0.030 (100)
OBX control
0.530±0.025a (329.2) 0.023±0.003a (26.7) 770.8±13.96a (247.1) 0.211±0.024a (30.0)
OBX+ C1
0.442±0.035 (274.5) 0.031±0.002 (36) 683.5±11.84 (199.8) 0.233±0.045 (35.9)
OBX+ C2
0.379±0.020b,c (235.4) 0.045±0.005b (52.3) 532.6±9.23b,c (170.7) 0.363±0.060b,c (51.6)
OBX+ C3
0.263±0.018c,d (163.4) 0.064±0.015c,d (74.4) 428.8±10.47c,d (150.3) 0.498±0.030c,d (58.0)
OBX+ P
0.506±0.030 (314.3) 0.028±0.0055 (32.6) 712.8±12.45 (228.5) 0.224±0.024 (31.9)
OBX+ C1+P
0.365±0.015c,e (226.7) 0.044±0.0025c,e (51.2) 545.4±15.78c,e (174.8) 0.354±0.043c,e (50.4)
OBX+ C2+P
0.274±0.015d,e (170.2) 0.062±0.0045d,e (67.4) 422.5±13.12d,e (154.6) 0.486±0.063d,e (60.6)
OBX+ I
0.176±0.022b (109.3) 0.079±0.0039b (91.9) 373±8.62b (119.6) 0.573±0.027b (77.2)
Values are expressed as mean ± SEM. For statistical significance,
a P<0.05 as compared to sham group;
b P<0.05 as compared to OBX control;
c P<0.05 as compared to OBX+C1;
d P<0.05 as compared to OBX+C2;
e P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effects of Curcumin, Piperine and their Co-administration on Mitochondrial Enzyme Complex Activity
There was a significant alteration in mitochondrial enzyme complex (I, II) activities (Fig. 3) and decreased number of viable cells (complex III) and levels of cytochrome C oxidase enzyme (complex IV) (Fig. 4) in OBX rats as compared to the sham group. Curcumin (200, 400 mg/kg) treatment significantly restored mitochondrial enzyme complex I, II activities and improved number of viable cells and levels of cytochrome C oxidase enzyme as compared to OBX control. However, curcumin (100 mg/kg) did not produce any significant effect on mitochondrial enzyme complex activities. Further, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) significantly potentiated its protective effects which was also significant as compared to their effects alone. The efficacy of the combination was comparable to that of imipramine (10 mg/kg). In addition, piperine (20 mg/kg) alone did not produce any significant effect on mitochondrial enzyme complex I [F(11, 54) = 30.88 (p<0.01)], II [F(11, 54) = 42.32 (p<0.05)], III [F(11, 54) = 38.0 (p<0.05)] and IV [F(11, 54) = 17.4 (p<0.05)] activities as compared to control.
10.1371/journal.pone.0061052.g003Figure 3 Effect of curcumin, piperine and their co-administration on mitochondrial enzyme complex I and II activities.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
10.1371/journal.pone.0061052.g004Figure 4 Effect of curcumin, piperine and their co-administration on mitochondrial enzyme complex III and IV activities.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effect of Curcumin, Piperine and their Co-administration on Serum Corticosterone (CORT) Levels
OBX group of animals showed significant increase in serum CORT levels as compared to sham group. However, chronic treatment with curcumin (200 and 400 mg/kg) significantly attenuated the serum CORT levels as compared to OBX control. In-addition, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) significantly attenuated serum CORT levels which was also significant as compared to their effects alone. The efficacy of the combination was comparable to that of imipramine (10 mg/kg) [F(11, 54) = 177.0 (p<0.01)] (Fig. 5).
10.1371/journal.pone.0061052.g005Figure 5 Effect of curcumin, piperine and their co-administration on serum CORT levels.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effect of Curcumin, Piperine and their Co-administration on Brain Tissue Necrosis Factor (TNF-α) and Caspase 3 Level
OBX rats showed significant increase in TNF-α and caspase 3 levels as compared to sham group. Curcumin treatment (200 and 400 mg/kg) significantly attenuated the levels of TNF-α and caspase 3 as compared to OBX control. However, curcumin (100 mg/kg) did not produce any significant effect on TNF-α and caspase 3 levels as compared to OBX group. Further, co-administration of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) potentiated its protective effect (lowered TNF-α and caspase 3 levels) which was significant as compared to their effects alone. Further, the efficacy of the combination was comparable to that of imipramine (10 mg/kg). In addition, piperine (20 mg/kg) alone did not produce any significant effect on TNF-α [F(11, 54) = 47.14 (p<0.05)] (Fig. 6) and caspase 3 levels [F(11, 54) = 38.93 (p<0.01)] (Fig. 7) as compared to control.
10.1371/journal.pone.0061052.g006Figure 6 Effect of curcumin, piperine and their co-administration on TNF- α activity.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
10.1371/journal.pone.0061052.g007Figure 7 Effect of curcumin, piperine and their co-administration on caspase 3 activity.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effects of Curcumin, Piperine and their Combination on Brain Derived Neurotrophic Factor (BDNF)
There was a significant reduction in BDNF levels of OBX animals as compared to sham group. Curcumin (200 and 400 mg/kg) treatment significant restored BDNF level as compared to OBX control. However, curcumin (100 mg/kg) treatment did not produce any significant effect on BDNF levels as compared to OBX control. Besides, combination of piperine (20 mg/kg) with curcumin (100, 200 mg/kg) significantly potentiated the protective effect (elevated BDNF level) as compared to their effects alone. Further, the efficacy of the combination was comparable to that of imipramine (10 mg/kg) [F(11, 54) = 50.99 (p<0.01)] (Fig. 8).
10.1371/journal.pone.0061052.g008Figure 8 Effect of curcumin, piperine and their co-administration on BDNF levels.
Values are expressed as mean ± SEM. For statistical significance, a
P<0.05 as compared to sham group; b
P<0.05 as compared to OBX control; c
P<0.05 as compared to OBX+C1; d
P<0.05 as compared to OBX+C2; e
P<0.05 as compared to OBX+P (One-way ANOVA followed by Tukey’s test). OBX, Olfactory Bulbectomy; C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (400 mg/kg); P, piperine (20 mg/kg); I, imipramine (10 mg/kg).
Effects of curcumin, piperine and their combination on histopathological changes in cerebral cortex and hippocampal CA1 region
Brain histology of sham animals showed typical histopathological structures of neurons in the cerebral cortex and CA1 region of the hippocampus. Removal of bulbs significantly enhanced levels of neuroinflammatory cells along with their degeneration (apoptosis) resulting in decreased cell density in both cerebral cortex and hippocampal CA1 region as compared with the sham group. However, treatment with curcumin (200 mg/kg) and (400 mg/kg) (not shown) attenuated these histological abnormalities. Combination of piperine (20 mg/kg) with curcumin (200 mg/kg) further restored histological alterations of neuronal cells as compared to curcumin (200 mg/kg) alone (Fig. 9).
10.1371/journal.pone.0061052.g009Figure 9 Representative photomicrographs of cerebral cortex (A) and hippocampal CA1 region (B) of rat brain sections.
Sections were stained with Haematoxylin and Eosin. Black arrows indicate neuroinflammation along with apoptotic cells 1: sham control: neurons are intact 2: OBX control: mild-moderate infiltration of inflammatory cells with large number of apoptotic cells 3: treated with curcumin (200 mg/kg): slight inflammation of neurons with less apoptotic cells 4: treated with curcumin (200 mg/kg) +piperine (20 mg/kg): neurons are preserved. (HE stain×250).
Discussion
Olfactory bulbectomy (OBX) is a well established and widely used experimental model for studying the pathogenetic-mechanism of depression [2]. Ablation of olfactory bulbs generate many biochemical and cellular alterations that are similar to clinically depressed patients [36] and can be restored by several pharmaco-therapeutic interventions [2].
In the present study, OBX rats exhibited a characteristic hyperactivity in the open field paradigm and increased immobility time in the forced swim test, predicting symptoms associated with model of depression. These behavioral findings are very much in conformity with the previous studies on OBX rats [2]. Further, chronic administration of curcumin significantly and dose dependently reduced the immobility time in OBX treated rats. Similarly, curcumin also attenuated the hyperactivity associated with open field in OBX rats. The protective effect of curcumin was comparable to that of imipramine, which served as a positive control in the experiment. These behavioral observations are in agreement with the previous work examining the effects of chronic antidepressant treatment in OBX rats [37]. OBX model may also mimic different psychiatric symptoms found in clinically depressed patients. One of such symptoms i.e., anhedonia (loss of interest or pleasure) is a characteristic feature of endogenous depression [3]. Sucrose preference is regarded as an indicator of anhedonia-like condition [38]. In the present investigation OBX rats showed significant reduction in sucrose preference when compared to sham group, indicating a state of anhedonia. Further, curcumin in a dose dependent manner significantly restored the decrease in sucrose preference; thereby showing its antidepressant-like effects. This result is consistent with the reports from previous studies [4]. All these behavioral tests respond selectively to chronic curcumin treatment thus mimicking the clinical time course of antidepressant action.
Chronic stress activates hypothalamic–pituitary–adrenal (HPA) axis [39] and increases the levels of blood corticosterone in rats [40], similar to the human cortisol [41]. In our study, chronic stress induced by removal of olfactory bulbs resulted in significant rise in the serum corticosterone levels indicating hyperactivity of the HPA-axis [42]. Since increase in the corticosterone levels may lead to the behavioral alterations including depression-like symptoms [43], therefore it is possible to suggest that OBX-induced behavioral alterations observed in present study may be due to the increased levels of the serum corticosterone. However, chronic treatment with curcumin restored the elevated levels of serum corticosterone in OBX rats. These results are in accordance with the previous finding which showed that chronic curcumin administration attenuates stress-associated increased serum corticosterone levels [18].
Oxidative stress is an emerging focus of research, and plays a crucial role in the pathogenesis of depression [44]. Ablation of olfactory bulbs is reported to be associated with production of oxygen reactive species and saturation of antioxidant enzymes [45]. A significant increase in lipid peroxidation and marked decrease in the activity of reduced glutathione and antioxidant enzymes in the brain of OBX rats were found [45]. Studies on patients suffering from depression have shown decreased lipid peroxidation and antioxidant enzymes levels which returned to normal after antidepressant treatment [46]. Similarly, in the present study OBX animals also showed a significant increase in lipid peroxidation and a marked decrease in the activity of reduced glutathione and catalase enzyme. Curcumin attenuated lipid peroxidation [47] and restored endogenous antioxidant profile [48] showing its powerful radical scavenging property [49]. In this study, OBX rats showed a significant increase in nitrite levels thereby implicating endogenous brain nitric oxide in the neurobiology of depression [50]. This is further confirmed from clinical reports of depressed patients showing an elevated plasma nitrate levels [51]. Further, curcumin significantly reduced nitrosative stress by inhibiting elevated nitrite levels in brains of OBX rats. Curcumin has also been reported to inhibit iNOS expression [49] and particularly scavenge NO-based radicals [52], thereby signifying its potential against increased nitrite levels. The beneficial effects of curcumin against both oxidative and nitrosative stress are in accordance with the previous findings from our laboratory [53].
Mitochondrial oxidative damage is based on the fact that mitochondrial respiratory chain is the major sources of superoxide anion (O2
+) generation [54]. Energy production in mitochondria is catalysed by protein complexes, namely NADH-ubiquinol oxidoreductase (complex-I), succinate-ubiquinol oxidoreductase (complex-II), ubiquinol cytochrome c oxidoreductase (complex-III) and cytochrome C oxidase (complex IV) [54]. In the present study OBX caused impairment in different mitochondrial enzymes complex activities. These results are in accordance with the previous findings from our laboratory [15]. Besides, mitochondrial impairment may generate excess of nitric oxide (NO) which further leads to oxidative damage [55]. Moreover, evidence suggests that mitochondria progressively gets damaged and loses their functional integrity due to reactive oxygen species (ROS) which thereby enhances oxidative damage [56]. This suggests that mitochondria dysfunction might be the key factor for the production of ROS which further leads to oxidative damage in OBX induced depression.
In addition to oxidative and nitrosative stress, OBX induced depression is also linked to the generation of inflammatory cytokines like TNF-α [57]. In our study we found elevated levels of TNF-α following OBX suggesting inflammatory reaction accompanied by neuronal damage [58]. This was later attenuated by chronic curcumin treatment in a dose-dependent manner which attributes to its potent anti-inflammatory properties [59]. Our findings are supported by observations from Cho et al. [16] who found a significant reduction of inflammatory cytokines (TNF-α) on treatment with curcumin. Apart from increased neuroinflammation, we also found significant enhancement in levels of apoptotic factor, caspase-3 in OBX animals, suggesting a role of apoptotic pathway in OBX-induced depression. Our findings are in concurrence with Hall and Macrides [60], who found neuronal cell death in different brain regions following olfactory bulbectomy. We found that treatment with curcumin significantly inhibited caspase-3 activity in OBX rats, which is further supported by studies from Bharti et al. [61]. These results are further evidenced through histopathological studies which show presence of a large number of inflammatory and apoptotic cells leading to mild-moderate neurodegeneration in OBX rats. Studies from Nesterova et al. [62] reported a significant degeneration in neurons of the temporal cortex and hippocampus following olfactory bulbectomy. Curcumin later showed marked improvement in the histopathology of neurons in both cortex and hippocampal neurons of OBX animals thereby displaying its neuroprotective effects.
The neurotrophin hypothesis of depression predicts that a downregulation of brain-derived neurotrophic factor (BDNF) is involved in the pathogenesis of depression [63]. Furthermore, studies from Koo et al. [5] have demonstrated a decreased brain neurogenesis in OBX animals. In the present investigation, OBX rats showed a significant decrease in BDNF levels, thereby showing a reduced neurogenesis, a putative pathogenic mechanism in depression. However, curcumin treatment stimulated neurogenesis and expression levels of the neurotrophic factors BDNF in OBX rats. These results are consistent with findings from other laboratories [64]. Thereby it can be suggested that behavioral alterations observed after OBX (decreased sucrose preference, hyperactivity in open field and increased immobility period) are due to neurodegeneration of various brain structures [65], [66].
Poor oral bioavailability of curcumin limits its therapeutic utility. Curcumin undergoes extensive reduction through alcohol dehydrogenase, followed by conjugations at various tissue sites mainly in liver and intestine [67]. Since piperine is a well known inhibitor of hepatic and intestinal glucuronidation therefore it is reported to increase the bioavailability of several drugs including curcumin [20]. In the present investigation, we witnessed a profound increase in neuroprotection effects of curcumin in combination with piperine, in olfactory bulbectomized animals indicating that piperine might have increased the bioavailability of curcumin. In addition to its inhibitory effect on hepatic and intestinal glucuronidation, piperine has also been reported to have many other actions viz; anti-depressant [68], anti-apoptotic [69], anti-inflammatory [70], anti-oxidative [71] and neuroprotectant [72]. However, in our study piperine per se did not produce any neuroprotective effect which might be due to difference in the dose, duration of treatment and pathologies of different experimental models involved in depression. Further, beneficial effects of piperine per se treatment against severe neurodegeneration and behavioral deficits associated with olfactory bulbectomy rat model have not been reported so far. To the best of our knowledge, this is also the first study which reports the inhibitory effect of piperine on hepatic and intestinal glucuronidation in olfactory bulbectomy rat model. In this study we unleashed the benefits of the combination by assessing significant changes in behavioral, biochemical, mitochondrial, molecular and histopathological parameters. The strength of this study lies in the fact that the combination of piperine and curcumin were strongly neuroprotective in assessment of different parameters of the study.
In conclusion, the findings of the present study raised the possibility that oxidative-nitrosative stress-mediated inflammatory cascade may have resulted in activation of apoptotic signaling pathways and contributed to the neurodegeneration associated with depression like symptoms in rat model of olfactory bulbectomy. Curcumin on the other hand showed anti-apoptotic, neuroprotective actions due to its multiple effects viz strong anti-inflammatory, radical scavenging and neuromodulating properties. Piperine on the other hand proved to be a potent bioavailability enhancer and to a greater extent has resolved the problems of intestinal degradation related to curcumin. However, further studies are required to fully understand the curcumin’s action in neurodegenerative process associated with depression and to establish its clinical effectiveness in patients suffering from depression and related disorders. Together, co-administration of curcumin and piperine may provide a useful natural adjuvant in the antidepressant therapy.
==== Refs
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==== Front
Front Cell Neurosci
Front Cell Neurosci
Front. Cell. Neurosci.
Frontiers in Cellular Neuroscience
1662-5102
Frontiers Media S.A.
23616746
10.3389/fncel.2013.00043
Neuroscience
Original Research
Suppression of epileptogenesis-associated changes in response to seizures in FGF22-deficient mice
Lee Clara H. 1
Umemori Hisashi 1 2 *
1 Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School Ann Arbor, MI, USA
2 Department of Biological Chemistry, University of Michigan Medical School Ann Arbor, MI, USA
Edited by: Juan P. Henríquez, Universidad de Concepcion, Chile
Reviewed by: Michele Simonato, University of Ferrara, Italy; Ursula Wyneken, Universidad de los Andes, Chile
*Correspondence: Hisashi Umemori, Department of Biological Chemistry, Molecular and Behavioral Neuroscience Institute, University of Michigan Medical School, Room 5065, BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA. e-mail: [email protected]
18 4 2013
2013
7 4326 12 2012
29 3 2013
Copyright © 2013 Lee and Umemori.
2013
https://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.
In the developing hippocampus, fibroblast growth factor (FGF) 22 promotes the formation of excitatory presynaptic terminals. Remarkably, FGF22 knockout (KO) mice show resistance to generalized seizures in adults as assessed by chemical kindling, a model that is widely used to study epileptogenesis (Terauchi et al., 2010). Repeated injections of low dose pentylenetetrazol (PTZ) induce generalized seizures (“kindled”) in wild type (WT) mice. With additional PTZ injections, FGF22KO mice do show moderate seizures, but they do not kindle. Thus, analyses of how FGF22 impacts seizure susceptibility will contribute to the better understanding of the molecular and cellular mechanisms of epileptogenesis. To decipher the roles of FGF22 in the seizure phenotype, we examine four pathophysiological changes in the hippocampus associated with epileptogenesis: enhancement of dentate neurogenesis, hilar ectopic dentate granule cells (DGCs), increase in hilar cell death, and formation of mossy fiber sprouting (MFS). Dentate neurogenesis is enhanced, hilar ectopic DGCs appeared, and hilar cell death is increased in PTZ-kindled WT mice relative to PBS-injected WT mice. Even in WT mice with fewer PTZ injections, which showed only mild seizures (so were not kindled), neurogenesis, hilar ectopic DGCs, and hilar cell death are increased, suggesting that mild seizures are enough to induce these changes in WT mice. In contrast, PTZ-injected FGF22KO mice do not show these changes despite having moderate seizures: neurogenesis is rather suppressed, hilar ectopic DGCs do not appear, and hilar cell death is unchanged in PTZ-injected FGF22KO mice relative to PBS-injected FGF22KO mice. These results indicate that FGF22 plays important roles in controlling neurogenesis, ectopic migration of DGCs, and hilar cell death after seizures, which may contribute to the generalized seizure-resistant phenotype of FGF22KO mice and suggests a possibility that inhibition of FGF22 may alleviate epileptogenesis.
fibroblast growth factor 22
temporal lobe epilepsy
neurogenesis
hilar ectopic dentate granule cells
hilar cell death
mossy fiber sprouting
==== Body
pmcIntroduction
Epilepsy is one of the most common neurological disorders, which is characterized by recurrent seizures. Currently, the only available pharmacological treatments for epilepsy are seizure suppressants. These anticonvulsants may control seizures with various success rates among the different forms of epilepsy. Temporal lobe epilepsy (TLE), which is the most common type of epilepsy in adults, is one of the most refractory epilepsies—about one-third of patients are resistant to pharmacological treatments (Semah et al., 1998; Brodie, 2005; French, 2007). Not only are seizure suppressants often ineffective against intractable epilepsy, they only address the symptoms of the disease and neither prevent its initial development nor stop its progression (McNamara, 1994; Brodie, 2005; French, 2007). In order to develop effective treatment of epilepsy, further understanding of the epileptogenic mechanisms is required.
The hippocampus is one of the epileptogenic regions (foci) in TLE and has been a target region for studies on cellular mechanisms of epileptogenesis (McNamara, 1994; Morimoto et al., 2004). Brain insults such as trauma, seizure, stroke, and infection may cause various changes in the hippocampus, which may eventually result in TLE (Pitkänen and Lukasiuk, 2011). Many possible mechanisms have been proposed that might be involved in the process of epileptogenesis (Simonato et al., 2006; Pitkänen and Lukasiuk, 2011), including four changes in the hippocampus that have been widely accepted to be associated with epileptogenesis (Dudek and Sutula, 2007; Parent, 2007): (1) increased neurogenesis in the dentate gyrus (DG), (2) hilar ectopic dentate granule cells (DGCs), (3) loss of hilar cells (interneurons and mossy cells), and (4) formation of mossy fiber sprouting (MFS). These changes are proposed to be induced by brain insults, which may result in the rewiring of the hippocampal network to establish a possible epileptic circuitry (McNamara, 1994; Coulter, 2001; Morimoto et al., 2004; Rakhade and Jensen, 2009; Kokaia, 2011). Increased neurogenesis and hilar ectopic DGCs may contribute to abnormal incorporation of DGCs into the circuitry (Parent et al., 2006; Jessberger et al., 2007; Walter et al., 2007; Kron et al., 2010), loss of hilar cells may disrupt existing physiological balance in the network (Dudek and Sutula, 2007; Jiao and Nadler, 2007), and MFS, where sprouted mossy fibers form synapses onto DGCs themselves, may induce recurrent excitation of the DGCs (Sutula et al., 1989; Koyama and Ikegaya, 2004; Morimoto et al., 2004). All of the four mechanisms could cause hyperexcitation of the brain by rearranging the circuitry of the hippocampus. Therefore, understanding of the molecular mechanisms that are responsible for these epileptogenesis-associated events may help identify possible drug targets for the treatment of epilepsy.
Growth factors such as neurotrophins and fibroblast growth factors (FGFs) are suggested to be involved in epileptogenesis (Simonato et al., 2006). For example, overexpression of FGF2 increases seizure susceptibility and reduces seizure-induced cell death (Zucchini et al., 2008). Application of BDNF and FGF2 reduces MFS and spontaneous seizures (Paradiso et al., 2009, 2011). MFS arises in the absence of FGF7 (Lee et al., 2012). Thus, growth factors may be crucial molecules to regulate epileptogenesis. We have recently identified a member of FGFs, FGF22 as a molecule that organizes excitatory presynaptic differentiation in the hippocampus (Umemori et al., 2004; Terauchi et al., 2010). Remarkably, we found that FGF22 knockout (KO) mice are resistant to generalized seizures induced by chemical kindling with repeated injections of low dose pentylenetetrazol (PTZ), a GABA receptor antagonist. We therefore asked whether the four epileptogenesis-associated changes—increased DG neurogenesis, hilar ectopic DGCs, hilar cell loss, and MFS—are induced in the hippocampus of FGF22KO mice in response to PTZ injections, in order to get insight into the cellular basis of their seizure-resistant phenotype. We here show that in FGF22KO mice, these changes do not occur, even with moderate seizures after prolonged PTZ injections. Our results suggest that inactivation of FGF22 may increase resistance to epileptogenesis-associated changes in response to seizures in the hippocampus and that FGF22 could be a potential target for treatment of epilepsy.
Materials and methods
Animals
FGF22KO mice were described previously (Terauchi et al., 2010). FGF22KO mice have been backcrossed with C57BL/6 for more than 15 generations. Five-month-old FGF22KO mice and C57BL/6 mice [wild type (WT)] were used in this study. Genotypes of the mice were verified by genomic PCR. Male mice were used. The numbers of animals used in this study are shown in Figure 1B. All animal use and care was in accordance with the institutional guidelines and was approved by the University Committee on Use and Care of Animals.
Figure 1 Characteristics of the three animal groups used in this study: non-kindled WT mice with mild seizures, kindled WT mice, and non-kindled FGF22KO mice with moderate seizures. WT and FGF22KO mice were injected with a GABA receptor antagonist pentylenetetrazol (PTZ; 35 mg/kg) every 48 h to induce seizures. Control animals received PBS. (A) Typical time course of seizure development and three animal groups. Group 1: WT mice were injected until they first showed a seizure greater then level 1 (~14 PTZ injections); Group 2: WT mice were injected until they were kindled (~24 PTZ injections); Group 3: FGF22KO mice were injected with PTZ for 37 times—they showed moderate seizures but did not kindle. (B) Summary of characteristics of three animal groups. For each mouse group, the genotype, the highest seizure level, kindled or not, the cumulative seizure score, and the number of animals are shown. The highest seizure level indicates the most severe seizure level observed over the entire injection period. Group 1 mice showed only mild seizures (one level 3 seizure), Group 2 mice showed severe seizures (level 6 = kindled), and Group 3 mice showed mild to moderate seizures (level 3–4). The cumulative seizure score is the sum of seizure levels that the mouse has experienced throughout the injection period. Group 1 mice received considerably less cumulative seizures relative to Groups 2 and 3, which received comparable amount of cumulative seizures to each other.
Kindling and animal groups
PTZ (35 mg/kg), a GABA receptor antagonist, was injected intraperitoneally every 48 h in WT and FGF22KO mice to induce seizures. Control animals received PBS. The mice were observed for an hour after each PTZ injection, and the maximum level of seizure within the hour was recorded. The seizure responses were noted on a revised Racine's scale for PTZ kindling (Lüttjohann et al., 2009) as follows: Stage 0: No seizure observed; Stage 1: Sudden behavioral arrest and/or motionless staring; Stage 2: Facial jerking with muzzle or muzzle and eye; Stage 3: Neck jerks; Stage 4: Clonic seizure in a sitting position; Stage 5: Convulsions including clonic and/or tonic–clonic seizures while lying on the belly and/or pure tonic seizures; Stage 6: Convulsions including clonic and/or tonic–clonic seizures while lying on the side and/or wild jumping. Mice were defined as “kindled” when they displayed either one class 6 seizure or two successive class 5 seizures. We prepared three animal groups (the number of mice in each group is shown in Figure 1B) that are different in genotype and seizure levels for the study (see Figure 1). Group 1: WT mice with mild seizures—WT mice were injected until they first showed a seizure greater then level 1 (average PTZ injections = 14); Group 2: WT mice with kindled seizures—WT mice were injected until they were kindled (average PTZ injections = 24); Group 3: FGF22KO mice with moderate seizures (not kindled)—FGF22KO mice were injected with PTZ 37 times; the mice showed moderate seizures but did not kindle.
Brain sectioning
The mice were perfused with 4% paraformaldehyde (PFA). The brains were dissected and post-fixed in 4% PFA for 24 h. The brains were then placed in 30% sucrose solution for 48 h, frozen, and stored at −80°C. The sections from the same region of the hippocampus were used (4–6 sections/mouse) in this study in order to standardize quantification of the cell numbers. Sections between 2.5 and 3.5 mm from the midline were used for the study. Each section was 20 μm thick.
Immunohistochemistry
The sections were blocked in 2% BSA, 2% normal goat serum, and 0.1% TritonX-100 for 30 min, followed by the incubation with primary antibodies for 2 h at room temperature. Secondary antibodies were then applied for 1 h at room temperature, and slides were mounted with p-phenylenediamine. Antibodies used were: DCX (Abcam, ab18723; dilution 1:500), Ki67 (Abcam, ab15580; dilution 1:200), Prox1 (Millipore, MAB5256; dilution 1:250), NeuN (Millipore, MAB377; dilution 1:1000), and ZnT3 (Synaptic Systems, #197002, dilution 1:200). DAPI was added to each section as a nuclear stain. The stained sections were observed under the Olympus BX61 fluorescent microscope. The images were taken with the F-View Soft Imaging System camera.
Neurogenesis
Neurogenesis within the DGC layer was assessed using immunohistochemistry for doublecortin (DCX), a marker for immature neurons, and Ki67, a marker for proliferating cells. DCX and Ki67 positive cells were counted from the entire DGC layer on a section and divided by the total number of DGCs stained with DAPI.
Hilar ectopic DGCs
Ectopic DGCs in the hilus were assessed using immunohistochemistry for Prox1, a marker for DGCs. Ectopic location was defined as greater than or equal to two cell body widths outside the granule cell layer (Kron et al., 2010). The number of ectopic Prox1-positive cells was quantified from the entire hilar region of a hippocampal section.
Hilar cell death
Cell death in the hilus was evaluated by quantifying the number of surviving neurons with immunohistochemistry for NeuN, a marker for mature neurons. The number of NeuN-positive cells was quantified from the entire hilar region of a hippocampal section. The number of hilar ectopic DGCs was subtracted from the number of NeuN-positive cells to evaluate hilar cell death.
Mossy fiber sprouting
MFS was examined by staining hippocampal sections for ZnT3, a zinc transporter enriched in mossy fibers. The presence of MFS in the inner molecular layer of the DG was examined.
Results
FGF22KO mice show moderate seizures but do not kindle with prolonged injections of PTZ
We have previously found that FGF22KO mice are resistant to generalized seizures induced by PTZ kindling (Terauchi et al., 2010), in which we injected animals with PTZ every 48 h to induce generalized (kindled) seizures. In that experiment, when about half of WT mice were kindled (level 6 seizure; after about 21 PTZ injections), no FGF22KO mice were kindled. Here, we first tested if prolonged PTZ injections can make FGF22KO mice kindled. With additional PTZ injections, FGF22KO mice did show mild to moderate (levels 1–4) seizures (Figure 1A). However, even after 37 PTZ injections, they did not kindle. We stopped after 37 PTZ injections, because the cumulative seizure score (the sum of all the seizure levels that the mouse has experienced) for the FGF22KO mouse became ~55 (Figure 1B), which was similar to the one for the kindled WT mouse (~53) (see below).
Preparation of three animal groups to understand the role of FGF22 in epileptogenesis
Since FGF22KO mice do not kindle even with prolonged PTZ injections, we designed experiments to address the cellular basis of their kindled seizure-resistant phenotype. We focused on four epileptogenesis-associated changes: enhanced DG neurogenesis, hilar ectopic DGCs, hilar cell loss, and MFS. For our experiments, it was important to consider the possibility that the levels of these changes may correlate with the amount of seizure overtime that the animal experienced in response to PTZ injections. Because FGF22KO mice do not kindle, direct comparison between kindled WT mice and non-kindled FGF22KO mice may not be conclusive. Therefore, to understand the role of FGF22 in the epileptogenesis-associated changes apart from the effects of seizure activities on them, we injected FGF22KO mice with PTZ until their cumulative seizure score becomes equivalent to that of kindled WT mice so that FGF22KO mice experience similar overall amount of seizure activity to kindled WT mice. Yet, these two groups of animals still differ in the highest seizure level experienced (level 3–4 moderate seizures for FGF22KO mice as opposed to level 6 kindled seizures for WT mice). Thus, we prepared another group of WT mice with only mild, non-kindled seizures. Together, we prepared three groups of animals for our experiments (Figure 1B)—Group 1, WT mice with mild seizures (not kindled with just one level 3 seizure; cumulative seizure score = 8.4); Group 2, kindled WT mice (with level 6 seizures; cumulative seizure score = 52.5); and Group 3, FGF22KO mice with moderate seizures (not kindled with up to level 4 seizures; cumulative seizure score = 55.4). By comparing epileptogenesis-associated changes happening in these three groups, we can evaluate the contribution of both the genotype and the seizures that animals experienced. For each group, we also prepared a control group of mice, which received PBS instead of PTZ for the same durations.
Dentate neurogenesis increases in WT mice with mild or kindled seizures, but decreases in FGF22KO mice with moderate seizures
Enhanced neurogenesis in the DG is implicated in epileptogenesis because it may lead to aberrant integration of DGCs (Parent et al., 2006; Jessberger et al., 2007; Walter et al., 2007; Kron et al., 2010). We first assessed the level of cell proliferation in the DGC layer by staining hippocampal sections prepared from the three groups of mice with Ki67, a marker of proliferating cells (Figure 2). The sections from the same region of the hippocampus were used in order to standardize quantifications of cell numbers. At the basal level (PBS-injected mice), there was no significant difference in the number of Ki67-positive cells in the DG between WT and FGF22KO mice (Figure 2B). The number of Ki67-positive cells was increased in kindled WT mice relative to PBS-injected controls. It was increased more dramatically in non-kindled WT mice with mild seizures, suggesting that mild seizures enhance cell proliferation in the DG in WT mice. In contrast, FGF22KO mice showed a decrease in the number of Ki67-positive cells in the DG when injected with PTZ, despite having moderate seizures (Figure 2).
Figure 2 The number of proliferating cells in the DG increases in WT mice but decreases in FGF22KO mice upon PTZ injections. Region-matched sagittal hippocampal sections were prepared from PBS- and PTZ-injected WT and FGF22KO mice (3 groups of mice as shown in Figure 1) and stained for Ki67, a marker for proliferating cells. (A) Ki67 positive cells (pink) with DAPI (blue) in the DG of PBS- or PTZ-injected WT and FGF22KO mice. (B) Quantification of the numbers of Ki67-positive cells in the DGC layer per section in PBS-injected WT and FGF22KO mice (n.s. = not significant). (C) Quantification of the percentages of Ki67-positive cells in DGCs. Data are normalized against corresponding PBS controls. The percentage of Ki67-positive cells was significantly increased in WT mice with mild and kindled seizures, but was decreased in FGF22KO mice with moderate seizures upon PTZ-injections. Bars are mean ± s.e.m. Data are from 4–6 sections from 3–10 mice (see Figure 1B) per each condition. Student's t-test P-values are indicated.
Similar but slightly different results were observed with staining for DCX, a marker of immature neurons (Figure 3). At the basal level (PBS-injected mice), we found less DCX-positive cells in the DG in FGF22KO mice than in WT mice (Figure 3B), suggesting that FGF22 is involved in neuronal differentiation. The number of DCX-positive cells was increased in WT mice with kindled seizures, which is consistent with previous reports (Parent et al., 2006; Jessberger et al., 2007; Yin et al., 2007; Buga et al., 2012). It was similarly increased in WT mice with mild seizures. These, together with the Ki67 results suggest that seizures induce neurogenesis, but mild seizures affect more on proliferation than kindled seizures do. In contrast, the number of DCX-positive cells was decreased in FGF22KO mice with moderate seizures. These results suggest that FGF22 is involved in seizure-induced increases in neurogenesis in the DG.
Figure 3 The number of immature neurons in the DG is increased in WT mice with mild and kindled seizures, but is decreased in FGF22KO mice with moderate seizures. Region-matched sagittal hippocampal sections were prepared from PBS- and PTZ-injected WT and FGF22KO mice (3 groups of mice as shown in Figure 1) and stained for doublecortin (DCX), a marker for immature neurons. (A) DCX positive cells (green) with DAPI (blue) in the DG of PBS- or PTZ-injected mice. (B) Quantification of the numbers of DCX-positive cells in the DGC layer per section in PBS-injected WT and FGF22KO mice. (C) Quantification of the percentages of DCX-positive cells in DGCs. Data are normalized against corresponding PBS controls. Neurogenesis, as reflected by the percentage of DCX-positive cells, was significantly increased in WT mice with mild and kindled seizures, but was decreased in FGF22KO mice with moderate seizures upon PTZ-injections. Bars are mean ± s.e.m. Student's t-test P-values are indicated.
Hilar ectopic DGCs appear in WT mice with mild or kindled seizures, but not in FGF22KO mice with moderate seizures
Hilar ectopic DGCs may lead to aberrant integration of DGCs and contribute to epileptogenesis (Parent et al., 2006; Jessberger et al., 2007; Walter et al., 2007; Kron et al., 2010). We next examined the number of hilar ectopic DGCs by staining hippocampal sections for Prox1, a marker of DGCs (Figure 4). There was no hilar ectopic DGC in the basal condition (PBS injected) in both WT and FGF22KO mice. The number of hilar ectopic DGCs dramatically increased in WT mice with kindled seizures (Figure 4B). It was also substantially increased in WT mice with mild seizures. However, there was no significant increase in FGF22KO mice with moderate seizures. These results suggest that FGF22 is involved in ectopic migration of DGCs in response to seizures.
Figure 4 The number of hilar ectopic DGCs is increased in WT mice with mild and kindled seizures, but not in FGF22KO mice with moderate seizures. Region-matched sagittal hippocampal sections were prepared from PBS- and PTZ-injected WT and FGF22KO mice (3 groups of mice as shown in Figure 1) and stained for Prox1, a marker for DGCs. Ectopic location was defined as greater than or equal to two cell body widths outside the granule cell layer. (A) Prox1 positive cells in the DG and hilus of PBS- or PTZ-injected mice. (B) Quantification of the numbers of ectopic Prox1-positive cells in the hilus. The number of hilar ectopic DGCs was significantly increased in WT mice with mild and kindled seizures, but not in FGF22KO mice with moderate seizures upon PTZ-injections. Bars are mean ± s.e.m. Student's t-test P-values are indicated.
Hilar cell death increases in WT mice with mild or kindled seizures, but is unchanged in FGF22KO mice with moderate seizures
An increase in hilar cell death is another change that is associated with epileptogenesis (Dudek and Sutula, 2007; Jiao and Nadler, 2007). We next evaluated the level of hilar cell death in PTZ-injected WT and FGF22KO mice by quantifying the number of surviving mature neurons in the hilar region with NeuN staining (Figure 5). We have calculated the number of hilar cells by subtracting the number of hilar ectopic DGCs (Figure 4) from the number of NeuN-positive cells. The number of hilar cells was decreased in kindled WT mice relative to PBS-injected mice, which is consistent with previous reports (Sloviter, 1983; Dudek and Sutula, 2007; Jiao and Nadler, 2007; Naseer et al., 2013). It was also decreased in non-kindled WT mice with mild seizures, suggesting that similar to the case of neurogenesis (Figures 2, 3), mild seizures are enough to increase hilar cell death in WT mice. However, the number of hilar cells was not significantly changed in PTZ-injected FGF22KO mice with moderate seizures relative to PBS-injected controls. These results suggest that FGF22 plays important roles in increasing hilar cell death in response to seizures.
Figure 5 Hilar cell death is increased in WT mice with mild and kindled seizures, but is unchanged in FGF22KO mice with moderate seizures. Region-matched sagittal hippocampal sections were prepared from PBS- and PTZ-injected WT and FGF22KO mice (3 groups of mice as shown in Figure 1) and stained for NeuN, a marker for mature neurons. (A) NeuN staining of the hilar sections from PBS- or PTZ-injected mice. (B) Quantification of the numbers of NeuN-positive cells in the hilus per section in PBS-injected WT and FGF22KO mice. (C) Quantification of the numbers of hilar cells. To evaluate hilar cell number, the number of hilar ectopic DGCs (Figure 4) was subtracted from the number of NeuN-positive cells. Data are normalized relative to the corresponding PBS control. After PTZ injections, the number of hilar cells significantly decreased in WT mice both with mild and with kindled seizures, but not in FGF22KO mice with moderate seizures. Bars are mean ± s.e.m. P-values by Student's t-test are indicated.
MFS is induced only in kindled WT mice
In TLE patients and animal models of TLE, mossy fibers (axons of DGCs) often abnormally sprout to the dentate molecular layer and form excitatory synapses with the dendrites of DGCs (MFS). Accordingly, MFS may induce recurrent excitation of DGCs and contribute to epileptogenesis. We next examined whether WT and FGF22KO mice display MFS after seizures. To examine MFS, we stained DG sections with ZnT3, a zinc transporter that is abundant in mossy fiber terminals (Wenzel et al., 1997). MFS in the dentate molecular layer was detected in kindled WT mice (Figure 6), as observed previously with Timm staining (Sutula et al., 1989; Cavazos et al., 1991; Parent et al., 1999). In contrast, MFS was not detected in non-kindled WT mice with mild seizures, suggesting that more severe or prolonged seizures may be required to form MFS in WT mice. MFS was not observed in FGF22KO mice with moderate seizures either. This implies that either FGF22 is required for the formation of MFS or that more severe seizures are necessary to induce MFS.
Figure 6 MFS is only induced in kindled WT mice. Region-matched sagittal hippocampal sections were prepared from PBS- and PTZ-injected WT and FGF22KO mice (3 groups of mice as shown in Figure 1) and stained for ZnT3, a zinc transporter enriched in mossy fiber terminals, to examine MFS in the DG. Kindled WT mice show MFS in the dentate molecular layer (arrows), while non-kindled WT and FGF22KO mice do not.
Discussion
FGF22KO mice are more resistant to generalized seizures than WT mice as assessed by PTZ kindling (Terauchi et al., 2010). Here we addressed the possible cellular basis for the phenotype by focusing on four changes in the hippocampus associated with epileptogenesis: enhancement of dentate neurogenesis, appearance of hilar ectopic DGCs, increase in hilar cell death, and formation of MFS. These changes were indeed observed in kindled WT mice. Even in non-kindled WT mice with only mild seizures, three changes, enhanced neurogenesis, hilar ectopic DGCs, and increased hilar cell death, occurred. In contrast, PTZ-injected FGF22KO mice did not show any of these four changes, despite having moderate seizures after prolonged injections of PTZ. From these results, we conclude that FGF22 plays critical roles in increasing neurogenesis, ectopic DGCs, and hilar cell death in response to seizures. As for MFS, since MFS was not detected in WT mice with mild seizures either, we conclude that either FGF22 is involved in the formation of MFS or that more severe seizures are necessary to induce MFS. Our results suggest that inhibition of FGF22 suppresses neurogenesis, hilar ectopic DGCs, and hilar cell death (and possibly MFS) in response to seizures.
Since FGF22KO mice did show moderate seizures in our experiments, suppression of epileptogenesis-associated changes in FGF22KO mice is not due to lack of seizures, but rather due to FGF22-deficiency. Thus, our results indicate that FGF22 is involved in seizure activity-induced modifications in the hippocampus. We have previously shown that FGF22 from CA3 pyramidal neurons promotes the organization of excitatory synapses formed onto them (Terauchi et al., 2010). FGF22 from CA3 may also send signals to DGCs to regulate neurogenesis and hilar cell survival in an activity-dependent manner. FGF22-dependent signals may induce other growth factors, neurotrophins, morphogens, and cytokines that are implicated in dentate neurogenesis and cell survival (Simonato et al., 2006; Parent, 2007; Zhao et al., 2008). We are currently indentifying FGF22 receptors and downstream signals mediating these epileptogenesis-associated changes using FGF receptor mutants and are analyzing the roles of FGF22-target genes in dentate neurogenesis.
Another interesting finding is that in FGF22KO mice with moderate seizures, neurogenesis is decreased, rather than unchanged as was seen in the case of hilar cell death. One possible interpretation for this finding is that seizure activities might eventually be generating two signals, positive signals resulting in reactive neurogenesis and negative signals for negative-feedback/homeostatic-control, to regulate dentate neurogenesis and that FGF22 is preferentially involved in the positive signals. Further analysis should reveal the activity-dependent signaling pathways that regulate neurogenesis.
Most importantly, our results suggest a possibility that FGF22 inactivation could suppress the enhancement of neurogenesis, ectopic DGCs, and hilar cell death (and possibly MFS) in response to seizures. Thus, FGF22 may join FGF2 as the FGFs that are involved in epileptogenesis or epileptogenesis-associated changes. It has been shown that aberrant adult-born DGCs can cause epilepsy (Pun et al., 2012), hilar cell death causes DGC hyperexcitability (Jinde et al., 2012), and MFS is likely to contribute to seizure susceptibility (Morgan et al., 2007). Thus, targeting FGF22 could offer a novel strategy to prevent epileptogenesis or epileptogenesis-associated changes. For this reason, we are currently performing small molecule screening for FGF22 inhibitors, hoping to identify candidate drugs to treat or alleviate TLE.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the Ester A. and Joseph Klingenstein Fund, the March of Dimes Foundation, and NIH grant NS070005 (Hisashi Umemori). We thank A. Dabrowski, M. Korn, and J. Parent for comments; and A. Terauchi, P. Lee, D. Javed, and M. Zhang for technical assistance.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23630579PONE-D-12-3871010.1371/journal.pone.0061079Research ArticleBiologyMolecular cell biologySignal transductionSignaling cascadesERK signaling cascadeCell DeathMathematicsStatisticsBiostatisticsMedicineOncologyBasic Cancer ResearchMetastasisCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaHyperthermia-Induced NDRG2 Upregulation Inhibits the Invasion of Human Hepatocellular Carcinoma via Suppressing ERK1/2 Signaling Pathway HT Induced NDRG2 Inhibits the Invasion of HCC CellGuo Yan
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*
1
Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Shaanxi, China
2
The State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, the Fourth Military Medical University, Shaanxi, China
3
Experiment Teaching Center, School of Basic Medicine, the Fourth Military Medical University, Shaanxi, China
4
Department of Neurosurgery, Xijing Hospital, the Fourth Military Medical University, Shaanxi, China
Guan Xin-Yuan Editor
The University of Hong Kong, China
* E-mail: [email protected] (JZ); [email protected] (WL)Competing Interests: The authors have declared that no competing interests exist.
Critical revision of the manuscript for important intellectual content and study supervision: WL. Study concept and design, obtained funding, critical revision of the manuscript for important intellectual content and study supervision: JZ LY. Conceived and designed the experiments: JZ WL. Performed the experiments: YG LW QW. Analyzed the data: Xia Li Xiaoming Li YZ. Contributed reagents/materials/analysis tools: JM JZ. Wrote the paper: JZ.
2013 22 4 2013 8 4 e6107910 12 2012 5 3 2013 © 2013 Guo et al2013Guo et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Hyperthermia (HT) has been proven to be able to alter the invasion capacity of cancer cells. However, the detailed mechanisms responsible for the anti-metastasis effects of HT have not been elucidated. N-myc downstream-regulated gene 2 (NDRG2), as a member of the NDRG family, has been suggested to be highly responsive to various stresses and is associated with tumor suppression. The present study aimed to investigate the biological role of NDRG2 in the invasion of human hepatocellular carcinoma (HCC) cells exposed to HT. We found that NDRG2 could be induced by HT at 45°C. In addition, NDRG2 overexpression inhibited the expression of matrix metallo proteinases-2 (MMP-2) and MMP-9 as well as the invasion of HCC cells, whereas knockingdown NDRG2 reversed the anti-invasion effect of HT in vivo. Further investigation revealed that the phosphorylation level of ERK1/2, but not that of JNK and p38MAPK, was reduced in NDRG2 overexpressing cells. Moreover, the knockdown of NDRG2 expression resulted in increased cell invasion, which was rescued by treating the HepG2 cells with the ERK1/2 inhibitor PD98059, but not with the p38MAPK inhibitor SB203580 or the JNK inhibitor SP600125. Finally, the synergistic cooperation of HT at 43°C and NDRG2 expression effectively reduced cytotoxicity and promoted the anti-invasion effect of HT at 45°C. Taken together, these data suggest that NDRG2 can be induced by HT and that it mediates the HT-caused inhibition of invasion in HCC cells by suppressing the ERK1/2 signaling pathway. The combined application of constitutive NDRG2 expression with HT may yield an optimized therapeutic benefit.
This work was supported by National Natural Science Foundation of China ((No. 30973437; 30700918; 81230043). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide, accounting for 85% to 90% of primary liver cancers [1], [2]. Conventional treatments of HCC include surgery, chemotherapy, radiation, percutaneous injection of ethanol (PEI) chemotherapy with anthracyclines or combinations of these treatments. Despite advances in therapeutic strategies, patients with HCC have a poor prognosis because of the propensity of HCC to metastasize [3], [4]. Therefore, the inhibition of invasion and metastasis has been the key factor for the successful treatment of HCC.
Hyperthermia, a minimally invasive treatment with few side effects, has recently been used for cancer therapy. A number of clinical and animal experiments have shown that HT exerts therapeutic effects not only by delaying tumor growth but also by inhibiting lymph node metastasis [5], [6], [7]. Nagashima et al. demonstrated that local HT inhibited the lymph node metastasis of hamster oral squamous cell carcinoma [8]. In vitro research has been carried out to understand the underlying mechanism for this effect. Most of these investigations have focused altering metastasis-related genes, such as vascular endothelial growth factor (VEGF) [9], urokinase type plasminogen activator receptor (uPAR) [10] and MMPs [11], [12]. Among MMPs, MMP-2 and MMP-9 are the critical enzymes that are known to degrade surrounding extracellular matrix components, thus resulting in tumor invasion during cancer metastasis [13]. Although some progress has been made in terms of assessing the biological effect of HT, the molecular mechanism that mediates the anti-metastatic effect of HT has not been elucidated.
N-myc downstream-regulated gene 2 (NDRG2) belongs to the NDRG family, a new family of differentiation-related genes that consists of four members: NDRG1, NDRG2, NDRG3 and NDRG4. Previous studies have reported that NDRG family members are associated with multiple cellular processes, such as proliferation, differentiation and stress responses. NDRG2 was first cloned from glioblastoma using polymerase chain reaction-based subtractive hybridization by our laboratory in 1999 [14]. Decreased expression of NDRG2 is found in a number of human cancers, including breast cancer [15], clear cell renal cell carcinoma [16], liver cancer and pancreatic cancer [17]. The ectopic expression of NDRG2 suppresses the proliferation of tumor cells [14], [18], [19]. In addition, accumulated evidence indicates that the absence of NDRG2 expression in a variety of carcinomas contributes to increased tumor metastatic potential via the regulation of MMP-2/MMP-9 production [20], [21], [22]. All of these findings suggest that NDRG2 has tumor suppressor role. In addition, increasingly more efforts have aimed to determine the role of NDRG2 under stress conditions. We previously reported that NDRG2 can be up-regulated following hypoxia or radiation-induced stress [23], [24]. Foletta et al. demonstrated that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle [25]. However, few studies have examined the response of NDRG2 to HT-induced heat stress and the influence of NDRG2 on the anti-metastatic effect of HT in cancer cells.
In the present study, we sought to clarify the biological role of NDRG2 during HCC invasion under HT conditions. We found that NDRG2 expression was upregulated by heat stress. The overexpression of NDRG2 enhanced the anti-invasion effects of HT in the HCC cell line HepG2, whereas the down-regulation of NDRG2 resulted in attenuated the inhibitory effects of HT on invasion of HCC cells in the xenograft mouse model. We also assessed the underlying intracellular signaling pathway and found that the NDRG2-mediated anti-invasion effect of HT occurs via the suppression of ERK1/2 signaling in human HCC cells. Moreover, the overexpression of NDRG2 synergized with HT to inhibit the invasiveness of HepG2 cells while decreasing spontaneous necrosis.
Materials and Methods
Cell lines and culture
Human HCC cell lines (HepG2 and Huh7) were obtained from the Chinese Academy of Sciences (Shanghai, China). All cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere of 5% CO2 at 37°C.
Hyperthermia treatment
The T-25 flasks or 6-well plates containing cells at approximately 70% confluence were sealed with parafilm and subjected to HT by immersion in a water bath at 37°C, 39°C, 41°C, 43°C and 45°C for 30 min. Cells subjected to the 37°C water bath served as the controls. All temperatures were maintained within ±0.05°C by testing for accuracy with a thermocouple (Fischer Scientific, Pittsburgh, PA). After the HT treatment, the parafilm was removed, and the flasks and plates were returned to a 37°C incubator for a period of time.
Lentivirus generation and infection
Recombinant lentiviral vectors were constructed with Invitrogen's ViraPower™ Lentiviral System in our laboratory. The cDNAs of human NDRG2 were cloned and subcloned into the vector pLenti6. Short hairpin RNAs (shRNA) against human NDRG2 were designed using a small interfering RNA design program and were then subcloned into the EcoR I/Age I sites of pLKO-TRC vector. The shRNA sequences specific for NDRG2 were as follows: (shNDRG2: forward, 5′-CCGGGAGGACATG CAGGAAATCATTCTCGAGAATGATTTCCTGCATGTCCTCTTTTTG-3′; reverse, 5′-AATTCAAAAAGAGGACATGCAGGAAATCATTCTCGAGAATGATTTCCTGCATGTCCTC-3′). The sequences for the control nonsense shRNA were as follows: (Scramble: forward, 5′-CCGGAAGGTCTTGTCCTCATCAACACTCGAGTGTTG ATGAGGACAAGACCTTTTTTTG-3′; reverse, 5′–AATTCAAAAAAAGGTCTT GTCCTCATCAACACTCGAGTGTTGATGAGGACAAGACCTT-3′). The HEK-293T cells were transfected with the pLenti6-Cherry/NDRG2, pLKO-Scramble/NDRG2-shRNA, PAX2 and PMD2G lentiviral vectors using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After 48 h, the lentiviral supernatants were collected, filtered (0.45 µm size filter; Millipore,Billerica, MA, USA), and added onto the HepG2 cells in the presence of 2 µg/ml Blasticidin (Sigma-Aldrich,USA) or 1 µg/ml Polybrene (Sigma-Aldrich,USA) for 6 to 8 h. Two rounds of infection were performed. After infection, the cells that survived this treatment were selected for a week before being analyzed for NDRG2 expression by Western blot.
In vitro tumor cell invasion assay
The invasive capacity of cells was evaluated using the transwell chamber assay. The upper and lower compartments of the chamber were separated with a polycarbonate filter (8-µm pore size) that was coated with 50 µg of reconstituted basement membrane Matrigel (Collaborative Biomedical Products, Bedford, USA). The cells were trypsinized and seeded in the upper chamber at 1×105 cells/well in serum-free medium. Medium supplemented with 10% FBS (used as a chemo-attractant) was placed in the bottom well, and the cells were then incubated for 24 h at 37°C in a humidified 5% CO2 atmosphere. After the incubation, the chambers were removed, and invading cells on the bottom side of the membrane were fixed with methanol for 15 min and stained with gentian violet for 10 min. Invasion was assessed by counting cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments.
Western blot analysis
Following treatment, cells were harvested and washed in PBS. The cells were homogenized in RIPA lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 2 µg/mL protease inhibitors (protease inhibitor cocktail, Cat No: 4693116001; Roche, Germany) and 0.1% phosphatase inhibitor (Phosphatase Inhibitor Cocktail II; Sigma-Aldrich,USA). Supernatants were collected and their protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Aliquots of the lysates (each containing 40 mg of protein) were boiled for 5 min and electrophoresed on a 10% SDS–polyacrylamide gel. The resolved proteins were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked by incubation in 5% bovine serum albumin at room temperature for 2 h and were then incubated with primary antibody for overnight at 4°C. The primary antibodies were as follows: anti-NDRG2 (Abnova, Taiwan, China), MMP-2, MMP-9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-ERK1/2, total-ERK1/2, phospho-p38/MAPK, total-p38/MAPK, phospho-JNK, and total-JNK (Cell Signaling, Bedford, MA, USA). The membranes were then incubated with the appropriate alkaline phosphatase-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 h. Antibody binding was finally detected using an enhanced chemiluminescence Western blot detection kit (Pierce Biotechnology, Rockford, IL, USA).
Establishment of the animal tumor model
BALB/C athymic nude mice (male, 6 weeks of age) from the Laboratory Animal Research Center of the Fourth Military Medical University (Xi'an, China) were used in compliance with the regulations of the Animal Ethics Committee of the Fourth Military Medical University of the People's Liberation Army. Resuspended HepG2 cells (5×106 cells in 200 µl) were infected separately with either the shRNA-control or shRNA-NDRG2 was injected into the hind legs of the mice. Each experimental group contained 5 mice. After 2 weeks of implantation, mice in the heat-treated groups were subjected to a 45°C water bath for 30 min. Four weeks later, the mice were sacrificed and their primary tumors were removed for further histological examination. All the experimental procedures were conducted in accordance with the Detailed Rules for the Administration of Animal Experiments for Medical Research Purposes issued by the Ministry of Health of China and received ethical approval by the Animal Experiment Administration Committee of the Fourth Military Medical University. All efforts were made to minimize the animals' suffering and to reduce the number of animals used.
Regional hyperthermia treatment in tumor-bearing mice
Tumors were heated by immersing the tumor-bearing leg in a thermostat-controlled waterbath (YGM Instrument, type DZKW-D-1, Beijing, China). The tumor-bearing leg was pulled down using a sinker, and the tumor was immersed at least 10 mm below the water surface. The temperature of water bath was set at 45°C for 30 min. The temperature at the center of the tumor nodule and the rectal temperatures were measured during the heat treatment with a thermocouple (Physitemp Instruments, type IT-18, USA). The mice were anaesthetized with an i.p. injection of pentobarbital sodium at a dose of 60 mg/kg before administering HT. To reduce systemic heating, the nude mice were cooled using a fan during treatment to prevent whole body HT [9].
Pathological analysis
Formalin-fixed, paraffin-embedded tumor sections were deparaffinized in xylene, followed by treatment with a graded series of alcohol and staining with hematoxylin-eosin (H&E). For immunohistochemistry, antigen retrieval for paraffin-embedded tissues was performed with sodium citrate, after which the samples were placed in boiling water for 20 min. Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide in methanol for 10 min. The samples were incubated with rabbit polyclonal antibodies to MMP-2 and MMP-9 (Santa Cruz Biotechnology, Santa Cruz, USA) or PBS (negative control) at 4°C overnight. The samples were then incubated for 30 min with the appropriate dilution of the secondary antibody (ZSGB Biotechnology, Beijing, China), followed by incubation with the HRP-linked streptavidin-biotin complex in a humidified chamber for 10 min at RT. Positive reactions were visualized by incubating the slides with DAB (ZSGB Biotechnology, Beijing, China) for 5 min. The sections were then counterstained with hematoxylin for 15 sec, dehydrated and cleared and observed under a photomicroscope (Olympus BX51, Tokyo, Japan).
Apoptosis analysis
The cells were collected at the indicated times, washed twice with PBS and incubated in the dark for 15 min with binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl and 2.5 mM CaCl2], Annexin V-FITC (200 mg/ml; BD Pharmingen, San Diego, CA, USA) and propidium iodide (PI, 1 mg/ml; Sigma-Aldrich, USA). The fluorescence of Annexin V-FITC and PI were measured using FCM (flow cytometry, Epics Elite flow cytometer; Coulter, Miami, FL, USA). The data were analyzed using Cell Quest software.
Statistical analysis
All experiments were repeated at least three times. Results are shown as the mean ± SD. The data were analyzed with the SPSS 13.0 software package, and statistical comparisons between groups were made using a one-way ANOVA followed by Student's t-test. P<0.05 was considered statistically significant.
Results
Inhibition of HCC invasion by HT is accompanied by NDRG2 upregulation
HT has been found to successfully inhibit the invasiveness of various cancer cells. Here, we examined the anti-invasive effect of HT on HepG2 and Huh7 cells by subjecting them to water baths at different temperatures (37°C, 39°C, 41°C, 43°C and 45°C) for 30 min. Matrigel invasion assays were performed to evaluate the effect of HT on metastatic activity. As shown in Fig. 1A, the invasiveness of HepG2 and Huh7 cells was not affected by heat treatment at temperatures of 37°C–43°C. Compared with the control group (37°C), heat treatment at 45°C reduced the percentage of invaded cells by 65.4% in HepG2 and 68.7% in Huh7 cells, respectively. There was no statistically significant difference in the percentage of invaded cells between HepG2 and Huh7 cells. To further confirm the anti-invasion effect of HT, we examined the expression of MMP-2 and MMP-9 in both HCC cell lines. As shown in Fig. 1B, the expression of MMP-2 and MMP-9 was significantly reduced by HT at 45°C. In addition, Western blot showed that the expression of NDRG2 was increased by heat shock at 45°C in both HepG2 and Huh7 cells, which was negatively correlated with the down-regulation of MMP-2 and MMP-9. These finding suggest that NDRG2 is induced by heat shock at 45°C and associated with inhibitory effect of HT (45°C) on HCC cells. Therefore, all experiments described below were performed at a heat shock temperature of 45°C in HepG2 cells, which is the most common cellular model for human liver tumors.
10.1371/journal.pone.0061079.g001Figure 1 HT retards the invasion of HCC with up-regulation of NDRG2.
(A) HepG2 and Huh7 cells were exposed to different temperatures (37°C, 39°C, 41°C, 43°C and 45°C) for 30 min. Matrigel invasion assays were performed after 24 h of heat treatment in order to evaluate the effect of HT on invasiveness. Cell invasion is shown in phase contrast images and graphical representations. Invasion was assessed by counting cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and calculated as the means ± SD. The invasion rate of cells at 37°C was defined as 100%, and *P<0.05 indicates the degree of statistical significance as compared to cells at 37°C. (B) Expression levels of NDRG2, MMP-2 and MMP-9 in heat-treated HepG2 and Huh7 cells were determined by Western blot after 24 h of heat treatment at different temperatures (37°C, 39°C, 41°C, 43°C and 45°C) for 30 min. Tubulin was used as the internal loading control.
Anti-invasion effect of HT is enhanced in NDRG2-overexpressing HCC cells
To elucidate whether NDRG2 is associated with the anti-invasion effects of HT, we established HepG2 cell lines that constitutively expressed NDRG2 protein by lentiviral infection (Fig. 2A, 2B). The overexpression of NDRG2 enhanced the inhibition of invasion in HepG2 cells exposed to HT, which is consistent with results reported previously. In NDRG2 overexpressing cells, heat shock at 45°C significantly reduced the invasive potential of the cells (Fig. 2C) compared with HepG2 cells treated by HT alone. These results indicate that NDRG2 strengthens the anti-invasive effect of HT. We next detected the expression of MMP-2 and MMP-9. Western blot revealed that the expression of MMP-2 and MMP-9 was decreased in NDRG2 overexpressing HepG2 cells and that the expression was even lower after the cells were treated by HT (45°C). These data indicate that NDRG2 might enhance the anti-invasion effect of HT by inhibiting the expression of MMP-2 and MMP-9 (Fig. 2D).
10.1371/journal.pone.0061079.g002Figure 2 NDRG2 overexpression enhances the anti-invasion effect of HT in HCC cells.
(A) Expression level of NDRG2 in Control, Cherry (transfected with overexpression control plasmid), and NDRG2 (transfected with NDRG2 overexpression plasmid) cells. (B) NDRG2 expression level in Cherry or NDRG2 cells was determined by Western blot after HT at 45°C for 30 min. (C) The anti-invasive effect of HT was studied in Cherry or NDRG2 cells after HT at 45°C for 30 min. Cell invasion is depicted in the phase contrast images and the graphical representation. Invasion was assessed by counting invasive cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and were calculated as the means ± SD. The invasion rate of Cherry-37°C cells was defined as 100%. *P<0.05 or **P<0.01 indicatesthe degree of statistical significance compared to Cherry-37°C cells. (D) Expression levels of MMP-2 and MMP-9 in 45°C heat-treated Cherry or NDRG2 cells were assessed by Western blot analysis. Tubulin was used as the internal loading control.
Down-regulation of NDRG2 attenuates the inhibitory effects of HT on invasion of HCC cells in vivo
To confirm the role of NDRG2 on the invasive capability of heat-treated HCC cells in vivo, we further examined the anti-invasion potential of NDRG2 in HCC-implanted mice. We first established stable NDRG2-deficient HepG2 clones by infecting cells with lentivirus-mediated NDRG2-specific shRNA (shNDRG2-HepG2). The Scramble shRNA sequence was used as a negative control (Scramble-HepG2). As shown in Fig. 3A, the NDRG2-specific shRNA decreased the expression of NDRG2 significantly compared with the Scramble shRNA. NDRG2-deficient HepG2 cells were injected into nude mice and treated as described in the Materials and Methods. H&E staining of histological sections from the mouse xenograft model revealed that HT significantly suppresses the invasion ability of HepG2 cells. As shown in Fig. 3B, malignant tumors slightly invaded into nearby tissues in HT-treated mouse model. In contrast, the suppression of NDRG2 facilitated the invasion of tumor nodules and reversed the anti-invasive effect of HT in HepG2 cells, which was accompanied by a significant destruction of the muscle layer. The expression of both MMP-2 and MMP-9 was also detected in tissue sections, and a representative immunohistochemical staining is presented in Fig. 3C. Consistent with the H&E staining results, we found that down-regulation of NDRG2 alleviated the repression of MMP-2 and MMP-9 expression by HT. These results imply that NDRG2 is involved in the HT-caused inhibition of invasion via the suppression of MMP-2 and MMP-9 expression.
10.1371/journal.pone.0061079.g003Figure 3 Down-regulation of NDRG2 affects the invasiveness of heat-treated HCC cells in vivo.
(A) Expression level of NDRG2 in Scramble-HepG2 (transfected with silencing expression control plasmid) and shNDRG2-HepG2 (transfected with NDRG2 silencing plasmid) cells. HepG2 cells were transfected separately with the Scramble or shNDRG2 and then individually injected into the hind legs of the mice. After two weeks of implantation, mice in the heat-treated groups were subjected to a 45°C water bath for 30 min. Each experimental group contained 5 mice. Four weeks later, mice were sacrificed, and primary tumors were removed for histological examination. (B) Representative H&E staining of histological sections revealed histological destruction in each group. Arrows point to invasion areas. Stars mark the locations of muscle. (C) Immunohistochemistry showed expression levels of NDRG2, MMP-2 and MMP-9 in the tumor tissues of each group. Bar = 30 µm (magnification 400×).
ERK1/2 signaling isreduced in NDRG2-overexpressing cells
Because NDRG2 was responsive to HT, we evaluated the role of NDRG2 in the anti-invasion effect of HT. The overexpression of NDRG2 has been found to inhibit the malignant potential of breast cancer cells in a MAPK-dependent manner. Therefore, we investigated the effect of NDRG2 on the downstream activation of the MAPKs signaling pathway by detecting phosphorylated p38MAPK, extracellular signal regulated kinase1/2 (ERK1/2), and c-Jun NH2-terminal kinase (JNK), which were induced by HT. The JNK and ERK1/2 pathways showed high intrinsic basal activation. Neither HT nor NDRG2 overexpression further enhanced JNK activation. On the other hand, the phosphorylation of ERK1/2 increased up to 1.25-fold within 2 h, rapidly declined to the basal level 4 h after heat shock treatment, and then reduced to even lower levels. Moreover, the overexpression of NDRG2 abrogated the intrinsic and HT-induced activation of the ERK1/2 pathway (Fig. 4). HT was found to increase the activation of p38MAPK in a time-dependent manner, whereas the phosphorylation level of p38MAPK remained unchanged in NDRG2-overexpressing cells. These findings indicate that ERK1/2 activation may be selectively inhibited by NDRG2 expression in HepG2 cells.
10.1371/journal.pone.0061079.g004Figure 4 NDRG2 inhibits HT-induced ERK1/2 activation in HCC cells.
Cherry (transfected with over expression control plasmid) and NDRG2 (transfected with NDRG2 expression plasmid) cells were subjected to a 45°C water bath for 30 min and then incubated at 37°C for the indicated periods of time. The cells were lysed and levels of ERK1/2, JNK, and p38MAPK were detected by Western blot. Levels of ERK1/2, p38MAPK and JNK phosphorylated proteins at the indicated time points after heat treatment were detected by Western blot and quantitated by measuring band intensities. The values of the fold activations were normalized to the total ERK1/2, p38MAPK or JNK values. The data are representative of three independent experiments and were calculated as the means ± SD. *P<0.05 indicates degrees of statistical significance as compared to Cherry cells. Tubulin served as the loading control.
NDRG2 mediates the anti-invasion effect of HT via inhibition of the ERK1/2 signaling pathway
To further confirm the role of NDRG2 on ERK1/2 activation, lentivirally infected short-hairpin RNA was employed to down-regulate endogenous NDRG2 expression in HepG2 cells. NDRG2-deficient cells were treated with the ERK1/2 inhibitor PD98059, the p38MAPK inhibitor SB203580 or the JNK inhibitor SP600125, respectively. As shown in Fig. 5A and 5B, silencing NDRG2 expression significantly increased the invasion ability of heat-shocked cells. In addition, PD98059 treatment significantly enhanced the anti-invasive effect of HT and decrease MMP-2/MMP-9 expression in NDRG2-deficient HepG2 cells. In contrast, no changes were observed in SB203580 and SP600125 treated cells. Therefore, our results suggest that ERK1/2 pathway inhibition, but not p38MAPK or JNK pathway inhibition, is responsible for the decreased invasiveness of NDRG2 knockdown HepG2 cells.
10.1371/journal.pone.0061079.g005Figure 5 NDRG2 mediates the anti-invasion effect of HT via the inhibition of ERK1/2 activation.
shNDRG2-HepG2 (transfected with NDRG2 silencing plasmid) cells were pre-incubated with 10 µmol/ml PD98059, 10 µmol/ml SB203580 or 50 µmol/ml SP600125 for 30 min before heat treatment at 45°C for 30 min. (A) Cell invasion is represented graphically. The data are representative of triplicate experiments and were calculated as the means±SD. *P<0.05 indicates the degree of statistical significance. (B)MMP-2 and MMP-9 expression levels were determined by Western blot. Tubulin served as the loading control.
NDRG2 synergizes with HT to inhibit the invasiveness of HCC cells and decrease spontaneous necrosis
HT leads to cell death by either apoptosis or necrosis depending on temperature [26]. Apoptosis, which is a naturally occurring cause of cellular death, often provides beneficial effects to the organism. In contrast, necrosis is almost always detrimental and can be fatal. We examined the type of cell death induced by HT treatment at different temperatures using double staining with Annexin V and PI. HepG2 cells treated at 45°C showed a significantly higher rate of cell apoptosis, compared with those treated at 43°C. Simultaneously, HT at 45°C caused a massive increase in the number of necrotic cells in comparison with cells treated at 43°C (Fig. 6A). Intriguingly, we found that the combination of NDRG2 expression and HT at 43°C resulted in increased apoptosis and reduced necrosis compared with HT at 45°C. Although heat shock at 43°C and NDRG2 expression each induced apoptosis to a certain degree, the effect of their combination exceeded the mere sum of the effect of each treatment alone, thus indicating a synergistic effect. Moreover, the capacity of heat shock and/or NDRG2 expression to affect the invasion of HepG2 cells was assessed. The cells treated at 45°C showed a significantly lower percentage of invaded cells (64.3%) compared with those treated at 43°C (81.5%). The combined treatment of heat shock at 43°C and NDRG2 expression decreased the expression of MMP-2/MMP-9 and reduced the invaded cell percentage by 65.2%. Simultaneously, exposure to the combination of HT at 43°C and NDRG2 expression resulted in less necrosis and reduced the invasiveness of HepG2 cells.
10.1371/journal.pone.0061079.g006Figure 6 NDRG2 enhances the efficacy of HT.
(A) The effect of HT on necrosis and apoptosis was studied in Cherry (transfected with over expression control plasmid) or NDRG2 (transfected with NDRG2 expression plasmid) cells after heat treatment at 43°C or 45°C for 30 min. Apoptosis was measured through FACS analysis of PI- or Annexin V-stained cells. Column data analysis of intact cells (Annexin V−, PI−), early apoptotic (Annexin V+, PI−) and late apoptotic/necrotic cells (Annexin V−/+, PI+) for each cell group. *P<0.05 indicates the degree of statistical significance compared to the Cherry-37°C group; #
P<0.05 indicates the degree of statistical significance as compared to the Cherry-45°C group. (B)The anti-invasive effect of HT was studied in Cherry or NDRG2 cells after HT at 43°C or 45°C for 30 min. Cell invasion is depicted in the phase contrast images and the graphical representation. Invasion was assessed by counting cells in five microscopic fields per well at 400×magnification. The data are representative of triplicate experiments and were calculated as the means ± SD. The invasion rate of Cherry-37°C cells was defined as 100%. *P<0.05 indicates the degree of statistical significance as compared to Cherry-37°C cells. (C) Expression levels of MMP-2 and MMP-9 in 43°C or 45°C heat-treated Cherry or NDRG2 cells were assessed by Western blot. Tubulin was used as the internal loading control.
Discussion
As a strong adjuvant treatment, HT has been extensively investigated for its propensity to induce apoptosis and alter the metastatic character of tumor cells. Here, our results revealed that HT at 45°C effectively inhibited the invasive capacity of HCC cells, which was accompanied by the decreased expression of MMP-2 and MMP-9. Further mechanistic investigation demonstrated that the NDRG2-mediated suppression of the ERK1/2 pathway accounted for the decreased invasiveness observed in HepG2 cells treated by HT. Moreover, our results suggest that the constitutive expression of NDRG2 optimized the therapeutic effect of HT by enhancing HT's anti-invasive effects and by decreasing spontaneous necrosisin HepG2 cells.
As a member of the NDRG family, the NDRG2 gene has been shown to be intimately involved in carcinogenesis and cancer progression. In addition, accumulating evidence has shown that the expression of human NDRG2 can be induced by a number of cell stress conditions. For instance, Wang et al. demonstrated that NDRG2 expression is significantly up-regulated by hypoxia or hypoxia-mimetic agents in several tumor cell lines [23]. Another previous study showed that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle [25]. Moreover, the increased expression of NDRG2 was observed during Adriamycin (ADR)-mediated DNA damage response [27]. In accord with these results, we determined that NDRG2 was up-regulated by HT-induced heat stress at 45°C in HCC cells (Fig. 1B and 2B). Therefore, our results together with previous reports support the idea that NDRG2 is stress responsive gene.
In contrast to the similar changes of NDRG2 expression, the biological effect of NDRG2 on tumor cell behaviour appeared paradoxical under different stress. In A549 cells exposed to hypoxic conditions, the ectopic expression of NDRG2 enhanced hypoxia-induced apoptosis. This phenomenon was confirmed by the observation that NDRG2, as a p53-inducible gene, is implicated in the p53-mediated apoptosis pathway in response to DNA damage [27]. Thus, in the above studies, NDRG2 appears to act as a stress-responsive gene in order to facilitate cell death. Contrary to these findings, NDRG2 was demonstrated to be involved in the AKT-mediated protection of β cells against lipotoxicity-induced apoptosis [28]. Moreover, NDRG2 contributes to the hypoxia-induced radio-resistance of Hela cells and the increased chemoresistance of Hela cells to cisplatin [24], [29]. Herein, our data showed that the constitutive expression of NDRG2 enhanced HT-induced apoptosis in HepG2 cells. However, the overexpression of NDRG2 alone did not result in cell apoptosis directly (Fig. 6A), and more important, we found that NDRG2 could potentiate the anti-invasive effect of HT (Fig. 1B, 2C and 2D). The down-regulation of NDRG2 by shRNA attenuated the inhibitory effects of HT on invasion in the nude mouse xenograft model (Fig. 3). We suspect that the alterations of cell responses induced by NDRG2 are complex and depend on the intensity or the type of stress. Another possible explanation is that NDRG2 has different effects on tumor malignancy in a cell context-dependent manner. Further investigations should be conducted to test this hypothesis.
NDRG2 has been reported to be a candidate suppressor of tumor metastasis based on a number of studies. Shon et al. reported that NDRG2 overexpression inhibited the metastatic potential of breast cancer cells through the BMP-4 mediated suppression of MMP-9 activation [21]. Moreover, a previous study showed that NDRG2 suppresses the metastatic potential of HT1080 human fibrosarcoma and B16F10 murine melanoma cells both in vitro and in vivo [30]. Recently, Kim et al. reported that the high expression level of NDRG2 is inversely correlated with tumor invasion depth and Dukes' stage of colon adenocarcinoma [31]. Very recently, NDRG2 expression was found to be down-regulated in gallbladder carcinoma, and the expression level was found to be closely correlated with deeper invasion depth and the TNM stage of the patients. Here, our data showed that the introduction of NDRG2 into HepG2 cells significantly suppressed the expression of MMP-2/MMP-9 and reduced the invasion of cells (Fig. 2C and 2D). These results are consistent with previous findings that NDRG2 plays important roles in suppressing tumor metastasis in HCC [17], [32], [33]. HT at 45°C was found to inhibit HCC invasion, which was accompanied by the upregulation of NDRG2. We speculated that a possible mechanism for the anti-invasion effect of HT is that up-regulated NDRG2, at least in part, contributes to decreased levels of MMPs, thereby resulting in the suppression of cell invasion.
Increasingly more studies have reported that MAPKs seemto play a pivotal role in tumor invasion andmetastasis [34], [35], [36]. Heat exposure, a kind of stress, is associated with the activation of the MAPK family [37], [38], [39]. Our data showed that HT did not alter the activation of JNK (Fig. 4), and a similar result was observed in MDA-MB-231 cells [40]. In addition, we observed that HT inhibited the activation of ERK1/2 and increased the phosphorylation of p38MAPK. More important, overexpression of NDRG2 abrogated the activation of the ERK1/2 pathway, whereas the activation of p38MAPK remained unchanged in NDRG2 overexpressing cells (Fig. 4). The signaling events involved in the heat shock-induced expression of MMPs and decreased invasion were then investigated in HT-treated HepG2 cells infected with shNDRG2. The inhibition of the ERK1/2 pathway using the specific inhibitor PD98059 reduced invasion in HT-treated NDRG2-deficient cells completely. However, the JNK inhibitor SP600125, and the P38MAPK inhibitor SB203580, only partly reversed the decreased cell invasion and expression of MMPs (Fig. 5). These data suggest that ERK1/2 pathway inhibition, but not p38MAPK or JNK pathway inhibition, is responsible for the decreased invasiveness of NDRG2-deficient HepG2 cells, which is consistent with the findings that NDRG2 antagonizes growth factor production via the selective inhibition of ERK1/2 activation in macrophages [41]. In our previous study, p38MAPK phosphorylation was increased by NDRG2 in HepG2 cells [42], which is not consistent with the unchanged p38MAPK activation observed in the present study. We speculate that the phosphorylation of p38MAPK was increased by HT to such a high level that NDRG2 cannot enhance it anymore.
Metastasis is the most frequent cause of death in patients with advanced liver cancer, and it still poses a challenge to the development of successful cancer therapeutics [43]. HT has recently been applied as a technique for raising the temperature locally to treat tumors. In general, the effect of HT has been shown to be markedly enhanced at temperatures above 43°C, whereas heat results in an independent cytotoxic effect on cultured cells in vitro at this temperature. The cytotoxicity of HT was clearly enhanced as the temperature increased. Xie et al. found that higher temperature HT at 45°C strengthened the anti-invasion effects when compared to lower temperature HT in MCF-7 cells, whereas the cytotoxicity of HT also increased [12]. A similar result was also demonstrated in our study. HepG2 cells treated at 45°C significantly attenuated invasiveness compared with those treated at 43°C, whereas the number of necrotic cells increased (Fig. 6A). The HT at 42°C–43°C was able to kill tumor cells and prevented normal cells from being destroyed. Thermal differences between tumor cells and normal cells disappear in temperatures above 45°C. Both tumor cells and normal cells are committed to necrosis under these circumstances [44]. Necrosis physiologically affects groups of contiguous normal cells and results in phagocytosis by macrophages and a significant inflammatory immune response. Thus, to optimize therapies and avoid or minimize side effects, the overexpression of NDRG2 combined with lower temperature HT at 43°C was used to treat HepG2 cells. As expected, HT at 43°C and NDRG2 appeared to concomitantly suppress invasiveness and decrease the necrosis of HepG2 cells. Because HT>43°C cannot be realistically achieved in a clinical setting, the constitutive expression of NDRG2 combined with HT at 43°C could be a promising approach for the treatment of HCC.
In summary, the present study demonstrates that NDRG2 can be upregulated by HT (45°C)-induced heat stress and that it enhances the anti-invasion effect of HT on HCC cells. Further mechanistic investigation revealed that HT-induced NDRG2 is able to suppress activity of ERK1/2 signaling pathway, which is mainly responsible for the attenuated invasiveness in HepG2. Finally, the synergistic cooperation of 43°C HT and NDRG2 effectively reduced cytotoxicity and promote the anti-invasion effects of HT at 45°C. Understanding the molecular mechanisms involved in HT may have valuable implications for developing optimized HT therapies for NDRG2-deficient cancers.
The authors are grateful to Dr. Yongchun Zhou forkindly reviewing the manuscript.
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23630583PONE-D-12-3791810.1371/journal.pone.0061343Research ArticleBiologyBiochemistryMetabolismLipid MetabolismGenomicsGenome Expression AnalysisImmunologyImmunityInflammationModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCell DeathMedicineMetabolic DisordersNephrologyUrologyKidney StonesEffect of Adiponectin on Kidney Crystal Formation in Metabolic Syndrome Model Mice via Inhibition of Inflammation and Apoptosis Adiponectin and Kidney Crystal in ObesityFujii Yasuhiro Okada Atsushi
*
Yasui Takahiro Niimi Kazuhiro Hamamoto Shuzo Hirose Masahito Kubota Yasue Tozawa Keiichi Hayashi Yutaro Kohri Kenjiro
Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Nakano Hiroyasu Editor
Juntendo University School of Medicine, Japan
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YF AO KK. Performed the experiments: YF AO. Analyzed the data: YF AO TY YH. Contributed reagents/materials/analysis tools: YF KN SH MH YK KT. Wrote the paper: YF AO. Instruction and advice: YH KK.
2013 22 4 2013 8 4 e613431 12 2012 7 3 2013 © 2013 Fujii et al2013Fujii et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The aims of the present study were to elucidate a possible mechanism of kidney crystal formation by using a metabolic syndrome (MetS) mouse model and to assess the effectiveness of adiponectin treatment for the prevention of kidney crystals. Further, we performed genome-wide expression analyses for investigating novel genetic environmental changes. Wild-type (+/+) mice showed no kidney crystal formation, whereas ob/ob mice showed crystal depositions in their renal tubules. However, this deposition was remarkably reduced by adiponectin. Expression analysis of genes associated with MetS-related kidney crystal formation identified 259 genes that were >2.0-fold up-regulated and 243 genes that were <0.5-fold down-regulated. Gene Ontology (GO) analyses revealed that the up-regulated genes belonged to the categories of immunoreaction, inflammation, and adhesion molecules and that the down-regulated genes belonged to the categories of oxidative stress and lipid metabolism. Expression analysis of adiponectin-induced genes related to crystal prevention revealed that the numbers of up- and down-regulated genes were 154 and 190, respectively. GO analyses indicated that the up-regulated genes belonged to the categories of cellular and mitochondrial repair, whereas the down-regulated genes belonged to the categories of immune and inflammatory reactions and apoptosis. The results of this study provide compelling evidence that the mechanism of kidney crystal formation in the MetS environment involves the progression of an inflammation and immunoresponse, including oxidative stress and adhesion reactions in renal tissues. This is the first report to prove the preventive effect of adiponectin treatment for kidney crystal formation by renoprotective activities and inhibition of inflammation and apoptosis.
This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 23249074, 23592374, 23592375, 23791770, 23791774, 23791775, 22591797, 22791481, 22791479, 22791484, 21791517, and 21791520) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The worldwide prevalence of kidney stone formation increased in the last quarter of the 20th century [1]. A change in diet, especially an increased intake of lipid and animal protein, is believed to be a cause of this increased prevalence of kidney stones. The prevalence of obesity, hypertriglyceridemia, and hypercholesterolemia shows a high correlation with that of kidney stones [2].
The major components of kidney stones are inorganic materials, with a small percentage of organic material called stone matrix; 90% of the inorganic components are calcium salts, of which the majority is calcium oxalate (CaOx) [3], [4]. Asplin et al. stated that excretion values and concentrations of urinary CaOx could not be identified as risk factors [5].
In our previous studies, we detected osteopontin (OPN) as a stone matrix protein [6] and reported an increase in renal OPN expression by using a crystal formation animal model [7]. We performed microarray analysis using the crystal forming mouse kidneys, and found increased expression of inflammation-related genes such as Monocyte Chemoattractant Protein-1 (MCP-1) and decreased expression of lipid metabolism-related genes such as adiponectin (APN) [8]. We also reported renal proximal tubular cell injury and oxidative stress caused by a mitochondrial disorder in the early phase of kidney crystal formation [9]. This mechanism of kidney crystal formation is very similar to that of atherosclerosis and was the basis of our hypothesis that kidney crystal formation is a metabolic syndrome (MetS)-related disease.
Recently, kidney stone formation has been recognized as a MetS-related disease [10]–[13]. Taylor et al. reported a positive relation between obesity, weight gain, and the risk of kidney stones in a prospective study of 3 large cohorts [10]. Moreover, researchers have shown an epidemiological association between atherosclerosis and kidney stones. Yasui et al. showed a high association between aortic calcification index and kidney stones, as detected by Computed Tomography [13].
A recent study has suggested that a decrease in APN, a fat-derived hormone, is involved in MetS pathology, and that APN reverses insulin resistance associated with both lipoatrophy and obesity [14]. Matsuda et al.
[15] reported that APN administration could prevent vascular stenosis. Yamauchi et al.
[16] showed that leptin-deficiency reversed insulin resistance in ob/ob mice and that APN administration could prevent atherosclerosis. Moreover, APN supplementation ameliorated renal fibrosis, diabetes nephropathy, and albuminuria in ob/ob mice [17]. Taken together, APN has antiatherosclerotic, renoprotective, anti-inflammatory, and antioxidative functions, and might be an ideal preventive medicine for kidney stones.
Here, we evaluated kidney crystal formation in a MetS mouse model, ob/ob mice. Moreover, we investigated the genetic environmental changes in the kidney using genome-wide analysis to explore the possible mechanism of kidney crystal formation in the MetS environment and the amelioration by APN.
Materials and Methods
Animals and genotyping
B6.V-Lepob/ob (designated here as ob/ob) and B6.V-Lep+/+ (designated here as +/+) (Charles River Japan, Yokohama, Japan) male mice were acclimated at 23±1°C on a 12-h light/dark cycle before initiation of experiments. All experiments were conducted using 8-week-old male littermates. All animals had free access to water and standard chow (Oriental Yeast Co., Tokyo, Japan). The genotypes of the progeny from heterozygous crossings were determined with TaqMan genotyping assays using genomic DNA extracted from tail tissue by using original, designed TaqMan probe sets (Applied Biosystems, Foster City, CA, USA). This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All experiments were approved by the Animal Care Committee of the Faculty of Medicine, Nagoya City University Graduate School of Medical Sciences (Permit Number: H19-34). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
CaOx crystal deposition was induced in male ob/ob mice (V-Lepob/ob, 8 wk of age, n = 12) and +/+ mice (V-Lep+/+, 8 wk of age, n = 12) by administering 50 mg/kg glyoxylate (GOx) for 6 days, as previously described [7].
APN treatment
Mice were treated with APN (Recombinant Mouse gAdiponectin/gAcrp30, R&D Systems, Inc., Minneapolis, USA) by subcutaneous injection in the back of each mouse for 6 days at the same time of administering glyoxylate. The amount of APN administered per day was 2.5 µg/ml in a total volume of 0.2 ml of PBS with a clean 27-gauge needle.
Serum and urine biochemistry and detection of kidney crystal formation
On days 0 and 6, kidneys were extracted, and the right unilateral kidney specimens were stored with RNAlate (Ambion, CA, USA) at −70°C until RNA preparation, and the contralateral specimens were fixed in 4% paraformaldehyde and embedded in paraffin. All mice were placed in metabolic cages on days 1 and 5. Before kidney extraction, 24-h urine samples were collected. Urinary volume, pH, calcium, phosphorus, magnesium, oxalate, and citrate were determined at each time point (SRL, Tokyo, Japan). Serum samples were collected just before kidney extraction, and calcium, phosphorus, creatinine, TG, TC, and FFA concentrations were determined at each time point (SRL, Tokyo, Japan). The amount of crystals was determined as previously described [7].
Microarray analysis and data mining
We prepared cRNA and conducted microarray analyses at Bio Matrix Research using the Affymetrix system (Santa Clara, CA, USA). Isolated total RNA (1 mg) was converted into double-stranded cDNA by using the One-Cycle cDNA Synthesis Kit (Affymetrix), which was purified using a GeneChip Sample Cleanup Module (Affymetrix). In vitro transcription reactions were performed using a GeneChip IVT Labeling Kit, which includes T7 RNA polymerase and biotin-labeled ribonucleotides. Biotin-labeled cRNA was purified using a GeneChip Sample Cleanup Module. The cRNA concentration was calculated from light absorbance at 260 nm using a UV spectrophotometer. Next, cRNA (15 mg) was fragmented at 94°C in the presence of a fragmentation buffer (Affymetrix). The labeled cRNA was purified, fragmented, and spiked with in vitro transcription controls. Fifteen micrograms of cRNA was hybridized using the GeneChip Mouse Genome 430 2.0 Array (Affymetrix). The array was incubated for 16 h at 45°C and automatically washed and stained with the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix) on an Affymetrix GeneChip Fluidics station. The probe array was scanned using a GeneChip Scanner 30007G.
The value of the transcript was calculated with the 11 values of perfect match (PM) and mismatch (MM) probes by using the GeneChip Operating Software (GCOS), in which the probabilities of the values of each transcript were indicated as “Flag” Present (p≥0 to <0.04), Marginal (p≥0.04 to <0.06), or Absent (p≥0.06 to <0.5), using one-sided Wilcoxon's signed rank test between the values of PM and MM. Analysis, normalizations, relative signal intensities, and fold changes between GOx-treated (day 6) and control samples (day 0) were calculated using GeneSpring GX 11.0 (Agilent Technologies, Loveland, Colorado, USA) data-mining software. For the extraction of kidney crystal formation-related genes, the Venn diagram function appended with the GeneSpring software was used to select genes whose expression changed by >2.0 fold and <0.5 fold. The sorted gene lists were analyzed with the GO ontology browser, one of the functions of GeneSpring GX 11.0, and the categories of biological process, cellular components, and molecular function were sorted on the basis of the annotation of listed genes. The p value of each category was calculated using Fisher's exact test. And we registered our data of microarray analysis data to the Gene Expression Omnibus of the National Center for Biotechnology Information (GEO accession number; GSE37173).
Quantitative PCR
Total RNA was isolated from frozen sections of mouse kidney samples with an RNeasy Midi Kit (Qiagen Co., Düsseldorf, Germany) according to the manufacturer's instructions. TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA,USA), 20× assay mix of primers, and TaqMan MG probes (FAM dye labeled; Applied Biosystems) were used for quantitative RT-PCR. This assay was designed to span exon–exon junctions so as not to detect genomic DNA. Validation experiments were performed to test the efficiency of the target amplification and reference amplification. The materials used for and genes analyzed by quantitative RT-PCR with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) are listed in Table S11. The primers and probe sequences were searched against the Celera database to confirm specificity. Validation experiments were performed to test the efficiency of the target amplification and reference amplification. Beta-actin (Actb, Mm00456425_m1) was used as the inner control for gene expression.
Immunohistochemical staining
Immunohistochemical staining was carried out on 4-µm-thick cross-sections. Sections were microwave treated for 15 min and blocked with 0.5% H2O2 in methanol for 30 min, washed in 0.01 M PBS, and further treated with skim milk in PBS for 1 h at room temperature. These slides were incubated overnight at 4°C with the antibodies listed in Table S12. The reacted antibodies were detected using a VECTASTAIN Elite ABC kit for rabbit or rat IgG (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. The staining of OPN, MCP-1, superoxide dismutase (SOD), APN were measured for 6 kidneys, and expressed as ratios (%) of the total tissue area of kidney cross-sections using Image-Pro Plus software (Medica Cybernetics Inc., Bethesda, MD, USA).
Western blot analysis
Whole-protein extracts from specimens were immersed in 1× lysis buffer and lysed by sonication on ice. The lysate was cooled on ice for 15 min and then clarified by low-speed centrifugation (1,000× g). The total protein concentration in the supernatant was spectrophotometrically quantified using an Ultrospec 3100 pro (GE Healthcare, Wallingford, CT, USA). Samples containing 30 µg total protein were mixed with loading buffer (Laemmli Sample Buffer; Bio-Rad Laboratories, Hercules, CA, USA) and boiled for 10 min. Proteins were resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon®-Polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Non-specific binding on the membranes was blocked for 1 h at room temperature by using Tris-buffered saline (pH 7.5)-Tween 20 containing 5% skim milk, followed by an overnight incubation at 4°C with the antibodies listed in Table S12.
TUNEL-positive cells counting
Images of sections were scanned and expressed as number of the total cortico-medulla junction of kidney cross-sections by using Image-Pro Plus software (Medica Cybernetics Inc., Bethesda, MD, USA). Cell counts were done by two researchers in a blinded fashion. To count cells, the slides were projected onto a grid comprising 12 fields. Six randomly chosen fields were used. These six fields were used for all study slide counts [18]. Magnification is ×400.
Statistical analysis
The amount of kidney crystal deposition and immunohistochemical staining deposition and the results of serum and urine tests are shown as means ± SD. The values of mRNA expression of all genes, measured by quantitative RT-PCR, are shown as means ± SE. The statistical significance of differences between groups was examined using the Mann-Whitney U test, and a probability of 0.05 was taken as significant.
Results
Serum and urine biochemistry
At day 0, the total cholesterol (TC) values in ob/ob mice were significantly higher than those in +/+ mice (Table S1). None of the other determined values were significantly different between the 2 groups. At day 6, the triacylglyceride (TG) and TC levels in ob/ob mice were substantially higher than those in wild-type mice. Moreover, the TC and free fatty acid (FFA) values in ob/ob mice were significantly higher than those in wild-type mice. At day 0, the serum APN value in ob/ob mice was lower than that in wild-type mice. Moreover, APN was decreased by glyoxylate administration. The plasma APN levels after recombinant APN injection were similar to physiological levels (Table S1). APN treatment did not result in any significant changes in serum biochemistry results.
Kidney crystal depositions
No crystal formation could be detected in +/+ kidneys at days 0 and 6. CaOx crystal depositions were detected in the renal proximal tubular lumen located at the region between the renal cortex and the medulla only in ob/ob kidneys at day 6 (Fig. 1A). APN treatment ameliorated crystal aggregation and scattered the crystals throughout the entire kidney region. A quantitative evaluation of kidney crystal formation at day 6 revealed the amount of the crystals in ob/ob kidneys was significantly higher than that in +/+ kidneys (p = 0.0004) and APN-treated ob/ob kidneys (p = 0.005) (Fig. 1B).
10.1371/journal.pone.0061343.g001Figure 1 Detection and quantification of calcium oxalate kidney crystal formation.
A. Kidney sections of wild type (+/+), obesity (ob/ob), and adiponectin-treated ob/ob (ob/ob+APN) mice at day 6. The upper images show calcium oxalate crystal deposits in Pizzolato-stained sections. The lower images show non-stained sections observed by polarized light optical microphotography (PLOM). Magnification is ×20 (in box: ×400). B. Quantification of kidney crystals in +/+, ob/ob, and ob/ob+APN mice. Data are indicated as the mean ± SD. a; p = 0.0004, b; p = 0.005.
Microarray analysis and data mining
A genome-wide expression assay using microarrays indicated 45,101 genes were significantly changed in expression. Based on the schema of Fig. 2A, the number of gene groups with >2.0-fold increased and <0.5-fold decreased expression between the relevant groups was as follows: ΔA (gene expression involving GOx administration), 157 and 159 genes; ΔB (gene expression involving obesity), 186 and 218; ΔC (gene expression involving GOx administration and kidney crystal formation), 621 and 627; ΔD (gene expression involving obesity and kidney crystal formation), 662 and 817; ΔE (gene expression involving obesity, GOx administration, and kidney crystal formation), 668 and 895; and ΔF (gene expression involving APN administration and kidney crystal prevention), 190 and 154.
10.1371/journal.pone.0061343.g002Figure 2 The scheme of the microarray analysis design.
A. Experimental design and gene expression changes between groups. Δ refers to a gene group with >2.0- or <0.5-fold expression change between 2 experimental groups. ΔA means gene expression involving GOx administration. ΔB means gene expression involving obesity. ΔC means gene expression involving GOx administration and kidney stone formation. ΔD means gene expression involving obesity and kidney stone formation. ΔE means gene expression involving obesity, GOx administration, and kidney stone formation. ΔF means gene expression involving APN administration and kidney stone prevention. +GOx, glyoxylate administration; +APN, adiponectin treatment. B. The selection scheme for kidney stone formation-related gene groups (Venn diagram). The gray-stained area is represented by (ΔC∩ΔD∩ΔE)\(ΔA∪ΔB) and is the kidney stone formation-specific gene group under a MetS environment.
Among the genes with increased expression (Table S2) were inflammatory-related genes such as lipocalin 2 (Lcn2) and cell cycle-related genes such as minichromosome maintenance deficient 5 (Mcm5). Among the genes with decreased expression (Table S4) were those from the solute career family (Slc12a1, Slc7a13). GO analyses were performed for the genes showing increased and decreased expression during kidney crystal formation (Tables S3 and S5, respectively). The upregulated genes belonged to the categories related to inflammation and immunoresponse, collagen production, and cell proliferation with DNA replication. Meanwhile, the downregulated genes belonged to the categories related to oxidoreductase and oxygen transporter activity and to chromatin and nucleosome assembly.
Among the kidney crystal-related genes, those that showed a reversal in their expression through exogenous APN treatment were selected, i.e., the genes among the >2.0-fold upregulated genes related to kidney crystal formation that showed a <0.5-fold reduction in expression with APN treatment [((ΔCi∩ΔDi∩ΔEi)\(ΔAi∪ΔBi))∩ΔFd], and vice versa for the genes among the <0.5-fold downregulated genes related to kidney crystal formation [((ΔCd∩ΔDd∩ΔEd)\(ΔAd∪ΔBd))∩Δfi]. The number of genes belonging to each group was 154 and 190, and the top 10 genes with the highest and lowest expression changes between the ob/ob and ob/ob+APN groups at day 6 are listed in Tables S6 and S8. The downregulated genes belonged to the categories related to immunoreactions with T-cell proliferation via IL-11 binding (Table S7); the upregulated genes, to the categories related to mitosis and the cell cycle and to oxidoreductase and oxygen transporter activity (Table S9).
Expression of kidney crystal-related genes (Spp1, Ccl2, Sod2) and Adipoq
The expression changes in Spp1, Ccl2, and Sod2, previously reported as kidney crystal formation-related genes, and Adipoq were evaluated by quantitative PCR (Fig. 3A). The expression of the encoded proteins (OPN, MCP-1, SOD, and APN, respectively) was subsequently examined using immunohistochemistry (Fig. 3B).
10.1371/journal.pone.0061343.g003Figure 3 Expression analyses of Spp1, Sod2, Ccl2, and Adipoq.
A. The results of quantitative PCR for the expression of Spp1, Sod2, Ccl2, and Adipoq. Expression levels are expressed relative to Actb (actin-beta) transcript levels. Spp1, secreted phosphoprotein 1; Ccl2, C-c chemokine ligands 2; Sod2, superoxide dismutase 2; Adipoq, adiponectin. Data are indicated as the mean ± SE. *, p<0.05 between groups at the same time point. †, p<0.01 compared with the same group at day 0. B. Immunohistochemical staining for OPN, MCP-1, SOD, and APN expression. OPN, osteopontin; MCP-1, monocyte chemotactic protein-1; SOD, superoxide dismutase; APN, adiponectin. Magnification is ×20 (in box: ×400). C. The quantification of immunohistochemical staining. Data are indicated as the mean ± SD. *, p<0.05 between groups at the same time point. †, p<0.05 compared with the same group at day 0.
Spp1 in ob/ob mice displayed relatively higher expression than in +/+ mice at day 0. This difference became significant at day 6 (2.60-fold, p = 0.020). Spp1 expression in +/+ mice showed a relative increase after GOx administration (2.10-fold, p = 0.070). Meanwhile, Spp1 in ob/ob mice increased significantly with kidney crystal formation (1.91-fold, p = 0.030), and the expression value did not change following exogenous APN treatment. Ccl2 expression demonstrated no significant difference between ob/ob and +/+ mice at day 0. However, its expression in ob/ob mice increased significantly at day 6 (14.59-fold, p = 0.004) compared to day 0 but showed no remarkable change in +/+ mice. APN treatment did not change Ccl2 expression values in ob/ob mice. Sod2 expression at day 0 showed no significant difference between both genotypes; however, its expression in ob/ob mice decreased significantly at day 6 (0.31-fold, p = 0.008). Further, the decreased Sod2 expression could not be recovered by exogenous APN treatment. The expression of Adipoq in ob/ob mice at day 0 was significantly lower than that of +/+ mice (0.17-fold, p = 0.046), and the expression in +/+ mice was significantly decreased by GOx administration at 6 days (without crystal formation) (0.25-fold, p = 0.049).
The expression of stone related protein and endogenous APN demonstrated by immmunohistochemistry (Fig. 3B) followed the result of quantitative PCR. OPN was focally present at the renal cortical proximal tubular cells for both genotypes at day 0. Its expression expanded in a somewhat diffuse manner and centered around the cortico-medullar border region at day 6, especially in the ob/ob and ob/ob+APN groups with crystal formation. MCP-1 expression was clearly detected, especially in the cortical proximal tubular cells at day 0, and its expression in ob/ob mice showed a remarkable increase at the cortico-medullar border regions. MCP-1 expression in the ob/ob+APN group showed a similar distribution but appeared to be weaker than that of the ob/ob group. For both genotypes, SOD was expressed in the proximal tubular cells at the cortico-medullar junction area at day 0. Its expression intensity at day 6 was almost unchanged in the +/+ group but decreased remarkably in the ob/ob and ob/ob+APN groups. At day 0, endogenous APN in the +/+ mice could be detected strongly in the interstitial cells at the renal papilla and also in the renal parenchyma. However, the ob/ob mice at day 0 showed a remarkably lower number of APN-positive interstitial cells than +/+ mice. All groups at day 6 showed a reduced number of APN-positive interstitial cells. The results of the quanitification of immunohistochemical staining were similar to the expression of stone related genes and APN.
Confirmation of the selected genes detected by microarray analysis
Among the selected genes with significant expression changes in the microarray analysis, the expression of several characteristic genes belonging to 5 or 6 categories based on GO analysis was evaluated by quantitative PCR (Fig. 4A–E), identified several genes that were affected by APN treatment, and they were evaluated by immunohistochemistry (Fig. 5A–F). and western blotting (Fig. 6)
10.1371/journal.pone.0061343.g004Figure 4 Confirmation that selected genes detected by microarray analysis showed differential expression between different treatment groups.
The expression of several characteristic genes belonging to 5 categories based on GO analysis was evaluated by quantitative PCR. Expression levels are expressed relative to Actb. Data are indicated as the mean ± SE. *, p<0.05 between the groups at the same time point. †, p<0.01 compared with the same group at day 0. 5 categories are as follows. A. Inflammation and immune-related gene group: Lcn2, lipocalin2; Cd44, CD44 antigen; Lyz1, lysozyme1. B. Apoptosis-related gene group: Stat3, signal transducer and activator of transcription 3; Aurka, aurora kinase A. C. Cell repair and proliferation-related gene group: Mcm5, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: Vcam1, vascular cell adhesion molecule 1; Col3a1, collagen, type III, alpha 1. E. Transporter-related gene group: Slc12a1, solute carrier family 12, member 1; Slc7a13, solute carrier family 7, member 13.
