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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22536352PONE-D-12-0197510.1371/journal.pone.0035142Research ArticleAgricultureAquacultureAlgacultureBiofuelsBiologyBiochemistryMetabolismCarbohydrate MetabolismLipid MetabolismMetabolic PathwaysGeneticsGene FunctionGenomicsGenome Analysis ToolsTranscriptomesPlant SciencePlantsAlgae De Novo Transcriptomic Analysis of an Oleaginous Microalga: Pathway Description and Gene Discovery for Production of Next-Generation Biofuels Transcriptomic Profile of Eustigmatos cf. polyphemWan LingLin 1 Han Juan 1 Sang Min 1 Li AiFen 1 Wu Hong 2 Yin ShunJi 2 Zhang ChengWu 1 * 1 Institute of Hydrobiology, Jinan University, Guangzhou, People's Republic of China 2 State Key Laboratory of Coal-Based Low Carbon Energy, Xinao Scientific & Technological Developmental Co. Ltd., Langfang, People's Republic of China Zhang Baohong EditorEast Carolina University, United States of America* E-mail: [email protected] and designed the experiments: LLW AFL CWZ. Performed the experiments: LLW JH MS. Analyzed the data: LLW. Contributed reagents/materials/analysis tools: LLW HW SJY AFL CWZ. Wrote the paper: LLW CWZ. 2012 20 4 2012 7 4 e3514214 1 2012 8 3 2012 Wan et al.2012This 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 Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. Results We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. Conclusions Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock. ==== Body Introduction Interest in biodiesel that can be used as an alternative to petroleum diesel fuel has grown significant recently due to the soaring oil prices, diminishing world oil reserves, emissions of greenhouse gas, and the reliance on unstable foreign fuel resources [1], [2]. In contrast to oil crops, the greatly minimized acreage estimates, efficiently use of CO2, an enormous variety of high oil contents, and biomass production rates may make microalgae a high potential feedstock to produce cost-competitive biofuels [3]–[7]. However, there are a number of obstacles to overcome for microalgae to be economically used as bioenergy. A key challenge is the choice of microalgal strains [7], [8]. By now only a few microalgal species show potential for industrial production, e.g. the eustigmatophyte Nannochloropsis oculata [9]. Nannochloropsis is a robust industrial microalga that can be extensively grown in outdoor ponds and photobioreactors for aquaculture [10], [11]. Numerous studies reported that some microalgae could accumulate high quantities of neutral storage lipids, mainly triacylglycerols (TAGs), the major feedstock for biodiesel production, in response to environmental stresses, such as nitrogen limitation, salinity, high light intensity or high temperature [12]–[16]. E. cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte [17]. We could obtain >9 g L−1 dry weight of E. cf. polyphem with oil exceeding 60% and β-carotene achieving 5% of its biomass on a dry cell-weight basis under nitrogen limited conditions (unpublished results). Furthermore, under nitrogen replete conditions, E. cf. polyphem cells could accumulate an amount of eicosapentaenoic acid (EPA, 20:5ω3) (unpublished results), an omega-3 fatty acid with numerous health benefits [18]. Based on the high biomass and considerable production of lipids, E. cf. polyphem is thus referred to as an oleaginous microalga. And it could be employed as a cell factories to produce oils for biofuels and other bio-products [19], [20]. The high production of valuable co-products, such as EPA and β-carotene, may allow biofuels from E. cf. polyphem to compete economically with petroleum [21], [22]. In theory, microalgae could be bioengineered, allowing improvement of specific traits [23], [24] and production of valuable products. However, before this concept can become a commercial reality, many fundamental biological questions relating to the biosynthesis and regulation of fatty acids and TAG in oleaginous microalgae need to be answered [20], [25]. Thus, understanding how microalgae respond to physiological stress at molecular level as well as the mechanisms and regulations of carbon fixation, carbon allocation and lipid biosynthetic pathways in biofuel relevant microalgae is very important for improving microalgal strain performances. The lack of sequenced genomes of oleaginous microalgae hampered investigation of the transcribed gene, the pathway information and the genetic manipulations in these microalgae. However, analysis of whole transcriptome can provide researchers with greater insights into the complexity of gene expression, biological pathways and molecular mechanisms in the organisms without the reference genome information. Next generation high-throughput sequencing platform, such as Solexa/Illumina sequencing by synthesis (SBS) technology, has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data which can be effectively assembled and used for gene discovery and comparison of gene expression profiles [26]–[29]. In this study, we determined the general patterns of carbohydrate, fatty acids, TAG and carotenoid synthesis and accumulation in the E. cf. polyphem which may have potential for production of biofuels and valuable co-products. We further conduct a transcriptome profiling analysis of E. cf. polyphem without the prior genome information to discover genes that encode enzymes involved in these biosynthesis and to describe the relevant metabolic pathways. Results and Discussion Illumina sequencing and reads assembly To obtain an overview of the gene expression profile and metabolic pathways involved in E. cf. polyphem, pure cultures were grown under nitrogen replete, nitrogen limited and nitrogen free conditions. Cells were harvested in the log and stationary growth phases. The normalized cDNA libraries of cells grown under the above conditions were pooled and sequenced using Solexa/Illumina RNA-seq deep sequencing analysis platform. After cleaning and quality checks, we obtained 29.1 million 75-bp pair end (PE) raw reads of sequencing. To facilitate sequence assembly, these raw reads were assembled using SOAPdenovo program [30], resulting in 132,357 contigs with an average contig length of 306 bp and an N50 of 487 bp, ranging from 100 bp to >3,000 bp (Table 1, Figure 1). Furthermore, TGICL [31] was used to assemble 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp (Table 1). Out of the 75,632 unigenes, 34,966 unigenes were ≥500 bp, 9,979 were ≥1,000 bp and 51 were >3000 bp. The unigene distribution followed the contig distribution closely (Figure 1). To demonstrate the quality of sequencing data, we randomly selected 10 unigenes and designed 10 pairs of primers for RT-PCR amplification. In this analysis, 9 out of 10 primer pairs resulted in a band of the expected size and the identity of all nine PCR products were confirmed by Sanger sequencing (data not shown). 10.1371/journal.pone.0035142.g001Figure 1 Statistics of Illumina short read assembly quality. The length distribution of de novo assembly for contigs and Unigenes is shown. 1, 200; 2, 300; 3, 400; 4, 500; 5, 600; 6, 700; 7, 800; 8, 900; 9, 1,000; 10, 1,100; 11, 1,200; 12, 1,300; 13, 1,400; 14, 1,500; 15, 1,600; 16,1,700; 17, 1,800; 18, 1,900; 19, 2,000; 20, 2,100; 21, 2,200; 22, 2,300; 23, 2,400; 24, 2,500; 25, 2,600; 26, 2,700; 27, 2,800; 28, 2,900; 29, 3,000; 30, >3,000. 10.1371/journal.pone.0035142.t001Table 1 Summary for the E. cf. polyphem transcriptome. Total number of reads 29,199,432 Total base pairs (bp) 2,335,954,560 Total number of contigs 132,357 Mean length of contigs 306 bp Total number of unigenes 75,632 Mean length of unigenes 503 bp Functional annotation For annotation, 75,632 unigenes were further searched using BLASTx against the non-redundant (nr) NCBI nucleotide database with a cut-off E-value of 10−5, resulting 44,477 unigenes sequences. Sequence orientations were determined according to the best hit in the database. Using ESTScan [32] to predict the orientation and coding sequences (CDS) of sequences have no hit in blast. BLASTx and ESTscan software analysis revealed that about 14,982 sequences have reliable CDS. These sequences have high potential for translation into functional proteins and most of them translated to proteins with more than 100 amino acids. Annotation of the these sequences using Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases yielded good results for approximately 9,597 consensus sequences and 6,561 putative proteins (Table 2). GO-annotated consensus sequences belonged to the biological process, cellular component, and molecular function clusters and distributed about 37 categories (Figure 2). Similarly, COG-annotated putative proteins were classified functionally into at least 25 molecular families (Figure 3). 10.1371/journal.pone.0035142.g002Figure 2 GO annotations of non-redundant consensus sequences. Best hits were aligned to the GO database, and 9,597 transcripts were assigned to at least one GO term. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function. 10.1371/journal.pone.0035142.g003Figure 3 COG annotations of putative proteins. All putative proteins were aligned to the COG database and can be classified functionally into at least 25 molecular families. A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton. 10.1371/journal.pone.0035142.t002Table 2 Annotation of non-redundant consensus sequences. Database Number of annotated consensus sequences Percentage of annotate consensus sequences Swissprot 5309 35.4% Nr 6898 46.0% GO 9,597 64.1% KEGG 9098 60.7% COG 6561 43.8% All 14,982 CDS sequences generated by ESTscan were annotated though Swissprot, Nr, GO, KEGG, and COG databases. To reconstruct the metabolic pathways involved in E. cf. polyphem, the assembled unigenes were annotated with corresponding enzyme commission (EC) numbers against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the Blast2Go program [33]. By mapping EC numbers to the reference pathways, a total of 9,098 unigenes were assigned to 113 known metabolic or signalling pathways including calvin cycle, glycolysis, pentose phosphate, citrate cycle, fatty acid biosynthesis and carotenoid biosynthesis (Table 2– 6 and Table S1, S2, S3, and S4). However, the annotation of E. cf. polyphem transcriptome did not identify the major genes encoding enzymes involved in starch biosynthesis and catabolism. Comparative analysis of enzyme-coding sequences between E. cf. polyphem and model organisms, Chlamydomonas reinhardtii, Phaeodactylum tricornutum and Thalassiosira pseudonana using BLASTx analysis revealed relatively low homology between E. cf. polyphem and these organisms for the enzymes described in this study (Table 4, 5, 6). These differences indicate that functional genomics and metabolic engineering of E.cf. polyphem cannot be fully based on the sequence information obtained from model organisms. Because of high production of lipids, TAG, and β-carotene in E. cf. polyphem cells, the metabolic pathways associated with biosynthesis and catabolism of lipids, carbohydrate and carotenoid were given further treatment below. 10.1371/journal.pone.0035142.t003Table 3 Essential metabolic pathways annotated in the E. cf. polyphem transcriptome. Pathway Enzymes found Known enzymes Photosynthetic carbon fixation (Calvin cycle) 12 13 Glycolysis/Gluconeogenesis 10 10 Pentose phosphate 5 5 Citrate cycle 10 10 Fatty acid biosynthesis 6 6 TAG biosynthesis 4 4 Carotenoid biosynthesis 4 4 10.1371/journal.pone.0035142.t004Table 4 Enzymes involved in fatty acid biosynthesis and metabolism identified by annotation of the E. cf. polyphem transcriptome. Enzyme Symbol EC Number Number of transcripts 1%Sequence alignment with corresponding enzymes in model organisms (Accession #) C. reinhardtii P. tricornutum T. pseudonana Fatty acid biosynthesis Biotin carboxylase BC 6.3.4.14 4 2NM 64(XP_002185458.1) 72(XP_002287470.1) Acetyl-CoA carboxylase ACCase 6.4.1.2 7 NM 66(XP_002184364.1) NM AMP-activated kinase AMPK 2.7.11.1 8 NM NM NM Malonyl-CoA-ACP transacylase MAT 2.3.1.39 1 NM 61(XP_002181767.1) 64(XP_002290601.1) 3-Ketoacyl ACP synthase I KAS I 2.3.1.41 1 NM NM NM 3-Ketoacyl ACP synthase II KAS II 2.3.1.179 9 NM 56(XP_002181453.1) 54(XP_002290056.1) 3-Ketoacyl ACP synthase III KAS III 2.3.1.180 3 52(XP_001703101.1) NM 58(XP_002295320.1) 3-Ketoacyl ACP reductase KAR 1.1.1.100 10 58(XP_001691899.1) 60(XP_002180902.1) 59(XP_002287667.1) 3-Hydroxy acyl-CoA dehydratase HD 4.2.1.- 1 NM NM NM Enoyl-ACP reductase (NADH) EAR 1.3.1.9 1 NM 78(XP_002177931.1) 77(XP_002288236.1) Oleoyl-ACP thioesterase OAT 3.1.2.14 2 NM NM NM Acyl-ACP thioesterase A FATA 3.1.2.14 3.1.2.- 0 NM NM NM Acyl-ACP thioesterase B FATB 3.1.2.14 3.1.2.- 0 NM NM NM Fatty acid desaturation Δ9 Acyl-ACP desaturase AAD 1.14.19.2 1 NM 58(XP_002181794.1) 59(XP_002290033.1) Δ12(ω6)-Desaturase Δ12D 1.4.19.6 1 NM 32(XP_002185498.1) NM Δ15(ω3)-Desaturase Δ15D 1.4.19.- 1 NM NM NM Δ5- Desaturase Δ5-D 1.14.99.- 1 NM NM NM Δ6- Desaturase Δ6-D 1.14.99.- 2 NM NM NM Fatty acid elongation 3-Hydroxyacyl-CoA dehydrogenase CHAD 1.1.1.35 5 NM 43(XP_002182878.1) NM Δ6-Elongase Δ6-E 6.21.3.- 3 NM 56(XP_002184740.1)52(XP_002184657.1) 57(XP_002293395.1) Long-chain-3-hydroxyacyl-CoA dehydrogenase LCHAD 1.1.1.211 5 NM NM NM Enoyl-CoA hydratase ECH 4.2.1.17 10 NM 44(XP_002180629.1) NM Trans-2-enoyl-CoA reductase (NADPH) TER 1.3.1.38 6 NM NM NM Palmitoyl-CoA hydrolase PCH 3.1.2.22 2 NM NM NM Fatty acid catabolism long-chain acyl-CoA synthetase ACSL 6.2.1.3 27 NM 51(XP_002185164.1) 53(AAW58006.1)51(XP_002287843.1)48(XP_002291500.1) Acyl-CoA oxidase AOx 1.3.3.6 5 NM 39(XP_002179644.1) 34(XP_002293157.1) Acyl-CoA dehydrogenase ACADM 1.3.99.3 2 NM 59(XP_002186235.1) 58(XP_002296341.1) Acetyl-CoA acyltransferase ACAT 2.3.1.16 2 60(XP_001697225.1) NM 54(XP_002291097.1) Acetyl-CoA C-acetyltransferase thiL 2.3.1.9 2 52(XP_001694888.1) 56(XP_002185228.1) NM Alcohol dehydrogenase ADH 1.1.1.1 10 48(XP_001693170.1) 73(XP_002176667.1) 72(XP_002286578.1) Aldehyde dehydrogenase (NAD+) ALDH 1.2.1.3 10 NM NM NM Ferredoxin-NAD+reductase FNR 1.18.1.3 0 NM NM NM 1 In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported. 2 NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms. 10.1371/journal.pone.0035142.t005Table 5 Enzymes involved in TAG biosynthesis identified by annotation of the E. cf. polyphem transcriptome. Enzyme Symbol EC Number Number of transcripts 1%Sequence alignment with corresponding enzymes in model organisms (Accession #) C. reinhardtii P. tricornutum T. pseudonana Acyl-CoA synthetases ACSL 6.2.1.3 25 2NM 47(XP_002179636.1)57(XP_002185164.1)39(XP_002180281.1)39(XP_002186275.1) 55(AAW58006.1)39(XP_002291517.1) Glycerol kinase GK 2.7.1.30 4 NM NM NM Glycerol-3-phosphate O-acyltransferase GPAT 2.3.1.15 2 33(XP_001694977.1) NM 45(XP_002292905.1) Acyl-sn-glycerol-3-phosphate O-acyltransferase AGPAT 2.3.1.51 5 NM NM NM Phosphatidate phosphatase PP 3.1.3.4 1 NM NM NM Diacylglycerol O-acyltransferase DGAT 2.3.1.20 9 33(XP_001693189.1) NM NM Phospholipid: diacyglycerol acyltransferase PDAT 2.3.1.158 6 NM NM 50(XP_002286433.1) 1 In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported. 2 NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms. 10.1371/journal.pone.0035142.t006Table 6 Enzymes involved in chrysolaminarin biosynthesis and metabolism identified by annotation of the E. cf. polyphem transcriptome. Enzyme Symbol EC Number Number of transcripts 1%Sequence alignment with corresponding enzymes in model organisms (Accession #) C. reinhardtii P. tricornutum T. pseudonana Chrysolaminarin biosynthesis UDP-glucose pyrophosphorylase UGPase 2.7.7.9 1 57 (XP_001692246.1) 69 (XP_002185375.1) 58(XP_002289637.1) β-1,3-glucan glycosyltransferase UDPG 2.4.1.34 3 2NM NM NM Chrysolaminarin metabolism exo-1,3-β-Glucanase exo-Glu 3.2.1.58 2 NM NM NM endo-1,3-β-Glucanase endo-Glu 3.2.1.39 1 NM NM NM β-Glucosidase BGL 3.2.1.21 27 NM 57(XP_002185317.1)34(XP_002179173.1) 58(XP_002290406.1)44(XP_002290406.1) 1 In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported. 2 NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms. Detection of sequences related to the fatty acid biosythesis and metabolism Microalgae synthesize fatty acids as building blocks for the formation of various types of lipids [20]. Understanding microalgal lipid metabolism is of great interest for the ultimate production of diesel fuel surrogates and other valuable bio-products. Both the quantity and the quality of diesel precursors from a specific microalgal strain are closely linked to how lipid metabolism is controlled. Under optimal conditions of growth, algae synthesize fatty acids principally for esterification into glycerol-based membrane lipids. Under unfavorable environmental or stress conditions for growth, however, some species can rapidly accumulate significant amounts of storage neutral lipids, especially TAG, the major feedstock for biodiesel production [8]. The basic pathway of fatty acid and TAG biosynthesis in microalgae is generally believed to be directly analogous to those demonstrated in higher plants. Based on the functional annotation of the transcriptome, we have successfully identified the genes encoding for key enzymes involved in the biosynthesis and catabolism of fatty acids in E. cf. polyphem (Table 4). The reconstructed pathway based on these identified enzymes is depicted in Figure 4. In microalgae, the de novo synthesis of fatty acids occurs primarily in the chloroplast, and produces 16- and 18-carbon fatty acid, which could be used as the precursors for the synthesis of cellular membranes, long-chain polyunsaturated fatty acids (LC-PUFAs) and storage neutral lipids (mainly TAGs). Fatty acid biosynthesis in E. cf. polyphem starts with the conversion of acetyl CoA to malonyl CoA, catalyzed by acetyl CoA carboxylase (ACCase, EC: 6.4.1.2). ACCase inhibition via phosphorylation can be catalyzed by AMP-activated kinase (AMPK, EC:2.7.11.1). Then, malonyl-CoA, the central carbon donor for fatty acid synthesis, is transferred next to an acyl carrier protein (ACP) catalyzed by malonyl-CoA ACP transacylase (MAT, EC: 2.3.1.39). All elongation reactions of the pathway involve malonyl-ACP with acyl ACP (or acetyl-CoA) acceptors that are catalyzed by the multiple isoforms of the condensing enzyme, ketoacyl-ACP synthase (KAS) until the finished products are ready for transfer to glycerolipids or export from the chloroplast. The first condensation reaction catalyzed by 3-ketoacyl ACP synthase III (KAS III, EC: 2.3.1.180) forms a 3-ketoacyl ACP (a four-carbon product) [34]. Another condensing enzyme, 3-ketoacyl ACP synthase I (KAS I, EC: 2.3.1.41), produces varying chain lengths (6 to 16 carbons). To form a saturated fatty acid, the 3-ketoacyl ACP product is reduced by the enzyme 3-ketoacyl ACP reductase (KAR, EC: 1.1.1.100), dehydrated by 3-hydroxy acyl-CoA dehydratase (HD, EC: 4.2.1.-) and then reduced by the enoyl-ACP reductase (EAR, EC: 1.3.1.9). A sequence of reduction, dehydration and reduction again results in the formation of palmitic acid (PA, 16:0) and stearic acid (SA, 18:0) bound to ACP. 10.1371/journal.pone.0035142.g004Figure 4 Fatty acid biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome. Identified enzymes are shown in boxes and include: ACCase, acetyl-CoA carboxylase (EC: 6.4.1.2); MAT, malonyl-CoA ACP transacylase (EC: 2.3.1.39); KAS, 3-ketoacyl ACP synthase (KAS I, EC: 2.3.1.41; KASII, EC: 2.3.1.179; KAS III, EC: 2.3.1.180); KAR, 3-ketoacyl ACP reductase (EC: 1.1.1.100); HD, 3-hydroxy acyl-CoA dehydratase (EC: 4.2.1.-); EAR, enoyl-ACP reductase (NADH) (EC: 1.3.1.9); AAD, Δ9 Acyl-ACP desaturase (EC: 1.14.19.2); OAT, oleoyl-ACP thioesterase (EC: 3.1.2.14); Δ12D, Δ12(ω6)-desaturase (EC: 1.4.19.6); Δ15D, Δ15(ω3)-desaturase (EC: 1.4.19.-); Δ5D, Δ5- desaturase(EC: 1.14.99.-), Δ6D, Δ6- desaturase(EC: 1.14.99.-) and Δ6E, Δ6-elongase (EC: 6.21.3.-). The fatty acid biosynthesis pathway in E. cf. polyphem produces saturated, PA, palmitic acid (16:0) and SA, stearic acid (18:0), and unsaturated fatty acids OA, oleic acid (18:1ω9); LA, linoleic acid (18:2ω6); ALA, α-linolenic acid (18:3ω3); SDA, stearidonic acid (18:4ω3); ETA, eicosatetraenoic acid (20:4ω3) and EPA, eicosapentaenoic acid (20:5ω3). To produce an unsaturated fatty acid, the introduction of double bonds into the acyl chain is catalysed by a soluble enzyme Δ9 Acyl-ACP desaturase (AAD, EC: 1.14.19.2). The elongation of fatty acids is terminated either when the acyl group is removed from ACP by an acyl-ACP thioesterase, oleoyl-ACP hydrolase (OAT, EC: 3.1.2.14), that hydrolyzes the acyl ACP and releases free fatty acid or when acyl transferases in the chloroplast transfer the fatty acid directly from ACP to glycerol-3-phosphate (G-3-P) or monoacylglycerol-3-phosphate [35]. The released free oleic acid (OA,18:1ω9) could be desaturated by a desaturation enzyme, Δ12(ω6)-desaturase (Δ12D, EC: 1.4.19.6) to form linoleic acid (LA, 18:2ω6), and further desaturated by Δ15(ω3)-desaturase (Δ15D, EC: 1.4.19.-), resulting in α-linolenic acid (ALA,18:3ω3). LA and ALA are essential fatty acids because they serve as important precursors for the synthesis of further longer and higher unsaturated polyunsaturated fatty acids (PUFAs). We have also identified key desaturation and elongation enzymes associated in the biosynthetic pathway of EPA, which is known to be cardiovascular-protective components of the human diet [36]. According to the position of the last double bond to the terminal methyl group of EPA, there are two possible biosynthetic pathways: the ω3 and ω6-pathway [37]. In the ω6 pathway, LA is desaturated to γ-linoleic acid (GLA, 18:3ω6) by Δ6-desaturase (Δ6-D, EC: 1.14.99.-), elongated to dihomo-γ-linoleic acid (DGLA, 20:3ω6) by Δ6-elongase (Δ6-E, EC: 6.21.3.-), and subsequently desaturated to arachidonic acid (ARA, 20:4ω6) by Δ5-desaturase (Δ5-D, EC: 1.14.99.-). Δ17-desaturase (Δ17-D) is responsible for the conversion of ARA to EPA. In the ω3 pathway, LA is first desaturated to ALA by Δ15D, and then sequentially converted to stearidonic acid (SDA, 18:4ω3), eicosatetraenoic acid (ETA, 20:4ω3) and EPA, presumably by the activity of Δ6-D, Δ6-E and Δ5-D, respectively (Figure 4). We speculate that the biosynthetic pathway of EPA is the ω3-pathway because of the lack of transcripts encoding Δ17-D in the annotation of E. cf. polyphem transcriptome. The annotation of E. cf. polyphem transcriptome has also identified all the genes encoding enzymes involved in fatty acid catabolism (Table 4). The pathway of fatty acid catabolism in microalgae involves four key enzymes: acyl-coA oxidase (AOx, EC: 1.3.3.6), enoyl-CoA hydratase (ECH, EC: 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (CHAD, EC: 1.1.1.35) and acetyl-CoA acyltransferase (ACAT, EC: 2.3.1.16). The acetyl-CoA resulting from fatty acid catabolism is then used to produce energy for the cell via the citrate cycle or participate in the synthesis of TAG. The E. cf. polyphem transcriptome presented here contains most of the enzymes required for the biosynthesis and metabolism of fatty acids (Table 4). These findings contribute to the biochemical and molecular information needed for metabolic engineering of fatty acid synthesis in microalgae. Under lipid-accumulating conditions, up-regulation of ACCase and down-regulation of AMPK have been observed in some oleaginous microalgae [38], [39], [40]. Thus, overexpression of ACCase, a major milestone in fatty-acid biosynthesis, is believed to be the most commonly stated strategy for improving fatty acid biosynthesis. Nevertheless, overexpression of the ACCase gene in the genetic transformed diatom cells failed to significantly increase lipid accumulation [19]. AMPK is proposed to serve as a fatty acid β-oxidation “metabolic master switch", which play a critical role in driving the equilibrium between acetyl-CoA and malonyl-CoA in the reverse direction, ultimately slowing the rate of fatty acid biosynthesis and increasing the rates of fatty acid β-oxidation [40]. The activity of AMPK under nitrogen-replete and nitrogen-deplete conditions is needed further investigation. TAG biosynthesis and catabolism E. cf. polyphem is capable of producing and accumulating high amounts of storage neutral lipids, mainly TAGs, under high light and nitrogen limited conditions (unpublished results). Unlike the glycerolipids found in membranes, TAGs do not perform a structural role but instead serve as a storage form of carbon and energy [20]. TAGs can serve as precursors for production of biodiesel and other bio-based products such as plastics, cosmetics, and surfactants [8]. Although the global pathway for TAG biosynthesis are known, the existing knowledge on the pathways and enzymes involved in TAG synthesis in microalgae is limited [41], [42]. Based on the KEGG pathway assignment of the functionally annotated sequences, transcripts coding for all enzymes involved in TAG biosynthesis were identified in E. cf. polyphem. These enzymes are presented in Table 5, and the suggested pathway for TAG synthesis in E. cf. polyphem is shown in Figure 5. TAG biosynthesis in algae has been proposed to occur via the direct glycerol pathway, as the three sequential acyl transfers from acyl CoA to a glycerol backbone [43]. G-3-P, as the precursor for TAG biosynthesis, is produced by the catabolism of glucose (glycolysis) or to a lesser extent by the action of the enzyme glycerol kinase (GK, EC: 2.7.1.30) on free glycerol. We identified four transcripts coding for GK in E. cf. polyphem transcriptome library. Fatty acids produced in the chloroplast are sequentially transferred from CoA to form acyl-CoA, another precursor for TAG synthesis. The first two steps of TAG biosynthesis involve sequential esterification of acyl chains from acyl-CoA to positions 1 and 2 of G-3-P to yield phosphatidic acid (PA), catalyzed by G-3-P acyl transferase (GPAT, EC: 2.3.1.15) and lyso-phosphatidic acid acyl transferase (AGPAT, EC: 2.3.1.51), respectively. Two and seven transtripts encoding for GPAT and AGPAT were identified in the E. cf. polyphem transcriptome library respectively. Dephosphorylation of PA catalyzed by a specific phosphatase, phosphatidate phosphatase (PP, EC: 3.1.3.4), releases diacylglycerol (DAG). Only one transcript was annotated as coding for this enzyme in the E. cf. polyphem transcriptome. PA and DAG can also be used directly as a substrate for synthesis of polar lipids, such as phospholipid, and phosphatidylcholine (PC). In the final step of TAG synthesis, a third fatty acid is transferred to the vacant position 3 of DAG, and this reaction is catalyzed by diacylglycerol acyltransferase (DGAT, EC: 2.3.1.20) using acyl CoA as an acyl-donor to form TAG. This enzymatic reaction is believed to be the main pathway for TAG synthesis [20], [44]. We identified nine genes coding for DGAT in the transcriptome of E. cf. polyphem. Besides this main pathway for TAG synthesis, Dahlqvist [45] reported an acyl CoA-independent mechanism for TAG synthesis in some plants and yeast. In this pathway, the final step of TAG synthesis is catalyzed by phospholipid: diacylglycerol acyltransferase (PDAT, EC: 2.3.1.158) using PC, a major polar lipid, as acyl donors [42], [46]. There are six transcripts coding for PDAT in E. cf. polyphem transcriptome. In the yeast, PDAT can catalyze a breakdown of the major membrane lipids (PC and PE), which act as acyl donors in the synthesis of TAG. Thus, PDAT could channel the bilayer-disturbing fatty acids from PC into the TAG pool [45]. Under stress conditions, some microalgae including E. cf. polyphem, usually undergo rapid degradation of the photosynthetic membrane with concomitant occurrence and accumulation of cytosolic TAG-enriched lipid bodies (unpublished results). Identification of PDAT in E. cf. polyphem suggests that the acyl CoA-independent synthesis of TAG catalyzed by PDAT could provide insight into the connection between rapid degradation of membrane lipids with concurrent accumulation of TAGs in response to various stress and growth conditions [20]. However, the in vivo function of PDAT still remains to be determined via gene-knockout experiments and analysis of lipid profiles. 10.1371/journal.pone.0035142.g005Figure 5 Triacylglycerol biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome. Identified enzymes are shown in boxes and include: GK, glycerol kinase (EC: 2.7.1.30); GPAT, glycerol-3-phosphate acyl transferase (EC: 2.3.1.15); AGPAT, lyso-phosphatidic acid acyl transferase (EC:2.3.1.51); PP, phosphatidate phosphatase (EC: 3.1.3.4); DGAT, diacylglycerol O-acyltransferase (EC: 2.3.1.20) and PDAT, phopholipid: diacyglycerol acyltransferase (EC 2.3.1.158). G-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; PC, phosphatidylcholine and TAG, triacylglycerol. Carbohydrate products–synthesis and degradation To investigate the main assimilatory product of photosynthesis, the carbohydrate content of E.cf. polyphem were measured quantitatively. Under nitrogen replete (N-replete, 17.7 mM NaNO3) conditions, the total carbohydrate content gradually increased from 18.7% to a maximum level of 42.31% of cell dry weight (DW) on day 3, and decreased to 27.39% DW on day 15. Similarly, the total carbohydrate content of E. cf. polyphem cells grown under nitrogen limited (N-limited, 5.9 mM NaNO3) conditions increased from 20.08% to 44.8% of DW on day 3, and decreased to 25.78% DW on day 15 (Figure 6A). We didn't found any starch content in this microalgal cells under N-replete or N-limited conditions. However, we detected a significant accumulation of chrysolaminarin in E. cf. polyphem cells (data not shown). Under N-limited conditions, the amount of chrysolaminarin could constitute 59.6% of total carbohydrate and 26.69% of DW on day 3 (Figure 6B). Chrysolaminarin is the principal energy storage polysaccharide of diatoms, that generally comprises between 10 and 20% of the total cellular carbon in exponentially growing cells but can accumulate to up to 80% of the total carbohydrate in cells under nitrogen limited conditions [47], [48]. Thus, chrysolaminarin is the primary carbon storage compound in E. cf. polyphem. 10.1371/journal.pone.0035142.g006Figure 6 Carbohydrate accumulation properties of E. cf. polyphem. (A) and (B) representative total carbohydrate and chrysolaminarin content for E. cf. polyphem cultured under nitrogen-replete (grey) and nitrogen-limited (black) conditions respectively. The biochemical pathways leading to chrysolaminarin synthesis and degradation have not been elucidated. The synthesis of most storage polysaccharides involves the condensation of nucleoside diphosphate sugars. For example, starch is formed in plants from ADP glucose, and UDP glucose is used to form sucrose in plants and glycogen in mammalian cells [49], [50], [51]. These reactions are catalyzed by nucleoside diphosphate sugar pyrophosphorylases, such as UDPglucose pyrophosphorylase (UGPase), which catalyzes the reversible transfer of an uridylyl group from UDP-glucose to pyrophosphate (PPi), producing glucose-1-phosphate (G-1-P) and UTP [52]. Based on enzyme activity assays of Cyclotella cryptica, Roessler [53] demonstrated the important role of UGPase in chrysolaminarin synthesis in diatoms. Subsequent studies identified a second enzyme, β-(1,3)-glucan-β-glucosyltransferase (UDPG, also known as chrysolaminarin synthase) associated with the synthesis of chrysolaminarin [54]. Furthermore, exo-1,3-β-glucanase (exo-Glu) activity was detected in several planktonic diatoms and upregulation of this activity coincided with chrysolaminarin degradation in the diatom Skeletonema costatum [47]. So we focused on exo-Glu and endo-1,3-β-glucanase (endo-Glu) and β-glucosidase (BGL) as the primary enzymes involved in digesting chrysolaminarin. Based on the KEGG pathway assignments, we identified numerous transcripts coding for enzymes involved in the biosynthesis and degradation of chrysolaminarin in E. cf. polyphem (Table 6 and Figure 7). A single transcript encoding for UGPase (EC: 2.7.7.9) involed in the chrysolaminarin synthesis was identified, which uses G-1-P and UTP to generate UDP-glucose. We also found three transcripts of UDPG (EC: 2.4.1.34), which catalyzes the synthesis of β-1,3-glucan using UDP glucose as substrate. The degradation of chrysolaminarin involves the enzymes exo-Glu (EC: 3.2.1.58), endo-Glu (EC: 3.2.1.39) and BGL (EC: 3.2.1.21) (Table 6). There were two transcripts coding exo-Glu in E. cf. polyphem, which hydrolyzes the chrysolaminarin by sequentially cleaving glucose residues from the non-reducing end, releasing free glucose [55]. A single endo-Glu was found, which digests the principle β-1,3-linkages at random sites of chrysolaminarin, releasing smaller oligosaccharides. Small amounts of these oligosaccharides dominated with β-1,6-linkages derived from surviving chrysolaminarin branch points, could be further hydrolyzed by BGL to free glucose. Twenty-seven putative BGLs in E. cf. polyphem transcriptome were identified, all belonging to glycosyl hydrolase family 3. The free glucose generated from complete chrysolaminarin degradation could subsequently participate in the glycolysis pathway (Figure 8). 10.1371/journal.pone.0035142.g007Figure 7 Chrysolaminarin biosynthesis and degradation pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome. Identified enzymes are shown in boxes and include: UGPase, UDP glucose pyrophosphorylase (EC: 2.7.7.9); UDPG, chrysolaminarin synthase (EC: 2.4.1.34); exo-Glu, exo-1,3-β-glucanase (EC: 3.2.1.58); endo-Glu, endo-1,3-β-glucanase (EC: 3.2.1.39) and BGL, β-glucosidases (EC: 3.2.1.21). G-1-P, glucose-1-phosphate; PPi, pyrophosphate. 10.1371/journal.pone.0035142.g008Figure 8 Glycolysis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome. Identified enzymes are shown in boxes and include: HK, hexokinase (EC:2.7.1.1); GCK, glucokinase (EC: 2.7.1.2); G6PI, glucose-6-phosphate isomerase (EC: 5.3.1.9); PFK, phosphofructokinase-6 (EC: 2.7.1.11); FBA, fructose-bisphosphate aldolase (EC:4.1.2.13); TPI, triose-phosphate isomerase (EC: 5.3.1.1); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.9, 1.2.1.12); GPDH, glycerol-3-phosphate dehydrogenase (EC:1.1.1.8); PGK, phosphoglycerate kinase (EC: 2.7.2.3); PGAM, phosphoglycerate mutase (EC: 5.4.2.1); ENO, enolase (EC: 4.2.1.11); PK, pyruvate kinase (EC: 2.7.1.40); PDC, pyruvate decarboxylase (EC: 4.1.1.1); ADH, alcohol dehydrogenase (EC: 1.1.1.1); PDHC, the pyruvate dehydrogenase complex consisting of PDHB, pyruvate dehydrogenase (acetyl-transferring) (EC: 1.2.4.1), DLAT, dihydrolipoamide acetyltransferase (EC: 2.3.1.12), DLD, dihydrolipoyl dehydrogenase (EC: 1.8.1.4). G-6-P, glucose-6-phosphate; F-6-P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; GA3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; G-3-P, glycerol-3-phosphate; 1,3BPG, 1, 3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate. We did not identify any transcripts encoding enzymes involved in the biosynthesis and catabolism of starch, such as ADP-glucose pyrophosphorylase (AGPase), which produces ADP-glucose, the substrate for starch synthesis [56]. E. cf. polyphem cells do not possess these genes, which is consistent with the deficiency of starch in this microalgal cells. The absence of genes encoding AGPase is similar to the lack of a plastidic AGPase in diatom cells, which export all carbohydrates immediately from the plastids and store them as chrysolaminarin in cytosolic vacuoles [48], and further supports the fact that UDP glucose serve as the substrate to the synthesis of chrysolaminaran in E. cf. polyphem cells. Carotenoid biosynthesis Carotenoids are important for photosynthetic organisms, from bacteria and microalgae to higher plants, where they play crucial roles in photosystem assembly, light-harvesting, and photoprotection, and thus their function and biosynthesis have been reviewed extensively [57]–[63]. Carotenoid pigments also provide substrate precursors for the biosynthesis of phytohormones such as abscisic acid (ABA), which may explain an apparent role in mediating the adaptation of the plant to stress [64]. Carotenogenesis pathways and their enzymes are mainly investigated in cyanobacteria [65] and land plants [66]. Microalgae have common pathways with land plants and also additional microalgae-specific pathways and carotenoids. β-carotene, vaucheriaxanthin and violaxanthin are the main carotenoid pigments in the chloroplast of the eustimatophyceae [17], [67], [68]. Under N-limited conditions, E. cf. polyphem cells accumulate an amount of β-carotene, violaxanthin and vaucheriaxanthin (unpublished results). β-carotene serves as the precursors for vitamin A, retinal, and retinoic acid in mammals, thereby playing essential roles in nutrition, vision, and cellular differentiation, respectively [69], which could be further used for industrial production of bio-pharmaceutical. Based on the functional annotation of the transcriptome, we have successfully identified the genes encoding for key enzymes involved in the carotenogenesis of E. cf. polyphem (Table 7 and Figure 9). In the initial step of carotenogenesis, an isopentenyl pyrophosphate (IPP, C5) is added to farnesyl pyrophosphate by geranylgeranyl pyrophosphate synthase (GGPS, EC: 2.5.1.1, 2.5.1.10, 2.5.1.29), resulting in the formation of geranylgeranyl pyrophosphate (GGPP, C20). There are two biosynthetic pathways of IPP, the mevalonate (MVA) pathway for biosynthesis of isoprenoids from acetyl-CoA in cytoplasm, and an alternate nonmevalonate pathway that is operative in the plastids from glyceraldehyde-3-phosphate (GA3P) and pyruvate to IPP [70], [71], [72] (Table S5 and Figure S1). In a head-to-head condensation of the two GGPP compounds, the first carotene, phytoene (C40), is formed by phytoene synthase (PSY, EC: 2.5.1.32) using ATP [73], [74]. We identified two transcripts and one unigene coding for GGPS and PSY in E. cf. polyphem transcriptome library respectively. Next, four desaturation steps are catalyzed by two enzymes: phytoene dehydrogenase (PDS, EC: 1.14.99.-), ζ-carotene desaturase (ZDS, EC: 1.14.99.30) to form lycopene from phytoene. PDS catalyzes the first two desaturation steps, from phytoene to ζ-carotene through phytofluene. The additional two desaturation steps, from ζ-carotene to lycopene through neurosporene is catalyzed by ZDS. During desaturation by ZDS, neurosporene and lycopene are isomerized to poly-cis forms, and then carotenoid isomerase (CrtH, EC: 5.-.-.-) isomerizes to all-trans forms [75]. The number of the transcripts coding for the enzymes involving in these four desaturation reaction is three for PDS, two for ZDS and four for CrtH in the transcriptome library of E. cf. polyphem. Subsequently, lycopene is cyclized to be dicyclic carotenoids, as either β-carotene or α-carotene. Lycopene beta-cyclase (CrtY, EC: 1.14.-.-), exhibiting lycopene β-cyclase activity, catalyzes the dicyclic reaction of lycopene to β-carotene through γ-carotene. Distribution of α-carotene is limited in some algae classes, which possess lycopene epsilon-cyclase (CrtL-e, EC:1.14.-.-), a bifunctional enzyme having both lycopene ε-cyclase and lycopene β-cyclase activities. In these algae, lycopene is first converted to δ-carotene by CrtL-e, and then to α-carotene by CrtY [65], [76]–[78]. We identified one transcript coding for CrtY, but none genes coding for CrtL-e in E. cf. polyphem transcriptome. The lack of transcripsts coding for CrtL-e is consistent with the deficiency of α-carotene in E. cf. polyphem cells. Additionally, the β-end groups of β-carotene is hydroxylated by beta-carotene hydroxylase (CrtZ, EC: 1.14.13.-) to form zeaxanthin through β-cryptoxanthin. Epoxy groups are introduced into zeaxanthin by zeaxanthin epoxidase (ABA1, EC: 1.14.13.90) to produce violaxanthin through antheraxanthin. Under high light conditions, violaxanthin is conversed to zeaxanthin by violaxanthin de-epoxidase (VDE, EC: 1.10.99.3) for dispersion of excess energy from excited chlorophylls. Furthermore, one end group of violaxanthin is changed to an allene group of neoxanthin by neoxanthin synthase (NSY, EC: 5.3.99.9). Neoxanthin might be further hydroxylated to vaucheriaxanthin, but the pathway and enzymes is still unknown [78]. By cis-isomerase, violaxanthin and neoxanthin could be transformed to 9-cis-epoxycarotenoid (9-cis-violaxanthin and 9-cis-neoxanthin), which can be further used as the precursors for ABA. 10.1371/journal.pone.0035142.g009Figure 9 Carotenoid biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome. Identified enzymes are shown in boxes and include: GGPS, geranylgeranyl pyrophosphate synthase (EC: 2.5.1.1 2.5.1.10 2.5.1.29); PSY, phytoene synthase (EC: 2.5.1.32); PDS, phytoene dehydrogenase (EC: 1.14.99.-); ZDS, ζ-carotene desaturase (EC: 1.14.99.30); CrtY, lycopene beta-cyclase(EC: 1.14.-.-); CrtZ, β-carotene hydroxylase (EC: 1.14.13.-); ABA1, zeaxanthin epoxidase (EC: 1.14.13.90); VDE, violaxanthin de-epoxidase (EC: 1.10.99.3) and NSY, neoxanthin synthase (EC: 5.3.99.9). GA-3-P, glyceraldehyde-3-phosphate; IPP, isopentenyl pyrophosphate (C5); GGPP, geranylgeranyl pyrophosphate (C20). 10.1371/journal.pone.0035142.t007Table 7 Enzymes involved in carotenoid biosynthesis identified by annotation of the E. cf. polyphem transcriptome. Enzyme Symbol EC Number Number of transcripts 1%Sequence alignment with corresponding enzymes in model organisms (Accession #) C. reinhardtii P. tricornutum T. pseudonana Geranylgeranyl pyrophosphate synthase GGPS 2.5.1.1 2.5.1.10 2.5.1.29 2 2NM NM 70(XP_002288339.1) Phytoene synthase PSY 2.5.1.32 1 NM 55(XP_002178776.1) NM phytoene dehydrogenase PDS 1.14.99.- 3 67(XP_001690859.1) 37(XP_002183881.1) NM Zeta-carotene desaturase ZDS 1.14.99.30 2 NM NM NM Carotenoid isomerase CrtH 5.-.-.- 4 NM 32(XP_002176863.1)55(XP_002179244.1)54(XP_002182606.1) 63(XP_002295888.1) Lycopene beta cyclase CrtY 1.14.-.- 1 NM 44(XP_002176612.1) 45(XP_002287870.1) Beta-carotene hydroxylase CrtZ 1.14.13.- 1 NM NM NM Zeaxanthin epoxidase ABA1 1.14.13.90 4 NM 38(XP_002180238.1) 39(XP_002287317.1) Violaxanthin de-epoxidase VDE 1.10.99.3 2 41(XP_001695042.1) 43(XP_002180051.1) 42(XP_002289140.1) Neoxanthin synthase NSY 5.3.99.9 0 NM NM NM 9-cis-epoxycarotenoid dioxygenase NCED 1.13.11.51 5 NM NM NM Xanthoxin dehydrogenase ABA2 1.1.1.288 12 NM NM NM Abscisic-aldehyde oxidase AAO3 1.2.3.14 1 NM NM NM 1 In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported. 2 NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms. The annotation of E. cf. polyphem transcriptome has identified all the genes encoding enzymes involved in the ABA biosynthesis. It is proposed that ABA could be produced from the cleavage of carotenoids in an “indirect pathway" in the plants [79], [80]. The first committed step for ABA synthesis is the oxidative cleavage of a 9-cis-epoxycarotenoid to produce xanthoxin by 9-cis-epoxycarotenoid dioxygenase (NCED, EC: 1.13.11.51). Next, xanthoxin is oxidized by an NAD-requiring enzyme, xanthoxin dehydrogenase (ABA2, EC: 1.1.1.288) to form abscisic aldehyde. Finally, abscisic aldehyde is oxidized to ABA by abscisic-aldehyde oxidase (AAO3, EC: 1.2.3.14). Pathways interactions Our KEGG pathway assignments revealed that the metabolic pathways associated with biosynthesis and degradation of carbohydrate, fatty acids, TAGs and carotenoids in E. cf. polyphem are closely linked. Chrysolaminarin catabolism provides the metabolites for biosynthesis of other valuable products through the glycolysis pathway (Figure 8). The global pathway of glycolysis has been reviewed extensively [81]–[84]. We identified transcripts coding for all enzymes that involved in this pathway (Table S2). These enzymes include hexokinase (HK, EC: 2.7.1.1) and glucokinase (GCK, EC: 2.7.1.2), which phosphorylated the free glucose generated from the degradation of chrysolaminarin, resulting in glucose-6-phosphate (G-6-P). Additionally, a single transcript encoding for G-6-P isomerase (G6PI, EC: 5.3.1.9) was identified, which catalyzes the reversible transfer between G-6-P and fructose 6-phosphate (F-6-P). F-6-P was converted to fructose-1,6-bisphosphate (FBP) by the action of phosphofructokinase-6 (PFK, EC: 2.7.1.11). There were ten transcripts coding fructose-bisphosphate aldolase (FBA, EC:4.1.2.13), which catalysed the reversible aldol cleavage or condensation of FBP into dihydroxyacetone-phosphate (DHAP) and GA3P. The reduction of DHAP catalyzed by glycerol-3-phosphate dehydrogenase (GPDH, EC:1.1.1.8) resulted in G-3-P, the precursor for TAG biosynthesis. Pyruvate, the ultimate metabolite of cytosolic glycolysis, can be transported into the chloroplast and enter into a variety of central metabolic pathways, such as de novo biosynthesis of fatty acid [85], [86], and nonmevalonate pathway for synthesis of IPP, the precursor for carotenoid biosynthesis [70], [71] (Figure S1). There were 44 transcripts in E. cf. polyphem transcriptome coding for a pyruvate dehydrogenase complex (PDHC) (EC: 1.2.4.1, 2.3.1.12, 1.8.1.4) which transforms pyruvate into acetyl-CoA through pyruvate decarboxylation. Acetyl-CoA may then be used in the fatty acid synthesis pathway or involved in the MVA pathway for biosynthesis of isoprenoids [13], [70], [72]. Furthermore, we identified 3 transcripts coding for pyruvate decarboxylase (PDC, EC: 4.1.1.1), which generates acetaldehyde and CO2 from pyruvate, and 10 genes encoding for alcohol dehydrogenase (ADH, EC: 1.1.1.1), which uses acetaldehyde and NADH+H+ to generate ethanol, an important liquid biofuel. These finding demonstrated that biosynthesis and degradation of chrysolaminarin may direct the photosynthetic carbon flow into different storage compounds. Over expression of genes may increase the accumulation of lipids and carotenoids. Further investigations are warranted to determine the relative importance of these pathways in E. cf. polyphem. Conclusions With this study, we present a rapid and cost-effective method for transcriptome annotation of a non-model oleaginous microalga that has potential for production of biofuels and valuable co-products using Solexa/Illumina sequencing technology. The substantial amount of transcripts obtained provides a strong basis for future genomic research on oleaginous microalgae and supports in-depth genome annotation. Transcripts encoding key enzymes have been successfully identified and metabolic pathways involved in biosynthesis and catabolism of carbohydrate, fatty acids, TAGs and carotenoids in E. cf. polyphem have been reconstructed. These findings provide a substantial contribution to genetically manipulate this organism to enhance the production of feedstock for commercial microalgae-biofuels. Materials and Methods E. cf. polyphem culturing E. cf. polyphem was obtained from CAUP Culture Collection of Algae and deposited in our laboratory. Standard axenic cultures were maintained in the modified BG-11 medium (17.7 mM NaNO3, 0.22 mM K2HPO4, 0.3 mM MgSO4·7H2O, 0.24 mM CaCl2·2H2O, 31.2 µM Citric acid, 22.2 µM FeCl3·6H2O, 2.69 µM EDTA disodium salt, 0.19 mM Na2CO3, and 1 mL A5 trace elements solution) at 23±1°C, with continuous (24 hr) white fluorescent light illumination (300 µmol photons/m2·s), and agitated with air containing 5% (v/v) CO2. Experiments were performed using the Φ3×60 cm cylindrical glass photobioreactor at a cell density of approximately 2.7×105 cells/mL. Cultures were cultivated in N-replete (17.7 mM NaNO3), N-limited (5.9 mM NaNO3) and nitrogen free BG-11 medium. Analysis of carbohydrates and chrysolaminarin Cells from axenic cultures under N-replete and N-limited conditions at different growth phase are harvested by centrifugation, dried in a freeze drier and stored at −20°C until analysis, respectively. 50 mg freeze-dried algae powder was placed in a Teflon capped glass tube and extracted lipid according to Goldberg et al. [87]. Lipid-removal residues were then used for the extraction of total carbohydrate by hydrolysed with 4 mL of 0.5 M H2SO4 at 100°C for 4 hr [88]. Chrysolaminarin (β-1,3-glucan) was extracted according to Granum and Myklestad [89]. 50 mg freeze-dried algae power was extracted with 5 mL 0.05 M H2SO4 at 60°C for 10 min. Aliquots of the hydrolysates were assayed quantitatively for carbohydrate and chrysolaminarin by the phenol-sulphuric acid method of Dubois et al. [90]. RNA extraction and library preparation for transcriptome analysis Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol from pure axenic cultures of E. cf. polyphem grown under N-replete, N-limited and nitrogen free conditions which were snap-frozen and stored at −70°C until processing. RNA integrity was confirmed using the Agilent 2100 Bioanalyzer with a minimum integrity number value of 8. The samples for transcriptome analysis were prepared using Illumina's kit following manufacturer's recommendations. Briefly, mRNA was purified from 6 µg of total RNA using oligo (dT) magnetic beads. Following purification, mRNA is fragmented into small pieces using divalent cations under elevated temperature and the cleaved RNA fragments were used for first strand cDNA synthesis using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNaseH. These cDNA fragments then went through an end repair process and ligation of adapters. These products were purified and enriched with PCR to create the final cDNA library. Illumina sequencing and De novo assembly The cDNA library was sequenced from both of 5′ and 3′ends on the Illumina GA IIx platform according to the manufacturer's instructions. The fluorescent images process to sequences, base-calling and quality value calculation were performed by the Illumina data processing pipeline (version 1.4), in which 75 bp paired-end reads were obtained. The transcriptome datasets are available at the NCBI Sequence Read Archive (SRA) with the accession number SRA049088.1. Before assembly, the raw reads were filtered to obtain the high-quality clean reads by removing adaptor sequences, duplication sequences, the reads containing more than 10% ‘N’ rate (the ‘N’ character representing ambiguous bases in reads), and low-quality reads containing more than 50% bases with Q-value≤5. The Q-value is the quality score assigned to each base by the Illumina's base-caller Bustard from the Illumina pipeline software suite (version 1.4), similar to the Phred score of the base call. De novo assembly of the clean reads was performed using SOAPdenovo program (version1.03, http://soap.genomics.org.cn) which implements a de Bruijn graph algorithm and a stepwise strategy. Briefly, the clean reads were firstly split into smaller pieces, the ‘k-mers’, for assembly to produce contigs using the de Bruijn graph. The resultant contigs would be further joined into scaffolds using the paired-end reads. Gap fillings were subsequently carried out to obtain the complete scaffolds using the paired-end information to retrieve read pairs that had one read well-aligned on the contigs and another read located in the gap region. To reduce any sequence redundancy, the scaffolds were clustered using the Gene Indices Clustering Tools (http://compbio.dfci.harvard.edu/tgi/software/). The clustering output was passed to CAP3 assembler for multiple alignment and consensus building. Others that can not reach the threshold set and fall into any assembly should remain as a list of singletons. Functional annotation and classification All Illumina assembled unigenes longer than 200 bp were annotated by the assignments of putative gene descriptions, conserved domains, GO terms, and putative metabolic pathways to them based on sequence similarity with previously identified genes annotated with those details. For assignments of predicted gene descriptions, the assembled unigenes were compared to the plant protein dataset of NR, the Arabidopsis protein dataset of NR, and Swiss-Prot/Uniprot protein database respectively using BLASTALL procedure (ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.18/) with a significant threshold of E-value≤10−5. To parse the features of the best BLASTX hits from the alignments, putative gene names, ‘CDS’, and predicted proteins of corresponding assembled sequences can be produced. At the same time, the orientation of Illumina sequences which failed to be obtained directly from sequencing can be derived from BLAST annotations. For other sequences falling beyond the BLAST, ESTScan program (version 3.0.1, http://www.ch.embnet.org/software/ESTScan.html) was used to predict the ‘CDS’ and orientation of them. And then, since a large portion of assembled unigenes have not yet been annotated, conserved domains/families were further identified in the assembled unigenes using the InterPro database (version 30.0, HMMpfam, HMMsmart, HMMpanther, FPrintScan, ProfileScan, and BlastProDom), Pfam database (version 24.0) and COG database at NCBI (as of December 2009, ftp://ftp.ncbi.nih.gov/pub/wolf/COGs/). Domain-based comparisons with the InterPro, Pfam and COGs databases were performed using InterProScan (version 4.5, ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/), HMMER3 (http://hmmer.janelia.org) and BLAST programs (E-value threshold: 10−5), respectively. Functional categorization by GO terms (GO; http://www.geneontology.org) was carried out based on two sets of best BLASTX hits from both the plant and Arabidopsis protein datasets of NR database using Blast2GO software (version 2.3.5, http://www.blast2go.de/) with E-value threshold of 10−5. The KEGG pathways annotation was performed by sequence comparisons against the Kyoto Encyclopedia of Genes and Genomes database using BLASTX algorithm (E-value threshold: 10−5). Supporting Information Table S1 Enzymes involved in Calvin cycle identified by annotation of the E. cf. polyphem transcriptome. (DOC) Click here for additional data file. Table S2 Enzymes involved in glycolysis identified by annotation of the E. cf. polyphem transcriptome. (DOC) Click here for additional data file. Table S3 Enzymes involved in pentose phosphate pathway identified by annotation of the E. cf. polyphem transcriptome. (DOC) Click here for additional data file. Table S4 Enzymes involved in Citrate cycle (TCA cycle) identified by annotation of the E. cf. polyphem transcriptome. (DOC) Click here for additional data file. Table S5 Enzymes involved in biosynthetic pathways of isopentenyl pyrophosphate identified by annotation of the E. cf. polyphem transcriptome. (DOC) Click here for additional data file. Figure S1 Biosynthetic pathways of isopentenyl pyrophosphate. Identified enzymes are shown in boxes and include: DXS, 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7); DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC:1.1.1.267); IspD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (EC:2.7.7.60); IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC:2.7.1.148); IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC:4.6.1.12); IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (EC:1.17.7.1); IspH, 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase (EC:1.17.1.2); PDHA, pyruvate dehydrogenase (EC: 1.2.1.51); AtoB, acetoacetyl-CoA thiolase (EC:2.3.1.9); HMGS, hydroxymethylglutaryl-CoA synthase (E2.3.3.10); HMGR, hydroxymethylglutaryl-CoA reductase (NADPH)(EC:1.1.1.34); MK, mevalonate kinase (EC:2.7.1.36); PMK, phosphomevalonate kinase (EC:2.7.4.2); MVAD, diphosphomevalonate decarboxylase(EC:4.1.1.33); PDHC, the pyruvate dehydrogenase complex consisting of PDHB, pyruvate dehydrogenase (acetyl-transferring) (EC: 1.2.4.1), DLAT, dihydrolipoamide acetyltransferase (EC: 2.3.1.12), and DLD, dihydrolipoyl dehydrogenase (EC: 1.8.1.4). GA3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methylerythritol; CDP-MEP, CDP-ME 2-phosphate; MEC, 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate; HMG-CoA, hydroxymethylglutaryl-CoA; MVA, mevalonate; MVAP, mevalonate-5-phosphate; MVAPP, mevalonate-5-diphosphate; IPP, isopentenyl pyrophosphate (C5); GGPP, geranylgeranyl pyrophosphate (C20). (TIF) Click here for additional data file. We acknowledge the Beijing Genomics Institute (BGI) Shenzhen for its assistance in original data processing and related bioinformatics analysis. Competing Interests: The authors have declared that no competing interests exist. Funding: The project was supported by the National High Technology Research and Development Program of China (2009AA06440), the National Basic Research Program of China (2011CB2009001), the National Natural Science Foundation of China (31170337) and the Fundamental Research Funds for the Central Universities (21611309). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Hirsch RL Bezdek R Wendling R 2006 Peaking of world oil production and its mitigation. AIChE J 52 2 8 DOI:10.1002/aic.10747 2 Dinh LTT Guo Y Mannan MS 2009 Sustainability evaluation of biodiesel production using multicriteria decision-making. Environ Prog Sustain 28 38 46 DOI:10.1002/ep.10335 3 Yun Y-S Lee SB Park JM Lee C-I Yang J-W 1997 Carbon dioxide fixation by algal cultivation using wastewater nutrients. 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==== Front BMC Syst BiolBMC Syst BiolBMC Systems Biology1752-0509BioMed Central 1752-0509-6-202243343710.1186/1752-0509-6-20Research ArticlePredicting new molecular targets for rhein using network pharmacology Zhang Aihua [email protected] Hui [email protected] Bo [email protected] Xijun [email protected] National TCM Key Lab of Serum Pharmacochemistry, Heilongjiang University of Chinese Medicine, Heping Road 24, Harbin 150040, China2 Key Pharmacometabolomics Platform of Chinese Medicines, Heping Road 24, Harbin 150040, China2012 21 3 2012 6 20 20 12 1 2012 21 3 2012 Copyright ©2012 Zhang et al; licensee BioMed Central Ltd.2012Zhang 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 Drugs can influence the whole biological system by targeting interaction reactions. The existence of interactions between drugs and network reactions suggests a potential way to discover targets. The in silico prediction of potential interactions between drugs and target proteins is of core importance for the identification of new drugs or novel targets for existing drugs. However, only a tiny portion of drug-targets in current datasets are validated interactions. This motivates the need for developing computational methods that predict true interaction pairs with high accuracy. Currently, network pharmacology has used in identifying potential drug targets to predicting the spread of drug activity and greatly contributed toward the analysis of biological systems on a much larger scale than ever before. Methods In this article, we present a computational method to predict targets for rhein by exploring drug-reaction interactions. We have implemented a computational platform that integrates pathway, protein-protein interaction, differentially expressed genome and literature mining data to result in comprehensive networks for drug-target interaction. We used Cytoscape software for prediction rhein-target interactions, to facilitate the drug discovery pipeline. Results Results showed that 3 differentially expressed genes confirmed by Cytoscape as the central nodes of the complicated interaction network (99 nodes, 153 edges). Of note, we further observed that the identified targets were found to encompass a variety of biological processes related to immunity, cellular apoptosis, transport, signal transduction, cell growth and proliferation and metabolism. Conclusions Our findings demonstrate that network pharmacology can not only speed the wide identification of drug targets but also find new applications for the existing drugs. It also implies the significant contribution of network pharmacology to predict drug targets. ==== Body Background Developing a new drug is an expensive and time-consuming process that is subject to a variety of regulations such as drug toxicity monitoring and therapeutic efficacy. Lengthy development procedures and the high risk of unexpected side-effects in advanced-stage clinical trials reduce the ability of the drug development process to be innovative. However, the organization of our rapidly growing knowledge on diseases, disease-related genes, drug targets and their structures, and drugs and their chemical structures gives us another exciting way to discover novel areas of drug development. Several networks have recently been constructed to help drug discovery [1]. Meanwhile, finding the potential application in other therapeutic categories of drugs by predicting their targets is an efficient and time-saving method in drug discovery [2]. Additionally, predicting interactions between drugs and target proteins can help decipher the underlying biological mechanisms. Therefore, there is a strong incentive to develop powerful statistical methods that are capable of detecting these potential drug-protein interactions. Various methods have been proposed to address the drug-target prediction problems. One common method is to predict the drugs interacting with a single given protein based on the chemical structure similarity in a classic classification framework. Nevertheless, all the methods did not utilize a wealth of information to assist prediction. Despite the dramatic increase of global spending on drug discovery and development, the approval rate for new drugs is declining, due chiefly to toxicity and undesirable side effects. Simultaneously, the growth of available biomedical data in the postgenomic era has provided fresh insight into the nature of drug-target pathways. This stagnation in drug approval can be overcome by the novel concept of network pharmacology, which is built on the fundamental concept that drugs modulate the multiple targets. Network pharmacology can be studied with molecular networks that integrate multidisciplinary concepts including cheminformatics, bioinformatics, and systems biology. Network analysis has become a cornerstone of fields as diverse as systems biology. Many studies have successfully reported interesting biological findings from these networks, including the relationships between various statistical properties of a gene and its function at the molecular level based on networks [3]. Network pharmacology can make an impact at several points in the drug-development process: target identification, lead discovery and optimization, mode of action, preclinical efficacy and safety assessment. Therefore, it could facilitate the systematic characterisation of drug targets, thereby helping to reduce the typically high attrition rates in discovery projects. Various approaches have been proposed for this task such as Bayesian Networks, models based on information theory, regression based models, and differential equation models [4,5]. Software to integrate and analyse the interactions and their attributes plays an increasingly important role. The most widely used open source network visualization workbench is Cytoscape, a popular bioinformatics package for biological network visualization and data integration, for screening the central nodes of the network, exploiting functional study of the central node genes [6]. Cytoscape software, a web-based network visualization tool, is an open-source software platform for visualizing molecular interaction networks and integrating these interactions with gene expression profiles and other functional genomics data. Data from various sources can be imported into this tool to build networks, and to highlight specific node or edge features. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. Cytoscape's main functionality is focused on the construction of networks. Currently, the network model is based on genes, proteins and functional relationships between them such as protein-protein, protein-dna regulatory interactions and gene-protein coding relationship [7]. Rhubarb, one of popular traditional Chinese herbal medicine, has been widely used for the treatment of diseases in traditional Chinese medicine. Rhein (Figure 1) is one of the most bioactive components from rhubarb. Earlier studies have identified rhein as a potent inhibitor of hepatic fibrosis induced by carbon tetrachloride, which is capable of reducing SMA expression, collagen synthesis and deposition in TGF-stimulated hepatic stellate cells. A recent study showed that rhein contributes to induce apoptosis of human cancer, including lung adenocarcinoma A549, myelogenous leukemia HL-60, lung squamous carcinoma CH27, cervical carcinoma HeLa cells, neuroblastoma IMR-32, bladder cancer T24, and hepatoma HepG2 cells [8]. It can inhibits cellular proliferation, induces apoptosis, and prevents metastasis through activation of tyrosine kinases, phosphoinositol 3-kinase, protein kinase C, NF-kappa B, and mitogen-activated protein kinase signaling cascades. It has antitumor properties through the p53 and its downstream p21 pathway, also reduce tumor size, prolong survival, decrease incidence of tumor invasion and neovascularization. Interestingly, rhein blocks the uptake of glucose in tumor cells, causing changes in membrane associated functions to trigger cell death. Although these results suggest that rhein may be a potent antitumor drug, and its therapeutic mechanism were still completely unclear. Figure 1 Chemical structure of rhein. ChemSpider ID: 9762; Systematic name: 4,5-Dihydroxy-9,10-dioxo-9,10-dihydro-2-anthracenecarboxylic acid; Molecular Formula: C15H8O6; Mass: 284.032074 Da. Reconstructing networks of biological system entities such as genes, transcription factors, proteins, compounds and other regulatory molecules are very important for understanding the biological processes. Identifying drug targets is a critical step in network pharmacology. Recent years network pharmacology has influenced all areas of life sciences including that of drug mechanism and development, new target discovery [9]. Efficient identification of drug targets is one of major challenges for drug discovery and drug development. Computational integration of different knowledge sources is a more effective approach and wealth of SysBiomics data provides unprecedent opportunities for drug target identification [10]. Although a number of computational approaches have been developed to integrate data from multiple sources for the purpose of predicting or prioritizing candidate disease genes, relatively few of them focus on identifying or ranking drug targets. To address this deficit, we construct a biological network to provide a global view of drug interactions and prioritize drug candidate targets. We demonstrate the applicability of integrative network pharmacology approaches to identify potential drug targets and candidate genes by employing information extracted from public databases. In the present investigation, we give an illustrative example to show that the potential drug target identification problem can be solved effectively by our method, which may become an effective strategy for the discovery of new drugs. Methods Interaction information was retrieved from NCBI's Entrez Gene in December 16, 2011. Genes/proteins molecular function, biological processes and cellular component is imported from the Gene Ontology project; while information on biochemical pathways is taken from KEGG. Additional information includes links to databases, such as Reactome, BioCyc, NCI Nature PID, DIP, BIND, HPRD, BioGRID, MINT, and Intact, which represent the major repositories of interacions from multiple organisms for further bioinformatics analysis. The associations between the diseases and genes were from the OMIM (Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/omim). The most stable one is the Entrez GeneID, which is the unique identifier for a gene in NCBI's Entrez Gene database. Cytoscape has being developed for reconstruction and visualization of networks. Nodes (represented as circles) in the interactome correspond to genes, and edges (connecting lines) represent documented interactions. Cytoscape is a desktop Java application and source code for Cytoscape 2.8 are available for download from http://cytoscape.org. Its default annotations are parsed from the GO information available from NCBI http://www.ncbi.nlm.nih.gov/Ftp/. Results Module annotation and visualization Functional annotation for predicted bindingtargets in our models by looking for enriched terms from multiple functional databases. Since modules often show strong functional coherence, the diverse set of annotations provide a complementary overview of module function. For pathway visualization and analysis of networks, we used open-source Cytoscape version 2.8 software. In Cytoscape, networks are represented as graphs where the nodes are the entities (e.g. genes, proteins) and the edges their interactions (e.g. reactions). For the visualization in the context of biological networks, we developed the different node attribute files and visual style files that can easily be imported into Cytoscape. This benchmark was run on a standard desktop computer (4 GHz Pentium intel with 2 GB of memory running Windows XP). Constructing regulatory network We used Cytoscape version 2.8 to model the signaling network. Taking the closely-connected and co-expressed differentially expressed genes, for the network shown in Figure 2 and 3, with 99 nodes and 153 edges. Four of them are significant frequencies and are likely to be responsible for driving the initiation, progression, or maintenance of disease. We find that 3 genes considered linked because they had close relationship and suggested as novel susceptibility genes using the Cytoscape visualization software. The 3 perturbed genes in the network that has strong connections to the genes. This is indicated by the thick edges between these nodes. Figure 2 Solid map on huge interactome of rhein-targets networks, built and visualized with Cytoscape. Edges: interactions. Nodes: specific proteins or genes. Central nodes (Yellow) of the interaction network were used to illustrate the gene expression obtained and represents significant change in expression. The connections between molecules show molecular interactions identified in the interactome. Gene expression illustrated in colored nodes was selected with a p < 0.05 value. Figure 3 Illustration and visualization of the interactome network (Circle Layout). Drugs and proteins are linked as per the known drug-target network. Discrete network map with a customized visual Cytoscape Web style. Yellow nodes refer to the significant ontologies of the target. This subnetwork represents a coregulated unit containing 99 nodes and 153 edges. Predicting drug targets Another issue clearly meriting further study is the performance of our proposed method in identifying drug targets. We believe these results are indicative of the multi-level nature of key perturbations, where direct interactions often take place at the protein and/or metabolite level, and therefore do necessarily affect expression level of the encoding genes. Main pathway information, 3 targets are observed in the Cytoscape-based network with significant P-values. Figure 4 denotes the identification of novel molecular targets using network analysis. These closely connected and coexpressed differentially expressed genes and proteins in the networks are regarded as the signatures of the rhein underlying targets. Regulatory sub-network of rhein targets through network pharmacology was constructed, and shown in Figure 5. The detail information of the novel molecular targets were shown in additional Table 1. Figure 4 Identification of novel molecular targets using network analysis. A: MMP2; B: MMP9; C: TNF. Figure 5 Regulatory sub-network of differentially expressed TNF of rhein through network pharmacology. Table 1 Information of the novel molecular targets GeneID Entry Gene name Disease Pathway 4313 MMP2 Cancer GnRH signaling pathway Choriocarcinoma Leukocyte transendothelial migration Torg-Winchester syndrome Pathways in cancer 4318 MMP9 Penile cancer Pathways in cancer Metaphyseal dysplasias Leukocyte transendothelial migration Transcriptional misregulation in cancer 7124 TNF Asthma MAPK signaling pathway Systemic lupus erythematosus Cytokine-cytokine receptor interaction Malaria Natural killer cell mediated cytotoxicity Pertussis NOD-like receptor signaling pathway Type I, II diabetes mellitus Toll-like receptor signaling pathway Alzheimer's disease RIG-I-like receptor signaling pathway Amoebiasis Natural killer cell mediated cytotoxicity Tuberculosis T cell receptor signaling pathway Hepatitis C Fc epsilon RI signaling pathway Influenza A Apoptosis HTLV-I infection TGF-beta signaling pathway Herpes simplex infection Natural killer cell mediated cytotoxicity Cardiovascular Diseases; Hematopoietic cell lineage Discussion Network pharmacology approach seeks to comprehend the complexity of organisms by combining many different kinds of data (protein-protein and protein-DNA interactions, protein modifications, biochemistry, etc.) to create predictive models. In the era of SysBiomics, the focus on understanding complex organisms is shifting from studying individual genes and proteins towards the relationships between them [11,12]. These relationships are usually expressed in terms of various kinds of biological networks that are the focus of many functional genomics studies. Systems biology is characterized by a focus on interaction networks--the biomolecules involved in a particular biological system or process, as well as the relationships between these components. Network pharmacology is used for visualizing and understanding these interactions, interpreting high-throughput experimental data, generating hypotheses and sharing results [13]. These diagrams can be difficult for a user to explore with currently available network display tools--the networks are often too large, on the order of thousands of nodes, and many tools do not provide biological context to the diagram. The increasing complexity of functional genomics data drives the development of methods and tools for data integration and visualization [14]. Interactions network models are crucially important for disease processes [15]. Many of the important properties of biological systems emerge as a result of the interactions among genes and among their protein products. Genes and the proteins they encode participate in gene-gene, gene-protein, and protein-protein interactions to mediate a wide variety of biological processes. Molecular interaction networks can be efficiently studied using network visualization software. Cytoscape that can generate a putative protein-protein interaction network for target genomes, make the creation of protein-protein interaction network predicting tools possible. Its central organizing principle is a network graph, with biological entities (e.g. genes, proteins) represented as nodes and biological interactions represented as edges between nodes. Data are integrated with the network using attributes, which map nodes or edges to specific data values such as gene expression levels or protein functions. Taken together, these features provide a mechanism for expressing relationships between sets of data while simultaneously visualizing the integrated results. In this study, we applied Cytoscape to explore targets expression data in the context of biological network information. Of note, Cytoscape successfully provided us with valuable clues for identification of drug-target interactions on a large scale. Rhein, a classic natural product, has been efficiently used for cancer relief in Asia, although its mechanism remains unclear. A promising approach in drug target discovery involves the integration of available metabolites data through mathematical modeling and data mining. Significant work has been done on drug discovery, however, few papers were discussed with the interaction network. This study was designed to further elucidate the underlying mechanism of rhein from the network pharmacology. Of note, 3 differentially expressed genes were observed. The characteristic functions of the differentially expressed proteins were based on biological processes such as immunity, cellular apoptosis, transport, signal transduction, cell growth and proliferation and metabolism. The detection of these proteins with distinct regulatory patterns provides evidence that novel biomarkers are actively involved in multifunctional pathways. Proteins of the matrix metalloproteinase (MMP2 and 9) family are involved in the breakdown of extracellular matrix in normal physiological processes, such as reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. This gene encodes an enzyme which degrades type IV collagen, the major structural component of basement membranes. The enzyme plays a role in endometrial menstrual breakdown, regulation of vascularization and the inflammatory response. Tumor necrosis factor (TNF) encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor superfamily. This cytokine is mainly secreted by macrophages. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. This cytokine has been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer. The dominant paradigm in drug discovery is the concept of designing maximally selective drug targets. However, many effective drugs act via modulation of multitargets rather than single targets. Advances in systems biology are revealing that integrated network biology holds the promise of expanding the current opportunity space for drug targets [16]. Identification of drug targets is one of the major tasks in drug discovery [17]. Under these circumstances, there is an urgent need to integrate phenotypic and chemical indexes together and develop new methods to predict drug-target interactions on a large scale. With the development of systems biology and the emergence of network pharmacology approach, it has been possible to integrate multidimensional information and heterogeneous data in drug studies [18]. Our method benefits from current knowledge such as the known drug-target interactions, more importantly, extends the candidate target proteins to a genome-wide scale, which greatly enlarges the number of known targets. Together with known drug-target interactions, such information makes it possible to relate pharmacological space with genomic space. Thus, we believe that combining the integration of multi-dimensional information in pharmacological space and genomic space gains advantages in target identification information could help to generate further drug discovery. Drug target is a key molecule involved in a signaling pathway that is specific to a disease condition [19-23]. Drugs can be designed to modify the functioning of the pathway in the diseased state by inhibiting a key molecule, or to enhance the normal pathway by promoting specific molecules that may have been affected in the diseased state and can influence the whole biological system by targets. Identification of drug target is the essential first step in new drug discovery and development [24]. Discovery of drug targets through network pharmacology analysis promises to be a useful and novel approach in this direction. Of note, we have characterized 3 specific genes relevant for drug target discovery and found drug-target interaction networks involve receptors, neurotransmitter, enzymes, signal transduction. These results suggest that network analysis can be an effective means to prioritize drug target interactions for further study. Conclusions System networks that are a central paradigm in biology will help us identifying new drug targets which in turn will generate more in-depth understanding of the mechanism of diseases. Network-based pharmacology is emerging as an important paradigm for analysis of biological systems. In this paper, we presented a integrated approach to predict targets for rhein by exploring network pharmacology, integrating information from chemical space, genomic space and drug-protein interaction network space. Furthermore, network interactions allowed us to confirm some strongly-predicted drug-target interactions on the data sets obtained using our method. Analyzing the topology of the network, we have detected 3 potential drug targets and predicted the major interactome by using validated Cyoscape method. The identified targets were found to encompass a variety of biological processes related to immunity, cellular apoptosis, transport, signal transduction, cell growth and proliferation and metabolism. Perturbed proteins tend to be highly coexpressed and functionally coherent and we have used this property for predicting drug targets and associating novel functions to drug. The findings demonstrate that the network target-based methods are of importance for elucidating the inter-relationship between complex diseases and drug interventions through the network target paradigm estimating. Competing interests The authors declare that they have no competing interests. Authors' contributions XJW and AHZ conceived of the study. AHZ and HS carried out the data analysis, simulations, and drafted the manuscript. HS analyze the results. BY carried out extensive revisions to the manuscript. All authors read and approved the final content. Acknowledgements This work was supported by grants from the Key Program of Natural Science Foundation of State (Grant No. 90709019), the National Specific Program on the Subject of Public Welfare (Grant No. 200807014), National Key Subject of Drug Innovation (Grant No. 2009ZX09502-005), and National Program on Key Basic Research Project of China (Grant No. 2005CB523406). ==== Refs Gilchrist M Thorsson V Li B Rust AG Korb M Roach JC Kennedy K Hai T Bolouri H Aderem A Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor Nature 2006 441 173-178 16688168 Hopkins AL Network pharmacology: the next paradigm in drug discovery Nat Chem Biol 2008 4 682 690 10.1038/nchembio.118 18936753 Li S Zhang B Zhang N Network target for screening synergistic drug combinations with application to traditional Chinese medicine BMC Syst Biol 2011 5 Suppl 1 S10 10.1186/1752-0509-5-S1-S10 21689469 Fang K Zhao H Sun C Lam CM Chang S Zhang K Panda G Godinho M Martins dos Santos VA, Wang J. 