10.1371/journal.pone.0061343.g005Figure 5 Confirmation of selected genes detected by microarray analysis.
A–F. Among the selected genes with significant expression changes in the microarray analysis, the expression of several characteristic genes belonging to 6 categories based on GO analysis was evaluated by immunohistochemical staining. Magnification is ×20 (in box: ×400). 6 categories are as follows. A. Inflammation and immune-related gene group: LYZ1, Lysozyme 1.CD44, CD44 antigen; MHC-class 2, major histocompatibility complex-class2. B. Apoptosis-related gene group: STAT3, signal transducer and activator of transcription 3; AURKA, aurora kinase A, Thymidine kinase 1, C. Cell repair and proliferation-related gene group: MCM5, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: Fn, Fibronectin. E. Oxidative stress-related gene group: 8OHdG, 8-Hydroxydeoxyguanosine. F: transporter-related gene group; SLC12A1, solute carrier family 12, member 1.
10.1371/journal.pone.0061343.g006Figure 6 Confirmation of selected genes detected by microarray analysis.
Western blot analysis for OPN, MCP-1, SOD, APN, LYZ1, AURKA, TK1, MCM5, NGAL, CD44, STAT3, SLC12A1, SLC7A13, VCAM1, COL3A1 and FN protein expression. Each molecular weight demonstrated as the bands were as follows. OPN, 75 and 55 kDa; MCP-1,17 kDa; SOD, 37 kDa; APN, 64 kDa; LYZ1, 17 kDa; AURKA, 40 kDa; TK1, 25 kDa; MCM5, 90 kDa; NGAL, 25 kDa; CD44, 80–95 kDa; STAT3, 92 kDa; SLC12A1, 47 kDa; SLC7A13, 52 kDa; VCAM1, 100 kDa; COL3A1, 138 kDa; FN, 212 kDa and β-actin, 37 kDa.
GOx administration resulted in the significant upregulation of Lcn2, Cd44, and Lyz1 in ob/ob and +/+ mice at day 6. For Cd44 and Lyz1, kidney crystal formation further enhanced their expression, as their transcript levels were significantly higher in ob/ob mice than in +/+ mice. APN treatment resulted in a relative decrease in Lcn2 and Cd44 expression and a significant decrease in Lyz1 expression in ob/ob mice (Fig. 4A).
Stat3 expression in ob/ob mice was significantly higher at day 6 than day 0 (Fig. 4B). APN treatment did not induce a remarkable change in Stat3 (Fig. 4B). Aurka significantly increased at day 6 in both genotypes and significantly increased with exogenous APN treatment. In ob/ob mice, Tk1 expression showed significant higher expression at day 6 relative to day 0 and significantly increased with exogenous APN treatment.
Meanwhile, Mcm5 expression significantly increased at day 6 in both genotypes and significantly increased with exogenous APN treatment (Fig. 4C).
Following GOx treatment, Vcam1 and Col3a1 were significantly upregulated. Kidney crystal formation further enhanced Vcam1 expression in ob/ob mice at day 6 when compared to wild-type mice. APN treatment resulted in relatively decreased Vcam1 and Col3a1 expression (Fig. 4D).
Slc12a1 and Slc7a13 were significantly downregulated in the presence of kidney crystal formation in ob/ob mice at day 6, showing significantly reduced levels compared to wild-type mice (Fig. 4E). Moreover, APN treatment did not induce a remarkable change in the expression of Slc12a1 and Slc7a13 (Fig. 4E).
Fig. 5A shows inflammation and immune-related protein expression for NGAL, CD44, LYZ1, F4/80, mouse macrophage surface marker, and major histocompatibility complex-class 2 (MHC-class 2). NGAL was barely detectable at day 0 and was observed in the proximal tubular cells from the cortex to the papilla. LYZ1 and CD44 were detected at the interstitium and at the proximal tubular cells from the cortex to the papilla in a diffuse manner at day 0. At day 6, these expressions strengthened focally at the cortico-medullary junction where kidney crystals were formed. This tendency was also particularly enhanced in ob/ob and ob/ob+APN mice. F4/80 was barely detectable at day 0 and was observed in interstitial cells from the cortex to the papilla. In ob/ob mice at day 6, MHC-class 2 showed particularly strong expression around the cortico-medulla junction area. MHC-class 2 expression indicates macrophage activity. MHC-class 2 could not be detected at day 0. However, it localized to interstitial and proximal tubular cells at cortico-medullary junction and in ob/ob kidneys at day 6. MHC-class 2 expression levels were lower in ob/ob+APN kidneys than in ob/ob ones at day 6.
Fig. 5B shows apoptosis-related protein expression for signal transducer and activator of transcription 3 (STAT3), aurora kinase A (AURKA), Thymidine kinase 1 (TK1), as well as TUNEL staining as an apoptosis index. STAT3 at day 0 was present in a diffuse manner in renal proximal tubular cells from the cortex to the medulla for both genotypes. In ob/ob kidneys at day 6, focal staining at the crystal formation area of the cortico-medullary junction was observed. At day 6, the expression in ob/ob+APN kidneys compared to ob/ob ones. AURKA expression was detected in the proximal tubular cells at the area of the cortex-medulla, and ob/ob and ob/ob+APN kidneys at day 6 showed strong expression at the cortico-medullary junction area. TK1 at day 0 was present in a diffuse manner in renal proximal tubular cells from the cortex to the medulla for both genotypes. In ob/ob kidneys at day 6, focal staining at the crystal formation area of the cortico-medullary junction was observed. At day 6, the expression in ob/ob+APN kidneys compared to ob/ob ones.
MCM5 was hardly detected at day 0 but showed strong expression in the nuclei of proximal tubular cells in the area from the cortex to the papilla at day 6, particularly in ob/ob+APN kidneys (Fig. 5C).
Fig. 5D shows the expression of the adhesion and fibrosis-related protein fibronectin (Fn) as a fibrosis index. Fn expression showed a diffuse localization pattern in the interstitial cells from the cortex to the papilla. At day 6, both ob/ob and ob/ob+APN kidneys showed strong expression around the crystal-forming area, with the latter showing relatively weaker expression than the former.
Fig. 5E shows an oxidative stress-related index for 8-hydroxydeoxyguanosine (8-OHdG). 8OHdG was detected in the nuclei of the proximal tubular cells in the area from the cortex to the papilla, with ob/ob and ob/ob+APN kidneys at day 6 showing strong expression. The former showed weaker staining than the latter.
Transporter-related solute career family 12, member 1 (SLC12A1) was expressed diffusely through the entire kidney in both genotypes at day 0 (Fig. 5F). At day 6, ob/ob and ob/ob+APN kidneys showed diminished expression at the cortico-medullary junction area where kidney crystals were formed.
By TUNEL staining, ob/ob and ob/ob+APN kidneys at day 6 showed stained nuclei in the area from the cortex to medulla, with the cortico-medullary junction showing particularly strong staining. At day 6, ob/ob+APN kidneys showed weak staining compared to ob/ob ones. At day6, TUNEL-positive cells increased significantly in ob/ob mice. In ob/ob+ APN mice, the number of TUNEL-positive cells was lower than that in ob/ob mice (Fig. 7).
10.1371/journal.pone.0061343.g007Figure 7 TUNEL staining and the number of TUNEL-positive cells.
Data are indicated as the mean ± SD. *, p<0.05 between groups at the same time. **, p<0.01 between groups at the same time point. †, p<0.01 compared with the same group at day 6.
The results of western blot analysis were similar to immunohistochemical staining in OPN, MCP-1, SOD, APN, LYZ1, AURKA, STAT3, MCM5 and SLC12A1 and were similar to the expression in OPN, MCP-1, SOD, APN, LYZ1, AURKA, TK1, MCM5 and SLC12A1.(Fig. 6). All of our findings by RT-PCR, immunohistochemical staining and western blotting were summalized in Table S10.
Discussion
In the present study, we investigated the following 3 points to elucidate the mechanism of kidney crystal formation under the MetS environment as well as the efficacy of exogenous APN treatment as an MetS prophylactic agent for kidney crystal prevention: (i) mineral environmental changes in blood and urine, (ii) expression changes and renal distribution of previously reported kidney crystal-related genes and endogenous APN, and (iii) novel genetic environmental changes in the kidney by using a genome-wide analysis.
Serum and urine biochemistry data indicated a high concentration of serum TC and triglyceride TG in MetS model mouse (ob/ob mouse). However, as previously reported, a decrease in urinary pH or citrate [19] or an increase in urinary calcium, uric acid, or oxalate concentration was not observed [20].
Next, changes in the expression of previously reported kidney crystal-related genes were investigated. Before kidney crystal formation, the kidneys in ob/ob mice showed significantly higher expression of OPN and significantly lower expression of endogenous APN than kidneys from +/+ mice. At day 6, the +/+ kidneys without crystals showed a slight increase in OPN expression and a decrease in SOD expression, but the expression of MCP-1 did not change remarkably. However, the expression of endogenous APN in the +/+ mice significantly decreased. This change might be the result of factors involved in the mechanism linking GOx administration with crystal formation. The lower value for endogenous APN expression at day 0 probably caused kidney crystal formation in ob/ob mice. Thus, the suppression of APN expression resulted in the progression of kidney crystal formation. In this study, we found that APN expression was localized in the renal interstitial cells, which possess lipid drops that produce prostaglandins and prostacyclins in the cytoplasm [21]. It is reported that APN is mainly produced from the adipose tissue [22]. We observed APN expression was decreased in ob/ob mouse and the kidney crystal formation kidneys in this study, but it is unclear whether the kidney-specific APN was more important for kidney crystal formation than adipocytes-derived one. The details regarding the physiological functions of the cells remain unclear.
Previous studies indicate that hypertrophy of adipocytes decreased exogenous APN expression. Tsuchida et al.
[23] reported that the control of APN receptor sensitivities via the Insulin/Foxo1 pathway might be involved in the progression of oxidation and inflammation via deficiency of APN. Recently, Shibata et al.
[24] reported a protective effect for APN in mitochondrial injury of cardiac myocytes. In the present study, we showed that exogenous APN treatment could prevent kidney crystal formation. However, APN treatment could not induce significant changes in the expression of “traditional” crystal-related genes such as Spp1, Ccl2, and Sod2. These results indicate that the crystal-preventive effect of APN treatment did not involve suppression of SOD-related oxidative stress, MCP-1-related inflammation, or OPN-related crystal matrix expression. Commonly, inflammation suppressed APN excretion from adipocytes via macrophage-derived Il-6 and TNF-alpha expression. Our previous study showed that kidney crystal formation was accompanied by inflammation involving OPN and MCP-1 expression, and induced macrophage migration to renal interstitium [25]. Our genome-wide study using stone model mice indicated increased expression of Il-6 and TNF-alpha during kidney crystal formation [26]. This might be the reason of decreased APN expression during kidney crystal formation.
In the microarray analysis, among the extracted functional categories by GO analysis, inflammation and immune-related genes were noticeable. Among the inflammation-related genes, Lyz1 showed significantly decreased expression following APN treatment. Lyz1, which was also detected by our previous genome-wide analysis using a crystal formation mouse model, is a macrophage-derived hydrolase and a component of the crystal matrix [26].
Based on the results of the TUNEL staining, APN treatment may improve apoptosis; however, STAT3 expression did not change with APN treatment. In contrast, AURKA, an antiapoptotic agent in the p73-dependent apoptosis pathway [27], was upregulated significantly at day 6, while the expression of this protein in ob/ob kidneys was significant lower than that in +/+ kidneys. Hence, the MetS environment may suppress the antiapoptosis ability of Aurka, and APN treatment may significantly recover its expression. Thymidine kinase 1 (TK1) is a cytoplasmic enzyme functioning in phosphorylation and DNA synthesis and acts in renoprotection against apoptosis [28]. In this study, TK1 might play important roles in the repair of injured proximal tubular cells by GOx, and APN treatment may improve this ability, leading to renoprotection against crystal formation.
For examining cell repair and proliferation, we investigated MCM5 expression during kidney crystal formation. As suggested by this study, MCM5 might play important roles in repairing proximal tubular cells injured following GOx, and APN treatment may improve this ability, leading to renoprotection from crystal formation.
We investigated the production of 8-OHdG as an index of oxidative stress, 8-OHdG is one of the major forms of DNA damage induced by reactive oxygen species [29]. The pattern of 8-OHdG production was similar to Sod2 expression, and APN was unable to inhibit the oxidative stress.
On the basis of these research findings, the decrease of serum and renal APN was thought to be one of the factors in process of crystallization. However, only by a decrease of APN, we think another formation mechanism under MetS.
Conclusions
This study provided compelling evidence indicating that the mechanism of kidney crystal formation in the MetS environment involved renal biomolecular environmental changes, including increased expression of inflammation molecules such as OPN, and decreased transporter activities due to accumulated oxidative stress and cell injury. The genome-wide analysis suggests that the mechanism of kidney crystal formation in the MetS environment does not involve changes in urinary mineral composition. Moreover, we showed that exogenous APN treatment significantly prevented kidney crystal formation and that the mechanism involved renoprotective functions via anti-inflammatory and antiapoptotic effects and injured cell repair functions. Therefore, APN might be an effective prophylaxis not only for atherosclerosis but also for kidney crystal formation.
Supporting Information
Table S1
Serum and urine biochemistry. Serum and urine biochemistry in +/+ and ob/ob mice (n = 6) on days 0 and 6. Each value is the mean ± SD value, n = 6. *p<0.05 versus +/+ on day 0. **p<0.01 versus +/+ on day 0. †p<0.05 versus +/+ on day 6.
(TIF)
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Table S2
Ten genes showing the greatest increase in expression in the kidney stone formation-related gene group. Listed genes showed a >2-fold change. Fold change compared with the controlled raw value of +/+ mice on day 0. Gray-toned genes are followed by quantitative PCR or immunohistochemistry.
(TIF)
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Table S3
GO analysis of overexpressed genes in the kidney stone formation-related gene group. The 259 genes were clustered based on their GO biological process, cellular component, and molecular function ontology. Within the top 10 of each category, the GO with the lowest p-value was selected.
(TIF)
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Table S4
Ten genes with the greatest decrease in expression in the kidney stone formation-related gene group. Listed genes showed a <0.5-fold change. Fold change compared with the controlled raw value of +/+ mice on day 0. Gray-toned genes are followed by quantitative PCR or immunohistochemistry.
(TIF)
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Table S5
GO analysis of genes with suppressed expression in the kidney stone formation-related gene group. The 243 genes were clustered based on their GO biological process, cellular component, and molecular function ontology. Within the top 10 of each category, the GO with the lowest p-value was selected.
(TIF)
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Table S6
Twelve genes with the greatest expression decrease in the >2.0-fold upregulated genes related to kidney stone formation and the <0.5-fold downregulated gene group by APN treatment. Fold change compared with the controlled raw value of ob/ob mice on day 6. Gray-toned genes are followed by quantitative PCR or immunohistochemistry.
(TIF)
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Table S7
GO analysis of genes with suppressed expression in the >2.0-fold upregulated genes related to kidney stone formation, <0.5x downregulated gene group by APN treatment. The 154 genes were clustered based on their GO biological process, cellular component, and molecular function ontology. Within the top 10 of each category, the GO with the lowest p-value was selected.
(TIF)
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Table S8
Ten genes from the <0.5-fold downregulated genes related to kidney stone formation, with the highest expression increase on APN treatment, >2.0-fold upregulated gene group. Fold change compared with the controlled raw value of ob/ob mice on day 6. Gray-toned genes are followed by quantitative PCR or immunohistochemistry.
(TIF)
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Table S9
GO analysis of genes from the <0.5-fold downregulated genes related to kidney stone formation that were overexpressed on APN treatment, >2.0-fold upregulated gene group. The 190 genes were clustered based on their GO biological process, cellular component, and molecular function ontology. Within the top 10 of each category, the GO with the lowest p-value was selected.
(TIF)
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Table S10
The expression changes of genes and proteins by GOx administration, obesity, and APN administration. ↑↑; more increased, ↑; increased, →; no remarkable change, ↓; decreased, ↓↓; more decreased. Gray-toned genes are followed significantly changes by APN administration.
(TIF)
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Table S11
Gene title, Protein name and Taqman MG probe ID TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA,USA), 20× assay mix of primers, and TaqMan MG probes (FAM dye labeled; Applied Biosystems) were used for quantitative RT-PCR. This assay was designed to span exon–exon junctions so as not to detect genomic DNA. Validation experiments were performed to test the efficiency of the target amplification and reference amplification.
(TIF)
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Table S12
The antibodies' name.
(TIF)
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We thank Ms. N. Kasuga, Ms. Kawamura, and Ms. Ichikawa for their secretarial assistance.
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74 : 1004 –1007 .19616291 | 23630583 | PMC3632593 | CC BY | 2021-01-05 17:25:57 | yes | PLoS One. 2013 Apr 22; 8(4):e61343 |
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10.1371/journal.pone.0062669
Research Article
Biology
Molecular Cell Biology
Signal Transduction
Signaling Pathways
Neuroscience
Molecular Neuroscience
Signaling Pathways
Neurochemistry
Neuroendocrinology
Neurophysiology
Central Nervous System
Homeostatic Mechanisms
Neural Homeostasis
Neurotransmitters
Hypothalamic Inhibition of Acetyl-CoA Carboxylase Stimulates Hepatic Counter-Regulatory Response Independent of AMPK Activation in Rats
Hypothalamic ACC Modulates Glucose Production
Santos Gustavo A. 1
Pereira Vinícius D. 1
Roman Erika A. F. R. 1
Ignacio-Souza Leticia 1
Vitorino Daniele C. 2
de Moura Rodrigo Ferreira 1
Razolli Daniela S. 1
Torsoni Adriana S. 3
Velloso Licio A. 1
Torsoni Marcio A. 3 *
1 Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil
2 Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil
3 Faculdade de Ciências Aplicadas, Universidade Estadual de Campinas, Limeira, São Paulo, Brazil
Bacurau Reury FP. Editor
University of Sao Paulo, Brazil
* E-mail: [email protected]
Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: LAV MAT. Performed the experiments: GAS VDP EAFRR DCV LI RFM DSR. Analyzed the data: GAS AST LAV MAT. Contributed reagents/materials/analysis tools: LAV MAT. Wrote the paper: AST MAT.
2013
23 4 2013
8 4 e6266911 9 2012
22 3 2013
© 2013 Santos et al
2013
Santos et al
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Background
Hypothalamic AMPK acts as a cell energy sensor and can modulate food intake, glucose homeostasis, and fatty acid biosynthesis. Intrahypothalamic fatty acid injection is known to suppress liver glucose production, mainly by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels. Since all models employed seem to involve malonyl-CoA biosynthesis, we hypothesized that acetyl-CoA carboxylase can modulate the counter-regulatory response independent of nutrient availability.
Methodology/Principal Findings
In this study employing immunoblot, real-time PCR, ELISA, and biochemical measurements, we showed that reduction of the hypothalamic expression of acetyl-CoA carboxylase by antisense oligonucleotide after intraventricular injection increased food intake and NPY mRNA, and diminished the expression of CART, CRH, and TRH mRNA. Additionally, as in fasted rats, in antisense oligonucleotide-treated rats, serum glucagon and ketone bodies increased, while the levels of serum insulin and hepatic glycogen diminished. The reduction of hypothalamic acetyl-CoA carboxylase also increased PEPCK expression, AMPK phosphorylation, and glucose production in the liver. Interestingly, these effects were observed without modification of hypothalamic AMPK phosphorylation.
Conclusion/Significance
Hypothalamic ACC inhibition can activate hepatic counter-regulatory response independent of hypothalamic AMPK activation.
This work was funded by grants from Fundação de Amparo a Pesquisa do Estado de Sao Paulo (Grant n. 2006/05660-5 and 2009/12523-2) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
The hypothalamus actively participates in energy expenditure, satiety signals and counter-regulatory response [1]–[3]. Neuropeptides such as NPY, AGRP, POMC and CART are known to be expressed in the hypothalamic nucleus, to participate in the regulatory mechanism of energy expenditure, satiety signals and counter-regulatory response, and be modulated by hormones and nutrients.
AMP-activated protein kinase (AMPK) can integrate signaling circuits between peripheral tissues and the hypothalamus to regulate food intake and whole-body energy expenditure [3], [4]. This important cell energy sensor can activate the catabolic pathways that produce ATP when energy availability is low. On the other hand, when energy is sufficient for cellular activity, it shuts down pathways that produce energy [5]. Additionally, hypothalamic AMPK has an important role in the expression of hypothalamic neuropeptides [6]–[8] and counter-regulatory response [2], modulating energy expenditure and plasma concentrations of corticosterone, glucagon, and catecholamines.
Acetyl-CoA carboxylase (ACC) is responsible for catalyzing the reaction that produces malonyl-CoA, an intermediate in the biosynthesis of fatty acids. ACC is an AMPK target, which phosphorylates on Ser72 and thereby inactivates ACC under conditions of energy surplus [9]. Several studies have shown that pharmacological activation of AMPK, which promotes the inhibition of ACC and a decrease in hypothalamic levels of malonyl-CoA, leads to an increase in food intake [10], [11]. Furthermore, recently Kinote and colleagues showed that fructose activates hypothalamic AMPK and stimulates hepatic PEPCK and gluconeogenesis [12]. On the other hand, refeeding and fatty acid synthase inhibitor increase the hypothalamic availability of malonyl-CoA and decrease food intake [13]–[16]. The hypothalamic level of malonyl-CoA increases (4.0-fold) in response to transition from fasted to fed state. In fasted rats, the reduction of hypothalamic level of malonyl-CoA occurs even in the presence of acetyl-CoA [17].
Hypothalamic lipid metabolism is important for the control of energy metabolism [18]–[20]. Obici and colleagues demonstrated that oleic acid [21] and inhibition of carnitine palmitoyltransferase-1 [22] decrease food intake and liver glucose production. More recently, Ross and collaborators demonstrated differential effects of hypothalamic long-chain fatty acid infusion on the glucose production [23]. In this study, they showed that a low dose of oleic acid administered to the medium basal hypothalamus is sufficient to markedly reduce liver glucose production, whereas a polyunsaturated fatty acid (linoleic acid) and a saturated fatty acid (palmitic acid) did not show any effect or only in high dose, respectively. Mammalian cells are not capable of producing polyunsaturated fatty acids, but the biosynthesis of saturated and monounsaturated fatty acids that occurs in the cytoplasm is very important for this pathway.
We hypothesized that inhibition of ACC independent of nutritional status and AMPK activation has an important role in the positive modulation of hepatic counter-regulatory response. To test this hypothesis, intracerebroventricular injection of antisense oligonucleotide (ASO) to acetyl-CoA carboxylase (ACC) was performed in rats with free access to food; the analyses were performed at noon because it is a time when the animal has no counter-regulatory stimulus resulting from long fasting.
Materials and Methods
Ethics Statement
This study was carried out in strict accordance with the recommendations of the COBEA (Brazilian College of Animal Experimentation) guidelines, which was approved by the Ethical Committee for Animal Use (ECAU) (ID protocol: 1970–1) of the Universidade Estadual de Campinas (UNICAMP), Campinas, São Paulo, Brazil.
Animals and Surgical Procedures
Male Wistar rats (12 wk old, 250–280 g) were taken from the University’s central breeding colony and maintained in polypropylene cages in a room at 24±1°C with lights on from 6∶00 to 18∶00 h and fed diets and water ad libitum. The rats were chronically instrumented with an ICV cannula and kept under controlled temperature and light-dark conditions in individual metabolic cages. Surgery was performed under anesthesia, and all efforts were made to minimize animal suffering. Briefly, the animals were anesthetized with 50 mg/kg ketamine and 5 mg/kg diazepam (ip) and positioned onto a Stoelting stereotaxic apparatus after the loss of cornea and foot reflexes. A stainless steel 23 gauge guide cannula with an indwelling 30 gauge obturator was stereotaxically implanted into the lateral cerebral ventricle at pre-established coordinates, anteroposterior, 0.2 mM from bregma, lateral, 1.5 mM; and vertical, 4.2 mM, according to a previously reported technique [24]. The cannulas were considered patent and correctly positioned by dipsogenic response elicited after injection of angiotensin II (2 µL of solution 10−6 M) [25]. After test for cannula function and position, rats were randomly assigned to one of the experimental groups.
Food intake and Body Weight Measures
Body weight was evaluated from the first to the fourth days of treatment with ASO. Food intake was evaluated on the third day after the ACC-ASO administration was started. Pre-weighed food was provided in individual cages 10 min before the start dark period. Cumulative food intake was measured after 12 h by weighing the residual food in the cages. The amounts of food left over on the bottom of the cages were recorded. Intake was calculated as the weight (g) of food provided less that recovered.
AICAR Injection
The fed rats received 3 µL of bolus injection of 2 mmol/L 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) into the lateral ventricle.
Sense (SO) and Antisense Oligonucleotide (ASO) Injection
Phosphorothioate-modified sense and antisense oligonucleotides (produced by IDT, Munich, Germany) were diluted to a final concentration of 1 nmol/µL in dilution buffer containing 10 mmol/L Tris–HCl and 1.0 mmol/L EDTA. The oligonucleotides sequences consisted of 5′-GCC AGT CAG TAA GAG CAG-3′ (sense) and 5′-TGA GAT CTG CAA TGC A-3′ (antisense). Wistar rats were injected into the lateral ventricle with two daily doses of 4 nmoles oligonucleotides in dilution buffer containing either sense (ACC-SO) or antisense oligonucleotides (ACC-ASO) for three days. Fragments of liver and hypothalamus were obtained at about 12∶00 h on the fourth day of treatment with oligonucleotide. The animals were provided free access to water and rat chow. Control animals received saline solution.
Pyruvate Challenge
Rats with free access to food were injected intraperitoneally with sodium pyruvate (0.5 g/kg). Blood samples were collected from the tail vein immediately before and at various time points (0–120 min) after the pyruvate load to measure blood glucose.
Tissue Extraction and Immunoblotting
The rats were anesthetized after specific treatments and tissue samples were obtained and homogenized in freshly prepared ice cold buffer (1% Triton X 100, 100 mM TRIS, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, and 0.01 mg aprotinin.mL−1). The insoluble material was removed by centrifugation (10,000 g) for 25 min at 4°C. The protein concentration in the supernatant was determined by the Bradford dye-binding method. The supernatant was resuspended in Laemmli sample buffer and boiled for 5 min before separation in SDS PAGE using a miniature slab gel apparatus (Bio Rad, Richmond, CA). Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant). The nitrocellulose transfers were probed with specific antibodies. The phophoenolpyruvate carboxykinase (PEPCK) and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. The total AMP activated protein (AMPK), phospho-AMPK (p-AMPK), total ACC (ACC) and phospho-ACC antibodies were obtained from Cell Signaling Technology, Inc., Danvers, MA. Subsequently, the blots were incubated with HRP-conjugate antibodies (KPL, Gaithersburg, MD- USA). The results were visualized by autoradiography with preflashed Kodak XAR film. Band intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software, ScionCorp) and the intensities of the bands were normalized to those of either total protein or GAPDH to correct for protein loading in the case of cellular lysate extracts.
Immunofluorescence Staining
For histological evaluation, hypothalamic tissue samples were fixed in 4% formaldehyde and processed routinely for embedding in paraffin block. The samples were dehydrated (alcohol at 70, 80, 90, 95%, and absolute), diaphanized by immersion in xylol, and embedded in paraffin. Hydrated (alcohol at absolute, 95, 90, 80, and 70%) 5.0-mm paraffin sections were processed for immunofluorescence staining. The expression of ACC (Cell Signaling Technology, Inc., Danvers, MA) was analyzed employing goat anti-rabbit-FITC was used as secondary. The images were obtained using a Leica FW 4500 B microscope, software LAS V3.8.
Real Time PCR Analysis
Hypothalamic total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations. Total RNA was rendered genomic DNA-free by digestion with Rnase-free Dnase (RQ1, Promega, Madison, WI, USA). Reverse-transcription was performed using total RNA from hypothalamic samples. Intron-skipping primers for NPY, POMC, CART, TRH, and CRH mRNAs were obtained from Applied Biosystems. Real-time PCR analysis of gene expression was performed in an ABI Prism 7700 sequence detection system (Applied Biosystems, California). The optimal cDNA and primer concentrations, as well as the maximum efficiency of amplification, were obtained through five-point, two-fold dilution curve analysis for each gene. Each PCR contained 20 ng of reverse-transcribed RNA and was run according to the manufacturer’s recommendation using the TaqMan PCR Master Mix (Applied Biosystems, California). Target mRNA expression was normalized to GAPDH expression and expressed as a relative value using the comparative threshold cycle (Ct) method (2−ΔΔCt) according to the manufacturer’s instructions.
Biochemical and Hormonal Measurements
Blood glucose was determined using a glucometer. Serum insulin, glucagon, and corticosterone were analyzed simultaneously and measured in duplicate at all time points using a commercially available rat endocrine Linco-plex kit (Rendo-85 K, Linco Research, St Charles, MO, USA). All blood samples were collected from the tail vein.
For the determination of the level of hepatic glycogen, a tissue fragment was evaluated as described by Burant and colleagues [26].
Data Presentation and Statistical Analysis
All numerical results are expressed as means ± SE of the indicated number of experiments. Blot results are presented as direct band comparisons in autoradiographs and quantified by densitometry using the Scion Image software (ScionCorp). Student’s t-tests of unpaired samples and variance analyses (ANOVA) for multiple comparisons were used as appropriate. Post hoc test (Tukey) was employed when required at significance level of p<0.05.
Results
AMPK Phosphorylation and ACC Expression
Initially, we evaluated the levels of hypothalamic phosphorylation of AMPK and the expression of ACC. As expected, fasted rats showed higher AMPK phosphorylation than fed rats. The ICV treatment of fed rats with either antisense or sense oligonucleotide (ASO and SO, respectively) to ACC proteins did not affect the hypothalamic phosphorylation of AMPK (Fig. 1A). In addition, we evaluated the effect of intracerebroventricular (ICV) injection of ACC-ASO (antisense oligonucleotide) on the hypothalamic, hippocampal and brainstem expression of ACC. For this purpose, rats were treated with either antisense or sense oligonucleotide (4 nmol/animal) twice a day for three days. As shown in Fig. 1B, the ACC-ASO treatment reduced ACC expression in the hypothalamus by 50%, when compared to both the control group (treated with saline) and the ACC-SO group (sense nucleotide). As expected, the treatment with ACC-SO did not affect the expression of ACC. ACC expression was also evaluated in the brainstem and hippocampus of control and ACC-ASO rats. As shown in Fig. 1C, ACC-ASO rats presented reduced ACC expression when compared to control rats in both investigated areas. Furthermore, to evaluate whether the ACC-ASO treatment affected the neuronal populations in the arcuate nucleus (ARC), paraventricular nucleus (PVH) and lateral hypothalamus (LH) differently, we performed immunoflorescence staining of hypothalamus samples from control and ACC-ASO rats. ACC expression was detected in all studied nuclei. However, as expected, it decreased in the ARC, PVH and LH after administration of ACC-ASO (Fig. 1D).
10.1371/journal.pone.0062669.g001 Figure 1 Modulation of AMPK/ACC pathway in central nervous system of ACC-ASO and ACC-SO mice.
Representative western blot of hypothalamic p-AMPK (A) and ACC (B) in fasted and fed rats, ACC-ASO (ASO) and ACC-SO (SO). Representative western blot of hippocampus and brainstem area of ACC (C). Representative ACC immunoflorescence staining (green) of samples from hypothalamus (ARC, LH and PVH) of Wistar rats. Cell nuclei were counterstained with DAPI (blue) (D). Bars show quantification of total p-AMPK and ACC proteins normalized by either total GAPDH or AMPK. Data are means ± SEM of five rats. (A)*p≤0.05 vs. control fed, ASO and SO. (B)*p≤0.05 vs. control and SO.
Food intake, Body Weight and Epididymal Fat Mass
Food intake was measured on the third night after the treatment with ACC-ASO had been started. As can be observed in Fig. 2A, rats treated with ACC-ASO presented higher food intake (20.0±0.7 g/12 h) than control (saline) and ACC-SO (15.9±1.3 g/12 h and 16.0±1.0 g/12 h, respectively). At the end of the experimental period, the epididymal fat mass and body weight gain were measured. Figure 2B shows that the treatment with ACC-ASO diminished epididymal fat mass (1.5±0.4 g), when compared to control (5.0±0.8 g) and ACC-SO animals (4.5±0.7 g). Furthermore, we evaluated body weight gain. Although, body weight gain was lower in ACC-ASO than in control and ACC-SO rats, the difference was not significant (Fig. 2C).
10.1371/journal.pone.0062669.g002 Figure 2 Anthropometric evaluation and feeding behavior in ASO-ACC and ACC-SO mice.
Food intake (A), epididymal fat mass (B) and body weight (C) in control, ACC-ASO (ASO) and ACC-SO (SO) rats. Data are means ± SEM of 8–10 rats. (A)*p≤0.05 vs. control and SO rats.
Real Time Analysis of Gene Expression
CART, TRH, and CRH mRNA were measured in the hypothalamus by real time PCR. As shown in Figure 3, the treatment with ACC-ASO significantly decreased gene expression of neuropeptides CART (50%), TRH (70%), and CRH (40%) (Figs. 3A, B, and C, respectively). On the other hand, NPY expression increased (2.7-fold) in ACC-ASO rats when compared to control rats. POMC mRNA was not different among the evaluated groups. Interestingly, the analysis of gene expression in fasted rats revealed a behavior similar to that of rats treated with ACC-ASO. ACC-SO treatment did not alter the expression of any of the hypothalamic neuropeptides evaluated.
10.1371/journal.pone.0062669.g003 Figure 3 mRNA level of neuropeptides in hypothalamus from ASO-ACC and ACC-SO mice.
Analyses of CART, TRH, CRH and NPY mRNA expression in the hypothalamus of control, ACC-ASO (ASO) and ACC-SO (SO) rats by RT-PCR. Data are means ± SEM of 8–10 rats. #p≤0.05 and *p≤0.01.
Serum Hormone Level
Serum glucagon, insulin, and corticosterone levels were quantified on the fourth day. The animals had previous free access to chow and blood samples were collected at 12∶00 h. The serum corticosterone level was not affected by the treatments with ACC-ASO, ACC-SO, or saline ICV (data not shown). However, the serum level of glucagon in the ACC-ASO group (14.9±2.7) was higher than in the control and ACC-SO groups (6.2±1.5 and 4.0±2.3 pmol.L−1, respectively), but it was similar in the fasted group (11.8±4.0 pmol.L−1) (Fig. 4A). On the other hand, the serum level of insulin for ACC-ASO was similar to that of fasted animals (204±38 and 194±102 pmol.L−1, respectively), but smaller than the control and ACC-SO treatment values (459±215 and 648±114 pmol.L−1, respectively) (Fig. 4B). Interestingly, the blood level of ketone bodies increased in the ACC-ASO group relative to the control and ACC-SO groups (0.47±0.11, 0.29±0.07 and 0.32±0.09 mmol.L−1, respectively) (Fig. 4C). The hepatic glycogen store was reduced in ACC-ASO (20.1±5.0 mg.g−1 of tissue) when compared to control and ACC-SO rats (35±7 and 34±3 mg.g−1 of tissue, respectively) (Fig. 4D).
10.1371/journal.pone.0062669.g004 Figure 4 Biochemical parameters in ASO-ACC and ACC-SO mice.
Serum glucagon (A) and insulin level (B), blood ketone bodies (C), and hepatic glycogen (D) in control (fed), ACC-ASO (ASO), ACC-SO (SO) and fasted rats. Data are means ± SEM of 8–10 rats. (A) *p≤0.01 to ASO and fasted vs. control and SO. (B) *p≤0.05 vs. ASO and fasted. (C and D) *p≤0.05 vs. control and SO.
Hepatic Gluconeogenic Profile
To assess whether the liver presented counter-regulatory activity, we evaluated the expression of PEPCK and the capacity of the liver to produce glucose after administration of pyruvate, a gluconeogenic substrate. As can be observed in Figure 5A, the expression of hepatic PEPCK increased significantly (by seven-fold) after treatment with ACC-ASO when compared to control animals, and so did the phosphorylation of AMPK and ACC (Fig. 5B). The treatment with ACC-SO, as expected, did not exert any effect on PEPCK expression and AMPK and ACC phosphorylation relative to control and ACC-SO.
10.1371/journal.pone.0062669.g005 Figure 5 Liver PEPCK expression and AMPK/ACC phosphorylation in ASO-ACC and ACC-SO mice.
Representative western blot of PEPCK (A). Bars show quantification of total PEPCK protein normalized by total GAPDH. Representative western blot of p-AMPK and p-ACC protein normalized by total GAPDH (B). Data are means ± SEM of 4–6 rats. *p≤0.05 vs. control and ACC-SO rats.
To evaluate liver glucose production, rats received intraperitoneal injection of sodium pyruvate and blood glucose was measured (Fig. 6A and B). As can be observed in Figures 6A and 6B, the glycemic curve and the area under the curve (AUC) were greater in the group treated with ACC-ASO than with ACC-SO and control (3960±700, 2800±400, 2340±450, respectively). Additionally, we evaluated the glycemic curve and AUC in rats previously ICV treated with AICAR, a pharmacological activator of AMPK. As expected, the increase in blood glucose was higher if compared to ACC-SO and control rats.
10.1371/journal.pone.0062669.g006 Figure 6 Liver glucose production after challenge with pyruvate in ASO-ACC and ACC-SO mice.
Blood glucose during pyruvate test (A) and area under curve (AUC) (B). Data are means ± SEM of 8–10 rats. *p≤0.05 vs. control and SO rats.
Discussion
The results presented in this study demonstrate the role of hypothalamic acetyl-CoA carboxylase (ACC) in the control of hepatic glucose production. The studies performed so far have demonstrated the participation of AMPK in the modulation of food intake and glucose homeostasis by different mechanisms in the hypothalamus [1]–[3], [16], [18], [21]–[23], [27]. A common feature to mechanisms proposed is the modulation of the hypothalamic level of malonyl-CoA. The biosynthesis of malonyl-CoA is controlled by ACC, a key enzyme in the control of biosynthesis of fatty acid and widely expressed in different tissues. Activated AMPK phosphorylates (at ser79) and inhibits ACC, leading to a reduction in the level of malonyl-CoA. The hypothalamic activity of AMPK increases during fasting and decreases during refeeding [4]. To investigate the hypothesis that the hypothalamic level of malonyl-CoA can modulate liver glucose production, we initially evaluated the hypothalamic phosphorylation of AMPK in fasted and freely fed rats (light cycle). As expected, fasted rats showed greater AMPK phosphorylation than fed rats. Reduced AMPK phosphorylation is linked to an increase in ACC activity and biosynthesis of malonyl-CoA. Tokutake and colleagues showed that the hypothalamic level of malonyl-CoA is modulated by the fasting/feeding transition in rats [17]. Interestingly, although the hypothalamic level of malonyl-CoA was modified by the fasting/feeding state, the acetyl-CoA level was not altered. To test the hypothesis that the reduction of hypothalamic ACC activity in fed rats would be enough to activate a counter-regulatory response, we injected ICV ACC-ASO in the light cycle in freely fed rats. Although, ACC-ASO injection did reduce hypothalamic ACC protein, it did not affect the hypothalamic phosphorylation of AMPK (Figs. 1A and B). Interestingly, the level of phospho-ACC (inactive form) was not different between groups (data not shown). This result reinforces the idea that treatment with ACC-ASO diminished the availability of active ACC and, consequently, of malonyl-CoA to the cells.
In an elegant study in Kahn’s Lab, Minokoshi and colleagues demonstrated that constitutively active-AMPK mice ate more and had increased expression of NPY and AGRP mRNA in ARC [4]. The increase in the AMPK activity was linked to diminished ACC activity, mimicking a fasting condition. In our study, ACC-ASO rats also presented reduced hypothalamic ACC expression (green), if compared to control rats (free access to food) and ACC-SO (Fig. 1B and 1D) in all nuclei studied (ARC, PVH and LH). Furthermore, ACC-ASO also decreased ACC expression in the brainstem and hippocampus (Fig. 1C). Interestingly, lower hypothalamic CART, CRH, and TRH mRNA levels and increased NPY mRNA, an orexigenic neuropeptide, accompanied this effect. CART, CRH, and TRH mRNA present reduced hypothalamic expression in response to fasting and leptin [28], [29]. Furthermore, it is important to point out that the analyses were performed in the light cycle, a period of reduced food intake [30]. Although ACC-ASO-treated rats had free access to food, they presented gene expression similar to those of fasted rats (higher NPY level). Additionally, they did not show difference in body weight (Fig. 2C) but epididymal fat mass was reduced (Fig. 2B), due to reduced expression of CART, TRH, and CRH, three neuropeptides linked to pro-thermogenic metabolism and anorexigenic behavior [31], [32]. Therefore, these results reinforce the role of malonyl-CoA as a hypothalamic indicator of energy homeostasis. Although, reduced epididymal fat mass seems to be contradictory, since ACC-ASO rats presented higher food intake than control rats (Fig. 2A), the increased lipolysis observed, as indicated by reduced epididymal fat pad, may be due to increased serum levels of glucagon observed in ACC-ASO rats. Furthermore, although the body weight gain was similar, we believe that three days were insufficient to affect body weight gain.
In fasting state, the hepatic metabolism shifts toward fat oxidation and synthesis of glucose as part of a counter-regulatory hormonal response. This shift in metabolism is important for the energy homeostasis, in which the hypothalamus has a fundamental role. Han and colleagues showed that pharmacological inhibition of hypothalamic AMPK or ARC/VMH DN-AMPK overexpression attenuated hypoglycemia-induced increases in plasma concentrations of corticosterone, glucagon, and catecholamine [2]. They concluded that systemic hypoglycemia causes hypothalamic activation of AMPK, which is important for counter-regulatory hormonal responses. In addition, recently, Kawashima and colleagues showed that hypothalamic AMPK activation by glucopenia occurs via a CaMKK-independent pathway [33] and that another AMPK upstream kinase might be involved in 2DG activation of AMPK, such as LKB1 [34]. Considering that ACC-ASO-treated rats mimic fasting condition, we compared their hormonal profile to that of fasted rats. ACC-ASO-treated rats presented reduced serum insulin and hepatic glycogen accompanied by increased serum glucagon, ketone bodies, and AUC in the pyruvate test (Figs. 4 and 6), typical hormonal and biochemical responses to fasting condition. Therefore, these results suggest that modulation of the hypothalamic activity of ACC may be able to trigger a counter-regulatory response, independent of the availability of nutrients, and hypothalamic phosphorylation of AMPK.
In recent years, many studies have shown that the hypothalamus participates in the modulation of hepatic glucose production by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels and parasympathetic signals delivered by the vagus nerve [35]–[38], which is associated with reduced hepatic expression of gluconeogenic genes, glucose-6-phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase (PEPCK). The liver is known to play an important role in glucose homeostasis through gluconeogenesis [39]. In ACC-ASO-treated rats, liver expression of PEPCK increased (Fig. 5A) corroborating the results of pyruvate tolerance test. These effects were accompanied by increase in liver AMPK and ACC phosphorylation in ACC-ASO-treated rat (Fig. 5B). Liver activation of AMPK is linked to diminished expression of gluconeogenic enzymes [40] and phosphorylation of glycogen synthase 1 [41], which reduces the glucose output from the liver. Furthermore, the increase in the blood level of ketone bodies (Fig. 4C), as well as ACC inactivation in the liver (Fig. 5B), suggests that fatty acid oxidation is increased under ACC-ASO, when compared to control and ACC-SO groups.
Thus, we believe that the hypothalamic levels of ACC protein and malonyl-CoA are important signals to control liver glucose production by an AMPK-independent mechanism. Although AMPK surely is an important nutrient and energy sensor that maintains energy homeostasis, many proteins can be modulated by kinase activity of AMPK. Thus, in the cell, it can modulate the pathway related to protein synthesis, mitochondrial biogenesis, fatty acid and glucose metabolism, and autophagia [42]. Therefore, ACC may be a better target to control the hepatic metabolism than AMPK.
The authors thank Laerte J. Silva for the English language editing.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23637748PONE-D-13-0028110.1371/journal.pone.0060369Research ArticleBiologyBiochemistryNucleic AcidsRNAMolecular Cell BiologyNucleic AcidsRNAMedicineDiagnostic MedicineOncologyCancers and NeoplasmsGastrointestinal TumorsGastric CancerBasic Cancer ResearchCancer Detection and DiagnosisCancer Risk FactorsCancer TreatmentMicroRNA-219-2-3p Functions as a Tumor Suppressor in Gastric Cancer and Is Regulated by DNA Methylation MiR-219-2-3p as a Tumor SuppressorLei Huizi
1
2
Zou Dongling
2
3
Li Zheng
4
Luo Min
2
Dong Lei
2
Wang Bin
2
Yin Haixin
2
Ma Yanni
2
Liu Changzheng
2
Wang Fang
2
Zhang Junwu
2
Yu Jia
2
*
Li Yu
1
*
1
Department of Pathology, Chongqing Medical University, Chongqing, People's Republic of China
2
Department of Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People's Republic of China
3
Department of Gynecologic Oncology, Chongqing Cancer Institute, Chongqing, People's Republic of China
4
Department of Orthopedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College, Beijing, People's Republic of China
Rishi Arun Editor
Wayne State University, United States of America
* E-mail: [email protected] (YL); [email protected] (JY)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: HL ML YM FW JZ JY YL. Performed the experiments: HL. Analyzed the data: HL LD. Contributed reagents/materials/analysis tools: DZ ZL BW HY CL. Wrote the paper: HL JY YL.
2013 23 4 2013 8 4 e6036914 12 2012 26 2 2013 © 2013 Lei et al2013Lei et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background & Aims
Gastric cancer is the most frequent gastrointestinal tumor in adults and is the most lethal form of human cancer. Despite of the improvements in treatments, the underlying mechanism of gastric carcinogenesis is not well known. To define novel modulators that regulate susceptibility to tumorgenesis, we focused on miR-219-2-3p.
Methods
Quantitative RT-PCR was employed to investigate the level of miR-219-2-3p in gastric cancer (GC) tissues (n = 113) and their matched adjacent normal tissues (n = 113). In vitro cell proliferation, apoptosis assays, cell migration, and invasion assays were performed to elucidate biological effects of miR-219-2-3p. Since silencing of miRNA by promoter CpG island methylation may be an important mechanism in tumorgenesis, GC cells were treated with 5-aza-2′-deoxycytidine and trichostatin A, and expression changes of miR-219-2-3p were subsequently examined by quantitative RT-PCR. Finally, the methylation status of CpG island upstream of miR-219-2-3p was analyzed by methylation-specific PCR in GC tissues (n = 22).
Results
miR-219-2-3p was down-regulated in GC and cell lines. In addition, the experiments documented the lower expression of miR-219-2-3p in GC specimens with higher grade and later stage tumors. Meanwhile, miR-219-2-3p exerted antiproliferative, proapoptotic, and antimetastatic roles and reduced levels of p-ERK1/2 in GC cells. Furthermore, 5-aza-2′-deoxycytidine and trichostatin A increased the expression (∼2 fold) of miR-219-2-3p in GC cells. By methylation-specific PCR, DNA methylation in the upstream region of miR-219-2-3p was detected in both adjacent normal tissues and cancer tissues. As expected, the methylation level was considerably higher in the miR-219-2-3p down-regulated group than up-regulated group.
Conclusions
miR-219-2-3p is potentially involved in gastric cancer progression and metastasis by regulating ERK1/2-related signal pathways, which may provide a novel therapeutic strategy for treatment of gastric cancer. Methylation mechanism may be involved in modulating the expression level of miR-219-2-3p in gastric cancer.
This work was supported by grants from the National Natural Science Foundation of China [2012, 91129716, to JY] and the Beijing Municipal Science & Technology Commission [2010B071, to JY]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Gastric cancer (GC) is the 4th most common cancer and the second-highest cause of cancer death worldwide. Nowadays, patients with late-stage GC are with an overall 5-year survival of approximately 20%[1]. Cancer develops as a result of an accumulation of various endogenous and exogenous causes. Eating habits and a increase in Helicobacter pyloriinfection are important exogenous causes for GC[2], while genetic, as well as dietary, levels of the hormone gastrin[3], and other chronic gastric inflammation-causing factors are found to be associated with predisposition to cancer development. Gene alterations play an important role in GC, and alterations in a large number of oncogenes and tumor suppressor genes have already been reported in GC.
Some prognostic tumor biomarkers in GC such as human epidermal growth factor receptor 2 (HER2), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), have been associated with disease characteristics and can therefore be used to inform patient management. For example, patients with tumors that test positive for HER2 can be treated with trastuzumab plus chemotherapy[4], and patients with tumors that test positive for VEGF can be treated with bevacizumab plus chemotherapy[5]. However, the molecular mechanisms underlying the development of GC remain a challenge, thus additionally informative biomarkers are urgently needed.
MicroRNAs (miRNA) are a class of small RNA molecules involved in regulation of translation and degradation of mRNAs[6]. MiRNAs bind to complementary sequences in the 3′ untranslated regions (UTR) of their target mRNAs and induce mRNA degradation or translational repression[7]. Most known functions of miRNAs are related to negative gene regulation: miRNAs silence gene expression, usually by interfering with mRNA stability or protein translation. In recent years, miRNAs were believed to act as oncogene or tumor suppressor gene, and contribute to cancer initiation and progression by regulating gene expression[8]. The discovery of cancer-specific upstream region hypermethylation of numerous miRNAs has demonstrated an epigenetic mechanism for aberrant miRNA expression[9], [10].
In human and mice, there are two genomic loci (miR-219-1 and miR-219-2) which encode miR-219 precursor transcripts. miR-219-1 is located on chromosome 6 (MI0000296) and mir-219-2 is located on chromosome 9 (MI0000740)[11] (
Fig. 1A
). Processing of the precursor transcripts by dicer generates three mature miRNAs: miR-219-5p from the 5′ ends of both precursors, and miR-219-1-3p and miR-219-2-3p from the 3′ end of pre-miR-219-1 and pre-miR-219-2, respectively. Since the seed region of these three mature products is unique, each miRNA is predicted to regulate unique targets. Although miR-219-5p is known to be down-regulated in multiple cancer such as malignant astrocytoma[12] and hepatocellular carcinoma[13], the expression of miR-219-1-3p and miR-219-2-3p has not been studied. Interestingly, miR-199b and miR-219-2-3p genes are located at proximity to a segment of chromosome 9q34.11 (
Fig. 1A
). A previous study shown that miR-199b-5p was down-regulated in medulloblastoma by methylation of a CpG island 3 kb upstream of the 5′-site of miR-199b-5p promoter[14]. Since DNA methylation can affect large regions of chromatin and regulate the transcription of distant genes, it is necessary to investigate whether miR-219-2-3p is down-regulated and regulated by methylation as miR-199b-5p in cancer. In this study, we found that miR-219-2-3p was down-regulated in GC tissues and associated with progressive phenotypes of GC. Moreover, re-introduction of miR-219-2-3p reduced the viability of GC cells and induced cell apoptosis, suggesting that miR-219-2-3p was a candidate tumor suppressor in GC. Further methylation analysis of miR-219-2-3p promoter indicated that its expression was regulated by methylation of correlated CpG islands to some extent. Finally, we found that miR-219-2-3p acted as a tumor suppressor through inhibiting the activity of ERK1/2 signal pathway in GC cells.
10.1371/journal.pone.0060369.g001Figure 1 The expression of miR-219-2-3p in gastric cancer tissues and cell lines.
(A) Schematic showing human mir-219-1 and mir-219-2 genomic structures. Processing of the precursor transcripts generates the same miR-219-5p and two unique miRNAs, 219-1-3p and 219-2-3p. (B) MiR-219-2-3p was detected in 113 GC patients by real-time -PCR. Data is presented as log 2 of fold change of GC tissues relative to non-tumor adjacent tissues. (C) Expression levels (as log 2 of fold change of GC tissues relative to non-tumor adjacent tissues) of miR-219-2-3p in I–II stages (n = 39) versus III–IV stages (n = 71) of the cancer patients. (D) The four patients who were diagnosed as gastric cancer in H&E staining (original magnification, x100). (E) Expression levels of miR-219-2-3p were examined by real-time PCR in four patients as described in fig.1D and four GC cell lines. Experiments were performed three times. All data uses independent t test and is shown as mean ± SD. *, P<0.05.
Results
miR-219-2-3p was differentially expressed in GC and GC cell lines
To assess the expression of miR-219-2-3p in GC, TaqMan RT-PCR analysis was conducted in 113 pairs of GC tissues and matched adjacent normal tissue samples. As compared with normal tissue samples, more than half of the primary tumors exhibited low levels of miR-219-2-3p (58%, 65 of 113;
Fig. 1B
). Furthermore, four patients whose expression of miR-219-2-3p were significant down-regulated were chosen (
Fig. 1D
). The miR-219-2-3p expression in those patients and four GC cell lines (MGC-803, HGC-27, MKN-45, SGC-7901) was analyzed to reveal that miR-219-2-3p was also down-regulated in GC cells (
Fig. 1E
). These results suggested that down-regulated miR-219-2-3p was a frequent event in human GC and might be involved in gastric carcinogenesis. Because of the universal down-regulation of miR-219-2-3p in tested GC cell lines, MGC-803 and HGC-27 were randomly chosen for further study.
Relationship between clinicopathological factors and miR-219-2-3p expression in GC
This study included 113 GC patients. To evaluate the correlation between miR-219-2-3p expression and clinicopathological characteristics, patients were divided into groups with down-regulation and up-regulation. As shown in
table 1
and
fig.1C
, a statistically significant association was observed between the expression of miR-219-2-3p and GC clinical stage. The patients with lower levels of miR-219-2-3p expression seemed to be associated with high-grade and late-stage tumors (p = 0.047, independent-samples t test). These data suggested that alterations of miR-219-2-3p could be involved in GC progression.
10.1371/journal.pone.0060369.t001Table 1 Clinicopathologic characteristics of patients with GC.
Parameter Total Percentage 95%CI of mean log2 fold P
samples change±SEM
Age(years)
≥60 112 47.3% −0.307 (−0.969–0.355) 0.847
<60 52.7% −0.216 (−0.877–0.444)
Gender
Male 112 84.8% −0.413 (−0.918–0.091) 0.118
Female 15.2% 0.601 (−0.561–1.762)
Location
Proximal 113 30.9% −0.124 (−0.956–0.708) 0.765
Body 41.6% −0.45 (−1.125–0.225)
Distal 27.4% −0.085 (−1.097–0.927)
Lauren's classification
Intestinal 72 18.1% 0.765 (−0.999–2.528) 0.397
Diffuse 68.1% −0.213 (−0.912–0.485)
Mixed 13.9% −0.508 (−2.283–1.268)
Grade
Well and Moderate 62 21.0% 0.434 (−1.324–2.192) 0.366
differentiated
Poorly differentiated 79.0% −0.297 (−1.010–0.416)
Tumor size
T1–T2 111 17.1% 0.282 (−0.701–1.264) 0.286
T3 21.6% 0.174 (−0.985–1.334)
T4 61.3% −0.539 (−1.136–0.058)
Lymphatic invasion
N0–N1 111 67.6% −0.143 (2.436–0.281) 0.533
N2–N3 32.4% −0.457 (−1.323–0.410)
Nodal involvement
Negative 110 90.9% −0.314 (−0.806–0.178) 0.306
Positive 9.1% −0.008 (−1.526–1.510)
Stage
I–II 110 35.5% 0.387 (−0.420–1.195) 0.047*
III–IV 64.5% −0.591 (−1.165–0.018)
Perineural Invasion
Negative 81 56.8% −0.174 (−0.838–0.490) 0.634
Positive 43.2% −0.424 (−1.265–0.417)
Ki67
1+2 27 25.9% 0.925 (−2.863–4.713) 0.163
3+4 74.1% −0.724 (−1.789–0.341)
Her2 amplification
Negative 41 48.8% −0.227 (−1.309–0.855) 0.348
Positive 51.2% 0.039 (−1.044–1.121)
Top2a
0+1 42 42.9% −0.0988 (−1.120–0.923) 0.957
2+3 57.1% 0.134 (−1.014–1.283)
CA199
<37 29 75.9% 0.092 (−0.859–1.042) 0.258
>37 24.1% −1.115 (−4.032–1.802)
CA724
<6.1 28 67.9% −0.424 (−1.226–0.378) 0.538
>6.1 32.1% 0.135 (−2.724–2.994)
Overexpression of miR-219-2-3p in GC cells inhibits cell proliferation and cell survival
The remarkable reduction of miR-219-2-3p expression in GC samples promoted us to explore the possible biological significance of miR-219-2-3p in tumorgenesis. Given that miR-219-2-3p played a role in the regulation of cell proliferation[15], MGC-803 and HGC-27 cells were transfected with miR-219-2-3p and scramble mimics and analyzed for cell growth, cell apoptosis and cell cycle progression respectively. First of all, RT-PCR was used to measure the level of miR-219-2-3p after overexpression experiments. We found that miR-219-2-3p increased by more than 100 folds in miRNA transfected MGC-803 and HGC-27 cells (
Fig.2A
). Furthermore, the CCK-8 proliferation assay shown that cell growth rate was reduced in miR-219-2-3p mimics-transfected MGC-803 and HGC-27 cells when compared with scramble-transfected cells or untreated cells (
Fig. 2B
). After transfection, the inhibition ratio was 26% (48 h) and 28% (96 h) in the MGC-803 cells and 13% (72 h) and 14% (96 h) in HGC-27 cells. These results suggested that miR-219-2-3p was indeed involved in the negative regulation of cell growth. However, there was no significant effect on cell cycle arrest in miR-219-2-3p treated GC cells (Fig. S1). To address whether up-regulation of miR-219-2-3p would induce GC cell apoptosis and cell death, the number of early apoptotic MGC-803 and HGC-27 cells following treatment with miR-219-2-3p mimics was examined. As expected, few early apoptotic cells (10% in MGC-803 or 2.9% in HGC-27) were detected in the scramble-treated cells, whereas miR-219-2-3p mimics treatment increased the percentage of early apoptotic cells (17.5% in MGC-803 or 8.3 in HGC-27) as judged by Annexin V staining (
Fig. 2C
). Therefore, we concluded that miR-219-2-3p could affect cell survival in GC cells.
10.1371/journal.pone.0060369.g002Figure 2 Overexpression of miR-219-2-3p inhibits gastric cancer cell growth and affects cell apoptosis.
(A) Expression levels of miR-219-2-3p were examined by real-time PCR after transfection with 50 nmol/L of miR-219-2-3p mimics or scramble or no transfection. (B) Growth of MGC-803 and HGC-27 cells was shown after transfection with 50 nmol/L of miR-219-2-3p mimics or scramble or no transfection. The growth index was assessed at 1, 2, 3, 4, and 5 d. (C) MGC-803 and HGC-27 cells were stained with PE Annexin V and 7-AAD 72 h after treatment with miR-219-2-3p mimics or scramble. Early apoptotic cells are shown in the right quadrant. *, P<0.05; ***, P<0.001.
Overexpression of miR-219-2-3p in GC cells inhibits cell migration and invasion
To further detect whether miR-219-2-3p is associated with progression of GC, wound healing and transwell assay were performed to analyze the effect of miR-219-2-3p expression on the migratory and invasive behavior of MGC-803 and HGC-27 cells. We found that introduction of miR-219-2-3p into MGC-803 and HGC-27 cells resulted in a significant reduction of cell migration during the closing of an artificial wound created over a confluent monolayer (
Fig. 3A
). These cells were maintained in serum-free medium during the course of wound healing to ensure that any augmented migratory behavior could not be affected by altered cell proliferation. In addition, restoration of miR-219-2-3p dramatically inhibited the normally strong invasive capacity of MGC-803 and HGC-27 cells, which carried low endogenous level of miR-219-2-3p (
Fig. 3B
). These results indicated that miR-219-2-3p overexpression contributes to regulation of GC cell motility and progression in vitro.
10.1371/journal.pone.0060369.g003Figure 3 Overexpression of miR-219-2-3p inhibits gastric cancer cell migration and invasion.
(A) MGC-803 and HGC-27 cells were not transfected or transfected with 50 nmol/L of miR-219-2-3p mimics or scramble for 24 hours, and wounds were made. The relative ratio of wound closure per field is shown. (B) MGC-803 and HGC-27 cells were not transfected or transfected with 50 nmol/L of miR-219-2-3p mimics or scramble for 24 hours, and transwell invasion assay was performed. The relative ratio of invasive cells per field is shown. Magnification for identification of migration is ×400 and invasion is ×40. All data is shown as mean ± SD.
MiR-219-2-3p expression is epigenetically regulated
Based on the above findings, we conclude that miR-219-2-3p was an important regulator in GC. However, the regulatory mechanisms of miR-219-2-3p expression were still unknown. Since many miRNAs were identified as targets of methylation regulation, such as miR-9, miR-34b/c and miR-148a in metastatic carcinomas[16], and miR-137 and miR-193a in oral cancer[17], miR-193b and miR-145 in prostate cancer[18], [19], we decided to analyze the regulatory mechanism of miR-219-2-3p expression from its genomic methylation. After analyzing the genomic region spanning the miR-219-2-3p gene, we identified a large CpG island (
Fig. 4A
). To investigate whether miR-219-2-3p was epigenetically regulated in GC, MGC-803, HGC-27 cells were treated with demethyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-Aza-CdR) and the histone-deacetylase inhibitor trichostatin A (TSA). Then the expression of miR-219-2-3p by RT-PCR was analyzed (
Fig. 4B
). The results shown that the expression of miR-219-2-3p was up-regulated in two situations: for the 5-Aza-CdR treatment, the expression of miR-219-2-3p was up-regulated in MGC-803 (5-Aza-CdR 1.5 µmol/L; 2.14-fold) and HGC-27 (5-Aza-CdR 0.5 µmol/L; 3.07-fold) compared with DMSO treated control group; for the 5-Aza-CdR and TSA combination treatment, the expression of miR-219-2-3p was much higher in MGC-803 (5-Aza-CdR 1.5 µmol/L; 1.98-fold) and HGC-27 (5-Aza-CdR 1.5 µmol/L; 1.28-fold) compared with TSA control group. These results indicated that epigenetic factors could affect miR-219-2-3p expression in GC. Synergy of demethylation and histone deacetylase inhibition led to the re-expression of miR-219-2-3p in GC. To further detect whether miR-219-2-3p was associated with methylation of GC, we examined the methylation status of the miR-219-2-3p upstream region using methylation-specific PCR (MSP;
Fig. 4C
). 22 pairs of tissues (primary tumors and their matched adjacent normal tissues) in the 113 pairs were chosen, including 11 patients who possessed lower miR-219-2-3p levels (down-regulation group) and 11 patients who possessed higher miR-219-2-3p levels (up-regulation group). We found that DNA methylation in upstream regions of miR-219-2-3p existed in both adjacent normal tissues and cancer tissues. However, the hypermethylation ratio of upstream region of miR-219-2-3p gene in the down-regulation group was 63.6% (7 of the 11), which was higher than the up-regulation group (36.3%, 4 of the 11). These results suggested that the methylation level of the upstream CpG region of miR-219-2-3p was higher in the miR-219-2-3p down-regulated group than in the up-regulated one.
10.1371/journal.pone.0060369.g004Figure 4 Down-regulation of miR-219-2-3p in gastric cancer cells is associated with methylation of miR-219-2-3p upstream region.
(A) A schematic illustration of deletion of a segment (90 M–141 M) of chromosome 9q34.11 where the miR-219-2-3p genes located. (B) Effect of 5-Aza-CdR and TSA on miR-219-2-3p expression in MGC-803 and HGC-27 gastric cancer cell lines. 5-Aza-CdR or 5-Aza-CdR combination with TSA significantly increased miR-219-2-3p levels. (C) Representative MSP results of miR-219-2-3p methylation in primary gastric cancer tumors and normal adjacent tumor tissues. Case numbers are shown on top. M: methylated primers; U: unmethylated primers. Cases with hypermethylation were marked.
Overexpression of miR-219-2-3p dampens ERK1/2 signaling pathway
Activation of ERK1/2 pathway was well documented in various tumor types, such as GC[20], pancreatic cancer[21] and breast cancer[22]. Previous studies have shown the importance of ERK1/2 signaling pathway in the regulation of migration, invasion and metastasis of cancer cell lines[23]. To investigate whether miR-219-2-3p affects cell activities through ERK1/2 pathway, the phosphorylation level of ERK1/2 in MGC-803 and HGC-27 cells was examined after miR-219-2-3p overexpression. Cellular levels of p-ERK1/2 significantly decreased in miR-219-2-3p mimics–transfected cells as compared with scramble-transfected or untreated cells. However, no obvious difference was observed in total ERK1/2 level (
Fig. 5A
). These findings suggested that the accelerated GC cell growth might be partially due to activated ERK1/2 pathways.
10.1371/journal.pone.0060369.g005Figure 5 miR-219-2-3p inhibits activation of p-ERK1/2.
(A) MGC-803 and HGC-27 cells untreated and transfected with scrambled or miR-219-2-3p mimics were subjected to western blot analysis of phosphorylated ERK1/2, total ERK1/2 and GAPDH (as a loading control). The data shown is representative of three individual western blot analyses. (B) The candidate onco-targets of miR-219-2-3p forecast by TargetScan Release 5.2, and miRDB. (C) Bioinformatically and functionally implicated miR-219-2-3p in GC.
Bioinformatics approach to search for potential targets of miR-219-2-3p
MiRNAs modulate gene expression by interacting with their target mRNAs resulting in mRNA degradation or translational repression. To further investigate the mechanism of miR-219-2-3p in GC, we bioinformatically (TargetScan Release 5.2 and miRDB) and functionally implicated miR-219-2-3p in GC, and found the genes targeted by miR-219-2-3p (Table S2). Among the 371 predicted targets of miR-219-2-3p, 31 of them shown high potential since they were predicted by both programs, while others were only predicted by one of the programs. Of these 31 genes, ERBB3, MAPK8, SCL7A11, YOD1, TBK1, SOX4 were found to be oncogene or apoptosis-related genes by previous published papers. (
Fig. 5B and 5C
).
Discussion
In recent years, accumulated evidence has led oncologists to speculate that unrevealed molecular factors, particularly non-coding RNAs previously classified as “junk,” play important roles in tumorigenesis and tumor progression. Depending on their mRNA targets, miRNAs can function as tumor suppressors or promoters of oncogenesis. However, the mechanisms that dysregulated miRNAs have not been widely studied, including aberrant miRNA biogenesis and transcription[24], [25], epigenetic alteration[26], [27], and amplification or loss of genomic regions that encode miRNAs[28].
As shown in this report, we analyzed the expression of miR-219-2-3p in 113 GC patients and found that the levels seem to be lower in GC. Although miR-219-2-3p has been reported to be closely related to diabetic retinopathy[29], oligodendrocytes[15], alzheimer disease [30]and glioblastoma[12], its function in GC remains to be determined. Furthermore, we shown that re-expression of miR-219-2-3p in GC cells resulted in the induction of cell apoptosis and reduced cell viability. These results allowed us to speculate that down-regulation of miR-219-2-3p might provide a survival advantage to GC cells. However, the mechanism responsible for miR-219-2-3p down-regulation in GC is still unknown. Because the loss at 9q34.11, where miR-219-2-3p is located, is rarely detected in GC [31], it is unlikely that allelic loss is responsible for its down-regulation. On the other hand, we found that miR-219-2-3p was markedly up-regulated when GC cells, MGC-803 and HGC-27, were treated with both 5-Aza-CdR and TSA. In addition, computational analysis reveals that miR-219-2-3p is located in a CpG island on chromosome 9q34.11. Therefore, it seems possible that DNA methylation and histone deacetylation may be associated with miR-219-2-3p regulation. By MSP, samples methylation frequencies detected in the upstream region of miR-219-2-3p was higher in the miR-219-2-3p down-regulated group than in the up-regulated group. This specificity furnished the hypothesis of a relationship between miR-219-2-3p expression and DNA methylation. Overall, the results suggested that methylation was an important mechanism for miR-219-2-3p down-regulation in GC.
We performed prediction by TargetScan and miRDB programs and found that 6 genes could be potential targets of miR-219-2-3p. Among the candidate targets of miR-219-2-3p, the receptor tyrosine kinases ERBB3 (epidermal growth factor receptor family) drew our attention. High levels of ERBB3 is strongly associated with tumor progression and poor prognosis of patients with GC[32]-[34] and the EGFR kinase inhibitors gefitinib could prevent EGFR and ERBB2 activation of ERBB3. Meanwhile, ERBB3 expression also serves as an effective predictor of sensitivity to gefitinib[35]. It is known that repressed ERBB3 transcription inhibits signaling cascades from ERK1/2 pathways[36]. However, the predicted target genes need to be further experimentally validated. Moreover, miRNAs may function according to a combinatorial circuits model, in which a single miRNA may target multiple mRNAs, and several coexpressed miRNAs may target a single mRNA. Recent studies have suggested that the biological concept of ‘one hit–multiple targets’ could be used in clinical therapeutics[37]. It is likely that a specific miRNA may function through cooperative down-regulation of multiple targets and miRNAs function also by suppressing the translation of their target genes. To explore the full impact of a miRNA, genome-wide proteomic studies should be done.
In conclusion, our expression and functional studies suggested that miR-219-2-3p was differentially expressed by methylation mechanism and had a tumoral suppression function by regulating ERK1/2-related signal pathways in GC. Meanwhile, the lower expression of miR-219-2-3p in GC specimens was correlated with higher grade and later stage. Reintroducing expression of miR-219-2-3p on GC cells suppressed cell proliferation, migration, invasion and induced apoptosis indicated that miRNA-based theraputic pattern might serve as a basis for the development of novel potential therapies in gastric cancer.
Materials and Methods
Tissue Specimens
Gastric tumors and their morphologically normal tissues (located >3 cm away from the tumor) were obtained between November 2009 and November 2011 from 113 GC patients undergoing surgery at Cancer Hospital of Chinese Academy of Medical Sciences(CICAMS, n = 21), Chinese PLA General Hospital (301 hospital, n = 31), and The First Affiliated Hospital of Shanxi Medical University (n = 61). The use of the tissue samples for all experiments was approved by the ethical board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All participants provided their verbal informed consent to participate in this study, and their verbal informed consents were written down. This consent process was also approved by the ethics board. Tissue samples were cut into two parts, one was fixed with 10% formalin for histopathological diagnosis, and the other was immediately snap-frozen in liquid nitrogen, and stored at −196°C in liquid nitrogen until RNA extraction. This group consisted of 95 males, 17 females and one without gender information with a median age of 58 years (range, 31–82 years).Formalin-fixed paraffin-embedded tissue blocks of GC were collected from the Cancer Hospital of Chinese Academy of Medical Sciences (CICAMS, n = 4) between 2009 and 2011. Due to individual differences between patients, we lacked information of some clinicopathologic data. The use of the tissue samples for all experiments was approved by all the patients and by Ethics Committee of the institution. The characteristics of patients are described in
Table 1
.
Cell Cultures and Transfection
A total of 4 human GC cell lines MGC-803 (mucinous gastric cancer, poorly differentiated), HGC-27 (metastatic lymph node, undifferentiated carcinoma), MKN-45 (Signet ring carcinoma poorly differentiated), SGC-7901 (adenocarcinoma, moderately differentiated) were examined in this study. The MGC-803 HGC-27, MKN-45, SGC-7901 cell line was purchased from the Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). MGC-803 was propagated in Dulbecco's modified Eagle medium (Gibco; Invitrogen; Life Technologies, Germany), supplemented with 10% fetal bovine serum (FBS; PAA, Pasching, Austria) and streptomycin (100 µg/ml), penicillin (100 U/ml). The HGC-27, MKN-45, SGC-7901 were maintained in RPMI 1640 medium (PAA) supplemented with 10% FBS (PAA). The human GC cell lines MGC-803, HGC-27 were transfected with miR-219-2-3p mimics and negative control miRNA mimics (GenePharma; Shanghai, China, Table S1) at a final concentration of 10 nmol/L using Dharmafect 1(Thermo Fisher; IL, USA) in accordance with the manufacturer's instructions.
TaqMan RT-PCR for miRNA Expression
Total RNA was extracted from the cells and tissues with Trizol reagent (Invitrogen, Calsbad, CA, USA). MiRNAs were quantitated by real-time PCR using TaqMan MicroRNA assay (Invitrogen, USA). First-strand complementary DNA (cDNA) synthesis was carried out from 1 µg of total RNA in 12 µl of final volume containing 2 M stem-loop primer, 10 mM dNTP Mix (Invitrogen, USA). The mix was plate at 65°C for 5 min, and then mixed with 5×RT buffer, 0.1 M DTT, 200 U/µl MultiScribe reverse transcriptase and 40 U/µl RNase inhibitor (Invitrogen, USA). The mix was plate at 37°C for 55 min, 70°C for 15 min and then held at −20°C. Real-time PCR was performed using a standard TaqMan PCR protocol. The 20 µl PCRs reactions included 1 µl of RT product, 1× Universal TaqMan Master Mix and 1× TaqMan probe/primer mix (Invitrogen, USA, Table S1). All RT reactions including no-template controls were run in triplicate. All mRNA quantification data was normalized to U6. The relative amount of transcript was calculated using the comparative Ct method.
5-Aza-CdR and Trichostatin A treatment of cell lines
GC cell lines MGC-803 were treated with 5-aza-2′-deoxycytidine(5-Aza-CdR;Sigma-Aldrich, USA) at 0.7 µmol/L,1.5 µmol/L,3 µmol/L and HGC-27 were treated with 5-Aza-CdR at 0.5 µmol/L,1 µmol/L,1.5 µmol/L for 3 days or 300 nmol/L trichostatin A (TSA; Sigma-Aldrich, USA) for 24 hours. For the combination treatment, cells were treated with 5-Aza-CdR for 48 hours firstly. Then TSA (300 nmol/L) was added, and the cells were treated for an additional 24 hours. Culture medium containing drug was replaced every 24 hours. RNA of cell lines was purified with TRIzol reagent following the instructions from the manufacturer. cDNA synthesis was carried out as described earlier, and 1 ml of the diluted cDNA for each sample was amplified by RT-PCR using the protocol previously described.
DNA isolation and bisulfite modification
Genomic DNA was obtained from −196°C in liquid nitrogen primary tumors, and their matched adjacent normal tissues (n = 22, include 11 patients which expression of miR-219-2-3p were down-regulated and the others were up-regulated) and used Biomed DNA Kit (Biomed, Beijing, China) according to the manufacturer instructions. Bisulfite treatment and recovery of samples were carried out with the Epitect Bisulfite kit (Qiagen; Hilden,Germany). Genomic DNA (2 µg) in 20 µl water was used for each reaction and mixed with 85 µl bisulfite mix and 35 µl DNA protect buffer. Bisulfite conversion was performed on a thermocycler as follows: 99°C for 5 min, 60°C for 25 min, 99°C for 5 min, 60°C for 85 min, 99°C for 5 min, 60°C for 175 min and 20°C indefinitely. The bisulfite-treated DNA was recovered by Epitect spin column and subsequently sequenced to confirm the efficiency of bisulfite conversion.
Methylation analysis
MSP was used to analyze methylation of miR-219-2-3p upstream region in cell lines and tissues. Methprimer was used to design MSP primer (Table S1). MSP reactions on new primers were optimized using Methylated positive control (M-DNA), which using normal human peripheral lymphocyte DNA treated in vitro with Sss I methyltransferase (New England Biolabs, Beverly, MA). The DNA of two normal human peripheral lymphocytes was used as normal control. Touchdown PCR consisted of two phases: phase 1 included an initial denaturation of 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s, annealing at variable temperatures for 30 s, and extension at 72°C for 40 s. In the first cycle, the annealing temperature was set to 58°C and, at each of the 10 subsequent cycles, the annealing temperature was decreased by 0.6°C. Phase 2 consisted of 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 40 s. MSP products were analyzed on 3% polyacrylamide gels.
Cell proliferation, apoptosis, and cell cycle assay
Cells were incubated in 10% CCK-8 (Dojindo; Kumamoto, Japan) diluted in normal culture medium at 37°C until visual color conversion occurred. Proliferation rates were determined at 0, 24, 48, 72, 96 hours after transfection. The absorbance of each well was measured with a microplate reader set at 450 nM and 630 nM. All experiments were performed in quadruplicate. The apoptosis assay was performed on MGC-803 and HGC-27 cell lines 72 hours after transfection using the PE Annexin V Apoptosis Detection Kit I (BD Pharmingen; San Diego, CA, USA) and analyzed by fluorescence-activated cell sorting (FACS). Cell cycle analysis was performed on MGC-803 and HGC-27 cell lines 48 hours after transfection with miR-219-2-3p mimics and scramble respectively. Cells were harvested, washed twice with cold PBS, fixed in ice-cold 70% ethanol, and incubated with propidium iodide (PI) and RNase A, then analyzed by FACS. Each sample was run in triplicate.
Cell migration and invasion assays
MGC-803 and HGC-27 cells were grown to confluence on 12-well plastic dishes and treated with scramble or miR-219-2-3p mimics. 24 hours after transfection, linear scratch wounds (in triplicate) were created on the confluent cell monolayers using a 200 µl pipette tip. To remove cells from the cell cycle prior to wounding, cells were maintained in serum-free medium. To visualize migrated cells and wound healing, images were taken at 0, 12, 24, 36, 48, 60 and 72 h hours. A total of ten areas were selected randomly from each well and the cells in three wells of each group were quantified. For the invasion assays, after 24 hours transfection, 1×105 cells in serum-free media were seeded onto the transwell migration chambers (8 µm pore size; Millipore, Switzerland) which were coated with Matrigel(Sigma-Aldrich; St Louis, MO, USA) on the upper chamber. Media containing 20% FBS was added to the lower chamber. After 24 hours, the non-invading cells were removed with cotton wool, invasive cells located on the lower surface of the chamber were stained with May-Grunwald-Giemsa stain (Sigma Diagnostics; St Louis, Missouri, USA) and counted using a microscope (Olympus; Tokyo, Japan). Experiments were independently repeated three times.
Protein isolation and western blotting
At the indicated times, MGC-803 cells and HGC-27 cells were harvested in ice-cold PBS and lysed on ice in cold preparation of modified radioimmunoprecipitation buffer supplemented with protease inhibitors. Protein concentration was determined by the BCA Protein Assay Kit (Bio-Rad, Milan, Italy) and equal amounts of proteins were analyzed by SDS–PAGE (10% acrylamide). Gels were electroblotted onto nitrocellulose membranes (Millipore, Bedford, MA, USA). For immunoblot experiments, membranes were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20, and incubated at 4°C over night with primary antibody. Detection was performed by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system. Primary antibodies used were: GAPDH from Zhong-Shan JinQiao(Beijing, China); ERK1/2 (rabbit anti–ERK1/2, New England Biolab, NEB) and phospho-ERK1/2 (rabbit anti-phospho-ERK1/2, New England Biolab, NEB)
Histology
Tissues were fixed overnight in buffered formalin, embedded in paraffin, cut to 3-µm thickness, and stained with hematoxylin-eosin (H&E) staining.
Bioinformatics and Statistical analyses of data
The miRNA targets predicted by computer-aided algorithms were obtained from miRDB (http://mirdb.org/miRDB/), targetscan5.2 (http://www.targetscan.org) and Statistical analysis were performed using SPSS 15.0. Data was presented as the mean ± standard deviation. Statistical analyses were done by analysis of variance (ANOVA) or Student's t test and statistical significance level was set at α = 0.05 (two-side).
Supporting Information
Figure S1
Overexpression of miR-219-2-3p has no significant effect on cell cycle in gastric cancer.
(TIF)
Click here for additional data file.
Table S1
Primer/mimics/probe sequence.
(TIF)
Click here for additional data file.
Table S2
Potential targets of miR-219-2-3p.
(TIF)
Click here for additional data file.
The authors thank Wenting Yan and Hualu Zhao for technical assistance.
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==== Front
PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23637790PONE-D-12-3075410.1371/journal.pone.0061135Research ArticleBiologyBiotechnologyBionanotechnologyMaterials ScienceMaterial by AttributeNanomaterialsNanotechnologyMedicineClinical ImmunologyImmunityVaccinationVaccinesVaccine DevelopmentInfectious DiseasesBacterial DiseasesTuberculosisEnhanced Immune Response and Protective Effects of Nano-chitosan-based DNA Vaccine Encoding T Cell Epitopes of Esat-6 and FL against Mycobacterium Tuberculosis Infection Nano-Chitosan-Based Vaccine Encoding Esat-6Feng Ganzhu
1
Jiang Qingtao
1
Xia Mei
1
Lu Yanlai
1
Qiu Wen
1
Zhao Dan
1
Lu Liwei
2
Peng Guangyong
3
Wang Yingwei
1
*
1
Department of Microbiology and Immunology, Nanjing Medical University, Nanjing, China
2
Department of Pathology, Hong Kong University, Hong Kong, China
3
Division of Infectious Diseases, Allergy and Immunology, Department of Internal Medicine, Saint Louis University School of Medicine, St. Louis, Missouri, United States of America
Doherty T. Mark Editor
Glaxo Smith Kline, Denmark
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: YW. Performed the experiments: GF QJ MX DZ. Analyzed the data: YL WQ DZ. Contributed reagents/materials/analysis tools: LL GP. Wrote the paper: GF YW.
2013 23 4 2013 8 4 e611352 10 2012 5 3 2013 © 2013 Feng et al2013Feng et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Development of a novel and effective vaccine against Mycobacterium tuberculosis (M.tb) is a challenging for preventing TB infection. In this study, a novel nanoparticle-based recombinant DNA vaccine was developed, which contains Esat-6 three T cell epitopes (Esat-6/3e) and fms-like tyrosine kinase 3 ligand (FL) genes (termed Esat-6/3e-FL), and was enveloped with chitosan (CS) nanoparticles (nano-chitosan). The immunologic and protective efficacy of the nano-chitosan-based DNA vaccine (termed nano-Esat-6/3e-FL) was assessed in C57BL/6 mice after intramuscular prime vaccination with the plasmids DNA and nasal boost with the Esat-6/3e peptides. The results showed that the immunized mice remarkably elicited enhanced T cell responses and protection against M.tb H37Rv challenge. These findings indicate that the nano-chitosan can significantly elevate the immunologic and protective effects of the DNA vaccine, and the nano-Esat-6/3e-FL is a useful vaccine for preventing M.tb infection in mice.
This work was supported by funding from Natural Scientific Fund of Jiangsu Province (09KJA310002, DG216D5016, 12KJB310007, 2011NJMU263 and 11JC005) in China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Tuberculosis (TB) infected by Mycobacterium tuberculosis (M.tb) has plagued humankind worldwide for many years [1]. Although bacillus Calmette-Guerin (BCG) is an effective vaccine for childhood TB, it produces incomplete and variable protection in adult TB [2]. Furthermore, BCG is also an attenuated vaccine that cannot be safely given to the persons infected with human immunodeficiency virus (HIV). Thus, a new and efficient vaccine against TB is urgently required.
Control of M.tb infection greatly depends on T cell-mediated immune responses [3]. The 6 kDa early secretory antigenic target (Esat-6), which presents in virulent mycobacterial strains and absents in BCG [4], is critical for M.tb immunogenicity and virulence. Because Esat-6 protein can be recognized by T cells and B cells [5], and it has attracted intense interests in vaccine development. Given that M.tb Esat-6 gene is located in virulent M.tb
[6], avoiding Esat-6 adverse reaction in the vaccine studies is also indeed important.
It has been reported that the DNA vaccines based on T cell epitopes of M.tb antigens can induce efficient immune responses, and decrease side effects and increase safety and stability of vaccines [7], [8]. However, DNA poor delivery to antigen presenting cells (APCs) and the rapid inactivation mediated by DNase results in low immunogenicity of DNA vaccines [9]. Thus, elevating DNA vaccine delivery efficiency of DNA vaccine and protecting DNA from degradation are important for enhance DNA vaccine potency.
Several documents have reported that fms-like tyrosine kinase 3 ligand (FL) plays an important role in proliferation and differentiation of myeloid and lymphoid progenitor cells as well as dendritic cells (DCs) [10]. Moreover, FL has a strong adjuvant effects that augment DC recruitment and expansion, as well as increase antigen uptake and cross presentation [11], [12].
Chitosan (CS) is a non-toxic biodegradable and biocompatible polycationic polymer, which can not only bind DNA and protect it from nuclease degradation [13], [14], [15], [16], but also exhibit potential adjuvant properties, such as promoting endocytotic uptake and elevating immune responses. Additionally, chitosan nanoparticles (nano-chitosan) possess lower cytotoxicity and more stability [17], [18], [19], [20], [21].
In order to evaluate the immunologic and protective efficacy of CS nanoparticle-based DNA vaccine expressing T cell epitopes of Esat-6 and FL against M.tb infection, in the present study, the three T cell epitopes of Esat-6 (Esat-6/3e) were predicted and selected based on the pitope prediction software analyses. Then, a recombinant plasmid DNA vaccine that contains the genes of Esat-6 three T cell epitopes (Esat-6/3e) was constructed and identified. Subsequently, the recombinants were enveloped with nano-chitosan, and the nano-chitosan-based recombinants were identified, and validated the expression in rat glomerular mesangial cells (GMCs). More importantly, the immunologic and protective effects against M.tb infection mediated by the nano-chitosan-based DNA vaccines were evaluated in vivo in mouse models.
Results
Prediction of T cell epitopes and identification of recombinant Esat-6/3e-FL plasmid
Based on MHC-binding scores of the MHCpre software analyses (http://www.syfpeithi.com/scripts/MHCSr.dll/home.htm; http://tools.immuneepitope.org) and literatures [22], the three T cell epitopes of Esat-6 were selected for the DNA vaccine development as follow: Esat-64–18 (QQWNFAGIEAAASAI), Esat-622–36 (VTSIHSLLDEGKQSL) and Esat-656–70 (QKWDATATELNNALQ). To protect the epitopes against amalgamation and enhance efficacy of intracellular protease incising [23], the Ala-Ala-Tyr (AAY) linker and a his tag were inserted into pIRES (vector) and pIRES-FL plasmids, named pIRES-Esat-6/3e (Esat-6/3e) and pIRES-Esat-6/3e-FL (Esat-6/3e-FL), respectively (Figure 1A.). To verify whether the recombinant plasmids can express the corresponding protein, the plasmids were transfected into rat GMCs for 60 h, and the expression of his tag and FL protein was determined by Western blot (Figure 1B).
10.1371/journal.pone.0061135.g001Figure 1 Construction and identification of pIRES-Esat-6/3e-FL (namely Esat-6/3e-FL).
A. Schematic representation of Esat-6/3e-FL. B. The expression of his tag and FL proteins from Esat-6/3e or Esat-6/3e-FL plasmids was determined using Western blot after transfection of the plasmids into rat GMCs for 60 h (1: pIRES-Esat-6/3e. 2: pIRES-Esat-6/3e-FL. 3: pIRES-FL. 4: pIRES). C. The irregular solid spheres of nano-chitosan and nano-Esat-6/3e-FL plasmids under EM. D. The expression of his or FL proteins from the Esat-6/3e-FL plasmids enclosed with chitosan nanoparticle (nano-Esat-6/3e-FL) was confirmed using Western blot with anti-his or anti-FL antibody at 60 h after transfection into rat GMCs (1: MEM. 2: nano-pIRES. 3: nano-Esat-6/3e-FL).
Diameter, zeta potential and shape of nano-chitosan and nano-Esat-6/3e-FL plasmids
The diameter and dispersing degree of nano-chitosan particles were 311.2±34 nm and 0.35 respectively. Their zeta potential was 30.6 mV. After the recombinant Esat-6/3e-FL plasmids were coated with nano-chitosan (nano-Esat-6/3e-FL), the diameter and zeta potential of the nano-plasmids were 314.8±38 nm and 33.6 mVseparately, and their dispersing degree was 0.39 (Figure S1A, and S1B). Moreover, the particles of nano-chitosan and nano-Esat-6/3e-FL displayed an irregular solid sphere, and their diameters were from 280 to 330 nm (Figure 1C).
Stability, transfection efficacy and expression of nano-Esat-6/3e-FL plasmid
The nano-Esat-6/3e-FL plasmids were first digested by different concentrations of DNase I. When agarose gel electrophoresis was performed, the Esat-6/3e-FL plasmid DNAs departed from the sample well, but the corresponding nano-plasmids stayed in the well (Figure S2A). The different volume ratio of nano-chitosan and Esat-6/3e-FL mixture was digested by the same dose of DNase I. The results manifested that Esat-6/3e-FL plasmids were well digested when the ratio was at 1∶4 (Figure S2B). In addition, the transfection efficiency and cytopathic effect of pGCsi-GFP enveloped by nano-chitosan (nano-chitosan-GFP) or liposome-2000 were observed in rat GMCs. The results confirmed that the CPE of GMCs transfected with nano-chitosan-GFP at 48 h or 72 h was markedly lower than that of GMCs transfected with pGCsi-GFP coaded by liposomes-2000 (Figure S3). Moreover, the nano-Esat-6/3e-FL plasmids could also express the corresponding proteins (Figure 1D).
Production of cytokines and expression of T-bet, Gata-3 from the splenocytes of the nano-Esat-6/3e-FL-immunized mice
In order to evaluate T cell immune responses elicited by the nano-Esat-6/3e-FL vaccination, splenocytes from the immunized mice were collected and cultured. The amounts of IFN-γ, IL-12, IL-4 and IL-10 in the supernatant were detected by ELISA. The levels of IFN-γ and IL-12 in the mice vaccinated with nano-Esat-6/3e-FL or nano-Esat-6-FL were obviously higher than those with other plasmids, which even exceed the levels in BCG treatment group.Furthermore, mice immunized with nano-Esat-6/3e-FL elicited much higher levels of IFN-γ and IL-12 than those immunized with Esat-6/3e-FL. However, the levels of IL-4 and IL-10 from the mice vaccinated with BCG were the highest among all the vaccinated mice (Figure 2A). In addition, the expression levels of T-bet mRNA and protein in nano-Esat-6/3e-FLimmunized mice were obviously higher than those in the other groups treated with other plasmids or BCG (Figure 2B). However, Gata-3 mRNA and protein expressions did not show obvious changes among all the groups. These results suggested that the mice immunized with nano-Esat-6/3e-FL and nano-Esat-6-FL could induce a stronger Th1-polarized response.
10.1371/journal.pone.0061135.g002Figure 2 Splenocytes of the mice immunized with different plasmids were cultured and stimulated with Esat-6/3e (10 µg/ml) for 72 h, and the production of IFN-γ, IL-12, IL-4 and IL-10 in the splenocyte supernatants and expression of T-bet and Gata-3 in the splenocytes were measured by ELISA and Western blot, respectively.
A. The levels of Th1 type cytokines (IFN-γ and IL-12) and Th2 type cytokines (IL-4 and IL-10). B. The expression levels of T-bet or Gata-3 mRNA and protein. The representative graph and histograms in the immunized mice were showed. The experiments were performed in three times. All data are from one representative of three experiments, and presented as the mean value ± SD (n = 6). The statistics were performed with one-way ANOVA. NS: P>0.05; *P<0.05 and ***P<0.001 versus the mice with nano-chitosan, nano-pIRES and nano-FL immunization respectively. The other significant differences among the mice vaccinated with nano-Esat-6/3e-FL, nano-Esat-6/3e, Esat-6/3e-FL, nano-Esat-6, nano-Esat-6-FL and BCG were displayed on the figure directly.
Proliferation of spleen cells and numbers of IFN-γ+ T cells from the nano-Esat-6/3e-FL immunized mice
To verify the efficacy of immunologic reaction induced by nano-Esat-6/3e-FL vaccination, proliferation of spleen cells from the immunized mice was detected. As shown in Figure 3A, the SI increased more conspicuously in the mice with nano-Esat-6/3e-FL vaccination than that in other groups. The SIs of the mice treated with nano-Esat-6-FL and BCG were also significantly elevated, but no marked difference, compared with the nano-Esat-6/3e-FL immunization group.
10.1371/journal.pone.0061135.g003Figure 3 The proliferation of the splenocytes and number of IFN-γ+ T cells of the mice immunized by nano-Esat-6/3e-FL.
A. Splenocytes were cultured and stimulated with the Esat-6/3e (10 µg/ml) for 72 h. The stimulation index (SI) of splenocytes in the mice treated with different vaccines was calculated to determine the proliferation activity. B. The numbers of IFN-γ+ T cells from the splenocytes were quantified by ELISPOT assays. Data are one representative of three experiments and presented as mean value ± SD (n = 6). The statistics were performed with one-way ANOVA. NS: P>0.05; ***P<0.001 versus the mice treated with nano-chitosan, nano-pIRES and nano-FL plasmids. The differences among other treatments were shown directly on the figure.
To further determine whether the nano-Esat-6/3e-FL can trigger high T cell responses, IFN-γ –producing T cells of immunized mice were evaluated using ELISPOT assays. IFN-γ+ T cell numbers (spot forming cell, SFC) in the mice with nano-Esat-6/3e-FL vaccination were much higher than those in the mice immunized with Esat-6/3e-FL. Although IFN-γ+ T cell numbers in the mice immunized with nano-Esat-6-FL were the highest, there was no significant difference in comparison to the mice immunized with nano-Esat-6/3e-FL vaccine (Figure 3B).
CTL activity of mice triggered by nano-Esat-6/3e-FL immunization
To assess the cytolytic capability of CTLs induced by nano-Esat-6/3e-FL vaccination, an in vivo cytotoxicity assay was performed. As shown in Figure 4, a significantly increased lyses were founded in the mice immunized with nano-Esat-6/3e, Esat-6/3e-FL, nano-Esat-6 or nano-Esat-6-FL separately (Figure 4). In addition, much higher cytolytic activity was observed in the mice immunized with nano-Esat-6/3e-FL or nano-Esat-6-FL, though there was no significant difference between these different groups, which also overtopped markedly BCG vaccination.
10.1371/journal.pone.0061135.g004Figure 4 The CTL activity of the mice elicited by nano-Esat-6/3e-FL or other plasmids.
Splenocytes from naive mice pulsed with (CFSEhigh) or without (CFSElow) peptides were transferred into the immunized mice. The representative histograms (A) and percentages (B) of specific lysis in the immunized mice were compared. Data are one representative results from three performed experiments, and presented as the mean ± SD (n = 6). NS: P>0.05; *P<0.05; ***P<0.001 versus the mice treated with nano-chitosan, nano-pIRES or nano-FL plasmids; the other differences among nano-Esat-6/3e-FL, nano-Esat-6/3e, Esat-6/3e-FL, nano-Esat-6, nano-Esat-6-FL and BCG treatments were showed on the figure directly.
Protective effects against M.tb H37Rv challenge from the mice immunized with nano-Esat-6/3e-FL
To determine the protective potential of nano-Esat-6/3e-FL vaccination, one week after the last boost with Esat-6/3e peptides, the vaccinated mice were intratracheally challenged with 1×106 bacilli M.tb H37Rv. The bacterial burdens in the lungs and spleens, including pulmonary injury were observed at 4 weeks post challenge. As shown in Figure 5, the reduction of M.tb colonies was significantly observed both in the lungs (Figure 5A) and spleens (Figure 5B) in the mice with nano-Esat-6/3e-FL immunization, and the value of log10 CFU was 0.403±0.023 and 0.324±0.062, respectively, although the log10 CFU value in the mice vaccinated with nano-Esat-6/3e-FL plasmids was similar to that of the mice treated with nano-Esat-6-FL plasmids. Meanwhile, the burden of log10 CFU in the lungs from these two groups was sharply decreased compared with that from BCG vaccination. These findings indicated that immune responses of the mice induced by nano-Esat-6/3e-FL (including nano-Esat-6-FL) plasmid DNA vaccine could indeed lead to an enhanced defense.
10.1371/journal.pone.0061135.g005Figure 5 The protection efficacy against M.tb infection of the mice immunized with different vaccines.
Mice vaccinated with different plasmids and boosted with Esat-6/3e peptides for two times were infected with 1×106 bacilli of M.tb H37Rv for 4 weeks. Bacterial loads in the lungs (A) and spleens (B) of these mice were examined and the sections of the lung tissues from these mice were performed HE staining. Representative histologic changes (100×) depicted the lung tissue of the infected mice (C).
On the other hand, the lung tissues of the mice vaccinated with nano-chitosan, nano-pIRES and nano-FL showed widespread and severe interstitial pneumonia including larger diffuse granuloma and infiltration of inflammatory cells after M.tb H37Rv infection, while the mice immunized with nano-Esat-6/3e-FL (including nano-Esat-6-FL or BCG) displayed moderate damage with relatively fewer cell infiltration, smaller granulomas and better lung structure (Figure 5C).
Additionally, to determine whether the mice immunized with nano-Esat-6/3e-FL affect the influx of T cells and macrophages, the lung sections were stained with anti-CD3 or anti-F4/80 antibody. The results displayed that the numbers of CD3 (Figure S4A) and F4/80 (Figure S4B) positive cells in the mice immunized with nano-Esat-6/3e-FL were much more than the mice from other treatment groups. Furthermore, the mice treated with nano-Esat-6/3e-FL possessed more intact alveolar tissue, implicating the inflammatory changes mediated by M.tb were attenuated (Figure S4C). These results suggest that the nano-chitosan-based DNA vaccine could induce a strong and effective protection against M.tb challenge in the immunized mice.
Discussion
Previous studies have reported that one of major contributors to the virulence and intercellular spread of M.tb is Esat-6 secretion system 1 (ESX-1) that is lost in BCG [24], [25], [26], [27], [28]. In addition, Esat-6 also contains a large number of antigen epitopes recognized by T and B cells. Immunization with Esat-6 in mice not only induces highlevels of IFN-γ and low bacterial loads after M.tb challenge [29], [30], [31], [32], [33], [34]. Since epitope vaccines can overcome safety problem and induce potent immune responses [35]–[37], in our previous [38] and present studies, three T cell epitopes of Esat-6 (Esat-64–18, Esat-622–36 and Esat-656–70), which contain Th1 and/or CTL epitopes [39]–[42], were selected according to higher scores via prediction software analyses.
In view of relatively low immunogenicity of DNA vaccine [13], the strategy of increasing delivery efficiency and protecting DNA from degradation may elevate the protective effects. FL is used as an effective adjuvant due to its enhancing antigen presentation [21]. Furthermore, in our previous experiments [38], co-delivery of FL for ESAT-6 epitopes DNA vaccines was confirmed to remarkably increase the efficacy of immunity and protection in mice.
It has been known that chitosan (CS) can effectively bind to DNA and protect it from nuclease degradation, and DNA packaged with nano-chitosan can improve cell uptake and vaccine delivery [13], [14], [15], [16], [41], [43], [44], [45], [46], [47]. In the present study, the recombinant candidate plasmid that contained 3 T cell epitopes and FL (pIRES-Esat-6/3e-FL), as well as the recombinant control plasmids i.e. pIRES-Esat-6/3e, pIRES-Esat-6 (containing full length of ESAT-6) and pIRES-FL (only containing FL) were first constructed and then enclosed with nano-chitosan. Our results showed that the nano-chitosan-based plasmids could transfect into GMCs and the corresponding protein was effectively expressed. Although the mice immunized with the control plasmids and BCG obviously elevated T cell responses and CTL activities, much more responses and effects were observed in the mice vaccinated with nano-Esat-6/3e-FL. Moreover, those immunized mice displayed remarkably protective efficacy, including the inhibition of M.tb growth and lung damage, but the better effects were seen in the mice immunized with nano-Esat-6/3e-FL and nano-Esat-6-FL, implicating that FL and nano-chitosan generated a markedly synergistic role as immune adjuvant for the DNA vaccine. Since lower cytotoxicity of nano-Esat-6/3e plasmid was demonstrated in rat GMCs, thus we think that the nano-Esat-6/3e plasmid DNA might be a better vaccine.
On the other hand, the mice vaccinated with BCG could not only induce high levels of IFN-γ and IL-12, increased T-bet expression and IFN-γ+ T cells and augmented CTL activity, but also provide strong protection. However, the immune response efficiency of the mice with BCG immunization were relatively lower than that of the mice with nano-Esat-6/3e-FL (including nano-Esat-6-FL) vaccination, indicating that the nano-Esat-6/3e-FL DNA vaccine can mediate more powerful T cell responses and may be superior to BCG. Furthermore, since the pathological changes of mice vaccinated with nano-Esat-6/3e-FL were markedly lower than those of the mice treated with nano-whole Esat-6 and FL (nano-Esat-6-FL), we consider that the plasmid DNA vaccine encapsulated with nano-chitosan (nano-Esat-6/3e-FL) is relatively better than nano-Esat-6-FL plasmid DNA.
Reportedly, Bennekov' study [48] showed that the adenoviral construct induced a strong CD8 response predominantly targeted to the epitope Esat-615–29, but there was no significant protection against M.tb infection. Thus, in our current experiment, we selected the three epitopes (Esat-64–18, Esat-622–36 and Esat-656–70) that based on the computer prediction. Our results demonstrated that the mice immunized with nano-Esat-6/3e-FL plasmid DNA vaccine not only elicited enhanced T cell responses, but also remarkably elevated the protection against M.tb challenge.
Additionally, it is worth mentioning that in vitre experiment, the splenocytes had also been stimulated with PPD, but the data (not shown) were similar to data of the splenocytes with Esat-6/3e peptides. Ergo, to demonstrate the increased antigen-specific immune responses, we used the data from the epitope peptides stimulation in the paper. In terms of such amount of IL-10 and IL-4 (look higher) in nano-chitosan, nano-pIRES and nano-FL (see Fig. 2), here we explain that IL-10 and IL-4 have a basic level in mice, while the relatively lower levels of IL-10 and IL-4 in the mice treated with the plasmid vaccines might be related with elevated Th1 and decreased Th2 responses. However, the mechanisms need to be explored in the future.
In summary, our data demonstrated that the selected three T cell epitopes of Esat-6 were dominant epitopes for Th1 cells and CTLs which can significantly increase Th1 polarization reactions and CTL activity. Moreover, FL is a critical immunostimulatory adjuvant that promoted more strong immune responses and protection against M.tb induced by DNA vaccines. More importantly, nano-chitosan could also significantly enhance the vaccination effects of Esat-6/3e-FL DNA vaccine, such as strong Th1 and CTL immune responses and inhibition of M.tb growth. Collectively, our findings indicate that the nano-chitosan-based DNA vaccine (nano-Esat-6/3e-FL) is a novel and useful tool for preventing M.tb infection.
Materials and Methods
Animals
Eight-week-old female C57BL/6 mice (H-2b) were purchased from the central animal laboratory of Yangzhou University (China), and maintained under specific pathogen-free conditions (eight in each group). All the experiments were conducted according to the Institutional Ethical Guidelines for Animal Experiments of Nanjing Medical University (permit number: 2010257).