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==== Front Clin Transl AllergyClin Transl AllergyClinical and Translational Allergy2045-7022BioMed Central 2045-7022-1-92240982910.1186/2045-7022-1-9ResearchVitamin C and asthma in children: modification of the effect by age, exposure to dampness and the severity of asthma Hemilä Harri [email protected] Mohammed [email protected] Ahmed A [email protected] Department of Public Health, University of Helsinki, Helsinki, Finland2 Department of Paediatrics, Faculty of Medicine, Tanta University, Tanta, Gharbia Governorate, Egypt2011 25 8 2011 1 9 9 26 5 2011 25 8 2011 Copyright ©2011 Hemilä et al; licensee BioMed Central Ltd.2011Hemilä 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 We previously found a significant benefit of vitamin C supplementation in asthmatic children. Purpose To test whether the effect of vitamin C on asthma is heterogeneous over the participant population. Methods Egyptian asthmatic children between 7 and 10 years of age (n = 60) were included in the cross-over trial. They were administered 0.2 grams per day of vitamin C and placebo for separate 6-week periods. The variation in the vitamin C effect on two clinically relevant outcomes was analyzed: the childhood asthma control test (C-ACT), which measures the severity of asthma symptoms (the scale ranges from 0 to 27 points, < 20 points indicating unsatisfactory asthma control), and FEV1. We used linear modeling to examine the variation of the vitamin C effect in the subgroups. Results The effect of vitamin C on the C-ACT was significantly modified by age and baseline C-ACT levels. In the children aged 7.0-8.2 years with a baseline C-ACT of 18 to 19 points, vitamin C increased the C-ACT score by 4.2 points (95% CI: 3.3-5.3); whereas in the children aged 8.3-10 years who had a baseline C-ACT of 14 to 15 points, vitamin C increased the C-ACT score by only 1.3 points (95% CI: 0.1-2.5). The effect of vitamin C on the FEV1 levels was significantly modified by age and exposure to dampness. In the children aged 7.0-8.2 years with no exposure to dampness, vitamin C increased the FEV1 level by 37% (95% CI: 34-40%), whereas in the children aged 8.3-10 years with exposure to dampness or mold in their bedroom more than one year prior to the study, vitamin C increased the FEV1 level by only 21% (95% CI: 18-25%). Conclusions We found strong evidence that the effect of vitamin C on asthmatic children is heterogeneous. Further research is needed to confirm our findings and identify the groups of children who would receive the greatest benefit from vitamin C supplementation. age factorsascorbic acidasthmachildeffect modifiersforced expiratory volumeobstructive lung diseasesquality of lifecontrolled trials ==== Body Background Proposals that vitamin C might be beneficial in the treatment of asthma date back to the 1940s [1,2]. Nevertheless, the role of vitamin C is still undefined. A study of Nigerian asthmatics reported a 78% lower incidence of asthma attacks in those administered vitamin C [3], whereas a study of British asthmatics found no effect of vitamin C on the symptoms or on the FEV1 levels [4]. Three trials found that vitamin C reduces bronchoconstriction caused by exercise in subjects who suffer from exercise-induced bronchoconstriction (EIB) [5-7]. Although these three EIB studies imply that vitamin C may have an effect on lung function, the findings cannot be generalized to patients with other variants of asthma. There is no well-defined mechanism whereby vitamin C may have an effect on asthma. Nevertheless, vitamin C influences the production of various prostanoids in lung tissues [8-11]. Indomethacin reverses the effect of vitamin C on bronchoconstriction in guinea pigs [10-13] and humans [14,15]. Thus, the effect of vitamin C on airways might be, at least partly, mediated by influences on the prostanoid metabolism. Furthermore, in asthmatic patients, the level of vitamin C is low in plasma [1,16-18] and bronchoalveolar fluid [19]. Although such a correlation does not imply a causal relationship, it encourages research on vitamin C and asthma. We have previously carried out a placebo-controlled cross-over trial in which we examined the effect of vitamin C, zinc, omega-3 fatty acids and their combination in Egyptian asthmatic children [20]. Vitamin C significantly decreased asthma symptoms and increased the FEV1 levels [20]. We reasoned that the effect of vitamin C might be greater in children who had low baseline FEV1 levels and found that the effect was modified by baseline FEV1 (unpublished). Therefore we decided to carry out a formally planned subgroup analysis of the data. In this subgroup analysis, we planned to use two clinically relevant outcomes: asthma symptoms as measured by the childhood asthma control test (C-ACT) [21,22] and pulmonary function as measured by FEV1. We planned to examine the effect of six baseline variables: the C-ACT, the FEV1/FVC ratio, gender, paternal smoking, exposure to dampness or mold in the bedroom, and residential neighborhood. In this subgroup analysis, we decided to use the baseline FEV1/FVC ratio instead of the baseline FEV1 since the former adjusts for the variation in the size of lungs. Methods Participants and study design The design and methods of the trial have been described earlier [20]. In brief, 72 Egyptian children between 7 and 10 years of age, who were diagnosed of having moderate persistent asthma (see [21] for the diagnostic criteria), were included in the trial. Twelve children were lost at follow-up due to change in their residence. This study reports the findings of the remaining 60 children (Table 1). The study was a randomized, double-blind, placebo-controlled cross-over trial carried out over 38 weeks. After a pre-trial assessment period of 2 weeks, the children entered the study on their normal diet, after which they entered five different 6-week therapeutic phases in a random sequence, with observers, participants and families blinded to the treatment: placebo, vitamin C, zinc, omega-3 fatty acids, and a combination of the three. Each phase was followed by a 2-week washout period before the next phase. Thus, by the end of the study, all the children had been exposed to the five treatment phases (placebo included) but in different sequences. In the vitamin C phase, the children were administered 0.2 g per day of ascorbic acid in capsules that appeared identical to the placebo capsules. The trial was approved by the Department Council and the Faculty Ethical committee. The current subgroup analysis is restricted to the comparison of the vitamin C and placebo phases. Table 1 Demographic data of the asthma patients Number of children who started the trial 76 Number of children who completed the trial 60 Mean (SD) Age (yr) 8.4 (1.0) Weight (kg) 32 (18) Height (cm) 124 (21) BMI (kg/m2) 17.2 (1.3) No. of children Boys/Girls 38/22 Urban/Rural 34/26 Associated nasal allergy 12 Asthma medications:  Long acting β2-agonist alone 0  Moderate daily dose inhaled corticosteroid 16  Long acting β2-agonist + inhaled corticosteroid 20  β2-agonist + inhaled corticosteroid + intermittent short acting β2-agonist 18  β2-agonist + inhaled corticosteroid + sodium cromoglycate 6 Allergy medications:  Antihistamine 24  Intermittent nasal decongestant 24  Nasal corticosteroid 16 Background data and outcomes At the beginning of the study and at the end of each treatment phase, the severity of asthma was assessed using the childhood asthma control test (C-ACT), and a pulmonary function test was performed. The C-ACT is a questionnaire for asthmatic children and their parents for identifying children aged 4-11 years whose asthma is inadequately controlled: the scale ranges from 0 to 27 points, < 20 points indicating unsatisfactory asthma control [22,23]. Spirometry was performed in a sitting position by all the children (spirometer: Morgan TLC Test Mk 11, Morgan Scientific, Haverhill, MA, USA). Participants performed three acceptable FVC maneuvers, and the highest FEV1 value was recorded. Information on the presence of dampness or mold in the bedroom was obtained with a questionnaire based on four items confirmed by parent reporting [24]: 1) mold odor (n = 3), 2) visible mold (n = 29), 3) moisture (n = 19), 4) water damage (n = 21). Dampness or mold in the bedroom was defined as one or more positive responses. The question about the time of exposure to dampness had two alternatives: 1) "during the past 12 months" and 2) "only earlier" (i.e. more than one year prior to the study); only one child chose both the recent and earlier exposure alternatives. As the outcome for the FEV1 change, we calculated the percentage increment in the FEV1 value between the end of the vitamin C and placebo phases. As the primary outcome for the C-ACT change, we calculated the arithmetic difference in the C-ACT between the end of the vitamin C and the placebo phases. Since we found that the vitamin C effect on the C-ACT was greater on participants with high baseline C-ACT values (Table 2), in Table 3 we also calculated the percentage increment in the C-ACT scores as a secondary outcome. In the normal plot, the distribution of the changes in the C-ACT and FEV1 were quite close to the normal distribution. Table 2 Effect of vitamin C on the symptoms of asthmatic children Subgroup No. of Children C-ACT (mean) Difference in C-ACT Test for interaction (P) Placebo Vitamin C Estimate 95% CI All 60 16.57 19.60 3.03 2.53-3.54 C-ACT at baseline 13-15 30 15.8 18.1 2.3 1.7-3.0 0.004a) 16-19 30 17.3 21.1 3.7 3.0-4.5 FEV1/FVC (%) < 59 28 16.4 19.3 2.9 2.3-3.6 0.7a) ≥59 32 16.8 19.9 3.1 2.3-3.9 FEV1 at baseline (L/s) < 1.1 29 16.7 20.3 3.6 3.0-4.3 0.013a) ≥1.1 31 16.5 18.9 2.5 1.7-3.2 Age (yr) 7.0-8.2 30 16.6 20.3 3.7 3.1-4.3 0.004a) 8.3-10 30 16.5 18.9 2.3 1.5-3.1 Weight (kg) 23-28 29 16.6 19.3 2.7 1.9-3.5 0.2a) 29-37 31 16.6 19.9 3.3 2.7-4.0 Gender Girl 22 16.5 19.5 3.0 2.2-3.9 1.0 Boy 38 16.6 19.6 3.0 2.3-3.7 Dampness exposure Never 25 16.8 19.2 2.4 1.4-3.4 0.1b) During past 1 yr 20 16.3 19.7 3.4 2.6-4.2 Earlier 14 16.5 20.1 3.6 2.8-4.4 Smoking by the father Never 21 16.5 19.0 2.5 1.6-3.5 0.4c) Current 22 16.8 19.8 3.0 2.2-3.9 Ex-smoker 17 16.4 20.0 3.6 2.7-4.6 Residential area Urban 34 16.5 19.5 3.0 2.4-3.7 1.0 Rural 26 16.7 19.7 3.0 2.2-3.9 a) The test of interaction was calculated by using dichotomized variables for the C-ACT, FEV1/FVC ratio, FEV1, age, and weight (cut points at the medians). The test of interaction by using the continuous variables gives P = 0.0002 for the baseline C-ACT and P = 0.005 for age. b) The test of interaction is restricted to the "Never" and "During past 1 yr" subgroups. One child who was exposed to dampness both "During past 1 yr" and "Earlier" is excluded from this subgroup comparison. c) The test of interaction is restricted to the "Never" and "Current" subgroups. Table 3 Vitamin C and asthma symptoms: effect modification by age and baseline asthma severity Age (yr) C-ACT at baseline No of. Children Symptoms (C-ACT) Mean Arithmetic increase in C-ACTa) Percentage increment in C-ACTb) Placebo Vitamin C Mean 95% CI Mean 95% CI 7.0-8.2 18-19 12 17.6 21.8 4.2 3.3-5.2 24% 18-31% 16-17 10 16.3 20.0 3.7 2.4-5.0 23% 15-32% 13-15 8 15.5 18.5 3.0 2.4-3.6 19% 16-23% 8.3-10 18-19 8 17.2 21.0 3.7 1.6-5.8 23% 9-36% 16-17 10 16.8 19.2 2.4 1.5-3.3 14% 9-20% 14-15 12 15.8 17.2 1.3 0.1-2.5 9% 1-17% a) Arithmetic increase in the C-ACT. When age and baseline C-ACT as continuous variables were included in the linear model explaining the effect of vitamin C, the model was improved by χ2(2 df) = 19.1, P = 0.00007 (model parameters are shown in Table 4). There was no second order interaction between the baseline C-ACT and age in their modification of the vitamin C effect (χ2(1 df) = 2.1, P = 0.2). b) Percentage increment in the C-ACT. When age and baseline C-ACT as continuous variables were included in the linear model explaining the effect of vitamin C, the model was improved by χ2(2 df) = 14.0, P = 0.0009. There was no second order interaction between the baseline C-ACT and age in their modification of the vitamin C effect (χ2(1 df) = 2.6, P = 0.1). Statistical methods To minimize the multiple comparison problem associated with subgroup analysis, we wrote a protocol in which we planned this study (Additional file 1). We decided to focus on two clinically relevant primary outcomes, the C-ACT difference and the FEV1 ratio (above), and to examine the possible modification of the vitamin C effect by six variables: the baseline C-ACT, the baseline FEV1/FVC ratio, gender, paternal smoking, dampness or mold in the bedroom, and residential neighborhood (urban/rural) (see details in the Additional file 1). This series of subgroup analyses were motivated by our finding that the baseline FEV1 modified the vitamin C effect on the FEV1 level. In the subgroup analysis protocol, we considered that the baseline FEV1/FVC ratio would be a better variable for subgroup division as it adjusts for the size of the lungs. Unexpectedly, we did not find a modification of the vitamin C effect by the baseline FEV1/FVC ratio. The baseline FEV1 has a close correlation with age (r = 0.95), whereas the correlation between the baseline FEV1 and the baseline FEV1/FVC ratio is weak (r = 0.12). Therefore, we added FEV1 and age to the tables to examine whether the divergence between the baseline FEV1/FVC ratio and the FEV1 levels might be explained by age. Furthermore, we also added weight to the tables to test whether it is the young age per se or the low weight (higher dose per weight unit) that better explains the greater effect on the younger children. We divided the children into subgroups by C-ACT, FEV1/FVC ratio, FEV1, age, and weight with the cut points at the medians. Effect modification by paternal smoking was tested by comparing never smokers with current smokers, so that ex-smokers were excluded. Similarly, effect modification by dampness or mold in the bedroom was tested by comparing never exposed to those children who were only recently exposed (< 1 year), so that those exposed to dampness in their earlier childhood (> 1 year prior to the study) were excluded. The excluded groups are shown in the tables, but they were not included in the test of interaction. We tested the interaction between vitamin C effect and the subgroup variables by using linear models. To test whether the vitamin C supplementation effect is different between the subgroups, we first added a uniform vitamin C effect to all the children. Then we added an interaction term between vitamin C and the subgroup variable. The improvement of the linear model fit was thereafter calculated from the change in -2 × log (likelihood), which follows the χ2 distribution with degrees of freedom defined by the number of interaction terms. Although the C-ACT difference was close to the normal distribution, the values were integers in a range of -2 to +6 points. Therefore, we confirmed the most essential subgroup differences in Table 2 by a nonparametric test. The Wilcoxon test gave similar P-values for the interaction test between vitamin C effect on C-ACT and the dichotomous baseline C-ACT (P = 0.010) and age (P = 0.005), consistent with the t-test results in Table 2. The linear models, the t-test-based 95% confidence intervals (95% CI) of the effects, and the Wilcoxon tests were calculated using the R-package [25]. Two-tailed P-values are shown. Results The essential characteristics of the 60 children are described in Table 1. On average, vitamin C supplementation increased the asthma symptom score, C-ACT, by 3.0 points (Table 2). This effect was modified by the baseline C-ACT so that vitamin C was more effective in those children who had less severe asthma symptoms. The evidence of effect modification was stronger when the baseline C-ACT was included in the statistical model as a continuous variable (P = 0.0002) than as a dichotomous variable (P = 0.004), which indicates that the effect modification was better captured by the continuous baseline C-ACT. The baseline FEV1/FVC ratio did not modify the effect of vitamin C on the C-ACT (Table 2). This was inconsistent with the modification caused by the baseline FEV1, which gave us the motivation for this subgroup analysis. Because of this discrepancy, we considered that the modification by the baseline FEV1 might be explained by the close correlation between age and FEV1. Since age significantly modified the vitamin C effect, whereas baseline FEV1/FVC ratio did not, we concluded that the modification by the baseline FEV1 was simply reflecting the effect of age on FEV1 (Table 2). There was no substantial difference between including age as a dichotomous or a continuous variable in the statistical model. Gender and residential area did not modify the effect of vitamin C. There was also no significant difference between the children who were currently or had never been exposed to dampness in the bedroom, or between the children whose fathers were current smokers or had never smoked (Table 2). Given that the baseline C-ACT and age modified the effect of vitamin C, we analyzed the combined effect of these two variables (Tables 3 and 4). When both of these variables were simultaneously included in the linear model, it was substantially improved (P = 0.0001), so that the proportion of variance in the vitamin C effect explained by these two variables was 27% (R2 = 0.27). There was no second order interaction between these two variables in their influence on the vitamin C effect (Table 3). The greatest effect of vitamin C on the C-ACT was seen in the younger children who had mild asthma symptoms (4.2 point increase), whereas the smallest effect was seen in the older children who had severe asthma symptoms (1.3 point increase). The estimated influence of the baseline C-ACT and age on the vitamin C effect is shown in Table 4. Table 4 Effect of vitamin C on the C-ACT level: parameters estimating the effect modification by age and baseline C-ACT Variable Effect of vitamin C on the C-ACT levela) Age 7.0 yr, baseline C-ACT 13 points +2.12 (SE 0.65) Age (per year over 7.0 yr) -0.52 (SE 0.22) Baseline C-ACT (per point over 13) +0.46 (SE 0.13) a) These parameters are from the linear model for the arithmetic increase in C-ACT described in the footnote of Table 3. SE, standard error. This model predicts, for example, that a 9-yr old child with baseline C-ACT of 19 would have 3.84 point increase in C-ACT by vitamin C administration which is close to that observed (3.7) for the older children with baseline C-ACT 18 or 19 in Table 3. In our analysis of the C-ACT change, we used the absolute difference as the primary outcome. However, as we found a greater effect in those children who had a high initial C-ACT score, we also analyzed Table 3 heterogeneity by using the percentage increment in the C-ACT score. With this secondary outcome, we also found strong evidence of heterogeneity in vitamin C effect between the children (P = 0.001). On average, vitamin C increased the FEV1 level by 29% (Table 5). This effect was modified by age, and continuous age was better than dichotomous age in capturing the interaction (Table 5). The effect of vitamin C on FEV1 was also modified by dampness in the bedroom. Our test of interaction was restricted to the children who were currently or had never been exposed to dampness in the bedroom. However, the effect of vitamin C was smallest in the children who were exposed to dampness in their earlier childhood. Other tested baseline variables did not modify the effect of vitamin C (Table 5). Table 5 Effect of vitamin C on the FEV1 levels of asthmatic children Subgroup No. of Children FEV1 Mean (L/s) Change in FEV1 Test for interaction (P) Placebo Vitamin C Estimate 95% CI All 60 1.125 1.446 28.9% 27.3-30.4% C-ACT at baseline 13-15 30 1.16 1.48 27.5% 25.4-29.6% 0.07a) 16-19 30 1.09 1.41 30.3% 28.0-32.6% FEV1/FVC (%) < 59 28 1.11 1.43 29.4% 27.0-31.9% 0.5a) ≥59 32 1.14 1.46 28.4% 26.3-30.5% FEV1 at baseline (L/s) < 1.1 29 1.00 1.31 31.1% 28.6-33.6% 0.004a) ≥1.1 31 1.24 1.58 26.8% 25.1-28.5% Age (yr) 7.0-8.2 30 1.00 1.31 31.1% 28.7-33.5% 0.003a) 8.3-10 30 1.25 1.58 26.7% 24.9-28.4% Weight (kg) 23-28 29 1.12 1.44 29.7% 27.1-32.3% 0.3a) 29-37 31 1.13 1.45 28.1% 26.2-30.0% Gender Girl 22 1.07 1.37 28.6% 25.6-31.6% 0.8 Boy 38 1.16 1.49 29.0% 27.2-30.9% Dampness exposure Never 25 1.16 1.53 32.4% 30.2-34.6% 0.001b) During past 1 yr 20 1.11 1.42 27.6% 25.9-29.4% Earlier 14 1.07 1.34 25.0% 21.2-28.8% Smoking by the father Never 21 1.17 1.52 30.1% 28.0-32.2% 0.4c) Current 22 1.08 1.38 28.5% 25.3-31.7% Ex-smoker 17 1.12 1.43 27.9% 24.9-30.9% Residential area Urban 34 1.09 1.40 29.2% 27.2-31.3% 0.6 Rural 26 1.17 1.50 28.4% 25.8-31.0% a) The test of interaction was calculated by using dichotomized variables for the C-ACT, FEV1/FVC ratio, FEV1, age, and weight (cut points at the medians). The test of interaction by using the continuous variables gives P = 0.2 for the baseline C-ACT and P = 0.001 for age. b) The test of interaction is restricted to the "Never" and "During past 1 yr" subgroups. One child who was exposed to dampness both "During past 1 yr" and "Earlier" is excluded from this subgroup comparison. c) The test of interaction is restricted to the "Never" and "Current" subgroups. When both age and exposure to dampness were included in the same statistical model to explain FEV1 changes, the model was significantly improved (P = 10-10) (Tables 6 and 7). The proportion of variance in the vitamin C effect explained by the two variables was 58% (R2 = 0.58). There was no second order interaction between age and exposure to dampness in their influence on the vitamin C effect. The greatest effect of vitamin C on FEV1 was seen in the younger children who had never been exposed to dampness or mold in their bedroom (37% increase in FEV1), whereas the smallest effect was seen in the older children who had been exposed to dampness more than one year prior to the study (21% increase in FEV1)(Table 6). The estimated influence of age and exposure to dampness on the vitamin C effect is shown in Table 7. Table 6 Vitamin C and FEV1: effect modification by age and exposure to dampness Age (yr) Exposure to dampness in the bedroom No of. Childrena) FEV1 (L/s) Percentage increment in FEV1 b) Placebo Vitamin C Mean 95% CI 7.0-8.2 Never 10 0.98 1.34 37.0% 33.9-40.2% During past 1 yr 11 1.04 1.34 29.0% 27.4-30.6% Earlier 9 0.99 1.25 27.1% 21.5-32.6% 8.3-10 Never 15 1.28 1.66 29.3% 27.6-31.1% During past 1 yr 9 1.21 1.52 25.9% 22.5-29.4% Earlier 5 1.23 1.49 21.3% 18.0-24.5% a) Since the goal of this table is to compare the effect of "During past 1 yr" and "Earlier" exposure to dampness, one child who had both modes of exposure was excluded (age 8.4 yr), making the number of children in this analysis 59. b) When age as a continuous variable and the three categories of dampness were included in the linear model explaining the effect of vitamin C on the FEV1 increase, the model was improved by χ2(3 df) = 51.8, P = 10-10 (model parameters are shown in Table 7). There was no second order interaction between age and dampness in their modification of the vitamin C effect (χ2(2 df) = 2.3, P = 0.3). Table 7 Effect of vitamin C on the FEV1 level: parameters estimating the effect modification by age and exposure to dampness Variable Effect of vitamin C on the FEV1 levela) Age 7.0 yr, no exposure to dampness +37.9% (SE 1.2%) Age (per year over 7.0 yr) -3.37% (SE 0.52%) Exposure to dampness during past 1 yr -5.8% (SE 1.2%) Exposure to dampness earlier -9.6% (SE 1.4%) a) These parameters are from the linear model described in the footnote of Table 6. SE, standard error. This model predicts, for example, that a 9-yr old child with exposure to dampness earlier than 1 year before the study would have 21.5% increase in FEV1 by vitamin C administration which is close to that observed (21.3%) for the older children with early dampness exposure in Table 6. Since exposure to dampness was composed of four indicator items, we explored whether there might be differences between the indicators; mold odor was reported only by 3 children, and it was excluded from this comparison. Within the accuracy of the confidence intervals, there were no differences between the three other indicators in the modification of the vitamin C effect on the FEV1 level (data not shown). Discussion We found that age modified the effect of vitamin C on asthma symptoms (C-ACT) and on the FEV1 level in this group of Egyptian children. In addition, the vitamin C effect on asthma symptoms was modified by baseline C-ACT, and the vitamin C effect on the FEV1 level was modified by exposure to dampness in the bedroom. Previously, an age-dependent variation in the vitamin C effect on common cold duration was noted, but it was not evident whether the greater effect on children than on adults was caused by age per se or by a higher dose per weight unit since children weigh less [26,27]. In the current study, we found a greater vitamin C effect on younger children, and this was not explained by weight differences (Tables 2 and 5). Still, it is possible that the heterogeneity over age might be caused by some factors closely correlated with age; however, this possibility does not challenge the strong evidence indicating that substantial heterogeneity exists across this group of children. When planning this subgroup analysis, we reasoned that the effect of vitamin C might be greater in children who had the lowest baseline C-ACT level and FEV1/FVC ratio, and in children who had been exposed to dampness (Additional file 1). However, we found the opposite direction for the modification by C-ACT, namely the effect of vitamin C was greater in those who had a high baseline C-ACT level. We also found that the relation between the baseline FEV1 and the vitamin C effect, which gave us the motivation for this study, was explained by age and not by the baseline FEV1/FVC ratio. In addition, contrary to our expectation, exposure to dampness in the bedroom was associated with a decreased effect of vitamin C. Thus, although the baseline C-ACT and dampness modified the vitamin C effect, the modification was in a direction opposite to our expectation. Gender differences have been found in the vitamin C effects on the common cold [28-30], but in this study we did not find any differences between boys and girls. Urban and rural neighborhoods differ in the type of outdoor air pollution, and passive smoking causes irritation of the airways, but we found no modification of the vitamin C effect by residential neighborhood or paternal smoking. We found substantial heterogeneity in the effect of vitamin C, over two-fold variation in the effect between the extremes of the subgroups in Tables 3 and 6. Thus, the effect of vitamin C on asthma seems to be context dependent. This heterogeneity in the vitamin C effect seems important since it indicates that no universal effect should be sought. Instead, the characteristics and living conditions of asthma patients who would get the greatest benefit from vitamin C should be targeted. The heterogeneity we found within these children also has implications for the interpretation of previous studies. Two randomized, double-blind, placebo-controlled trials found divergent effects of vitamin C in asthmatic patients. In Nigeria, Anah et al. found a 78% reduction in the incidence of asthma attacks in 15 to 46 year-old patients administered 1 g/day of vitamin C [3]. In the UK, Fogarty et al. did not find any effect of 1 g/day of vitamin C on asthma symptoms or FEV1 levels in 18 to 64 year-old asthmatics [4]. Since differences in nutrition or lifestyle, or other differences between the participants of the two trials may explain the divergent findings, the newer trial [4] should not be considered a refutation of the older trial [3]. Asthma is a collection of different phenotypes, rather than a single disease [31,32]. These phenotypes are categorized under the broad umbrella of "asthma" because they meet the criteria for the clinical diagnosis of the disease. Allergic sensitization that triggers asthma may be the largest phenotype. There is also evidence that molds are an important environmental trigger for asthma exacerbations, and the effects of molds are possibly caused, at least partly, by their mycotoxins [24,33-35]. Furthermore, the mechanisms behind EIB seem to be different from those of ordinary asthma [36]. Given the variety of mechanisms causing asthma-type symptoms, it seems plausible that vitamin C has different effects on different types of asthma. Thus, although three trials consistently found a benefit of vitamin C against EIB [5-7], those studies cannot be extrapolated to other types of asthma. In this study, we found that the effect of vitamin C on the FEV1 level of asthmatic children was significantly modified by exposure to dampness or mold in the bedroom. A number of subgroup comparisons were carried out in our study, and therefore the multiple comparison problem might be of concern. However, the particularly low P-values seen in Tables 2 and 5 are not easily explained by the 18 subgroup comparisons in these two tables. Furthermore, the proportion of variance in the vitamin C effect explained by the statistical models (R2) in Tables 3 and 6 is high. Therefore, we do not consider that the differences identified might be easily explained by multiple testing. Our study was randomized, double blind and placebo controlled. Nevertheless, our study has various limitations. Our study subjects were Egyptian children, and it is not clear whether the same modifying factors might apply to children in industrialized countries or in other developing countries or to adults. There may have been inaccuracy in the measurement of mold exposure; however, nondifferential misclassification would move the estimate of interaction effect towards the null value (of no interaction) and cannot generate an artificial difference between the exposed and unexposed [37]. The duration of vitamin C administration was only 6 weeks, and it is not evident whether the observed effect lasts substantially longer. In a cross-over study, the carry-over effect from the intervention phase to the placebo phase could reduce the difference between the two phases, but cannot bias in the direction of greater effect. The dose of vitamin C was rather low, 0.2 g/day, and our study does not give any information about dose dependency: whether higher doses might cause a greater effect or whether similar effects might be caused by even lower doses. Such issues should be considered in future studies on vitamin C and childhood asthma. Conclusions We found strong evidence that the effect of vitamin C on asthmatic Egyptian children is heterogeneous. The highest effects observed, the 37% increase in the FEV1 level and the 4.2 point increase in the C-ACT level, are substantial and clinically important. It would seem important to carry out further research to confirm our findings and more accurately identify the groups of children who would receive the greatest benefit from vitamin C supplementation. Competing interests This study was not funded by external sources. We have no conflicts of interest. Authors' contributions HH and MB wrote the protocol for the subgroup analysis. MB and AB carried out of the trial which is analyzed in this subgroup analysis. HH wrote the first version of the manuscript and MB and AB participated in the critical revision of the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Additional file contains the protocol that was written before the subgroup analysis was initiated. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22567126PONE-D-11-2249010.1371/journal.pone.0036093Research ArticleBiologyGeneticsCancer GeneticsGene ExpressionGene FunctionGenetics of DiseaseMolecular Cell BiologyCell DivisionCell GrowthMedicineOncologyBasic Cancer ResearchTumor PhysiologyCancers and NeoplasmsNeurological TumorsSPARC Overexpression Inhibits Cell Proliferation in Neuroblastoma and Is Partly Mediated by Tumor Suppressor Protein PTEN and AKT SPARC Expression Inhibits Neuroblastoma GrowthBhoopathi Praveen 1 Gorantla Bharathi 1 Sailaja G. S. 1 Gondi Christopher S. 1 Gujrati Meena 2 Klopfenstein Jeffrey D. 3 Rao Jasti S. 1 3 * 1 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America 2 Department of Pathology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America 3 Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America Li Jun EditorSun Yat-sen University Medical School, China* E-mail: [email protected] and designed the experiments: PB JSR. Performed the experiments: PB BG GSS. Analyzed the data: PB CSG MG JDK JSR. Contributed reagents/materials/analysis tools: JSR. Wrote the paper: PB. 2012 2 5 2012 7 5 e360937 11 2011 29 3 2012 Bhoopathi et al.2012This 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.Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell–cell and cell–matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo. ==== Body Introduction Neuroblastoma is the most common extracranial solid tumor in children under the age of five years. When neuroblastoma is first diagnosed, 50 percent of the patients are considered to be high-risk patients. Neuroblastoma originates from neural crest cells and these tumors, which are normally dissimilar both clinically and biologically, are found in the adrenal medulla or near the sympathetic chain [1]. Neuroblastoma in infants may regress spontaneously while tumors in older patients may settle into benign ganglioneuromas. Neuroblastoma is caused by rapid cell production by the neuroblast during fetal growth, which causes a growth or a tumor to develop. Usually, these tumors cannot be removed completely through surgery due to metastasis at the time of diagnosis and have a very poor prognosis. Metastasis is a complex process mainly dependent on cell adhesion to the extracellular matrix (ECM) and basement membrane and takes place through a multi-step process that includes cell infiltration from the primary tumor, intravascular invasion, and eventually proliferation to the metastatic site [2], [3] Although intensive multimodality therapies have produced some developments in the overall cure rate of these tumors, the therapies have considerable short- and long-term toxicities. Thus, a detailed knowledge of mechanisms controlling proliferation and differentiation may lead to a better understanding of the molecular pathogenesis of neuroblastoma, which may result in novel biologically-based therapies that are less toxic and more effective. While the ECM is classically thought to instruct cell behavior primarily through biochemical recognition by cell adhesion receptors, signals encoded in the ECM may play a significant role in guiding neuroblastoma differentiation and proliferation. Overall, the mechanical stringency of the ECM can intensely alter cellular behavior, including morphology, motility, and proliferation [4]–[6]. The matricellular proteins are extracellular proteins and do not contribute structurally to the extracellular environment as do the classical extracellular matrix proteins, but instead they modulate interactions between the extracellular matrix and cells. One such matricellular protein is Secreted Protein Acidic and Rich in Cysteine (SPARC), also known as osteonectin or BM-40, a 34 kDa, calcium-binding glycoprotein shown to associate with the cell membrane and membrane receptors [7], [8]. SPARC is known to not only modulate cell–cell and cell–matrix interactions, but also to influence de-adhesive and growth regulatory properties [9]. In cancers, SPARC may elicit different actions, showing the complexity of the protein [10], [11]. In certain types of cancers, like melanomas and gliomas, SPARC is associated with a highly aggressive tumor phenotype [9], whereas in other cancers, mainly ovarian, neuroblastomas, colorectal and PNET tumors, SPARC may function as a tumor suppressor [9], [12]. Recent studies show that SPARC modulates cellular functions and proliferation through modulation of different growth factor signaling [13]. In addition, we have shown that SPARC inhibits medulloblastoma tumor growth both in vitro and in vivo by inducing autophagy-mediated cell death and causing neuronal differentiation [14]. The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a phosphatase, which is deleted or mutated in a variety of human cancers [15]–[17]. PTEN plays an important role in keeping the processes of cell migration and proliferation under control. PTEN is a negative regulator of phosphatidylinositol-3-kinase (PI3K) signaling by dephosphorylating phosphatidylinositol- 3–5-triphosphate (PIP3). However, very little is known about the existence of other substrates for PTEN, with the exception of PtdIns-3,4,5-P3 and PtdIns-3,4-P2, which are required for the phosphorylation and activation of the AKT protein kinase, a survival factor that fuels the progression of the cell cycle [18]–[20] and also prevents cells from undergoing apoptosis by inhibiting pro-apoptotic factors as well as nuclear translocation of the forkhead transcription factors [21]–[23]. Earlier studies from our laboratory and others have shown that high levels of SPARC correlate with inhibited proliferation in many cancer types. We have shown that SPARC overexpression by an adenoviral vector induced autophagy-mediated apoptosis in PNET tumor cells. In the present study, we sought to further characterize the mechanism by which SPARC is capable of inhibiting proliferation in neuroblastoma cells. Results Overexpression of SPARC in neuroblastoma cells in vitro SPARC, a prototype of the matricellular protein family, has been shown to play an important role in various aspects of tumorigenesis including tumor invasion, angiogenesis and tumor growth [12]. To elucidate the effect of SPARC overexpression using a genetic approach and to observe its effects on neuroblastoma tumor growth in vitro and in vivo, we subcloned a human SPARC cDNA in a pcDNA3.1 mammalian expression vector and transfected it into SK-N-AS, NB-1691 and IMR-32 neuroblastoma cells. Figure S1A shows that SPARC protein levels were increased in the three cell lines when compared to mock or empty vector-transfected cells. We observed a ∼3- to 4-fold increase in SPARC protein levels in pSPARC-transfected cells compared to controls. To confirm that this upregulation of SPARC mRNA translated into increased levels of SPARC protein, we assessed mRNA transcript levels in the pSPARC-transfected cells. SPARC-overexpressed neuroblastoma cells showed a 3- to 4-fold increase in mRNA levels when compared to mock or empty vector-transfected cells (Fig. S1B). As assessed by immunofluorescence microscopy, intense staining for SPARC was observed in all three cell lines transfected with pSPARC when compared to mock or empty vector-transfected cells (Fig. S1C). We compared the SPARC levels in tumor cells with HMEC cells (as control cells) and did not find much change in SPARC levels (Fig. S1D). X-ray radiation inhibits SPARC expression in neuroblastoma cells SPARC has been shown to be a therapy-resistant reversal gene whose expression was significantly decreased in resistant cancer cells [24]. To determine whether there was a dose-dependent radiation effect on these cells we performed an in vitro clonogenic assay to characterize the survival of neuroblastoma cells, after exposure to ionizing radiation. SK-N-AS, NB1691 and IMR-32 cells were given a single dose of radiation (from 2 Gy to 12 Gy) and assayed for survival. Irradiated cells showed a dose-dependent decrease in survival fraction with a 27.6% survival rate at 8 Gy for SK-N-AS, a 30% survival rate at 8 Gy for NB1691 and a 25% survival rate at 4 Gy for IMR-32 cells when compared to non-radiated cells (Fig. S1E). To examine the effect of radiation on SPARC expression, we determined SPARC protein levels in SK-N-AS, NB1691 and IMR-32 neuroblastoma cells. Figure 1A indicates that SPARC expression levels were inhibited with radiation in a dose-dependent manner when compared to non-irradiated cells. Densitometric analysis revealed about 30–40% inhibition in SPARC levels when cells were treated with 8 Gy (SK-N-AS and NB1691) and 4 Gy in IMR-32 cells as compared to non-irradiated cells. 10.1371/journal.pone.0036093.g001Figure 1 Irradiation inhibits SPARC expression and inhibits proliferation of neuroblastoma cells. (A) SK-N-AS, NB1691 and IMR-32 cells were irradiated (IR) with X-ray (0–12 Gy), incubated for 12 hours, and cells were collected. (A) SPARC expression was determined by western blot analysis in cell lysates. Results are representative of three independent experiments. GAPDH served as a loading control. Columns, mean of three experiments; bars, SD. (B) Neuroblastoma cell lines SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or pSPARC for 24 hours, and SK-N-AS and NB1691cells were irradiated with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation. Left panel : SPARC levels were determined by Western blot analysis using a SPARC-specific antibody. GAPDH served as a loading control. Middle panel : cDNA was produced from total RNA extracted from the mock and infected cells. RT-PCR was performed for SPARC. Results are representative of three independent experiments. GAPDH served as a control for RNA quality. Right panel : Densitometric analysis showing levels of SPARC protein and mRNA levels. Columns, mean of three experiments; bars, SD. * p<0.01 vs pEV; ** p<0.01 vs IR+pEV. (C) SK-N-AS, NB-1691 and IMR-32 cells were transfected with mock, pEV or pSPARC and at the indicated time points, the plates were incubated by adding MTT reagent for a further 6 hours. The developed purple color Formazan crystals were solubilized using DMSO, and color intensity was measured using a spectrophotometer at 570 nm. Data were plotted as absorbance at 570 nm. Results are representative of three independent experiments. Points, mean of three experiments. H = hours. (D) SK-N-AS, NB1691 and IMR-32 neuroblastoma cells were transfected with mock, pEV or pSPARC. After 24 hours of incubation, SK-N-AS and NB1691 cells were irradiated with 8 Gy; IMR-32 cells were irradiated with 4 Gy and clonogenic assay was performed as described in Materials and Methods. The cells were cultured and colonies larger than 50 cells were counted. Columns: mean of triplicate experiments; bars: SD. (E) SK-N-AS, NB1691 and IMR-32 neuroblastoma cells were transfected with mock, pEV or pSPARC. After 24 hours of incubation, SK-N-AS and NB1691 cells were irradiated with 8 Gy and cultured for another 16 hours. TUNEL assay was performed as per the manufacturer's procedure. (F) SK-N-AS neuroblastoma cells were transfected with mock, pEV or pSPARC. After 24 hours of incubation, cells were irradiated with 8 Gy and cultured for another 16 hours. Western blot analysis was performed for Caspase3 and PARP specific antibodies and GAPDH served as loading control. SPARC overexpression inhibits proliferation in neuroblastoma cells We next examined the possible role of SPARC in radiation response. Inhibition of SPARC levels by radiation was restored using a plasmid vector encoding the SPARC full-length gene. SPARC overexpression in neuroblastoma cell lines prior to irradiation exhibited increased SPARC protein and transcript levels in neuroblastoma cell lines (Fig. 1B) when compared to mock or empty vector-treated cells prior to irradiation. Densitometric analysis for SPARC protein and transcript levels showed a 3- to 4-fold increase (Fig. 1B) in the pSPARC treatment prior to irradiation. Further, we assessed the sensitivity of neuroblastoma cells to SPARC overexpression in combination with radiation using the MTT proliferation assay. The results revealed that SPARC-overexpressed cells had increased sensitivity to radiation, and their proliferation rate was less than that of cells treated with radiation alone or combined with mock or empty vector treatment (Fig. 1C). We also assessed the impact of the combination treatment on neuroblastoma cells using clonogenic survival assay and found that combining SPARC and ionizing radiation resulted in increased cell death (Fig. 1D). To further confirm the effect of SPARC overexpression on neuroblastoma cell growth, we performed TUNEL assay. SPARC overexpression in neuroblastoma cell lines prior to irradiation exhibited increased TUNEL positive cells compared to that of cells treated with radiation alone or combined with mock or empty vector treatment (Fig1E). To further confirm this result, we also analyzed cleavage of PARP and caspase3 by western blot analysis. Western blot analysis revealed that SPARC overexpression in neuroblastoma cell line (SK-N-AS) prior to irradiation exhibited increased cleavage of capspase3 and PARP when compared to that of cells treated with radiation alone or combined with mock or empty vector treatment (Fig. 1F). SPARC overexpression abates irradiation-induced cell cycle arrest in neuroblastoma cells When cells are exposed to radiation, they initiate a complex response that includes the arrest of cell cycle progression [25]. DNA is an important subcellular target of ionizing radiation, but oxidative damage to plasma membrane lipids initiates signal transduction pathways that activate apoptosis and may play a role in cell cycle regulation [26]. Moreover, irradiation-induced G2 arrest was shown to require inhibitory phosphorylation of the kinase Cdc2 [27]. To identify whether the growth inhibitory effect in cells that received the combined treatment of radiation and SPARC overexpression was caused by specific perturbation of cell cycle-related events, DNA contents of neuroblastoma cells were measured by flow cytometric analysis. Flow cytometric analyses using neuroblastoma cells treated with mock, empty vector (pEV), pSPARC with and without radiation (IR) demonstrated a significant increase in the proportion of G2/M cells and a reduced number of G1 cells after IR treatment when compared to controls. However, SPARC treatment prior to IR increased the number of sub-G1 cells as compared to SPARC treatment alone (Fig. 2A). As shown in Figure 2A, treatment of neuroblastoma cells with radiation resulted in an increase in the percentage of G2/M cells (from 10% to ∼50%) as compared to control, non-irradiated cells. In cells transfected with pSPARC prior to radiation, the percentage of cells in the sub-G1 phase increased to a maximum of 20%. 10.1371/journal.pone.0036093.g002Figure 2 SPARC overexpression sensitizes neuroblastoma cells to radiation by abating irradiation-induced cell cycle arrest. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC for 24 hours, and SK-N-AS and NB1691cells were irradiated (IR) with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation. (A) Cells were collected and subjected to FACS analysis with propidium iodide staining for DNA content and represented in a graphical manner. Results are representative of three independent experiments. Columns: mean of triplicate experiments. (B) Cells were collected and the cell lysates were subjected to western blotting for Chk1, Chk2, Cdc25C, Cyclin B1, Cdc2 and pCdc2. Results are representative of three independent experiments. Densitometric analysis for Chk1 and Cdc25C is shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SD. * p<0.01 vs pEV; ** p<0.01 vs IR+pEV. Functional defect in DNA damage checkpoint pathways showed increased sensitivity to radiation and other DNA damage agents [28], [29]. This observation suggests a possibility that components of these DNA damage checkpoint pathways may serve as potential therapeutic targets for enhancing radiosensitivity of tumor cells [30]. To test this hypothesis, we evaluated the protein levels of Chk1 and phospho-Cdc25C in SPARC-overexpressed neuroblastoma cells. Irradiated neuroblastoma cell lines exhibited a significant increase in Chk1 levels but not in Chk2 levels. Further, irradiation-induced Chk1 levels were increased in cells treated with pSPARC prior to IR (Fig. 2B). Densitometric analysis for Chk1 and Cdc25C indicated 3- to 4-fold increase in SPARC-overexpressed cells when compared to mock or empty vector-treated cells (Fig. 2B). Recent studies showed that Chk1, a serine-threonine kinase, is critical for G2/M arrest in response to DNA damage and is also known to modulate Cdc25C. We next determined the levels of Cdc25C by western blotting and found that irradiation increased the levels of Cdc25C protein levels when compared to non-irradiated cells. This increase was further enhanced by SPARC overexpression, thereby suggesting that SPARC overexpression in neuroblastoma cells abates cell cycle arrest and leads to decreased proliferation. Cyclin B is known to be one of the regulatory proteins involved in mitosis and forms a complex with Cdc2. Here, we sought to determine the levels of these proteins by western blotting. Our results demonstrated that Cyclin B and Cdc2 levels were increased in the combination treatment (Fig. 2B), indicating that irradiation-induced G2/M cell cycle arrest was abating by SPARC overexpression in neuroblastoma cells. SPARC overexpression decreases radiation-induced PI3K-AKT and PTEN signaling Ionizing or ultraviolet radiation-induced cellular survival signaling pathways induce development of cancer and insensitivity of tumor cells to radiation therapy [31]. Collecting evidence suggests that the phosphatidylinositide 3-kinase (PI3K)/AKT signal pathway is an important contributor to radioresistance [31]. In many cell types, PI3K/AKT signaling is a key cytoprotective response downstream of the EGFR family receptors and mediates carcinogenesis [31]. The phosphatase and tensin homologue (PTEN) is also a negative regulator of proliferation in many cancer types. Furthermore, AKT activity is elevated in cell lines with the mutated PTEN tumor suppressor gene. Therefore, we sought to characterize the effects of physiologic and genetic manipulation of AKT signaling on combined treatments of IR and SPARC overexpression on neuroblastoma cell proliferation. We first assessed the expression levels of EGFR and PI3K/AKT signaling molecules in the combination treatment. Radiation-induced EGFR phosphorylation and PI3K/AKT levels were inhibited and PTEN levels were increased by SPARC overexpression prior to irradiation in neuroblastoma cells (Fig. 3A). SPARC overexpression prior to irradiation decreased pAKT levels by 60–70% when compared to mock or empty vector-treated controls (Fig. 3B); PTEN levels were increased up to 3- to 4-fold in SPARC-overexpressed neuroblastoma cells when compared to mock or empty vector-treated cells (Fig. 3B) as determined by densitometry analysis. 10.1371/journal.pone.0036093.g003Figure 3 SPARC overexpression inhibits AKT phosphorylation and induces PTEN. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC for 24 hours, and SK-N-AS and NB1691cells were irradiated (IR) with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation for further 16 hours. (A) Cell lysates were assessed for EGFR, AKT and their phosphorylations, PI3K, mTOR, PTEN and c-Myc by western blotting. (B) Protein levels were quantified by densitometric analysis for pAKT and PTEN is shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SD. Results are representative of three independent experiments. * p<0.01 vs pEV; ** p<0.01 vs IR+pEV. PTEN is capable of modulating the c-Myc gene and has been implicated in the control of cell proliferation, differentiation, and pathogenesis of malignant diseases. We next determined the levels of c-Myc in the combination treatment and observed that c-Myc levels were inhibited by SPARC overexpression, thereby indicating a role of PI3K/AKT signaling in neuroblastoma cell proliferation. AKT has been shown to play an important role in several cellular functions such as cell survival, growth, proliferation, migration, metabolism and angiogenesis [32]. It is clear that AKT has the capacity to act on the substrates affecting various cellular signaling pathways and it was of interest to us to check the role of AKT overexpression in the SPARC-mediated effect of neuroblastoma cell proliferation. To confirm the role of PI3K/AKT pathway in SPARC-overexpressed neuroblastoma cell proliferation, studies were performed using myristoylated (constitutively active) AKT (myr-AKT) overexpression. Earlier reports showed that decreased Chk1 can inhibit the G2 cell cycle arrest [33]. Therefore, we first determined the levels of Chk1 in AKT overexpressed pSPARC-transfected neuroblastoma cells. Activation of AKT signaling by myr-AKT in the SPARC-overexpressed neuroblastoma cells led to decreased cell cycle check point Chk1 (Fig. 4A). In parallel, activation of AKT inhibits SPARC-induced PTEN in neuroblastoma cells (Fig. 4A). To confirm the role of collective inhibition of Chk1 and PTEN on proliferation, we assessed the proliferation rate using MTT assay in AKT overexpressed, pSPARC-transfected cells with or without radiation. The results clearly demonstrate that overexpression of constitutively active AKT led to increased proliferation in irradiated and non-irradiated cells with pSPARC transfection (Fig. 4B). 10.1371/journal.pone.0036093.g004Figure 4 Constitutively active AKT overexpression blocks the radiosensitization capability of SPARC in neuroblastoma cells. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with pSPARC alone or in combination with plasmid overexpressing constitutively active AKT (myrAKT; pAKT) for 24 hours, and SK-N-AS and NB1691cells were irradiated with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation and incubated for further 16 hours. (A) Cell lysates were assessed for SPARC, pAKT, EGFR, pEGFR, PTEN, c-Myc and Chk1 by western blotting. Protein levels were quantified by densitometric analysis and shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SE. Results are representative of three independent experiments. (B) At the indicated time points, the plates were incubated by adding MTT reagent for a further 6 hours. The developed purple color formazan crystals were solubilized using DMSO, and color intensity was measured by spectrophotometer at 570 nm. Data are plotted at an absorbance of 570 nm. Results are representative of three independent experiments. Points, mean of triplicate experiments. H = hours. SPARC overexpression induces c-Jun activation and leads to increased PTEN In certain cancers SPARC is known to increase the levels and activity of the transcription factor c-Jun [34]. We sought to determine the levels of JNK activation as this can potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73 [35]. Western blot analysis revealed that SPARC overexpression increased the phosphorylation of JNK by about 2–3 fold in all the cell lines when compared with mock or empty vector-treated neuroblastoma cells (Fig. 5). We evaluated the levels of phosphorylation of c-Jun at serine 63 and 73 upon SPARC overexpression. SPARC overexpression led to increased p-c-Jun (Ser-63) and p-c-Jun (Ser-73) when compared to mock and empty vector-transfected cells (Fig. 5). As JNK is a known modulator of PTEN [36], we further tested whether inhibition of JNK activation will suppress PTEN in neuroblastoma cells. We found that inhibition of JNK using the JNK activation inhibitor reduced the level of PTEN in SPARC overexpressed cells when compared with non-treated SPARC overexpressed neuroblastoma cells (Fig. 5). 10.1371/journal.pone.0036093.g005Figure 5 SPARC overexpression activates JNK and JNK in turn activates PTEN. SK-N-AS, NB1691 and IMR-32 cells were transfected for 24 hours with mock (PBS control), empty vector (pEV) or with pSPARC and treated with or without JNK activation inhibitor (JNK-I) for another 12 hours. Cell lysates were assessed for pJNK, JNK, c-Jun, p-c-Jun (ser-63), p-c-Jun (ser-73) and PTEN by western blotting. Protein levels were quantified by densitometric analysis and shown in the corresponding bar graph. Columns, mean of triplicate experiments; bars, SE. Results are representative of three independent experiments. * p<0.01 vs pEV; ** p<0.01 vs pEV+JNK-I. PTEN overexpression inhibits proliferation in SPARC-inhibited neuroblastoma cells Direct inhibition of signaling pathways that are negatively regulated by PTEN suppress proliferation and migration in SPARC-expressing GBM cells in vitro [37]. PTEN is a negative regulator of AKT that is often mutated or deleted in AIPC, resulting in AKT-mediated survival signaling, which confers chemotherapeutic resistance in AIPC [38]. To examine the effect of PTEN on the phosphorylation status of AKT cell survival signaling pathway, we overexpressed PTEN in SPARC-inhibited neuroblastoma cells. The neuroblastoma cells (SK-N-AS, NB-1691 and IMR-32) were transfected with either SPARC siRNA or with non-specific siRNA for 24 hours, and then treated with PTEN-overexpressing plasmid for a further 24 hours. The results suggested a significant reduction of phosphorylated AKT in SPARC-inhibited neuroblastoma cell lines when PTEN was overexpressed, suggesting that PTEN plays an important role in inhibition of proliferation with the combination treatment (Fig. 6A). MTT assay of the PTEN-overexpressed neuroblastoma cells showed decreased proliferation at the 72-hour time point in SPARC-inhibited cells to 70%, 62% and 75% in SK-N-AS, NB1691 and IMR-32 cells, respectively (Fig. 6B) 10.1371/journal.pone.0036093.g006Figure 6 PTEN overexpression inhibits neuroblastoma cell proliferation in SPARC-inhibited cells. SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or with siRNA against SPARC (pSP-siRNA) alone or in combination with plasmid overexpressing PTEN (pPTEN) for 24 hours. SK-N-AS and NB1691cells were irradiated (IR) with 8 Gy and IMR-32 cells were irradiated with 4 Gy dose of radiation. (A) Cell lysates were assessed for SPARC, pAKT, PTEN, c-Myc and Chk1 by western blotting. (B) SK-N-AS, NB-1691 and IMR-32 cells were transfected and irradiated as above, and at the indicated time points, the plates were incubated by adding MTT reagent for a further 6 hours. The developed purple color formazan crystals were solubilized using DMSO, and color intensity was measured using a spectrophotometer. Data are plotted as absorbance at 570 nm. Results are representative of three independent experiments. Points, mean of triplicate experiments. H = hours. SPARC overexpression inhibits in vivo growth capacity of neuroblastoma cells in SCID mice To assess the effect of SPARC overexpression on sensitizing tumors to radiotherapy, NB1691 neuroblastoma cells were orthotopically injected into the right adrenal gland of SCID mice. After one week of treatments, tumors were removed and fixed in 10% phosphate-buffered formaldehyde. Significant reduction in tumor growth was observed in mice treated with SPARC alone or with SPARC in combination with radiation as compare to mice treated with controls with or without radiation (Fig. 7A). H&E staining for tumor sections showed decreased tumor volume in SPARC-overexpressed mice when compared to mock or empty vector-treated mice with or without radiation (Fig. 7B). To determine SPARC overexpression in vivo, tumor sections were stained with a monoclonal antibody for human SPARC. Figure 7C indicates that tumor sections from pSPARC alone or in combination with radiation treatment showed intense staining for SPARC as compared to radiation alone or in combination with mock or the pEV treatment. To assess whether reduced tumor growth was due to inhibition of tumor cell proliferation, we analyzed the levels of Ki67 in the tumor sections. Ki67 staining was found to be higher in mock or pEV treatment alone or in combination with radiation as compared to SPARC treatment alone or in combination with radiation, thereby indicating that SPARC overexpression in in vivo reduced tumor cell proliferation (Fig. 7C). To determine whether inhibited proliferation in SPARC-overexpressed tumors was due to decreased AKT and increased PTEN levels, we analyzed AKT and PTEN levels in these tumor sections using specific antibodies against pAKT and PTEN. Tumor sections from either SPARC alone or in combination with radiation showed moderate staining for pAKT. In contrast, we found intense staining for PTEN when compared to mock or pEV treatment alone or in combination with radiation (Fig. 7C). These results are consistent with a role for AKT-PTEN in SPARC-mediated inhibition of proliferation as observed in vitro. 10.1371/journal.pone.0036093.g007Figure 7 SPARC overexpression alone and in combination with radiation inhibits neuroblastoma cell proliferation in vivo through increased expression of PTEN and inhibited phosphorylation of AKT. Neuroblastoma orthotopic tumor sections from mice injected with mock, pEV or pSPARC plasmids alone or in combination with radiation (IR) were analyzed as described in Materials and Methods. (A) Tumor photographs from representing mice, (B) Hematoxylin and Eosin (H&E) staining for the tumors (Magnification at 4× and 40×), and (C) immunohistochemical analysis for SPARC, Ki-67, pAKT and PTEN were carried out as described in Materials and Methods. All results are representative of multiple tumors taken from five separate mice in each treatment group (Magnification at 60×). Discussion Earlier reports suggested that SPARC negatively regulates cell proliferation in several cancers without stimulating metastasis [39]. In certain cancers, such as melanomas and gliomas, SPARC is associated with a highly aggressive tumor phenotype. In other cancers, mainly ovarian, neuroblastoma, and colorectal, SPARC may function as a tumor suppressor [9]. These opposing effects on cell growth, cell migration and tumor formation suggest that the functions of SPARC are cell-specific and may be dependent upon concentration as well as regulation of ECM components. Earlier studies [40] were done to investigate the effects of downregulated SPARC expression on the radiosensitivity of human glioma U-87MG cells and its possible mechanism. With a small-interfering RNA (siRNA) expression plasmid vector targeting SPARC, these authors obtained the stably transfected cells in which the expression of SPARC was successfully downregulated. The cells were then irradiated and analyzed by several methods, including clonogenic assay, flow cytometry, comet assay, and western blotting. Clonogenic assay showed that downregulation of SPARC expression enhanced cell survival after radiation. Flow cytometry analysis indicated that SPARC siRNA decreased cell apoptosis responding to irradiation. Analysis of signaling molecules with western blotting showed that the level of AKT phosphorylation was increased in irradiated U-87MG/SPARCsiRNA cells. Further, cell-cycle analysis by flow cytometry showed enhanced G2 accumulation in U-87MG/SPARCsiRNA cells after irradiation. The data suggest that inhibition of SPARC expression may diminish the radiosensitivity of human glioma U-87MG cells. One of the mechanisms for this effect may be associated with the reduced cell apoptosis responding to radiation, which may be contributed by the phosphoinositide 3-kinase/AKT pathway activation. Moreover, the authors hypothesized that enhanced G2 accumulation and increased DNA repair may also account for the decreased radiosensitivity. SPARC is also known for sensitizing therapy-resistant tumors for either chemotherapy or radiation. Neuroblastoma is the most common pediatric solid tumor. This aggressive embryonal malignancy of neural crest origin has a peak age of onset of 22 months, and accounts for ∼11% of all pediatric cancers and 15% of all pediatric cancer deaths. With current treatment protocols, including chemotherapy, stem cell transplantation, radiation, and surgery, ∼80% of high-risk patients go into remission, although the majority relapse and succumb to therapy-resistant tumors. The role of SPARC in cell survival and death is complex. SPARC was originally identified as a stress response gene [41] and subsequently described as a c-Jun-responsive target gene that can be repressed or induced depending on cell type [42], [43]. On the other hand, there is evidence in some contexts that SPARC induces apoptosis in ovarian cancer cells [44] and modulates sensitivity to chemotherapy in colon cancer cells by enhancing apoptosis [45]. Therefore, the focus of this investigation was to determine the role of SPARC in neuroblastoma cell proliferation. Our earlier findings revealed that proliferation, adhesion, migration, invasion, and MMP-9 activity in medulloblastoma (data published earlier) [12], could be inhibited by SPARC. We have also shown in an earlier study that SPARC induced autophagy-mediated apoptosis in PNET tumor cells [12]. Here, we sought to determine the mechanism by which SPARC could inhibit neuroblastoma cell proliferation. Moreover, SPARC overexpression increased the levels of tumor suppressor protein PTEN and inhibited pro-proliferating protein AKT. These results demonstrate that SPARC has the potential to inhibit neuroblastoma cell proliferation, thereby leading to reduced tumor growth. In the present study, we used a plasmid encoding the SPARC full-length gene for SPARC overexpression in the neuroblastoma cell lines to determine proliferation rate and tumor growth. The cells transfected with pSPARC showed a 3- to 4-fold increase in protein levels (as determined by western blotting and immunocytochemical analysis) and in gene transcript levels (as determined by RT-PCR analysis) when compared to mock or empty vector-transfected cells. SPARC overexpression inhibited neuroblastoma cell proliferation as determined by MTT assay. Previously, SPARC was shown to be an anticipated resistance-reversal gene as evidenced by low SPARC expression in refractory human MIP101 colon cancer cells [24]. Restoration of cells radiosensitivity and chemosensitivity was achieved by re-expression of SPARC in tumor xenografts of colon cancer. Additionally, mice treated with SPARC showed increased sensitivity to chemotherapy, which led to significant regression of xenografted tumors. Earlier reports showed that modulation of SPARC expression affected colorectal cancer sensitivity to radiation and chemotherapy [24]. In the present study, SPARC expression levels were reduced with radiation in a dose-dependent manner in neuroblastoma cell lines. SK-N-AS and NB1691 cells showed radiation resistance until 8 Gy radiation, whereas IMR-32 cells showed resistance only until 4 Gy. It is well known that when cells are exposed to radiation, they cause a complex response that includes cell cycle arrest in G1 and G2 phases [26], [46]. DNA is an important subcellular target of ionizing radiation, but oxidative damage to plasma membrane lipids initiates signal transduction pathways that activate apoptosis, which may play a role in cell cycle regulation [47]. In this study, we illustrate that SPARC alters the cell cycle and sensitizes neuroblastoma cells to radiation by altering Chk1 and Cdc25C. Earlier reports [48] show that cyclin B increase can also occur in the absence of spindle inhibition if c-Myc deregulation is combined with inactivation of the p53 tumor suppressor. Under these conditions, cyclin B1 protein is induced but retains its normal cell cycle regulation. The authors show that c-Myc and the loss of p53 cooperate to induce cyclin B1 mRNA and protein. The central role of cyclin B1 in the maintenance of genomic integrity is underscored by the consequences of its deregulated expression. This may be one of the reasons for the increase in the cyclin B levels in the SPARC treatment MAPK families are known to play an important role in key cellular processes including proliferation, differentiation, development, transformation, and apoptosis [49]. We evaluated the levels of JNK in SPARC-overexpressed neuroblastoma cells and found increased activation of JNK. Earlier reports showed that the treatment of the colon cancer cell line HT29 with the differentiating agent sodium butyrate (NaBT) increased PTEN protein and mRNA expression and also induced c-Jun NH2-terminal kinase (JNK) activation. Inhibition of JNK by chemical or genetic methods attenuated NaBT-induced PTEN expression [36]. We observed that SPARC overexpression increased the activation of JNK and led to increased PTEN levels which were inhibited by the JNK specific inhibitor. Based on the these results, we hypothesize that SPARC activates JNK and activated JNK leads to increased PTEN levels in SPARC-overexpressed neuroblastoma cells. SPARC overexpression in neuroblastoma cells prior to radiation inhibited AKT phosphorylation and increased levels of the PTEN tumor suppressor protein. Accumulating evidence suggests that the phosphatidylinositide 3-kinase (PI3K)/AKT signal pathway is a major contributor to radioresistance [31]. Earlier reports suggested that activation of the PI3K/AKT pathway is also associated with tumorigenesis and resistance to apoptosis [50], [51]. To confirm the role of the AKT pathway in sensitizing SPARC capabilities towards radiation in neuroblastoma, we overexpressed activated AKT using myr-AKT. The results suggest that activation of AKT in the presence of SPARC overexpression led to increased proliferation in neuroblastoma cells prior to radiation. We further determined that phosphorylation of EGFR was inhibited with SPARC overexpression prior to radiation in these cells. In many cell types, PI3K/AKT signaling is a key response downstream of the EGFR family of receptors and mediates carcinogenesis [31]. Moreover, AKT activity is commonly dysregulated in a variety of human tumors because of frequent inactivation of the PTEN tumor suppressor gene, which negatively regulates phosphatidylinositol 3 phosphate levels [52], [53]. Moreover, alterations of this gene have been identified in a large fraction of cancers [36], [54], [55]. In light of this, we postulate that increased PTEN function with subsequent inhibition of SPARC prior to radiation reduced AKT phosphorylation, thereby leading to decreased proliferation in neuroblastoma cells. PTEN reduced AKT signaling in both control and SPARC-overexpressing cells, suggesting that PTEN signaling works as a downstream effector for SPARC overexpression in neuroblastoma cells. In the current study, we found that SPARC overexpression increased PTEN levels both in vitro and in vivo. One possible explanation is that when PTEN is expressed, the resulting suppression in cell growth may induce the cells to become more dependent on growth factor signaling, which can be antagonized by SPARC. Our in vitro data indicated that SPARC inhibited proliferation and sensitized neuroblastoma cells to radiation via reducing the phosphorylation levels of AKT, thereby leading to increased PTEN. Moreover, our in vitro data also showed that both SPARC and PTEN were expressed at the same time and followed inhibition of pAKT in tumor cells when treated with pSPARC. We expected the same results in vivo. To test this hypothesis, we orthotopically grafted mice with NB1691 neuroblastoma cells and treated them with mock, pEV or pSPARC alone or in combination with radiation. The animals treated with pSPARC showed increased SPARC levels as compared to mock or empty vector-treated tumors. Immunohistochemical analysis for PTEN and pAKT demonstrated that SPARC and PTEN were overexpressed when treated with pSPARC and at the same time, pAKT was inhibited, leading to reduced proliferation in vivo. Other studies support our findings since SPARC-overexpressing glioma cells have been reported to inhibit cell proliferation upon PTEN induction [37]. Accordingly, the addition of SPARC was shown to modulate the proliferation of many primary cells including endothelial cells [56], [57], skeletal myoblasts [58], mesangial cells [59], mesenchymal cells [60], mesothelial cells [44], and epithelial cells [61]. The effect that changes in SPARC levels have on tumor cell proliferation is more complex and debatable. Earlier studies [62] showed that SPARC expression is inversely correlated with the degree of malignant progression in neuroblastoma tumors. Knockdown of SPARC in neuroblastoma cells may increase the malignant progression in neuroblastoma tumors. SPARC has also been shown to have a role in growth rate modulation as demonstrated by SPARC knockout mice having an increased rate of tumor growth than those mice with intact SPARC [63], [64]. Based on the literature available, it may be possible that forced knockdown of SPARC in these conditions may lead to more tumor growth. Said and Motamed [65] evaluated the effect of host-derived SPARC on ovarian cancer growth in vivo, demonstrating more rapid and aggressive tumor growth in SPARC-deficient animals. In summary, we have shown that overexpression of SPARC decreases proliferation and sensitizes neuroblastoma cells to irradiation. We hypothesized that overexpression of SPARC resulted in inhibition of pAKT and increased PTEN, leading to cell cycle abate that subsequently caused the inhibition of proliferation and sensitized cells to radiation in vitro and in vivo. On the basis of these observations, we conclude that endogenous overexpression of SPARC can decrease neuroblastoma cell proliferation and tumor growth and therefore act as a tumor suppressor in human neuroblastoma. Future studies in human neuroblastoma should focus on the design of treatment strategies that specifically target SPARC–PTEN interactions. Materials and Methods Ethics statement The Institutional Animal Care and Use Committee of the University of Illinois College of Medicine at Peoria, Peoria, IL, USA, approved all surgical interventions and post-operative care. The consent was written and approved. The animal protocol number is 872, dated May 20th 2010 and renewed on May 20th 2011. Cell culture SK-N-AS and IMR-32 cells were obtained from ATCC (Manassas, VA) and NB-1691 cells were obtained from Dr. Houghton of St. Jude Children's Research Hospital (Memphis, TN). Cells were cultured in RPMI medium with 10% fetal bovine serum, 2 mM/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained in a humidified atmosphere containing 5% CO2 at 37°C. Antibodies and reagents Antibodies against SPARC, pAKT, AKT, c-Myc, PTEN, EGFR, pEGFR, Cyclin B, Cyclin D1, Cdc2, pCdc2, Cdc25C, Chk1, Chk2, mTOR, PI3K, JNK, pJNK, c-Jun, p-c-Jun (Ser-63), p-c-Jun (Ser-73), caspase-3, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), Antibodies against PARP (Cell Signaling Technology, Beverly, MA) were also used in this study. Plasmids encoding myr-AKT delta4–129 (Addgene plasmid 10841) and HA PTEN wt (Addgene plasmid 10750) were obtained from Addgene Inc. (Cambridge, MA). Construction of pcDNA3.1-SPARC and transfection An 1100-bp cDNA fragment of human SPARC was amplified by PCR using synthetic primers and sub-cloned into a pcDNA3.1 vector (Invitrogen, San Diego, CA) in the “sense” orientation. Neuroblastoma cells were transfected with full-length cDNA SPARC-containing vector using FuGene HD (Roche, Indianapolis, IN) as per the manufacturer's protocol. siRNA design and transient transfection SPARC siRNA sequences were designed with the help of a siRNA designer program (Imgenex, Sorrento Valley, CA). The siRNA was complementary to an exonic sequence of the target mRNA and compatible with the pcDNA3.1 vector (Invitrogen, San Diego, CA). The following siRNA sequence was used to construct SPARC siRNA and designated as SP-siRNA: 5′-TCGAGGGTGTGCAGCAATGACAA CAAGAGTCGTCGTTGTTGTCATTGCTGCACACCG-3′. A control vector containing siRNA with a scrambled sequence was constructed and designated as control siRNA. We used the following scrambled sequence: 5′-CACGGAGGTTGCAAAGAATAATCGATTATTCTTT GCAACCTCC GTGC-3′. Transfection with plasmids All transfection experiments were performed using FuGene HD transfection reagent according to the manufacturer's protocol (Roche). Briefly, plasmid/siRNA was mixed with FuGene HD reagent (1∶3 ratio) in 500 µL of serum-free medium and left for 30 min to allow for complex formation. The complex was then added to the 100-mm plate, which had 2.5 mL of serum-free medium (2 µg plasmid per ml of medium). After 6 hours of transfection, complete medium was added and cells were cultured for another 36 hours. Immunocytochemistry We used a previously described protocol with minor changes [66]. Briefly, the cells were cultured on 8-well chamber slides and fixed with 4% paraformaldehyde (w/v) in PBS, permeabilized with 0.1% Triton X-100 (w/v) in PBS and blocked with 1% BSA (w/v) in PBS for 1 hour at 4°C. Cells were incubated overnight at 4°C with anti-SPARC antibody followed by corresponding Alexa fluor-594-conjugated secondary antibody for 1 hour and counterstained with DAPI. For negative controls primary antibody was replaced by non-specific IgG. Slides were washed and mounted with anti-fade mounting solution (Invitrogen, San Diego, CA) and analyzed with an inverted microscope. Western blotting Western blot analysis was performed as described previously [67]. Briefly, 36 hours after transfection, cells were collected and lysed in RIPA buffer. Equal amounts of protein were resolved on SDS-PAGE and transferred onto a PVDF membrane. The blot was blocked with 5% non-fat dry milk and probed overnight with primary antibodies followed by HRP-conjugated secondary antibodies. An ECL system was used to detect chemiluminescent signals. All blots were re-probed with GAPDH antibody to confirm equal loading. RT-PCR Neuroblastoma cells were transfected with mock, pEV or pSPARC for 36 hours. Total RNA was extracted from these cells and cDNA synthesized using poly-dT primers as described earlier [68]. PCR was performed using the following primers: SPARC: 5′-GGAAGAAACTGTGGCAGAGG-3′ (sense), and 5′-ATTGCTGCACACCTTCTCAA-3′ (antisense); GAPDH: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ (sense), and 5′-CATGTGGGCCATGAGGTCCACCAC-3′ (antisense). Quantification of SPARC mRNA levels was determined based on densitometry. Flow cytometry For assessment of DNA content, neuroblastoma cells were plated overnight in 100-mm tissue culture plates and transfected for 36 hours as described above. We used FACS analysis that utilizes propidium iodide staining of nuclear DNA to characterize hypo-diploid cells [69]. Briefly, cells were harvested by trypsinization and stained with propidium iodide (2 mg/mL) in 4 mM sodium citrate containing 3% (w/v) Triton X-100 and RNase A (0.1 mg/mL; Sigma, St. Louis, MO). Suspensions of 2×106 cells were analyzed by FACS Caliber System (Becton Dickinson Bioscience, San Jose, CA) with laser excitation at 488 nm and using an emission 639 nm band pass filter to collect the red propidium iodide fluorescence. The percentages of cells in the various phases of the cell cycle (sub-G1, G1/S, and G2/M) were assessed using Cell Quest software (Becton Dickinson Bioscience). Cell proliferation assays Cell growth rate was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a measurement of mitochondrial metabolic activity as described earlier [70]. Cells were transfected with indicated plasmids and incubated at 37°C. After 0 to 72 hours, MTT reagent was added to the cells and incubated for 6 hours at 37°C. The rate of absorbance of formazan (a dye produced by live cells) was measured with a microplate reader at A550. Clonogenic assay Cells were transfected with mock, pEV, and pSPARC for 24 hours and irradiated with different Gy of radiation. Cells (5×102 cells) were trypsinized and seeded in 100 mm Petri dishes. On day 10 after irradiation, cells were fixed in cold methanol and stained with Giemsa and colonies (>50 cells) were counted. The plating efficiency (PE) is defined as the number of colonies observed/the number of cells plated. Surviving fraction (SF) is the colonies counted divided by the number of colonies plated with a correction for the plating efficiency. Intra-adrenal tumor model and immunohistochemistry The Institutional Animal Care and Use Committee at the University of Illinois College of Medicine at Peoria approved all experimental procedures involving the use of animals. Orthotopic, localized neuroblastoma tumors were established in C.B-17 SCID mice by injection of 1×106 NB-1691 cells in 100 µL PBS into the retroperitoneal space as described earlier [71]. After 2 weeks of tumor cell implantation, the mice were separated into six groups containing 6 animals per group, and each group was injected intravenously with PBS (mock) or pEV or pSPARC (100 µL volume) and was given three doses on alternate days. Between the first and the second injections, and the second and the third injections, one group was radiated with a dose of 5 Gy each time. Mice were euthanized when animals had lost >20% of body weight or had trouble ambulating, feeding, or grooming. The tumors were removed and either fixed in 10% phosphate-buffered formaldehyde or snap frozen and maintained at −70°C until sectioning. Briefly, all tumors were serially sectioned and tissue sections (7 µm thick) obtained from the paraffin blocks were stained with hematoxylin and eosin (H&E) using standard histologic techniques. For immunohistochemical analysis, sections were incubated with mAb (1 hour at room temperature) followed by the appropriate secondary antibody. For HRP-conjugated secondary antibodies, we used DAB solution as the chromogen. Negative control slides were obtained by nonspecific IgG. Sections were mounted with mounting solution and analyzed with an inverted microscope. Statistical analysis All data are expressed as mean ± SD. Statistical analysis was performed using the student's t test or a one-way analysis of variance (ANOVA). A p value of less than 0.05 was considered statistically significant. All experiments were performed in triplicate to obtain consistent results. Supporting Information Figure S1 Overexpression of SPARC in neuroblastoma cells. The human full-length SPARC cDNA was subcloned into the pDNR-CMV mammalian expression vector and termed as pSPARC. pDNR-CMV was the vector without the SPARC gene and termed as empty vector (pEV). SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or SPARC full-length gene inserted vector (pSPARC) for 36 hours. (A) SPARC levels were determined by Western blotting using a SPARC-specific antibody. GAPDH served as a loading control. Columns, mean of three experiments; bars, SD.* p<0.01 vs pEV. (B) cDNA was produced from total RNA extracted from the mock and infected cells. RT-PCR analysis was performed for SPARC. Results are representative of three independent experiments. GAPDH served as a control for RNA quality. Columns, mean of three experiments; bars, SD. * p<0.01 vs pEV. (C) SK-N-AS, NB1691 and IMR-32 cells were transfected with mock (PBS control), empty vector (pEV) or pSPARC for 36 hours and immunocytochemical analysis for SPARC was performed. Representing images were shown taken from 5 different microscopic fields of three independent experiments. (D) SK-N-AS, NB1691 neuroblastoma cells and HMEC cells were culture for 24 hours and Western blot analysis was performed for SPARC using specific antibody. GAPDH served as loading control. (E) Clonogenic survival assay for irradiated neuroblastoma cells. SK-N-AS, NB1691 and IMR-32 neuroblastoma cells were irradiated (IR) with 2 Gy to 12 Gy doses of radiation and clonogenic assay was performed as described in Materials and Methods. The cells were culture and colonies larger than 50 cells were counted. Points: mean of triplicate experiments. (TIF) Click here for additional data file. The authors thank Shellee Abraham for manuscript preparation and Diana Meister and Sushma Jasti for manuscript review. We also thank Dr. Richard A. Roth (Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA) for providing plasmid myr-AKT delta4–129, Dr. William R. Sellers (Department of Adult Oncology, Dana–Farber Cancer Institute and Harvard Medical School, Boston, MA) for providing pSG5L HA PTEN wt, and Dr. P. Houghton (St. Jude Children's Research Hospital, Memphis, TN) for providing NB1691 neuroblastoma cell line. Competing Interests: The authors have declared that no competing interests exist. Funding: This project was supported by award number CA147792 (to JSR) from the National Institutes of Health. Contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Ho R Eggert A Hishiki T Minturn JE Ikegaki N 2002 Resistance to chemotherapy mediated by TrkB in neuroblastomas. Cancer Res 62 6462 6466 12438236 2 Kurschat P Mauch C 2000 Mechanisms of metastasis. Clin Exp Dermatol 25 482 489 11044183 3 Poste G Fidler IJ 1980 The pathogenesis of cancer metastasis. Nature 283 139 146 6985715 4 Discher DE Janmey P Wang YL 2005 Tissue cells feel and respond to the stiffness of their substrate. Science 310 1139 1143 16293750 5 Janmey PA Winer JP Murray ME Wen Q 2009 The hard life of soft cells. 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PLoS One. 2012 May 2; 7(5):e36093