T cell epitope prediction, plasmid construction and identification
The plasmids of pIRES-Esat-6-FL (containing full length of Esat-6 and FL) and pIRES-Esat-6 (only containing full length of Esat-6) as well as pIRES-FL (only containing full length of FL) have been previously described [7] and kept in our lab. The primary structure of Esat-6 antigen was analyzed by epitope prediction software online (http://www.syfpeithi.com/scripts/MHCSr.dll/home.htm; http://tools.immuneepitope.org). Three T cell epitopes, namely Esat-64–18 (QQWNFAGIEAAASAI), Esat-622–36 (VTSIHSLLDEGKQSL) and Esat-656–70 (QKWDATATELNNALQ) were sorted based on their scores [38]. A his tag, the three peptides and Ala-Ala-Tyr (AAY) linker were synthesized and inserted into pIRES or pIRES-FL plasmids, which were termed pIRES-Esat-6/3e (Esat-6/3e) and pIRES-Esat-6/3e-FL (Esat-6/3e-FL). Thereafter, these plasmids were prepared and purified with endoFree plasmid maxi kit (Qiagen, Germany) and transfected into rat GMCs. Subsequently, the expression of his and FL protein of these plasmids was detected using Western blot [38].
Chitosan nanoparticle, nano-plasmid DNA preparation and identification
Chitosan (CS) was purchased from Treechem. Ltd (Shanghai, China), and dissolved in 1% acetic acid and mixed with 0.1% pentasodium triphosphate (TPP), then the formation of chitosan-TPP nanoparticles started spontaneously via the TPP initiated ionic crosslink and coacervation [21]. To get mixture of nano-chitosan and plasmid DNA, both prepared nano-chitosan and plasmid DNA suspension heated and mixed in different volume. Thereafter, the particle size, zeta potential and polydispersity of nano-chitosan and nano-Esat-6/3e-FL plasmids were measured by a Malvern Zetasizer 3000-HS at 633 nm. The morphological shape of the nanoparticles was observed by electron microscopy (EM).
The suspensions of nano-chitosan and nano-plasmid DNA were separated by ultra centrifugation. The amount of free-DNA in the supernatant was detected at 595 nm, and the DNA encapsulation efficiency (EE) was calculated using Eq: EE = (total amount DNA–free amount DNA in supernatant)/total amount DNA×100%. Meantime, different gradient amount of DNase I (0.02U, 0.04U and 0.1U) was added into the naked plasmid DNA and nano-plasmid DNA with the same quantity for 15 min. The mixture was electrophoresed with 2% agar. Furthermore, these plasmids were transfected into glomerular mesangial cells (GMCs) separately and cultured for 60 h. Thereafter, the lysate of GMCs was electrophoresed on SDS-PAGE gels, and stained with anti-his or anti-FL mAb (Santa Cruz, USA).
Esat-6 peptide, BCG and M.tb H37Rv preparation
The Esat-6 peptides (Esat-6/3e) comprising the 3 T cell epitopes were synthesized by Sangon Biotech Co. Ltd (Shanghai, China), and confirmed to be of >90% purity by high-performance liquid chromatography and mass spectrometry profiles. BCG (Denmark strain 1331) was provided by Center for Disease Control of Jiangsu Province in China. A virulent M.tb H37Rv strain (ATCC 27294) supplied by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) was grown in 7H9 liquid medium supplemented with albumin-dextrose-catalase enrichment.
Vaccine immunization and M.tb H37Rv challenge
All female C57BL/6 mice housed in specific pathogen free conditions, and were randomly assigned to 9 groups (eight in each group) as shown in Table 1. To compare the different effects on T cell immune response in the mice immunized various relative materials, the mice of candidate group were treated with nano-Esat-6/3e-FL plasmid DNA. And the mice of control groups treated with nano-Esat-6/3e, nano-Esat-6-FL, nano-Esat-6, nano-FL, nano-chitosan, nano-pIRES, Esat-6/3e-FL and BCG, respectively. In nano-plasmids treatment, the mice were injected intramuscularly three times at 3-week intervals in both quadriceps muscles with 100 µl nano-chitosan or 100 µl different nano-plasmids (containing 100 µg DNA, separately). In Esat-6/3e-FL treatment, the mice were vaccinated with 100 µl Esat-6/3e-FL plasmids (containing 100 µg DNA) without nano-chitosan envelopment. In BCG treatment, the mice were injected subcutaneously with 106 BCG only once at equal pace. Three weeks after two immunizations, the mice were dripped via airway with 25 µg Esat-6/3e peptides two times at 1-week interval. In order to induce higher immune responses, the animals were boosted [49], and 2 weeks after the last peptides boost, parts of mice were euthanized for testing immune response, and the remaining mice were challenged intratracheally with 100 µl (106 bacilli) of M.tb H37Rv per animal. Four weeks post M.tb challenge, these mice were sacrificed for bacterial burdens and histological assessment [48]. The whole schedule of vaccination in mice was displayed in Figure 6.
10.1371/journal.pone.0061135.g006Figure 6 Sketch drawing of immunization procedure and challenge by M.tb H37Rv.
A. C57BL/6 mice were injected intramuscularly three times at 3-week intervals in both quadriceps muscles with different nano-plasmids, nano-chitosan, Esat-6/3e-FL or BCG. Those mice were then boosted with Esat-6/3e peptides through intranasal administration once a week for 2 weeks, then sacrificed at 1 week after the last peptide boost. B. Mice vaccinated with different nano-plasmids for 3 times and boosted with Esat-6/3e peptides for 2 times as above mentioned time schedules were infected with M.tb H37Rv the airway at 1 week after the last peptide boost, and then sacrificed at 4 weeks post M.tb H37Rv challenge.
10.1371/journal.pone.0061135.t001Table 1 The groups of mice given with different treatments.
Esat-6/3e Esat-6 FL pIRES nano-chitosan BCG
Candidate group
nano-Esat-6/3e-FL + − + + + −
Control group
nano-Esat-6/3e + − − + + −
nano-Esat-6-FL − + + + + −
nano-Esat-6 − + − + + −
nano-FL − − + + + −
nano-chitosan − − − − + −
nano-pIRES − − − + + −
Esat-6/3e-FL + − + + − −
BCG − − − − − +
Cytokines, T-bet and Gata-3 detection
The spleen cells from immunized mice were cultured on plates at 2.5×105 per well, and then incubated with the Esat-6/3e peptides (10 µg/ml) for 72 h. The levels of IFN-γ, IL-12, IL-4 and IL-10 in the supernatants were determined by ELISA (Biolegend, USA). In addition, total RNA from the spleen cells was extracted, and the cDNA was subjected to PCR with specific primers for mouse T-bet (up: GGAGCGGACCAACAGCATC, down: CCACGGTGAAGGACAGGAAT), and Gata-3 (up: TCTGGAGGAGGAACGCTAATGG, down: GAACTCTTCGCACACTTGGAGACTC). The ratios for T-bet/β-actin and Gata-3/β-actin mRNA were also calculated for each sample. Meanwhile, the expression of T-bet and Gata-3 from the spleen cells were detected using Western blot.
Splenocyte proliferation and IFN-γ+ T cell measurement
The spleen cells isolated from the mice were cultured and incubated with the Esat-6/3e peptides or medium alone [50]. The proliferation response of splenocytes were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) [51]. Besides, ELISPOT experiment was performed with the assay kit (DAKEWEI, China). And the splenocytes (5×105 cells/well) were plated and treated as treated as above mentioned. Thereafter, the biotinylated anti-IFN-γ mAb was added for 2 h, and the plate was supplemented with streptavidin-alkaline phosphates, and the color was shown by AP-colorimetric substrate.
CTL assay in vivo
CTL assay in vivo was performed as described procedure [52]. Briefly, CTL target cells were prepared from the spleen of naïve mice and pulsed with the mixture of the Esat-6/3e peptides (10 µg/ml) or no peptides overnight at 4°C. Peptides-pulsed splenocytes were then labeled with a high concentration of CFSE (5 µM) or unpulsed splenocytes with a low concentration of CFSE (0.5 µM). 2.5×106 pulsed and 2.5×106 unpulsed splenocytes (1∶1 ratio) were mixed and transferred to the immunized mice. The splenocytes of mice at 18 h after treatments were isolated for acquisition on a FACS caliber instrument (BD Biosciences, USA). Finally, the ratio of CFSEhigh/CFSElow in the vaccinated mice was compared.
M.tb H37Rv challenge
Fore weeks after M.tb H37Rv challenge, the left lungs and spleen of mice were homogenized. Then, 100 µl of diluted samples (10−3, 10−4, and 10−5) were plated on agar plates and incubated for 8 weeks. The number of CFU was then counted, and the data were expressed as mean of log10 CFU value ± standard deviation for each group.
Besides, the lung samples of mice were subjected to hematoxylin-eosin (HE) staining for histological analysis. To score lung inflammation and damage, the sections of entire lung were observed under light microscope (LM) with software Image plus 6.0. And meanwhile, the immunohistochemistry (IHC) of the lung sections were performed with anti-CD3 or anti-F4/80 antibody staining, and the influx of T cells and macrophages (anti-CD3 and anti-F4/80 positive cells) was determined.
Statistical analysis
Data were presented by mean ± standard deviation (mean ± SD), and analyzed using one-way ANOVA as well as multiple comparisons between groups by student's t test adjusted by Bonferroni method. A probability of P≤0.05 was considered statistically significant. All analysis were conducted under SAS 9.2 (SAS Institute Inc., Cary, NC, USA).
Supporting Information
Figure S1
The particle size distributions and zeta potential of nano-chitosan or nano-Esat-6/3e-FL plasmids were displayed.
A. The size and ploy dispersity of nano-chitosan and nano-Esat-6/3e-FL plasmids. B. The zeta potential of nano-chitosan and nano-Esat-6/3e-FL plasmids respectively.
(TIF)
Click here for additional data file.
Figure S2
The nano-Esat-6/3e-FL showed high stability against DNase I.
A. The image showed different degradation reaction of nano-Esat-6/3e-FL and Esat-6/3e-FL with DNase I (U) digestion (1: nano-Esat-6/3e-FL+0.1U. 2: Esat-6/3e-FL+0.1U. 3: nano-Esat-6/3e-FL+0.04U. 4: Esat-6/3e-FL+0.04U. 5: nano-Esat-6/3e-FL+0.02U. 6: Esat-6/3e-FL+0.02U. 7: nano-Esat-6/3e-FL. 8: Esat-6/3e-FL). B. The image displayed that ratios of different volume mixture in nano-chitosan and Esat-6/3e-FL was digested by same dose of DNase I (0.1U). The lane 1 and 6 were Esat-6/3e-FL, and lane 2, 3, 4 and 5 were nano-Esat-6/3e-FL plasmids in different volume ratio of nano-chitosan and Esat-6/3e-FL (namely 1∶1, 1∶2, 1∶3 and 1∶4, respectively).
(TIF)
Click here for additional data file.
Figure S3
The detection of cytopathic effect (CPE) on rat GMCs at 48 h and 72 h after transfection of nano-pGCsi-GFP and liposome-pGCsi-GFP.
A and D showed the GMCs condition after transfection of liposome-2000-pGCsi-GFP. B and E displayed the GMCs condition transfected with nano-pGCsi-GFP. C and F showed the GMCs condition after pGCsi-GFP transfection alone (magnification: 200×).
(TIF)
Click here for additional data file.
Figure S4
Cell infiltration of lung sections from the mice challenged with
M.tb
was observed. The lung sections were stained by immunohistochemistry (IHC) with anti-CD3 (A) or anti-F4/80 (B) antibody (magnification: 200×). The percentage of lung inflammation account for the entire lung sections of these mice was scored by morphometric analysis (C). All experiments were conducted three times, with the representative photos. The data are shown as mean ± SD (n = 6). *P<0.05, **P<0.01, ***P<0.001 versus the mice immunized with nano-chitosan or nano-pIRES or nano-FL plasmid, and the other obvious differences of the mice with different treatments were directly displayed on the figures.
(TIF)
Click here for additional data file.
We thank Jun Dou (Department of Microbiology and Immunology, Medical College of Southeast University, Jiangsu Province, China) for providing the pIRES plasmid and Zuhu Huang (Division of Infection of the People's Hospital, Jiangsu Province, China) for offering the FL plasmid.
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EcancermedicalscienceEcancermedicalscienceecancermedicalscienceecancermedicalscience1754-6605Cancer Intelligence 10.3332/ecancer.2013.312can-7-312Case ReportPrimary extraosseous Ewing sarcoma of the lung in children Alsit Nidal 1Fernandez Clara 2Michel Jean Luc 3Sakhri Linda 4Derouet Audrey 5Pirvu Augustin 61 Department of Thoracic, Vascular, and Cardiac Surgery, University Hospital Felix Guyon, Reunion, France2 Department of Pathological Anatomy, University Hospital Felix Guyon, Reunion, France3 Department of Pediatrics Surgery, University Hospital Felix Guyon, Reunion, France4 Department of Onco-pneumology, University Hospital Grenoble, France5 Department of Pediatrics, University Hospital Felix Guyon, Reunion, France6 Department of Thoracic, Vascular, and Endocrine Surgery, University Hospital Grenoble, FranceCorrespondence to: Augustin Pirvu. [email protected]; [email protected] 30 4 2013 7 31204 3 2013 © the authors; licensee ecancermedicalscience.2013This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.We report a case of primary extraosseous Ewing sarcoma (EES) of the lung in a four-year-old child. In the literature, there are only a few case reports of EES located in the thorax.
lung tumor in childrenextraosseous Ewing sarcoma
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Background
Extraosseous Ewing sarcoma (EES) is an uncommon malignant neoplasm in which pulmonary localisation is exceptionally rare.
Case Report
A four-year-old girl, without any medical history, was referred to our department for a lung mass (Figure 1). An initial thoracic computed tomography (CT) scan revealed a large cystic tumour in the middle of the left lung (Figure 2). The diagnosis of intrathoracic EES was made by punction under CT control. The patient was subsequently treated with six chemotherapy courses (vincristin, ifosfamid, doxorubicin, and etoposide).
At the end of chemotherapy, after a negative search for metastasis, we performed a radical resection, which consisted of a left pneumonectomy. The pathologic examination confirmed the need for a complete resection and also confirmed the initial diagnosis. During an interdisciplinary meeting, it was decided that postoperatively the patient would receive seven courses of chemotherapy (vincristin, actinomycin, and ifosfamid) without radiotherapy. The patient is currently receiving postoperative chemotherapy.
Discussion
In our case, the patient was a four-year-old girl, which was unusually young when compared with the previously reported cases [1–6]. The most common CT finding of EES reported is a heterogeneous mass [1–3], but in the present case, the CT showed the EES as a cystic structure.
The chemotherapy treatment was done according to the Eurowing 99 protocol and was also followed by a very aggressive surgery justified by the size, location, as well as the aggressive character of the tumour [2–5]. According to most of the authors, this kind of tumour should be resected as an attempt to obtain complete control of the disease [1–6].
Conclusion
Intrathoracic EESs are extremely rare and complex conditions requiring a pluridisciplinary collaboration. This case highlights the importance of preoperative evaluation and strategy in aggressive tumours.
Conflicts of interest
The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
Figure 1: CT scan: coronal reconstruction of a left lung mass (arrow)
Figure 2: CT scan: (A) cystic tumour in the middle of left lung between the superior and the lower bronchus with maximum diameter of 10/8 cm (arrow); (B) close contact of the tumour (arrow) with the left pulmonary artery
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References
1. Siddiqui MA Akhtar J Shameem M Baneen U Zaheer S Shahid M Giant extraosseous Ewing sarcoma of the lung in a young adolescent female–a case report Acta Orthop Belg 2011 77 2 270 3 21667743
2. Halefoglu AM Extraskeletal Ewing’s Sarcoma Presenting as a Posterior Mediastinal Mass Arch Bronconeumol 2012 epub ahead of print 10.1016/j.arbres.2012.02.020 22575810
3. Xie CF Liu MZ Xi M Extraskeletal Ewing’s sarcoma: a report of 18 cases and literature review Chin J Cancer 2010 29 4 420 4 10.5732/cjc.009.10402 20346219
4. Hancorn K Sharma A Shackcloth M Primary extraskeletal Ewing’s sarcoma of the lung Interact Cardiovasc Thorac Surg 2010 10 5 803 4 10.1510/icvts.2009.216952 20067987
5. Lee YY Kim do H Lee JH Choi JS In KH Oh YW Cho KH Roh YK Primary pulmonary Ewing’s sarcoma/primitive neuroectodermal tumor in a 67-year-old man J Korean Med Sci 2007 22 S159 63 10.3346/jkms.2007.22.S.S159 17923745
6. Kara IO Gonlusen G Sahin B Ergin M Erdogan S A general aspect on soft-tissue sarcoma and c-kit expression in primitive neuroectodermal tumor and Ewing’s sarcoma. Is there any role in disease process? Saudi Med J 2005 26 8 1190 6 16127511 | 23653672 | PMC3634713 | CC BY | 2021-01-04 22:39:36 | yes | Ecancermedicalscience. 2013 Apr 30; 7:312 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23637992PONE-D-12-3659310.1371/journal.pone.0062170Research ArticleBiologyBiochemistryNucleic AcidsRNAGeneticsGene FunctionMolecular Cell BiologySignal TransductionSignaling in Cellular ProcessesApoptotic SignalingSignaling in Selected DisciplinesOncogenic SignalingMedicineObstetrics and GynecologyBreast CancerOncologyCancers and NeoplasmsBreast TumorsMedicine and Health SciencesOncologyCancers and NeoplasmsBreast TumorsBreast CancerBiology and life sciencesGeneticsGene expressionGene regulationMicroRNAsBiology and life sciencesBiochemistryNucleic acidsRNANon-coding RNAMicroRNAsBiology and Life SciencesCell BiologyCell ProcessesCell DeathApoptosisBiology and Life SciencesCell BiologyCell ProcessesCell Cycle and Cell DivisionBiology and Life SciencesBiochemistryProteinsCytoskeletal ProteinsVimentinBiology and Life SciencesCell BiologyCell ProcessesCell ProliferationBiology and Life SciencesCell BiologyCell ProcessesCell Cycle and Cell DivisionCell Cycle InhibitorsResearch and Analysis MethodsImmunologic TechniquesImmunoassaysEnzyme-Linked ImmunoassaysmiR-221 Promotes Tumorigenesis in Human Triple Negative Breast Cancer Cells miR-221 Promotes Tumorigenesis in TNBCsNassirpour Rounak Mehta Pramod P. Baxi Sangita M. Yin Min-Jean
*
Oncology Research, Pfizer Worldwide Research and Development, San Diego, California, United States of America
Ahmad Aamir Editor
Wayne State University School of Medicine, United States of America
* E-mail: [email protected] Interests: All authors are current full-time employees of Pfizer Inc. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: RN MJY. Performed the experiments: RN PPM SMB. Analyzed the data: RN MJY. Contributed reagents/materials/analysis tools: RN PPM SMB MJY. Wrote the paper: RN MJY.
2013 24 4 2013 10 4 2017 8 4 e6217020 11 2012 18 3 2013 © 2013 Nassirpour et al2013Nassirpour et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis. TNBCs are characterized by their resistance to apoptosis, aggressive cellular proliferation, migration and invasion, and currently lack molecular markers and effective targeted therapy. Recently, miR-221/miR-222 have been shown to regulate ERα expression and ERα-mediated signaling in luminal breast cancer cells, and also to promote EMT in TNBCs. In this study, we characterized the role of miR-221 in a panel of TNBCs as compared to other breast cancer types. miR-221 knockdown not only blocked cell cycle progression, induced cell apoptosis, and inhibited cell proliferation in-vitro but it also inhibited in-vivo tumor growth by targeting p27kip1. Furthermore, miR-221 knockdown inhibited cell migration and invasion by altering E-cadherin expression, and its regulatory transcription factors Snail and Slug in human TNBC cell lines. Therefore, miR-221 functions as an oncogene and is essential in regulating tumorigenesis in TNBCs both in vitro as well as in vivo.
The authors have no support or funding to report.
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Introduction
microRNAs (miRNAs) are non-coding, single-stranded ∼22 nucleotides long small RNAs that act as agents of the RNA interference pathway and negatively regulate the translation by either cleaving or degrading their targeted transcripts [1]. Because miRNAs usually bind to their targets with incomplete complementarity, a single miRNA can potentially regulate the translation of multiple target genes involved in various cellular processes [2], [3]. In fact, miRNAs have been implicated in the regulation of a variety of biological functions, including cellular proliferation, differentiation, and apoptosis [4], [5]. Growing evidence indicates that miRNAs can function as tumor suppressors or oncogenes [4], and miRNA expression profiling analyses have revealed characteristic miRNA signatures in a variety of human cancers [6], [7]. Furthermore, miRNAs are frequently located at fragile genomic regions susceptible to amplification, deletion, or translocation during tumor development [8]. Since miRNAs are believed to be pivotal players in tumor development, investigations of differential expression of miRNAs and their corresponding targets might prove to be instrumental for the diagnosis and treatment of various cancers.
Molecular profiling has allowed us to classify breast cancers to five subtypes based on their distinctive gene expression signatures [9]. The five subtypes are luminal A, luminal B, Human Epidermal Growth Factor Receptor 2 (HER2) positive, basal-like, and normal-like breast cancers. Basal-like tumors are characterized by the expression of genes specific for basal epithelial cell proliferation, inhibition of apoptotic pathways, and aggressive migration and invasion [9]–[11]. Basal-like breast cancers (BLBCs) are often stained negative by immunochemistry for estrogen receptor (ER), progesterone receptor (PR), and HER2 and thus are called triple negative breast cancers (TNBCs). Although BLBC and TNBC share numerous clinical and pathological features, they are not identical [12], [13]. In the majority of cases, however, these two categories share similar clinical characteristics, prognosis and treatment options and thus, the term “TNBC” will be used in this study to collectively describe BLBC and TNBC cell lines and patient populations. Clinical studies have shown that TNBCs are the most aggressive breast cancer type and TNBC patients are frequently faced with poor prognosis and high mortality [14], [15]. Thus, the development of targeted therapies for TNBCs is urgently and critically needed for this patient population.
miR-221 and miR-222 are encoded in tandem from a gene cluster located on chromosome X and have been shown to be up-regulated in a panoply of cancer types. Due to their seed sequence similarity, both miRNAs have been shown to directly target p27kip1, Bmf, PTEN, Mdm2, PUMA, and TRPS1 [16]–[22]. In breast cancer, miR-221/miR-222 have been shown to be involved in regulation of ERα expression, suppression of ERα-mediated signaling, as well as drug resistance mechanisms [23]–[27]. More recently miR-221/miR-222 have been shown to be over-expressed in triple-negative primary breast cancers or cell lines [28]–[30]. In this study, we investigated the molecular mechanisms of cellular transformation regulated by miR-221/222 specifically in a panel of human TNBC cell lines compared to other breast cancer types and validated our findings in vivo. We show that miR-221 is an oncogene and modulates cell proliferation and tumor progression via targeting p27kip1 and EMT transition in TNBCs both in vitro as well as in vivo.
Materials and Methods
Cell Culture
All cell lines were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with L-glutamine and grown in a humidified atmosphere of 5% CO2 in air at 37°C.
Stable Cell Line Generation
System Bioscience’s miRZip™ anti-sense miRNAs are stably expressed RNAi hairpins that inhibit miRNA activity. The miRZip shRNAs produce short, single-stranded anti-miRNAs that competitively bind their endogenous miRNA target and inhibit its function. The miRZip short hairpin RNAs are cloned into SBI’s pGreenPuro™ shRNA expression vector, an improved third generation HIV-based expression lentivector. The lentiviral vector contains the genetic elements responsible for packaging, transduction, and stable integration of the viral construct into genomic DNA, inducing expression of the anti-miRNA effector sequence. For production of a high titer of viral particles, we used the ViraPower™ Lentiviral Support Kit (Invitrogen) employing Lipofectamine™ 2000 (Invitrogen) for transfecting the miRzip vectors into HEK-293T cells. Because infected cells stably express cop-GFP and puromycin, as well as the anti-miRNA cloned into the miRZip™ vector, we successfully used puromycin to select for the infected cells harboring the miRzip.
RT-PCR
TaqMan miRNA assays (Life Technologies, CA) were used to quantify the expression levels of mature miR-221 and miR-222 as well mRNAs p27, Snail, Slug, vimentin and E-Cadherin. Total RNA extracted by mirVana (Life Technologies) was reverse transcribed in a reaction mixture containing miR-specific stem-loop RT primers. Quantitative polymerase chain reaction (qPCR) was performed in triplicate with reactions containing amplified cDNA and TaqMan primers in Universal Master Mix without AmpErase UNG (Applied Biosystems). The qPCR was conducted at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds in 7900 HT Real Time PCR system (Applied Biosystems) and threshold cycles (C
T) were calculated using Sequence Detection Software (SDS v2.2.1, Applied Biosystems). All mRNA quantification data were normalized to GAPDH. All miRNA data are expressed relative to a RNU48 small nuclear (sn) RNA TaqMan PCR performed on the same samples, unless otherwise specified. Fold expression was calculated from the triplicate of C
T values following the 2−ΔΔCt method.
Immunoblotting
Cells were lysed in buffer composed of 150 mM NaCl, 1.5 mM MgCl2, 50 mM HEPES, 10% glycerol, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40 supplemented with 1 mM Na3VO4, 1 mM PMSF, 1 mM NaF, 1 mM β-glycerophosphate, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche) added prior to use. Protein concentration was determined using the BCA Protein Assay (Pierce/Thermo Fisher Scientific) per manufacturer’s instructions. Protein (30–50 µg) was resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Blots were probed with primary antibodies to detect proteins of interest. After incubation with secondary antibodies, membranes were visualized by chemiluminescence (Pierce/Thermo Fisher Scientific). All antibodies were purchased from Cell Signaling Technology, with the exception of GAPDH (Santa Cruz Biotechnology).
Cell Proliferation, Apoptosis Assay, Migration, and Invasion Assays
Resazurin Fluorescent Assay was used for the proliferation studies. Briefly, Cells were seeded at 3000–5,000 cells/100ul/well in DMEM +10% FBS in a 96 well plate, and incubated overnight at 37°C in 5% CO2. Resazurin (Sigma) fluorescent dye was added (1∶100) to each well. The cells were incubated at 37°C in 5% CO2 for 4 hours at which point the plate was read for fluorescence at 530/590 nm on the HTS 7000 plate reader. Cell Signaling Technologies PathScan® Apoptosis Multi-Target Sandwich ELISA Kits were used in the apoptosis assays. Briefly, antibodies for cleaved caspase 3 and phosphorylated BAD had been coated onto microwells. After incubation with the cell lysates, the target protein was captured by the coated antibody. Following extensive washing, a detection antibody was added to detect the captured target protein. An HRP-linked secondary antibody was then used to recognize the bound detection antibody. HRP substrate, TMB, was added for color development which was measured to quantity the level of bound target protein. Cell Biolab’s CytoSelect™ Cell Migration Assay Kit containing polycarbonate membrane inserts (8 µm pore size) in a 24-well plate was used in our migration assays. Migratory cells were able to extend protrusions towards FBS (used as the chemo-attractant), and pass through the pores of the polycarbonate membrane. The non-migratory cells were removed from the top of the membrane and the migratory cells were stained and quantified. Similarly, Cell Biolabs CytoSelect™ Cell Invasion Assay Kit containing basement membrane-coated inserts were used to assay the invasive properties of the cells. The upper surface of the insert membrane is coated with a uniform layer of dried basement membrane matrix solution. This basement membrane layer serves as a barrier to discriminate invasive cells from non-invasive cells. The non-invasive cells were removed from the top of the membrane and the invading cells were stained and quantified.
Cell Cycle Analysis
MDA-MB-231, BT-20, and MDA-MB-468 parental cells and cells stably expressing miR-221-ZIP, or scramble miRNA-ZIP were cultured to 70–90% confluency and allowed to adhere overnight. Cells were collected, fixed, and permeabilized using the Cell Cycle Phase Determination Kit (Cayman Chemical) following the manufacturer’s protocol. Samples were stored at −20°C until DNA stained with propidium iodide and read on a BD FACSCalibur (BD Biosciences). Data analysis was done with FCS Express (De Novo Software). All experiments were repeated at least 3 times and representative data set is shown.
Animal Studies
Six- to eight-week-old nu/nu athymic female mice were obtained from Jackson Labs. The mice were maintained in pressurized ventilated caging at the Pfizer La Jolla animal facility. All animal studies were done under the ethical approval by Pfizer Institutional Animal Care and Use Committees. Tumors were established by injecting 5×106 cells suspended 1∶1 (v/v) with reconstituted basement membrane (Matrigel, BD Biosciences). Tumor dimensions were measured with Vernier calipers, and tumor volumes were calculated using this formula: π/6×(larger diameter)×(smaller diameter)2. Tumor growth inhibition percentage (TGI %) was calculated as 100×(1−ΔT/ΔC). One way ANOVA Statistical analysis were performed and noted as *** depicting p-value less than or equal to 0.001.
Results
miR-221 is Specifically Up-regulated in Human TNBC Cell Lines
The expression level of miR-221 and miR-222 were examined by qRT-PCR in 4 TNBC lines: MDA-MB-231, Hs-578T, BT-20, and MDA-MB-468; 2 HER2 positive lines: SKBR3 and MDA-MB-361; and 3 ER positive lines: T47D, ZR75–1, and MCF-7. In comparison to the expression level in normal breast tissue (RNA acquired from Applied Biosystems), miR-221 is up-regulated in all the TNBC lines while down-regulated in the non-TNBC lines (Figure 1A). Surprisingly, although clustered with miR-221, the expression level of miR-222 is only up-regulated mildly (1–1.5 fold) in Hs-578-T and BT-20 (compared to normal breast tissue), but down regulated in MDA-MB-468 and the other non-TNBC lines tested (Figure 1B). These results indicate that although miR-221/miR-222 are both down-regulated in non-TNBC cells, miR-221 is specifically over-expressed in the TNBC cell lines in comparison to normal breast tissue. Therefore, over-expression of miR-221 may be important in maintaining the characteristics of triple negative breast cancer cells.
10.1371/journal.pone.0062170.g001Figure 1 miR-221 is over expressed in TNBCs.
qRT-PCR was performed to quantitatively measure RNA expression levels of miR-221 (A) and miR-222 (B) in a panel of breast cancer cell lines. All expression levels are displayed as fold changes normalized to the expression level in normal breast tissue.
miR-221 Targets p27kip1 to Regulate Cell Cycle Progression in TNBCs
p27kip1, an inhibitor of cyclin dependent kinase involved in the regulation of the cell cycle, has previously been shown to be a potential target of miR-221 in a variety of cancers [17], [24], [31], [32]. Since highly activated cell proliferation is one of the major characteristics of TNBCs, we investigated whether miR-221 also targets p27kip1in this particular breast cancer subtype. If so, we would expect an inverse relationship between the miR-221 and p27 levels. We thus measured the expression level of p27 in a variety of breast cancer cell lines as shown in Figure 2A. As expected, p27 is expressed at much lower levels in TNBCs than in other types of breast cancer cell lines. In fact, p27 expression level was inversely correlated to miR-221 expression level in most the breast cancer cell lines tested as shown in Figure 1A and Figure 2A. Since miR-221 and miR-222 are highly homologous and contain identical seed sequences, one might expect them to regulate the same target genes and play similar biological functions in cancer cells [17]. However, the relative expression level of miR-221 versus normal breast tissue is higher in the comparison to miR-222, and since miR-221, but not miR-222, was specifically over expressed in the TNBCs tested, we focused our experiments on miR-221. Next, we successfully and stably knocked down miR-221 using a miR-ZIP lentiviral vector in various breast cancer cells, as shown in Figure 2B. Briefly, miRZip are short, single-stranded anti-miRNAs in a lentivirus backbone that can be stably expressed to specifically target miRNAs of interest and alter translation. Knockdown of miR-221 in MDA-MB-231, BT-20, and MDA-MB-468 TNBC cell lines induced significant increases of p27kip1 both in mRNA expression and protein levels as shown in Figure 2C and Figure 2D, confirming that p27kip1 is a target of this miRNA in TNBCs. Since p27kip1 is involved in cell cycle regulation by modulating cyclin-dependent kinase (CDK) activity [33], we also investigated the effects of miR-221 inhibition on cell cycle progression in TNBC cell lines. miR-221 knockdown induced a G1 arrest as evidenced by observing a higher number of cells in G1 phase compared to S phase (as shown in Figure 2E). Furthermore, cell cycle profile analysis demonstrated that miR-221 knockdown in MDA-MB-231, BT-20, and MDA-MB-468 cells exhibited higher sub-G1 cell population, again suggesting that miR-221 knockdown restored p27kip1 levels and subsequently induced apoptosis probably by blocking the aggressive cell cycle progression in these TNBC cell lines.
10.1371/journal.pone.0062170.g002Figure 2 miR-221 modulates cell cycle progression by targeting p27kip1.
(A) mRNA expression level of p27kip1 was measured in a panel of breast cancer cell lines. Data are displayed as fold changes normalized to the expression level in normal breast tissue. (B) TNBC lines, MDA-MB-231, BT-20, and MDA-MB-468 cells were established to stably express anti-miR-221 (miR-221-ZIP) or a control scramble miRNA (Scramble-ZIP). miR-221 expression level was measured and is displayed as fold changes normalized to the expression level of parental cell lines. (C) Transcript expression level of p27kip1 was measured and is displayed as fold changes normalized to the expression level of parental cell lines. (D) Western blot analysis of MDA-MB-231, BT-20, and MDA-MB-468 cells stably expressing anti-miR-221 or scramble miR-ZIP depicting changes in p27 protein level. GAPDH was used as loading control. (E) MDA-MB-231, BT-20, and MDA-MB-468 cells stably expressing anti-miR-221 or scramble miR-ZIP were cultured for 72 hours to reach 80–90% confluency before harvesting and cell cycle analysis was performed and displayed as percentages of each cell cycle stage. These experiments were repeated at least three times, and representative data is shown.
Down Regulation of miR-221 in TNBCs Inhibits Cell Proliferation and Tumor Growth in Mice
Since cell cycle analysis demonstrated that miR-221 knockdown was able to block cell cycle progression and induce higher sub-G1 cell population, we next investigated whether miR-221 knockdown also induced apoptosis by direct measurement of apoptosis markers: cleaved caspase 3 and BAD phosphorylation (pBAD). As shown in Figure 3A, down regulation of miR-221 induced significantly higher levels of cleaved caspase 3 in MDA-MB-231 and BT-20 cells, and slightly higher levels of cleaved caspase 3 in MDA-MB-468 cells. Knocking down miR-221, however, did decrease phosphorylation of Bcl-2-associated death promoter (BAD), a pro-apoptotic member of the Bcl-2 gene family whose activity is regulated by survival kinases such as AKT, in all three TNBC cell lines (Figure 3A). We next investigated the cell proliferation rates of TNBCs with miR-221 knockdown. Down-regulation of miR-221 in all three TNBC lines (MDA-MB-231, BT-20, MDA-MB-468) decreased cell proliferation rates (Figure 3B), likely due to the cell cycle block and increased apoptosis induced by miR-221 knockdown. Although we did not observe a significant increase of cleaved caspase 3 in MDA-MB-468 cells, miR-221 knockdown indeed was able to decrease pBAD (Figure 3A) and induced G1 arrests (Figure 2E) and subsequently resulted in cell proliferation inhibition probably through mitochondrial apoptosis regulated by BCL2 family [34], [35].
10.1371/journal.pone.0062170.g003Figure 3 miR-221 knockdown induces apoptosis, inhibits cell proliferation and supresses tumor growth in mice.
MDA-MB-231, BT-20, and MDA-MB-468 cells stably expressing miR-221-ZIP or scramble-ZIP were used to perform apoptosis, cell proliferation, and in-vivo tumor growth assays. (A) Cleaved caspase 3 and phosphorylation of BAD were measured as apoptosis markers. All cell lines were seeded at similar density as the parental cell lines and cultured for 72 hours until the parental cell lines reached 80–95% confluencey, before cell lysates were prepared and subject to cleaved caspase 3 assays. (B) Cell proliferation measurements were normalized to the readings in parental cells at day 1. (C) Nude mice were implanted subcutaneously with MDA-MB-231 parental cells, and MDA-MB-231 cells stably expressing miR-221-ZIP or scramble-ZIP. Tumor measurements were recorded and tumor growth inhibition was calculated as described in Materials and Methods. T-test was performed in (A) and (B) and one way ANOVA was performed in (C) to compare the differences between parental cells versus miR-221-ZIP cells. *denotes p-value ≤0.05. ***denotes p-value ≤0.005.
To expand on our in vitro results, we also investigated whether miR-221 is required for in vivo tumor growth. miR-221 stably knocked down MDA-MB-231 cells were implanted in nude mice and tumor growth was measured and plotted to compare with the tumor growth of MDA-MB-231 parental cell line and cells infected with the control ZIP vector alone as shown in Figure 3C. Our results indicated that miR-221 knockdown also inhibited in vivo tumor growth in TNBC cell line MDA-MB-231. Therefore, both the in
vitro assays and in vivo studies confirm that miR-221 functions similar to an oncogene and is essential in mediating cell proliferation and tumor progression in TNBC.
miR-221 Modulates Cell Migration and Invasion by Regulating Epithelial-mesenchymal Transition
Relative to luminal subtypes, TNBCs, having undergone an epithelial to mesenchymal transition (EMT), express higher levels of vimentin and low levels of E-cadherin which allow for their characteristic high migration and invasion capabilities through the basement membrane to promote metastasis [36]. Since miR-221 knockdown can inhibit cell proliferation and tumor growth in mice (Figure 3), we wanted to investigate the molecular mechanism for the miR-221 mediated cell transformation activity in TNBC human cell lines. Therefore, we next examined the levels of EMT markers and performed cell migration and invasion assays. The levels of E-cadherin and vimentin in a variety of breast cancer cells were quantified relative to the normal breast tissue as shown in Figure 4A. As expected, E-cadherin is highly expressed in luminal and HER2 positive cells but not in TNBC cell lines. Conversely, vimentin is expressed in higher levels in TNBC cell lines compared to non-TNBC cells. E-cadherin and vimentin levels were measured at both the transcript and protein levels in parental, vector control and miR-221 knocked down MDA-MB-231, BT-20, and MDA-MB-468 cells. Results indicate that knocking down miR-221 in these TNBCs significantly increased both the mRNA and protein levels of E-cadherin as shown in Figure 4B. Interestingly, vimentin levels were not altered by knocking down miR-221 in these cell lines. These data suggest that although suppression of E-cadherin is regulated by miR-221, the vimentin level in TNBCs is probably regulated by other mechanisms. Since E-cadherin lacks a miR221 binding site and is likely not a direct target, we next investigated if this regulation is mediated by any of the transcription factors that have previously been reported to directly regulate E-cadherin expression [37]. Figure 4C outlines the effects of miR-221 knockdown on some of the EMT transcription factors known to regulate E-cadherin levels. We observed a robust decrease in the expression levels of mesenchymal markers Snail and Slug by miR-221 knockdown in MDA-MB-231, BT20 and MDA-MB-468 (Figure 4C). As previously reported however, the expression level of Slug in MDA-MB-468 was much lower than the other two TNBC cell lines tested [38].
10.1371/journal.pone.0062170.g004Figure 4 Down regulation of miR-221 increases E-cadherin levels and decreases the expression levels of Snail and Slug.
(A) The RNA expression level of E-cadherin and vimentin was measured in a panel of breast cancer cell lines. Fold changes are recorded as normalized to normal breast tissue levels. (B) E-cadherin and vimentin expression levels were measured in MDA-MB-231, BT-20, MDA-MB-468 parental cells, as well cells harboring miR-221-ZIP, or scramble-ZIP. Data are normalized to the expression level in parental cells. Western blot analysis was also performed to examine the protein levels of E-cadherin and vimentin. (C) Snail and Slug expression levels were also examined in MDA-MB-231, BT-20, and MDA-MB-468 cells. Data were normalized to the expression level in parental cells and fold changes were plotted. ***denotes p<0.005. **denotes p<0.01.
We next investigated the effects of miR-221 knock down on cell migration and invasion of TNBC cell lines. As expected, MDA-MB-231, BT-20, and MDA-MB-468 showed high migratory and invasive properties in the migration and invasion assays performed upon stimulation with 10% FBS. Knocking down miR-221 decreased the FBS stimulated migration and invasion in all three cell lines as shown in Figure 5A and Figure 5B. Our data thus indicate that miR-221 alters the migration and invasion properties of TNBCs by suppressing E-cadherin expression. miR-221 knockdown in TNBCs restored E-cadherin expression and the increased E-cadherin in these TNBC cells was sufficient to block the activity of cell migration and invasion. Interestingly, although vimentin levels did not change with miR-221 knock down and high vimentin levels were maintained in these transduced TNBCs, it was not sufficient to maintain their migration and invasion capabilities. Therefore, these results suggest that the modulation of E-cadherin by miR-221 plays a critical role in maintaining the triple negative cell phenotype and knockdown of miR-221 can lead to increased E-cadherin and subsequently inhibit cell migration and invasion independent of vimentin levels.
10.1371/journal.pone.0062170.g005Figure 5 Down regulation of miR-221 inhibits cell migration and invasion in TNBC lines.
(A) Migration and (B) Invasion assays were performed in the absence or presence of 10% FBS after 72 hours in culture. Data are displayed as the percentage of cells migrated or invaded. T-test was performed to compare the differences between parental cells versus miR-221-ZIP cells, ***denotes p<0.005.
Discussion
miR-221 has been reported to be dysregulated in a variety of tumor types and has previously been shown to be involved in suppression of ERα expression in luminal breast cancer cells and EMT transition in basal-like breast cancers [23]–[26], [29], [30], [39]. Here we demonstrate that miR-221 is specifically over expressed in TNBCs and that miR-221 knockdown induces G1 arrest and apoptosis, inhibits cell proliferation and tumor growth (probably by altering expression levels of p27kip1), and suppresses the migratory phenotypes of TNBCs by restoring E-cadherin levels. These results suggest that miR-221 is essential in regulating the aggressive characteristics of triple negative or basal like of breast cancer cells, including cell proliferation, suppressed apoptosis, high migratory and invasive abilities, as well as accelerated in-vivo tumor growth. Therefore, our results provide direct evidence that overexpression of miR-221 leads to in vitro and in vivo tumorigenesis in TNBCs.
Therefore, miR-221 regulates two key mechanisms to promote the aggressive tumorogenic characteristics observed for TNBC: it promotes cell cycle progression by inhibiting p27kip1 and it promotes EMT transition by inhibiting the expression of E-cadherin. Both of these mechanisms may account for the aggressive cellular proliferation, suppression of apoptosis, as well as higher cell migration and invasion characteristics associated with BLBCs and TNBCs [9]–[11]. Although, we could not rule out alternative targets of miR-221 that may explain the cellular phenotypes observed, knockdown of miR-221 alone is sufficient to induce in vitro and in vivo anti-tumor activities in the TNBC cell lines tested. Hence, the translational suppression of miR-221 targets is crucial in maintaining the aggressive tumor progression of TNBCs. Previously, miR-221 over expression has been shown to alter E-cadherin/vimentin levels in an EMT-induced MCF-7 cell line, which normally does not express endogenous miR-221 [39]. Additionally, EMT transcription factor Slug has previously been shown to decrease both miR-221 and E-cadherin/vimentin levels in MDA-MB-231 cells [30]. In this study, although we did observe changes in Slug and Snail, we did not detect changes in vimentin expression after stable miR-221 knockdown in MDA-MB-231, BT20, and MDA-MB-468 cell lines. Although vimentin levels were not altered, E-cadherin expression changed significantly with miR-221 knockdown. Our data suggest that increasing E-cadherin while maintaining vimentin levels in TNBCs seems to be sufficient for inhibiting cell migration and invasion. Therefore, E-cadherin seems to be critical in regulating cell motility, at least in the TNBC cell lines, MDA-MD-231, BT-20, and MDA-MB-468. Although miR-221 was able to regulate E-cadherin expression, we were unable to identify the complementary sequence in the 3′UTR of this protein. Therefore E-cadherin may not be a direct target of miR-221, and its expression is likely affected by alteration in EMT transcription factors Snail and Slug.
In conclusion, we have demonstrated that miR-221 is a potential oncomiR and functions as an oncogene to mediate tumor progression of TNBCs via targeting p27kip1 and inhibiting E-cadherin levels to mediate EMT transition. These results may prove useful for therapeutic options for TNBCs when systemic delivery of anti-miR-221 becomes feasible.
The authors would like to thank Tod Smeal, Timothy S Fisher, and Lars Engstrom for their help and support.
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==== Front
Mol Cell Biochem
Mol Cell Biochem
Molecular and Cellular Biochemistry
0300-8177
1573-4919
Springer US Boston
23456481
1604
10.1007/s11010-013-1604-z
Article
Role of CC-chemokine receptor 5 on myocardial ischemia–reperfusion injury in rats
Shen Bo 12
Li Jun 3
Gao Ling 12
Zhang Jieyu 12
Yang Bo [email protected]
12
1 Department of Cardiology, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan, 430060 People’s Republic of China
2 Cardiovascular Research Institute, Wuhan University, Wuhan, People’s Republic of China
3 Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China
3 3 2013
3 3 2013
2013
378 1 137144
7 11 2012
23 2 2013
© The Author(s) 2013
https://creativecommons.org/licenses/by/2.0/ Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
The expression level of CC-chemokine receptor 5 (CCR5) is enhanced post inflammatory stimulations and might play a crucial role on inflammatory cells infiltration post myocardial ischemia. The purpose of this study was to evaluate the role of CCR5 on myocardial ischemia–reperfusion (I/R) injury in rats. Adult male rats were randomized to sham group, I/R group (I/R, 30 min coronary artery occlusion followed by 2-h reperfusion), ischemic preconditioning (I/R + Pre), CCR5 antibody group [I/R + CCR5Ab (0.2 mg/kg)], and CCR5 agonist group [I/R + CCR5Ago, RNATES (0.1 mg/kg)], n = 12 each group. The serum level of creatine kinase (CK) and tumor necrosis factor α (TNF-α) were measured by ELISA. Myocardial infarction size and myeloperoxidase (MPO) activity were determined. Myocardial protein expression of CCR5 and intercellular adhesion molecule-1 (ICAM-1) were evaluated by Western blotting and immunohistochemistry staining, respectively. Myocardial nuclear factor-kappa B (NF-κB) activity was assayed by electrophoretic mobility shift assay. Myocardial CCR5 protein expression was significantly reduced in I/R + Pre group (P < 0.05 vs. I/R) and further reduced in I/R + CCR5Ab group (P < 0.05 vs. I/R + Pre). LVSP and ±dP/dt max were significantly lower while serum CK and TNF-α as well as myocardial MPO activity, ICAM-1 expression, and NF-κB activity were significantly higher in I/R group than in sham group (all P < 0.05), which were significantly reversed by I/R + Pre (all P < 0.05 vs. I/R) and I/R + CCR5Ab (all P < 0.05 vs. I/R + Pre) while aggravated by I/R + CCR5Ago (all P < 0.05 vs. I/R). Our results suggest that blocking CCR5 attenuates while enhancing CCR5 aggravates myocardial I/R injury through modulating inflammatory responses in rat heart.
Keywords
Ischemia–reperfusion injury
CC-chemokine receptor 5
Antibody
Agonist
Nuclear factor-kappa B
issue-copyright-statement© Springer Science+Business Media New York 2013
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Introduction
Reperfusion strategies have substantially contributed to the effective treatment of ischemic heart diseases despite the potential negative impact of ischemia/reperfusion (I/R) injury[1]. I/R injury is characterized by robust local and systemic inflammatory responses which may aggravate tissue injury and adversely affect left ventricular (LV) recovery [2]. A variety of studies in the last decades has shown that the extent of postischemic tissue damage strongly correlates with the number of leukocytes recruited to the reperfused tissue [3–5]. It is known that increased chemokine expression post I/R could promote the adhesion of neutrophils and increase leukocyte infiltration in the cardiac tissue [6], which could serve as one of the important mechanisms mediating the ischemic myocardial damage. Over the past years, chemokines and their receptors have become the subject of intensive investigations and there is a growing body of evidence that chemokines and their receptors are also critically involved in the pathogenesis of I/R. Chemokines are small, secreted proteins that are produced constitutively or in an inducible manner by most cell types and that induce directed cell migration [7]. CC-chemokine receptor 5 (CCR5) is expressed on T-lymphocytes with memory/effector phenotype, macrophages, monocytes, as well as the immature dendritic cells [8]. The expression levels of CCR5 are very low in the mononuclear cells and T cells of human peripheral blood under normal conditions, but could increase significantly after the inflammatory stimulation both in vivo and in vitro [9]. Previous study showed that TAK-779, a small-molecule, nonpeptide compound that selectively binds to a certain subtype of the CC-chemokine receptor, CCR5, with high affinity [10], could effectively reduce leukocyte infiltration of the reperfused tissue and attenuate subsequent postischemic organ failure in mouse models of focal cerebral ischemia [11]. In this study, we tested the hypothesis that myocardial I/R injury could be attenuated by CCR5 antibody or aggravated by CCR5 agonist RANTES in rats.
Materials and methods
Animals and reagents
Healthy adult male Wistar rats (200–250 g) were purchased from Vital River Laboratories, Beijing, China. All experiments were approved by the Institutional Animal Care and Use Committee of Wuhan University. CCR5 antibody and CCR5 agonist RANTES were purchased from Sigma (USA); creatine kinase (CK) and myeloperoxidase (MPO) kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). TNF-α ELISA kit was purchased from Wuhan Boster Bioengineering Co. Ltd. (China). Electrophoretic mobility shift assay (EMSA) kit was purchased from Promega Corp. (Madison, WI, USA).
Surgical preparation
The surgical protocol was performed as described previously [12]. The rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg). After endotracheal intubation with a 14 gauge tube, the rats were then connected to a rodent respirator (TKR-400H, Jiangxi, China, 70 breaths per minute, tidal volume was set to 1.0 ml/100 mg body weight). Body temperature was measured by a rectal thermometer and maintained between 36 and 37 °C by infrared heating lamp. Hemodynamics (left ventricular systolic pressure (LVSP); left ventricular end-diastolic pressure (LVEDP); and heart rate (HR), +dP/dt max and −dP/dt max) were measured through a short segment of saline-filled PE50 tubing which was advanced to left ventricle through right carotid artery and connected to a multi-channel physiological monitoring system (LEAD 2000, Sichuan, China) before left anterior descending coronary artery (LAD) ligation or sham operation. Hemodynamic parameters were obtained immediately after 2-h reperfusion. Electrocardiograph (ECG) leads were connected to the chest and limbs for continuous ECG monitoring throughout the experiment (LEAD 2000, Sichuan, China). Then, the chest was opened via left thoracotomy through the fourth or fifth intercostal space, and the ribs were gently retracted to expose the heart. A 7-0 prolene suture was placed under left anterior descending coronary artery (LAD) after pericardiotomy.
Experimental protocol
Sixty rats were randomly divided into five groups: sham operation group (SHAM, n = 12), I/R group (I/R, n = 12), ischemic preconditioning group (I/R + Pre, n = 12), CCR5 antibody group (I/R + CCR5Ab, n = 12), and CCR5 agonist (RANTES) group (I/R + CCR5Ago, n = 12). Each group was subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion except sham group. (1) SHAM group: 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and LAD was not ligated; (2) I/R group: LAD ligation for 30 min followed by 2-h reperfusion, 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia; (3) I/R + Pre group: two cycles of 5-min ischemia followed by 5-min reperfusion and one cycle of 10-min ischemia followed by 10-min reperfusion, 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein before the 3rd circle reperfusion; (4) I/R + CCR5Ab group [13]: LAD ligation for 30 min followed by 2-h reperfusion, 0.2 mg/kg CCR5 antibody diluted in 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia; (5) I/R + CCR5Ag group [14]: LAD ligation for 30 min followed by 2-h reperfusion, 0.1 mg/kg RANTES diluted in 0.1 ml of anhydrous ethanol was bolus injected through the external jugular vein after thoracotomy and after 20-min ischemia. For each individual group, six rats were assigned for myocardial MPO activity determination and measurement of infarct zone and risk area and another 6 rats were assigned for myocardial inflammatory cells counting in HE-stained slices, myocardial CCR5 and ICAM-1 expression, and NF-κB activity determination.
Measurement of infarct zone and risk area
Immediately after hemodynamic measurements, LAD was re-occluded with a 7-0 prolene suture which was used previously at the same place for rats assigned for myocardial MPO activity determination and measurement of infarct zone and risk area, and Evans blue dye (2 ml of a 1 % solution) was injected via the external jugular vein to delineate the area at risk (AAR). The rats were sacrificed under deep pentobarbital anesthesia (60 mg/kg, i.p.) after blood sampling. The heart was then rapidly excised and washed in 0.9 % saline. After removal of the atrium, the ventricle was cut into transverse slices of equal thickness (3 mm) from the apex to the base. The slices were then incubated for 20 min in phosphate-buffered 1 % 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C, and then fixed in 10 % formalin solution. The AAR was defined as the area not stained with Evans blue dye. The area not stained by TTC was defined as the infarcted zone (AI). The border zones (Evans blue stained area neighboring Evans blue-unstained area), infarcted zones [TTC and The border zones (TTC-stained), infarcted zones (TTC and Evans blue-unstained)], and the nonischemic zones (Evens blue-unstained area remote from Evans blue-unstained area) were photographed and analyzed by the software program Image J 1.36. The AAR, AI, and ventricle size (VS) were assessed by a technician who was blinded to the experimental protocol using computer-assisted planimetry (NIH Image 1.57 software).
The infarct zone, border zones, and risk area were digital recorded. The percentage of the ischemic region (AR) in the whole LV (AR/LV) represents the severity of myocardial ischemia. The percentage of the infarcted region (IS) in the whole ischemic region (AR) (IS/AR) represents the extent of myocardial infarction. The three parts of LV samples (nonischemic zone, border zone, and infract zone) were stored in −80 °C refrigerator for determining MPO activity.
Blood collection of tissue sampling
After hemodynamic measurements, 4-ml blood was obtained from the carotid artery of all rats. Blood samples were placed static for 30 min at room temperature, and then centrifuged at 4,000 r/min for 10 min at 4 °C. The upper serum was removed into new EP tubes and stored in −80 °C refrigerator for detection of CK and TNF-α.
For rats assigned myocardial inflammatory cells counting in HE-stained slices, myocardial CCR5 and ICAM-1 expression, and NF-κB binding activity determination, rats were sacrificed under deep pentobarbital anesthesia (60 mg/kg, i.p.) after blood sampling, hearts were excised and washed with ice-cold saline solution. Two transversal sections (3-mm thick) from the middle part of each heart were prepared and stained with hematoxylin–eosin (HE) for evaluation of the inflammatory response in the cardiac tissues. Severity of inflammatory cell infiltration on HE staining was scored using the following scale [15]: 0 = no inflammation; 1 = cellular infiltrates only around blood vessel and meninges; 2 = mild cellular infiltrates in parenchyma (1–10/section); 3 = moderate cellular infiltrates in parenchyma (11–100/section); 4 = serious cellular infiltrates in parenchyma (100/section).
The remaining LV free wall was divided into three parts. One portion of LV free wall was used for the determination of myocardial ICAM-1 and fixed in 4 % paraformaldehyde. The second portion of LV free wall was stored in −80 °C refrigerator until use for NF-κB binding activity determination. The third portion was stored in −80 °C refrigerator until use for myocardial CCR5 protein expression determination.
Determination of Serum CK, TNF-α, and myocardial MPO activity
Serum CK level was determined by chemical colorimetric method. The serum level TNF-α was determined by rat TNF-α ELISA kit, referring to the manual. Tetramethyl benzidine method was applied for the determination of myocardial MPO activity.
Immunohistochemistry
Immunohistochemical staining of ICAM-1 was performed by the Strept Avidin Biotin Complex (SABC) method. Mouse monoclonal ICAM-1 antibody sc-107 (Santa Cruz Biotechnology, CA) was diluted at 1:100 as primary antibodies. The streptavidin–biotin complex kit was purchased from Wuhan Boster Biological Technology, Ltd. Wuhan, China. All the procedures were carried out according to the manufacturer’s manual. The data of the extent and intensity of staining were obtained using Image Pro Plus Version 6.0 (Media Cybernetics, Bethesda, MD). Five fields of each slice were photographed and analyzed by mean optical density.
Electrophoretic mobility shift assay
EMSA method was used to detect the DNA-binding activities of NF-κB in nuclear extracts. NF-κB oligonucleotide’s sequence was 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 5′-GCCTGGGAAAGTCCCCTCAACT-3′. Protein-DNA binding assays were performed with 20 μg of nuclear protein. In order to block the unspecific binding, 1 μg of poly (dI-dC) • poly (dI-dC) was added to the samples; then apply the binding medium containing 5 % glycerol, 1 % NP40, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 2 mM DTT, and 10 mM Tris/HCl, and with its pH around 7.5. In each reaction, 20,000 cpm of a radiolabeled probe was included. Samples were incubated at room temperature for 20 min. In order to separate the nuclear protein oligonucleotide complex labeled with 32P from free 32P-labeled oligonucleotide, the samples were subjected to electrophoresis through a 5 % native polyacrylamide gel for 2 h in a running buffer containing 50 mM Tris, pH 8.0, 45 mM borate, and 0.5 mM EDTA. After the separation was achieved, the gel was vacuum-dried for autoradiography and exposed to Fuji X-ray film for 24–48 h at −80 °C. The results were analyzed by medical image analysis system, with its gray-scale value representing the activity of NF-κB [16].
Western blotting
Western blotting was conducted to determine protein levels of CCR5 from myocardial tissues. Total cellular membrane proteins were extracted by Plasma Membrane Protein Extraction Kit (Catalog #K268-50) according to Membrane Protein Extraction Protocol. Supernatants were boiled for 10 min in loading buffer, and then separated by SDSPAGE and transferred onto nitrocellulose membranes. After blockage with 5 % skim milk in Tris-buffered saline (TBS) for 1 h at room temperature, the membranes were incubated with primary antibody at 4 °C overnight. After three washings with TBST, (HRP)-labeled secondary antibodies were added and incubated for another 1.5 h on the shaker at room temperature; the blots were then washed two times with TBST. The developed signal was detected using ECL as per the manufacturer’s instructions and exposed to Hyperfilm.
Antibodies against CCR5 were obtained from Cell Signaling Technology (Boston, MA, USA). The antibody against β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-rabbit and anti-mouse HRP-labeled antibodies and the ECL detection reagents were from Santa Cruz Biotechnology. The X-ray film used for Western-blot analysis was from Kodak (Rochester, NY, USA). Other chemicals and reagents were of analytical grade.
Statistical analysis
Data are presented as mean ± standard deviation (SD). Normality of distribution of all continuous variables was explored by examining skewness, kurtosis, and Q–Q plots. Differences on continuous data among groups were compared using one-way analysis of variance (ANOVA) followed by either Tukey’s or Games-Howell multiple comparison post-hoc tests as appropriate. Variables with non-normal distribution were compared using the non-parametric Mann–Whitney U-statistic test. A P value <0.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS, version 20 for Windows.
Results
Mortality
Five rats died post LAD ligation due to malignant arrhythmias and the remaining 55 rats were used in the study and 5–6 rats were examined in each individual group.
Cardiac function
The cardiac function indexes after 2-h reperfusion or sham operation are shown in Table 1. LVSP, +dP/dt max, and −dP/dt max were all significantly lower in I/R group than those in sham group and were significantly higher in I/R + Pre group and I/R + CCR5Ab group while significantly lower in I/R + CCR5Ago group as compared to I/R group. LVEDP was increased in I/R group than those in sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab group while significantly higher in I/R + CCR5Ago group as compared to I/R group.Table 1 Cardiac function in vivo
n HR (beats/min) LVSP (mmHg) LVEDP (mmHg) +dP/dt max (mmHg/s) −dP/dt max (mmHg/s)
SHAM 12 322 ± 17 139 ± 14 2.6 ± 0.2 7999 ± 772 −5005 ± 711
I/R 11 367 ± 14* 93 ± 6* 7.4 ± 0.5* 4868 ± 525* −2556 ± 444*
I/R + Pre 10 358 ± 9* 116 ± 6*,† 4.1 ± 0.3*,† 6316 ± 603*,† −3512 ± 551*,†
I/R + CCR5Ab 12 352 ± 10*,† 126 ± 5†,‡ 4.3 ± 0.38*,† 7077 ± 445*,†,‡ −4253 ± 666*,†,‡
I/R + CCR5Ago 10 370 ± 21*,§ 77 ± 6*,†,‡,§ 7.5 ± 0.5*,‡,§ 2987 ± 685*,†‡,§ −1705 ± 515*,†‡,§
* P < 0.05 vs. SHAM; † P < 0.05 vs. I/R; ‡ P < 0.05 vs. I/R + Pre; § P < 0.05 vs. I/R + CCR5Ab
Serum levels of CK and TNF-α
Similarly, serum levels of CK and TNF-α were both significantly higher in I/R group compared to sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab groups while significantly increased in I/R + CCR5Ago group compared to I/R group (Fig. 1).Fig. 1 Serum levels of CK and TNF-α. Serum levels of CK and TNF-α were both significantly higher in I/R group compared to sham group and were significantly reduced in I/R + Pre group and I/R + CCR5Ab groups while significantly increased in I/R + CCR5Ago group compared to I/R group
Myocardial infarct size
As shown in Fig. 2, the ischemia region (AR/LV) was similar among group. The myocardial infarct size was significantly smaller in I/R + Pre group and I/R + CCR5Ab group while was significantly larger in I/R + CCR5Ago group than those in I/R group, and was significantly smaller in I/R + CCR5Ab group as compared to I/R + Pre group (P < 0.05).Fig. 2 Myocardial infarct size. The ischemia region (AR/LV) was similar among group. The myocardial infarct size was significantly smaller in I/R + Pre group and I/R + CCR5Ab group while was significantly larger in I/R + CCR5Ago group than those in I/R group, and was significantly smaller in I/R + CCR5Ab group as compared to I/R + Pre group (P < 0.05)
MPO activity in myocardial tissues
MPO activities were significantly increased in I/R group compared to sham group in normal zone; MPO activities were significantly lower in I/R + Pre group and I/R + CCR5Ab group while was significantly higher in I/R + CCR5Ago group in normal, risk and infract zone than in I/R group (Fig. 3).Fig. 3 MPO activity in myocardial tissues. MPO activities were significantly increased in I/R group compared to sham group in normal zone, MPO activities were significantly lower in I/R + Pre group and I/R + CCR5Ab group while was significantly higher in I/R + CCR5Ago group in normal, risk, and infract zone than in I/R group
Inflammatory levels in myocardial tissues
The extent of inflammation of myocardial tissues from LV free wall in different groups was examined in HE-stained transversal myocardial slides. Histopathological images and histological score are listed in Fig. 4. Myocardial tissue in I/R and I/R + CCR5Ago groups presented massive inflammatory cell infiltration as compared to sham group indicating that I/R injury could trigger the inflammatory response which was reduced in I/R + Pre and I/R + CCR5Ab group. Accordingly, histological score was significantly lower in I/R + Pre and I/R + CCR5Ab groups than in I/R group and higher in I/R + CCR5Ago group than in I/R + Pre and I/R + CCR5Ab groups.Fig. 4 Inflammatory levels in myocardial tissues staining by HE (magnification ×400). Representative sections from rats in I/R and I/R + CCR5Ago groups presented massive inflammatory cell infiltration (arrows) as compared to sham group indicating that I/R injury could trigger the inflammatory response which was reduced in I/R + Pre and I/R + CCR5Ab group. Accordingly, histological score was significantly lower in I/R + Pre and I/R + CCR5Ab groups than in I/R group and higher in I/R + CCR5Ago group than in I/R + Pre and I/R + CCR5Ab groups
Myocardial ICAM-1 expression
Brown particles indicated the expression levels of ICAM-1 in myocardial tissues from LV free wall (Fig. 5). Myocardial ICAM-1 expression was significantly upregulated in I/R group compared to sham group and was significantly attenuated in I/R + Pre group and I/R + CCR5Ab group while was significantly increased in I/R + CCR5Ago group compared to that in I/R group.Fig. 5 Myocardial ICAM-1 expression. Brown particles indicated the expression levels of ICAM-1 in myocardial tissues from LV free wall. Myocardial ICAM-1 expression was significantly upregulated in I/R group compared to sham group and was significantly attenuated in I/R + Pre group and I/R + CCR5Ab group while was significantly increased in I/R + CCR5Ago group than in I/R group. (Color figure online)
Myocardial NF-κB binding activity
The myocardial DNA-binding activity of NF-κB was significantly higher in I/R group than in sham group, and was significantly lower in I/R + Pre and I/R + CCR5Ab groups while higher in I/R + CCR5Ago group as compared to I/R group (Fig. 6).Fig. 6 Myocardial NF-κB binding activities. The myocardial DNA-binding activity of NF-κB was significantly higher in I/R group than in sham group, and was significantly lower in I/R + Pre and I/R + CCR5Ab groups while higher I/R + CCR5Ago group as compared to I/R group
CCR5 protein level of myocardial tissues
We also determined the membrane protein levels of CCR5 in myocardial tissue from LV free wall (Fig. 7). The specific protein expression levels of CCR5 were normalized to β-actin. There is no difference in expression levels of membrane protein CCR5 between sham and I/R groups which was downregulated in I/R + Pre and I/R + CCR5Ab and I/R + CCR5Ago groups.Fig. 7 CCR5 protein level of myocardial tissues. The specific protein expression levels of CCR5 were normalized to β-actin. There is no difference in expression levels of membrane protein CCR5 between sham and I/R groups which was downregulated in I/R + Pre and I/R + CCR5Ab and I/R + CCR5Ago groups
Discussion
Our study showed that blocking CCR5 attenuates while enhancing CCR5 aggravates myocardial I/R injury through modulating inflammatory responses in rat heart. Thus, strategies modulating CCR5 might serve as potential therapeutic modalities to reducing I/R injury.
Although the precise mechanism of I/R injury has not been fully revealed, a series of studies have demonstrated that I–R could activate the intrinsic inflammatory network. The adhesion and aggregation of neutrophils in the cardiac tissue might be one of the important factors mediating myocardial I/R injury [17]. CCR5 is a receptor for various proinflammatory chemokines. Blocking CCR5 has been proposed as a novel therapeutic approach for cardiovascular conditions by interfering with systemic inflammation. This concept is supported by an animal study by Veillard et al. [18] in which treatment of hypercholesterolemic mice with the CCR5 antagonist Met-RANTES reduced progression of atherosclerosis and CCL5/RANTES inhibition attenuated myocardial reperfusion injury in atherosclerotic mice [14]. Moreover, treatment of apoE-deficient mice with Met-RANTES reduced neointimal plaque area and macrophage infiltration [19] and treatment with TAK-799, a CCR5 chemokine receptor antagonist, reduced lesion development in a collar-induced carotid artery atherosclerosis model [20]. Finally, TAK-779 treatment also reduced leukocyte infiltration and ischemic injury in a mouse model of focal cerebral ischemia [11]. In line with the above findings, we demonstrated that CCR5 antibody effectively reduced myocardial inflammatory cell infiltration and myocardial infarct size in this rat I/R model. It is to note that CCR5 activation was not evidenced in this I/R model; however, our results showed that CCR5 antibody treatment reduced myocardial injury in this model by reducing inflammatory responses. The exact mechanism responsible for the CCR5 antibody treatment effects in this model warrants further studies. Previous studies found that stimulation of increased TNF-α activity could upregulate ICAM-1 expression which then could function as an adhesion molecule promoting neutrophils infiltration [9]. Treatment with specific antibody of ICAM-1 resulted in coronary vascular and myocardial protection as shown by the decrease of myocardial infarct size [21]. Similarly, we showed that treatment with CCR5 antibody significantly reduced the myocardial expression of ICAM-1. In addition, MPO activity (an indicator of neutrophil accumulation in tissue) decreased significantly in both the risk area and infarcted area in I/R + Pre and I/R + CCR5Ab groups while increased in I/R + CCR5Ago group compared with I/R group. Thus, CCR5 antibody reduced while CCR5 agonist enhanced the inflammation in ischemic hearts by down- or upregulating the expression of TNF-α and ICAM-1. Taken together, treatment with CCR5 antibody that reduced infiltration of neutrophils in the ischemic myocardium might contribute to the reduced infarcted size and serum level of CK in this rat I/R model.
Conclusion
In conclusion, our study provides the first evidence that CCR5 antibody could reduce cardiac inflammation and protect the heart from I/R injury via inhibition of the activity of NF-κB, ICAM-1 expression, and MPO activities in this rat I/R model. We propose that targeting CCR5 might serve as a potential novel promising strategy for the treatment of ischemic myocardial disease.
Conflict of interest
None.
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BMC CancerBMC CancerBMC Cancer1471-2407BioMed Central 1471-2407-13-1822356573610.1186/1471-2407-13-182Research ArticleCorrelation of CD44v6 expression with ovarian cancer progression and recurrence Shi Jun [email protected] Zhou [email protected] Wen [email protected] Ningli [email protected] Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, People’s Republic of China2 Department of Obstetrics and Gynecology, Ren ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China2013 8 4 2013 13 182 182 27 10 2012 3 4 2013 Copyright © 2013 Shi et al.; licensee BioMed Central Ltd.2013Shi et al.; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Previously some groups demonstrated that CD44 variant 6 (CD44v6) is correlated with progression and metastasis of ovarian cancer. However, a number of other groups failed to find such an association. Moreover, epithelial ovarian cancer is known to easily metastasize to distinct sites such as the pelvic and abdominal cavities, but the potential association of CD44v6 expression with site-specific metastasis of ovarian cancer has not been explored. This study sought to evaluate the expression of CD44 standard (CD44s) and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence.
Methods
Tumor specimens were procured from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. CD44s and CD44v6 expression in the tumor tissues was evaluated by real-time RT-PCR and Western blot. Moreover, serum soluble CD44s or CD44v6 concentrations of early stage (FIGO I, G1), advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma patients were determined by enzyme-linked immunosorbent assays (ELISA). CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) and the correlation of CD44v6 expression with clinicopathologic features was analyzed. Finally, the effects of knockdown of CD44v6 in SKOV3 cells on cell adhesion, invasion and migration were assessed.
Results
The expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous cancer compared to advanced primary tumor. CD44v6 expression is also preferentially increased in the tumor at the abdominal cavity metastasis site of advanced diseases. Consistently, serum soluble CD44v6 levels of recurrent ovarian cancer were higher than those of early stage and advanced primary diseases. The IHC data demonstrate that CD44v6 expression is correlated with clinicopathologic features and tumor progression. Lastly, knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells.
Conclusions
CD44v6 expression levels are associated with epithelial ovarian cancer progression, metastasis and relapse. Moreover, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.
Ovarian cancerCD44v6Tumor progressionAbdominal cavity metastasisRecurrence
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Background
Epithelial ovarian cancer is the most lethal gynecological cancer and one of the common causes of cancer-related deaths in women worldwide
[1-3]. Epithelial ovarian cancer is characterized by frequent development of pelvic and abdominal cavity metastases in its asymptomatic stage
[2]. As a result, over 75% of ovarian cancer patients had already developed metastases when they were first diagnosed. Despite initial chemosensitivity and improved surgical procedures, abdominal recurrence remains a serious clinical issue since it often results in poor prognosis
[4]. Thus, it is critical to identify and develop specific markers and novel therapeutic strategies for advanced and recurrent ovarian cancer.
CD44 is a transmembrane glycoprotein which functions primarily as an important cell surface adhesion molecule interacting with hyaluronan
[5]. CD44 is encoded by a single gene which contains 20 exons and located on the short arm of chromosome 11 (11p13)
[6]. Alternative splicing of exons 6–15 (variant exons 1–10) gives rise to numerous variant forms of CD44 (CD44v), in which an additional segment encoded by one or more of the variant exons is inserted in the extracellular domain of CD44s, which is encoded by exons 1–5 and exons 16–20
[5,7]. CD44s and CD44v are expressed in a wide variety of cell types including epithelial and hematopoietic cells
[8,9]. In addition, the soluble form of CD44s and CD44v also exits
[10], and the soluble proteins have been shown to derive from the proteolytic cleavage of the membrane-associated CD44s and CD44v
[11].
CD44s and CD44v have been shown to mediate the cell-extracellular matrix interaction in various biological processes such as lymphocyte homing
[12], hematopoiesis
[13], embryogenesis
[14], and wound-healing
[15]. However, aberrant expression of CD44v has been implicated in the initiation, progression and recurrence of various human cancers
[16-18]. Also, it has been demonstrated that CD44s and CD44v have the potential to be used as diagnostic and/or prognostic factors for a number of cancers including bladder cancer
[19], colorectal cancer
[20], breast cancer
[21], and lung cancer
[22].
Among numerous CD44v, CD44v6 was initially shown to be able to promote the metastatic potential of a rat pancreatic adenocarcinoma cell line in animal models
[23,24]. Since these seminal studies, it has been established that CD44v6 plays role in tumor development and progression in a variety of human cancers such as breast cancer
[25] and ovarian cancer
[26]. Specifically, CD44v6 has been shown to promote ovarian cancer metastasis by mediating ovarian tumor cell attachment to the peritoneum
[27]. Moreover, numerous studies have also been carried out to assess the correlation of CD44v6 with tumor development and progression to address the diagnostic and prognostic values of CD44v6 for ovarian cancer
[28-37]. However, these studies generated conflicting data.
In the current study, we further investigate the expression of CD44s and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence.
Methods
Patient samples
Between January 2010 and December 2011, tumor tissues and peripheral blood were obtained from 45 patients (mean age: 54.17±9.43 years) with advanced (FIGO III, G3) and 20 patients (mean age: 50.23±6.24years) with recurrent ovarian serous adenocarcinoma in the Department of Obstetrics and Gynecology, Ren ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. The recurrent tumor tissues were chosen from tumor nodes of pelvic recurrent site, abdominal metastasis specimens from omentum, and pelvic metastasis samples from any pelvic apparatus peritoneum tumor nodes. In addition, peripheral blood was collected from 10 patients with early-stage (FIGO I, G1) ovarian serous adenocarcinoma as control. The exclusion criteria were inadequate follow-up data, and chemotherapy/radiotherapy before operation. The tumor tissues were snap-frozen in liquid nitrogen and blood samples were frozen at -80°C. This study was approved by ethics committee of Ren ji Hospital, School of Medicine, Shanghai Jiao Tong University, and it was in compliance with the Helsinki Declaration. All the patients gave written informed consent for participation in the study.
Quantitative real-time RT-PCR
Total RNA was extracted from tumor tissues using Trizol (Invitrogen). The quantity and quality of RNA were assessed using a NanoDrop 1000 (Thermo). cDNA was synthesized using the cDNA Reverse Transcription kit (TAKARA) according to the manufacturer’s instructions. PCR reactions were performed using the Realtime PCR system (Applied Biosystems 7500) with the following conditions: 2 min at 50°C followed by 40 cycles of 95°C, 10 min; 95°C, 15 sec; 60°C, 1 min. Each 10 μl reaction contained SYBR Premix Ex Taq 5 μl, Primer mix 0.15 μl, cDNA 1.5 μg, ddH2O 3.85 μl. Primers for CD44s and CD44v: 5′-CCTTTGATGGACCAATTACCATAAC-3′ and 5′-TCAGGATTCGTTCTGTATTCTCCT-3′; Primers for CD44v6: 5′-GGCAACAGATGGCATGAGGG-3′ and 5′-AGTGGTATGGG-ACCCCCCACTGGGG-3′; Primers for GAPDH: 5′-CACATGGCCTCCAAGGAGTAA-3′ and 5′-TGAGGGTCTCTCTCTTCCTCTTGT-3′. The experiment was repeated once with triplicate measurements in each experiment. Relative CD44s/CD44v6 mRNA levels are calculated as ratios of CD44s/CD44v6 mRNA levels to GAPDH mRNA levels.
Western blot
100 μg tumor tissues were lysed using Mem-PER Eukaryotic Membrane Protein Extraction kit (PIERCE, Cat No: 89826). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membrane, which was blocked using 5% BSA in Tris-Buffered saline with Tween 20. The membrane was incubated overnight at 4°C with primary antibodies (Anti-CD44s, Cat Log#: ab119863, Abcam; anti-CD44v6, Cat Log#: MAB4073, Millpore; 1:5000). After incubation, the membrane was rinsed and incubated for 1 hour at room temperature in appropriate anti-mouse HRP-conjugated secondary antibody (1:10,000). The membrane was rinsed, developed and specific protein bands were detected with the infrared fluorescence scanning imaging system (odyssey Li-Cor). GAPDH served as loading control. Relative CD44s/CD44v6 expression levels are calculated as ratios of CD44s/CD44v6 band density to that of GAPDH.
Enzyme-linked immunosorbent assays
Enzyme-linked immunosorbent assay kits (CD44s, Cat Log#: KA0119; CD44v6, Cat Log#: KA0120, Abnova) for quantitatively detecting soluble CD44s or CD44v6 levels in patient sera were used according to the manufacturer’s instructions. Briefly, the sera were added in duplicate to the wells of the microtiter plate coated with an antibody against CD44s or CD44v6 with horseradish peroxidase-conjugate. Then, absorbance at the wave length of 450 nm in each microwell was measured using a spectrophotometer.
Immunohistochemistry
Ovarian Cancer Tissue Chip (Cat No: BC11115) was purchased from Xi’an Alena Biotechnology Ltd. The tissue chip contains 80 samples of epithelial ovarian cancer, 10 samples of metastasis lymph nodes and 10 samples of edge tissues of normal ovary. IHC staining of the tissue chip was performed using EliVision plus IHC Kit (Maixin Biological Ltd, Fuzhou, China) following the manufacturer’s instructions. The tissue chips were roasted at 60°C for 30 min for deparaffinization and then were placed in 10 mmol/L citrate buffer (pH 6.0) under the high pressure for 2 min. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide in absolute methanol at room temperature for 10 min. Then they were incubated with primary antibodies (Cat Log#: Anti-CD44v6, 1:500) overnight at 4°C. The reaction products were visualized with diaminobenzidine (DAB Kit, Maixin Biological, Fuzhou, China), and sections were counterstained with hematoxylin, dehydrated, and evaluated under light microscope. Tris-buffered saline solution was used as negative controls. The sections were all quantified by two pathologists in a blinded manner. The average number of stained cells in 5 visual fields was regarded as the percent ratio of positively stained cells in each section. Positive range score: 0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; 4, >75%. Positive extent score: 0, no staining; 1, light yellow; 2, brown; 3, dark brown. Judged by positive range score plus positive extent score: <2, negative (–); 2–3, slight positive (+); 4–5, moderately positive (++); 6–7, strongly positive (+++).
For statistics, all the samples that expressed CD44v6 form (+) to (+++) were regards as positive.
Cell culture
SKOV3 cells, a human serous ovarian cancer cell line from ATCC, were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (HyClone),100 U/mL penicillin and streptomycin, in a 5% CO2 atmosphere at 37°C.
Small interfering RNA (siRNA) knockdown of CD44v6
SKOV3 cells were transiently transfected with siRNA obtained from Shanghai Integrated Biotech Solutions Co., Ltd. siRNA-1 against CD44v6: sense 5′-UGAGGGAUAUCGCC-AAACATT-3′ and anti-sense 5′-UGUUUGGCGAUAUCCCUCATT-3′; siRNA-2 against CD44v6: sense 5′-GCAACUCCU-AGUAGUACAAT-T-3′ and anti-sense5′-UUGUACUA-CUAGGAGUUGCTT-3′; and siRNA-NC: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense5′-ACGUGACACGUUCGGA-GAATT-3′) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Following transfection, cells were incubated at 37°C in a CO2 incubator for 24 or 48 hours before being harvested for quantitative real time RT-PCR and Western blot analyses.
Cell-extracellular matrix (ECM) adhesion assay
96-well plates were pre-coated with 50 uL Matrigel (diluted 1:8) for 4 hours at 37°C. Matrigel became solidified and simulated the major components of ECM. The cells (5×104) were seeded (in triplicate for each condition) in coated wells with 100 uL of serum-free RPMI 1640, and allowed to adhere for 60 min at 37°C and 5% CO2. Then the non-adherent cells were washed with PBS. Adherent cells were counted with the Cell Counting Kit 8 (Dojindo, Tokoy, Japan) according to the manufacturer’s protocol. The adhesion rates were then calculated.
Cells invasion and migration assays
Transwell filters (Millipore) were coated with Matrigel (diluted 1:5) on the upper surface of the polycarbonic membrane (diameter of 12 mm, pore size 8 mm). After incubation at 37°C for 2 hours, the transfected (transfected for 48 h) and control cells (4×105) suspended in 200 uL of serum-free RPMI 1640 were added to the upper chamber and 500 uL contained 20% FBS RPMI 1640 were added to the low chamber. After 16 hours of incubation at 37°C in a 5% CO2 incubator, the cells that had invaded the lower surface of the filter were fixed with 4% paraformaldehyde and stained with crystal violet. The invaded cells were counted by light microscopy and the average numbers of cells of at least five fields from each well were calculated. In a similar fashion, the cells migration were evaluated using the same transwell filters without coated with the Matrigel, and other conditions and the analysis were the same as the invasion assay. Triplicate assays were performed for each group of cells in both the invasion and migration.
Statistical analysis
Statistical analysis was performed using SPSS version 13.0. Quantitative real time RT-PCR, Western blot, adhesion assay, migration assay, and invasion assay data were analyzed using Student’s t-test and expressed as mean ± SD. The correlation between CD44v6 positive expression and the clinicopathologic parameters was assessed by Chi-square test. Differences were considered statistically significant when P values are smaller than 0.05.
Results
CD44v6 expression is up-regulated in recurrent human ovarian serous tumors compared to primary tumors
First we determined the levels of CD44 (a mixed population of CD44s and CD44v) and CD44v6 transcripts in samples from primary and recurrent tumors of ovarian serous cancer using real-time RT-PCR. Our data reveal that while there is no significant difference in CD44 mRNA levels between primary and recurrent tumors, CD44v6 mRNA levels in the recurrent disease are significantly higher than those in the primary adenocarcinoma (Figure
1A). This finding was further supported by Western blot analysis demonstrating that primary and recurrent tumors express similar levels of CD44s proteins but the recurrent disease expresses significantly higher levels of CD44v6 protein than the primary tumor (Figure
1B-C). These data indicate that the expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous tumors compared to primary tumors.
Figure 1 Expression of CD44 and CD44v6 in advanced primary and recurrent epithelial ovarian cancer tissues, and serum concentrations of soluble CD44s (sCD44s) and CD44v6 (sCD44v6) in patients with early-stage, advanced-stage and recurrent epithelial ovarian cancer. (A) Real-time RT-PCR analysis of mRNA levels of CD44 (using a pair of primers complementary to common regions of CD44s and CD44v) and CD44v6 (using a pair of primers specific to CD44v6) in 45 advanced primary tumor samples and 20 tissues from recurrent diseases. **: P<0.01. (B) Western blot analysis of CD44s and CD44v6 protein levels in the same set of advanced primary and recurrent tumor samples as in A. *: P<0.05. (C) Representative images of Western blots performed in B. (D) Serum levels of sCD44s and sCD44v6 in peripheral blood samples from patients with early-stage, advanced-stage and recurrent epithelial ovarian cancer were determined by ELISA as described in Methods. The ELISA was repeated three times. **: P<0.01.
To further investigate the association of CD44v6 with progression and recurrence of ovarian serous adenocarcinoma, we measured serum soluble CD44s and CD44v6 concentrations of early-stage, advanced and recurrent ovarian serous adenocarcinoma patients by ELISA. The results demonstrate that soluble CD44s levels among early-stage, advanced-stage and recurrent diseases had the trend of gradual decreases without significant differences among the three groups. However, serum soluble CD44v6 levels of the recurrent disease were markedly increased compared to those of early-stage and advanced diseases (Figure
1D). This finding suggests that the serum soluble CD44v6 concentration may be used as a potential marker for recurrent ovarian serous tumors.
CD44v6 expression is increased in tumor tissues from the abdominal cavity metastasis compared to those from the primary and pelvic metastasis sites
Next, we sought to examine and compare CD44s and CD44v6 expression in tumors at the primary site versus the pelvic and abdominal cavity metastasis sites of the same patients. To this end, we selected 28 out of the 45 cases of the advanced ovarian serous adenocarcinoma since we had obtained tumor specimens from the primary site (primary site), the abdominal cavity metastasis site (Met-1), and the pelvic cavity metastasis site (Met-2) for each of these 28 advanced diseases. As in the previous study (Figure
1), we carried out both real-time RT-PCR and Western blot analysis to assess the expression of CD44s and CD44v6 in tumor samples from the different tumor sites. Our data show that whereas tumors at the primary and two metastasis sites exhibit similar levels of CD44s expression both at the mRNA level (Figure
2A) and at the protein level (Figure
2B-C), tumors at the abdominal cavity metastasis site expresses significantly higher levels of CD44v6 both at the mRNA level (Figure
2A) and at the protein level (Figure
2B-C) than those at the primary site or the pelvic cavity metastasis site. Thus, these findings indicate that CD44v6 expression is preferentially increased in tumors at the abdominal cavity metastasis site of the advanced ovarian serous adenocarcinoma.
Figure 2 Expression of CD44 and CD44v6 in tumor samples from different locations in the same patients. (A) Real-time RT-PCR analysis of mRNA levels of CD44 (using a pair of primers complementary to common regions of CD44s and CD44v) and CD44v6 (using a pair of primers specific to CD44v6) in tumor tissues from the primary site, the abdominal cavity metastasis (Met-1) and the pelvic cavity metastasis (Met-2) of 28 patients with advanced ovarian cancer. **: P<0.01. (B) Western blot analysis of CD44s and CD44v6 protein levels in the same set of tumor samples as in A. *: P<0.05. (C) Representative images of Western blots performed in B.
CD44v6 expression is correlated with clinicopathologic features and tumor progression
To further investigate the association of CD44v6 expression with progression and metastasis of ovarian cancer, we examined CD44v6 expression in tumor tissues on an ovarian cancer tissue chip by IHC. This tissue chip contains 80 samples of epithelial ovarian cancer, 10 samples of metastasis lymph nodes and 10 samples of edge tissues of normal ovary. We categorized these samples according to four different clinicopathologic parameters: age (<50 years or ≥50 years), histological types (Mucinous or Serous), FIGO stage (I, II or III), Differentiation grade (G1, G2, G3) (Table
1). Representative IHC staining images of grade 3 ovarian serous adenocarcinoma, lymph node metastasis, edge tissues of normal ovary and corresponding negative controls are shown in Figure
3 to demonstrate the efficient detection of CD44v6 expression in the tumor samples on the tissue chip.
Figure 3 IHC analysis of CD44v6 expression in tumor tissues on an Ovarian Cancer Tissue Chip. Representative IHC staining images of grade 3 ovarian serous adenocarcinoma (A) and corresponding negative control (D), grade 2 lymph node metastatic serous adenocarcinoma (B) and corresponding negative control (E), and edge tissues of normal ovary (C) and corresponding negative control (F).
Table 1 Correlation of the clinicopathologic features and CD44v6 expression positive rate
CD44v6 expression
Clinicopathologic parameters n — + ++ +++ Positive rate
Age
<50y 45 20 12 9 4 55.56
≥50y 45 16 11 14 4 64.44
Pathological type
Mucinous 10 5 4 0 1 50.00
Serous 59 16 20 17 6 72.88
FIGO stage
I 34 19 9 4 2 44.12
II 12 2 2 7 1 83.33
III 20 3 11 4 2 85.00
Differentiation grade
G1 17 11 3 2 1 35.29
G2 26 3 12 7 4 88.46
G3 34 5 15 11 3 85.29
Correlation of CD44v6 expression positive rate with the clinicopathologic features.
The IHC data show that while there is no difference in the percentage of CD44v6 positive tumor cells between the two age groups and two pathological type groups, significant differences in the percentage of CD44v6 positive tumor cells exist in other clinicopathologic parameters. The positive CD44v6 expression rates of FIGO stage II and III tumors were 83.33% and 85.00%, respectively, which both are higher than that of FIGO I tumor (44.12%), (p<0.05, Table
1). Moreover, the positive CD44v6 expression rates of G2 and G3 tumors were 88.46% and 85.29%, respectively, which both are significantly higher than that of G3 tumors (35.29%), (p<0.05, Table
1). This indicates that the poorer the tumor differentiation, the higher the CD44v6 expression. These data reveal that CD44v6 expression is correlated with the clinicopathologic features and tumor progression.
Knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells
The data obtained from the analysis of the clinical samples above suggest that CD44v6 may play a role in mediating tumor metastasis. To explore this possibility, we extended our study to knock down CD44v6 expression in SKOV3 cells, a human serous ovarian cancer cell line, and assess the impact of knockdown of CD44v6 on the adhesion, invasion and migration capacities of SKOV3 cells. Transient transfection of SKOV3 cells with two distinct siRNAs (siRNA-1 and siRNA-2) led to a significant reduction in CD44v6 mRNA levels (Figure
4A) and protein levels (Figure
4B and
4C). Then, we used parental SKOV3 cells and SKOV3 cells transfected with control siRNA (siRNA-NC), siRNA-1 or siRNA-2 to perform cell adhesion, migration and invasion assays as described in Methods. The data indicate that SKOV3 cells with CD44v6 knockdown showed a decreased cell adhesion (Figure
5A) and migration (Figure
5C) compared with the cells transfected with siRNA-NC control groups. However, we found that knockdown of CD44v6 in SKOV3 cells did not affect the invasion capacity of this human serous ovarian cancer cell line (Figure
5B). Taken together, these findings suggest that CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.
Figure 4 Efficient knockdown of CD44v6 in SKOV3 cells by transfected siRNA against CD44v6. (A) Quantitative real-time RT-PCR analysis of mRNA levels of CD44v6 expression in parental SKOV3 cells and SKOV3 cells transiently transfected with control RNA, siRNA-NC, siRNA-1 and siRNA-2. **: P<0.01. (B) Western blot analysis of CD44v6 protein levels in the same set of SKOV3 cell as in A. *: P<0.05. (C) Representative images of Western blots performed in B.
Figure 5 Assessment of the adhesion, invasion and migration capacities of SKOV3 cells with CD44v6 knockdown. (A) Cell adhesion assays with parental SKOV3 cells and SKOV3 cells transiently transfected with siRNA-NC, siRNA-1 and siRNA-2. (B) Cell invasion assays with parental SKOV3 cells (a) and SKOV3 cells transiently transfected with siRNA-NC (b), siRNA-1 (c) and siRNA-2 (d). (C) Cell migration assays with parental SKOV3 cells (a) and SKOV3 cells transiently transfected with siRNA-NC (b), siRNA-1 (c) and siRNA-2 (d).
Discussion
Recently, numerous studies have focused on investigating the expression of CD44v6 in malignancy to address the association of CD44v6 with tumor progression, metastasis and recurrence. So far, CD44v6 has been shown to be a useful prognostic factor for a variety of cancers including those of the stomach
[38], head and neck
[39], prostate
[40], and lung
[22]. As expected, a number of groups have also investigated the expression of CD44v6 in ovarian cancer to examine the potential correlation of this CD44 variant with ovarian cancer development and progression. However, the data from these studies are not consistent. While several studies demonstrated that soluble CD44v6 is not associated with the development and metastasis of ovarian cancer
[28,30,33], other investigations showed that CD44v6 is correlated with tumor progression and metastasis
[32,35-37]. These studies are further in conflict with one study showing that CD44v6, together with CD44s and CD44v3, are inversely associated with the ovarian carcinoma FIGO stage
[31]. Furthermore, several questions regarding the correlation of CD44v6 with ovarian cancer metastasis remain to be answered. For example, epithelial ovarian cancer is known to metastasize to distinct sites such as the pelvic and abdominal cavities; it is still not clear whether CD44v6 expression is associated with site-specific metastasis of ovarian cancer.
In light of the controversy and unanswered questions, we carried out independent studies to further address the association of CD44v6 with development and progression of ovarian cancer. Most previous studies assessed CD44v6 expression using one or two methods
[28,30,32,33,35-37]. To obtain accurate and reliable data, we assessed CD44v6 expression in large numbers of tumor specimens collectively by three methods: quantitative real-time RT-PCR, Western blot analysis and IHC analysis. Our first study aimed to examine CD44v6 expression in tumor tissues from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. Our data indicate that CD44v6 expression is associated with ovarian serous cancer recurrence (Figure
1), which is consistent with the finding of a previous study in which CD44v6 expression was assessed by IHC
[29]. We then extended our study to investigate CD44v6 expression in tumor samples from the primary site and two different metastasis sites. Interestingly, we found that CD44v6 expression is correlated with the abdominal cavity metastasis of epithelial ovarian cancer (Figure
2). Furthermore, CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) (Figure
3). The IHC data demonstrated that CD44v6 expression is correlated with clinicopathologic features and tumor progression (Table
1).
More importantly, we found that serum soluble CD44v6 levels are significantly higher in patients with recurrent diseases than in those with advanced primary diseases (Figure
1D). Soluble CD44 and CD44v are derived from the proteolytic cleavage of the membrane-associated CD44 and CD44v
[11]. Soluble CD44 and CD44v can be detected in human sera and other body fluids and thus they have the potential to be used as diagnostic factor for tumor burden and metastasis
[41]. Elevated serum concentrations of soluble CD44s and CD44v6 have been shown to correlate with tumor progression and metastasis of gastric carcinoma, colon carcinoma or breast cancer
[41-43]. Our current study has revealed that soluble CD44v6 levels are correlated with ovarian cancer recurrence, indicating that serum soluble CD44v6 also has the potential to be used as a diagnostic marker for monitoring ovarian tumor recurrence.
Liottta et al initially proposed the theory of tumor metastasis process, which consists of three steps: adhesion, degradation and metastasis
[44]. These investigators demonstrated that the adhesion was the first and the most important step of tumor metastasis. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. Extracellular matrix molecules including versican and hyaluronan which interacts with CD44 have been shown to play key roles in ovarian cancer metastasis
[26]. Our results indicate that CD44v6 expression is only up-regulated in tumor tissues from the recurrent disease and abdominal cavity metastasis site, suggesting that CD44v6 may be an adhesion molecule during the process of ovarian cancer cell adhesion and metastasis. Moreover, SKOV3 cell adhesion and migration were decreased after knocking down CD44v6 expression by siRNA, indicating that CD44v6 may play a role in mediating tumor cell adhesion and migration during the metastasis process. Therefore, we propose that CD44v6 may also have the potential to serve as an effective therapeutic target for preventing and treating the recurrence and abdominal cavity metastasis of ovarian cancer.
Conclusions
In conclusions, our current work demonstrates that CD44v6 expression levels are associated with progression, metastasis and relapse of epithelial ovarian cancer. Furthermore, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration. Significantly, these studies have not only provided more convincing evidence supporting the correlation of CD44v6 expression levels with ovarian cancer progression, metastasis and relapse but have also laid a foundation for future investigations to further explore the potential of CD44v6 as a diagnostic marker for monitoring ovarian tumor recurrence and as an effective therapeutic target for preventing and treating the recurrence and abdominal cavity metastasis of ovarian cancer.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JS developed the idea, performed the experiments, analyzed the data, and prepared the manuscript. Zhou Zhou provided technical assistance. WD and NL both initially conceived the idea, and participated in the experimental design and manuscript preparation. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2407/13/182/prepub
Acknowledgements
The work is supported by the Foundation of the Shanghai Committee of Science and Technology, China (Grant No. 10dz2212100).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23638106PONE-D-13-0404510.1371/journal.pone.0062529Research ArticleBiologyBiochemistryProteinsProtein SynthesisRecombinant ProteinsBiophysicsProtein FoldingBiotechnologyApplied MicrobiologyGeneticsGene ExpressionProtein TranslationMicrobiologyApplied MicrobiologyPhysicsBiophysicsProtein FoldingProkaryotic Ubiquitin-Like ThiS Fusion Enhances the Heterologous Protein Overexpression and Aggregation in Escherichia coli
ThiS Fusion for Heterologous Expression in E. coliYuan Sujuan Xu Jian Ge Ying Yan Zheng Du Guohua Wang Nan
*
Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Materia Medica, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Beijing, People’s Republic of China
Uversky Vladimir N. Editor
University of South Florida College of Medicine, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: NW. Performed the experiments: SY JX YG NW. Analyzed the data: SY JX YG NW. Contributed reagents/materials/analysis tools: ZY GD. Wrote the paper: NW.
2013 25 4 2013 8 4 e6252924 1 2013 21 3 2013 © 2013 Yuan et al2013Yuan et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Fusion tags are commonly employed to enhance target protein expression, improve their folding and solubility, and reduce protein degradation in expression of recombinant proteins. Ubiquitin (Ub) and SUMO are highly conserved small proteins in eukaryotes, and frequently used as fusion tags in prokaryotic expression. ThiS, a smaller sulfur-carrier protein involved in thiamin synthesis, is conserved among most prokaryotic species. The structural similarity between ThiS and Ub provoked us into expecting that the former could be used as a fusion tag. Hence, ThiS was fused to insulin A and B chains, murine Ribonuclease Inhibitor (mRI) and EGFP, respectively. When induced in Escherichia coli, ThiS-fused insulin A and B chains were overexpressed in inclusion bodies, and to higher levels in comparison to the same proteins fused with Ub. On the contrast, ThiS fusion of mRI, an unstable protein, resulted in enhanced degradation that was not alleviated in protease-deficient strains. While the degradation of Ub- and SUMO-fused mRI was less and seemed protease-dependent. Enhanced degradation of mRI did not occur for the fusions with half-molecules of ThiS. When ThiS-tag was fused to the C-terminus of EGFP, higher expression, predominantly in inclusion bodies, was observed again. It was further found that ThiS fusion of EGFP significantly retarded its refolding process. These results indicated that prokaryotic ThiS is able to promote the expression of target proteins in E. coli, but enhanced degradation may occur in case of unstable targets. Unlike eukaryotic Ub-based tags usually increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins.
This work was supported by grants from China National Science & Technology Major Project “New Drug Innovation and Production Program” (General platform construction, No: 2012ZX09301002-001-002 and 2012ZX09301002-002-006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Recombinant production of bioactive proteins plays a major role in developing biopharmaceutical agents. High-level expression of recombinant proteins, especially those from eukaryotes, is often difficult to achieve in Escherichia coli. Poor expression of proteins can be attributed to many factors, such as inefficient transcription or translation or rapid breakdown of the mRNA or protein by the host. Fusion protein technology is often used to enhance protein expression and solubility, chaperone proper folding, reduce protein degradation, and facilitate purification.
Fusion tags of prokaryotic origin, including widely used maltose-binding protein (MBP) [1], [2], NusA [3] and thioredoxin (TRX) [4], usually provide the high-level expression of recombinant proteins. But the high molecular weights of these tags reduce the productivity of target proteins.
Ubiquitin (Ub) and related polypeptides (Ubl) are highly conserved small single-domain proteins found in all eukaryotic cells. Through covalent attachment to other proteins, they regulate numerous important cellular processes such as apoptosis, transcription and the progression of the cell cycle. The proteins modified by ubiquitination might have different fates depending both on the specific Ubl used, and on the type of modification they undergo [5]. It is well known that Ub modification directs proteins to the proteasome for degradation, while sumoylation prevents some proteins from proteasomal degradation [6]. They function as a unique protein modification system which does not exist in prokaryotes except for Mycobacterium tuberculosis [7]. Eukaryotic Ub and SUMO are among the favorable fusion tags frequently used for prokaryotic expression. They can be easily cleaved off by deubiquitinases, leaving a native N-terminus in target protein. They enhance the fused expression, increase the solubility and stability, and protect the peptides from proteolytic degradation in prokaryotes [8], [9], regardless of their contradicting effects on protein degradation in eukaryotes.
Prokaryotic ThiS, a 66 amino acid small sulfur carrier involved in the thiamin biosynthesis, displays a high degree of structural similarity although sharing limited sequence homology to Ub [10], [11]. It interacts with correlating enzymes in a similar way as Ub [11] and is suggested as prokaryotic antecedent of Ub [12].
In this work, we observed the effect of fusion of ThiS to heterologous proteins on their expression, and on the solubility, stability and foldability of target proteins in E. coli. ThiS showed different characteristics from eukaryotic Ubl in these aspects.
Results
1. ThiS Enhanced the Expression of Insulin A and B Chains
In the initial attempt [13] to express recombinant human insulin in E. coli, insulin chain A and B had to be fused to an E. coli β-galactosidase to provide the stable chain products separately. When the gene encoding insulin chain A was fused downstream to the gene of ThiS or Ub and cloned into prokaryotic expression vector pET28a, the fused insulin chain A protein (with His-tag fused further at the upstream) was successfully expressed in E. coli BL21 (DE3) pLysS, predominantly in inclusion bodies, by IPTG induction (Fig. 1A, left panel). The yield of ThiS fusion product (38.90 mg/L bacterial culture, averaged from 2 batches) was higher than Ub fusion (11.45 mg/L, averaged from 2 batches) in large scale expression. Anti-His-tag immunoblot (Fig. 1A, right panel) of the proteins revealed the overexpressed bands as the target proteins. Trace amounts of soluble products were observed in Western blot, both for Ub fusion and ThiS fusion at similar level. Molecular weights of the expressed fusion proteins were as expected and confirmed by MALDI-TOF MS (Fig. S1).
10.1371/journal.pone.0062529.g001Figure 1 Expression of insulin chains with ThiS or Ubiquitin fusion?
Insulin A chain (A) or B chain (B) fused with ubiquitin (Ub) or ThiS, were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cells were electrophoresed on 15% SDS-PAGE, shown in left panels. Marker proteins are shown in lane M with sizes at left. Expressed proteins were verified by Western blot probed with anti His-tag antibody, shown in right panels. Arrows highlight expressed proteins at expected positions.
Likewise, when insulin chain B was fused with ThiS or Ub, the fused proteins were also expressed predominantly in inclusion bodies, by IPTG induction (Fig. 1B). The yield of ThiS fusion product (33.15 mg/L, mean of 2 batches) was also higher than Ub fusion (20.45 mg/L, mean of 2 batches) in large scale production. The identities of the overexpressed proteins were confirmed by Anti-His-tag immunoblot (Fig. 1B, right panel) and MALDI-TOF MS (Fig. S1). Trace amounts of soluble products were observed in Western blot, at a higher level for Ub fusion than ThiS fusion.
Ub and ThiS, although sharing same secondary structure of β-grasp domain, showed differential efficiency on enhancing the protein expression. This difference may not be attributed to the coden bias due to the prokaryotic origin of ThiS, since the coding gene of Ub used for fusion was synthesized according to the coden bias of E. coli.
2. Half-molecule of ThiS Fusion Enhanced the Expression
Half-protein molecules of Ub were used as fusion tags [14]. The splitted N- and C-terminal half-proteins are incapable of fast folding to a compact stable structure of the whole molecule of Ub. Fig. 2 showed the effect of fusion by the C-terminal and N-terminal half-ThiS to insulin A and B chains. Insulin A fusion to the N-terminal half of Ub gave a protein yield of 31.58±3.52 mg/L (three batches), and the N-terminal and C-terminal half-ThiS fusions gave yields of 20.62±3.09 and 13.61±6.48 mg/L, respectively. The overexpressed proteins were confirmed by Anti-His-tag immunoblot (Fig. 2A, right panel) and MALDI-TOF MS (Fig. S1).
10.1371/journal.pone.0062529.g002Figure 2 Expression of Insulin chains fused with half-molecules of ThiS or Ubiquitin.
Insulin A chain (A) or B chain (B) fused with the N-terminal half (ThN-) or C terminal half (ThC-) of ThiS or the N-terminal half of ubiquitin (UbN-), were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cell were resolved on 15% SDS-PAGE, shown in left panels. M indicates Marker proteins. Western blot probed with anti His-tag antibody were shown in right panels. Arrowheads highlight observed positions of expressed proteins.
The N-terminal and C-terminal half-ThiS fusions to insulin B chain had similar results to that of insulin A chain (yields of 22.62±1.92 and 25.24±3.42 mg/L, respectively, Fig. 2B), while the N-terminal half-Ub fusion gave a lower yield. MALDI-TOF MS (Fig. S1) indicated that the N-terminal and C-terminal half-ThiS fusions of insulin B were expressed at molecular weights as expected. While the N-terminal half-Ub fusion product had a major peak about 1 kD smaller than expected. This may suggest a partial degradation of the target protein which was responsible for the lower expression level of the half-Ub fusion.
3. ThiS Fusion Expression of Murine Ribonuclease Inhibitor
We suspected if ThiS fusion enhanced the target expression by improving its stability in vivo. Since murine Ribonuclease Inhibitor (mRI) was shown as an unstable protein when expressed in E. coli
[15], we tried to observe the effect of fusion of ThiS on the stability of mRI, and compared with that of Ub and SUMO. When mRI was coded as Ub and SUMO fusions in expression vector pVI (E. coli trc promoter driven, with hexa-His-tag at the N-terminus), they were expressed predominantly as full length products (Fig. 3A, upper panel, and confirmed by MS) in inclusion bodies, with degradation as fast migrating smaller fragments in Western blot (Fig. 3A, lower panel). Like the His-tag fusion shown previously [15], degradation of Ub and especially SUMO fusions was alleviated somewhat in the Lon protease deficient E. coli strain BL21 (DE3) pLysS (Fig. 3B), compared with that in native strain TG1.
10.1371/journal.pone.0062529.g003Figure 3 Expression of mRI with different tag fusion.
mRI with ubiquitin (Ub) or SUMO fusion were expressed in (A) E. coli TG1 or (B) E. coli BL21 (DE3) pLysS. (C) ThiS fusion of mRI was expressed in E. coli TG1, and protease-deficient strains BL21 (DE3) pLysS, KY2966 and JW3903. (D) mRI with the N-terminal half (ThN-mRI) or C-terminal half (ThC-mRI) of ThiS fusion were expressed in E. coli BL21 (DE3) pLysS. Total cell lysate from uninduced (−) or induced (+) cells with IPTG, and the soluble (S) or insoluble fraction (I) of induced cells were resolved on 10% SDS-PAGE, shown in each upper panel. Western blot probed by anti His-tag antibody was shown in each lower panel. Expressed products migrating at the expected molecular weight are indicated by arrows.
Quite unexpectedly, when ThiS-fused mRI was induced in E. coli TG1 strain, the expressed fusion product was indiscernible at range of 50 to 66 kD in SDS-PAGE (Fig. 3C). An overexpressed band was noticed at around 25 kD in the inclusion bodies. Except the intense bands of smaller fragments as degradated products, only trace amount of product at the expected molecular weight was shown in Western blot (Fig. 3C, lower panel). Since Lon was involved in mRI degradation for His, Ub and SUMO fusions, we explored the role of Lon, as well as HslV, another ATP-dependent protease in E. coli, in the breakdown of ThiS fusion of mRI. In all the protease-deficient hosts (BL21 for Lon deficiency, JW3903 [16] and KY2966 [17] for HslV deficiency), degradation was not blocked or alleviated, as observed on immunoblot (Fig. 3C).
Questions may be raised respecting the specificity of immunoblots, hence the possibility arises that immuno-reactive bands came from non-specific proteins rather than the degradated target protein. It seems unlikely since all the blots over this study showed clear background for cells without chemical induction, except that a small amount of leaky expression was exclusively observed for some target fusions. The overexpressed band of ThiS-mRI at around 25 kD was subjected to in-gel trysinization and MS analysis. It was identified as an N-terminal fragment of ThiS-mRI (Fig. S2), thus verified as the degradated target protein instead of non-specific proteins.
Ubl from both eukaryotes and prokaryotes share similar tertiary structure with different primary structure. It was possible that a specific sequence or motif in ThiS, which is not present in other Ubl, was responsible for ThiS-directed breakdown of fusion target. We further explored which part of ThiS protein was involved in the target degradation. The result in Fig. 3D indicated that both the N-terminal and C-terminal half-proteins conferred much less degradation than full length ThiS. It suggested that the whole structure of ThiS rather than a single fragment was responsible for the protein degradation.
4. ThiS Fusion Enhanced the Expression of EGFP
We further explored the effect of fusion on Green Fluorescent Protein (GFP) expression. GFP is a highly stable protein that can be easily expressed in E. coli. We fused the gene encoding EGFP in frame but at upstream to the gene of ThiS and cloned into prokaryotic expression vector pQE30 (with His-tag fused further at the upstream of EGFP). This EGFP in fusion with ThiS at the C-terminus, was expressed in E. coli TG1 in inclusion bodies at 37°C (Fig. 4A), the same as EGFP protein alone without fusion. SDS-PAGE (Fig. 4B, upper panel) of cell lysates indicated that the ThiS fusion product was expressed more abundantly and induced at earlier time than EGFP alone. Anti-His-tag immunoblot (Fig. 4B, lower panel) of the proteins revealed the overexpressed bands as the target proteins. Series of fast migrating smaller fragments were seen for both proteins in immunoblot but not in gel staining, that indicated a mild degradation of expressed products, which was more prominent for ThiS fusion than EGFP alone.
10.1371/journal.pone.0062529.g004Figure 4 Enhanced expression of EGFP fused with ThiS.
(A) The recombinant EGFP proteins without (EGFP) or with ThiS-tag (ThiS-EGFP) fused at C-terminus, were induced by 1 mM IPTG for 4 h at 37°C. Total cell lysate (T) and the soluble (S) or insoluble (I) fraction were resolved on 12% SDS-PAGE. Expressed proteins are highlighted by arrows. (B) Expression in total cell lysate from cells at different time after IPTG induction at 37°C were analyzed on 12% SDS-PAGE (upper panel) and immunoblot (lower panel). (C) Cell growth (open circle for EGFP, solid circle for ThiS-EGFP) was recorded by measuring absorbance at 600 nm; the fluorescence of expressed products (open triangle for EGFP, solid triangle for ThiS-EGFP) was measured (excitation 488 nm; emission 509 nm), on different time point after induction by IPTG, for 4 hours at 37°C (the upper panel) or for 20 hours at room temperature (25°C, the lower panel). Each point represents mean and SD of 3 independent experiments. *P<0.05; **P<0.01.
Since both the cells expressing EGFP with and without ThiS fusion were fluorescent, suggesting that even the folded active proteins aggregated in inclusion bodies [18], we wondered if the enhanced expression of ThiS fusion was correlated with its improved foldability of EGFP in vivo. The fluorescence of cells expressing EGFP with or without ThiS fusion was measured after IPTG induction. Fig. 4C showed that the intensity of fluorescence increased steadily at 37°C. ThiS fusion bearing cells had lower fluorescence than cells bearing EGFP alone, although not statistically significant due to big variations. It suggested that ThiS-fused EGFP was accumulated as less active protein in inclusion bodies, although with larger amount than EGFP without fusion. Indeed, the intensity of fluorescence reached to a higher and similar level, for cells expressing EGFP proteins with or without ThiS fusion at room temperature, as the inclusion body formation is usually disfavored at lower temperature. Fig. 4C also indicated that both cells grown at the same rate, as measured by OD600.
EGFP proteins with and without ThiS fusion induced at room temperature were further investigated. Native fluorescent EGFP proteins purified from supernatant, when not heat denatured before SDS-PAGE, migrated faster than the heat-denatured samples. The later migrated at the same rate as proteins collected from inclusion bodies with or without heat denaturation (Fig. 5, left panel). Only purified native proteins without heat denaturation retained fluorescence in the gel before staining (Fig. 5, right panel). This also indicated that aggregated EGFP with or without fusion was in non-native forms in inclusion bodies without proper folding, possibly as the folding intermediates, although with fluorescent in vivo.
10.1371/journal.pone.0062529.g005Figure 5 Folded EGFP proteins with or without ThiS fusion retained their native states during SDS-PAGE.
Purified EGFP and ThiS-EGFP from supernatants (expressed at room temperature), as well as the proteins solubilized from inclusion bodies (I), were resolved on 12% SDS-PAGE, with (+) or without (−) boiling the samples. The gel was photographed under UV illumination (right panel), then stained by Coomassie blue (left panel). Purified native proteins migrated at faster rate and retained fluorescence after SDS-PAGE, if not heat denatured.
The EGFP proteins were noticeably expressed in soluble portion at room temperature (Fig. 6A). Similar amount of native EGFP was expressed for both proteins, which remained fluorescent in the gel before staining (Fig. 6B). At the same time, a large amount of ThiS-fused EGFP was expressed as denatured form (identity confirmed by Western blot, Fig. 6C); while expressed EGFP without fusion was largely soluble, with less amount of denatured form (Fig. 6D). This would suggest either that ThiS fusion slowed down the EGFP folding in vivo to enhance the aggregation of denatured proteins, or that folding capacity of the cells was overridden by the quickly expressed ThiS fusions which was then aggregated to inclusion bodies. Western blot indicated a mild degradation of ThiS fusion but not of EGFP alone (Fig. 6C).
10.1371/journal.pone.0062529.g006Figure 6 Soluble expression and in vitro refolding of EGFP proteins with or without ThiS fusion.
(A) Three independent clones of E. coli TG1 bearing plasmids expressing EGFP or EGFP fused with ThiS, were induced with IPTG at room temperature for 20 h. Unboiled total cell lysates were resolved on 12% SDS-PAGE. Soluble native form and insoluble denatured form were separated, as indicated by arrows. (B) The native form was verified by UV illumination indicating retained fluorescence of corresponding bands. (C) The Western blot with His-tag antibody further confirmed the identities of overexpressed products. (D) The ratio of native form to unfolded form of ThiS-EGFP was compared to that of EGFP. ** P<0.01. (E) The refolding kinetics of both proteins was compared in vitro. Left panel represents a typical result of the short term refolding curves, with fluorescence (normalized to the respective final fluorescence recovered) plotted against time. In the right panel, kinetics of an initial fast refolding phase, the following slow refolding phase, and the percentage of refolding at final stage (15 h) were compared between two proteins from 3 independent experiments.
Purified native fluorescent EGFP with or without fusion to ThiS was denatured and renatured in vitro. Upon dilution, both proteins refolded gradually with an increase in fluorescence, and the fluorescence recovery did not increase after 15 h. In comparison with EGFP alone, ThiS-fused EGFP had a higher final recovery of fluorescence (Fig. 6E). This was the same case as in vivo fluorescent measurements at longer expression time (Fig. 4C, lower panel). But ThiS-fused EGFP refolded at a significantly slower rate in both fast and slow refolding phases. It conformed to the prediction that enhanced in vivo aggregation resulted from slower EGFP folding for ThiS fusion.
Discussion
Many foreign proteins expressed in bacteria fail to accumulate owing to their improper folding. They are considered as abnormal products by cells and subjected to proteolytic degradation [19]. On the other hand, the misfolded proteins or folding intermediates during overexpression are deposited as insoluble aggregated form in inclusion bodies. Inclusion bodies afford protection from proteolytic degradation and favor the production in a larger quantity and rapid isolation from the cells. But they impose the disadvantages of solubilization and tedious refolding process.
Prokaryotic Ubl protein ThiS increased heterologous protein expression in E. coli. At mRNA level, it was suggested that the mRNA folding near the ribosomal binding site is more responsible for the variation in protein expression levels [20]. In our experiments, all the fused expression vectors had the same sequence near ribosomal binding site as their respective control vectors. For the stability of mRNA in bacteria, the susceptivity to degradation is more correlated with the sequence at 5′ terminus of the mRNA [21]. But when the fused sequence was placed at downstream of the target gene of EGFP, the obviously increased expression was still noticed. Thus enhanced expression by ThiS fusion probably is not attributed to an facilitated transcription or higher stability of mRNA. It may not either be attributed to an efficient translation due to its favorite coden bias because of its bacterial origin, when compared to the coden bias optimized Ub as fusion tag. Enhanced expression of insulin chains was less for half-molecules of ThiS fusion than the whole molecule fusion. That conformed to an enhancing mechanism at protein level, rather than at mRNA level.
At protein level, fusion tags usually act as solubility enhancers and chaperones or are designed to promote proper folding and to enhance the solubility of the protein of interest [3], [22], [23], ThiS-tag showed an opposite effect to its eukaryotic counterparts. ThiS did not improve the solubility of insulin A or B chain when fused at their N-termini, and that of EGFP when fused at its C-terminus. EGFP refolded more slowly in vitro when fused with ThiS, and was expressed in relatively less amount of native fluorescent EGFP than the accumulation of non-native EGFP in vivo, in comparison to EGFP alone.
Decreased foldability of ThiS fusion may account for the slightly higher degradability of the stable protein EGFP. Although not having the Ub-proteasome pathway in eukaryotes, E. coli has evolved an elaborate proteolytic machinery to destroy misfolded proteins rapidly [24], [25]. Misfolded target proteins are subjected to rapid proteolytic degradation before aggregated to inclusion bodies. The enhanced degradation of unstable mRI, was also probably attributed to the misfolding induced by fusion with full length ThiS. Since neither the N-terminal nor C-terminal half-ThiS fusion conferred the enhanced degradation. Aggregation of misfolded ThiS fusion to inclusion bodies competed with the proteolysis. Promoted expression was achieved by accumulation of misfolded aggregates that were protected from further proteolysis due to inclusion body formation.
This passive destruction model for the misfolded ThiS fusions could not fully explain the enhanced degradation, especially of the unstable protein mRI. Ub and SUMO fusion products were also misfolded and aggregated to inclusion bodies, but the degradation was not greatly enhanced. ThiS fusion of mRI showed a different protease sensitivity from Ub and SUMO fusions. Half-ThiS fusions were also misfolded and present in the form of inclusion bodies, while they were spared from enhanced degradation. An active degradation mechanism by ThiS fusion remains as a possible explanation.
Ub is required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic Ub-like protein (Pup) in Mycobacterium tuberculosis is the only functional analogue to Ub found in prokaryotes [7]. ThiS protein itself was able to be overexpressed in E. coli and purified as reported [26], which was confirmed in our Lab. ThiS is an extraordinarily conserved small protein across all kind of bacteria and an ancestor of Ub. Does it play a physiological role in delivering misfolded or damaged polypeptides to the prokaryotic proteases for destruction, to ensure the quality of intracellular proteins in bacteria? It seems far fetching to discuss this issue now without further in-depth experiment.
It should be noted that ThiS fusion significantly improved the final refolding yield of EGFP in spite of a retardation for the refolding in vitro. Although soluble expression of active proteins is preferred in prokaryotic system as it avoids the tedious renaturation process, it is usually unachievable for most of the heterologous proteins, in reasonable amount even in fusion with well-developed fusion tags. Expression in inclusion bodies is a practically alternative option, since recombinants could be produced in larger quantities and isolated rapidly from bacteria. That’s a reason why much effort has been devoted to the regeneration studies and various techniques are developed to improve the refolding process. Combining with its small size and enhanced fusion overexpression, fusion with ThiS could find a practical application in production of some heterologous proteins.
In conclusion, as one of the smallest Ubl, prokaryotic ThiS can be fused in either upstream or downstream to enhance the expression of some target proteins in E. coli. Unlike the eukaryotic Ub-based tags which are used to increase the solubility and folding of proteins, ThiS fusion enhances the expression by augmenting the formation of inclusion bodies, probably through retardation of the folding of target proteins. ThiS fusion induces enhanced degradation of certain targets, especially of unstable proteins.
Materials and Methods
Materials
Oligonucleotides were synthesized from Invitrogen (Shanghai, China). All restriction enzymes and T4 DNA ligase were from TaKaRa (Dalian, China). M-MLV reverse transcriptase, Pfu DNA Polymerase and LA Taq DNA Polymerase were from Vigorous Biotechnology (Beijing, China). Ni-IDA agarose affinity resin was from Vigorous Biotechnology.
E. coli Strains
E. coli TG1 cells were used for cloning, maintenance and propagation of plasmids and also for expression. Protease Lon deficient BL21 (DE3) pLysS cells were used as host for expression studies. Protease HslV deficient E. coli strains JW3903 [16] and KY2966 [17] were from National BioResource Project (NIG, Japan), and used for expression studies. E. coli cells were cultivated in Luria broth under appropriate selective conditions.
Construction of Expression Vectors
Standard molecular biology techniques were used for cloning [27]. Total RNA was extracted from cells and subjected to reverse transcription and PCR amplification. All clones were verified by sequencing (Invitrogen, Shanghai, China). All primers used can be found in Table S1.
ThiS gene was amplified from genomic DNA of E. coli strain TG1. Human ubiquitin cDNA, cDNA of human insulin chain A and B were synthetic genes with coden bias for E. coli (gifts from Vigilance Biotechnology, Beijing, China). Human Sumo1 cDNA was amplified from reversed transcripts from breast cancer MCF-7 cell line (Cell Resource Center, Peking Union Medical College, Beijing, China). A cDNA of mRNH coding mRI (with 456 amino acid residues) [15] was also used for gene fusions. EGFP coding gene was from vector pEGFP-C1 (Clontech, Palo Alto, CA, USA). Gene fusions were made by restricted fragment ligation.
The expression constructs were based on the backbone of pQE30 (Qiagen, Hilden, Germany) with hexa-His at 5′ fusion, pVI (E. coli trc promoter based expression vector, Vigilance Biotechnology, Beijing, China) with sept-His at 5′ fusion, or pET28a (Novagen, Madison, WI, USA) with hexa-His at 5′ fusion. All the expression plasmids and their expected products were listed in Table S2.
Expression and Purification of Recombinant Proteins
Overnight cultures of E. coli were subcultured at 1∶100 into Luria broth containing ampicillin or kanamycin and grown to a mid-exponential phase, usually at 37°C (or at 25°C as indicated). Protein expression was induced by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, with a further 4 h growth (or the time as indicated). Five to six colonies of bacteria for each protein were screened for their expression level and the highest one was used for further experiments. The harvested cells were subjected to freezing and thawing and then lysed by sonication. The soluble protein fraction was separated from insoluble one by centrifugation at 4°C (10 min at 14,000 g).
Soluble fraction of His-tagged recombinant proteins were purified by nickel-affinity chromatography under native conditions based on the supplier’s instructions.
Electrophoresis and Western Blot
The insoluble fraction and the total cells were solubilized in PBS containing 8 M urea. The samples of total cells or the protein fractions were mixed with Laemmli buffer, heated by boiling for 5 min (or not heated, as indicated) and analyzed by reducing SDS-PAGE, as described by Laemmli [28], using a 5% stacking gel and a 10% to 15% separating gel run in a Mini-Protean II electrophoresis system (BioRad, Hercules, CA, USA). The gels were stained with Coomassie blue, or electroblotted onto nitrocellulose or PVDF membranes. For fluorescent EGFP detection, the gels were photographed under ultraviolet illumination before staining. His-tagged fusions were detected by immunoblot using anti-His antibody and goat anti-mouse HRP labelled antibody (CoWin Biotech, Beijing, China). Chemiluminescence was detected using the reagents according to supplier’s protocol (CoWin Biotech, Beijing, China).
Protein Expression Quantification
The expressed samples were subjected to SDS-PAGE. The target bands were determined by densitometric analysis using QuantiScan Software (Biosoft, Cambridge, UK), with predefined amount of Marker proteins as standards. Recombinant productivity was estimated from large scale expression (300–1000 ml culture in shaking flasks). The results from batches of independent production of the same protein were averaged for the estimation, presented as mean±SD for 3 batches, and only mean for 2 batches.
Fluorescence Determination of EGFP
For bacteria expressing EGFP proteins, cultured media containing live whole cells was aliquoted and the fluorescence was measured immediately using EnSpire Multimode Reader (Perkin-Elmer, Waltham, MA, USA), with excitation wavelength at 488 nm and emission wavelength at 509 nm. The bacteria concentration of same sample was also measured by absorbance at OD600. The fluorescence of purified soluble EGFPs was measured the same way.
Denaturation and Refolding of EGFP
Purified ThiS-EGFP and EGFP were denatured in PBS containing 8 M urea and 5 mM DTT for 5 min at 100°C. Urea-denatured samples were renatured at room temperature by 10-fold dilution into PBS with 5 mM DTT. Fluorescence recovery was monitored with an interval of 5 s for 50 min. Data were fitted with Sigma Plot (Systat Software, San Jose, CA, USA) and kinetics for fast and slow refolding phases obtained as described [29]. Final refolding was measured at 15 h. The percentage of refolding was calculated on the basis of the final constant amount of fluorescence, corresponding to the amount of fluorescence before denaturation.
Mass Spectrometry
Protein samples were diluted in water and mixed with 30 mg/mL solution (70% acetonitrile and 30% methanol, with 0.1% TFA) of α-cyano-4-hydroxycinnamic acid (CHCA) or ferulic acid (FA), at a ratio of 1∶1(v/v) and spotted onto the sample plate and air-dried. The MALDI-TOF mass spectra of the samples were acquired using a MALDI-TOF/TOF Analyzer 4800 Plus (Applied Biosystem, Foster City, CA, USA) in reflector or linear mode.
Statistical Analysis
The results were derived from three independent experiments. The Student’s t-test for two samples was used to calculate the p values. The statistical analyses were performed using SPSS 13.0 (IBM SPSS, Armonk, NY, USA), and p values smaller than 0.05 were considered statistically significant.
Supporting Information
Figure S1
Mass spectra of expression products.
(TIF)
Click here for additional data file.
Figure S2
Identification of a degradated fragment of ThiS-mRI by Mass Spectrometry.
(DOC)
Click here for additional data file.
Table S1
Primers used in this study.
(DOC)
Click here for additional data file.
Table S2
Strains and plasmids used in this study.
(DOC)
Click here for additional data file.
We thank the National BioResource Project [National Institute of Genetics (NIG), Japan] for providing HslV knockout strains of E. coli from the KEIO and ME Collections. We also thank the Vigilance Biotechnology (Beijing, China) for providing the genes and plasmid. We are grateful to Professor Xiaoming Yu of this Institute for critical reading and amendment of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23658642PONE-D-10-0602910.1371/journal.pone.0062705Research ArticleBiologyDevelopmental BiologyMolecular DevelopmentSignalingMorphogenesisHeart DevelopmentEmbryologyGeneticsGenetic MutationMutagenesisModel OrganismsAnimal ModelsMouseMolecular cell biologySignal transductionMechanisms of Signal TransductionCrosstalkSecond Messenger SystemMembrane Receptor SignalingHormone Receptor SignalingSignaling cascadesCalcium signaling cascadePKA signaling cascadeProtein kinase signaling cascadeSignaling in cellular processesG-protein signalingGTPase signalingProtein kinase C signalingSignaling in Selected DisciplinesDevelopmental SignalingSignaling PathwaysAdrenergic Signal TransductionMedicineCardiovascularCardiomyopathiesHeart FailureAKAP13 Rho-GEF and PKD-Binding Domain Deficient Mice Develop Normally but Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy Mutant AKAP13 in Mouse Development and HypertrophySpindler Matthew J.
1
2
*
Burmeister Brian T.
3
Huang Yu
1
Hsiao Edward C.
4
Salomonis Nathan
5
Scott Mark J.
1
Srivastava Deepak
1
6
7
Carnegie Graeme K.
3
Conklin Bruce R.
1
2
8
9
1
Gladstone Institute of Cardiovascular Disease, San Francisco, California, United States of America
2
Graduate Program in Pharmaceutical Sciences and Pharmacogenomics, University of California San Francisco, San Francisco, California, United States of America
3
Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, United States of America
4
Department of Medicine in the Division of Endocrinology and Metabolism and the Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America
5
California Pacific Medical Center Research Institute, San Francisco, California, United States of America
6
Department of Pediatrics, University of California San Francisco, San Francisco, California, United States of America
7
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America
8
Department of Medicine, University of California San Francisco, San Francisco, California, United States of America
9
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, United States of America
Klymkowsky Michael Editor
University of Colorado, Boulder, United States of America
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: M. Spindler BTB DS GKC BRC. Performed the experiments: M. Spindler BTB YH ECH NS M. Scott. Analyzed the data: M. Spindler BTB YH DS GKC BRC. Wrote the paper: M. Spindler BRC.
2013 26 4 2013 8 4 e627051 12 2010 28 3 2013 © 2013 Spindler et al2013Spindler et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.
Methodology/Principal Findings
To determine if these AKAP13 protein domains are required for development, we used gene-trap events to create mutant mice that lacked the Rho-GEF and/or the protein kinase D-binding domains. Surprisingly, heterozygous matings produced mutant mice at Mendelian ratios that had normal viability and fertility. The adult mutant mice also had normal cardiac structure and electrocardiograms. To determine the role of these domains during β-adrenergic-induced cardiac hypertrophy, we stressed the mice with isoproterenol. We found that heart size was increased similarly in mice lacking the Rho-GEF and PKD-binding domains and wild-type controls. However, the mutant hearts had abnormal cardiac contractility as measured by fractional shortening and ejection fraction.
Conclusions
These results indicate that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, these domains regulate aspects of β-adrenergic-induced cardiac hypertrophy.
This work was supported by the National Institutes of Health grants RO1 HL60664 and UO1 HL100406 (to BRC), 7 K08 AR056299-02 (to ECH), and T32 Training Grant 5T32HL072742-09 through the University of Illinois at Chicago Department of Cardiology (BTB). Fellowship support was provided by the American Heart Association Western States Predoctoral Fellowship 0715027Y (to MJSpindler). GKC received funding from the American Heart Association Grant 11SDG5230003 and the National Center for Advancing Translational Science-University of Illinois at Chicago Center for Clinical and Translational Sciences Grant UL1TR000050. DS received funding from the National Institutes of Health grant P01HL089707, the California Institute of Regenerative Medicine, the Younger Family Foundation, the L.K. Whittier Foundation and the Eugene Roddenberry Foundation. The J. David Gladstone Institutes received support from a National Center for Research Resources Grant RR18928. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
A-kinase anchoring proteins (AKAPs) organize multi-protein signaling complexes to control a wide range of signaling events, including those important for development [1], [2], fertility [3], [4], learning and memory [5]–[7], and cardiac structure and physiology [8]–[11]. The diverse AKAP family members all bind protein kinase A (PKA) and many other signaling proteins, such as protein kinase C (PKC) and D (PKD), to create unique signaling complexes [12], [13]. Many of these signaling proteins are activated by common intracellular second messengers (e.g., cyclic AMP (cAMP) or calcium), which activate PKA and PKC, respectively. If the activated signaling proteins are left uncontrolled, they could nonspecifically affect multiple downstream proteins. However, AKAPs provide signaling specificity by anchoring multi-protein complexes close to specific downstream substrates. Thus, AKAPs integrate multiple upstream signals into specific downstream events by organizing multi-protein signaling complexes at specific cellular locations.
In the heart, the signaling events coordinated by AKAPs control aspects of cardiac growth, remodeling [9], [14], [15], and physiology, including excitation/contraction (EC) coupling and calcium regulation [16], [17]. The physiological roles of several AKAPs in coordinating EC coupling have been studied in isolated cardiomyocytes and whole organisms [18]. However, the roles of AKAPs in coordinating cellular growth and remodeling during cardiac hypertrophy have been limited to studies in isolated cardiomyocytes [9], [14], [19], [20]. Interestingly, many of the signaling pathways involved in cardiac remodeling are also important in the developing heart.
We studied AKAP13 in mice because of its expression pattern, published knockout phenotype, and the well-characterized signaling pathways it coordinates in isolated cardiomyocytes. We first identified AKAP13 because its expression is up-regulated during mouse fetal development [21] and mouse embryonic stem (ES) cell differentiation [22] (Information S1). In addition, AKAP13 is highly expressed in the adult heart [23], [24]. Second, a null allele of AKAP13 causes embryonic death and exhibits cardiac defects [11]. Finally, AKAP13 coordinates a signaling complex that transduces cardiac remodeling signals induced by G protein-coupled receptors (GPCRs) into hypertrophic responses in isolated cardiomyocytes [14], [20].
AKAP13 is a large gene that encodes for three main transcripts, AKAP-Lbc [23], Brx [24], and Lbc [25], through the use of alternative promoters. The protein isoforms encoded by these three transcripts share a common carboxyl-terminal region that contains a guanine nucleotide exchange factor (GEF) domain and PKD binding domains (Fig. 1). The unique amino-terminus of AKAP-Lbc encodes the PKA binding domain [23], [26], [27]. The roles these AKAP13 protein domains play during hypertrophic signaling have been well studied in isolated rat cardiomyocytes. Several GPCR ligands that signal through the G-protein pathways G12/13 and Gq activate the GEF domain of AKAP13 and AKAP13-bound PKC, respectively [14], [20]. Once activated, the GEF domain activates RhoA, which leads to cardiomyocyte hypertrophy [20]. Activated PKC activates co-bound PKD, which, through several additional steps, activates the transcription factor MEF2C and leads to hypertrophy [14], [26], [28].
10.1371/journal.pone.0062705.g001Figure 1 Gene-traps disrupt AKAP13 in multiple locations.
(A) Schematic of the AKAP13 genomic locus. Exons are depicted with black bars, cassette exons with a grey box, and alternative promoters with arrows. The three gene-trap insertions are indicated. (B) Diagram of the gene-trap constructs (blue boxes) integrated between AKAP13 exons (open boxes with exon numbers). The gene-trap vector contains a strong splice acceptor (SA), βGeo cassette (β−galactosidase and neomyocin resistance genes), and stop codon, as well as a polyadenylation (pA) sequence. The splicing events indicated were confirmed by RT-PCR and sequencing. Primers used to genotype the wild-type and gene-trap alleles are shown (black arrows). (C) Resulting protein fusions of AKAP-Lbc, Brx, and Lbc isoforms with βGeo for the gene-trap mutational series. PKA = protein kinase A binding domain, GEF = Rho-guanine nucleotide exchange factor domain, PKD = protein kinase D binding domain, LZ = leucine zipper domain. (D) Sample genotyping of mouse tail clips for the AKAP13 gene-trap mutations using primers in (B). WT = Wild-type, Het = Heterozygote, Hom = Homozygote.
The same signaling pathways coordinated by AKAP13 to regulate isolated cardiomyocyte hypertrophy could be required for cardiac development. Despite the finding that AKAP13-null embryos die, likely from cardiovascular defects [11], the protein domains and coordinated signaling pathways of AKAP13 required for development are unknown. Both the G12/13 and Gq signaling pathways, which can signal upstream of AKAP13, are required for development of the mouse cardiovascular system [29], [30]. In addition, proteins downstream of AKAP13 are required for proper development since mutant MEF2C and PKD mouse embryos die from heart formation defects and unknown causes, respectively [31], [32].
In this study, we asked if the signaling events coordinated by AKAP13 in isolated cardiomyocytes were important for cardiac development and hypertrophic remodeling in mice. We hypothesized that the AKAP13 protein domains for Rho-GEF activity and PKD binding are required for mouse development. To test this hypothesis, we mated AKAP13 gene-trap mutant mouse lines and assessed them for viable offspring. Unexpectedly, we found that mice lacking the Rho-GEF and PKD-binding domains had normal viability. These mice also had normal cardiac electrical activity, as assessed by 6-lead electrocardiograms (ECGs), and cardiac structure.
We then hypothesized that the Rho-GEF and PKD-binding domains of AKAP13 are important for cardiac remodeling in response to β-adrenergic-induced cardiac hypertrophy. To test this hypothesis, we treated mice with isoproterenol for 14 days, measured cardiac structural and functional changes by echocardiography, and analyzed heart size and structure by morphology and histology. Surprisingly, we found that AKAP13 Rho-GEF and PKD-binding deficient mice induced cardiac hypertrophic remodeling but had abnormal cardiac contractility as measured by fractional shortening (FS) and ejection fraction (EF).
Results
Gene-Trap Events Disrupt AKAP13 in Multiple Locations
An AKAP13 knockout allele causes embryonic death in mice, possibly from cardiac defects [11]. However, AKAP13 contains multiple protein domains, and it is unclear which domains are required for development. In addition, the AKAP13 gene locus utilizes alternative promoters to drive expression of at least three different isoforms, AKAP-Lbc, Brx, and Lbc.
To determine if the AKAP13 Rho-GEF and PKD-binding domains are required for mouse development, we generated AKAP13 mutant mice from gene-trapped ES cells. The gene-trap construct uses a strong splice acceptor to create a fused mRNA of the upstream AKAP13 exons with the trapping cassette [33]. The resulting fusion protein contains the amino-terminus of AKAP13 fused to βGeo, which confers β-galactosidase activity and neomyocin resistance. These fusion proteins create truncation mutants that can be used to dissect the role of AKAP13 protein domains in vivo.
We used the International Gene Trap Consortium (IGTC) database (at www.genetrap.org) [34] and the IGTC Sequence Tag Alignments track on the UCSC Genome Browser [35] to select three gene-trap events at different positions of the AKAP13 gene, ΔBrx (from ES cell line AG0213), ΔGEF (CSJ306), and ΔPKD (CSJ288), for further analysis (Fig. 1A). We confirmed the splicing of upstream AKAP13 exons into the gene-trap cassette (Fig. 1B) by RT-PCR and sequencing from total ES cell RNA. We also identified the insertion site of each gene-trap event by long-range PCR and designed genotyping strategies for these mutant lines (Fig. 1B, D). These three gene-trap events create a mutational series that affects specific AKAP13 isoforms and protein domains (Fig. 1C). The ΔBrx mutation creates a fusion of the AKAP-Lbc and Brx isoforms with βGeo that disrupts the Rho-GEF and PKD-binding domains for these two isoforms. However, the Lbc isoform should be normally expressed. The ΔGEF mutation is expected to be the most severe as it creates a fusion of all three isoforms that disrupts the Rho-GEF and PKD-binding domains. Finally, the ΔPKD mutation disrupts the PKD-binding domain of all three isoforms while the Rho-GEF domain remains intact. Male chimeric mice were generated from these three gene-trap ES cell lines and crossed to female C57Bl/6 mice to generate heterozygotes. We used these mice to study the roles of AKAP13 Rho-GEF and PKD-binding domains in vivo.
To verify that the gene-trap events disrupt the expected AKAP13 protein domains, we generated corresponding V5-tagged AKAP-Lbc truncation constructs and expressed them in HEK293 cells (Fig. 2A). To determine the effect of these truncations on Rho-GEF activity, we immunoprecipitated the AKAP-Lbc truncation mutants and performed in vitro Rho-GEF assays. As expected, both AKAP-Lbc-ΔGEF and -ΔBrx had disrupted Rho-GEF activity (Fig. 2B
top panel). Western blot analysis confirmed that all the AKAP-Lbc truncation constructs were expressed and immunoprecipitated to an equivalent extent (Fig. 2B
bottom panels). We next tested these AKAP-Lbc truncations for their ability to bind PKD by immunoprecipitation of the AKAP-Lbc protein complexes, followed by in vitro kinase assays and immunoblotting. As expected, the AKAP-Lbc-ΔPKD, -ΔGEF, and -ΔBrx protein complexes all lacked PKD activity and binding (Fig. 2C). Finally, we confirmed that these AKAP-Lbc truncation mutants could immunoprecipitate PKA and that PKA activity was unaffected (Fig. 2D). These results show that the AKAP-Lbc-ΔGEF and -ΔBrx truncations disrupt AKAP13 Rho-GEF activity and PKD binding. Furthermore, the AKAP-Lbc-ΔPKD truncation disrupts PKD binding but still contains Rho-GEF activity. Thus, these results indicate that the gene-trap events will disrupt the expected AKAP13 protein domains.
10.1371/journal.pone.0062705.g002Figure 2 The gene-trap induced truncations of AKAP13 disrupt the expected protein domains.
(A) Expression constructs corresponding to the AKAP13 gene-trap events were generated using V5-tagged AKAP-Lbc truncation mutants. (B-D) These expression constructs were transfected into HEK293 cells and protein complexes were co-immunoprecipitated using anti-V5 antibody. (B) Rho-GEF activity was measured after immunoprecipitation (IP). Both AKAP-Lbc-ΔGEF and -ΔBrx had disrupted Rho-GEF activity, compared to AKAP-Lbc-WT and -ΔPKD. Immunoblotting (IB) for AKAP-Lbc-V5 with anti-V5 antibody confirmed that the AKAP-Lbc truncation mutants were expressed and immunoprecipitated at an equivalent extent. (C) Protein kinase D (PKD) activity was measured following IP. The AKAP-Lbc-ΔPKD, -ΔGEF, and -ΔBrx protein complexes lacked PKD activity compared to AKAP-Lbc-WT. Immunoblotting for GFP-PKD1 with anti-GFP antibody confirmed that only AKAP-Lbc-WT bound PKD1. The bottom gel image confirmed that GFP-PKD1 was expressed at the same level in all conditions. (D) Protein kinase A (PKA) activity was measured after IP. All AKAP-Lbc truncation mutants immunoprecipitated PKA activity and bound PKAc. The means and standard deviations are graphed for three independent experiments. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). *, p<0.05; ***, p<0.001.
AKAP13 Is Broadly Expressed During Mouse Development and in Adult Tissue
Despite the requirement of AKAP13 for mouse development, its expression pattern during this process is unknown. In addition to disrupting the AKAP13 protein, the gene-trap events report the expression pattern of AKAP13 because the endogenous AKAP13 promoters drive expression of the AKAP13-βGeo fusion proteins.
To determine the expression of AKAP13 during mouse development, we conducted X-Gal staining of AKAP13+/ΔGEF embryos at E8.5, E9.5, E10.5, and E14.5 (Fig. 3). We found X-Gal staining in the head folds, notochord, and somites of E8.5 embryos but little to no staining in the looping heart (Fig. 3A, B). At E9.5, the staining pattern was broadly expanded with higher levels of expression in the heart (Fig. 3C). There was also staining in the vasculature, eye, ear, somites, gut and brain. E10.5 embryos had a staining pattern similar to that of E9.5 embryos (Fig. 3D). However, there was stronger staining throughout the heart (Fig. 3D, E). E14.5 embryos had high levels of staining in the atrial and ventricular myocardium and endocardium, trabeculae, and outflow tract (Fig. 3F–H). There was also staining in skeletal muscle, tongue, gut, kidney, lung, urinary system, and the choroid plexus of the brain (Fig. 3F). Finally, the yolk sac and umbilical cord of mouse embryos stained positive with X-Gal (Fig. 3I). We found the same staining patterns in AKAP13+/ΔBrx and AKAP13+/ΔPKD embryos, and no staining in wild-type embryos was detected. These results show that AKAP13 is broadly expressed during mouse development with increasing levels of expression in the heart and outflow tract. They also show that AKAP13 is expressed in skeletal and smooth muscle throughout the developing embryo.
10.1371/journal.pone.0062705.g003Figure 3 AKAP13 is broadly expressed during mouse development.
(A–D) Whole-mount AKAP13+/ΔGEF embryos stained with X-Gal (in blue) to identify AKAP13-βGeo expression at (A&B) E8.5, (C) E9.5, and (D) E10.5. (A&B) E8.5 embryos showed expression in the head folds, notochord, and somites. (C) Right side view of E9.5 embryo showed expression in the heart (ht), brain, eye (arrow), otic pit (arrowhead), gut, and somites. (D) Right side view of E10.5 embryo showed similar expression as in (C) with higher expression in the heart (ht). (E) Frontal view of an E10.5 heart showed high levels of expression in the ventricle (v), bulbous cordis (bc), and outflow tract (oft). (F) Sagittal and (H) transverse sections of E14.5 embryos stained with X-Gal and nucleofast red. E14.5 embryos showed expression in the heart (ht), tongue (t), lung (l), gut (g), kidney (k), skeletal muscle, brain (arrow), and urogenital region (arrowhead). (G&I) Close ups of the hearts boxed in F and H, respectively, showed expression in atrial (at), and ventricular (v) myocardium, endocardium and trabeculae. The right and left atria (ra & la) and ventricles (rv & lv) all showed expression with higher levels in the left ventricle (lv). There was also expression in the aorta (a). (J) X-Gal staining of E9.5 embryos with the yolk sac attached showed expression in the yolk sac (ys). Black scale bars are 0.5 mm.
Previous studies using northern blot analysis found AKAP13 to be highly expressed in human heart tissue with less expression in other tissues, including the lung and kidney [23], [24]. However, the expression patterns of AKAP13 within these organs remain unknown. To determine the expression pattern of AKAP13 within adult mouse organs, we conducted X-Gal staining of AKAP13+/ΔGEF heart, kidney, and brain samples (Fig. 4). We found X-Gal staining throughout the entire heart and in the pulmonary arteries and aorta (Fig. 4A). In the kidney, the cortex, arteries and ureter stained positive (Fig. 4C). The vasculature of the brain, olfactory bulb, and part of the cerebellar cortex stained positive (Fig. 4D). The same staining patterns were seen in kidney and brain from AKAP13+/ΔBrx and AKAP13+/ΔPKD adult mice. Surprisingly, AKAP13+/ΔPKD hearts lacked staining in the ventricles; however, there was still staining in the atria, pulmonary arteries, aorta, and ventricular vasculature (Fig. 4B). These results show that AKAP13 is highly expressed in the adult heart and vasculature and is expressed in specific regions of additional organs, including the kidney and brain.
10.1371/journal.pone.0062705.g004Figure 4 AKAP13 is expressed in adult heart, kidney, and brain.
Adult AKAP13+/ΔGEF organs were bisected and stained with X-Gal (in blue) to determine AKAP13-βGeo expression in heart (A), kidney (C) and brain (D). (A) The AKAP13-ΔGEF hearts showed strong staining throughout the entire heart, including the left (la) and right (ra) atria, left (lv) and right (rv) ventricles, pulmonary artery, and aorta. (B) AKAP13-ΔPKD hearts had staining in the atria pulmonary artery, and aorta, as expected, but lacked staining in the ventricles. The blood vessels of the ventricles stained positive. (C) The kidney cortex (c), ureter (u), and arteries (ar) stained positive. (D) The interior of the right hemisphere of the brain showed staining of the olfactory bulb (ob), vasculature (arrow), and part of the cerebellum (cbx). Black scale bars are 1 mm.
AKAP13 Rho-GEF and PKD-Binding Domains Are Not Required for Mouse Development
Recently, an AKAP13-null mouse was reported to die at E9.5–E10.5 during embryonic development, and it was proposed that this was due to a loss of Rho-GEF signaling [11]. Since AKAP13 also encodes for PKA and PKD binding domains, we asked whether the AKAP13 Rho-GEF and PKD-binding domains were required for mouse development. To answer this question, we conducted heterozygote crosses for the three mutant mouse lines and assessed the matings for viable offspring. We found that all of these matings produced homozygous mutant offspring at the expected Mendelian ratios (Table 1). In addition, the homozygous mutant mice lacked gross abnormalities, were fertile, and had normal viability.
10.1371/journal.pone.0062705.t001Table 1 Genotypes of pups from heterozygous AKAP13 mutant matings.
Genotype Expected Mendelian Ratio % Observed Ratios % (Number of Pups)
ΔBrx ΔGEF ΔPKC
WT
25 23 (n = 39) 25 (n = 52) 25 (n = 64)
Het
50 54 (n = 91) 56 (n = 116) 54 (n = 141)
Hom
25 23 (n = 39) 19 (n = 39) 21 (n = 55)
WT = Wild-type, Het = Heterozygote, Hom = Homozygote.
To verify that the gene-trap mutations disrupt full-length AKAP13 expression, we conducted quantitative PCR on total RNA from newborn pup heart and lung tissue (Fig. 5). We used TaqMan probes to measure relative expression of the E4-5, Brx-9, and E37-38 exon-exon junctions (Fig. 5A). As expected, we found that none of the gene-trap mutations changed the expression of the AKAP13 E4-5 junction, which lies upstream of the three gene-trap insertion sites (Fig. 5B). The expression of the Brx-9 junction was reduced in a dose-dependent manner only in ΔBrx mice, and AKAP13ΔBrx/ΔBrx mice completely lacked expression at this exon-exon junction (Fig. 5C). These results were also expected because the ΔBrx insertion site lies between the Brx specific exon and exon 9, and the other two gene-trap insertions are downstream of this exon-exon junction. Finally, all three gene-trap mutations decreased expression of the E37-38 junction in a dose-dependent manner, as expected (Fig. 5D). The ΔGEF mutation was particularly effective at reducing expression, as the AKAP13ΔGEF/ΔGEF mice completely lacked expression of this exon-exon junction.
10.1371/journal.pone.0062705.g005Figure 5 Full-length AKAP13 mRNA levels are reduced by the gene-trap events.
(A) TaqMan gene expression assays were used to measure the expression of AKAP13 transcripts at the indicated exon-exon junctions (E4-5, Brx-9, & E37-38). (B) Quantitative PCR analysis of wild-type (WT), heterozygote (Het) and homozygote (Hom) neonatal mouse heart and lung RNA for AKAP13 showed that none of the gene-trap mutations affected expression of the E4-5 exon-exon junction. The ΔBrx gene-trap dose dependently decreased expression of the Brx-9 exon-exon junction. Expression of the Brx-9 junction was eliminated in the AKAP13ΔBrx/ΔBrx mice. All three gene-traps decreased expression of the E37-38 exon-exon junction in a dose-dependent manner. Expression of the E37-38 junction was eliminated in the AKAP13ΔGEF/ΔGEF mice. The means and standard deviations are graphed for six mice per genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p<0.10; *, p<0.05; **, p<0.01.
Contrary to our expectations, these results indicate that the AKAP13 gene-trap mutations do not affect development or viability. Specifically, the ΔBrx mutation eliminates expression of the Brx-9 exon-exon junction indicating that the Brx isoform of AKAP13 is not required for development or viability. Likewise, the ΔGEF mutation completely eliminates expression of E24-25 (data not shown) and E37-38. Additionally, we showed that the ΔGEF truncation disrupts the AKAP13 Rho-GEF and PKD-binding domains (Fig. 2). Thus, these results show that the AKAP13 Rho-GEF and PKD-binding domains are not required for mouse development or viability.
Cardiac Electrical Activity and Structure Is Normal in AKAP13 Mutant Mice
Since AKAP13 is highly expressed during cardiac development and throughout the adult heart (Fig. 3 & 4) and regulates cardiomyocyte physiology [14], [20], we asked whether the ΔGEF mutation affected adult cardiac electrical activity or structure. To address this, used 6-lead ECG to analyze heart activity and then harvested the hearts from 16–18-week-old male homozygous mutant and wild-type control mice.
ECG analysis showed that heart rate (HR), PR interval, P wave duration, QRS interval, and corrected QT interval (QTc) of AKAP13ΔGEF/ΔGEF mice were indistinguishable from wild-type littermates (Table 2). Gross morphology showed that the ΔGEF hearts had normal atrial and ventricular structures (Fig. 6A) and a properly formed pulmonary artery and aorta. Additionally, the wild-type and ΔGEF hearts were the same size as assessed by the heart weight to tibia length (HW/TL) ratios (Fig. 6B). Hearts from AKAP13ΔBrx/ΔBrx and AKAP13ΔPKD/ΔPKD mice also had normal morphology and size (data not shown). Histological analysis of ΔGEF hearts by hematoxylin and eosin (H&E) staining showed proper cardiomyocyte organization and structure (Fig. 6C). Finally, the ΔGEF hearts had normal levels of Masson’s trichrome staining, indicating no change in cardiac fibrosis (Fig. 6D). These results indicate that the loss of AKAP13 Rho-GEF and PKD-binding domains does not affect cardiac electrical activity or structure under normal physiological conditions.
10.1371/journal.pone.0062705.g006Figure 6 AKAP13-ΔGEF mutant mice had normal cardiac structure.
(A) Hearts isolated from six wild-type (WT) and six AKAP13ΔGEF/ΔGEF (ΔGEF) adult male mice at 16–18 weeks of age had normal gross morphology; representative images shown. White scale bar is 1 mm. (B) WT and ΔGEF hearts were the same size as measured by heart weight to tibia length (HW/TL) ratios (in milligrams per millimeter). Means and standard deviations are graphed for six hearts of each genotype. Hearts were sectioned for histology and stained with (C) H&E or (D) Masson’s trichrome. The bottom panels of C&D are higher magnifications of the boxed regions in the top panels. (C) Cardiac structure was normal in ΔGEF hearts (top), and cardiomyocytes had proper organization (bottom). (D) ΔGEF hearts had normal levels of fibrosis as assessed by Masson’s trichrome staining. Black scale bars in C&D are 1 mm (top), 50 µm (C bottom), and 250 µm (D bottom).
10.1371/journal.pone.0062705.t002Table 2 Six-Lead ECG analysis of AKAP13-ΔGEF mutant mice.
Genotype Heart Rate PR (ms) P (ms) QRS (ms) QTc (ms)
WT
462.3±30.6 38.4±3.2 9.16±1.14 11.3±1.3 52.2±3.5
ΔGEF
437.1±17.9 39.1±2.3 9.30±0.61 11.5±1.0 55.4±5.7
Heart rate is in beats per minute, ms = milliseconds.
Values are given as the mean ± standard deviation for six mice in each genotype.
AKAP13 ΔGEF Mice Have an Abnormal Response to β-Adrenergic-Induced Cardiac Hypertrophy
AKAP13 coordinates many signaling processes to mediate the cellular response to cardiac hypertrophic signals [14], [20], [36], [37]. Specifically, the AKAP13 Rho-GEF and PKD-binding domains transduce hypertrophic signaling events in isolated cardiomyocytes [14], [20]; however, it is unclear if they are required for the hypertrophic response in mice. Thus, we asked whether the AKAP13 Rho-GEF and PKD-binding domains are required for a β-adrenergic-induced cardiac hypertrophic response in mice. To answer this, we implanted mini-osmotic pumps into 22–32-week-old wild-type and AKAP13ΔGEF/ΔGEF littermate mice to infuse PBS vehicle (Veh) or isoproterenol (Iso; 60 mg/kg per day) for 14 days [38]. Iso activates β-adrenergic receptors to induce cardiac hypertrophy [39] partially through PKD signaling [31]. To assess the cardiac structural and functional response to β-adrenergic-mediated cardiac hypertrophy, we conducted echocardiography on mice in a blinded fashion. We recorded echocardiograms before pump implantation to obtain a baseline value and on day 13 of treatment. We then isolated the hearts from these mice on day 14 of treatment to further analyze cardiac structural changes.
M-Mode echocardiogram recordings on day 13 showed that Iso treatment increased left ventricular wall thickness in wild-type and AKAP13ΔGEF/ΔGEF mice. However, the degree of cardiac contraction was lower in the Iso-treated ΔGEF mice than wild-type mice (Fig. 7A). Cardiac structural and functional changes were quantified from the echocardiogram recordings (Fig. 7B–E). Iso treatment increased left ventricular mass (LV Mass) in both wild-type (51%) and ΔGEF (60%) mice from baseline values (Fig. 7B). Left ventricular anterior wall thickness at diastole (LVAW;d) increased in both wild-type (43%) and ΔGEF (34%) mice treated with Iso (Fig. 7C). Left ventricular posterior wall thickness was increased similarly to LVAW (data not shown). There was no difference in LV Mass or LVAW;d between the wild-type and ΔGEF mice at baseline or after Iso treatment. These results show that the ΔGEF mice induce structural changes associated with cardiac hypertrophy.
10.1371/journal.pone.0062705.g007Figure 7 AKAP13-ΔGEF mutant mice undergo cardiac remodeling but have abnormal contractility in response to β-adrenergic-induced hypertrophy.
(A) Representative M-Mode echocardiogram images showed a thicker left ventricular wall in wild-type (WT) and AKAP13ΔGEF/ΔGEF (ΔGEF) male mice treated with isoproterenol (Iso; 60 mg/kg per day for 13 days) than in those treated with PBS vehicle (Veh). Iso treatment increased the magnitude of contraction in WT mice but not in ΔGEF mice. The horizontal black scale bar is 200 ms; the vertical black scale bars are 1 mm. (B–E) Quantification of echocardiography data for left ventricle structural and functional changes in response to Iso treatment. Echocardiograms were recorded the day before mini-osmotic pumps were implanted for baseline levels (0) and after 13 days of Iso (+) or Veh (-) treatment. (B) Both WT and ΔGEF mice increased left ventricular (LV) mass to the same level with Iso treatment. (C) LV anterior wall thickness at diastole (LVAW;d) was increased to the same level in both WT and ΔGEF mice treated with Iso. (D) The percent of fractional shortening (FS) was greater in wild-type mice treated with Iso compared to baseline or Veh treatment. FS was not different in ΔGEF mice treated with Iso compared to baseline or Veh controls. However, ΔGEF mice treated with Iso tended to have reduced FS compared to wild-type controls. (E) The percent ejection fraction (EF) also was greater in wild-type mice treated with Iso than baseline or Veh treatment. Again, EF was not different in ΔGEF treated with Iso compared to baseline or Veh controls, but tended to be less than wild-type controls. The means and standard deviations are graphed in B–E for seven WT and nine ΔGEF mice at baseline (0), three WT and three ΔGEF mice with Veh treatment, and four WT and six ΔGEF mice with Iso treatment. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted (Prism 5; GraphPad). †, p<0.10; *, p<0.05; **, p<0.01; ***, p<0.001.
We next assessed cardiac contractility by calculating left ventricular FS and EF from echocardiogram recordings (Fig. 7D, E). At day 13 of Iso treatment, wild-type mice had 15% greater FS (Fig. 7D) and 22% greater EF (Fig. 7E) than Veh-treated controls. However, ΔGEF mice treated with Iso showed no differences in FS or EF as compared to vehicle controls. Moreover, ΔGEF mice treated with Iso tended to have reduced FS and EF as compared to wild-type controls that trended towards significance (p<0.1). We also found that Iso treatment increased heart rate for both wild-type and ΔGEF mice (Table 3). These results show that despite similar hypertrophic structural changes, the ΔGEF mice have an abnormal functional response to chronic Iso treatment as measured by cardiac contractility.
10.1371/journal.pone.0062705.t003Table 3 Heart rate changes with Iso treatment.
Genotype Baseline Vehicle Isoproterenol
WT
431.0±31.1 (n = 7) 446.7±62.8 (n = 3) 554.3±17.9 (n = 4)
ΔGEF
439.9±43.6 (n = 9) 477.7±57.9 (n = 3) 569.2±20.1 (n = 6)
Heart rate is in beats per minute.
Values are given as the mean ± standard deviation.
Morphological analysis of whole hearts verified that Iso treatment induced cardiac hypertrophy in both wild-type and AKAP13ΔGEF/ΔGEF mice to a similar extent (Fig. 8A). HW/TL increased in wild-type mice treated with Iso from a Veh-treated value of 11.97±0.81 (mean ± SD, n = 3) to 16.07±2.01 mg/mm (n = 4; p = 0.022). Similarly, HW/TL increased in ΔGEF mice from a Veh-treated value of 12.47±3.49 (n = 3) to 15.58±2.12 mg/mm (n = 6; p = 0.133). H&E staining of histological sections of these hearts showed that Iso treatment increased left ventricular wall thickness in both sets of mice (Fig. 8B, top). Closer examination of the cardiomyocytes at the top of the left ventricular wall showed increased interstitial cells between the myocytes and a looser myocyte configuration in Iso-treated than Veh-treated hearts (Fig. 8B, bottom). Iso treatment also increased fibrosis in the myocardium of both wild-type and ΔGEF hearts as assessed by Masson’s trichrome staining (Fig. 8C). This fibrosis was interspersed within the myocardium. Qualitative analysis of these heart sections suggested that there was more fibrosis in the ΔGEF than wild-type hearts. Quantification of Masson’s trichrome staining also suggested a trend for increased fibrosis in the ΔGEF hearts (10.11±8.42%, n = 6, for ΔGEF vs. 5.63±2.10%, n = 4, for wild-type; p = 0.336). Interestingly, one of the Iso-treated ΔGEF hearts had a large area of fibrosis at the top of the right and left ventricular walls (>25% of myocardial area).
10.1371/journal.pone.0062705.g008Figure 8 AKAP13-ΔGEF mutant mice induced cardiac hypertrophy in response to chronic isoproterenol treatment.
(A) Hearts from wild-type (WT) and AKAP13ΔGEF/ΔGEF (ΔGEF) male mice showed hypertrophy with isoproterenol (Iso) treatment (60 mg/kg per day for 14 days). Three WT and three ΔGEF mice were treated with PBS vehicle (Veh), and four WT and six ΔGEF mice were treated with Iso; representative images are shown. White scale bar is 1 mm. Hearts were sectioned for histology and stained with (B) H&E or (C) Masson’s trichrome. (B) WT and ΔGEF left ventricular walls were thickened by Iso treatment (top). Higher magnification of the upper left ventricular wall (box) showed disruption of myocyte organization in Iso-treated hearts (bottom). (C) Fibrosis increased throughout the WT and ΔGEF hearts as assessed by Masson’s trichrome staining. Iso-treated ΔGEF hearts appeared to have more fibrosis than Iso-treated WT hearts. Higher magnification of the left ventricular wall (box) showed fibrosis within the myocardium (bottom). Black scale bars in B&C are 1 mm (top), 50 µm (B bottom), and 250 µm (C bottom).
The echocardiography and morphological results showed that AKAP13ΔGEF/ΔGEF mice induce cardiac hypertrophy in response to chronic β-adrenergic stimulation. However, the ΔGEF mice had lower levels of cardiac contractility than wild-type mice. Moreover, the ΔGEF mice also appeared to have increased fibrosis. These results indicate that the AKAP13 Rho-GEF and PKD-binding domains are not required for β-adrenergic induced cardiac hypertrophy. However, the results indicate that these AKAP13 domains do regulate aspects of cardiac hypertrophy.
Discussion
In this study, we investigated the roles of the Rho-GEF and PKD-binding domains of AKAP13 in mouse development, adult cardiac physiology, and hypertrophic remodeling. Contrary to our expectations, our results show that these AKAP13 domains are not required for mouse development, normal adult cardiac architecture, or β-adrenergic-induced cardiac hypertrophy. However, the AKAP13 Rho-GEF and PKD-binding domains may regulate the compensatory response to cardiac hypertrophy. In developing mice, AKAP13 was broadly expressed with high levels in the cardiovascular system, and in the adult heart, expression remained high. Despite the disruption of the AKAP13 Rho-GEF and PKD-binding domains in AKAP13ΔGEF/ΔGEF mice, we found that these mice were born at a normal Mendelian ratio, had normal viability, and were fertile. Additionally, the mutant adult mice had normal cardiac structure and function. The ΔGEF mice induced cardiac remodeling in response to chronic isoproterenol treatment. However, these mice had abnormal cardiac contractility and slightly increased fibrosis in response to chronic isoproterenol treatment.
Contrary to our expectations that the AKAP13-ΔGEF mutation would phenocopy AKAP13-null mice, we found that AKAP13ΔGEF/ΔGEF mice developed normally. A previous study reported that AKAP13-null embryos die at E9.5–10.5, display a thinned myocardium and loss of trabeculation, and have decreased expression of cardiac developmental genes [11]. The authors proposed that these phenotypes were due to the loss of AKAP13 Rho-GEF activity in the heart [11]. However, AKAP13 also coordinates a PKC-PKD signaling pathway, and both the Rho-GEF and PKC-PKD pathways regulate cardiomyocyte hypertrophic growth [14], [20]. We expected that eliminating both the Rho-GEF and PKD-binding domains of AKAP13 would cause embryonic lethality and phenocopy the AKAP13-null mutation. However, our results show that AKAP13-mediated Rho-GEF and PKD signaling are not required for mouse development. These results, combined with the published AKAP13-null mouse phenotype, indicate that other AKAP13 protein domains are required for mouse development.
The PKA-binding domain of AKAP13 is an intriguing candidate for the developmentally required AKAP13 protein domain. The AKAP13-ΔGEF mutation used in this study fuses the amino-terminus of AKAP13, including the PKA binding domain, to the βGeo cassette. We confirmed that this mutation eliminates full-length AKAP13 mRNA but maintains expression of mRNA upstream of the gene-trap insertion. Thus, the AKAP13 region upstream of the ΔGEF mutation seems to be sufficient for mouse development, possibly through binding PKA. AKAP13-bound PKA inhibits AKAP13-Rho-GEF activity [40] and enhances PKD signaling [14], [26] in isolated cardiomyocytes. If PKA binding to AKAP13 were required for development, it would suggest a novel AKAP13-mediated signaling pathway. The requirement for AKAP13-PKA binding during development would not be unprecedented since proper regulation of PKA signaling is required for mouse development [41]. Moreover, the cardiac-specific disruption of a regulatory subunit of PKA, which holds the kinase in an inactive state until cyclic AMP activation, results in a thinning of the myocardium and loss of trabeculation [42]. Interestingly, the phenotype observed after cardiac disruption of PKA regulation [42] is very similar to the phenotype described for the AKAP13-null mouse [11]. Alternatively, an unappreciated AKAP13 protein domain could be required for development. Additional mutational analysis of the AKAP13 gene locus is required to fully investigate these possibilities.
AKAP13 is expressed in many tissues during mouse development, and we were surprised that the AKAP13ΔGEF/ΔGEF mice had no obvious developmental phenotypes. This suggests that additional proteins might compensate for the loss of AKAP13-mediated Rho and PKD signaling. Several additional AKAP family members are expressed during mouse development. Two that might have compensatory roles are AKAP6 (mAKAP) and AKAP12 (Gravin). AKAP6 is expressed developmentally and becomes highly expressed in cardiac and skeletal muscle [43] to coordinate PKA, small GTPases [19], and calcium signaling events [44], [45]. AKAP12 is broadly expressed in mouse embryos and in the adult heart [46] and is required for gastrulation in zebrafish [2]. AKAP12 coordinates PKA, PKC, and Raf signaling events to regulate cellular shape changes and movement [47]. Additionally, Rho signaling may be compensated for by the large Rho-GEF containing structural protein, Obscurin, which is required for proper cardiac, muscle, and brain development in zebrafish [48]. The roles of AKAP6, AKAP12, and Obscurin during mouse development are unknown, and disruption of these proteins may produce developmental defects. It would also be interesting to determine if these scaffolds provide functional redundancy for the loss of AKAP13 protein domains by creating double mutant mice.
AKAP13ΔGEF/ΔGEF mice had normal viability, and their adult cardiac structure and electrical activity were indistinguishable from wild-type littermates despite high levels of AKAP13 expression in the heart. These results indicate that AKAP13 Rho-GEF and PKD-binding domains are not required for mouse survival or normal cardiac physiology. This suggests that additional proteins provide redundancy in controlling Rho and PKD signaling during heart maturation and normal physiology. The scaffolding molecules AKAP6 and AKAP12, as well as Obscurin, could again provide this redundant function. Additional Rho-GEF proteins, including p115RhoGEF and p63RhoGEF, are expressed in cardiomyocytes and could provide redundancy for RhoA signaling [49]. AKAP13 is also expressed in other organs, such as the vasculature, kidney, lung, gut and brain. Since we did not detect gross phenotypes in these tissues, other proteins might compensate for the loss of AKAP13 Rho-GEF and PKD signaling in these tissues as well. Alternatively, AKAP13 may not regulate normal physiology but may specifically regulate cellular stress responses.
We then decided to test the role of the Rho-GEF and PKD-binding domains for cardiac remodeling in response to β-adrenergic-mediated cardiac hypertrophy. AKAP13 transduces multiple upstream signaling events including α- and β-adrenergic, angiotensin, and endothilin receptor signaling during cardiomyocyte hypertrophy [14], [20], [36], [37]. The AKAP13 Rho-GEF and PKD-binding domains are important for the induction of isolated cardiomyocyte hypertrophy in response to many of these signaling [14], [20]. Additionally, PKD is required for the cardiac hypertrophic response to several stresses, including isoproterenol activation of β-adrenergic receptors in vivo
[31]. Thus, we were surprised that AKAP13ΔGEF/ΔGEF mice induced cardiac remodeling to a similar extent as wild-type controls upon chronic β-adrenergic stimulation. This indicates that the Rho-GEF and PKD-binding domains of AKAP13 are not required for β-adrenergic induced cardiac hypertrophy in mice and that another AKAP regulates this process. AKAP6 could regulate cardiac remodeling in vivo because it transduces adrenergic signaling events, such as isoproterenol stimulation, into cardiomyocyte hypertrophy in vitro
[9]. Despite the cardiac hypertrophic response to isoproterenol, the AKAP13 Rho-GEF and PKD-binding domains might be important for regulating phenylephrine, angiotensin II, and endothelin-1-induced cardiac remodeling. The pathways activated by these molecules signal through AKAP13 to induce hypertrophy in isolated cardiomyocytes [14], [20]. Thus, the series of mutant mice described in this study provide a great resource to investigate the role of specific AKAP13 protein domains in regulating cardiac hypertrophy induced by these molecules in vivo.
Even though AKAP13ΔGEF/ΔGEF mice induced cardiac hypertrophy, they had abnormal cardiac FS and EF in response to isoproterenol treatment. Both FS and EF tended to be lower in ΔGEF mice treated with Iso than in wild-type controls on day 13 of treatment (p<0.1). In addition, FS and EF were increased in wild-type mice but not in ΔGEF mice treated with Iso (Fig. 7D, E). The increased contractility in the wild-type mice treated with Iso indicates that, at this time, the mice are still in the compensatory phase of hypertrophy and have not yet reached cardiac dysfunction [50], [51]. These results indicate that the AKAP13 Rho-GEF and PKD-binding domains are important for regulating aspects of the cardiac hypertrophic response to chronic β-adrenergic stimulation. There are several possible models why the ΔGEF mouse hearts have abnormal cardiac contractility, compared to wild-type controls. One likely model is that the AKAP13-ΔGEF mice might reach cardiac dysfunction more quickly than the wild-type mice. In agreement with this, the mutant mice undergo cardiac hypertrophic remodeling and tend to have slightly higher fibrosis than wild-type mice after chronic isoproterenol treatment. Our study examined cardiac function at a single time point during chronic isoproterenol treatment. To determine if AKAP13 coordinates a cardioprotective role during hypertrophy, future experiments will require continual monitoring of cardiac function from the initiation of hypertrophy until full heart failure is reached. An alternative model is that AKAP13 directly mediates increased cardiac contraction in response to isoproterenol treatment. The AKAP13-coordinated signaling complex that includes PKA, PKC, and RhoA could mediate this direct regulation of cardiac contractility. This model could be tested using acute isoproterenol treatment of mutant mice or isolated cardiomyocytes. Finally, the AKAP13 Rho-GEF and PKD-binding domains might be required for signaling through compensatory pathways, including additional adrenergic or angiotensin pathways, activated during cardiac hypertrophy. Measuring cardiac contractility during acute stimulation of α- and β-adrenergic and angiotensin pathways in AKAP13 mutant mice could help determine the direct pathways AKAP13 regulates.
The regulatory elements that control expression of AKAP13 isoforms in specific tissues remain unknown. ΔPKD mice lacked AKAP13-βGeo expression specifically in ventricular cardiomyocytes of adult hearts. This suggests that the ΔPKD mutation disrupts a cis-regulatory element required for AKAP13 expression in ventricular cardiomyocytes. Furthermore, there are several conserved elements within the ΔPKD-disrupted intron that could function as ventricular myocyte enhancer elements. A detailed analysis of these possible enhancer elements would be required to test this possibility. Additionally, a more detailed characterization of the AKAP13 isoforms expressed during development and in adult tissues could aid in designing future studies. Evidence of additional splicing events from GenBank cDNAs and ESTs suggests alternative termination and cassette exons that could result in functionally important protein isoforms for development or adult physiology. In fact, the main AKAP13 isoforms appear to localize to different subcellular sites with AKAP-Lbc localizing to the cytoplasm and cytoskeleton and Brx localizing to the cytoplasmic and nuclear compartments [11], [24], [52]. A closer examination of all the transcripts expressed from the AKAP13 gene locus is needed to better understand the effects of certain mutations on AKAP13 protein structure. Since AKAP13 undergoes extensive alternative splicing to produce multiple protein isoforms, it may be necessary to add back specific transcripts in an AKAP13-null background to identify the unique roles played by each isoform during mouse development and disease.
Finally, the mice created in this study should prove valuable for investigating AKAP13 functions in additional tissues and diseases. Since AKAP13 is highly expressed in the vasculature, it may transduce angiotensin II, or endothelin-1 signals into vascular responses. Genome-wide studies have linked AKAP13 to corneal thickness of the eye [53] and Alzheimer’s disease-associated tau phosphorylation [54]. Since we found AKAP13 expression in the eye and specific regions of the brain during development, further investigation into the role of AKAP13 in these processes is warranted. Additionally, AKAP13 may function in regulating immunity as it mediates glucocorticoid signaling in lymphocytes [55] and Toll-like receptor 2 signaling in epithelial and leukemia cell lines [52]. Finally, AKAP13 has been associated with several types of cancer, including leukemia [25], breast cancer [24], [56], [57], and colorectal cancer [58]. From these studies, AKAP13 appears to have diverse functions in a multitude of tissues. Despite this, we do not see an obvious phenotype in unstressed mice that lack the Rho-GEF and PKD-binding domains of AKAP13. Thus, we propose that these domains function to transduce acute signaling events in response to stresses.
In summary, we found that the Rho-GEF and PKD-binding domains of AKAP13 are not required for mouse development, normal adult cardiac architecture, or β-adrenergic-induced cardiac hypertrophic remodeling. However, we found that the AKAP13 Rho-GEF and PKD-binding domains regulate aspects of β-adrenergic-induced cardiac hypertrophy possibly through cardioprotective roles. These findings suggest that additional AKAP13 protein domains are sufficient for regulating normal mouse development, but that AKAP13 is critical for transducing signaling events that regulate stress responses, such as regulating cardiac function during hypertrophy. The mice generated in this study provide an ideal system to investigate the roles of specific AKAP13 protein domains in mediating these stress responses. They could also be used to investigate the roles of AKAPs in pathological responses to injury, particularly in tissues expressing AKAP13, such as blood vessels, the eye, and the brain.
Materials and Methods
Ethics Statement
All mouse studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee and the Laboratory Animal Research Center at the University of California, San Francisco. Protocol ID: AN080925-02B.
Expression Analysis of the AKAP Gene Family
Publicly available microarray datasets were analyzed by GC-RMA to determine expression profiles during mouse development [21] and ES cell differentiation [22]. Gene expression during mouse development was compared to expression in a blastocyst (GEO series GSE1133). Gene expression during mouse ES cell differentiation was compared to pluripotent mouse ES cells (GSE3749). The largest fold change was reported when greater than an absolute fold change of 1.8. The data set containing mouse developmental time points also included a large number of adult tissues. We considered a gene to be present (P) during mouse development if its expression was twofold higher than the minimum expression across all samples.
Characterization of AKAP13 Gene-Trap ES Cells
Gene-trap events within AKAP13 were identified from the International Gene Trap Consortium (IGTC) database (at www.genetrap.org) and the IGTC Sequence Tag Alignments track on the UCSC Genome Browser [34], [35]. From the sequence tag alignments, we identified ten uniquely trapped exons for AKAP13. We mapped these trapping events onto the AKAP13 protein to identify the domains affected by the traps. The following cell lines were obtained from the Mutant Mouse Regional Resource Centers: AG0213 (for AKAP13-ΔBrx), CSJ306 (for AKAP13-ΔGEF), & CSJ288 (for AKAP13-ΔPKC) (Fig. 1). The feeder-free gene-trap ES cell lines were cultured in normal growth media supplemented with murine leukemia inhibiting factor as described [33]. Correct splicing of AKAP13 exons into the gene-trap construct was verified by RT-PCR and sequencing. Total RNA was extracted from ES cells with Trizol (Invitrogen), and RT-PCR was conducted using the SuperScript III One-Step RT-PCR kit (Invitrogen). Forward primers for RT-PCR were designed using Primer3 (Table 4A) [59]. The resulting products were sequence verified and confirmed the expected AKAP13–gene-trap splicing events.
10.1371/journal.pone.0062705.t004Table 4 Primer Sequences.
A. RT-PCR primers for AKAP13-gene trap splicing
Primer Name Location (Mutant) Sequence (5'-3') Size (bp)
MJS218 Exon 8 (ΔBrx)
ACACCCAAGATGAAGCAAGG
441
MJS219 Exon Brx (ΔBrx)
AATTTCGGACCTGTGTGAGC
573
MJS220 Exon 21–22 (ΔGEF)
TGGAGTTGGCAATGATGAGA
674
AKAPlbc-F1_MS Exon 27 (ΔPKC)
TGAAGAGCACAACAGGAAGG
432
MJS213 Gene Trap (Univ. Rev)
TAATGGGATAGGTCACGT
B. Long-Range PCR primers in the gene trap construct
Primer Name
Location
Sequence (5'-3')
MJS236 βGal (Rev)
CCCTGCCATAAAGAAACTGTTACCC
MJS237 Neo
GTGGAGAGGCTATTCGGCTATGACT
C. Genotyping primers
Primer Name
Allele Identified
Sequence (5'-3')
Size (bp)
MJS299 Univ. ΔBrx (For)
TGGCATCTACCCAGGATCTC
MJS390 WT ΔBrx (Rev)
CAAAGGCCATCTGCACACC
1697
MJS284 GT ΔBrx (Rev)
GTGAGGCCAAGTTTGTTTCC
1275
MJS274 Univ. ΔGEF (For)
TACCAAATAACAGTGCCTGCTCTCC
MJS253 WT ΔGEF (Rev)
ATCTTGAGTGTGCGGATGTGATGTA
1533
MJS214 GT ΔGEF (Rev)
AGTATCGGCCTCAGGAAGATCG
1182
MJS339 WT ΔPKC (For)
TGTCTCTGGCCTGTTTGTGA
1112
MJS340 WT ΔPKC (Rev)
TCGGAAGAGGTTAAGGGACA
MJS272 GT ΔPKC (For)
ACATTTCCCCGAAAAGTGC
435
MJS260 GT ΔPKC (Rev)
GGCTCACACTGGGTTCAATC
(A) RT-PCR primers for verifying AKAP13 gene-trap splicing events are listed. The primer locations and mutant line verified are indicated. The size of the RT-PCR product is given in base pairs (bp). (B) The common long-range PCR primers within the gene-trap construct are listed. These primers were used with AKAP13 specific genomic DNA primers to identify the gene-trap insertion. (C) The genotyping primers used to identify the wild-type and mutant allele for the three mutant mouse lines are listed. The primer direction is also given: forward (For) and reverse (Rev). The size of the PCR product is given in base pairs (bp).
The genomic insertion sites for the gene-trap events were identified by long-range PCR of genomic DNA using Phusion High-Fidelity DNA Polymerase (Finnzyme). In summary, Primer3 was used to design ∼25mer forward and reverse primers with melting temperatures of 62–68°C throughout the introns containing the gene-trap insertions. These designed primers were used with common primers within the gene-trap construct to amplify genomic DNA (Table 4B). The PCR products were cloned into pCR-XL-TOPO (Invitrogen) and sequenced to identify the genomic insertion sites.
In Vitro Co-Immunoprecipitations
Full-length and truncation mutants for AKAP-Lbc were cloned into pcDNA3.1 with C-terminal fusion to V5. HEK293 cells were transfected with the AKAP-Lbc-V5 and pEGFP-PKD1 constructs and lysed as described [26]. Lysates were incubated on ice for 10 min and centrifuged at 20,000×g for 15 min at 4°C. Cleared lysates were incubated with Anti-V5 antibody (Invitrogen) for 1 h at 4°C with rocking, followed by precipitation of antibody-antigen complexes with protein A-agarose (Millipore). Immunoprecipitates were washed 5×1 ml in lysis buffer, eluted in SDS-PAGE sample buffer, and separated by SDS-PAGE. Antibodies used for immunoblotting were: anti-V5 (mouse; 1∶5000) from Invitrogen, anti-GFP (mouse; 1∶000) from Clontech, and anti-PKAc (rabbit; 1∶1000) from Cell Signaling.
In Vitro Kinase Assays
After immunoprecipitation of AKAP-Lbc-V5, immune complexes were washed five times with IP buffer (10 mM sodium phosphate, pH 6.95, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100) and then resuspended in kinase assay buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2). Assays were performed as described [60]. PKD activity assays were carried out in a total reaction volume of 50 µl, including 100 µM Syntide-2, 5 µM ATP, and 5 µCi of [γ-32P]-ATP in kinase assay buffer. Reactions were incubated for 20 min at 30°C, starting with the addition of ATP. Reactions were terminated by centrifugation and the reaction mix (40 µl) was spotted onto P81 phosphocellulose paper (Whatman). The phosphocellulose papers were washed three times with 75 mM phosphoric acid, once with acetone and then dried. Kinase activity was determined by liquid scintillation counting. PKA activity assays were performed as described for PKD. Before the assay, PKA catalytic subunit was eluted from AKAP-Lbc immune complexes by adding 50 µl of 10 mM cAMP and incubating for 20 min. PKA assays were carried out at 30°C for 20 min in a total reaction volume of 50 µl, using 20 µl of eluted PKA catalytic subunit, 200 µM Kemptide, 5 µM ATP, and 5 µCi of [γ-32P]-ATP in kinase assay buffer.
In Vitro Rho-GEF Assays
After immunoprecipitation of AKAP-Lbc-V5, immune complexes were washed five times with IP buffer (10 mM sodium phosphate buffer, pH 6.95, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100) and incubated with RhoA (40 pmol) in binding buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.5 mM EDTA, 50 mM NaCl, 5 mM MgCl2, 0.05% polyoxyethylene-10-lauryl ether (C12E10), and 10 µM GTPγS with ∼500 cpm/pmol [35S]GTPγS) in a final reaction volume of 50 µL. Reactions were terminated after 20 min incubation at 30°C by addition of wash buffer. GTPγS binding to RhoA was determined as described [61]. [35S]-GTPγS (specific activity = 1,250 Ci/mmol) was obtained from PerkinElmer Life Sciences.
Mouse Studies
Chimeric mice were generated by the Gladstone Transgenic Gene-Targeting Core by injecting C57Bl/6 blastocysts with the gene-trapped ES cell lines AG0213, CSJ306 and CSJ288. Male chimeric mice (N0) were backcrossed to C57Bl/6 (National Cancer Institute, National Institutes of Health) females and the resulting progeny (N1) were genotyped to identify heterozygotes carrying the gene-trap allele. Mice were genotyped from tail clips with a REDExtract-N-Amp Tissue PCR Kit (Sigma Aldrich) and the primer pairs listed in Table 4C. Heterozygous mice were inter-crossed to obtain homozygous mice, AKAP13ΔBrx/ΔBrx (from AG0213), AKAP13ΔGEF/ΔGEF (from CSJ306), AKAP13ΔPKC/ΔPKC (from CSJ288), for the three gene-trap mutational events, and litters were analyzed for Mendelian ratios at 3 weeks of age. All studies performed in this report used littermate and age-matched control and mutant mice generated from heterozygous crosses.
These mouse lines will be available through the Mutant Mouse Regional Resource Center (MMRRC).
X-Gal Staining of Gene-Trap Embryos and Adult Tissue
To identify AKAP13 expression patterns during development, whole-mount embryos at embryonic day (E)8.5, E9.5, and E10.5 and cryosectioned E14.5 embryos were stained with X-Gal. To determine embryonic ages, the morning a post-coital plug was identified was designated as E0.5. Whole embryos (E8.5, E9.5, and E10.5) were fixed in 2% formaldehyde (Sigma), 0.2% glutaraldehyde (Sigma), 0.02% sodium deoxycholate (Sigma), and 0.01% Nonidet P-40 substitute (Fluka) in PBS (Mediatech) for 15 to 45 min, depending on age, at 4°C. Embryos were permeabilized in 0.02% sodium deoxycholate and 0.01% Nonidet P-40 substitute in PBS at 4°C overnight. Embryos were stained in 5 mM potassium ferricyanide (Sigma), 5 mM potassium ferrocyanide (Sigma), 2 mM MgCl2 (Sigma), 1 mg/ml X-Gal (Fermentas, AllStar Scientific, or Invitrogen), 0.02% sodium deoxycholate and 0.01% Nonidet P-40 substitute in PBS at 37°C for 5 hours. Embryos were post-fixed in 4% paraformaldehyde (PFA) at 4°C overnight. Images were obtained on a Leica MZ16F dissecting microscope with a Leica DFC500 camera and Leica Application Suite software.
E14.5 embryos were bisected and fixed in 4% PFA and 0.2% glutaraldehyde in PBS for 1 hour at 4°C. The embryos were sucrose protected and frozen in Tissue-Tek OCT (Sakura Finetek). Cryostat sections were stained with X-Gal and mounted. Mosaic images of entire sagittal and transverse sections were obtained using an inverted Axiovert 200 M microscope and AxioCam HRc (Carl Zeiss) camera. Individual images were stitched together to create a mosaic image using AxioVision Software. Higher magnification images of specific regions of interest were obtained using an upright Leica DM4000B microscope with a QImaging Retiga EXi Fast 1394 camera and Image-Pro Plus software.
Adult organs were obtained from euthanized 17–18-week-old mice. Mice were perfused with 10 mM KCl (Sigma), followed by PBS, and finally with 4% PFA. Heart, kidney, and brain samples were bisected and organs were fixed in 4% PFA for 1 hour at 4°C. Organs were permeabilized in 2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% Nonidet P-40 substitute in PBS at 4°C overnight. They were stained in 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 1 mg/ml X-Gal, 0.02% sodium deoxycholate and 0.01% Nonidet P-40 substitute in PBS at 37°C for 5 hours. Organs were post-fixed in 4% PFA at 4°C overnight. Images were obtained on a Leica MZ FLIII dissecting microscope with an AxioCam (Carl Zeiss) camera and Openlab 4.0.4 software.
Quantitative PCR Analysis
Gene expression analysis was performed on total RNA isolated from neonatal mouse heart and lung tissue. Wild-type, heterozygous, and homozygous samples were collected from six mice each for the three mouse lines. Heart and lung samples were homogenized (4.5 mm Tissue Tearor, Research Products International) in Trizol (Invitrogen). cDNA was generated from 1 µg of TurboDNAse-treated (Ambion) total RNA with the SuperScript III First Strand Synthesis kit and random hexamers (Invitrogen) as described by the manufacturer. Expression was assessed using TaqMan probesets (Applied Biosystems) for AKAP13 exon-exon junctions E4-5 (Mm01320101_m1), Brx-9 (Mm01318390_m1), and E37-38 (Mm01320099_m1) as well as GAPDH (Mm99999915_g1) and β-actin (Mm00607939_s1). Reactions were run on an Applied Biosystems 7900HT real-time thermocycler. Samples were assayed in technical triplicates and average AKAP13 expression levels were determined from GAPDH and β-actin normalized values. Relative expression was calculated against wild-type mouse samples. Means ± standard deviations were reported for six mice of each genotype. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted to determine significant differences (Prism 5; GraphPad).
Electrocardiographic Analysis
Six-lead ECG analysis was conducted on 16–18-week-old wild-type and AKAP13ΔGEF/ΔGEF (ΔGEF) littermate male mice anesthetized with inhaled Isoflurane, USP (Baxter and Phoenix Pharmaceutical) [62]. In brief, anesthetized mice were placed on a heating pad, and body temperature was continually monitored to maintain at 36–37°C. Needle electrodes were implanted subcutaneously at each limb and ECGs were recorded for leads I, II, III, aVR, aVL, and aVF using the AD Instruments system: Dual BioAmp (ML135), PowerLab 4/30 (ML866) and Chart5 Pro (v5.4.2). ECG data were acquired for 15–45 seconds for each lead. The ECG recordings were analyzed using the mouse preset option in Chart5 Pro. The ECG signals were averaged within each lead and the temporal locations of P Start, P Peak, P End, QRS Start, QRS Max, QRS End, T Peak, and T End were identified and manually adjusted as needed. Values were calculated for heart rate, PR interval, P wave duration, QRS interval, and corrected QT interval (using the provided Mitchell et. al calculation). These calculated values were averaged across all leads for a given mouse. Means ± standard deviations were reported for six mice of each genotype. Two-tailed student’s t-test was conducted to determine significant differences (Excel).
Cardiac Structural Analysis
Hearts were isolated from the six wild-type and six ΔGEF littermate mice used for ECG analysis. Mice were weighed and euthanized and their hearts were collected and weighed. Hearts were washed with heparin (5 µg/ml) and PBS to remove the blood and incubated in 25 mM KCl to relax the cardiac muscle. The hearts were fixed in 4% PFA at 4°C overnight. The right tibia was removed and the length was measured using calipers (Scienceware). Hearts were imaged using a Leica MZ FLIII dissecting microscope with an AxioCam (Carl Zeiss) camera and Openlab 4.0.4 software. The hearts were then embedded in paraffin for sectioning. Five-micron sections were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) and Masson’s trichrome following standard protocols. Mosaic images of entire heart sections were obtained using an inverted Axiovert 200 M microscope and AxioCam HRc (Carl Zeiss) camera. Individual images were stitched together to create a mosaic image using AxioVision Software. Higher magnification images of specific regions of interest were obtained using an upright Leica DM4000B microscope with a QImaging Retiga EXi Fast 1394 camera and Image-Pro Plus software.
Isoproterenol-Induced Cardiac Hypertrophy
Cardiac hypertrophy was induced in 22–32-week-old wild-type and AKAP13ΔGEF/ΔGEF (ΔGEF) littermate mice [38]. Mice were treated for 14 days with isoproterenol (60 mg/kg per day; Sigma) diluted in PBS (Iso) or PBS alone (vehicle; Veh) using mini-osmotic pumps (Alzet Model 2002) implanted subcutaneously into the peritoneum. Three wild-type and three ΔGEF mice were Veh-treated, four wild-type and six ΔGEF mice were Iso-treated. On day 14 after initiating treatment, mice were weighed and euthanized. Their hearts were collected, weighed, and processed for structural analysis as described above. Sections were stained with H&E or Masson’s trichrome. Fibrosis was quantified from mosaic images of Masson’s trichrome stained sections using Image-Pro Plus software. Means ± standard deviations were reported. Two-tailed student’s t-test was conducted to determine significant differences (Excel and Prism 5; GraphPad).
One additional ΔGEF mouse was treated with Iso and died on the fourth day of treatment. Baseline echocardiography indicated that this mouse had enlarged right and left atria.
Echocardiography
Baseline (before implantation of mini-osmotic pumps) and end-point (day 13) echocardiograms were recorded for isoflurane-anesthetized mice as described [63]. M-Mode and B-Mode echocardiograms were recorded using the Vevo 770 Imaging System and RMV707B probe (VisualSonics). M-Mode measurements were taken for diastolic and systolic left ventricular anterior wall (LVAW;d & LVAW;s), internal diameter (LVID;d & LVID;s), and posterior wall (LVPW;d & LVPW;s). Corrected left ventricular mass (LV Mass; mg) was calculated from these measurements:
.
Left ventricle fractional shortening (FS) was also calculated from these measurements:
Measurements were made on three separate heartbeats for each mouse.
B-Mode measurements were taken for endocardial area and major axis at diastole and systole (End Area;d, End Area;s, & End Major;d, End Major;s respectively). These B-Mode measurements were used to calculate endocardial volume at diastole and systole (End Vol;d & End Vol;s), left ventricular stroke volume (End SV), and left ventricular ejection fraction (EF):
One set of B-Mode measurements were made per mouse.
Means ± standard deviations were reported. One-way ANOVA and Bonferroni’s multiple comparison tests were conducted to determine significant differences (Prism 5; GraphPad).
Statistical Analysis
Two-tailed student’s t-tests were performed using Excel or Prism 5 (GraphPad) software. One-way ANOVA followed by Bonferroni’s multiple comparison tests were performed using Prism 5 software (GraphPad).
Supporting Information
Information S1
Analysis of cardiac and developmental expression and gene-trap events for the AKAP family. A literature review was conducted to identify AKAPs expressed in cardiac tissue and that have cardiac phenotypes in cell culture or animals. The type of cardiac phenotype is indicated: Hyper = hypertrophy, Ca2+ = calcium regulation, K+ = potassium regulation, Dev = development. Publicly available microarray data sets were mined to determine the expression of AKAPs during mouse development and embryonic stem (ES) cell differentiation. Absolute fold changes are reported when greater than 1.8, and expressed, but unchanged, genes are marked as present (P). The number of uniquely trapped exons for each AKAP gene is indicated.
(DOC)
Click here for additional data file.
The authors would like to thank Mark von Zastrow, Benoit Bruneau, Silvio Gutkind, Oren Shibolet, James Segars, Joshua Wythe, Trieu Nguyen, Jennifer Ng, Faith Kreitzer, Jill Dunham and Gary Howard for valuable discussions and technical advice. We would also like to thank Paul Swinton of the Gladstone Institutes Transgenic Gene-Targeting Core for microinjection of ES cells and Jo Dee Fish and Caroline Miller of the Gladstone Institutes Histology Core for histological sectioning and staining.
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292 : H1187 –1192 .17028161 | 23658642 | PMC3637253 | CC BY | 2021-01-05 17:26:13 | yes | PLoS One. 2013 Apr 26; 8(4):e62705 |
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Mol CancerMol. CancerMolecular Cancer1476-4598BioMed Central 1476-4598-12-172349725610.1186/1476-4598-12-17ResearchEffects of HDM2 antagonism on sunitinib resistance, p53 activation, SDF-1 induction, and tumor infiltration by CD11b+/Gr-1+ myeloid derived suppressor cells Panka David J [email protected] Qingjun [email protected] Andrew K [email protected] James W [email protected] Division of Hematology-Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA2 Division of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, China3 330 Brookline Avenue, RW-571, Boston, MA, 02215, USA4 330 Brookline Avenue, RW-563A, Boston, MA, 02215, USA2013 5 3 2013 12 17 17 11 9 2012 27 2 2013 Copyright © 2013 Panka et al; licensee BioMed Central Ltd.2013Panka et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The studies reported herein were undertaken to determine if the angiostatic function of p53 could be exploited as an adjunct to VEGF-targeted therapy in the treatment of renal cell carcinoma (RCC).
Methods
Nude/beige mice bearing human RCC xenografts were treated with various combinations of sunitinib and the HDM2 antagonist MI-319. Tumors were excised at various time points before and during treatment and analyzed by western blot and IHC for evidence of p53 activation and function.
Results
Sunitinib treatment increased p53 levels in RCC xenografts and transiently induced the expression of p21waf1, Noxa, and HDM2, the levels of which subsequently declined to baseline (or undetectable) with the emergence of sunitinib resistance. The development of resistance and the suppression of p53-dependent gene expression temporally correlated with the induction of the p53 antagonist HDMX. The concurrent administration of MI-319 markedly increased the antitumor and anti-angiogenic activities of sunitinib and led to sustained p53-dependent gene expression. It also suppressed the expression of the chemokine SDF-1 (CXCL12) and the influx of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) otherwise induced by sunitinib. Although p53 knockdown markedly reduced the production of the angiostatic peptide endostatin, the production of endostatin was not augmented by MI-319 treatment.
Conclusions
The evasion of p53 function (possibly through the expression of HDMX) is an essential element in the development of resistance to VEGF-targeted therapy in RCC. The maintenance of p53 function through the concurrent administration of an HDM2 antagonist is an effective means of delaying or preventing the development of resistance.
p53HDM2HDMXMI-319Renal cell carcinomaMyeloid-derived suppressor cells (MDSC)SDF-1EndostatinCollagen prolyl hydroxylase
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Background
One of the major determinants of the response to angiogenesis inhibitors is the p53 status of the tumor cells. Yu et al, for example, showed in 2002 that tumors derived from p53(+/+) HCT116 colorectal carcinoma cells were far more sensitive to VEGF receptor targeted therapy than tumors generated from isogenic p53(-/-) cells [1]. This differential sensitivity to treatment correlated with the in vitro susceptibility of the tumor cells to the pro-apoptotic effects of hypoxia. Since the publication of these data over a decade ago, the known range of biologic effects regulated by p53 has expanded well beyond cell cycle control and the expression of pro-apoptotic genes to include such diverse functions as the suppression of angiogenesis [2]. It is possible that the differential sensitivity of p53(-/-) and p53(+/+) HCT116 tumors to VEGF receptor-targeted therapy is due to an ability of p53 to complement the effects of VEGF receptor inhibition on the tumor microcirculation.
Although the advent of small molecule inhibitors of VEGFR2 has vastly improved the treatment of patients with renal cell carcinoma (RCC), the response to these agents is generally short-lived [3]. The mechanisms by which tumors ultimately manage to evade the effects of these agents are numerous and only partly understood [3-5]. One such mechanism involves the production of chemokines (e.g. SDF-1, CSF-1, IL-8) that either drive angiogenesis directly or recruit macrophages and other myeloid lineage cells, including CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs), from the bone marrow into tumor tissue [5-11]. These cells produce a variety of factors that promote tumor growth, invasiveness, angiogenesis, and immunosuppression [10-13]. p53 has been shown to suppress the expression of SDF-1 [14,15]. Otherwise, little is known about how the p53 status of a tumor might affect the extent to which tumors are infiltrated by MDSC or the facility with which they develop resistance to VEGF-targeted therapy.
Another mechanism by which p53 suppresses angiogenesis is through the induction of genes that modify the extracellular matrix (ECM). Angiogenesis is negatively regulated, for example, by several ECM-resident peptides (e.g. endostatin, canstatin, arresten) which interact with integrin receptors on the surface of endothelial cells and suppress their proliferation, survival, and motility [16,17]. These peptides are all derived from the noncollagenous (NC1) domains of certain types of collagen through the action of proteases such as MMP9. The genes encoding the collagen α chains (e.g. COL4A1) from which these angiostatic peptides are derived as well as that encoding the prolyl hydroxylase needed for the post-translational modification and stabilization of collagen [i.e. α(II) PH] are direct p53 transcriptional targets [18,19]. p53 activation might therefore be expected to suppress the tumor microvasculature through the enhanced production of these peptides. As an illustration of this point, the production of arresten, an angiostatic collagen fragment processed from α1 collagen IV, is markedly diminished in p53(-/-) tumor cells and its overexpression has been shown to retard tumor growth and limit angiogenesis [19]. The role played by these collagen-derived peptides in the regulation of angiogenesis in RCC and the extent to which their production is regulated by p53 is unknown.
p53 levels are generally low in unstressed cells as a result of HDM2-dependent ubiquitination and proteasomal degradation [20]. p53 can be activated as a result of phosphorylation of any of several sites in its N-terminal domain, which dissociates p53 from HDM2 and enhances its stability [21]. Several of the kinases capable of phosphorylating p53 (e.g. ATM) are redox-sensitive and capable of activating p53 in the setting of hypoxia [22]. The p53 gene is intact (i.e. neither deleted, mutated, nor methylated) in most RCC [23]. One might therefore expect p53 to be activated in RCC subjected to the stress of angiogenesis inhibition. Several factors, however, limit the extent, duration, and biological consequences of p53 activation in these cells. RCC, for example, generally fail to express p53-dependent genes in response to DNA damage, presumably due to high constitutive NF-κB activity [24-26]. The transcriptional activity of p53 is also limited by a member of the POK family (KR-POK) frequently overexpressed in RCC [27]. This protein physically interacts with p53 and with the transcriptional corepressors NCoR and BCoR, resulting in reduced histone H3 and H4 acetylation at the promoters of certain p53-dependent genes (e.g. p21waf1/CDKN1A). These signaling aberrations suggest that p53 might not be able to contribute to the suppression of angiogenesis or any other biological process in RCC, despite the integrity of the p53 gene. Hammond et al, however, have pointed out that many of the functions of p53 in the setting of hypoxia are due to transcriptional repression rather than activation [28-31]. The anti-angiogenic effects of p53, for example, are in part due to the repression of the miR-17-92 microRNA family [32] and possibly to SDF-1 [14,15] and it is unclear how these functions would be affected by constitutive NF-κB activity or KR-POK expression.
Several drugs that inhibit HDM2 are in preclinical or Phase I trials [33-35]. These drugs offer distinct advantages over conventional chemotherapy in that they are able to activate p53 in genetically permissive tumor cells without inducing DNA damage. The studies described in this paper were undertaken to assess the effects of HDM2 blockade alone and in conjunction with VEGF-targeted therapies on p53 function, tumor growth, and angiogenesis in RCC.
Results
Sunitinib-induced p53 activation in RCC xenografts
To assess the effects of sunitinib treatment on tumor cell p53 levels and transcriptional activity, 1 × 107 786-0 or A498 cells were implanted subcutaneously into the flanks of nude/beige mice and the resulting tumors allowed to grow to a diameter of 10 mm, at which point sunitinib treatment (50 mg/kg daily) was begun. The growth of 786-0 xenografts is typically arrested by sunitinib for a period of only 7-10 days, after which growth resumes despite the continued administration of the drug [36]. In the case of A498 xenografts, sunitinib-induced growth arrest extends to approximately 40 days, after which the tumors become resistant to treatment. With each xenograft model, the tumor-bearing mice were randomly divided into three groups and sacrificed at one of three time points, after which the tumors were promptly excised and frozen in liquid N2. One-third of the tumor-bearing mice were untreated and sacrificed when the tumors reached 16 mm in diameter. Half of the remaining mice were sacrificed at a point when tumor measurements were stable on treatment (day 3), and the other half were sacrificed at a point when sunitinib resistance had developed (tumor size 16 mm).
Tumors were thawed, lysed, and the lysates analyzed by western blot for p53, and the p53 dependent genes p21waf1, HDM2, HDMX, and NOXA. As shown in Figure 1, p53 levels increased markedly in response to sunitinib administration and remained elevated throughout the course of treatment in both 786-0 and A498 xenografts. The p53-dependent genes encoding p21waf1 and HDM2 were also induced early during treatment but this effect was transient in that the levels of both proteins reverted to baseline with the emergence of drug resistance, despite persistent expression of p53. NOXA was undetectable in untreated 786-0 and minimally expressed in A498 xenografts. However, in both xenografts, levels rose significantly early during treatment only to decline with the development of resistance. The p53 antagonist HDMX was also constitutively present in A498 and 786-0 xenografts and in both models, HDMX disappeared from the tumor lysates early during treatment only to reappear with the development of resistance. These data suggest that although p53 is stably induced by sunitinib treatment, its function as a transcription factor becomes impaired at some time point during treatment. The data also establish a temporal link between this loss of p53 function, the induction of HDMX, and the development of sunitinib resistance.
Figure 1 p53 activation in 786-0 and A498 RCC xenografts during sunitinib treatment. Lysates were from control (vehicle only), sunitinib, day 3 (sunitinib responding) and sunitinib, day 21 (suntinib resistant) mice. Lanes represent data from individual tumors for each treatment group. Blots were probed for p53, and the p53 dependent genes noxa, hdm2 and p21, as well as hdmx and vinculin.
Effect of HDM2/HDMX inhibition on tumor growth and p53 function
To assess the effect of HDM2/HDMX inhibition on tumor growth, 786-0 and A498 tumors generated as described above and in Methods were allowed to reach a diameter of 10 mm. Tumor-bearing mice were then divided into four treatment groups and treated with either sunitinib (50 mg/kg), the HDM2/HDMX antagonist MI-319 (200 mg/kg), both drugs, or saline daily by gavage. As shown in Figure 2A, sunitinib and MI-319 had only a modest growth-retarding effect on 786-0 xenografts when the drugs were administered individually. However, the combination of both drugs actually induced tumor regression (p < 0.0001 combination vs suntinib alone; p < 0.0002 vs MI-319 alone). Sunitinib as a single agent had a more pronounced effect on A498 than on 786-0 xenografts. MI-319 likewise had single agent activity in this model and augmented that of sunitinib (p < 0.006 combination vs suntinib alone; p < 0.0187 vs MI-319 alone). The basis for the different responses of these two VHL-deficient RCC cell lines to treatment is unknown.
Figure 2 A). Effects of sunitinib and MI-319 on the growth of 786-0 and A498 xenografts. Tumor volume was normalized to the initial volume when treatment began for each individual tumor for each treatment group. Each growth curve represents the mean from 6 mice in each treatment group. B). p53 activation in RCC xenografts. Lysates were from tumors on day 21 after the start of treatment. Lanes represent data from individual tumors for each treatment group. Blots were probed for p53, and the p53 dependent genes hdm2 and p21, as well as hdmx and vinculin.
In this study, all tumors were removed on day 21 or when the untreated tumors reached a diameter of 20 mm. Excised tumors were then divided and one half frozen for biochemical analysis and the other half paraffin-embedded for IHC. As shown in Figure 2B, p21waf1 was undetectable in the 786-0 tumors from sunitinib alone-treated mice (despite abundant p53) but readily seen in the tumors from the dually treated xenografts. HDM2 was detectable in the tumors from mice treated with MI-319 alone or the drug combination, but not in those from mice that received sunitinib alone. In the A498 xenografts, both p21waf1 and HDM2 were absent from the sunitinib alone-treated tumors but abundant in the tumors excised from mice treated with either MI-319 alone or the sunitinib/MI-319 combination. HDMX was present in all tumors except those from the untreated (control) mice. These data indicate that the concurrent administration of MI-319 is able to maintain the expression of the p53-dependent genes p21waf1 and HDM2 despite the presence of HDMX, suggesting that MI-319 has significant activity against both HDM2 and HDMX.
Proapoptotic, antiproliferative and antiangiogenic effects of MI-319
To assess the ability of MI-319 and sunitinib treatment to induce tumor cell apoptosis, TUNEL assays were performed on histologic sections of tumors obtained from mice in the various treatment groups. Sunitinib (but not MI-319) treatment resulted in a significant increase in the number of TUNEL-positive cells in both tumor models (p < .001 vs control for both 786-0 and A498) However, MI-319 increased the pro-apoptotic effect of sunitinib only in 786-0 (p <0.021), but not A498 xenografts (Figure 3A).