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22574218PONE-D-12-0443710.1371/journal.pone.0036717Research ArticleBiologyBiochemistryProteinsProtein ChemistryMolecular Cell BiologySignal TransductionSignaling CascadesProtein Kinase Signaling CascadeMedicineAnatomy and PhysiologyCell PhysiologyCardiovascularAortic DiseasesAtherosclerosisCardiovascular PharmacologyVascular BiologyRegulation of the Proteasome by AMPK in Endothelial Cells: The Role of O-GlcNAc Transferase (OGT) AMPK Regulates Proteasome via OGTXu Jian 1 Wang Shuangxi 2 Viollet Benoit 3 4 5 Zou Ming-Hui 2 * 1 Division of Endocrinology, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America 2 Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America 3 Inserm, U1016, Institut Cochin, Paris, France 4 Cnrs, UMR 8104 Paris, France 5 Université Paris Descartes, Paris, France Xu Aimin EditorUniversity of Hong Kong, China* E-mail: [email protected] and designed the experiments: JX MHZ. Performed the experiments: JX SW. Analyzed the data: JX SW. Contributed reagents/materials/analysis tools: BV. Wrote the paper: JX MHZ. 2012 4 5 2012 7 5 e3671713 2 2012 12 4 2012 Xu et al.2012This 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.26S proteasome is a macromolecular multi-subunit complex responsible for recognizing, unfolding, and ultimately destroying proteins. It remains poorly understood how 26S proteasome activity is regulated. The present study was to investigate if AMP-activated protein kinase (AMPK) functions as a physiological suppressor of the 26S proteasome in endothelial cells. 26S proteasome assembly, activity, and O-GlcNAcylation of P700 were assayed in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from C57BL6 wild type and AMPKα2 knockout mice with or without being exposed to selective AMPK activators or inhibitors. Pharmacological and genetic activation of AMPK effectively suppresses 26S proteasomes in endothelial cells. Conversely, inactivation of AMPK either pharmacologically or genetically increases 26S proteasome activity; furthermore, the inactivation decreases the O-GlcNAcylation of PA700/S10B (the regulatory complex in 26S proteasomes) and increases the assembly of 26S proteasomes. In contrast, AMPK activation increases levels of O-GlcNAcylated PA700/S10B, likely through enhanced association of PA700 with O-GlcNAc transferase (OGT), the enzyme that catalyzes protein O-GlcNAcylation. Finally, aortas from AMPK-KO vs wild type mice exhibit elevated 26S proteasome activity in parallel with decreased PA700/S10B O-GlcNAcylation and PA700/S10B-OGT association. Taken together, we conclude that AMPK functions as a physiological suppressor of 26S proteasomes. ==== Body Introduction The ubiquitin proteasome system (UPS) is the major non-lysosomal degradative machinery for most intracellular proteins [1], [2]. A key component of this machinery is the 26S proteasome [3], a macromolecular multi-subunit complex that is responsible for recognizing, unfolding, and ultimately destroying proteins. To be degraded, most target proteins must first be tagged with polyubiquitin chains, generally at the -NH2 group of an internal lysine residue [4], [5]. The 26S proteasome (a 2-MDa complex) is made up of two sub-complexes: the catalytic particle (or 20S proteasome) and the regulatory particle (19S proteasome) [3]. The 20S proteasome is a cylindrical protease complex consisting of 28 subunits configured into four stacks of heptameric rings. On the other hand, the 19S (or PA700) consists of at least 18 subunits, including 6 putative ATPases and 12 non-ATPase subunits [3], [6]. The 26S proteasome is known to require ATP hydrolysis to degrade ubiquitinated substrates and for its assembly [7]. Over the past few years, it has become clear that deregulation of the UPS leads to inappropriate destruction or accumulation of specific proteins and ensuing pathological consequences [1]. The UPS is now recognized as a regulator of the cell cycle and cell division [8], [9], immune responses and antigen presentation [10], [11], apoptosis [12], and cell signaling [13], [14]. The UPS has been shown to be either activated in certain cancers (e.g., multiple myeloma) [15], [16] or dysfunctional in neurodegenerative disorders (e.g., Alzheimer's disease, Huntington's disease [17], and amyotrophic lateral sclerosis [18], [19]). AMPK was initially identified as a sensor of cellular energy [20], [21] and is also likely a sensor of cellular redox status [22], [23]. As a phylogenetically conserved enzyme, AMPK is present in all mammalian cells. AMPK is a heterotrimeric enzyme comprised of a catalytic (α) subunit and two regulatory (β and γ) subunits [24], [25]. AMPK is activated by at least three distinct signals: a Ca2+-dependent pathway mediated by calcium calmodulin-dependent kinase kinase-β (CaMKK-β) [26], an AMP-dependent pathway mediated by LKB1 [27], and TGF-β-activated kinase-1 (Tak1) [28] via phosphorylation at Thr172 on the α-subunit. Binding of AMP to the γ-subunit leads to allosteric activation of AMPK, a change that also protects the Thr172 site from dephosphorylation [29]. Once activated, AMPK switches on catabolic pathways that generate ATP, while switching off ATP-consuming processes (e.g., biosynthesis, cell growth, and proliferation). In this way, it functions as “energy gauge” [29], [30]. This has been regarded as a fundamental feature of multiple AMPK-mediated biological processes. AMPK is generally quiescent under normal conditions but is activated in response to hormonal signals and stresses sufficient to increase the AMP/ATP ratio, such as hypoglycemia, strenuous exercise, anoxia, and ischemia. In contrast to traditional N- and O-glycosylation, which occurs in secretory pathways (endoplasmic reticulum and Golgi) [31], O-GlcNAcylation is defined as the O-linked attachment of N-acetylglucosamine (O-GlcNAc) onto Ser/Thr residues of cytosolic and nuclear proteins [32]. O-GlcNAcylation is an important regulatory mechanism for signal transduction [32], [33], [34], [35]. OGT mediates O-GlcNAcylation, while removal of O-GlcNAc is catalyzed by the complementary β-N-acetylhexosaminidase (O-GlcNAcase, or OGlcNAc hydrolase as OGA). To date, more than 80 different proteins including transcription factors, kinases, phosphatases, cytoskeletal proteins, nuclear hormone receptors, nuclear pore proteins, signal transduction molecules, and actin regulatory proteins [34], [35] have been shown to undergo O-GlcNAcylation [35] although the mechanisms underlying OGT regulation are not completely understood. Endothelial dysfunction, which is characterized by impaired endothelium-dependent vasorelaxation [36], [37], [38], is a common feature of CVD. During CVD, the endothelium losses its homoeostatic potential to inhibit the disease process [38]. We [39], [40], [41] and others [42] have shown that the UPS can contribute to the development of endothelial dysfunction, which is reflected as enhanced 26S proteasome activity, an accelerated degradation of endothelial-protective molecules (such as the guanosine 5-triphosphate cyclohydrolase I or GTPCH I, the rate limiting enzyme essential for the de novo synthesis of an eNOS co-factor [39], [40], [43]), and consequent impaired endothelium-dependent vasorelaxation. Most importantly, we have discovered that such an impairment can be restored by activation of AMPK [43]. Interestingly, we recently found that global knockout of both AMPK and ApoE genes enhances NAD(P)H oxidase expression as well as exacerbates atherosclerosis and endothelial dysfunction by influencing 26S proteasome activation [44]. Further, we found that AMPK activation alleviates diabetic endothelial dysfunction by suppressing the 26S proteasome [43]. However, how AMPK regulates 26S proteasome activity in these cases remains unknown. Here we report that AMPK suppresses 26S proteasome activity by promoting PA700/S10B-OGT association and PA700/S10B O-GlcNAcylation. Materials and Methods Materials and animal model Mouse-derived PA700/S10B antibody was from Abcam (Cambridge, MA); MG132 from BioMol (Plymouth Meeting, PA); fluorogenic proteasome substrates from Calbiochem (San Diego, CA); Proteasome inhibitor MG132 was purchased from BioMol (Plymouth Meeting, PA). 1% protease inhibitor cocktail were obtained from Sigma (St Louis, MO). 1% phosphatase inhibitor cocktail was purchased from Pierce. PUGNAc (O-GlcNAcase inhibitor) was from Sigma (St Louis, MO). Anti-O-GlcNAc antibody (CTD110.6 antibody, sc-59623) and negative control siRNA or target-specific siRNA duplex against OGT were from Santa Cruz Biotechnology (Santa Cruz, CA). Control siRNA duplex and PA700-specific siRNA duplex were from Ambion (Austin, TX). Protein-A Sepharose CL-4B was purchased from Amersham Biosciences (Piscataway, NJ). All other chemicals were obtained from Fisher Scientific (Pittsburgh, PA), unless otherwise noted, were of the highest possible grade. Human umbilical vein endothelial cells (HUVEC) were obtained from Cascade Biologics (Walkersville, MD). Control and AMPKα2−/− male mice were studied at 12 weeks of age. The establishment and characterization of AMPKα2−/− mice were previously reported [45]. Animals were housed under controlled temperature (21°C) and lighting, with 12 hours of light and 12 hours of dark, and had free access to water and a standard mouse chow diet. All procedures were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC Protocol: 10-005). 26S proteasome activity assay 26S proteasome function was assayed as described previously but with minor modifications [46]. Chymotrypsin-like activity was measured using SucLLVY-7-amido-4-methylcoumarin (AMC), trypsin-like activity with Bz-VGR-AMC, and caspase-like activity with Z-LLE-AMC. These fluorogenic proteasome substrates were added to the cell lysate at a final concentration of 80 µM in 1% DMSO. ATP-dependent cleavage activity was monitored continuously by detection of free 7-amido-4-methylcoumarin using a fluorescence plate reader (Gemini, Molecular Devices) at 380/460 nm at 37°C. Adenoviral infection and siRNA transfection Confluent HUVEC were infected with adenovirus encoding AMPK-CA, AMPK-DN, or GFP (as adenoviral infection control), as described previously [43]. SiRNA transfection was done, as described previously [40]. Identification of O-GlcNAc-modified proteins in mouse aortas Aortas isolated from mice were weighed and homogenized in lysis buffer containing 25 mmol/L HEPES (pH 7.0), 1 mmol/L EDTA, 1 mmol/L EGTA, 1% NP-40, 0.1% SDS, 1% protease inhibitor cocktail (Sigma, St Louis, MO), 1% phosphatase inhibitor cocktail, and 1–100 µmol/L PUGNAc (O-GlcNAcase inhibitor). Proteins modified by O-linked GlcNAc will be immunoprecipitated with anti-O-GlcNAc antibody. Proteins were released from the beads by boiling in Laemmli buffer containing 50 mmol/L dithiothreitol and separated by 12% SDS-PAGE. Separated proteins were subjected to western blot analysis using anti-PA700 antibodies. Statistical analysis Statistical comparisons of all results were analyzed using a one-way ANOVA. Values are expressed as mean ± SEM. p<0.05 is considered as significant. Results AMPK inactivation increases 26S proteasome activity in HUVEC To determine whether AMPK inactivation alters proteasome activity, we tested the effect of modulating AMPK activity on proteasome activities in HUVEC. Compound C (0.5 µmol/L for 15 min), the nonselective inhibitor of AMPK [47], but not vehicle (DMSO), significantly increased 26S proteasome activity, and this effect could be reversed by co-incubation with MG132 (proteasome inhibitor, 0.5 µmol/L) or AICAR (5-aminoimidazole-4-carboxamide ribose, an AMPK activator) (Fig. 1A). As expected, MG132 alone significantly inhibited proteasome activity (Fig. 1A). However, AICAR alone for 6 h had no significant effect on 26S proteasome activity (Fig. 1A), although prolonged incubation with AICAR (overnight) did suppress 26S proteasome activity in HUVEC [43]. 10.1371/journal.pone.0036717.g001Figure 1 AMPK suppression increases 26S activity in HUVEC. AMPK suppression in HUVEC either by (A) compound C (10 µmol/L for 2 hour) or by (B) overexpression of AMPK-dominant negative mutant (DN) increases 26S proteasome activities, as demonstrated by ATP-dependent increased chymotrypsin-like, trypsin-like and caspase-like activity on fluorescent proteasome substrates. MG132 (0.5 µmol/L for 1 hour) or AICAR (2 mmol/L for 6 hours) treatment blocks the increased chymotrypsin-like activity in AMPK-suppressed HUVEC. * indicates P<0.05 vs vehicle (DMSO) or GFP, n = 3 per group. Next, we investigated whether genetic inhibition of AMPK had a similar effect as pharmacological AMPK inhibition by compound C. Overexpression of an AMPK dominant negative mutant (AMPK-DN), but not of GFP (control), also induced 26S proteasome activation, while MG132 could block the activation (Fig. 1B). Interestingly, unlike MG132, AICAR failed to inhibit AMPK-DN promoted 26S proteasome activation (Fig. 1B), suggesting that the suppressive effect of AICAR on proteasome was mediated, at least in part, by AMPK activation. Together, these results suggest that AMPK inactivation contributes to 26S proteasome activation in HUVEC. Of note, AMPK inactivation increased all 3 major protease-like activities at various degrees (Fig. 1A and B). However, only chymotrypsin-like activity could be significantly reversed or suppressed by the treatment with AICAR (Fig. 1A) or MG132 (Fig. 1A and B) at indicated concentrations and duration of the treatment. Therefore, we only used chymotrypsin-like activity to demonstrate AMPK dependent effects on 26S proteasome activity in subsequent cell experiments. AMPK inhibition-induced 26S proteasome activation is accompanied by increased 26S assembly To confirm that AMPK responds to its activator or inhibitor as reported, we first detected AMPK activation in challenged HUVEC. As expected, AICAR promoted, but compound C suppressed AMPK phosphorylation (Thr172), a marker of AMPK activation, so as its downstream target ACC phosphorylation (Ser79), based on quantified data that had been normalized with the total AMPK or ACC protein levels (Fig. 2A). To begin to understand how AMPK inactivation may activate the 26S proteasome, we first determined if pharmacological or genetic inactivation of AMPK affects 26S assembly (association), an event known to be crucial for 26S proteasome activation [48], [49], [50]. Western blot analysis of PA700 immunoprecipitates using 20S proteasome-specific antibody (against its β7 subunit) revealed that a greater association occurred between PA700 and the 20S proteasome in compound C-treated cells than in vehicle-treated cells (Fig. 2B). However, co-incubation of cells with AICAR abolished the effect of compound C. None of these treatments altered either PA700 or β7 protein levels (Fig. 2B). To confirm the 26S proteasome assembly, we further separated the same samples under non-reducing condition on a native gradient PAGE (3–14%) and performed Western blot staining with antibodies to the subunits of either 19S (PA700/S10B) or 20S proteasome sub-complex (β7) on duplicate blots. As shown in Fig. 2C, on the same molecular weight site (above the native protein marker 1236 kD) of the duplicated blots, both staining for PA700/S10B and β7 were increased in the presence of compound C (Fig. 2C left), pre-incubation with AICAR blocked the increase (Fig. 2C), suggesting 26S proteasome assembly enhancement did occur in the cells. The enhanced 26S assembly was accompanied by an increase in 26S proteasome activity (Fig. 2D). 10.1371/journal.pone.0036717.g002Figure 2 Increased 26S proteasome activity in AMPK-suppressed HUVEC is correlated with the enhanced association of 19S and 20S sub-complexes. Compound C (10 µmol/L for 2 hour)-treated HUVEC present (A) AMPK inactivation, (B) an increase in association of PA700/S10B (from 19S complex) with β7 (from 20S complex), which can be blocked by AICAR pre-incubation (2 mmol/L for 6 hours), (C) 26S proteasome assembly (same samples were run on 3–14% native-PAGE under non-reducing condition followed by conventional Western blot on duplicated blots with PA700/S10B and β7 antibodies, respectively), and (D) an increase in 26S proteasome activity (chymotrypsin-like) (n = 3). The increased association of proteasome sub-complex is also observed in (E) HUVEC overexpressing AMPK-DN but not AMPK-CA or GFP. All of the blots shown are representative of 3 independent experiments. NS represents not significant. Finally, given the potential non-selective effects of compound C [47], we tested if AMPK-DN could replicate the effect of compound C. HUVEC overexpressing AMPK-DN showed enhanced 26S assembly compared to those overexpressing GFP (control) or constitutively active AMPK (AMPK-CA) (Fig. 2E). Of note, none of these treatments affected PA700 or the β7 protein levels (Fig. 2E). Like in compound C-treated cells, the increased 26S proteasome assembly (association) in AMPK-DN overexpressing HUVEC was accompanied with an increase in 26S proteasome activity (Fig. 1B), in contrasting to the suppressive effect on 26S proteasome activity detected in AMPK-CA over-expressing HUVEC [43]. The fact that 26S activation could always be detected no matter how AMPK was inactivated suggests that AMPK regulates 26S proteasomes. AMPK activation increases OGT-PA700 association and PA700 O-GlcNAcylation, while AMPK inactivation decreases these outcomes Next, we investigated whether modulation of AMPK activity could alter the OGT-PA700 association and PA700 O-GlcNAcylation. Western blot analysis of OGT immunoprecipitates using specific PA700 antibody showed that compound C reduced OGT-PA700 association, and this effect was reversed by AICAR (Fig. 3A), consistent with the ability of compound C to decrease PA700 O-GlcNAcylation in an AICAR-reversible manner (Fig. 3A). 10.1371/journal.pone.0036717.g003Figure 3 AMPK suppression is accompanied by the decreased association of OGT with proteasome. AMPK suppression in HUVEC either by (A) compound C (10 µmol/L for 2 hour) or by (B) overexpression of AMPK-dominant negative mutant (DN) decreases both the OGT association with proteasome and the O-GlcNAcylation of PA700/S10B (vs. controls), which can be reversed by AICAR pre-incubation (2 mmol/L for 6 hours) in AMPK-present but not AMPK-DN cell. In contrast, constitutive activation of AMPK (overexpression of AMPK-CA vs GFP) (B) increases OGT-PA700 association. All of the blots shown are representative of 3 independent experiments. NS reprents not significant. We also tested if genetic inhibition of AMPK through overexpression of AMPK-DN produced a similar effect as compound C. Compared to GFP overexpression, AMPK-DN overexpression elicited a dramatic reduction in OGT-PA700 association (Fig. 3B), which might contribute to enhanced 26S proteasome assembly (Fig. 2C) and activation (Fig. 2D). Importantly, although AICAR increased the association of inhibitory OGT with PA700 in cells overexpressing GFP (Fig. 3B), this effect was absent in cells overexpressing AMPK-DN (Fig. 3B), indicating that AMPK participated in enhanced OGT-P700 association. Conversely, constitutive activation of AMPK (overexpression of AMPK-CA) resulted in greater OGT-PA700 association than GFP overexpression (Fig. 3B). However, none of the treatments altered the protein levels of PA700 or OGT (Fig. 3B). An OGT activator, but not an OGA inhibitor, blocks 26S proteasome activation induced by AMPK inhibition To determine if OGT mediates AMPK suppression of proteasome activity, we compared 26S proteasome assembly in the presence of UDP-GlcNAc, a specific OGT substrate and activator. Western blot analysis of PA700/S10B immunoprecipitates by using specific 20S proteasome antibody (to its β7 subunit) revealed that activation of OGT by UDP-GlcNAc blocked compound C-induced increases in 26S proteasome assembly (Fig. 4A) and activation (Fig. 4B). OGT treatment also inhibited the decrease of PA700/S10B O-GlcNAc modification (Fig. 4A). Two dynamic enzymes directly control O-GlcNAcylation: OGT, which catalyzes O-GlcNAcylation, and its negative regulator OGA, which removes O-GlcNAc. Inhibition of OGA has been shown to have synergistic effects on OGT activation [34]. We investigated whether OGA, like OGT, participates in AMPK regulation of the 26S proteasome. Surprisingly, inhibition of OGA with PUGNAc did not prevent compound C-enhanced 26S proteasome assembly, reduction of PA700/S10B O-GlcNAcylation (Fig. 4A) or activation (Fig. 4B). To confirm the functional outcomes of altered 26S proteasome activity, we measured the protein levels GTPCH I, which degradation is enhanced by 26S proteasome activation [39], [40], [43]. As depicted in Fig. 4, 26S proteasome activation by compound C reduced GTPCH I protein levels (Fig. 4); pre-incubation of OGT activator UDP-GlcNAc, but not OGA inhibitor PUGNAC, blocked the reduction of GTPCH I (Fig. 4C). Collectively, these data indicate that OGT plays a major role in AMPK regulation of the proteasome. 10.1371/journal.pone.0036717.g004Figure 4 Activation of OGT prevents AMPK-inhibition induced 26S proteasome activation. AMPK suppression by compound C (10 µmol/L for 2 hour) in HUVEC (A) increases the association of PA700/S10B (from 19S complex) with β7 (from 20S complex) accompanied by a decrease of PA700/S10B O-GlcNAc modification, and (B) increases 26S proteasome activity, which can be prevented by pre-incubation of UDP-GlcNAc (25 µmol/L for 30 min), but not by PUGNAc (14 µmol/L for 30 min), the inhibitor of O-GlcNAcase. The blots shown are representative of 3 independent experiments. siRNA-mediated knockdown of OGT enhances 26S proteasome assembly and activity To determine whether OGT is required for 26S proteasome regulation by AMPK, we performed knockdown experiments using siRNA. We repeated the experiments previously shown in cells infected with siRNA. As shown in Fig. 5, OGT protein levels were more than 90% lower in cells transfected with OGT siRNA than in control siRNA-treated cells. Surprisingly, knockdown of OGT significantly increased 26S assembly, as reflected by an increase in PA700 and β7 association (Fig. 5A), and 26S proteasome activity (Fig. 5B). However, compound C did not further enhance 26S proteasome assembly (Fig. 5A) and activity (Fig. 5B) in OGT siRNA-transfected cells as it did in control siRNA cells (Fig. 5A and B). The absence of this effect in OGT knockdown cells further suggests that OGT is indispensable for AMPK regulation of the 26S proteasome. As shown for other treatments, these treatments did not alter PA700 or β7 protein levels (Fig. 5A). Cumulatively, these results suggest that, under basal conditions, AMPK serves to keep the 26S proteasome at the minimal required activation level by maintaining the association of OGT with PA700 and in turn, keeping O-GlcNAcylation of PA700 in a constitutive state. 10.1371/journal.pone.0036717.g005Figure 5 Loss of OGT increases 26S proteasome assembly. HUVEC transfected with OGT siRNA but not control siRNA show an increase in (A) association of PA700/S10B (from 19S complex) with β7 (from 20S complex) and (B) 26S proteasome activity, mimicking the effect of AMPK-suppression by compound C (10 µmol/L for 2 hour). The blots shown are representative of 3 independent experiments. * represents p<0.05 vs the control (without compound C treatment). Aorta from AMPK-KO mice exhibit a decrease in OGT-PA700/S10B association along with an increase in 20S complex formation and 26S proteasome activity We next explored if AMPK regulates the 26S proteasome in vivo. We compared aortic proteins prepared from AMPKα2-KO and WT (C57BL/6J) mice. Western blot analysis revealed that, although AMPKα2 protein was undetectable in AMPKα2-KO aortas (Fig. 6A), levels of β-actin (Fig. 6A), OGT (Fig. 6B), β7 (Fig. 6C) and PA700/S10B (Fig. 6) did not significantly differ between WT and AMPK-KO aortas. However, compared to WT aortas, AMPK-KO aortas exhibited a decrease in OGT-PA700/S10B association (Fig. 6B), an increase in 26S assembly as seen by an increase in PA700/S10B-β7 association (Fig. 6C), and an increase in all three major protease-like activities of the 26S proteasome (Fig. 6D). 10.1371/journal.pone.0036717.g006Figure 6 AMPK depletion is associated with decreased association of OGT with proteasome and increased 26S proteasome assembly and activity in AMPK-KO mice. Gender (male) and age (12 weeks) matched mice (n = 8/group) with the genotypes of wild type (C57BL/6J) and AMPKα knockout were used. Compared to aortas from wild type (C57BL/6J) mice, (A) aortas from AMPKα knockout mice exhibit (B) a decrease in the association of OGT with proteasome (PA700/S10B), (C) an increase in proteasome assembly (PA700/S10B-β7 association), and (D) an increase in 26S proteasome activity, without alteration in the expression levels of proteasome (β7 or PA700/S10B) or OGT. * represents p<0.05 vs wild type (n = 8). Discussion The present study has demonstrated that inactivation of AMPK elevates 26S proteasome activity and that this effect is associated with decreased PA700/S10B O-GlcNAcylation and increased 26S proteasome assembly. Conversely, AMPK activation increases GlcNAcylated PA700/S10B by stabilizing its association with OGT, the key enzyme responsible for protein O-GlcNAcylation. OGT appears to be crucial for this event, as AMPK-mediated 26S proteasome inhibition is blocked by siRNA-mediated OGT knockdown. The most conclusive evidence that AMPK suppresses the 26S proteasome comes from analysis of aortas from AMPK-knockout (AMPK-KO) mice. We found that, compared to wild type (WT) aortas, aortas from AMPK-KO mice exhibit elevated 26S proteasome activity with decreased PA700/S10B O-GlcNAcylation and PA700/S10B-OGT association. Importantly, in spite of different mechanisms underlying AMPK inactivation by compound C and AMPK-DN, both treatments in HUVEC induce 26S proteasome activation, indicating the essential role of AMPK activity in 26S proteasome regulation. Furthermore, all the promoted 26S proteasome activations through AMPK suppression in HUVEC are attenuated by MG132, a potent proteasome inhibitor, suggesting AMPK suppression is truly associated with 26S proteasome activation. Overall, our results support the notion that AMPK, a sensor of cellular energy, maintains low basal 26S proteasome activity by keeping PA700/S10B O-GlcNAcylated and in turn, suppressing 26S proteasome activity and likely preventing 26S proteasome-mediated degradation of endothelial protective molecule(s). In contrast to traditional N- and O-glycosylation, which occurs in secretory pathways (endoplasmic reticulum and Golgi) [31], O-GlcNAcylation is defined as the O-linked attachment of N-acetylglucosamine (O-GlcNAc) onto Ser/Thr residues of cytosolic and nuclear proteins [32]. O-GlcNAcylation is an important regulatory mechanism for signal transduction [32], [33], [34], [35]. OGT mediates O-GlcNAcylation, while removal of O-GlcNAc is catalyzed by the complementary β-N-acetylhexosaminidase (O-GlcNAcase, or OGlcNAc hydrolase as OGA). To date, more than 80 different proteins including transcription factors, kinases, phosphatases, cytoskeletal proteins, nuclear hormone receptors, nuclear pore proteins, signal transduction molecules, and actin regulatory proteins [34], [35] have been shown to undergo O-GlcNAcylation [35] although the mechanisms underlying OGT regulation are not completely understood. The most important finding of this paper is that we demonstrate that OGT and its connection to the AMPK-mediated regulation of 26S proteasome in endothelial cell. The present study demonstrates that AMPK, as a physiological suppressor of 26S proteasome, regulates proteasome function by controlling OGT-mediated proteasome O-GlcNAcylation. Indeed, there is evidence that 19S subunit can be subjected to O-GlcNAcylation with consequent 26S proteasome inhibition [51]. Hence, O-GlcNAc is considered an endogenous inhibitor of the 26S proteasome [51]. A proteomic study revealed that several other proteins in the 26S proteasome can also be extensively O-GlcNAcylated [52]. Hence, O-GlcNAc is considered an endogenous inhibitor of the 26S proteasome [51] and O-GlcNAc modification links a nutritional sensor to modulation of proteasome function [53]. Intriguingly, AMPK, a cellular energy sensor and a major component of the nutrient pathway [54], has recently been linked to O-GlcNAc modification through OGT upregulation [55]. This effect is likely independent of a direct association between OGT and AMPK [56], though co-immunoprecipitation of these proteins is possible [57]. In line with this relationship, we found that OGT is essential in mediating AMPK-dependent 26S proteasome suppression both in cultured cells and aortas of AMPK2α-KO mice, likely through the control of PA700/S10B modification. These data suggest that a novel functional link exists between AMPK and OGT that regulates 26S proteasome function in endothelial cell. It is worthy of note that compared to the effect of AMPK-DN, AMPK-CA alone did not generate a robust changes in 26S proteasomes assembly. This is in line with the observations that AMPK-CA alone has either marginal [43], [44] or not significant effects on 26S proteasome activity. All these data might imply that basal AMPK activity is essential in keeping 26S proteasome activity in check; further studies are warranted. The concept that AMPK activation could be used as a strategy to promote vascular health, including overcoming endothelial dysfunction, has only recently emerged [22], [58], [59]. Endothelial dysfunction, characterized by impaired endothelium-dependent vasorelaxation, is a common feature of CVD including diabetes and hypertension. We have recently shown that link exists between 26S proteasome activation and endothelial dysfunction through accelerated proteasomal degradation of GTP-CH I and consequent BH4 deficiency [39], [40]. More importantly, we have demonstrated that activation of AMPK by Metformin reverses endothelial dysfunction in a 26S proteasome-dependent fashion in streptozotocin-induced diabetic mice [43]. Thus, our data suggest that AMPK-dependent OGT-mediated 26S proteasome suppression might operate in vivo contributing to the protective effect of AMPK on endothelial function in our previous studies [43], [44] In summary, this is the first report of a novel function for AMPK-dependent 26S proteasome regulation in endothelial cells, a mechanism that may bridge endothelial function with both the energy (AMPK) and metabolic (OGT) sensors. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by National Institutes of Health grants (HL079584, HL074399, HL080499, HL105157, and HL110488), a research award from the American Diabetes Association (ADA), and funds from the Warren Chair in Diabetes Research of the University of Oklahoma Health Sciences Center (all to MHZ). MHZ is a recipient of the National Established Investigator Award of American Heart Association (AHA). JX is supported by a Scientist Development Grant (AHA, 10SDG2600164), a Center of Biomedical Research Excellence (COBRE) grant (NIH/NCRR: 5P20RR024215-5), a Junior Faculty Award (ADA, 1-12-JF-58), and a research award from the Oklahoma Center for Advancement of Science and Technology (HR11-200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Schwartz AL Ciechanover A 1999 The ubiquitin-proteasome pathway and pathogenesis of human diseases. 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==== Front PLoS MedPLoS MedPLoSplosmedPLoS Medicine1549-12771549-1676Public Library of Science San Francisco, USA 22589704PMEDICINE-D-11-0279410.1371/journal.pmed.1001214EssayMedicineGlobal HealthSocial and Behavioral SciencesEconomicsDoes Development Assistance for Health Really Displace Government Health Spending? Reassessing the Evidence Batniji Rajaie * Bendavid Eran Stanford University, Stanford, California, United States of America* E-mail: [email protected] the first draft of the manuscript: RB. Contributed to the writing of the manuscript: RB EB. ICMJE criteria for authorship read and met: RB EB. Agree with manuscript results and conclusions: RB EB. The Essay section contains opinion pieces on topics of broad interest to a general medical audience. 5 2012 8 5 2012 9 5 e1001214Batniji, Bendavid.2012This 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.Rajaie Batniji and Eran Bendavid dispute recent suggestions that health aid to developing countries leads to a displacement of government spending and instead argue that current evidence about aid displacement cannot be used to guide policy. ==== Body Summary Points At the core of the current aid debate is the question of whether development assistance for health provided to developing country governments increases health expenditures. It has recently been suggested that development assistance for health to governments leads to a displacement of government spending, reinforcing skepticism about health aid. Here we examine a database of public financing for health from 1995 to 2006 and demonstrate that prior conclusions drawn from these data are unstable and driven by outliers. While government spending may be displaced by development assistance for health in some settings, the evidence is not robust and is highly variable across countries. We recommend that current evidence about aid displacement cannot be used to guide policy. Re-thinking Aid Displacement in the Health Sector There has been considerable concern in the international development community about aid displacement in the health sector. That is, a concern that foreign aid to the health sector leads to a displacement or diversion of government funds from the health sector. Foreign aid, also known as development assistance, includes funding from international development agencies in donor countries, multilateral agencies like the World Bank and Global Fund, and private sources. The concern about displacement penetrates questions about the strings attached to aid, the monitoring of aid, and whether aid should be given to governments in the first place. A core question being asked is, does development assistance for health increase health spending? Or, do aid funds merely displace government funding for health? Questioning the Argument That Health Aid Leads to Reduced Government Investment in Health Concerns about aid displacement are as old as development assistance. As a leading World Bank economist famously said in 1947, “When the World Bank thinks it is financing an electric power station, it is really financing a brothel.” [1] More recently, experts commenting in the press have suggested, “When an aid official thinks he [sic] is helping a low-income African patient avoid charges at a health clinic, in reality, he is paying for a shopping trip to Paris for a government minister and his wife [2].” The concerns—and the cynicism— about foreign aid being displaced and diverted for less-than-noble purposes have been in place the last 60 years. Some analysis has been done to assess the scale of aid displacement in different sectors and countries. In one analysis, World Bank economists examined aid to 18 African countries from 1971 to 1995, and found that for every US dollar of aid received, government spending increased by US$0.90 [1]. That study also found that some aid intended for capital improvements, like the building of hospitals, went to operations and to the repayment of past loans. But across sectors and regions, the evidence for aid displacement is mixed. For example education aid was found to have no discernible effect on education spending worldwide; however, each US dollar in education aid in Africa led to nearly US$1.00 increase in education spending [1]. Country analyses have also identified great variation in the extent of displacement. One study showed that aid to India “merely substitutes for spending that the government would have undertaken anyway,” concluding that “funds freed by aid are spent on non-development activities [3].” In contrast, a study of aid to Vietnam's transportation sector found that aid stayed within that sector [4]. Recently, attention has turned to whether health aid increases government health spending. In 2009, Farag and colleagues found that from 1995 to 2006, each US dollar increase in development assistance for health (DAH) to low-income countries was associated with a US$0.14 decrease in government health spending [5]. A subsequent analysis by Lu and colleagues made the case that for every additional US dollar of DAH from 1995 to 2006, government health expenditures from domestic sources fell by at least US$0.43 [6]. This latter study is referenced frequently in conversations with decision-makers at aid agencies as a cautionary note about DAH. The analysis appears to have reinforced skepticism about health aid. A closer look at the analysis by Lu and colleagues, using data made available in May 2011 [7], shows that the association between DAH and displacement of government health expenditures is not robust after exclusion of a small subset of data. The trends are driven by outliers, and country data cluster and follow widely divergent trends (Figure 1). The primary finding by Lu and colleagues, which we are challenging here, is the negative relationship between government health expenditure (GHE-S)/gross domestic product (GDP) and DAH-Gov/GDP. GHE-S/GDP is government health spending from domestic sources as a percentage of GDP for a country in each year from 1995 to 2006. DAH-gov/GDP is development assistance for health disbursed to government as a percentage of GDP for each country in each year from 1995 to 2006. Using Lu and colleagues' data on government health spending and Institute for Health Metrics and Evaluation (IHME) data on DAH [8], we replicated their results and found that the linear relationship between DAH and government health expenditure from domestic sources is lost when country-years in which World Health Organization (WHO) and International Monetary Fund (IMF) estimates differ by 10-fold or more are removed, when a small set of implausible data points are removed, and when restricting the sample to eliminate those countries that receive very little DAH as a percentage of GDP. In sum, any linear relationship that exists among the data is too tenuous to be a basis for policy. 10.1371/journal.pmed.1001214.g001Figure 1 Primary data scatter of the relationship between DAH and GHE-S in all countries. Both DAH and GHE-S are presented here as a percentage of GDP, with GHE-S based on IMF data. Each point on the above plot represents a country-year observation used in the analysis. Data source: IHME [2]. Re-evaluating the Data: Inconsistencies and Omissions Much debate surrounded the initial publication of the paper by Lu and colleagues, but those engaged in the correspondence acknowledged that they were challenging neither the overall findings nor the data [9],[10]. Since publication, the data's reliability and heterogeneity have been called into question: nearly half of the observations are missing for low-income countries [11], making a reliance on modeled estimates and imputation essential. As Lu and colleagues acknowledge, there is only a 65% correlation between the two main data sources, WHO and IMF. In 29 country-year observations, the ratio between WHO and IMF estimates is greater than 10 (Table 1). In addition to questionable data, Lu and colleagues leave out of the analysis 51 countries that IHME previously analyzed as recipients of DAH, including Russia and much of Eastern Europe, Iraq, Afghanistan, the occupied Palestinian territory, Somalia, and several small Island states (Box 1) [12]. 10.1371/journal.pmed.1001214.t001Table 1 Observations where the ratio of WHO to IMF estimates of government health expenditure from domestic sources as a percentage of GDP (GHE-S/GDP) is greater than 10. Country Ratio of WHO to IMF GHE-S Central African Republic, 2002 1,600.98 Suriname, 2001, 2004, 1996, 2005, 2003, 1999 108.98, 104.12, 84.15, 35.08, 32.21, 15.52 Laos, 1998 54.20 Rwanda, 1996, 1995 44.57, 15.83 Cambodia, 2006, 2000 37.55, 23.27 Guinea, 2003, 2000 34.75 Burundi, 2002 33.84 Mozambique, 2004 24.78 Costa Rica, 1995, 1997, 2006, 2003, 2004, 2002, 1999 19.56, 18.16, 17.80, 16.47, 16.10, 15.94, 13.05 Malawi, 2000, 2003 13.43, 10.15 Niger, 1996 12.81 Laos, 1997 12.41 Fiji, 1998 11.58 Equatorial Guinea, 2000 10.93 WHO estimates exceed IMF estimates in about two-thirds of observations. However, there are also notable country-years in which WHO estimates are more than 10-fold lower than IMF estimates, including: Guinea-Bissau, 1995 (ratio of 0); Eritrea in 2003 (ratio of 0.047). Box 1. DAH-Receiving Countries Omitted from Study by Lu and Colleagues [6] Afghanistan Albania Belarus Bosnia and Herzegovina Bulgaria Cook Islands Croatia Cuba Dominica Estonia Falkland Islands Gibraltar Grenada Honduras Iraq Kiribati Korea, Democratic People's Republic Latvia Lithuania Macedonia Marshall Islands Mayotte Micronesia Moldova Montenegro Montserrat Myanmar Nauru Niue Northern Mariana Islands Palau Palestinian Territory, occupied Poland Romania Russian Federation Saint Helena Saint Kitts and Nevis Saint Lucia Saint Vincent and the Grenadines Sao Tome and Principe Serbia Seychelles Somalia Timor-Leste Tokelau Tonga Turks and Caicos Islands Tuvala Ukraine Wallis and Fortuna Yugoslavia Even if we accept the concerns about data consistency and accept the highly imputed data, the relationship between DAH and GHE-S is not stable to the exclusion of a few data points. We replicated the author's fixed effects model (Arellano-Bover/Blundell Bond model). We confirmed their main results, but we further explored the sensitivity of the results to the exclusion of questionable data. The associations failed significance testing at p = 0.05 when excluding the lowest 10% of GHE-S using the IMF data and the lowest 20% of the WHO data. Given the concerns about Lu and colleagues' model choice, we repeated the analysis using an alternative statistical model to assess the robustness of the relationship between DAH and GHE-S. We used ordinary least squares regression with country fixed effects, clustered by country, for the main model estimation of the association between DAH and GHE-S. We avoided random effects estimation because the aid literature suggests that the differences in the manner in which countries handle aid funds are structural (“fixed”) and idiosyncratic, so an exchangeability assumption seemed inappropriate. Countries have different means of interacting with donors: some require donors to buy into and contribute toward a national plan (e.g., India), while others allow donors freedom to implement projects with few constraints (e.g., Tanzania) [13]. Countries also have differences in national institutions that collect, disseminate, and report on foreign assistance. These fixed differences may be beneath the wide variation seen in country trends (Figure 2). Furthermore, donors take widely varying strategies toward liaising with government. For example, while World Bank funding largely goes through official government channels, the United States Government's President's Emergency Plan for AIDS Relief (PEPFAR) had largely bypassed recipient country budget-planning procedures in its effort to achieve a rapid scale-up of HIV/AIDS programs [14]. 10.1371/journal.pmed.1001214.g002Figure 2 Widely divergent trends in the relationship between DAH and GHE-S in select countries. Both DAH and GHE-S are presented here as a percentage of GDP, with GHE-S based on IMF data. In Rwanda, GHE-S is effectively zero, regardless of DAH. In Lesotho, GHE-S appears to rise with DAH (aid is associated with additional GHE-S). Eritrea exhibits erratic response, while Zambia generally follows the predictions of Lu and colleagues about decreasing government expenditure with increasing DAH. Data source: IHME [2]. We tested the sensitivity of the results to the exclusion of observations from years where recipient governments were calculated to spend less than 0.01% of GDP from domestic sources on health. In a country with a GDP per capita of US$1,000, this means that the government is spending less than US$0.10 of non-donor money on health per capita. The IHME data show that GHE-S is less than 0.01% of GDP in 47 (out of over 1,200) country-year observations using IMF data and in 8 country-year observations using WHO data (Table 2). The linear association between DAH and GHE-S as percentage of GDP is not significant after excluding these observations. According to IMF data, Rwanda has had such near-complete displacement every year from 1997 to 2006. Cambodia, Ethiopia, and Guinea-Bissau follow a very similar trend to Rwanda, all showing near-complete displacement every year. If real, such complete displacement suggests alternative national priorities, and donors could seek alternative approaches to aligning their priorities with those of the recipient government. If we exclude these observations where GHE-S is less than 0.01%, there is no longer a statistically significant linear relationship between GHE-S and DAH. 10.1371/journal.pmed.1001214.t002Table 2 Country-years in which GHE-S/GDP <0.0001 in IMF and WHO data. Country Years Using IMF Data Years Using WHO Data Burundi 2002–2006 2003–2006 Cambodia 1995, 1998, 2003–2005 — Central African Republic 2002, 2004 — Comoros 2005 — Ethiopia 2003, 2005, 2006 — Guinea 2004–2006 — Guinea-Bissau 2001–2004, 2006 1995, 2001, 2006 Laos 1999, 2005 2005 Madagascar 2004 — Malawi 2004 — Mozambique 1995–1997 — Rwanda 1997–2006 — Suriname 1995 — Tanzania 2004–2006 — The Gambia 2005 — Zambia 2003–2004 — If these data points are excluded, the relationship between GHE-S and DAH is no longer statistically significant. Furthermore, linear regressions examining the association between DAH and GHE-S as a percentage of GDP when DAH to government is greater than or equal to 0.5% of national GDP are not significant. That is, countries that receive a substantial amount of DAH show little evidence of displacement. Notably, just under half of DAH is given to countries receiving greater than or equal to 0.5% of GDP as DAH (Table 3). DAH to governments is not displaced when aid makes a large (greater than or equal to 0.5% of GDP) contribution to health spending. This calls into question the argument that governments displace aid because they are not able to absorb it. It appears that aid displacement trends, even if we accept all the flawed data, are driven by those countries that receive very small amounts of aid for health, as the relationship is absent if we look at country-years in which aid makes up 0.5% of GDP or greater (Table 4). 10.1371/journal.pmed.1001214.t003Table 3 Country-years in which DAH equals or exceeds 0.5% of GDP. Country Years Angola 1995, 2001 Armenia 2000 Benin 1999–2001, 2003, 2004 Bhutan 1997–2000 Bolivia 2003–2004 Burkina Faso 2000, 2001, 2005 Burundi 2002–2006 Cambodia 1995–1998, 2000–2006 Cape Verde 2001, 2003, 2005, 2006 Central African Republic 1999–2002, 2004 Chad 1997–2004 Comoros 1995–2000, 2002 Congo, Democratic Republic 2000, 2001, 2003–2006 Djibouti 1997–2000, 2005, 2006 Equatorial Guinea 2003–2004 Eritrea 1995–2006 Ethiopia 2001–2006 Fiji 2003 Ghana 2000–2006 Guinea 2006 Guinea-Bissau 1996–1998, 2000–2006 Guyana 2003–2006 Haiti 1995–2004 Kenya 2001–2006 Kyrgyzstan 2000, 2003–2006 Lao People's Democratic Republic 1999–2000, 2003–2006 Lesotho 2001–2006 Madagascar 2004–2005 Malawi 1995–2006 Mali 1995–2006 Mongolia 1999, 2001 Mozambique 1995–2006 Namibia 2002, 2006 Nepal 1998–2004 Nicaragua 1997–2006 Niger 1995–1997, 2004–2006 Papua New Guinea 1996, 2000–1005 Rwanda 1999–2006 Samoa 1998, 2001–2005 Senegal 1999–2005 Sierra Leone 2003–2006 Suriname 1995–1999, 2000–2005 Swaziland 2003, 2005 Tajikistan 2003–2004 Tanzania 1995–2006 The Gambia 2005, 2006 Trinidad and Tobago 2006 Uganda 1996, 1998–2006 Zambia 1996–2006 Zimbabwe 2003–2006 Limiting the analysis to these country-years (comprising 47% of all DAH) reveals no significant relationship between DAH and GHE-S. 10.1371/journal.pmed.1001214.t004Table 4 Summary of regressions, with GHE-S/GDP as dependent variable, DAH to governments/GDP as independent variable. Models IMF GHE-S Data WHO GHE-S Data Coefficient (SE) p-Value Coefficient (SE) p-Value All observations Arellano-Bover/Blundell Bond model −0.40 (0.07) 0 −0. 45 (0.04) 0 Linear, country clustered −0.39 (0.11) 0.001 −0.20 (0.19) 0.15 Exclude if WHO to IMF GHE-S estimate >10 (flawed data) Arellano-Bover/Blundell Bond model −0.40 (0.07) 0 −0.46 (0.05) 0 Linear, country clustered −0.17 (0.14) 0.232 −0.20 (0.18) 0.27 Exclude if GHE-S <0.0001 (outlier and improbable data) Arellano-Bover/Blundell Bond model −0.32 (0.03) 0 −0.39 (0.03) 0 Linear, country clustered −0.23 (0.14) 0.103 −0.20 (0.20) (0.34) Exclude if DAH to government <0.5% of GDP (policy relevant) Arellano-Bover/Blundell Bond model −0.22 (0.10) 0.835 −0.25 (0.07) 0.001 Linear, country clustered −0.23 (0.13) 0.103 −0.01 (0.25) 0.966 Note that linear regressions fail with modest exclusions. SE, standard error. The Current Evidence on Aid Displacement Cannot Guide Policy First, even if displacement does exist, there is no evidence that it is a bad thing. A large-scale empirical analysis found no evidence that non-fungible sectoral aid (that is, aid earmarked or otherwise dedicated to its intended purposes) works better than fungible aid, when “better” is understood as economic growth, spending in pro-poor sectors, or reductions in infant mortality [15]. In Vietnam's health sector, Wagstaff found that project-level outcomes are not harmed by displacement of government funding, suggesting that governments aim to shift spending to support projects where additional investments provide the greatest improvements [16]. Furthermore, as a recent analysis of health financing in Honduras, Rwanda, and Thailand showed that these countries increased their domestic spending from domestic sources in response to increases in donor funding, a finding based on close examination of country-spending that is at sharp odds with the cross-country conclusions [17]. This study also found that donors were likely to shift funds in the face of increasing resources from the Global Fund. This finding raises the possibility that some of the measured “displacement” is exogenous; that is, countries are shifting resources in response to anticipated, promised, or real changes in DAH. Aid displacement may be a reasonable approach for governments to improve the societal benefits of resource allocation decisions when development assistance is volatile or threatened. Even if we accept Lu and colleagues finding that DAH to NGOs undergoes less displacement (and thus, increases government health expenditure), this likely reflects the fact that NGOs are less likely to be burdened by the risks of aid volatility; and, since NGO salaries tend to be higher, they may drive up public sector health wages and in turn government expenditures (see Text S1 for an analysis of the vulnerability of this finding). Given the concerns raised over data plausibility and completeness, conclusions about the mean relationship between DAH and government health spending should be called into question. While there does appear to be an association, it is too tenuous, too dependent on problematic model selection, and inconsistent (even among individual countries) to be used for policy or resource-allocation decisions. We show that there is no significant aid displacement when government health expenditures from domestic sources exceed 0.01% of GDP, and no evidence of aid displacement when DAH exceeds 0.5% of GDP (Table 4). No statistical model can adequately compensate for systematically wrong and missing data. While Lu and colleagues have gathered the best available data, and have been fully transparent in sharing their datasets and methods, the reality is that we still lack a sufficient accounting of public financing on health to make any conclusions on overall trends. Of course, some displacement of aid from the health sector may occur. It would be rational for governments seeking to improve the distribution of limited national resources, and seeking to avoid interruptions in health service provision with annual fluctuations in aid to avoid a rapid rise in health sector spending. However, our findings should relieve donors of the need to make unrealistic demands on recipient governments, and of the pressure to divert resources to NGOs. While in some settings aid likely is displaced from the health sector, we call into question the assertions that donor health funds are being systematically displaced and misused. Supporting Information Text S1 Statistical supplement: aid displacement. (DOC) Click here for additional data file. The authors have declared that no competing interests exist. Funding from the Stanford University Freeman Spogli Institute's ‘Global Underdevelopment Action Fund’ supported this work. See http://fsi.stanford.edu/news/request_for_proposals_global_underdevelopment_action_fund_at_fsi_20100712. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Provenance: Not commissioned; externally peer reviewed. Abbreviations DAHin development assistance for health GDPgross domestic product GHEhealth expenditure IHMEInstitute for Health Metrics and Evaluation IMFInternational Monetary Fund WHOWorld Health Organization ==== Refs References 1 Devarajan S Rajkumar AS Swaroop V 1999 What does aid to Africa finance? Washington (D.C.) World Bank 2 Cheng M 2010 April 10 Health aid made some countries cut budgets. The Guardian 3 Swaroop V Jha S Rajkumar AS 2000 Fiscal effects of foreign aid in a federal system of governance The case of India. J Public Econ 77 307 330 4 Van de Walle D Mu R 2007 Fungibility and the flypaper effect of project aid: micro-evidence for Vietnam. J Dev Econ 84 667 685 5 Farag M Nandakumar A Wallack SS Gaumer G Hodkin D 2009 Does funding from donors displace government spending for health in developing countries? Health Affair 28 1045 1055 6 Lu C Schneider MT Gubbins P Leach-Kemon K Jamison D 2010 Public financing of health in developing countries: a cross-national systematic analysis. Lancet 375 1375 1387 20381856 7 Institute for Health Metrics and Evaluation (IHME) 2010 Public financing of health (developing country) estimates 1995–2006 Seattle (Washington) Institute for Health Metrics and Evaluation (IHME) 8 Institute for Health Metrics and Evaluation (IHME) 2010 Development assistance for health database 1990–2008 Seattle (Washington) Institute for Health Metrics and Evaluation (IHME) 9 Ooms G Decoster K Miti K Rens S Van Leemput L 2010 Crowding out: are relations between international health aid and government health funding too complex to be captured in averages only? Lancet 375 1403 1405 20381858 10 Sridhar D Woods N 2010 Are there simple conclusions on how to channel health funding? Lancet 375 1326 1328 20381855 11 Stuckler D Basu S McKee M 2011 International Monetary Fund and aid displacement. Int J Health Serv 41 67 76 21319721 12 Ravishankar N Gubbins P Cooley RJ Leach-Kemon K Michaud CM 2009 Financing of global health: tracking development assistance for health from 1990 to 2007. Lancet 373 2113 2124 19541038 13 GEG 2008 Preliminary report of a high-level working group, 11–13 May 2008 Oxford Global Economic Governance Programme 14 Sridhar D Batniji R 2008 Misfinancing global health: a case for transparency in disbursements and decision making. Lancet 372 1185 1191 18926279 15 Pettersson J 2007 Foreign sectoral aid fungibility, growth and poverty reduction. Journal of International Development 19 1074 1098 16 Wagstaff A 2011 Fungibility and the impact of development assistance: evidence from Vietnam's health sector. 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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2259367510.1100/2012/345983Research ArticleFlavonoid-Deficient Mutants in Grass Pea (Lathyrus sativus L.): Genetic Control, Linkage Relationships, and Mapping with Aconitase and S-Nitrosoglutathione Reductase Isozyme Loci Talukdar Dibyendu *Department of Botany, R.P.M. College, University of Calcutta, Uttarpara, West Bengal, Hooghly 712 258, India*Dibyendu Talukdar: [email protected] Editors: K. Chakravarty, E. Olmos, and K. Shoji 2012 19 4 2012 2012 34598325 10 2011 25 12 2011 Copyright © 2012 Dibyendu Talukdar.2012This 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.Two flavonoid-deficient mutants, designated as fldL-1 and fldL-2, were isolated in EMS-mutagenized (0.15%, 10 h) M2 progeny of grass pea (Lathyrus sativus L.). Both the mutants contained total leaf flavonoid content only 20% of their mother varieties. Genetic analysis revealed monogenic recessive inheritance of the trait, controlled by two different nonallelic loci. The two mutants differed significantly in banding patterns of leaf aconitase (ACO) and S-nitrosoglutathione reductase (GSNOR) isozymes, possessing unique bands in Aco 1, Aco 2, and Gsnor 2 loci. Isozyme loci inherited monogenically showing codominant expression in F2 (1 : 2 : 1) and backcross (1 : 1) segregations. Linkage studies and primary trisomic analysis mapped Aco 1 and fld 1 loci on extra chromosome of trisomic-I and Aco 2, fld 2, and Gsnor 2 on extra chromosome of trisomic-IV in linked associations. ==== Body 1. Introduction Flavonoids are secondary metabolites derived from phenylalanine and acetyl CoA that perform a variety of important functions in plant growth, reproduction, and survival and also serve as important micronutrients in human and animal diets [1, 2]. The pigmented flavonoid metabolites have been used as phenotypic markers in many model plant species [3, 4] and have proven to be an excellent tool to study the genetic, molecular, and biochemical processes [4, 5]. One of the functional tools in this regard is the genetic characterization of mutants, exhibiting significantly altered flavonoid compounds. A good number of mutants with altered flavonoid levels have been utilized in Arabidopsis, maize, grape, and Petunia to reveal biosynthetic pathway of different flavonoids and their diverse roles [6–10]. Although very rich in flavonoid components [11], no reports are available regarding the genetic analysis of flavonoid mutant in leguminous plants. Plant flavonoids play pivotal role in protection/tolerance against different types of abiotic stress [12]. Evidences are accumulating about functional interplay between flavonoid metabolism and thiol-based (glutathione/thioredoxin) antioxidant defense system in plants during stress response [13, 14], where nitric oxide (NO) functions as signaling molecule [15]. The enzyme aconitase (ACO) is known to be responsible in iron homeostasis and in regulating resistance to oxidative stress [16], but its activity is inhibited by NO [17]. On the other hand, the S-nitrosoglutathione reductase (GSNOR) activity has been described to be associated with the enzyme glutathione-dependent formaldehyde dehydrogenase [18]. GSNOR uses GSNO as its substrate which is formed by the reaction of reduced glutathione with NO molecule. GSNOR is extremely important in maintenance and turnover of cellular NO pool and modulation of hormonal response such as jasmonic acid and salicylic acid, responsible for alteration of stress-induced phenylpropanoid pathway [14, 19]. Grass pea (Lathyrus sativus L.), an annual winter legume crop, possesses high level of bioactive compounds including flavonoids [20]. The potential of this hardy crop has been extensively utilized in recent years through isolation and genetic analysis of novel mutants for plant habit [21], flower and seed coat colour [22, 23], pod indehiscence [24], seed size [25], and so forth. Some of these mutant lines are now being tested for their fitness to different abiotic stresses including salinity [26, 27] and arsenic [28], and very recently, a novel ascorbate-deficient mutant has been detected [29]. Linkage mapping and chromosomal assignment of desirable mutations are now being accomplished through establishment of a functional cytogenetic stocks including aneuploids [30, 31], polyploids [32], and translocation lines [33, 34]. Perusal of literature cites only limited information regarding inheritance and linkage association of morphological, biochemical (isozyme), and other molecular markers in grass pea [35]. Although isozyme markers are widely used in gene mapping of different crops and have advantages over other markers due to their codominant expression, lack of sufficient number of polymorphic isozymes loci possesses problems in existing germplasms of grass pea [36]. Creation of additional variability in esterase and root peroxidase isozyme systems through induced mutagenesis has recently been successfully explored in dwarf mutant population of this crop, and genetic control of their allozyme variants has been studied [37]. During screening of desirable mutations in EMS-mutagenized population, two variant plants with white flower color was isolated. The mutants were later found to be highly deficient in total flavonoid content in their leaves. Despite immense importance of flavonoids in legume crops and its relation with enzymes involved in stress responses, no reports in these regards are available in grass pea. Keeping all these in mind, a genetic approach has been taken to investigate the basis of flavonoid deficiency in the preset materials of grass pea and its association with isozymes of ACO and GSNOR enzymes. The main objectives of the present work are to (1) trace the mode of inheritance of flavonoid deficiency and the zymogram phenotypes of both enzymes, (2) investigate the segregation pattern and linkage associations between different isozymes loci and loci controlling flavonoid deficiency, and (3) ascertain their possible chromosome location through primary trisomic analysis. 2. Methods 2.1. Plant Materials Altogether eleven parents have been used in the present study of which four were diploid (2n = 14) and rest seven were primary trisomic (2n + 1 = 15) types. Among the diploid parents, two varieties “BioL-212” and “Hooghly Local” were used as mother control throughout the experiment. Fresh and healthy seeds of these two varieties presoaked with water (6 h) were treated with freshly prepared 0.15% aqueous solution of EMS (Sigma-Aldrich) for 10 h with intermediate shaking at 25 ± 2°C. M1 seeds were sown treatment-wise in completely randomized block design as reported earlier [25]. Two variant plants showing white flowers and absence or modified stipule morphology were distinguished from usual occurrence of blue flower and typical papilionaceous stipules in EMS-treated M2 progeny. During screening of antioxidant activities of different mutant lines, these two plants exhibited abnormally low foliar flavonoid contents. The levels were again confirmed at M3 generation, and on the basis of stipule characters the progeny of the two plants was primarily designated as fld L-1 (flavonoid-deficient Lathyrus type 1 mutant, white flower, estipulate) and fld L-2 (flavonoid deficient Lathyrus type 2 mutant, white flower, linear-acicular stipule). Both the mutants bred true for their phenotypes, and no significant change in leaf flavonoid content was found in M3 generation. Chromosome location of different loci was performed by utilizing a set of primary trisomics, isolated and characterized earlier in grass pea [30, 38]. 2.2. Determination of Total Flavonoid Content Total flavonoid content from leaves of mutants and their control varieties were determined spectrophotometrically in both ethanol and aqueous extracts, based on the formation of a flavonoid-aluminium complex [39]. An amount of 2% ethanolic AlCl3 solution (0.5 mL) was added to 0.5 mL of sample. After 1 h at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of flavonoids. Extract samples were evaluated at a final concentration of 0.1 mg mL−1. Total flavonoid contents were calculated as rutin (mg g−1 of extract) (Table 1). 2.3. Isozyme Analysis: Gel Electrophoresis and Nomenclature Horizontal 10% starch-gel (Sigma) electrophoresis was carried out to analyse the banding profile of aconitase (ACO, EC 4.2.1.3) in mutants (M4) and control varieties and trisomic lines. Crude extracts were prepared by macerating young leaf tissues of 4-d-old seedlings in ice-cold extraction buffer containing 20% sucrose, 5% PVP-40, 0.1 M KH2PO4, 0.05% triton X-100 (Sigma), and 14 mM 2-mercaptoehanol (Sigma) at pH 7.0. Triton X-100 and 2-mercaptoehanol were added just before use. After extraction, sample was stored at −20°C for future use. ACO isozymes were separated using the electrode and gel buffer system (pH 6.5) of Cardy et al. [40]. Bands of ACO systems were stained according to the recipes (0.1 M Tris-HCL, pH 8.0, cis-Aconitic acid, MgCl2, Isocitrate dehydrogenase, MTT, PMS, and NADP) of Cardy and Beversdorf [41]. For GSNOR (EC 1.2.1.1) activity, native PAGE was done using 6% acrylamide gels in TRIS-boric-EDTA buffer (pH 8.0). For staining of GSNOR activity, gels were soaked in 0.1 M sodium phosphate, pH 7.4, containing 2 mM NADH for 15 min in an ice bath. Excess buffer was drained, and gels were covered with filter paper strips soaked in freshly prepared 3 mM GSNO. After 10 min, the filter paper was removed, and gels were exposed to UV-light and analysed for the disappearance of the NADH fluorescence, indicating GSNOR activity [42]. Based on the observed variations, isozyme bands were assigned to putative loci following the principles of Weeden [43]. The isozymes were designated as all letters capitals (ACO and GSNOR) but the loci controlling these two isozymes had only the first letter capitalized and presented in italics (Aco and Gsnor). When two or more isozymes, coded by different loci in an enzyme, were visualized on gel, they were numbered sequentially according to their mobility relative to the anode with the most anodal isozyme being number one, and subsequent isozymes were assigned sequentially higher numbers. Likewise, the most anodal allele producing allozyme (fastest variant) of a particular locus was termed as “a” and progressively slower forms “b”, “c”, and so on. Only clearly visible bands for both enzyme systems were scored in the present study. 2.4. Inheritance and Linkage Analysis Inheritance and linkage of loci controlling flavonoid deficiency and different isozymes were traced in segregating populations of F2 and backcross generations derived from single locus as well as joint segregation of two loci in different cross-combinations. Following two generations of selfing, intercrosses including reciprocals were made among control varieties and the mutant lines (M4) to raise F1 and, subsequently, backcross (BC1) and F2 progenies (Table 2). Measures were taken at every stage from sowing to harvesting to prevent any type of outcrossing pollination and intermixing. For allelism test, intercrosses were made among fld L-1 and fld L-2. Chi-square test was employed to test the goodness of fit between observed and expected values for all crosses (Table 2). Zymogram phenotypes of both ACO and GSNOR were studied in selfed and intercrossed (F2 and backcross) progenies of different parents. Linkage associations of the segregating isozyme markers along with flavonoid deficiency trait were examined for pair-wise combinations of different isozyme loci and also between pairs of isozyme loci and loci controlling flavonoid deficiency for the expected ratio of 1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 : 1 and 3 : 1 : 6 : 2 : 3 : 1, respectively, in the F2 progeny. Testcross population was raised by crossing F1 plant with the parent showing comparatively slow moving allozymes in case of isozyme loci and with recessive lines in segregation of flavonoid deficiency. Chi-square test was employed to test the goodness of fit, and significant deviation from the expected ratio was considered as linkage between the markers. Recombination fraction (r) was calculated from testcross data and was converted to map distance in centiMorgans (cM) through Kosambi's mapping function [44]. Data from different families was pooled when homogeneous for analysis (Table 3). 2.5. Mapping Flavonoid-Deficient Mutant and Isozyme Loci by Primary Trisomic Analysis The seven primary trisomic types were crossed as female parent with the four different homozygous diploid genotypes (two controls, two mutants), and F1 population was obtained in each case. The trisomic F1 plants could be readily identified at early seedling stage on the basis of their specific leaflet phenotypes [30]. Trisomic F1 plant was self-fertile and subsequently selfed to obtain F2 progeny and also backcrossed to the respective diploid parent to produce BC1 population. In the segregating F2 progeny, banding patterns were analysed by means of chi-square test for a fit to a normal disomic ratio. Significant deviation from the expected disomic ratio of 1 : 2 : 1 in F2 and 1 : 1 in BC1 was further tested with the expected trisomic ratios of 4 : 4 : 1 in F2, 2 : 1 in BC1 in diploid portion and 2 : 7 : 0 (F2) in trisomic portion of the progeny to locate possible chromosome/s, bearing gene/s of different isozyme loci (Table 4). Necessary cytological confirmation of trisomy was performed at meiosis-I following Talukdar and Biswas [30]. To save space, only segregation of trisomics carrying concerned loci has been presented in Table 4. 2.6. Statistical Analysis Total flavonoid contents in leaves of mother and mutants are presented as mean ± standard error (SE) with 20 plants in each of the four genotypes. Significant differences between mother and mutant plants for total flavonoids were determined by simple “t-test.” A probability of P < 0.05 was considered significant. 3. Results 3.1. Total Flavonoid Contents and Morphology of Mutant and Mother Plants Total flavonoid contents as determined in aqueous and ethanol extract in leaves of mutant and mother leaves were significantly (P < 0.05) different. Leaves of both the mutants contained total content (mg g−1 extract) only 20% of mother plants (Table 1). However, flavonoid content was nearly normal in aqueous extract of fldL-1 mutant, but reduced by about 5-fold in ethanol extract. In contrast, flavonoid content reduced marginally in ethanol extract of fldL-2 leaves but had reduced by nearly 5-fold in aqueous extract (Table 1). Both the mutants produced characteristic white flower and modification in stipule characters. While fldL-1 was completely estipulate, a linear-acicular type of stipule was observed in fldL-2 plants. Furthermore, fldL-1 showed normal pollen fertility (98.77%) like mother plants, while it reduced (66%) in fldL-2 plants. Root formation in both mutants, however, was quite normal. 3.2. Inheritance and Allelic Relationship of Gene/s Controlling Flavonoid Deficiency Reciprocal crosses between fldL-1 as well as fldL-2 and mother control varieties yielded F1 plants with normal level of flavonoids (Table 2). Segregation of normal and flavonoid-deficient plant type showed good fit to 3 : 1 in F2 and 1 : 1 in backcross (Table 2). Flavonoid content from leaves of every genotype was tested and verified with parents. The recessive mutants recovered in F2 generation of the above two crosses were also self-pollinated, and in F3 all the 210 plants exhibited only marginal variations in total flavonoid content compared with their respective parents (data not in table). In order to study the allelic relationships of genes governing flavonoid deficiency in grass pea, fldL-1 and fldL-2 were reciprocally crossed. All the F1 plants derived from the crosses contained normal flavonoid level like mother control plants. In F2, four types of plants: normal type, fldL-1, fldL-2, and a variant type appeared in the progeny showing good fit to 9 : 3 : 3 : 1 ratio (Table 2). Gene symbols of Fld for normal type and fld 1 and fld 2 for fldL-1 and fldL-2 were assigned, respectively. The normal plant type, thus recovered, manifested usual phenotypes such as blue flower, papilionaceous stipules, and normal level of foliar flavonoids. The variant plant type exhibited extreme reduction in total flavonoid contents, containing only 10% of that in mother control, and this feature was accompanied with reduced root length, absence of stipules, abnormal elongation of leafless stem, and much higher pollen sterility (79.33%) than either of its parents. 3.3. Inheritance of Isozyme-Banding Pattern in Selfed and Intercrossed Progenies 3.3.1. ACO Two mutant lines and the control varieties bred true for their respective single-banded phenotypes in successive selfed generations (M2-M4). Two zones of enzyme activity were conspicuous of which the most anodal one, designated as ACO-1 contained a total of three bands exhibiting two different types of migration (Figures 1 and 4). The fast moving allozyme (ACO-1a) was unique to bothmutants (lane 1 and 3), whereas relatively slower variant (ACO-1b) was common in mother control variety (lane 2). In the ACO-2 zone, three different types of mobility were manifested by a total of three bands; one of them was fast moving (ACO-2a) and developed only in fldL-1 (lane 1). It was followed by a unique slower band (ACO-2b) generated in fldL-2  mutant (lane 3). The slowest band in this zone (ACO-2c) was found specific to mother variety, BioL-212 (Figures 1 and 4, lane 2). The other variety “Hooghly Local” produced identical zymograms of var. BioL-212 (not shown in figure). Two types of allozyme activity in ACO-1 zone have been confirmed in segregating populations of different F2 and backcross (Figures 2 and 5). Segregation of Aco-1a/Aco-1b was found in mutant (Aco 1a/Aco 1a) × control variety (Aco 1b/Aco 1b) (Table 2). In the Aco 2 locus, heterozygous individuals for the alleles Aco 2a/Aco 2b were detected in the F2 progeny of crosses involving fld L-1 (Aco 2a/Aco 2a) and fld L-2 (Aco 2b/Aco 2b) mutants. Similarly, crosses between fld L-2 (Aco 2b/Aco 2b) and two control varieties (Aco 2c/Aco 2c) and between fld L-1 (Aco 2a/Aco 2a) and control varieties yielded homozygous individuals parental types and heterozygous individuals for Aco 2b/Aco 2c in former cross and for Aco 2a/Aco 2c in case of latter. F1 phenotype and one parental phenotype were observed in backcross progeny. In case of both loci, three types of phenotypes-two single-banded homozygous parental and one double-banded heterozygous phenotypes of fast and slow allozymes in F2 and two phenotypes: one heterozygous and one respective parental type in corresponding backcrosses segregated and conformed well with 1 : 2 : 1 in F2 and 1 : 1 in backcrosses, respectively (Table 2). 3.3.2. GSNOR Two control varieties and two induced mutant lines together generated 4 bands which could be clearly resolved in two separate zones of enzyme activity tentatively designated as GSNOR1 and GSNOR 2 from anodal side of the gel in the present material. All the four parents bred true in successive selfed (M2-M4) generations for the single-banded pattern corresponding to different allozymes in these two zones, and only representative zymograms showing differences in banding pattern have been shown. The mutant line fld L-2 was conspicuously different from control and also from fld L-1 by possessing a unique band in zymogram (lane 2). The GSNOR 1 zone was monomorphic with same mobility and intensity of bands in all four parents. By contrast, in GSNOR 2 zone, the fastest band at lane 1 (GSNOR 2a) was present in control variety and fld L-1 mutant, while the slower one at lane 2 (GSNOR 2b) was visualized as unique band in fldL-2 line (Figure 3). F2 progeny revealed allelic segregation (single locus) in GSNOR 2 zones of enzyme activity in crosses between fld L-2 mutant and other three parents (Figures 3 and 6). Allozymes in this zone segregated into three phenotypic classes: two homozygotes for respective parental alleles (lanes 1 and 2) and one heterozygote of these two alleles (lanes 3 and 4) showing good agreement with the expected 1 : 2 : 1 ratio in F2 generation (Figures 3 and 6; Table 2), and no segregation distortion was found. The F1 hybrid was backcrossed to parents slowing slow allozyme and zymogram phenotypes agreed well with 1 parental  : 1 hybrid ratio in each cross (Table 2). Segregation of allozymes could not be detected in F2 progeny of two control varieties in this zone. No segregation of banding pattern was observed in GSNOR 1 zone also. 3.4. Linkage Analysis between Isozyme Loci, fld 1, and fld 2 Genetic linkage relationship was analyzed on the basis of joint segregation of zymogram phenotype and flavonoid level in F2 and backcrosses (Table 3). Individual locus in Aco and Gsnor loci (except Gsnor 1) as well as fld 1 and fld 2 exhibited normal mendelian segregation (1 : 2 : 1/3 : 1 in F2 and 1 : 1 in backcrosses) of alleles, but their joint segregation in different cross-combinations showed significant deviations (P < 0.05) from the expected ratios of independent assortment in different F2 families and respective backcross progenies (Table 3). In each case, recombination fraction (r) calculated from backcross data was put into Kosambi's mapping function, and map distance between loci was estimated. Aco 1 and fld1 was linked with a map distances of 9.75 cM, whereas fld 2 and Aco 2 were mapped 11.80 cM apart. A linked association with 15.48 cM and 26.19 cM map distance was found between fld 2 and Gsnor 2 and between Aco 2 and Gsnor 2, respectively (Table 3). Gsnor1 could not be mapped due to absence of segregating alleles. 3.5. Chromosome Location of fld1, fld2, and Isozyme Loci by Primary Trisomic Analysis Chromosomal association of fld1 and fld2 loci controlling different phenotypes of flavonoid deficiency in grass pea was traced in crosses between seven trisomics as female parents and the diploid mutant lines as their male counterpart. For trisomic-I and IV, the segregation of leaf flavonoid content as normal, recessive mutant phenotype derived from fldL-1 × trisomic-I and from fldL-2 × trisomic-IV, respectively, exhibited a large and significant 𝒳 2 value (P < 0.05) for 3 : 1 in F2 and 1 : 1 in backcrosses but agreed well with expected trisomic ratios of 8 : 1 in F2 and 2 : 1 in testcross progenies (Table 4). On the other hand, segregation of normal and recessive mutant type in rest of the crosses involving other trisomics showed good fit with expected disomic ratio of 3 : 1 ratio in F2 and 1 : 1 ratio in corresponding testcrosses in diploid population, and a good number of recessive homozygotes in 2n + 1 portion of these crosses were cytologically detected as trisomic plants (data not in table). Among the four isozyme loci visualized in gel, linkage was detected only between Aco 2 and Gsnor 2. Presumably, these two isozyme loci were on the same chromosome. To confirm this assumption and to localize them on chromosomes, the trisomics were crossed as female parent with diploid control and two mutant lines, and F1 progeny in each case was raised. The rationale of the trisomic analysis in the present material involved trisomic segregation of different phenotypes in diploid portion of F2 and BC1. A significant (P < 0.05) departure of allozyme segregation coded by Aco 1 from normal disomic ratios in F2 (1 : 2 : 1) as well as backcross (1 : 1) ratios was manifested in the progenies involving only trisomic-I. Similar situation was encountered for Aco 2 and Gsnor 2 loci in trisomic-IV (Table 4). For both cases, segregation of allozymes in respective trisomics agreed well with the expected trisomic ratio of 4 : 4 : 1 in F2 and to 2 : 1 in BC1 generations of diploid portion and to 2 : 7 : 0 (F2) in trisomic portion of the progeny (Table 4). Segregation was disomic for all other trisomics in F2 and corresponding backcrosses (data not presented). Linkage studies and trisomic segregation pattern in F2 as well as BC1 generations revealed that Aco1 and fld1loci were linked with each other on extra chromosome of trisomic-I, whereas fld 2 and two isozyme loci Aco 2 and Gsnor 2 were carried by extra chromosome of trisomic-IV in linked conditions. Based on the result, the map positions (in cM) among different loci are as shown in Figure 7. 4. Discussion Both fldL-1 and fldL-2 mutants, isolated in EMS-treated M2 progeny, exhibited huge deficiency in total foliar flavonoid contents, containing only 20% of normal level as measured in mother controls. However, the mutants differed from each other in the type of extract, where the flavonoid content reduced, ethanol extract for fldL-1 and aqueous extract for fldL-2 leaves. Flavonoid deficiency was found associated with modification of usual blue colour of flower into white and stipule characters in both type of mutants, and also rising level of pollen sterility in fldL-2 plants. The modification of flower colour was ascribed to the deficiency of anthocyanin biosynthesis and is of considerable assistance in plant breeding [45]. In Petunia, flavonoid deficiency resulted in male sterility [46], while in maize male fertility was not affected at all [47]. Both the phenomena, however, were found in the present mutants, supporting differential behavior of the two mutants and deficiency of total flavonoids might be due to reduction of ethanol-dissolved and water-soluble compounds. Flavonoid deficiency in Arabidopsis was found associated with modifications in seed testa colour [7] and UV-sensitivity [6]. Both fldL-1 and fldL-2 in the present study are nonlethal and provided easily detectable phenotypes such as flower color. No significant variation of flavonoid content, however, was found in two mother plants, suggesting lack of its variation in common genotypes of grass pea. Mode of inheritance of flavonoid deficiency was traced in self-pollinated as well as in intercrossed population, involving two mutants and two mother controls. In all four parents, marginal variation in flavonoid content was found in advanced generations, indicating true breeding nature of the mutant traits. Inheritance studies in intercrossed progeny obtained from control × mutant plants revealed monogenic recessive nature of the low flavonoid content in both the mutant types with dominant allele that was always with mother plants. The result is in agreement with monogenic recessive nature of different flavonoid mutants in plants including Arabidopsis [7]. Interestingly, flower colour and stipule characteristics appeared unmodified in the respective recessive mutant type, confirming their true breeding nature in the present material. A completely different result, however, was obtained when the two mutants were crossed reciprocally. Occurrence of F1 plants with normal level of flavonoids and usual presence of blue flower and papilionaceous stipules and its segregations into four different plant types: normal, fldL-1, fldL-2, and a double-mutant type, consistent with 9 : 3 : 3 : 1 ratio in F2, suggested involvement of two independent nonallelic loci Fld1/fld 1 (for fldL-1 mutant) and Fld2/fld 2 (for fld L-2 mutant) in controlling flavonoid deficiency in two mutant types under study. Both the genes (Fld 1 and Fld 2) exhibited dominance over their respective recessive alleles (fld1 and fld 2). In presence of both the genes in dominant form (Fld 1–Fld 2-), normal phenotype appeared whereas presence of fld 2 gene in double recessive form (fld 2 fld 2 Fld 2-) produced phenotypes characteristic of fldL-2 type. On the other hand, fldL-1 type occurred in the presence of double recessive nature of fld 1 gene (Fld1-fld 1 fld1). In homozygous recessive condition of both the genes (fld1 fld1 fld2 fld2) variant plant type showing leaf flavonoid content only 10% of mother control plants and high pollen sterility (79.33%) resulted in the F2 progeny. This type bred true in advanced generations and tentatively designated as “flavonoid-deficient double mutant type” in grass pea. Recovery of fldL-1 and fldL-2 phenotypes in F2 and occurrence of the double mutant type strongly indicated possibility of multiple blockages in flavonoid biosynthesis pathway which was different in two mutant types, but combined in double mutant plants, leading to further depletion of its flavonoid content in relation to fldL-1 and fldL-2 levels. The double mutants have immense significance as it provides valuable clues in functional biology of glutathione, NO and thioredoxin-mediated redox signaling in plants [14, 48]. The differences in genetic constitution of flavonoid deficiency between two mutant plant types were also manifested by banding profiles of aconitase and S-glutathione reductase isozymes. Inheritance pattern in the present study revealed that fldL-1 and fldL-2 were not only different from control varieties but also differed from each other due to variant banding profiles that were heritable and bred true for all the four loci resolved here. The distinct zones of enzyme activity are mostly coded by different loci, and the variants within a particular zone are usually due to presence of different alleles or their interaction as heterozygotes [43]. In the present material, consistency in two zones of enzyme activity for both ACO and GSNOR enzymes was confirmed in successive self-pollinated and intercrossed populations of four parents. Quite remarkably, the “loss-of-function” mutation in flavonoid content led to gain of isozyme functions in the present mutants. Allozyme variation is essential for construction of saturated linkage map with other markers in grass pea [35, 36]. Although control varieties showed monomorphic banding pattern, consistent occurrences of mutant specific bands in the present Aco and Gsnor loci indicated evolution of variant alleles which inherited as recessive gene mutations in the present material of grass pea. Both the mutant lines possessed some unique bands coded by specific alleles: Aco 2a in fld L-1 and Gsnor 2b and Aco 2b in fld L-2. Obviously, Aco 2 was triple allelic while Aco 1 and Gsnor 2 both were double allelic, resulting in increased polymorphism in the present mutants over their control plants. Presence of more than two alleles was also reported in Aco loci of grass pea [35] and lima bean [49]. Like Gsnor 1, single zone of activity was reported in GSNOR enzyme of Pisum sativum L. [15]. Segregation pattern of different allozymes in the present F2 and backcross-population indicated involvement of codominant alleles in monogenic segregation of Aco 1, Aco 2 loci of ACO system, and Gsnor 2 locus of GSNOR enzyme. Presence of double-banded phenotypes in heterozygotes suggested monomeric nature of aconitase in grass pea, and no distorted segregation was apparent in F2 generation. The GSNOR, on the other hand, was functionally dimeric as confirmed by the presence of four-banded phenotypes in the heterozygotes. However, single-banded phenotype was exhibited in heterozygotes of F2 and backcross-populations obtained from crosses involving mutant and control parents for Gsnor 1 locus, confirming its monomorphic nature in the present material. In F2 population of crosses between different subaccessions of Lathyrus sativus L., Chowdhury and Slinkard [35] also detected polymorphism in both Aco 1 and Aco 2 loci with occurrence of codominant alleles, while, in regenerated plants of soybean, a rare mutation in Aco2b locus was detected as a null allele [50]. Polymorphisms displaying segregation ratios close to those expected for single locus traits suggested involvement of different alleles at the structural loci in generation of variation in different isozyme loci [51]. Among the closely related genera of Lathyrus, Aco 1 was monomorphic, but Aco 2 was polymorphic in Vicia faba L. [52]. Polymorphic Aco loci showing codominant expression of different alleles were also studied in pea [43], lens [53], and Cicer arietinum L. [54, 55]. Absence of distortion in F2 single locus and joint segregation was another interesting feature in the present study consisting of four true breeding parental lines of Lathyrus sativus L. for the concerned traits. The result was in contrast with earlier reports of distorted segregation of other isozyme loci in grass pea [35]. Helentjaris et al. [56] explained that intraspecific cross-minimized genetic distortion and other errors than wide crosses to establish linkage maps. The true breeding nature of isozyme phenotypes in selfed progeny and simple segregation in F2 population of different intercrossed progenies confirmed their stability in the present material. Mutation has been identified as one of the main sources of isozyme variation in higher plants [57]. For the first time, allelic variations in Aco and Gsnor loci have been generated in two stable mutant lines, deficient in flavonoid contents, of grass pea through induced mutagenesis. Consistent presence of polymorphism in isozymes of both enzymes indicated origin of different molecular forms of allozymes. Induction of variant allele in leaf isozyme system has been reported in different legumes including Glycine max [50] and Trifolium resupinatum [58]. However, in some accessions of Lathyrus sativus L. and Centrosema occurrences of higher number of alleles per locus have been attributed to heterozygosity induced by significant outcrossing rate in these crops [36]. In the present study, in addition to using outcrossing preventive measure during hybridization, effective isolation between lines and populations has been maintained throughout the experiment to prevent intermixing, and inheritance studies were carried out in advanced selfed generation (M4) of different true breeding parental lines. It seemed likely that the occurrences of new alleles in Aco and Gsnor loci resulted from the action of the recessive genes induced by EMS treatments in the present materials. Linkage analysis involving Aco 1, Aco 2, and Gsnor 2 isozyme loci, and fld 1 and fld 2 mutations revealed independent assortment between two Aco loci, of which Aco 1 was linked tightly with fld 1 whereas Aco 2 was mapped with fld 2 and Gsnor 2 loci in linked states showing a distance of 11.80 cM and 26.19 cM, respectively. Absence of linkage between different Aco loci was also reported in different genotypes of grass pea, soybean, and Phaseolus vulgaris [35, 59], and this was confirmed in the present study also. However, for the first time, a Gsnor locus was mapped in linked association with a flavonoid-deficient locus and also with an Aco locus in any leguminous crop. The aconitase is exquisitely sensitive to NO and other ROS [17], while Gsnor reportedly showed reduced band intensity in cadmium-treated Pisum sativum L. [15]. Altered expression of the present Aco and Gsnor loci indicated modulation of enzyme activities under flavonoid-deficient conditions, and the mapping of their isozyme loci with fld 1 and fld 2 genes in closely linked state confirmed this assumption. The importance of any mutant trait as a potential tool in functional biology enhanced once it was assigned to a particular chromosome. Primary trisomic has been used as an excellent tool in legume crops to confirm possible chromosomal location of various traits [60]. When the loci under study were located on a particular chromosome in trisomy, the normal disomic segregation ratio was modified due to presence of an extra chromosome. Trisomic segregation of electrophoretic phenotypes of different isozymes in the present zymogram strongly indicated possible location of Aco 1 on extra chromosome of trisomic-I and Aco 2 and Gsnor 2 on extra chromosome of trisomic-IV. Similarly, a good fit of fld 1 and fld 2 to trisomic segregation strongly indicated possible location of fld 1 gene on extra chromosome of trisomic I and that of fld 2 gene on extra chromosome of trisomic IV. The deviations from the normal segregation ratio are ascribed to the phenomenon of primary trisomy. Furthermore, no recessive homozygote plant in trisomic portion was recovered in these crosses, and all the recessive homozygotes in population were cytologically confirmed as diploids (data not presented). Segregating phenotypes in F2 and BC1 generations involving other trisomic types in respective crosses were consistent with normal mendelian disomic ratios and confirmed the above observation. In grass pea, primary trisomic has been successfully utilized to assign genes of agronomic interest on specific chromosomes [21, 24, 61] and to study gene-dosage effect of aneuploidy on antioxidant defense enzymes [62]. The isolation of two different flavonoid-deficient mutants and their mapping with closely linked isozyme markers of two prominent enzymatic systems on specific chromosomes may provide vital clues in understanding the role of flavonoid in integrated antioxidant defense system and their genetic basis in grass pea. Conflict of Interests No conflict of interests is involved in any way with the present work. Figure 1 Zymogram phenotype of aconitase isozymes; lane 1-fldL-1, lane 2-mother variety BioL-212, lane 3-fldL-2 mutant, small letters indicate alleles of respective loci in Lathyrus sativus L. Figure 2 Segregation of phenotypes in F2 derived from crosses between BioL-212 × fldL-1 in Aco 1 and Aco 2 loci; F-Fast allele, S-slow allele, H-heterozygotes in different lanes, in Lathyrus sativus L. Figure 3 Segregation of S-nitrosoglutathione reductase loci, Gsnor 2 in F2 generation of fldL-2 × mother plant; F-Fast allele, S-slow allele, H-heterozygote. No segregation was observed in Gsnor 1 locus in Lathyrus sativus L. Figure 4 Zymogram phenotype of aconitase isozymes; lane 1-fldL-1, lane 2-mother variety BioL-212, and lane 3-fldL-2 mutant. Small letters indicate alleles of respective loci in Lathyrus sativus L. Figure 5 Segregation of phenotypes in F2 derived from crosses between BioL-212 × fldL-1 in Aco 1 and Aco 2 loci; F-Fast allele, S-slow allele, H-heterozygotes in different lanes, in Lathyrus sativus L. Figure 6 Segregation of S-nitrosoglutathione reductase loci, Gsnor 2 in F2 generation of fldL-2 × mother plant; F-Fast allele, S-slow allele, H-heterozygote. No segregation was observed in Gsnor 1 locus in Lathyrus sativus L. Figure 7 Table 1 Total foliar flavonoid contents (mg g−1 extract) in aqueous and ethanol extract of grass pea (Lathyrus sativus L.) mutants (fld L-1 and fld L-2) and mother plants (BioL-212 and Hooghly Local). Genotype Aqueous extracts Ethanol extract BioL-212 160.55 ± 3.6 354.37 ± 3.9 Hooghly Local 148.59 ± 3.2 346.40 ± 3.1 fld L-1 150.53 ± 3.2 70.13 ± 2.2* fld L-2 30.77 ± 1.4* 337.27 ± 2.9 *Significantly different from mother plants at P < 0.05. Table 2 Single locus segregation of fld 1 and fld 2 mutations, two aconitase (Aco1 & 2), and S-nitrosoglutathione reductase 2 (Gsnor 2) isozyme loci in F2 and backcross (BC1) populations of different intercrosses among four parents in Lathyrus sativus L. aFF-Homozygote of fast alleles, SS-Homozygote of slow allele, FS-Heterozygotes. *, **, and *** consistent with 1 : 2 : 1, 1 : 1, and 3 : 1 ratios, respectively, at 5% level of significance, ++parent/s showing slow allozyme used in testcross with F1 and pooled data of several crosses presented. Cross++ Locus Phenotype (F1) F2/BC1 phenotypea N 𝒳 2 Normal Deficient (3 : 1/1 : 2 : 1/1 : 1) Flavonoid mutant BioL-212 × fldL-1 fld 1 Normal flavonoid 61 — 23 84 0.25*** F1 × fldL-1 fld 1 — 54 — 43 97 1.25** HL × fldL-1 fld 1 Normal flavonoid 81 — 28 109 0.02*** F1 × fldL-1 fld 1 — 37 — 30 67 0.72** BioL-212 × fldL-2 fld 2 Normal flavonoid 118 — 41 159 0.05*** F1 × fldL-2 fld 2 — 33 — 27 60 0.60** HL × fldL-2 fld 2 Normal flavonoid 120 — 43 163 0.16*** F1 × fldL-2 fld 2 — 48 — 42 90 0.04** Normal fldL-1 fldL-2 Double 𝒳 2 type type recessive (9 : 3 : 3 : 1) fldL-1 × fldL-2 fld 1/fld 2 Normal flavonoid 153 50 57 20 0.96 Isozyme loci Locus Alleles FF FS SS N 𝒳 2 (3:1/1:2:1/1:1) BioL-212/HL × fldL-2 Gsnor 2 ab 47 101 51 199 0.20* F1 × fldL-2 Gsnor 2 ab — 54 61 115 0.43** fldL-1 × fldL-2 Gsnor 2 ab 60 118 54 232 0.36* F1 × fldL-2 Gsnor 2 ab — 50 46 96 0.16** fldL-1/fldL-2 × BioL-212/HL Aco 1 ab 25 54 25 104 0.15* F1 × BioL-212/HL Aco 1 ab — 37 44 81 0.60** fldL-1 × fldL-2 Aco 2 ab 51 92 44 187 0.57* F1 × fldL-2 Aco 2 ab — 44 38 82 0.44** BioL-212/HL × fldL-2 Aco 2 bc 46 88 39 173 0.62* F1 × BioL-212/HL Aco 2 bc — 40 34 74 0.49** BioL-212/HL × fldL-1 Aco 2 ac 23 38 20 81 0.53* F1 × BioL-212/HL Aco 2 ac — 17 13 30 0.53** Table 3 Joint segregation of pairs of four isozyme loci and fld1 and fld 2 genes exhibiting significant deviations from expected F2 and backcross (BC1) ratios of random assortment in Lathyrus sativus L. aH1-Heterozygous for alleles at “X” locus, H-heterozygous for alleles at “Y” locus. r-recombinant value. *, **, and *** significant at 5% level for 3 : 1 : 6 : 2 : 3 : 1, 1 : 1 : 1 : 1, and 1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 : 1, respectively. Number of progeny with designated phenotypesa (F2/BC1 generation) Loci Progeny XY XH Xy H1Y H1H H1y xY xH xy Total 𝒳 2 r Map (X)-(Y) distance (cM.) Aco 1-fld 1 F2 27 — 11 07 — 28 02 — 39 114 205.55* — — Aco 1-fld 1 BC1 — — — — 49 08 — 05 73 135 96.68** 0.0963 9.75 Aco 2-fld 2 F2 22 — 13 10 — 03 02 — 17 67 86.39* — — Aco 2-fld 2 BC1 — — — — 57 06 — 10 65 138 82.56** 0.116 11.80 Gsnor 2-fld 2 F2 39 — 09 07 — 15 11 — 30 111 126.78* — Gsnor 2-fld 2 BC1 — — — — 62 11 — 07 40 120 67.12** 0.150 15.48 Gsnor2-Aco-2 F2 18 01 02 03 10 07 03 05 21 70 137.61*** — — Gsnor2-Aco-2 BC1 — — — — 52 17 — 14 46 129 35.49** 0.240 26.19 Table 4 Segregations of fld 1 and fld 2 along with Aco 1, Aco 2, and Gsnor 2 isozyme loci in F2 and BC1 generations obtained from several crosses (data pooled) between two different primary trisomics (Tr I and IV) and four diploid parents. Data of only critical trisomics carrying isozyme loci presented here. aF- Homozygous for fast/dominant allele, S-Homozygous for slow/recessive allele, H-Heterozygous; (2n+1)b-consistent at 5% level; *, **, and *** consistent with 4 : 4 : 1, 8 : 1 in F2, and 2 : 1 in BC1 at 5% level of significance, respectively. F2 and BC1 phenotypesa Trisomic types Progeny Loci 2n 𝒳 2 (2n+1)b FF FS SS Total (1 : 2 : 1/3 : 1) (1:1) (4 : 4 : 1/8 : 1) (2: 1) X2 (2 : 7 : 0) Tr-I F2 fld 1 129 — 17 146 13.89 — 0.04** — — Tr-I BC1 fld 1 — 68 37 105 — 9.15 — 0.17*** — Tr-IV F2 fld 2 118 — 14 132 14.08 — 0.03** — Tr-IV BC1 fld 2 — 45 25 70 — 5.71 — 0.18*** — Tr-I F2 Aco-1 50 60 14 124 21.04 — 0.912* — 2.53 Tr-I BC1 Aco-1 — 31 25 56 — 15.68 — 0.51*** — Tr-IV F2 Aco-2 47 52 11 110 23.90 — 0.39* — 0.51 Tr-IV BC1 Aco-2 — 71 30 101 — 16.64 — 0.60*** — Tr-IV F2 Gsnor 2 35 29 08 72 22.98 — 0.56* — 0.008 Tr-IV BC1 Gsnor 2 — 44 19 63 — 9.92 — 0.29*** — ==== Refs 1 Taylor LP Grotewold E Flavonoids as developmental regulators Current Opinion in Plant Biology 2005 8 3 317 323 15860429 2 Miyagi Y Om AS Chee KM Bennink MR Inhibition of azoxymethane-induced colon cancer by orange juice Nutrition and Cancer 2000 36 2 224 229 10890034 3 Holton TA Cornish EC Genetics and biochemistry of anthocyanin biosynthesis Plant Cell 1995 7 7 1071 1083 12242398 4 Chopra S Hoshino A Boddu J Iida S Grotewold E Flavonoid pigments as tools in molecular genetics The Science of Flavonoids 2006 Columbus, Ohio, USA The Ohio State University 147 173 5 Koes R Verweij W Quattrocchio F Flavonoids: a colorful model for the regulation and evolution of biochemical pathways Trends in Plant Science 2005 10 5 236 242 15882656 6 Li J Ou-Lee TM Raba R Amundson RG Last RL Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation Plant Cell 1993 5 2 171 179 12271060 7 Shirley BW Kubasek WL Storz G Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis Plant Journal 1995 8 5 659 671 8528278 8 Albert S Delseny M Devie M Banyuls, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat Plant Journal 1997 11 2 289 299 9076994 9 Bieza K Lois R An Arabidopsis mutant tolerant to lethal ultraviolet-B levels shows constitutively elevated accumulation of flavonoids and other phenolics Plant Physiology 2001 126 3 1105 1115 11457961 10 Sharma M Cortes-Cruz M Ahern KR McMullen M Brutnell TP Chopra S Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize Genetics 2011 188 1 69 79 21385724 11 Dixon RA Sumner LW Legume natural products: understanding and manipulating complex pathways for human and animal health Plant Physiology 2003 131 3 878 885 12644640 12 Filkowski J Kovalchuk O Kovalchuk I Genome stability of vtc1, tt4, and tt5 Arabidopsis thaliana mutants impaired in protection against oxidative stress Plant Journal 2004 38 1 60 69 15053760 13 Alfenito MR Souer E Goodman CD Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S -transferases Plant Cell 1998 10 7 1135 1149 9668133 14 Bashandy T Taconnat L Renou J-P Meyer Y Reichheld J-P Accumulation of flavonoids in an ntra ntrb mutant leads to tolerance to UV-C Molecular Plant 2009 2 2 249 258 19825611 15 Barroso JB Corpas FJ Carreras A Localization of S -nitrosoglutathione and expression of S -nitrosoglutathione reductase in pea plants under cadmium stress Journal of Experimental Botany 2006 57 8 1785 1793 16595575 16 Moeder W Del Pozo O Navarre DA Martin GB Klessig DF Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana Plant Molecular Biology 2007 63 2 273 287 17013749 17 Navarre DA Wendehenne D Durner J Noad R Klessig DF Nitric oxide modulates the activity of tobacco aconitase Plant Physiology 2000 122 2 573 582 10677450 18 Sakamoto A Ueda M Morikawa H Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase FEBS Letters 2002 515 1–3 20 24 11943187 19 Díaz M Achkor H Titarenko E Martínez MC The gene encoding glutathione-dependent formaldehyde dehydrogenase/GSNO reductase is responsive to wounding, jasmonic acid and salicylic acid FEBS Letters 2003 543 1–3 136 139 12753920 20 Pastor-Cavada E Juan R Pastor JE Girón-Calle J Alaiz M Vioque J Antioxidant activity in Lathyrus species Grain Legumes 2009 54 10 11 21 Talukdar D Dwarf mutations in grass pea (Lathyrus sativus L.): Origin, morphology, inheritance and linkage studies Journal of Genetics 2009 88 2 165 175 19700854 22 Talukdar D Biswas AK Inheritance of flower and stipule characters in different induced mutant lines of grass pea (Lathyrus sativus L.) 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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2259370810.1100/2012/849632Research ArticleChanges of Radial Diffusivity and Fractional Anisotopy in the Optic Nerve and Optic Radiation of Glaucoma Patients Engelhorn Tobias 1 *Michelson Georg 2 Waerntges Simone 2 Otto Marlen 1 El-Rafei Ahmed 3 Struffert Tobias 1 Doerfler Arnd 1 1Department of Neuroradiology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany2Department of Ophthalmology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany3Department of Computer Science, University of Erlangen-Nuremberg, Martensstrasse 3, 91058 Erlangen, Germany*Tobias Engelhorn: [email protected] Editors: M. Rosa, G. Tedeschi, E. J. Thompson, and C. Yu 2012 19 4 2012 2012 84963212 12 2011 2 1 2012 Copyright © 2012 Tobias Engelhorn et al.2012This 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.Purpose of this study was to evaluate with diffusion-tensor imaging (DTI) changes of radial diffusivity (RD) and fractional anisotropy (FA) in the optic nerve (ON) and optic radiation (OR) in glaucoma and to determine whether changes in RD and FA correlate with disease severity. Therefore, glaucoma patients and controls were examined using 3T. Regions of interest were positioned on RD and FA maps, and mean values were calculated for ON and OR and correlated with optic nerve atrophy and reduced spatial-temporal contrast sensitivity (STCS) of the retina. We found, that RD in glaucoma patients was significantly higher in the ON (0.74 ± 0.21 versus 0.58 ± 0.17·10−3 mm2 s−1; P < 0.05) and OR (0.79 ± 0.23 versus 0.62 ± 0.14·10−3 mm2 s−1; P < 0.05) compared to controls. Aside, FA was significantly decreased (0.48 ± 0.15 versus 0.66 ± 0.12 and 0.50 ± 0.20 versus 0.66 ± 0.11; P < 0.05). Hereby, correlation between changes in RD/FA and optic nerve atrophy/STCS was observed (r > 0.77). In conclusion, DTI at 3 Tesla allows robust RD and FA measurements in the ON and OR. Hereby, the extent of RD increase and FA decrease in glaucoma correlate with established ophthalmological examinations. ==== Body 1. Introduction Glaucoma is responsible for approximately 10% of the cases of blindness throughout the world and thus is the third leading cause of blindness with more than 8 million cases each year [1]. Glaucoma is considered a nervous-system-based degenerative disease that is only partially influenced by ocular factors [2]. Moreover, neuronal degeneration involving all parts of the central visual pathways was documented at autopsy in a patient who had advanced open-angle glaucoma and severe visual field losses in both eyes [3]. Central neuronal degeneration can be assessed noninvasively using diffusion tensor imaging (DTI) with calculation of radial diffusivity (RD) and fractional anisotropy (FA). The parameter fractional anisotropy (FA) measures the orientation coherence of diffusion and provides information about the fiber integrity. The radial diffusivity (RD) is the apparent water diffusion coefficient in the direction perpendicular to the axonal fibers. It represents a parameter of demyelination or glia cell impairment [4]. Hence, goal of this study was to evaluate whether 3 Tesla DTI with calculation of RD and FA in different sections of the visual pathways can assess the spread of glaucomateous damage within the central nervous system and to determine whether these functional MRI-derived parameters correlate with disease severity in 22 severely ill patients with glaucoma aged 37 to 86 years and 20 age-matched control subjects aged 45 to 83 years. 2. Materials and Methods The study was conducted in accordance with the Declaration of Helsinki on Biomedical Research Involving Human Subjects. The institutional Clinical Investigation Ethics Committee approved the study protocol, and written informed consent was obtained from all subjects prior to the study after explanation of the nature and possible consequences of the study. Eyes were assessed in full ophthalmologic examinations. Available additional examinations were referred to for evaluation, including Heidelberg Retinal Tomography (Heidelberg Engineering, Heidelberg, Germany), automated perimetry (Octopus 101 dG2; Interzeag, Schlieren, Switzerland), spatial-temporal contrast sensitivity (STCS, frequency doubling test; Carl Zeiss Meditec AG, Jena, Germany), nonmydriatic fundus images (nonmyd-alpha 45; Kowa Optimed, Inc, Torrance, CA), and measurement of intraocular pressure. MRI was performed on a 3T high-field scanner (Magnetom Tim Trio, Siemens Healthcare AG, Erlangen, Germany) with gradient field strength up to 45 mT/m (72 mT/m effective). The anatomical data were obtained in a T1-weighted 3D-MPRAGE sequence (TR = 900 ms, TE = 3 ms, FoV = 23 × 23 cm, acquisition matrix size = 512 × 256 reconstructed to 512 × 512, reconstructed axial plans with 1.2 mm slice thickness). DTI was performed in the axial plane with 4 mm slice thickness using a single-shot, spin echo, echo planar imaging (EPI) diffusion tensor sequence thus covering the whole visual pathway (TR = 3400 ms, TE = 93 ms, FoV = 23 × 23 cm, acquisition matrix size = 256 × 256 reconstructed to 512 × 512, number of signal averages = 7, partial Fourier acquisition = 60%). Diffusion weighting with a maximal b-factor of 1000 s/mm2 was carried out along 15 icosahedral directions complemented by one scan with b = 0. Datasets were automatically corrected for imaging distorsions and coregistered in reference to T1-weighted MPRAGE images. DTI data were interpolated in the Log-Euclidean framework to avoid the swelling effect. Then, the data were further processed and the estimated segmented optic radiation was checked for concordance with anatomical knowledge. The slice, which soonest included the lateral geniculate nucleus (LGN), was chosen for further proceeding and calculations. Analysis of the segmentation errors showed that additionally, anatomical structures were segmented, which are not belonging to the optic radiation. These structures were manually removed. Predominantly, parts of the optic tract, the forceps major of the corpus callosum, which proceeds medial of the optic radiations and all tracts, which do not connect to the visual cortex were concerned. The special anatomy in the area of the LGN has to be considered in more detail. In axial slices of this region the axons of the optic radiation, which derive from the LGN, overlap with the distal part of the optic tract, which connects to the LGN. Exclusion of this area would effectuate a loss of information from the LGN and origin of the optic radiation. An inclusion would produce acquisition of the distal part of the optic tract. As we decided in favour of the latter solution, some information from the distal part of the optic tract was included in our calculations of the mean values of the radial diffusivity (RD) and fractional anisotropy (FA). These and further calculations such as determining the independent elements of the diffusion tensor, deriving the corresponding eigenvalues and eigenvectors, and calculation of RD and FA maps were performed with a dedicated software package (Neuro 3D, Siemens Healthcare AG, Erlangen, Germany). Drawing the regions of interest (ROIs) of approximately 9 mm2 on the RD and FA maps superimposed over the T1-weighted 3D-MPRAGE sequence was done by two experienced neuroradiologists (T. S. and A. D.), who were blinded to the patients' clinical data. The following ipsi- and contralateral ROIs were used for evaluation of RD and FA:    ROI 1: the intracranial part of the ON directly before the optic chiasm (Figure 1(b));    ROI 2: the optic radiation (OR) directly after the lateral geniculate nucleus (Figure 1(e));    ROI 3: the OR on the level of the posterior horn of the lateral ventricle (Figure 1(e));    ROI 4: the OR directly before its cortical spread (Figure 1(f)). These ROIs were chosen to examine the 3rd neuron (optic nerve) as well as the 4th neuron (optic radiation) of the visual pathways. Hereby, the intracranial part and not the intraorbital part of the ON was chosen to avoid movement artefacts that affect the intraorbital part caused by eye motion during the MRI examination. Statistical analyses were performed using the PASW software (release 18.0, SPSS Inc. Chicago, IL, USA). 3. Results The ophthalmological examination revealed no significant affection of the eyes in all 20 controls. Aside, MRI revealed no space occupying lesions or infarction along the extra- and intracranial visual pathway in all 42 test persons. Measurement of RD and FA in ipsi- and contralateral ROIs was feasible in all test persons. Mean evaluation time was 32 minutes per patient. Cronbach-α at the 95% confidence interval for RD was 0.89 (right side, F = 2.54, P = 0.19) and 0.86 (left side, F = 1.65, P = 0.27) while for FA it was 0.99 (right side, F = 2.34, P = 0.15) and 0.88 (left side, F = 1.08, P = 0.32). Thus, the correlation coefficients of reliability of the RD and FA measurements done by two neuroradiologists were within a very good range of reliability. Hence, the results of both RD and FA measurements were averaged. In controls, there was good correlation between an increase in RD of both sides with increasing age (r = 0.71). The overall percentage change in RD in control subjects between 45 and 83 years was 15.3 ± 5.0%. This difference was significant (P = 0.04). Aside, there was good correlation between a decrease in FA of both sides with increasing age (r = 0.78). The overall percentage change in FA in control subjects between 45 and 83 years was 33.7 ± 10.7%. This difference was also significant (P = 0.02). Right and left side RD and FA measurements in controls and glaucoma patients were similar and differed only in small ranges up to five percent (no significant differences in all test persons). Hence, the measurements for both sides were averaged and are displayed in Tables 1 and 2. In glaucoma patients, good correlation between an increase in RD as well as a decrease in FA measured in the intracranial part of the ON and in the OR and the extent of optic nerve atrophy and STCS of the retina was observed (r > 0.77 and r > 0.81, resp.). 4. Discussion Physiological changes of white matter tracts in the brain show different regional patterns dependent on age, and circulatory disorders [5–7]. The compromised fiber microstructure may at least in part participate in mechanisms of functional degradation [5]. An anterior-posterior gradient of impairment in the brain was described, which is detectable by diffusion tensor imaging (DTI) [8]. This examination method is based on magnetic resonance imaging (MRI) and characterizes the structure of brain tissue based on underlying water diffusivity [4]. Water diffusion is highly anisotropic in white matter of the nervous system because the overall mobility of the diffusing molecules is limited by intracellular and extracellular compartments, by neurons, glial cells, and by axons [9]. The DTI-derived parameter fractional anisotropy (FA) measures the orientation coherence of diffusion and provides information about the fiber integrity [4, 5], while the radial diffusivity (RD) is the apparent water diffusion coefficient in the direction perpendicular to the axonal fibers. It represents a parameter of demyelination or glia cell impairment [10]. In a recently published study [11], DTI was shown to enable quantitative assessment of the optic radiation by volume rendering of the reconstructed fiber tracts. Hereby, the DTI-derived volume of the optic radiation of glaucoma patients was significantly decreased compared to age-matched controls. To our best knowledge, this is the first study to report qualitative measurements of DTI-derived radial diffusivity (RD) and fractional anisotropy (FA) correlated with established ophthalmological tests in different sections of the 3rd neuron (optic nerve) and 4th neuron (optic radiation) of the visual pathways of glaucoma patients and normal seeing controls using a high-field 3 Tesla MR scanner. We could demonstrate that (1) measurement of RD and FA in different sections of the visual pathways is feasible, and (2) there is a significant increase in RD as well as a significant decrease in FA in healthy controls with increasing age. (3) Compared to age-matched controls, RD is significantly increased and FA is significantly decreased in the optic nerve and in all examined sections of the optic radiation in glaucoma patients, and (4) there is good correlation between the increase in RD and the decrease in FA and the severity of optic nerve atrophy and retinal impairment quantified with established ophthalmological tests. Thereby, (5) FA is superior to RD in detecting glaucoma-induced intracranial damage. Hereby, the strong influence of age-related processes on RD and FA is not surprising. Different patterns of RD and FA, suggesting mechanisms of functional degradation, were shown to compromise at least in part the fiber microstructure [5]. Age-related increases in RD as well as decreases in FA, that reflect the axonal integrity including myelination, were seen throughout the brain and several studies [12–14]. In this study, the overall percentage change in RD and FA in control subjects between 45 and 83 years was plus 15.3 ± 5.0 and minus 33.7 ± 10.7%, respectively. As result, RD and FA in different sections of the visual pathways of glaucoma patients were compared to age-matched controls. Hereby, we found a significant change in RD and FA in the optic nerve (3rd neuron of the visual pathways) and in all three sections of the optic radiation (4th neuron of the visual pathways), thus in all sections with a more or less straight axonal direction before the cortical spread. In contrast, there was no significant change in RD and FA in the intraorbital optic nerve, the optic chiasm, and the lateral geniculate nucleus (RD and FA values not shown). The explanation for this finding is most likely the crossing of axons in the optic chiasm and the reconnection of axons in the lateral geniculate nucleus resulting in a change of the straight axonal direction with subsequent change in RD and FA [15], whereby ineluctably movement of the eyeball subsequently results in movement of the intraorbital optic nerve downgrading the quality of RD and FA assessment in this section of the visual pathways by motion artefacts. This limitation could be overcome with retrobulbar anesthesia or by simply using the intracranial part of the optic nerve as done in this study. As most important finding, we could demonstrate that there is correlation of MRI-derived changes in RD and FA in the optic nerve and the optic radiation of glaucoma patients with established ophthalmological scores to determine the degree of optic nerve atrophy and reduced spatial-temporal contrast sensitivity of the retina. Hereby, changes in FA with r > 0.81 seem to be slightly superior to changes in RD with r > 0.77. In conclusion, there is degeneration of the visual pathways with subsequent characteristic changes in RD and FA along the 3rd and 4th neuron in glaucoma patients. Hereby, the extent of degeneration can be assessed noninvasively by 3 Tesla DTI with robust measurement of RD and FA with correlation to optic nerve atrophy and retinal impairment. Authors' Contribution T. Engelhorn and G. Michelson contributed equally to this work and thus share first authorship. Figure 1 Different regions of interest (red circles in the right visual pathways) that were used for evaluation of radial diffusivity (RD) and fractional anisotropy (FA) in glaucoma patients and controls in the left and right visual pathways: intraorbital part of the optic nerve (a), intracranial part of the optic nerve (b), optic chiasm (c), lateral geniculate nucleus (d), and different sections of the optic radiation (e, f). All regions of interest are drawn in a fused dataset of FA-weighted DTI and 3D-MPRAGE sequences (a–c: coronal slides; d–f: axial slides). Table 1 Averaged RD values in different ROI of glaucoma patients and controls. Localization Mean RD ± standard deviation (glaucoma patients versus controls) ROI 1 0.74 ± 0.21 versus 0.58 ± 0.17·10−3 mm2 s−1∗ ROI 2 0.70 ± 0.26 versus 0.55 ± 0.16·10−3 mm2 s−1∗ ROI 3 0.76 ± 0.22 versus 0.60 ± 0.14·10−3 mm2 s−1∗ ROI 4 0.79 ± 0.24 versus 0.65 ± 0.14·10−3 mm2 s−1∗ *Significant lower RD values compared to controls (P < 0.05). Table 2 Averaged FA values in different ROIs of glaucoma patients and controls. Localization Mean FA ± standard deviation (glaucoma patients versus controls) ROI 1 0.48 ± 0.15 versus 0.66 ± 0.12* ROI 2 0.40 ± 0.16 versus 0.57 ± 0.13* ROI 3 0.48 ± 0.17 versus 0.64 ± 0.11* ROI 4 0.44 ± 0.22 versus 0.53 ± 0.20* *Significant lower FA values compared to controls (P < 0.05). ==== Refs 1 Quigley H Broman AT The number of people with glaucoma worldwide in 2010 and 2020 British Journal of Ophthalmology 2006 90 3 262 267 16488940 2 Gupta N Yücel YH What changes can we expect in the brain of glaucoma patients? Survey of Ophthalmology 2007 52 6 S122 S126 17998036 3 Gupta N Ang LC De Tilly LN Bidaisee L Yücel YH Human glaucoma and neural degeneration in intracranial optic nerve, lateral geniculate nucleus, and visual cortex British Journal of Ophthalmology 2006 90 6 674 678 16464969 4 Basser PJ Mattiello J LeBihan D MR diffusion tensor spectroscopy and imaging Biophysical Journal 1994 66 1 259 267 8130344 5 Sullivan EV Rohlfing T Pfefferbaum A Quantitative fiber tracking of lateral and interhemispheric white matter systems in normal aging: relations to timed performance Neurobiology of Aging 2010 31 3 464 481 18495300 6 Xu J Sun SW Naismith RT Snyder AZ Cross AH Song SK Assessing optic nerve pathology with diffusion MRI: from mouse to human NMR in Biomedicine 2008 21 9 928 940 18756587 7 Dong Q Welsh RC Chenevert TL Clinical applications of diffusion tensor imaging Journal of Magnetic Resonance Imaging 2004 19 1 6 18 14696215 8 Bennett IJ Madden DJ Vaidya CJ Howard DV Howard JH Age-related differences in multiple measures of white matter integrity: a diffusion tensor imaging study of healthy aging Human Brain Mapping 2010 31 3 378 390 19662658 9 Beaulieu C The basis of anisotropic water diffusion in the nervous system—a technical review NMR in Biomedicine 2002 15 7-8 435 455 12489094 10 Song SK Sun SW Ju WK Lin SJ Cross AH Neufeld AH Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia NeuroImage 2003 20 3 1714 1722 14642481 11 Engelhorn T Michelson G Waerntges S Struffert T Haider S Doerfler A Diffusion tensor imaging detects rarefaction of optic radiation in glaucoma patients Academic Radiology 2011 18 6 764 769 21377906 12 Abe O Aoki S Hayashi N Normal aging in the central nervous system: quantitative MR diffusion-tensor analysis Neurobiology of Aging 2002 23 3 433 441 11959406 13 Hsu JL Leemans A Bai CH Gender differences and age-related white matter changes of the human brain: a diffusion tensor imaging study NeuroImage 2008 39 2 566 577 17951075 14 Gunning-Dixon FM Brickman AM Cheng JC Alexopoulos GS Aging of cerebral white matter: a review of MRI findings International Journal of Geriatric Psychiatry 2009 24 2 109 117 18637641 15 Staempfli P Rienmueller A Reischauer C Valavanis A Boesiger P Kollias S Reconstruction of the human visual system based on DTI fiber tracking Journal of Magnetic Resonance Imaging 2007 26 4 886 893 17896363
22593708
PMC3349161
CC BY
2021-01-05 11:53:16
yes
ScientificWorldJournal. 2012 Apr 19; 2012:849632
==== Front BMC Res NotesBMC Res NotesBMC Research Notes1756-0500BioMed Central 1756-0500-4-5472218548310.1186/1756-0500-4-547Research ArticleEffect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa Ashrafi Iraj [email protected] Hamid [email protected] Hamid [email protected] Majid [email protected] Hamid [email protected] Young Researchers Club, Science and Research Branch, Islamic Azad University, Tehran, Iran2 Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran3 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran4 Animal Breeding Center of Iran2011 20 12 2011 4 547 547 15 4 2011 20 12 2011 Copyright ©2011 Ashrafi et al; licensee BioMed Central Ltd.2011Ashrafi 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 Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters. Results Ejaculates were collected using the artificial vagina from four Chal rams and three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen for 15 min and plunged into liquid nitrogen. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics. Conclusions Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing. ==== Body Background Considerable effort has been directed towards developing techniques for artificial insemination (AI) using frozen ram semen. Its potential in sheep breeding has become evident following the development of controlled reproduction procedures and more intensive management systems. The widespread application of AI depends largely on the use of frozen semen and thus on the availability of techniques that result in acceptable fertility in a selective breeding control programme. AI with frozen semen dispensed through the cervix gives quite low fertility rates in ram and use of laparoscopy with thawed semen is more costly and time-consuming but it improve the fertility rate significantly. The need to prepare a large number of doses of ram's semen each year requires the development of a rapid and effective method for freezing semen [1]. Spermatozoa are not adapted to survive cryopreservation, and therefore have variable responses to cooling and rewarming depending both on individual male and species [2,3]. Ram spermatozoa are susceptible to various stresses during cryopreservation [4]. The physiological and functional changes that occur in spermatozoa such as an irreversible reduction in motility, viability and acrosome integrity [5-7] cause changes similar to capacitation and acrosome reaction in the surviving population [8]. Damage to sperm membranes primarily occurs during the freezing and thawing process over the temperature range -15°C to -60°C and not during storage in liquid nitrogen [9]. In the case of ram spermatozoa, most damage occurs between -10°C and -25°C, the region of ice crystallization [6]. The process of cell dehydration that accompanies slow freezing is potentially beneficial for cell survival, whereas rapid freezing rates are considered more likely to cause cell death [5]. Of considerable importance in the cooling regime is the cooling rate through the critical temperature range, defined as the range when ice crystal formation and consequent cell dehydration is occurring. The cooling rate determines whether the cells remain in equilibrium with their extracellular environment or become progressively super cooled with the increasing possibility of intracellular ice formation [10]. Freezing of ram spermatozoa in cell freezer has been commonly carried out from 5°C after precooling of straws up to 5°C in the cold chamber [11-17]. Computer-controlled cooling machine was built in the laboratory to achieve rapid cooling of samples in straws under controlled conditions. A protocol based on controlled-rate cooling and freezing of ram semen in straws has been reported to improve ram semen freezing technique but the post-thaw attributes of spermatozoa were evaluated by subjective assessment [10]. Computer-aided semen analysis (CASA) technique provides precise and validated objective assessment of sperm motion characteristics [18-20] and has been applied for short-term [21] and long-term preservation of ram. The aim of this study was to determine the effects of two cooling methods (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters. Methods Animals and semen collection Semen samples were collected from four mature Chal rams (3-4 years) maintained at the Animal Breeding Center Farm of IRAN. After collection, the ram semen samples were transferred within a minute to the laboratory and kept in a water bath at 37°C for examination. The rams were fed 0.91 kg of concentrate daily and good quality hay and water were supplied ad libitum. Ejaculates were collected from the rams using the artificial vagina twice a week during the breeding season (autumn to early winter). Cryobiological procedures General aspects Some general procedures were established for the two methods. The volume of ejaculates was measured in a conical tube graduated at 0.1 ml intervals. The sperm concentration was determined by means of a haemocytometer and sperm motility was estimated using phase contrast microscopy (400 × ). Only ejaculates containing a semen volume varying between 1 and 2 ml, spermatozoa with >80% forward progressive motility and concentrations higher than 2.5 × 109 spermatozoa/ml were mixed in a pool balancing the sperm contribution of each male to eliminate individual differences [22]. Tris-citrate modified solution (Tris 27.1 g/l, citric acid 14.0 g/l, fructose 10.0 g/l, egg yolk 10% (v/v) glycerol 5% (v/v): 300 mOsM, pH 6.8, was used as the base extender (freezing extender). Each mixed ejaculate was split into two equal aliquots and diluted at 37°C with the base extender to a final concentration of approximately 4 × 108 spermatozoa per milliliter, in one step, in a 10 ml-glass centrifuge tube. Diluted samples were aspirated into 0.25 ml (medium-sized) French straws and sealed with polyvinyl alcohol powder. The sample processed according to the following methods and evaluated pre-freezing and post -thawing. Semen cooling and freezing methods Method 1: straws cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built), and equilibrated at 5°C for 80 min. After equilibration, the straws were frozen in same machine at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C, then the straws were plunged into liquid nitrogen for storage. The Thermocouples of the custom-built cool in machine record the chamber temperature via a computer program and drives a motor to open and close the entrance of the nitrogen vapor source. Method 2: straw transferred to refrigerator and maintained at 5°C for 3 h, then the straws were frozen in liquid nitrogen vapor, 4 cm above the liquid nitrogen, for 15 min and plunged into liquid nitrogen [23]. Semen thawing Frozen straws were thawed individually at 37°C for 20 s in a water bath for evaluation immediately after thawing [23]. Evaluation of percentage of motile cells, recovery rate and kinetic parameters A computer-assisted sperm motility analysis (CASA, Version 12 IVOS, Hamilton-Thorne Biosciences, Beverly, MA, USA) was used to analyze sperm motion characteristics. CASA was set up as follows: phase contrast; frame rate - 60 Hz; minimum contrast - 70; low and high static size gates - 0.6 to 4.32; low and high intensity gates - 0.20 to 1.92; low and high elongation gates 7 to 91; default cell size - 10 pixels; default cell intensity - 80. Semen was diluted (5 μl semen +95 μl extender) in a Tris-based extender (without egg yolk) and evaluated immediately after dilution. A 4 μl sample of diluted semen was put onto a pre-warmed chamber slide (Leja 4, Leja Products, Luzernestraat B.V., Holland) and sperm motility characteristics were determined with a 10 × objective at 37°C. Percentages of total motility and progressive motility were recorded. For both parameters the recovery rate of motility after thawing with respect to before freezing was calculated (post-thaw motility/pre-freeze motility). For thawed semen, VSL (straight linear velocity, μm/s), VCL (curvilinear velocity, μm/s), VAP (average path velocity, μm/s) and LIN = VSL/VCL × 100 (linearity, %) were noted as specific parameters of sperm motility. For each evaluation, 10 microscopic fields were analyzed to include at least 300 cells. Analysis The results were analyzed by Analysis of Variance with semen samples as replicates, using the following model:Y = μ+αi+eij Results As shown in Tables 1 controlled rate of freezing (method 1) significantly improve the pre-freezing and post-thaw total and progressive motility of ram spermatozoa compared to uncontrolled rate of freezing (method 2). The changes observed in motility are reflected in the recovery rate which indicates that method 1, preserve sperm motility better, showing significant difference with respect to the method 2. In general, it may be noted that the total motility is preserved better than progressive motility with recovery rates of 0.89 and 0.74, respectively. Table 1 Kinetic parameters (CASA) of pre-freezing and post-thawing ram sperm for two freezing methods Pre-freezing Post-thawin RR. for TM RR. for PM TM (%) PM (%) TM (%) PM (%) VSL (μm/s) VAP (μm/s) VCL (μm/s) LIN (%) Method 1 83.2 ± 8.2a 73.1 ± 13.5a 74.6 ± 7.8a 53.9 ± 10.5a 96.1 ± 44.4a 107.3 ± 40.5 158.6 ± 51.8a 60.59 ± 19 0.89 0.74 Method 2 79.1 ± 8.0b 64.8 ± 12.5b 61.2 ± 11.1b 46.1 ± 6.7b 78.4 ± 18.4b 91.1 ± 20.3 133.0 ± 28.3b 58.9 ± 9 0.77 0.71 a,b Different superscripts within columns are significantly differ. VSL (straight linear velocity, μm/s), VCL (curvilinear velocity, μm/s), VAP (average path velocity, μm/s), LIN = VSL/VCL × 100 (linearity, %), RR (recovery rate), TM (total motility) and PM (progressives motility). In specific kinetic parameters, method 1 gives significantly higher value for VSL and VCL in comparison with method 2. There are no significant differences between the two methods for VAP and LIN (additional file 1). Discussion The cryopreservation cycle for semen samples includes the entire process from sperm preparation and dilution through to the post-thawing maintenance of functional capacity. At each of these stages, spermatozoa need to maintain a range of functional attributes that ensure their fertilizing capacity [5]. Maintenance of sperm function during freezing and thawing depends upon several interrelated factors that include cooling rate, equilibration period and freezing method [4,8,13,24] but their adverse effects are manifested on thawing. The degree of cryo-damage also depends on several factors [5,25] which limit the survival of spermatozoa during incubation. Under the best experimental conditions about half of the population of motile sperm survives the freeze-thaw process [5,24,26]. In the present study it was observed that controlled rate of cooling and freezing resulted in significantly higher sperm total and progressive motility, compared to uncontrolled rate of cooling and freezing. The overall good post-thaw may be attributed to the efficacy of controlled rate freezing protocol and the criteria of processing only those ejaculates for cryopreservation which have thick consistency, >80% initial motility, >2.5 × 109 spermatozoa per ml. The recovery rate indicates that motility is better preserved with Method 1. The fact that progressive motility is more affected by the freezing process than individual motility implies that these parameters measure different aspects of cell physiology and in particular, that the physiological basis for the progressive motility parameter is more sensitive to cryobiological damage [1]. The controlled-rate cooling protocol, besides providing complete automation in the cryopreservation process, might also protect spermatozoa against some adverse effect caused by minor fluctuation in temperature imposed by the transfer of cooled straws from cold cabinet to cell freezer as done in the uncontrolled cooling rate ram semen freezing protocol. Apart from identifying motile and static spermatozoa CASA can also categorize spermatozoa on the basis of velocity of each motile sperm, measure the mean sperm velocity and related sperm track dimensions [20]. The measurement of sperm velocity has been considered as an indirect indicator of mitochondrial function in spermatozoa. During cryopreservation spermatozoal mitochondria undergo damages [27,28] resulting in the decrease of respiratory rate of frozen-thawed ram spermatozoa [29]. In the present study, the mean VCL and VSL of post-thaw spermatozoa were significantly higher in samples cooled at a controlled rate (method 1), compared to samples cooled at an uncontrolled-rate (method 2), VAP of post-thaw was higher in sample cooled at method 1 but the effect was not significant thereby implying that the magnitude of mitochondrial damage was almost similar under both the cooling treatments. Ram spermatozoa can tolerate a wide range of freezing rates [10,13,30]. In this study, the overall cooling rate of straws achieved under uncontrolled conditions was approximately at the rate of 0.15°C/min from 25 to 5°C, which was close to the approximate cooling rate of 0.14°C/min on cooling straws from 30 to 5°C in the cold chamber. However, under uncontrolled conditions, cooling over the period of 135 min was not at a linear rate, commencing at the rate of 0.4°C/min from 25°C for 15 min, and continuing at the rate of 0.2°C/for 15 min, 0.13°C/min for 60 min and thereafter progressed at the rate of 0.06°C/min for 45 min up to 5°C. Kumar et al. (2003) observed optimal cryosurvival of ram spermatozoa when cooled at the rate of 0.2°C/min from 22 to 5°C over a period of 90 min followed by freezing at the rate of 30°C/min from 5 to -50°C and concluded that careful control of the cooling and freezing rates are essential for maximal recovery of viable and functional cells. Conclusions The results indicated that controlled rate of cooling had significant effect on percentage of motile cells (total and progressive motility) pre-freezing and post-thawing and kinetic parameters of post-thawing ram spermatozoa, compared to uncontrolled rate of cooling prior to programmable freezing. Further research efforts are needed to comparatively assess the fertilizing ability of ram semen frozen by controlled and uncontrolled cooling rate cryopreservation protocols. Competing interests The authors declare that they have no competing interests. Authors' contributions IA carried out the design of the study and performed the cryopreservation process and analyze the sample. HK participated in its design and coordination and helped to draft the manuscript. HN helped to sample collection and cryopreservation process. MB helped to the cryopreservation process. HM make the programmable cooling machine. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Tubular data. Click here for file Acknowledgements The authors thank Animal Breeding Center of IRAN for supplying the rams and instrument of cryopreservation. ==== Refs Anel L de Paz P Alvarez M Chamorro CA Boixo JC Manso A Gonzalez M Kaabi M Anel E Field and in vitro assay of three methods for freezing ram semen Theriogenology 2003 60 1293 1308 10.1016/S0093-691X(03)00140-7 14511783 Holt WV Fundamental aspects of sperm cryobiology: the importance of species and individual differences Theriogenology 2000 53 47 58 10.1016/S0093-691X(99)00239-3 10735061 Thurston LM Siggins K Mileham AJ Watson PF Holt WV Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation Biol Reprod 2002 66 545 554 10.1095/biolreprod66.3.545 11870056 Anel L Alvarez M Martinez-Pastor F Garcia-Macias V Anel E de Paz P Improvement strategies in ovine artificial insemination Reprod Domest Anim 2006 41 Suppl 2 30 42 16984467 Watson PF Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function Reprod Fertil Dev 1995 7 871 891 10.1071/RD9950871 8711221 Salamon S Maxwell WM Storage of ram semen Anim Reprod Sci 2000 62 77 111 10.1016/S0378-4320(00)00155-X 10924821 Medeiros CM Forell F Oliveira AT Rodrigues JL Current status of sperm cryopreservation: why isn't it better? Theriogenology 2002 57 327 344 10.1016/S0093-691X(01)00674-4 11775978 Bailey JL Bilodeau JF Cormier N Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon J Androl 2000 21 1 7 10670514 Mazur P Causes of Injury in Frozen and Thawed Cells Fed Proc 1965 24 S175 182 14314563 Kumar S Millar JD Watson PF The effect of cooling rate on the survival of cryopreserved bull, ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines Cryobiology 2003 46 246 253 10.1016/S0011-2240(03)00040-3 12818214 Fiser PS Fairfull RW Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa Theriogenology 1986 25 473 484 10.1016/0093-691X(86)90057-9 16726139 Fiser PS Fairfull RW Marcus GJ The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws Cryobiology 1986 23 141 149 10.1016/0011-2240(86)90005-2 3698643 Pontbriand D Howard JG Schiewe MC Stuart LD Wildt DE Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa Cryobiology 1989 26 341 354 10.1016/0011-2240(89)90058-8 2766782 Soderquist L Madrid-Bury N Rodriguez-Martinez H Assessment of ram sperm membrane integrity following different thawing procedures Theriogenology 1997 48 1115 1125 10.1016/S0093-691X(97)00344-0 16728200 Byrne GP Lonergan P Wade M Duffy P Donovan A Hanrahan JP Boland MP Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro Anim Reprod Sci 2000 62 265 275 10.1016/S0378-4320(00)00121-4 10924829 Gil J Soderquist L Rodriguez-Martinez H Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen Theriogenology 2000 54 93 108 10.1016/S0093-691X(00)00328-9 10990351 Bag S Joshi A Naqvi SM Mittal JP Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws Theriogenology 2004 62 415 424 10.1016/j.theriogenology.2003.10.018 15225998 Holt WV Palomo MJ Optimization of a continuous real-time computerized semen analysis system for ram sperm motility assessment, and evaluation of four methods of semen preparation Reprod Fertil Dev 1996 8 219 230 10.1071/RD9960219 8726859 Verstegen J Iguer-Ouada M Onclin K Computer assisted semen analyzers in andrology research and veterinary practice Theriogenology 2002 57 149 179 10.1016/S0093-691X(01)00664-1 11775967 Joshi A Naqvi SM Bag S Dang AK Sharma RC Rawat PS Mittal JP Sperm motion characteristics of Garole rams raised for a prolonged period in a semi-arid tropical environment Trop Anim Health Prod 2003 35 249 257 10.1023/A:1023347514476 12797414 Kasimanickam R Kasimanickam V Pelzer KD Dascanio JJ Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C Anim Reprod Sci 2007 101 60 73 10.1016/j.anireprosci.2006.09.001 17014975 Gil J Lundeheim N Soderquist L Rodriiuez-Martinez H Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen Theriogenology 2003 59 1241 1255 10.1016/S0093-691X(02)01177-9 12527072 Bucak MN Atessahin A Varisli O Yuce A Tekin N Akcay A The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen Microscopic and oxidative stress parameters after freeze-thawing process Theriogenology 2007 67 1060 1067 10.1016/j.theriogenology.2006.12.004 17280711 Curry MR Cryopreservation of semen from domestic livestock Rev Reprod 2000 5 46 52 10.1530/ror.0.0050046 10711735 Watson PF The causes of reduced fertility with cryopreserved semen Anim Reprod Sci 2000 60-61 481 492 10.1016/S0378-4320(00)00099-3 10844218 Sanchez-Partida LG Windsor DP Eppleston J Setchell BP Maxwell WM Fertility and its relationship to motility characteristics of spermatozoa in ewes after cervical, transcervical, and intrauterine insemination with frozen-thawed ram semen J Androl 1999 20 280 288 10232663 Gillan L Maxwell WM Evans G Preservation and evaluation of semen for artificial insemination Reprod Fertil Dev 2004 16 447 454 10.1071/RD04034 15315743 Peris SI Morrier A Dufour M Bailey JL Cryopreservation of ram semen facilitates sperm DNA damage: relationship between sperm andrological parameters and the sperm chromatin structure assay J Androl 2004 25 224 233 14760008 Windsor DP Mitochondrial function and ram sperm fertility Reprod Fertil Dev 1997 9 279 284 10.1071/R96109 9261876 Colas G Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen J Reprod Fertil 1975 42 277 285 10.1530/jrf.0.0420277 235025
22185483
PMC3349620
CC BY
2021-01-04 21:03:28
yes
BMC Res Notes. 2011 Dec 20; 4:547
==== Front Case Rep SurgCase Rep SurgCRIM.SURGERYCase Reports in Surgery2090-69002090-6919Hindawi Publishing Corporation 2260660210.1155/2012/457272Case ReportFitz-Hugh-Curtis Syndrome in a Male Patient: A Case Report and Literature Review Saurabh Shireesh 1 *Unger Eric 1 Pavlides Constantinos 2 1Drexel University College of Medicine, Philadelphia, PA 19102, USA2Hahnemann University Hospital, Philadelphia, PA 19102, USA*Shireesh Saurabh: [email protected] Editors: S. Gourgiotis and M. Rangarajan 2012 26 3 2012 2012 45727218 12 2011 4 2 2012 Copyright © 2012 Shireesh Saurabh et al.2012This 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.Fitz-Hugh-Curtis syndrome is a condition characterized by inflammation of the liver capsule with concomitant pelvic inflammation without involvement of liver parenchyma. It is classically seen in young women who present with sharp, pleuritic right upper quadrant pain, usually but not always accompanied by symptoms of pelvic inflammatory disease (PID), and is frequently confused with biliary tract disease. Rarely the syndrome has been reported in males, and hematogenous and lymphatic spread to liver is thought to be the underlying mechanism. Serological tests and computed tomography (CT) scan may aid in diagnosis of Fitz-Hugh-Curtis syndrome. Definitive diagnosis is made by laparoscopy, which provides both diagnostic and therapeutic benefits. We report a case of Fitz-Hugh-Curtis syndrome in a young male patient, which was diagnosed and treated by laparoscopy. We also include a review of the literature. ==== Body 1. Introduction Fitz-Hugh-Curtis syndrome is an extrapelvic manifestation of pelvic inflammatory disease and is characterized by perihepatic adhesions between liver capsule and diaphragm or anterior peritoneal surface [1–3]. Most Fitz-Hugh-Curtis syndrome patients are women of child bearing age and rarely has the syndrome been reported in males. The predominant symptom is pain in the right upper quadrant, which may be confused with biliary disease. Pelvic manifestation of pelvic inflammatory disease (PID) may not be present in male patients. An abdominal computed tomography (CT) scan may reveal subcapsular enhancement of the liver in arterial phase [4]. We herein report a case of Fitz-Hugh-Curtis syndrome in a male patient that was diagnosed via laparoscopy. 2. Case Report A 29-year-old African American male with Russell-Silver Dwarfism presented with one-day history of diarrhea, nausea, vomiting, right side abdominal pain, and abdominal distention. The pain was constant, gradually increasing in severity, and not related to food intake. The patient denied fever and other gastrointestinal or genitourinary complains. His medical history was significant for Russell-Silver Dwarfism, calcium deficiency, cardiomegaly, and bilateral testicular implants for undescended testicles. The patient was sexually active only with his girlfriend and denied any history of sexually transmitted disease. On examination patient was afebrile and his vitals were stable. His abdomen was soft, mildly distended and diffusely tender on right side. There was no abdominal guarding, rigidity, or rebound tenderness. Laboratory workup revealed white blood cell (WBC) count of 14,000/μL with normal liver function tests. Chest and abdominal radiographs appeared normal. CT scan of the abdomen and pelvis showed a small amount of free fluid in pelvis; the proximal appendix appeared normal, however, the distal appendix was not visualized. The liver capsule appeared normal and there was no subcapsular fluid collection as seen in Figure 1. The patient was admitted to the surgical service. He was made nil per os (NPO) and placed on intravenous fluids and pain medication. His symptoms of anorexia, diarrhea, and nausea remained unchanged. His right-sided abdominal pain worsened, while the WBC count normalized. He was refusing any surgical intervention at this point. On hospital day 5, a repeat abdominal CT scan demonstrated a normal appearing liver, small bowel, large bowel, and appendix, with a mild increase in pelvic free fluid. The clinical diagnosis at this point was abdominal pain of unknown etiology. As the patient's symptoms did not improve with conservative management, he ultimately agreed to undergo a diagnostic laparoscopy and was taken to the operating room on hospital day 9. The cecum and the appendix appeared normal. The small bowel was run in a retrograde fashion starting at the cecum, and no stricture, mass, or perforation was noticed. The large bowel also appeared normal. There were extensive adhesions between the liver and anterior abdominal wall, as seen in Figure 2. These adhesions were lysed using the electrocautery and Endo Shears. Cultures were also obtained from the pelvic free fluid. Appendectomy was not performed as per patient's wishes. Following the procedure, the patient reported complete resolution of his symptoms. His diet was gradually advanced, which he tolerated well, and was discharged on postoperative day 2. Pelvic free fluid cultures were negative. 3. Discussion Fitz-Hugh-Curtis syndrome was first described in 1920 by Carlos Stajano. In the 1930s Thomas Fitz-Hugh and Arthur Curtis also described the syndrome and made a connection between right upper quadrant pain following a pelvic infection and violin-string like perihepatic adhesions [5]. The first case of gonococcal perihepatitis in a male was reported by Kimball and Knee in 1970 [6]. The incidence ranges from 4% to 14% in women with PID, but is as high as 27% in adolescents with PID, whose less mature genitourinary tract anatomy makes them more susceptible to infection [1]. There are very few reported cases in male patients. Neisseria gonorrhoeae and Chlamydia trachomatis are thought to be the primary causative agents. There is no reported relation with Russell-Silver Dwarfism. The pathogenesis of Fitz-Hugh-Curtis syndromes is poorly understood. In women, the inflammation of the liver capsule has been attributed to the direct bacterial spread from an infected fallopian tube via the right paracolic gutter. In men, hematogenous and lymphatic spread to liver has been postulated as the underlying mechanism of spread [1, 3]. The predominant symptoms are right upper quadrant pain, tenderness, and pleuritic right-sided chest pain [2]. These symptoms can pose diagnostic challenges as they may be confused with biliary tract symptoms. In a clinical setting, the diagnosis is adequately established by excluding other possible causes of right upper quadrant pain. On laboratory examination, white blood cell count can be elevated in nearly half of the patients, while liver function tests are normal in most patients. Because urethral cultures frequently fail to demonstrate the presence of gonorrhea and Chlamydia, the serologic microimmunofluorescence antibody test is helpful in diagnosis [2]. CT scan may show subcapsular fluid collection, thickening of hepatic capsule in the arterial phase, and wedging enhancement of the involved liver parenchyma in more than 50% of patients. In our patient, CT scan showed a normal hepatic capsule [4]. CT scan is more sensitive than ultrasound in diagnosis of Fitz-Hugh-Curtis syndrome [7]. Definitive diagnosis requires invasive procedures like laparoscopy or laparotomy. Most cases of Fitz-Hugh-Curtis syndrome are managed with antibiotics against Gonorrhea and Chlamydia. In our case if the diagnosis had been made preoperatively, a trial of antibiotics may have been beneficial. If symptoms persist, then surgical lysis of adhesions should be considered. Laparoscopy has both diagnostic and therapeutic benefits. It provides a less invasive therapy than laparotomy. Mechanical lysis of adhesions can provide complete resolution of symptoms [8, 9]. 4. Conclusion Fitz-Hugh-Curtis syndrome is inflammation of liver capsule associated with genital tract infection. It occurs mostly in premenopausal women; however, cases in males have also been reported. Diagnosis is made by clinically eliminating other causes of right upper quadrant pain. Laparoscopy has both diagnostic and therapeutic benefits. Mechanical lysis of adhesions can provide complete resolution of symptoms. Conflict of Interests The authors have no financial or personal interest and no conflict of interests. Authors' Contribution S. Saurabh was responsible for conception and design (Group 1), drafting the paper and critical revision of the paper (Group 2), final approval of the version to be published (Group 3). E. Unger was responsible for acquisition, analysis, and interpretation of data (Group 1); drafting the paper and critical revision of the paper (Group 2); final approval of the version to be published (Group 3). C. Pavlides was responsible for conception and design (Group 1); critical revision of the paper (Group 2); final approval of the version to be published (Group 3). Figure 1 Normal appearing liver capsule and no perihepatic fluid collection. Figure 2 Demonstrates extensive adhesions between liver and anterior abdominal wall (violin-string appearance). ==== Refs 1 Peter NG Clark LR Jaeger JR Fitz-Hugh-Curtis syndrome: a dignosis to consider in women with right upper quadrant pain Cleveland Clinic Journal of Medicine 2004 71 3 233 239 15055246 2 Yang HW Jung SH Han HY Clinical feature of Fitz-Hugh-Curtis syndrome: analysis of 25 cases The Korean Journal of Hepatology 2008 14 2 178 184 18617765 3 Baek HC Bae YS Lee KJ A case of Fitz-Hugh-Curtis syndrome in a male The Korean Journal of Gastroenterology 2010 55 3 203 207 20357533 4 Wang CL Guo XJ Yuan ZD Shi Q Hu XH Fang L Radiologic diagnosis of Fitz-Hugh-Curtis syndrome Chinese Medical Journal 2009 122 6 741 744 19323945 5 Fitz-Hugh T Jr. Acute gonococcic peritonitis of the right upper quadrant in women JAMA 1934 102 2094 2096 6 Kimball MW Knee S Gonococcal perihepatitis in a male. The Fitz-Hugh-Curtis syndrome The New England Journal of Medicine 1970 282 19 1082 1084 4245224 7 Nishie A Yoshimitsu K Irie H Fitz-Hugh-Curtis syndrome: radiologic manifestation Journal of Computer Assisted Tomography 2003 27 5 786 791 14501371 8 Wu HM Lee CL Yen CF Wang CJ Soong YK Laparoscopic diagnosis and management of Fitz-Hugh-Curtis syndrome: report of three cases Chang Gung Medical Journal 2001 24 6 388 392 11512371 9 Owens S Yeko TR Bloy R Maroulis GB Laparoscopic treatment of painful perihepatic adhesions in Fitz-Hugh-Curtis syndrome Obstetrics and Gynecology 1991 78 3, part 2 542 543 1831253
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Case Rep Surg. 2012 Mar 26; 2012:457272