Figure 3 Effects of treatment on (A and B) apoptosis (tunel) and (C and D) proliferation in RCC xenografts. In both 786-0 (A) and A498 (B) models, sunitinib induced apoptosis as shown by an increase in tunel positive cells. The addition of MI-319 increased apoptosis in 786-0 but not A498 xenografts. Data is presented as a bar graph showing the mean percent tunel positive cells from six tumors in each treatment group. Sunitinib treatment increased Ki-67 nuclear staining in 786-0 (C) but not A498 (D) xenografts. The Ki-67 staining in 786-0 xenografts from suntinib treatment was suppressed in the presence of MI-319. Data is presented as a bar graph showing the mean percent Ki-67 positive cells from six tumors in each treatment group.
The effects of the two drugs on proliferation were assessed by Ki-67 staining. Sunitinib treatment increased the number of cycling cells only in 786-0 xenografts (p < 0.05) and this proliferative effect was blocked by MI-319 (p < 0.004). As a single agent, MI-319 had no discernible antiproliferative effect in either 786-0 or A498 xenografts (Figure 3B).
The antiangiogenic effects of sunitinib and MI-319 were assessed by IHC using an anti-CD31 antibody. As shown in Figure 4, both drugs individually induced a marked decline in microvessel density (MVD) (p < 0.0001 for either drug vs untreated) in both xenograft models and in 786-0 xenografts, the effects of the two drugs were additive (p < 0.0007 for both drugs vs sunitinib).
Figure 4 Effects of treatment on microvessel density (MVD) in RCC xenografts. In both (A) 786-0 and (B) A498 models, sunitinib and MI-319 suppressed tumor angiogenesis. In 786-0, the effects of the two drugs were additive. Data is presented as a bar graph showing the mean MVD from six tumors in each treatment group. A representative tumor section stained for CD31 from each treatment group for 786-0 and A498 xenografts are in C and D, respectively.
Effect of MI-319 on sunitinib-induced tumor infiltration by CD11b+/Gr-1+ MDSC
Tumor-infiltrating myeloid-derived suppressor cells (MDSC) dually expressing CD11b and Gr-1 have been shown to contribute to the development of resistance to several forms of treatment, including antiangiogenic agents that target VEGF receptor signaling [5-11]. To assess the effects of sunitinib and MI-319 on the accumulation of these cells in tumor tissue, tumors of mice from the various treatment groups were analyzed by immunofluorescence. Photographs of the 786-0 slides are shown in Figure 5A and bar graphs of the data from both 786-0 and A498 tumors are shown in Figure 5B. As shown in the figure, very few CD11b+/Gr-1+ MDSC were detected in untreated 786-0 or A498 xenografts. However, in both xenografts, sunitinib treatment induced an influx of these cells (p < 0.0001 for 786-0, sunitinib vs untreated; p < 0.021 for A498) which was markedly attenuated by the concurrent administration of MI-319 (p < 0.0001 for 786-0, both drugs vs sunitinib; p < 0.036 for A498). In the 786-0 model, this suppression of MDSC tumor infiltration was essentially complete. Of note, MI-319 did not suppress tumor infiltration by all myeloid cells as indicated by the persistence of red (but not magenta) cells in the dually treated tumors. The total number of CD11b+ cells present within the tumors was essentially the same in the sunitinib and sunitinib/MI-319 treatment groups, suggesting that the suppressive effects of MI-319 were directed at specific subpopulations of CD11b+ myeloid cells.
Figure 5 Effects of treatment on CD11b+/Gr-1+ MDSC infiltration into 786-0 and A498 xenografts. A). Immunofluoresence data from 786-0 xenografts. In this study, CD11b = red, Gr-1 = blue. Dually expressing cells are magenta colored; tumor nuclei are yellow. B). Bar graph generated from the manual counting of CD11b+/Gr-1+ cells from five individual tumors from each treatment group. Data from both 786-0 and A498 xenografts are shown. C). SDF-1 levels in RCC xenografts. Lysates were from tumors on day 21 after the start of treatment. Lanes represent data from individual tumors for each treatment group.
The accumulation of CD11b+/Gr-1+ MDSC within tumor tissue is driven by several chemokines (e.g. SDF-1) produced by tumor and associated stromal cells [5-11]. The production of the SDF-1 is known to be hypoxia-induced and negatively regulated by p53 [14,15]. One would therefore predict that treatment with an angiogenesis inhibitor such as sunitinib would induce the expression of SDF-1 and the concurrent administration of an HDM2 antagonist such as MI-319 might block this induction. To test this hypothesis, tumor lysates from the various treatment groups were analyzed by western blot for SDF-1. As shown in Figure 5C, SDF-1 was not detected in 786-0 tumor lysates from untreated mice. Sunitinib treatment induced SDF-1 expression, however, and this induction was completely suppressed by the concurrent administration of MI-319. In A498 xenografts, SDF-1 was present constitutively but increased with sunitinib treatment. As with the 786-0 xenografts, this induction was suppressed by MI-319.
α(II) prolyl hydroxylase induction by sunitinib: effects of treatment on endostatin and arresten deposition
α(II) PH is essential for the proper post-translational modification and stabilization of collagen α chains and for the production of angiostatic peptides (e.g. endostatin, canstatin, arresten) from their non-collagenous NC1 domains [18,19]. The gene encoding this enzyme is p53-dependent. To determine the extent to which p53 activation regulates the deposition of endostatin and arresten in the ECM of RCC, mice bearing xenografts generated from 786-0 stably transfected with a tetracycline-regulable p53 shRNA (see Methods) were treated with sunitinib with or without the inclusion of doxycycline in the drinking water. The mice were then sacrificed and the tumors excised. As shown in Figure 6A, sunitinib treatment was less effective in the absence of p53, especially during the first few days of treatment (p < 0.025 at day 7, sunitinib alone vs sunitinib + doxycycline). In fact, the growth curve of the sunitinib + doxycycline-treated mice overlapped with that of the control mice. Analysis of tumor lysates showed a complete suppression of endostatin and arresten production by the tumors that failed to activate p53 in response to sunitinib (Figure 6B). These data suggest that p53 activation is essential for the deposition of endostatin and arresten triggered by the administration of sunitinib in RCC xenografts.
Figure 6 A). Effect of p53 knockdown on the response of 786-0 xenografts to sunitinib treatment. The expression of a p53 shRNA markedly reduced the antitumor effect of sunitinib. Each growth curve represents the mean tumor volume from 6 mice in each treatment group. B). p53 knockdown blocked the deposition of endostatin and arresten otherwise induced by sunitinib treatment. Lysates were from tumors on day 28 after the start of treatment. Lanes represent data from individual tumors for each treatment group. Blots were probed for p53, p21, endostatin, arresten and vinculin.
To determine if the variable p53 function observed during the course of treatment with sunitinib affected the levels of α(II) PH, endostatin and arresten, the tumor lysates from Figure 1 were analyzed by western blot for these proteins. As shown in Figure 7A and B, low levels of α(II) PH, endostatin and arresten were detectable in untreated 786-0 and A498 xenografts, but all three proteins were up regulated by sunitinib treatment. However, in contrast to p21, Noxa, and HDM2, which nearly disappeared with the development of resistance (Figure 1), endostatin and arresten persisted at nearly the same level with the onset of drug resistance. These data suggest that sustained p53 transcriptional activity is not required to maintain endostatin and arresten levels in the tumor ECM and that the development of resistance cannot be due to a reduction in the level of these angiostatic peptides.
Figure 7 The effects of treatment on α(II) PH, endostatin, and arresten levels in RCC xenografts. A, B). The expression of all three proteins was induced by sunitinib treatment and levels did not diminish with the onset of drug resistance. C, D). Levels of endostatin and arresten were increased by treatment with either sunitinib or MI-319 but the inductive effects of the two drugs were not additive. Lanes represent data from individual tumors for each treatment group.
To determine if HDM2 blockade could increase endostatin or arresten levels beyond those achieved with sunitinib alone, 786-0 xenografts were treated with sunitinib, MI-319, or both drugs and the tumors examined by western blot. As shown in Figure 7C and D, the levels of neither endostatin nor arresten were further increased by the concurrent administration of MI-319 in either 786-0 or A498 xenografts. These data suggest that the transient activation of p53 induced by sunitinib treatment in genetically permissive RCC is sufficient to maximize the deposition of endostatin and arresten in the ECM. The data also suggest that the superior antitumor and anti-angiogenic effects of the sunitinib/MI-319 combination cannot be explained by an increase in the abundance of these angiostatic collagen fragments in the ECM. Of note, single agent MI-319 increased p21waf1 and arresten levels in A498 xenografts, the only model of the two evaluated in which the drug had single agent antitumor activity.
Discussion
Despite the numerous constraints on p53 function in RCC [24-27], sunitinib treatment does induce the expression of several p53-dependent genes (e.g. NOXA, HDM2, p21waf) in RCC xenografts. The induction of these genes is, however, limited to the interval during which tumor growth is suppressed and is attenuated once resistance develops. Although several factors have been shown to block p53 transcriptional activity in RCC, these are for the most part stable genetic alterations (e.g. KR-POK expression) that are not known to be subject to regulation by hypoxia or other metabolic changes that occur during treatment with angiogenesis inhibitors.
The factor(s) responsible for the transient activation and subsequent inactivation of p53 transcriptional activity during the course of treatment with sunitinib are unknown but at least one well-characterized p53 transcriptional suppressant (i.e. HDMX) appears to be temporally linked to p53 function in our xenograft models and may therefore be a candidate. Unlike its binding partner HDM2, HDMX is not regulated by p53 [37]. HDMX is constitutively expressed in RCC xenografts but vanishes with the initiation of sunitinib treatment – along with the appearance of p21waf. HDMX reappears with the development of resistance, in association with the down modulation of p21waf. These temporal associations strongly implicate HDMX as the factor responsible for the failure of p53 to maintain p21waf1 expression. The reappearance of HDMX during sunitinib treatment also explains why the suppression of p53 with an shRNA affected the response of 786-0 xenografts to sunitinib only during the first few days of treatment. As shown in Figure 6A, the xenografts in which p53 activation is not impeded characteristically stall for several days during sunitinib treatment but subsequently catch up with those in which p53 expression is suppressed. These data are consistent with the inactivation of p53 function by HDMX.
HDMX is physically associated with HDM2 and drugs that block the interaction between HDM2 and p53 such as MI-319 also interfere to some extent with the ability of HDMX to suppress p53 transcriptional activity. The fact that MI-319 maintains p21waf levels during sunitinib treatment suggests that the factor responsible for limiting p53 nuclear function most likely interacts with HDM2. This consideration, in addition to the temporal linkage between HDMX expression and the absence of p21waf, supports the hypothesis that HDMX is the dominant regulator of p53 nuclear function during sunitinib treatment and possibly a major factor in the development of drug resistance.
Despite the induction of p21 (Figure 2B), MI-319 treatment does not have a consistent effect on tumor cell proliferation as determined by Ki67 staining (Figure 3). In A498 xenografts, for example, MI-319 neither retards proliferation when administered as a single agent or when given concurrently with sunitinib. In 786-0 xenografts, however, the addition of MI-319 suppresses the increase in proliferation induced by sunitinib treatment. The enhanced tumor cell proliferation induced by sunitinib is presumably the result of tumor hypoxia, which has been reported to enhance proliferation in other tumor models [38].
The anti-angiogenic effect of sunitinib is augmented by the concurrent administration of MI-319. One mechanism by which MI-319 might further limit angiogenesis is the suppression of the influx of MDSC that generally occurs in response to sunitinib treatment. This suppressive effect is particularly obvious in 786-0 xenografts, from which MDSC are virtually excluded by MI-319 treatment. The means by which MI-319 (and p53) inhibit MDSC trafficking from the bone marrow to tumor tissue is not known but may involve the suppression of chemokine (e.g. SDF-1) production by tumor cells and stromal elements that were rendered hypoxic by the disruption of the tumor vasculature. The ability of MI-319 to suppress both baseline and sunitinib-induced SDF-1 expression in our RCC xenografts is consistent with the known ability of p53 to suppress SDF-1 expression [14,15]. Although our data suggest that the suppression of SDF-1 may account for the diminution in the influx of MDSC observed in the tumor infiltrates of mice treated with MI-319, it is possible that other factors that are both hypoxia-inducible and suppressed by p53 will be identified that might contribute to the anti-angiogenic effects of the drug.
Our observation that sunitinib treatment increases MDSC infiltration of RCC xenografts is at odds with several previous reports showing that the drug limits the expansion of these cells and enhances immune function [39-43]. Most of these earlier reports, however, were based on analyses of peripheral blood or splenocytes. Ko et al, for example, showed that RCC patients have increased numbers of MDSC in the peripheral blood and that sunitinib treatment results in a decline in their numbers [39]. Sunitinib treatment consistently reduces MDSC accumulation in the spleens of tumor-bearing mice in several tumor models [40]. However, this effect on splenic MDSC did not extend to the tumor microenvironment, where MDSC continued to accumulate with the expected deleterious effect on T cell function, regardless of treatment. These site-specific effects may be attributable to the cytokine GM-CSF, which is capable of rendering MDSC resistant to the effects of sunitinib [40].
Our studies suggest that sunitinib can actually increase the influx of MDSC into tumor tissue in some circumstances. This result may be unique to VHL-deficient RCC and dependent on the severity of the hypoxia induced in these tumors by VEGF-targeted agents. To the extent that this is the case, one would expect that these tumors would abundantly produce SDF-1 and other HIF-dependent chemokines (which recruit MDSC) in response to sunitinib treatment. The suppressive effects of sunitinib on MDSC accumulation and function are thought to be mediated through the inhibition of STAT3 and c-kit [41,42]. It is possible that hypoxia-induced chemokine production within tumor tissue may in some circumstances trump these inhibitory effects of sunitinib, resulting in an increase in MDSC infiltration.
Another mechanism by which p53 regulates angiogenesis is through the induction of α(II) PH and the deposition of anti-angiogenic collagen fragments (e.g. arresten, endostatin, canstatin) in the ECM [16-19]. Several previous studies have in fact suggested that this is one of the dominant mechanisms by which tumor angiogenesis and growth are suppressed by p53 [19]. Our data clearly establish that p53 activation is essential for the deposition of endostatin and arresten in 786-0 xenografts (Figure 6A) and in this respect, our results corroborate the results of Assadian et al, who demonstrated a similar requirement for p53 in the production of arresten by HCT116 cells [19]. Despite the absolute requirement for p53, however, sustained p53 activation does not appear to be essential to maintain endostatin and arresten levels in RCC xenografts during sunitinib treatment. We have not been able to demonstrate a significant decline in endostatin or arresten levels after the initial induction despite the apparent loss of p53 transcriptional activity (i.e. the disappearance of p21waf1) during treatment. Indeed, endostatin and arresten levels remain nearly unchanged with the development of sunitinib resistance, when p21waf1 is no longer detectable. Nor have we been able to demonstrate any enhancement in endostatin or arresten deposition by the addition of MI-319 to the treatment regimen, although HDM2 antagonism is essential for the maintenance of p21waf1 expression. Collectively, these data suggest that although the failure to express α(II) PH and to deposit angiostatic collagen fragments (e.g. endostatin, arresten) in the ECM might account for the faster growth and more vigorous angiogenesis observed in p53(-/-) tumors, changes in endostatin or arresten levels are not a factor in the development of sunitinib resistance in p53-WT RCC nor in the enhanced suppression of angiogenesis and tumor growth resulting from the concurrent administration of MI-319 with sunitinib.
We have demonstrated that treatment of mice bearing RCC xenografts with VEGF-targeted agents results in p53 activation, the biological effects of which are quickly undermined with the onset of drug resistance, possibly due to the induction of the p53 antagonist HDMX. We have further shown that the HDM2/HDMX antagonist MI-319 maintains p53 function during treatment and delays/prevents the emergence of resistance. These data suggest that the evasion of p53 function is an essential element in tumor escape from the effects of VEGF-targeted therapy. The effects of MI-319 appear to be at least in part due to the ability of the drug to suppress the influx of MDSC into the tumor, which may in turn be due to its ability to block the production of chemokines such as SDF-1 that are otherwise induced in the setting of hypoxia. The potential utility of a combination of an HDM2 antagonist with sunitinib may not be limited to RCC. For example, in a recent study by Henze et al, the HDM2 antagonist Nutlin-3 was shown to augment the apoptotic response of imatinib-resistant gastrointestinal stromal tumor (GIST) cell lines to sunitinib [44]. In this case, however, the effects of sunitinib were most likely attributable to its ability to inhibit c-kit rather than its antiangiogenic effects. Collectively, these data provide a strong rationale for the concurrent use of HDM2 antagonists as adjuncts to VEGF receptor inhibitors in the management of metastatic RCC and other tumor types.
Materials and methods
Cell lines and reagents. The human RCC cell lines 786-0 and A498 were obtained from ATCC and maintained in RPMI-1640 (Lonza) and Eagle minimal essential medium (ATCC), respectively containing 10% fetal bovine serum (USA Scientific), 2 mM glutamine and 50 μg/ml gentamycin at 37°C in 5 percent CO2. The MI-319 was provided by Ascenta Therapeutics (Malvern, PA) and Sanofi-Aventis (Paris, France).
Western blots
Cells were treated as described in Results and then lysed in Lysis Solution (Cell Signaling) supplemented with sodium fluoride (10 μM, Fisher Scientific, Hampton, NH) and phenylmethylsulfonyl fluoride (100 μg/ml, Sigma-Aldrich, St Louis, MO). Lysates were fractionated in 8-16% gradient SDS-polyacrylamide gels as indicated and the separated proteins were transferred to nitrocellulose. The blots were probed for the proteins of interest with specific antibodies followed by a second antibody-horse radish peroxidase conjugate and then incubated with SuperSignal chemiluminescence substrate (Pierce, Rochford, IL). The blots were then exposed to Kodak X-Omat Blue XB-1 film. The p21waf1, noxa, SDF-1, collagen type XVIII (endostatin) and collagen type IV (arresten) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the p53 antibody was purchased from Cell Signaling (Beverly, MA); the HDMX and HDM2 antibodies were obtained from ABCAM (Cambridge, MA). The vinculin antibody was obtained from Sigma (St. Louis, MO). The CD11b antibody conjugated to Alexa 488 and the Gr-1 antibody conjugated to Alexa 647 were purchased from Biolegend (San Diego, CA). The α(II) PH antibody was obtained from Bethyl Laboratories (Montgomery, TX).
Xenograft model
All animal studies were conducted according to an Institutional Animal Care and Use Committee (IACUC)-approved protocol at the Beth Israel Deaconess Medical Center. Six to eight week old athymic nude/beige female mice (Charles River Labs) were implanted subcutaneously with 1.0 × 107 RCC cells. When the tumors reached 10 mm in diameter, the mice were divided into 4 treatment groups of 6 mice each and treated daily for 21 days by gavage with sunitinib (50 mg/kg), MI-319 (200 mg/kg), sunitinib + MI-319, or saline (control). The doses of sunitinib [36,45] and MI-319 [46,47] were as previously reported. Tumors were measured bidimensionally daily. Tumor tissue from the sacrificed mice was frozen in liquid N2 for western blot analysis as described in Results or fixed in formalin for paraffin embedding.
Immunohistochemistry and immunofluorescence microscopy
The paraffin-embedded tumor tissue was sectioned at 5 microns using a Leica RM 2125 rotary microtome. The sections were dewaxed at 60°C, serially immersed in solutions of decreasing alcohol concentration, and then boiled in 10 mM sodium citrate, pH 6.2, for 30 minutes to unmask antigens. The tissue was then incubated in 3% hydrogen peroxide for 5 minutes, blocked with 1% BSA and 5% goat serum, and incubated overnight at 4°C with an antibody to Ki-67 (Dako, Carpinteria, CA). The Ki-67 epitope was detected using a biotinylated anti-mouse Ig antibody and an avidin-horseradish peroxidase conjugate (Vector Laboratories, Burlingame, CA). Similarly, sections were stained for endothelial cells with an antibody to CD 31 (ABCAM), followed by a biotinylated anti-rabbit Ig antibody (Vector Laboratories, Burlingame, CA). Slides were then counterstained with hematoxylin, dehydrated, and mounted. Tissue staining was quantitated using IMAGE Pro 6.0 software (MediaCybernetics, Inc, Bethesda, MD).
The sections were assayed for apoptosis using the TUNEL method (Millipore, Billerica, MA) in accordance with an established protocol [48]. The tissue was hydrated and treated sequentially with proteinase K and hydrogen peroxide, and then blocked as described above for the Ki-67 staining. The sections were then exposed to a solution containing mixed nucleotides, some of which were digoxygenin-labeled, and terminal deoxynucleotidyl transferase (TdT). The slides were developed with an anti-digoxigenin antibody-peroxidase conjugate and DAB substrate.
Immunofluorescence microscopy was utilized to image the infiltration of the CD11b+/ Gr-1+ MDSC cells with each paraffin embedded tissue. The protocol followed the procedure outlined above for Ki-67 and CD31 staining for dehydration to hydration and unmasking followed by blocking with 5% normal goat serum in PBS/0.05% triton ×-100. Antibodies to CD11b antibody conjugated to Alexa 488 and the Gr-1 antibody conjugated to Alexa 647 were added concurrently at 1:200 dilution in PBS/1% BSA/0.05% triton ×-100 and incubated overnight at 4°C. After several washings with PBS, nuclei were stained with Bisbenzimide H33342 (Alexis Biochemicals, San Diego, CA). Immunofluoresence microscopy was carried out with a Nikon TE-2000E microscope at 20× magnification and a Hamamatsu Orca ER camera. The data was acquired with Nikon’s NIS-Elements and analyzed with ImageJ software.
Design and construction of tet-inducible p53 shRNA-transfected 786-0 cell line
To generate 786-0 cells expressing a tetracycline inducible shRNA to p53, the shRNA sequence selector and shRNA hairpin oligonucleotide sequence designer software provided by BD Clontech was used to select optimal sequences. Three shRNAs were generated for each gene to be silenced. To produce tetracycline-regulable shRNAs, the oligonucleotides selected were cloned into the pSingle-tTS-shRNA vector (BD Clontech). This vector is a tet-on vector. The three shRNA constructs were transfected as a group into 786-0 cells and stable transfectants obtained by selection in G418. Clones were screened individually for inducible expression of the shRNA (i.e. the suppression of doxorubicin-induced p53 expression as determined by Western blot) and 2-3 representative clones were selected for each shRNA based on the degree to which tetracycline exposure suppressed p53 expression.
Statistical analysis
In vitro data depicted as bar graphs represent mean values from at least 3 separate experiments +/- standard error. For most of the studies shown, the significance of an apparent difference in mean values for any parameter (e.g. the percent of cells staining with propidium iodide) was validated by a Student’s unpaired t test and the difference considered significant if p <0.05. For the xenograft studies, the growth curves of the different treatment groups were statistically compared using one-way ANOVA.
Abbreviations
SDF-1: Stromal cell-dervived factor-1; VEGF: Vascular endothelial growth factor; PH: Prolyl hydroxylase; RCC: Renal cell carcinoma; ECM: Extracellular matrix; ATM: Ataxia telangiectasia mutated; HDM2: Human double minute 2.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QL and AG carried out many of the xenograft experiments, immnuohistochemistry, wide field fluorescence and western blots. JM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. DP also conceived of the study, and participated in its design and coordination and helped to draft the manuscript. In addition, DP performed all in vitro experiments including the generation of tet-regulable shRNA cell lines and their implementation, immnuohistochemistry, wide field fluorescence and western blots. All authors read and approved the final manuscript.
Acknowledgments
This work was supported by a developmental project from the NCI SPORE in Renal Cancer 5P50CA101942 and by the 2012 AACR-Kure-It Grant for Kidney Cancer Research (12-60-36 to JWM).
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Cancer MedCancer MedcamCancer Medicine2045-76342045-7634Blackwell Publishing Ltd Oxford, UK 10.1002/cam4.51Clinical Cancer ResearchExpression of adrenomedullin in human colorectal tumors and its role in cell growth and invasion in vitro and in xenograft growth in vivo Nouguerède Emilie 1Berenguer Caroline 1Garcia Stéphane 2Bennani Bahia 3Delfino Christine 1Nanni Isabelle 4Dahan Laetitia 5Gasmi Mohamed 6Seitz Jean-François 5Martin Pierre-Marie 14Ouafik L'Houcine 141 Inserm, UMR 911-CRO2Marseille, F-13000, France2 Laboratoire d'Anapathologie, CHU Nord (AP-HM)Marseille, F-13000, France3 Laboratoire de Biologie du Cancer, Faculté de Médecine et de PharmacieBP 1893, Route de Sidi Harazem, Fès, Maroc4 Laboratoire de Transfert d'Oncologie Biologique (AP-HM)Marseille, F-13000, France5 Service d'oncologie digestive, CHU la Timone (AP-HM)Marseille, F-13000, France6 Service de Gastro-entérologie, CHU Nord (AP-HM)Marseille, F-13000, FranceCorrespondence L'Houcine Ouafik, Inserm UMR 911-CRO2, Faculté de Médecine Timone, Bd Jean Moulin, 13385 Marseille Cedex 05, France. Tel: (33) 491324443; Fax: (33) 49254232; E-mail: [email protected] 2013 29 1 2013 2 2 196 207 04 6 2012 08 11 2012 09 11 2012 © 2013 Published by John Wiley & Sons Ltd.2013This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). In this study, real-time quantitative reverse transcription demonstrated a significant expression of AM mRNA in tumor samples from colorectal cancer (CRC) patients in clinical stage II, III, and IV when compared with normal colorectal tissue. AM, CLR, RAMP2, and RAMP3 proteins were immunohistochemically localized in the carcinomatous epithelial compartment of CRC tissue. Tissue microarray analysis revealed a clear increase of AM, CLR, RAMP2, and RAMP3 staining in lymph node and distant metastasis when compared with primary tumors. The human colon carcinoma cells HT-29 expressed and secreted AM into the culture medium with a significant increase under hypoxia. Treatment of HT-29 cells with synthetic AM stimulated cell proliferation and invasion in vitro. Incubation with anti-AM antibody (αAM), anti-AM receptors antibodies (αAMR), or AM antagonist AM22–52 inhibited significantly basal levels of proliferation of HT-29 cells, suggesting that AM may function as an autocrine growth factor for CRC cells. Treatment with αAM significantly suppressed the growth of HT-29 tumor xenografts in vivo. Histological examination of αAM-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial cells and pericytes, and increased tumor cell apoptosis. These findings highlight the potential importance of AM and its receptors in the progression of CRC and support the conclusion that αAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting that AM may be a useful therapeutic target.
AdrenomedullinAM1 and AM2 receptorsangiogenesiscolorectal cancertumor growth
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Introduction
Colorectal cancer (CRC) remains a leading cause of cancer-related death worldwide despite recent advances in adjuvant chemotherapeutic regimens. CRC progresses through a series of clinical and histopathological stages ranging from single crypt lesions through small benign tumors (adenomatous polyps) to malignant cancers (carcinomas) 1. The model of colorectal tumorigenesis includes several genetic changes that are required for cancer initiation and progression 2. The earliest and most prevalent genetic event yet identified in colorectal tumorigenesis is the disruption of a functional adenomatous polyposis coli (APC) complex due to a mutation of the APC gene or the β-catenin gene, leading to inappropriate activation of the Wnt signaling pathway 3,4. These results in nuclear accumulation of β-catenin, which associates with the T-cell factor-4, leading to transcription of genes involved in dedifferentiation, cell proliferation, and survival events involved in carcinogenesis.
Adrenomedullin (AM) is a multifunctional peptide with properties ranging from inducing vasorelaxation to acting as a regulator of cellular growth and angiogenesis 5–7. AM binds to and mediates its activity through the G-protein-coupled receptor calcitonin receptor-like receptor (CLR), with specificity for AM being conferred by the receptor activity-modifying protein-2 (RAMP2) and -3 (RAMP3) 8. The ability of CLR/RAMP2 and CLR/RAMP3 to respond with high affinity to AM implies the existence of two molecularly distinct AM receptors, respectively, referred as AM1 and AM2 receptors 9. Two other AM receptors (L1 and RDC1) with different affinities have already been described 6. AM is widely expressed in a variety of tumor types 10 and was shown to be mitogenic for human cancer cell lines including lung, breast, colon, glioblastoma, kidney, and prostate lineages in vitro 11–14. Several in vivo studies have shown a reduction of tumor angiogenesis and growth upon the treatment with neutralizing AM antibodies 11, AM receptor antagonist 15,16, or AM receptor RNA interference 17.
In the gastrointestinal tract, it has been reported that the expression of AM in the small intestine was localized in the mucosa and submucosa, but not in the muscularis or serosa 18. A very weak expression in the rodent colon at a distal site of the small intestine has been reported 18. In a normal colon, AM is able to regulate blood supply and protect the gut mucosa. In addition, it has been demonstrated that AM expressed in epithelial neuroendocrine cells and epithelial microvilli could exert effects such as antibacterial activity or may influence the absorption of nutrients 19,20. Immunoreactive AM has been reported in the human stomach, small intestine, and colon 21.
Although the expression of AM was reported in colorectal carcinoma cell lines Snuc 1 and DLD-1 22,23, little is known about AM and its receptors as well as its role in CRC. The frequency and distribution of AM and AM receptors expressions in tumors have not been previously reported and the role of AM in CRC is not completely understood. In this study, we investigated the expression of AM and its receptors in a large cohort of human CRC (primary tumors, lymph node, and distant metastasis) and used colorectal carcinoma cell line HT-29 to investigate the role of AM in cultured cells in vitro and in HT-29 xenografts in vivo.
Materials and Methods
Patient data and colon tumor samples
Two series of patients were enrolled in this study. They were consecutively treated for CRC in CHU Nord between 1992 and 2000. The eligibility criterion was a proven metastatic colorectal adenocarcinoma by histological biopsy. All patients signed an informed consent to participate and were over 18 years old. Surgical samples taken from CRC tumors or nonneoplastic tissue were obtained from the AP-HM Tumor Tissue Bank (ISO 9001:2008) at the CHU in Marseilles. Tumors were classified according to the International Union against Cancer (UICC) into different classical clinical stages 24. In the first series, frozen samples of colorectal tissues (n = 91) conserved in the AP-HM tumor bank from 45 women and 46 men were classified according to their clinical stages as follows: normal tissue (n = 30), stage I (n = 8), stage II (n = 32), stage III (n = 12), and stage IV (n = 9) and used to quantitate the AM mRNA levels.
The second series included CRC samples (n = 147) embedded in paraffin with clinical stage I (n = 21), stage II (n = 41), stage III (n = 44), and stage IV (n = 41) from 68 women and 79 women between 33 and 88 years old (mean = 65.7 years; SD = 13 years). These samples were used for tissue microarray (TMA) analysis and immunohistochemistry. Primary tumors and lymph node samples from 47 patients were also recovered in the second series.
Cell culture
Human CRC cell line HT-29 was obtained from American Type Culture Collection (Rockville, MD) and maintained in minimum essential medium containing penicillin (50 U/mL), streptomycin (50 μg/mL), and glutamine (1 mg/mL), and supplemented with 10% fetal bovine serum. Cells were cultured under a moist 5%-CO2/95%-air atmosphere, and fed with fresh medium every 2 days, being routinely monitored for mycoplasma contamination (Roche Diagnostics, Meylan, France). Cells growing exponentially were harvested and prepared for RNA analysis and protein extracts. All culture media components were purchased from Invitrogen Life Technologies (Paris, France).
RNA preparation and real-time quantitative RT-PCR
Total RNA was prepared from frozen CRC tumors, HT-29 cells, and HT-29 tumor xenografts, reverse transcribed to cDNA, and quantified as described 25.
Development and characterization of polyclonal anti-human AM antibody
The polyclonal antibody against human AM was developed by use of the synthetic peptide corresponding to the entire AM1–52 amide peptide (Bachem, Weil am Rhein, Germany) as described 11. Female New Zealand rabbits received injections at multiple subcutaneous sites with 300 μg of synthetic peptide emulsified with complete Freund's adjuvant. The rabbits were subsequently further immunized at 2.5 week intervals with 120 μg of AM1–52 amide emulsified with incomplete Freund's adjuvant. The antisera obtained after the fourth booster injection were screened for anti-AM activity, and then affinity purified on rProtein A Sepharose Fast Flow columns (GE Healthcare, Vélizy-Villacoublay, France). The anti-AM polyclonal antibody (purified IgG) showed very low cross-reactivity (<7%) with AM-related peptides such as AM22–52 amide, AM26–52 amide, and AM13–37. Calcitonin, CGRP1–37 amide, CGRP8–37 amide, and amylin showed insignificant anti-AM antibody binding (<0.1%) despite some homology with AM. We also demonstrated that anti-AM antibody blocked the binding of 125I-AM to its cell-surface receptor on HT-29 cells in a dose-related manner.
Immunohistochemistry of AM, CLR, RAMP2, and RAMP3 proteins
Tumor specimens were frozen on dry ice/butane, and stored at −80°C. Frozen sections (6 μm) were cut on a Leica cryostat. Sections of each specimen were stained using hematoxylin and eosin (H&E). Immunohistochemistry was carried out using the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA). Optimal dilution for rabbit anti-AM polyclonal antibody (referred here as αAM), anti-CLR (αCLR), anti-RAMP2 (αRAMP2), and anti-RAMP3 (αRAMP3) developed and characterized following the same protocol described for the generation of anti-AM antibody 5,15 were respectively used at dilution of 1:1000, 1:3000, 1:2000, and 1:1500. Detection was carried out using DAB chromogen. As a control for immunostaining, the antibodies preabsorbed by human synthetic AM peptide (50 μmol/L; Bachem), CLR, RAMP2, and RAMP3 peptides (50 μmol/L synthesized in the laboratory) were used instead of the primary antibodies.
Tissue microarray construction, immunohistochemistry, and image analysis
Tissue microarray construction and analysis were performed as previously described 26. Sections of paraffin-embedded samples of human CRC specimens were analyzed using the automate image analyzer as described 27.
Cell proliferation and invasion assays
The effects of AM, AM22–52, αAM (purified IgG), αCLR, αRAMP2, and αRAMP3 on cell proliferation were examined at the indicated time points as described 11. The invasion was assessed using a modified Boyden chamber assay as previously described 5.
Peptide extraction and AM radioimmunoassay
Extraction of proteins and immunoreactive-AM (ir-AM) radioimmunoassay (RIA) were performed as previously described 12.
Experimental protocol for animals
Animal work was carried out in the animal facility of the School of Medicine according to the institutional animal welfare guidelines. Athymic NMRI (nu/nu) nude mice (Harlan, Gannat, France) were maintained in a sterile environment with a daily 12 h light/12 h dark cycle. HT-29 cells (3 × 106) were s.c. injected in the right flanks of nude mice. Tumors were measured with a dial-caliper, and volumes were determined using the formula width × length × height × 0.5236 (for ellipsoid form). After 20 days, when the primary tumors were 300–500 mm3 in size, animals were randomly divided into two groups. One group (n = 20) received intraperitoneal injection of the αAM (350 μg of purified IgG) every 3 days. The amount of αAM was determined based on the data of preliminary experiments in which increasing amounts of αAM (100, 200, 350, 500, 800 μg) were used to determine the best concentration of αAM inhibiting xenograft growth in vivo. As control, one group (n = 10) received a rabbit control-IgG (350 μg) of irrelevant specificity. Mice were sacrificed at the indicated time.
Immunohistochemical analysis
All tumor xenografts were excised, fixed in 10% (v/v) formalin, and processed for immunohistochemical analysis. Paraffin blocks were cut to 6 μm sections and stained with H&E for morphology evaluation. Immunohistochemistry was carried out using the Vectastain Elite ABC Kit (Vector Laboratories). Sections were incubated with anti-von Willebrand factor (anti-vWF) (1:400; Dako, Trappes cedex, France), or anti-α-Smooth Muscle Action (anti-α-SMA) (1:80; Dako), and subsequently with fluorochrome (Alexa 488 or Alexa 647)-conjugated secondary antibodies (Invitrogen Life Technologies). To assess a programmed cell death, tissue sections were evaluated using Mab F7-26 to detect single-strand DNA (AbCys, Paris, France). For nonimmunofluorescence staining, detection was carried out using a DAB chromogen, which resulted in a positive brown staining. Sections were counterstained with hematoxylin. As a control for immunohistochemistry, normal rabbit or mouse serum was used instead of the primary antibodies.
Statistical analysis
Data are expressed as mean ± SEM from at least three independent experiments. One-way analysis of variance (ANOVA) or Fisher's protected least significant difference test (Statview 512; Brain Power Inc., Calabasas, CA) was used for statistical analysis. P < 0.05 was considered significant and is indicated with asterisk in the figures. A double and triple asterisk, respectively, indicates P < 0.01 and P < 0.001.
Results
Expression of AM mRNA in human CRC
Total RNA from human colorectal normal tissue and human CRC was prepared to assess steady-state levels of AM mRNA transcript. Real-time quantitative RT-PCR analysis was performed on colorectal stage 0 fragments (n = 30) and tumor fragments of the 61 CRC that present samples with clinical stage I (n = 8), stage II (n = 32), stage III (n = 12), and stage IV (n = 9). Quantification of AM mRNA transcripts revealed high levels of AM mRNA in CRC clinical stages II, III, and IV, compared with colon stage 0 tissue and CRC clinical stage I (Fig. 1). The mean level of AM mRNA expression was 364 ± 46 fg/pg GAPDH mRNA in colorectal normal tissue (mean ± SEM), whereas it was, respectively, 457 ± 133, 810 ± 156, 825 ± 151, and 1072 ± 265 fg/pg GAPDH mRNA for CRC stages I, II, III, and IV (Fig. 1). AM mRNA levels determined in CRC stages II, III, and IV were significantly higher when compared with colorectal stage 0 tissue (P < 0.0078, P < 0.0009, P < 0.0005, respectively). Omission of the reverse transcriptase eliminated the signal, indicating that it was not linked to contaminating genomic DNA (not shown).
Figure 1 AM mRNA levels in normal and tumor colorectal tissues. Total RNA, DNA free, prepared from normal colon tissue and colorectal cancer (CRC) at different clinical stages (I–IV) were transcribed to cDNA and subjected to real-time quantitative RT-PCR for the estimation of relative AM mRNA to GAPDH mRNA ratios. Each bar represents the mean ± SEM of two independent assays in triplicate. The asterisk indicates that the values for CRC (clinical stages, II, III, and IV) are significantly different from the values of normal tissue and stage I of CRC (*P < 0.05; **P < 0.01; ***P < 0.001).
Immunohistochemistry of AM, CLR, RAMP2, and RAMP3 proteins
Adrenomedullin, CLR, RAMP2, and RAMP3 proteins immunodetections are shown in Figure 2. AM labeling was slightly detectable in the majority of the epithelial compartment of the crypts (Fig. 2a). Positive stained cells are observed in the stroma that could be macrophages, mast cells, and leukocytes (Fig. 2a). Well-differentiated cancer specimens displayed overt and strong AM labeling of epithelial cells, but not stromal cells (Fig. 2b). A higher degree of labeling was observed in poorly differentiated specimen with a clear increase in distant metastasis tissue (Fig. 2c and d). CLR, RAMP2, and RAMP3 immunostaining was barely detectable in the colonic epithelia of the crypts in normal tissue (Fig. 2e, i, and m). Cancer specimens displayed a strong staining for CLR, RAMP2, and RAMP3 of the epithelial compartment of the crypts in the well-differentiated adenocarcinomas (Fig. 2f, j, and n). The same labeling is observed in poorly differentiated (Fig. 2g, k, and o) and distant metastasis tissues (Fig. 2h, l, and p). Positive AM staining was completely abolished by preabsorption of the antibody with 50 μmol/L synthetic AM (Fig. 2q, r, s, and t) or CLR, RAMP2, RAMP3 peptides (not shown).
Figure 2 Immunohistochemistry for AM, CLR, RAMP2, and RAMP3 of the normal and tumor colorectal tissues. In normal tissue, AM immunoreactivity is observed in the cytoplasm of the epithelial cells and some cell types in the stroma (a). Positive immunoreactivity against CLR (e), RAMP2 (i), and RAMP3 (m) in epithelial cells is very weak in normal tissue. In well-differentiated colorectal cancer specimens, a far more intense labeling of epithelial cells is observed. In poorly differentiated and distant metastasis, a heavily labeled adenocarcinomatous structure can be observed. AM immunoreactivity is completely canceled by the antibody preabsorbed with 50 μmol/L AM peptide (q, r, s, and t). Magnification: ×10. AM, adrenomedullin; CLR, calcitonin receptor-like receptor; RAMP2, receptor activity-modifying protein-2; RAMP3, receptor activity-modifying protein-3.
Adrenomedullin immunoreactivity was revealed in the apical side cytoplasms of surface columnar epithelia of the human colonic mucosa and in the musculus (Fig. 3a). Interestingly, in the normal colon, AM immunoreactivity was also detected in neuroendocrine cells (Fig. 3b) as previously reported 14.
Figure 3 Adrenomedullin (AM) immunoreactivity of the human colonic mucosa. Immunoreactivity for AM is observed in the apical side cytoplasm of the surface epithelia and at the luminal surface (a). Positive immunoreactivity for AM is also noted in neuroendocrine cells (b). Magnification ×20.
TMA of AM, CLR, RAMP2, and RAMP3 proteins
To further investigate the prognostic value of AM, CLR, RAMP2, and RAMP3 expression, we used automatized quantitative image analysis on TMA. The data demonstrate that AM is expressed in all colorectal primary tumors and the staining quantification showed no significant changes between patients with different grades (Fig. 4a), either in the absence of negative (N0) or in the presence of positive (N+) lymph node (Fig. 4b); nor between clinical stages (I–IV) (Fig. 4c). Interestingly, an increase in AM staining was found in lymph node and distant metastasis when compared with primary tumors (Fig. 4d). Similarly, a significant increase in the staining was obtained for CLR, RAMP2, and RAMP3 proteins (P < 0.001; Fig. 4e). There was no difference between lymph node and distant metastasis. On the contrary, TMA analysis of primary tumor tissue and associated lymph node (N+) for each individual patient (n = 47) showed a highly significant increase of AM as well as CLR, RAMP2, and RAMP3 staining in lymph node when compared with primary tumor tissue (P < 0.001; Fig. 4f and g).
Figure 4 Statistical analysis of αAM and αAMR staining on TMA slices. ANOVA analysis of TMA-1 staining of cohort of patients (n = 147) according to histological grade T1, T2, T3, T4 (a); node status associated (N+) or not (N0) with metastatic process (b); and the clinical stages (I, II, III, IV) (c). TMA analyses for the expression of AM and AMR (CLR, RAMP2, and RAMP3) were quantified in tumor (T), node (N), and metastasis (M) (d, e). Expression of AM (f) and AMR (g) obtained with TMA analysis with tumors and nodes samples issued from the same patients (n = 47). Data demonstrate a clear increase of AM, CLR, RAMP2, and RAMP3 expression in nodes versus tumors (***P < 0.001). TMA, tissue microarray; AM, adrenomedullin, αAM, anti-AM; αAMR, anti-AM receptors, CLR, calcitonin receptor-like receptor; RAMP2, receptor activity-modifying protein-2; RAMP3, receptor activity-modifying protein-3.
Expression of AM and regulation by hypoxia in HT-29 cells
The expression of AM and its receptors in colorectal carcinomas suggest that the AM system might play a role in promoting tumor growth in situ. Accordingly, we used HT-29 cells to have more insight in the role of AM system in CRC. Quantitative RT-PCR analysis demonstrated that HT-29 cells express AM mRNA (Fig. 5a) and interestingly, sixfold increase of AM mRNA expression was observed under hypoxia (Fig. 5a). RIA demonstrated a clear increase of secreted immunoreactive-AM (ir-AM) by HT-29 cells under hypoxia versus normoxia (48 ± 2 pg/mL/h vs. 26 ± 7 pg/mL/h). Accordingly, the intracellular ir-AM showed an increase under hypoxia versus normoxia (12.32 ± 0.61 pg/μg protein/h vs. 7.41 ± 1.28 pg/μg protein/h).
Figure 5 AM stimulates proliferation and invasion of HT-29 cells in vitro. (a) Expression of AM mRNA in HT-29 cells. Total RNA (1 μg) DNA-free prepared from HT-29 cells was reverse transcribed into cDNA and subjected to quantitative RT-PCR for the estimation of relative AM to GAPDH mRNA ratios. Error bars indicate the SEM. The asterisks indicate that the value in HT-29 cells under hypoxia is significantly different from the HT-29 cells under normoxia in vitro (***P < 0.001). (b) For proliferation assays, tumor cells were seeded at the density of 2 × 103 per well in 12 multiwell plates in the presence of ITS medium. AM at 10−7 mol/L, αAM (30 μg/mL), combined αCLR/αRAMP2 or αCLR/αRAMP3 (30 μg/mL), AM22–52 at 10−6 mol/L, and control-IgG (30 μg/mL) were added for 6 days treatment. For each treatment, six wells were prepared for MTT assays. Bars represent SEM of three independent experiments. **P < 0.01; ***P < 0.001. (c) for invasion, HT-29 cells (3 × 104) were seeded on a Matrigel layer in a Boyden chamber assay. HT-29 cells were preincubated for 30 min with 15 μg/mL each of αCLR/αRAMP2 or αCLR/αRAMP3, AM22–52 at 10−6 mol/L or control-IgG. AM at indicated concentrations was added in the lower wells. Cells migrating through the filter were counted after 24 h. Data are expressed as the percentage of migrated cells in 10 high-power fields and are the means of three independent experiments each performed in triplicate. Bars represent SEM. ***P < 0.001 compared to control. AM, adrenomedullin; αAM, anti-AM; αCLR, anti-calcitonin receptor-like receptor; αRAMP2, anti-receptor activity-modifying protein-2; αRAMP3, anti-receptor activity-modifying protein-3.
AM stimulates HT-29 cells growth and invasion in vitro
The expression of AM and its receptors in CRC tissues as well as in cell lines suggests that AM might be involved in CRC cell growth by an autocrine/paracrine mechanism. AM at 10−7 mol/L stimulated the proliferation of HT-29 cells by 20% (P < 0.01) after 6 days of treatment (Fig. 5b). To test the autocrine hypothesis, HT-29 cells were treated with 30 μg/mL of αAM, αCLR/αRAMP2, or αCLR/αRAMP3 (purified IgG) to determine their effect on the in vitro growth of the HT-29 cells. The inhibition of proliferation reached up to 70% (P < 0.001) and 80% (P < 0.001) by 6 days of treatment, respectively, when compared to control (Fig. 5b). In contrast, control IgG (30 μg/mL) of irrelevant specificity (Fig. 5b) showed no inhibition of proliferation. To confirm that endogenous hAM produced by the HT-29 cells acts as an autocrine growth factor, HT-29 cells were incubated for up to 6 days with the AM antagonist AM22–52 at 10−6 mol/L. In the presence of AM22–52, inhibition of cell growth reached up to 70% (P < 0.001) when compared with control cells (Fig. 5b). Taken together, these observations support that AM acts as an autocrine growth factor via AM1 and AM2 receptors to stimulate HT-29 cells proliferation.
To further investigate the influence of AM on CRC cell function, the invasive capacity of HT-29 cells in response to AM was investigated by measuring the invasion of a Matrigel layer in a Boyden chamber assay. AM induced a dose-dependent increase in HT-29 cells invasion through Matrigel coating of the membrane (P < 0.005) (Fig. 5c), with a threefold increase in the number of migrated cells was obtained with AM at 10−14 mol/L (Fig. 5c). Preincubation of HT-29 cells with αCLR/αRAMP2, αCLR/αRAMP3, or AM22–52, completely blocked the invasion induced by AM indicating that the AM1 and AM2 receptors are involved in the response to AM (Fig. 5c). Preincubation of the cells with control IgG did not block the induced effect of AM on invasion (Fig. 5c). These observations demonstrate that AM induces HT-29 cells invasion via the AM1 and AM2 receptors.
αAM inhibits growth of HT-29 tumor xenograft
To assess the potential therapeutic value of αAM, athymic nude mice bearing established HT-29 tumor xenografts (>300 mm3) were treated with αAM or a rabbit control IgG of irrelevant specificity. Treatment was administered by i.p. injection every 3 days and tumor growth was monitored as a function of tumor volume through the time of therapy. The growth of HT-29 xenografts was significantly inhibited by the αAM when compared with control group (Fig. 6a). After a 40 day treatment period, a group of animals (n = 8) were sacrificed, and tumor size and vascularity were assessed. Tumors from αAM-treated animals appeared pale with diminished vasculature, whereas large tumors with extensive vascularization were observed in control groups. The mean tumor weights in the control and in the αAM-treated groups were 2.8 g versus 0.8 g at 60 days of treatment.
Figure 6 Treatment with anti-adrenomedullin (αAM) inhibits growth of HT-29 xenograft in vivo. HT-29 tumor xenografts were established by injecting 3 × 106 HT-29 cells subcutaneously into the right flank of athymic (nu/nu) nude mice. (a) Tumors were allowed to reach 300–500 mm3 in size, mice received i.p. injections of αAM (350 μg/mouse) every 3 days. Control mice were treated with 350 μg/mouse of nonimmune control-IgG. Tumor size was measured every 3 days. In animals treated with αAM, the size of tumors was significantly smaller. (b and d) Microphotographs of immunofluorescence and immunohistochemical-stained tumor sections for von Willebrand factor (vWF) (red), α-SMA (green), and single-strand DNA(ssDNA) in control and αAM-treated tumors. DAPI-stained nuclei are blue. Scale bars = 30 μm. (c) Quantitative assessment of the cells density that stained positive for vWF and α-SMA through a microscope. NIH image 1.62 software was used for analysis. Values are means ± SD; n = 8. ***P < 0.001. (e) Cells undergoing apoptosis were determined using Mab F7-26, which stained ssDNA. Values are means ± SD; n = 6, ***P < 0.001.
Histological examination of αAM-treated tumors for 60 days showed a significant decreased vessel density when compared with control IgG-treated group (Fig. 6b). Immunostaining of tumors with antibodies for vWF demonstrated that αAM-treated tumors were noticeably less vascular than control tumors (Fig. 6b). Costaining with anti-vWF and anti-α-SMA antibodies demonstrated that both cell types are sparse, and the vascularization is deeply disrupted and characterized by an overall reduction of endothelial cells and pericytes (Fig. 6b). In contrast, control IgG-treated tumors showed a well-organized vascularization (Fig. 6b). Quantification of vWF-stained endothelial cells demonstrates a clear decrease of vWF-positive cells in αAM-treated tumors when compared with control IgG-treated tumors (P < 0.001; Fig. 6c). Interestingly and despite the fact that the apoptosis labeling is heterogeneous among the tumors, the apoptotic index of the αAM-treated tumors was two- to threefold higher than the control tumors (P < 0.01; Fig. 6d and e).
Discussion
In this study, we investigated the expression of AM in normal and CRC tissues and its potential role as an autocrine/paracrine growth factor to promote CRC growth in vitro and in vivo. In the normal colon tissue, our immunohistochemical study demonstrates that AM is located in the cytoplasm of epithelia of the crypts and in the colonic surface epithelia in addition to neuroendocrine cells. The surface epithelium serves as a protective barrier between the host and the luminal environment. An immunopositive reaction to AM was observed at the luminal surface, which could indicate that AM was actually released from the colonic epithelia into the lumen. These lines of evidence suggest that AM may play some functional roles such as contribution to the mucosal defense system without ruling out some unrecognized roles in the digestive system. Many authors have reported that AM has bactericidal activity against Gram-positive and -negative bacterial strains 19,20.
The expression of AM and its receptors in CRC tissue strongly suggest that AM might be involved in CRC progression. Real-time quantitative RT-PCR demonstrated a significant increase in the expression of AM mRNA levels in late clinical stages compared with stage I of CRC. Immunohistochemical analysis localized a high staining of AM and AM receptors in the carcinomatous epithelial compartment of colorectal tumors when compared to a detectable staining in epithelial cell from normal tissue localized in the crypts. The expression of AM, CLR, RAMP2, and RAMP3 in a large number of samples of human CRC was assessed by TMA analysis. The data demonstrated the presence of AM and its receptors in the vast majority of colorectal adenocarcinomas of different clinical stages. The amount of AM staining in colorectal tumor tissues represents a balance among synthesis, storage, degradation, and secretion. The dissociation between levels of AM mRNA and AM staining in late clinical stages may reflect an increase of secretion rate of ir-AM and/or altered translational efficiency for AM mRNA. Interestingly, a higher expression of AM and its receptors was found in the CRC-associated node (n = 47) when compared with a primary colorectal tumors, strongly suggesting that AM might be involved in the colorectal metastasis.
The degree of AM expression has been associated with lymph node metastasis in breast cancer 28,29. In pancreatic adenocarcinoma, the median AM mRNA expression levels were higher in lymph node-positive compared with lymph node-negative patients 30. Lymph node metastasis is a sensitive indicator of poor prognosis in patients with CRC 31,32,33, but even in node-negative patients, approximately 10–20% patients suffer from relapse in <5 years 34. Recently, Uemura et al. 35 reported that AM was a novel independent prognostic factor for CRC with no correlations between AM expression and lymph node metastasis.
Previous studies have demonstrated the ability of reduced oxygen tension to mediate elevations in AM message/protein expression in several animal and cell systems 23. Nakayama et al. 23 have demonstrated that hypoxia stimulates AM expression in human colorectal carcinoma cell line, DLD-1. This study demonstrates that hypoxic conditions induced an increase of AM expression in HT-29 cells. These data suggest that the resulting reduction in tissue oxygen tension may lead to an increased expression of AM mRNA in colorectal tumors. Interestingly, a recent study reported that the expression of AM correlated with hypoxia-inducible factor-1α in vivo samples from a cohort of 373 CRC patients 35.
The presence of AM and AM receptors (AM1 and AM2) opens up the possibility for AM to be an autocrine/paracrine growth factor in CRC. This hypothesis is supported by the data showing that exogenous AM stimulates HT-29 cell growth. Proliferation assays revealed that a neutralizing αAM and αAMR could significantly suppress cell growth of HT-29 cells. The inhibition by αAM could be reversed by the addition of the exogenous AM, thus, the inhibition of cell growth by αAM and αAMR in vitro was most likely the result of blocking the autocrine/paracrine effects of immunoreactive AM synthesized and secreted by the colorectal carcinoma cells. These observations indicate that colorectal carcinoma cells are able to respond to AM in ways that would be expected to further promote cell and tumor growth. Our conclusion that AM can act as an autocrine/paracrine growth factor in colorectal carcinomas is in agreement with the previously reported data in other forms of cancer where AM may act in the same manner 11,12–17,30.
To extend the in vitro observations, in vivo experiments were performed. Our results demonstrated that the αAM significantly suppresses the growth of established HT-29 tumor xenografts. After 2 months of treatment, tumors in mice treated with control-IgG grew progressively to a size that led to sacrifice, whereas the volume of the αAM-treated tumors decreased by 95% after 80 days of treatment. The immunohistological analysis of tumors from αAM-treated animals showed a clear decrease in microvessel density with 80% reduction of endothelial cells and pericytes within the tumor, confirming the role of AM in endothelial cells and pericytes activation and/or recruitment. These data demonstrate that AM is involved in neovascularization and/or vessel stabilization. AM signaling is of particular significance in endothelial cell biology because the peptide protects these cells from apoptosis 36,37, promotes angiogenesis and vascular stabilization 5,38, and affects vascular tone and permeability 39. Our findings are consistent with the previously reported data involving AM in tumor angiogenesis through CLR/RAMP2 and CLR/RAMP3 5,15,38. Previously, our group reported that targeting AM receptors with systemic delivery of neutralizing antibodies inhibits tumor angiogenesis and suppresses growth of colorectal carcinoma HT-29, U87 (glioblastoma), and A549 (lung cancer) tumor xenografts in mice 15.
In summary, our study demonstrates the relevance of AM and its receptors CLR/RAMP2 and CLR/RAMP3 in CRC. Our data strongly suggest that AM is involved in the progression of colon cancer. Targeting AM system by αAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting strongly that AM may be a useful therapeutic target.
We thank Françoise Boudouresque for RIA assays and Mylène Cayol for assistance in animal handling. Inserm, AP-HM, and the ARTC Sud supported this work.
Conflict of Interest
The authors declare that they have no conflict of interest to disclose.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23658789PONE-D-12-3469510.1371/journal.pone.0062942Research ArticleBiologyBiochemistryProteinsTransmembrane Transport ProteinsMolecular Cell BiologySignal TransductionSignaling CascadesCalcium Signaling CascadeCell DeathToxicologyNeurotoxicologyMedicineAnesthesiologyRegional AnesthesiaNeurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells T Type Calcium Channel and Local AnestheticsWen Xianjie
1
2
*
Xu Shiyuan
1
*
Liu Hongzhen
2
Zhang Quinguo
1
*
Liang Hua
2
Yang Chenxiang
2
Wang Hanbing
2
1
Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China
2
Department of Anesthesiology, The First People’s Hospital of Foshan & Foshan Hospital of Sun Yat-sen University, Foshan, Guangdong Province, China
Ceña Valentin Editor
Universidad de Castilla-La Mancha, Spain
* E-mail: [email protected] (XW); [email protected] (SX); [email protected] (QZ)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: XW. Performed the experiments: XW SX HL QZ. Analyzed the data: HL HW. Contributed reagents/materials/analysis tools: CY. Wrote the paper: XW.
2013 2 5 2013 8 5 e629422 11 2012 27 3 2013 © 2013 Wen et al2013Wen et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation.
This study was supported by the national natural science foundation of China (number 81100831) and the medical research foundation of Guangdong Province (number B2011303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Regional anesthetics have been used widely in clinical settings and as postoperative analgesics, because of their reduced systemic effects [1], [2]. However, local anesthetics may cause neurotoxicity, such as transient neurological syndrome (TNS), and cauda equina syndrome, which has raised concerns about their use [3], [4]. One multicenter study found that the incidence of TNS was approximately 8.1%, which resulted in pain or sensory abnormalities in the lower back, buttocks, or lower extremities, with symptoms beginning after spinal anesthesia and lasting for hours to 4 days [5]. Although there is low incidence of anesthetic-induced cauda equina syndrome, it results in severe damage to neurons [6]–[8].
Local anesthetics can cause cell apoptosis, induce the release of reactive oxygen species and lactate dehydrogenase (LDH) [9], [10]. Several studies have shown that lidocaine, bupivacaine, tetracaine, dibucaine, and procaine can induce apoptosis [11]. The underlying mechanisms of local anesthetic neurotoxicity are not clearly understood. Previous studies indicated that intracellular calcium overload is involved in local anesthetic-induced neurotoxicity [12], [13]. Extracellular calcium influx and intracellular calcium store release are the most important factors for local anesthetic-induced calcium overload. Also, an influx of extracellular calcium can induce calcium-dependent release of intracellular calcium stores [14], [15].
The main route of extracellular calcium influx into cells is via voltage-dependent calcium channels (VDCCs) [16]. Currents arising from VDCCs are subdivided into two major classes based on the membrane potential at which they become activated: high-voltage activated (HVA), which are further divided into L-, P-, Q-, N- and R-subtypes, and low-voltage activated (LVA) or transient (T-type) Ca2+ currents, which are further divided into Cav3.1, Cav3.2 and Cav3.3 [17]. The T subtype of VDCCs are known to perform several roles in neurons, such as lowering the threshold for action potentials, promoting burst firing, oscillatory behavior, and enhancing synaptic excitation [17]. With electrophysiological characteristics, such as activation at resting potential, T-type calcium channels act as pacemakers in many pathological and physiological conditions [18], [19]. This pacemaker-like activity of T-type calcium channels allows them to regulate the excitability of neurons. T-type calcium channels can be activated at the resting potential, and then extracellular calcium ions enter into the cells by T-type calcium channels. On the one hand, cell membrane depolarization induced by T-type currents activates the HVA channels and promotes extracellular calcium ion entry into the cell. On the other hand, T-type currents prime calcium-induced calcium release (CICR) [20].
Although calcium channel blockers (CCB) can cause cancer cell growth, they can inhibit the neuronal apoptosis in several neuron injury models [21]–[23]. For example, the L-type voltage-gated calcium channel blocker, nifedipine, lowered the intracellular Ca2+ concentration of the cerebellar granule cells treated with kainate from 1543 nM to 764 nM and reduced kainate neurotoxicity. Yagami and colleagues found that S-312-d, another L-type voltage sensitive calcium channel blocker, rescued cortical neurons from apoptosis induced by beta amyloid and human group II A secretory phospholipase A2. The neuroprotective effects of CCB were shown by lowering the intracellular Ca2+ concentration. We conjectured that T-type calcium channels, with the pacemaker-like activity, may be involved with the calcium overload of local anesthetic-induced neurotoxicity. In this study, we hypothesize that neurotoxicity induced by bupivacaine involves T-type calcium channels. Therefore, we employed an in vitro model of cytotoxicity using SH-SY5Y cells treated with bupivacaine. In addition, we monitored the effect of NNC 55-0396 dihydrochloride, a highly selective T-type calcium channel blocker that does not significantly alter currents mediated by other subtypes of calcium channels [24], on cell viability, LDH release, cytosolic Ca2+ ([Ca2+]i), apoptosis, and caspase-3 expression, following bupivacaine treatment.
Materials and Methods
Materials
The SH-SY5Y cell line was purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). Goat polyclonal anti-caspase-3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), bupivacaine hydrochloride and NNC 55-0396 dihydrochloride were purchased from Sigma (St. Louis, MO, USA) and Boston Biochem (Boston, MA, USA), respectively. Other reagents used in this study were as follows: DMEM/F12 medium and fetal bovine serum (Gibco, Grand Island, NY, USA), 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT; Beyotime, Nantong, China), Quest Fluo-8 AM ester (AAT Bioquest Inc., Sunnyvale, CA, USA), Hoechst 33258 (Beyotime), annexin V-FITC and propidium iodide (KeyGEN, Nanjing, China), and the LDH cytotoxicity detection kit (Beyotime). All other reagents were from commercial suppliers and of standard biochemical quality.
Cell Culture
SH-SY5Y cells were cultured in DMEM/F12 medium with 15% (v/v) fetal bovine serum, 100 units/mL of penicillin and 100 µg/mL of streptomycin, and maintained in a humidified 5% CO2 incubator at 37°C. The medium was replaced every 2 days.
Viability of the Cell Treated with Different Concentration Bupivacaine
To investigate the effects of different bupivacaine concentrations on SH-SY5Y cell viability, we treated SH-SY5Y cells with 0.1, 0.5, 0.75, 1, 2, 5, or 10 mM bupivacaine for 24 h. The effects of the different bupivacaine concentrations on SH-SY5Y cell viability were evaluated by the MTT assay.
Experimental Classification
Bupivacaine hydrochloride in powder form was dissolved in medium with serum resulting in the final concentration of bupivacaine being 1 mM, based on the results from the cell viability experiment. Cells were pretreated with 10, 50, or 100 µM NNC 55-0396 dihydrochloride (NNC) 30 min prior to treatment with either the culture medium containing 1 mM bupivacaine or an equivalent amount of medium alone for 6, 12, or 24 h. The experimental groups were: (1) NNC 55-0396 dihydrochloride 100 µM (S+NNC 100 group), (2) bupivacaine (S+B group), (3) bupivacaine+NNC 10 μΜ (S+B+NNC 10 group), (4) bupivacaine+NNC 50 μΜ (S+B+NNC 50 group), (5) bupivacaine+NNC 100 µM (S+B+NNC 100 group), and (6) non-pretreated (S group).
MTT Assay
Cell viability was measured using the MTT assay as previously described [25]. The cells were seeded into 96-well plates at a concentration of 5×103 cells/well with 100 µL culture medium per well. The cells were exposed to either 1 mM bupivacaine or an equivalent amount of medium for 6, 12, or 24 h. MTT (20 µL) was added to each well and incubated at 37°C for 4 h. The optical density of the homogenous purple solution was measured using a spectrophotometer (Bio-Tek, Winooski, VT, USA). The control group without bupivacaine treatment was set as 100% cell survival and all other groups were normalized to the corresponding control values.
LDH Assay
LDH activity was determined using an LDH cytotoxicity detection kit after cells were exposed to 1 mM bupivacaine, or an equivalent amount of medium for 6, 12, or 24 h [26]. The incubation solution was collected from the 12-well plates at the end of each experiment, and then centrifuged at 13,000×g for 10 min. The supernatant (100 µL) was transferred to 96-well plates and incubated with the same amount of reaction mixture. LDH activity was determined using a colorimetric assay at an absorbance wavelength of 492 nm and a reference wavelength of 655 nm using a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). Background absorbance from the cell-free buffer solution was subtracted from all absorbance measurements. After removal of the buffer from 12-well plates, 1% (v/v) Triton X-100 solution was applied to the remaining cells. The percentage of LDH released into the incubation buffer was calculated as follows: spontaneously released LDH into the buffer/(spontaneously released LDH into the buffer+intracellular LDH released by Triton X-100).
Measurements of Cytosolic Ca2+
Cytosolic Ca2+ ([Ca2+]i) from each group after treatment, with or without drugs for 24 h, was measured with Quest Fluo-8 AM ester. Briefly, a 5 mM stock solution of Quest Fluo-8 AM ester was prepared in high-quality anhydrous DMSO and a 10 µM working solution was prepared in Hanks and HEPES buffer (HHBS). The Quest Fluo-8 AM ester reagent concentration was 5 uM. The cells were incubated with the Quest Fluo-8 AM ester for 20 min at room temperature. Cells were washed twice in HHBS to remove excess probe. The experiments were analyzed at excitation and emission wavelengths of 490 and 525 nm, respectively. To determine either the free calcium concentration in the solution ([Ca2+]i) or the Kd of a single-wavelength calcium indicator, the following equation was used: [Ca2+]i = Kd[F−Fmin]/Fmax−F]. Where F is the fluorescence of the indicator at experimental calcium levels, Fmin is the fluorescence in the absence of calcium and Fmax is the fluorescence of the calcium-saturated probe. The dissociation constant (Kd) is a measure of the affinity of the probe for calcium, which is provided in the kit manual.
Detection of Apoptosis by Flow Cytometry
After cells were treated as described above for 24 h, the cells were seeded into 24-well plates at a concentration of 5×105 cells/well, with 500 µL culture medium per well. Cells were rinsed with phosphate buffered saline (PBS) and collected. Each pellet was resuspended in 500 µL binding buffer. In addition, 5 µL annexin V-FITC and 5 µL propidium iodide were added to each well. After a 5 min incubation, apoptotic cell death was measured by flow cytometry.
Apoptotic Cell Death Detected with Hoechst 33258
Cells in 24-well plates were rinsed 3 times with PBS and stained with Hoechst 33258. Subsequently, the cells were examined and photographed under a fluorescence microscope (Nikon ECLIPSE TE2000-u, Tokyo, Japan) with a UV excitation wavelength of 300–500 nm. Apoptotic cells were defined on the basis of nuclear morphology changes: chromatin condensation and fragmentation. The number of apoptotic and normal cells was counted manually by researchers blinded to the treatment schedule. For each well, at least 5 different fields were examined and the apoptosis rate was expressed as the percentage of apoptotic cells to the total number of cells counted.
Detection of Caspase-3 Protein Expression by Western Blotting
Culture flasks or plates were quickly rinsed with chilled PBS. Cells were collected using a plastic cell scraper, removed, and lysed in lysis buffer A (20.0 mmol/L Tris-HCl, 1.0 mmol/L Na3VO4, 1.5 mmol/L MgCl2, 10.0 mmol/L KCl, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1 mmol/L ethylene glycol tetraacetic acid (EGTA), 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 0.02% (w/v) protease inhibitor cocktail (pH 7.9)). After addition of 90 µL NP-40 (10% (v/v)), samples were shaken for 30 sec and then centrifuged at 800×g for 15 min at 4°C. The supernatants were centrifuged at 10 000×g for 1 h at 4°C. The samples were then homogenized in lysis buffer B (20.0 mmol/L Tris-HCl, 0.03 mmol/L Na3VO4, 2.0 mmol/L MgCl2, 10.0 mmol/L KCl, 2.0 mmol/L EDTA, 2.0 mmol/L EGTA, 2.0 mmol/L PMSF, 0.1% (v/v) Triton X-100, 5.0 mmol/L NaF, and 0.02% (w/v) protease inhibitor cocktail). The samples were centrifuged at 10,000×g for 1 h at 4°C, and the supernatants were used for western blot analysis. Protein concentration was determined using the Bradford method, and protein samples were stored at −80°C. Protein samples were dissolved in 4× sample buffer (250 mmol/L Tris-HCl, 200 mmol/L sucrose, 300 mmol/L dithiothreitol, 0.01% (w/v) Coomassie brilliant blue-G, and 8% (w/v) SDS, pH 6.8), and were subsequently denatured at 95°C for 5 min. Equivalent amounts of protein were separated on a 7.5% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4°C with either goat polyclonal anti-caspase-3 antibody or anti-β-actin (1∶500; Santa Cruz Biotechnology, Santa Cruz CA, USA). The membranes were washed extensively with Tris-buffered saline/Tween-20 and incubated for 2 h in peroxidase-conjugated rabbit anti-goat IgG secondary antibody (1∶500; Santa Cruz Biotechnology) at room temperature. The immune complexes were detected by enhanced chemiluminescence, and membranes were then exposed to X-ray film. Quantification of protein bands was conducted by scanning the films and importing the images into Adobe Photoshop software (Adobe, San Jose, California, USA). Scanning densitometry was used for semi-quantitative analysis of the data. Caspase-3 protein was normalized to β-actin levels.
Statistical Analysis
Results are presented as the mean±SD. Factorial design ANOVA (SPSS 11.0 statistical software, IBM, USA) was used to analyze the data from the MTT assay, LDH assay, apoptosis assay and western blot assay. Multiple comparisons tests were performed by LSD. A probability value of P<0.05 was considered to be statistically significant.
Results
Cell Viability
Viability of SH-SY5Y cells dose-dependently decreased with increasing concentrations of bupivacaine. Treatment with 0.1, 0.5, 0.75, 1, 2, 5, and 10 mM bupivacaine for 24 h resulted in 58±5%, 47±6%, 39±5%, 27±4%, 15±3%, 5±2%, and 2±1% viability, respectively (Fig. 1).
10.1371/journal.pone.0062942.g001Figure 1 The effect of increasing concentrations of bupivacaine on SH-SY5Y cell viability.
SH-SY5Y cells were exposed to different concentrations of bupivacaine (0.1, 0.5, 0.75, 1, 2, 5, and 10 mM). The viability of the cells declined with increasing bupivacaine concentration.
Compared with the S group, cell viability of SH-SY5Y cells in the S+NNC 100 group was not significantly different at 6, 12, and 24 h. However, 1 mM bupivacaine caused marked cell injury, and cell viability in the S+B group was 47±7%, 36±5% and 26±5% at 6, 12, and 24 h, respectively. Compared with the S+B group, NNC 55-0396 dihydrochloride pretreatment with the three different concentrations protected SH-SY5Y cells against bupivacaine-induced cell injury at 6, 12, and 24 h. Viability of SH-SY5Y cells treated with NNC 55-0396 dihydrochloride improved to 60±8%, 48±6% and 35±4% in the S+B+NNC 10 group, 70±7%, 61±7%, and 45±4% in the S+B+NNC 50 group, and 67±7%, 62±7% and 46±4% in the S+B+NNC 100 group, respectively. Although there was a significant difference between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+ NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups (Fig. 2).
10.1371/journal.pone.0062942.g002Figure 2 SH-SY5Y cell viability following treatment with 1 mM bupivacaine (%, mean±S.D, n = 6).
aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group; eP<0.05 vs. time point of 6 hours; fP<0.05 vs. time point of 12 h.
LDH Activity
SH-SY5Y cells in the S group showed marked LDH release, with extracellular LDH being 8.8±1.9%, 9.2±1.6%, and 10.1±1.2% of total LDH at 6, 12, and 24 h, respectively. In addition, there was no significant change in LDH release in SH-SY5Y cells pretreated with 100 μΜ NNC 55-0396 dihydrochloride for 6, 12, and 24 h. However, incubation with 1 mM bupivacaine increased extracellular LDH release to 20.7±2.1%, 27.1±2.8% and 31.3±2.9% at 6, 12, and 24 h, respectively. Interestingly, SH-SY5Y cells pretreated with NNC 55-0396 dihydrochloride resulted in a reduction in LDH release following bupivacaine treatment. Extracellular LDH at 6, 12, and 24 h was 17.3±1.6%, 22.2±2.7% and 25.3±1.6%, respectively, in the S+B+NNC 10 group; was 14.3±1.8%, 16.7±1.6% and 20.1±1.7% in the S+B+NNC 50 group; and was 13.5±1.9%, 16.5±2.1% and 20.8±1.9% in the S+B+NNC 10 group. Although there was a significant difference between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups (Fig. 3).
10.1371/journal.pone.0062942.g003Figure 3 Bupivacaine treatment leads to the release of LDH.
SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. LDH release was determined by the level of LDH activity present in the culture media. (%, mean±S.D, n = 6). aP<0.05 vs. S group; bP<0.05 vs. S+NNC100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group; eP<0.05 vs. time point of 6 h; fP<0.05 vs. time point of 12 h.
Changes in Cytosolic Ca2+
[Ca2+]i in SH-SY5Y cells in the S group and S+NNC 100 group was 358±25 nM and 372±32 nM, respectively. However, [Ca2+]i in the S+B group increased dramatically after treatment with 1 mM bupivacaine for 24 h to 715±35 nM. SH-SY5Y cells pretreated with NNC 55-0396 dihydrochloride resulted in a reduction of [Ca2+]i following bupivacaine exposure. [Ca2+]i of the cells in the S+B+NNC 10, S+B+NNC 50 and S+B+NNC 100 groups was 657±29 nM, 619±37 nM and 585±39 nM, respectively (See Fig. 4).
10.1371/journal.pone.0062942.g004Figure 4 Bupivacaine treatment leads to an increase in cytosolic Ca2+ ([Ca2+]i).
SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. [Ca2+]i levels were measured by Quest Fluo-8 AM ester (mean±SD, n = 6)). A: Representative image of Quest Fluo-8 AM ester flow cytometry analysis. B: [Ca2+]i levels in the different treatment groups. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.
Apoptotic Cell Death Measured by Flow Cytometry
The rate of apoptosis in SH-SY5Y cells from the S and S+NNC 100 group was 12.5±2.7% and 12.9±2.3% respectively. After treatment with 1 mM bupivacaine for 24 h, the rate of apoptosis in the S+B group dramatically increased to 41.6±2.3%. NNC 55-0396 dihydrochloride pretreatment reduced the amount of apoptotic cell death following bupivacaine exposure, and the rates of apoptosis in the S+B+NNC 10, S+B+NNC 50 and S+B+NNC 100 groups were 36.2±3.9%, 28.7±3.2% and 25.1±2.8%, respectively. Although there was a significant difference between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+NB+NC 100 groups (Fig. 5).
10.1371/journal.pone.0062942.g005Figure 5 NNC 55-0396 dihydrochloride protects SH-SY5Y cells from bupivacaine-induced apoptosis.
Cells were either treated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. Apoptosis was measured by Annexin-V staining with flow cytometry (%, mean±SD, n = 6). A: Representative image from the flow cytometric analysis. B: Rates of apoptosis in the different treatment groups. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.
Detection of Apoptosis Using Hoechst 33258
Nuclear alterations of apoptotic cells were observed using Hoechst 33258 nuclear staining. As seen in Figure 6, apoptotic cells were observed to have condensed or segmented nuclei accompanied by bright blue fluorescence. Data analysis revealed similar results to that of flow cytometry (Table 1).
10.1371/journal.pone.0062942.g006Figure 6 NNC 55-0396 dihydrochloride protects SH-SY5Y cells from bupivacaine-induced nuclear alterations during apoptosis.
Cells were either treated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine treatment for 24 h. Nuclear morphology was evaluated by Hoechst 33258 staining (×200). Apoptotic cells were observed to have condensed or segmented nuclei accompanied by bright blue fluorescence.
10.1371/journal.pone.0062942.t001Table 1 Apoptosis measured by Hoechst 33258 staining (%, mean±S.D, n = 6).
Group Apoptosis
S 7.5±1.9
S+NNC 100 7.5±2.3
S+B 49.2±3.0ab
S+B+NNC 10 36.3±2.2abc
S++B+NNC 50 25.5±2.7abcd
S+B+NNC 100 24.5±2.9abcd
a
P<0.05 vs. S group;
b
P<0.05 vs. S+NNC 100 group;
c
P<0.05 vs. S+B group;
d
P<0.05 vs. S+B+NNC 10 group.
Detection of Caspase-3 Expression by Western Blotting
The expression of cleaved caspase-3 (active form) and procaspase-3 (inactive form) were measured. The expression of procaspase-3 in SH-SY5Y cells in the S group and S+NNC 100 group was markedly higher than in the other groups. After treatment with 1 mM bupivacaine for 24 h, the expression of procaspase-3 in SH-SY5Y cells decreased and the expression of caspase-3 dramatically increased. However, NNC 55-0396 dihydrochloride pretreatment prevented the bupivacaine-induced reduction in procaspase-3. Therefore, NNC 55-0396 dihydrochloride pretreatment inhibited caspase-3 cleavage. Although the effects of NNC 55-0396 dihydrochloride were significantly different between SH-SY5Y cells in the S+B+NNC 10 group and the S+B+NNC 50 and S+B+NNC 100 groups, there were no significant differences between SH-SY5Y cells in the S+B+NNC 50 and S+B+NNC 100 groups (Fig. 7).
10.1371/journal.pone.0062942.g007Figure 7 Inhibition of T-type calcium channels prevents bupivacaine-induced cleavage of caspase-3.
SH-SY5Y cells were either pretreated with the indicated concentrations of NNC 55-0396 dihydrochloride or left untreated prior to 1 mM bupivaine exposure for 24 h. Procaspase-3 (inactive form) and cleaved caspase-3 (active form) expression was measured by western blot analysis (mean+S.D, n = 6). Lane 1 = S group; Lane 2 = S+NNC 100 group; Lane 3 = S+B group; Lane 4 = S+B+ NNC 10 group; Lane 5 = S+B+NNC 50 group; Lane 6 = S+B+NNC 100 group. aP<0.05 vs. S group; bP<0.05 vs. S+NNC 100 group; cP<0.05 vs. S+B group; dP<0.05 vs. S+B+NNC 10 group.
Discussion
Generally, nerve damage resulting from local anesthetic exposure is related to the dose, concentration, and the time of exposure to the local anesthetic [27]. The precise mechanism of local anesthetic-induced nerve damage remains unclear. An intracellular overload of calcium may be a contributing factor to local anesthetic-induced nerve injury [12], [13]. Calcium is an important mineral essential for cellular function. Calcium can serve as a chemical signal in cells, and its levels are carefully regulated. One intriguing role of calcium is its ability to trigger apoptosis, a controlled form of cell death. Extracellular calcium ions can enter cells through voltage-dependent calcium channels or ligand-gated calcium channels, and activate calcium-dependent enzymes. Over-activation of these enzymes can cause nerve damage. At the same time, calcium ions entering cells can produce calcium-induced calcium release (CICR), causing an overload of intracellular calcium, and subsequently apoptosis and nerve damage [14].
In the present study, we detected the intracellular Ca2+ concentration with Fluo-8, with absorption and emission peaks at 490 nm and 514 nm, respectively. They can be excited with an argon ion laser at 488 nm, and their emitted fluorescence increases with increasing concentrations of Ca2+.Compared with Fluo-3 or Fluo-4, Fluo-8 is an excellent probe to use with high sensitivity. In this study, we found intracellular Ca2+ concentrations of SH-SY5Y cells treated with 1 mM bupivacaine for 24 h increased sharply and NNC, inhibited the rise of the intracellular Ca2+ concentration and prevented the apoptosis induced by bupivacaine.
The Cav3 family T-type calcium channels generate low-voltage-activated Ca2+ currents, and play an important role in many physiological and pathological processes, such as the regulation of cellular excitability, neurotransmitter secretion and release, motor coordination and function, learning and memory, epilepsy, and neuropathic pain [18], [28]. In our previous study, we monitored the protein and mRNA expression of T-type calcium channels in SH-SY5Y cells [29]. In this study, we found that 1 mM bupivacaine induced apoptosis in SH-SY5Y cells, activated caspase-3, and increased LDH release. However, NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels, reduced bupivacaine-induced cell injury. Therefore, T-type calcium channels may be involved in the neuronal injury observed following local anesthetic administration.
We found that NNC 55-0396 dihydrochloride protection against bupivacaine-induced apoptosis was dose-dependent. Although 10 μΜ NNC 55-0396 dihydrochloride significantly protected SH-SY5Y cells from 1 mM bupivacaine-induced cell death, the effects of 50 μΜ NNC 55-0396 dihydrochloride were notably enhanced. However, there was no significant difference between 50 μΜ and 100 μΜ NNC 55-0396 dihydrochloride pretreatment, demonstrating that the protection of NNC 55-0396 dihydrochloride exhibited a ceiling effect.
One limitation of this study was that NNC 55-0396 dihydrochloride is not subtype specific, and may have acted on Cav3.1, Cav3.2 and Cav3.3, which are all expressed in SH-SY5Y cells [26]. To investigate the subtypes of T-type calcium channels involved in bupivacaine toxicity, we would like to have employed Cav3.1, Cav3.2 or Cav3.3 specific antagonists. However, to our knowledge, there are currently no drugs available that specifically block Cav3.1, Cav3.2 or Cav3.3. Therefore, genetic engineering to silence subtype gene expression may be necessary to understand the role of specific subtypes in local anesthetic toxicity.
In summary, we found that treatment of SH-SY5Y cells with 1 mM bupivacaine for 24 h resulted in apoptosis, activation of caspase-3 and release of LDH. Interestingly, inhibition of T-type calcium channels with NNC 55-0396 dihydrochloride reduced bupivacaine-induced cell death, suggesting a novel role for these calcium channels in local anesthetic toxicity.
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Diabetol Metab SyndrDiabetol Metab SyndrDiabetology & Metabolic Syndrome1758-5996BioMed Central 1758-5996-5-222363494910.1186/1758-5996-5-22ReviewHistorical facts of screening and diagnosing diabetes in pregnancy Negrato Carlos Antonio [email protected] Marilia Brito [email protected] Bauru’s Diabetics Association, Department of Internal Medicine, Bauru, São Paulo, 17012-433, Brazil2 Department of Internal Medicine, Diabetes Unit, State University Hospital of Rio de Janeiro, Rio de Janeiro, Brazil2013 1 5 2013 5 22 22 24 2 2013 22 4 2013 Copyright © 2013 Negrato and Gomes; licensee BioMed Central Ltd.2013Negrato and Gomes; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Diabetes is the most common metabolic disorder affecting pregnancy. Its prevalence seems to be growing in parallel with the epidemics of overweight and obesity. Recognizing and treating diabetes or any degree of glucose intolerance in pregnancy results in lowering maternal and fetal complications. These patients present higher risk for excessive weight gain, preeclampsia, cesarean sections, a high risk of developing type 2 diabetes and cardiovascular disease in the future. Infants born to these mothers are at higher risk for macrosomia and birth trauma, and after delivery, these infants have a higher risk of developing hypoglycemia, hypocalcemia, hyperbilirubinemia, respiratory distress syndrome, polycythemia and subsequent obesity and type 2 diabetes. Despite several international workshops and a lot of research there is still no unique approach to diagnose and treat diabetes in pregnancy. Who, when and how to screen and diagnose diabetes in pregnancy has been debated in the literature for so many decades and this debate seems to be endless. We present the evolution that screening and diagnosing diabetes in pregnancy has had over time. Besides many evidence of the benefits these procedures bring, health care providers still often prefer to use alternate criteria for this purpose. The myriad of maternal and fetal complications that could be avoided with an appropriate and simple screening procedure are ignored. Robust clinical trials such as the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study have shown how harmful can even slightly altered blood glucose levels be, but it has been found a resistance in the adoption of the new criteria proposed after this and other trials by many diabetes organizations. These organizations state that these new criteria would increase the incidence of diabetes in pregnancy, would imply in longer term follow-up of these patients and would pose an economic problem; they also state that alerting too many people in order to benefit a relatively few potential diabetics would arise psychologic ill-effects. We think that health care providers should look for an uniformity in the screening and diagnosing diabetes in pregnancy based on evidence based medicine and not on specialists consensus.
History of diabetesDiabetes in pregnancyScreening diabetes in pregnancyDiagnosing diabetes in pregnancy
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Introduction
Diabetes mellitus is a very ancient disease first described in the Egyptian Ebers papyrus around 1500 BC; written records of human pregnancy are still older. Although, the first description of diabetes in pregnancy was done by Bennewitz in 1824 in Germany [1]. He described a clinical case of a woman with intensive thirst and recurrent glycosuria in three successive pregnancies. One of her babies weighted almost 5,5 kg. Her urine contained a big amount of saccharine matter (about 60 g per 0,20 L). In 1846 a similar case was reported by Lever [2].
Before the discovery of insulin in 1922, infertility was well recognized in women with diabetes. The suggested causes of infertility present in these women were amenorrhea, uterus and Graafian follicles atrophy and malnutrition [3,4]. Few reports of conceptions in women with diabetes existed then; seven pregnancies in 114 women with diabetes [3], 55 in 427 women with diabetes of childbearing age [5], and four in 190 married women with diabetes [6]. After the discovery of insulin and its use initiation in 1923, the pregnancy rate increased by seven-fold in women with a short duration of the disease; women with a long duration of diabetes still remained sterile or with low conception rates for a long time.
In 1882, Duncan in London, reported the outcomes of pregnancy in 16 women with 22 pregnancies. High rates of maternal and perinatal mortality were present: more than 60% for the mothers and 47% for the newborns. The observations and the findings he had following these pregnancies allowed him to several conclusions such as: that diabetes may develop during pregnancy; diabetes may occur only during pregnancy, being absent in other times; diabetes may cease with the termination of pregnancy, recurring some time after birth; diabetes may develop soon after parturition; diabetes may not return in a pregnancy occurring after its cure; pregnancy may occur in the presence of diabetes; pregnancy and delivery may be apparently unaffected in its healthy progress by diabetes and finally that pregnancy is likely to be interrupted frequently by the fetus death [7].
Williams, Professor of Obstetrics in Baltimore, in 1909, reported 66 cases from the literature. Fifty-five patients had diabetes previous to the conception; nine patients developed diabetes after conception and in two the time of onset was not certain. The mortality rates were still high, being 27% at the time of delivery with around an additional 23% dying within two years after delivery. The perinatal mortality rates varied from 27-53% [8]. The principal focus of his manuscript was on the interpretation and the diagnostic role of glycosuria in pregnancy, because at that time the diagnosis of diabetes was based on the presence of sugar in the urine. He showed that if a woman’s urine had between 1 and 3 g /L of sugar, it was most likely to be a physiologic condition, but a higher concentration was suggestive of diabetes, particularly if present in early pregnancy or in the presence of symptoms. His study might have been the first prospective screening program for diabetes in pregnancy [8].
The presence of glycosuria during pregnancy and its role as diagnostic of asymptomatic diabetes was a strongly debated subject by that time. In 1856, the presence of physiological glycosuria in pregnancy and lactation was first described [9], and in 1877 different types of sugar were identified in the urine, being lactose the most frequently type of sugar found in the urine of pregnant women [10]. The incidence of true glycosuria varied from 10% to 100%; some authors believed that it represented true diabetes and others that it was a benign condition. The believe that it might represent true diabetes was based on the fact that many women that presented glycosuria had typical symptoms of diabetes such as polydpsia, polyuria, polyhydramnios and even adverse fetal outcomes [8].
The cause of glycosuria was unknown. Some suggested that it was alimentary (caused by a greater absorption of carbohydrates) or toxemic (caused by liver abnormalities). Brocard, in 1898 demonstrated by the first time that pregnant women were less tolerant to sugar compared to non-pregnant women; he has found the presence of glycosuria 2 hours after the ingestion of 50 g of glucose in 50% of pregnant women compared to 11% found in non-pregnant women [11]. Glycosuria was also found to be recurrent in successive pregnancies [11].
In order to solve this problem of classifying a woman as having or not diabetes based on the glycosuria levels, J.W.Williams recommended a follow-up of all mothers that presented glycosuria for possible complications of diabetes. He concluded that it was important to distinguish if the patient had a transient or a persistent glycosuria, to distinguish whether the sugar was lactose or glucose. If glycosuria appears late in pregnancy, is less than 2%, and not accompanied by symptoms, it is probably transient, of slight clinical significance and indicates the patient should be carefully watched. However, if glycosuria appears early in pregnancy and in large amounts, it represents a more serious condition, because it will make it difficult to diagnosis diabetes until after delivery. In cases of diabetes it will persist; the course of diabetes in pregnancy is variable, pregnancy may occur in women with diabetes or diabetes may withdrawn manifest during pregnancy and finally he suggested that if the amount of glycosuria is large and cannot be controlled, the induction of abortion or premature labor should be indicated, even if the patient does not show severe symptoms [8].
Between 1920 and 1930 many reports have described the presence of pancreatic abnormalities in stillborn infants from mothers with diabetes, mainly hypertrophy of Langerhans’ islands; which was suggested to occur as a consequence of glucose transfer from the mother to the fetus, to a poor maternal glucose control and the possible cause of severe neonatal hypoglycemia, that could be fatal in a few days after birth [4,12].
After the discovery of insulin, and its use during pregnancy, many reports of its efficacy were done by many authors [6,13-16]. Lambie in Edinburgh, in 1926, pertinently concluded that when diabetes appears for the first time in pregnancy, it usually manifests in the fifth or sixth month and exceptionally before the fourth or after the eighth month of gestation. He also suggested the 50 g oral glucose challenge test (OGCT) for calculating the ketogenic-antiketogenic balance [16]. Skipper in 1933, published a vast review of the literature in the use of insulin in pregnancy and found a dramatic improvement in maternal mortality and a modest impact on fetal and neonatal outcomes and survival [6]. He then concluded that the use of insulin has led to lower maternal mortality, but no reduction in fetal mortality; women with diabetes usually present glucose intolerance during the latter months of pregnancy; hypoglycemia is common in the puerperium and may have serious consequences including coma; if a woman with diabetes receives an adequate treatment, pregnancy should not be harmful; ketonuria is common in badly treated cases; the most important cause of fetal death is poor metabolic control, overdevelopment of the fetus and presence of congenital malformations; every pregnant woman with glycosuria should be investigated as possibly having diabetes because it may appear during pregnancy; a rigid control of diabetes through the evaluation of glycosuria is of great importance; cesarean section may be indicated when the fetus is overdeveloped; breast feeding should always be tried and sterilization should be considered in women with unstable diabetes and according to the number of children the patients already had [6]. Many of his conclusions are applied with some modifications and adaptations until today.
Miller in 1945 reported the first observations on obstetrical complications in the prediabetic period [17]. In the 1950s many risk factors for the development of abnormalities in carbohydrate metabolism in pregnancy were defined and the term gestational diabetes mellitus (GDM) became accepted [18-21]. Soon after, screening programs were proposed for the early detection of diabetes in pregnancy.
In 1949, Dr. Priscilla White working at the Joslin Clinic in Boston, wrote a paper and proposed the “White's Classification” that became a hallmark in the classification of diabetes and pregnancy. This classification was revised many times in order to separate those patients with GDM from those with pre-existing diabetes. An alphabetic list was added to the original classification that took into account the age of diabetes onset, diabetes duration and the presence of diabetes-related complications [22].
Screening for hyperglycemia in pregnancy
In the 1960s, the screening for GDM was done by taking patients’ history alone. The increased obstetrical risk associated with GDM, was first described by Hoet in 1954 [23]. Soon after that the National Institutes of Health developed a program in the epidemiology of chronic diseases, and a center for their study was established in Boston, Massachusetts, under Hugh Wilkerson [24]. At that time the role of glycosuria in pregnancy was controversial, although most investigators agreed of the possibility that it could be the first indicator of the presence of diabetes mellitus.
Based on Hoet’s study [23] and in the observation of a large group of women who were followed-up by Dr. John B. O’Sullivan, Wilkerson and Remein [19] in 1957 proposed offering a 3-hour oral glucose tolerance test (OGTT) for patients presenting risk factors for diabetes such as family history of diabetes, gestational glycosuria and overdeveloped infants at birth. For women without known risk factors, they proposed determining a 1-hour blood glucose value after the ingestion of a 50 g glucose load. A value of 130 mg or more was considered abnormal and a 3-hour OGTT should be performed afterwards [19].
Jackson in 1960, wrote a review article stating that a temporary abnormality in glucose tolerance during pregnancy indicated a potentially permanent diabetes state in the mother [25]. He also defined different stages in the development of diabetes: prediabetes (patients with a retrospective diagnosis of diabetes and with significant presence of risk factors for its development); chemical diabetes (asymptomatic patients with abnormal glucose tolerance) and finally overt diabetes (symptomatic patients) [25].
In order to detect any degree of carbohydrate metabolism disorder early in pregnancy, several modifications were proposed for the OGTT like the use of intravenous tolbutamide by Unger and Madison in 1958 [26] and of cortisone some hours before the oral glucose load by Conn and Fajans in 1961 [27]. The obtained curves presented much higher values than those performed without any drug and the methodology was abandoned.
There was a great confusion with the criteria for the definition of diabetes during pregnancy and even outside of pregnancy, based on glucose tolerance tests, hampered by different methodology to determine blood glucose levels (Somogyi-Nelson and Folin-Wu in the USA and Henederman in Europe), by the concentration of glucose in the solution to be ingested and whether the determination of glucose levels should be done in venous or capillary blood [4]. The Somogyi-Nelson, Folin-Wu and Henederman assays, are not specific for glucose; they measure all reducing substances present in the whole blood and give results that are 15–20 mg/dl higher than assays that measure only glucose [4].
The controversy on how to screen and diagnose GDM was great. Using the OGTT criteria for nonpregnant subjects, in pregnant women, the incidence of diabetes was about one-third of the entire pregnant population. In order to solve this problem, O’Sullivan performed 100-g OGTTs in 752 mainly second- and third-trimester pregnant women and found with the statistician Claire Mahan the first, second, and third standard deviation upper limits for these glucose values [28]. These were the first statistically based criteria for assessing glycemic normality in pregnancy. Compared to those found in normal individuals they had higher upper-limit values at the 2nd and 3rd hour, consistent with an impaired glucose tolerance in pregnant compared with nonpregnant individuals. The O’Sullivan and Mahan criteria, became the standard for diabetes detection in pregnancy for the next decades, although they were originally formulated to predict type 2 diabetes in the future and not to predict maternal and fetal problems in the index pregnancy [28]. The values proposed by O’Sullivan and Mahan were: fasting, 110 mg/dl; 1-hour, 170 mg/dl; 2-hours, 120 mg/dl and 3-hours, 110 mg/dl (for Somogyi-Nelson method and venous blood). Two or more abnormal values were enough to diagnose an abnormal test [28].
These values selected by O’Sullivan and Mahan represented the mean plus two standard deviations because they believed that the more lenient the test, for example mean plus one standard deviation, the greater would be the prevalence of diabetes, resulting in a long term follow-up of these patients what would pose an economic problem. They also believed that alerting too many people in order to benefit a relatively few potential diabetics would arise psychologic ill-effects [28].
Many studies such as those conducted by Pedersen and Priscilla White, showing the importance of maternal glucose levels in the outcomes of pregnancy in women with diabetes have changed the focus of the importance of diagnosing and treating these women as early as possible, not only aiming to predict the risk of type 2 diabetes in the future, but also to prevent adverse outcomes with the mother and the fetus in the current pregnancy [22,29].
In 1979, the National Diabetes Data Group (NDDG) converted the values of whole blood glucose thresholds to those approximately 14% higher plasma glucose values, as most of the laboratory instruments started to report plasma glucose values instead of whole blood glucose [30].
Gradually antenatal screening for hyperglycemia in pregnancy became established but also different screening and diagnostic procedures became proposed, even in the same country. The increasing incidence of diabetes in the background population, the altering demographic changes in human reproduction, the increasing prevalence of obesity among many other factors, resulted in important variation in the reported prevalence. For this reason, a series of International Colloquia on Carbohydrate Metabolism in Pregnancy were conducted between 1973 and 1988, four of them held in Aberdeen in Scotland. All of them failed to reach a worldwide consensus on the dose of glucose to use, how it should be given and when blood glucose should be measured [31].
Diagnosing hyperglycemia in pregnancy
The international workshops on gestational diabetes mellitus
Because of a growing disagreement in the best way to screen and diagnose diabetes in pregnancy Norbet Freinkel organized the First International Workshop on GDM in October 1979 in Chicago; another four would still come and also take place in Chicago. GDM was then defined as “carbohydrate intolerance of variable severity recognized for the first time in pregnancy”. The criteria used for the diagnosis were those established by O’ Sullivan and Mahan in 1964 [28,32,33]. It was then recommended reinforcement in research to achieve more accurate diagnosis, precisely define outcomes criteria, correlate outcomes with maternal variables and finally find more effective therapy alternatives to control blood glucose levels. It was also proposed that if a patient was at risk for glucose intolerance, a fasting plasma glucose or a random glucose at least 2 hours postprandial should be done. All women who had not been identified as having glucose intolerance before 24 weeks gestation, should be screened for GDM between 24–28 weeks gestation. A special attention should be given to those patients with a fasting plasma glucose level > 105 mg/dl. In cases of GDM, a close surveillance of the mother and the fetus should be done; nutritional counseling, including advice to limit intake of concentrated sweets and insulin therapy should be established if diet alone failed to maintain a fasting plasma glucose < 105 mg/dl and a 2-hour postprandial < 120 mg/dl; oral hypoglycemic agents were not recommended. A strict control of blood glucose levels showed to be important in reducing fetal and perinatal morbidity and mortality [33].
The Second International Workshop on GDM was held in October 1984. The definition of GDM developed at the First Workshop was confirmed, adding that “the definition applies irrespective of whether or not insulin is used for the treatment or if the condition persists after pregnancy. It does not exclude the possibility that the glucose intolerance may have antedated the pregnancy” [34]. In terms of GDM detection, it was determined that all pregnant women should be screened for glucose intolerance, since selective screening based on clinical attributes or past obstetric history is inadequate. This screening should be performed by glucose measurement between 24–28 weeks gestation in women not identified as having glucose intolerance before the 24th gestation week. A 50 g oral glucose challenge test (OGCT) for screening was proposed, regardless of last meal or time of the day, and a venous plasma glucose cutoff of ≥ 140 mg/dl on a sample obtained one hour after the glucose load was considered abnormal. For diagnostic purposes it was recommended to continue the utilization of 100 g OGTT and its interpretation according to diagnostic criteria of O’Sullivan and Mahan. Capillary blood measurement should not be used for diagnostic purposes. The measurement of glycated hemoglobin was also not considered a sensitive diagnostic indicator for GDM. In terms of management of GDM, those patients with fasting and potprandial hyperglycemia should be considered at greater risk for intrauterine death or neonatal mortality; they must undergo careful antepartum fetal surveillance. It was also mentioned by the first time that impaired carbohydrate tolerance may develop in macrosomic offspring. The nutritional counseling comprehended limitation of sucrose intake, monitoring of maternal weight, a caloric intake equivalent to that of nondiabetic women of normal weight but not so restrictive in calories. A program of moderate exercise was also recommended [34].
The blood glucose levels should be monitored, and if dietary management does not consistently maintain fasting plasma glucose < 105 mg/dl and/or a 2-hour postprandial plasma glucose < 120 mg/dl on two or more occasions within a two week interval, insulin should be initiated, accompanied by self-monitoring of blood glucose; breastfeeding should be encouraged. Finally, it was reinforced that more than half of women with GDM will develop permanent diabetes. In order to detect diabetes early, an evaluation with a 2-hour, 75 g OGTT should be performed at the first postpartum visit. Regular physical activity should be encouraged to these patients [34].
The Third International Workshop on GDM happened in November 1990. The previous definition of GDM was confirmed. Screening and diagnostic criteria were also confirmed but with some modifications: Plasma glucose levels ≥ 200 mg/dl outside of formal OGTT, or fasting glucose ≥ 140 mg/dl suggests a diabetic state, warranting further investigation. A proportion of patients who meet recommended criteria for GDM have screening levels < 140 mg/dl and consequently the detection of GDM requires a substantial increase in the number of full OGTT performed. Also for diagnostic purposes, it is to mention that adjustments for conversion of whole blood glucose concentrations to equivalent plasma glucose values may overcorrect glucose levels; for this reason it is inadvisable to introduce minor corrective modifications. One single abnormal OGTT value may merit further evaluation since it may be associated with increased morbidity. Fixed diagnostic criteria were suggested for all populations. Macrosomia could be clinically estimated by fetal size and asymmetric growth identified by ultrasonography, and an earlier intervention could improve this outcome. In terms of long-range implications, it was emphasized that babies born to mothers with GDM present an increased risk of overt diabetes later in life and an increased likelihood of obesity, glucose intolerance and neurobehavioral and developmental abnormalities at birth and during childhood [35].
In March 1997 happened the Fourth International Workshop on GDM. The previous definition of GDM was confirmed. It was proposed and recommended a screening strategy to identify women at low-risk (belonging to an ethnic group with a low prevalence of GDM, having no known diabetes in first-degree relatives, aged < 25 years, presenting normal weight before pregnancy and at birth, no history of abnormal glucose metabolism or of poor obstetric outcome) who would not need evaluation; average risk (should perform blood glucose testing at 24–28 weeks using either a two-step procedure with a 50 g OGCT followed by a diagnostic OGTT in those meeting the threshold value in the OGCT of ≥ 140 mg/dl, or a one-step procedure with an OGTT performed on all subjects) as shown in Table 1, and high risk (those with at least one or more of these risk factors: marked obesity, strong family history of type 2 diabetes, previous history of GDM, impaired glucose metabolism, glucosuria or belonging to high-risk ethnic groups such as Hispanic, African, Native American, South or East Asian and from Pacific Islands, or of Indigenous Australian ancestry) it was recommended universal screening or diagnostic testing using the Carpenter and Coustan criteria for interpretation of the 100 g OGTT, with new cutoff values: fasting 95 mg/dL, 1h 180 mg/dL, 2h 155 mg/dL, 3 h 140 mg/dL, and also a 75 g 2-hours OGTT with the above criteria. They should perform blood glucose testing as soon as feasible. If GDM is not diagnosed, blood glucose testing should be repeated at 24-28 weeks or at any time a patient has symptoms or signs suggestive of hyperglycemia [36]. For the first time it was recommended the use of ultrasound to detect congenital anomalies in patients with GDM diagnosed in the first trimester or who presented with fasting glucose concentrations > 120 mg/dL [36].
Table 1 Screening and diagnosis of GDM in a one or two-step approach according to ADA
SCREENING
Single step OGTT 100 or 75g For average and high risk pregnant 24th-28th gestation weeks
Two steps OGCT 50g If 1-hour≥ 140 mg/dl Perform an OGTT 100g For all pregnant women 24th-28th gestation weeks
DIAGNOSIS OGTT 100g OGTT 75g
FASTING 95 mg/dl 95 mg/dl
1-hour 180 mg/dl 180 mg/dl
2-hours 155 mg/dl 155 mg/dl
3-hours 140 mg/dl -
Criteria for GDM diagnosis= 2 altered values.