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22606231PONE-D-11-0959410.1371/journal.pone.0035232Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyImmune CellsBiochemistryGeneticsMolecular GeneticsGene RegulationGene ExpressionImmunologyImmune ResponseMedicineObstetrics and GynecologyPregnancyMiscarriage and StillbirthPlacental Expression of CD100, CD72 and CD45 Is Dysregulated in Human Miscarriage CD100, CD72, and CD45 in Human MiscarriageLorenzi Teresa 1 * Turi Angelo 2 Lorenzi Maria 1 Paolinelli Francesca 1 Mancioli Francesca 2 La Sala Lucia 3 Morroni Manrico 1 Ciarmela Pasquapina 1 Mantovani Angelo 4 Tranquilli Andrea Luigi 2 Castellucci Mario 1 * Marzioni Daniela 1 1 Division of Neuroscience and Cell Biology, Department of Experimental and Clinical Medicine, School of Medicine, Università Politecnica delle Marche, Ancona, Italy 2 Unit of Obstetrics and Gynecology at "G. Salesi" Hospital, Department of Clinical Sciences, Università Politecnica delle Marche, Ancona, Italy 3 Department of Clinical and Molecular Sciences, School of Medicine, Università Politecnica delle Marche, Ancona, Italy 4 Unit of Obstetrics and Gynecology, San Severino Marche Hospital, San Severino Marche, Italy Milstone David S. EditorBrigham and Women’s Hospital, United States of America* E-mail: [email protected] (TL); [email protected] (MC)Conceived and designed the experiments: TL AT ML MC DM. Performed the experiments: TL ML FP FM LL DM. Analyzed the data: TL ML FP FM LL DM PC. Contributed reagents/materials/analysis tools: AT MM MC DM. Wrote the paper: TL DM. Collection of samples: AM ALT. Final approval of the version to be published: MM AM ALT MC. 2012 11 5 2012 7 5 e3523226 5 2011 13 3 2012 Lorenzi et al.2012This 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.Context and Objective The etiology of miscarriage is often multifactorial. One major cause, immunological rejection of the fetus, has not been clearly elucidated. Our aim was to establish whether the semaphorin CD100, its natural receptor CD72, and the glycoprotein CD45, implicated in immune mechanisms, are involved in pregnancy loss by examining their placental expression with real-time PCR, immunohistochemistry and western blotting techniques. Patients Placenta tissue from 72 Caucasian women undergoing surgical uterine evacuation due to early spontaneous pregnancy loss between the 8th and 12th week of gestation was divided into four groups based on miscarriage number. Gestational age-matched placentas from 18 healthy women without a history of miscarriage undergoing voluntary pregnancy termination were the control group. Placenta from 6 Caesarean deliveries performed at 38–40 weeks of gestation was also studied. Results CD100, CD72 and CD45 were expressed in placenta and exhibited different mRNA and protein levels in normal pregnancy and miscarriage. In particular, protein levels were highly dysregulated around 10 weeks of gestation in first and second miscarriage placentas. The CD100 soluble form was produced and immediately shed from placental tissue in all samples. Conclusions Fetal CD100, CD72 and CD45 seem to play a role in miscarriage. The present data support the involvement of the fetal immune system in pregnancy maintenance as well as failure. ==== Body Introduction Miscarriage (fetal death before 24 weeks of gestation, w.g.) is a frequent event in human pregnancy. Indeed as many as one in five clinical pregnancies results in miscarriage [1]; recurrent miscarriage (three or more consecutive miscarriages) accounts for ≅ 10% of all cases. The etiology of miscarriage is often multifactorial; established risk factors include parental chromosomal and uterine anatomical abnormalities [2], advanced maternal age [3], a history of miscarriage [4], and infertility [5]. Several behavioral and social risk factors, such as alcohol [6] and caffeine consumption [7], [8] and cigarette smoking [7], have been reported to increase the risk. An additional cause is immunological rejection of the fetus due to disruption of the mechanisms that normally prevent maternal immune system activation by the paternal antigens expressed by the developing fetus [9]. In a normal pregnancy the maternal immune system is not suppressed; on the contrary, it is capable of efficiently recognizing and reacting against foreign antigens of the “fetal transplant” [10]. The goal of the maternal response is to avoid extravillous trophoblast cell over-invasion [11], balancing womb integrity and fetal nutrition [12]. Such balance is realized by the development by maternal leukocytes of tolerance for the antigens expressed in the semi-allogeneic/allogeneic fetal cells. Specific fetal mechanisms also provide for acceptance of the mother’s cells, since some cell surface characteristics are not inherited [13]. Shao L. et al. [14] noted that human placental trophoblasts activate a particular type of T cells that modulate T cell-dependent B cell responses, resulting in efficient suppression of Ig secretion. A further insight into the mechanisms underpinning fetal tolerance to maternal cells is the recent discovery of a mechanism by which the mother trains the fetus’s budding immune system: the mother’s foreign antigens cross the placenta [15], [16] to lodge in fetal lymph nodes, which are populated by T cells as early as 10 w.g. [17], inducing development of antigen-specific Tregs that suppress antimaternal immunity and persist at least until early adulthood [18]. However, the fetal mechanisms that circumvent the maternal immune response in the pregnant uterus are still unclear. Semaphorins are transmembrane proteins implicated in many processes including neural development [19], tumor progression [20], and cardiovascular development [21]. Recently, several roles for semaphorins have been identified in the immune system [22]; 4D (CD100), the first semaphorin to be found and characterized in the immune system, is sometimes cleaved from the cell surface to release a soluble semaphorin [23]. CD100 is constitutively expressed on T cells, where it is up-regulated as a result of T cell activation [24]; it is also expressed, albeit to a lesser extent, on macrophages, B cells, natural killer (NK) cells and neutrophils [24]. CD100 acts on immune cells–such as B cells and dendritic cells (DCs)–through CD72, its main receptor in lymphoid tissue [25], [26]. CD72 is a well-known B cell antigen expressed on the surface of antigen-presenting cells (APCs), mainly B cells and, to a lesser extent, DCs and macrophages [24], [27]. It seems to function as a negative regulator of immune cell responses [25], [26]. CD100 induces tyrosine dephosphorylation of the CD72 cytoplasmic domain, turning off its inhibitory signaling and enhancing immune cell responses [25], [26]. CD100 is physically associated to the transmembrane glycoprotein CD45 (common leukocyte antigen), an association that becomes closer during immune cell activation [28], [29]. In addition, CD45 triggers generation of the soluble form of CD100 which, similarly to other diffusible factors, augments the immune response by acting on remote immune cells [30]. 10.1371/journal.pone.0035232.t001Table 1 Characteristics of the primers used for SYBR green Q-PCR assays. Target gene Primera Primer sequence (5′–3′) Tm (°C)b % GC Ampliconlength (bp) Accession no. Human CD100 hCD100_F hCD100_R GAGAAGCAGCATGAGGTGTA ATGACGGATGTGTAGCTGTG 57,3 57,3 50 50 266 NM_006378 NM_001142287 Human CD72 hCD72_F hCD72_R CTGAGCAACATGGAGAACAG GCATAAGTCCTAGTGCGTTG 57,3 57,3 50 50 323 NM_001782 Human CD45 hCD45_F hCD45_R CTGACATCATCACCTAGCAG TGCTGTAGTCAATCCAGTGG 57,3 57,3 50 50 257 NM_002838 NM_080921 NM_080922 NM_080923 Human SDHA hSDHA_F hSDHA_R AGCATCGAAGAGTCATGCAG TCAATCCGCACCTTGTAGTC 57,3 57,3 50 50 398 NM_004168 a The letters F and R at the end of the primer name indicate forward and reverse orientations, respectively. b Theoretical melting temperature (Tm) calculated using the MWG Oligo Property Scan (MOPS). Given the active role of CD100, CD72 and CD45 in immune response modulation and the growing interest in the immunological causes of spontaneous miscarriage, we investigated for the first time whether these molecules are expressed in placental tissue and whether they could be involved in pregnancy loss. Real-time PCR, immunohistochemistry and quantitative western blotting techniques allowed documenting significant dysregulation in their expression in miscarriage tissue, supporting the potential involvement of the fetal immune system in pregnancy maintenance as well as failure. 10.1371/journal.pone.0035232.t002Table 2 Antibodies used in the study. Antibody Specificity Ab dilutionfor IH‡ µg of Ab/samplefor IP§ Ab dilutionfor WB∥ Reference mAb* MCA1269 Human soluble CD100 1∶50 2 µg 1∶1000 AbD Serotec, Oxford, UK mAb 610670 Human membrane-bound CD100 1∶5 / 1. 500 BD Transduction Laboratories™, Milan, Italy mAb MCA2501 Human CD72 1∶25 2 µg 1∶1000 AbD Serotec, Oxford, UK Rabbit pAb† ab10558 Human CD45 1∶10 / 1∶500 Abcam, Cambridge, UK mAb A5316 Human β-actin / / 1∶5000 Sigma-Aldrich, Milan, Italy mAb M 0814 Human CD68 1∶80 / / DAKO Cytomation, Glostrup, Denmark * mAb, monoclonal antibody; †pAb, polyclonal antibody; ‡IH, immunohistochemistry; §IP, immunoprecipitation; ∥WB, western blotting. Materials and Methods Ethics Statement All patients provided their informed consent to participate in the study, which was approved by the Ethics Committee of Università Politecnica delle Marche. We used placenta and tonsil tissue collected in surgical bins for disposal as rubbish. Since the tissues did not require histopathological examination and the study did not expose subjects to any risk, an oral authorization in lieu of a written consent was obtained from patients and from children’s parents (Department of Clinical Sciences, 8/1 February 2009). A verbal consent form, one per patient, reporting the study summary; the subject’s comprehension and ability to consent; voluntariness (freedom from coercion or undue influence, real or imagined); and the opportunity to ask questions and consider their decision was signed and dated by the operator (T.L.) to document the donor’s verbal authorization and filed in the Department archives. A power analysis was performed to establish how many cases were needed. The methods used to achieve the study aims were documented. A data collection procedure was devised to obtain an anonymous database ensuring blind sample-result correlation and standardization. Sample Types and Tissue Processing Placental tissue was obtained during surgical uterine evacuation from women with early spontaneous pregnancy loss, defined as miscarriage, between the 8th and 12th w.g., the period when the fetal immune system begins to develop. Gestational age (GA) was calculated from the last menstrual period. Patients were evaluated by ultrasonography (US) when they presented with vaginal bleeding. Uterine evacuation was performed within 24 h of US documentation of fetal death. All women undergoing surgical uterine evacuation due to miscarriage at the Unit of Obstetrics and Gynecology, “G. Salesi” Hospital, Ancona, from February 2008 to December 2010, were invited to participate. Exclusion criteria were antiphospholipid syndrome, endocrine disorders, infection, chromosomal aberrations, uterine structure abnormality, cigarette smoking, use of cocaine or alcohol and caffeine overuse. A total of 72 women (mean age 35.7 years, range 28–44) were enrolled and divided into first, second, third and fourth miscarriage; all had had singleton pregnancies. The control group included GA-matched placental tissue from 18 healthy women without a history of miscarriage, who underwent termination of pregnancy in the first trimester for psychological or social reasons. The mean age of this group was 33 years (range 25–40). All these tissue samples were subdivided into 3 w.g. subgroups (8≤ w.g. <9; 9≤ w.g. ≤10; and w.g. >10), each containing 6 specimens. Finally, we studied normal term placenta from 6 Caesarean deliveries performed at 38–40 w.g. The mean age of these subjects was 34 years (range 31–40). All subjects were Caucasian. Tonsils from children undergoing tonsillectomy were used as a positive control [30], [31], [32]. Two randomly selected samples from each placenta and tonsil specimen were washed with saline; one was frozen in liquid nitrogen within 5 min and stored at − 80°C until use for molecular and biochemical analysis, the other was fixed for 24 h in 4% neutral buffered formalin at 4°C and embedded in paraffin for immunohistochemistry. Preparation of cDNA for Real-time PCR Total RNA was extracted from 10 mg of frozen placenta and tonsil using the Total RNA purification kit and then cleaned up and concentrated using the CleanAll RNA/DNA Clean-Up and Concentration kit (both from Norgen, Biotek Corp., Thorold, Ontario, Canada) according to the manufacturer’s instructions. The quality (A260/A280) and quantity (A260) of extracted RNA were tested with a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Celbio, Milan, Italy) and 1 µg of RNA was reverse transcribed using the high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 µl using random primers. Real-time PCR The sequences of the real-time PCR (Q-PCR) primers targeting CD100, CD72 and CD45 genes, comprising known transcript variants (2 variants for CD100 and 4 variants for CD45) are summarized in Table 1. SDHA (succinate dehydrogenase complex subunit A) was used as the housekeeping gene for data normalization, to correct for variations in RNA quality and quantity. Q-PCR was performed in a reaction mixture containing 10 µl of 2X iQ SYBR Green Supermix (Bio-Rad Laboratories, Milan, Italy), 0.1 µM of each primer, 15 ng of sample template and RNase-free sterile water to reach a final volume of 20 µl. Amplification was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories) as follows: i) an initial denaturation step at 95°C for 15 min; ii) 45 cycles, with 1 cycle consisting of denaturation at 95°C for 10 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. Q-PCR assays with CT values >40 were considered negative. For each PCR run, a negative (no template) control was used to test for false-positive results or contamination. The absence of non-specific products or primer dimers was confirmed by the observation of a single melting peak in melting curve analysis. For each Q-PCR assay, genes were run in duplicate and all samples tested in three separate experiments. In addition, the standard curve for each gene was constructed using serial dilutions of the cDNA obtained from the tonsil sample. Since PCR efficiencies were close to 100%, the 2-ΔΔCt (Livak) method was used to compare data from miscarriage and term placentas to normal first trimester placentas (control group). The results were expressed as “fold changes” in relative gene expression compared to the control group. Immunohistochemistry Each paraffin-embedded placenta and tonsil sample was cut into 3 µm serial sections that were then deparaffinized and rehydrated through xylene and a graded series of ethyl alcohol. The first section was stained with hematoxylin-eosin for morphological examination. To inhibit endogenous peroxidase activity, sections were incubated for 30 min with 3% hydrogen peroxide in deionized water. Then, they were washed in 50 mM Tris/HCl, pH 7.6 and pretreated at 98°C in 10 mM sodium citrate, pH 6.0 for 45 min (for membrane-bound and soluble CD100, CD72 and CD45) and for 25 min (for CD68, used as a macrophage marker). To block non-specific background, sections were incubated for 1 h at room temperature (RT) with normal horse serum diluted 1∶75 (for membrane-bound and soluble CD100, CD72 and CD68); or with normal goat serum diluted 1∶75 (for CD45) (Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with the primary antibody (listed in Table 2), overnight at 4°C. In particular, we used two CD100 antibodies, one identifying only the (free) soluble form and another identifying the (membrane-bound) intracytoplasmic portion of CD100, which recognizes both the native and the truncated form. After several washes in 50 mM Tris/HCl, pH 7.6, slides were incubated with biotinylated horse anti mouse antibody (CD100, CD72 and CD68) or biotinylated goat anti rabbit antibody (CD45) diluted 1∶200 for 1 h at RT (both from Vector Laboratories). The peroxidase ABC method (Vector Laboratories) was applied for 1 h at RT using 3′,3′ diaminobenzidine hydrochloride (DAB; Sigma, St Louis, MO, USA) as the chromogen. Sections were counterstained in Mayer’s hematoxylin, dehydrated and mounted with Eukitt solution (Kindler GmbH and Co., Freiburg, Germany). For negative controls, the primary or the secondary antibody was omitted. Further negative controls were performed using non-immune murine or rabbit serum. Preparation of Lysates for Biochemical Analysis Tissue lysates of tonsil and miscarriage, term and first trimester placenta were obtained after complete potter homogenization (Ultra-Turrax T8, IKA®-WERKE, Lille, France) in cold lysis buffer containing 20 mM Tris/HCl, pH 8.0, 1% NP-40, 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,10% glycerol, 5 mM EGTA, 10 mM EDTA, and freshly added protease inhibitors (Protease Inhibitor Cocktail, Sigma, Milan, Italy). Extracts were cleared by centrifugation (20,000×g) and protein concentrations assessed with the Bradford protein assay (Bio-Rad Laboratories) [33]. Samples were then immunoprecipitated for analysis of expression of soluble CD100 and CD72, or directly subjected to western blotting for quantitative determination of membrane-bound CD100 and CD45. Immunoprecipitation Immunoprecipitation was performed using a 50% slurry of washed GammaBind G Sepharose beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in homogenization buffer; 2 µg of specific antibody (see Table 2) was added to 50 µl of the bead suspension on ice, using a microcentrifuge tube for each sample. Incubation was carried out overnight at 4°C under rotary agitation. The beads were washed five times with lysis buffer to remove unbound antibody, then gently resuspended in 50 µl of the same buffer, and finally pipetted into a test tube containing 2 mg of protein extract (the protein concentration of samples was equalized to 2 mg/ml with lysis buffer). The lysate-beads mixture was incubated overnight at 4°C under rotary agitation and subsequently centrifuged at 1,000×g for 2 min to remove the unbound fraction. The beads were then washed three times with 1 ml of homogenization buffer and centrifuged at 13,000×g for 2 min to remove any residual supernatant. Bound proteins were eluted from the beads by incubation with 40 µl glycine 100 mM, pH 2.5, for 30 min, under agitation at RT. Elutes were neutralized with NaOH 0.1 M. Western Blotting Immunoprecipitated proteins (soluble CD100 and CD72) or 100 µg of tissue extract (membrane-bound CD100 and CD45) were denatured in 1X loading denaturing buffer [34]. Samples were boiled for 5 min and fractionated on 10% sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). All blots, except membrane-bound CD100 blots, were incubated with 5% bovine serum albumin (BSA, Sigma) in TBS-T 0.1%; membrane-bound CD100 blots were incubated with 5% non-fat dry milk (Bio-Rad Laboratories) in TBS-T 0.1%; then all blots were incubated overnight with the antibodies listed in Table 2. After washing, blots were treated with appropriate secondary antibodies conjugated to horseradish peroxidase and processed with the ECL-Western blotting detection kit (both from Amersham Italia srl, Milan, Italy) according to the manufacturer’s instructions. The levels of CD100, CD72 and CD45 were assessed in at least three separate experiments per molecule by densitometric analysis using Bio-Rad’s ChemiDoc and Quantity-One software. Relative quantities were expressed as the ratio of the densitometric reading of each protein to the β-actin content of each sample. Statistical Analysis Results were expressed as mean + sd (for gene expression) or mean ± sd (for protein expression). A two-tailed t test (PRISM software, version 4 for Windows: GraphPad Software Inc, San Diego, CA) was used to analyze gene and protein expression data. The significance level was considered as low (p≤0.05), medium (p≤0.01) or high (p≤0.001). Results CD100 Expression Q-PCR analysis was applied to establish whether CD100 is transcribed in placental tissue. As shown in Fig. 1a, CD100 mRNA levels never showed significant differences: differences among the four miscarriage groups and differences between any of the miscarriage groups and first trimester or term placenta were not significant, even when comparisons were made with each of the 3 w.g. subgroups (not shown). In contrast, CD100 transcripts showed highly significant down-regulation in term compared with first trimester placenta. 10.1371/journal.pone.0035232.g001Figure 1 Quantitative RT-PCR of normal pregnancy and miscarriage placenta. Data are expressed as “fold changes” in relative gene expression measured in miscarriage and term placentas compared with control tissue (first trimester placenta). mRNA expression profiles of a) CD100, b) CD72 and c) CD45 show transcript down-regulation in pregnancy at term (p<0.001) and the highest values in second miscarriage placenta (p>0.05). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). Immunostaining for soluble CD100 was negative in all placenta samples (not shown). Membrane-bound CD100 was expressed in a subpopulation of Hofbauer cells [Fig. 2a, 3a], identified using CD68 [Fig. 2b, 3b], although these cells were scarcely detectable in first trimester placenta [Fig. 3a]. The positive control (tonsil) for membrane-bound CD100 and for CD68 yielded a positive result on immunohistochemistry [Fig. 4a, d], confirming the quality of the method used. 10.1371/journal.pone.0035232.g002Figure 2 Tissue from a representative first miscarriage, 9th week of gestation. Paraffin serial sections. A subpopulation of Hofbauer cells residing in the villous stroma, identified by CD68 (panel b,d,f), are positive for CD100 (panel a, red asterisks), CD72 (panel c, red asterisk) and CD45 (panel e, red asterisks). a,b,c,d,e,f,: Bar = 60 µm. 10.1371/journal.pone.0035232.g003Figure 3 Representative first trimester placenta (control). Paraffin serial sections of representative first trimester placenta (9th week of gestation) from a voluntary termination of pregnancy. A subpopulation of Hofbauer cells, identified by CD68 (panel b), are positive for CD100 (panel a, red asterisks). Staining for CD72 (panel c) and CD45 (panel d) is largely negative. a,b,c,d: Bar = 60 µm. 10.1371/journal.pone.0035232.g004Figure 4 Positive and negative control tissue. Tonsil tissue was used as a positive control for CD100 (panel a), CD45 (panel b), CD72 (panel c) and CD68 (panel d). A positive reaction for CD100 (panel a, see inset), CD45 (panel b, see inset) and CD72 (panel c, see inset) was detected in lymphocytes, and a positive reaction for CD68 was seen in macrophages (panel d, see inset). Negative control is shown in panel e. a,b,c,d,e: Bar = 30 µm; insets: Bar = 300 µm. Analysis of western blots failed to disclose soluble CD100 expression in miscarriage, first trimester or term placenta (not shown), whereas membrane-bound CD100 (either the native and the truncated form) was expressed in all samples. The relative abundance of membrane-bound CD100 protein made it possible to analyze its expression by directly loading the protein extracts onto SDS-PAGE, skipping the immunoprecipitation step. Western blot analysis [Fig. 5] revealed that the expression of native CD100 was significantly (p<0.01) lower in first trimester placenta compared with first and second miscarriage and with term placenta [Fig. 5, 6a], and changed with w.g. [Fig. 5, 6b]. When comparisons were made on the basis of w.g., differences were more significant [Fig 5, 6c]. In first trimester placenta and in all miscarriage groups, native CD100 protein expression peaked between the 9th and the 10th week of pregnancy [Fig. 5, 6b]. 10.1371/journal.pone.0035232.g005Figure 5 Native CD100 protein expression in normal pregnancy and in miscarriage. Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein. nCD100: native CD100. 10.1371/journal.pone.0035232.g006Figure 6 Densitometric analysis of native CD100 protein expression relative to β-actin in normal pregnancy and in miscarriage. a) Native CD100 protein expression is significantly (p<0.01) lower in the first trimester of gestation than at term as well as in first trimester vs first and second miscarriage (both p<0.01). Its expression level is highest in first and second miscarriage and decreases subsequently. b) In the first trimester and in all miscarriage groups, native CD100 expression increases from the 8th to the 10th week of gestation; these differences are significant only for third (p<0.01) and fourth (p<0.05) miscarriage. A reduction in native CD100 expression after the 10th week is significant for first trimester and for third miscarriage (both p>0.05). Different levels of native CD100 are seen in all miscarriage groups between the 8≤ w.g. <9 and the w.g. >10 subgroups, but not in first trimester. c) In all weeks of gestation, native CD100 expression is significantly lower in first trimester vs first and second miscarriage (both p<0.001). Before the 9th week of pregnancy, it is significantly greater in second vs third and fourth miscarriage (both p<0.001). Between the 9th and the 10th week, native CD100 expression is significantly lower in first trimester placenta vs third and fourth miscarriage (both p<0.01), and significantly higher in second and third miscarriage vs fourth miscarriage (both p<0.05). After the 10th week, its expression is significantly lower in first trimester vs fourth miscarriage (p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). The truncated form of CD100 was expressed in placenta specimens and shared a very similar expression profile with native CD100 (not shown). Tonsil, used as a positive control, showed a strong signal for membrane-bound CD100 on western blotting [Fig. 5]. CD72 Expression Q-PCR analysis showed very similar expression profiles for CD72 mRNA [Fig. 1b] and protein [Fig. 7, 8a]. However, there were no significant differences in mRNA levels among miscarriage groups or between any of these four groups and first trimester or term placenta. As in the case of CD100, even when comparisons were made with each of the 3 w.g. subgroups, differences were not significant (not shown). In contrast, CD72 transcripts were highly significantly down-regulated in term compared with first trimester placenta [Fig. 1b]. 10.1371/journal.pone.0035232.g007Figure 7 CD72 protein expression in normal pregnancy and in miscarriage. Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein. 10.1371/journal.pone.0035232.g008Figure 8 Densitometric analysis of native CD72 protein expression relative to β-actin in normal pregnancy and in miscarriage. a) CD72 is more abundant in first and second miscarriages and absent in term placenta. Significant differences in CD72 expression are detected only in first trimester vs first miscarriage (p<0.05) and in first vs third miscarriage (p<0.01). b) In first trimester and in first, second and third miscarriage, CD72 expression shows a significant increase from the 8th to the 10th week of gestation and subsequently a significant reduction after the 10th week (both p<0.001). Similar CD72 expression levels are found in first trimester and in first, second and third miscarriage between the 8≤ w.g. <9 and the w.g. >10 subgroups. c) Trend of CD72 expression in first trimester and in the four miscarriage groups at approximately the same gestational week. Before the 9th and after the 10th week, CD72 expression is significantly greater in first miscarriage vs first trimester (p<0.001) and significantly lower in second, third and fourth miscarriage vs first miscarriage (p<0.001). Between the 9th and the 10th week, CD72 expression is significantly lower in first trimester vs first and second miscarriage (both p<0.001) and significantly higher in second miscarriage vs third and fourth miscarriage (both p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). CD72 immunostaining was detected in all four miscarriage groups, in a subpopulation of Hofbauer cells [Fig. 2c], identified using CD68 [Fig. 2d], but not in first trimester placenta [Fig. 3c]. Western blot analysis documented CD72 expression [Fig. 7] in all placenta samples. It was significantly (p<0.05) increased in first miscarriage compared with first trimester placenta, and significantly (p<0.01) decreased in third miscarriage compared with first miscarriage [Fig. 7, 8a]. When comparisons were made on the basis of w.g. [Fig.7, 8b], differences among placental groups were more significant [Fig. 7, 8c]. Interestingly, CD72 expression peaked between the 9th and the 10th w.g. in several sample groups (first trimester placenta and first, second and third miscarriage) [Fig.7, 8b]. Tonsil, used as a positive control, exhibited strong CD72 and CD68 protein expression on immunohistochemistry [Fig. 4b, d] and western blotting [Fig. 7]. CD45 expression Q-PCR analysis showed very similar expression profiles for CD45 mRNA [Fig. 1c] and protein [Fig. 9, 10a] except in term placentas, where transcript levels were considerably lower. The differences in mRNA levels among the four miscarriage groups and differences between any of the miscarriage groups and first trimester or term placenta were not significant, even when comparisons were made with each of the 3 w.g. subgroups (not shown). In contrast, CD45 transcripts were highly significantly down-regulated in term compared with first trimester placenta [Fig. 1c]. 10.1371/journal.pone.0035232.g009Figure 9 CD45 protein expression in normal pregnancy and in miscarriage. Representative immunoblot. MC = miscarriage. 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). β-actin: housekeeping protein. In miscarriage placenta, CD45 immunopositivity was detected in a subpopulation of Hofbauer cells [Fig. 2e] identified by CD68 [Fig. 2f] and in syncytiotrophoblasts, whereas most specimens from normal first trimester pregnancies showed no macrophage reactivity and weak heterogeneous syncytial staining [Fig. 3d]. Since CD45 protein is abundant in placenta, its expression was analyzed by directly loading the protein extracts onto SDS-PAGE, as in the case of CD100. Representative western blots are shown in Fig. 9. CD45 expression was significantly (p<0.01) lower in first trimester than in second miscarriage and term placenta [Fig. 9, 10a]. As in the case of both CD100 and CD72, its expression changed markedly with w.g. [Fig. 9, 10b]. When comparisons were made on the basis of w.g., differences were more significant [Fig 9, 10c]. In first trimester placenta and in all miscarriage groups, CD45 protein expression peaked between the 9th and the 10th week of pregnancy [Fig. 9, 10b]. Tonsil, used as a positive control, revealed strong CD45 and CD68 expression on immunohistochemistry [Fig. 4c, d] and western blotting [Fig. 9]. 10.1371/journal.pone.0035232.g010Figure 10 Densitometric analysis of native CD45 protein expression relative to β-actin in normal pregnancy and in miscarriage. a) CD45 expression is significantly (p<0.01) lower in the first trimester of gestation than at term. Its expression is highest in second miscarriage and declines subsequently. Differences are significant only in first trimester vs second miscarriage (p<0.01). b) In the first trimester and in all miscarriage groups, CD45 expression increases from the 8th to the 10th week; these differences are significant only for first (p<0.05), second (p<0.05) and third (p<0.01) miscarriage. A reduction in CD45 expression after the 10th week is significant for first trimester (p<0.01) and for first (p<0.05), second (p<0.05) and third (p<0.001) miscarriage. Similar CD45 expression levels are found in all miscarriage groups between the 8≤ w.g. <9 and the w.g. >10 subgroups, with the exception of third miscarriage and first trimester, where they are significantly lower in the samples with gestational age >10th week (both p<0.01). c) In all weeks of gestation, CD45 expression is significantly lower in first trimester vs first (p<0.001) and second (p<0.001) miscarriage and in first vs second miscarriage (p<0.05). Before the 9th and after the 10th week, it is significantly greater in second miscarriage vs third and fourth miscarriage (both p<0.001). Between the 9th and the 10th week, CD45 expression is significantly decreased not only in second vs third (p<0.05) and fourth miscarriage (p<0.001), but also in third vs fourth miscarriage (p<0.001). MC = miscarriage. Asterisks indicate the significance level: low (*), medium (**), high (***). 8–9 weeks (8≤ w.g. <9); 9–10 weeks (9≤ w.g. ≤10); >10 weeks (w.g. >10). Discussion To the best of our knowledge, this is the first study investigating the expression of CD100, CD72 and CD45 mRNA and protein in placenta from miscarriage, the commonest complication of pregnancy. The three molecules are known to play a role in the immune system, which has come to be recognized as an important factor in miscarriage. We found similar CD100, CD72 and CD45 mRNA and protein profiles, but different expression levels, in miscarriage compared to normal pregnancy. The western blotting data were confirmed by the immunohistochemical findings. In particular, tissue from the majority of normal first trimester pregnancies showed no CD100 and CD72 staining and weak heterogeneous syncytial staining for CD45, whereas in all miscarriage groups the three molecules were expressed by subpopulations of Hofbauer cells (placental macrophages) residing in the villous placental stroma. Absence of native CD100, which is normally expressed by macrophages, results in reduced macrophage recruitment; therefore, the CD100 found in Hofbauer cells, independently of its effects on the adaptive immune response, could have a role in macrophage recruitment, as described in other organs [35]. We also found that part of the CD100 expressed by Hofbauer cells was cleaved from their surface, giving rise to the soluble form [30]. This form results from phosphorylation of cytoplasmic CD100, which is associated to serine and/or threonine kinase activities [36]. CD100 and CD45 form a functional association in immune cells; their levels increase during immune system activation [28], promoting the maturation and differentiation of these cells [29]. CD45 is endowed with intrinsic protein tyrosine phosphatase (PTPase) activity [37], which is responsible for initial activation of the serine and/or threonine kinase associated to CD100. CD45 was abundant in placental macrophages and in syncytiotrophoblasts from miscarriage tissue and shared a similar expression profile with CD100. This finding corroborates the production of the soluble CD100 form, which is immediately shed from placental tissue, as shown by its absence in placental villi of all samples. Moreover, since soluble CD100 release from the immune cell surface is well regulated and strictly dependent on cell activation [38], this form enhances the immune response by acting on remote immune cells in a similar manner to other diffusible factors, such as cytokines and chemokines. CD100 could therefore be involved in immune response stimulation in miscarriage, supporting the hypothesis of high levels of fetal immune system activity during pregnancy loss [18]. It is thus reasonable to speculate that membrane-bound CD100 might constitute a reservoir of soluble CD100, enabling a stronger immune response [39]. A role for CD45 in fetal immunotolerance may also be hypothesized. The present findings suggest that high CD45 expression could be associated to fetal rejection of maternal cells. Excessive amounts of CD45 protein in placenta tissue probably enhance release of soluble CD100, which fetal mechanisms are unable to break down. Interestingly, soluble CD100 levels in the sera of patients with autoimmune diseases correlate with autoantibody titre, suggesting that its determination could help monitor disease activity [38]. These data lend support to the hypothesis that soluble CD100 is released in maternal blood and acts on maternal cells. Notably, CD45 expression was higher in term compared with first trimester placenta despite exhibiting lower mRNA levels in the former specimens. Increased CD45 protein expression at the end of pregnancy is an index of strong fetal immune system activation; high activation levels may exceed immunotolerance by the fetus, precipitating labor. CD72 immunostaining was also detected in a subpopulation of Hofbauer cells. The molecule functions as a negative regulator, blocking immune cell responses in normal conditions. Immune activation by soluble CD100 turns off its inhibitory signal [26]. Interestingly, CD72 protein was higher in first trimester than in term placentas, as shown by western blot analysis, suggesting its involvement in pregnancy maintenance. The almost overlapping expression patterns of CD72 and CD45 proteins found in all miscarriage groups suggest a link between CD45 over-expression and CD72 up-regulation. The expression of CD100, CD72 and CD45 seemed to be finely regulated, because small gestational week increments induced strong changes. An outstanding question is why their expression was most altered in first and second miscarriage placenta compared with third and fourth miscarriage tissue, particularly around the 10th w.g. It is indeed surprising that their levels increase on this particular w.g., when progesterone production switches from the corpus luteum to the placenta. The fact that progesterone has a role in modulating macrophage activation [40] suggests that it can exert an influence on macrophage expression of CD100, CD72 and CD45 precisely on the 10th week of gestation. Competing Interests: The authors have declared that no competing interests exist. Funding: This study was supported by grants from Università Politecnica delle Marche 2008-2010 to AT, MM, MC, ALT, DM and a grant from Fondazione Cassa di Risparmio di Fabriano e Cupramontana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 García-Enguídanos A Calle ME Valero J Luna S Domínguez-Rojas V 2002 Risk factors in miscarriage: a review. 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Blood 97 3498 3504 11369643 39 Kikutani H Kumanogoh A 2003 Semaphorins in interactions between T cells and antigen-presenting cells. Nat Rev Immunol 3 159 167 Review 12563299 40 Menzies FM Henriquez FL Alexander J Roberts CW 2011 Selective inhibition and augmentation of alternative macrophage activation by progesterone. Immunology 134 281 291 21977998
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PLoS One. 2012 May 11; 7(5):e35232
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22615844PONE-D-11-2257010.1371/journal.pone.0036914Research ArticleBiologyMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesOncogenic SignalingMedicineGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BInfectious diseasesViral diseasesHepatitisHepatitis BOncologyBasic Cancer ResearchTumor PhysiologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaHBsAg Inhibits the Translocation of JTB into Mitochondria in HepG2 Cells and Potentially Plays a Role in HCC Progression HBsAg Inhibits the JTB in HepG2 CellsLiu Yun-Peng 1 Yang Xiao-Ning 1 Jazag Amarsanaa 3 Pan Jin-Shui 1 Hu Tian-Hui 2 Liu Jing-Jing 1 Guleng Bayasi 1 2 * Ren Jian-Lin 1 * 1 Department of Gastroenterology, Zhongshan Hospital affiliated to Xiamen University, Xiamen, Fujian Province, China 2 Medical College of Xiamen University, Xiamen, Fujian Province, China 3 National Institute of Medical Research, 3rd General Hospital, Ulaanbaatar, Mongolia Andre Frederic EditorAix-Marseille University, France* E-mail: [email protected] (BG); [email protected] (JLR)Conceived and designed the experiments: BG JLR. Performed the experiments: YPL XNY JSP JJL. Analyzed the data: THH. Contributed reagents/materials/analysis tools: AJ. Wrote the paper: BG YPL. 2012 15 5 2012 7 5 e3691411 11 2011 10 4 2012 Liu et al.2012This 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 and Aims The expression of the jumping translocation breakpoint (JTB) gene is upregulated in malignant liver tissues; however, JTB is associated with unbalanced translocations in many other types of cancer that suppress JTB expression. No comprehensive analysis on its function in human hepatocellular carcinoma (HCC) has been performed to date. We aimed to define the biological consequences for interaction between JTB and HBsAg in HCC cell lines. Methods We employed the stable transfection to establish small HBsAg expressing HepG2 cell line, and stably silenced the JTB expression using short hairpin RNA in HepG2 cell line. The effects of JTB and small HBsAg in vitro were determined by assessing cell apoptosis and motility. Results Silencing of JTB expression promoted cancer cell motility and reduced cell apoptosis, which was significantly enhanced by HBs expression. Expression of HBsAg inhibited the translocation of JTB to the mitochondria. Furthermore, silencing of the JTB resulted in an increase in the phosphorylation of p65 in HepG2 cells and HepG2-HBs cells, whereas HBsAg expression decreased the phosphorylation of p65. The silencing of JTB in HepG2-HBs cells conferred increased advantages in cell motility and anti-apoptosis. Conclusion HBsAg inhibited the translocation of JTB to the mitochondria and decreased the phosphorylation of p65 through the interaction with JTB, After JTB knockdown, HBsAg exhibited a stronger potential to promote tumor progression. Our data suggested that JTB act as a tumor suppressor gene in regards to HBV infection and its activation might be applied as a therapeutic strategy for in control of HBV related HCC development. ==== Body Introduction Complications of chronic HBV (CHB), including liver failure and hepatocellular carcinoma (HCC), which is one of the greatest risk factors for the development of HCC and the 10th leading cause of mortality worldwide [1], [2], [3]. In the past two decades, a number of strong findings have shown that the X protein (HBX) acts as a transactivation factor and is clearly associated with tumorigenesis [4], [5], [6]. Functions in the carcinogenesis of other proteins, such as HBs, that are encoded by HBV are also related to liver tumor development [7], [8], [9], [10], [11]. HBV encodes three envelope proteins in the pre-S/S open reading frame, which are named the large, middle, and small surface proteins [7]. A number of truncated surface gene mutants with a partially deleted pre-S region have been identified; one of the major mutant types is the deletion of the pre-S2 region (pre-S2D). These pre-S2D mutants have become increasingly prevalent in the serum and liver tissues of patients with chronic HBV infection and HCC [12], [13]. The overexpression of pre-S2D (S2 characterized by the deletion of the pre-S2 region) large surface proteins has been demonstrated in the induction of endoplasmic reticulum (ER) stress [14], oxidative stress, DNA damage [15], COX-2 expression [16], cyclin A expression [17] and the degradation of p27Kip1 [18]. These results suggest that the expression of the HBV large surface protein, especially the pre-S2D mutant, might be important for hepatocarcinogenesis. However, determining putative additional roles for the S protein require further investigation. Current cytogenetic evidence indicates the important role of the 1q21-q22 region in drug resistance [19], tumor metastasis [20] and a shorter duration of patient survival. Therefore, the function of the genes that are located proximal to this region may be associated with the process that accounts for the frequent translocation of the region in many types of tumors. A comparative genomic hybridization analysis of HCC indicates frequent gains of 1 q and an amplicon at 1q21-q22 [20]. Jumping translocation breakpoint (JTB) is a gene that is located on human chromosome 1 at q21 and undergoes an unbalanced translocation. Although JTB expression is suppressed in many cancers of different organs [21], [22], some studies have reported the overexpression of JTB in cases of hepatocellular carcinoma [20]. Therefore, the biological function of JTB remains unclear. Previous studies have raised the possibility that aberrations in the structure or expression of JTB induce neoplastic changes in cells, such as deregulated cell growth and/or death through mitochondrial dysfunction [22]. Our previous studies have suggested that there is an interaction between HBsAg and JTB, such as a recombination event [23], and that JTB may play a critical role in oncogenesis in the liver. However, the role of the interaction between HBs and JTB in liver tumorigenesis remains unknown. In this study, we aimed to investigate the functional changes in HepG2 cells by evaluating the interaction between JTB and HBs. Materials and Methods Cell culture, treatment and transfection HepG2, L-02, HuH-7 and GES cells were grown in DMEM medium, SGC7901 cell was in RPMI1640, and AGS cell was in F12K medium, respectively. All cells were cultured in mediums with 10% fetal calf serum at 37°C in a 5% CO2 humidified atmosphere. The HCC cell lines HepG2, L-02 and HuH-7 were provided by the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. The HCC cells were treated with 0.6 mM H2O2 for 8 h. The HepG2 cells were transfected with 2 μg of the pCMV-HBsAg vector using FuGENE HD (Roche, Indianapolis, IN) in six-well plates. After 48 hours of transfection, the cells were split 1∶10 and selected with 600 μg/mL G418 (Sigma, St Louis, MO) for 2 weeks. The G418-resistant colonies were pooled together, and the HBs-expressing clones were identified by immunoblotting and maintained with 600 μg/mL G418. HepG2 cells were transfected with siJTB (pcPUR+U6-siJTB) or a PU6 (pcPUR+U6-siRenilla) plasmid using the FuGENE HD (Roche, Indianapolis, IN). Puromycin (2.0 μg/ml) was used to screen stably transfected clones. The expression of the JTB protein was examined by Western blotting analysis using an antibody against JTB (these experiments were repeated three times) to validate the efficiency of the constructs to inhibit target gene expression. Stable knockdown of JTB A plasmid containing an RNA interference sequence that targeted HepG2 was constructed as previously described [24], [25], and a line of stable HepG2 knockdown cells were also established as previously described [26], [27]. One pair of the siJTB sequence was Forward primer, 5′- CACCGCGGAAGAGTGTTCTTCATA CGTGTGCTGTCCGTATGGAGAGCACTCTTCTGCTTTTT -3′, and Reverse primer, 5′- GCATAAAAAGCAGAAGAGTGC TCTCCATACGGACAGC ACACGTATGAAGAACACTCTTCCGC -3′. HepG2 cells were transfected with pcPUR+U6-siJTB or pcPUR+U6-siRenilla (control) and selected as puromycin-resistant pools. Quantitative reverse transcription-PCR (qRT-PCR) was performed to confirm the JTB mRNA suppression using the primers 5′- CGGGCTAAAACTACCCCTGAG -3′ and 5′-TGAGCGGCAGCTTTTGAACT-3′. Western blot analysis and co-immunoprecipitation Cells were re-suspended in lysis buffer containing a protease inhibitor cocktail, and the extracted proteins were separated using 10–15% SDS–PAGE gels. β-actin was used as a loading control. Antibodies against β-actin, tubulin, coxIV, bcl-XL, caspase-9, cytoC, MMP-2, PARP, p-p65 (ser536) and p65 were obtained from Cell Signaling Technology; JTB antibody was obtained from Sigma Aldrich. Co-immunoprecipitation was performed using whole-cell lysates and antibodies against JTB and HBsAg. The immunoprecipitation was performed with A/G agarose beads that were coated with anti-JTB or anti-HBs, and the proteins were detected using anti-JTB and anti-HBs antibodies via western blot analysis. Mouse or rabbit immunoglobulin IgGs were used as negative controls. Flow cytometric and apoptosis assays HCC cells were used for the apoptosis assays. The HCC cells were seeded into a 24-well culture dish and treated with 0.6 mM H2O2 for 8 h. The treated cells were washed with PBS three times followed by trypsinization. The harvested cells were stained with the Annexin V-FITC apoptosis assay kit (Kaiji Corp., Nanjing, China) according to the manufacturers' manual. Finally, the cells were assessed for apoptosis ratios using flow cytometry. The experiments were performed in triplicate. RNA extraction, reverse transcription and real-time PCR Briefly, first-strand cDNA was synthesized using the ImProm-II reverse transcription system (Promega, Madison, WI) and was subjected to quantitative PCR using a Light Cycler 480 system (Roche, Indianapolis, IN). The primer sequences were as follows: MMP-2, forward 5′-TGATCTTGACCAGAATACCATCGA- 3′ and reverse 5′-GGCTTGCGAGG GAA G AAGTT- 3′; GAPDH: forward 5′-CAAGGTCATCCATG ACAACTTTG- 3′ and reverse 5′-GTC CACCACCCTGTTGCTGTAG-3′. Bcl-XL: forward 5′- GTGAATGGAGCCACTGCGC A- 3′ and reverse 5′-CCCCATCCCGGAAGAGTTCA- 3′. Transwell invasion and wound healing Transwell invasion assays were performed with 8.0-μm pore inserts in a 24-well transwell plate. For this assay, the HCC cell lines were added to the upper chamber of a Transwell with 0.5 mg/mL collagen type l (BD Bioscience, San Jose, CA)-coated filters for the invasion assay. DMEM with 10% fetal bovine serum and 1% of each antibiotic was added to the lower chamber, and the cells were allowed to incubate for 16 hours. Invading cells were quantified after Gentian violet staining. Each experiment was performed in triplicate, and the data were expressed as mean values. A wound-healing assay was performed in 24-well plates. Tumor cells in medium containing 10% FBS were seeded into 24-well plates (Corning, CA). After the cells grew to confluence, made the wounds using sterile pipette tips, then, cells were washed with PBS and refreshed with medium with 10% FBS. The photographs were token at 0 and 24 h. Isolation of cytosolic and mitochondrial fractions The isolation of cytosolic and mitochondrial fractions was performed using the ApoAlert kit. After H2O2 treatment, the cells were plated in 75-cm2 flasks and harvested by trypsinization. A total of 5×107 cells were centrifuged at 600× g for 5 min at 4°C. The pellet was resuspended in 0.8 ml of ice-cold fractionation BufferMix, incubated on ice for 15 min, and homogenized with a Dounce tissue grinder on ice. The homogenate was subsequently transferred to a microcentrifuge tube and centrifuged at 700× g for 10 min at 4°C. The supernatant was collected and transferred to a fresh microcentrifuge tube and centrifuged at 10,000× g for 25 min at 4°C. The cytosolic fraction was collected, and the pellet, representing the mitochondria-containing fraction, was resuspended in 100 μL of fractionation BufferMix. The protein concentration was determined using the BCA method with bovine serum albumin as standard. The cytochrome c in the cytosolic and mitochondrial fractions was analyzed using western blot analysis. Cell immunofluorescence analysis For cell immunofluorescence, 5×104 HepG2 and HepG2-HBs cells were plated onto coverslips in 24-well plates for 24 hours. The cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 10% BSA for 30 minutes. Next, the cells were incubated at room temperature with or without primary anti-JTB, anti-HBs or anti-coxIV antibodies for 1 hour followed by the respective secondary antibodies Texas Red goat anti-mouse antibodies and fluorescein isothiocyanate (FITC). The nuclei were counterstained with DAPI dye before the slides were mounted with the FluorSave reagent. The slides were observed under an Olympus FV-500 fluorescent microscope. Luciferase reporter gene assays The transcriptional activity of the p65-responsive element was assessed using the p65 reporter assay. Briefly, the p65 and negative control reporter plasmids were separately transfected into HCC cells in replicate wells using Lipofectamine 2000 reagent (Invitrogen). Two days after transfection, the luciferase activity was determined using the dual-luciferase reporter assay system (Promega). Statistical analysis The data were expressed as the mean ± standard error of the mean (SEM). Experimental differences were analyzed using SPSS v11 with the 2-tailed t-test and with P<0.05 defined as the selected level of significance. Results JTB binds HBs, and HBs expression reduces the mitochondrial localization of JTB To explore the expression of JTB in different liver cancer cell lines, we examined the nomal liver cells and several liver cancer cell lines on JTB expression by western blotting. Meanwhile we established HepG2 stable cell line expressing HBsAg and silenced JTB expression using short hairpin RNA stable produced in HepG2 cell line. The JTB expression in the HCC cell lines recapitulated the pattern of expression in the human HCCs. The cell lines expressed high (HepG2 and Huh7 cells), intermediate (L-02 cells), or low-to-undetectable (HepG2-JTBi and HepG2-HBs-JTBi cells) levels of JTB. In addition, gastric cancer SGC7901 and AGS cell lines showed the lower expression level of JTB compared with normal gastric GES cell (Figure 1 A). We have previously shown that the JTB protein binds to the HBV surface antigen (HBsAg). However, we used JTB delivered exogenously by transfection. To further illustrate the relationship between the combination of JTB and HBsAg. Recently, we used the endogenous JTB in HepG2 cells to detect whether a combined effect of the endogenous JTB protein and HBs existed. The cell lysates were subjected to immunoprecipitation with the anti-JTB antibody followed by western blot analysis with anti-JTB and anti-HBs antibodies; another set of cell lysates were also subjected to immunoprecipitation with the anti-HBs antibody followed by western blot analysis with anti-JTB and anti-HBs antibodies. The whole-cell lysate was used as a positive control, and immunoglobulin IgG was used as a negative control. The results showed that the anti-JTB antibody precipitated HBs and that the anti-HBs antibody precipitated JTB (Figure 1B). 10.1371/journal.pone.0036914.g001Figure 1 JTB interaction with HBs in HepG2 cells. (A) The expression levels of JTB in HCC cell lines and normal liver cell, JTB in gastric cancer cells and normal gastric cell were determined using western blot analysis. HBs was detected for stable transfected cells. β-actin was used as a loading control. (B) Immunoprecipitation was performed as described in the Materials and Methods section. JTB and HBs were detected by western blot analysis. The whole-cell lysate was used as a positive control. (C) A western blot showing the distribution of JTB in the cytosolic and mitochondrial subfractions. Tubulin and coxIV were used as cytosolic and mitochondrial markers, respectively. (D) The effects of JTB on the mitochondrial localization and co-localization of JTB and HBs. Cellular localization by the immunocytochemical analysis of JTB and HBs in HepG2 cells using (a, b) FITC-labeled anti-JTB (green) and Texas red-labeled anti-coxIV (red) antibodies, (d, e) FITC-labeled anti-JTB (green) and Texas red-labeled anti-HBs (red) antibodies, (g, h) and FITC-labeled anti-JTB FITC (green) and Texas red-labeled anti-coxIV (red) antibodies under confocal microscopy. To label the nuclei, 4′,6-diamidino-2-phenylindole (DAPI) was used (blue). Bars, 10 μm. Furthermore, we applied immunofluorescence confocal technology to prove the combination of HBsAg and JTB. We determined the localization of JTB in HBs-transfected HepG2 cells using western blot analysis (Figure 1C) and immunocytochemistry (Figure 1D). JTB was localized to the mitochondria (Figure 1D a, b and c), and HBs co-localized with JTB (Figure 1D d, e and f). The expression of HBs decreased the translocation of JTB to the mitochondria and caused the cytoplasmic accumulation of JTB, which inhibited the co-localization of JTB with the mitochondria marker cox IV (Figure 1D g, h and i). The change of JTB cellular localization could make effect on cell function. In the next experiments, we will explore the cell function involved apoptosis and motility after the change of cellular localization. JTB knockdown suppresses H2O2-induced apoptosis by increasing Bcl-XL The exposure of cells to H2O2 induces apoptosis through pathways that involve the disruption of normal mitochondrial function. One key mechanism involves the opening of the mitochondrial permeability transition pores (PTPs) that allow the passage of some small pro-apoptotic molecules, such as cytochrome c into the cytoplasm [28], [29]. Previous studies have reported that the opening of PTPs increases when the mitochondrial membrane potential decreases [30]. To explore the role of JTB and interaction with HBsAg on cell apoptosis, we used H2O2 to induce oxidative stress which cause the cell apoptosis. The response to H2O2 -induced apoptosis in different liver cancer cell lines was examined by Flow cytometric and apoptosis assays. As shown in Figure 2A and B, the Annexin V signal significantly decreased in cells knocked down for JTB compared to cells transfected with the control pU6-sh plasmid. HBs expression enhanced the effect of decreasing the number of Annexin V-positive cells in the population of HepG2 cells. Our results show that JTB and HBs act cooperatively to reduce apoptosis. HBs expression was expected to reduce the sensitivity of a cell to H2O2-induced apoptosis after JTB knockdown, and HBs did not exhibit anti-apoptotic effects in the presence of JTB. Knockdown of JTB induced Bcl-XL upregulation(Figure 2C and D). Cytochrome c accumulated in the cytosol of vector-transfected cells and was markedly abundant compared to that found in the cytosol of cells knocked down for JTB (Figure 2E). In the cytoplasm, cytochrome c binds to APAF-1 to form an apoptosome, which activates caspase 9 (Figure 2F). H2O2-induced cell apoptosis was reduced in the cells with JTB knockdown, and the disruption of the mitochondrial transmembrane potential (MTP) and cytochrome c leakage were reduced. The anti-apoptotic gene Bcl-XL was also highly expressed. In the hepatoma cells, JTB expression protected the cells against H2O2 injury. This effect may be associated with the preservation of mitochondria. Upregulation of Bcl-XL plays a crucial confrontation to apoptosis. JTB regulate the Bcl-XL expression through some unknown pathway. 10.1371/journal.pone.0036914.g002Figure 2 Stable knockdown of JTB in HCC cells decreases cell apoptosis. HepG2 cells were treated with 0.6 mM H2O2 for 8 h. (A) The percentage of apoptotic cells in HepG2 cells. (B) Quantification of apoptosis cells in different HepG2 cells. The percentage of apoptotic cells was determined using cyto-fluorimetric analysis with Annexin-V and PI labeling. The data were obtained from three analyses (*P<0.05). (C and D) The level of the Bcl-XL mRNA in HepG2 cells as measured using qRT-PCR. The results represent the mean of three amplifications and were calculated using the 2 exp(−△△Ct) formula. β-actin mRNA in HepG2 cells was used as a control (*P<0.05). (E) Western blot analysis of the cytosolic and mitochondrial cytochrome c levels in HepG2 cells induced by H2O2 after 8h. (F) Uncleaved (p47) and activated caspase-9 (p37) were detected by western blot analysis in HepG2 cells induced by H2O2 after 8 h. Silencing of JTB increases cell motility, which is enhanced by HBs To further explore the biological function of JTB, We determined the effects of HBs and JTB silencing on cell invasion and wound healing (Figure 3A–D). As expected, JTB silencing significantly promoted both of these processes, whereas HBs did not induce migration or invasion in HepG2 cells. To show the effect of JTB on HBs, we stably silenced JTB using its shRNA in the HepG2-HBs cells, which strongly express JTB. Invasion assays were performed to determine the effect of JTB silencing on cancer cell motility, and the cell numbers were counted (Figure 3C) and recorded (Figure 3A) using a microscope. As shown in Figure 3A and B, JTB knockdown increased cell migration and invasion, which was significantly enhanced by HBs expression. Furthermore, to explore the molecular mechanisms in cell migration and invasion, we examined whether JTB regulates cell motility through the MMP-2 protein and mRNA expression. We found that the level of MMP-2 expression was significantly enhanced in HBs+JTBi cells in comparison to pU6-JTBi cells (Figure 3E–F). These results suggest that JTB could regulate the expression of MMP2 and affect the cell motility, while the presence of HBsAg play has an important biological synergy function with JTB. 10.1371/journal.pone.0036914.g003Figure 3 Transient knock-down of JTB promotes migration and invasion in the HCC cells expressing high levels of JTB. (A) Images and graphs (C, *P<0.05) from invasion assay at 16 h; (B) Images and graphs (D, *P<0.05) from migration assay the migrating ability of control cells at 24 h was considered as 1 in (D); The MMP-2 protein expression was detected using western blot analysis (E), and the mRNA expression was quantified using qRT-PCR (F). Silencing of JTB increases p65 activity, and HBs enhances this effection Summarize the previous results, JTB and HBsAg have new biological functions on cell apoptosis and motility. We initially explore the possible associated signal transduction pathway. We examined the effect of JTB and HBs on NF-κB activation. Canonical NF-κB activation was assessed using phospho-immunoblot analysis (Figure 4A) and luciferase activity assay (Figure 4B). Immunoblot analysis for phospho-p65 revealed an increased phosphorylation of p65 in pU6-JTBi and HBs+JTBi cells, although the level of phospho-p65 was significantly increased in HBs+JTBi cells compared to that in pU6-JTBi cells. The same trends were demonstrated in luciferase activity assay. These results show the biological activity of HBs in HCC progression. The presence of JTB masked the function of HBs. In addition, we are trying to find more upstream signaling molecules. We used the protein chip technique to detect changes in the major protein kinase activities in different HCC cell lines, and the results of this experiment revealed that several protein kinases, such as AKT, ERK, RSK1, GSK-3β, showed dramatic changes in their level of phosphorylation (Figure 4C). Thus, we detected the phosphorylation level of three protein kinases using western blot analysis (Figure 4D), and the results were consistent with those obtained with the protein chip technique. 10.1371/journal.pone.0036914.g004Figure 4 Activity of p65 and other protein kinases in HCC cell lines. The transcriptional activity of p65 was measured indirectly using luciferase reporter assays and normalized against TK-renilla. (A) Immunoblot analysis for phospho-p65 and total p65 protein expression. Increased phosphorylation of p65 was detected in both pU6-JTBi and HBs+JTBi cells, whereas HBs inhibited p65 phosphorylation. (B) Transcriptional activity of the p65 promoter in HepG2 cells using the luciferase activity assay was consistent with the level of p65 phosphorylation. The data were expressed as the mean of three experiments and as a percentage of the p65 promoter activity compared to that in the HepG2-vector cells (100%). (C) The results of the Human Phospho-MAPK Array Kit (R&D Systems, Inc.) In addition, we determined the relative levels of phosphorylated mitogen-activated protein kinases (MAPKs) and other serine/threonine kinases. (D) The phosphorylation levels of AKT, ERK and GSK-3β were detected by western blot analysis. Discussion The hepatitis B surface antigen (HBsAg) was the Nobel Prize discovery that identified the hepatitis B virus (HBV) approximately 40 years ago. Currently, HBsAg remains the hallmark of overt HBV infection [3]. Hildt et al. found that the Pre-S2 members of the regulatory protein family, including L-HBs and truncated M-HBs, are transcriptional activators that trigger the activation of tumor promoters [31]. The small (S) protein is expressed at the highest levels and is predominant in virions and subviral particles. However, the role of S-HBs in HBV-induced oncogenesis is not well understood. Therefore, the present study was designed to verify the function of HBs via the interaction of HBs with JTB. Our previous study had shown that the viral envelope protein HBs interacts with JTB. In addition, S-HBs and L-HBs functionally bind JTB, which presumably indicates that the binding site may be located in the S region of viral HBs [23]. JTB is upregulated in liver cancer cells [20], and previous research has demonstrated that JTB is downregulated in other cancer tissues [22]. It has been shown that the elevation of Sp1 protein expression is a critical factor in tumor development and growth and metastasis, and it is the promoter of JTB contains a sp1-binding site [21]. Therefore, JTB expression may be elevated in human tumors. In the current study, the overexpression of JTB reduced neoplastic changes in cells, such as regulated cell death via mitochondrial dysfunction. We also found that overexpression of JTB lead to apoptosis in normal liver L-02 cell line induced by H2O2, JTB can increase the activity of caspase9 and promote the cleavage of PARP (Figure S1 and data S1). When the JTB gene undergoes an unbalanced translocation, its expression is suppressed; therefore, we concluded that JTB is a tumor suppressor gene. The jumping translocation of the JTB gene eventually leads to the development of tumors to promote tumor differentiation and staging. When we stably transfected the HepG2 cells with HBs, the JTB protein interacted with HBs and inhibited the translocation of JTB to the mitochondria. The interaction between JTB and its specific binding protein, HBs, mediates several cellular functions. Our data indicate the possibility that blocking JTB may increase the metastatic potential and reduce the apoptotic potential of cancer cells. However, transfection of HBs alone into HepG2 cells did not perturb apoptosis or metastasis. Surprisingly, blocking JTB in pCMV-HBs cells seems to enhance anti-apoptotic and metastastic activities compared to that in HepG2 cells. These results suggest that HBs has a biological activity that is JTB-independent. We showed that JTB inhibited the phosphorylation and reduced the biological activity of p65; thus, we speculate that the presence of HBs may sequester JTB in the cytoplasm and decrease the phosphorylation of p65. Blocking or knockdown of JTB may promote the HBs-mediated phosphorylation of p65. NF-kB plays a well-known function in the regulation of immune responses and inflammation, but growing evidences support a major role in oncogenesis. In vitro, neutralizing the interaction of JTB and HBs significantly impaired the metastasis of liver cancer cells and cell apoptosis. This result suggests that JTB is important for the function of HBs in HCC cells and indicates the possibility that the strengthening of JTB/HBs interactions may be a strategy to prevent cancer cell metastasis and antiapoptosis. JTB expression reduces tumor metastasis and antiapoptosis by decreasing p65 activity. p65 regulates the expression of genes involved in many processes that play a key role in the development and progression of cancer such as proliferation, migration and apoptosis. The Bcl-XL protein plays an important role in maintaining the intergrity of mitochondrial thereby inhibiting the release of factors which activate the pro-apoptotic caspase cascade [32]. MMP-2 is known to play a crucial role in tumor invasion via its ability to degrade basement membrane collagens. It plays important roles in cancer development and aggression. p65 directly activates expression of the apoptosis inhibitor Bcl-XL [33] and matrix-modifying enzyme MMP-2 [34]. The activation of HBs following JTB knockdown may protect cell from apoptosis and trigger the degradation of extracellular matrix substrates. To investigate the mechanism of regulation in p65 activity, we used protein chip technology to detect changes in signaling molecules that are associated with the MAPK pathways. We determined that HBs may phosphorylate AKT and ERK for activation. However, the pCMV-HBs cells were not protected from apoptosis and did not exhibit increased migration. We speculate that HBs activity was inhibited due to JTB. GSK-3 beta is phosphorylated in vitro at serine 9 by RSK1, resulting in its inhibition [35]. The activation of RSK1, which is involved in regulating cell survival and proliferation, lays at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases [36]. HBs activated ERK signal pathways. But HBs may decrease p65 activity following the translocation of JTB into the cytoplasm. After JTB knockdown, HBs exhibited a stronger potential to promote tumor progression. We observed that there is a lower level of GSK-3βphosphorylation and higher level of p65 phosphorylation in HBs+JTBi cells. It has been reported that GSK-3β can phosphorylate the C-terminal domain (residues354–551) of p65 in vitro [37]. However, the role of the interaction between JTB and HBs in GSK-3βphosphorylation is unclear. This issue will be our research focus in the future. JTB protein and HBs play different roles in human HCC. HBs may act as an oncogene that is integrated into the human genome, whereas JTB is presumably a tumor suppressor gene. Therefore, therapeutic approaches that are aimed at eliminating HBs and/or reactivating JTB might be highly useful in the treatment of human liver cancer. Supporting Information Figure S1 Caspase9 activity and western blot analysis in L-02 cells induced by H2O2. (TIF) Click here for additional data file. Data S1 Supplementary data. (DOC) Click here for additional data file. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by National Natural Science Foundation of China (No. 30971362 and 81072013); Fundamental Research Funds for the Central Universities in China (No. 2010111082); Key Projects for Technology Plan of Fujian Province in China (No. 2009D020); and Foundations of Health Bureau of Fujian and Xiamen in China (No. 2007CXB8 & No. 3502z20077046). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 World Health Organization 2000 Hepatitis B Fact Sheet NO 204 2 Liu J Fan D 2007 Hepatitis B in China. Lancet 369 1582 1583 17499584 3 Lok AS McMahon BJ 2007 Chronic hepatitis B. Hepatology 45 507 539 4 Wang WH Hullinger RL Andrisani OM 2008 Hepatitis B virus X protein via the p38MAPK pathway induces E2F1 release and ATR kinase activation mediating p53 apoptosis. 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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2262922110.1100/2012/897867Research ArticleProduction of Gymnemic Acid Depends on Medium, Explants, PGRs, Color Lights, Temperature, Photoperiod, and Sucrose Sources in Batch Culture of Gymnema sylvestre Ahmed A. Bakrudeen Ali 1, 2 *Rao A. S. 3 Rao M. V. 2 Taha Rosna Mat 1 1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia2Department of Plant Science, Bharathidasan University, Tamil Nadu, Tiruchirappalli 620 024, India3Department of Biotechnology, Bharathidasan University, Tamil Nadu, Tiruchirappalli 620 024, India*A. Bakrudeen Ali Ahmed: [email protected] Editor: Béla Tóthmérész 2012 2 5 2012 2012 89786727 10 2011 8 12 2011 Copyright © 2012 A. Bakrudeen Ali Ahmed et al.2012This 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. Gymnema sylvestre (R.Br.) is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA). The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L) and KN (0.5 mg/L). Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w) was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w). Maximum GA production (58.28 mg/g d.w) was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors. ==== Body 1. Introduction In vitro techniques are very useful in ensuring sustainable, optimized sources of plant-derived natural products. However, ex situ cultivation should be preceded by proper evaluation of the plants for their ability to produce the required bioactive constituents before commencing cultivation or introducing the technology to potential growers. The ability of plants to produce certain bioactive substances is largely influenced by the physical and chemical environments in which they grow. Plants also produce certain chemicals to overcome abiotic stresses [1]. Plants use light not only as an energy source for photosynthesis but also as an important environmental signal. Plants can detect almost all facets of light such as direction, duration, quantity, and wavelength by using three classes of photoreceptors: the red/far-red (600–750 nm) absorbing phytochromes, and the blue/UV-A (320–500 nm) absorbing cryptochromes. These photoreceptors perceive, interpret, and transducer light signals, via distinct intracellular signaling pathways, to mediate a broad range of physiological responses to light in addition to cell growth and development [2]. Light can affect morphogenesis and the formation of plant metabolites as a signal and stress factor from phytohormones. In most plant cell cultures, secondary metabolism, including the production of phenolic terpenoid and alkaloid compounds, is stimulated by light [3]. On the other hand, light had an inhibitory effect on the accumulation of secondary metabolites in the case of nicotine and shikonin [4]. The literature shows that the majority of pharmacologically important compounds of plant origin are products of defense and secondary metabolism [1, 5]. This ability of plants to respond to physical and/or chemical stimuli can be used for elicitation of pharmacologically active substances by subjecting an intact plant to stress factor(s) [1]. Growing a plant outside its natural environment under ideal conditions may therefore, result in it being unable to produce the desired bioactive substance, hence the need for prior evaluation. Gymnema sylvestre is an important medicinal climber belonging to the family Asclepiadaceae. This climber is extensively used in almost all the Indian systems of medicine as a remedy for rheumatism, cough, ulcer, and pain in the eyes. It is also useful for inflammations, dyspepsia, constipation, jaundice, and so forth. The roots of this plant have been reported as a remedy for snakebite [6]. The plants occur mainly in the Deccan peninsula of western India, Tropical Africa, Vietnam, Malaysia, SriLanka and is widely available in Japan, Germany, and the USA as a health food [7]. The extract of G. sylvestre plays a major role in blood glucose homeostasis through increased serum insulin level through regeneration of the endocrine pancreas [8]. We have reported that leaf and callus extracts of G. sylvestre maintained the blood glucose and lipid profiles in alloxan-induced diabetic Wistar rats [9]. Several research studies in the early 2000s described G. sylvestre callus culture for the production of GA. Physical stress was established in which MS medium supplemented with PGRs was used for callus growth, and modifying the shaking speed, pH and medium play an important role in the production of GA [10]. Furthermore, Lee et al. (2006) tried the addition of sucrose, inoculum density, and aeration factors suitable for GA production by using bioreactors [11]. Veerashree et al. (2012) reported that yeast extract, pectin, and chitin elicitor enhanced the GA via cell suspension culture [12]. We have recently reported the G. sylvestre in vivo and in vitro callus extracts regenerated the damaged pancreatic β cells in alloxan-induced diabetic Wistar rats [13]. The present study highlights the evaluation of GA content in: (i) callus derived from different plant parts of G. sylvestre, namely leaves, stem and petioles; (ii) in vitro elicitation of GA employing physical-chemical factors of light, temperature, photoperiod, and sucrose concentration; (iii) isolation and characterization of GA from callus, namely, HPTLC and HPLC. 2. Materials and Methods 2.1. Chemicals Standard GA was gifted by Professor Kazuko Yoshikawa, Department of Pharmaceutical Science, Kyoto Pharmaceutical University, Japan. Medium salt bases and adenine sulphate were purchased from Sigma Chemical Co., USA. Other medium components and phytohormones were of tissue culture grade or equivalent. All solvents used for chromatographic purposes were HPLC grade. Other solvents were reagent grade or equivalent. 2.2. Plant Material and Sterilization G. sylvestre was collected from the Pachamalai hills (attitude: 1000–1200 m), Tamil Nadu, India, and maintained in the Department of Plant Science garden of the Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Healthy, young leaf, stems (shoot internodal segment without buds), and petiole explants (Figure 1(a)) were washed thoroughly in running tap water 3–5 times, including 2% (v/v) Teepol (Reckitt Benckiser, India) for 10 min, then washed with 70% ethanol for 30 sec followed by another wash with 0.1% HgCl2 for 2 min. Prior to inoculation, the explants were washed three times with distilled water. 2.3. Callus Induction and Culture Conditions Leaf, stem, and petiole explants of G. sylvestre were grown in MS medium [14], SH medium [15], B5 medium [16], and WPM [17] (woody plant medium) supplemented with 0.5–5.0 mg/L of IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), NAA (1-naphthaleneacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid); 0.2–2.0 mg/L of BA (6-benzylaminopurine) and KN (6-Furfurylaminopurine) were used for callus induction. The callus culture was maintained at 25 ± 2°C, 16/8 h (light/dark) of photoperiod with 25 μmol m−2 s−1 of light intensity. The pH of the medium was adjusted to 5.7–5.8 and gelled with 0.8% agar (w/v) (Bacteriological grade, Hi-media, India). Sucrose 30 g/L (Hi-media, India) served as the carbon source. The culture medium was sterilized by autoclaving at 1.06 kg cm−1 and 121°C for 20 min. The role of media on the nature and biomass of callus was studied in leaf, stem, and petiole explants of G. sylvestre. 2.4. Measurement of Callus Growth For all callus growth measurement experiments, 40 mg fresh weight of leaf (in vivo) was inoculated on to 20 mL of the fresh agar MS solid medium in the culture tube (25 × 150 mm), and the biomass gain was monitored at 10 day intervals over a 0–55-days cultured cycle. By treating with various combinations of auxins and cytokinins, fresh and dry weights of the calli were determined at 0–15, 15–25, 25–35, 35–45, and 45–55 days. At regular intervals for all treatments, each callus was harvested by careful separation from the media using metal spatulas, and fresh and dry weight was promptly recorded. A minimum of 3 replicates were run for all the treatments, and the experiments were repeated thrice. 2.5. Physical-Chemical Stress Condition For improvement of GA, G. sylvestre callus cells, 15–25-day-old cells, were placed in the different physical stress of lights (blue, red, green, and white fluorescent tube, Phillips, India); temperature (20°C, 25 ± 2°C, 30°C, and 35°C); photoperiod (4 h/20 h, 8 h/16 h, 12 h/12 h, and 20 h/4 h light/dark) and chemical stress of sucrose (2%, 4%, 5%, and 6%), respectively. OPGRs [MS + 2,4-D (1.5 mg/L) + KN (0.5 mg/L)], 25 ± 2°C, 16 h/8 h (light/dark), 3% sucrose kept under white fluorescent tube used as control. All treatment callus biomass was determined using growth curve analysis, in all the physical-chemical treatments. After treatment, callus cells were harvested at time intervals, washed twice with 100 mL water on a porous glass funnel with filter paper (Whatman No-1), then frozen in liquid nitrogen, and stored in the deep freezer for further investigation and analysis. 2.6. GA Extraction and Phytochemical Analyses The extraction, sample preparation, and chromatographic analyses (HPTLC and HPLC) were performed. Briefly, in vivo leaf and in vitro callus (500 mg d.w) were extracted with methanol 5 times as described by Rehman et al. [18]. The collected methanol extract was centrifuged at 5000 × g for 10 min at room temperature, and then the methanol supernatant was carefully pipetted out into fresh eppendorf tubes without disturbing the inter-phase residues. Green-color methanol supernatant (4 mL) was evaporated and dried. The residue (ca. 6 mg) was dissolved in MeOH (5.0 mL) and 20 μL injected on HPTLC and HPLC with standard GA. HPTLC system (Camag, Switzerland) assisted with sample applicator Linomat IV for quantification of GA. 10 samples were applied on each plate at a start line 8 mm from the bottom, including nine lanes of in vitro callus and in vivo leaves with standard GA (20 μL). The mobile phase of isopropyl alcohol : chloroform : methanol : acetic acid (5 : 3: 1 : 0.5; v/v/v/v) was allowed to run up to 80 mm for separation of GA at a wavelength of 200 nm by the use of TLC scanner III, integration and quantification was performed using CAT 4.0 software. Methanol extracts of in vivo leaf and in vitro callus were further analyzed via HPLC (Shimadzu, Kyoto, Japan). This system consisted of a two 510 Pumps, a 7725 Rheodyne auto injector, a DUG-12 A degasser, SCL-10Avp system controller, C18 (ODS) reverse-phase column (150 mm × 4.6 mm i.d., 5 μM particle size), and a Spectromonitor 486 variable wavelength UV/VIS detector. The analog detector output was acquired and digitized by an Advanced Computer Interface and then processed by AI-450 Chromatography Automation Software (Dionex Corp., Sunnyvale, CA, USA). The flow rate used was 1.0 mL/min, and GA was detected by UV absorption at 230 nm with a mobile phase of 0.1% acetic acid, 35% water, and 65% methanol (HPLC grade). Each injection volume was 20 μL. For quantification of GA, the respective retention time (R T) and peak area were calculated. 2.7. Method Validation 2.7.1. Linearity In this study, each calibration curve was analysed three to times with three to four different concentrations using the same HPLC condition as described above. The calibration graphs were plotted based on linear regression analysis on the integrated peak areas (y) versus concentrations (x). The regression equation was calculated in the form of y = ax + b, where y and x were the values of peak area and sample amount, respectively. The standard solution was diluted with methanol to provide appropriate concentrations. The limit of detection (LOD) was defined as the lowest concentration level resulting in a peak area of three times the baseline. The limit of quantification (LOQ) was defined as the lowest concentration level resulting in a peak area of ten times the baseline. 2.7.2. Precision The precision test was evaluated by the intra-day and inter-day variability. Three different concentration solutions (low, medium and high) of the standards were prepared in methanol. Three replicates of the samples at each concentration were evaluated on the same day for intra-day precision, whilst repeated analysis at each concentration of the samples three times per day over three consecutive days for inter-day precision. The quantity of each analyte was obtained from corresponding calibration curve. The relative standard deviation (R.S.D) was taken as a measure of precision. 2.7.3. Recovery In order to check the accuracy of the developed method, the recovery experiments were carried out as follows: three different quantities (low, medium and high) of the authentic standards were spiked into the samples in form of solution. The quantity of each analyte was subsequently obtained from the corresponding calibration curve. 2.8. Statistical Analysis All the experiments were repeated thrice and 30 replicates were used. The effect of different treatments was quantified as mean ± SE and the data were subjected to statistical analysis using Duncan's multiple range test (DMRT) at 5% level significance [19]. 3. Results and Discussion 3.1. Influence of PGRS, Medium and Explants on Callus Induction In all the media tested, the callus initiation did not occur without PGRs (control) in leaf explants of G. sylvestre. The suitable callus induction was obtained in 2,4-D and NAA on MS, B5, SH and WPM medium which induced GCC (green compact callus), GFC (green friable callus), WFC (white friable callus), WWC (white watery callus) and BFC (brown friable callus) in leaf, petiole and stem explants. For successful callus induction, factors such as type of explants, PGRs, culture media and cultural conditions are very important [20]. MS medium callus induction, proliferation and biomass (fresh and dry weight) was better than B5, SH and WPM medium (Figures 1(b)–1(e); 2(a)–2(h)). The leaf explants produced GCC with higher induction frequency (94.5%) than petiole (79.1%) and shoot tip explants (72%) cultured on MS medium (Figures 1(f)–1(h); 3(a)–3(d)). Among the various concentrations of auxins tried in callus induction, 2,4-D (1.5 mg/L; 93.5%) and NAA (1.0 mg/L; 86.8%) significantly induced GC natured callus with a maximum biomass at 35–45 days in terms of fresh and dry weight (Figures 2(a)–2(d)). In our study, callus biomass progressively increased with an increase in the 1.5 mg/L of 2,4-D (GCC; DW-121 mg/L; Figure 1(i)) and 1.0 mg/L of NAA (GCC; DW-115 mg/L; Figure 1(j)) concentration (>0.5–1.5 mg/L) especially when the culture contains NAA concentration of 2.5–5.0 mg/L, WFC and WWC were induced significantly. In addition, it was observed that 1.5 mg/L of IBA (DW-87 mg/L; Figure 1(k)) and 2.0 mg/L of IAA (DW-63 mg/L; Figure 1(l)) and drastically reduced the callus biomass resulting in WFC natured calli all the media. These results are well authenticated with previous reports of Rani et al. (2010), who demonstrated that NAA and 2,4-D were suitable for the callus induction of G. sylvestre [21]. Successful biomass was obtained by the use of 2,4-D (1.5 mg/L) with BA (0.5 mg/L) (DW-113 mg/L; Figure 1(m)) and 2,4-D (1.5 mg/L) and KN (0.5 mg/L) (DW-144 mg/L; Figure 1(n)), which increased GC nature at 35–45 days. However, 2,4-D + BA and 2,4-D + KN (<3.0–5.0 mg/L) combinations drastically reduced the callus biomass and showed the GFC, BFC, and WFC (data not shown). Auxins and cytokinins regulate plant cell division, which influenced the different phases of the growth cycle and regulates the signalling pathway [22]. In addition, various combinations of NAA with BA, and KN were tried for callus biomass, which resulted in less biomass and GC nature than 2,4-D with BA, and KN (data not shown). During the callus biomass, the batch callus culture was continuously examined by taking a subculture at weekly intervals to prevent cell death and browning of media. 3.2. Measurement of Callus Growth Curve G. sylvestre callus growth curve was sigmoid, and four growth phases can be distinguished in the MS medium supplemented with OPGRs [2,4-D (1.5 mg/L) + KN (0.5 mg/L)] at different days (0–15, 15–25, 25–35, 35–45, and 45–55 days). In the lag phase (0–15 days; DW-49 mg/L), in vitro callus was slowed at the initial stage; the callus biomass was drastically reduced over the other phase, and the GA content was absent (data not shown). In the lag phase (15–25 days; DW-92 mg/L), callus initiation and proliferation were observed by profound cell division [23]. At 25–35 days (exponential phase), biomass (DW-105 mg/L) of the GC was significantly increased. The MS medium supplemented with OPGRs induced the high level of callus biomass in the stationary phase (35–45 days; DW-144 mg/L; Figure 1(o)) of the callus growth curve suggests the cellular membrane stabilization. It has been previously reported that the stationary phase callus evidently demonstrated an increase in the accumulation of gagaminine in the callus (GC) of Cynanchum wilfordii [24]. At the decline phase (45–55 days; DW-119 mg/L), the callus biomass was drastically reduced as compared to other phases. 3.3. Influence of Color Light on Callus Induction Light is an important physical factor, which influences growth, development, and the formation of primary and secondary metabolites [25]. In the present study, color light was also one of the most important factors in inducing and maintaining GC nature of the cells on G. sylvestre callus (Figure 4(d)). The analysis of G. sylvestre callus growth in batch culture under blue light conditions showed that GC callus yielded higher biomass (DW-172 mg/L) at the stationary phase of 35–45 days of culture (Figure 4(a)). In general, the optimal flux of blue light for leaf explants is about 10–15% of the total photosynthetically active radiation. Moreover, the higher flux of blue light is essential for normal carbohydrate metabolism, photosynthetic assimilating, and transport from leaves. Cryptochrome wave length (450 nm), was closed to the blue light, and in this condition more Pr transformed into Pfr for phenylethanoid glycoside production [26]. Figure 4(b) callus growth on red light was, however, extremely slow, and growth curve values of only 31 mg/L, 59 mg/L, 85 mg/L, 122 mg/L and 102 mg/L of dry weight could be registered after 15, 25, 35, 45, and 55 days of the growth. In comparison, MS medium + OPGRs with green light supported maximum GA accumulation in callus harvested on 35–45 days of incubation, respectively, (DW 116 mg/L; Figure 4(c)). 3.4. Influence of Temperature on Callus Induction Temperature has many effects on the mechanisms of metabolic regulation, permeability, nutritional needs, and the rate of intracellular reactions in plant cell cultures [27]. 30°C increased the callus biomass and decreased the GA content (2.9 mg/g d.w) than the control (25 ± 2°C) in 35–45 days of stationary phase. Thus, changing the culture temperature may change the physiology and metabolism of cultured cells and subsequently affect callus growth and secondary metabolite production. The callus culture exposed to the lower temperature (20 ± 2°C) showed decrease in callus biomass (DW-45 mg/L) and GA content (0.03 mg/g d.w) (Figure 4(e)). MS medium supplemented with OPGRs kept under 30°C induced GFC (Figure 4(f)) and biomass (DW-150 mg/L) in G. sylvestre. However, each plant species may favour a different temperature. For strawberry callus culture, maximum anthocyanin content was obtained at 15°C and it was about 13 fold higher than that obtained at 35°C [27]. The cultures incubated at 35 ± 2°C had lower callus biomass (DW-125 mg/L) and GA (1.4 mg/g d.w) (Figure 4(g)), respectively. For Asclepiadaceae species, callus biomass increased at 31°C, more than at 25°C, hence gagaminine content was higher at 25°C in Cynanchum wilfordii [24]. 3.5. Influence of Sucrose on Callus Induction GA productivity at the growth stage (35–45 day) was also assessed at seven concentrations of sucrose (1–6% w/v) under continuous illumination. Data showed that the OPGRs biomass increase in selected callus line was highest at 5% level of sucrose after 35–45 day of culture (DW-164 mg/L; GA content 33.3 mg/g d.w; Figure 4(h)). Kintzios et al. (2003) reported that MS medium supplemented with auxins and cytokinins in combination to treatment of 5% sucrose induced the rosmarinic acid in Ocimum basilicum [28]. Cultures grown on MS medium with 1.0% sucrose indicated a further drastic fall in biomass in all the growth phases (data not shown). GA production, on the other hand, was found to be positively correlated with increasing sucrose concentration in the medium up to 3.0% after 45 days (control) and GA content (12.77 mg/g d.w). We have found that GA content depends on the callus biomass in sucrose treatment. The lowest GA content was recorded in tissue cultured on 2.0% (biomass DW-89 mg/L; GA-5.4 mg/g d.w; Figure 4(i)), 4.0% (biomass DW-152 mg/L; GA-17.6 mg/g d.w; Figure 4(j)), 6.0% (biomass DW-108 mg/L; GA-24.1 mg/g d.w; Figure 4(k)) at 35–45 days of stationary phase, respectively. When data on biomass gain and GA contents were extrapolated in terms of net GA yield, it was found that cultures grown in presence of 1.0–6.0% sucrose supplementation had almost different levels of GA until day 55 of the culture cycle. However, if the culture cycle was extended to 65 days, then cultures on medium with 7-8% sucrose were lesser yielders of the GA and callus biomass (data not shown). 3.6. Influence of Photoperiod on Callus Induction Influence of photoperiod on callus growth and GA accumulation was also studied under seven sets of lights and dark regimes: (a) 4 h light/20-h dark, (b) 8 h light/16 h dark, (c) 12 h light/12 h dark, (d) 16 h light/8 h dark (control), (e) 20 h light/4 h dark, (f) 24 h light, (g) 24 h dark. A time course study at 10 day intervals under the 12 h light/12 h dark photoperiod conditions indicated that selected OPGRs callus attained maximum biomass gain (DW-159 mg/L; GA-26.27 mg/g d.w; Figure 4(l)) on the 35–45th day of culture under continuous light conditions. Zhang et al. (1995) succeeded in 12 h light/12 h dark photoperiod with MS medium increased the triterpenoids of harringtonine, homoharringtonine, and isoharringtonine via batch culture in Cephalotaxus fortunei [29]. Rapid biomass gain in these cultures became evident between 15th and 45th day, followed by slight decline around the 55th day of the culture cycle. In the present study, less callus biomass was produced in 4-h light/20-h dark (DW-59 mg/L; Figure 4(m)), 24-h light (DW-35 mg/L; Figure 4(n)), and 24-h dark (DW-45 mg/L; Figure 4(o)), when compared to 8-h light/16-h dark (DW-132 mg/L; Figure 4(p)). Incubation in complete darkness resulted in poor callus growth during the initial 15–55 days of the culture cycle. 3.7. Screening and Quantification of Gymnemic Acid For TLC separation, the intact leaf and callus extracts reaction mixtures were applied to the plates after concentrating. IAA and IBA induced callus extracts that did not show the brown band in TLC and HPTLC analysis, when sprayed with the vanillin sulphuric acid reagent. PGRs (combination of auxins and cytokinins) methanol extracts showed brown bands and the Rf value close to the standard GA. When the auxins and cytokinins concentrations were increased; the plant cells induced the brown friable, white friable, and white compact callus extracts Rf values were slightly higher than the green compact callus (data not shown). The developed TLC plates were scanned for several times with the same parameters. The concentrates (x 10) callus extract samples were analyzed in one run, this method proves to be very sensitive, relatively fast, inexpensive, and suitable for therapeutic drug monitoring and pharmacokinetic studies [30]. The chromatography developing time was shorter in HPTLC (6 min) than in TLC (40 min) of the mobile phase of Isopropyl alcohol: chloroform: methanol: acetic acid (5 : 3 : 1 : 0.5; v/v/v/v). The gifted GA purity was confirmed in the leaf and callus extracts by recording the absorption spectra at the start, middle, and end of the peak. Standard GA had shown the single peak at different time intervals of the experiment. The intact leaf and callus extracts sample curve was linear; the correlation coefficients had good linearity between concentration and area, it could be helpful to calculate the GA amount in the respectable sample. Green friable callus was induced in MS medium supplemented with NAA (1.0 mg/L) and 2,4-D (1.5 mg/L), and the GA content was drastically reduced with the combination of auxins and cytokinins. When, NAA and 2,4-D are combined with cytokinins, the callus extracts increased the GA content. This controversial MS medium with OPGRs only has produced the maximum biomass and GA compared to the combinations of auxins and cytokinins in 35–45 days of the stationary phase. GA was significantly increased in the MS medium combined with auxins and cytokinins where concentrations derived from leaf explants of G. sylvestre were determined in HPTLC [31]. For the HPLC analysis, leaf and callus methanol extracts (20 μL) were uploaded in the HPLC system to quantify GA under retention time (5 min) with help of UV spectrophotometer where peak area data was compared with standard GA. Secondary metabolites were increased in callus culture of G. sylvestre [12, 13, 21, 32]. Maximum GA production was observed in MS medium supplemented with OPGRs under blue light-induced 4.4-fold as compared with white fluorescent light and out of which 2.8-fold is found in intact leaves determined by HPLC analysis. We have recently published a paper of pharmacological activities, a phytochemical investigation and in vitro studies of G. sylvestre [33]. In the HPLC mobile phase [0.1% acetic acid; water/methanol (v/v) (35 : 65, HPLC grade)], purity was analyzed, without sample and with standard GA (Figure 5(a)). Standard GA stability and impurity were characterized through single peak at initial, middle, and end of the HPLC experiment. Imoto et al. (1991) reported that GA content was confirmed through HPLC in methanol leaf extracts of G. sylvestre [34]. Figure 5(b) describes the GA content of in intact leaf explants (19.52 mg/g d.w), which was increased compared to Figure 5(c) of in vitro callus culture of MS medium supplemented with OPGRS (12.22 mg/g d.w). Many authors had isolated and identified GA earlier in leaf explants of methanol extracts. In 1989, Yoshikawa and coworkers isolated GAs from a hot water extract of G. sylvestre, which they named GA I, II, III, IV, V, VI, and VII, respectively, and evaluated using HPLC [35, 36]. For GA enhancement, OPGRs culture was kept under physical-chemical stress conditions determined by growth curve analysis. Blue light with OPGRs induced the maximum GA (53.94 mg/g d.w) (Figure 5(d)) rather than 5% sucrose treatment (33.39 mg/g d.w). However, with other physical stress conditions, the GA was reduced in this order 12 h photoperiod (26.27 mg/g d.w), red light (8.90 mg/g d.w), green light (5.72 mg/g d.w), and 30°C (2.9 mg/g d.w). In case of dark treatment, GA content was absent. We have reported, that in vitro callus of G. sylvestre significantly increased the pancreatic β-cells and maintained the body weight, pancreas weight, liver weight and liver glycogen level in alloxan-induced diabetic Wistar rats [13]. 3.8. Linearity, Precision, Recovery, and Robustness in HPLC As per the ICH guidelines, the method validation parameters checked were linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. The linearity of the method was determined at three concentrations (10–30 ug/mL) of GA. 20 ug/mL GA results show that an excellent correlation exists between response factor and concentration of GA within the concentration range indicated above. The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 80, 100, and 120%, and the percentage recovery was calculated. Our studies recovery was within the range of 100 ± 2% which indicates accuracy of the method. The precision of the method was demonstrated by interday and intraday variation studies. In the intraday studies, 3 repeated injections of standard and sample solution were made in a day and the response factor of GA peaks and percentage were calculated. In the interday variation studies, 3 repeated injections of standard and sample solutions were made on 3 consecutive days, and response factor of GA peak and percentage were measured. Intra- and interday accuracy were established from quality control standards by evaluating nominal and mean measured concentrations of quality control standards which were compared and expressed as % difference (diff %). Diff % was calculated using the formula: Diff % = [(mean measured concentration − nominal concentration)/nominal concentration] × 100. Wave length (200 nm) GA compound was studied showing that a sufficient absorption and an overloading of the column can be avoided. Adding 0.2% acetic acid gave a rather good separation of GA. In order to shorten the analytical time and improve the sensitivity and peak shape of GA a gradient, characterized by an decreased amount of acetic acid (0.1%), was applied before the elution of GA. However, GA is eluted isocratically in order to guarantee robustness. In conclusion, the present research has provided new information about in vitro secondary metabolites, especially the effects of light, temperature, sucrose, and photoperiod. Enhancement of bioactive compounds through different physical and chemical factors has been achieved at all levels. (i) Our study on batch culture of leaf explants of G. sylvestre has shown that both the biomass and GA accumulation were influenced by the OPGRs with blue light stress. (ii) Growth curve analysis, GA production and biomass were higher in the stationary phase of all treatments at 35–45 days. Although precise mechanism as to how these factors affect GA remains to be determined, a combination of these factors used for production of valuable compounds via in vitro abiotic stresses in the future is a promising strategy. In addition, a simple, reliable, and accurate HPLC assay method of simultaneous determination of GA from G. sylvestre was successfully established. The above results will be useful in designing systems for the large-scale cultivation for the production of GA. Acknowledgments The authors are thankful to Professor Kazuko Yoshikawa, Kyoto Pharmaceutical University, Kyoto, Japan, for providing gymnemic acid standard; Mr. S. Govindu, Technician, Central Electrochemical Research Institute, Karaikudi, India, for carrying out the HPLC analysis; Dr. Manimaran, Lecturer, JSS College of Pharmacy, Ooty, India for the help rendered during HPTLC analysis. Figure 1 Effect of culture media, explants, and PGRs on callus induction in Gymnema sylvestre after 45 days. (a) Habit; (b) B5 medium (1.2x); (c) SH medium (1.3x); (d) MS medium (1.3x); (e) WPM medium (1.4x); (f) petiole (1.2x); (g) stem (1.3x); (h) leaf explants (1.2x); (i) 2,4-D treatment (2.1x); (j) NAA treatment (1.5x); (k) IBA treatment (1.5x); (l) IAA treatment (2.5x); (m) 2,4-D + BA (1.5x); (n) 2,4-D + KN (1.5x); (o) mass callus culture. Figure 2 Effect of different media (B5, MS, SH, and WPM) supplemented with 2,4-D and NAA on induction of biomass (fresh and dry weight) on leaf explants after 45 days of incubation. Figure 3 Effect of MS medium supplemented with auxins and petiole, leaf, stem explants role on callus induction (%) of Gymnema sylvestre after 45th day of incubation. Figure 4 MS medium supplemented with OPGRs, physical and chemical treatments on leaf explants of Gymnema sylvestre induced callus after 45 days incubation. (a) Blue light (1.8x); (b) red light (1.8x); (c) green light (1.6x); (d) color light setup; (e) 30°C (1.7x); (f) 20°C (1.8x); (g) 35°C (1.6x); (h) 5% (1.2x); (i) 2% (1.4x); (j) 4% (1.4x); (k) 6% (1.2x); (l) 12 h/12 h (light/dark, 1.9x); (m) 4 h/20 h (light/dark, 1.7x); (n) 24 h light (1.5x); (o) 24 h dark (1.8x); (p) 8 h/16 h (light/dark, 1.6x). Figure 5 (a) Standard gymnemic acid; (b) in vivo leaf; (c) OPGRs MS + 2,4-D (1.5 mg/L) with KN (0.5 mg/L); (d) blue light treatment with OPGRs. ==== Refs 1 Kuzel S Vydra J Triska J Vrchotova N Hruby M Cigler P Elicitation of pharmacologically active substances in an intact medical plant Journal of Agricultural and Food Chemistry 2009 57 17 7907 7911 19663425 2 Wang HY Deng XW Phytochrome signaling mechanism The Arabidopsis Book 2002 American Society of Plant Biologists 1 35 3 Weisshaar B Jenkinst GI Phenylpropanoid biosynthesis and its regulation Current Opinion in Plant Biology 1998 1 3 251 257 10066590 4 Tabata M Mizukami H Hiraoka N Konoshima M Pigment formation in callus cultures of Lithospermum erythrorhizon Phytochemistry 1974 13 6 927 932 5 Andrew RL Wallis IR Harwood CE Henson M Foley WJ Heritable variation in the foliar secondary metabolite sideroxylonal in Eucalyptus confers cross-resistance to herbivores Oecologia 2007 153 4 891 901 17593399 6 Nadkarni KM Indian Materia Medica 1993 1 Bombay, India Popular Prakashan 7 Ye WC Zhang Q Liu X Che C Zhao S Oleanane saponins from Gymnema sylvestre Phytochemistry 2000 53 8 893 899 10820799 8 Shanmugasundaram ERB Leela KG Radha KS Rajendran VM Possible regeneration of the islets of Langerhans in streptozotocin-diabetic rats given Gymnema sylvestre leaf extracts Journal of Ethnopharmacology 1990 30 3 265 279 2259215 9 Ahmed ABA Rao AS Rao MV Role of in vivo leaf and in vitro callus of Gymnema sylvestre in maintaining the normal levels of blood glucose and lipid profile in diabetic Wistar rats Biomedicine 2008 28 2 134 138 10 Devi CS Murugesh S Srinivasan VM Gymnemic acid production in suspension cell cultures of Gymnema sylvestre Journal of Applied Sciences 2006 6 10 2263 2268 11 Lee EJ Mobin M Hahn EJ Peak KY Effects of sucrose, inoculum density, auxins, and aeration volume on cell growth of Gymnema sylvestre Journal of Plant Biology 2006 49 6 427 431 12 Veerashree V Anuradha CM Kumar V Elicitor-enhanced production of gymnemic acid in cell suspension cultures of Gymnema sylvestre R. Br Plant Cell, Tissue and Organ Culture 2012 108 1 27 35 13 Ahmed ABA Rao AS Rao MV In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate β -cells regeneration and anti-diabetic activity in Wistar rats Phytomedicine 2010 17 13 1033 1039 20537514 14 Murashige T Skoog F A revised medium for rapid growth and bioassays with tobacco tissue culture Plant Physiology 1962 15 473 497 15 Schenk RV Hildebrandt AC Medium and techniques for induction of growth of monocotyledonous and dicotyledonous plant cell cultures Canadian Journal of Botany 1972 50 166 204 16 Gamborg OL Miller RA Ojima K Nutrient requirements of suspension cultures of soybean root cells Experimental Cell Research 1968 50 1 151 158 5650857 17 Lloyd G Mc Cown B Commercially feasible micropropagation of Mountain Laurel, Kalmia latifolia by use of shoot tip culture Proceedings of the International Plant Propagator's Society 1981 30 421 427 18 Rehman RU Israr M Srivastava PS Bansal KC Abdin MZ In vitro regeneration of witloof chicory (Cichorium intybus L.) from leaf explants and accumulation of esculin In Vitro Cellular and Developmental Biology 2003 39 2 142 146 19 Gomez KA Gomez AA Statistical Procedures for Agricultural Research with Emphasis on Rice 1976 Los Banos, Laguna, Philippines International Rice Research Institute 20 Yeoman MM Yeoman CL Manipulating secondary metabolism in cultured plant cells New Phytologist 1996 134 4 553 569 21 Rani MSA Chandrasekaran S Vijayakumar M In-vitro production of secondary metabolites in Gymnema sylvestre Indian Journal of Horticulture 2010 67 1 85 89 22 Coenen C Lomax TL The diageotropica gene differentially affects auxin and cytokinin responses throughout development in tomato Plant Physiology 1998 117 1 63 72 9576775 23 Leticia CA Paiva PDO Paiva R Garciano HP Growth curve and biochemical analyses of callus of IPE-BRANCO (Tabebuia roseo alba .) Naturalia 2010 33 45 56 24 Shin G Chio M Lee D Comparative study of the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture of Cynanchum wilfordii Biological and Pharmaceutical Bulletin 2003 26 9 1321 1325 12951479 25 Rout GR Samantaray S Das P In vitro manipulation and propagation of medicinal plants Biotechnology Advances 2000 18 2 91 120 14538112 26 Ouyang J Wang X Zhao B Wang Y Light intensity and spectral quality influencing the callus growth of Cistanche deserticola and biosynthesis of phenylethanoid glycosides Plant Science 2003 165 3 657 661 27 Zhang W Seki M Furusaki S Effect of temperature and its shift on growth and anthocyanin production in suspension cultures of strawberry cells Plant Science 1997 127 2 207 214 28 Kintzios S Makri O Panagiotopoulos E Scapeti M In vitro rosmarinic acid accumulation in sweet basil (Ocimum basilicum L.) Biotechnology Letters 2003 25 5 405 408 12882562 29 Zhang W Bai XF Bu Z Wang J Yu X Yuan Q Enhanced production of harringtonine and homoharringtonine in Cephalotaxus fortunei callus culture by periodic temperature oscillation Biotechnology Letters 1995 20 1 63 66 30 Marston A Millard M Hostettmann K The role of TLC in the investigations of medicinal plants of Africa, South America and other tropical regions GIT Laboratory Journal 1997 1 36 39 31 Raju VSR Kannababu S Subbaraju GV Standardisation of Gymnema sylvestre R.Br. by high-performance thin-layer chromatography: an improved method Phytochemical Analysis 2006 17 3 192 196 16749427 32 Kanetkar PV Singhal RS Laddha KS Kamat MY Extraction and quantification of gymnemic acids through gymnemagenin from callus cultures of Gymnema sylverstre Phytochemical Analysis 2006 17 6 409 413 17144249 33 Ahmed ABA Komalavalli N Muthukumar M Pharmacological activities, phytochemical investigations and in vitro studies of Gymnema sylvestre R.Br.—a historical review Comprehensive Bioactive Natural Products Vol 1 Potential and Challenges 2009 75 99 34 Imoto T Yamamoto F Miyasaka A Hatano H High-performance liquid chromatography-atmospheric pressure ionization mass spectrometry of gymnemic acids Journal of Chromatography 1991 557 1-2 383 389 35 Yoshikawa K Amimoto K Arihara S Matsuura K Structure studies of new antisweet constituents from Gymnema sylvestre Tetrahedron Letters 1989 30 9 1103 1106 36 Sugihara Y Nojima H Matsuda H Murakami T Yoshikawa M Kimura I Antihyperglycemic effects of gymnemic acid IV, a compound derived from Gymnema sylvestre leaves in streptozotocin-diabetic mice Journal of Asian Natural Products Research 2000 2 4 321 327 11249615
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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22615843PONE-D-11-2074610.1371/journal.pone.0036909Research ArticleBiologyMicrobiologyVirologyViruses and CancerMolecular Cell BiologyCell GrowthMedicineInfectious DiseasesSexually Transmitted DiseasesHuman Papillomavirus InfectionViral DiseasesHuman Papillomavirus InfectionOncologyCancer Risk FactorsViral and Bacterial Causes of CancerCancer TreatmentCytokine TherapyInterferon-β Induces Cellular Senescence in Cutaneous Human Papilloma Virus-Transformed Human Keratinocytes by Affecting p53 Transactivating Activity IFNβ-Induced Senescence and p53 ActivityChiantore Maria V. 1 Vannucchi Serena 1 Accardi Rosita 2 Tommasino Massimo 2 Percario Zulema A. 3 Vaccari Gabriele 4 Affabris Elisabetta 3 Fiorucci Gianna 1 5 Romeo Giovanna 1 6 * 1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy 2 Infections and Cancer Biology Group, International Agency for Research on Cancer-WHO, Lyon, France 3 Department of Biology, University of Rome 3, Rome, Italy 4 Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy 5 Institute of Molecular Biology and Pathology, CNR, Rome, Italy 6 Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University, Rome, Italy Fusco Alfredo EditorConsiglio Nazionale delle Ricerche (CNR), Italy* E-mail: [email protected] and designed the experiments: MVC GF GR. Performed the experiments: MVC SV ZAP RA. Analyzed the data: MVC SV ZAP GV EA GF GR. Contributed reagents/materials/analysis tools: RA MT. Wrote the paper: MVC GF GR. 2012 16 5 2012 7 5 e3690919 10 2011 10 4 2012 Chiantore et al.2012This 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.Interferon (IFN)-β inhibits cell proliferation and affects cell cycle in keratinocytes transformed by both mucosal high risk Human Papilloma Virus (HPV) and cutaneous HPV E6 and E7 proteins. In particular, upon longer IFN-β treatments, cutaneous HPV38 expressing cells undergo senescence. IFN-β appears to induce senescence by upregulating the expression of the tumor suppressor PML, a well known IFN-induced gene. Indeed, experiments in gene silencing via specific siRNAs have shown that PML is essential in the execution of the senescence programme and that both p53 and p21 pathways are involved. IFN-β treatment leads to a modulation of p53 phosphorylation and acetylation status and a reduction in the expression of the p53 dominant negative ΔNp73. These effects allow the recovery of p53 transactivating activity of target genes involved in the control of cell proliferation. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. This additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears useful in aiding further promising development of biomolecular strategies in the IFN therapy of cancer. ==== Body Introduction The group of cellular proteins known as Interferons (IFNs) appeared to be expressed in infected cells as an early response to viral infection but, in addition to their antiviral activity, IFNs also have a profound effect on cell growth [1]. IFN-α2 was the first human protein shown to be effective for cancer treatment and the first economically viable clinical product developed from recombinant DNA technology in cancer therapy. Antitumor activity of IFNs is probably exerted through direct and indirect mechanisms. It is conceivable that numerous direct effects play a central role in the overall antitumor response, such as down-regulation of oncogene expression, induction of differentiation, inhibition of cell cycle progression and induction of tumor suppressor genes, and programmed cell death [2]. However, additional understanding of IFN antitumor action and mechanisms influencing tumor responsiveness or resistance appears necessary to aid further promising development of biomolecular strategies in IFN therapy of cancer. The most extensively studied anticancer treatment-induced mechanism is apoptotic programmed cell death. Nevertheless, the correlation between the induction of apoptosis and drug response cannot explain the overall tumor cell sensitivity [3]. Numerous recent studies have shown that in cells where apoptosis is blocked, non-apoptotic cell death or irreversible cell growth arrest, namely senescence, can be activated as potential tumor-suppressor mechanisms [4]. The concept of senescence is applied in general to the irreversible proliferative arrest of cells caused by various stresses [5] including oxidative damage, telomere dysfunction, and DNA damage. One particularly relevant source of stress in tumor cells is derived from the aberrant proliferative signals of oncogenes which may trigger senescence through a process known as oncogene-induced senescence, functioning as a potential tumor suppressor mechanism. Senescent cells are identified in vitro by distinctive morphological changes, such as large cell size, flat vacuolated morphology, the inability to synthesize DNA, the formation of domains of heterochromatin called Senescence-Associated Heterochromatin Foci (SAHF) and the expression of a Senescence-Associated β-galactosidase activity (SA-β-gal) [6], [7]. One of the earliest steps in the senescence programme is the translocation of histone chaperone HIRA (Histone Repression factor A) into promyelocytic leukemia (PML) nuclear bodies (NBs), but the role played by HIRA localization into PML bodies has not yet been identified. PML bodies are nuclear structures known to serve as sites of protein modification and the assembly of macromolecular regulatory complexes, and have been extensively implicated in the induction of senescence and apoptosis [8], [9]. Consistent with its role in tumor suppression, the critical senescence pathways converge on the two major tumor suppressor genes p53 and pRb, whose mutations or inactivation are most common in all cancers [10]. Abrogation of senescence can be achieved by SV40 large T, a combination of HPV oncoproteins E6 and E7, E1A and MDM2 coexpression or small interfering RNA against pRb and p53 [11]. Tumors initiated by loss of p53 can be eliminated by senescence induced by p53 restoration, tumor regression being achieved through an innate immune response that leads to the clearance of senescent cells [12], [13]. Cellular senescence results in altered gene expression including IFNs and their related genes [14]. PML is known to be regulated by the interferon pathway via the STAT transcription factors [15]. IFN also regulates many other components of PML nuclear bodies, suggesting that in conjunction they mediate the antiviral and antiproliferative activities of this cytokine [9]. Cellular senescence is induced in human fibroblasts by prolonged IFN-β treatment through DNA damage signaling and a p53-dependent pathway [16]. IFN-α also induces replicative senescence in endothelial cells after continuous stimulation [17]. Taken together, these studies suggest that signaling through the IFN pathway might play an important role in cellular senescence. Human Papilloma Viruses (HPVs) are small DNA viruses involved in the development of both benign and malignant lesions localised in different anatomic districts, that are able to replicate exclusively in the stratified squamous cutaneous and mucosal epithelium. More than 100 different HPV types have been isolated so far, and they can be sub-grouped into cutaneous or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts [18]. To date, the causative association between mucosal high risk-HPV and cervical carcinoma has been clearly demonstrated. It is well known that the expression of E6 and E7 viral oncoproteins is a common feature of cervical cancer cells and is strongly implicated in the process of cancer development, E6 and E7 principally targeting and inhibiting p53 and pRb tumor suppressor proteins, respectively [19]. Emerging lines of evidence support the involvement of the cutaneous HPV types belonging to the beta genus in non melanoma skin cancer (NMSC). However, although the role of beta HPV types in NMSC in Epidermodysplasia verruciformis (EV) patients is well accepted, their involvement in skin carcinogenesis in the general population is not entirely proven. The transforming properties of the majority of cutaneous HPV types have been poorly investigated. Tommasino and co-workers [20] have shown that HPV38 E7 appears to act similarly to HPV16 E7, by binding to pRB and promoting its degradation via the proteasome pathway. On the contrary, HPV38 E6 oncoprotein differs from mucosal high risk HPV E6 proteins in the mechanism by which it counteracts p53 activity. The expression of HPV38 E6 and E7 in human keratinocytes induces the stabilization of p53, which in turn selectively activates transcription of ΔNp73, a p53 inhibitor [21]. High ΔNp73 levels have been found in a number of human malignancies including cancers of the breast, prostate, liver, lung and thyroid [22]. Recently, it has been shown that IFN-α reduces ΔNp73 levels in Huh7 hepatoma cells, and this effect correlates to increased susceptibility to IFN-α triggered apoptosis [23]. Here, we show that prolonged treatment with IFN-β induces senescence in cutaneous HPV38-transformed keratinocytes. PML is essential in IFN-β induction of senescence in HPV38-transformed keratinocytes, and both p53 and p21 pathways contribute to the execution of the phenomenon. p53 colocalyzes with IFN-β-induced PML into PML Nuclear Bodies. By recruitment of p53 into NBs, IFN-β can modulate p53 post-translational modification at specific phosphorylation and acetylation sites and downregulate ΔNp73 expression, leading to the recovery of p53 transactivating activity of selected target genes involved in cell proliferation control. Results IFN-β Inhibits Cell Proliferation of K38 and K16 Cells Keratinocytes expressing E6 and E7 proteins of HPV-38 and HPV-16, referred to as K38 and K16 respectively, were obtained as described [20]. To test whether IFN-β could affect proliferation of both transformed keratinocytes, K-38 and K-16 cells were treated with IFN-β for several time points and the amounts of viable cells were revealed. As shown in Fig. 1A, proliferation of both cell lines was strongly affected by IFN-β. 10.1371/journal.pone.0036909.g001Figure 1 IFN-β affects cell proliferation in K16 and K38 cells. (A) IFN-β inhibits cell proliferation in K16 and K38 cells. Cells were seeded in triplicate at 105 cells per 35 mm dish and, after 24 h, IFN-β was added to the cultures for the indicated times. IFN-β treated and control cells were counted in a hemocytometer and viability was evaluated by trypan blue exclusion. Data represent means ± s.d. of three independent experiments. ** = p<0.01; *** = p<0.001. (B) IFN-β treatment differently affects cell cycle progression of K16 and K38 cells. K16 and K38 cells were treated with IFN-β for the indicated time points. DNA staining was performed by incubating cells in PBS containing 0.18 mg/ml propidium iodide and 0.4 mg/ml DNase-free RNase. Cells were analysed on a FACScan flow cytometer. DNA profiles, derived from one representative experiment of three performed, are shown. (C) IFN-β treatment differently affects DNA synthesis in K16 and K38 cells. To determine the number of S-phase nuclei, cells were plated in triplicate at 105 cells per 35 mm dish, treated with IFN-β for different time points and incubated with 50 µM BrdU for the last 5 hours. BrdU treated samples were then fixed and stained with an anti-BrdU monoclonal antibody followed by a rhodamine conjugated goat anti-mouse antibody. BrdU-positive cells were counted under a fluorescence microscope. Data represent means ± s.d. of three independent experiments. To exclude that the antiproliferative effect of IFN-β could be due to downregulation of E6/E7 expression, we checked HPV-38 and HPV-16 E6 and E7 mRNA levels by RT-PCR. No significant variations were observed in either E6 or E7 expression in K38 and K16 cells upon treatment with IFN-β for different time points (data not shown). We previously demonstrated that IFN-β exerts its antiproliferative effect on high-risk HPV-positive cell lines by lengthening cell cycle S-phase progression [24]. We analyzed cell cycle distribution of K38 and K16 cells after treatment with IFN-β for several time points. Both cell lines showed a significant augment of S-phase cell amount starting from 48 h of treatment. Interestingly, in K16 cells S-phase cell accumulation increased with time whereas in K-38 cells the S-phase increase was followed by an augment of G1 population (Fig.1B). K16 and K38 cells were pulse-labelled with BrdU for 5 h and analysed for BrdU incorporation. As with what was observed in SiHa and other mucosal high-risk HPV-positive cell lines [24], an increased number of BrdU-positive cells reflecting an S-phase cell accumulation was revealed in IFN-β treated K16 populations. On the contrary, in K-38 samples the number of cells incorporating BrdU upon IFN-β treatment appeared clearly reduced (Fig. 1C). IFN-β Induces Cellular Senescence in K38 but Not in K16 Cells To study apoptosis and senescence induction, specific assays were performed. Annexin-V externalization assay showed no significant increase of apoptosis in either cell types after IFN-β treatment (data not shown). Senescent cells were quantified by counting cells displaying β-galactosidase activity at pH 6.0 (SA-βgal). This lysosomal hydrolase is elevated in senescent cells as a result of lysosomal activity at suboptimal pH, which is detectable only in senescent cells due to an increase in lysosomal content. Interestingly, increasingly high percentages of senescent cells were observed exclusively in K38 cells transformed by E6 and E7 proteins of cutaneous HPV genotype, starting from 4 days of IFN-β treatment, compared to control keratinocytes (LXSN), K16 cells and mucosal high risk HPV-positive cell lines (Fig. 2A, B, and C). 10.1371/journal.pone.0036909.g002Figure 2 IFN-β induces senescence in K38 cells. (A) Control keratinocytes (LXSN), K16, K38 cells and high risk HPV-positive squamous carcinoma cell lines ME-180, Caski, HeLa and SiHa were treated with IFN-β for 4 days and senescent cells were quantified by counting cells displaying senescent-associated β-galactosidase (SA-βgal) activity at pH 6.0. (B) Percentage of senescent cells in K16 and K38 cells treated with IFN-β for different time points. (C) SA-βgal-positive K38 blue cells observed under a light microscope after 4 days of IFN-β treatment. * = p<0.05; *** = p<0.001. Involvement of PML, p53 and p21 in Cell Senescence Induced by IFN-β in K-38 Cells It is known that important senescence regulators are found in IFN-inducible genes. In addition, it has been reported that prolonged IFN-β stimulation can induce senescence in normal cells through the activation of a DNA damage response triggered by an ATM-chk2-p53 pathway [16]. We asked whether IFN-β could induce the senescence phenotype in K38 cells through the involvement of PML and the activation of p53, thus counteracting the inhibitory action exerted on p53 by HPV-38 E6/E7 expression. We analysed the protein levels of PML, p53 and p21, upon IFN-β treatment, and the respective involvement in senescence through RNA silencing (siRNA) technique. Three different siRNAs were used for PML, p53 and p21 genes. Upon IFN-β treatment, PML was up-regulated as well as p21 (Fig. 3). On the other hand in K16 cells, even if IFN-β treatment induces PML expression, it is not detectable any increase in p21 protein levels (data not shown). 10.1371/journal.pone.0036909.g003Figure 3 IFN-β affects the expression of proteins involved in senescence in K38 cells. Western blot analysis of PML, p53 and p21 expression in K38 treated with IFN-β for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed as reported in M&M. PML seems to be an essential component of senescence response in K38 cells, since, when PML expression is inhibited by specific siRNAs (Fig. 4A), IFN-β-induced senescence is strongly reduced (Fig. 4D). p21 silencing (Fig. 4C) partially affects IFN-β-induced senescence (Fig. 4D), while p53 silencing (Fig. 4B) appears much more effective (Fig. 4D), suggesting that different p53 targets may be involved in IFN-β-induced senescence in K38 cells and different pathways may cooperate towards this phenomenon. 10.1371/journal.pone.0036909.g004Figure 4 Effect of PML, p53 and p21 silencing on senescence induction by IFN-β in K38 cells. PML (A), p53 (B) and p21 (C) were silenced by specific small interfering RNAs and protein expression was analyzed by Western blot in cells treated with IFN-β for 4 days. (D) Senescence induction by IFN-β (4 days treatment) was evaluated by SA-βgal staining. * = p<0.05; ** = p<0.01. PML Target Proteins Colocalyze in PML Nuclear Bodies It is known that PML recruits into NBs p53 and different proteins involved in p53 post-translational modifications that are critical for the activation of p53 and for the selection of target genes [25]. We studied colocalization of PML with p53 and ΔNp73 through confocal microscopy analyses of K38 cells treated with IFN-β for different time points. Fig. 5 shows that p53 (A) and ΔNp73 (B) colocalyze with IFN-β-induced PML into PML NBs. On the other hand, colocalization is not detectable after PML silencing (data not shown). 10.1371/journal.pone.0036909.g005Figure 5 p53 and ΔNp73 co-localyze with IFN-β-induced PML into PML Nuclear Bodies. (A,B) For confocal microscopy analysis, K38 cells were cultured on glass bottom dishes (MatTek Corporation) and treated with IFN-β for 4 days. Cells were then fixed in PBS 4% paraformaldehyde for 30 min on ice, immuno-fluorescence labelling was performed as described in Materials and Methods and sample were analyzed using confocal microscope (Leica TCS SP5). Post-translational Modification of p53 by IFN-β The expression of HPV38 E6 and E7 in human keratinocytes induces the stabilization of p53, as shown by WB analysis of p53 in K38 cells compared with control keratinocytes (LXSN), K16 cells and high risk HPV-positive cell lines SiHA and ME-180 (Fig. 6A). This p53 stabilization can be related to increased phosphorylation [21] and acetylation (Fig. 6C). 10.1371/journal.pone.0036909.g006Figure 6 IFN-β effect on p53 post-translational modification and expression of its target genes. (A) WB analysis of p53 in control keratinocytes (LXSN), K16 and K38 cells and in high risk HPV-positive squamous carcinoma cell lines SiHa and ME-180 treated with IFN-β for 48 h. (B) WB analysis of p53 phosphorylated at different phosphorylation sites. (C) WB analysis of acetylated p53. (D) WB analysis of phosphorylated and acetylated p53 and ΔNp73 in K38 cells silenced by PML siRNA and treated with IFN-β for 48 h. (E) WB analysis of ΔNp73 in K38 cells treated with IFN-β for different time points. Whole cell extracts were resolved on SDS-PAGE and transferred onto PVDF membrane. Immunoblotting was performed as reported in M&M. (F) Real time PCR analysis of Bax and Pig3 was carried out on K38 cells treated with IFN-β for 48 h, also in the presence of PML siRNA. NT  =  not transfected. * = p<0.05; ** = p<0.01. In this respect, we may hypothesize that IFN-β, through PML up-regulation, can lead to the recovery of p53 transactivating activity of target genes involved in cell proliferation control. Therefore p53 phosphorylation and acetylation status was analyzed in K38 cells treated with IFN-β. IFN-β modulates p53 phosphorylation status at different phosphorylation sites (Ser-6, Ser-15, Ser-46, Ser-392, Fig. 6B ) while acetylation is mainly downregulated in Lys-320 (Fig. 6C). Ser-6, Ser-392, and Lys-320 seem to be the most important p53 post-translational modifications involved in IFN-β-induced senescence in K38 cells. In fact, when PML expression is silenced, IFN-β is not able to modulate p53 Ser-6, Ser-392, and Lys-320 status (Fig. 6D). Accardi et al. [21] reported that p53 stabilization in K38 cells leads to transcriptional activation of ΔNp73, a p53 inhibitor, able to inhibit p53 transactivation of genes involved in cell growth suppression. It has been shown that IFN-α reduces ΔNp73 levels in Huh7 hepatoma cells and this effect correlates to increased susceptibility to IFN-α triggered apoptosis [23]. We observed that in K38 cells, IFN-β treatment downregulates ΔNp73 mRNA levels (data not shown). The ΔNp73 protein expression appears to be reduced upon IFN-β treatment (Fig. 6E), probably as a result of the p53 post-translational modifications induced by IFN-β. In fact, when PML expression is silenced, ΔNp73 protein levels are not downregulated by IFN-β (Fig. 6D). Real time PCR array results indicate that some genes involved in senescence and growth control are IFN-β-upregulated (Fig.6F). In particular, the observed induction of p53 target genes Bax and Pig3 indicates that IFN-β treatment leads to the recovery of p53 transactivating activity of selected target genes involved in the control of cell proliferation. In fact, it has been reported that modification of specific p53 phosphorylation and acetylation sites may correlate to the transactivation of growth related genes, suggesting a tissue and promoter-specific p53 activity regulation [26]. PML depletion reduces IFN-β induction of Bax and Pig3 in K-38 cells (Fig.6F), indicating the role of PML in the ability of IFN-β to recover p53 transactivation activity of specific target genes. Discussion IFNs were the first successful biological therapy for human malignancy and currently there are several approved IFN cancer therapies. Clinical effectiveness of different IFN subtypes in treatment of various forms of cancer has been extensively reviewed [1]. Better definition of therapeutic molecular targets appears to be critical to fully realize the potential of IFNs in oncology and further understand the mechanisms of antitumor action of the IFN family. Senescence is a permanent cell cycle arrest that is resistant to growth factors and other signals that induce cell proliferation. It has been proposed that senescence prevents cancer in the early stages of its development [6]. Tumor suppressors such as p53, pRb and PML are critical regulators of senescent programme [10], [25], and genes required for senescence are often found to be mutated in human cancers. Cellular senescence is induced in human fibroblasts by prolonged IFN-β treatment through DNA damage signaling and a p53-dependent pathway [16]. IFN-α also induces replicative senescence in endothelial cells after continuous stimulation [17], and treatment with IFN-γ induces cellular senescence in young human umbilical vascular endothelial cells [27]. However, whether induction of senescence is sufficient to repress tumor in vivo is controversial. Recent reports showed that conditional restoration of p53 in mice with hepatocarcinomas, sarcoma or lymphoma is able to promote tumor regression [13], [12]. In addition, it has been reported that HeLa cells cease proliferation and undergo senescence by introduction of the bovine papillomavirus E2 gene that inhibits the expression of the HPV18 E7 gene [28]. Antisense sequences directed against HPV16 E6 and E7 genes transfected in SiHa cells contributed to apoptosis and senescence [29]. In contrast to mucosal high-risk HPV types, the involvement of cutaneous HPV types in human carcinogenesis is still unclear. Cutaneous HPV types that belong to the beta genus of the HPV phylogenetic tree were first isolated in patients suffering from EV, a rare autosomal recessive cancer-prone genetic disorder, and are consistently detected in NMSC from EV, immunocompromised and normal individuals [30]. The transforming properties of the majority of the cutaneous HPV types have been poorly investigated. It has been reported that cutaneous HPV5 E6 protein targets and abrogates Bak function by promoting its proteolitic degradation both in vitro and in regenerated epithelium [31]; however, regulation of Bax has also been reported [32]. The E6 protein of HPV5 compromises the repair of UV-induced thymine dimers [33] and E6 of HPV7 forces keratinocytes into the S1-phase by inhibiting p53-activated, pro-apoptotic genes [34]. HPV8 E6 is able to bind XCRR1 that functions in a single strand DNA repair [35] and it has been shown that UV-irradiated cutaneous HPV8 E2-transgenic mice develop invasive carcinomatous lesions more rapidly than non-irradiated counterparts [36]. Moreover, E6/E7 expression of HPV20 influences proliferation and differentiation of the skin of UV-irradiated transgenic mice [37]. The anti-apoptotic activity and the delay of the DNA repair mechanism may lead to the persistence of UV-damaged keratinocytes, suggesting that cutaneous HPV types may be involved in the early stages of carcinogenesis. A different mechanism behind the lack of cell cycle arrest in cutaneous HPV expressing cells is the up-regulation of ΔNp73 as a result of p53 accumulation observed in HPV38 E6 and E7 expressing human keratinocytes. ΔNp73 in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression [20], [21]. This observation, together with the efficiency of pRb binding and degradation by HPV38 E7, the HPV38 E6/E7-induced suboptimal activation of telomerase and the HPV38 E6/E7 transforming properties in vivo [38], seems to indicate that HPV38 E6 and E7, differently from proteins of other cutaneous HPV types, may be involved in the maintenance of oncogenic transformation. We have previously reported that type I IFNs inhibit cell proliferation in high risk mucosal HPV-positive Squamous Carcinoma Cell (SCC) lines by inducing a significant accumulation of cells in S-phase [24]. The S-phase deregulation triggers apoptotic cell death specifically mediated by the pro-apoptotic factor TRAIL [39]. The present study shows that IFN-β affects cell proliferation in keratinocytes expressing E6 and E7 proteins of cutaneous HPV-38 to a greater extent than in E6 and E7 mucosal HPV-16 transformed cells. In particular, K38 cells undergo senescence upon prolonged IFN-β treatment. IFN-β appears to induce senescence by up-regulating the expression of the tumor suppressor PML. Indeed, experiments of gene silencing via specific siRNAs have shown that PML is essential in the execution of senescence programme and that both p53 and p21 pathways are involved in senescence induction by IFN-β in K38 keratinocytes. P53 and PML are critical mediators of senescence. PML is essential for the formation of discrete protein assemblages in the nucleus known as Nuclear Bodies (NBs) [40]. PML recruits into NBs p53 and proteins involved in p53 post-translational modifications that are essential for the activation of p53 and for the selection of target genes, such as the DNA damage responsive kinases ATM and ATR [25]. ATM kinase phosphorylates p53 at Ser-15, a senescence-inducible modification [41], in IFN-β-induced cellular senescence in human fibroblasts [16]. Over-expression of PML is capable of inducing premature senescence by stabilizing p53 via p53 acetylation on Lys-382 and phosphorylation on Ser-15 and Ser-46 [42]. In contrast, deacetylation of p53 antagonizes PML-induced premature senescence [43]. It has been shown that PML interacts with CBP/p300 acetyltransferase and stabilizes p53 through Lys-382 acetylation [42]. PML also recruits the tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) which induces p53Ser46 phosphorylation [44]. It has been reported that HIPK2-mediated phosphorylation of p53Ser46 is required for the CBP-induced p53 acetylation at Lys-382 [45]. PML has also been recently identified as a direct target of p53 revealing a regulatory positive feedback loop between p53 and PML [46]. Our results indicate that in K38 cells p53 colocalyzes with IFN-β-induced PML into PML NBs. IFN-β can significantly modulate p53 phosphorylation at Ser-6,-15,-46, and -392 and acetylation status mainly at Lys-320. It has been also shown that p53 acetylation in Lys-320 is the first step in IFN-β-induced senescence in human fibroblasts [16]. DNA damage response is required for the activation of p53 in response to oncogenes. Oncogene induced senescence is accompained by DNA replicative stress, including prematurely terminated DNA replication forks and DNA double-strand breaks caused by hyper-DNA replication [47]. Consistent with this, Ras-induced senescence is associated with activation of DNA damage response effectors, such as ATM/ATR and Chk2/Chk1, and inactivation of these DNA damage effectors by RNA interference attenuates oncogene induced senescence [48], [49]. We observed that the inhibition of ATM and ATR prevents IFN-β induction of senescence in K38 keratinocytes, suggesting that IFN-β might induce senescence through a p53-dependent DNA damage pathway (data not shown). It has been reported that HPV16 E6 mediates resistance to IFN-induced senescence through inhibition of p53 acetylation by binding to CBP/p300. Conversely, treatment of HPV16 E7-expressing cells with IFN ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by CBP/P300 [50]. Moreover, HPV16 E7 up-regulates SIRT1, thus attenuating p53 activity via its deacetylation [51]. It has been shown that HPV16 E6 can induce multiple site phosphorylation of p53 [52]. HPV38 E6 and E7 expression in human keratinocytes induces phosphorylation of p53, which leads to the up-regulation of ΔNp73 and the inhibition of p53 transcriptional induction of genes involved in growth suppression [21]. All together these observations indicate that p53 post-translational modifications are critical for p53 involvement in senescence programme induced by IFN-β and that modulation of p53 activity could be a common strategy utilized by both mucosal and cutaneous HPV to inhibit p53 function. We report that by recruitment of p53 into NBs via PML induction, IFN-β may modulate p53 phosphorylation and acetylation status and downregulate ΔNp73 expression in K38 keratinocytes, leading to the recovery of p53 transactivating activity of selected target genes involved in cell proliferation control. Real time PCR array confirms that some genes involved in senescence and growth control are IFN-β-upregulated, in agreement with the reported observations [25] that modification of specific p53 phosphorylation and acetylation sites may correlate to the transactivation of growth related genes, suggesting a tissue and promoter-specific p53 activity regulation. Our results contribute to one of the most interesting current research questions about the exact contribution of post-translational modification sites to the selectivity of the global transcriptional programme of p53. Other important questions remain to be answered to clarify the multitude and redundancy of p53 covalent post-translational modifications required for p53-dependent senescence. It is possible that no single specific post-translational modification leads to specific p53 gene transactivation activity, but each modification might help to regulate p53 function in a tissue and promoter-specific manner. Materials and Methods Cell Cultures and Treatments Primary human foreskin keratinocytes were transduced with empty retrovirus pLXSN (control), or with pLXSN38E6E7 or pLXSN16E6E7 as described by Caldeira et al., 2003 [20] and were grown in KBM BulletKit (Lonza). HPV16-positive cell line Caski and SiHa, HPV18-positive cell line HeLa, and HPV68-positive cell line ME180, obtained from the American Type culture Collection, were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. Cells were maintained in a humidified atmosphere of 5.5% CO2 at 37°C. Human recombinant IFN-β (Rebif; 3×108 IU/mg of protein; Ares-Serono) was added to the medium at the concentration of 200 IU/ml. Measurement of Cell Proliferation Transformed keratinocytes were seeded in triplicate at 105 cells per 35 mm dish. After 24 h, IFN-β was added to the cultures for the indicated times. Adherent cells were detached with 0.05% trypsin-0.02% EDTA in PBS, suspended in growth medium, and counted in a hemocytometer. Viability was evaluated by trypan blue exclusion. Flow Cytometry For cell cycle analysis, cells were fixed in 70% ice-cold ethanol for at least 30 min. DNA staining was performed by incubating cells in PBS containing 0.18 mg/ml propidium iodide and 0.4 mg/ml DNase-free RNase. Cells were analysed on a FACScan flow cytometer (Becton and Dickinson). BrdU Incorporation To determine the number of S-phase nuclei, cells were plated in triplicate at 105 cells per 35 mm dish, treated with IFN-β for different time points and incubated with 50 µM BrdU for the last 5 hours. Samples were fixed with 95% ethanol, 5% acetic acid, treated with 1.5 M HCl and stained with an anti-BrdU monoclonal antibody (Amersham) followed by a rhodamine conjugated goat anti-mouse antibody (Cappel). Senescence-associated β-galactosidase Staining Cells were plated in 12 multi-well, 0,5×105 cells per well, and treated with IFN-β at different time points. Senescent cells were quantified by counting cells displaying senescent-associated β-galactosidase activity at pH 6.0, assayed through Senescent Detection Kit (Calbiochem) following manufacturer’s instruction. Western Blot Analysis To analyse protein expression, control and IFN-β treated cells were lysed in SDS reducing sample buffer. Total cell extracts were clarified by centrifugation and boiled in the presence of 5% 2-Mercaptoethanol and 0.01% bromophenol blue. Protein concentration was determined (Bio-Rad Protein Assay) and 30 mg of total proteins were resolved on SDS-PAGE and transferred onto PVDF membrane (Amersham). The membranes were blocked with 5% skim milk dissolved in PBS-T and incubated with primary antibodies (mouse anti-p53; rabbit anti-p21 (Santa Cruz); rabbit anti-PML (Bethyl); rabbit anti-phospho-p53 Ser6, anti-phospho-p53 Ser15, anti-phospho-p53 Ser46, anti-phospho-p53 Ser392 (Cell Signaling); rabbit anti-acetyl-p53 Lys320, anti-acetyl-p53 Lys373/382 (Millipore), and anti-human β tubulin mouse IgG1 antibody (ICN), as an internal control. Immune complexes were detected with horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antiserums (ICN) followed by enhanced chemiluminescence reaction (Millipore). PML, p53 and p21 silencing Small interfering RNAs (siRNAs) targeted to PML, p53 and p21 were designed and validated by Qiagen and a non-silencing siRNA (Qiagen) served as control. PML siRNAs were: 1) Sense:5′-CGUCUUUUUCGAGAGUCUGtt-3′;Antisense:5′-CAGACUCUCGAAAAAGACGtt-3′; 2)Sense:5′-CCCGCAAGACCAACAACAUtt-3′; Antisense: 5′-AUGUUGUUGGUCUUGCGGGtg-3′; 3)Sense:5′-GGGACCCUAUUGACGUUGAtt-3′; Antisense: 5′-UCAACGUCAAUAGGGUCCCtg-3′. p53 siRNA were: 1) Sense:5′–GGAAAUUUGCGUGUGGAGUtt-3′; Antisense: 5′–CUCCACACGCAAAUUUCCtt-3′; 2) Sense: 5′-GCAUCUUAUCCGAGUGGAAtt-3′; Antisense: 5′-UUCCACUCGGAUAAGAUGCtg-3′; 3) Sense: 5′-GCAGUUAAGGGUUAGUUUAtt-3′; Antisense: 5′-UAAACUAACCCUUAACUGCaa-3′. p21 siRNA (Dharmacon) was: Sense:5'-GAUGGAACUUCGACUUUGUUU-3': Antisense:5'-PACAAAGUCGAAGUUCCAUCUU 3'. Shortly before transfection, 2×105 cells per dish were seeded in 35 mm dishes in 1 ml of fully supplemented culture medium. siRNA was diluted in 50 µl culture medium without supplements to a final concentration of 10 nM. 3.5 µl of HiPerfect Transfection Reagent (Qiagen) were added to the diluted siRNA and mixed by vortexing. After an incubation of 10 min at room temperature, the transfection complex was added drop-wise onto the cells. 24 hrs post-transfection cells were treated with IFN-β for the indicated time points. Immunofluorescence Immunofluorescence staining of cells was performed on cells grown on Glass Bottom Culture Dishes 14 mm Microwell poly-d-lysine Coated from Mat Tek Corporation (Ashland, MA 01721 U.S.A.) and fixed with 4% formaldehyde, permeabilized with PBS/0.1% Triton-X, and stained using the following primary antibodies: anti-PML; ap53 (Santa Cruz), anti-ΔNp73 (Imgenex). Anti-mouse-Fitc (Cappel), anti-mouse-Alexa 546 (Molecular Probe # A11030), and anti-rabbit-Alexa 610 (Molecular Probe # A31551) were used as secondary antibodies. Sample were analyzed using confocal microscope (Leica TCS SP5). Software: LAS AF version 1.6.3 (Leica Microsystem). Real-time PCR Real-time PCR was carried out by using the MESA GREEN MasterMixes Plus, Low ROX (Eurogentec). The primer sequences are: Bax F: 5' TTT GCT TCA GGG TTT CAT CC 3', R: 5' ATC CTC TGC AGC TCC ATG TT 3'; Pig3 F: 5' GCT TCA AAT GGC AGA AAA GC 3', R: 5' AAC CCA TCG ACC ATC AAG AG 3'. We thank Roberto Gilardi for excellent editorial assistance. Competing Interests: The authors have declared that no competing interests exist. Funding: Research in our laboratory is currently funded in part by “Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 2008) del Ministero dell’Istruzione, dell’Università e della Ricerca” and Progetti Ateneo, Sapienza University, 2009 and 2011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Vannucchi S Chiantore MV Mangino G Percario ZA Affabris E 2007 Perspectives in biomolecular therapeutic intervention in cancer: from the early to the new strategies with type I interferons. 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PLoS One. 2012 May 16; 7(5):e36909
==== Front Skelet MuscleSkelet MuscleSkeletal Muscle2044-5040BioMed Central 2044-5040-2-62254164410.1186/2044-5040-2-6ResearchMyoD-dependent regulation of NF-κB activity couples cell-cycle withdrawal to myogenic differentiation Parker Maura H [email protected] Maltzahn Julia [email protected] Nadine [email protected] Ban [email protected] Jeff [email protected] Denis [email protected] Michael A [email protected] Faculty of Health Sciences Graduate Programme, McMaster University, Hamilton, Ontario, Canada2 Ottawa Hospital Research Institute, Molecular Medicine Program, Ottawa, Ontario, Canada3 Current address: Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA, USA4 The Ohio State University College of Medicine, Columbus, Ohio, USA5 Ottawa Hospital Research Institute, 501 Smyth Rd, Ottawa, ON, K1H 8L6, Canada2012 19 5 2012 2 6 6 3 1 2012 27 4 2012 Copyright ©2012 Parker et al; licensee BioMed Central. 2012Parker et al; licensee BioMed Central. 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 Mice lacking MyoD exhibit delayed skeletal muscle regeneration and markedly enhanced numbers of satellite cells. Myoblasts isolated from MyoD-/- myoblasts proliferate more rapidly than wild type myoblasts, display a dramatic delay in differentiation, and continue to incorporate BrdU after serum withdrawal. Methods Primary myoblasts isolated from wild type and MyoD-/- mutant mice were examined by microarray analysis and further characterized by cell and molecular experiments in cell culture. Results We found that NF-κB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. As a result, expression of cyclin D is maintained during serum withdrawal, inhibiting expression of muscle-specific genes and progression through the differentiation program. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Importantly, this deficit was rescued by forced expression of IκBαSR, a non-degradable mutant of IκBα, indicating that inhibition of NF-κB is sufficient to induce terminal myogenic differentiation in the absence of MyoD. Conclusion MyoD-induced cytoplasmic relocalization of NF-κB is an essential step in linking cell-cycle withdrawal to the terminal differentiation of skeletal myoblasts. These results provide important insight into the unique functions of MyoD in regulating the switch from progenitor proliferation to terminal differentiation. Skeletal muscleMyoblastsMyoDNF-κBIKKIκBDifferentiationMyogenesis ==== Body Background Cell survival and differentiation is regulated by NF-κB, a family of ubiquitously expressed transcription factors comprising RelA/p65, c-Rel, RelB, p50 (processed form of p105), and p52 (processed form of p100) [1]. NF-κB proteins function as homo- or heterodimers, the most common of which is the p50/p65 heterodimer. All family members contain a DNA-binding domain, a protein-protein dimerization domain, and a nuclear localization sequence (NLS). However, only RelA/p65, c-Rel, and RelB have a transactivation domain [2]. Sub-cellular localization of NF-κB is regulated by ‘inhibitor of κB’ proteins: IκBα, IκBβ, and IκBϵ [3]. IκB proteins bind NF-κB, mask the nuclear localization signal, and sequester NF-κB in the cytoplasm as an inactive protein. Upon induction, IκB kinases (IKKs) phosphorylate IκB, releasing NF-κB and targeting IκB for degradation. The released NF-κB enters the nucleus, binds DNA, and regulates gene transcription. This process is normally activated by molecules such as cytokines, growth factors, and bacterial products [4]. Myogenic specification and differentiation requires the myogenic regulatory factors (MRFs), namely MyoD, Myf5, myogenin, and MRF4/Myf6 [5]. The MRFs share a highly homologous basic helix-loop-helix (bHLH) domain, which is required for DNA binding and dimerization with the E-protein family of transcription factors. MRF-E-protein heterodimers bind to the consensus E-box sequence, CANNTG, in gene promoters and regulate transcriptional activation. In particular, MyoD and Myf5 are essential for murine skeletal muscle development [6]. However, mice lacking MyoD are viable and fertile, and display no overt phenotype [7]. This indicates that myogenic specification and differentiation during embryonic and fetal development occurs in the absence of MyoD, due to the presence of other myogenic regulatory factors, such as Myf5. Analysis of regeneration in MyoD-/- muscle established an essential role for MyoD in regulating adult myogenesis. In particular, increased numbers of satellite cells and a deficient muscle regenerative process in mice lacking MyoD (MyoD-/-), or MyoD and dystrophin (MyoD-/-:mdx), suggests that in the absence of MyoD, satellite cells have an increased propensity for self-renewal [8]. Analysis of the differentiation kinetics of cultured MyoD-/- satellite cell derived myoblasts revealed a marked delay in differentiation, characterized by reduced expression of differentiation specific markers such as myosin heavy chain, myogeninMRF4α-actins and acetylcholine receptor-δ[9-11]. Although a portion of MyoD-/- cells express myosin heavy chain (MyHC), these myocytes fail to fuse and remain primarily mononuclear. Moreover, the majority of MyoD-/- myoblasts display continued incorporation of bromodeoxyuridine (BrdU) into DNA after serum withdrawal, indicating DNA synthesis is maintained in the absence of mitogen stimulation. In this study, we examined the role MyoD plays in regulating cell-cycle withdrawal during terminal differentiation in adult myogenesis by undertaking a closer investigation of the molecular phenotype of MyoD-/- myoblasts. We observed that MyoD-/- myoblasts maintained nuclear localization of NF-κB after serum withdrawal, and displayed altered expression of NF-κB target genes. In particular, MyoD-/- myoblasts failed to down-regulate cyclin D1, an NF-κB target gene and key mediator of cell-cycle withdrawal and differentiation in myoblasts [12,13]. Importantly, inhibition of NF-κB activity, through expression of a mutant form of IκBα (IκB-SR), rescued the differentiation of MyoD-/- myoblasts. Therefore, we conclude that MyoD controls cell-cycle withdrawal by regulating the subcellular localization of the NF-κB family of transcription factors. Methods Myoblast isolation and cell culture Myoblasts were isolated from 6 to 8 week old wild type (WT) Balb/C mice and MyoD-/- mice and cultured as previously described [9]. To induce differentiation, the cells were washed once with PBS and transferred to differentiation medium (DMEM supplemented with 5% horse serum (Invitrogen), and 2X penicillin/streptomycin). C2C12 murine myoblasts were cultured and differentiated as previously described [14]. Transfections and luciferase assay C2C12 and MyoD-/- myoblasts were transfected in low serum Opti-MEM using Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer. Reporter and expression plasmids were previously described [12], and all transfections were normalized to CMV-βGAL expression. For luciferase assays, cells were lysed in MPER (Pierce) and assays were performed as previously described [12]. MyoD siRNA was obtained from SantaCruz and transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). RNA isolation and RNase protection assay RNA was isolated using TriZol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNase protection assay was performed using the RiboQuant kit according to the manufacturer’s instructions (BD Bioscience, Franklin Lakes, NJ, USA). The relative amount of radioactivity present in each band was quantitated using a Phosphorimager (GE Healthcare, Buckinghamshire, England), and the values obtained for each cyclin were normalized to the value for the GAPDH control. Immunoblotting and antibodies Proteins were separated on 10% or 12% SDS-polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA) according to established protocols. The antibodies used were all from Santa Cruz Biotechnology (Santa Cruz. CA, USA): anti-cyclin D1 (C-20), anti-cyclin D2 (H-289), anti-cyclin D3 (C-16), anti-cdk4 (C-22), anti-cyclin A1 (C-19), anti-cyclin E (C-19), anti-cdk2 (H-298), anti-cyclin H (C-18), anti-cdk7 (C-19), anti-NF-κB p65 (C-20), anti-Myf5 (C-19), anti-MyoD (C-20), and anti-IKKγ (FL-419). For immunoblotting, all antibodies were used according to the manufacturer’s instructions, normally at a dilution of 1:500 or 1:1,000. Goat anti-mouse and goat anti-rabbit secondary antibodies were used at 1:2,000 (BioRad, Hercules, CA, USA). Antibody-bound proteins were detected using enhanced chemiluminescence (ECL; GE Healthcare, Buckinghamshire, England) and X-OMAT 5 X-ray film. Kinase assays Each 10-cm plate from a differentiation time course of WT and MyoD-/- primary myoblasts were lysed with 300 μL of NP-40 Lysis/IP buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 10% glycerol, 0.1 mM Na2VO3, 50 mM NaF, 20 mM β-glycerophosphate, 50 μg/mL PMSF, 2 μg/mL leupeptin, 1 μg/mL aprotinin, 10 μg/mL pepstatin). Immunoprecipitated cdk2 was incubated with 1 μg of histone H1 (Invitrogen) and 5 μCi of γ-32P-ATP (GE Healthcare, Buckinghamshire, England) in kinase buffer, and incubating at 30°C for 20 min. IKK kinase assays were performed as previously described using a GST-IκBα substrate [15]. Gene expression analysis Mouse U74Av2 GeneChip microarrays (Affymetrix, Santa Clara, CA, USA) were used to analyse gene expression in wild type and MyoD-/- primary myoblasts [16]. A list of genes related to the NF-κB pathway was defined with reference to commercial microarrays (Panomics; Superarray Bioscience, Frederick, MD, USA) and the literature. Microarray data is available from StemBase (http://www.scgp.ca:8080/StemBase/) under experiment number E223 (samples S361-4) and E59 (S78-9) and from the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under series accession number GSE3245 (GSM73053…64) and GSE3244 (GSM73026, -29, -32, -35, -38, and -41). Virus production and transduction To generate ecotrophic retroviral supernatant, Phoenix-eco packaging cell lines were obtained from ATCC, cultured in DMEM with 4.5 g/L glucose (Invitrogen Life Technologies), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, and 10% FBS (Hyclone, Logan, UT, USA), and transfected either with pBabe/Iκ (expressing IKBα-SR) or pBabe (empty vector) using lipofectamine 2000 (Invitrogen Life Technologies). Retroviral supernatant was harvested 48 h after transfection and filtered through a 0.45 μm syringe filter (Millipore). Primary proliferating MyoD-/- cells were infected with diluted viral supernatant plus 8 μg/mL polybrene (hexadimetherine bromide, Sigma Aldrich, ST. Louis, MO, USA). Drug selection was conducted using 2 μg/mL Puromycin (Sigma Aldrich) for 7 days; uninfected controls were obliterated after 4 days of selection. Differentiation was induced by replacing growth medium with DMEM + 5% horse serum (Hyclone). Animals All animal procedures conform with the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals, the Animals for Research Act, and were approved by the Animal Care Committee at University of Ottawa. Results Sustained activation of NF-κB during differentiation of MyoD-/- myoblasts MyoD-/- myoblasts express myosin heavy chain upon serum withdrawal, yet fail to fuse into multinucleated myotubes [9]. This phenotype is recapitulated in MyoD-expressing IκBα-/- fibroblasts, in which NF-κB is persistently nuclear localized [17,18]. Given that NF-κB is normally relocalized to the cytoplasm during myogenic differentiation, we sought to determine if NF-κB remains in the nucleus during induction of differentiation in myoblasts lacking MyoD. Nuclear and cytoplasmic protein extracts from a differentiation time course of WT and MyoD-/- myoblasts were subjected to western blot analysis using an antibody specific for p65/RelA. Strikingly, MyoD-/- myoblasts displayed continued nuclear localization of NF-κB (RelA/p65) after mitogen withdrawal, whereas RelA/p65 was appropriately relocated to the cytoplasm in WT myoblasts (Figure 1A). Furthermore we conducted immunofluorescence analyses of WT and MyoD-/- cells under growth and differentiation conditions. We observed continued nuclear localization of NF-κB (RelA/p65) in MyoD-/- cells during differentiation (Figure 1B) while WT cells displayed the expected shift in localization of NF-κB (RelA/p65) during differentiation. Furthermore, MyoD-/- myoblasts appear to have less cytoplasmic NF-kB than WT myoblasts in growth conditions, suggesting that NF-κB signalling is aberrant in the absence of MyoD in proliferation as well as differentiation. Figure 1 NF-κB remains localized to the nucleus after serum withdrawal inMyoD-/-myoblasts.(A) Protein was isolated from a differentiation time course of WT and MyoD-/- primary myoblasts and a western blot using the nuclear (N) and cytoplasmic (C) fractions was performed with an antibody specific for the p65 subunit of NF-κB. A western blot with an antibody specific for α-tubulin was performed using the cytoplasmic fractions. (B) Immunofluorescence analysis of WT and MyoD-/- myoblasts and myotubes demonstrate that NF-κB remains localized in the nucleus in MyoD-/- myoblasts even after differentiation. Staining for NF-κB in red, nuclei are counterstained with DAPI (in blue). Scale bar: 20 μm. To determine if continued nuclear localization of p65 correlates with increased transcriptional activity, an NF-κB specific luciferase reporter (3xκB-Luc) was transfected into C2C12 cells lacking MyoD expression. C2C12 myoblasts transfected with MyoD-specific siRNA (siMyoD-C2C12) exhibited a significant decrease in MyoD expression, and resembled MyoD-/- myoblasts (Figure 2A, data not shown). Luciferase activity in 3xκB-Luc-transfected siMyoD-C2C12 myoblasts was increased relative to control cells, indicating that in the absence of MyoD, RelA/p65 continued to be transcriptionally active after induction of differentiation (Figure 2B). Co-transfection with exogenous p65 enhanced activation of the 3xκB-Luc reporter, yet siMyoD cells continued to exhibit elevated NF-κB activity (Figure 2C). Figure 2 Loss of MyoD activates NF-κB.(A) Protein isolated from C2C12 myoblasts transfected with control siRNA or siRNA specific for MyoD were analyzed by western blot using antibodies specific to MyoD and α-tubulin. (B) C2C12 cells transfected with control siRNA (siCont) or MyoD siRNA (siMyoD) were co-transfected with an NF-kB reporter vector (3xκB-Luc). Bars represent average luciferase activity (relative light units (RLUs)) (n = 3). Error bars represent standard deviation. (C) Same as B, with the addition of a p65 (NF-κB) expression vector. To determine if expression of NF-κB target genes, or genes that participate in the NF-κB signalling pathway, were altered in the absence of MyoD, the expression profiles of MyoD-/- myoblasts and WT myoblasts were compared using an Affymetrix microarray (Tables 1 and 2) [16]. Known NF-κB target genes were selected from genes displaying differences in expression (see http://www.bu.edu/nf-kb/gene-resources/target-genes/ for an overview). Genes that were specifically up-regulated in myoblasts lacking MyoD are tabulated in Table 1, whereas genes that were down-regulated are documented in Table 2. NF-κB target genes such as MCP-1/CCL2/GRO-αMIP-2/CXCL1, matrix metalloproteinases (MMP3 and MMP13), VCAM1IL-6BCL-2IL-11IGFBP-2, and JunB are represented in both Tables 1 and 2, indicating that nuclear localized NF-κB was transcriptionally active in MyoD-/- myoblasts. Furthermore we investigated genes involved in myogenesis. We found the following genes to be down-regulated in MyoD-/- myoblasts compared to WT myoblasts: myogenin, Myf5, Mef2C, embryonic myosin heavy chain, MRF4/Myf6, as well as Six1. Table 1 NF-κB-related genes up-regulated inMyoD-/-myoblasts Gene RefSeq Fold Gene name Ccl2 NM_011333 166.7 Chemokine (C-C motif) ligand 2 Bgn NM_007542 73.5 Biglycan Mmp3 NM_010809 34.0 Matrix metallopeptidase 3 Cxcl1 NM_008176 33.0 Chemokine (C-X-C motif) ligand 1 Thbs2 NM_011581 16.9 Thrombospondin 2 Mmp13 NM_008607 9.0 Matrix metallopeptidase 13 Vcam1 NM_011693 8.2 Vascular cell adhesion molecule 1 Il6 NM_031168 8.0 Interleukin 6 Ier3 NM_133662 5.6 Immediate early response 3 Penk1 NM_001002927 4.6 Preproenkephalin 1 Pcaf NM_020005 4.3 p300/CBP-associated factor H2-T23 NM_010398 3.2 Histocompatibility 2, T region locus 23 Stat6 NM_009284 2.1 Signal transducer and activator of transcription 6 Ptgs2 NM_011198 2.8 Prostaglandin-endoperoxide synthase 2 Abcb1b NM_011075 2.2 ATP-binding cassette, sub-family B (MDR/TAP), 1B Gja1 NM_010288 2.7 Gap junction membrane channel protein alpha 1 Tnfaip3 NM_009397 2.6 Tumor necrosis factor, alpha-induced protein 3 Bcl2 NM_009741 2.5 B-cell leukemia/lymphoma 2 Table 2 NF-κB-related genes down-regulated inMyoD-/-myoblasts Gene RefSeq Fold Gene name Il11 NM_008350 -23.2 Interleukin 11 Igfbp2 NM_008342 -9.1 Insulin-like growth factor binding protein 2 Tlr6 NM_011604 -3.3 Toll-like receptor 6 Fos NM_010234 -3.1 FBJ osteosarcoma oncogene Ccnd3 NM_007632 -2.7 Cyclin D3 Cd80 NM_009855 -2.3 CD80 antigen Junb NM_008416 -2.3 Jun-B oncogene Selp NM_011347 -1.9 Selectin, platelet MyoD-/- myoblasts maintain expression of cyclin D1 and D2 after serum withdrawal MyoD-null myoblasts continue to proliferate in the absence of mitogens, indicating that cell-cycle modulators, such as cyclins, may be aberrantly expressed and/or regulated. Given that RelA/p65 specifically regulates expression of cyclin D1 in myoblasts we hypothesized that persistent activation of RelA/p65 would result in continued expression of cyclin D1 in MyoD-/- myoblasts, even after serum withdrawal [12]. In order to investigate changes in important cell-cycle genes in the absence of MyoD, RNA was isolated from a differentiation time course of MyoD-/- and WT primary myoblasts, and the relative expression level of a variety of cyclins was determined using an RNase protection assay. The relative amount of mRNA present for each cyclin was quantitated using a phosphorimager, and normalized to a GAPDH control. Figure 3A and 3C show resulting autoradiographs of polyacrylamide gels, and Figure 3B and 3D graphically depicts quantitation of the results. MyoD-/- myoblasts exhibited a higher level of cyclin D1 and D2 mRNA, and a lower level of cyclin D3 and cyclin G1 mRNA when compared to WT myoblasts (Figure 3A, 3B, and 3F). However, neither cyclin E nor cyclin H mRNA was affected by the absence of MyoD (Figure 3C and 3D). Figure 3 MyoD-/-myoblasts maintain cyclin D expression after serum withdrawal.