75 g OGTT does not include the 3-hour value.
A carbohydrate-rich diet with more than 150 g/ day/ 3 days before the test.
Patients should be seated and do not smoke during the test.
Some new therapeutic interventions were proposed during pregnancy with GDM such as: ideal glycemic targets to prevent fetal risk should be lowered to capillary glucose levels of fasting ≤ 95 mg/dL, 1 h ≤ 140 mg/dL, and/or 2 h ≤ 120 mg/dL; the weight gain should be of ~ 7 kg for obese patients (BMI > 29 kg/m2) and greater weight gain of up to 18 kg for underweight patients (BMI < 19.8 kg/m2); it was recommended the use of glucose meters that store results electronically including postprandial testing as well as fasting and/or premeal testing; measurement of pre-breakfast urine ketone for patients on hypocaloric or carbohydrate-restricted diets. Insulin therapy was recommended with minimally antigenic insulin preparations for patients who fail glycemic goals or show signs of excessive fetal growth. Physical activity should be performed three times per week for at least 15 minutes [36].
In order to monitor fetal wellbeing it was recommended maternal fetal movement counting during the last 8–10 weeks of pregnancy; in patients requiring insulin, non stress testing from 32 weeks onward and at or near term in those requiring only dietary management. More complex fetal monitoring such as the biophysical profile or doppler assessment of umbilical cord blood flow may be considered when excessive or poor fetal growth are noted or there are complicating medical problems, such as preeclampsia. Fetal abdominal circumference measurements by ultrasound at 29–33 weeks gestation are useful in identifying a large subset of patients with maternal fasting glucose levels < 105 mg/dL who are at low risk for fetal macrosomia at term when managed with dietary therapy alone [36].
In terms of long-range implications and management after pregnancy, it was noted that the progression to type 2 diabetes within 5 years after the diagnosis of GDM was related to gestational age, severity of GDM at diagnosis, level of glycemia at first postpartum assessment, impairment of β-cell function, obesity, and further pregnancy. It was then recommended an evaluation of glucose tolerance in the mother 6–12 weeks postpartum with a 75 g OGTT; if postpartum testing does not indicate diabetes, fasting plasma glucose should be evaluated annually and in preparation for any future pregnancy. Patients should be instructed in lifestyle behaviors to reduce weight and increase physical activity to reduce the risk of subsequent diabetes; preconception counseling should be given to address appropriate contraception and women contemplating a future pregnancy should be advised to take supplementary folic acid to avoid risks of congenital malformations. An increased risk of obesity and abnormal glucose tolerance by puberty in offspring of women with GDM was identified, and lifestyle measures aimed at reducing or preventing obesity may decrease these risks. Breastfeeding should also be encouraged to reduce the risk of obesity and possibly diabetes in the offspring [36].
The Fifth International Workshop on GDM was held in November 2005, under the sponsorship of the American Diabetes Association (ADA). The meeting provided a forum for review of new information concerning GDM in the areas of pathophysiology, epidemiology, perinatal outcomes, long-range implications for the mother and her offspring, and management strategies as did the previous four International Worshops on GDM [37]. The issues regarding strategies and criteria for the detection and diagnosis of GDM were not reviewed or discussed in detail, since it was anticipated that the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study [38] would provide data that would adopt criteria for the diagnosis of GDM that would be based on perinatal outcomes. Thus, a motion to continue use of the definition, classification criteria, and strategies for detection and diagnosis of GDM that were recommended at the Fourth International Workshop was endorsed [36]. Minor modifications were done mainly in relation to metabolic assessments recommended after GDM. These assessments should be done as follows: a fasting or random plasma glucose 1–3 days after delivery, to detect persistent, overt diabetes; around the time of postpartum visit, a 75-g 2-hour OGTT for the postpartum classification of glucose metabolism; the OGTT should be repeated one year postpartum, then tri-annually and before another pregnancy in order to assess glucose metabolism. It was also recommended to measure a fasting plasma glucose annually [37].
After so many international workshops and several decades of research, there is still no unified global approach for GDM diagnosis [38-43], as seen in Table 2.
Table 2 Existing criteria for GDM diagnosis
Diagnostic test Blood glucose values (mg/dl) Diagnostic criteria
fasting 1-hour 2-hours 3-hours
National Diabetes Data Group, 1979[30] 100g OGTT-whole blood 105 190 165 145 2 or more values above limit
Carpenter e Coustan,1982[38] 100g OGTT- plasma glucose 95 180 155 140 2 or more values above limit
World Health Organization, 1998[39] 75g OGTT- plasma glucose 126 - 140 - 1 value above limit
Brazilian Health Ministry, 2002[40] 75g OGTT- plasma glucose 110 - 140 - 1 value above limit
ADA, 2004[41] 100g OGTT 95 180 155 140 2 or more values above limit
American Diabetes Association, 2009[42] 100g OGTT/ 75gOGTT 95 180 155 - 2 or more values above limit
Brazilian Health Ministry, 2002[43] 75g OGTT- plasma glucose 95 180 155 - 2 or more values above limit
ADA, 2011 (46) and IADPSG,2010[45] 75g OGTT- plasma glucose 92 180 153 -
The hyperglycemia and adverse pregnancy outcomes study
The objective of the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study was to clarify the risk of adverse outcomes associated with different degrees of maternal glucose intolerance less severe than overt diabetes during pregnancy. Glucose tolerance was measured in approximately 25,000 women from nine different countries and fifteen different centers, in a heterogeneous, multicultural, ethnically diverse cohort of women at 24–32 gestation weeks. Positive associations were found between higher fasting, 1- and 2-h OGTT plasma glucose concentrations and birth weight >90th percentile and cord serum C-peptide >90th percentile, primary cesarean delivery, clinical neonatal hypoglycemia, preterm delivery, shoulder dystocia or birth injury, intensive neonatal care, hyperbilirubinemia, and preeclampsia, as well as with newborn adiposity [44].
The associations of maternal glycemia with perinatal outcomes were continuous with no obvious thresholds at which risks increased, it was evident that a consensus was required to translate these results into clinical practice. Many other issues had then to be addressed such as the importance to have all three OGTT glucose measurements (fasting, 1-, and 2-h-post load values) in the OGTT since the individual OGTT glucose measures were not highly correlated, and no single measure was clearly superior to each other in predicting the primary outcomes. It was necessary to find out the threshold at or above which the risk of adverse outcomes was too high.
The international association of diabetes and pregnancy study groups
The International Association of Diabetes and Pregnancy Study Groups (IADPSG) was formed in 1998 to facilitate collaboration between the various regional and national groups that have a primary or significant focus on diabetes and pregnancy. The IADPSG sponsored an “International Workshop Conference on Gestational Diabetes Diagnosis and Classification” in Pasadena, CA on June 2008 to initiate the process of a consensus development based on the data found in the HAPO study, associating maternal glycemia with perinatal and long-term outcomes in the offspring. Under the coordination from the Consensus Panel Steering Committee/Writing Group, the Panel reviewed further HAPO Study results provided by the HAPO Study Data Coordinating Center and the Consensus Panel has formulated the “Recommendations on the Diagnosis and Classification of Hyperglycemia in Pregnancy” which was soon after published, with new thresholds for the diagnosis of GDM [45]. It was then expected that this report would be considered by all of the major diabetes organizations and would serve as the basis for internationally uniform criteria for the diagnosis and classification of diabetes in pregnancy [45].
The new diagnostic criteria were soon adopted by many pre-eminent diabetes organizations such as the American Diabetes Association [46] and the Brazilian diabetes Society [47]. Despite the clear evidences that the HAPO study has shown on how harmful can even slightly altered blood glucose levels be, it has been found a resistance in the adoption of the new criteria by many diabetes organizations stating that these new criteria would increase the incidence of GDM from around 7% to 18.7%, and that the greater the prevalence of diabetes diagnosis, a longer term follow-up of these patients would be needed and would pose an economic problem, and also that alerting too many people in order to benefit a relatively few potential diabetics would arise psychologic ill-effects. These are the same arguments used by O’Sullivan and Mahan to select the upper standard deviation when they first described the OGTT in 1964. It seems that we did not have too much progress in the last decades.
Conclusions
After several international workshops and many decades of research, there is still no unified global approach to GDM, as seen in Table 2. Most countries have their own diabetes associations each one with 1 to 3 diabetes societies as an International Diabetes Federation member [48]. These societies often have their own guidelines for GDM, which may be very similar or markedly different, and often, no guideline is proposed. The problem of GDM is the lack of an international consensus among these diabetes organizations. There is a wide diversity in the methods used in most countries due to multiple reasons. Health providers often prefer to use alternate criteria, follow the recommendation of a diabetes or health organization from another country and often there is disagreement between the country’s national diabetes organization, its local health society, and its regional obstetric organization, with each one recommending different approaches for screening and diagnosing GDM. It would be of interest of pregnant patients the formulation of unified universal guidelines for GDM. A consensus could be achieved with the evidence based gained from the data obtained in recent trials. It is time to an agreement about one global guideline for GDM.
Abbreviations
HAPO study: The Hyperglycemia and Adverse Pregnancy Outcomes Study; OGCT: Oral glucose challenge test; GDM: Gestational diabetes mellitus; OGTT: Oral glucose tolerance test; NDDG: National Diabetes Data Group; IADPSG: The International Association of Diabetes and Pregnancy Study; ADA: American Diabetes Association.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Carlos Antonio Negrato and Marilia Brito Gomes drafted, reviewed and edited the manuscript. Both authors read and approved the final manuscript.
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Mediterr J Hematol Infect DisMediterr J Hematol Infect DisMediterranean Journal of Hematology and Infectious DiseasesMediterranean Journal of Hematology and Infectious Diseases2035-3006Università Cattolica del Sacro Cuore 10.4084/MJHID.2013.028mjhid-5-1-e2013028Original ArticlesRecurrent/Persistent Pneumonia among Children in Upper Egypt Saad Khaled 1Mohamed Sherif A. 2Metwalley Kotb A. 11 Department of Pediatrics, Assiut University, Assiut 71516, Egypt.2 Chest Diseases Department, Assiut University, Assiut 71516, Egypt.Correspondence to: Khaled Saad, Department of Pediatrics, Faculty of medicine, University of Assiut, Assiut 71516, Egypt, Tel +20-106-080-182*, Fax +20-88-236-8371. E-mail: [email protected] 18 4 2013 5 1 e201302816 12 2012 10 4 2013 2013This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Objectives
Recurrent/persistent pneumonia in children continues to be a major challenge for the pediatricians. The aim of our study was to establish the prevalence and underlying causes of recurrent/persistent pneumonia in children in Upper Egypt.
Settings
Assiut University Children Hospital, Assiut, Egypt.
Patients and Methods
Patients, admitted for pneumonia to the hospital during 2 years, were investigated with microbiological, biochemical, immunological and radiological tests in order to establish the prevalence of recurrent/persistent pneumonia and to find out its underlying causes.
Results
113 out of 1228 patients (9.2%) met the diagnosis of recurrent/persistent pneumonia. Identified causes were; aspiration syndrome (17.7%), pulmonary TB (14.0%), congenital heart disease (11.5%), bronchial asthma (9.7%), immune deficiency disorders (8.8%) and vitamin D deficiency rickets (7.0%). Other causes included; congenital anomalies of the respiratory tract, interstitial lung diseases, bronchiectasis, and sickle cell anemia. No predisposing factors could be identified in 15% of cases.
Conclusion
Approximately 1 out of 10 children with diagnosis of pneumonia in Assiut University Children Hospital had recurrent/persistent pneumonia. The most frequent underlying cause for recurrent/persistent pneumonia was aspiration syndrome, followed by pulmonary TB.
Recurrent/persistent pneumoniaaspirationpulmonary TBasthmaimmune deficiency
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Introduction
Pneumonia is a major problem in children, especially those younger than 5 years, accounting for up to 4 million deaths each year in developing countries. 1–2 A small subset of these children develop recurrent or persistent pneumonia, which is one of the most common reasons for referral to the paediatric physicians and continues to be a major challenge.3 There are limited data on the underlying causes predisposing to persistence or recurrence of pneumonia in children. Moreover, only few reports had studied this problem in developing countries.4–8
The aim of this study is to address the prevalence and underlying causes of recurrent/persistent pneumonia in children attending a tertiary care pediatric hospital; Assiut University Hospital, Upper Egypt; considering this as an important issue in our locality.
Material and Methods
This is a prospective hospital-based study. Children younger than 18 years admitted with a hospital diagnosis of pneumonia to Assiut University Children’s Hospital, from June 2009 to May 2011 were included. This hospital is the largest referral paediatric hospital in Upper Egypt. Diagnosis of pneumonia was based on cough, chest wall in-drawing and/or difficult breathing and tachypnea (respiratory rate ≥ 50 cycles/minute in infants 3 to 12 months old; ≥ 40/min in children 12 to 60 months old; ≥ 30/min in children older than 60 months), fever and lobar or bronchopneumonic infiltration demonstrated by X-ray. 1 Recurrent pneumonia was defined as two episodes of pneumonia occurring in 1 year or three episodes of pneumonia occurring in any time frame.3,6 Persistent pneumonia was defined as features of lower respiratory tract infection (i.e., cough, tachypnea and fever with or without chest retractions) with radiological evidence of infiltrates or consolidation in the lungs persisting for 30 days or more, despite receiving antibiotics for a minimum period of 10 days.6,7 The study protocol was approved by the ethical committee of Assiut University Children’s Hospital, Egypt. Written informed consents were obtained from the parents of patients. Studied cases were subjected to the following:
History
Thorough history including age, sex, duration of symptoms and treatment given, if any, contact with tuberculosis patients, immunization status, developmental milestones, and history of foreign body (FB) aspiration.
Clinical Examination
Complete physical examination including anthropometric measurement, presence of clubbing, pallor, dextrocardia, abdominal distension, eczema, signs of heart failure, signs of rickets and full neurological examination.
Investigations were tailored for every case according to the clinical presentation and suspected clinical diagnosis, and included:
Laboratory Tests
Complete hemogram including hemoglobin, total and differential leukocyte count, absolute eosinophil count, and peripheral smear.
Erythrocyte sedimentation rate (ESR).
Blood culture and the antimicrobial susceptibility of the isolated microorganisms.
Sputum, gastric lavage, or bronchoalveolar lavage (BAL) for culture for tuberculosis in selected cases.
Tracheal aspirate for culture in cases admitted in paediatric intensive care unit (PICU) and intubated.
Enzyme-linked immunosorbent assay (ELISA) for human immunodeficiency virus (HIV) infection.
Serum electrolytes and arterial blood gases.
Sweat chloride testing.
Quantitative serum immunoglobulins (IgG, IgA, and IgM)
flow cytometry was used to assess the number and subsets of lymphocytes.
Radiological and Endoscopic Examination
Chest roentgenograms; posteroanterior view in older children and anteroposterior view in younger children and a lateral view were taken, to document the presence of infiltrates or consolidation.
X ray paranasal sinuses.
Computed tomogram of thorax was done whenever necessary.
Chest radiographs and computed tomography images were interpreted by three independent observers.
Laryngoscopy & flexible fiberoptic bronchoscopy and BAL were done in selected cases. Fiberoptic bronchoscopy was performed in cases of suspected FB inhalation and in those with radiologic evidence of atelectasis. BAL fluid was subjected to microbiological and cytological assessment.
Echocardiography.
Barium swallow & esophageal pH manometry.
Others
Mantoux test was done for tuberculin sensitivity using 1 TU PPD with RT Tween 80 which was administered intradermally and reading was taken after 48–72 hours. Gastric aspirate for young children and sputum for older children were sent for acid fast staining on three consecutive days.
Pulmonary function tests; were carried out at the Department of Chest Diseases, Assiut University Hospital.
Statistical Analysis
Collected data were coded, analysed and computed, using the Statistical Package for Social Sciences (SPSS) version 16 (SPSS Inc., Chicago, IL, USA). Simple statistics such as frequency, arithmetic mean and standard deviation were used.
Results
Demographic and Clinical Data
From June 2009 to May 2011, 1228 patients were admitted to the hospital with the diagnosis of pneumonia; 113 (9.2%) of them met the definition for recurrent and/or persistent pneumonia. They were 74 (65.0%) males and 39 (35.0%) females. Their age ranged from 2 months to 14 years with a mean age of 3.2 ±3.8 years. Fourteen children (12.0%) had onset of symptoms before 3 months of age, 22 (19.0%) between 3 and 12 months, 45(40.0%) between 1 and 5 years and 32 (29.0%) after the age of 5 years. With regards to the number of pneumonia episodes, 23 patients had 2–3 episodes, 56 patients had 4 episodes and 7 patients had 5 or more episodes. The presenting symptoms included cough in all cases, respiratory distress in 76 (67.0%) cases, fever in 89 (79.0%) cases, wheezing in 15 (13.2%) cases, and pallor in 9 (8.0%) cases. On examination, 12 (10.6%) patients had clubbing, 8 (7%) had advanced rickets with chest deformities in 3 of them. (Table 1)
Radiographic and Endoscopic Findings
The chest radiograph showed consolidation in all patients cohort, atelectasis in 13 (11.5%) patients, para-pneumonic effusion in 9 (8.0%) cases, while 4 (3.5%) children had findings suggestive of bronchiectasis. CT chest was performed in 49 cases; it revealed bronchiectasis in 4 (8.2%) cases, interstitial lung disease in 5 (10.2%) cases, congenital cystic adenomatoid malformation in 3 (6.1%) cases and 2 (4.0%) patients had congenital lobar emphysema. On the other hand, chest radiographs of patients with recurrent pneumonia revealed recurrence in the same location in 28 (32.5%) patients while in 58 (67.5%) cases, it was at different locations. Rigid & fiberoptic bronchoscopies were done in 22 patients; with extraction of the forgein bodie(s) in 16 (72.7%) cases. Gastro-esophageal reflux disease (GERD) was diagnosed in 4 children by clinical suspicion and confirmed by barium swallow and esophageal pH manometry; all cases with GERD were younger than 2 years of age.
Etiologic Factors in Recurrent Pneumonia
Eighty-six patients had recurrent pneumonia. Aspiration syndromes were found in 13 (15.1%) patients; 4 of them were secondary to GERD, while 9 patients had definite history of foreign body (FB) aspiration. This history was confirmed by endoscopic extraction in 4 patients. All cases of foreign body aspiration had the recurrence of pneumonia at the same site, whereas it was the case in only one patient with GERD. The rest of GERD cases had a recurrence in different lung regions. Six patients (7%) had immune deficiency disorders, 4 of them had hypogammaglobulinemia and 2 had selective IgA deficiency. Congenital heart disease was confirmed by echocardiography in 9 patients (10.4%). This included 6 patients with ventricular septal defect (VSD), 2 with transposition of great arteries (TGAs), and one with complex anomalies. Eleven patients (9.7%) had bronchial asthma. Eight patients (9.3%) had vitamin D deficiency rickets. They had radiological evidence for rickets including two or more of the following signs: generalised osteopenia, fraying and cupping of the distal ends of the radius or ulna and widening of the costochondral junction. Interstitial lung disease (ILD), sickle cell anemia, and cystic fibrosis were diagnosed in 5 (5.8%), 3 (3.5%), and one patient (1.2%); respectively. All these patients had recurrence that involved more than one lung lobe.
Pulmonary TB was collectively diagnosed in 16 (14%) patients; 10 with recurrent pneumonia and 6 with persistent pneumonia. All cases had positive tuberculin by Mantoux test. Gastric lavage/sputum for acid fast bacilli were positive in 3 patients with recurrent pneumonia. The remaining 13 patients had clinical and radiographic findings strongly suggestive of pulmonary tuberculosis, and 10 of them were contacts with known adult cases of tuberculosis. Among these 13 cases four had positive culture of BK in gastric lavage/sputum. All of these 13 cases did not respond to relevant antibiotics and responded to anti-tuberculous therapy.
Four patients (4.7%) had congenital anomalies of the respiratory tract; 2 had congenital lobar emphysema, and 2 had congenital cystic adenomatoid malformation. Two (2.3%) patients had bronchiectasis. In the later two groups, recurrence was in the same lung region. No predisposing factors could be identified in 14 (16.3%) cases. (Table 2)
Etiologic Factors in Persistent Pneumonia
Twenty-seven patients had persistent pneumonia. Aspiration syndromes were found in 7 (26.0%) patients; all of them were due to FB aspiration, confirmed by endoscopic extraction. Four (14.8%) patients had immune deficiency disorders, all of them had hypogammaglobulinemia. Congenital heart diseases were found in 4 (14.8%) patients; one with TGAs, one with total anomalous pulmonary venous return, and two with VSD. Other causes for persistent pneumonia are shown in Table 3. No predisposing factors could be identified in 3 (11.1%) cases.
Discussion
Pneumonia is an important cause of morbidity and mortality in children, especially those younger than 5 years of age in developing countries. 1 A subgroup of children with pneumonia suffer from recurrent/persistent pneumonia, raising the question of whether there is an underlying cause(s) for such recurrence or persistence. The lack of epidemiological studies from developing countries makes it difficult to plan even national/local strategies for prevention and treatment. 2, 8 Recurrent and/or persistent pneumonia pose a significant challenge to the pediatricians; particularly in developing countries. Therefore, we aimed to address the prevalence of recurrent/persistent pneumonia in Upper Egypt, and to identify its possible causes. There are few studies of recurrent/persistent pneumonia in children (Table 4). Notably, most of the literatures describe the etiology of recurrent/persistent pneumonia. 7–16 In the current study; 9.2% of patients with pneumonia met the definition of recurrent/persistent pneumonia, 7% for recurrent and 2.2% for persistent pneumonia. Similarly; 1–9% of patients met the criteria for recurrent/persistent in previous studies.7–16
We had observed that, the most frequent underlying cause for recurrent/persistent pneumonia in children was aspiration syndrome (17.7%), followed by pulmonary TB (14%), congenital heart disease (11.5%), bronchial asthma (9.7%), immune deficiency disorders (8.8%) and nutritional rickets (7%). Other causes included; congenital anomalies of the respiratory tract, interstitial lung diseases, bronchiectasis, sickle cell anemia and cystic fibrosis. No predisposing factors could be identified in 15% of the patients (Table 2 and 3).
Chronic aspiration is the most common cause of recurrent pneumonia in childhood. Aspiration pneumonia arises after inhalation of oropharyngeal contents into the lungs. It may be an acute event or occurring on a chronic recurrent basis. Also, aspiration of foreign bodies into the lung represents an important cause of intraluminal airway obstruction in the pediatric population. Retained foreign bodies occur most commonly in the 6-month to 3-year age group. Foreign body inhalation should be suspected in the presence of sudden-onset cough, dyspnea, and recurrent pneumonia with a history of choking episodes. However, there may be no definite history and this can lead to long delays in diagnosis that increase the risk of long-term complications such as bronchiectasis. A physical examination may reveal respiratory distress, localised pulmonary hypoventilation, wheezing, ronchi and metallic sounds. Radiography may show atelectasis or areas of hyperinflation, although the findings are normal in about 20–40% of children with foreign body inhalation. 6,17,18
On the other hand, recurrent pneumonia may be a complication of GERD due to aspiration of gastric contents. Certain conditions in children put them at risk for higher incidence, relapse and chronicity of GERD symptoms. These include children with chronic neurologic impairment, repaired esophageal atresia, hiatal hernia, chronic respiratory diseases like cystic fibrosis (CF) and genetic conditions like Down syndrome. GERD should always be considered a possible cause of recurrent pneumonia when children complain of typical symptoms (i.e. heartburn, regurgitation and dysphagia). 17–19
Aspiration pneumonia accounted for 17.7% of our patients; 13 patients had recurrent pneumonia and 7 had persistent pneumonia, 4 of them were secondary to GERD, and 16 had FB aspiration. This is in agreement with many reports 7, 9, 11, 20, 24 who observed that aspiration disorders were the most frequent cause of recurrent/persistent pneumonia. Interestingly, 16 of our cases had FB aspiration. Lack of aspiration history can’t rule out the diagnosis of FB aspiration; therefore in questionable cases bronchoscopy is advised. The triad of coughing, wheezing and decreased breath sound should point to a diagnosis of FB aspiration. The occurrence of these symptoms specially in times where the patient has no history of aspiration sometimes results in misdiagnosis as bronchitis, asthma and pneumonia and the patients undergo treatment with antibiotics, bronchodilators, and corticosteroids which itself result in the changing of clinical manifestations and chronicity of the disease. 7, 9, 17, 18
Tuberculosis is one of the most common infectious diseases among children in the world. TB is suspected when an ill child has a history of chronic illness of usually more than 3 weeks of duration that includes a cough and a fever, weight loss or failure to thrive, history of contact with an adult case of pulmonary TB and a non response of symptoms to potent antibiotics.21 Tuberculosis is a common cause of extraluminal compression of the airways associated with recurrent lung infections. 18 In the current study, pulmonary TB was diagnosed in 16 (14%) patients; 10 with recurrent pneumonia and 6 with persistent pneumonia. All cases had positive tuberculin by Mantoux test, while only 13 cases were diagnosed on strong clinical and radiographic findings suggestive of pulmonary tuberculosis, and history of contacts with adult cases of tuberculosis in 10 cases. Interestingly, among those 13 cases, cultures of gastric lavage/sputum were positive for tuberculosis in 4 cases only. These findings are in agreement with those of Strake 21; who observed that, even under optimum conditions for collecting gastric aspirates; three gastric aspirates yield M. tuberculosis in < 50% of cases. So, negative cultures never exclude the diagnosis of tuberculosis in a child. He concluded that, the need for culture confirmation is usually low. If the child has a positive tuberculin skin test, clinical or radiographic findings suggestive of tuberculosis, and known contact with an adult case of tuberculosis, the child should be treated for tuberculosis disease. 21
Lodha et al 9 reported pulmonary TB as a cause of recurrent pneumonia in 7.1% of patients in addition; Çelebi 20 and his colleagues reported 4.8%. In previous studies with persistent pneumonia; Kumar7 and Lodha12 reported pulmonary TB as a cause in 19.2% and 31.5% of patients, respectively. This relatively high prevalence of pulmonary TB should alarm the physicians and health authorities in our locality to take more intensive measures for prevention and control of this disease.
Congenital heart diseases are important causes for recurrent/persistent pneumonia in children. Dilated blood vessels or chambers of the heart may compress the bronchi, causing impaired drainage of pulmonary segments. Also patients with congenital lesions causing left-to-right shunting and an increased pulmonary blood flow have an increased susceptibility to respiratory infections.5 Previous studies have reported congenital heart disease in 1.2–25.4 % of cases.7,8,11,14,15,16,20,24 In agreement with these figures, our results demonstrated that congenital heart diseases were identified in 11.5% of cases. Among the various shunt lesions that present in infancy, ventricular septal defect is the most common. Other defects include atrial septal defect, patent ductus arteriosus, and atrioventricular septal defect. In these diseases, the blood is shunted through an abnormal opening from the left to the right side of the heart, with increase in pulmonary blood flow and increased cardiac workload (including ventricular strain, dilation, and hypertrophy). A left-to-right shunt can adversely affect lung function, and superimposed lower respiratory tract infections cause additional compromise and might lead to respiratory failure.18
Bronchial asthma was diagnosed among 9.7% of our patients. Notably, children with asthma presented with episodes of pneumonia but were otherwise healthy. Growth, development, and physical examination, were all within normal reference limits. These children were diagnosed as having asthma clinically and/or functionally. Children who have a history of nocturnal cough, cough or wheezing with exercise or protracted coughing after upper respiratory illnesses should undergo spirometry and assessment of bronchodilator responsiveness, or they should receive an empiric trial of inhaled corticosteroids and bronchodilators.23 Bronchial asthma is the most important underlying illness for recurrent and persistent pneumonia in children reported by different researchers8,12,14,16 accounting for 15%–69 % of cases. Our study and previous reports emphasize that asthma is a common cause of recurrent and persistent pneumonia and that pneumonia may occur as the initial symptom, even in the absence of wheezing. In contrast to our study and previous studies; Hoving and Brand;24 reported that asthma was not diagnosed as an underlying cause of recurrent pneumonia in their study. They believed that asthma is a rare cause of recurrent pneumonia in children, and if occurs this seems to be confined to very unusual and complicated cases of asthma.24,25
Remarkably, immune deficiency disorders were identified in 10 patients (8.8%) of our cohort; 8 patients had hypogammaglobulinemia and 2 had selective IgA deficiency. Our results are similar to those studies8,9,11,13,20,24 demonstrated immune deficiency disorders in 7.7–17.75 % of cases. Children with immune defects usually present with highly recurrent and/or severe bacterial infections of the respiratory tract without any seasonality, recurrent gastrointestinal infections and recurrent skin infections. Lymphadenopathy and a failure to thrive are also common features. The family history is often characterized by recurrent infections and early deaths. There is often a delay of years between the onset of symptoms and the diagnosis being made: this delay increases the risk of bronchiectasis and irreversible lung damage occurring before appropriate treatment is given.18, 26 From the clinical point of view, Screening for immunodeficiency is useful in evaluating recurrent pneumonia, it should be suspected in children with infections that are especially severe and recurrent, that are caused by unusual organisms, or that involve multiple sites in addition to the lungs.25–27 Immunoglobulin replacement therapy has significantly reduced the frequency and severity of acute bacterial infections in primary immunodeficiencies, although long-term pulmonary complications such as chronic lung disease do occur.18
Studies in developing countries have suggested an association between nutritional rickets and pneumonia.28–30 In the present study 7% of studied cases were attributed to rickets. Rickets is a commonly recognized disease in Egypt, the factors responsible for occurrence of rickets in Egypt are repeated poorly spaced pregnancies with lack of maternal vitamin D supplementation, the dusty atmosphere especially during winter and spring, lack of health and nutritional education, the habit of excessive wrapping of infants and keeping them indoors without exposure to sunlight, poor housing and faulty weaning.29,30 Recurrence of pneumonia among children with rickets may be attributed to many factors first: generalized hypotonia including chest wall muscle with difficulty to clear secretion, also vitamin D has immunoprotective role. It acts upon T and B cells and can modulate functions of lymphocytes that produce cytotoxins and antibodies. Early treatment with vitamin D and calcium can prevent the recurrence or persistence of pneumonia.28–31
In the present study, 5 patients (4.4%) had congenital anomalies of the respiratory tract; (4 patients with recurrent pneumonia; 2 patients had congenital lobar emphysema, 2 had congenital cystic adenomatoid malformation and one had persistent pneumonia with congenital cystic adenomatoid malformation). Previous studies have reported congenital anomalies of the respiratory tract in 3.7–8.5% of cases.9–11,20,24 Recurrent or persistent chest infections are often the presenting feature of congenital abnormalities of the airways, lung parenchyma and pulmonary vasculature. For example, repeated episodes of pneumonia are often the presenting feature of lobar sequestration, bronchial stenosis and bronchomalacia, and cystic adenomatoid malformations of the lung. Such an abnormality should be suspected if one lobe is repeatedly infected or if there is incomplete resolution after treatment. Computerised tomography and magnetic resonance scanning are helpful in defining the anomaly prior to surgical excision.27,32,33
Our results showed that 3 patients (2.6%) had sickle cell anemia. This was suspected secondary to anemia residence and/or family history. Owayed and coworkers11 reported 4% of cases had sickle cell anemia. It has long been recognized that children with homozygous sickle cell anemia are at increased risk for pneumonia relative to other children, even with penicillin prophylaxis. 34 Moreover, it was shown that penicillin prophylaxis is not effective in the absence of strict compliance and even then, protection against bacterial infections may not be absolute.34–36
The current study showed that only one case had CF, diagnosed by sweat chloride test. Although the prevalence of CF is rare in our community, not only due to rarity of the disease in our community but also due to non-availability of advanced and specific diagnostic tools for diagnosis CF. A history of neonatal jaundice, poor weight gain, steatorrhea and highly recurrent pneumonia may suggest cystic fibrosis, although atypical cases may present with recurrent pneumonia alone, in the absence of malabsorption. Also recovery of pseudomonas aeruginosa from the respiratory tract, especially the mucoid form, is highly suggestive of CF.18,27,33
The present study is not without limitations. Despite being a prospective study; our results included only hospitalized patients, thus we might have underestimated number of patients with recurrent/persistent pneumonia in the community. In addition; we were not able to do all the necessary immunological investigations as a result of financial constrains.
Finally, the results of this study will have an important impact on the differential diagnosis and management of recurrent and or persistent pneumonia in children in our locality. The most encountered known etiologies were aspiration, pulmonary TB, and congenital heart disease. Proper health education for preventing aspiration in children should be carried out. Bronchoscopy is advisable in questionable cases of FB aspiration even in the absence of aspiration history. The relatively high prevalence of pulmonary TB should alarm the physicians and health authorities in our locality to take more intensive measures for prevention and control of such a communicable disease. Lastly, congenital heart diseases should be carefully looked for when managing a case of recurrent/persistent pneumonia and relevant clinical and investigatory approaches should be carried out.
Conclusion
Approximately 1 in 10 children with pneumonia in our locality had recurrent/persistent pneumonia. The most frequent underlying cause for recurrent/persistent pneumonia in children in Upper Egypt is aspiration syndrome, followed by pulmonary TB. The results of this study would help the pediatricians identify and hence prevent and manage the most common aetiologies of recurrent/persistent pneumonia in our locality.
Competing interests: The authors have declared that no competing interests exist.
Abbreviation
BALBronchoalveolar lavage
CFcystic fibrosis
ELISAEnzyme-linked immunosorbent assay
FBforeign body
GERDGastro-oesophageal reflux disease
HIVhuman immunodeficiency virus
ILDinterstitial lung disease
PICUpaediatric intensive care unit
TGAstransposition of great arteries
VSDventricular septal defect
Table 1 Demographic and clinical data among patients with recurrent/persistent pneumonia
Characteristic Patients No (%)
Age at diagnosis
Range 2 months–14 years
(mean ± SD) 3.2 ± 3.8 years
Gender
Male/female 74/39
M:F ratio 1.9:1
Presenting symptoms
Cough 113 (100%)
Respiratory distress 76 (67.0%)
Fever 89 (79.0%)
Wheezing 15 (13.2%)
Pallor 9 (8.0%)
Clinical signs
Clubbing 12 (10.6%)
Advanced rickets 8 (7.0%)
Table 2 Etiologic factors in cases with recurrent pneumonia
Underlying illness Recurrent Pneumonia
N %
Aspiration syndrome
GERD 4 4.7
FB aspiration 9 10.4
Congenital heart disease 9 10.4
Immune deficiency 6 7
Bronchial asthma 11 9.7
Pulmonary TB 10 11.6
Vitamin D deficiency Rickets 8 9.3
Intersitial lung diseases 5 5.8
Anomalies of respiratory tract:
Congenital lobar emphysema 2 4.7
Congenital cystic adenomatoid malformation 2
Sickle cell anemia 3 3.5
Bronchiectasis 2 2.3
Cystic fibrosis 1 1.2
Unknown cause 14 16.3
Total 86 100%
N: Number of patients
Table 3 Etiologic factors in cases with persistent pneumonia
Underlying illness Persistent pneumonia
N %
Aspiration syndromes
FB aspiration 7 26.0
Pulmonary TB 6 22.2
Congenital heart disease 4 14.8
Immune deficiency 4 14.8
Bronchiectasis 2 7.4
Anomalies of respiratory tract:
Congenital cystic adenomatoid malformation 1 3.7
Unknown cause 3 11.1
Total 27 100%
N: Number of patients
Table 4 Previous studies of children with recurrent/persistent pneumonia
Study Paulina Owayed Lodha 2002 Lodha 2003 Adam Ozdemir Çiftçi Kumar Cabezuelo Eigen Çelebi Hoving Current study
Type of pneumonia recurrent recurrent recurrent persistent Recurrent and persistent recurrent recurrent persistent recurrent Recurrent and persistent recurrent recurrent recurrent and persistent
Reference number 10 11 9 12 13 8 14 7 15 16 20 24 -
Number of cases 121 238 70 19 18 62 71 41 106 81 185 62 113
Bronchial asthma 19 19 10 5 1 19 23 - 28 56 16 - 11
Aspiration syndromes 13 127 21 3 3 11 13 13 25 8 58 16 20
Immune deficiency disorder 8 34 11 1 6 11 7 3 9 5 10 10 10
Pulmonary TB - - 5 6 - - - 8 - - 9 - 15
Anomalies of respiratory tract 8 18 6 - - 3 4 2 2 3 6 5 5
Congenital heart disease - 22 - - - 7 6 2 27 1 32 3 13
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23675406PONE-D-12-3088210.1371/journal.pone.0060727Research ArticleMathematicsStatisticsBiostatisticsMedicineClinical ImmunologyImmunologic SubspecialtiesTransplantationClinical Research DesignCohort StudiesRetrospective StudiesGastroenterology and HepatologyBiliary DisordersSurgeryTransplant SurgeryIntraoperative Cryoprecipitate Transfusion and Its Association with the Incidence of Biliary Complications after Liver Transplantation-A Retrospective Cohort Study Cryoprecipitate Transfusion Was Associated with BCLiu Shuang Fan Junwei Wang Xiaoliang Gong Zijun Wang Shuyun Huang Li Xing Tonghai Li Tao Peng Zhihai
*
Sun Xing
*
Department of General Surgery, Shanghai First People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
Hills Robert K. Editor
Cardiff University, United Kingdom
* E-mail: [email protected] (ZP); [email protected] (XS)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: XS ZP. Performed the experiments: SL JF XW. Analyzed the data: SL ZG TL. Contributed reagents/materials/analysis tools: LH TX. Wrote the paper: SL XS. Modified the article: XS SW.
2013 10 5 2013 8 5 e607279 10 2012 1 3 2013 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Cryoprecipitate is largely used for acquired hypofibrinogenemia in the setting of massive hemorrhage in liver transplantation (LT). However, the influence of intraoperative cryoprecipitate transfusion on biliary complications (BC) after LT has not been studied in detail.
Study Design and Methods
In a series of 356 adult patients who received their first LT, the causes of BC were retrospectively studied by multivariate logistic regression analysis. The clinical relationship between intraoperative cryoprecipitate transfusion and BC occurrence was studied through a retrospective cohort study in patients. All patients received follow-ups for one year, and, during the follow-up period, the time of BC occurrence and liver biopsies were recorded.
Results
Intraoperative cryoprecipitate transfusion (RR = 3.46, 95% CI [1.72–6.97], P<0.001), cold ischemia time >8 h (RR = 4.24, 95% CI [2.28–7.92], P<0.01), and high-level Child-Pugh ( RR = 1.71, 95% CI [1.11–2.63], P = 0.014) are independent risk factors to predict BC after LT according to time-to-event analysis. One year BC-free survival probability of patients received intraoperative cryoprecipitate transfusions was significantly lower when compared to the group that received no cryoprecipitate(P<0.001). Moreover, BC patients in the cryoprecipitate transfusion group owned different liver pathological feature, pathological micro-thrombus formation and cholestasis were seen more often (41.4% vs 0%, 62.1% vs 12.5%, respectively) than no cryoprecipitate transfusion group.
Conclusion
These findings suggested that intraoperative cryoprecipitate transfusion was associated with BC after LT. The mechanism of BC occurrence might involve micro-thrombus formation and immune rejection.
This work is supported by the National Natural Science Foundation of China (81170445). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Improvement of surgical techniques and immunosuppression, as well as better organ preservation have brought great improvements in success rate of liver transplantation (LT). However, postoperative biliary complications (BC) remain the weakest part of LT, and have been referred to as the “Achilles' heel” of the procedure [1]. According to the literature, the incidence of BC after LT varies from 10% to 30% [2], [3].
Although BC is a significant source of patient morbidity and mortality after LT, the mechanism of BC remains unclear and identifying risk factors of BC might give insight into the pathogenesis and reduce graft loss. Due to improvement in surgical techniques and perioperative care, the incidence of biliary anastomosis strictures and leaks decreased, however the incidence of non-anastomotic bile duct stricture became more apparent [4], [5]. Previous studies in LT have found that apart from the obvious life-saving benefits, an increase in blood loss and subsequent transfusion of blood products has been associated with substantial side effects, such as increased risk of BC [6]
[7].
Cryoprecipitate provided a major therapeutic advantage for patients in LT, as it does not require blood cross- matching, and is convenient to obtain. Currently, the most common indication for the use of this product is in hypofibrinogenemia in the setting of massive hemorrhage. With its increased usage in surgery, there has also been a high incidence of inappropriate use of cryoprecipitate which may range from 24%–62% [8], [9]. The influence of intraoperative cryoprecipitate transfusion on BC after LT, however, has not been studied in detail.
We retrospectively reviewed our LT patients in a single center to address two issues. Firstly, whether or not intraoperative cryoprecipitate transfusion is associated with BC after LT. If this was the case, our second objective was to detect the possible mechanism of BC occurrence. This information would allow the implementation of specific measures in high-risk patients to minimize BC occurrence.
Materials and Methods
Patients
389 patients who underwent LT at Shanghai First People's Hospital between January 1, 2005 and December 31, 2010 were initially selected. All transplants were from cardiac death donors. We excluded Data from children (n = 1), patients who required re-transplantation (n = 23), paients with primary biliary cirrhosis (n = 7) and primary sclerosing cholangitis (n = 2), and the remaining 356 consecutive adult cases formed the analysis population.
We defined BC on the basis of a 2-fold increase of serum bilirubin greater than normal levels occurring within the first year after LT. The increased serum bilirubin levels also needed to be sustained for at least 1 week. The final diagnosis of BC was then verified through “gold standard” protocols as follows : magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangio-pancreatography(ERCP), percutaneous transhepatic cholangiography(PTC), liver biopsy or intraoperative observation. The biliary anastomotic strictures and vascular anastomotic stenosis were not included in this study.
In our center, we apply cryoprecipitate transfusion to patients with fibrinogen <1.0 g/l or patients with significant coagulation problems (prothrombin time >6 s), to compensate fibrinogen or coagulation factors. We'll stop transfusion when the thrombelastography during surgery recovers to normal range or blood exudation is significantly improved. For patients with large amount of blood loss (>2500 ml), we'll have to transfuse blood cells which will very often lead to decreased fibrinogen (<1 g/l) and lead to cryo transfusion. For some patients with coagulation problems, even when the blood loss is less than 2500 ml, we need to do cryo transfusion to improve their coagulation function to facilitate the operation.
Clinical characteristics of these patients, including donor and recipient variables, as well as surgical factors were obtained from the China Liver Transplantation Registry (CTLR) computer database. When necessary, the original patient notes were reviewed for missing information. Risk factors determined to be meaningful predictors of BC were selected based on a review of the literature. National legislation and the ethical committee of Shanghai First People's Hospital approved this retrospective study.
Surgical techniques and perioperative care
Grafts from cardiac death donors were used for all patients. The orthotropic LT technique was used for implantation. In our center, duct-to-duct reconstruction for biliary anastomosis using 7/0 prolene sutures was performed with microsurgical technique without a stent or T-tube. All surgical procedures were supervised by one of the two most experienced surgeons, who assume responsibility for the surgical team. Color Doppler sonographer was used for detecting abnormalities of the hepatic vasculature in patients with LT in the first 3 days. All patients were treated with standard immunosuppressive therapy as described elsewhere [10]. Anesthetic management during LT was performed using a common protocol previously decided by consensus. During the research period, the anesthetic protocol had little substantive change.
Follow up
Follow-ups were performed every week in the first three months, then fortnightly until six months post-transplant. Beyond this, follow-ups were performed monthly. All cases were followed routinely by our outpatient department for at least one year until the patient was lost to follow-up due to death, emigration to another district , or re-transplantation, whichever occurred first. Once serum bilirubin increased up to 2-fold of normal level, the patient was immediately admitted to hospital for further treatment.
Statistics
Firstly, univariate analysis of risk factors which might be associated with BC was performed. Categorical variables were compared using the Chi-squared test. Mann-Whitney u-test was used to rank data and non-normal distribution continuous variables. According to univariate analysis and previous literature, all variables might be associated with BC were subjected to stepwise logistic regression analysis with 0.1 level for entry into the model . Odds ratios and 95% confidence intervals were calculated to filter out independent risks for BC.
Using cryoprecipitate transfusions as an exposure factor, the patients with intraoperative blood product transfusions were divided into two groups. A retrospective cohort study was carried out, and baseline fractures were compared between the two groups. Intraoperative blood loss was treated as the stratified factor of the two groups given it maybe a confounding factor., MH Chi-squared test was performed when analysis combined RR. Multivariate stepwise COX regression was also performed with 0.1 level for entry into the model, to enhance the risk evidence of BC. The BC-free survival curves were calculated according to the Kaplan–Meier method and compared using the log-rank test. The time between BC occurrence and obtaining of liver biopsies from BC patients were compared to detect the possible mechanism of BC occurrence.
Continuous data are presented as mean±standard deviation. All tests were two-sided. Statistical analysis was performed using the SPSS/PC Advanced Statistics Package, Version 19.0 (SPSS, Chicago, IL).Statistical tests were assumed to have reached significance at the conventional level of 0.05.
Results
Characters of BC patients and case-control analysis
In 356 patients, a total of 40 patients had developed the BC in the first year after LT, the overall incidence of BC was 11.2% (40/356). 356 patients were divided into two groups depending on whether they had developed BC or not. Pre-, intra- and postoperative factors, blood product transfusion are summarized in
TABLE 1
. The mean requirement of cryoprecipitate for the BC group was 13.5±12.2, while the non-BC group was 5.4±9.0 U (P<0.001). The need for RBC transfusion was significantly higher in BC group (10.2±8.9) than in non-BC group (8.1±10.6, P = 0.013). Univariate analysis also indicated that MELD (P = 0.017) and cold ischemia time (P<0.001), were statistically significant factors between the two groups. Furthermore, according to univariate analysis and previous literature, all variables might be associated with BC were subjected to stepwise logistic regression analysis to evaluate independent risk factors associated with BC. Multivariate logistic regression analysis demonstrated that cold ischemia time >8 h (OR = 6.30, 95% CI [2.97–13.3]), intraoperative cryoprecipitate transfusion (OR = 4.23, 95% CI [1.95–9.17]), and Child-Pugh (OR = 1.79, 95% CI [1.09–2.94]) were independent risk factors predicting BC after LT (
TABLE 2
).
10.1371/journal.pone.0060727.t001Table 1 Patient characteristics and demographics by study groups.
Variables Total Non-BC BC P
N = 356 N = 316 (88.8%) N = 40 (11.2%)
Age(year)
48±10 48±9 46±10 0.171
Gender
Male 304 (85.4%) 269 (85.1%) 35 (87.5%) 0.689
Female 52 (14.6%) 47 (14.9%) 5 (12.5%)
Diagnosis
FHF*
45(12.6%) 40(12.7%) 5 (12.5%) 0.577
Cirrhosis 282(79.2%) 250 (79.1%) 32 (80.0%)
Carcinoma 24 (6.7%) 22 (7.0%) 2 (5.0%)
Others 5(1.4%) 4(1.3%) 1(2.5%)
Child-Pugh
0.073
Grade A 117 (32.9%) 113(35.8%) 4 (10.0%)
Grade B 142 (39.9%) 121 (35.4%) 21 (52.5%)
Grade C 97 (27.2%) 82 (26.9%) 15 (37.5%)
ABO blood group
Compatible 306 (86.0%) 272 (86.1%) 34 (85.0%) 0.854
Incompatible 50 (14.0%) 44 (13.9%) 6 (15.0%)
MELD
0.017
MELD≤10 53 (14.9%) 50(15.8%) 3 (7.5%)
11≤MELD≤18 190 (53.4%) 175 (55.4%) 15 (37.5%)
18<MELD≤24 20 (5.6%) 13 (4.1%) 7 (17.5%)
MELD≥25 93 (26.1%) 78 (24.7%) 15 (37.5%)
Surgical factors
WIT* (min) 3.4±0.8 3.4±0.8 3.3±0.8 0.497
CIT* (min) 449±111 440±108 516±107 <0.001
Anhepatic phase (min) 58±10 58±10 58±7 0.578
Operation time (min) 392±95 389±90 411±130 0.851
Blood loss(ml) 3371±3419 3288±3451 4023±3121 0.025
Intravenous infusion(ml) 5890±2852 5893±2881 5869±2646 0.915
Blood products transfusion
RBC(U) 8.3±10.4 8.1±10.6 10.2±8.9 0.013
FFP*(U) 2.0±4.7 1.9±4.4 2.8±6.5 0.86
PLT(U) 0.66±0.96 0.66±0.95 0.65±1.08 0.735
Cryo* (U) 6.3±9.7 5.4±9.0 13.5±12.2 <0.001
Whole blood(U) 0.40±2.07 0.38±2.10 0.55±1.92 0.172
Cell saver(ml) 573±1396 550±1391 754±1439 0.091
Postoperative factors
Immunesuppressor
FK506+MMF 305(85.7%) 270(85.4%) 35(87.5%) 0.595
CysA+MMF 43(12.1%) 38(12.0%) 5(12.5%)
Others 8(2.2%) 8(2.5%) 0(0.0%)
Steroid
195(54.8%) 172(48.3) 23(6.5%) 0.713
Acute rejection
7(2.0%) 6(1.7%) 1(0.3%) 0.569
Chronic rejection
9(2.5%) 6(1.7%) 3(0.8%) 0.068
* FHF = fulminant hepatic failure; FFP = fresh frozen plasma; WIT = warm ischemia time; CIT = cold ischemia time; Cryo = cryoprecipitate.
10.1371/journal.pone.0060727.t002Table 2 Independent risk factors associated with BC (case-control analysis).
Variables OR*
95% C.I. P
Lower Upper
Child-Pugh 1.79 1.09 2.94 0.022
CIT(>8 h) 6.30 2.97 13.3 <0.001
Cryo transfusion 4.23 1.95 9.17 <0.001
* OR were derived from multivariate stepwise logistic regression analysis. These factors were adjusted in the multivariate regression analysis: age, diagnosis, child-pugh, MELD, CIT, blood loss, RBC, Cryo transfusion, Cell saver transfusion, chronic rejection.
Retrospective cohort analysis
We analyzed the incidence of BC by a retrospective cohort study. Stratified analysis was performed depending on whether a patient had blood loss of ≤2500 ml or >2500 ml (
TABLE 3
). Of the 221 patients with intraoperative blood loss of less than 2500 ml, the 60 patients that received intraoperative cryoprecipitate transfusions showed a higher BC incidence than the 161 patients who had no cryoprecipitate transfused (15.0% vs. 4.97%, RR = 3.02; 95% CI [1.22–7.46]; P = 0.013). Similarly, of the 135 patients with intraoperative blood loss more than 2500 ml, the 86 patients with cryoprecipitate transfused had significantly higher BC occurrence compared to the patients who did not receive cryoprecipitate (23.3% vs. 6.12%, RR = 3.80; 95% CI [1.19–12.1]; P = 0.011). When treated blood loss as the stratified factor, the combined RR of BC (Cryo vs no-Cryo) is 3.38(95% CI [1.62–7.05]; P<0.001). We then stratified patients according to the blood loss amount and compared the characteristics of the patients between intraoperative cryoprecipitate transfusion (cryo) or no-cryo groups (
TABLE 4
). For patients with less than 2500 ml blood loss, gender, diagnosis, MELD, RBC, FFP and Child-Pugh were found to be significantly different between cryo and no-cryo groups. However, for patients with more than 2500 ml blood loss, none of these characters were found to be significantly different.
10.1371/journal.pone.0060727.t003Table 3 Incidence of BC in Cryo group and No-Cryo group.
BC RR*
95% C.I. P
Cryo vs No-cryo Lower Upper
Blood loss≤2500
3.02 1.22 7.46 0.013
Cryo 9/60(15.00%)
No-Cryo 8/161(4.97%)
Blood loss>2500
3.80 1.19 12.1 0.011
Cryo 20/86(23.3%)
No-cryo 3/49(6.12%)
Combined
3.38 1.62 7.05 <0.001
* Univariate analysis was performed, the combined RR is computed when blood loss as the stratified factor.
10.1371/journal.pone.0060727.t004Table 4 Baseline in No-cryo group versus cryo group.
Blood loss≤2500 ml Blood loss>2500 ml
Variables Total Cryo No-cryo P Total Cryo No-cryo P
N = 221 N = 60 N = 161 N = 135 N = 86 N = 49
Age(year)
47.0±9.8 47.4±10.0 46.9±9.7 0.904 49.0±9.2 49.2±8.9 48.5±9.7 0.773
Gender
0.029 0.943
Male 197(89.1%) 49(81.7%) 148(91.9%) 107(79.3%) 68(79.1%) 39(79.6%)
Female 24(10.9%) 11(18.7%) 13(8.1%) 28(20.7%) 18(30.9%) 10(20.4%)
Diagnosis
0.035 0.322
FHF 13(5.9%) 5(8.3%) 8(5.0%) 32(23.7%) 22(16.3%) 10(20.4%)
Cirrhosis 186(84.2%) 54(90.0%) 132(82%) 96(71.1%) 61(45.2%) 35(71.4%)
Carcinoma 20(9.0%) 0(0%) 20(12.4%) 4(3.0%) 1(1.2%) 3(6.2%)
Others 2(0.9%) 1(1.7%) 1(0.6%) 3(2.2%) 2(2.3%) 1(2.0%)
Child-Pugh
0.001 0.105
Grade A
95(43.0%) 16(26.7%) 79(49.1%) 22(16.3%) 17(19.8%) 5(10.2%)
Grade B
83(37.6%) 26(43.3%) 57(35.4%) 59(43.7%) 40(46.5%) 19(38.8%)
Grade C
43(19.5%) 18(30.0%) 25(15.5%) 54(40.0%) 29(33.7%) 25(51.0%)
ABO blood group
0.198 0.759
Compatible 192(86.9%) 55(91.7%) 137(85.1%) 114(84.4%) 72(83.7%) 42(85.7%)
Incompatible 29(13.1%) 5(8.3%) 24 (14.9%) 21(15.6%) 14(16.3%) 7(14.3%) 0.810
MELD
0.041 0.079
MELD≤10 46(20.8%) 8(13.3%) 38(23.6%) 7(5.2%) 7(8.14%) 0(0%)
11≤MELD≤18 122(55.2%) 31(51.7%) 91(56.5%) 68(50.4%) 45(52.3%) 23(46.9%)
18<MELD≤24 12(5.4%) 3(5.0%) 9(5.6%) 8(5.9%) 6(7.0%) 2(4.1%)
MELD≥25 41(18.6%) 18(30.0%) 23(14.3%) 52(38.5%) 28(32.6%) 24(49.0%)
Surgical factors
WIT (min) 3.4±0.8 3.3±0.8 3.4±0.8 0.510 3.4±0.8 3.3±0.8 3.4±0.8 0.654
CIT (min) 442±113 445±96 441±119 0.525 458±107 452±108 471±106 0.250
Anhepatic phase (min) 57.2±10.9 57.7±10.1 57.0±11.2 0.316 59.2±8.6 60.6±8.8 58.4±8.4 0.112
Operation time (min) 360±66 363±72 359±64 0.803 445±111 446±122 443±89 0.547
Intravenous infusion(ml) 4927±1359 4872±1402 4940±1347 0.915 7476±3800 7147±3738 8053±3878 0.079
Blood products
RBC(U) 3.3±3.2 4.8±3.3 2.8±3.1 <0.01 16.5±12.7 17.2±12.1 15.2±13.7 0.132
FFP(U) 0.8±2.1 1.3±2.9 0.6±1.6 0.045 4.0±6.7 4.5±7.4 3.1±5.3 0.299
PLT(U) 0.4±0.8 0.4±0.8 0.4±0.8 0.859 1.2±1.1 1.1±1.1 1.1±1.1 0.910
Whole blood(U) 0.3±1.3 0.50±2.0 0.2±1.0 0.445 0.6±2.9 0.2±1.7 1.2±4.3 0.107
Cell saver(ml) 607+1350 594±1348 628±1358 0.659 1296±2038 1346±2184 1207±1772 0.749
Postoperative factors
Immunesuppressor
0.060 0.093
FK506+MMF 188(85.1%) 55(91.7%) 133(82.6%) 117(86.7%) 75(87.2%) 42(85.7%)
CysA+MMF 29(13.1%) 3(5.0%) 26(16.2%) 14(10.4%) 7(8.1%) 7(14.3%)
Others 4(1.8) 2(3.33%) 2(1.2%) 4(3.0%) 4(4.7%) 0(0%)
Steroid
118(53.4%) 35(58.3%) 83(51.6%) 0.808 77(57.0%) 45(52.3%) 32(65.3%) 0.143
Acute rejection
4(1.8%) 2(3.3%) 2(1.2%) 0.297 3(2.2%) 3(3.5%) 0(0.0%) 0.553
Chronic rejection
2(0.9%) 2(3.3%) 0(0%) 0.073 7(5.2%) 3(3.5%) 4(8.2%) 0.255
Further multivariate COX regression were performed to fix the difference of characteristics. This analysis showed that there was independent higher risk of BC when cold ischemia time >8 h (RR = 4.24, 95% CI [2.28–7.92], P<0.01), intraoperative cryoprecipitate transfusion (RR = 3.462, 95% CI [1.721–6.966], P<0.01), and high-level Child-Pugh (RR = 1.71, 95% CI [1.11–2.63], P = 0.014) after LT (
TABLE 5
). Therefore, the independent risk factors derived from cohort analysis were in accordance with case-control analysis.
10.1371/journal.pone.0060727.t005Table 5 Independent risk factors associated with BC (cohort analysis).
Variables RR*
95% C.I. P
Lower Upper
Cryo vs No-cryo 3.46 1.72 6.97 <0.01
CIT(>8 h) vs CIT(≤8 h) 4.24 2.28 7.92 <0.01
Child-Pugh increased one grade 1.71 1.11 2.63 0.014
Chronic rejection vs No chronic rejection 3.23 0.99 10.6 0.052
* RR were derived from multivariate stepwise COX regression.
Pathological feature
All 37 liver biopsy specimens submitted from the BC patients were evaluated in this study. The biopsies showed pathological findings that ranged from nearly normal to severe cholestasis (
TABLE 6
). Varying degrees of inflammatory cell infiltration was seen in both groups. In most of the no-cryoprecipitate patients, the structure of the hepatic lobule was integrated (7/8), hepatic cells showed vacuolar degeneration (4/8), and some degree of epithelial hyperplasia was seen (4/8) (
Figure 1 A,C
). In comparison, in a majority of biopsies from the cryoprecipitate transfusion group, the integrity of the lobular structure was lost (21/29), part of the intrahepatic bile duct disappeared (19/29), and varying degrees of cholestasis and micro-thrombosis were observed in various sized portal area vessels (18/29, 16/29 respectively) (
Figure 1 B,D
).
10.1371/journal.pone.0060727.g001Figure 1 Liver biopsies from BC patients.
Representative histopathology images of BC patients liver biopsies, A,C are from No-cryo group and B,D are from Cryo group. In the No-cryo group, the structure of hepatic lobule was integrated, normal hepatic plates were observed, hepatic cells occurred vacuolar degeneration, bile duct epithelium hyperplasia and a few inflammatory cells infiltration, cholestasis was seen (A, C). The integrity of the lobular structure was loss. Intrahepatic bile ducts proliferated significantly, part of the bile duct disappeared. Bile duct epithelial deformation, atrophy, shedding, ?the portal area shows infiltration of lymphocytes, the intrahepatic seen varying degrees of cholestasis, micro-thrombosis was observed in various sized portal area vessels (B, D).
10.1371/journal.pone.0060727.t006Table 6 Histopathological Features of Liver Biopsies from 37 BC patients.
Features No-cryo Cryo
N = 8 N = 29
Knodell HAI score
*
Minimal (1–3) 3 ( 37.5%) 10 (34.5%)
Mild (4–6) 2 (25.0%) 12 (41.4%)
Moderate (7–9) 1 (12.5%) 4 (13.8%)
Marked (10–12) 0 ( 0%) 2 (6.9%)
Hepatic lobule structure
Integrated 7 ( 87.5%) 8 (27.6%)
Disappeared 1 ( 12.5%) 21 (72.4%)
Hepatic cells Vacuolar degeneration
4 ( 50%) 9 (31.0%)
Bile duct
Epithelial hyperplasia 4 ( 50%) 6 (20.7%)
Bile duct disappeared 1 ( 12.5%) 19 (65.5%)
Cholestasis
1 ( 12.5%) 18 (62.1%)
Micro-thrombosis
0 ( 0%) 12 (41.4%)
* Total of Knodell Histology Activity Index scores for periportal injury, parenchymal injury, and portal inflammation.
Analysis of one year BC-free survival
The BC-free survival curve for post-transplant patients is shown in
Figure 2
. It shows that the BC-free survival rate for patients received intraoperative cryoprecipitate transfusions was significantly lower than the group that received no cryoprecipitate (P<0.001). According to previous literature, BC can be classified as early or late based on whether they occur ≤90 days or >90 days after LT [11]. The analysis of 40 patients who developed BC showed that, in the group receiving cryoprecipitate transfusion, BC occurred 111±84 days after LT and 18 out of 29 patients developed late BC. In the no-cryoprecipitate transfusion group, the mean time to BC occurrence was 72±59 days (P = 0.226) and 5 in 11 patients developed late BC (P = 0.477) (
TABLE 7
). Neither of these parameters was significantly different, indicating that the cryoprecipitate transfusion didn't alter the basic pathological properties of BC.
10.1371/journal.pone.0060727.g002Figure 2 One year BC-free survival curve for live-transplant patients.
One year BC-free survival curve for live-transplant patients according to the Kaplan–Meier method. BC-free survival probability of Patients received intraoperative cryoprecipitate transfusions(red line) was statistically significant lower when compared to the group that received no cryoprecipitate (blue line)(P<0.001).
10.1371/journal.pone.0060727.t007Table 7 Analysis of BC occurrence.
Variables Total No-cryo Cryo P
N = 40 N = 11 N = 29
Occurrence time (days)
101±79 72±58.5 111±84 0.226
Periods
*
0.477
Early BC 17 (42.5%) 6( 54.5%) 11 (37.9%)
Late BC 23 (57.5%) 5 (45.5%) 18 (62.1%)
* Periods classified based on the time of BC occurrence (Early BC occurred≤90 days after the operation, Late BC occurred >90 days after the operation).
Discussion
Although BC is a major cause of graft loss and re-transplantation in patients who survive the early post-operative period after LT, the pathogenesis of BC is uncertain. In the last few years, multiple studies have found an association between intraoperative blood products transfusion and subsequently poor outcomes for patients who underwent LT [12]–[15]. Cryoprecipitate, which can control bleeding in LT through supplementation of fibrinogen and some pro-coagulant factors, was widely used in many centers. However, our study has shown that intraoperative cryoprecipitate transfusion was associated with the incidence of BC after LT. This result implies that cryoprecipitate transfusion during LTx should be performed only after careful consideration. Although our study did not provide a causal relationship between cryoprecipitate transfusion and BC, our analysis (case-control and cohort analysis) all indicated that cryoprecipitate transfusion is a significant risk factor of BC. Furthermore, it should be noted that while several baseline characters between cryo and no-cryo groups were significantly different in patients lost less than 2500 ml blood, none of these baseline characters were significantly different in patients lost more than 2500 ml blood (TABLE 4). This further supported the notion that cryoprecipitate transfusion is a significant risk factor, especially for patients lost more than 2500 ml blood.
The preparation of cryoprecipitate was first described by Judith Graham Pool in 1964 [16]. Cryoprecipitate is comprised of plasma coagulation proteins, in particular factor VIII, fibrinogen, von Willebrand factor, and Platelet microparticles (PMPs) [17]. The PMPs concentration of the cryoprecipitate has been shown to be 265-fold greater than that of the original plasma sample [18]. Isolated PMPs have been reported to activate both the extrinsic and intrinsic coagulation cascades [19], and provide a surface for thrombin formation [20]. Generally, PMPs have 50- to 100-fold higher specific procoagulant activity than activated platelets [21]. Therefore, cryoprecipitate might have a strong potential ability of promoting thrombosis. In the current study, micro-thrombus formation was observed more frequently in various sized portal area vessels in the massive cryoprecipitate transfusion group. This indicated that cryoprecipitate transfusion in LT might mediate the formation of micro-thrombosis.Large number of micro-thrombosis result in biliary microcirculation disturbance, leading to the occurrence of BC.
Small amounts of immunogenic components, such as immunoglobulin (IgG, IgM), are also present in cryoprecipitate [22]. In addition, previous research reported that PMPs are also capable of stimulating antigen-specific IgG production and activating adaptive immune cells [23]. When cryoprecipitate was transfused in patients underwent LT, the effects of these immune components were not take into account. In the current study, liver biopsy specimens from BC patients showed that bile ducts disappeared more frequently in cryoprecipitate transfusion group. As this pathological finding was one of the typical manifestations in organ rejection, it is strongly suggested that immunogenic components existed in cryoprecipitate, played an important role in the development of BC.
Despite the complex composition of cryoprecipitate, in end-stage liver disease patients, the criteria for when and how to use cryoprecipitate in LT has not been established. The British Committee for Standards in Hematology, Blood Transfusion Task Force and College of American Pathologists(CAP) have published guidelines for the use of cryoprecipitate, and is considered appropriate to use when the fibrinogen level was less than 1.0 g/L [24]–[26]. However, compared to healthy individuals, the coagulation system in patients with end-stage liver disease has its own characteristics. Both prohemostatic drivers and antihemostatic drivers in these patients are relatively reduced, and the entire coagulation system is a low-equilibrium state [27]. Moreover, end-stage liver disease patients are characterized by increased levels of factor VIII and von Willebrand factor combined with decreased levels of most other procoagulant factors [28]. Therefore, inappropriate cryoprecipitate transfusion to replenish fibrinogen can tip this fragile balance toward thrombosis simultaneously, due to high concentration of factor VIII, and von Willebrand factor contained in cryoprecipitate [17]. Once the thrombus formation affected the blood supply of biliary tract, the incidence of BC increased significantly. Furthermore , the fibrinogen content in a unit of cryoprecipitate varied widely, ranging from 120 to 796 mg [22], this was another important cause increased the degree of difficulty to use cryoprecipitate correctly.
In the current study, cold ischemia time more than 8 h is also associated with the incidence of BC. Many previous study have showed the same conclusion [29]–[31]. Although this finding was not new, it strengthened a concept that minimizing cold ischemic time is essential to reduce the risk of BC. Another study stressed that high MELD score might also contribute to the risk of BC [32], but our multivariate logistic regression analysis indicated that Child-Pugh classification, not MELD score, could be associated with the risk for BC. Previous study had also stressed certain MELD components (bilirubin level and international normalized ratio) to be risk factors for BC [33]. Therefore, recipient preoperative liver function trends might have effects on BC following LT, but further confirmation is required.
Previous literature has mentioned many other risk factors that may affect the incidence of BC, such as cytomegalovirus infection [34], Rh-incompatible [35], hepatitis C virus infection [36], and hepatic artery thrombosis [37]. However, these factors were relatively minimized in this research, and the relationship between the above factors and BC could not be further determined. In addition, some studies have also proposed that T tube drainages increased the incidence of BC [38], [39]. In our center, a randomized and controlled multicenter trial in 2001 reached the conclusion that T-tube drainages increased the incidence of BC [40], after which the T-tube drainages in LT had been abandoned. According to previous literature [11], we analyzed the time of BC occurrence, but there was no significant difference between cryoprecipitate transfusion group and no-cryoprecipitate transfusion group.
The limitation of the study was its retrospective nature. We did not evaluate factors that were not found for BC in the analysis. Surgical factors also contributed to the BC, but the role of these factors is becoming less significant due to the improvement of surgical techniques and perioperative care. Furthermore, we have no effective methods to carry out the assessment of the impact of surgical factors. So in this study, we excluded cases with biliary anastomosis strictures and vascular anastomotic stenosis, which were mainly caused by surgical techniques, and then the overall BC rate reduced to 11.2%.
The findings of our study suggested that cryoprecipitate transfusion, which was not noticed in other multiple analysis programs, was associated with the incidence of BC after LT. The possible risks of cryoprecipitate transfusions could be due to the complex ingredients in the product, which may cause micro-thrombus formation and immune rejection injury of bile ducts. To evaluate the exact role of cryoprecipitate transfusion playing in patients with end-stage liver diseases undergone LT, more randomized, controlled multicenter trials and further research needs to be performed to develop a reasonable, standard application regarding the use of cryoprecipitate in LT. Before this research is conducted, it is necessary to be cautious while using cryoprecipitate transfusions during LT. If the use of cryoprecipitate was solely for fibrinogen supplement, it is better to adopt the protocol carried out in some European countries, where cryoprecipitate was substituted by virally inactivated fibrinogen concentrate with more standardized concentration and a safer profile (effectively reducing the risk of pathogen and immune-related complications) [41], [42].
==== Refs
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Biomed Res IntBiomed Res IntBMRIBioMed Research International2314-61332314-6141Hindawi Publishing Corporation 10.1155/2013/867537Research ArticleMicroRNA-124 Regulates the Proliferation of Colorectal Cancer Cells by Targeting iASPP
Liu Kuijie Zhao Hua Yao Hongliang Lei Sanlin Lei Zhendong Li Tiegang Qi Haizhi *Department of General Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China*Haizhi Qi: [email protected] Editor: Wolfgang Arthur Schulz
2013 10 4 2013 2013 86753720 12 2012 24 2 2013 27 2 2013 Copyright © 2013 Kuijie Liu et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.MicroRNAs are a class of small, noncoding RNAs that function as critical regulators of gene expression by targeting mRNAs for translational repression or degradation. In this study, we demonstrate that expression of microRNA-124 (miR-124) is significantly downregulated in colorectal cancer tissues and cell lines, compared to the matched adjacent tissues. We identified and confirmed inhibitor of apoptosis-stimulating protein of p53 (iASPP) as a novel, direct target of miR-124 using target prediction algorithms and luciferase reporter gene assays. Overexpression of miR-124 suppressed iASPP protein expression, upregulated expression of the downstream signaling molecule nuclear factor-kappa B (NF-κB), and attenuated cell viability, proliferation, and colony formation in SW480 and HT-29 colorectal cancer cells in vitro. Forced overexpression of iASPP partly rescued the inhibitory effect of miR-124 on SW480 and HT29 cell proliferation. Taken together, these findings shed light on the role and mechanism of action of miR-124, indicate that the miR-124/iASPP axis can regulate the proliferation of colorectal cancer cells, and suggest that miR-124 may serve as a potential therapeutic target for colorectal cancer.
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1. Introduction
Colorectal cancer (CRC) still is the common cancer, the third leading cause of cancer deaths in the whole world [1], and generally has a very poor prognosis due to its high propensity for tumor invasion and migration. Although invasion and migration have been acknowledged as the most lethal attributes of solid tumors, knowledge of the molecular mechanisms underlying these processes is still limited.
Recently, growing evidence has supported cancer-related roles for microRNAs (miRNAs), a newly discovered class of small, noncoding RNAs which negatively regulate the expression of a variety of genes. MicroRNAs affect multiple cellular pathways through inducing the mechanism of RNA interference at the posttranscription level. Primary miRNAs (pri-miRNAs) are transcribed by RNA polymerase, then processed into smaller precursor hairpin structures (pre-miRNAs) in the nucleus, and exported to the cytoplasm. They are further processed by the nuclease Dicer to mature functional miRNAs approximately 21 nucleotides in length. Mature miRNAs induce mRNA degradation or inhibit translation by integrating into an RNA-inducing silencing complex (RISC) and binding to specific complementary sites within the 3′ untranslated regions (3′ UTR) of their target mRNAs [2–6]. Bioinformatic algorithms indicate that human miRNAs perhaps regulate up to 30% of all human genes, which represent the majority of cellular pathways [7, 8]. Due to the complicated combination relationships between miRNAs and mRNA 3′ UTRs, several online tools have been developed for miRNA target prediction. These resources are used to microRNA target predictions based on sequence complementarity, including TargetScan [5], PicTar [7], and TargetRank [8], which provide precise base pairing of the miRNA to the seed region and sequence conservation.
Inhibitor of apoptosis stimulating protein of p53 (iASPP), also known as RelA-associated inhibitor (RAI), is one of the most ancient members of the ASPP protein family, and an evolutionarily conserved inhibitor of p53. As a binding partner, iASPP negatively regulates the RelA subunit (p65RelA) of the nuclear factor-kappa B (NF-κB), which plays a pivotal role in the inflammatory response and apoptosis [9, 10]. Therefore, it is likely that the expression level or activity of iASPP would influence the availability of NF-κB, and thus modify the regulation of cell growth. iASPP may serve as an independent prognostic marker of tumor proliferation in some other cancer types. Li et al. [11] reported that knockdown of iASPP significantly inhibited cell growth and proliferation in U251 cells. Zhang et al. [12] reported that the expression of iASPP in tumor tissues was higher than the adjacent tissues of prostate cancer. Downregulation of the expression of iASPP by shRNA inhibited proliferation and induced apoptosis of p53-defective prostate cancer cells. Existing data regarding the involvement of iASPP in tumor metastasis also suggests thatit may have potential as a therapeutic target [6, 13].
In order to identify novel microRNAs which could specifically target iASPP, several candidate microRNAs (miR-124, miR-506, miR-182, miR-19a, and miR-19b) were initially predicted by software analysis programme. We therefore challenged their expression in patient samples and also explored iASPP inhibition in colorectal cancer cells by introduction of exogenous miRNA expression. miR-124 expression was decreased significantly in CRC tissues compared to the adjacent normal colonic tissues in a panel of matched tissues from 17 CRC patients. In the present study, we confirmed the regulatory relationship between miR-124, a known tumor-suppressive miRNA, and an oncogene, iASPP. We provide evidence that miR-124 can inhibit CRC cell proliferation, at least in part by targeting iASPP. Ectopic overexpression of miR-124 downregulated iASPP protein expression and upregulated NF-κB protein expression. Force expression of iASPP rescued the effects of miR-124. These results provide novel insights into our understanding of the role and mechanism of miR-124 in the pathoetiology of CRC, which may provide a potential therapeutic strategy for treatment of CRC in the future.
2. Materials and Methods
2.1. Tissue Samples, Cell Lines, and Cell Transfection
A total of 17 paired primary CRC tissues and the matched adjacent normal colonic epithelial tissues were collected. All samples were obtained from patients who underwent surgical resection at The Second Xiangya Hospital of Central South University (Changsha, China). The tissues were snap-frozen in liquid nitrogen, and then stored at −80°C. This project was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University.
Human CRC cell lines, including SW480, SW620, and HT29 cells, were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) at 37°C in a humidified atmosphere with 5% CO2. Ectopic overexpression of miR-124 was achieved by transfection of miR-124 lentivirus (Genepharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen). Overexpression of iASPP was achieved using an iASPP ORF-expressing clone (GeneCopoecia, Guangzhou, China). Cells were plated in 6-well plates or 96-well plates, transfected, incubated for 24 h or 48 h, and used for further assays or RNA/protein extraction.
2.2. Construction of miR-124 and iASPP shRNA Expressing Lentivirus Vectors
To generate lentivirus expressing mature miR-124, the pre-miRNA sequence was synthesized; a control scrambled construct (control RNAi) with no homology to the human genome was also created (AAT GTA CTG CGC GTG GAG A). The sequences were cloned into the HpaI and XhoI sites of pGCSIL-GFP (GeneChem, Shanghai, China) to generate pGCSIL-GFP-miR-124 or pGCSIL-GFP-Ctr, respectively. Viral shRNA targeting iASPP was purchased from Auragene Bioscience Inc. (Changsha, China).
2.3. Lentivirus Production, Titration, and Infection
To generate miR-124 or control lentivirus, the plasmids encoding miR-124 or the control scrambled sequence were cotransfected into 293T cells together with the plasmids pHelper1.0 and pHelper 2.0 (Genechem, Shanghai, China) which contain the elements required for virus packaging, using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instructions. The culture supernatants containing lentivirus were harvested and concentrated by ultracentrifuge, and the viral titers were determined. To perform lentiviral infection, the target cells were plated at 40%–50% confluence and incubated overnight (16 h). On the day of infection, the culture medium was replaced with viral supernatant at an appropriate titer (1.5 mL/well), incubated at 37°C for 10 h, then the viral supernatant was replaced with fresh media. Forty-eight hours later, the infected cells were selected using puromycin (2 mg/mL). After 5 days of selection, shRNA knockdown efficiency was determined by real-time PCR and Western blot analysis.
2.4. RNA Extraction and Quantitative Real-Time RT-PCR
Total RNA was extracted from 10–20 mg of tumor samples and from 30–40 mg of normal tissues. Samples were mechanically disrupted and simultaneously homogenized in the presence of QIAzol Lysis reagent (Qiagen, Valencia, CA, USA), using a Mikrodismembrator (Braun Biotech International, Melsungen, Germany). RNA was extracted using the miRNeasy Mini kit (Qiagen) according to the manufacturer's instructions.
Approximately 1.0 × 106 SW480 or HT29 cells (uninfected or infected) were seeded into 6-well culture plates, cultured for 72 h, and harvested. Small RNAs (~200 nt) were isolated using the mirVanaTM PARIS TM Kit (Ambion, CA, USA) according to the manufacturer's instructions.
For RT reactions, 1 mg of small RNAs was reverse transcribed with the miScript Reverse Transcription Kit (Qiagen, CA, USA) at 37°C for 60 min followed by a final incubation at 95°C for 5 min. miRNA real-time RT-PCR was carried out using the miScript SYBR Green PCR kit (Qiagen) on an CFX96 real-time PCR machine (Bio-Rad, Hercules, CA, USA). PCR was conducted at 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s. The expression of each miRNA was normalized to U6 snRNA.
Expression of iASPP mRNA was detected by quantitative real-time RT-PCR (qRT-PCR) using the standard SYBR Green RT-PCR Kit (Bio-Rad, CA, USA) according to the manufacturer's instructions. Briefly, total RNA was extracted from the cells using TRIzol reagent (Invitrogen) and cDNA was synthesized using the RevertAid First-Strand cDNA Synthesis kit (Fermentas, CA, USA, according to the manufacturer's protocol. Each cDNA sample was used as a template for PCR in triplicate with iQTM SYBR Green Supermix (Bio-Rad, CA, USA) by denaturation at 94°C for 1 min; 30 cycles of 94°C for 40 and 60°C for 40 s; followed by extension at 72°C for 6 min. The specific primer pairs were iASPP (107 bp), sense: 5′-GGC GGT GAA GGA GAT GAA C −3′; antisense: 5′-TGA TGA GGA AAT CCA CGA TAG AG-3′; β-actin (202 bp) sense: 5′-GGC GGC ACC ACC ATG TAC CCT-3′; reverse: 5′-AGG GGC CGG ACT CGT CAT ACT-3′. The relative levels of iASPP mRNA were normalized to the internal control β-actin. Relative gene expression was quantified using CFX Manager software (Bio-Rad, CA, USA) and expressed as percentage of control cells.
2.5. Western Blotting
Cells cultured in 35 mm dishes were lysed in 0.2 mL lysis buffer (0.1% SDS, 1% NP-40, 50 mM HEPES, pH 7.4, 2 mM EDTA, 100 mM NaCl, 5 mM sodium orthovanadate, 40 μM p-nitrophenyl phosphate and 1% protease inhibitor mixture set I (Calbiochem, USA). Lysates were centrifuged at 12,000 rpm for 15 min, the supernatants were collected, denatured, separated using 10% SDS-PAGE gels, and blotted onto polyvinylidene difluoride membranes. The membranes were blocked in 5% albumin from bovine serum (BSA) for 1.5 h at room temperature, then probed with 1 : 1000 diluted rabbit polyclonal iASPP and NF-κB (p65) antibody (Abcam, MA, USA) at 4°C overnight, and the blots were subsequently incubated with HRP-conjugated secondary antibody (1 : 5000). Signals were visualized using ECL Substrates (Millipore, MA, USA). β-actin was used as an endogenous protein for normalization.
2.6. MTT Assay
Cell viability was evaluated using a modified MTT assay. The viability of SW480 or HT29 cells transfected with miR-124 or control was assessed at five time points (on days 1, 2, 3, 4, and 5) after seeding 2 × 103 transfected cells/well into 96-well culture plates. Briefly, quantification of mitochondrial dehydrogenase activity was achieved via the enzymatic conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, MO, USA) to a colored formazan product. MTT (10 μL, 10 mg/mL) was added to the cells, incubated for 4 h, and the reaction was terminated by removal of the supernatant and addition of 100 μL DMSO to dissolve the formazan product. After 0.5 h, the optical density (OD) of each well was measured at 570 nm using a plate reader (ELx808 Bio-Tek Instruments, City, ST, USA).
2.7. BrdU Incorporation Assay
DNA synthesis in proliferating cells was determined by measuring 5-Bromo-2-deoxyUridine (BrdU) incorporation. BrdU assays were performed at 24 h and 48 h after transfecting SW480 or HT29 cells with miR-124 or control vector. The infected cells were seeded in 96-well culture plates at a density of 2 × 103 cells/well, cultured for 24 h or 48 h, and incubated with a final concentration of 10 μM BrdU (BD Pharmingen, San Diego, CA, USA) for 2 h to 24 h. At the end of the incubation period, the medium was removed, the cells were fixed for 30 min at RT, incubated with peroxidase-coupled anti-BrdU-antibody (Sigma-Aldrich) for 60 min at RT, washed three times with PBS, incubated with peroxidase substrate (tetramethylbenzidine) for 30 min, and the absorbance values were measured at 490 nm. Background BrdU immunofluorescence was determined in cells not exposed to BrdU but stained with the BrdU antibody.
2.8. Colony Formation Assay
The effect of iASPP silencing on the colony formation ability of SW480 or HT29 cells was analyzed using the colony formation assay. SW480 or HT29 control, RNAi control, or iASPP RNAi cells were plated at 200 cells per well in 6-well culture plates and cultured in DMEM containing 10% FBS at 37°C and 5% CO2 for 2 weeks. The cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde (PFA) for 15 min, stained with Gimsa for 20 min, and washed twice with ddH2O. Individual clones with more than 50 cells were counted.
2.9. 3′ UTR Luciferase Reporter Assay
UTR luciferase reporter assays were performed in human embryonic kidney 293 (HEK293) cells. Vectors based on pMIR-REPORT harboring the wild-type (WT) 350 bp fragment of the iASPP 3′ UTR, or the same fragment in which the miR-124 binding site (199–194) was mutated (MUT), were inserted downstream of the luciferase reporter gene stop codon in pMIR-REPORT using HindIII and SpeI. The cells were cotransfected with (1) miR-124 lentivirus or miR-67-negative-control lentivirus (50 nM), (2) pMIR-REPORT vectors containing the WT or MUT miR-124 binding sites (400 ng), and (3) pRL-SV40 (Promega, Sunnyvale, CA, USA) expressing Renilla luciferase (400 ng) for normalization of transfection efficiency. Cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum, and luciferase activities were measured at 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega).
2.10. Statistical Analysis
Data were expressed as mean ± SD of three independent experiments and processed using SPSS 17.0 statistical software (SPSS, Chicago, IL, USA). The expression of miR-124 in CRC tissues and the paired adjacent normal colonic tissues were compared using Wilcoxon's paired test. The differences between groups in the migration and invasion assays were evaluated using the one-way ANOVA. P values of < 0.05 were considered statistically significant.
3. Results
3.1. miR-124 Is Frequently Downregulated in CRC Tissues and Cell Lines
Targetscan and microRNA.org [14, 15] predicted that miR-124, miR-506, miR-182, miR-19a, and miR-19b could bind to and target the iASPP 3′ UTR. In the present study, we first examined the expression levels of these miRNAs in clinical samples of CRC tissues and matched normal tissues using qPCR. Expression of miR-214 was found to be downregulated in tumor tissues compared with the matched normal tissues in 14/17 (82.3%) of samples. In 70.6%, (12/17) of samples, miR-506 was found to be downregulated. Figures 1(a) and 1(b) show the mean expression levels of miR-214 and miR-506, which were significantly lower in tumor tissues than in matched normal tissues. On the other hand, the expression levels of miR-182, miR-19a, and miR-19b were not significantly different between tumor and matched normal tissues (Figures 1(c)–1(e)). In addition, miR-124 was also expressed at significantly lower levels in the three human CRC cell lines compared to the normal human colon cell line (Figure 1(f)).
3.2. iASPP mRNA Is a Direct Target of miR-124
To determine whether the 3′-UTR of iASPP mRNA is a functional target of miR-214 in CRC cells, we created a WT-iASPP 3′ UTR luciferase reporter vector (WT-iASPP), as well as a MUT-iASPP 3′ UTR luciferase reporter vector (MUT-iASPP) by sequentially mutating the predicted 8-base pair miR-124 binding site in the iASPP 3′ UTR (Figure 2(a)). We co-transfected the WT-iASPP vector and miR-124 lentivirus or a scrambled control into HEK293 cells. The luciferase activity of the iASPP 3′ UTR luciferase reporter vector was significantly reduced in miR-124 transfected cells, compared to scrambled control cells (Figure 2(b)). Moreover, miR-124-mediated repression of iASPP 3′ UTR luciferase reporter activity was abolished by mutation of the putative miR-24 binding site in the iASPP 3′ UTR (Figure 2(b)).
3.3. Knockdown of iASPP or Overexpression of miR-124 Inhibit iASPP Expression and Increases NF-κB (p65) Expression
We infected SW480 or HT29 cells with miR-124 and miR-scramble lentivirus, iASPP shRNA or shRNA-control lentivirus. Quantitative PCR showed that, at 96 h after transfection, miR-124 was significantly overexpressed in SW480 or HT29 cells infected with miR-124 lentivirus, compared to cells infected with the scrambled control lentivirus (Figure 3(a)). Overexpression of miR-124 or knockdown of iASPP using shRNA both led to a moderate decrease in iASPP mRNA expression in SW480 or HT29 cells (Figure 3(b)), suggesting that iASPP is potentially regulated by miR-124. These results indicated that miR-124 can transcriptionally regulate and inhibit iASPP expression. Additionally, Western blotting demonstrated that infection of miR-124 lentivirus or iASPP shRNA reduced iASPP protein expression (Figure 3(c)) and increased NF-κB (p65) protein expression (Figure 3(d)). These results suggest that overexpression of miR-124 upregulated the expression of NF-κB (p65), at least in part, by reducing the expression of iASPP.
3.4. Overexpression of miR-124 or iASPP shRNA Attenuate CRC Cell Proliferation and Colony Formation
It has been reported that NF-κB played a role in the cell proliferation and apoptosis [12]. To investigate if miR-124 can regulate CRC cell viability by targeting iASPP and upregulating NF-κB, we transfected miR-124 lentivirus, control lentivirus, iASPP shRNA or shRNA-control into SW480 or HT29 cells, and performed the MTT assay. Overexpression of miR-124 or knockdown of iASPP significantly inhibited SW480 or HT29 cell viability, compared to cells transfected with scrambled control lentivirus (Figure 4(a)). Transfection of miR-124 lentivirus or iASPP shRNA also significantly suppressed SW480 or HT29 cell proliferation compared to the scramble control lentivirus, as indicated by the BrdU incorporation assay (Figure 4(b)). The colony formation assay indicated that cells transfected with iASPP shRNA (10%) or miR-124 lentivirus (30%) formed significantly lower numbers of colonies than shRNA-control-infected cells (100%; Figures 4(c) and 4(d)). These results suggest that overexpression of miR-124 attenuated cell proliferation by downregulating iASPP signaling.
3.5. Forced Expression of iASPP Restores the Effects of miR-124 in CRC Cells
As shown above, iASPP is a direct target gene of miR-124. Therefore, we wondered whether forced overexpression of iASPP could reverse miR-124-induced upregulation of NF-κB (p65). An iASPP ORF-expressing plasmid was transfected into miR-124- or miR-SCR-expressing cells. As shown in Figure 5, the reduced expression of iASPP in miR-124-overexpressing cells was rescued by the introduction of iASPP cDNA. Similarly, ectopic overexpression of iASPP also prevented miR-124-induced NF-κB (p65) upregulation in SW480 or HT29 cells, confirming that miR-124 upregulates NF-κB (p65) by targeting iASPP mRNA.
4. Discussion
In this research, we identified and confirmed that iASPP is a target gene of human miR-124. A luciferase reporter assay validated the binding and repressive effects of miR-124 on the iASPP 3′-UTR in HEK293 cells. Additionally, qPCR demonstrated that miR-124 is downregulated in CRC cell lines and tumor tissues. A shRNA targeting iASPP, or overexpression of miR-124, downregulated the expression of iASPP and reduced the viability, proliferation, and colony formation ability of the CRC cancer cell line SW480 or HT29. In addition, overexpression of miR-124 downregulated the expression of iASPP and upregulated the expression of NF-κB (p65), and overexpression of iASPP inhibited the ability of miR-124 to upregulate NF-κB (p65) expression.
These results are consistent with research on leukemia, breast cancer, and nonsmall cell lung cancer, which identified that iASPP is expressed at high levels [16]. Additionally, genotype mapping of tissues from breast cancer patients suggested that iASPP might act to regulate the p53 and NF-κB pathway, and therefore control the growth of cancer cells [17]. It was originally described as RelA-associated inhibitor (RAI), which binds to the NF-κB subunit p65 (RelA) and inhibits its transcriptional activity [18–21]. This evidence suggests that altered regulation of iASPP may be involved in tumorigenesis.
Recent studies found that the expression of iASPP in carcinoma tissues was higher than the normal colonic tissues from the same patients [22–24]. Knockdown of iASPP significantly inhibited cell proliferation and induced G0/G1 cell cycle arrest in U251 cells by regulating expression of p21Waf1/Cip1 and cyclin D1 [25]. Downregulation of iASPP in human hepatocellular carcinoma cells inhibits cell proliferation and tumor growth [26]. iASPP is important for bladder cancer cell proliferation [27]. These suggested that iASPP played an important role in cancer cell proliferation. However, it is unclear what cell signals iASPP directly regulated in cancer cell proliferation. It was reported that iASPP may maintain epithelial homeostasis by binding and inhibiting the activity of p65RelA [28]. Future genetic studies are needed to test whether iASPP as a key player in epithelial stratification, a function that is achieved through its ability to bind and inhibit p63's activities and suppress cellular senescence and terminal differentiation [9].
Here, we demonstrated that overexpression of miR-124 leads to upregulation of NF-κB, due to downregulation of the miR-124 target gene iASPP. Forced overexpression of miR-124 also attenuated SW480 or HT29 cell viability, proliferation, and colony formation. These findings indicate a novel role and mechanism of action for miR-124 in tumors and suggest that miR-124 may provide a potential target for cancer therapy.
miR-124 has been reported to play a role in glioblastoma differentiation, especially when intracellular growth factors are absent [29]. Likewise, miR-124 is upregulated in SW480 or HT29 cells, which can undergo androgen-independent growth in vitro [30]. It is possible that an intrinsic mechanism connects growth factor depletion and miR-124-regulated cell growth; this hypothesis requires further investigation in vivo.
In summary, we identified that miR-124 inhibits CRC cell viability, proliferation, and colony formation through targeting iASPP; these effects were due, at least in part, to upregulation of NF-κB. The miR-124/iASPP axis identified in this study may play a vital role in regulating the proliferation of CRC cells and may provide a potential diagnostic and therapeutic target for CRC.
Figure 1 miR-124 is downregulated in both primary CRC tissues and CRC cell lines. The expression of miRNAs in the CRC tissues and the matched normal tissues was detected by qRT-PCR and normalized to that of U6. Results showed that the expression of miR-124 (a) and miR-506 (b) were significant decreased in tumor tissue compared with the matched normal tissue; while there was no significantly difference in the expression of miR-182 (c), miR-19a (d), and miR-19b (e) between the two groups. Data are presented as individual samples (n = 17) with the line indicating the mean level. (f) miR-124 was expressed at significantly lower levels in three CRC cell lines in comparison with normal colonic mucosa pooled from three healthy individuals. The figure is representative of three experiments with similar results.
Figure 2 miR-124 directly targets iASPP by binding to its 3′ UTR. (a) The predicted miR-124 binding site within the 3′ UTR of iASPP and the mutated version generated by site mutagenesis are shown. (b) Repression of wild-type iASPP 3′ UTR luciferase reporter gene activity by miR-124 (**P < 0.01); miR-124 had no effect on the luciferase activity of the mutated iASPP 3′ UTR reporter vector compared to control HEK293 cells or cell transfected with scrambled miR lentivirus (without miR-124) (miR-SRC).
Figure 3 Knockdown of iASPP or overexpression of miR-124 inhibits iASPP expression and increases NF-κB(p65) expression. SW480 or HT29 or HT29 cells were infected with scrambled miR lentivirus (without miR-124) (miR-SRC), miR-124 lentivirus, iASPP shRNA, or control shRNA, lentivirus. (a) Real-time PCR analysis of miR-124 and U6 expression. (b) Real-time PCR quantification of iASPP mRNA expression; **P < 0.01 compared to SW480 or HT29 or HT29 cells transfected with scrambled miR-124 lentivirus (miR-SRC) or control shRNA lentivirus. (c) Western blot of iASPP protein expression. Expression of iASPP was inhibited by both iASPP shRNA and miR-124, compared to SW480 or HT29 or HT29 cells expressing Con-shRNA or miR-SCR. (d) Western blot of NF-κB(p65) protein expression. Expression of NF-κB(p65) was increased by both iASPP shRNA and miR-124, compared to SW480 or HT29 or HT29 cells expressing Con-shRNA or miR-SCR. The figure is representative of three experiments with similar results.
Figure 4 Effects of miR-124 and iASPP on the viability, proliferation, and clone forming ability of SW480 or HT29 or HT29 cells. SW480 or HT29 cells were transfected with scrambled miR lentivirus (without miR-124) (miR-SRC), miR-124 lentivirus, iASPP shRNA, or control shRNA. (a) MTT cell proliferation assay. Ectopic overexpression of miR-124 using an miR-124 lentivirus or iASPP shRNA significantly reduced the proliferation of SW480 or HT29 cells, compared to NC (**P < 0.01) (b) Cell viability, as determined by the BrdU incorporation assay. (c) Colony formation assay. Cells were seeded in soft agar as described in Section 2. (d) Number of colonies at two weeks after seeding. The figure is representative of three experiments with similar results.
Figure 5 Overexpression of iASPP inhibits the effects of miR-124 on iASPP and NF-κB(p65) expression. SW480 or HT29 cells stably expressing miR-SCR or miR-124 were transfected with iASPP ORF-expressing plasmid. After 48 h, the levels of specific proteins were analyzed by immunoblotting, and the band intensities were quantified using Image-Pro Plus and normalized to β-actin. (a and c) Overexpression of iASPP increased iASPP protein expression. (b and d) Overexpression of iASPP decreased NF-κB(p65) protein expression; **P < 0.01.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23691078PONE-D-13-0093210.1371/journal.pone.0063636Research ArticleBiologyBiochemistryProteinsRegulatory ProteinsMolecular Cell BiologySignal TransductionMedicineObstetrics and GynecologyBreast CancerOncologyCancers and NeoplasmsLung and Intrathoracic TumorsNon-Small Cell Lung CancerBasic Cancer ResearchIKBKE Phosphorylation and Inhibition of FOXO3a: A Mechanism of IKBKE Oncogenic Function IKBKE Phosphorylates/Inhibits FOXO3aGuo Jian-Ping
1
2
Tian Wei
1
2
Shu Shaokun
2
Xin Yu
2
Shou Chengchao
1
*
Cheng Jin Q.
2
*
1
Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital and Institute, Beijing, China
2
Department of Molecular Oncology, H. Lee Moffitt Cancer Center, Tampa, Florida, United States of America
Creighton Chad Editor
Baylor College of Medicine, United States of America
* E-mail: [email protected] (JQC); [email protected] (CCS)Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: JPG WT JQC. Performed the experiments: JPG WT SS YX. Analyzed the data: JPG WT CCS. Contributed reagents/materials/analysis tools: CCS. Wrote the paper: JPG JQC.
2013 14 5 2013 8 5 e636363 1 2013 4 4 2013 © 2013 Guo et al2013Guo et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival and growth. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications. Here we report that IKBKE regulates FOXO3a through phosphorylation of FOXO3a-Ser644. The phosphorylation of FOXO3a resulted in its degradation and nuclear-cytoplasmic translocation. Previous studies have shown that IKBKE directly activates Akt and that Akt inhibits FOXO3a by phosphorylation of Ser32, Ser253 and Ser315. However, the activity of Akt-nonphosphorytable FOXO3a-A3 (i.e., converting 3 serine residues to alanine) was inhibited by IKBKE. Furthermore, overexpression of IKBKE correlates with elevated levels of pFOXO3a-S644 in primary lung and breast tumors. IKBKE inhibits cellular function of FOXO3a and FOXO3a-A3 but, to a much less extent, of FOXO3a-S644A. These findings suggest that IKBKE regulates FOXO3a primarily through phosphorylation of SerS644 and that IKBKE exerts its cellular function, at least to some extent, through regulation of FOXO3a.
This work was supported by National Institutes of Health (NIH) grant CA137041, CA160455 and Florida James & Esther King Biomedical Research Program 1KG02 to JQC, 10KD-04 to JPG and National 973 Program of China (2009CB521805) to CCS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
IKBKE (Inhibitor of nuclear factor kappa-B kinase subunit epsilon, also called IKKε and IKKi) is an IκB kinase family member [1], [2]. Accumulating studies have shown that IKBKE and IKKα/β are activated by inflammatory factor, interferon and viral infection. The activation of IKBKE and IKKα/β subsequently induces NF-κB nuclear accumulation and DNA-binding activity by phosphorylation of IκB-Ser36 and –Ser32/Ser36, respectively, which leads to increase of transcription of cell growth/survival genes such as cyclin D1, Bcl-xL and Bcl2 etc [3], [4]. However, the kinase domain of IKBKE only exhibits 27% and 24% identity to IKKα and IKKβ, respectively [5], implying that IKBKE may regulate different molecules from IKKα/β. A recent study demonstrated that IKBKE but not IKKα/β phosphorylates CYLD [6], which is a deubiquitinase of several NF-κB regulators, including TRAF2, TRAF6, and NEMO, to activate the NF-κB pathway [7]–[10]. Moreover, in response to inflammatory factor and viral infection, IKBKE phosphorylates interferon response factors 3 and 7 (IRF3 and IRF7) and STAT1 [1], [11], [12], [13] as well as induces phosphorylation of p65/RelA [14]. We and others have independently shown IKBKE, but not IKKα/β, direct phosphorylation of Akt-Thr308/Ser473 [15], [16], leading to Akt activation independent PI3K, PDK1, mTORC2 and PH domain of Akt [15], [16]. Unlike IKKα/β, IKBKE has been shown to be frequently amplified/overexpressed in human malignancy and ectopic expression of IKBKE results in malignant transformation [15], [17]. We also showed that elevated expression of IKBKE is involved in chemo- and tamoxifen-resistance [18].
FoxO transcription factor family is a key player in an evolutionary conserved pathway, which consists of FOXO1, 3, 4 and 6 in mammals. Four members of FOXO share high similarity in their structure, function and regulation. They are involved in diverse cellular and physiological processes including cell survival, proliferation, cell cycle and metabolism as well as reactive oxygen species (ROS) response and longevity. A number of target genes of FOXOs have been identified which include Bim and FasL for inducing apoptosis [19], [20]; p27kip1 and cyclin D for cell cycle control [21], [22], GADD45a for DNA repair [23] and G6Pase for glucose metabolism [24], [25]. Accumulating studies demonstrated that these FOXOs are predominantly regulated by post-translational modifications, including phosphorylation, acetylation, methylation and ubiquitination [6], [26]–[28]. For instance, FOXO3a has been shown to be phosphorylated by IKKα/β at Ser644 [26], Akt at Ser32, Ser253 and Ser315 [29], and ERK1/2 at Ser294, Ser344 and Ser425 [30], [31], resulting in either decrease of FOXO3a DNA binding activity or/and protein stability.
In the present study, we show that IKBKE inhibits FOXO3a and FOXO3a-A3, an Akt-nonphosphorylatable form, function by direct phosphorylation of FOXO3a. While the kinase domain of IKBKE is distinct from IKKα and IKKβ [5], it also phosphorylates FOXO3a-Ser644. As a result, IKBKE induces FOXO3a degradation and nuclear-cytoplasmic translocation leading to abrogation of FOXO3a cellular function.
Materials and Methods
Cell Lines, Lung Tumor Specimens, Antibodies and Recombinant Protein
The non-small cell lung cancer (NSCLC) cell lines were provided by Moffitt Cancer Center Lung Cancer Cell Core. Breast cancer cell lines (MCF7, MDA-MB435 and T47D), HEK293 and HeLa were purchased from ATCC. These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Ikbke-knockout mouse fibroblast (MEF-Ikbke
−/−) and wild type MEF (MEF-W) were kindly provided by Tom Maniatis (Harvard University). Doxycycline-inducible IKBKE cell line was established by transfection of HeLa tet-on cell (Clontech) with pTRE-Tight-IKBKE.
Fifty primary human NSCLC and 57 breast tumor specimens were collected from 1998 to 2005 at the Moffitt Cancer Center, approved by the Institutional Review Boards (IRB) of University of South Florida (#17121). All participants provided written consent and data were de-identified. Consent forms were kept on file and the IRB approved of the consent procedure under Moffitt Cancer Center Total Cancer Control protocol. The tissues were snap frozen and stored at −70°C.
Antibodies against IKBKE, Myc and HA were purchased from Sigma. Anti-FOXO3a and -p27 antibodies were from Santa Cruz Biotechnology. Antibodies of Bim and GFP were from Cell Signaling. Anti-phospho-FOXO3a-S644 antibody was kindly provided by Mickey C.-T Hu (Stanford University). Recombinant protein IKBKE was purchased from Cell Signaling.
Plasmids
The pCMV-Myc tagged IKBKEand DN-IKBKE-K38A were described previously [18]. Myr-IKBKE was obtained from Dr. William Hahn at Harvard Medical School [7]. The pLKO1-shRNAs of IKBKE were from Open Biosystems. The GFP-FOXO3a, HA-FOXO3a, HA-FOXO3a-A3 and GST-FOXO3a were provided by Boudewijn M.T. Burgering (University Medical Center Utrecht). FOXO3a-S644A was generated with QuikChange® Site-Directed Mutagenesis Kit (Stratagene). The reporter plasmids pGL3-FHRE-Luc and pGL3-p27 were purchased from Addgene. The truncation mutants of GST-FOXO3a (GST-FOXO3a 1–300, 301–673, 301–391, 393–538, 532–578, 579–625, 625–673, 530–673) were provided by Mien-Chie Hung (M.D. Anderson Cancer Center).