(A, C) RNA was isolated from a differentiation time course of WT and MyoD-/- primary myoblasts and an RNase protection assay was performed with the indicated probes. The protected 32P-labeled fragments were separated on a polyacrylamide gel, the gel dried and exposed to X-ray film. (B, D) The amount of each protected fragment was quantitated using a phosphorimager. The values obtained were normalized relative to the GAPDH control and the L32 control, and graphed as a function of the number of days in differentiation medium (DM), n = 3. (E) Nuclear protein was isolated from a differentiation time course of WT and MyoD-/- primary myoblasts and a western blot was performed with the indicated antibodies. (F) Quantitative real-time PCR for Cyclin D3 of WT and MyoD-/- myoblasts, n = 3. To determine if changes in mRNA expression are translated into changes in protein levels, nuclear protein extracts were isolated from a similar differentiation time course and analyzed by western blot analysis. In accord with RNase protection assay results, MyoD-/- myoblasts displayed a higher level of cyclin D1 and D2 protein in the nucleus, and continued expression of both cyclins after mitogen withdrawal (Figure 3E). Expression of cyclin D3 protein, on the other hand, was not altered in the absence of MyoD. Taken together, these data imply that persistent activation of RelA/p65 is responsible for expression of cyclin D1 in MyoD-/- myoblasts after serum withdrawal. pRb is also phosphorylated by cyclin E-cdk2, and this phosphorylation is required for S-phase entry [19]. Moreover, forced expression of cyclin E bypasses the need for cyclin D/cdk4 activity in cell-cycle progression from G1 to S phase [20,21]. Expression of cyclin E mRNA was not altered in the absence of MyoD, indicating that the increase in cyclin D1 expression is not inducing cell-cycle progression through stimulation of E2F activity on the cyclin E promoter (Figure 3C and D). However, overexpression of cyclin D1 may also inhibit MRF activity and myogenic differentiation through a mechanism independent of pRb or MyoD phosphorylation [22-24]. Indeed, a hypophosphorylated mutant of pRb (pRbΔp35; EF), which induces cell-cycle withdrawal, is unable to rescue cyclin D-dependent inhibition of muscle-specific gene expression (MH Parker, MA Rudnicki, unpublished observations). Loss of MyoD stimulates S-phase entry Although expression of cyclin E mRNA was not altered (Figure 3C and 3D), western blot analysis of protein extracts from a differentiation time course of wild type and MyoD-/-myoblasts displayed an increased level of cyclin E protein in the nucleus (Figure 4A). This may be the result of increased stability of cyclin E protein, or of prolonged nuclear localization after mitogen withdrawal. The level of nuclear cdk2 protein was also elevated, and more importantly, appeared to be the slower migrating, activated form. In order to determine if cdk2 activity was maintained after mitogen withdrawal, a kinase assay was employed using protein extracts from the same differentiation time course of wild type and MyoD-/- myoblasts. As expected, elevated levels of cdk2 correlated with increased cdk2 kinase activity (Figure 4B). Figure 4 MyoD-/-myoblasts enter S-phase more readily than WT myoblasts.(A) Western blot was performed using the indicated antibodies and nuclear protein isolated from a differentiation time course of WT and MyoD-/- primary myoblasts. (B) Cdk2 was immunoprecipitated from protein extracts of a differentiation time course of WT and MyoD-/- primary myoblasts, and assayed for kinase activity using histone H1 and γ-32P-ATP as substrate. (C) Proliferating WT and MyoD-/- myoblasts were fixed in ethanol and DNA stained with propidium iodide (PI). Cells were analyzed for DNA content by FACS analysis. The percentage of cells in each phase of the cell cycle is indicated. To determine if increased cdk2 kinase activity forced cells to enter S-phase more readily, proliferating MyoD-/- and WT myoblasts were fixed and analyzed for DNA content using fluorescence activated cell sorting (FACS). As expected, MyoD-/- myoblasts displayed a two-fold increase in the number of cells in S-phase (23% of MyoD-/- myoblasts compared to 12% of WT myoblasts; Figure 4C). This increase in the proportion of MyoD-/- myoblasts in S-phase was accompanied by a decrease in the proportion of cells in the G1 phase of the cell cycle (68% of MyoD-/- cells compared to 84% of WT cells). Therefore, in the absence of MyoD, myoblasts enter into S-phase more readily, likely as a result of increased cyclin E-cdk2 activity. Inhibition of NF-κB restores differentiation of MyoD-/- myoblasts Skeletal muscle differentiation requires both cell-cycle withdrawal and muscle-specific gene expression. MyoD-/- myoblasts fail to withdraw from the cell cycle, as evidenced by continued DNA synthesis after induction of differentiation [9]. Given that MyoD-null myoblasts aberrantly maintain transcriptionally active RelA/p65 after serum withdrawal, we hypothesize that persistent activation of RelA/p65 is responsible for the failure to exit the cell cycle. Moreover, we propose that the inability to induce cell-cycle withdrawal is directly responsible for the delay in differentiation in MyoD-/- myoblasts. IκB kinases (IKKs) phosphorylate IκB, resulting in degradation of IκB and nuclear localization of RelA/p65 [3]. To determine if IKK activity is aberrantly maintained in MyoD-null myoblasts after induction of differentiation, IKK was immunoprecipitated from MyoD-/- and wild type myoblast protein extracts and kinase activity assayed using GST-IκBα and γ-32P-ATP as substrate. MyoD-null myoblasts displayed approximately 2-fold greater IKK activity, as compared to WT myoblasts (Figure 5A). Figure 5 Elevated IKK activity inMyoD-/-myoblasts is responsible for increased NF-κB activity.(A) Kinase assay (KA): IKK was immunoprecipitated from protein extracts of WT (MyoD+/+) and MyoD-/- primary myoblasts, and assayed for kinase activity using GST-IκBα and γ-32P-ATP as substrate. Immunoblot (IB): IKK was immunoprecipitated from protein extracts of WT (MyoD+/+) and MyoD-/- primary myoblasts, and assayed by western blot using an antibody specific for IKKγ. (B)MyoD-/- myoblasts were transfected with vector control, vector expressing a dominant negative mutant IKKβ (IKKβ DN) or a non-phosphorylatable mutant of IkBα (IκB-SR), in addition to an NF-κB reporter (3xκB-Luc). Bars represent average luciferase activity (relative light units (RLUs)) (n = 3). Error bars represent standard deviation. If increased IKK activity results in RelA/p65 activation, then inhibiting IKK should inhibit NF-κB transcriptional activity. Treating MyoD-/- myoblasts with a chemical inhibitor of IKK (data not shown), or expressing a dominant negative mutant of IKKβ (IKKβ DN), resulted in decreased activity of 3xκB-Luc, indicating that elevated IKK activity is in part responsible for persistent NF-κB transcriptional activity (Figure 5A). Therefore, to target RelA/p65 directly, a non-phosphorylatable mutant form of IκBα (IκBα-SR) was expressed, which is resistant to targeted degradation and prevents nuclear localization of NF-κB. Indeed, expression of IkBα-SR in MyoD-/- myoblasts inhibited 3xκB-Luc activity, indicating that NF-κB was directly repressed (Figure 5B). Moreover, expression of IκBα-SR in MyoD-/- myoblasts down-regulated expression of cyclin D1 and cdk2 upon initiation of differentiation (Figure 6C). This strongly suggests that sustained nuclear localization of RelA/p65 in the MyoD-null myoblasts is responsible for cellular proliferation after serum withdrawal. Figure 6 Nuclear localization of NF-κB inhibits terminal differentiation.(A)MyoD-/- myoblasts were infected with empty capsid (pBABE) or virus expressing IκBα-SR (pBABE-IkB). Protein extracts from proliferating infected cells and uninfected cells (control) were analyzed by western blot analysis using an antibody specific to IκBα. (B)MyoD-/- myoblasts were infected with empty capsid (pBABE) or virus expressing IκBα-SR (pBABE-IkB). Proliferating cells (GM), or cells induced to differentiate for 5 days, were fixed and assessed for myosin heavy chain (MyHC) expression (in green). Nuclei were visualized using DAPI (in blue). (C) Fusion index of myotubes from MyoD-/- cells either infected with a control (pBABE) or an IκBα-SR (pBABE-IkB) expressing virus, n = 5, ** = P < 0.01, *** = P < 0.001. (D)MyoD-/- myoblasts were infected with empty capsid (pBABE) or virus expressing IκBα-SR (pBABE-IkB). Protein extracts from a differentiation time course of infected and uninfected cells were analyzed by western blot analysis using the antibodies indicated Importantly, inhibition of NF-κB activity also induced expression of myogenin and myosin heavy chain one day after serum withdrawal, and resulted in the formation of multinucleated myotubes (Figure 6B to 6D). This is in contrast to MyoD-/- myoblasts infected with control virus, which up-regulated myogenin and myosin heavy chain to a much lower extent, even 5 days after serum withdrawal (Figure 6D, DM5). Additionally we examined the fusion index of myotubes of control and IκBα-SR expressing MyoD-/- cells at days 3 and 5 after serum withdrawal. Overexpression of IκBα-SR led to a significantly increased fusion index as well as to more differentiation in general (Figure 6B and 6C). It is interesting to note that IκBαSR-expressing MyoD-/- cells up-regulate expression of Myf5 during proliferation and early differentiation (Figure 6D). Increased expression of Myf5 may compensate for the lack of MyoD, and may be responsible for the induction of differentiation specific genes, such as myogenin and myosin heavy chain, after cell-cycle withdrawal. This is consistent with the fact that embryonic and fetal skeletal muscle development is able to occur in MyoD-/- mice [7]. Discussion Finding the specific MyoD-regulated gene product that links cell-cycle withdrawal and terminal myogenic differentiation has been hitherto elusive. In this study, we demonstrate that continued proliferation and inhibition of differentiation in MyoD-null myoblasts is due to persistent nuclear localization of RelA/p65. Expression of a non-phosphorylatable mutant of IκBα (IκBα-SR), which induces cytoplasmic retention of RelA/p65, down-regulated expression of cyclin D1 in MyoD-/- myoblasts, and resulted in the formation of multinucleated myotubes. Therefore, inhibition of RelA/p65 activation was able to substitute for MyoD expression during myogenic differentiation. Taken together, this indicates that RelA/p65 provides the link between MyoD-induced cell-cycle withdrawal and differentiation. MyoD is postulated to initiate expression of p21 and p57, inhibitors of cdk2 and cdk1 kinase activity [25-27]. Moreover, mice lacking p21 and p57 are phenotypically similar to myogenin knockout mice, in that they lack differentiated myofibers [28]. These data suggest that myogenic cell-cycle withdrawal and differentiation requires MyoD-dependent induction of cdk inhibitor expression. However, MyoD-/- myoblasts have a similar level of p21 mRNA as compared to WT myoblasts, and mice lacking p21 display no apparent muscle abnormalities [9,29]. Therefore, we propose that adult satellite cells utilize an alternate or additional mechanism for inducing cell-cycle withdrawal during terminal differentiation: down-regulation of RelA/p65 activity. Notably, MIP-2/CXCL1/GRO-α and IL-6, which were upregulated 33-fold and 8-fold, respectively, in MyoD-/- myoblasts, are associated with constitutive activation of NF-κB, and promote tumor growth and progression [30]. Importantly, IGFBP-2, which binds IGFs and specifically inhibits IGF-dependent myogenic cell proliferation, was down-regulated 9.1-fold in MyoD-/- myoblasts [31-35]. Therefore, in the absence of MyoD, myoblasts are programmed to proliferate as a result of maintaining growth factor signaling. This is consistent with data demonstrating that MyoD-/- myoblasts have an increased propensity for proliferation and self-renewal. Biglycan and thrombospondin 2, two genes that were up-regulated in MyoD-/- myoblasts (73.5-fold and 16.9-fold, respectively), are extracellular matrix components that play important roles in scaffolding and signal transduction during myogenic regeneration. In particular, biglycan binds TGF-β, and plays an important role in mediating TGF-β signaling in responding cells [36,37]. This is important given that TGF-β inhibits myogenic differentiation and induces expression of cyclin D1[22]. Taken together, these data strongly suggest that NF-κB regulates expression of genes important for inducing cell proliferation. During normal differentiation of myoblasts, NF-κB is relocalized to the cytoplasm and DNA-binding activity decreases within 24 h of serum withdrawal [12,18]. Differentiation is accelerated in myoblasts expressing a non-phosphorylatable form of IκBα (IκBαSR), which is unable to be degraded, thus inhibiting NF-κB (p65) nuclear localization [12]. Furthermore, these IκBαSR-expressing myoblasts proliferate less rapidly and down-regulate expression of cyclin D1. In contrast, IκBα-/- mouse embryonic fibroblasts (MEFs) infected with a MyoD-expressing retrovirus maintain NF-κB nuclear localization, resulting in the formation of fewer myotubes that are smaller and incorporate fewer nuclei [17]. Our experiments explain mechanistically why MyoD-null myoblasts display a phenotype similar to that of MyoD-infected IκBα-/- MEFs. During myogenic regeneration, satellite cells are activated and proliferate prior to initiating differentiation. Increased numbers of satellite cells and a deficient muscle regenerative process in MyoD-/- mice suggest that in the absence of MyoD, satellite cells have an increased propensity for self-renewal rather than differentiation [38]. In light of the data presented here, we conclude that RelA/p65 plays an important role during the myoblast to myotube transition during adult myogenesis. Indeed, treatment of mdx mice with a cell permeable peptide inhibitor of IKK, which specifically inhibits NF-κB activity, restores regeneration, as evidenced by a greater number of newly formed myofibers, and increased muscle tetanic force [39]. Failure to induce myogenic differentiation is also illustrated in rhabdomyosarcoma (RDS), one the most common childhood solid tumors. RDS is characterized by inhibition of MyoD activity and concomitant failure to withdraw from the cell cycle and differentiate. This study suggests that loss of MyoD activity in RDS cells may cause aberrant nuclear localization of NF-κB, resulting in sustained cyclin D1 expression. As such, inhibiting IKK and stabilizing IκBα may play a valuable role in inhibiting proliferation and inducing differentiation in RDS cells. During myogenesis, cytoplasmic re-localization of RelA/p65 after mitogen withdrawal plays a key role for down-regulating cyclin D expression, inducing cell-cycle withdrawal and activating differentiation-specific gene expression. Therefore, the regulation of NF-κB is essential in the induction of myogenic differentiation. Our experiments define the mechanistic link between MyoD and NF-κB that acts to couple cell-cycle withdrawal to terminal differentiation. Conclusion We have demonstrated that NF-κB, a key regulator of cell-cycle withdrawal and differentiation, aberrantly maintains nuclear localization and transcriptional activity in MyoD-/- myoblasts. Cyclin D is consequently maintained during serum withdrawal, inhibiting progression through myogenic differentiation. Sustained nuclear localization of cyclin E, and a concomitant increase in cdk2 activity maintains S-phase entry in MyoD-/- myoblasts even in the absence of mitogens. Forced expression of IκBαSR, a non-degradable mutant of IκBα, rescued the deficit indicating that inhibition of NF-κB is sufficient to induce terminal myogenic differentiation. Therefore, MyoD-induced cytoplasmic relocalization of NF-κB is an essential step in linking cell-cycle withdrawal to terminal differentiation. Abbreviations bHLH, Basic helix-loop-helix; BrdU, Bromodeoxyuridine; FACS, Fluorescence activated cell sorting; IKK, IκB kinase; MEF, Mouse embryonic fibroblast; MRF, Myogenic regulatory factors; MyHC, Myosin heavy chain; NLS, Nuclear localization sequence; WT, Wild type; siRNA, Small interfering RNA. Competing interests The authors declare no competing interests. Authors’ contributions MHP and NB carried out the experiments and drafted the manuscript. BAJ and JM carried out the rescue experiments. JI performed the microarray experiment. DG provided essential reagents and participated in the design and coordination of the study. MAR 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. Acknowledgements We thank Mark Gillespie for critical reading of the manuscript. MAR holds the Canada Research Chair in Molecular Genetics and is an International Research Scholar of the Howard Hughes Medical Institute. 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Skelet Muscle. 2012 May 19; 2:6
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22629437PONE-D-11-2586710.1371/journal.pone.0037647Research ArticleBiologyComputational BiologyMolecular GeneticsGene ExpressionDevelopmental BiologyMolecular DevelopmentGeneticsEpigeneticsChromatinGene ExpressionChromatinMolecular GeneticsMolecular Cell BiologyChromosome BiologyChromatinGene ExpressionChromatinThe Msx1 Homeoprotein Recruits G9a Methyltransferase to Repressed Target Genes in Myoblast Cells Msx1 Recruits G9a to Target GenesWang Jingqiang Abate-Shen Cory * Departments of Urology and Pathology & Cell Biology and Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York, United States of America Spilianakis Charalampos Babis EditorUniversity of Crete, Greece* E-mail: [email protected] and designed the experiments: JW CAS. Performed the experiments: JW. Analyzed the data: JW CAS. Wrote the paper: JW CAS. 2012 22 5 2012 7 5 e3764723 12 2011 27 4 2012 Wang, Abate-Shen.2012This 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.Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development. ==== Body Introduction Cellular differentiation during development involves the coordinated change in expression of many thousands of genes in appropriate spatial and temporal contexts. A principal mechanism by which this occurs is through modification of the core histones (H3, H4, H2A, and H2B) that comprise nucleosomes, which are the fundamental units of chromatin. There are at least 8 distinct types of histone modifications, of which the most critical for transcriptional repression is lysine methylation, the enzymatic transfer of one or more methyl groups from the donor S-Adenosylmethionine (SAM) onto the ε-nitrogen of lysine [1], [2]. Genome-wide mapping studies using ChIP-Chip and ChIP-Seq have shown that di-methylation of histone H3 at lysine 9 (H3K9me2) is widely found at repressed genes during development and in embryonic stem cells [3], [4] while perturbation of the H3K9me2 mark results in a profound change in the repression status [5], [6]. The relevant histone lysine methyltransferase enzyme is G9a, a member of the highly conserved SET domain family, which, as part of a complex containing GLP, is responsible for catalyzing the H3K9me2 mark [7]–[11]. Thus, mutant mice lacking G9a are seriously impaired in the H3K9me2 mark and display embryonic lethality reflecting the consequences of global perturbation of gene repression [12]. Notably, the H3K9me2 mark, which is associated with transcriptional repression (regulated inhibition of gene expression), is distinct from tri-methylation of H3K9, which is associated with transcriptional silencing (permanent inhibition of gene expression), and is mediated by another SET domain protein, Suv39H1 [10], [13]–[15]. The homeoprotein family comprises one of the major classes of sequence-specific DNA binding proteins, which regulate gene expression and cellular differentiation during development. Among its members is the Msx1 homeoprotein, which is expressed in diverse spatial and temporal domains during development but restricted to cells that have not yet begun to differentiate [16], [17]. In the myogenic lineage, for example, Msx1 is expressed in myogenic precursors during development as well as in adult myogenic satellite cells (i.e., stem cells), but not in differentiated myotubes [18]–[20]. Forced expression of Msx1 in myoblast cells inhibits their differentiation [21], [22], which is mediated by the actions of Msx1 as a transcriptional repressor. In particular, Msx1 represses MyoD, which is a principal regulator of myogenic differentiation, by binding to a key regulatory element, the Core Enhancer Region (CER) [20], [22]–[25] that regulates the timing of MyoD expression in vivo [26]. Previously, we have shown that regulation of myoblast differentiation and repression of MyoD expression by Msx1 is correlated with increased repressor marks at the CER of MyoD [25], which include increased tri-methylation of H3K27 (H3K27me3) [23]. We now show that in myoblasts as well as the developing limb genomic binding by the Msx1 homeoprotein promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of the G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark [7]–[11]. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes. Based on our findings on the recruitment of the H3K9me2 mark, in conjunction with our recently published findings regarding the role of Msx1 in recruiting H3K27me3 to target genes [23], we describe four distinct categories of Msx1 target genes that are distinguished by differential recruitment of the relevant histone methyltransferases. Our findings suggest that an important means of regulating gene expression during development involves the differential recruitment of histone modifying enzymes to regulatory regions of target genes to influence chromatin status. Results Msx1 is Associated with H3K9me2 and Binds to G9a Following from our previous study showing that repression of MyoD by Msx1 is correlated with increased repressor marks at a key regulatory element, the CER [25] and associated with increased tri-methylation of H3K27 (H3K27me3) [23], we looked more generally at how Msx1 may influence the modification status of core histones on target genes in myoblast cells. We found that Msx1 associates specifically with H3K9me2 but not H3K9me3 in co-immunoprecipitation assays using proteins immunopurified from C2C12 cells (Figure 1A). Notably, the H3K9me2 mark, which is associated with repression, is distinct from tri-methylation of H3K9, which is associated with transcriptional silencing [10], [13]–[15]. 10.1371/journal.pone.0037647.g001Figure 1 Msx1 binds to G9a/GLP via the homeodomain and the C-terminal region. (A) Co-immunoprecipitation assays were done using C2C12 cell protein extracts expressing Flag-Msx1 and immunoprecipitated with anti-Flag followed by immunoblotting for the indicated histone marks. (B) 293T cells were transfected with the indicated expression plasmids. Proteins were immunoprecipitated with Flag antibodies and immunoblotted with anti-Flag or Anti-Myc, as indicated. (C) (Left) C2C12 cell protein extracts expressing Flag-Msx1 were immunoprecipitated with anti-Flag following by immunoblotting with antibodies to detect the indicated proteins. (Right) Limb extracts (11.5 dpc) expressing endogenous Msx1 were immunoprecipitated with anti-Msx1 antibody (4F11) following by immunoblotting with antibodies to detect the indicated proteins. (D) Truncated Msx1 proteins expressed in 293T cells were immunopurified with anti-Flag followed by immunoblotting to detect G9a. Shown is the quantitative analyses of G9a, normalize to G9a input and Msx1 input. In each immunoprecipitation assays, 1 mg of protein was used and input was 1% of the total protein. (E) Schematic representation of Msx1 and truncated derivatives showing a summary of data. Considering that Msx1 is associated with H3K9me2, we next asked whether Msx1 interacts with G9a, which is the enzyme that is responsible for this methyl mark. We found that both exogenous Msx1 expressed in C2C12 myoblast cells and endogenous Msx1 expressed in the developing limb interacted strongly with G9a, regardless of whether co-immunoprecipitation assays were done using antibodies to pull down Msx1 or G9a (Figure 1B and 1C). Msx1 also associated with GLP (Figure 1C), which forms a complex with G9a, but it did not interact with Suv39H1 (Figure 1C), which is responsible for tri-methylation of H3K9 and associated with gene silencing rather than repression [14]. Msx1 has multiple functional domains that mediate interactions with protein partners, DNA binding, transcriptional repression and/or sub-nuclear location [23]–[25]. Analyses of truncated Msx1 proteins lacking these various functional domains revealed that the homeodomain of Msx1 is the primary domain required for its interaction with G9a (Figure 1D and 1E). In particular, a truncated Msx1 protein lacking the homeodomain [Msx1(1–172)] did not interact with G9a, while various other truncated Msx1 proteins that contained the homeodomain but lacked for example domains required for repression Msx1(139–303); Msx1(1–239); and Msx1(1–271)] interacted with G9a, albeit with varying degrees of efficacy (Figure 1D and 1E). Notably the homeodomain is required for DNA binding by Msx1 but also mediates interactions of Msx1 with other protein partners [16], [20], [23], [27]. Taken together, these findings indicate that Msx1 associates with G9a histone methyltransferase via the homeodomain, although the important caveat to these studies in the possible influence of these altered domains on the structure of the protein overall. Msx1 Genomic Binding Associates with Enrichment of the H3K9me2 Repressive Mark Having established that Msx1 interacts with G9a, we next asked whether genomic binding by Msx1 is associated with increased levels of H3K9me2 on its repressed target genes in myoblast cells. In particular, we examined the status of H3K9me2 as a consequence of Msx1 expression at several sites on MyoD as well as several other myogenic regulators that are repressed by Msx1 [23], namely Myf5, Angpt1, Myc, Six1, and Snai2 [28]–[34] (Figure 2A and 2B). Interestingly, we found that binding by Msx1 resulted in increased levels of H3K9me2 on certain but not all of these genomic binding sites. For example, Msx1 binding was associated with increased H3K9me2 on the CER regions of MyoD (MyoD-4; P = 4.4×10−5) and the −58 kb site of Myf5 (Myf5-2; P = 2.4×10−7), both of which are known homeoprotein regulatory elements that control expression of these respective genes in vivo [26], [33], [34]. In contrast, H3K9me2 was not increased at the Msx1 binding sites of other target genes, such as Snai2 (Figure 2B). Interestingly, for the target gene, Six1, where Msx1 binds to two sites (Six1–3 and Six1–6), only one of the sites (Six1–6) was enriched for H3K9me2 (Figure 2B); notably, we have found that the other site (Six1–3) displays an Msx1-dependent enrichment for an alternative methyl mark, H3K27me3 [23]. Furthermore, the Msx1-dependent enrichment of H3K9me2 was well correlated with recruitment of G9a binding to these sites (Figure 2C). In particular, ChIP-qPCR analyses revealed that G9a binding was significantly enriched at relevant Msx1 binding sites, including the CER (MyoD-4; P = 0.001) and the −58 kb site of Myf5 (Myf5-2; P = 5.8×10−5), but not on its binding site on Snai2, which is also not enriched for H3K9me2 (Figure 2C, compare with Figure 2B). In the case of Six1, G9a was bound at only at the Msx1 site that was enriched for H3K9me2 (Six1–6; Figure 2B). 10.1371/journal.pone.0037647.g002Figure 2 Msx1 genomic binding associates with enrichment of the H3K9me2 repressive mark in myoblast cells. (A) Diagram of six Msx1 repressed target genes [23] showing the positions of Msx1 binding sites and known regulatory regions as well as their overlap; also shown is a negative control site. DRR: Distal Regulatory Region. (B) ChIP-qPCR analyses showing the relative levels of H3K9me2 at Msx1 genomic binding sites in C2C12 cells. ChIP data are expressed as relative enrichment of H3K9me2 normalized to input in C2C12 cells expressing or lacking Msx1. (Inset) ChIP data expressed as fold enrichment of H3K9me2 in C2C12 cells expressing Msx1 versus the control cells lacking Msx1 (and normalized to input). (C) ChIP-qPCR assays were done using C2C12 cells expressing Flag-Msx1 or a control vector to evaluate binding of G9a to the indicated Msx1 target sequences. ChIP data are expressed as relative enrichment of G9a binding normalized to input in C2C12 cells expressing or lacking Msx1. (Inset) ChIP data are expressed as fold enrichment of G9a binding in C2C12 cells expressing Msx1 versus the control cells lacking Msx1 (and normalized to input). In B and C, the * indicate the following: ***P<0.0001, **P<0.001, *P<0.01. To investigate whether these findings were also relevant for endogenous Msx1 in vivo, we performed ChIP analysis using limb from wild-type or Msx mutant embryos. We found that the levels of H3K9me2 were significantly reduced in the Msx mutant versus wild-type limb at the MyoD CER (MyoD-4; P = 1.7×10−5) and the −58 kb region of Myf5 (Myf5-2; P = 4.9×10−5) but not at Snai2-1 (Figure 3). Notably, similar to our findings in the myoblast cells, levels of H3K9me2 on the Six1 gene were significantly reduced in the Msx mutant versus wild-type limb at the Six1–6, but not at Six1–3 (Figure 3). Taken together, these data suggest that, for the endogenous protein in vivo as well as the exogenous protein in myoblast cells, Msx1 recruits G9a to selected genomic targets where it promotes enrichment of the H3K9me2 repressive mark in the vicinity of its binding. 10.1371/journal.pone.0037647.g003Figure 3 Msx1 genomic binding associated with enrichment of the H3K9me2 repressive mark in the developing limb. ChIP-qPCR analyses show relative levels of H3K9me2 at Msx1 genomic binding sites in Msx1; Msx2 conditional mutant versus wild-type limb (13.5 dpc). ChIP data are expressed as relative enrichment of the H3K9me2 mark normalized to input. (inset) ChIP data expressed as fold enrichment in wild-type embryonic limb versus Msx1; Msx2 conditional mutant embryonic limb (and normalized to input). The * indicate the following: ***P<0.0001, **P<0.001, *P<0.01. Association of Msx1 with G9a is Required for Regulation of Myoblast Differentiation We next investigated the consequences of Msx1 association with G9a for regulation of myogenic differentiation by evaluating the consequences of G9a knock-down in C2C12 myoblast cells. We used two independent G9a siRNAs and verified their efficacy and specificity in C2C12 cells using q-PCR to evaluate G9a mRNA levels and Western blotting to detect levels of G9a protein or levels of histone marks (Figure 4A-C); for most assays, only one siRNA is shown. The consequences of G9a knock-down for regulation of differentiation by Msx1 were evaluated by the appearance of myotubes and by Western blot detection of markers of terminal muscle differentiation, namely myosin heavy chain (MHC) and Myogenin (Figure 4D and 4E). Without exogenous Msx1 expression, C2C12 cells (Vector) were differentiated by 3 days after induction regardless of whether they expressed the control or G9a siRNA as evident by myotubes formation (Figure 4D, top panel) and expression of MHC and Myogenin (Figure 4E, left panel). However, as we have shown previously [21], [24], [25], Msx1 completely abrogates differentiation of C2C12 cells, as evident by the absence of myotubes (Figure 4D, left bottom panel) and lack of expression of MHC and Myogenin (Figure 4E, right panel). In contrast, depletion of G9a reverted these inhibitory effects of Msx1 on myoblast differentiation, as evident from the appearance of myotubes (Figure 4D, right bottom panel) and expression of MHC and Myogenin (Figure 4E, right panel). 10.1371/journal.pone.0037647.g004Figure 4 Association of Msx1 with G9a is required for regulation of myoblast differentiation. (A) Quantitative PCR to determine the level of G9a mRNA following knock-down by siRNA, mRNAs were prepared 48 hours after transfection with the indicated siRNA. (B) Western blot assay for efficiency of G9a knock-down, C2C12 cells were transfected with the indicated siRNA, extracts prepared 72 hours after transfection and analyzed by western blotting for the indicated proteins. (C) Western blot assay for histone marks. C2C12 cells were transfected with the indicated siRNA, extracts prepared 72 hours after transfection and analyzed by western blotting for the indicated histone marks. (D) Differentiation assay of C2C12 cells expressing (+) or lacking (-) Msx1 along with the control or G9a siRNA. Micrographs of C2C12 cells show the absence of myotubes in Msx1-expressing cells but not in cells also expressing the G9a siRNA on Day 3 after differentiation. (E) Western blots analyses of markers of terminal muscle differentiation, MHC and Myogenin on Day 3 after differentiation. (F) Immunofluorescence assays done on Day 2 after differentiation as detected using antibody for MHC and the nuclear marker TOPRO3. The scale bars represent 50 µm. (G) Quantitative analyses of myogenic index on Day 2 after differentiation. The percentage of MHC+ cells in vector cells with control siRNA was given a value of 100%. In A and G, the * indicate the following: ***P<0.0001, **P<0.001, *P<0.01. We quantified the differentiation status of the C2C12 cells at 2 days after induction of differentiation using an antibody for MHC to detect the MHC+ cells and determine the myogenic index [35]. In vector cells, depletion of G9a resulted in a higher myogenic differentiation compared with those expressing the control siRNA (Figure 4F and 4G), consistent with a role G9a in myogenic differentiation as reported recently [35]. However, in Msx1-expressing cells, depletion of G9a restored the myogenic differentiation nearly the levels without Msx1 (Figure 4F and 4G). These findings demonstrate that G9a is essential for Msx1 to inhibit myogenic differentiation in these myoblast cells. Association of Msx1 with G9a is Required for Transcriptional Repression We next investigated the consequences of Msx1 association with G9a for transcriptional repression following knock-down of G9a in C2C12 myoblast cells. Depletion of G9a significantly reduced Msx1 binding to genomic sites of repressed genes in myoblast cells in ChIP assays, as exemplified for the CER (MyoD-4; P = 1.1×10−7) and the −58 kb element of Myf5 (Myf5-2; P = 3.8×10−4) (Figure 5A). Interestingly however, analyses of Msx1 binding in vitro in gel shift assays showed that G9a knock-down did not inhibit binding by Msx1 to these genomic target sequences, namely the MyoD CER (MyoD-4) or Myf5-1 (Figure 5B). Notably, we have previously shown a distinction between binding of Msx1 in vitro and its ability to bind with target sequences in vivo, which is dependent on interactions with its protein partners in vivo [23], [24]. Thus, while G9a may not directly affect the affinity of Msx1 for its binding sites (Figure 5B), considering that depletion of G9a impaired in Msx1 ability to interact with genomic binding sites in vivo (Figure 5A), G9a may affect the ability of Msx1 to access authentic target sites in myoblast cells. 10.1371/journal.pone.0037647.g005Figure 5 Association of Msx1 with G9a is required for transcriptional repression. (A) ChIP-qPCR analyses showing relative Msx1 binding in C2C12 cells expressing or lacking Msx1 as well as a control or G9a siRNA. ChIP data are expressed as fold enrichment of Msx1 binding in C2C12 cells expressing Msx1 versus control cells lacking Msx1. (B) Gel shift analyses were done using nuclear extracts from C2C12 cells expressing Flag-Msx1 as well as the control or G9a siRNA with DNA sequences corresponding to the MyoD CER (MyoD-4) and Myf5-1. The * indicate the protein-DNA binding bands, the arrowhead indicates the free probes. (C) Expression levels of Msx1 target genes in Msx1-expressing or lacking Msx1 C2C12 cells also expressing the control or G9a siRNA. Data are expressed as the fold change of mRNA relative to that of the control siRNA cells. The * indicate the following: ***P<0.0001, **P<0.001, *P<0.01. (D) Western blots analyses showing levels of MyoD and Myf5 protein following by G9a knock-down in Msx1-expressing or lacking Msx1 C2C12 cells. Notably, the diminished binding of Msx1 to repressed genes in myoblast cells as a consequence of G9a knock-down was accompanied by a partial abrogation of repression of its target genes, including MyoD, Angpt1, Myc and Six1 following knock-down of G9a in Msx1-expressing but not the cells without Msx1 (Vector) (Figure 5C and 5D). The notable exception was Myf5, which was increased in expression at the protein and mRNA levels in the vector cells (lacking exogenous Msx1) following G9a knock-down (Figure 5C and 5D). Although depletion of G9a in C2C12 cells does not affect MyoD mRNA and protein levels (see Figure 5C and 5D), it may inhibit its transcriptional activity, in turn leading to up-regulation of Myf5 (Figure 5C and 5D, top panel), which is consistent with a recent study showing that G9a interacts with MyoD to constrain its transcriptional activity [35]. Finally, following G9a knock-down in Msx1 expressing C2C12 cells, we observed a significant reduction in the H3K9me2 mark, but not the H3K9me3 mark, on Msx1 target genomic binding sites such as CER (MyoD-4; P = 7.8×10−5) and the −58 kb region of Myf5 (Myf5-2, P = 4.2×10−4), as well as on the genes that are regulated by G9a but not Msx1 such as MageA2, Major satellite, and Wfdc15a, but not on a gene (GAPDH) that is not regulated by either Msx1 or G9a (Figure 6A–H) [6], [12]. Taken together, these findings demonstrate that the interaction of Msx1 with G9a in myoblast cells is essential for Msx1 to bind and repress target genes in cells and for enrichment of the H3K9me2 repressive mark at specific genomic binding sites. 10.1371/journal.pone.0037647.g006Figure 6 Binding analyses of H3K9 methyl marks in G9a knock-down cells. ChIP-qPCR assays were done using C2C12 cells expressing Flag-Msx1 as well as the control or G9a siRNA using anti-H3K9me2 or anti-H3K9me3 or control IgG, as indicated. ChIP data are expressed as relative enrichment normalized to input in control siRNA cells or G9a knock-down cells. The G9a associated with Redistribution of H3K27me3 by Msx1 to Genomic Sites Finally, since we have shown previously that repression by Msx1 is correlated with increased tri-methylation of H3K27 (H3K27me3) [23], we asked whether Msx1 association with G9a affected the H3K27me3 mark on target genes. Since depletion of G9a did not reduce the total level of H3K27me3 (Figure 4C), we asked whether Msx1 association with G9a is required for the Msx1-dependent redistribution of H3K27me3 on its genomic bound sites. In the presence of control siRNA, Msx1 promotes enrichment of the H3K27me3 repressive mark on its genomic sites, such as MyoD-2, MyoD-4, Myf5-1 and Myf5-2 [23] (Figure 7A). In contrast, genes that were neither bound nor regulated by Msx1, such as Dkk1, En2, and Irx1 had reduced levels of H3K27me3 in Msx1-expressing C2C12 cells with control siRNA [23] (Figure 7B). However in Msx1 expressing C2C12 cells, knock-down of G9a dramatically reduced the H3K27me3 mark on Msx1 genomic sites (Figure 7A), while it resulted in reversed the H3K27me3 reduction at Dkk1, En2, and Irx1 associated with exogenous Msx1 expression (Figure 7B). Furthermore, since the association of Msx1 with the nuclear periphery is associated with the redistribution of the H3K27me3 mark [23], we asked whether G9a affects the sub-nuclear location of Msx1. Strikingly, we found that depletion of G9a dramatically interrupted Msx1 nuclear periphery localization (Figure 7C). These findings suggest that Msx1 association with G9a contributes to the Msx1-dependent redistribution of H3K27me3 genomic bound sites by affecting the nuclear periphery localization of Msx1. 10.1371/journal.pone.0037647.g007Figure 7 G9a is required for Msx1-induced redistribution of H3K27me3 by Msx1 and localization of Msx1 at the nuclear periphery in C2C12 myoblasts. (A) ChIP-qPCR analyses of H3K27me3 mark on Msx1 target genes in C2C12 cells lacking Msx1 or expressing exogenous Msx1 also expressing the control or G9a siRNA. ChIP data are expressed as relative enrichment of the H3K27me3 mark normalized to input. (B) ChIP-qPCR analyses of H3K27me3 mark on genes not bound by Msx1 in C2C12 cells lacking Msx1 or expressing exogenous Msx1 also expressing the control or G9a siRNA. ChIP data are expressed as relative enrichment of the H3K27me3 mark normalized to input. (C) Immunofluorescence assays were done on C2C12 cells expressing exogenous Msx1 together with the G9a siRNA or a control siRNA and detected using antibodies for Msx1 or by detection of the nuclear marker TOPRO3. Quantitative analyses of nuclear localization for Msx1 using ImageJ show representative data from 3 independent assays, each counting a minimum of 20 cells per variable. The scale bars represent 5 µm. Discussion The impact of Msx1 on cellular differentiation during development is dependent on its ability to repress the expression of regulatory genes in specific cellular contexts, such as occurs for MyoD in cells of the myogenic lineage [22], [24], [25]. Our current findings suggest that these activities of Msx1 are mediated by its ability to directly influence the methylation status of nucleosomes on selected regulatory elements to which it is bound in myoblast cells (Figure 8). Our findings further indicate that this reflects, in part, recruitment of the G9a histone methyltransferase to these regulatory sites, which in turn promotes methylation of core histones in the vicinity of Msx1 binding (Figure 8; Table S1). Notably, recruitment of the G9a histone methyltransferase by Msx1 is mediated via the homeodomain, which is the defining feature of this family of sequence specific developmental regulators [16], [20], [22], [24], [25]. Implicit in this observation is that other homeoproteins may also recruit methyltransferase enzymes to target genes as a means of regulating their expression. 10.1371/journal.pone.0037647.g008Figure 8 Working model. As described in the text, we have proposed that binding of the Msx1 homeoprotein to specific target genes brings G9a and/or Ezh2 to the regulatory regions of these genes to influence histone modifications. According G9a and Ezh2 bound status, the Msx1 bound and down-regulated target genes were categorized in 4 categories. (A) Category I, Msx1 brings G9a and Ezh2 to the same site on target genes. (B) Category II, Msx1 brings G9a and Ezh2 to the same target genes but at different sites. (C) Category III, Msx1 only brings Ezh2 to the Msx1 bound site on target genes. (D) Category IV, Msx1 do not brings ether G9a or Ezh2 to the target genes, but Msx1 may brings other factors to Msx1 bound site to repress target genes expression. However, G9a is not recruited to all Msx1 target genes. Moreover, Msx1 also interacts via the homeodomain with another histone methyltransferase, Ezh2, which leads to enrichment of its respective histone mark, namely H3K27me3, on certain target genes (Figure 8; Table S1) [23]. In fact, we can identify 4 scenarios in which Msx1 differentially recruits histone methyltransferases to target genes to regulate their expression. In particular, in the first case Msx1 recruits both H3K9me2 and H3K27me3 repressive marks at the same binding site, as is the case for target genes, such as, MyoD, Myf5, Myc, and Angpt1 [18], [26], [28], [31], [33], [34]. In the second scenario, Msx1 recruits both H3K9me2 and H3K27me3 repressive marks to target genes, but at the different sites. For instance, Msx1 binds to two sites on Six1 (Six1–3 and Six1–6), one of the sites (Six1–6) is enriched for H3K9me2 while the other site (Six1–3) displays an Msx1-dependent enrichment for H3K27me3 [23], [29], [30]. In the third case, Msx1 only recruits the H3K27me3 mark, as in the case for Snai2, Met, and Id3 [32], [36], [37]. The final case is target genes that are bound by Msx1, but are not enriched for either H3K9me2 or H3K27me3, such as Clcn3 and Fgf7 [38], [39]. Implicit in this description is the important but yet unanswered question regarding how Msx1 recruits multiple histone methyltransferases to distinct target genes in specific spatial contexts. Therefore, Msx1 may interact with multiple histone methyltransferases to influence the expression of target gene in dynamic spatial contexts. Furthermore, since the region of methyltransferases recruitment is the homeodomain, a conserved motif, our findings raise the possibility that other homeoproteins may also function by promoting the recruitment of histone modifying enzymes to target genes. Thus, our findings demonstrate a novel means by which homeoproteins can regulate gene expression during development by interacting with histone modifying enzymes to directly influence the chromatin status of target genes. Materials and Methods Plasmids Most of the expression plasmids used in this study has been described previously [24], [25]. As indicated, pcDNA3 expression plasmids were used for transient transfection and pLZRS-IRES-GFP plasmids for retroviral gene transfer. Flag-tagged G9a was generated using PCR amplification and cloned into BamH I – Xho I sites of pcDNA3. The complete sequences of all PCR-amplified constructs were confirmed. Cell Culture Analyses Cell culture studies were done using human 293T cells (ATCC) or mouse C2C12 myoblast cells (ATCC). Cells were maintained in DMEM supplemented with 10% fetal bovine serum in humidified atmosphere with 5% CO2 at 37C. For myoblast differentiation assays, undifferentiated C2C12 cells were grown in media containing 10% fetal bovine serum, and differentiation was induced by shifting cells to media containing 2% horse serum 21,24,25. Transient transfection was performed using Lipofectamine 2000 reagent (Invitrogen). For retroviral gene transfer, replication-defective retroviruses were made in ecotropic Phoenix retroviral packaging cells (ATCC) by transfection of the relevant pLZRS-IRES-GFP plasmid derivatives using Lipofectamine 2000 reagent (Invitrogen). C2C12 myoblast cells were seeded 1 day before infection and infected with viral supernatants for two consecutive days. For siRNA, C2C12 cells were first infected with viruses expressing Msx1 or the empty vector and then transfected with siRNA against G9a (Ambion) using the Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s recommendations. For the control siRNA using Silencer® Select negative control #1 siRNA (Ambion). The sequences of G9a siRNA (Ambion) used in these studies are provided in Table S2. Quantitative analyses of differentiation of the G9a knock-down C2C12 cells were done by immunofluorescence staining with MHC antibody (see the below). Quantitative analyses of myogenic index were preformed on Day 2 after differentiation. Myogenic index calculation was done as described in [35]. At least 3 independent assays were done per variable, counting a minimum of 500 cells per variable for each independent assay. The percentage of MHC+ cells in vector cells with control siRNA was given a value of 100%. Analyses of Msx1 Mutant Embryos All experiments using animals were performed according to protocols approved by the Institutional Animal Care and Use Committee at Columbia University Medical Center. The Msx1 mutant were performed using a compound Msx1; Msx2 conditional allele [40] crossed to a ubiquitously-expressed tamoxifen-inducible Rosa26CreERT2 allele [41] to generate mice of the genotype Rosa26CreERT2/+; Msx1lox/lox; Msx2lox/lox. Targeted deletion was induced by delivery of tamoxifen in corn oil (2 mg/40 grams; Sigma-Aldrich) by oral gavage at embryonic day 9.5 (9.5 days post-coitum, dpc); targeted deletion in the tissue of interest (i.e., the limb) was confirmed by PCR analyses. The analyses described herein focused on mid-gestation embryos, from days 10.5 to 13.5 dpc, at which developmental stages the limb buds are maturing and Msx1 expression is robust in the limb mesenchyme [16], [42]. Embryos were collected from timed mating with noon on the day of the plug considered to be embryonic day 0.5; embryos were genotyped from yolk sac DNA. For ChIP assays, freshly dissected limbs were collected in PBS with protease inhibitor cocktail and disrupted using a Polytron homogenizer. Tissue extracts were cross-linked with 1% formaldehyde for 30 minutes at room temperature, cross-linking was stopped with 0.1 M Glycine, and cross-linked limb was collected by centrifugation and processed for ChIP-qPCR analyses as below. Real-time PCR for Gene Expression Real-time PCR was done using RNA isolated from C2C12 cells, using Trizol reagent (Invitrogen), and purified using an RNeasy kit (Qiagen). First strand cDNA was synthesized using SuperScript III kit (Invitrogen) and quantitative real-time PCR was performed using SYBR green reagent (Qiagen) in the Realplex2 machine (Eppendorf). Expression values were normalized to GAPDH. At least three independent experiments were performed for each gene. The average values are given as the mean ± SD. The primer sequences for real-time PCR using in this study were provided in Table S2. Chromatin Immunoprecipitation (ChIP) ChIP analysis was performed as described previously [24], [25]. Briefly, C2C12 cells expressing or lacking Msx1 and/or siRNA for G9a were cross-linked using 1% formaldehyde and genomic DNA was sonicated and then the relevant protein-DNA complexes were isolated by immunoprecipitation. ChIP analysis from limb, the cross-linked limb tissue was sonicated and then the relevant protein-DNA complexes were isolated by immunoprecipitation. The antibodies were used for ChIP assays were provided in Table S3. Quantitative real-time PCR was performed in triplicate using SYBR green reagent (Qiagen) using a Realplex2 machine (Eppendorf). At least three independent experiments were done for each assay. Primer sequences for ChIP-qPCR analyses were provide in Table S2. Immunoprecipitation and Western Blotting Analyses For Western blotting, C2C12 cells were lysed in RIPA buffer and proteins were analyzed using ECL plus Western Blotting Detection (GE Healthcare) by indicated antibodies (Table S3). For immunoprecipitation assays, C2C12 cells were lysed in RIPA buffer and proteins were immunoprecipitated by addition of antibodies [24], [25]. Where indicated, nuclear extracts from embryonic limbs were obtained by homogenization in hypotonic buffer (20 mM HEPES [pH 7.4], 5 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 1 mM DTT, and protease inhibitor cocktail) to isolate nuclei followed by extraction of nuclear proteins in buffer BC1000 (25 mM HEPES [pH 7.9], 10% glycerol, 0.2 mM EDTA, 1000 mM KCl, protease inhibitor cocktail, and PMSF) containing 0.1% NP40. The samples were then sonicated, centrifuged at 16,000 g, and then dialyzed against BC200 (25 mM HEPES [pH 7.9], 10% glycerol, 0.2 mM EDTA, 200 mM KCl, protease inhibitor cocktail, and PMSF) containing 0.1% NP40. Immunoprecipitations were done in BC200 containing 0.1% NP40 and immunoprecipitated proteins were analyzed using ECL plus Western Blotting Detection (GE Healthcare). The antibodies were used for immunoprecipitation and immunoprecipitation Western blotting were provided in Table S3. Gel Shift Assay The probe of MyoD CER (MyoD-4) and Myf5-1 were generated fragment from ChIP DNA by using PCR method with ChIP-qPCR primer sets (Table S2). For nuclear extracts, the C2C12 cells were homogenized in hypotonic buffer. The nuclei were incubated for 10 min on ice and then pelleted by centrifugation at 8,000 g, and resuspended in BC1000 buffer containing 0.1% NP40 to extract nuclear proteins. The samples were then sonicated, centrifuged at 16,000 g, and dialyzed against BC200 containing 0.1% NP40. The binding reaction was perform in 1× binding buffer (10 mM Tris-HCl pH 7.5, 5 mM NaCl, 0.7 mM MgCl2, 0.1 mM EDTA, 5% Glycerol, 0.05% NP-40, and 50 µg/ml BSA, 2.5 mM DTT) with 2 µg poly d(I-C), 50 ng probe, and 5 µg of nuclear protein from C2C12 cells expressing Flag-Msx1 as well as the control or G9a siRNA in total volume of 20 µl. Binding was done at room temperate for 20 min. Protein-DNA binding complexes were resolved by electrophoresis. After finished running the gel, the gel was stained by SYBR Safe DNA gel stain (Invitrogen) and the digital imagines were captured by CCD camera. Immunofluorescence Analyses Immunofluorescence analyses were done as described previously [23]. Briefly C2C12 cells seeded on 1-well BD Falcon™ CultureSlide and transfected with the indicated Msx1 plasmids and, where indicated, also with the siRNA for G9a. Cells were fixed in 4% PFA in PBS with 1% sucrose and permeabilized by incubation in an isotonic solution, 0.5% Triton X-100 (10% sucrose, 50 mM NaCl, 6 mM MgCl2, 20 mM HEPES [pH 7.2], 0.5% Triton X-100). After blocking with 1% BSA (bovine serum albumin) in PBS, cells were incubated for 1.5 hours at room temperature with primary antibodies. Following incubation with primary antibodies, samples were washed in PBS containing 0.1% Tween 20 followed by incubation for 1 hour with TOPRO 3 and AlexaFluor 488 and/or AlexaFluor 555 secondary antibodies (Molecular Probes). Immunofluorescence staining was visualized using a Leica TCS SP5 inverted confocal microscope equipped with argon/krypton and helium/neon lasers capable of excitation wavelengths 488, 555, and 642 nm. Details of primary antibodies are provided in Table S3. The Msx1 sub-nuclear localization was quantified using ImageJ (http://rsb.info.nih.gov/ij/) [43]. A line was drawn from the nuclear periphery to the nuclear center, and along this line, the fluorescence intensity was recorded; the pixel values versus radial position were used to generate the quantitative plot. Statistical Analysis At least three independent experiments were performed for each assay. The average values of the parallel experiments are given as the mean ± SD. Comparison of differences among the groups was carried out by Student’s t-test. Significance was defined as p<0.01. (***p<0.0001, **p<0.001, *p<0.01). Supporting Information Table S1 Summary of ChIP data for Msx1 bound and down-regulated target genes. (DOCX) Click here for additional data file. Table S2 List of primers used for ChIP-qPCR, RT-PCR and RNA interference. (DOCX) Click here for additional data file. Table S3 List of antibodies used in this study. (DOCX) Click here for additional data file. We thank Drs. Danny Reinberg and Raphael Margueron for providing reagents and many helpful discussions. We are grateful to Dr. Robert Maxson for providing the Msx1 and Msx2 conditional mice, and Dr. Yoichi Shinkai for providing G9a antibodies. We thank members of the Abate-Shen laboratories and Dr. Michael Shen for many discussions and helpful comments on the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was supported by The T.J. Martell Foundation for Leukemia, Cancer and AIDS Research, http://www.tjmartellfoundation.org (C.A.-S.) and by grant HD029446, http://www.nichd.nih.gov (C.A.-S.). ==== Refs References 1 Kouzarides T 2007 Chromatin modifications and their function. Cell 128 693 705 17320507 2 Shilatifard A 2006 Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression. 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PLoS One. 2012 May 22; 7(5):e37647
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22629354PONE-D-11-2324810.1371/journal.pone.0037102Research ArticleBiologyBiochemistryProteinsProtein InteractionsRegulatory ProteinsTransmembrane ProteinsBiomacromolecule-Ligand InteractionsEnzymesMicrobiologyBacterial PathogensGram NegativeMolecular Cell BiologySignal TransductionMembrane Receptor SignalingChemistryChemical BiologyOrganic ChemistryStereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing Stereochemical Insignificance in Quorum SensingGarner Amanda L. 1 2 3 Kim Sook Kyung 1 2 Zhu Jie 1 2 Struss Anjali Kumari 1 2 Watkins Richard 4 Feske Brent D. 4 Kaufmann Gunnar F. 1 2 Janda Kim D. 1 2 3 * 1 Departments of Chemistry and Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, United States of America 2 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America 3 The Worm Institute for Research and Medicine, The Scripps Research Institute, La Jolla, California, United States of America 4 Department of Chemistry, Armstrong Atlantic State University, Savannah, Georgia, United States of America Johnson Eric A. EditorUniversity of Wisconsin, Food Research Institute, United States of America* E-mail: [email protected] and designed the experiments: ALG SKK JZ AKS BDF GFK KDJ. Performed the experiments: ALG SKK JZ AKS RW BDF. Analyzed the data: ALG SKK JZ BDF GFK KDJ. Wrote the paper: ALG KDJ. 2012 22 5 2012 7 5 e3710221 11 2011 17 4 2012 Garner et al.2012This 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.Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria. ==== Body Introduction Bacteria exploit many mechanisms to gain advantage over environmental competitors and confer protection on themselves to ensure their survival. In Gram-negative bacteria, a class of autoinducers, the N-acyl-homoserine lactones (AHLs), have been identified as key mediators for cell-to-cell signaling, or quorum sensing, necessary for virulence factor expression and biofilm formation [1]–[3]. For example, in Pseudomonas aeruginosa, quorum sensing is mediated by two AHLs, N-(3-oxododecanoyl)-L-homoserine lactone 1 (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone 2 (Figure 1A) [4]–[6]. Similar to P. aeruginosa, Acinetobacter baumannii is also a nosocomial opportunistic pathogen and accounts for ∼10% of hospital-acquired infections [7]–[9]. This species has been linked to numerous types of clinical manifestations including wound, bloodstream and urinary tract infections, ventilator-acquired pneumonia, septicemia and necrotizing fasciitis [7]–[9]. Importantly, A. baumannii has been found to be prevalent in a large number of infection cases in wounded military personnel returning from combat in Iraq and Afghanistan earning it the nickname “Iraqibacter" [8], [9]. A. baumannii has proved to be a particularly daunting challenge as the bacteria easily adapt to variable conditions and are able to survive even in dry, desiccated environments [9]. Moreover, many strains of this species are gaining multidrug resistant phenotypes, including resistance to β-lactam antibiotics, quinolones and colistin; thus, new therapies are needed to target such infections [7], [9]. 10.1371/journal.pone.0037102.g001Figure 1 N-Acyl-homoserine lactone autoinducers. A, P. aeruginosa autoinducers. B, A. baumannii autoinducer. Recently, an autoinducer synthase (AbaI) and corresponding AHL signaling molecules were reported for A. baumannii [10]. The primary AHL isolated from this species was identified as N-(3-hydroxydodecanoyl)-L-homoserine lactone (3, 3-OH-C12-HSL, Figure 1B) by mass spectrometry and confirmed with a synthetic sample [10]. Although little is known regarding factors required for biofilm formation and virulence factor expression in A. baumannii [11], this process was found to be impaired in an abaI::Km mutant incapable of producing AHLs [10] indicating that quorum sensing signaling molecules, including AHL 3, may play a role in this process. To date, 3-hydroxy-substituted AHLs have been identified in a limited number of bacterial species [12]–[16]. With respect to AHL 3, this autoinducer has previously been identified as a quorum sensing signaling molecule in Vibrio scophthalmi [17], Yersinia pseudotuberculosis [18] and Acidithiobacillus ferrooxidans [19], each of which is unrelated to A. baumannii. Interestingly, despite the presence of a stereocenter at the 3-hydroxy position in 3, chirality requirements at this site in AHL 3 produced by A. baumannii have remained cryptic; however, the stereochemistry of the 3-OH position has been defined in 3-OH AHLs from other species [15], [16], [20], [21]. In most of these prior cases, the (R)-stereoisomer was identified as the natural stereoisomer and was found to exhibit greater autoinducer activity than the corresponding (S)-stereoisomer [15], [16], [20], [21]. The detection of (R)-stereochemistry at this position is not surprising, as AHL biosynthesis is believed to be stereoselective with respect to the 3-OH stereocenter, and FabG, the β-ketoacyl acyl carrier protein reductase of the fatty acid biosynthetic pathway, has been shown to selectively produce (R)-hydroxy substituents following reduction of the corresponding β-ketoacyl moieties [22]–[24]. Moreover, lactone stereochemistry has already been shown to be critical for autoinducer activity [25]. Thus, since molecular recognition in biological systems is generally highly stereospecific, we were interested in deciphering the stereochemical requirements at this position. To complement the lactone-focused stereochemical explorations, herein, we describe our efforts to synthesize each diastereomer of AHL 3 and examine their impact on quorum sensing in A. baumannii. From our studies, we have uncovered a case where stereochemical integrity does not affect the biological signaling process, thus, providing an example where a stereochemical center appears non-critical for biological activity. Results Synthesis of AHL 3 Autoinducers Our studies commenced with the chemical syntheses of both diastereomers of AHL 3 (Figure 2) (see Supporting Information for experimental detail). The syntheses began with commercially available fatty acid 4, which was first activated with DCC followed by nucleophilic displacement of the resultant activated ester with Meldrum's acid and coupling with l-homoserine lactone 5 to yield AHL 1. Enantioselective reduction of the 3-oxo-position was performed using Corey-Bakshi-Shibata reduction conditions to obtain AHL diastereomers 3a and 3b [26]. Mosher ester analysis [27] was used to confirm the stereochemical integrities of AHLs 3a and 3b (see Supporting Information for experimental detail and assignment: Scheme S1, Table S1, Figures S1, S2, S3, S4, S5, and S6). Since it was previously demonstrated that the stereochemistry of the lactone ring is key for activity in P. aeruginosa [28] among other Gram-negative bacteria [29], only the l-homoserine lactone ring was examined at this position. 10.1371/journal.pone.0037102.g002Figure 2 Synthesis of (R)-3-OH-C12-l-HSL (3a) and (S)-3-OH-C12-l-HSL (3b). Characterization of AHL 3 Autoinducers in A. baumannii With the diastereomerically pure lactones in hand, we examined their impact as autoinducers in A. baumannii. Autoinducer assays were conducted using an abaI::lacZ mutant A. baumannii strain wherein the abaI promoter is fused with a lacZ gene [10]. In this assay, since the abaI gene is activated in a positive feedback loop by an AbaI-dependent AHL signal, successful autoinducers will promote expression of lacZ. Mutant A. baumannii were treated with varying concentrations of AHLs 3a and 3b (0–100 µM), and β-galactosidase activity was measured using a luminescence-based assay (see Supporting Information). Unexpectedly, as Figure 3A shows, AHL diastereomers 3a and 3b exhibited nearly the same autoinducer activity with EC50 values of 0.67 ± 0.06 µM and 0.82 ± 0.06 µM, respectively, and no statistical significance was found between these values (p  =  0.3471). Of further significance, the activities of 3a and 3b were also nearly identical at lower, more physiologically relevant concentrations (Figure 3C). This is important, as receptors have typically evolved to specifically recognize one stereochemical form of a molecule. To underscore the relevance of this finding, in Vibrio harveyi [16] and Rhizobium leguminosarum [20], [21], their corresponding 3-OH AHLs were identified as possessing (R)-3-OH stereocenters, and in each case, the unnatural (S)-isomer exhibited little to no autoinducing activity. Moreover, previous studies in the AHL biosensor strains E. coli MT102 (pJBA132) and Pseudomonas putida F117 (pKR-C12) showed a difference in potency between (R)-3-OH-C8-HSL and (S)-3-OH-C8-HSL, albeit only at concentrations beyond 10 µM [15]. A similar finding was also reported earlier in Erwinia carotovora, and a>3-fold difference in activity was noted between the two diastereomers of 3-OH-C6-HSL [29]. Thus, our findings in A. baumannii appear to be unique. 10.1371/journal.pone.0037102.g003Figure 3 Autoinducer activities of AHL derivatives in an abaI::lacZ mutant A. baumannii strain. Values shown are relative luminescence units normalized with respect to cell viability (OD600). A, Autoinducer activities from 0–100 µM. Closed circle  =  3a. Open circle  =  3b. Closed triangle  =  1. Square  =  6. Open triangle  =  3-oxo-C12-d-HSL. B, Structure of N-(3-dodecenoyl)-l-homoserine lactone (6). C, Enhanced view of autoinducer activity of 3a and 3b at lower concentrations. Closed circle  =  3a. Open circle  =  3b. EC50 and p values determined using GraphPad Prism v5.0b for Mac OS X. In addition to AHLs 3a and 3b, although not produced by AbaI, AHL 1 was also found to exhibit autoinducer activity, but to a lesser extent (Figure 3A). Stereochemistry of the lactone ring, as expected, was found to be vital, and 3-oxo-C12-d-HSL showed diminished autoinducer activity (Figure 3A) in comparison to AHL 1. We also examined the effect of treatment with N-(3-dodecenoyl)-l-homoserine lactone (6, Figure 3B), a possible elimination product common to both AHLs 3a and 3b; however, no activity was observed with this compound (Figure 3A). In sum, these results indicate that an oxygen heteroatom is required at the 3-position, possibly due to hydrogen bonding interactions within the AHL 3 binding site in AbaR, and lactone stereochemistry is critical as previously found in other bacterial species utilizing AHLs. To determine whether AHLs 3a and 3b are acting on the same or different receptors in A. baumannii, the mutant strain was treated with varying concentrations of a 1∶1 mixture of AHLs 3a and 3b. As Figure 4 shows, no change in autoinducer activity was observed with this epimeric mixture. These results indicate that AHLs 3a and 3b act on the same receptor in an equipotent manner. This finding adds to the curiosity of our study, and may demonstrate that the lactone configuration (i.e., the “head") is more crucial for receptor binding and that “tail" substitution may provide only minor interactions within the AbaR AHL binding site, as both 3-hydroxy configurations and the 3-oxo moiety showed activity. 10.1371/journal.pone.0037102.g004Figure 4 Autoinducer activity of a 1∶1 mixture of 3a and 3b. Values shown are relative luminescence units normalized with respect to cell viability (OD600). Square  =  1∶1 3a∶3b. Closed circle  =  3a. Open circle  =  3b. Stability Studies of AHL 3 Autoinducers To ensure that the added AHL compounds were not being chemically modified during the course of our assay, the stability and chemical integrity of AHL 3 was assessed. It has been well documented that AHLs undergo lactone hydrolysis over extended incubation periods under physiological conditions [30]. Additionally, our group uncovered an additional side reaction of AHL 1, namely the formation of a tetramic acid via an intramolecular Claisen-like condensation reaction [31]. To determine the stability and possible side products of AHL 3, a derivative of this compound containing an aromatic moiety at the tail end (see Supporting Information and Scheme S2 for experimental detail) was used to aid in HPLC and LC-MS analyses. The compound was assayed in phosphate buffered saline (PBS) (pH 7.4) at 37°C over a period of 36 h. From this study, the half-life of AHL 3 was found to be approximately 20 h, which is similar to that previously reported for AHL 1 [32], and the only detectable side product resulted from lactone hydrolysis. Moreover, no epimerization was observed upon similar treatment of AHLs 3a and 3b. Thus, AHLs 3a and 3b are stable during the assay conditions, and are AHL signals recognized by A. baumannii. Characterization of AHL 3 Autoinducers in P. aeruginosa We also assessed the activities of AHLs 3a and 3b in Pseudomonas aeruginosa. Although 3-hydroxy-AHLs have not been isolated from this species, a previous study found that a racemic mixture of AHLs 3 was active as an autoinducer in P. aeruginosa; however, this compound was approximately 8-fold less potent than AHL 1 [33]. Moreover, as AHL 1 exhibited partial activity in our A. baumannii biochemical assays, we were interested to examine if any crossover activity existed with AHLs 3 [34]. Autoinducer activity assays were conducted using P. aeruginosa luminescence reporter strain PAO-JP2, a PAO1 lasI/rhlI double mutant (see Supporting Information) [35], [36]. Although AHLs 3a and 3b exhibited >200-fold diminished autoinducer activities with respect to AHL 1 (EC50 values of 3.13 µM, 2.60 µM and 12.6 nM, respectively), the activities of AHLs 3a and 3b were again nearly identical (Figure 5). These results further confirm the insignificance of stereochemistry at the 3-hydroxy position for quorum sensing and possibly implicate some evolutionary stereochemical promiscuity for 3-OH-AHL quorum sensing signaling molecules. 10.1371/journal.pone.0037102.g005Figure 5 Autoinducer activities of AHLs 3a and 3b in comparison to AHL 1 in P. aeruginosa strain PAO-JP2. Values shown are relative luminescence units normalized with respect to cell viability (OD600). Closed square  =  1. Closed circle  =  3a. Open circle  =  3b. Attempt at the Determination of 3-OH Stereochemistry of AHL 3 from A. baumannii In order to determine the natural stereochemistry of the 3-OH substituent of AHL 3 produced by A. baumannii, we utilized the recently reported protocol from Schulz and co-workers. Secreted AHL extract was obtained from an overnight culture of A. baumannii by extraction with acidified ethyl acetate (0.1% formic acid in ethyl acetate) as previously described [10]. Upon concentration of the crude extract, LC-MS analysis was performed to confirm the presence of AHL 3. To determine the stereochemistry of the 3-OH position, the homoserine lactone was hydrolyzed to the corresponding methyl ester using acidified methanol for analysis using chiral gas chromatography (GC) as previously reported by Schulz and co-workers. However, even with pure samples of 3a and 3b, the reported conditions resulted in racemization of the hydroxyl group. Silylation using BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) and TMCS (trimethylchlorosilane) and acetylation of the 3-OH group were also attempted; however, in each case, the resulting product failed to be detected by chiral GC. Discussion To try and elucidate this stereochemical anomaly, we first examined the reported crystal structures of 3-oxo-AHLs bound to quorum sensing-facilitating LuxR-type receptors [37]. While the binding interactions for the lactone and 1-oxo moieties are well conserved and would also likely be conserved in AbaR, deviations have been observed within the chemical sphere of the 3-oxo group [37]. In LasR, the AHL receptor in P. aeruginosa, the 3-oxo group of AHL 1 hydrogen bonds with Arg61 via a water molecule [37], [38]. However, in TraR, which recognizes 3-oxo-C8-HSL in Agrobacterium tumefaciens, the 3-oxo group hydrogen bonds with Thr129 and the main chain of Ala38 [37]. These differences cause the acyl chains to adopt different orientations in the hydrophobic binding tunnels of these receptor proteins. Thus, it is plausible that no conservation exists in the binding of the 3-oxo functionality and therefore, the binding site in AbaR readily accepts either stereoisomer. The structures of β-hydroxy- and β-ketoamides have also been studied previously using NMR spectroscopy [39], [40]. β-Ketoamides largely exist in the keto tautomeric form unlike their β-ketoester counterparts due to resonance stabilization by the more electron-donating nitrogen heteroatom. This conformation is further stabilized due to reduced steric interactions between the groups flanking the β-ketoamide motif [39], [40]. To minimize possible electrostatic repulsion between the two carbonyl groups, however, the keto tautomer is conformed such that the carbonyls are in an opposed orientation [39]. In fact, a similar orientation was observed in the crystal structure of AHL 1 bound to LasR [38]. Thus, there is little “cross-talk" between the lactone and β-ketoamide portions of AHL 1. As for β-hydroxyamides, interestingly, by NMR no internal hydrogen bonds have been observed between the hydroxy group and amide carbonyl, both in previous studies [40] and by us (data not shown). This implies that β-hydroxyamides exist in a trans-like configuration, and as such, AHLs 3a and 3b may be structurally similar to AHL 1 within the receptor's microenvironment with little binding site reorganization required to accommodate either 3-hydroxy stereoisomer. While A. baumannii is classified as a γ-proteobacteria of the order Pseudomonadales similar to P. aeruginosa, its quorum sensing genes are more closely related to those of environmental strains rather than pathogenic strains and little similarity exists between LasR and AbaR [11]. Phylogenetic studies have indicated that these genes were likely acquired horizontally from Halothiobacillus neapolitanus, a sulfur-oxidizing bacterial species of the order Chromatidales [11]. Interestingly, however, A. baumannii shows no evolutionary relationship with H. neapolitanus [11]. As Acinetobacter have been shown to readily acquire foreign DNA, including many multidrug resistance genes [41], we would suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous AHL-mediated quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria. This is particularly evident in light of our data demonstrating that AHL 1, which is not produced by A. baumannii, is still ∼50% active as an autoinducer. Although mixed A. baumannii biofilms have not been reported such as those identified with P. aeruginosa and Burkholderia cepacia [34], future research aimed at probing the validity of this hypothesis would be important to determine if A. baumannii engages “eavesdropping" to exacerbate its virulence [42]. Additional support for this could stem from the fact that A. baumannii was found to produce up to six different AHL signals (Figure 6); however, only one receptor has been identified based on genome mining. It remains to be seen if AbaR is its only quorum sensing receptor and what role other AHL signaling molecules play in this species. 10.1371/journal.pone.0037102.g006Figure 6 AHLs produced by A. baumannii. With respect to our findings, we would like to frame it within the context of other groups working in this area. Firstly, although the observed stereochemical insignificance is unique to AHLs, a similar finding was observed for CAI-1, a non-AHL-based autoinducer produced by Vibrio cholerae (Figure 7) [43]. While (S)-CAI-1 is the natural autoinducer, both stereoisomers (Figure 7) were generated synthetically and examined in a V. cholerae reporter strain [43]. Interestingly, similar to our findings, both the (R)- and (S)-stereoisomers exhibited very similar autoinducer activities [43]. However, these compounds are not AHLs; thus, our findings are unique for this class of autoinducer. In these regards, for AHLs, a (R)-stereocenter at the 3-OH position was observed in all prior stereochemical studies on this class of autoinducer, and the unnatural (S)-stereoisomer was found to exhibit little to no activity [15], [16], [20], [21]. Although we were not successful in discerning the stereochemistry of the 3-OH substituent of AHL 3 from A. baumannii cell culture, it is likely that it is the (R)-stereoisomer in agreement with these previous studies, as 3-OH AHL biosynthesis is believed to be stereoselective [22], [23]. Secondly, we would also like to bring to light the difference between our study and the unnatural AHL analogue syntheses being performed by several laboratories [44]. In our study, we have synthesized a single stereochemical isomer to understand the impact that this position has on quorum sensing in the bacteria from which it is produced, while Blackwell and others [44] are interested in the synthesis of unnatural AHLs through modification of both “head" and “tail" moieties to generate modulators, both agonists and antagonists, of quorum sensing. Thus, we view these studies to be fundamentally different. 10.1371/journal.pone.0037102.g007Figure 7 Structure of (S)-CAI-1 produced by V. cholerae and its stereoisomer. In conclusion, we have synthesized both diastereomers of 3-OH-C12-HSL, an autoinducer isolated from A. baumannii, in order to discern the impact that the 3-position stereochemistry in this AHL has upon quorum sensing. We have discovered that stereochemistry at this position does not appear to play a role in this bacterial signaling process, thus, providing a unique example of stereochemical insignificance for signaling activity. Moreover, as this phenomenon was observed in both A. baumannii and P. aeruginosa, it is possible that similar findings may be uncovered among other Gram-negative bacteria employing 3-OH-AHLs. The research developed herein is expected to facilitate studies toward developing strategies for combating A. baumannii infection including antibodies raised to sequester this quorum sensing signaling molecule [32], [45]. Methods General Materials and Methods The relative amount of β-galactosidase expressed in each sample was determined using the chemiluminescence-based detection kit Beta-Glo® Assay System (Promega, Madison, WI). All luminescence and absorbance readings were measured on a SpectraMax M2e Microplate Reader (Molecular Devices). All data was analyzed using GraphPad Prism version v5.0b for Mac OS X (GraphPad Software, www.graphpad.com). Data are represented as normalized with respect to the negative control. A. baumannii β-Galactosidase Assay A β-Galactosidase assay for A. baumannii using a abaI::lacZ mutant A. baumannii strain, containing an abaI promoter fused with a lacZ gene, was employed and adapted to 96-well plate format. Briefly, 200 µL of 1∶1000 diluted overnight culture in modified M9 media (0.2% glucose and 0.5% casimino acids) was added to wells of a 96-well microtiter plate, which were subsequently treated with various concentrations of AHLs at 37°C with shaking until the culture reached mid-log phase. The relative amount of β-galactosidase expressed in each sample was then determined. Following the manufacturer's manual, after equilibrating the cell culture to 25°C for 10 min, 50 µL of cell suspension was transferred from each well to a new opaque 96-well plate and mixed with an equal amount of assay solution. After 1 h incubation at 25°C in the dark, the development of luminescence signal was recorded in relative light units (RLU) and normalized by the original OD600 in each well. The ratio of luminescence/OD was plotted against the concentration of compounds to compare the relative activation level of abaI promoter. Each concentration of the dose-response curve was analyzed in triplicate simultaneously (i.e. on the same 96-well plate). Supporting Information Scheme S1 Mosher ester synthesis. (TIF) Click here for additional data file. Scheme S2 Synthesis of AHL 3 derivative for stability studies. (TIF) Click here for additional data file. Figure S1 Conformations used for the analysis of the Mosher esters of 3b. (TIF) Click here for additional data file. Figure S2 1H NMR spectral data of 3b-(S)-MTPA ester. (TIF) Click here for additional data file. Figure S3 1H NMR spectral data of 3b-(R)-MTPA ester. (TIF) Click here for additional data file. Figure S4 Preparative HPLC spectra of a mixture of 3a and 3b. (TIF) Click here for additional data file. Figure S5 1H NMR analysis. •  =  (S)-Mosher acid chloride ((R)-Mosher acid); □  =  DMAP; ◊  =  Triethylamine; Δ  =  3−[(Diethylamino)propyl]amine. (TIF) Click here for additional data file. Figure S6 1H NMR analysis. •  =  (R)-Mosher acid chloride ((S)-Mosher acid); □  =  DMAP; ◊  =  Triethylamine; Δ  =  3−[(Diethylamino)propyl]amine. (TIF) Click here for additional data file. Table S1 Δδ ( = δS−δR) data for the (S)- and (R)-MTPA- Mosher esters of 3b. (TIF) Click here for additional data file. We are grateful to Professor P. N. Rather (Emory University) for donation of the abaI::lacZ A. baumannii mutant strain and Professor M. G. Surette (University of Calgary) for donation of P. aeruginosa strain PAO-JP2. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by the National Institutes of Health (Grants AI079503 and DE18452 to KDJ, and Grants AI080715 and AI085324 to GFK) and The Skaggs Institute for Chemical Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Fuqua C Greenberg EP 2002 Listening in on bacteria: acyl-homoserine lactone signalling. Nat Rev Mol Cell Biol 3 685 695 12209128 2 Shiner EK Rumbaugh KP Williams SC 2005 Inter-kingdom signaling: deciphering the language of acyl homoserine lactones. FEMS Microbiol Rev 29 935 947 16219513 3 Waters CM Bassler BL 2005 Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21 319 346 16212498 4 Pearson JP Gray KM Passador L Tucker KD Eberhard A 1994 Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. 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PLoS One. 2012 May 22; 7(5):e37102