In Vitro Kinase Assay and In Vivo [32P]Pi Cell Labeling
In vitro IKBKE kinase assay was performed as previously described [18], [32]. Briefly, recombinant IKBKE was incubated with GST-FOXO3a in the presence of 10 µCi of [γ-32P]ATP (NEN) and 3 µM cold ATP in a kinase buffer. After incubation at 30°C for 30 min, the reaction was stopped and separated in SDS-PAGE gels. Each experiment was repeated three times.
For in vivo labeling, H1299 cells were transfected with HA-FOXO3a-A3 or HA-FOXO3a-A3-S644A together with and without myr-IKBKE. After serum starvation overnight, cells werelabeled with [32P]Pi (0.5 mCi/ml) in phenol red-free MEM without phosphate for 4 hours. FOXO3a was immunoprecipitated with HA antibody, separated on SDS-PAGE and transferred to membrane. Phosphorylated FOXO3a was detected by autoradiography and quantified.
Western Blot, Co-immunoprecipitation (co-IP) and Immunofluorescence
Western blot, co-IP and immunofluorescence were performed as previously described [33]. Briefly, cell lysates were prepared in a lysis buffer and subject to immunoprecipitation and immunoblot analysis. To determine if the FOXO3a band shift is due to the phosphorylation, a part of cell lysate were treated with CIP (1 unit/µg protein) for 30 min at 37°C prior to Western blot. For immunofluorescence, MCF7 cells were transfected with myc-IKBKE and GFP-FOXO3a using Lipofectin® reagent (Invitrogen). After 48 hours, cells were stained with Alexa Fluor® 555 conjugated (red) Myc antibody and DAPI and observed under fluorescence microscope.
Luciferase Reporter, Reverse Transcription-PCR (RT-PCR) and Chromatin Immunoprecipitation (ChIP) Assay
The luciferase reporter assay was carried out as previously described [33]. RT-PCR was performed with gene specific primers of p27 (forward, 5′-GCAATGCGCAGGAATAAGGA-3′; reverse, 5′-TCCACAGAACCGGCATTTG-3′) and GAPDH (forward, 5′-CATGTTCGTCATGGGTGTGAACCA-3′; reverse, 5′-AGTGATGGCATGGACTGTGGTCAT-3′). ChIP assay was performed with anti-HA (FOXO3a) antibody and primers flanking FOXO3a binding site of p27 promoter: forward 5′-GTCCCTTCCAGCTGTCACAT-3′; reverse 5′-GGAAACCAACCTTCCGTTCT-3.
Cell Viability and Programmed Cell Death
Cell viability was evaluated using CellTiter-Glo® Luminescent Cell Viability Agent according to manufacturer’s protocol (Promega). Apoptosis was determined by caspase 3/7 activity and TUNEL assay [34]. Briefly, cells were plated into 96-well plate with 1×104 cells/well and then transfected with different constructs. Following incubation for 48 hours, caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay Systems (Promega). TUNEL assay was performed with the TUNEL Apoptosis Detection Kit (Millipore).
Statistic Analysis
For luciferase activity and cell survival, the experiments were repeated at least three times in triplicate. The data are represented by means ± SD. Differences between control and testing cells were evaluated by Student's t test; the correlation of IKBKE expression with phosphorylation of FOXO3a-S644 was analyzed by Chi-square test, all analyses were completed with SPSS software, version 10.0. P<0.05 was considered statistically significant.
Results
IKBKE Represses Transcription and DNA-binding Activity of FOXO3a and FOXO3a-A3
Previous studies showed that FOXO3a functions as a tumor suppressor and inhibits cell survival and growth [35]. Akt was identified as a key regulator of FOXO3a by phosphorylation of three serine residues, Ser32, Ser253 and Ser315 [29]. We have recently shown that IKBKE functions as an Akt-T308 and -S473 kinase and directly activates Akt independent of PI3K/PDK1 and mTORC2 [15]. These findings prompted us to examine whether IKBKE regulated FOXO3a function. As an initial step, we assessed the effect of IKBKE on the transcription activity of FOXO3a and FOXO3a-A3, an Akt-nonphosphorylatable form in which 3 serine residues were converted to alanine. In agreement with previous reports [29], [35], p27 promoter activity was induced by FOXO3a or FOXO3a-A3. Unlike Akt, which only inhibited FOXO3a, IKBKE abrogated both FOXO3a- and FOXO3a-A3-induced p27 promoter activities (Fig. 1A and Fig. S1). FOXO3a- and FOXO3a-A3-induced p27 mRNA levels were also inhibited by IKBKE (Fig. 1B). Moreover, ChIP assay showed that IKBKE inhibited DNA binding activity of FOXO3a and FOXO3a-A3 (Fig. 1C). In addition to p27 promoter, we examined the effect of IKBKE on FHRE-Luc, which is a luciferase reporter driven by the promoter containing three repeats of FOXO3a binding consensus motif [29]. Fig. 1D shows that constitutively active (Myr)-IKBKE reduced whereas DN-IKBKE enhanced FOXO3a transcription activity. Furthermore, knockdown of IKBKE increased FHRE-Luc activity (Fig. 1E). Together, these findings suggest that IKBKE inhibition of FOXO3a activity is independent of Akt.
10.1371/journal.pone.0063636.g001Figure 1 IKBKE represses FOXO3a and Akt-nonphosphorylatable FOXO3a-A3.
(A) Luciferase assay. H1299 cells were transfected with pGL3-p27-Luc together with indicated plasmids. Following incubation for 48 h, luciferase activity was measured and normalized to β-galactosidase. Results are the mean ± S.E. of three independent experiments performed in triplicate. The left open bar is relative basal p27-promoter activity (1.0). (B) FOXO3a- and FOXO3a-A3-induced p27 transcription was reduced by IKBKE. H1299 cells were transfected with indicated plasmids. The p27 mRNA levels were determined by semi-quantitative RT-PCR (upper panels). GAPDH was used as control. Western blot shows the expression of transfected plasmids (panels 3 and 4). Bottom panel is a loading control. (C) IKBKE inhibits FOXO3a and FOXO3a-A3 DNA-binding activity. H1299 cells were transfected with indicated plasmids. ChIP assay was performed as described in “Experimental Procedures”. Anti-HA antibody was used for chromatin IP. IgG was served as negative control. The DNA prior to the IP was used as positive controls (input). (D) IKBKE inhibition of FOXO3a depends on its kinase activity. MCF7 cells were transfected with pGL3-FHRE-Luc (e.g., 3 repeats of FOXO binding motif) and IKBKE or DN-IKBKE. Luciferase activity was determined after 48 h of the transfection. Western blot shows expression of transfected plasmids (bottom). (E) Knockdown of IKBKE increases FOXO3a transcription activity. H292 cells were transfected with 2 shRNA of IKBKE and pGL3-FHRE-Luc. After 48 h of incubation, cells were subjected to luciferase assay (upper panel) and Western blot (bottom panel).
Direct Phosphorylation of FOXO3a-S644 by IKBKE
We next investigated if IKBKE interacts with and phosphorylates FOXO3a. Co-immunoprecipitation revealed no interaction between IKBKE and FOXO3a (data not shown). However, when IKBKE and FOXO3a were co-expressed in H1299 cells, we observed remarkable mobility shift of FOXO3a as well as decrease of FOXO3a and p27 protein levels (Fig. 2A). The mobility shift was inhibited by treatment of cell lysate with calf intestinal alkaline protein phosphatase CIP but not by treatment of cells with Akt inhibitor MK2206 (Fig. 2B). These data suggest that IKBKE could phosphorylate FOXO3a via an Akt-independent manner.
10.1371/journal.pone.0063636.g002Figure 2 IKBKE phosphorylates FOXO3a.
(A and B) IKBKE induces FOXO3a mobility shift which is inhibited by protein phosphatase CIP but not MK2206. H1299 cells were transfected with indicated plasmids. Following treatment with and without MK2206, cells were lysed. A portion of cell lysate was treated with CIP at 37°C for 30 min prior to SDS-PAGE electrophoresis (lane 5 of B). Immunoblots were probed with indicated antibodies. (C) A diagram illustration of GST-FOXO3a fusion proteins. (D) C-terminal region of FOXO3a was phosphorylated by IKBKE. In vitro kinase was performed by incubation of recombinant IKBKE with indicated GST-FOXO3a fusion proteins (top). Bottom panel is coomassie blue staining (CBS) showing GST-FOXO3a fusion proteins used for in vitro IKBKE kinase assay.
To determine whether IKBKE directly phosphorylated FOXO3a, in vitro IKBKE kinase assay was performed by incubation of recombinant IKBKE and GST-FOXO3a fusion proteins. Figs. 2C and 2D showed that three C-terminal truncation GST-FOXO3a proteins (FO3-2, FO3-7 and FO3-8) were phosphorylated by IKBKE with minimal region FO3-7.
To define the phosphorylation site(s), we mutated individual serine/threonine residue into alanine within FO3-7 region. In vitro kinase assay revealed that phosphorylation of GST-FO3-7/S644A by IKBKE was significantly reduced compared to the wild-type and other mutant fusion proteins (Fig. 3A). Sequencing analysis revealed that FOXO3a-S644 partially fits a putative IKBKE phosphorylation consensus motif [7] and is also conserved among different species (Fig. 3B). To further examine if FOXO3a-S644 was phosphorylated by IKBKE, in vivo labeling was performed by transfection of wild type FOXO3a, FOXO3a-A3 and FOXO3a-A3-S644A together with and without myr-IKBKE. Fig. 3C showed that IKBKE phosphorylated FOXO3a-A3 and that the phosphorylation level was significantly reduced by mutation of Ser644 (FOXO3a-A3-S644A). Western blot analysis with specific antibody against phospho-FOXO3a-S644 further showed that IKBKE phosphorylated FOXO3a (Fig. 3D and 3E). Furthermore, expression levels of p27 and Bim, 2 representative targets of FOXO3a, were reduced by expression of IKBKE or myr-IKBKE but were increased by knockdown of IKBKE (Fig. 3E).
10.1371/journal.pone.0063636.g003Figure 3 Direct phosphorylation of FOXO3a-S644 by IKBKE in vitro and in vivo.
(A) Mutation of Ser644 into alanine reduced IKBKE phosphorylation of C-terminal region of FOXO3a. In vitro IKBKE kinase assay was carried out using C-terminal region of FOXO3a (e.g. GST-FO3-7) containing indicated Ser/Thr-Ala mutation as substrates. (B) Sequence alignment of FOXO3a-S644 with putative IKBKE phosphorylation consensus motif (25). (C) IKBKE phosphorylates FOXO3a-S644 in vivo. H1299 cells were transfected with indicated plasmids and labeled with [32P]-orthophosphate. Following immunoprecipitation with anti-HA antibody, the immunoprecipitates were separated by SDS-PAGE, transferred and then exposed (top). Expression of transfected plasmids is shown in panels 2 and 3. (D and E) IKBKE induces endogenous FOXO3a-S644 phosphorylation and reduces p27 and Bim expression Indicated cells were transfected with different forms of IKBKE and shRNA-IKBKE, and then immunoblotted with indicated antibodies. (F and G) Expression of IKBKE positively correlates with pFOXO3a-S644 level in NSCLC specimens. Representative tumors were lysed, immunoprecipitated and probed with indicated antibodies (F). Chi-square test analysis of IKBKE and pFOXO3a-S644 in 50 NSCLC specimens examined. The correlation is significant (p = 0.006; G). (H) MCF7 cells were transfected with indicated plasmids. Following incubation for 48 h, cells were treated with and without Akt inhibitor MK2206 for 2 h and then immunoblotted with indicated antibodies.
To determine if this event occurred in vivo, we examined 50 NSCLC specimens for protein expression of IKBKE and pFOXO3a-S644 (Fig. 3F). Of the 50 lung tumors, 27 had overexpression of IKBKE and 28 had elevated pFOXO3a-S644. Of the 28 tumors with elevated pFOXO3a-S644, 20 (71.4%) also had elevated IKBKE (p = 0.006; Fig. 3G). The other 8 cases with elevated pFOXO3a-S644 could result from activation of IKKα or/and IKKβ, which have been shown to also phosphorylate FOXO3a-S644 [26]. Similar results were obtained by evaluating additional 57 breast cancer specimens (Fig. S2). Collectively, these data suggest that FOXO3a-S644 is directly phosphorylated by IKBKE. In addition, we noted that IKBKE still induced FOXO3a-S644A mobility shift which was not affected by Akt inhibitor MK2206 (Fig. 3H). These data suggest that IKBKE-induced FOXO3a mobility shift could result from IKBKE regulation of other kinase(s) in addition to Akt.
IKBKE Phosphorylation of FOXO3a-S644 Results in FOXO3a Nuclear-cytoplasmic Translocation and Loss of Transcription Activity
We also examined the effects of phosphorylation of Ser644 on FOXO3a subcellular localization and transcription activity. H1299 cells were transfected with GFP-FOXO3a or GFP-FOXO3a-S644A together with constitutively active IKBKE. Following incubation for 48 hours, the subcellular localization of FOXO3a was determined and quantified. Figs. 4A and 4B showed that GFP-FOXO3a and GFP-FOXO3a-S644A were predominantly located in the nucleus. Expression of IKBKE led to FOXO3a translocation from the nucleus to cytoplasm. However, FOXO3a-S644A remained in the nucleus (Figs. 4A and 4B). In addition, p27 reporter assay was carried out to assess the effect of phosphorylation of Ser644 on FOXO3a transcription activity. As shown in Fig. 4C, expression of myr-IKBKE repressed FOXO3a-induced p27 promoter activity but had insignificant effect on p27 promoter activity induced by FOXO3a-S644A (e.g., 20% reduction; p>0.05). Based on these data, we concluded that IKBKE phosphorylation of Ser644 leads to FOXO3a nuclear-cytoplasmic translocation and loss of its transcription activity.
10.1371/journal.pone.0063636.g004Figure 4 Expression of IKBKE results in nuclear-cytoplasmic translocation and loss of transcription activity of FOXO3a but not FOXO3a-S644A.
(A and B) Expression of constitutively active IKBKE induces FOXO3a but not FOXO3a-S644A nuclear-cytoplasmic translocation. MCF7 cells were transfected with indicated plasmids. After 48 h of transfection, cells were stained with Alexa Fluor® 555 conjugated (red) Myc antibody and DAPI (A). Scale bar is 25 µm. Cellular localization of FOXO3a was quantified by counting 400 cells (B). (C) IKBKE inhibition of FOXO3a transcription activity depends on phosphorylation of Ser644. MCF7 cells were transfected with pGL3-p27-Luc, FOXO3a or FOXO3a-S644A together with and without myr-IKBKE. Luciferase assay was performed after 48 h of transfection as described in Figure 1.
IKBKEinduces FOXO3a Degradation in a p-Ser644-dependent Manner
We also noticed that expression of IKBKE decreased FOXO3a protein level (Figs. 2A, 2B and 3E). To further examine the effect of IKBKE on FOXO3a expression, we transfected H1299 cells with IKBKE and found that ectopic expression of IKBKE reduced FOXO3a protein but not mRNA level in a dose-dependent manner (Fig. 5A). Furthermore, increase of FOXO3a protein level was detected in IKBKE-depletion cells (Figs. 5B and 5C).
10.1371/journal.pone.0063636.g005Figure 5 IKBKE phosphorylation of FOXO3a-S644 induces FOXO3a degradation.
(A) Expression of constitutively active IKBKE reduces FOXO3a expression at protein but not mRNA levels. H1299 cells were transfected with an increasing amount of myr-IKBKE and subjected to immunoblot (upper panels) and RT-PCR (lower panels) analysis. (B) Knockdown of IKBKE increases FOXO3a protein levels. Two IKBKE-shRNAs were introduced into MDA-MB435 cells. Following incubation for 72 h, immunoblot (upper panels) and RT-PCR (lower panels) analyses were performed. (C) Immunoblot analysis of wild-type and Ikbke-knockout MEFs with indicated antibodies. (D–F) IKBKE induces FOXO3a protein degradation which depends on phosphorylation of Ser644. H1299 cells were transfected with FOXO3a or FOXO3a-S644A together with and without IKBKE. After 48 h of transfection, cells were treated with CHX for different times and then were immunoblotted with indicated antibodies (D and E). Degradation rates of FOXO3a and FOXO3a-S644A in the presence and absence of IKBKE were quantified (F). (G) IKBKE induces FOXO3a degradation more significantly than FOXO3a-S644A in IKBKE tet-on cells. The IKBKE tet-on HeLa cells were transfected with FOXO3a and FOXO3a-S644A. After 48 h of transfection, cells were treated with doxycycline for indicated times and then immunoblotted with indicated antibodies. (H) IKBKE-induced FOXO3a degradation was inhibited by proteasome inhibitor. T47D cells were transfected indicated plasmids, treated with and without MG132 and then were immunoblotted with indicated antibodies.
To determine if the observed IKBKE-promoted reduction in FOXO3a expression levels depends on the phosphorylation of FOXO3a-S644, we compared the degradation rate of FOXO3a and FOXO3a-S644A in the absence or presence of IKBKE. Following transfection of FOXO3a or FOXO3a-S644A together with and without IKBKE, H1299 cells were treated with cycloheximide (CHX) for different times. Immunoblot analysis unraveled that FOXO3a-S644A was more stable than FOXO3a (Figs. 5D and 5F). Expression of IKBKE induced more significant degradation of FOXO3a than FOXO3a-S644A (Figs. 5E and 5F). Similar result was observed in doxycycline-inducible IKBKE cell line (Fig. 5G). Moreover, IKBKE-induced FOXO3a degradation was largely abrogated by treatment with proteasome inhibitor MG132 (Fig. 5H). Based on these results, we conclude that IKBKE induces FOXO3a protein degradation primarily via phosphorylation of Ser644.
IKBKE Protects Cells from FOXO3a-induced Apoptosis Primarily through Phosphorylation of FOXO3a-S644
Since IKBKE directly phosphorylates FOXO3a-S644 leading to FOXO3a nuclear-cytoplasmic translocation and protein degradation, IKBKE could suppress FOXO3a cellular function via a Ser644 phosphorylation-dependent manner. To this end, cell viability was assessed in H1299 cells following transfection of FOXO3a, FOXO3a-S644A or FOXO3a-A3 together with and without myr-IKBKE. As shown in Fig. 6A, expression of FOXO3a alone induced cell death. The effects of FOXO3a and FOXO3a-A3 on cell death were largely abrogated by expression of constitutively active IKBKE. In addition, caspase 3/7 and TUNEL assays revealed that myr-IKBKE inhibited the programmed cell death induced by FOXO3a and FOXO3a-A3 (Figs. 6B and 6C). However, FOXO3a-S644A induced cell death was only inhibited by IKBKE at approximately 20%, which did not reach statistical significance (p>0.05; Figs. 6A and 6B). These data indicate that IKBKE inhibits FOXO3a largely through phosphorylation of Ser644 and, to a much less extent, through IKBKE-activated Akt phosphorylation of Ser32, Ser253 and Ser315 (Fig. 7).
10.1371/journal.pone.0063636.g006Figure 6 IKBKE inhibition of FOXO3a-induced cell death depends on phosphorylation of FOXO3a-S644.
H1299 cells were transfected with indicated plasmids. After incubation for 48 h, cell viability (A) and caspase 3/7 activity (B) as well as TUNEL (C; scale bar is 20 µm) assays were performed as described in “Experimental Procedure”.
10.1371/journal.pone.0063636.g007Figure 7 Proposed model of regulation of FOXO3a by IKBKE.
IKBKE regulates FOXO3a subcellular localization, protein stability and transcription activity predominantly through direct phosphorylation of Ser644. In addition, IKBKE could modulate FOXO3a function through activation of Akt and other kinase(s).
Discussion
IKBKE has essential role as a regulator of innate immunity by modulating interferon and NF-κB signaling [1], [2]. Recent studies have also implicated IKBKE in malignant transformation [15], [17]. We and others have shown IKBKE induction of cell survival, growth and chemoresistance [16], [18], [36]. However, underlying molecular mechanism remains elusive. In this study, we show that IKBKE directly mediates phosphorylation of FOXO3a-S644 and induces FOXO3a nuclear-cytoplasmic translocation and protein degradation. As a result, FOXO3a cellular function was inhibited by IKBKE. Furthermore, overexpression of IKBKE significantly correlated with phospho-FOXO3a-S644 in primary lung tumors examined. These findings indicate that FOXO3a is a bona fide substrate of IKBKE and that negative regulation of FOXO3a by IKBKE is a key mechanism for promoting cell survival.
Accumulating studies show that FOXO3a regulates a wide range of biological processes, including inhibition of cell survival and proliferation, protection against oxidative stress, and metabolism [37], [38], [39]. The biological activity of FOXO3a is regulated predominantly by post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Consistent with this notion, one of the first and potentially most important control mechanisms characterized for FOXO3a is its regulation by Akt, where the phosphorylation of FOXO3a at Ser32, Ser253 and Ser315 by Akt results in the cytoplasmic accumulation and subsequent degradation of this transcription factor [29]. We recently reported that IKBKE activated Akt by direct phosphorylation of Akt-T308 and -S473, which is independent of PDK1 and mTORC2 [15]. Thus, IKBKE could regulate FOXO3a through both indirect (e.g., Akt) and direct (e.g., phosphorylation of Ser644) mechanism. Interestingly, our data show that IKBKE phosphorylation of Ser644 residue, while it locates in transactivation domain (Fig. 2C), is sufficient to promote FOXO3a nuclear-cytoplasmic translocation and degradation (Figs. 4 and 5). Moreover, IKBKE inhibits Akt-nonphosphorylatable FOXO3a-A3 transcription and DNA binding activities as well as FOXO3a-A3-induced cell death. While IKBKE also suppresses FOXO3a-S644A function towards p27 and apoptosis (approximately ∼20%), it did not reach statistical significance (Figs. 4C and 6A/B). These findings suggest that IKBKE represses FOXO3a primarily through direct phosphorylation of Ser644 and that phosphorylation of FOXO3a by IKBKE-Akt axis has a less important role.
While IKBKE is a member of IKK family, its kinase domain shares ∼27% amino acid identity to IKKα and IKKβ [5]. Even though a majority of IKBKE substrates could not be phosphorylated by IKKα and IKKβ, which include Akt, IRF3/7 and STAT1 [13], [15], IKKα/β andIKBKEshare some common substrates including IκB. IKKα/β have been shown toactivate NF-κB by phosphorylation of IκB at Ser32/Ser36 whereas IKBKE induces NF-κB by phosphorylation of IκB-Ser36 [40]. Previous studies have shown that FOXO3a-S644 is phosphorylated by IKKα and IKKβ, which leads to FOXO3a cytoplasmic accumulation and degradation in a manner of independent of Akt and ERK signaling [26]. Thus, our study provided an additional substrate that is shared by IKBKE and IKKα/β. Furthermore, a recent study shows that IKKα and IKKβ inhibit Akt through phosphorylation of p85α in response to starvation [41]. However, we observed activation of Akt by IKBKE but not by IKKα/β in normal condition, which induces p-FOXO3a-S253, -S32 and -S315 (Fig. S3 and data not shown), while IKBKE and IKKα/β all phosphorylate FOXO3a-S644 (Fig. S3).
Based on our findings, we propose a model IKBKE-dependent repression of FOXO3a that promotes cell survival, growth and tumorigenesis. While IKBKE directly activates Akt, IKBKE phosphorylation of Ser644 plays a predominant role in repression of FOXO3a (Fig. 7). Our study suggests that restoration of FOXO3a activity could be an attractive therapeutic strategy for human tumors expressing elevated levels of IKBKE. In addition, a recent report showed that phosphorylation of FOXO3a by IKBKE regulates INFβ expression suggesting the role of FOXO3 in immune response control [42]. Further investigation is required for defining the mechanism by which Ser644 regulates FOXO3a subcellular localization as well as the significance of Ser644 in FOXO3a tumor suppressor function in animal model.
Supporting Information
Figure S1
Expression of transfected plasmids. Western blot analysis was performed in H1299 cells transfected with indicated plasmids.
(TIF)
Click here for additional data file.
Figure S2
Expression of IKBKE correlates with pFOXO3a-S644 in breast cancer. (A) Immunoblot analysis of representative breast tumors with indicated antibodies. (B) Chi-square test analysis of IKBKE and pFOXO3a-S644 in 57 breast cancer specimens examined. Elevated p-FOXO3a-S644 was observed in 24 of 36 specimens with high levels of IKBKE and the correlation is significant (p = 0.034).
(TIF)
Click here for additional data file.
Figure S3
IKBKE and IKKα/β regulate FOXO3a and Akt. MCF7 cells were transfected with Myc-IKBKE, Flag-IKKα and Flag-IKKβ and then immunoblotted with indicated antibodies.
(TIF)
Click here for additional data file.
We are grateful for Tissue Procurement, DNA Sequence, Proteomics and Image Core Facilities at H. Lee Moffitt Cancer Center for providing cancer specimens, sequencing and cell apoptosis analysis. We also thank Moffitt Cancer Center Lung Cancer SPORE for providing NSCLC cancer cell lines.
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ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XHindawi Publishing Corporation 10.1155/2013/684690Review ArticleRecent Advances in DENV Receptors Fang Shuyu Wu Yanhua Wu Na Zhang Jing An Jing *Department of Microbiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China*Jing An: [email protected] Editors: G. Borkow and E. J. Im
2013 23 4 2013 2013 68469027 2 2013 3 4 2013 Copyright © 2013 Shuyu Fang et al.2013This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Dengue is an old disease caused by the mosquito-borne dengue viruses (DENVs), which have four antigenically distinct serotypes (DENV1–4). Infection by any of them can cause dengue fever (DF) and/or a more serious disease, that is, dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). In recent decades, incidence of dengue disease has increased 30-fold, putting a third to half of the world's population living in dengue-endemic areas at high infection risk. However, the pathogenesis of the disease is still poorly understood. The virus binding with its host cell is not only a first and critical step in their replication cycle but also a key factor for the pathogenicity. In recent years, there have been significant advances in understanding interactions of DENVs with their target cells such as dendritic cells (DC), macrophages, endothelial cells, and hepatocytes. Although DENVs reportedly attach to a variety of receptors on these cells, consensus DENV receptors have not been defined. In this review, we summarize receptors for DENVs on different cells identified in recent years.
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1. Introduction
Dengue viruses (DENVs) belong to the Flaviviridae family. Four antigenically distinct serotypes of the viruses (DENV1–4) are transmitted to humans through the mosquito vector, Aedes aegypti. In the last century, dengue has escalated in geographic distribution and disease severity and therefore becomes an important public health concern worldwide. According to reports of the World Health Organization (WHO), dengue disease is endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, Southeast Asia, and the Western Pacific. With South-East Asia and the Western Pacific the most seriously affected, approximately 500,000 people with DHF require hospitalization each year, of whom 2.5% die.
DENVs are small (50 nm) enveloped particles with a single-stranded messenger (positive) sense RNA of approximately 11 kb in length. The single open reading frame is directly translated into a polyprotein precursor [1], which is subsequently glycosylated by cellular glycosyltransferases and cleaved by proteases from virus and host cell to release three structural proteins (envelope glycoprotein (E), membrane (M), and capsid (C)) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). Among them, E protein is the main structural protein with 55 kDa, which is a glycoprotein embedded in the viral membrane. It is known that E protein is not only a functional protein molecule that binds to receptors on the host cell membrane but also a major antigen, which can induce neutralizing antibody and host specific protective immunity [1–3]. As enveloped viruses, the DENVs enter the cells through receptor mediated endocytosis [4–7] and rearrange cell internal membranes to establish specific sites of replication [8–10]. As DENVs circulate between two hosts, humans and insects, they have to be adapted to replicate and infect both species. Recently there have been significant advances in understanding interactions of DENV with target cells such as dendritic cells (DCs), macrophages, vascular endothelial cells (VEC), and hepatocytes, and we would summarize receptors for DENVs on different cells identified in last several years in this review (Table 1).
2. DENV Receptors in DCs
After being bitten by an infected mosquito, immature DCs in the skin are believed to be the first target cells during DENV infection [11]. A C-type lectin mainly expressed by monocyte-derived DC [12, 13], named DC-specific ICAM 3-grabbing nonintegrin (DC-SIGN or CD209), mediates DENV infection. DC-SIGN is considered to be one of the most important receptors for DENVs [12–15]. Tassaneetrithep and his colleagues discovered that anti-DC-SIGN monoclonal antibodies could block DENV infection in DCs [13]. It was demonstrated biochemically that the interaction between DC-SIGN and DENV occurred through high-mannose N-linked glycans present in the viral envelope glycoproteins [16, 17]. Alen and his colleagues demonstrated that various carbohydrate-binding agents (CBAs) could block the replication of DENV1–4 in Raji/DC-SIGN+ cells as well as monocyte-derived DC (MDDC) [18]. MDDC, isolated from human donor blood, may not represent all DC subsets in vivo, but they express DC-SIGN, which made MDDC susceptible for DENV [12]. However, anti-DC-SIGN-specific antibodies could profoundly, but not completely, inhibit DENV infecting MDDC and other DCs. Till now complete inhibition of DENV infection is not achieved, indicating that other entry pathways are potentially involved in DCs.
3. DENV Receptors in Monocytes/Macrophages
As had been observed previously in the MDDC, the infection of macrophages such as mature MDMØ was also blocked by anti-DC-SIGN antibodies [13]. Tassaneetrithep and his colleagues found that THP-1 cells (human acute monocytic leukemia cell line) become susceptible to dengue infection after the transfection of DC-SIGN [13]. Therefore, DC-SIGN may be considered as a new target for dengue infection in macrophages. However, only anti-DC-SIGN antibodies could not completely inhibit DENV infection in macrophages, indicating that there are other receptors mediating DENV entry into macrophages. Recently, it was shown that the mannose receptor (MR, CD206) was associated with DENV infection in macrophages [19]. In 2008, using enzyme linked immunosorbent assay (ELISA) and blot overlay assays, Miller et al. further demonstrated that MR could bind to all four serotypes of DENV and specifically to the E glycoprotein via its carbohydrate recognition domains. This binding was abrogated by deglycosylation of E protein [19]. A recombinant MR fusion protein (CRD4–7-Fc) was also shown to recognize E protein of DENV in ELISA and blot overlays, and the binding was inhibited by mannose, fucose, and EDTA [19]. Expression of recombinant MR on the surface of NIH3T3 cells conferred DENV increased binding and human MR antibodies could block this process in macrophages [19]. Pretreatment of primary human monocytes with Th2 cytokines such as interleukin IL-4/IL-13, which upregulated MR expression, could cause increase in their susceptibility to DENV infection in vitro [19]. All the above strongly suggest that MR is a novel functional receptor contributing to DENV infection in macrophages. However, it was shown that single antibody to either MR or DC-SIGN could not completely inhibit DENV infection while the combination of anti-DC-SIGN and anti-MR antibodies (CD206) was even more effective in inhibiting DENV infection in this kind of cells [19], indicating that several molecules may be involved in the process of DENVs entry into macrophages.
More recently, it was reported that C-type lectin domain family 5 member A (CLEC5A) contributed substantially to the mortality associated with DENV infection by triggering excessive macrophage activation and blockade of CLEC5A improved survival in mice [20]. Both human and mouse CLEC5A have been reported to bind to DENV, and the interaction was inhibited by fucose and mannan in vitro. Further studies showed that the CLEC5A-virus interaction triggered macrophage activation with a marked proinflammatory cytokine release through the associated adapter molecule DNAX-activating protein (DAP12) [20]. In addition, heat shock protein 90 (HSP90) and HSP70 have been identified as part of a receptor complex required for DENV entry and as CD14-independent cell surface functional receptors for lipopolysaccharide (LPS) in human monocytes/macrophages [21]. Interestingly, it has been reported that DENV infection was inhibited by bacterial LPS in human monocytes [22]. And also, Chen et al. found that the “binding” of LPS to CD14 was critical for DENV attachment and/or entry in macrophages [23]. About the phenomenon, Jorge Reyes-Del Valle and his colleagues offered an explanation: when monocytes were incubated with LPS prior to DENV infection, HSP90 and HSP70 were clustered around CD14, which prevented them from interacting with DENV [24], further implying an importance of HSP90 and HSP70 in the entry of DENV into monocytes/macrophages.
4. DENV Receptors in Human VECs
The endothelium is the primary fluid barrier of the vasculature, and the edema or hemorrhage seen in DHF/DSS is mainly due to changes in permeability of VEC induced by DENV infection. But the involvement of DENV receptors on VEC have not been revealed completely. One report, using a continuous ECV304 cell line, suggested that DENV interacted with three undefined cellular proteins [25]. However, these interactions have not been confirmed further in primary human VECs [26–28]. Lately, Dalrymple and Mackow found that DENV efficiently and productively infected human VECs via the interaction with heparan sulfate on glycosaminoglycan, heparan sulfate, as a nonspecific receptor molecule responsible for DENV attachment in several cell lines [22, 29]. Heparan sulfate is expressed in almost all cell types and is composed of alternating hexuronic acid/D-glucosamine disaccharides, which contains different degrees and patterns of sulfation, forming a linear chain with a remarkable diversity in length and structural complexity. The contribution of heparan sulfate to DENV entry has been shown by (i) a significant decrease in the binding capacity of DENV after enzymatic removal of heparan sulfate [30–34], (ii) a dose-dependent binding inhibition, which was only observed in heparin-pretreated mammalian cells, but not insect cell lines [31, 32, 35–37] suggesting that heparan sulfate as receptor for DENV was limited to mammalian cells, and the initial interaction (binding) between heparan sulfate and DENVs was likely influenced by the target cell types and viral serotypes [34], and (iii) an obvious decrease in virus binding to a mutant target cell lacking heparan sulfate expression [33, 37]. Interestingly, it was also demonstrated that DENV could bind specifically to immobilized heparin and both heparin and heparan sulfate ligands blocked DENV infection [33]. These findings were consistent with previous reports in which heparan sulfate proteoglycans (HSPGs) had been shown to mediate DENV attachment to Vero E6 cells and hepatocyte cell lines [22, 30, 33, 36]. Additionally, it was reported that purified E protein domains of DENV could interact with heparan sulfate [22, 38, 39], and VEC could not be infected by DENV after treatment with heparinase III, which cleaves both heparin and heparan sulfate side chains from cell surface HSPGs [29]. Together, all above results suggest that HSPGs were important receptors of DENV on VEC. However, there are some discrepancies. For example, Mertens et al. found that syndecans and glypicans were the most abundant HSPGs on cell surfaces, with syndecan-3 and glypican-1 being highly expressed on VEC, but syndecan antibodies failed to block DENV infection of endothelial cells [40, 41], indicating, other moleculars may act as receptor or coreceptor for mediating DENV entry into endothelial cells. In addition, Zhang and his colleagues found that high expression of integrin β3, which colocalized with dengue antigen, was observed in human microvascular endothelial cells 1 (HMEC-1) after dengue infection [42]. And about 90% of virus entry was inhibited when the expression level of integrin β3 was downregulated by RNA interference, indicating that DENV infection could induce upregulating expression of integrin β3, and integrin β3 was required for DENV entry into HMEC-1 [42]. Therefore, integrin β3 may be considered as a new target for dengue infection.
5. DENV Receptors in Hepatocytes
In severe cases of dengue, the impact of the DENV on liver function is prominent as shown by hepatomegaly and elevated serum levels of liver enzymes. Although the nature of the target cells for DENV in the liver is somewhat unclear, several studies based upon autopsy specimens have suggested the involvement of both hepatocytes and Kupffer cells [43–45]. Thepparit and Smith [46] identified the 37/67 kDa high-affinity laminin receptor as a DENV1 receptor expressed by HepG2 cells, a human hepatoma cell line, using a combination of virus overlay protein binding assay (VOPBA) and mass spectroscopy. The study also indicated that there was an association between the 37/67 kDa high-affinity laminin receptor protein and other glycoproteins at the cell surface, including the DENV low-affinity binding molecule heparan sulfate and the prion protein, suggesting that DENV receptor might be a complex consisting of those three proteins: heparan sulfate, the 37/67 kDa high-affinity laminin receptor, and prion protein [47]. Additionally in 2004, Jindadamrongwech and Smith identified the 78 kDa band for DENV2 as the glucose-related protein, GRP78 (BiP) on membrane extracts of HepG2 [48]. Pretreatment with anti-GRP78 antibodies resulted in a partial inhibition of DENV2 infection suggesting that additional receptor elements were involved in the entry process of DENV. In 2007, Cabrera-Hernandez and coworkers again noted a modest but definite inhibition of DENV2 entry into HepG2 cells in the presence of specific antibody directed against GRP78 [49]. The reproducible inhibition about 40% of the viral wild-type entry clearly demonstrated that GRP78 acted as at least a minor receptor in DENV internalization [49]. Moreover, it was reported that liver/lymph node-specific ICAM-3-grabbing integrin (L-SIGN) [50], homolog of DC-SIGN, expressed on liver sinusoidal endothelial cells as well as a subset of endothelial cells in the paracortex zone of lymph nodes, had the ability to bind to DENVs [51, 52]. And its expression in THP-1 cells induced susceptibility to DENV infection [53]. Specific antibodies against L-SIGN could subsequently block the acquired susceptibility. The L-SIGN-dependent DENV infection of THP-1 cells offered an intriguing possibility for the participation of L-SIGN in DENV infection [49].
6. Fc Receptors
Generally, DENV infection can induce subtype-specific humoral and cellular immune responses. If a secondary infection is caused by another serotype of DENV in the same individual, the preexisting antibodies will mediate virus infecting monocytes more efficiently. The outcome may be an increase in the viral replication and a high risk of severe dengue. This situation is referred to as antibody-dependent enhancement (ADE) of DENV infection. One possibility explaining this phenomenon is that in secondary infections, the virus may enter cells through the primary receptor(s) or it may also form immune complexes with preexisting nonneutralizing antibodies and interact with an alternative receptor, such as the immunoglobulin G (IgG) receptor (Fc gamma receptor, FcγR), which exists in FcγR-bearing cells including monocytes/ macrophages [54, 55]. By this process, the antibody-virus complexes may increase the ability of the virus to bind to and internalize into host cells, leading to maximum productive infection, that is, ADE of infection.
7. DENV Receptors on Mosquito Cells
Generally more is known about the detail of DENV replication cycle in mammalian cells as compared with that in mosquito cells. An Aedes albopictus mosquito cell line (C6/36) was frequently used for almost all studies associated with DENV receptors in host of mosquito during recent years. In the earlier study, Salas-Benito and Del Angel [56] identified two membrane proteins about 40 and 45 kDa as DENV4 binding molecules expressed on the surface of C6/36 cells. Further study certified that the 45 kDa protein definitely was a DENV receptor protein and may be immunologically related to Hsp 90 [57]. Using the same method, Munoz and his group [58] identified two proteins of 67 and 80 kDa on C6/36 cells as putative DENV 2 receptor proteins. Following work found that these two proteins (67 and 80 kDa) acted as receptors for all four serotypes of DENVs [59]. In 2006, Sakoonwatanyoo and his colleagues identified two common bands of approximately 50 and 100 kDa. The protein of 50kDa could bind with DENV2, 3, and 4 and was supposed as laminin binding homologue that might play important roles in the internalization of DENV3 and DENV4 to C6/36 cells [60]. The band of 100 kDa could bind with DENV4 [61]. In 2010, Kuadkitkan indicated that prohibitin was specific for DENV2 in C6/36 cells, additionally shown to be significantly colocalized with E protein of DENV2, suggesting that the association of prohibitin-DENV2 interaction might be a multifunctional interaction occurring at several stages during the virus replication cycle [62]. In the same year, Paingankaret and his colleagues proposed a model for DENV2 entry and transport in mosquito cells: prohibitin, possibly in complex with an ATP synthase, Hsp70, actin, ava-1, and tubulin β chain, might serve to concentrate the virus particle at the cell membrane and activate a signal transduction pathway during infection, indicating that DENV2 may exploit an array of housekeeping molecules for its entry in C6/36 cells [63].
8. Others
The receptors used by flaviviruses are very complicated molecules. In addition to that mentioned above, recent studies have shown that the plasma membrane contains numerous microdomains, which are essential for cellular functions and are involved in viral infection. These lipid microdomains, also known as lipid rafts, were characterized by detergent insolubility, light density, and enrichment for cholesterol, glycosphingolipids, and GPI-linked proteins [24]. Cholesterol had a strong promoting effect on membrane binding and trimerization of DENV E protein [64]. To investigate the significance of lipid raft integrity for DENV entry, Jorge Reyes-Del Valle et al. [24] tested the infectious ability of DENV to human peripheral monocytes/macrophages pretreated with a lipid raft disrupter, such as methyl-cyclodextrin (MCD), an agent that depleted the cholesterol from the cells. They found that MCD treatment could inhibit DENV infection in a dose-dependent manner, suggesting that raft integrity was involved in DENV infection by clustering its receptor complex, which associated with membrane microdomains. Additionally, treatment with raft-disrupting drug could cause a significant inhibition in DENV infection, indicating that rafts may be a site for virus entry and thus had a profound impact on pathogenicity [24].
In summary, the receptors used by flaviviruses may defer very much, depending on cell types and viral serotypes [61]. Many findings strongly suggested that DENV probably bound to multiple molecules that might form complexes on host cells and that DENV used specific combinations of receptor candidates to enter different types of cells. Elucidation of the molecular mechanisms underlying the interaction of DENV with receptor(s) in humans and mosquitoes is not only essential for the understanding of dengue pathology but also crucial for the development of effective new therapies for treatment of dengue disease. Further investigations for understanding the nature of DENV receptor complex are required.
Authors' Contribution
Shuyu Fang and Yanhua Wu contributed equally to this work.
Acknowledgments
This work was supported by Grants from the National Key Programs on Basic Research of China (2011CB504703, http://www.973.gov.cn/English/Index.aspx), the National Natural Science Foundation of China (81271839, http://www.nsfc.gov.cn/), and the Funding Project of Beijing Municipal Commission of Education (SQKM201210025005, http://www.bjedu.gov.cn/publish/portal0/tab40/).
Table 1 Dengue virus receptors found in different types of cells.
Cell types Receptors
DC DC-SIGN/L-SIGN
Monocyte/macrophage DC-SIGN, MR, CRD4–7-Fc, CLEC5A, HSP90/HSP70, CD14-associated protein, and FcγR
VEC Heparan sulfate, HSPGs, and integrin β3
Hepatocyte Heparan sulfate, 37/67-kDa high-affinity laminin, prion protein, GRP78, and L-SIGN
C6/36 cell HSP90/HSP70, 40/45 kDa membrane proteins, 50, 67, 80, and 100 kDa proteins, and tubulin-like protein
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23696868PONE-D-12-2446710.1371/journal.pone.0064171Research ArticleBiologyMolecular cell biologyGene expressionProtein translationRNA stabilityNucleic AcidsNucleotidesRNASignal TransductionSignaling CascadesStress Signaling CascadeCell GrowthCellular Stress ResponsesEnhanced Translation of mRNAs Encoding Proteins Involved in mRNA Translation during Recovery from Heat Shock Regulation of TOP mRNADatu Andrea-Kaye Bag Jnanankur
*
University of Guelph, Department of Molecular & Cellular Biology, Guelph, Ontario, Canada
Tuller Tamir Editor
Tel Aviv University, Israel
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Performed the experiments: AD. Analyzed the data: AD. Wrote the paper: AD. Designed the experiments: AD. Conceived the project: JB. Helped write the manuscript: JB.
2013 16 5 2013 8 5 e6417116 8 2012 12 4 2013 © 2013 Datu, Bag2013Datu, BagThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
The mRNAs encoding poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) belong to the family of terminal oligo pyrimidine tract (TOP) containing mRNAs. Translation of the TOP mRNAs is regulated by growth signals and usually codes for proteins involved in mRNA translation. Previous studies from our laboratory showed that translation of PABP1 mRNA was preferentially enhanced during recovery of HeLa cells from heat shock. Presence of the 5′ TOP cis element was required for the observed increase of PABP1 mRNA translation. In the studies reported here we showed that translation of two additional TOP mRNAs such as, eEF1A and RPS6 was similarly enhanced during recovery. In addition, we showed by in vivo cross-linking experiments that the cellular nucleic acid binding protein ZNF9 binds to all three TOP mRNAs examined in these studies as well as to the β-actin mRNA that lacks a TOP cis element. Binding of ZNF9 to mRNAs was observed in both heat-shocked and non heat- shocked cells. However, depletion of ZNF9 by siRNA prevented the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect of ZNF9 depletion on the basal level of expression of either β-actin or PABP1, eEF1A and RPS6 in HeLa cells following recovery from heat shock.
Conclusion
Although the presence of ZNF9 was required for the translational stimulation of PABP1, eEF1A and RPS6 mRNAs, the mechanistic details of this process are still unclear. Since ZNF9 was shown to bind both TOP and non-TOP mRNAs, it is uncertain whether ZNF9 exerts its stimulatory effect on TOP mRNA translation following recovery from heat shock through the TOP cis-element. Perhaps additional factors or post-translational modification(s) of ZNF9 following heat shock are necessary for the preferred increase of TOP mRNA translation.
This work was supported by a discovery grant from the Natural Sciences & Engineering Research Council of Canada (grant number 3542-2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
PABP is a multifunctional and ubiquitous mRNA-binding protein in eukaryotes.
PABP is known to play a role in several cellular processes including stimulation of mRNA translation and control of mRNA stability [1]
[2]. As such, PABP is important for cell survival. PABP has also been seen to play a role in development and in cancer [3]
[4]
[5]. Almost all known functions of PABP are attributed to its ability to bind to the poly (A) tail and to act as a scaffold for protein-protein interactions [6]. The other members of cytoplasmic PABP family are t-PABP also known as PABP2 in mouse and PABP3 in humans, PABP4 (or inducible PABP) and PABP5 are separate gene products but are highly homologous to PABP1. All four cytoplasmic PABPs have four RNA binding domains at their N-terminal ends and with the exception of PABP5, have a proline rich linker and a PABPC domain [7]. PABP5 is a smaller polypeptide than all other cytoplasmic PABPs and is missing the entire C-terminal end. Very little information regarding the biochemical functions of these cytoplasmic PABPs are known. Antisense morpholino oligodeoxynucleotide mediated knock down experiments in Xenopus laevis suggests that both PABP1 and PABP4 are required for normal development [8]
[9]. In addition, rescue studies [8] showed that PABP4 could not restore the effect of PABP1 knock down on development. Therefore, it is likely that each PABP has distinct functions in vertebrates [8]. PABP1 mRNA is a member of the family of mRNAs containing a terminal oligo pyrimidine tract (TOP) at their 5′ ends [10]. TOP mRNAs are growth-dependent mRNAs and encodes various components of the translation apparatus such as several ribosomal proteins and the elongation factors eEF1A and eEF2 [11]. The TOP cis-regulatory element typically starts with a C residue at the cap site followed by a stretch of 4–14 pyrimidines and is generally composed of a similar proportion of C and U residues which is trailed by a CG rich sequence. In mammals, the activity of TOP is contained within the first 30 nucleotides of TOP mRNAs and is strictly dependent on its integrity adjacent to the cap structure; therefore it fails to have any effect on mRNA translation when it is located internally even when it comes before an A residue [12].
The translation of TOP mRNAs is sensitive to the cellular growth rate. Thus, arrest in cell growth results in inhibition of TOP mRNA translation. This is observed by their shift from polysomes in growing cells into free mRNP particles (sub-ribosomal fraction) in quiescent cells [12]. These TOP mRNAs are maintained in a repressed state for later use when better growth conditions return. This bimodal distribution of TOP mRNAs between mRNP and polysomes suggests that the translational repression results from an obstruction at the translational initiation step [11].
Translational control of TOP mRNAs allows cells to quickly repress the biosynthesis of translational machinery during episodes of amino acid shortage resulting in growth arrest and thus preventing unnecessary energy wastage [12]
[13]. The detailed mechanism of translational control of different TOP mRNAs may be different. It has been observed that there may be a relationship between the phosphorylation of ribosomal protein S6 (RPS6) by p70 ribosomal protein S6 kinase 1 and translational activation of TOP mRNAs [11]. RpS6 is phosphorylated by S6K (S6 kinase) 1 and 2 in response to mitogens. When S6K activity is blocked using the mTOR (mammalian target of rapamycin) inhibitor, rapamycin, there is repressed translation of TOP mRNAs in N1H 3T3 cell lines [14].
However this model remains controversial. A study using amino acid starved cells, which results in the translational repression of TOP mRNAs, observed that RPS6 phosphorylation is insufficient to relieve the translational repression of TOP mRNAs [15]. Another study using mice showed that translation of TOP mRNAs still occurs when mice lack both S6K genes [16]. It is however possible that different strategies are employed by cells to stimulate TOP mRNA translation under different circumstances.
It has been suggested that the 5′ TOP motif is recognized by specific trans-acting factors that have the ability modulate TOP mRNA translation. An example of a trans-acting factor is the La auto antigen protein which is an RNA-binding protein that is involved in initiation and termination of RNA polymerase III transcription. A study using Xenopus cell lines with inducible over-expression of wild-type La or a mutated La, suggested that the La protein also plays a positive role in translation of TOP mRNAs. It was shown that it had a stimulatory effect on the translation of TOP mRNAs including those encoding several ribosomal proteins and eEF1A [17]. Furthermore, another study investigated the translational control of TOP mRNA encoding eEF1A in rapamycin treated human BJAB B lymphocytes. Gel shift assays confirmed that La interacts with an RNA containing the eEF1A TOP element. However, using a functional in vitro assay, it was observed that recombinant La protein specifically repressed expression of a reporter mRNA that contained the EF1A TOP element [18]. Collectively, these results indicate that TOP mRNA translation may be either repressed or activated through La binding to the TOP element.
More recently, the presence of another TOP binding protein has been reported. Myotonic dystrophy 2 (DM2) is a disease of the skeletal muscle and is caused by (CCTG)n expansion in the introns 1 of the ZNF9 (Zinc finger factor 9) gene, also known as the cellular nucleic acid binding protein (CNBP). The ZNF9 protein contains 7 zinc finger domains and is believed to function as an RNA-binding protein [10]. Its absence in DM2 cells is thought to contribute to the disruption of RNA metabolism in these cells [19]. The biological function of ZNF9 in normal and DM2 cells is not entirely clear. ZNF9 structure is highly conserved suggesting that this protein plays a basic biological role [10]. The seven conserved Zinc-finger (CCHC) repeats are found commonly in transcription factors, ribosomal proteins, and proteins involved in the processing of mRNAs coding for ribosomal proteins [10]. Several studies suggested a role of ZNF9 in regulating both cap-dependent and cap-independent translation [19]
[20]. ZNF9 has been shown to bind the internal ribosome entry site (IRES) cis element of human ornithine decarboxylase (ODC) mRNA and forms an IRES trans-acting factor complex with another known IRES binding protein PCBP2. Mutational studies of IRES of ODC mRNA showed that mutations that abolished IRES function also reduced binding to ZNF9 and PCBP2 [20]. Both human and Xenopus laevis ZNF9 can bind to TOP elements of several mRNAs including eEF1A, eEF2, RPS17, RPL4 and PABP1 [10]
[19]. Binding of ZNF9 to these mRNAs is believed to stimulate their translation. It was reported that there is reduced expression of ZNF9 in myotonic dystrophy patients which leads to a decrease in translation of TOP mRNAs as well as of global mRNA translation [10]. It is not clear whether the decline of global mRNA translation was due to the effect of ZNF9 depletion on the reduced translation of several TOP mRNAs encoding various factors necessary for mRNA translation. Furthermore, as expected ectopic expression of ZNF9 in DM2 myoblasts resulted in an increased rate of protein synthesis, suggesting that stimulation of translation of TOP mRNAs was responsible for the observed effect on global protein synthesis in DM2 muscle cells [10]. The details, however, of how ZNF9 or any other novel TOP binding protein enhances translation remains to be investigated.
In our laboratory we have used heat shock treatment to study regulation of TOP mRNA translation [21]. It is well known that translation of normal cellular mRNAs undergoes rapid changes when cells are subjected to heat shock. Cells down regulate protein synthesis in order to cope with stress [22], because the presence of many unfolded proteins produced by heat shock may harm the survival of cells which could ultimately lead to cell death.
The precise mechanism of how general cap dependant cellular mRNA translation is inhibited in heat-shocked cells is not fully understood. Most likely, it takes place through inactivation of the eIF4F complex and other initiation factors. Studies have shown that there is decreased phosphorylation of eIF4E and eIF4B, increased phosphorylation of eIF2α, and insolubilization of eIF4G following heat shock [21]. Since PABP1 interacts with eIF4G, regulation of its expression is important in modifying gene expression in response to heat stress. Studies from our laboratory have shown that indeed there was a decline in the cellular abundance of PABP1 following 2 hours of heat shock in HeLa cells. However, during the recovery period in which the cells were placed back to their normal temperature following heat shock, PABP1 abundance increased 2.5 fold [21]. This increase in PABP1 abundance during recovery from heat shock was achieved by translating PABP1 mRNA more efficiently. It was postulated that the increase in PABP1 abundance may act as a signal for cells to stimulate global mRNA translation to meet protein synthesis requirements for the cell to recover completely from the heat induced stress. Results from our laboratory showed that the TOP cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation in cells undergoing recovery from heat shock [21].
In this report we examined whether translation of other TOP mRNAs such as RPS6 and eEF1A are also up regulated during recovery from heat shock and investigated the nature of the trans-acting factor responsible for regulating translation of TOP mRNAs in HeLa cells. We demonstrate here that expression PABP1, eEF1A and RPS6 mRNAs were enhanced during recovery from heat shock. We showed that ZNF9 interacts with both TOP+ and non TOP− cellular mRNAs. However, depletion of ZNF9 abolished the preferred increase of PABP1, eEF1A and RPS6 expression during recovery from heat shock. Our results suggest an essential role of ZNF9 in the stimulation of translation during recovery from heat shock of all three TOP mRNAs examined here.
Materials and Methods
Cell Culture and Heat Shock Treatment
HeLa cells were grown at 37°C with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for 2–3 days to desired confluence. Cells were subjected to heat shock at the indicated temperatures for different times, and returned to 37°C for recovery. Control cells were maintained at 37°C.
Western Blotting
Cells were washed three times with 1×PBS (125 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4 and 2.5 mM KCl) and lysed with 200 µL of 1× SDS gel-loading buffer (50 mM Tris-HCl, pH 6.8; 2% (w/v) SDS; 0.1% (w/v) bromophenol blue; 10% (v/v) glycerol and 5% (v/v) 2-mercaptoethanol). The samples were boiled for 5 minutes and 10 µL aliquots were separated by 10% SDS-PAGE. The separated polypeptides were electrophoretically transferred from the gel onto a nitrocellulose membrane. Afterwards, membranes were blocked with a blocking buffer (5% non fat dry milk, 0.2% Tween-20 in PBS) for 3 hours at room temperature. Proteins were detected by incubating the membrane with the appropriate specific primary antibody (Table 1) for 2–4 hours at room temperature, followed by incubation with the appropriate HRP-conjugated secondary antibody. The membrane bound antigen-antibody complex was developed with western lighting chemiluminescence reagent plus (PerKinElmer, LAS, Inc. Shelton, USA). The X ray film was scanned and quantified by using the Image J program and recorded in arbitrary units after subtracting the background.
10.1371/journal.pone.0064171.t001Table 1 Primary antibodies used for immuno blotting.
Type Name From (Company)
Primary β – actin Santa Cruz Biochemical, Santa Cruz, CA, USA
Primary PABP Santa Cruz Biochemical, Santa Cruz, CA, USA
Primary PABP 3 Abnova, Wal Nut, CA, USA
Primary PABP 4 Novus Biologicals, ON, Canada
Primary PABP 5 Santa Cruz Biochemical, Santa Cruz, CA, USA
Primary HSP 70 Santa Cruz Biochemical, Santa Cruz, CA, USA
Primary eIF2α Santa Cruz Biochemical, Santa Cruz, CA, USA
Primary Paip1 Abcam, SF, CA, USA
Primary eEF1A Cell Signaling Tech., Danvers, MA, USA
Primary RPS6 Abnova, Wal Nut, CA, USA
Primary ZNF9 Abcam, SF, CA, USA
Secondary Anti-mouse Santa Cruz Biochemical, Santa Cruz, CA, USA
Secondary Anti-rabbit Santa Cruz Biochemical, Santa Cruz, CA, USA
Secondary Anti-goat Santa Cruz Biochemical, Santa Cruz, CA, USA
RT PCR
Cells were washed three times with 1×PBS and total cellular RNA was isolated using the High Pure RNA Isolation Kit according to manufacture’s instructions (Roche Biochemical, Indianapolis, IN, USA). The quality and quantity of the RNA were determined by 1.5% agarose gel electrophoresis and spectrophotometric measurements respectively. The absence of contaminating DNA in RNA samples was tested by PCR using several mRNA specific primers. The levels of specific mRNAs were determined by RT-PCR. An aliquot of total RNA (500 ng) was reverse transcribed at 42°C for 1 hour in a total reaction volume of 20 µL using SuperScript II reverse transcriptase (Invitrogen, Burlington, Canada) and 150 ng of random primers. After the reaction, 2 µL of the cDNA sample was amplified by PCR in a total master mix (Fermentas, Amherst, NY, USA) reaction volume of 50 µL, which included 100 ng of primers specific for PABP1, β –actin, eEF1A1, eEF1A2 or RPS6 (Sigma, Oakville, ON, Canada) (Table 2). The amplification was performed using an initial denaturation step at 95°C for 4 min, and for the mRNAs measured in this study, was followed by 25 cycles of denaturation at 95°C for 20 s, annealing ranging from 58–66°C, depending on the primer for 20 s and extension at 72°C for 20 s. Samples from the PCR reactions were analyzed by 1% agarose gel electrophoresis and the band intensities of the scanned images of the print were quantified by using the Image J program. The relative expression values of all mRNAs were normalized to the β-actin mRNA level. For detecting any DNA contamination of our RNA, PCR reactions of each RNA samples were carried out without the reverse transcription step.
10.1371/journal.pone.0064171.t002Table 2 Primers used for RT-PCR. S, sense oligonucleotide; AS, antisense oligonucleotide.
mRNA Nucleotide Sequence (5′ to 3′)
Human β –actin (S)
CTCTTCCAGCCTTCCTTCCT
Human β –actin (AS)
CACCTTCACCGTTCCAGTTT
PABP1 (S)
GCACAGAAAGCTGTGGATG
PABP1 (AS)
TTTGCGCTTAAGTTCCGTC
eEF1A1 (S)
GGCATACCCGAGAGCATG
eEF1A1 (AS)
AGGCAT GTTAGCACTTGGC
eEFA2 (S)
GAGCCCTCCCCCAACATGCC
eEFA2 (AS)
ATGTTCACTGGCGCAAAGGTCAC
RPS6 (S)
GTTATCTGCTGACTGCCTTGGA
RPS6 (AS)
GGGAATCCCTGTTTGTCGTTT
Immunoprecipitation of RNA-Protein Complexes
RNA-protein cross-linking and immunoprecipitation assay was performed as previously described in [23]. Cells in a 35 mm dish were treated with 1% formaldehyde (Electron Microscopy Sciences) in PBS for 30 minutes at room temperature to crosslink the RNA and proteins of RNP complexes. Cells were then incubated in 0.25 M of glycine for 15 minutes which was removed before cell lysis. Cells were then removed from the plate using a scraper with 0.1 mL of RIPA buffer (50 mM Tris-HCl, 1% Ipegal CA6–30, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 0.5 mM PMSF, 10 µg/mL leupeptin, 2 µg/mL aprotinin, 100 U RNasin (Promega). The cell suspension was passed through a 20 gauge syringe several times until more than 90% of cells were lysed as judged by phase contrast microscopy. The cell lysate was centrifuged at 10, 000 rpm for 1 minute in a microfuge at 4° C. The supernatant was first pre-cleared by mixing with a 20 µL packed volume of Sepharose G and 100 µg of E.coli t-RNA in a rotator for 1 h at 4°C. It was then centrifuged at 4000 rpm for 5 minutes and the supernatant (cell lysate) was collected. ZNF9 (Abcam, San Francisco, CA, USA) antibody and 10 µg coated protein G-Sepharose beands (20 uL, packed volume) was incubated with the pre-cleared cell lysate (100 uL) in RIPA buffer at 4°C for 2 hours. The beads were washed extensively with high-stringency RIPA buffer (50 mM Tris-HCl, 1% Ipegal CA-630, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 M NaCl, 2.5 M Urea and 0.5 mM PMSF) five times for 10 minutes at room temperature. The RNA-protein cross-links were reversed by incubation at 70°C for 45 minutes in 100 µL of elution buffer (50 mM Tris-HCl, 5 mM EDTA, 10 mM DTT, 1% SDS). The RNA was then extracted with three volumes of a mixture of phenol/chloroform (50/50). The RNA was precipitated using one volume of ethanol, and left overnight at −20°C. The precipitated RNA s was collected by centrifugation at 6000 rpm for 10 minutes at 4°C and resuspended in RNAse free water. Contaminating DNA was removed from the samples by RQ1 RNase-free DNAse (Promega) treatment prior to being reverse transcribed using random primers as described above followed by PCR amplification using specific primers (Table 2). The samples were analyzed by electrophoresis in a 1% agarose gel and band intensities were determined by scanning and Image J program analysis and expressed as arbitrary unit after normalizing by the β-actin mRNA level.
Sub-cellular Fractionation of Polysomes and RNA Isolation
Samples corresponding to equal numbers of cells were used for analysis. The cells were lysed in 500 µL of polysomal buffer (10 mM MOPS, pH 7.2, 250 mM NaCl, 2.5 mM MgOAc, 0.5% Nonidet P-40, 0.1 mm phenylmethanesulfonyl fluoride, 200 µg mL−1 heparin, and 50 µg mL−1 cycloheximide) [24]. After removal of the nuclei and cell debris by centrifugation at 12,000 x g for 10 min, the supernatant fraction was centrifuged in a 12 mL 10–50% sucrose gradient, containing 25 mM Hepes (pH 7.0), 50 mm KCl, 2 mM MgOAc, 200 µg mL−1 heparin, 50 µg mL−1 cycloheximide and 15 mM 2-mercaptoethanol at 100,000 x g in a Beckman SW 41 Ti rotor for 3 h. Gradient fractions of approximately 1 mL each were collected using an Auto Densi-Flow IIC apparatus (Buchler Instruments, Fort Lee, NJ, USA) [24]. Fractions 1–3, 4–6 and 7–10 were pooled together and total RNA from each fraction was isolated using Trizol as described by the manufacturer (Roche), and precipitated with ethanol. Absence of contaminating DNA in RNA samples was examined by PCR using several mRNA specific primers.
Cell Culture and siRNA Transfection
HeLa cells were grown at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented by 10% fetal bovine serum (FBS) for 2–3 days, until the desired degree of cell confluence was obtained. One day before transfection, cells were trypsinized and suspended in 1 mL of DMEM medium. 100 µL of suspended cells were grown in a 35 mm tissue culture dish with 2 mL of DMEM medium including 1% glutamine (Sigma, Oakville, Canada) and 10% fetal bovine serum (VWR, Mississauga, ON, Canada) without antibiotics in a humidified 5% CO2 incubator at 37°C. Cells were either kept at 37°C as controls or were heat shocked for 3 hours at 44°C and allowed to recover for 4 hours at 37°C. Control and recovered cells were transfected at 30–50% confluence using Lipofectamine 2000™ (Invitrogen, Burlington, ON, Canada). For each transfection, 1.5 µL of siRNA duplex (20 µM annealed duplex from Invitrogen) was mixed with 250 µl of OPTI-MEM® I (Invitrogen, Burlington, ON, Canada). In a separate tube, 5 µL of Lipofectamine 2000™ was diluted with 250 µL OPTI-MEM® I and incubated for 5 minutes at room temperature. Afterwards, both solutions were mixed gently and incubated for 20 minutes at room temperature to form a complex. The solutions were then added to the 35 mm dish containing cells and 1 mL of medium. Cells were incubated at 37°C in a CO2 incubator in presence of the transfection complex for 24 hours, then the media was refreshed and kept at 37°C for 20 hours and then processed for further analyses. All cell samples were transfected with either anti CNBP siRNA (siZNF9) or a validated negative control siRNA from Qiagen (Cambridge, MA, USA). Additionally non-transfected cells and mock transfected cells were used as controls. The sequence of the ZNF9 siRNA and control siRNA are:
3′-ACAGCGAUAACAAAUAUCUgg-5′ and 5′-AGAUAUUUGUUAUCGCUGUtt -3′; 5′-UUCUUCGAACGUGUCACGUdTdT and 5′-ACGUGACACGUUCGGAGAAdTdT-3′. For mock transfection, cells were treated with the transfection reagent without the siRNA for the same length of time as transfection with siRNA.
Results
Expression of Polypeptides Involved in Translation During Heat Shock and Recovery
Previously we reported that translation of PABP1 mRNA is preferentially enhanced in HeLa cells during recovery from heat shock at 37°C [21]. PABP1 mRNA belongs to a family of TOP containing mRNAs that include mRNAs encoding various components of the cellular translation machinery such as eEF1A, and different ribosomal proteins. We therefore examined whether eEF1A and ribosomal protein S6 (RPS6) are co-coordinately regulated with PABP1 during recovery from heat shock. The abundance of three polypeptides, PABP1, eEF1A (both A1 and A2 isoforms) and RPS6 following heat shock and recovery was measured by western blotting (Figure 1A). Prior to these analyses the conditions of western blotting was optimized for linear dose response (Figure 1B). These polypeptides showed a differential effect of heat shock on their abundance. Abundance of PABP1 was significantly reduced by heat shock while that of eEF1A and RPS6 remained virtually unchanged. However, following recovery, levels of all of these polypeptides increased by almost 2 fold over what was observed in the non-heat shocked control cells. We also tested whether the abundance of another translation initiation factor such as eIF-2α and a translation regulatory protein Paip1 responds similarly to heat shock. The abundance of eIF-2α did not change after heat shock but that of Paip1 showed a decrease similar to what was observed for PABP1 (Figure 1A). Since Paip1 is a PABP1 interacting protein, it is not surprising that the cellular levels of both proteins are coordinately regulated. However, in contrast to PABP1, eEF1A and RPS6, the abundance of Paip1 and eIF-2α did not increase following recovery. In our studies cellular β –actin level was used as a control and its cellular level did not change significantly following heat shock and recovery.
10.1371/journal.pone.0064171.g001Figure 1 Changes in cellular level of polypeptides during heat shock and recovery.
A) Cells were grown to 40% confluence and heat shocked for 3 hours at 44°C and recovered at 37°C for 24 hours. The control cells were maintained at 37°C. For all experiments cells were directly lysed on the plate using gel loading buffer, and lysates were analyzed by SDS/PAGE. Individual polypeptides were detected by western blotting using appropriate antibodies. B) Shows dose response of western blotting of cell extract from exponentially growing cells with different antibodies. The sample volumes are indicated at the top of the panel. The abundance of specific polypeptides in both panels was determined by scanning the X-ray film and quantifying the images with Image J as described in materials and methods. The values in arbitrary scale are shown bellow each lane. All values were normalized by the using the β -actin levels as loading controls. The experiment was repeated three times, and the averages are shown in panel B as mean ± standard error. C) Western blotting and quantification was performed to measure the abundance of PABP4 in control, heat- shocked and recovered cells as described above. Data are mean ± SE, p<0.05 for those with an * and p>0.05 for those that are unmarked, n = 3.
In addition to PABP1, PABP4 is also expressed in HeLa cells [25]. Since PABP4 mRNA is not a TOP containing mRNA we examined whether the cellular abundance of PABP4 was influenced by heat shock and recovery. The results show (Figure 1A) that the cellular level of PABP4 remained unchanged by heat shock and did not increase following recovery from heat shock treatment.
Changes in Specific mRNA Levels following Heat Shock and Recovery
In order to assess whether the increase in the cellular levels of PABP1, eEF1A and RPS6 was due to an increase in the cognate mRNA level, different mRNA levels were measured by RT-PCR (Figure 2). To determine the optimum cDNA concentration and cycle time for a linear dose response, samples were subjected to 25 cycles of amplification with different concentrations of cDNA using different mRNA specific primers. The results show (Figure 2A), that 1.5–3 µL of cDNA samples and 25 cycles of PCR under these reaction conditions were within the linear range of dose response for PABP1, eEf1A1 and 2, RPS6 and β–actin mRNA. Furthermore, no product was observed when reverse transcription step was omitted before the PCR (-RT lane). Minus RT reactions were performed with the same RNA samples used in RT-PCR studies, and all mRNAs were measured using the same cDNA preparation, thus confirming that, the PCR products were derived from RNA. For subsequent studies we used 2 µL of cDNA and 25 cycles for PCR. The results show that the PABP1, eEF1A1, eEF1A2, and RPS6 mRNA levels were not altered by exposure to heat shock or following the subsequent recovery phase (Figure 2B & C). These results suggest selective enhancement of translation of PABP1, RPS6, eEF1A1 and eEF1A2 mRNAs during recovery from heat shock. Both eEF1A1 and eEF1A2 are TOP mRNAs encoded by different genes [26] with tissue specific patterns of expression. eEF1A1 is expressed in the brain, placenta, lung, liver, kidney, and pancreas, while eEF1A2 is expressed in brain, heart, and skeletal muscle [27]. However, in HeLa cells expression of both eEF1A1 and eEF1A2 was detected.
10.1371/journal.pone.0064171.g002Figure 2 mRNA levels of PABP1, eEF1A and RPS6.
Total cellular RNA from HeLa cells was isolated and the levels of specific mRNAs were determined by RT-PCR. Products of RT-PCR were analyzed by 1% agarose gel electrophoresis and quantified by scanning as described in materials and methods. Where applicable the relative expression values of all mRNAs were normalized by the β-actin mRNA level The values bellow each lane represent the relative abundance of each mRNA using an arbitrary scale where the level in control cells was considered to be 1.00. A) The dose response of the PCR reaction using 25 cycles of amplification for different mRNAs is shown. B) HeLa cells were grown to 40% cell confluence, heat shocked for 3 hours at 44°C and recovered for 24 hours at 37°C. Total cellular RNA from control, heat -hocked and following recovery from heat shock was analyzed. The C (-RT) lane indicates samples of amplification of RNA from control HeLa cells without reverse transcription. C) Experiments were repeated three times, and the averages are shown here as mean ± standard error.
The Polysomal Distribution of Different mRNA following Heat Shock and Recovery
Comparison of mRNA levels for PABP1, eEF1A and RPS6 between control, heat shocked and recovered cells suggest that expression of these proteins is regulated at the level of mRNA translation. Therefore, the polysomal distribution of these mRNAs was studied to examine whether translation of these mRNAs was activated following recovery from heat shock. Translationally active polysomes and non-translated sub-ribosomal populations of mRNAs were separated by means of sucrose gradient centrifugation, as described in the materials and methods section. For our analyses gradient fractions were pooled into three fractions. Fractions 1–3 represent the sub-ribosomal region of the gradient, fractions 4–6 represent the 80S initiation complexes, disomes, trisomes and small polysomes, while fractions 7–10 represent efficiently translated medium to large polysomes [21]. The results from our analyses show that nearly 40–50% of cytoplasmic PABP1, eEF1A and RPS6 mRNAs were present in the non-translated sub -ribosomal fractions of untreated exponentially growing cells. In contrast, only 10–15% of β-actin mRNA was present in this fraction. Following heat shock, the proportion of non translated mRNAs coding for all four polypeptides (PABP1, eEF1A, RPS6, and β-actin) increased to approximately 40–50% of cytoplasmic mRNA. This was most likely due to repression of global mRNA translation by heat shock [22]. However, the effect of reduced mRNA translation in heat-shocked cells did not affect the abundance of different polypeptides to the same extent (compare β-actin and Paip1, Figure 1A). This was probably due to differential stability of the polypeptides. Following recovery from heat shock translation resumed for all four mRNAs, and the majority of non translated sub-ribosomal constituents of PABP1, eEF1A, and RPS6 were transferred to the actively translated fractions (80S to polysomes) and represented almost 90–100% of the cytoplasmic population (Figure 3 panels A and B). Our results show that compared to β-actin mRNA, the PABP1, eEF1A, and RPS6 mRNAs are inefficiently translated in exponentially growing cells. Following recovery from heat shock, there was a preferential increase in the translation efficiency of all three TOP mRNAs.
10.1371/journal.pone.0064171.g003Figure 3 The polysomal distribution of mRNA following heat shock and recovery.
Cell lysates were prepared as described in materials and methods and the 12.000×g supernatant fraction of the cell lysate was centrifuged in a 12 mL 10–50% sucrose gradient at 100,000×g in a Beckman SW 41Ti rotor for 3 h. Gradient fractions of approximately 1 mL each were collected and fractions 1–3, 4–6 and 7–10 were pooled together. Total cellular RNA from each pooled fraction was isolated using the Trizol RNA isolation kit as described in materials and methods. A) The abundance of PABP1, eEF1A and RPS6 and β -actin mRNA levels in each fraction was measured by RT-PCR as described in materials and methods section. Each RNA fraction was also subjected to the PCR step without the prior reverse transcription step to determine DNA contamination in our samples, and none was found (results not shown). The amplicons were analyzed by 1% agarose gel electrophoresis, and quantified by scanning the image as described in materials and methods. B) Representative results of two separate experiments. The distribution of mRNA in the sub-polysomal region (fraction numbers 1–3) and the polysomal region (fraction numbers 7–10) of the gradient was determined by quantifying the mRNA level in each fraction using an arbitrary scale. The value of mean ± standard error was derived from two separate experiments.
Immunoprecipitation of RNA-ZNF9 Complex
ZNF9 is known to bind to the TOP cis element of mRNAs in Xenopus laevis and regulate both cap-dependent and cap-independent translation [19]
[20]. In addition, it was previously reported that ZNF9 deficiency results in reduced translation of several TOP containing mRNAs in muscle cells from a mouse model of myotonic dystrophy [10]. We therefore, examined whether ZNF9 interacts with PABP1, eEF1A and RPS6 RNAs during recovery from heat shock in a human cell culture model using HeLa cells. The RNA and proteins of cellular RNA-protein complexes were covalently cross-linked in vivo with formaldehyde. Following cell the RNA protein complexes were immunoprecipitated with an anti ZNF9 and after reversing the cross-link the RNA was extracted and analyzed for the presence of different mRNAs by RT-PCR.
The results (Figure 4A) show that the ZNF9 did interact with PABP1, eEF1A and RPS6 in untreated exponentially growing cells and remained bound to these mRNAs after heat shock and during recovery. The binding of ZNF9 to these mRNAs did not appear to be specific for TOP containing mRNAs as β-actin and GAPDH mRNAs were also present in the immunoprecipitated samples. Furthermore, there were insignificant differences in the levels of different mRNAs immunoprecipitated by ZNF9 antibody from control, heat shocked and recovered samples (Figures 4A & B). These results suggest that ZNF9 may act as a general RNA-binding protein and may not be directly involved in enhancing translation of PABP1, eEF1A and RPS6 mRNAs. Immunoprecipitation and subsequent RT-PCR experiments using non cross-linked samples show that none of the mRNAs examined were immunoprecipitated without cross-linking from exponentially growing cells (Figure 4C, top row). In addition, western blotting of immunoprecipitated samples from cross-linked control, heat-shocked and recovered samples show that ZNF9 was successfully immunoprecipitated (Figure 4C, bottom row).
10.1371/journal.pone.0064171.g004Figure 4 Immunoprecipitation of RNA-ZNF9 Complex.
A) HeLa cells were grown to 40% confluence; heat shocked for 3 hours at 44°C, and allowed to recover at 37°C for 24 hours. Cellular RNA and proteins were cross-linked with formaldehyde and lysed as described in materials and methods. Cell lysates were subjected to immunoprecipitation of RNA-ZNF9 complexes as described in the methods section. RNA from the immunoprecipitates was isolated and reverse-transcribed using random primers followed by PCR amplification using mRNA specific primers. PCR products were analyzed by electrophoresis in a 1% agarose gel and quantified by scanning as described in materials and methods. The levels of different mRNAs were normalized to the level of β-actin mRNA and expressed in arbitrary units below each lane. B) Each set of experiments was repeated three times, and the averages are shown here as mean ± standard error. C) First row; as a negative control immunoprecipitation of non cross-linked cell extract of exponentially growing HeLa cells was performed with ZNF9 antibody and RNA was extracted and used for RT-PCR as described in materials and methods. The results of analyses of RT-PCR products by 1% agarose gel electrophoresis with different mRNA specific primers are shown. The bottom row shows the results of western blotting experiment of immunoprecipitated samples prior to RNA isolation from the lysate of cross- linked control (C), heat shocked (HS) and recovered cells (Re). D) Exponentially growing HeLa cells were subjected to cross-linking as described above and the cell lysate was treated with either, non-immunized serum or GAPDH or PABP1 antibody and the RNA was extracted from the immunoprecipitates. The RNA samples were analyzed for the presence of either PABP1 or β-actin mRNA as described before.
Additional control experiments using PABP1, GAPDH antibodies and non-immunized serum for immunoprecipitation showed that β-actin mRNA was immunoprecipitated by PABP1 antibody (Figure 4D). In contrast, GAPDH antibody and non-immunized serum did not pull down β-actin or PABP1 mRNA. Therefore, immunoprecipitation of mRNAs by ZNF9 antibody suggest a specific interaction between these mRNAs and ZNF9.
Effect of ZNF9 Depletion on TOP mRNA Translation
In order to test whether ZNF9 has any effect on PABP1 expression, we knocked down ZNF9 expression in HeLa cells by using a ZNF9 specific siRNA. We initially tested the efficacy of siRNA on normal (at 37°C) cells using 24 and 48 hours of transfection. The results show that the designed siRNA was able to inhibit ZNF9 expression by approximately 70% (Figure 5A) within 24 hours in exponentially growing control cells. We then used cells that were heat shocked at 44°C for 3 hours for siRNA treatment. In addition we used normal non- transfected and heat-shocked mock-transfected cells as controls. Analyses of polypeptide levels in total cell extracts by western blotting show (Figure 5B & C) that there was approximately 80% reduction in ZNF9 abundance after 24 hours of incubation with the siRNA, in heat shocked cells. There was no reduction of ZNF9 abundance when cells were transfected with the control siRNA or in mock transfected cells. Cellular levels of β-actin were used as loading controls and its abundance was not affected by siRNA treatments. The results (Figure 5B & C) further show that the characteristic induction of PABP1, eEF1A and RPS6 expression during recovery from heat shock was ablated by depletion of ZNF9. The mock and control siRNA transfected cells, however did exhibit normal induction of expression all of these polypeptides during recovery from heat shock. These results, therefore, suggest that the presence of ZNF9 is required for the increased of abundance of PABP1, eEF1A and RPS6 during recovery from heat shock. However, whether this observed effect of ZNF9 depletion was related to the preferred increase of TOP mRNA translation or the presence of the TOP cis-element has not been determined yet.
10.1371/journal.pone.0064171.g005Figure 5 ZNF9 knock down with siRNA.
A) Exponentially growing HeLa cells were transfected with ZNF9 siRNA as described in materials and methods for 24 and 48 hours. Samples of total cell lysate from non-transfected (C) and transfected cells following 24 and 48 hours of transfection (SiZNF9) were analyzed for the abundance of ZNF9 and β-actin (loading control) by western blotting. B) Cells at 40% confluency were either kept at 37°C as controls or were heat shocked for 3 hours at 44°C and allowed to recover for 4 hours at 37°C before being transfected with either no RNA (M), ZNF9 siRNA or a validated negative control siRNA. Cells were incubated at 37°C in a CO2 incubator in the presence of the transfection complex for 24 hours and following lysis samples were processed for further analyses by SDS-PAGE and western blotting as described in materials and methods. C) The abundance of specific polypeptides was determined by scanning the images, and normalizing the values using the β -actin levels as loading controls as described in materials and methods.
Discussion
PABP1, eEF1A and RPS6 Abundance following Recovery from Heat Shock
Our studies not only showed preferential increase of PABP1 expression during recovery from heat shock, but the same was seen for the expression levels of eEF1A and RPS6. This is not surprising considering that previous studies found that the abundance of elongation factors and several ribosomal proteins increased when mRNA is reactivated for translation following growth stimulation of serum starved cells [12]. Interestingly, a number of other factors involved in mRNA translation such as eIF2-α and Paip1, which are coded by mRNAs lacking a TOP cis element did not show any change in their abundance in our studies. In addition, previous studies from our laboratory reported that the abundance of other initiation factors such as eIF4G and eIF4E also did not change following recovery from heat shock [21]. This is intriguing because PABP, Paip1 and eIF4G are partners of a multi-protein initiation complex; therefore our results suggest that expression of these proteins is not co-coordinately regulated. We also demonstrated that amongst the two different cytoplasmic PABPs, PABP1 and PABP4, expressed in HeLa cells, only PABP1 abundance increased following recovery from heat shock. Again, interestingly the mRNA encoding PABP4 lacks the TOP cis element (Figure 6).
10.1371/journal.pone.0064171.g006Figure 6 Nucleotide sequence of the 5′ end of different mRNA.
Sequences were obtained from the NCBI database The TOP cis elements are highlighted. The 5′ ends of PABP1, eEF1A1, eEF1A2, RPS6 and RPS17 are shown. Note that both RPS17 and eEF1A2 TOP elements contain a terminal G instead of a terminal C present in PABP1, EEF1A1 and RPS6 mRNAs.
Translational Control of TOP mRNAs
In an attempt to understand how the abundance of PABP1, eEF1A and RPS6 proteins was increased during recovery from heat shock we demonstrated that this increase in protein abundance occurs without a corresponding increase in the level of cognate mRNAs. This observation ruled out the possibilities of either transcriptional control or regulation of mRNA stability as a means of increasing protein abundance. Our analyses of distribution of mRNAs in sucrose gradients clearly showed that PABP1, eEF1A1, eEF1A2 and RPS6 mRNA translation was preferentially enhanced during recovery from heat shock. In an earlier report from our lab it was shown that the translational activation of PABP1 mRNA following recovery from heat shock was indeed mediated by the TOP element [21]. It was shown that the heat shock and recovery response can be transferred to a reporter mRNA by placing the TOP element for PABP1 mRNA at the 5′ end of the reporter mRNA. In this report we showed that RPS6 and eEF1A mRNAs also behave similarly to PABP1 mRNA in response to heat shock. It should be noted that although mRNAs encoding all three proteins examined here behaved similarly to heat stress, the precise nature of the TOP element differs considerably amongst these mRNAs (Figure 6). However, further studies will be necessary to demonstrate that the TOP cis element is also involved in the up-regulation of RPS6, eEF1A1 and eEF1A2 mRNA translation during recovery from heat shock.
Role of ZNF9 in TOP mRNA Translation
How the TOP cis-element regulates mRNA translation has remained elusive for more than a decade. Different laboratories have obtained conflicting results regarding the trans- acting factors involved in binding and regulating TOP mRNA translation. Two stress granule associated factors TIA-1 and TIAR have recently been shown to repress TOP mRNA translation by binding to the 5′ end of TOP element upon amino acid starvation [28]. However, repression and subsequent activation of the TOP mRNAs may involve different sets of factors under different conditions. Studies in Xenopus laevis suggest that translation of several TOP- containing ribosomal protein mRNAs is regulated by a complex interaction between three different proteins La, ZNF9, and an unidentified protease sensitive factor [17]
[19]. It has been proposed that ZNF9 acts as a repressor and following growth stimulation La relieves this repression [17]. In contrast, another recent study reported ZNF9 to be a stimulator of TOP as well as global mRNA translation [10]. We demonstrated here by RNA CHIP analyses that ZNF9 possibly binds globally to most mRNAs since β-actin mRNA which lacked the TOP element was also immunoprecipitated by the ZNF9 antibody. Our results also showed that ZNF9 does not dissociate from the TOP mRNAs studied here following heat shock, and remained bound to TOP and actin mRNAs during recovery. However, since these mRNAs were not immunoprecipitated when the cross-linking step was omitted suggest that binding of ZNF9 might be transient or requires other proteins to form a complex with mRNA. When we tested the effect of ZNF9 knock down on TOP mRNA translation using three different models, a similar effect was observed on the abundance of all three TOP mRNA encoded proteins. We demonstrated that depletion of ZNF9 prevented the accumulation of PABP1, eEF1A and RPS6 during recovery from heat shock. This change was most likely due to the inability of ZNF9 depleted cells to preferentially stimulate TOP mRNA translation following heat shock and recovery. Depletion of ZNF9 however, did not affect the basal level of expression of all four polypeptides including PABP1, eEF1A, RPS6 and β-actin during recovery from heat shock. Our results thus suggest that although the precise nucleotide sequence of the TOP cis element of three different mRNAs examined here are different, they are similarly regulated by ZNF9. Our results also suggest that stimulation of TOP mRNA translation by ZNF9 is not directly mediated by its binding to these mRNAs. It appears that a stimulus such as heat shock is necessary to fine tune ZNF9 interaction with TOP mRNAs. The fine-tuning might involve interaction with a TOP binding polypeptide. It is conceivable that a novel protein may bind to TOP elements following heat shock, which in turn interacts with ZNF9 to promote translation of TOP mRNAs. Additionally, post-translational modification(s) of a novel TOP binding protein or ZNF9 itself following heat shock could promote protein-protein interactions and stimulate TOP mRNA translation. It will therefore be important to further investigate whether ZNF9 is post translationally modified and/or interacts with different polypeptide partners following heat shock.
As discussed earlier PABP mRNA translation is positively regulated by TOP and negatively regulated by ARS cis elements [2]. Studies from our laboratory have shown that the TOP element from PABP1 mRNA is sufficient for the up-regulated translation of a reporter mRNA during recovery from heat shock. Furthermore, presence of the ARS, where PABP1 itself binds to repress translation [2], down-stream from the TOP of the reporter mRNA, did not influence the up-regulation of translation during recovery. This observation ruled out the possibility that ZNF9 functions by relieving PABP1 mediated repression of translation.
All known TOP containing mRNAs including PABP1, eEF1A and RPS6 are inefficiently translated in exponentially growing cells. Although we know that PABP1 mRNA translation is negatively regulated by a feed back mechanism by binding of PABP1 to the ARS [2], it is not known whether a similar feed back mechanism also regulates RPS6 and eEF1A translation. Interestingly, control of mRNA translation by its own translation product was first described for the bacterial ribosomal protein L10 mRNA more than three decades ago [29], it is therefore, a possibility worth investigating. In addition, different TOP mRNAs may have unique sets of additional control elements that respond to different growth signals. Therefore, a highly complex set of interactions between different trans-acting factors and cis-elements may determine the fate of TOP mRNA translation. Thus, regulation of the translation of TOP containing mRNAs is more complex than, for example, that of the ferritin mRNA, which lacks the TOP cis element. It is known that ferritin mRNA translation is controlled by modulating the interaction between its iron response cis element and a single polypeptide by cellular iron level [30]. This is however expected because ferritin mRNA translation needs to respond to one stimulus, namely the intracellular iron level, where as translation of TOP-containing mRNA must respond to many different stimuli.
The authors thank Dr. Richard Mosser for editorial help.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23696862PONE-D-12-3675310.1371/journal.pone.0063966Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologySignal TransductionSignaling CascadesApoptotic Signaling CascadeSignaling in Cellular ProcessesApoptotic SignalingCell DeathMedicineObstetrics and GynecologyGynecologic CancersOncologyBasic Cancer ResearchTumor PhysiologyCancers and NeoplasmsGynecological TumorsOvarian CancerNovel Insights into the Synergistic Interaction of a Thioredoxin Reductase Inhibitor and TRAIL: The Activation of the ASK1-ERK-Sp1 Pathway DTCD Sensitizes TRAIL-Induced ApoptosisLin Tingting
1
*
Chen Yong
2
Ding Zhiying
3
Luo Guimin
2
Liu Junqiu
4
Shen Jiacong
4
1
College of Instrumentation and Electrical Engineering, Jilin University, Changchun, P.R. China
2
College of Life Science, Jilin University, Changchun, P.R. China
3
School of Pharmaceutical Sciences, Jilin University, Changchun, P.R. China
4
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, P.R. China
Schneider-Stock Regine Editor
Institute of Pathology, Germany
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: TL JL. Performed the experiments: TL YC ZD. Analyzed the data: TL JS. Contributed reagents/materials/analysis tools: GL JL. Wrote the paper: TL YC. Software used and operation: GL JL. Breeding nude mice: ZD JL. Cell cultural supervision: GL.
2013 16 5 2013 8 5 e6396621 11 2012 9 4 2013 © 2013 Lin et al2013Lin et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death in various types of cancer cells but has little or no effects on normal cells. Unfortunately, not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. Here, we reported that 6-(4-N,N-Dimethylaminophenyltelluro)-6-deoxy-β-cyclodextrin (DTCD), a cyclodextrin-derived diorganyl telluride which has been identified as an excellent inhibitor of thioredoxin reductase (TrxR), could sensitize TRAIL resistant human ovarian cancer cells to undergo apoptosis. In vitro, DTCD enhanced TRAIL-induced cytotoxicity in human ovarian cancer cells through up-regulation of DR5. Luciferase analysis and CHIP assays showed that DTCD increased DR5 promoter activity via Sp1 activation. Additionally, DTCD stimulated extracellular signal-regulated kinase (ERK) activation, while the ERK inhibitor PD98059 blocked DTCD-induced DR5 expression and suppressed binding of Sp1 to the DR5 promoter. We further demonstrated that DTCD could induce the release of ASK1 from its complex with Trx-1, and recovered its kinase activity. Meanwhile, suppression of ASK1 by RNA interference led to decreased ERK phosphorylation induced by DTCD. The underlying mechanisms reveal that Trx-1 is heavily oxidized in response to DTCD treatment, in accordance with the fact that DTCD could inhibit the activity of TrxR that reduces oxidized Trx-1. Moreover, using an A2780 xenograft model, DTCD plus TRAIL significantly inhibited the growth of tumor in vivo. Our results suggest that Trx/TrxR system inhibition may play a critical role in apoptosis by combined treatment with DTCD and TRAIL, and raise the possibility that their combination may be a promising strategy for ovarian carcinoma treatment.
This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 21101071); Project supported by the China Postdoctoral Science Foundation (Grant No. 2012T50306; 20110491303); Outstanding Youth Foundation of Jilin Province, China (Grant No. 3D511B190537); Fundamental Research Funds for Jilin University (Grant No. 450060441140, 450060445216). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Ovarian carcinoma is the fifth most common cause of cancer deaths in women and accounts for the highest tumor-related mortality of gynecologic malignancies [1]. While the majority of ovarian cancer patients experience a clinical remission following surgical cytoreduction and adjuvant platinum/taxane-based chemotherapy, up to 80% of these patients’ cancers will recur within 18–24 months and only 20% of the people will survive 5 years from diagnosis [2]. As with other solid tumors, the focus of therapeutic research in ovarian cancer has shifted from more traditional cytotoxic drugs to biologic agents targeting specific cellular mechanisms.
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of cytokines. TRAIL exerts cytotoxic effects on malignant cells without any harm to normal cells [3]. TRAIL can bind to two death receptors (DR), DR4 and DR5, which contain a cytoplasmic functional death domain [4]. Following the engagement with DR, TRAIL triggers cell death through at least two fundamental apoptotic pathways, referred to as the extrinsic pathway and the intrinsic pathway [5]. The apoptosis initiated by the extrinsic pathway involves DR engagement, death-inducing signaling complex formation, and proteolytic caspase-8 activation [6]. Caspase-8 further activates Bid, which, in turn, translocates to the mitochondria and activates the intrinsic pathway [7]. Meanwhile, activated caspase-8 is also released into the cytoplasm and induces a protease cascade that stimulates effector caspases such as caspase-3 and caspase-7, which ultimately cut vital cellular substrates resulting in apoptosis [8]. DR not only give the apoptosis signal but also activate NF-κB, which regulates the expression of survival factors such as members of the inhibitor of apoptosis (IAP) family and Bcl-xL [9].
However, despite its promise, TRAIL resistance is well established and limits efficacy in many preclinical models, including ovarian cancers [10], [11]. The mechanism of the resistance has been attributed to dysfunction of different steps in the apoptosis pathways, as well as elevation of survival signals. The former include suppressed expression of the DRs or caspases by mutation or imprinting [12]. The survival signals consist of over expression of Bcl-2 and IAPs family [13]. Therefore, modulation of these points with a chemotherapeutic agent would sensitize TRAIL-induced apoptosis in ovarian cancer cells [14], [15].
6-(4-N,N-Dimethylaminophenyltelluro)-6-deoxy-β-cyclodextrin (DTCD; Figure 1A), a synthetic cyclodextrin-derived diorganyl telluride, has exhibited marked anti-inflammatory activity with very low toxicity [16], [17]. Recent studies reveal that DTCD also possesses anti-cancer activities against malignant cancer cells [18], [19]. The chemopreventive activity has been attributed to its ability to inhibit thioredoxin reductase (TrxR), which is overexpressed in primary tumors and becoming an attractive target for cancer therapy [20]–[22]. However, the anticancer mechanism of DTCD in TRAIL-mediated apoptosis has not yet been characterized.
10.1371/journal.pone.0063966.g001Figure 1 DTCD sensitizes human ovarian cancer cells to TRAIL-induced cytotoxicity in vitro
.
(A) The structural formula of DTCD. (B) Cytotoxic effects of TRAIL or DTCD on human ovarian carcinoma cells. Cells were treated with different concentrations of TRAIL (0–500 ng/mL, left) or DTCD (0–10 µM, right) for 24 h. Then cell viability was determined by MTT assay. *, P<0.05, *, P<0.01 versus vehicle control. (C) DTCD sensitize resistant ovarian cancer cells, but not HOSE cells to TRAIL induced cytotoxicity. Ovarian cancer cells and HEMC cells were treated simultaneously with DTCD (10 µM) and/or TRAIL (200 ng/mL) for 24 h. (D) Synergistic induction of cell death by DTCD and TRAIL. For combination experiments, five doses of DTCD and TRAIL were used from serial dilutions covering the IC50 (fractional affected 0.5) values. The combination index (CI) analysis was conducted according to the median-effect plot analysis of Chou and Talalay. CI <1, CI = 1, and CI >1 represent synergism, additivity, and antagonism of the two agents, respectively. (E) Effects of combined treatment with DTCD and TRAIL on cell apoptosis. Cells were treated with DTCD (10 µM) and/or TRAIL (200 ng/mL) for 24 h after 30 min pretreatment with (+)/without (−) z-VAD-fmk (25 µM), then assayed by AnnexinV/PI staining. (F) Representative images of DNA fragmentation and nuclear condensation in response to DTCD and/or TRAIL treatment as detected by TUNEL and DAPI staining assay (magnification, 200×).
In this study, we have examined whether DTCD and TRAIL interact to enhance their cytotoxicity towards ovarian carcinoma cells. Our results show that DTCD potentiates TRAIL-triggered apoptosis through DR5 up-regulation which is dependent on the activation of ASK1-ERK-Sp1 signaling pathway. Using the A2780 xenograft model, we further demonstrated that this combination significantly reduced tumor burden in vivo. Taken together, our findings suggest that DTCD is an ideal candidate for TRAIL-induced apoptosis in human ovarian carcinoma.
Materials and Methods
Cells and Materials
Human ovarian cell lines A2780, SKOV3, OVCAR3, CAOV3, PA-1, OV-4 and ES-2 cells obtained from American Type Culture Collection (ATCC) were grown in DMEM (Gibco) supplemented with 4.5 g/L of glucose, 4 mmol/L of L-glutamine, 100 units/mL of penicillin/streptomycin and 10% FCS (Invitrogen). DTCD was prepared as previously described [16]. Human ovarian surface epithelial cells (HOSEs, Cambres) were grown as described in the manufacturer’s instructions. Soluble Recombinant Human TRAIL was purchased from R&D Systems. 3,3-dihexyloxacarbocyanine iodide (DiOC6) and 4,6-diami-dino-2-phenyindole (DAPI) were obtained from Sigma. PD98059, SP600125, SB203580, z-VAD-fmk, and Z-IETD-fmk were purchased from Calbiochem.
Cell Viability and Apoptosis Assay
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT, Chemicon) colorimetric method. Annexin V assays were done using the Annexin V-FITC apoptosis detection kit (Clontech). For TUNEL & DAPI staining, apoptotic DNA fragmentation was examined by using an in situ cell death detection kit (Roche) following the manufacturer’s recommendations.
Cellular Fractionation
Cells were collected after 24 h of treatment and pretreated with a 25-µL protease inhibitor cocktail (Pierce). An NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce) was used to extract nuclear and cytoplasmic contents. Samples were concentrated with PEG 8000, and protein concentrations were estimated using a Micro BCA kit (Pierce). To separate cytosolic proteins from mitochondria, cells (106) were lysed in 30 µL ice-cold lysis buffer (80 mM KCl, 250 mM sucrose, 500 µg/mL digitonin and proteases inhibitors in PBS). Then, cell lysates were centrifuged for 5 min at 10000×g. Proteins from the supernatant (cytosolic fraction) and pellet (mitochondria fraction) were mixed with Laemmli buffer.
Western Blot Analysis
Samples (30 µg protein) were separated by SDS-PAGE and then transferred to nitrocellulose paper (Immobilon-NC, Millipore) soaked in a Tris (20 mM), glycine (150 mM) and methanol (20%) buffer at 55 V for 4 h. After washing, the blots were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-poly ADP ribosyl polymerase (PARP), anti-Sp1 (diluted 1∶1000 v/v, Santa Cruz); anti-caspase-3, anti-caspase-8, anti-caspase-9, anti-Cytochrome c, anti-Smac/DIABLO, anti-TRAIL-R2, anti-TRAIL-R1, anti-cIAP1, anti-cIAP2, anti-XIAP, anti-Bcl-xL, anti-Bid, anti-t-Bid, anti-Bax, anti-Bad (1∶2000 v/v, Stressgen) and mouse monoclonal anti-nucleolin, anti-β-actin, anti α-tubulin and anti-COX IV (1∶2000 v/v, Sigma). Antibodies against JNK, ERK, p38, ASK-1 and their phosphorylated products were purchased from Cell Signalling Technology. Following incubation, the secondary antibodies (diluted 1∶2000 v/v, Sigma) were added for 1 h at room temperature. Proteins were visualized using an enhanced chemiluminescence (ECL) system (Santa Cruz) and captured on X-ray films.
Flow Cytometry of Death Receptors
Following 24 h-incubation with DTCD or DTCD+TRAIL, adherent cells (1 ×106) were stained with 200 µL PBS containing saturating amounts of anti-DR4 or anti-DR5 antibody (R&D Systems) on ice for 30 min. Then, cells were reacted with FITC-conjugated rabbit anti-goat IgG (Sigma) on ice for 30 min. After washing with PBS, the expressions of these death receptors were analyzed by FACS Calibur (Becton Dickinson).
Real-time PCR
For quantitative PCR, 1 µL of gene primers with SYBR Green (Applied Biosystems) in 20 µL of reaction volume was applied. Primer sequences for each of the genes analyzed are as follows: DR4 forward primer: 5′-GCTCAGGTTGTTTGTTGCATCGGC-3′, reverse primer: 5′-GCCAGTTTTGTTGGAGGCGTTCCG-3′; DR5 forward primer: 5′-GAGACAACAAAACGGCGCCGAGGT-3′, reverse primer: 5′-CAGCAACTGTGAGACTACGGCTAC-3′ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, all purchased from Clontech. Temperature cycling and real-time fluorescence measurement were done using an ABI prism 7300 Sequence Detection System (Applied Biosystems).The relative expression level for each target gene mRNA was calculated using the following formula: [2∧-(CT target - CT GAPDH)] × 100%, where CT is the threshold cycle.
Luciferase Assays
The pDR5/−605 plasmid was prepared according to our previous work (Lin et al., 2011). For luciferase assay, A2780 cells were co-transfected with 1 µg of pDR5/605 plasmid construct and 0.2 µg of the pCMV-β-galactosidase plasmid by the Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer’s instructions. Luciferase activity was normalized by β-galactosidase activity in cell lysates and expressed as an average of three independent experiments.
Chromatin Immunoprecipitation Assay
The chromatin immunoprecipitation (ChIP) assay was done using the EZ-Chip assay kit according to the manufacturer’s protocol (Upstate Biotechnology). The primers used for the amplification of the Sp1-binding site of DR5 promoter region were 5′-GCCAGGGCGAAGGTTA-3′(sense) and 5′-GGGCATCGTCGGTGTAT-3′(antisense; 276-bp DNA product).
Electrophoretic Mobility Shift Assay
DNA-protein binding assays were carried out with a nuclear extract. Synthetic complementary Sp1 (5′-ATTCGATCGGGGCGGGGCGAGC-3′) binding oligonucleotides (Santa Cruz Biotechnology) were 3′-biotinylated using the biotin 3′-end DNA labeling kit (Pierce) according to the manufacturer’s instructions.
Small Interfering RNA
DR5, ASK1 siRNA, and control siRNA were purchased from Dharmacon, Inc. (Lafayette,CO). Cells were transfected with Lipofectamine 2000 (Invitrogen), according to the manufacturer’s recommendations in the presence of siRNAs. For siRNA-mediated silencing of JNK, p38 and ERK, transient transfections were performed using JNK, p38, ERK1 or ERK2 SMARTpool siRNA (Dharmacon RNA Technologies).
ASK1 in vitro Kinase Assay
Cell lysates were prepared under non-denaturing conditions as described previously [23], and 150 µg of protein was used for each kinase assay. The kinase in cell lysates was captured using the anti-ASK1 antibody and then incubated with MKK4 (Upstate Biotechnologies) in the presence of [γ- 32P] ATP, and MKK4 phosphorylation was detected using autoradiogram.
Assay for ASK1-Trx-1 Complex Formation
Immunoprecipitation of ASK1-Trx-1 complexes was performed using Protein A/G PLUS-Agarose beads as per the instructions of the manufacturer (Santa Cruz Biotechnology). Briefly, cells were lysed in IP assay buffer, and half of each cell lysate was incubated with 1 mmol/L DTT for 30 min. The lysate was then precleared by adding 40 µL Protein A/G PLUS-Agarose beads and incubating for 30 min at 4 °C. Cell lysates were then incubated with 6 µg anti-Trx-1 and 40 µL Protein A/G PLUS-Agarose beads for 4 h at 4 °C. The immunopercipitates were boiled in 1×electrophoresis sample buffer, and samples were subjected to SDS-PAGE analysis.
Trx Redox Status Assay
A2780 cells (106) were lysed in 6 mol/L guanidinium chloride, 50 mmol/L Tris/HCl (pH 8.3), 3 mmol/L EDTA, and 0.5% Triton-X-100 containing 50 mmol/L iodoacetic acid. After 30 min at 37 °C, the excess iodoacetic acid was removed using Microspin G-25 columns (GE Healthcare Life Sciences). Oxidized and reduced Trx-1 were separated by native PAGE. The gel was electroblotted onto a nitrocellulose membrane and probed with a Trx-1 antibody, followed by HRP-conjugated secondary antibody. Bands corresponding to Trx-1 were visualized by ECL.
Ethics Statement
The authors confirm that all animal studies were conducted according to the experimental practices and standards specifically approved by the Animal Welfare and Research Ethics Committee at Jilin University. Nude female mice were bred in rodent facility. The animals were kept in a specific pathogen-free environment, in positive pressure rooms with filtered and humidified air. The animals were kept under standard conditions, and food and water were supplied ad libitum.
In vivo Tumor Growth Model
A2780 cells (5×106) resuspended in 0.1 mL serum-free DMEM were subcutaneously (s.c.) injected into the right axilla of 6-week-old female Balb/c nu/nu mice (National Academy of Medical Sciences). When the tumor volume (TV) reached 150 mm3, mice were randomly divided into four groups (n = 8/group) to receive treatment of an intraperitoneal (i.p.) injection of vehicle control (100 µL of 0.9% NaCl), DTCD (60 mg/kg/2d – dose of 0.05LD50, based on preliminary experiments, 1.2 mg/2 d for a 20-g mouse in a maximal volume of 100 µL 0.9% NaCl), TRAIL (10 mg/kg/2d) and the combination of DTCD plus TRAIL (6 h after DTCD treatment). The volume of the tumors and the weight of the mice were measured every 2–3 days. Tumor volume was measured with a caliper and calculated by the following formula: (long axis×short axis2)/2.
The mice were treated for a period of 20 days. At the end of in vivo experiments, the animals were sacrificed under anesthesia using avertin, approximately 24 hours following TRAIL and/or DTCD treatment. Tumor tissues were then immediately removed, fixed in paraformaldehyde at room temperature and then embedded in paraffin for the further immunohistochemistry, which is beyond the scope of the present study.
Statistical Analysis
The mean and standard deviation (SD) were calculated for each experimental group. Differences between groups were analyzed by one-or two-way ANOVA followed by Bonferroni’s multiple comparison tests using PRISM statistical analysis software (GraphPad Software version 5.0). Significant differences among groups were calculated at P<0.05.
Results
DTCD Sensitizes TRAIL-induced Apoptosis Regardless of Cell type Specificity through Activation of Caspase
We first examined the cytotoxic effects of TRAIL sensitivity on a panel of human ovarian cancer cells using a standard MTT cell survival assay. Different concentrations (50,100,200, and 500 ng/mL) of recombinant human soluble TRAIL were used. As shown in Fig. 1B, only two (OVCAR3, CAOV3) out of seven ovarian cancer cell lines were efficiently killed by TRAIL in a dose-dependent manner. In contrast, the remaining cell lines were either partially resistant (40–80% cell death; ES-2, PA-1) or resistant (<20% cell death; SKOV3, A2780, OV-4) to TRAIL at concentrations up to 500 ng/mL. Hence, we choose 200 ng/mL of TRAIL for the experiment further on.
To investigate whether DTCD could exhibit anti-proliferative effects, TRAIL-resistant cell lines SKOV3 and A2780 were incubated with the increasing concentrations of DTCD (1.25, 2.5, 5.0, 10.0 µM) for 24 h and then also subjected to the MTT assay. DTCD alone induced a limited cell death (<10%) at concentrations up to 10 µM. We thus choose 10.0 µM dose of DTCD for the following characterization of cell death.
We next evaluated whether DTCD could cooperate with TRAIL to induce growth suppression of ovarian cancer cells. As shown in Fig. 1C, approximately 70% decrease of cell viability in A2780 and SKOV3 cells was observed. In contrast, the combination treatment induced minimal cytotoxic effects in normal human ovarian surface epithelial cells (HOSEs), indicating DTCD did not abrogate the potential tumor selectivity of TRAIL. Notably, exposure to the combination of DTCD and TRAIL exerts synergistic effects in A2780 and SKOV3 cells (CI<1), as determined by the median dose-effect isobologram analysis [24] (Fig. 1D).
We then investigated whether the combination treatment is dependent on apoptosis. As shown in Fig. 1E, the treatment of A2780 cells with a combination of DTCD and TRAIL for 24 h significantly increased the accumulation of apoptotic cells (AnnexinV–positive/propidium iodide–negative), whereas DTCD or TRAIL alone slightly induced apoptosis. Cell apoptosis induced by the combined treatment were further confirmed by TUNEL and DAPI staining assay which can detect early stage of DNA fragmentation in apoptotic cells prior to morphology changes (Fig. 1F). Finally, the apoptosis induced by DTCD in combination with TRAIL were significantly diminished when cells were pre-incubated for 1 h with 100 µM z-VAD-fmk, a broad-spectrum caspase inhibitor, indicating a caspase-dependent mechanism. Taken together, these results suggest that DTCD synergistically sensitize resistant breast cancer cells, not untransformed ovarian cells, to TRAIL-induced apoptosis in vitro.
Treatment with a Combination of DTCD and TRAIL Activates Extrinsic and Intrinsic Apoptosis Pathways
When using DiOC6, we found that marked reduction in the mitochondrial membrane potential had occurred in cells treated with DTCD plus TRAIL (Fig. 2A), indicating that the combinational treatment could suppress the inhibitory factors in mitochondria. While pretreatment with caspase-8 inhibitor z-IETD-fmk normalized mitochondrial membrane potential, confirming the involvement of caspase-8 activity in the DTCD-enhanced sensitization. It is known that TRAIL-induced caspase-8 activation can lead, via tBid and Bax, to cytochrome c and Smac/DIABLO release from mitochondria. Cytosolic cytochrome c enables apoptosome formation, which leads to caspase-9 activation that in turn processes and activates the executioner caspases. We then evaluated these proximal events in TRAIL-induced apoptotic pathways. As shown in Fig. 2B, treatment with TRAIL in combination with DTCD promoted robust caspase-8 processing, whereas, little or no change was observed in cells treated with a single agent. Moreover, the combinatorial group caused a time-dependent cleavage of BID protein and the formation of its truncated form of BID (tBID). As expected, during DTCD and TRAIL treatment, Bax levels were high in the cytosol at 6 h when co-incubated with TRAIL and DTCD, and then they declined thereafter. Meanwhile, Bax levels in the mitochondrial fraction were increased at 6 h post-drug exposure, and this process was accompanied by the release of cytochrome c and Smac/DIABLO, from the mitochondria into the cytosol (Fig. 2C). Similar to caspase-8 cleavage, caspase-3, caspase-9, and PARP were also activated after the combined treatment (Fig. 2D). Collectively, these results indicated that treatment with a combination of DTCD and TRAIL reduces the expression of multiple proteins associated with cell survival through the extrinsic and intrinsic apoptotic signal pathway.
10.1371/journal.pone.0063966.g002Figure 2 Treatment with a combination of DTCD and TRAIL activates apoptotic signal via the extrinsic and intrinsic pathways.
(A) Effects of DTCD plus TRAIL on mitochondrial membrane potential. A2780 cells were pretreated with(+)/without (-) z-IETD-fmk, and then were treated with DTCD (10 µM) and/or TRAIL (100 ng/mL). Mitochondrial membrane potential was measured by flow cytometry using DiOC6 dye. (B) Effects of TRAIL combined with DTCD on Caspase-8 and Bid cleavage. A2780 and SKOV3 cells were treated with DTCD (10 µM) and/or TRAIL (200 ng/mL) for the indicated time points, then subjected to Western blotting. (C) Levels of cytochrome c, Smac/DIABLO and Bax in cytosolic and pellet fractions were determined by Western blot. Following treatment, A2780 cells were lysed and cytosolic proteins were separated from mitochondria as described in Materials and Methods. COX IV and β-actin levels were used as internal controls for mitochondrial and cytosol specimen, respectively. (D) Effects of TRAIL combined with DTCD on levels of proapoptotic proteins. (E) DTCD combined with TRAIL regulates the expression of Bcl-2 and IAPs families.
DR5 Upregulation is important for DTCD Stimulated TRAIL-induced Apoptosis
Since TRAIL is known to trigger apoptotic signals via DR4 and DR5, we next examined whether the modulation of DR4 and/or DR5 protein levels by DTCD might be involved in its sensitizing effect on TRAIL-induced apoptosis. We found that treatment of A2780 cells with DTCD induces a time-dependent (left) and dose-dependent (right) increase in the protein levels of DR5 but did not affect the levels of DR4 (Fig. 3A). Flow cytometric analysis, considered as a more sensitive method, also showed that surface expression of DR5 but not DR4 was also notably increased by DTCD treatment (Fig. 3B). Consistent with this result, the expression of DR5 was significantly up-regulated not only in ovarian carcinoma cells [25] with wild-type p53 (A2780), but also in those with mutant p53 (OVCAR3 and ES2) and p53 null type (SKOV3), indicating that DTCD-induced DR5 up-regulation is not p53-dependent in ovarian cancer cells (Fig. 3C). Furthermore, suppression of DR5 expression by transfection of A2780 cells with DR5 siRNA also effectively inhibited DTCD stimulated TRAIL-induced growth inhibition (Fig. 3D), confirming the idea that DTCD-induced upregulation of DR5 is critical for the enhancement of TRAIL sensitivity in A2780 cells.
10.1371/journal.pone.0063966.g003Figure 3 DTCD increases DR5 but not DR4 levels in ovarian cancer cell lines.
(A) DTCD-induced DR5 upregulation in A2780 cells. Cells were treated with 10 µM DTCD for the indicated time or treated with indicated amount of DTCD for 24 h and then subjected to Western blot analysis. (B) Effects of DTCD on the surface expression levels of DR5. Cells were incubated with or without 10 µM DTCD for 24 h, then subjected to flow cytometry analysis. (C) DTCD-induced DR5 upregulation in other types of ovarian cancer cells. (D) Effect of DR5 siRNA on DTCD/TRAIL-induced cell death. A2780 cells were transfected with control siRNA or DR5 siRNA. 24 h after the transfection, cells were treated with 10 µM DTCD and 200 ng/mL TRAIL for another 24 h. Cellular viability was determined by MTT assay (left). The levels of DR5 were analyzed by Western Blotting (right). *, P<0.05 versus vehicle control.
DTCD Activates DR5 Transcription through the Activation of Sp1 in the DR5 Promoter Regions
To assess whether DTCD–induced DR5 up-regulation is tightly controlled at transcriptional level, we analyzed expression of DR5 mRNA by RT-PCR. We found that DTCD treatment increases DR5 mRNA expression in a dose-dependent manner (Fig. 4A), but not DR4 mRNA (data not shown). Next, the effects of DTCD on the promoter activities of reporter constructs containing 605-bp fragments of the DR5 gene promoter region (pDR5/−605) in A2780 cells were examined by luciferase assays. Results indicated that DTCD significantly increases the promoter activities of pDR5/605 in time- and dose-dependent manners (Fig. 4B). In particular, 10 µM DTCD enhanced the DR5 promoter activity approximately four fold greater than control values at 24 h, supporting the idea that DTCD-induced DR5 up-regulation is controlled at the transcriptional level. To further demonstrate the precise mechanism by which DTCD regulates DR5 expression, western blot and ChIP analysis were performed to quantify Sp1 activity on the DR5 promoter regions. As expected, treatment with 10 µM DTCD time dependently increased Sp1 nucleus translocation (Fig. 4C). Additionally, DTCD also increased Sp1 binding to the promoter regions of DR5 and significant Sp1 binding levels were observed at 6 h after DTCD treatment (Fig. 4D). These results show that DTCD up-regulates DR5 expression via Sp1 binding and activation on the DR5 promoter region.
10.1371/journal.pone.0063966.g004Figure 4 DTCD mediates transcription of DR5 through Sp1 activation.
(A) DTCD treatment increases the DR5 mRNA levels. Real-time PCR was used to quantify the DR5 mRNA levels following 24 h of DTCD treatment. (B) Effects of DTCD on DR5 promoter activity. pDR5/−605 plasmid was transfected into A2780 cells, which were then treated with DTCD (10 µM) for the indicated time points or indicated amounts of DTCD, lysed and assayed for luciferase activity. (C) DTCD activates Sp1 translocation to the nucleus. (D) In vivo binding of Sp1 on DR5 promoter regions by DTCD. ChIP assay was done using antibodies against Sp1 in both cells. Negative controls were done using antibody against rabbit IgG.
Transactivation of the DR5 Promoter Requires Activation of Mitogen-activated Protein Kinase by DTCD
The phosphorylation of Sp1 has been widely studied, with results showing that certain kinases that phosphorylate Sp1. Some of them affect its transactivation activity, while others, regulate its DNA binding affinity [26]. Serine or threonine residues could be phosphorylated by kinases, we then investigated whether DTCD-mediated Sp1 DNA binding is mitogen-activated protein kinase (MAPK) dependent. The results revealed that three major MAPKs, JNK, p38, and ERK, were activated by the treatment with 10 µM of DTCD, following a time-dependent pattern (Fig. 5A). In particular, JNK-MAPK activation displayed a rapid on set within 30 min of treatment, followed by a progressive decline, returning to basal levels after 3 h. Activation of p38 by DTCD was marginal, reaching maximum values within 30 min of treatment, decreasing thereafter and reaching control levels at 6 h. Activation of ERK1/2 was also observed at 30 min of DTCD treatment, followed by a consistently strong activated form within 24 h. The above results indicate that DTCD induces a differential activation of the three well-established MAPK subfamilies, in relation to time of exposure. These MAPKs were also activated by DTCD in a dose-dependent manner (5–50 µM) (Fig. 5B). Although phosphorylation of JNK and p38 slightly occurred at 50 µM of DTCD treatment, treatment with 5 µM of DTCD for 1 h was enough to activate ERK with a fixed concentration of TRAIL. The above results indicate that activation of ERK could play an essential role in DTCD-mediated potentiation of TRAIL-induced apoptosis.
10.1371/journal.pone.0063966.g005Figure 5 DTCD affects the levels of MAPK phosphorylation.
A2780 cells were incubated with 10 µM of DTCD for indicated times (A) or with indicated amounts of DTCD+TRAIL for 1 h (B) and the levels of -ERK, -JNK, - p38 and their phosphorylated forms were determined by western blotting. (C) MAPKs are involved in DTCD-mediated upregulation of Sp1 DNA binding activity. A2780 cells were stimulated with 10 µM DTCD for 24 h after pretreatment with 20 µmol/L PD98059, 20 µmol/L SP600125, and 10 µmol/L SB203580 for 1 h. Then, Sp1 DNA binding activity and nuclear translocation were analyzed by EMSA (left) and Western blotting (right), respectively. (D) Effects of PD98059 on DR5 promoter activity and expression assay. PD98059 inhibits DR5 luciferase activity induced by DTCD. pDR5/605 plasmid was transfected into A2780 cells, which were then pretreated with 20 µmol/L PD98059 for 1 h and further treated with 10 µM DTCD for 24 h, lysed and assayed for luciferase activity and Western blot assay, respectively. (E) Effects of PD98059 on the cytotoxicity induced by DTCD and TRAIL.A2780 cells were treated with DTCD (10 µM) and TRAIL (200 ng/mL) in the presence of indicated amount of PD98059 for 24 h. Cell viability was determined by MTT assay. (F) Cells were transfected with ERK1 siRNA, ERK 2 siRNA or control siRNA, respectively. 24 h after the transfection, cells were treated with 10 µM DTCD for another 24 h, and cell extracts were subjected to Western blotting. (G) Cells were transfected with JNK1/2 siRNA or p38 siRNA, then treated with 10 µM DTCD for 24 h. Western blotting was then used to analyze the extracts for JNK, and p38 MAPK expression.
To date, there is no evidence indicating how DTCD stress-induced MAPK activation affects Sp1 gene expression in cancer cells, therefore pharmacological inhibitors of various kinases were used to study these pathways. As shown in Fig. 5C, only inhibition of ERK by PD98059 significantly blocked DTCD-induced Sp1-binding activity and translocation of Sp1 to the nucleus. But SB203580 (a specific inhibitor of p38 kinases) and SP600125 (a specific inhibitor of JNK kinases) have no significant effect on Sp1 expression level. Meanwhile, pretreatment with PD98059 dose dependently attenuated the DTCD-mediated upregulation of both promoter activity and protein levels of DR5 (Fig. 5D). Cell viability assay further confirmed that inhibition of ERK by PD98059 reduced the cytotoxic effects of combined treatment with DTCD and TRAIL (Fig. 5E).
As inhibition of protein expression using RNA interference is often more specific than functional inhibition using small molecules. We then transfected A2780 cells with control siRNA and specific siRNA against ERK1 and ERK2. As shown in Fig. 5F, the reduction in ERK1/2 expression by the siRNA correlated with suppression of DTCD induced up-regulation of DR5 and Sp1. However, the JNK and p38 siRNA had minimal effects on DTCD-induced DR5 and Sp1 up-regulation, which further confirmed that ERK is required for death receptor induction (Fig. 5G).
DTCD-mediated ASK1 Induction Contributes to ERK Activation and Cell Apoptosis Induced by TRAIL
ASK1, also known as MAPK kinase kinase 5 (abbreviated as MAP3K5), is part of the MAPK cascade [27]. We, therefore, examined the effects of DTCD on TRAIL-induced activation of ASK1. As shown in Fig. 6A, DTCD could increase the phosphorylated levels of ASK1 (p-ASK1) and the kinase activity of ASK1. This observation raised the possibility that ASK1 induction by DTCD might lead to enhancement of ERK activity. We then determined whether ASK1 knockdown by siRNA could impair the activation of ERK targets. We found that depletion of ASK1 by siRNA not only prevented its own induction by DTCD but also substantially impair the induction of ERK and its down stream targets, Sp1 and DR5. These results confirm that DTCD-mediated rapid ASK1 phophorylation (within 1 h) could be responsible for ERK activation (Fig. 6B). To this end, we used ASK1 siRNA to reduce ASK1 expression in A2780 cells and observed a marked reduction in the level of apoptosis following DTCD treatment or both (Fig. 6C). The above observations suggest that the active engagement of ASK1-ERK is necessary in response to potentiation of TRAIL-induced apoptosis by DTCD. Reduced Trx is known to inhibit activation of ASK1, whereas when Trx is oxidized, it releases ASK1, permitting ASK1 to be phosphorylated and becoming an active MAPKKK. We, therefore, explored the status of the ASK-Trx-1 complex in A2780 cells in response to DTCD. As expected, DTCD dose-dependently decreased the amount of ASK1 associated with Trx-1 (Fig. 6D), indicating that Trx-1 might be one of the key players in regulating DTCD-mediated promotion of TRAIL-induced apoptosis. Incubation of the cell lysate with the reducing agent DTT can restore the integrity of the ASK-Trx-1 complex, suggesting that their dissociation could be resulted from DTCD-induced oxidation of Trx-1. We therefore determined whether DTCD could influence the cellular levels and the redox status of Trx-1. As shown in Fig. 6E, DTCD did not change the total protein level of Trx-1, however, a significant change in the oxidation status of Trx-1 in response to treatment with DTCD was observed. Furthermore, the redox status of Trx-1 changed in a concentration-dependent manner. At low concentrations, we observed both reduced and oxidized forms of Trx-1, whereas, at high concentrations, the reduced band was undetectable, and only two oxidized forms were present. Meanwhile, oxidation status of Trx-1 was also increased when cells were co-treated with DTCD plus TRAIL.
10.1371/journal.pone.0063966.g006Figure 6 DTCD synergizes with TRAIL in activation of ASK1/ERK pathway.
(A) Cells were treated with DTCD (10 µM) at indicated time points and the expression of ASK1 and p-ASK1 was determined by immunoblotting. For in vitro kinase activity assay, the ASK1 in the lysates was immunoprecipitated (IP) with the anti-ASK1 antibody and then incubated with MKK4 in the presence of [γ-32P] ATP, and MKK4 phosphorylation was detected via autoradiogram. (B) Suppression of ASK1 expression by RNA interference inhibits DTCD and/or TRAIL-induced apoptosis upon ERK activation. A2780 cells were transfected with ASK1 siRNA and treated with DTCD in the presence or absence of TRAIL. The levels of p-ASK1, ASK1, p-ERK, ERK, Sp1 and DR5 were analyzed by Western Blotting. Cell apoptosis were assayed by AnnexinV/PI staining (C). (D) Oxidation of Trx will bring on dissociation of the complex Trx-1-ASK1 and activation of MAPK system. A2780 cells were treated with DTCD for 1 h. Cell protein lysates were treated with or without DTT (1 mmol/L) for 30 min and then subjected to immunoprecipitation (IP) using an anti-Trx-1 antibody and the precipitates were immunoblotted for ASK1. (E) The level of Trx-1 was detected by Western blot in whole-cell lysates from A2780 cells treated with DTCD for 1 h in the absence or presence of TRAIL (200 ng/mL). Cell lysates were processed to identify Trx-1 redox forms denoted as follows: red, reduced form; ox, oxidized form, the topmost ox band is the one most oxidized.
The Combination of DTCD and TRAIL Inhibits Tumor Growth in Nude Mice Implanted with Human A2780 Xenografts
Based on the above findings, we next sought to evaluate whether the effect of DTCD alone and in combination with TRAIL could inhibit tumor growth in vivo. As expected, the combination therapy of TRAIL and DTCD notably inhibited the tumor growth of the A2780 xenografts (Fig. 7A), with T/C value 27.5% and inhibition rate 70.5%, which was prominently stronger than TRAIL-administrated group (T/C value: 83.3%, inhibition rate: 10.2%), and DTCD-treated group (T/C value: 61.7.6%, inhibition rate: 31.9%) (Fig. 7B). The relative tumor volume (RTV) of combination group was remarkably decreased from that of vehicle group (p<0.001); and more importantly, comparing with TRAIL-alone group or DTCD- alone group, combination group exerted significantly more potent activities. Thus the simultaneous administration of TRAIL and DTCD more distinctly arrested the growth of A2780 xenograft tumors, compared to either agent alone or vehicle control groups. Finally, the tumor growth suppression that was observed in the A2780 xenograft tumor model was also observed in all other xenograft (OVCAR3, ES2, and SKOV3) models tested (data not shown).
10.1371/journal.pone.0063966.g007Figure 7
In vivo efficacy of TRAIL in combination with DTCD.
(A) The tumor growth inhibitory effect of DTCD and TRAIL on A2780 human xenograft models. The relative tumor volume (RTV) on day n was calculated using RTV = TVn/TV0, where TVn is the TV on day n and TV0 is the TV on day 0. (B) Therapeutic effect of treatment was expressed in terms of T/C. The calculation formula is T/C (%) = mean RTV of the treated group/mean RTV of the control group×100%.
Discussion
Earlier, we reported that 2-Tellurium-bridged b-cyclodextrin (2-TeCD), one of TrxR inhibitors, could sensitize TRAIL-resistant human breast cancer cells and xenograft tumors to undergo apoptosis via Sp1-mediated DR5 up-regulation. However, we do not provide mechanistic evidence that how this effect achieved and how this effect could be correlated with the TrxR inhibitory activity [28]. In this study, we have examined whether DTCD (a novel synthetic TrxR inhibitor) and TRAIL interact to enhance their cytotoxicity towards ovarian carcinoma cells with the previous results, the present study show that DTCD could also potentiate TRAIL-triggered apoptosis through DR5 up-regulation. Additionally, we provided evidences to demonstrate the fact that the sensitization effects is heavily dependent on the activation of ASK1-ERK-Sp1 signaling pathway. These findings are accordance with the underlying mechanism that DTCD could inhibit activity of TrxR that reduces oxidized Trx-1.
Sp1 is well known to bind to G-rich elements such as GC-box (GGGGCGGGG) and GT-box (GGTGTGGGG) [29]. Recently, it has been evidenced that binding of Sp1 in the promoter regions tightly regulates DR5 transcription in a variety of cancer cells, and the required specific Sp1 site was mainly found at -605 to +3 to the transcription start site [30]. In the present study, we found that treatment with DTCD significantly induced expression of Sp1 in A2780 and SKOV3 cells that was correlated to the induction of DR5. Furthermore, the ChIP assay showed that Sp1 can directly bind to the DR5 promoter regions and regulated transcriptional expression.
Moreover, we explored the novel mechanism that ERK activation was involved in the DTCD-mediated Sp1 activation and cell death, which can be blocked significantly by PD98059, the inhibitor for ERK. According to our knowledge, the TrxR inhibitory activity of DTCD would be possible explanation for the observed results. Mammalian TrxR is a key enzyme for maintenance of intracellular reduced environment since it acts as a direct antioxidant of Trx-1. Impairment of TrxR will lead to increased levels of oxidized Trx-1. In the reduced form, Trx-1 associates with ASK-1, therefore, oxidation of Trx-1 results in ASK-1 dissociation and consequent activation of the MAPK/ERK pathway [31]. This hypothesis is consistent with our results: (a) the oxidation of Trx-1 seemed complete at the highest DTCD concentrations; (b) ASK1 is phosphorylated (within 1 h) before long-lasting ERK activation and cell death; (c) In the case of ERK activation, the release of ASK1 from its complex with Trx-1. Our findings are also consistent with recent publications indicating that in TrxR inhibitor-induced apoptosis, ERK phosphorlylation was activated by the ASK1-p38 MAPK pathway [32], [33]. Our results represent a convincing mechanistic link between Trx/TrxR system and ERK pathways. The mechanisms by which ERK induces apoptosis are not completely clarified yet, but it is believed that might occur at many different levels involving both the extrinsic and intrinsic apoptotic pathways [34]. For instance, inhibition of ERK phosphorylation decreases Bax expression [35] and in addition, ERK activation has been shown to be crucial in the regulation of Sp1 phosphorylation and consequently Sp1 dependent proapoptosis gene transcription [36], [37]. These facts rationalize the present observations indicating that DTCD could upregulate both of ERK and Sp1 phosphorylation and then potentiate cell death.
Based on these results, we proposed a working model for the mechanism of action of DTCD (Fig. 8). As already reported for some organotellurium compounds, DTCD can inhibit TrxR by irreversible covalent binding to its catalytic site [38]. This hampers the function of both mitochondrial and cytosolic TrxR that act as mediators of electron flow from NADPH to peroxiredoxins through Trx, and lead to an increase in the oxidized form of Trx and to the accumulation of hydrogen peroxide. Both of the events can improve the levels of phospho-ERK. Indeed, it has been reported that hydrogen peroxide accumulation can trigger ERK1/2 phosphorylation [39]. On the other hand, oxidation of Trx will bring on dissociation of the complex Trx-1-ASK1 and activation of MAPK system observed as subsequent ERK1/2 phosphorylation and Sp1 activation. These events are critical in DTCD-induced DR5 expression, and renders cells more sensitive to the cytotoxic activities of TRAIL.
10.1371/journal.pone.0063966.g008Figure 8 Proposed model for the molecular mechanisms underlying TRAIL and DTCD-induced apoptotic pathways.
In summary, here we have highlighted a novel function of DTCD: sensitizing human ovarian cancer cells to TRAIL-induced apoptosis via upregulation of DR5 which is dependent on activation of the ASK1-ERK-Sp1 signaling pathway. It deserves further assessment as a candidate mechanism for the pharmacologic control of cancer. Moreover, it is more reasonable to consider that the mechanism presented here may be shared by more compounds, and provide strong evidences that TrxR inhibitors in combination of TRAIL can be a promising strategy for cancer therapy.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23696886PONE-D-13-0540110.1371/journal.pone.0064373Research ArticleBiologyImmunologyImmunityInnate ImmunityMolecular Cell BiologySignal TransductionSignaling CascadesAkt Signaling CascadeSignaling in Selected DisciplinesImmunological SignalingMedicineClinical Research DesignObservational StudiesGastroenterology and HepatologyLiver DiseasesCirrhosisInfectious HepatitisHigh Mobility Group Box-1 Promotes the Proliferation and Migration of Hepatic Stellate Cells via TLR4-Dependent Signal Pathways of PI3K/Akt and JNK HMGB1 Promotes HSCs Proliferation and MigrationWang Fu-ping
1
Li Lei
1
Li Jing
2
Wang Ji-yao
1
Wang Ling-yan
3
Jiang Wei
1
*
1
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China
2
Department of Gastroenterology, Tongji Hospital, Tongji University, Shanghai, China
3
Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, China
Avila Matias A. Editor
University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), Spain
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: WJ FPW JYW. Performed the experiments: FPW LL JL LYW. Analyzed the data: FPW LL. Contributed reagents/materials/analysis tools: WJ JL. Wrote the paper: FPW LL WJ.
2013 16 5 2013 8 5 e643734 2 2013 12 4 2013 © 2013 Wang et al2013Wang et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The migration of hepatic stellate cells (HSCs) is essential to the hepatic fibrotic response, and recently High-mobility group box 1 (HMGB1) has been shown up-regulated during liver fibrosis. Nevertheless, whether HMGB1 can modulate the proliferation and migration of HSCs is poorly understood, as well as the involved intracellular signaling. In this study, we examined the effect of HMGB1 on proliferation, migration, pro-fibrotic function of HSCs and investigated whether toll-like family of receptor 4 (TLR4) dependent signal pathway is involved in the intracellular signaling regulation.
Methodology/Principal Findings
Modified transwell chamber system to mimic the space of Disse was used to evaluate the migration of human primary HSCs, and the protein expressions of related signal factors were evaluated by western blot. Cell proliferation was analyzed by MTT assay, the pro-fibrotic functions of HSCs by qRT-PCR and ELISA respectively. Recombinant human HMGB1 could significantly promote migration of HSCs under both haptotactic and chemotactic stimulation, especially the latter. Human TLR4 neutralizing antibody could markedly inhibit HMGB1-induced migration of HSCs. HMGB1 could enhance the phosphorylation of JNK and PI3K/Akt, and TLR4 neutralizing antibody inhibited HMGB1-enhanced phosphorylation of JNK and PI3K/Akt and activation of NF-κB. JNK inhibitor (SP600125) and PI3K inhibitor (LY 294002) significantly inhibited HMGB1-induced proliferation and migration of HSCs, and also reduced HMGB1-enhanced related collagen expressions and pro-fibrotic cytokines production.
Conclusions/Significance
HMGB1 could significantly enhance migration of HSCs in vitro, and TLR4-dependent JNK and PI3K/Akt signal pathways are involved in the HMGB1-induced proliferation, migration and pro-fibrotic effects of HSCs, which indicates HMGB1 might be an effective target to treat liver fibrosis.
This study is supported by grants from the National Nature Science Foundation of China (No. 30300151 &81070341, http://www.nsfc.gov.cn) and Shanghai Nature Science Foundation (No. 09ZR1406000, http://www.stcsm.gov.cn). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Currently, liver fibrosis caused by chronic liver diseases affects millions of people worldwide. Liver fibrosis, which is characterized by excessive deposition of extracellular matrix (ECM), is the hallmark feature associated with the failure of liver function, irrespective of different aetiological onsets [1], [2]. Therefore, a better understanding of the reversible steps in the fibrotic response may lead to the identification of new therapeutic targets.
Hepatic stellate cells (HSCs), which are located in the space of Disse between hepatocytes and sinusoidal endothelium, play a central role in the progression of liver fibrosis. Quiescent HSCs are mainly involved in Vitamin A metabolism, but they may proliferate, produce ECM and even migrate following activation [3]. It is increasingly recognized that HSC migration is essential for fibrosis owing to the observation that during cirrhosis HSCs migrate to and accumulate in fibrotic areas far from their usual location [1], [2], [4]. The motility of HSCs can be influenced by changes in their microenvironment, including extracellular matrix and growth factors [4]. In our previous research, we found transforming growth factor-β1 (TGF-β1) induced the migration and cytoskeletal remodeling of rat HSCs following RhoA activation, and the level of RhoA activation determined the motility of the HSCs [5].
High-mobility group box 1 (HMGB1) protein, originally described as a nuclear nonhistone protein with DNA-binding domains, has been implicated as an important endogenous danger signaling molecule and a potent pro-inflammatory cytokine [6]–[8]. HMGB1 can act as a chemoattractant for fibroblasts, endothelial cells and smooth muscle cells, which suggests that HMGB1 can directly stimulate fibroblast proliferation and participate in fibrogenesis [9], [10]. Recently, HMGB1 has been shown up-regulated during liver fibrosis and can promote the proliferation of HSCs [11]. However, specific extracellular and intracellular signals that regulate the proliferation and migration of HSCs are poorly understood.
Several membrane receptors are implicated in HMGB1 signaling, including the receptor for advanced glycation end-products (RAGE) and members of the toll-like family of receptors (TLRs) [12]. RAGE expression in fibrotic liver is restricted to HSCs and also is up-regulated during cellular activation and transition to myofibroblasts [13]. Silencing RAGE expression by specific siRNA can effectively suppress nuclear factor-kappaB (NF-κB) activity, HSCs activation and ECM deposition in the fibrotic liver [14]. Despite the expression of RAGE is up-regulated in activated HSCs, RAGE stimulation by advanced glycation end products (AGE) does not alter their fibrogenic activation [15]. Therefore, RAGE may not contribute directly to hepatic fibrogenesis. On the other hand, the the activation of HSCs with high expressions of TLR4 is closely associated with the progression of liver fibrosis [16]. Hepatic injury is associated with a barrier deficiency and increased hepatic exposure to bacterial products, and the functional TLR4, not TLR2, is required for hepatic fibrogenesis [17]. TLR4-mutant mice have less liver inflammation and fibrosis than TLR4-wild-type mice following bile duct ligation (BDL) and chronic treatment of carbon tetrachloride (CCl4), or thioacetamide [15]. Recently, the release of HMGB1 induced by liver ischemia has been reported to be involved in TLR4-dependent reactive oxygen species production and calcium-mediated signaling [18], and TLR-4 is also involved in HMGB1-induced vascular smooth muscle cells migration [19].So whether the interaction of HMGB1 with TLR4 can play a critical role in hepatic fibrosis and the related mechanism still need further investigation.
The ligation of HMGB1 to TLR4 results in the activation of diverse intracellular signaling pathways including Jun N-terminal kinase (JNK), phosphoinositide 3-kinase (PI3K) and its downstream serine/threonine kinase (Akt) [20], [21], whose activation is believed to play a major role in regulating the activation, proliferation and migration of HSCs [5], [22], [23]. And PDGF-mediated proliferation and migration of cultured HSCs are associated with the inhibition of Akt phosphorylation [24]. Activated Akt can phosphorylate a number of proteins including glycogen synthase kinase-3β (GSK-3β), 6-phosphofructo-2-kinase, and inhibitor kappa B (IκB) [25]. The phosphorylation of IκB frees NF-κB and allows it to translocate to the nucleus to bind and subsequently activate target genes [26]. Activation of the transcription factor NF-κB has been demonstrated in activated HSCs [27] and many drugs ameliorate liver fibrosis progression and influence fibrotic functions of HSCs through NF-κB signaling [28]. Based on these findings, the purpose of this study is to investigate whether HMGB1 can induce proliferation and migration of HSCs and whether TLR4-dependent signal pathway is involved in the mechanism.
Here, our results suggest that HMGB1 can significantly stimulate migration of HSCs in vitro, and TLR4-dependent JNK and PI3K/Akt signal pathways are involved in the HMGB1-induced proliferation, migration and pro-fibrotic effects of HSCs. To our knowledge, this is the first report on HMGB1- associated HSCs migration. These data further indicates a significant pro-fibrotic function of HMGB1 and its possibility of being an effective target to treat liver fibrosis.
Materials and Methods
Ethics Statement
The study protocol was approved by the Research Ethics Committee of Zhongshan Hospital (No. 2010-87) and written informed consent was obtained from each subject.
Regents
Recombinant human HMGB1 was purchased from R&D systems (Minneapolis, MN, USA). Human TLR4 neutralizing antibody was obtained from Invivogen (San Diego, CA, USA). JNK inhibitor (SP600125) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and ConA (10 µg/mL) and PI3K inhibitor (LY 294002) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-JNK, anti-phospho-JNK, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, anti-NF-κB, anti- IκB, anti-phospho-IκB and anti-GAPDH antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).TransAM kit was purchased from Active Motif (Carlsbad, CA, USA) and the NE-PER nuclear and cytoplasmic extraction kit was from Pierce (Rockford, IL). The Annexin V-FITC Apoptosis Detection Kit was obtained from eBioscience (San Diego, CA, USA).
Preparation of human primary hepatic stellate cells
Human primary HSCs were obtained from liver specimens of patients with hepatic hemangioma who had undergone surgical resections. HSCs were isolated using methods previously described in detail [28]. They were cultured at a concentration of 1×105 cells per well in high glucose Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) containing 20% FCS for 1–2 (quiescent), 3–5 (intermediate), or 7–10 (activated) days as described elsewhere. Cell viability was greater than 90% as assessed by trypan blue exclusion. The purity of the HSCs ranged from 90% to 95% as determined by glial fibrillary acidic protein (GFAP) staining and the typical microscopic appearance of the lipid droplets. On days 1–2, the HSCs were quiescent, round, had abundant lipid droplets, and lacked α-smooth muscle actin (α-SMA) expression. At day 7, the cells had become activated and expressed α-SMA. Cells from days 3–5, which had an intermediate appearance, were chosen for in vitro analyses in this study.
Cell viability assay
The cytotoxicity of HMGB1 toward HSCs was evaluated using a cell viability assay. In brief, after incubation of HSCs with HMGB1 (1–1000 ng/ml), the cells were exposed to 0.4% trypan blue solution for 5 minutes and viewed under a light microscope. Cell viability was defined as the ratio of unstained cells to the total number of cells.
Cell migration assay
During liver fibrosis, the basement membrane– like matrix is progressively replaced by fibrillar matrix and profibrogenic growth factors, such as PDGF-BB, TGF-β1, EGF, bFGF, and VEGF, which are released by hepatocytes, inflammatory cells, and activated HSCs. In the Boyden chamber system, the upper compartment mimics the normal space of Disse microenvironment, which is mainly comprised of a basement membrane–like matrix (represented by type IV collagen or Matrigel coating of the upper side of the polycarbonate membrane), and the lower compartment mimics inflamed areas of liver microenvironment which is characterized by fibrillar matrix (represented by type I collagen or fibronectin coating of the lower side of the polycarbonate membrane). To delineate different properties of growth factors in facilitating migration of activated HSCs, experiments were performed as follow to test the migratory behavior of cells after direct stimulation in the upper chamber (mimicking HSCs direct stimulation) or in the lower chamber (mimicking chemotactic stimuli from the injured lower compartment).
Polyvinyl/pyrrolidone–free polycarbonate membranes with 8 µm pores, which separate the upper and lower wells in a transwell chamber system (Corning, NY, USA), were coated with type IV collagen on the upper side (50 µg/ml) and type I collagen on the lower side (50 µg/ml), as previously described. The bottom wells of the chamber were filled with DMEM, and 2×104 cells/well, which had been serum starved for 24 h, were added into the upper chamber. HMGB1 (1–1000 ng/ml) was added into the upper chamber as a direct haptotactic stimulant, and into the lower chamber as an indirect chemotactic stimulant, to mimic the in vivo autocrine and paracrine mechanisms of cytokines respectively. The transwell chamber was incubated at 37°C for 4 h to allow the migration of cells through the membrane into the lower chamber. The migrated cells were stained with Hema3 according to the manufacturer's protocol (Biochemical Sciences Inc., NJ, USA) and counted in six random fields on a phase contrast microscope.
Western blot
HSCs were washed twice with ice-cold PBS and prepared with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate and 0.1% SDS) containing protease inhibitor mixture (Roche). The samples were separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) using SemiDry Transfer Cell (Bio-Rad, Hercules, CA, USA). The polyvinylidene difluoride membrane was blocked with 5% non-fat milk for 3 h followed by incubation with primary antibody in TBST (100 mM Tris–HCl, pH 7.5, 0.9% NaCl, 0.1% Tween 20) overnight at 4°C with gentle shaking: the specific primary antibodies against JNK, p-JNK, PI3K, p-PI3K, Akt, p-Akt, NF-κB, IκB and p-IκB. The blots were incubated with an HRP-conjugated anti-GAPDH antibody (1∶10,000) for 1 h at room temperature. The ratio of each protein to GAPDH was calculated as the relative quantification.
Inhibition experiments
First HSCs, which had been incubated with human TLR4 neutralizing antibody (10 µg/mL) for 1 h, were collected and added into the upper chamber of modified transwell chamber system, and then HMGB1 (100 ng/ml) was added into the upper chamber as a direct haptotactic stimulant or into the lower chamber as an indirect chemotactic stimulant to test whether the TLR4 is involved in HMGB1-induced HSCs migration. Second, TLR4 neutralizing antibody (10 µg/mL) was incubated with human primary HSCs for 1 h, and then HMGB1 (100 ng/ml) was added into the culture medium to determine whether the TLR4 is involved in HMGB1-induced HSCs proliferation and activation of JNK, PI3K/Akt and NF-κB. Third, JNK inhibitor (SP600125, 100 nM) and PI3K inhibitor (LY 294002, 25 mM) were incubated with human primary HSCs for 1 h, and then HMGB1 (100 ng/ml) was added into the culture medium to determine whether the JNK and PI3K/Akt signal pathways are involved in HMGB1-induced HSCs proliferation and pro-fibrotic effects. Finally, HSCs, which had been incubated with SP600125 and LY 294002 at above concentrations for 1 h, were then collected and added into the upper chamber of modified transwell chamber system and HMGB1 (100 ng/ml) was added into the upper chamber or the lower chamber to test whether the JNK and PI3K/Akt signal pathways are involved in HMGB1-induced HSCs migration.
Determination of NF-κB activity
NF-κB activity was determined using TransAM kit from Active Motif (Carlsbad, CA, USA), according to the manufacturer's instructions. Nuclear and cytosolic fractions were prepared using NE-PER nuclear and cytoplasmic extraction kit from Pierce (Rockford, IL), according to manufacturer's instructions. Briefly, nuclear extract from control and HMGB1-treated HSCs with or without TLR4 neutralizing antibody were added to 96-well plates pre-coated with the oligonucleotide containing NF-κB consensus sequence (5′-GGGACTTTCC-3′). Following incubation at room temperature for 1 h to facilitate the binding, a primary antibody, which recognizes only activated NF-κB/p65, was added to each well. The absorbance was read at 450 nm using a Lab System ELISA plate reader. This assay is specific for NF-κB/p65 activation and more sensitive than electrophoretic mobility shift assay.
HSCs proliferation assay
The HSCs, trypsinised from the cultures, were resuspended at 1×106 cells/ml and then inoculated into 96-well plates at 1000 cells per well. Cells were incubated with 20 µl methyl thiazolyl tetrazolium for 4 h. After centrifugation, 150 µl dimethyl sulfoxide was added to the precipitate and the absorbance of the enzyme was measured at 490 nm. Cell growth rates (average absorbance of each inhibited group/non-inhibited group) were then calculated. All groups of experiments were performed in triplicate.
HSCs apoptosis assay
To detect early apoptotic changes, staining with Annexin V–fluorescein isothiocyanate (FITC) was used, because of its known high affinity to phosphatidylserine. In the early phases of apoptosis, phosphatidylserine is translocated to the outer layer of the membrane (i.e. the external surface of the cell) and the cell membrane itself remains intact. In contrast to apoptosis, necrosis is accompanied by loss of cell membrane integrity and leakage of cellular constituents into the environment. To distinguish apoptosis and necrosis, propidium iodide, a common dye exclusion test, and annexin V–FITC were used in parallel to show membrane integrity after annexin V–FITC binding to cells. Stained cells were analyzed by FACSCalibur (Becton Dickinson) and FlowJo software 7.6.1 (Tristar, El Segundo, CA, USA).
Quantitative reverse transcriptase-polymerase chain reaction
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA). Following the manufacturer's instructions, reverse transcription was performed using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Beijing, China) and quantitative real-time PCR conducted with a SYBR reverse transcription-polymerase chain reaction Kit (Takara) using the following conditions: 30 seconds at 95°C, followed by a total of 40 two-temperature cycles (5 seconds at 95°C and 30 seconds at 60°C). Each assay was performed in triplicate. For analysis, the expression of target genes was normalized by the housekeeping gene GAPDH. Based on the ΔΔCt method, relative amounts of mRNA were expressed as 2−△△Ct. The primer sequences used were as follows: GAPDH sense 5′-TGTGTCCGTCGTGGATCTGA-3′; GAPDH antisense 5′-TTGCTGT TGAAGTCGCAGGAG-3′; collagen type I alpha 1 (COL1a1) sense 5′-TGCTGGCCCCAAGGGTCCTT-3′; antisense 5′-GGCTGCCAGGACTGCCAGTG-3′; collagen type III alpha 1 (COL3a1) sense 5′-CTTAGAAGCTGATGGGATC-3′; antisense 5′-TTGCCTTGCGTGTTTGT-3′; α-SMA sense 5′-AAGAGCATCCGACACTGCTGAC-3′; antisense 5′-AGCACAGCCT GAATAGCCACATAC-3′.
Detection of cytokines
The pro-fibrotic of cytokines including TGF-β1, platelet derived growth factor (PDGF)-BB, connective tissue growth factor (CTGF) and epidermal growth factor (EGF) principally produced by HSCs in the supernatant were also evaluated using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer's instructions.
Statistical analysis
Results are presented as mean ± standard error of the mean (SEM), in triplicate. Statistical analyses were performed using the GraphPad Software Version 5.01 (CA, USA). Student's t-test, one-way ANOVA, χ2 test and Pearson's rank correlation were performed as appropriate, and p values of less than 0.05 (two-tailed) were considered statistically significant.
Results
HMGB1 promoted the migration of primary human HSCs by both chemotactic and haptotactic mechanisms
To examine the effects of HMGB1 on the migration of primary human HSCs, we employed the modified Boyden Chamber system mimicing the space of Disse in vivo. To mimic both the autocrine and paracrine activities of cytokines in vivo, HMGB1 was either added to the upper transwell chamber containing the cells (haptotactic stimulation) or to the lower chamber not containing cells (chemotactic stimulation) respectively.
As shown in Figure 1A, chemotactic stimulation with 1 ng/ml HMGB1 significantly enhanced the migration of primary human HSCs, whereas a similar haptotactic effect on their migration occurred at or above 10 ng/ml HMGB1. The motility of primary HSCs was not further enhanced by either chemotactic or haptotactic stimulation with HMGB1 at concentrations higher than 100 ng/ml, suggesting that the pro-migratory effect of HMGB1 on primary HSCs peaked at 100 ng/ml. Therefore, a HMGB1 concentration of 100 ng/ml was chosen as the optimal concentration at which to perform subsequent experiments. Furthermore, at all HMGB1 concentrations, chemotactic stimulation proved to be more effective than haptotactic stimulation in the promotion of HMGB1-induced cell migration (P<0.05). Furthermore, HMGB1 did not cause any cytotoxic effects at any concentrations (Figure 1B).
10.1371/journal.pone.0064373.g001Figure 1 HMGB1 promoted the migration of primary human HSCs.
(A) HMGB1 was either added to the upper transwell chamber containing the cells (haptotactic stimulation) or to the lower chamber not containing cells (chemotactic stimulation). HMGB1 promoted the migration of primary human HSCs by both chemotactic and haptotactic mechanisms. Data are expressed as the mean ± SEM (n = 6). *P<0.05, compared to 0 ng/ml group; #P<0.05, chemotactic stimulation compared to the haptotactic stimulation in the same concentration. (B) HMGB1 did not cause any cytotoxic effects. Cell viability was detected by trypan blue staining. Data are expressed as mean ± SEM (n = 6).
HMGB1 induced the activation of JNK and PI3K/Akt through TLR4 signaling in HSCs
Firstly, we found the protein expression of TLR4 elevated after the stimulation of HMGB1 especially at the highest concentration (Figure 2A). To investigate the potential mechanisms for HMGB1 to regulate HSCs migration, we assessed the protein levels of JNK, PI3K/Akt in HSCs after the HMGB1 stimulation. We incubated the primary human HSCs with HMGB1 at different concentrations(0,10,100 ng/mL)for 24 h and detected the protein levels of JNK, PI3K, and Akt and their respective active forms(p-JNK, p-PI3K and p-Akt) by western blot. We found the proteins of p-JNK, p-PI3K and p-Akt on HSCs significantly increased in response to HMGB1 stimulation; however no change of JNK, PI3K, and Akt were detected (Figure 2A).
10.1371/journal.pone.0064373.g002Figure 2 HMGB1 induced the activation of JNK and PI3K/Akt through TLR4.
(A) Representative of significantly elevated expressions of both TLR4 and active forms of JNK, PI3K/Akt (p-JNK, p-PI3K and p-Akt) in response to HMGB1 stimulation at different concentrations. However no change of JNK, PI3K, and Akt were detected. Relative quantification of the active and total forms of JNK, PI3K, and Akt proteins are presented in the right panel. #p<0.05, compared with 0 ng/ml HMGB1; NS, not significant. (B) After pretreated with TLR4 neutralizing antibody (10 µg/mL) for 1 h, HSCs were cocultured with HMGB1 (100 ng/ml) for 24 h and then HMGB1-enhanced expression of p-JNK, p-PI3K and p-Akt were markedly decreased. Relative quantification of the active and total forms of JNK, PI3K, and Akt proteins are presented in the right panel. *p<0.05, compared with 100 ng/ml HMGB1; NS, not significant.
Secondly, to further investigate the possible involvement of JNK and PI3K/Akt signaling in HMGB1-induced migration of HSCs, we tested the expressions of JNK, p-JNK, PI3K/p-PI3K, and Akt/p-Akt by western blot, when HSCs were pretreated with TLR4 neutralizing antibody (10 µg/mL) for 1 h and then HMGB1 (100 ng/ml) was added into the culture medium for 24 h. As shown in Figure 2B, the pretreatment with TLR4 neutralizing antibody pretreatment markedly decreased HMGB1-enhanced expression of p-JNK, p-PI3K and p-Akt, which indicated HMGB1 could induce the activation of JNK and PI3K/Akt pathways through TLR4 in HSCs.
TLR4 also took part in HMGB1-induced activation of NF-κB
Increased NF-kB activity has been demonstrated in cell proliferation and NF-kB is retained in the cytoplasm in association with inhibitor protein IkBα [29], [30]. Upon phosphorylation on serine residues, IkBα is degraded allowing NF-kB to translocate to the nucleus and activate transcription of genes responsible for cell growth [26]. Employing western blot analysis, we investigated the effect of TLR-4 neutralizing antibody pretreatment on the levels of constitutively expressed NF-kB protein in HSCs stimulated with HMGB1. As shown in Figure 3A, compared to the HMGB1 stimulation, TLR-4 neutralizing antibody pretreatment resulted in a decrease in NF-kB protein level in the cytosolic as well as nuclear fraction. Notably, a decrease in NF-kB protein level was correlated with a decrease in phospho-IkBα while a concomitant increase in the cytosolic IkBα protein level.
10.1371/journal.pone.0064373.g003Figure 3 TLR4 took part in HMGB1-induced activation of NF-
κB. A:With or no pretreatment of TLR4 neutralizing antibody, HSCs were stimulated by HMGB1 for 24 h to be analyzed the protein levels of NF-kB, phospho-IkBα and IkBα by western blot. Compared to the HMGB1 stimulation, TLR-4 neutralizing antibody pretreatment resulted in a decrease in NF-kB protein level as well as in phospho-IkBα level, while a concomitant increase in the cytosolic IkBα protein level. *p<0.05, compared with 100 ng/ml HMGB1. B: Effect of HMGB1 with or without TLR4 neutralizing antibody on NF-kB/p65 activity determined by an ELISA-based assay as described in Materials and Methods. *p<0.05, compared with 100 ng/ml HMGB1.
To determine if HMGB1 with or without TLR-4 neutralizing antibody pretreatment induced changes in the levels and /or phosphorylation of NF-kB/p65, the effect of HMGB1 on DNA-binding activity of NF-kB was determined and the results are shown in Figure 3B. The NF-kB activity was enhanced by HMGB1 stimulation, whereas the blockage of TLR-4 significantly inhibited that NF-kB activity enhancement.
The pathways of TLR4-dependent JNK and PI3K/Akt were involved in HMGB1-induced the proliferation and migration of HSCs
First, to investigate whether PI3K/Akt signaling is involved in HMGB1-induced HSCs proliferation, HSCs pretreated with SP600125 or LY294002 were stimulated with HMGB1 and subsequently subjected to the MTT assay separately to examine their proliferation. The proliferation of HSCs stimulated only with HMGB1 was enhanced to about 200% compared with those without any stimualtion (P<0.05). And after pretreated with SP600125 or LY294002, the HSCs proliferation was markedly decreased compared with those stimulated only with HMGB1 (P<0.05) (Figure 4A). Second, pretreated HSCs were added to the upper chamber of modified transwell chamber system and then HMGB1 was either added to upper or the lower transwell chamber respectively exactly like the previous performance. We found the HSCs migration induced by both chemotactic and haptotactic stimulation of 100 ng/ml HMGB1 were markedly inhibited after pre-blockage of JNK or PI3K/Akt signal pathway (Figure 4C). Considering the changes of p-JNK and p-PI3K/p-Akt brought by TLR4 neutralizing antibody, we further incubated HSCs with TLR4 neutralizing antibody ahead of HMGB1 to test HSCs proliferation and migration. The results showed that pre-blockage of TLR4 significantly inhibited HSCs proliferation and migration compared with those stimulated only with HMGB1, which was consistent with the outcomes of JNK and PI3K/Akt inhibitor experiments (Figure 4A & 4C).
10.1371/journal.pone.0064373.g004Figure 4 TLR4-dependent JNK and PI3K/Akt were involved in HMGB1-induced HSCs proliferation and migration.
A: The proliferation of HSCs pretreated with SP600125 (JNK inhibitor) or LY294002 (PI3K/Akt inhibitor) or TLR4 neutralizing antibody for 1 h was analyzed after their incubations with HMGB1 for 24 h by MTT assay. # P<0.05, compared with the HMGB1group. B: The apoptosis of HSCs was analyzed by flow cytometry and Annexin V-FITC+PI- represented the HSCs apoptosis as shown in the upper part. The apoptosis percent was calculated and shown by the bar chart. C: After pretreatment with SP600125 (JNK inhibitor) or LY294002 (PI3K/Akt inhibitor) or TLR4 neutralizing antibody, the HSCs migration was significantly inhibited compared with those stimulated only with HMGB1 no matter by haptotactic or chemotactic mechanism. *P<0.05, compared with the HMGB1 group stimulated in the same way (haptotactic or chemotactic stimulation). Data in subfigures A–B are both presented as mean ± SEM (n = 6).
Based on the reports that inhibiting the activation of JNK pathway could accelebrate HSCs apoptosis[31], so we decided to investigate whether the preblockage of TLR4 or JNK or PI3K signalings could affect HSCs apoptosis except for their influence on HSCs proliferation. It turned out that HMGB1 decreased the HSCs apoptosis level slightly whereas the preblockage of TLR4, PI3K/Akt and JNK increased cell apoptosis, all of which had no significant difference (Figure 4B).
Integrated with our previous findings, these results suggest TLR4-dependent JNK and PI3K/Akt signal pathways are involved in HMGB1-induced HSCs proliferation and migration.
The pathways of TLR4-dependent JNK and PI3K/Akt were also involved the pro-fibrotic effects of HMGB1 on HSCs
To investigate whether JNK and PI3K/Akt signaling are involved in the pro-fibrotic effects of HMGB1 on HSCs, the cells which were pretreated with SP600125 or LY294002 were stimulated with HMGB1 and subsequently subjected to q RT-PCR to test gene expressions including Col I, Col III and α-SMA, and also subjected to ELISA to assess the pro-fibrotic cytokines including TGF-β1, PDGF-BB, CTGF and EGF produced by HSCs in the supernatant. The gene expression of Col I and Col III and pro-fibrotic cytokines production of HMGB1-stimulated HSCs were significantly enhanced compared with those without any stimulation, but when pretreated with SP600125 or LY294002, the pro-fibrotic effects of HSCs aggravated by HMGB1 were markedly decreased (P<0.05) (Figure 5). Similarly, whether TLR4 is involved in the pro-fibrotic effects of HMGB1 on HSCs needs further study. And the results of pretreatment with TLR4 neutralizing antibody indicated that preblockage of TLR4 obviously decreased the enhancement of pro-fibrotic effects caused by HMGB1 stimulation, no matter the Col I, Col III and α-SMA expressions or the pro-fibrotic cytokines production.
10.1371/journal.pone.0064373.g005Figure 5 TLR4-dependent JNK and PI3K/Akt were involved in HMGB1-induced HSCs pro-fibrotic effects.
A: HMGB1 enhanced the level of α-SMA, collagen I, collagen III mRNA in primary human HSCs, whereas SP600125, LY294002 and TLR4 neutralizing antibody decreased the level. α-SMA, collagen I, collagen III/GAPDH were considered as the relative mRNA level. Data are expressed as mean ± SEM (n = 6). #P<0.05, compared with the control group;*P<0.05, compared with the HMGB1-stimulated group. B: HMGB1 enhanced the level of TGF-β1 produced by HSCs, whereas SP600125, LY294002 and TLR4 neutralizing antibody decreased that enhancement markedly. C: HMGB1 enhanced the level of PDGF-BB produced by HSCs, whereas SP600125, LY294002 and TLR4 neutralizing antibody decreased that enhancement markedly D: HMGB1 enhanced the level of CTGF produced by HSCs, whereas SP600125, LY294002 and TLR4 neutralizing antibody decreased that enhancement markedly E: HMGB1 enhanced the level of EGF produced by HSCs, whereas SP600125, LY294002 and TLR4 neutralizing antibody decreased that enhancement markedly Data in subfigures B–E are all presented as mean ± SEM (n = 6). # P<0.05, compared with the control group at the same time; * P<0.05, compared with the HMGB1 group at the same time.
Discussion
Liver fibrosis represents a transitional and reversible stage between chronic hepatitis and cirrhosis [1]. During liver fibrogenesis, the normal basement membrane-like matrix, which consists largely of type IV and type VI collagens, can be replaced by fibrillar matrix such as collagens type I and type III. Also, cytokines and reactive oxygen species released from injured cells can directly or indirectly act on HSCs [1]–[3]. The key event during liver fibrosis is that HSCs become activated and transform into myofibroblast–like cells, enabling them to proliferate aggressively, produce large amounts of ECM, migrate in a similar manner to tumor cells, and finally accumulate in injured sites to regulate the fibrotic process [4], [5], [22].
Cell migration usually begins in response to extracellular stimuli such as cytokines, ECM and surrounding cells and may activate transmembrane receptors to promote intracellular signal transduction [4], [8]. During liver fibrosis, the migratory features of activated HSCs are responsible for their accumulation in inflammatory regions to interact with adjacent parenchyma cells and non-parenchyma cells. Our findings confirm that HMGB1 can promote the migration of primary human HSCs through both chemotactic and haptotactic mechanisms, as well as the proliferation of HSCs. Furthermore, chemotactic stimulation is proved to be more effective than haptotactic stimulation in inducing the migration of HSCs, suggesting that HMGB1 exerts its pro-migratory effect through paracrine rather than autocrine mechanisms. HMGB1 can be released from both active secretion of various cells, including activated monocytes/macrophages, neutrophils, and endothelial cells, and passive release of necrotic cells [30], [32]–[34]. Therefore, the migration of HSCs may be regulated primarily by intercellular chemokine activity, and the influence of cell-cell interactions on their migration mechanisms should also be addressed in future researches.
TLR4, as a novel receptor for HMGB1, is capable of evoking the immune and inflammatory response through its intra-cellular signal pathways. TLR4 enhances TGF-β signaling and hepatic fibrosis, and LPS-mediated signaling through TLR4 has been identified as key fibrogenic signal in HSCs [35], [36]. PI3K/Akt, which has been shown as activated downstream of TLR4 [37], [38], is critically needed for the regulation of cells growth, migration, and proliferation [22]–[24]. In vivo, inhibition of PI3K signaling inhibits extracellular matrix deposition and reduces expression of profibrogenic factors including TGF-β, tissue inhibitor of metalloproteinase 1 (TIMP-1), and CTGF [23]. In vitro, inhibition of PI3K signaling in HSCs not only decreases the proliferation, collagen expression and several profibrogenic gene expressions of HSCs, but also promotes cell death [39]. However in this experiment, inhibiting PI3K did not increase HSCs apoptosis level, nor did JNK inhibitor. It can be explained by the different HSCs status (intermediate) partly, and why the ability of JNK inhibitor to enhance the HSCs sensitization to induced apoptosis[31] did't display probably is that HMGB1 actually didn't induce apoptosis. Till now,HMGB1 has been found to modulate functions of many cell types, such as human airway epithelial cells, leukemia cells, lung adenocarcinoma cells, through PI3K/Akt signal pathway [40]–[42]. On the other hand, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-κB and JNK and up-regulate chemokines and adhesion molecules [36]. As to other cell line like Kuffer cells, HMGB1 can induce proinflammatory cytokines production after sever burn injury, largely dependent on TLRs-dependent MAPKs/NF-kB signal pathway[43]. In our previous research, JNK signaling had been shown activated following RhoA activation, which determined the motility of the HSCs [5]. Moreover, activated Akt can phosphorylate IκB, which frees NF-κB to allow it to translocate to the nucleus to bind and subsequently activate target genes [44], and NF-κB activity is essential for PI3K/Akt-induced oncogenic transformation [45]. Thus, it will be interesting to determine whether the signal pathways of JNK and PI3K/Akt are involved in HMGB1-induced HSCs migration via TLR4.
First, we found the HSCs migration in response to HMGB1 stimulation was markedly inhibited by pretreatment with TLR4 neutralizing antibody, which indicated TLR4 was involved in HMGB1-induced HSCs migration. Second, we demonstrated that HMGB1-enhanced phosphorylate expressions of JNK, PI3K/Akt and activity of NF-κB in HSCs were significantly suppressed by TLR4 neutralizing antibody, which indicated HMGB1 could induce the activation of JNK and PI3K/Ak through TLR4 in HSCs. Third, by using JNK inhibitor (SP600125) and PI3K inhibitor (LY294002) to block the signal pathway of JNK and PI3K/Akt, we demonstrated that blockage of JNK and PI3K reduced HMGB1-induced activation of NF-κB in HSCs. Fourth, by using modified Boyden Chamber system, HMGB1- induced migration of HSCs were markedly inhibited after pre-blockage of JNK and PI3K/Akt signal pathways. Integrating all these findings, we confirm that TLR4-dependent signal pathways of JNK and PI3K/Akt are involved in HMGB1-induced migration of HSCs. Furthermore, after the pre-treatment with specific inhibitors of JNK and PI3K/Akt, HMGB1-enhanced proliferation and related pro-fibrotic cytokines production of HSCs were markedly inhibited, which indicated the signal pathways of JNK and PI3K/Akt were involved in the pro-fibrotic effects of HMGB1 on HSCs.
Nevertheless, the suppression of HMGB1-induced cells proliferation, migration and pro-fibrotic effects induced by blocking TLR4, JNK and PI3K/Akt signal pathways were often incomplete, indicating other signal pathways might be involved in the regulatory mechanisms. First, TLR4 inhibitor even at much higher concentration could not completely abolish HSCs migration mediated by HMGB1, which could be explained by that other membrane receptors especially RAGE could also participate in this regulatory process [46]. As mentioned previously, RAGE expression in fibrotic livers is restricted to HSCs and its expression is up- regulated during cellular activation and transition to myofibroblasts [13], [14]. Second, ligation of HMGB1 to TLR4 can also activate other intracellular signal pathways besides JNK and PI3K/Akt signal pathway. For instance, MAPK / ERK signaling is involved in the HSCs proliferation and TGF-β1 can mediate the migration of HSCs possibly by Smad2/3 phosphorylation and MAPK pathway [47], [48]. Novo et al. showed that mitochondrial-dependent ROS-mediated activation of ERK and JNK participated in hypoxia-induced migration of HSCs [49]. Our previous study also showed that following RhoA activation TFG-β1 induced the activation of Smad and p38, which determined the motility of the HSCs [5]. Therefore, it is necessary to further explore the intracellular signaling mechanisms underlying the chemotactic action of HMGB1 in HSCs.
Taken together, our results have demonstrated that HMGB1 promotes the proliferation and migration of HSCs via TLR4-dependent signal pathways of JNK and PI3K/Akt, which indicates a significant functional role of HMGB1 in the development of liver fibrosis and HMGB1 may be an effective target to treat liver fibrosis. But whether HMGB1 interacts with other TLRs to modulate the functions of HSCs, whether RAGE-mediated signaling also participates in the modulation of HSCs and whether other intracellular signal pathways are involved in HMGB1-induced proliferation and migration of HSCs, require further investigation.
The authors are grateful for all the patients enrolled in this study for their kindly understanding and supporting.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23696846PONE-D-12-3705210.1371/journal.pone.0063680Research ArticleBiologyDevelopmental BiologyMorphogenesisSexual DifferentiationModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCellular TypesGerm CellsSignal TransductionSignaling CascadesApoptotic Signaling CascadeSignaling in Cellular ProcessesApoptotic SignalingCell DeathCell GrowthProteomicsProtein InteractionsSUMOylation of Mouse p53b by SUMO-1 Promotes Its Pro-Apoptotic Function in Ovarian Granulosa Cells SUMOylation of Mouse p53b in Granulosa CellLiu Xiao-Ming Yang Fei-Fei Yuan Yi-Feng Zhai Rui Huo Li-Jun
*
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, People’s Republic of China
Sun Qing-Yuan Editor
Institute of Zoology, Chinese Academy of Sciences, China
* E-mail: [email protected] Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: XML LJH. Performed the experiments: XML FFY YFY RZ. Analyzed the data: XML LJH. Wrote the paper: XML LJH.
2013 16 5 2013 8 5 e6368021 11 2012 5 4 2013 © 2013 Liu et al2013Liu et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Follicular atresia is a process of spontaneous degradation of follicles, hindering growth and development in the mammalian ovary. Previous studies showed that follicular atresia was caused by apoptosis of granulosa cells, for which a number of apoptosis-related genes have already been identified. The roles of p53 in apoptosis of mouse granulosa cells and its post-translational modification are still unclear. The main objective of this study was to explore the roles of p53 in mouse granulosa cells. We found that mouse p53b, but not p53a, could be SUMOylated by SUMO-1 at lysine 375, which was essential for the protein stability of p53b in a dose-dependent manner. Immunofluorescent staining showed that wild p53b was located in the nucleus of granulosa cells, while its mutation of SUMOylated site (K375R) was localized in both nucleus and cytoplasm, implying that SUMOylation was necessary for the nuclear localization of p53b in granulosa cells. Overexpression of wild-type p53b, but not the mutation of SUMOylation site (K375R), significantly induced the expression of apoptosis-related gene, Bax, and increased the level of apoptosis in granulosa cells. This suggested that SUMO-1 modification of p53b was essential for inducing apoptosis in granulosa cells. Our results provide strong evidences that modification of p53b by SUMO-1 at lysine 375 was necessary for its activity to induce apoptosis in mouse granulosa cells, and it was involved in the regulation of p53b protein stability and nuclear localization. This implies that modification of p53b by SUMO-1 might regulate follicular atresia by inducing the apoptosis of ovarian granulosa cells in mice.
This study was supported by National Natural Science Foundation of China (Grant No.31071273), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20100146110011), and the Fok Ying-Tong Education Foundation, China (Grant No. 121029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
During the process of follicular atresia, follicles spontaneously degrade in the mammalian ovary, hindering growth and development. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells [1]–[6]. Although atresia can occur at any time during follicular development, the majority of follicles become atretic during the early antral stage of development [7]. The transition from preantral to antral follicles occurs after the granulosa cells are exposed to gonadotropin. The gonadotropin initiates differentiation of the granulosa cells making them susceptible to apoptosis.
Apoptosis in granulosa cells is characterized by nucleosome DNA fragmentation, cell shrinkage, membrane blistering, and the formation of apoptotic bodies [8]. Many apoptosis-related factors have been implicated in follicular atresia, including death ligands and receptors, intracellular pro- and anti-apoptotic molecules, cytokines, growth factors, and a number of apoptosis-related genes, including the p53 gene [9]–[11].
The p53 protein is an antiproliferative transcription factor that increases the rate of transcription of various genes involved in mitosis and apoptosis [9], [12]–[15]. It plays a critical role in cell cycle regulation (G1/S transition), DNA repair, and induction of apoptosis [14], [16]–[18]. In granulosa cells, p53 content is correlated with contents of Fas and FasL, death ligands that are involved in the induction of apoptosis and are regulated by gonadotropins [19]. When gonadotropin induced, p53 becomes markedly elevated, suggesting that induction of atresia is p53-dependent [20]. Furthermore, overexpression of p53 resulted in increased Fas content and apoptosis [16]. However, mouse p53 gene contains only a promoter, which differs from the selective promoter of the human p53 gene; the alternatively spliced RNA species of tumor-suppressor gene p53 (which contains an additional 96 bases derived from intron 10) is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal cells, carcinoma cells, and mouse liver and testis cells [21]. Precisely because of this alternative splicing, there are two types of p53 protein: p53a encodes 390 amino acids and p53b encodes 381 amino acids. The main difference between p53a and p53b is the C-terminal sequence. However, the biological properties of different types of the p53 protein in mice have not yet been determined.
The half-life of the p53 protein in normal cells is increased when the cells are exposed to various kinds of external stimuli. These stimuli influences the post-translational events and the stability of the protein, including acetylation, methylation, phosphorylation, ubiquitination, neddylation, and SUMOylation [22]–[24]. The small ubiquitin-related modifier-1 (SUMO-1) is an ubiquitin-related protein that was discovered in the yeast Saccharomyces cerevisiae in 1995 [25], [26]; SUMO-1 is involved in many cellular processes, including cell proliferation, differentiation, and apoptosis [25], [27]. Studies have reported that human p53 can be modified by SUMO-1 and the SUMOylation site is lysine386 [28]–[30]. Conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53 [28], [30]. However, SUMOylation has no effect on mutant p53 transcriptional activity [29], [31]. In addition to comparing wild-type and SUMOylation-deficient p53 for transactivation, studies analyzed potential differences in localization and growth inhibition or apoptosis. Mutating the p53 SUMO-acceptor site lysine386 to arginine had no obvious effect on p53 localization [29], but one study generated p53-SUMO-1 fusion protein as a model for the effect of SUMO modification on the localization and function of p53, showing that p53-SUMO-1 fusion protein significantly redistributed from the nucleus to the cytoplasm when the SUMOylation site lysine386 was destroyed [32]. Studies showed the SUMOylation of p53 enhanced the apoptosis in Saos-2 cells [33]. In addition, SUMO modification of Drosophila p53 is required for its pro-apoptotic activity [34].
While it is not very clear whether p53 is involved in regulating follicular atresia and granulosa cells apoptosis, and its regulatory mechanism is also unclear. Furthermore, there are two types of p53 protein (p53a and p53b) in mice, and the roles of these p53 forms in mouse granulosa cells and whether they can be SUMOylated have not been reported. In this study, the main objective is to explore the roles of p53 in mouse granulosa cells and the effects of SUMOylation.
Materials and Methods
Experimental Animals
We obtained immature 21 to 23 d-old Kunming White female mice from the Centre of Laboratory Animals of Hubei Province (Wuhan, PR China). All animal treatment procedures were approved by the Ethical Committee of the Hubei Research Center of Experimental Animals (Approval ID: SCXK (Hubei) 2008-0005). Mice were housed under controlled temperature (24°C) and lighting (12 h light/12 h darkness) with food and water ad libitum. Follicle development was primed by injection of each mouse with 10 IU pregnant mare serum gonadotropin (PMSG; SanSheng, Ningbo), and mice were killed by cervical dislocation 44–48 h later.
Plasmid Construction
A 1310-bp mouse p53a and a 1213-bp mouse p53b cDNA sequence were amplified using polymerase chain reaction (PCR) from mouse ovary cDNA using the following primers: p53a- Forward 5′-CGGGATCCGGCAGGGTGTCACGCTTCT-3′; p53a- Reverse 5′-CGGAATTCCG AGGGACCGGGAGGATTGT-3′; p53b- Forward 5′-CGGGATCCGGCAGGGTGTCACGCTTCT-3′; and p53b- Reverse 5′- GCGAATTCGGAGGGATGAAGTGATGGGA-3′. Both included BamHI and EcoRI restriction sites. We subcloned into pCMV-N-HA to generate a HA-tagged p53a and p53b cDNA.
To generate the p53b lysine 375 arginine mutant, two pairs of primers were used for PCR: Pair 1- Forward 5′- CGGGATCCGGCAGGGTGTCACGCTTCT-3′; Pair 1- Reverse 5′- GGGCTTTCCTCCCTGATCAAGGCTTGG -3′; Pair 2- Forward 5′- CGGGATCCGGCAGGGTGTCACGCTTCTCCGAAGACTGGATT-3′; and Pair 2- Reverse 5′-GCGAATTCGGAGGGATGAAGTGATGGGAGCTAGCAGTTTGGGCTTTCCTCCCTG -3′. Amino acid of p53b was mutated from lysine to arginine using the p53b cDNA sequence as the template for the first reaction and the product as the template for the second reaction. The results were confirmed by sequencing.
HA-tagged sumo-1 or ubc9, or Flag-tagged sumo-1 was preserved in our laboratory.
In vitro Culture of Granulosa Cells and DNA Transfection
Granulosa cells from pre-ovulatory follicles (pre-GCs) were obtained from ovaries of 21 to 23 d-old Kunming White female mice injected with 10 IU PMSG (SanSheng, Ningbo) 44–48 h. Granulosa cells were cultured in 6-well culture plates in Dulbecco’s Modified Eagle’s Medium/Nutrient F-12 (DMEM/F12; Gibco) medium with 10% fetal bovine serum (FBS; Invitrogen), 60 mg/mL penicillin, and 50 mg/mL streptomycin. All cultures were maintained in DMEM/F12 medium at 37°C in a humidified atmosphere of 5% CO2. After being cultured 24 h, granulosa cells were washed by phosphate-buffered saline (PBS), and cultured for 24 h in fresh serum-free DMEM/F12 medium before DNA transfections.
For DNA transfection or co-transfection, granulosa cells were plated and then transfected with 4 µg of the desired plasmids for 24 h with Lipofectamine LTX (Invitrogen) according to manufacturer’s instructions. Desired plasmids and LTX were diluted in Opti-MEM (Gibco) medium and incubated for 5 min. This solution was then mixed with granulosa cells at a proportion of 1∶1.5 for 30 min; 24 h after transfection, granulosa cells were collected for protein extraction or apoptosis analysis.
To study the effect of SUMO-1 mediated modification of p53b on its protein level in granulosa cells, different dosages of pCMV-Flag-sumo-1 plasmids (2 µg, 3 µg, 4 µg, and 6 µg) were co- transfected with a fixed proportion of pCMV-HA-p53b or pCMV-HA-p53bK375R (2 µg) plasmids into granulosa cell.
Protein Extraction, Immunoprecipitation and Western Blot Analysis
Subconfluent cells seeded on 6-well culture plates were transfected with the expression vectors indicated. At 24 h after transfection, cells were washed twice with ice-cold PBS and harvested in 80 µL of ice-cold RIPA buffer (Santa Cruz), containing 10 µM phenylmethylsulfonyl fluoride (PMSF; DingGuo, Beijing) and 10 mg/mL protease inhibitors cocktail (Santa Cruz). Protein lysis was performed on ice for 20 min. Then, the lysates were centrifuged at 12000 rpm for 5 min, and the supernatant was collected and stored at −80°C.
The concentration of total protein was determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, USA), and 20 µg of total protein was subjected to gel electrophoresis. The cell lysates mixed with 2× SDS gel-loading buffer were loaded on 4% stacking gel and 10% separating gel, and were then transferred to 0.2 µm polyvinylidene fluoride (PVDF) membrane (Milli-pore, Bedford, MA). After blocking in TBST [10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% Tween 20] containing 5% skimmed milk (Sigma-Aldrich), membranes were incubated with the corresponding primary antibody diluted in blocking buffer overnight at 4°C. Polyclonal rabbit anti-p53 IgG (1∶500 dilution; Boster, Wuhan), monoclonal mouse anti-β-actin IgG (1∶500 dilution; Santa Cruz), and monoclonal mouse anti-HA IgG (1∶750 dilution; CWBIO, Beijing) were used as the primary antibody, respectively. After incubation with the primary antibody, the membrane was washed three times in TBST, and incubated with HRP-conjugated secondary antibodies diluted in TBST for 1 h at room temperature, then washed three times in TBST. Chemiluminescent detection was performed using ECL Western blot detection reagents (Amersham Biosciences, Piscataway, NJ). To analyze the SUMO-1 modification of p53 protein, membranes were probed with the HA antibody or p53 antibody, respectively, while the membrane was probed with β-actin antibody for normalization. The band intensities were measured with Gel-Pro analyzer 4.0 (Media Cybernetics, USA).
Immunoprecipitation (IP) was conducted to detect if p53 could be modified by SUMO-1 in granulosa cells in vivo. Briefly, after transfection with pCMV-Flag-sumo-1 plasmid or pCMV-Flag plasmid (as a negative control), granulosa cells (106−107) were harvested in 1 mL lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 10 µM PMSF (DingGuo, Beijing) and 10 mg/mL protease inhibitors cocktail (Santa Cruz). The extract was centrifuged at 12,000 g for 15 min at 4°C, and the supernatant were collected. 800 ul of the supernatant were incubated with ANTI-FLAG M2 Affinity beads (Sigma-Aldrich) overnight at 4°C with continual shaking. After recovery by centrifugation at 8,000 g for 30 s, the beads were washed four times with ice cold TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4). Then Flag-fusion proteins were eluted by 100 µL 3× FLAG peptide (Sigma-Aldrich) at 4°C with gentle shaking for 30 min. After elution, the beads were centrifuged at 8,000 g for 30 s and the supernatant was collected. For Western blot, the supernatant was mixed with equal volume of 2× SDS loading buffer and heated for 5 min at 100°C before being loaded on a SDS-PAGE gel. Polyclonal rabbit anti-p53 IgG was used as the primary antibody.
In order to confirm which type of p53 (p53a or p53b) could be modified by SUMO-1, granulosa cells were co-transfected with pCMV-Flag-sumo-1 plasmid and pCMV-HA-p53a plasmid or pCMV-HA-p53b plasmid. After transfection, Flag tagged SUMO-1 were pulled down by Flag antibody and then HA antibody were used to detect the expression of p53a or p53b by Western blot.
Immunofluorescence Cytochemistry
In order to detect the subcellular localization pattern of p53b and p53bK375R, pre-GCs were plated on coverslips and transfected with pCMV-N-HA-p53b or pCMV-N-HA-p53bK375R vector respectively. After 24 h transfection, cells were washed twice with PBS and were fixed in 4% paraformaldehyde (Santa Cruz) for 15 min. Next, the cells were washed in PBS three times and then permeabilized in PBS containing 0.5% Triton-100 (Santa Cruz) at room temperature for 20 min. After blocking in PBS containing 5% bovine serum albumin (BSA) (Santa Cruz), cells were washed in PBS three times. After that, cells were incubated with anti-HA primary antibody (1∶100 dilution, CWBIO, Beijing) at 37°C for 2 h, followed by FITC-conjugated secondary antibody (1∶100 dilution, Boster, Wuhan) in the dark. The nucleus of cells was stained in 10 µg/mL propidium iodide (PI; Santa Cruz) for 5 min. Slides were mounted in DABCO (Sigma-Aldrich,) and viewed under a Zeiss- LSM 510 Meta confocal microscope.
For co-localization study of p53b or p53bK375R with SUMO-1 in granulosa cells, pre-GCs were co-transfected with pCMV-Flag-sumo-1 plasmid and pCMV-HA-p53b plasmid or pCMV-HA-p53bK375R plasmid. After 24 h transfection, SUMO-1 and p53b or p53bK375R were detected by polyclonal rabbit anti-SUMO-1 antibody (1∶200 dilution; Santa Cruz) or monoclonal mouse anti-HA antibody (1∶100 dilution, CWBIO, Beijing), respectively. Cy3-conjugated anti-rabbit IgG antibody (1∶100 dilution, Boster, Wuhan) or FITC-conjugated anti-mouse IgG antibody (1∶100 dilution, Boster, Wuhan) were used as the secondary antibody, respectively. The nucleus was stained in 10 µg/mL 4′,6′,-diamidino-2-phenylindole (DAPI; Santa Cruz).
Cell Apoptosis Assay
Pre-GCs were transfected with pCMV-N-HA-p53b, pCMV-N-HA-p53bK375R, or negative control pCMV-N-HA for 24 h. Cells were washed twice in PBS, then trypsinized and collected for apoptosis assay. Apoptosis was performed by using the AnnexinV kit (AntGene, Wuhan) according to manufacturer’s instructions. Cells were incubated in AnnexinV–FITC and PI solution at room temperature in the dark for 15 min, then 300 µL of 1 × binding buffer was added to each sample. Flow cytometric analysis was conducted using a BD FACSCalibur (Becton, Dickinson and Company, USA; Ex, 488 nm and Em, 530 nm).
Real-time RT-PCR Analysis
To determine genes regulated by SUMOylation of p53b, at 24 h after transfection, total RNA was prepared using the RNAiso Plus (TaKaRa, Dalian), and in vitro transcription was carried out using PrimeScript RT reagent Kit With gDNA Eraser (TaKaRa, Dalian). Real-time PCR was then conducted to quantify the steady-state mRNA levels of the tested genes using SsoFast™ EvaGreen® Supermix (Bio-Rad, USA) on the Bio-Rad CFX96 Real-time PCR System. The threshold cycle (Ct) was used to determine the relative expression level of each gene by normalizing to the Ct of β-actin mRNA. The method of 2–ddCt was used to calculate the relative fold change of each gene. To ensure that only target-gene sequence-specific, non-genomic products were amplified by real-time PCR, careful design and validation of each primer pair, as well as cautious manipulation of RNA, were undertaken to quantify the steady-state mRNA levels of Bax and housekeeping gene β-actin (internal control). The primers used were Bax- forward: 5-CCAGGATGCGTCCACCAA-3; Bax- reverse: 5-CAAAGTAGAAGAGGGCAACCAC-3; β-actin- forward: 5- CCCATCTACGAGGGCTAT-3; and β-actin- reverse: 5-TGTCACGCACGATTTCC-3. Calculation of the relative fold change of Bax was done using the method of 2−ΔΔCt. In each experiment, levels of Bax mRNA were presented as relative changes to a specific group (control), in which its expression level was set at 1.
Data Analysis and Statistics
All experiments were performed independently at least three times, and data are presented as mean ± SD. Differences between groups were analyzed by one-way ANOVA followed by Tukey’s Honesty Significant Difference (HSD) test using SPSS (Version 17.0; SPSS, Chicago, IL, USA). P<0.05 was considered significantly different, and P<0.01 was extremely significantly different.
Results
SUMOylation Increased the Expression Level of Mouse p53 Protein
At 24 h after transfection, total proteins were extracted from the cells and analyzed by Western blot using the HA-specific antibody or p53-specific antibody. After transfection with p53a or p53b plasmid, the expression of HA-p53 fusion protein or increased level of p53 protein could be observed (Fig. 1, lanes 3 and 4). Interestingly, after transfection with HA-sumo-1 or HA-ubc9 plasmid, a shifting-up band detected by using anti-HA antibody was also visible (Fig. 1, lanes 5 and 6), which was consistent with a form of protein that was covalently modified by HA-SUMO-1 or HA-UBC9. More importantly, the expression of p53 protein was significantly increased by transfection with sumo-1 or ubc9 plasmid (Fig. 1, lanes 5 and 6). Thus we predicted that the shifting band detected by HA antibody represented HA-SUMO-1-p53 fusion protein.
10.1371/journal.pone.0063680.g001Figure 1 SUMOylation increased the expression level of mouse p53 protein.
Pre-GCs were seeded in 6-well culture plates and transfected with 2 µg of HA-p53a, HA-p53b, HA-sumo-1, HA-ubc9, or empty HA expression plasmids. 20 µg total extracts were prepared and resolved by SDS–PAGE and analyzed by Western blot using anti-HA (the top group) or anti-p53 (the middle group) specific antibody. Positions of molecular weight markers, free HA-p53 or putative p53/HA–SUMO-1 and p53/HA-UBC9 conjugates are indicated. β-actin is a loading control.
SUMO-1 Conjugation to p53b in vivo Requires Lysine 375
Based on the results above, we speculated that the mouse p53 protein could also be modified by SUMO-1. Immunoprecipitation (IP) study was designed to confirm whether mouse p53 could be SUMO-1 modified or not in granulosa cells. Pre-GCs were transfected with pCMV-Flag-sumo-1 plasmid or pCMV-Flag plasmid (as a negative control) for 24 h. Flag-tagged protein were pulled down by anti-Flag affinity beads and then analyzed by Western blot using anti-p53 antibody. A visible band of p53 was observed in the sample of pCMV-Flag-sumo-1 transfected granulosa cells, but not in the control sample (Fig. 2A). This study clearly showed that mouse p53 protein could be SUMO-1 modified in granulosa cells in vivo. However, there are two types of p53 in mouse, and which type of p53 that could be modified was unknown. Previous studies showed lys386 of human p53 was required for SUMO-1 modification in Saos-2 cells [29], [35], [36]. To identify which type of mouse p53 could be modified by SUMO-1 in mouse, we aligned the protein sequence of mouse p53a and p53b to human p53 around the SUMOylation site at lys386 (Fig. 2B). The lysine 375 of 374IKEE377 in mouse p53b was aligned to the lysine within the consensus SUMOylation site of human p53, 385FKTE388 (Fig. 2B). The region 374IKEE377 of mouse p53b was also confirmed as a high probability SUMOylation site, with a score of 2.943 predicted by SUMOsp2.0.4 software. However, there are no conserved amino acids in mouse p53a with mouse p53b at lys375 or human p53 at lys386, and there are no SUMOylated sites in mouse p53a predicted by SUMOsp2.0.4 software. In order to further confirm if only p53b, but not p53a, could be SUMO-1 modified in granulosa cells, pre-GCs were co-transfected with Flag-tagged sumo-1 plasmid and HA-tagged p53a or p53b plasmid. After transfection, the Flag-tagged fusion protein were pulled down by Flag antibody and then detected by western blot using HA antibody. The results showed that only p53b, but not p53a, could be co-immunoprecipitated by Flag-tagged sumo-1 (Fig. 2C).
10.1371/journal.pone.0063680.g002Figure 2 Identification of SUMOylation of p53b in vivo at Lys 375.
(A) Immunoprecipitation analysis of SUMOylation of p53 in vivo. Pre-GCs were transfected with pCMV-Flag-sumo-1 plasmid or pCMV-Flag plasmid. Flag-tagged protein were pulled down by anti-Flag affinity beads and then analyzed by Western blot using anti-p53 antibody. A visible band of p53 was detected in the sample of pCMV-Flag-sumo-1 transfected granulosa cells, but not in the control sample. (B) SUMOylation consensus site aligned in sequences of p53 from human and mouse. The SUMOylation consensus site is ψKXE with a hydrophobic residue, such as Phenylalanine (F), Isoleucine (I), or Leucine (L). Sequences of human p53 and mouse p53b were aligned. Lys386 in human p53 is SUMO-1 modified and the mouse p53b has a highly homologous sequence to this SUMOylation site. (C) Co-immunoprecipitation of SUMO-1 with p53a or p53b. Pre-GCs were co-transfected with Flag-tagged sumo-1 plasmid and HA-tagged p53a or HA-tagged p53b plasmid. Flag-tagged proteins were pulled down by anti-Flag affinity beads and then analyzed by Western blot using anti-HA antibody. A band of p53 (p53b) was detected in the sample of Flag-sumo-1 plasmid and HA-p53b plasmid co-transfected granulosa cells, but not in the HA-p53a plasmid and Flag-sumo-1 plasmid co-transfection group or control sample. (D) p53b is SUMOylated at lys375. Pre-GCs were co-transfected with 2 µg of wild type HA-p53b or mutant p53b (K375R) plasmid and 2 µg of Flag-sumo-1 plasmid. 20 µg total extracts were prepared and resolved by SDS–PAGE and analyzed by Western blot using the anti-HA-specific antibody. Positions of molecular weight markers, free p53 or p53-SUMO-1 conjugates are indicated. β-actin is a loading control. After transfection, two bands could be detected in the group with co-transfection of wild type p53b and sumo-1, the lower molecular weight below was considered as free p53b and the higher molecular weight above was p53b-SUMO-1 as the SUMOylated p53b, but only one band (mutant p53b) could be observed in the group with co-transfection of mutant p53b and sumo-1 plasmids, while the p53b (K375R)-SUMO-1- band was not observed.
Although Lys375 of p53b were predicted by SUMOsp2.0.4 software as a putative SUMO-1 modified site, it is still unknown whether this Lys375 is necessary for p53b’s modification by SUMO-1. Therefore, we mutated lysine 375 of mouse p53b to arginine and generated a mutant, HA-tagged p53b (K375R) expression plasmid. Wild-type p53b plasmid or mutant p53b plasmid, with sumo-1 plasmid, were co-transfected into mouse pre-GCs and analyzed by Western blot to examine the expression and SUMOylation. When cells were co-transfected with wild-type p53b plasmid and sumo-1 plasmid, two bands were detected (Fig. 2D, lane 3), but only one band could be observed when co-transfected with mutated p53b (K375R) plasmid and sumo-1 plasmid (Fig. 2D, lane 4). The lower band (55 kDa) was considered p53b protein and the band above was considered SUMO-1 covalently conjugated p53b protein, SUMOylated p53b. These results supported our hypothesis that mouse p53b could be SUMO-1 modified, and lysine 375 of p53b was necessary for SUMO-1 modification. The SUMOylation of mouse p53b at lys375 by SUMO-1 raised another interesting question: what regulatory roles of SUMOylation of p53b plays on the function of p53b in mouse ovarian granulosa cells. Further experiments were conducted to explore the effects of SUMOylation modification of p53b on its protein stability, subcellular localization, and biological activity in mouse granulosa cells.
SUMO-1 Modification Enhanced the Protein Stability of p53b in a Dose-dependent Manner
Our results above showed that transfection with sumo-1 or ubc9 plasmid could enhance the expression level of p53 in granulosa cells. To further explore the patterns of SUMO-1 modification to enhance the stability of p53b, pre-GCs were co-transfected with HA-wild-type p53b or HA-p53b (K375R) plasmid and different amounts of Flag–sumo-1 plasmids. Total proteins were extracted and subjected to Western blot analysis with HA antibody (Fig. 3). As predicted, increasing the amount of Flag-sumo-1 plasmid for transfection resulted in a significant increase of both SUMOylated p53b and free p53b in a dose-dependent manner (Fig. 3A and B, compare lanes 3, 4, 5, and 6), but did not increase p53b (K375R) (Fig. 3C). These results demonstrated that SUMO-1 modification was responsible for increasing the stability of p53b in a dose-dependent manner, and further confirmed that Lys 375 was required for SUMOylation of p53b by SUMO-1.
10.1371/journal.pone.0063680.g003Figure 3 SUMO-1 modification enhanced the stability of p53b in a dose-dependent manner.
(A) Pre-GCs were co-transfected with 2 µg HA-p53b plasmid and different amounts (2 µg, 3 µg, 4 µg, and 6 µg) of Flag-sumo-1 plasmids. 20 µg total proteins were prepared and resolved by SDS–PAGE and analyzed by Western blot using anti-HA-specific antibody. Positions of molecular weight markers, free p53b and p53b-SUMO-1 conjugates are indicated. β-actin is a loading control. (B) Relative expression quantity of p53b and SUMO-1-p53b were determined by densitometric scans. The total amount of β-actin present in the lower set of lanes was used to standardize the amount of p53b and SUMO-1-p53b present in the upper set of lanes. The value expressed by each bar represents the mean ± SD (n = 3). Different letters indicated statistical difference (P<0.05). (C) Pre-GCs were co-transfected with 2 µg HA-p53b (K375R) and different amounts (2 µg, 3 µg, 4 µg, and 6 µg) of Flag-sumo-1 plasmids. HA antibody were used to detect the expression of p53b (K375R) after transfection. The results showed that elevating the amount of transfected Flag-sumo-1 resulted in a simultaneous increase in the level of SUMOylated p53b and free p53b, but not mutant p53b (K375R).
SUMO-1 Modification was Required for the Nuclear Accumulation of p53b in Granulosa Cells
In order to study whether SUMO-1 modification of p53b at Lys375 is involved in the regulation of p53b’s subcellular localization in granulosa cells, pre-GCs were transfected with HA-p53b plasmid or mutant HA-p53b (K375R) plasmid, immunofluorescence cytochemistry was used to detect the subcellular localization of p53b protein by HA antibody. Wild type p53b proteins were accumulated only in the nucleus of pre-GCs, while mutant p53b (K375R) were localized in both nucleus and cytoplasm of pre-GCs (Fig. 4A). Meanwhile, the co-localization of SUMO-1 with p53b was observed in the nucleus of granulosa cells after co-transfection with sumo-1 plasmid and p53b plasmid. Mutant p53b (K375R) was still localized in both nucleus and cytoplasm of granulosa cells after co-transfection with sumo-1 plasmid and p53b (K375R) plasmid. Overall, the results indicated that SUMO-1 modification of p53b at Lys 375 was required for its nuclear accumulation in mouse granulosa cells.
10.1371/journal.pone.0063680.g004Figure 4 SUMO-1 modification of p53b at Lys 375 was required for its nuclear accumulation in granulosa cells.
(A) Pre-GCs were transfected with HA-tagged p53b or mutant p53b (K375R) plasmid, respectively. Then immunofluorescence cytochemistry was used to detect the subcellular localization of p53b or p53b (K375R) by HA antibody. The immunostaining signal of p53b were observed in nucleus of granulosa cells, but p53b (K375R) were seen in both nucleus and cytoplasm of granulosa cells (green). The nucleus was stained by PI (red). (B) Pre-GCs were co-transfected with HA-tagged p53b or mutant p53b (K375R) plasmid with Flag-tagged sumo-1 plasmid. Wild type p53b and mutant p53b (K375R) were detected by HA antibody with FITC-conjugated secondary antibody (green) and SUMO-1 was detected by SUMO-1 antibody with Cy3-conjugated secondary antibody (red), and the nucleus was stained by DAPI (blue). Co-localization of SUMO-1 and p53b in the nucleus were observed, but p53b (K375R) were still localized in both nucleus and cytoplasm of granulosa cells.
p53b-induced Cell Apoptosis was Enhanced by SUMO-1 Modification
As p53 is a cellular gatekeeper [14], [37], one of its roles is to survey cellular stress and induce apoptosis, if necessary. Therefore, we investigated the roles of mouse p53b in inducing apoptosis of pre-GCs. Cells were transfected with wild-type p53b or mutant p53b (K375R) for 24 h and then collected for apoptosis analysis by flow cytometer. The apoptosis rate was significantly increased by transfection with wild-type p53b compared with the control group and mutant p53b (K375R; Fig. 5A and 5B; P<0.05). The mutant p53b (K375R) transfected cells also showed significant increases in apoptosis compared with the control group (p<0.05), but were significantly lower in apoptosis compared with wild p53b transfected group (p<0.05). In addition, RT-PCR was used to detect the expression of a marker of apoptosis (Bax). Overexpression of either wild type p53b or mutant p53b (K375R) could significantly increase the expression of Bax mRNA, compared with control group. However, overexpression of wild type p53b could induce much higher expression level of Bax mRNA, compared with mutant p53b (Fig. 5C). These results were consistent with our other findings related to the apoptosis rate of granulosa cells. All these results implied that SUMO-1 modification of p53b enhanced the ability of p53b to induce apoptosis in pre-GCs. Mutating the SUMOylation site of p53b could significantly weaken the activity of p53b to induce apoptosis and apoptosis-related gene, Bax. On the other hand, in addition to inducing apoptosis, the cell death ratio was increased by mutation of p53b (K375R), but not by wild-type p53b (Fig. 5A).
10.1371/journal.pone.0063680.g005Figure 5 SUMOylation of p53b induced apoptosis in granulosa cells.
(A) Representative flow cytometric analysis of apoptotic cells stained with Annexin V-FITC and PI after transfection with 2 µg wild type p53b, mutant p53b (K375R) plasmid, control plasmid or non-transfection control group for 24 h. In each panel, the lower right quadrant contains apoptotic cells (positive for Annexin V and negative for PI). (B) The apoptosis rate of granulosa cells. The value expressed by each bar represents the mean ± SD (n = 3). Different superscripts denote statistical difference at a P<0.05. (C) Expression level of Bax mRNA. 24 h after transfection with 2 µg wild type p53b, mutant p53b (K375R), control plasmid or non-transfection control group, expression level of Bax mRNA was detected by quantitative real-time PCR. Fold changes were calculated from β-actin normalized Ct values. The value expressed by each bar represents the mean ± SD (n = 3). Different superscripts denote statistical difference at a P<0.05.
Discussion
In normal cells, p53 is maintained at a low level, which is partly due to the short half-life of the protein [38]. However, in response to a variety of stress signals, p53 is stabilized, causing protein to accumulate and activating p53-dependent transcription. Related factors, including lower levels of the MDM2-mediated poly-ubiquitination of p53 and others, improved post-translational modifications [22]–[24].
Although human p53 had been reported to be modified by SUMO-1 [11], [28], [39], there are two kinds of p53 (p53a and p53b) in mouse; whether mouse p53 could be SUMOylated was unclear. In this study, overexpression of sumo-1 or ubc9 could increase the protein level of p53 in granulosa cells, and a specific migrating band was also observed by using HA antibody, implying that SUMOylation is involved in the stability of p53 protein by post-translational modification. Based on the prediction results of SUMOsp2.0.4 software, there is a conserved SUMOylated site at lys375 of p53b, but not in p53a. The results of IP confirmed that mouse p53b, but not p53a, could be SUMO-1 modified at lys375. Further experiments showed that lys375 mutation of p53b could result in its nucleus-cytoplasm translocation and decrease its ability to induce apoptosis, confirming that p53b modified at lys375 by SUMO-1 plays crucial roles for p53 functions in granulosa cells.
The SUMO-1 modification increased the stabilization of mouse p53b in a dose-dependent manner (Fig. 3A); these results were similar to the results in U20S cells, in which SUMO-1 modified form of p53 accumulated after UV irradiation [36]. However, several researchers have delineated a conserved pathway, in which SUMOylation and ubiquitination cooperate in protein degradation [40]. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase [41]. Polo-like kinase 1 (Plk1) mediated phosphorylation of Topors inhibited Topors-mediated SUMOylation of p53, whereas p53 ubiquitination was enhanced, leading to p53 degradation [42], whether SUMOylation and ubiquitination of p53 cooperate in its degradation or the SUMOylation of p53 enhances the stabilization of p53 is still unclear. One research group proposed a hypothesis about an involvement of SUMOylation in p53 degradation [43], while there was some experimental evidences that SUMOylation may indeed facilitate p63 and p73 degradation [44], [45]. Considering that in our results, the stabilization of p53b was increased by SUMOylation (perhaps because it prevented ubiquitination), SUMOylation of p53b may inhibit its degradation by competing for the same lysine residue that was required for p53 ubiquitination or by interfering with conjugation of ubiquitin molecules to neighboring sites [46]. However, further studies will be required to address this issue.
Another issue to be addressed is the role of the SUMO-1 modification pathway in the subcellular distribution of p53b. In normal cell circumstances, p53 protein is present in the nucleus, and p53 nuclear export is critical to determine their degradation; we can speculate that the distribution of p53 in the nucleus and cytoplasm is a key factor in determining its stability. Interestingly, it has been reported that p53-SUMO-1 fusion protein localize to PML bodies. While damages the use of the fusion proteins by mutating the C-terminal glycines in the SUMO-1 portion (ΔGG) to inhibit the formation of isopeptide bonds between SUMO-1 and target lysines, the localization of p53-SUMO-1ΔGG is predominantly at the nuclear envelope with some cytoplasmic staining [32]. Similarly, SUMOylation helped recruit Drosophila p53 to nuclear dot-like structures that could be marked by human PML and the Drosophila homologue of Daxx [34]. In our study, we also found that the localization of p53b was in the nucleus; after mutation, the nucleus localization was lost compared to wild p53b and part of the p53b was localized in the cytoplasm (Fig. 4 A and B). So our results directly demonstrated that SUMOylation was required for the nuclear localization of p53b, which provided a novel platform for explaining why the SUMOylation of p53b increased its stabilization.
Research has firmly established that p53, is the cellular gatekeeper for growth, division, apoptosis, tumor suppression, and reproductive regulation [14], [38], [47]. Loss of the p53 gene in female mice leads to a significant decrease of fertility. The p53 gene product regulates maternal reproduction at the implantation stage of the embryo [48]. In addition, p53 is required for the induction of apoptosis [49]. Apoptosis of granulosa cells is an essential component of ovarian follicular atresia [50], and it has been reported that p53 protein is mainly expressed in the apoptotic granulosa cells of atretic follicles in the ovary [51], [52] and p53 decreases the expression of the apoptosis-suppressing gene bcl-2 while simultaneously increased the expression of Bax, a gene that encodes a dominant inhibitor of bcl-2 protein [53], [54]. Previous studies had shown that human p53 could be SUMO-1 modified, and when mutated the SUMOylation site lys386, the ability of p53 to induce apoptosis was weakened or even disappeared [33]. Mutation of both SUMOylation sites of Drosophila p53 dramatically reduced the transcriptional activity of p53 and its ability to induce apoptosis in transgenic flies [34]. In our study, SUMOylation of p53b enhanced the apoptosis in pre-GCs; mutating the SUMOylation site of p53b could significantly weaken the activity of p53b in inducing cells apoptosis (Fig. 5A and 5B). Actually, we also tested the effect of sumo-1 or p53/sumo-1 co-transfection on apoptosis of granulosa cells, the results showed that sumo-1 transfection could also induce apoptosis of granulosa cells, but co-transfection with both p53 and sumo-1 has much more significant effect of inducing apoptosis of granulosa cells (data not shown). Meanwhile, overexpression of p53b significantly increased the expression of Bax (Fig. 5C). Interestingly, destruction of the SUMOylation site simultaneously decreased the expression of Bax (Fig. 5C). To some extent, p53b was regulated by SUMO-1 modification at the transcriptional level. However, further studies will be required to address whether SUMO-1 modification could enhance mouse p53b-dependent transactivation. Moreover, FSH is a potent survival factor of granulosa cells during follicular development. The future study about how FSH affects the SUMOylation of p53b-induced apoptosis during folliculogenesis will help us in better understanding of follicular development.
In conclusion, our data demonstrated that mouse p53b, but not p53a, can be SUMO-1 modified, and the SUMOylation site is lys375. The SUMOylation modification is clearly important for the functions of mouse p53b, regulating its stabilization, nucleus-cytoplasm translocation, and pro-apoptosis ability. These results provide important information about the roles and regulatory pathways of p53 in follicular granulosa cell apoptosis, follicular atresia, and ovarian cancer.
This work was conducted in the Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University. We are also grateful to Yao Hang (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University) for providing technical assistances for confocal microscopy.
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