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22649503PONE-D-12-0763310.1371/journal.pone.0036941Research ArticleBiologyAnatomy and PhysiologySkinSkin AnatomyBiochemistryProteinsDNA-binding proteinsDevelopmental BiologyCell DifferentiationMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesDevelopmental SignalingGene ExpressionSuppression of AP1 Transcription Factor Function in Keratinocyte Suppresses Differentiation TAM67 Impact on AP1 SignalingHan Bingshe 1 Rorke Ellen A. 1 Adhikary Gautam 1 Chew Yap Ching 1 Xu Wen 1 Eckert Richard L. 1 2 3 * 1 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America 2 Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America 3 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, Maryland, United States of America Slominski Andrzej T. EditorUniversity of Tennessee, United States of America* E-mail: [email protected] and designed the experiments: BH EAR GA YCC RLE. Performed the experiments: BH EAR GA YCC WX. Analyzed the data: BH EAR GA YCC RLE. Contributed reagents/materials/analysis tools: BH EAR GA YCC RLE. Wrote the paper: BH RLE. 2012 23 5 2012 7 5 e3694114 3 2012 16 4 2012 Han et al.2012This 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 study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression. ==== Body Introduction Activator protein one (AP1) transcription factors are a family of jun and fos proteins that form jun-jun and jun-fos homo- and heterodimers, and these complexes interact with AP1 factor DNA binding sites to regulate gene expression [1]–[4]. The AP1 factor family includes c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2. These proteins are implicated in control of keratinocyte proliferation [5]–[7], differentiation [8]–[10], apoptosis [11], [12], and transformation [13]–[16]. The importance of these proteins is confirmed by in vivo studies [13], [17]–[25]. Analysis of the role of these proteins in epidermis is complicated because AP1 proteins display context-dependent functions and because multiple family members are expressed. An altered form of c-jun, which is truncated to remove the N-terminal transactivation domain, has been used to study AP1 factor function [26]. Deletion of the c-jun transactivation domain creates a dominant-negative form of the protein (TAM67) that inhibits AP1 factor function [26]. TAM67 has been used in a number of systems. TAM67 expression in lung cancer in mice [27], [28] and in nasopharyngeal carcinoma inhibits cell growth by altering cell cycle protein expression [29]. TAM67 inhibits growth of MCF-7 breast cancer cells [30], and halts HT-1080 cell proliferation in G1 phase [31]. TAM67 has also been used to study the impact of AP1 factor signaling on cell differentiation. Inhibition of AP1 factor function in neuroblastoma cells suppresses nerve growth factor-dependent differentiation [32]. In melanoma cells, induction of the melanoma differentiation associated genes is increased by AP1 factors and inhibited by TAM67 [33], and TAM67 also inhibits differentiation in monocytic leukemia cells [34]. We [35], [36] and others [37]–[43] have used TAM67 to study AP1 factor function in keratinocytes. These studies show that TAM67 inhibits keratinocyte differentiation [35], [36]. Cell culture based studies in human primary foreskin keratinocytes show that AP1 factors are required for expression of markers of terminal differentiation and that inhibition of AP1 factor function with TAM67 suppresses these responses [10], [36], [44]. We have also recently shown that expression of TAM67 in vivo in suprabasal mouse epidermis results in delayed and incomplete epidermal differentiation [35]. However, the molecular mechanism of TAM67 action in these models is not fully understood. In the present study we examine the mechanism of TAM67 action on AP1 factor function in epidermal keratinocytes. These studies indicate that TAM67 homodimer binds to AP1 factor DNA binding sites in human keratinocytes to inhibit jun and fos factor binding, and also reduces the mRNA and protein level of endogenous jun family members. In the case of c-jun this is via inhibition of transcription. Moreover, TAM67 binding to the AP1-5 binding site of the involucrin (hINV) promoter reduces expression of involucrin, a keratinocyte differentiation marker, in cultured keratinocytes. We further show that TAM67 in murine epidermis reduces involucrin (and loricrin) gene expression and reduces binding of endogenous AP1 factors to AP1 factor binding elements. Results TAM67 is a truncated form of c-jun that lacks the amino terminal transactivation domain and is not transcriptionally active [26] ( Fig. 1A ). In the present study we utilize TAM67 as a tool to study AP1 factor function in normal human keratinocytes. To initiate these studies, we monitored TAM67-FLAG expression. Fig. 1B shows that TAM67-FLAG is expressed in keratinocytes and Fig. 1C shows that, as expected of a nuclear transcriptional regulator, TAM67-FLAG accumulates in the nucleus. 10.1371/journal.pone.0036941.g001Figure 1 TAM67-FLAG expression in keratinocytes. A Comparison of c-jun and TAM67 structure. The numbers are indicated in amino acids. The transactivation, DNA binding and leucine zipper domains are indicated. The TAM67 truncated protein is FLAG epitope tagged as indicated. B/C TAM67-FLAG is expressed in keratinocytes. Normal human keratinocytes were infected with 10 MOI of tAd5-EV or tAd5-TAM67-FLAG with 5 MOI of Ad5-TA. After 24 h the cells were fixed for immunostaining and extracts were prepared for immunoblot with anti-FLAG. Similar results were observed in each of three repeated experiments. AP1 factors are key regulators of function in keratinocytes [45]–[47]. To understand the impact of TAM67 on AP1 factor function, we monitored endogenous AP1 factor level in TAM67-expressing cells. Fig. 2A shows a reduction in c-jun, junB and junD but no change in Fra-1, Fra-2 or c-fos level in TAM67 expressing cells, suggesting that TAM67 reduces the level of a subset of AP1 factors. To assess the mechanism causing c-jun, junB and junD reduction, we monitored mRNA level using quantitative RT-PCR. The level of c-jun, junB and junD encoding mRNA is reduced in TAM67 expressing cells, indicating that part of the reason for loss of these factors is a reduction in mRNA level ( Fig. 2B ). In contrast, the level of RNA encoding fos family members (Fra-1, Fra-2, c-fos) is not altered. We next examined the ability of TAM67-FLAG to interact with other AP1 factors by testing the ability of TAM67-FLAG to co-precipitate individual AP1 factors in keratinocytes. As shown in Fig. 2C , anti-FLAG precipitation of TAM67-FLAG co-precipitates Fra-1, Fra-2 and c-fos. In contrast, junB and junD did not co-precipitate, which is expected considering that these proteins are reduced in level in TAM67-expressing cells ( Fig. 2A ). In spite of the reduction in total c-jun level ( Fig. 2A ), sufficient c-jun appears to remain and interacts with TAM67-FLAG ( Fig. 2C ). We next monitored the impact on nuclear AP1 factor level. Fig. 2D shows that TAM67 expression is associated with reduced nuclear c-jun, junB and junD. In contrast, nuclear c-fos, Fra-1 and Fra-2 levels are not affected. 10.1371/journal.pone.0036941.g002Figure 2 Impact of TAM67-FLAG on AP1 factors. Keratinocytes were infected with empty (EV) or TAM67-FLAG encoding adenovirus and after 24 h cells were harvested and extracts prepared. A Total extracts were electrophoresed for immunoblot detection of the indicated proteins. B TAM67-FLAG suppresses jun factor mRNA level. At 24 h post-infection with EV or TAM67-FLAG encoding virus, mRNA was prepared for detection by quantitative PCR. The bars are mean ± SD and the asterisks indicate a significant reduction (p<0.005, n = 3). C Total extract (200 µg) was immunoprecipitated with anti-FLAG and the immunoprecipitate was electrophoresed for immunoblot to detect the indicated jun and fos proteins. D Nuclear extract was prepared and the level of each indicated protein was measured by immunoblot. Similar results were observed in three experiments. TAM67-FLAG Impact on c-jun Gene Expression As shown above, c-jun, junB and junD protein and mRNA levels are reduced in TAM67-expressing cells. To gain insight regarding the mechanism, we used c-jun as a model. We examined the impact of TAM67 on c-jun mRNA level and promoter activity in a side-by-side comparison. Fig. 3A shows that TAM67 reduces c-jun encoding mRNA by more than 50%. To gain insight into the mechanism, we monitored the impact of TAM67 on activity of a c-jun promoter construct in which nucleotides −1780/+731 is linked to luciferase ( Fig. 3B ). Our studies show that TAM67 reduces promoter activity by 50% in keratinocytes. In contrast, the same promoter in which the key AP1 factor binding sites are mutated, −1780/+731(AP1m) [48], displays basal activity and is not regulated by TAM67. Fig. 3C shows the structure of the promoter constructs. These findings suggest that c-jun level is reduced by a transcriptional mechanism that requires AP1 factor binding sites in the c-jun promoter upstream regulatory region. 10.1371/journal.pone.0036941.g003Figure 3 TAM67 suppresses c-jun promoter activity. A TAM67 reduces c-jun mRNA. Keratinocytes were infected with empty (EV) or TAM67-FLAG encoding adenovirus (10 MOI) and after 24 h mRNA was prepared and c-jun mRNA level was measured by quantitative PCR. B TAM67 suppresses c-jun promoter activity. Keratinocytes were transfected with 1 µg of the indicated c-jun promoter luciferase reporter construct and 1 µg of pcDNA3 (EV) or pcDNA3-TAM67-FLAG (TAM67-FLAG). After 24 h the cells were harvested and assayed for luciferase activity. The values in both plots are mean + SD and the asterisks indicate a significant reduction (p<0.005, n = 3). C Map of c-jun promoter region. The promoter constructs encode nucleotides −1780 to +731 with the transcription start site at +1. c-jun(−1780/+731) is the wild-type intact promoter and c-jun(−1780/+731)-AP1m is a construct in which the critical AP1 sites are eliminated by mutation [48]. LUC indicates the luciferase gene. The numbers are given in nucleotides. TAM67-FLAG Inhibits AP1 Factor Binding to AP1 Consensus DNA Binding Element Gel mobility shift and supershift analysis, using a consensus AP1 binding probe, was performed to investigate the effect of TAM67-FLAG on AP1 factor interaction with DNA. Fig. 4A shows a gel shift using 32P-labeled AP1 consensus binding site oligonucleotide and human foreskin keratinocyte nuclear extract. Shifted bands appear at increased intensity and free probe appears at reduced intensity in nuclear extracts prepared from TAM67-FLAG expressing cells (lanes 3, 5 and 9). This is caused by an increase in total cellular AP1 site binding capacity due to the presence of TAM67. As expected, addition of anti-FLAG results in appearance of a supershifted band only in extracts from TAM67-FLAG expressing cells (lane 5, asterisk). We next incubated nuclear extract from control and TAM67-expressing cells with anti-c-jun, junB, junD, Fra-1, Fra-2, c-fos or fosB. Supershifted bands are observed for each AP1 factor. The most obvious supershifts were observed for c-jun, junD, Fra-1, Fra-2 and c-fos ( Fig. 4B ). The amount of supershifted DNA is reduced in TAM67-expressing cells, demonstrating that TAM67 competes for endogenous AP1 factor binding to the AP1c element ( Fig. 4B ). We also examined the ability of TAM67 to form dimers. Nuclear extract from TAM67-FLAG expressing cells was crosslinked with disuccinimidyl suberate (DSS) prior to denaturing gel electrophoresis. Fig. 4C shows the presence of a TAM67 dimer, in DSS-treated extracts, migrating at 54 kDa. There are also higher molecular weight forms (brackets) which most likely represent TAM67 crosslinking to endogenous AP1 factors. However, the major band appears as a TAM67 homodimer. These findings suggest that TAM67 homodimers interact with AP1 binding sites to inhibit binding of endogenous AP1 factors. It also suggests that a TAM67 homodimer may be the major AP1 site interacting species in these cells. 10.1371/journal.pone.0036941.g004Figure 4 TAM67-FLAG inhibits AP1 factor binding to AP1 consensus DNA binding element. Keratinocytes were infected with 10 MOI tAd5-EV or tAd5-TAM67-FLAG and after 24 h nuclear extracts were prepared. A AP1 factors interact with AP1 consensus DNA element. Nuclear extracts were incubated with AP1c-P32 without or with a 50-fold molar excess of Sp1c or AP1c oligonucleotides, or anti-FLAG antibody and electrophoresed on a 6% acrylamide non-denaturing gel. FP indicates free probe and NE is nuclear extract. The arrow indicates the major shifted band and asterisks indicate migration of supershifted complexes. AP1c and Sp1c encode consensus AP1 and Sp1 binding elements. B TAM67-FLAG reduces AP1 factor binding to DNA. Nuclear extracts were incubated with AP1c-P32 in the absence or presence of c-jun, junB, junD, Fra-1, Fra-2, c-fos, or fosB antibodies, and electrophoresed on a 6% acrylamide non-denaturing gel. Arrows indicate shifted band and asterisks supershifted bands. FP indicates free probe. C TAM67-FLAG forms homodimers and heterodimers. Nuclear extracts were treated with or without DSS crosslinker prior to electrophoresis on a denaturing 8% polyacrylamide gel and TAM67-FLAG was detected by anti-FLAG immunoblot. Identical results were observed in three repeated independent experiments. TAM67-FLAG Inhibits hINV Gene Expression We next investigated the impact of TAM67 on AP1-regulated gene expression using involucrin (hINV) as an AP1 responsive gene [10]. Involucrin is a keratinocyte differentiation marker and is known to be increased by AP1 transcription factor signaling [25], [47], [49]–[51]. Control and TAM67-FLAG expressing keratinocytes were harvested and the level of hINV protein and mRNA was measured. Fig. 5A shows a TAM67-dependent reduction in hINV protein and mRNA. To assess the mechanism, we monitored the impact on hINV promoter activity. Three promoter constructs were used and keratinocytes were treated with TPA, a strong inducer of AP1-dependent hINV promoter activity [47], [49]. The hINV basal promoter, which encodes 41 nucleotides upstream of the transcription start site [47] and no AP1 sites, is not regulated by TAM67 or TPA ( Fig. 5B ). In contrast, pINV-241, which encodes the proximal regulatory region, and pINV-2473, which encodes both the proximal and distal regulatory regions [23], [47], are responsive to TPA and basal and TPA-stimulated promoter activity is inhibited by TAM67. The proximal and distal regulatory regions encode regulatory elements required for promoter activity in cultured keratinocytes [47] and involucrin expression in vivo [47], [49]. These experiments indicate that TAM67 inhibits differentiation-associated AP1-dependent transcriptional events in keratinocytes. 10.1371/journal.pone.0036941.g005Figure 5 TAM67-FLAG inhibits hINV gene expression. A TAM67 reduces hINV protein and mRNA level. Keratinocytes were infected with indicated MOI of tAd5-EV or tAd5-TAM67-FLAG and after 48 h extracts were prepared to detect hINV protein by immunoblot and mRNA by quantitative PCR. The values are mean ± SD and the asterisks indicate a significant reduction using student’s t-test, n  = 3 (p<0.001). B TAM67 suppresses AP1 factor-dependent promoter activity. Keratinocytes were transfected with the indicated hINV reporter constructs in the presence of empty pcDNA3 vector or pcDNA3-TAM67-FLAG and treated 24 h with or without 50 ng/ml TPA prior to preparation of extracts and assay of luciferase activity. The values are mean ± SEM and the asterisks indicate a significant reduction using student’s t-test, n = 3 (p<0.001). TAM67-FLAG Binds to the AP1-5 Site of hINV Gene Promoter AP1 factors regulate transcription of hINV via binding to the proximal and distal AP1 sites in the hINV promoter [25], [47], [49]–[51]. In particularly, the AP1-5 site in the distal promoter is absolutely required for involucrin gene expression in vivo [22]–[25]. We therefore examined the impact of TAM67 on AP1 factor interaction at the AP1-5 site. Fig. 6 shows gel mobility shift analysis of AP1 factor binding to the hINV promoter AP1-5 site. Fig. 6A shows that the presence of TAM67-FLAG markedly increases the intensity of the gel shifted band (compare lanes 2 and 3) and that incubation with anti-FLAG produces a strong supershifted band only in cells expressing TAM67-FLAG (compare lanes 4 and 5). Moreover, the binding is competed by incubation with a 50-fold molar excess of radioinert AP1-5 oligonucleotide (see lanes 6 and 7), but a 50-fold excess of AP1-5 m does not compete (lane 8). We next examined the impact of TAM67 on endogenous AP1 factor interaction with AP1-5. As shown in Fig. 6B , supershifted bands (asterisks) are observed when extracts are incubated with anti-c-jun, junB, junD, Fra-1, Fra-2 and c-fos, and this interaction is reduced in the presence of TAM67-FLAG. Fos-B was not detected. The low signal intensity of the shifted bands is consistent with previous reports [47]. 10.1371/journal.pone.0036941.g006Figure 6 TAM67 binds to the AP1-5 site of hINV gene promoter. Keratinocytes were infected with 10 MOI tAd5-EV or tAd5-TAM67-FLAG and after 24 h nuclear extracts were prepared for gel shift. A TAM67 interaction with hINV promoter AP1-5 site. Nuclear extracts were incubated with AP1-5-P32 with or without a 50-fold molar excess of AP1-5 or AP1-5 m oligonucleotide, or anti-FLAG antibody, and electrophoresed on a 6% acrylamide non-denaturing gel. FP indicates free probe and NE is nuclear extract. The arrow indicates the major shifted bands and asterisks indicate supershifted bands. AP1-5 is an oligonucleotide encoding the AP1-5 site of hINV promoter. AP1-5 m is an AP1-5 mutant that does not bind AP1 transcription factors [47]. B TAM67 inhibits AP1 factor interaction with AP1-5. Nuclear extracts were incubated with AP1-5-P32 in the absence or presence of c-jun, junB, junD, Fra-1, Fra-2, c-fos, or fosB specific antibodies, and electrophoresed on a 6% acrylamide non-denaturing gel. Arrows indicate major shifted band and asterisks indicate supershifted bands. FP is free probe. C ChIP analysis reveals TAM67 presence at the hINV upstream regulatory region AP1-5 site in vivo. Nuclear extracts were prepared for ChIP analysis and incubated with anti-IgG or anti-FLAG and the precipitated DNA was analyzed for AP1-5 site encoding sequences. The values are mean ± SD (n = 3, p<0.001) and the asterisk indicates a significant increase compared to all other groups. Nucleotides −2218/−2055 encodes the AP1-5 site and nucleotides −1040/−919 is a region of the hINV upstream regulatory region that lacks an AP1 site. To further assess the in vivo impact of TAM67 we used chromatin immunoprecipitation. Nuclear extracts from TAM67-FLAG positive and negative keratinocytes were prepared for ChIP analysis using a primer set that targets the AP1-5 binding site (−2218/−2005) and a second primer set that targets a region of the promoter lacking an AP1 factor binding site (−1040/−919). TAM67-FLAG and associated chromatin was precipitated with anti-FLAG. Fig. 6C shows that TAM67-FLAG is substantially enriched at the AP1-5 binding site (nucleotides −2218/−2055) as compared to the control DNA segment that lacks an AP1 binding site (nucleotides −1040/−919), suggesting TAM67 interaction at the hINV promoter AP1-5 site in vivo. TAM67 Impact on AP1 Factors in vivo We previously described TAM67-rTA mice in which TAM67-FLAG expression can be induced in the suprabasal epidermis by addition of doxycycline to the drinking water [35]. Expression of TAM67 in this tissue would be expected to reduce expression of AP1 factor-regulated genes. To assess this, we compared expression of two AP1-factor regulated genes, involucrin and loricrin [10], [52], [53]. TAM67-rTA mice were treated for three days with doxycycline and total epidermal extracts were prepared to detect involucrin and loricrin. Consistent with the finding that involucrin expression is reduced in TAM67-expressing cultured keratinocytes, we find that involucrin level is reduced in TAM67 expressing mouse epidermis ( Fig. 7A ). We also show that loricrin protein level is reduced. Loricrin expression is also AP1 factor signaling dependent [52]. 10.1371/journal.pone.0036941.g007Figure 7 Impact of TAM67 on AP1 factors in vivo. TAM67-rTA mice were treated with (+) or without (−) 2 mg/ml doxycycline in drinking water for 3 days. A Murine epidermis was collected free of the dermis by high temperature separation as previously described [35]. Total extract was prepared for immunoblot to detect the indicated proteins. TAM67-FLAG was detected with anti-FLAG. B Interaction of TAM67 with AP1 site consensus element. Nuclear extracts were prepared from epidermis and incubated with AP1c-P32 and other probes as indicated. FP indicates free probe, NE indicates nuclear extract. Similar results were observed in each of three experiments. C Impact of TAM67 on interaction of endogenous AP1 factors with AP1 site element. Nuclear extracts were prepared from TAM67-negative and TAM67-expressing epidermis and incubated with the AP1c-P32 and antibodies as indicated. The complexes were then separated on a non-denaturing 6% polyacrylamide gel. FP indicates free probe and NE is nuclear extract. Note the reduction in jun factor binding in the presence of TAM67-FLAG (left panel). We did not observe a significant reduction in fos factor interaction in the presence of TAM67 (right panel). We next examined the impact of TAM67 on endogenous AP1 factor DNA binding in mouse epidermis nuclear extracts. Fig. 7B shows an increase in the quantity of shifted AP1c-P32 probe in extract prepared from TAM67-expressing epidermis. This binding is specifically reduced by addition of excess radioinert AP1c, but is not competed by Sp1 consensus sequence. Moreover, TAM67-FLAG binding to AP1c-P32 is confirmed by anti-FLAG supershift ( Fig. 7B ). We also examined the impact of TAM67 on endogenous AP1 factor binding to DNA. The supershift analysis in Fig. 7C shows that TAM67 binding to the AP1 consensus element reduces c-jun, junB and junD interaction, with a strong reduction observed for junD. In contrast, Fra-2 and c-fos interaction is not altered by TAM67 and interaction of Fra-1 and FosB is below the limits of detection. Discussion We recently expressed dominant-negative c-jun in murine epidermis and observed significant changes in epidermal phenotype [35]. These changes included increased cell proliferation, delayed differentiation and reduced tumor formation [35]. We presume that TAM67 is impacting AP1 target genes in this tissue and so in the present study we examine the TAM67 mechanism of action in more detail. We studied the role of dominant-negative c-jun (TAM67) in human epidermal keratinocytes and in an in vivo murine keratinocyte model of differentiation. In cultured human keratinocytes TAM67-FLAG was detected in punctate foci in the center of the nucleus. Expression of TAM67 produced profound changes in AP1 transcription factor function. The first change we observed was a reduction in c-jun, junB and junD protein and mRNA level. The decrease in mRNA encoding the jun factors could be due to a reduction in mRNA stability or to a reduction in transcription. Further study with the c-jun promoter upstream regulatory region revealed a TAM67-dependent reduction in promoter activity. This reduction required the presence of AP1 transcription factor binding sites within the c-jun promoter. These findings are consistent with previous reports indicating that AP1 factor auto-regulate via a feedback loop [48], [54]–[57]. Our findings suggest that TAM67 binds to these elements, displacing other AP1 factors, and thereby suppresses c-jun transcription. In contrast, it is interesting that level of the fos family members (Fra-1, Fra-2 and c-fos) is not altered by TAM67. The loss of jun factors is also reflected in co-precipitation experiments. Fra-1, Fra-2 and c-fos co-precipitate with TAM67-FLAG, but junB and junD do not. Presumably, the reduction in junB and junD co-precipitation is due to reduced expression of these proteins. Surprisingly, c-jun, which is markedly reduced in level, does co-precipitate with TAM67. Perhaps c-jun homodimer formation is favored and TAM67, which retains the leucine zipper domain required for dimerization [26], may seek out and interact with residual c-jun in the cells. An interesting finding is that the population of jun family transcription factors is highly depleted in TAM67-positive keratinocytes. This feature has not been previously appreciated. Since AP1 factor signaling requires jun family members as dimerization partners for jun and fos, the absence of jun factors is expected to severely limit AP1 factor signaling. An equally interesting feature is that expression of fos family members is not reduced. This suggests that fos family proteins are not regulated by an AP1 factor-dependent feedback loop in this system. Second, we examined the impact of TAM67 on AP1 factor interaction with DNA. DNA gel shift experiments indicate that TAM67-FLAG interacts with the AP1 consensus DNA sequence, and that TAM67, at the level we achieve in these experiments, substantially reduces interaction of endogenous AP1 factors with DNA binding sites. Previous studies suggest that TAM67 forms transcriptionally inactive heterodimers with jun and fos family members [26]. These factors bind to the promoter of target genes, but are not able to activate transcription. This mechanism, called transcriptional quenching, leads to reduced target gene expression [26]. Our findings also suggest an additional mechanism. Protein-protein crosslinking and gel shift experiments strongly suggest that TAM67-FLAG homodimers are preferentially formed in these cells, and we suspect that this homodimer is a major AP1 factor complex that interacts with DNA. This would suggest that a major mechanism whereby TAM67 inhibits AP1 target gene expression in keratinocytes is TAM67 homodimer interaction with DNA to block endogenous AP1 factor access to these sites. It is also clear, as reported previously [26], that TAM67 forms heterodimers with jun and fos proteins, to form inactive complexes that quench activity of the complex. Thus, two mechanisms are possible: a blocking action wherein the TAM67 homodimer binds to DNA to block endogenous AP1 factor interaction with AP1 sites, and a quenching action wherein TAM67 inhibits the transactivation potential of endogenous AP1 factors by forming inhibitory TAM67:jun and TAM67:fos heterodimers ( Fig. 8 ). Our studies favor the blocking mechanism involving TAM67 homodimers. 10.1371/journal.pone.0036941.g008Figure 8 Mechanism of TAM67 action in keratinocytes. A Wild-type regulation involves the binding of fos:jun heterodimers (and jun:jun hetero and homodimers, not shown) to AP1 response element to drive differentiation-associated gene expression. Blocking occurs when the concentration of TAM67 present in the cells is high enough that TAM67 homodimers comprise the major complex bound to DNA and this complex blocks interaction of endogenous AP1 factors with the element. Quenching occurs when TAM67 complexes with endogenous jun and fos factors and this complex, which is transcriptionally inactive, binds to DNA. We propose that blocking is a major mechanism of TAM67 action in our system, but that quenching is also important. B TAM67 interaction at the promoter elements leads to blocking and quenching to reduce AP1 factor interaction and activity at AP1 binding sites. This leads to reduced expression of jun factors and ultimately reduced target gene (involucrin, loricrin) expression. In addition, we examined the impact of TAM67 on an important AP1 transcription factor-regulated target, involucrin. Involucrin is a marker of suprabasal differentiation in epidermis that is regulated by a MAP kinase cascade [36], [47]. Activation of this cascade leads to AP1 factor interaction with specific DNA binding elements on the hINV promoter to drive expression [23]–[25]. A key DNA binding site that is required for involucrin expression, both in cultured keratinocytes and in vivo, is the AP1-5 DNA binding site located in the distal regulatory region of the hINV gene promoter [36], [47], [50]. We show that TAM67 reduces hINV mRNA and protein level in cultured keratinocytes. Moreover, hINV promoter activity is also reduced, suggesting that TAM67 is inhibiting AP1 factor-dependent transcription. We confirmed TAM67 interaction with AP1-5 transcription factor binding site in the hINV promoter by gel mobility shift and chromatin IP. These findings confirm the important role of the AP1-5 binding site in driving hINV gene expression [22]–[25]. The fact that this is associated with reduced binding of AP1 factor at this site, as measured by gel mobility supershift assay, suggests that TAM67 is displacing these factors by competition. We also examined the impact of TAM67 expression on involucrin protein level in TAM67-expressing murine epidermis. We compared control mice (lacking TAM67 expression) and TAM67-expressing mice. These studies reveal a substantial reduction in murine involucrin protein in TAM67-expressing epidermis. This is associated with a 2 to 3-fold increase in transcription factor binding to the AP1 site in extracts prepared from TAM67-expressing epidermis. This increase is directly associated with increased TAM67 level, suggesting that TAM67 is a major factor interacting with the AP1 binding elements in the epidermis of these mice. TAM67 appears to readily compete jun family factors off from this site, but appears less efficient at competing fos family factors. We suspect that this is due preferred interaction with jun factors and to the somewhat lower level of TAM67 expression in mouse epidermis as compared to cultured keratinocytes. In summary, we describe several findings regarding the mechanism of TAM67 action in keratinocytes and in TAM67-expressing murine epidermis. First, our findings suggest that AP1 transcription factors regulate c-jun, junB, junD mRNA and protein level. Moreover, we show that TAM67 inhibits activity of the c-jun promoter, suggesting a transcriptional mechanism of regulation. Second, we show that blocking AP1 factor access to the hINV gene promoter AP1 factor binding site inhibits transcription, both in cultured human cells and in vivo in mouse epidermis. Third, this inhibition appears to be mediated by a “blocking” mechanism where a TAM67 homodimer interacts with the AP1 response element to suppress transcription by preventing endogenous jun and fos factor binding to the element ( Fig. 8 ). Crosslinking experiments suggest the presence of TAM67 homodimers as the major species present in keratinocytes. We suspect that the balance of TAM67 homodimers versus TAM67 heterodimerzation with endogenous jun and fos factors is depend upon the concentration of TAM67 expressed. At higher concentrations we would expect TAM67 homodimers to be the major species and that these factors will block endogenous AP1 factor interaction with DNA. An alternate mechanism, quenching, where TAM67 forms heterodimers with fos and jun proteins to produce a transcriptionally inactivate complex at AP1 DNA binding sites, is also likely. Crosslinking and co-immunoprecipitation experiments suggest some formation of TAM67 heterodimers with endogenous AP1 factors. An additional mechanism, called squelching (not shown), is also possible [26], [58], [59]. In this mechanism an inhibitor protein interacts with endogenous factors involved in transcription regulation that are not bound to DNA [26], [58], [59]. Although this may also be a mechanism of TAM67 inhibition, wherein TAM67 sequesters co-activator proteins away from the AP1 binding sites, we suspect that the major mechanisms whereby TAM67 suppresses gene expression in keratinocytes are AP1 site blocking and quenching ( Fig. 8A ) leading to suppression of jun factor and target gene (hINV, loricrin) expression ( Fig. 8B ). Our results suggest that these mechanisms are active both in cultures keratinocytes and in TAM67-expressing murine epidermis. Materials and Methods Cell Culture and Virus Infection Primary cultures of human newborn foreskin keratinocytes were cultured in keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor and bovine pituitary extract (10724, Gibco, Invitrogen, Carlsbad, CA). These are obtained as discarded tissue samples and their use was reviewed and approved in writing by the University of Maryland Human Subjects Institutional Review Board. For virus infection, cells were plated at 40% confluence (0.5 million cells per 21 cm2 dish) and infected with 0, 2 or 10 MOI of tAd5-EV or tAd5-TAM67-FLAG in the presence of 5 MOI of Ad5-TA virus in KSFM containing 6 µg/ml polybrene (H9268, Sigma, St. Louis, MO). After 6 h the cells were washed and shifted to fresh virus-free medium. Immunological Methods and Antibodies For immunoblot, keratinocytes were washed twice with phosphate-buffered saline (PBS), drained, and 0.5 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4 and 1 µg/ml leupeptin) supplemented with protease inhibitors was added to each 21 cm2 (60 mm) dish. After 5 min on ice, the cells were collected by scraping, and pelleted and sonicated. After centrifugation at 4°C and 15,000×g for 10 min, protein aliquots containing 30 µg of protein were separated on denaturing and reducing Laemmli [60] 8% polyacrylamide gels and transferred to nitrocellulose. The membrane was blocked in PBS containing 5% milk powder and 0.1% Tween 20, and incubated at 4°C overnight with primary antibody and for 1 h at 25°C with horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized using chemiluminescence detection reagent [61]. For anti-FLAG immunoprecipitation, keratinocytes were infected with 10 MOI of tAd5-EV or tAd5-TAM67-FLAG with 10 MOI of Ad5-TA. At 24 h, 200 µg of total cell extract was diluted to final volume of 500 µl in lysis buffer and pre-cleared by addition of 25 µl of protein A/G-agarose for 1 h at 4°C. The samples were then incubated with 20 µl of anti-FLAG M2 affinity gel (Sigma, A2220) overnight, and the antibody complex was washed three times with lysis buffer and boiled in 40 µl of Laemmli sample buffer for electrophoresis. Immunofluorescence Keratinocytes, growing on coverslips, were rinsed with PBS and fixed with 1∶1 acetone:methanol for 10 min at −20°C. Cells were washed three times in PBS for 5 min, and the coverslips were blocked in 3% bovine serum albumin in PBS for 1 h at room temperature and then incubated with monoclonal anti-FLAG M2 antibody (F3165, Sigma, diluted 1∶1000) for 1 h at room temperature. Coverslips were washed three times in PBS for 5 min each and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (A11029, Invitrogen, Eugene, OR, diluted 1∶1000) for 45 min at room temperature. Cells were further co-stained with 2 µg/ml Hoechst 33258 (H3569, Invitrogen) for 5 min, rinsed in PBS and place in mounting medium (M1289, Sigma). Fluorescence was visualized using an Olympus OX81 spinning-disc confocal microscope. No signaling was detected in the absence of primary antibody. Antibodies Rabbit polyclonal antibodies including anti-c-jun (sc-1694, diluted 1∶1000), anti-jun D (sc-74×, diluted 1∶500), anti-Fra-1 (sc-605×, diluted 1∶1000) and anti-Fra-2 (sc-604×, diluted 1∶1000), and mouse anti-junB (sc-8051, diluted 1∶300) and goat anti-fosB (sc-482, diluted 1∶300) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-c-fos (ab7963, diluted 1∶500) was from Abcam (Cambridge, UK). Monoclonal anti-FLAG M2-Peroxidase (A8952, diluted 1∶3000) and monoclonal anti-β-Actin (A1978, diluted 1∶3000) were purchased from Sigma (St. Louis, MO). Rabbit anti-human involucrin (hINV) serum (diluted 1∶2000) was produced in our laboratory [62]. Donkey anti-rabbit (NA934, diluted 1∶3000) and sheep anti-mouse HRP-conjugated secondary antibody (NA931, diluted 1∶3000) were from GE Healthcare (GE Healthcare, Piscataway, NJ). Donkey anti-goat HRP-conjugated secondary antibody (sc-2033, diluted 1∶3000) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-loricrin (PRB-145P, diluted 1∶1000) was obtained from Covance (Princeton, NJ). Nuclear Extract Preparation Keratinocytes (5×106 cells) were harvested with trypsin-EDTA, collected by centrifugation at 500×g and washed several times with PBS. Nuclear pellet and cytoplasmic fractions were prepared using Nuclear and Cytoplasmic Extraction Kit (78833, Pierce Biotechnology, Rockford IL) and stored at −80°C. For protein crosslinking, the pellet (nuclear fraction, 5×106 cell equivalents) was suspended in 100 µl of PBS (pH 8.0) containing 1 mM disuccinimidyl suberate (DSS, 21555, Pierce, Rockford, IL) and incubated for 10 min at room temperature. Tris-HCl (1 M, pH 7) was added to a final concentration of 10 mM to stop the reaction, and the protein samples were used for gel electrophoresis and immunoblot. To prepare nuclear extract from mouse epidermis, skin was removed and placed on ice and the epidermis was removed by scraping with a razor blade. Nuclear extract was prepared from the epidermal tissue using the nuclear and cytoplasmic extraction kit (78833, Pierce Biotechnology, Rockford, IL) and stored at −80°C. Chromatin Immunoprecipitation Assay (ChIP) ChIP assay was performed as described [63] with minor modification. Keratinocytes (5×105 cells) from a 35 mm dish were crosslinked with 1.42% formaldehyde at room temperature for 15 min followed by quenching with 125 mM glycine and then washed with ice cold PBS containing histone deacetylase inhibitors. The cells were then lysed in 150 µl of lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, 1 mM PMSF, 20 mM sodium butyrate, and protease inhibitors). Samples were chilled on ice and DNA was sheared using a Branson Sonifier (three 30-sec pulses on ice at 40% amplitude with 30 seconds between pulses to produce fragments of 1,000 bp). Four hundred microliters of RIPA buffer containing protease inhibitors and histone deacetylase inhibitors was added followed by a centrifugation of 12,000×g for 10 min. Aliquots of supernatant containing sheared chromatin were used for immunoprecipitation. Mouse monoclonal anti-FLAG (2 µg, F3165, Sigma, St. Louis, MO) was added to Dynabeads Protein A and incubated for 2 h at 4°C with rotation at 40 rpm. Sheared chromatin was added and mixture was incubated at 4°C overnight with rotation. The chromatin-antibody complex was washed twice with RIPA buffer and 40 µl of Chelex 100 slurry (10% wt/vol) was added to the washed beads prior to boiling for 10 min. The samples were then treated with proteinase K for 30 min at 55°C and boiled for 10 min. Enrichment of TAM67-FLAG-associated DNA sequences in immunoprecipitated samples and input samples were detected by quantitative RT-PCR using sequence specific primers and LightCycler 480 SYBR Green I Master mix. ChIP primers included hINV promoter AP1-5 (nucleotides −2218/−2055) forward: 5′-TCAGCTGTATCCACTGCCCTCTTT-3′ and reverse: 5′-TCACACCGGTCTTATGGGTTAGCA-3′ primers, and hINV promoter control (nucleotides −1040/−919) forward: 5′-CCTCTCAGGGAGAGATTGACATGA-3′ and reverse: 5′-CAACAGTGCACCAGCACACTTGAA-3′ primers [23]. Gel Mobility Shift Assays Cells were washed with PBS for preparation of nuclear extract using NE-PER Nuclear and Cytoplasmic Extraction Reagent (78833, Pierce Biotechnology, Rockford, IL). Binding of transcription factors to double-stranded AP1 consensus (AP1c) oligonucleotide 5′-CGCTTGATGAGTCAGCCGGAA-3′ (E320A, Promega, Madison, WI, AP1 site in bold) or hINV AP1-5 probe which encodes the AP1-5 binding site in upstream regulatory region of human involucrin promoter, 5′-CTTAAGGCTCTTATTATGCCGTGAGTCAGAGGGCGGGAGGCAGATCT-3′ (AP1 site in bold, Sp1 site underlined) [50], was monitored by gel mobility shift assay. Three micrograms of nuclear extract was incubated for 30 min at room temperature in a volume of 20 µl containing 20 mM HEPES, pH 7.5, 10% glycerol, 50 mM KCl, 2 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 1 µg/ml poly(dI:dC), 0.1 mg/ml bovine serum albumin, and 40,000 cpm radioactive double-stranded AP1c-P32 or AP1-5-P32 oligonucleotide. For competition studies, a fixed molar excess of non-radioactive competitor oligonucleotide was added to the DNA binding reaction. These competitors included Sp1 consensus oligonucleotide (Sp1c) 5′-ATTCGATCGGGGCGGGGCGAGC-3′ (E323, Promega, Madison, WI, Sp1 site underlined), AP1c and AP1-5 m/Sp1 m, 5′-CTTAAGGCTCTTATTATGCCGTGAGCCAGAGTCA AGGAGGCAGATCT-3′ (AP1 site in bold, Sp1 site underlined, mutant nucleotides shaded) [50]. For gel mobility supershift assay, 2 µg of antibody specific for FLAG (F3165, Sigma, St. Louis, MO) or c-jun (sc-45×), junB (sc-46×), junD (sc-74×), Fra1 (sc-605×), Fra-2 (sc-604×), c-fos (sc-253×) and fosB (sc-48×) was added to the reaction mixture and incubated 1 h at 25°C. The 32P-labeled probe was then added and the incubation was continued for an additional 30 min at 25°C. Protein-DNA complexes were resolved in 6% polyacrylamide gels under nondenaturing conditions [8], [61]. Quantitative RT-PCR Total RNA was extracted using Illustra RNAspin Mini Isolation kit (25-0500-70, GE Healthcare) according to instructions. One microgram of total RNA was reverse-transcribed to cDNA using Superscript III reverse transcriptase (18080-093, Invitrogen Inc.) and random primers (10814270001, Roche, Indianapolis, IN). Gene expression was measured by quantitative PCR using Roche LightCycler 480 System and SYBR Green reagents (LightCycler 480 SYBR Green I Master, 04 707 516 001, Roche). RNA level was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Relative mRNA level was analyzed by the comparative CT method. The primers, designed to detect the indicated genes in mRNA isolated from human keratinocytes, include (forward/reverse) glyceraldehyde-3-phosphate dehydrogenase 5′-TCCACTGGCGTCTTCACC-3′/5′-GGCAGAGATGATGACCCTTT-3′; c-fos 5′-TGTCTGTGGCTTCCCTTGATCTGA-3′/5′-TGGATGATGCTGGGAACAGGAAGT-3′; Fra-1 5′-CTGTGCTTGAACCTGAGGCA-3′/5′-GGTGAAAGGAGTTAGGGAGGGT-3′ [64]; Fra-2 5′-CCCTGCACACCCCCATCGTG-3′/5′-TGATTGGTCCCCGCTGCTACTGCTT-3′ [65]; c-jun 5′-GTACCTGATGAACCTGATGC-3′/5′-GGTCACAGCACATGCCACTT-3′ [66]; junB 5′-GTCACCGAGGAGCAGGAGG-3′/5′-TCTTGTGCAGATCGTCCAGG-3′ [66]; junD 5′-AAGACCCTCAAGAGTCAGAACACG-3′/5′-TGTTGACGTGGCTGAGGACTTTCT-3′; and involucrin 5′-CCTCAGCCTTACTGTGAG-3′/5′-GGGAGGCAGTGGAGTTGG-3′. hINV and c-jun Promoter Activity Human involucrin hINV reporter plasmids, encoding various lengths of hINV promoter upstream regulatory region fused to the luciferase reporter gene have been described [36], [47]. We used hINV promoter constructs, pINV-2473, pINV-241, and pINV-41, which include nucleotides −2473/−7, −241/−7 and −41/−7, respectively, of the hINV promoter linked to the luciferase reporter gene [47]. TAM67 expression plasmid was pcDNA3-TAM67-FLAG. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained from Sigma (St. Louis, MO). For experiments, 2×105 cells were seeded into 35 mm dishes 24 h before transfection. For transfection, 6 µl of Fugene-6 reagent (11 814 443 00, Roche, Indianapolis, IN) was mixed with 94 µl of KSFM, and incubated at 25°C for 10 min. This mixture was then added to 1 µg of hINV plasmid and 1 µg of TAM67-FLAG expression plasmid and incubated at 25°C for 20 min followed by direct addition to cultures containing 2 ml of KSFM. The final DNA concentration in all groups was maintained constant by addition of empty expression vector. At 24 h after transfection, 2 ml of fresh medium was added containing 0 or 50 ng/ml TPA. After an additional 24 h, the cells were washed with PBS and scraped into 200 µl of cell lysis buffer, and luciferase activity was assayed immediately. All assays were performed in triplicate, and each experiment was repeated a minimum of three times. Luciferase activity was normalized per microgram of protein. Promoter activity experiments were also performed in keratinocytes using c-jun promoter luciferase reporter constructs c-jun(−1780/+731) and c-jun(−1780/+731)-AP1 m which encode nucleotides −1780/+731 of the human c-jun promoter and upstream regulatory region [48]. The latter construct is identical except that the AP1 sites in the c-jun upstream regulatory region are mutated [48]. TAM67-rTA Transgenic Mice The TAM67-rTA mice are maintained in the genetic background as previously described [35]. These mice harbor a transgene that encodes TAM67-FLAG linked to a tetracycline-inducible promoter. Epidermis-specific TAM67-FLAG expression is induced by addition of 2 mg/ml doxycycline in drinking water and expression is maximal within two day [35]. A FLAG epitope is included at the carboxyl terminus of TAM67 so that expression can be easily monitored. For the experiments outlined in the present study we utilize 20 wk old female mice from TAM67-44 strain [35]. Epidermal extracts were prepared for gel mobility shift or immunoblot after a three day treatment with doxycycline. Mice were maintained in the University of Maryland School of Medicine animal facility in compliance with NIH regulations with laboratory chow and water accessible ad libitum. The study was approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. We thank Dr. Wayne V. Vedeckis, Department of Biochemistry and Molecular Biology, Louisiana State Medical Center, for providing the c-jun promoter constructs. Competing Interests: Richard L. Eckert is a PloS ONE Editor Board member, but this does not alter his adherence to all of the PloS ONE policies on sharing data and materials, as detailed online in the guide for authors. The other authors have declared that no competing interests exist. Funding: This work was supported by National Institutes of Health (NIH) grant R01 AR046494 awarded to Richard L. Eckert. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Angel P Szabowski A Schorpp-Kistner M 2001 Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene 20 2413 2423 11402337 2 Karin M Liu Z Zandi E 1997 AP-1 function and regulation. Curr Opin Cell Biol 9 240 246 9069263 3 Shaulian E Karin M 2002 AP-1 as a regulator of cell life and death. Nat Cell Biol 4 E131 E136 11988758 4 Shaulian E Karin M 2001 AP-1 in cell proliferation and survival. 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PLoS One. 2012 May 23; 7(5):e36941
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22662229PONE-D-12-0558610.1371/journal.pone.0037804Research ArticleBiologyComputational BiologyGenomicsGenome SequencingEvolutionary BiologyPopulation GeneticsGenetic PolymorphismMutationMicrobiologyVector BiologyLiceEmerging Infectious DiseasesPopulation BiologyEpidemiologyInfectious Disease EpidemiologyEvidence for an African Cluster of Human Head and Body Lice with Variable Colors and Interbreeding of Lice between Continents Study of Human Lice Phenotypic and Genotypic DataVeracx Aurélie 1 Boutellis Amina 1 Merhej Vicky 1 Diatta Georges 2 Raoult Didier 1 * 1 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UMR CNRS 6236, IRD 198, IFR48, Faculté de Médecine, Université de la Méditerranée, Marseille, France 2 Institut de Recherche pour le Développement, Campus International de Recherche IRD/UCAD, Hann, UMR 198 URMITE, Dakar, Senegal Knight Matty EditorBiomedical Research Institute, United States of America* E-mail: [email protected] and designed the experiments: DR. Performed the experiments: AV AB GD. Analyzed the data: AV VM DR. Wrote the paper: AV AB VM GD DR. 2012 25 5 2012 7 5 e3780413 2 2012 24 4 2012 Veracx et al.2012This 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 head lice and body lice have been classified based on phenotypic characteristics, including geographical source, ecotype (preferred egg laying site hair or clothes), shape and color. More recently, genotypic studies have been based on mitochondrial genes, nuclear genes and intergenic spacers. Mitochondrial genetic analysis reclassified lice into three genotypes (A, B and C). However, no previous study has attempted to correlate both genotypic and phenotypic data. Materials and Methods Lice were collected in four African countries: Senegal, Burundi, Rwanda and Ethiopia and were photographed to compare their colors. The Multi-Spacer-Typing (MST) method was used to genotype lice belonging to the worldwide Clade A, allowing a comparison of phenotypic and genotypic data. Results No congruence between louse color and genotype has been identified. Phylogenetic analysis of the spacer PM2, performed including lice from other sources, showed the existence of an African cluster of human lice. However, the analysis of other spacers suggested that lice from different areas are interbreeding. Conclusions We identified two geotypes of Clade A head and body lice including one that is specifically African, that can be either black or grey and can live on the head or in clothing. We also hypothesized that lice from different areas are interbreeding. ==== Body Introduction Humans are infested by two genera of lice: Pthirus and Pediculus. The Pediculus genus has been studied for decades and is classified based on its ecology, shape and color [1]–[14]. Two ecological forms of Pediculus lice may be distinguished: head lice and body lice. The head louse, Pediculus humanus capitis, lives and lays its eggs on hairs, whereas the body louse, Pediculus humanus humanus, lives and lays its eggs in clothing [7]. Differences in shape between head and body lice have also been described, but these criteria have not been shown to be relevant enough to divide the two into distinct species [12]. Also, louse coloration was described in the beginning of the 20th century, and it was noted that lice have different colors depending on their geographic region and the color of their host's skin [3], [8], [9], [11]. A series of gradations between the black head or body louse of West Africa and the light dirty grey head or body louse of Europe was described [10], [13], [14]. Later, researchers began performing genetic studies on lice [15]–[23]. First, a study based on the 18S ribosomal RNA gene reported that head and body lice were not phylogenetically distinct. In fact, two phylogenetic groups were described: the Sub-Saharan African lice and other lice that are distributed elsewhere worldwide. Each of these groups contained two distinct subgroups: head and body lice [23]. The divergence of human head and body lice was considered to be a recent event occurring within each of the geographical groups. A second phylogenetic study was based on mitochondrial genes [21], [22]. This allowed the description of three phylogenetically different clades of lice: the “most common worldwide clade”, which comprises both head and body lice (called Clade A), the “head lice only clade”, found in America, Europe and Australia (called Clade B), and “another head lice only clade”, which was first found in Nepal and Ethiopia [21] but was also recently found in Senegal [24] (called Clade C). Finally, another phylogenetic study of Clade A lice based on intergenic spacers (Multi-Spacer-Typing method or MST) reported two clusters of lice: Non-African lice (which we will call A1) and African lice (which we will call A2) [20]. Since then, there have been no studies aiming to correlate phenotype and genotype in human lice. Therefore, we wanted to examine both aspects to determine whether there is any correlation among the color, geographical source, ecotype (head and body) and phylogeny of lice. For this approach, we used Clade A lice collected in Senegal, Burundi, Rwanda and Ethiopia. They were photographed and then genotyped with the MST method [20]. To complete the phylogenetic analysis of our data, we also used previously genotyped Clade A lice from African and Non-African regions [20]. Materials and Methods Ethics statement Informed verbal consents were obtained from all the participants involved in our study. All participants of this study were illiterate. It is why a verbal informed consent procedure was preferred over written consent. The Ethic Committee of the Institute Fédératif de Recherche approves this verbal consent procedure as it is in accordance with the French Bioethics law N° 2004-800 60 (06/08/2004). Dr Oleg Mediannikov participated in the collection and was a witness of the participant's consents. Local authorities (village chief) approved, and were also present. This study was approved (Agreement #12-004), by the Ethic Committee of the Institute Fédératif de Recherche 48, Marseilles, France since this study was a non-interventional epidemiological research study as a part of the French Bioethics law N° 2004-800 (06/08/2004). The poorest districts of Dakar were chosen because of the highest possibility to find lice. Girls are more likely to have long hairs (boys are often shaved). Lice were anonymized before processing for genetic analysis. The study In this study, we had four parameters to compare: ecology, geographical origin, phylogeny based on 4 spacers and color. So we decided to sequence only Clade A lice because this is the only clade that comprise both head and body lice and therefore is a specific problem of public health as body lice are vectors of outbreaks. Lice from four countries were genotyped with the MST method including head lice from Senegal collected in October and November 2010 [24], body lice from Southern Ethiopia collected in 2010 [25], body lice from Rwanda collected in 2011 (three lice per individual) and both head and body lice from Burundi collected in 2008 (three lice of each ecotype per individual). Altogether, 19 Clade A lice were used in this study (Table 1). All lice were stored at −20°C before DNA extraction with the QiAamp Tissue kit (QIAGEN, Hilden, Germany). Senegalese and Rwandan lice were photographed with a camera (Olympus DP71) fixed on a low power stereo microscope (Olympus SZX16). The lice from Ethiopia and France were photographed in the field (with a Nikon D90) either in the hand of one of the authors (Ethiopia) or directly on the clothes of the infested person (France). Four intergenic spacers (S2, S5, PM1, PM2) known to be very polymorphic [20] were used in this study. The sequencing was performed following the protocol previously described [20] with some minor modifications. Briefly, the PCR reactions were prepared on ice and contained 3 µl of the DNA template, 4 µl of Phusion HF Buffer, 250 µM of each nucleotide, 0.5 µM of each primer, 0.2 µl of Phusion DNA Polymerase (Ozyme) and water to a final reaction mixture volume of 20 µl. The reactions were performed in a PTC-200 automated thermal cycler (MJ research, Waltham, MA, USA). The cycling conditions were 98°C for 30 sec; 35 cycles of 5 sec at 98°C, 30 sec at 56°C, 15 sec at 72°C; and a final extension time of 5 min at 72°C. The success of the PCR amplification was then verified by migration of the PCR product on an agarose gel. The NucleoFast 96 PCR Plates (Macherey-Nagel EURL, France) and BigDye Terminator version 1.1 cycle sequencing-ready reaction mix (Applied Biosystems, Foster City, CA) were then used to purify the PCR products before sequencing in both directions with the same primers used in the PCR amplification. The ABI 3100 automated sequencer (Applied Biosystems) resolved the sequenced products. The program ChromasPro was used to analyze, assemble and correct the sequences. When forward and reverse sequences could not be assembled, they were analyzed separately and resolved. Each sequence was aligned with genotypes published in Genbank [20]) for identification. When less than 100% homology was obtained, the new genotype was recorded, a new number was assigned to it and sequences were deposited in Genbank under accession numbers from JQ652371 and JQ652455. When the chromatogram indicated possible heterozygotic sequences, these samples were cloned to identify the different allelic sequences. The PCR products were cloned into pGEM-T-Easy vector (Promega, Madison, WI) following the manufacturer's instructions with some minor modifications. Before ligation, A-overhangs were added to the PCR product. This was performed by incubating 4.2 µl of purified PCR product with 1 U of DyNAzyme II DNA polymerase, 0.5 µl of Optimized DyNAzyme Buffer and 0.2 mM dATP with a final volume of 5 µl for 20 min at 72°C. Then, each reaction was ligated with 5 µl of 2X Rapid Ligation Buffer, 3 µl of purified A-overhangs-PCR product, 1 µl of T4 DNA ligase and 1 µl of pGEM®-T Easy Vector and incubated overnight at 15°C. Each ligation reaction was transformed into 50 µl of JM109 High Efficiency Competent Cells by letting them incubate together on ice for 20 min before a 1 min heat shock in a 42°C water bath. 950 µl of LB broth was then added to cells before incubation on a 37°C shaker for 1.5 hours. 300 µl of transformed cells was plated onto LBagar/ampicillin/IPTG/X-Gal plates and these were incubated overnight at 37°C. Eight white colonies per sample were then resuspended in 100 µl of RNase/DNase free water and subsequently PCR amplified and sequenced using the M13 universal and M13 reverse primers. Phylogenetic analysis was done using our data and data from a previous study performed in 2010, which included lice from France, Portugal, Mexico, Russia, Burundi and Rwanda (these lice were given names beginning with “li-” in the trees) [20]. Two phylogenetic methods, Maximum Parsimony (MP) and Maximum Likelihood (ML), were used to infer the trees for each individual spacer. For each spacer, the louse nucleic sequences were aligned with the genotype 1 found in Genbank (EU928781.1, EU928804.1, EU913096.1, EU913178.1 for PM1, PM2, S2 and S5, respectively) using the MUSCLE algorithm [26]. Then, trees were drawn within the MEGA 5 software with complete deletion [27]. A tree was also constructed with the concatenated sequences of the four spacers (S2, S5, PM1, PM2). Because the louse genome is diploid, in instances where there were two different alleles per locus, all possibilities of concatenation were made for each louse and all of them were taken into account in the tree (the possibilities were labeled from a to h in the tree). 10.1371/journal.pone.0037804.t001Table 1 Results of the Multi-Spacer-Typing of African lice. Country Type Host ID Louse ID Spacer S2 Spacer S5 Spacer PM1 Spacer PM2 Senegal 2010 HEAD 1 12 83 36/42 13 60/61 13 83/84 42 13 60/62 2 14 NA 36/42 13 NA 3 48 85/86 8/36 13 63/64 4 94 87 8 13 65/66 95 87 8/42 13 66 5 181 89 47 13 43 6 214 NA 35 13 65 7 223 90 8/36 13 67 8 226 91/92 36/62 13 62/68 Ethiopia 2010 BODY 9 2408 100 36 4/5 85/87 10 2409 96 54/59 18 83/92 Burundi 2008 HEAD 11 2394 97/101 35/53 25/34 82 12 2395 93 51 35 76 BODY 13 2396 76 35 17 80/74 14 2397 76 32/36 17 69 Rwanda 2011 BODY 15 2400 103 36/54 4 69/70 16 2401 108 50 4 92 17 2402 104 60 3/4 86 In case of heterozygocy, the numbers of the two genotypes were mentioned. NA, not available. New genotypes in bold. Results Clade A lice collected from Senegal, Burundi and Rwanda (head or body) were all black. Clade A body lice collected in southern Ethiopia and lice collected in France were grey (Figure 1). Head lice collected in Ethiopia were black, belonged to genotype C and were not included here [25]. Photographs of genotyped lice from other Non-African regions (Russia, Portugal, Mexico) and from Burundi were not available. After sequencing (Table 1), many new genotypes were found, especially for S2 and PM2 spacers, but there were also new genotypes for spacers S5 and PM1. In Senegal, all 10 tested lice had genotype 13 for spacer PM1. The other spacers had higher variation. We found that some lice collected from the same human host have the same genotype (lice 12 and 13 and lice 94 and 95). There is a lack of data for lice 14 and 214 due to the failure of spacer S2 to amplify, despite three attempts and a good positive control. Moreover, for spacer PM2 of louse 14, the amplification was non-specific on three attempts. 10.1371/journal.pone.0037804.g001Figure 1 Pictures of lice. Phylogenetic analysis was performed including lice from other sources [20], and trees were drawn for each of the four spacers. Altogether, 55 Non-African lice and 40 African lice were included in the analysis. The same tree topologies were found with Maximum Likelihood (ML) and Maximum Parsimony (MP) methods. However, the topologies of the trees constructed with the different spacers were not congruent and had low bootstrap support (Figures 2B and 3). The tree from spacer PM2 showed two distinct clusters: African lice separated from Non-African lice, which was supported by a 54% bootstrap (Figure 2B). The analysis of the sequence alignment of the spacer PM2 showed two positions at which single-base substitutions allowed us to distinguish African lice from lice coming from other regions: positions 26 (T in Africa and A out of Africa) and 31 (A or C in Africa and T out of Africa) and may have caused the observed clustering (Figure 2A). The only exception is the Senegalese louse 181 that clustered with Non-African lice in the tree (Figure 2B) and has the same signature than Non-African lice have at these positions (not shown). For spacers PM1, S2 and S5, no such clustering was observed (Figure 3, A to C). When concatenated sequences of the four intergenic spacers were used (Figure 3D), the resulting tree had the same topology as the PM2 spacer tree, but with a 43% bootstrap. Given that phylogenetic analysis of spacer PM2 sequences showed a correlation between geography and genotype, the proportion of each genotype found in African and/or Non-African countries (Geotyping) was compared for each spacer (Figure 4). We represented in the circles only genotypes found in two or more lice; the others genotypes were grouped and called “others.” Spacers PM2 and S2 (67 and 55 different genotypes) were more variable than PM1 and S5 (25 and 34 different genotypes). In several cases, genotypes were shared between African and Non-African lice in each spacer. However, in the PM2 spacer, this was very rare, as only one louse from Senegal (louse 181) and one louse from Russia had the same genotype (genotype 43). All other genotypes were not shared by African and Non-African countries. In spacer S5, two genotypes were shared between the two geographic areas: genotype 8 (Senegal and Moscow) and 42 (Senegal, Rwanda, France, Mexico and Moscow). In spacer S2, one genotype was shared: genotype 39 (Burundi and Mexico). However, it was in spacer PM1 that the biggest proportion of lice were observed to have genotypes shared by the two areas. Indeed, genotypes 13 and 18 were the most prevalent genotypes (respectively 30 and 33% of genotyped lice) and were found both in African and non-African countries: genotype 13 was found in Senegal, France and Portugal and genotype 18 was found in Rwanda, Ethiopia, Mexico and Russia (Figure 5). 10.1371/journal.pone.0037804.g002Figure 2 Spacer PM2 analysis. Phylogenetic analysis of African (black) and Non African lice (blue) (A) The first 69 bp of the alignment of a subset of PM2 spacer sequences. Two polymorphisms (shown with arrows) discriminate between African and Non-African lice (B) Phylogenetic tree based on PM2 sequences using Maximum likelihood method. For lice being heterozygote in PM2 spacer, the two alleles were included in the tree and they were called the same with one letter “a” or “b” to distinct them. 10.1371/journal.pone.0037804.g003Figure 3 Analysis of spacer PM1, S2, S5 and concatenation of the four spacers. Phylogenetic analysis of African (black) and Non African lice (blue) using Maximum likelihood method based on spacer PM1 (A), spacer S2 (B), spacer S5 (C), the concatenation of the four spacers (D). For lice being heterozygote, the two alleles were included in the trees and they were called the same with one letter “a” or “b” to distinct them. 10.1371/journal.pone.0037804.g004Figure 4 Proportion of each genotype among African (black) and Non-African (blue) lice. The name (ID numbers) of the genotypes found in both African and Non African countries were framed. Genotypes (either African and Non African) found in only one louse were not represented and were grouped as “others”. 10.1371/journal.pone.0037804.g005Figure 5 Geographic repartition of the four spacers genotypes. Genotypes found in at least two countries are colored (one color per genotype) and genotypes found in only one country are in white. The size of the symbols vary with the number of lice found for each genotype. Only genotypes found in at least two lice were represented. Altogether, these data show a correlation between geography and genotype but only in spacer PM2. Moreover, among the four spacers studied, none showed a correlation between the ecotype and the genotype, as head and body lice were not separated in the phylogenetic trees. Finally, no correlation has been observed between louse color and genotype as we did not find black and grey lice separately clustered in the trees. Discussion In this study, MST based on the four intergenic spacers S2, S5, PM1 and PM2 was confirmed to be a very sensitive method that is able to discriminate among individuals. The observation of identical genotypes from the sequences of two lice collected on the same person strengthens our faith in the reliability of the method. This method therefore appears to be well adapted for the study of human lice and able to address population-level questions. Our first observation was that the PM1 genotype 13 appeared in all Senegalese lice, but not in lice from Burundi, Rwanda and Ethiopia. This genotype was also found in lice from France and Portugal [20]. This observation that lice from Senegal and France have the same genotype on spacer PM1 suggested that interbreeding has occurred between lice from these two countries. As the French have been in Senegal since the XVIII century, this hypothesis is historically logical. Genotype 18 is also prevalent in African and Non-African countries (Figure 4), supporting the possibility that lice from different continents are interbreeding. When the frequency of the genotypes of the PM2 spacer were observed, it was noted that this spacer was the most variable among those tested and that only one louse from Senegal had the same genotype as a louse from Russia. In all other cases, African and Non-African lice harbored distinct genotypes, as confirmed by the phylogenetic tree that showed a cluster of African lice (54% extra support), regardless of the method used. This topology was similar to that of a previous study based on 18S rRNA [23]. However, with the MST method, head and body lice did not cluster separately inside their geographic cluster, as opposed to the results observed when using 18S-based phylogeny. Finally, when concatenated sequences of the four spacers were used, the resulting tree conserved the same topology as the PM2 tree but with lower bootstrap values. This showed the importance of first drawing separated trees for each spacer before drawing concatenated trees when using the MST method. This allows researchers to check if the different spacers tell the same story. In conclusion, a clear correlation between genotype and phenotype could not be shown. First, a correlation between genotype and geographic origin was observed, but only with spacer PM2. Second, there was no correlation between color and genotype or ecotype (head and body). Moreover, contrary to results from previous studies [3], [8], [9], [11], the color of lice may not be linked to the color of the host's skin as grey body lice were found on black hosts in Ethiopia. We acknowledge Pr Pierre Pontarotti for the helpful comments that improved the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: These authors have no support or funding to report. ==== Refs References 1 Alpatov WW 1955 Transformation of the head form of Pediculus humanus L. into the body form under the influence of changed living conditions. Bull Soc Nat 60 79 92 2 Bacot AW 1917 A contribution to the bionomics of Pediculus humanus (vestimenti) and Pediculus capitis. Parasitology 9 228 258 3 Burgess IF 1995 Human lice and their management. 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PLoS Biol 2 23 Yong Z Fournier PE Rydkina E Raoult D 2003 The geographical segregation of human lice preceded that of Pediculus humanus capitis and Pediculus humanus humanus. C R Biol 326 24 Boutellis A Veracx A Angelakis E Diatta G Mediannikov O 2011 Bartonella quintana in head lice from Senegal. Vector-Borne and Zoonotic Diseases In press 25 Angelakis E Diatta G Abdissa A Trape JF Mediannikov O 2011 Altitude-dependent Bartonella quintana Genotype C in Head Lice, Ethiopia. Emerg Infect Dis 17 2357 2359 10.3201/eid1712.110453 [doi] 22172306 26 Edgar RC 2004 MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32 1792 1797 10.1093/nar/gkh340 [doi];32/5/1792 [pii] 15034147 27 Tamura K Peterson D Peterson N Stecher G Nei M 2011 MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28 2731 2739 msr121 [pii];10.1093/molbev/msr121 [doi] 21546353
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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2266614710.1100/2012/736730Research ArticleProcess Evaluation of a Positive Youth Development Program in Hong Kong Based on Different Cohorts Law Ben M. F. 1 *Shek Daniel T. L. 2 3 4 5 6 1Department of Social Work and Social Administration, The University of Hong Kong, Hong Kong2Department of Applied Social Sciences, The Hong Kong Polytechnic University, Hong Kong3Public Policy Research Institute, The Hong Kong Polytechnic University, Hong Kong4Department of Social Work, East China Normal University, Shanghai, China5Kiang Wu Nursing College of Macau, Macau, China6Division of Adolescent Medicine, Department of Pediatrics, Kentucky Children's Hospital, University of Kentucky College of Medicine, Lexington, KY, USA*Ben M. F. Law: [email protected] Editor: Joav Merrick 2012 22 5 2012 2012 7367306 11 2011 25 12 2011 Copyright © 2012 B. M. F. Law and D. T. L. Shek.2012This 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.There are only a few process evaluation studies on positive youth development programs, particularly in the Chinese context. This study aims to examine the quality of implementation of a positive youth development program (the Project P.A.T.H.S.: Positive Adolescent Training through Holistic Social Programmes) and investigate the relationships among program adherence, process factors, implementation quality, and perceived program success. Process evaluation of 97 classroom-based teaching units was conducted in 62 schools from 2005 to 2009. Findings based on different cohorts generally showed that there were high overall program adherence and implementation quality. Program adherence and implementation process were highly correlated with quality and success of the program. Multiple regression analyses further showed that both implementation process and program adherence are significant predictors of program quality and success. Theoretical and practical implications of the findings are discussed. ==== Body 1. Introduction Program evaluation is systematic assessment of the process and outcomes of a program with the aim of contributing to program improvement, such as deciding whether to adopt the program further, enhancement of existing intervention protocols, and compliance with a set of explicit or implicit standards [1]. This paper documents the process evaluation of the Project P.A.T.H.S. (Positive Adolescent Training through Holistic Social Programmes), a large-scale positive youth development program in Hong Kong. Outcome evaluation focuses mainly on the results of the program, whereas process evaluation is concerned with how the program is actually delivered [2, 3]. Process evaluation is widely adopted in prevention science, such as nursing care [4, 5], chronic illness prevention programs [6–9], smoking cessation programs [10–12], dietary programs [13–17], and AIDS rehabilitation programs [18–22]. In social work practice, process evaluation has been used in family programs [23, 24], but is not commonly used in youth programs [25–27]. Process evaluation consists of five components, namely, program adherence, implementation process, intended dosage, macrolevel implication, and process-outcome linkage [28]. Program adherence deals with whether the program is being delivered as intended according to the original program design. It is an important factor affecting the quality of program implementation [3, 29]. True program fidelity is not easily achieved because program implementers often change or adapt the program content during actual implementation, whether intentionally or otherwise. Studies have shown that a number of preventive programs do not follow the prescribed program content entirely and adaptation is often made for specific target groups [30, 31]. One study found tension between the desire of the program implementer to adhere to the manualized plan and the desire to make adaptations in accordance with the needs of clients [32]. Although it is not an easily resolved issue, program fidelity is generally encouraged, especially when programs are designed with vigorous trial runs and repeated success rates [27, 33, 34]. Process factors are elements that are contingent to implementation quality or success and can be observed during the implementation process. There is a variety of process factors in terms of program characteristics and program developers' needs. Some programs are even designed with their own process measurements [35]. Common process factors in prevention programs include program receivers' engagement, program implementers' use of feedback, goal attainment, and program implementers' familiarity with the program receivers. Program dosage, which refers to the effort by the program implementer to follow the required time prescribed for a program, is considered another process factor because inadequate time affects the quality of program implementation [27, 36]. Dosage also refers to the group size of program receivers. A discrepancy between the intended and actual program receiver to program implementer ratio affects the program delivery process as well [26]. Process evaluation can provide important findings with macrolevel program implications, such as the importance of the engagement of different community stakeholders [37, 38], client needs [11], assessment of the environment [39, 40], and challenges of the programs for a particular context [41]. Finally, process evaluation and outcome evaluation are strongly linked. Process evaluation sheds light on which types of intervention strategies or processes are related to the program success [5, 11]. These factors can be amplified during program reimplementation. The components of process evaluation point towards its importance. First, outcome evaluation provides inadequate hints on the quality of program implementation. Process evaluation demystifies the “black box” of intervention and aids in the understanding of the elements of program success or failure [42]. Process evaluation facilitates the program developers to understand fully the strengths and weaknesses of the developed programs. Program implementers can follow the suggestions from the process evaluation for further program delivery. This is one essence of evidence-based practice, and also the foundation of bridging the gap between research and practice [43, 44]. Second, process evaluation can inform program developers about whether the program is delivered according to the standardized manual. The existence of other activities different from those intended by the program developers will not truly reflect the effectiveness of the prescribed program. Third, different human organizations and communities arrange the program in various settings, with different levels of involvement by the stakeholder and support. Perceptions of the program also vary across program implementers and program receivers. Process evaluation can document the variety of implementations in real human service settings based on the same manualized plans. Finally, process evaluation provides insights for program developers and implementers into the linkage between process and outcome. These insights allow both program developers and implementers to delineate areas that are either successful or require improvement during the process and connect them with the program outcomes. Many primary prevention programs and positive youth development programs have been developed in the West to address the growing adolescent development problems, such as substance abuse, mental health problems, and school violence [45]. However, in Hong Kong, there are very few systematic and multiyear positive youth development programs. To promote holistic development among adolescents in Hong Kong, the Hong Kong Jockey Club Charities Trust approved the release of HK$750 million (HK$400 million for the first phase and HK$350 million for the second phase) to launch a project entitled “P.A.T.H.S. to Adulthood: A Jockey Club Youth Enhancement Scheme.” The trust invited academics from five universities in Hong Kong to form a research team in order to develop a multiyear universal positive youth program [46]. The project commenced in 2004 and is targeted to end by 2012. There are two tiers of programs in this project. The Tier 1 Program is a universal positive youth development program where students from Secondary 1 (Grade 7) to Secondary 3 (Grade 9) participate in a classroom-based program, normally with 20 h of training in the school year in each grade. Around one-fifth of adolescents with more psychosocial needs join the Tier 2 Program, which consists of intensive training on volunteer service, adventure-based counseling camp, and other experiential learning activities. The project was designed according to 15 constructs conducive to adolescent healthy development [47]: promotion of bonding, cultivation of resilience, promotion of social competence, promotion of emotional competence, promotion of cognitive competence, promotion of behavioral competence, promotion of moral competence, cultivation of self-determination, promotion of spirituality, development of self-efficacy, development of a clear and positive identity, promotion of beliefs in the future, provision of recognition for positive behavior, provision of opportunities for prosocial involvement, and promotion of prosocial norms. The overall objective of the Tier 1 Program is to promote holistic development among junior secondary school students in Hong Kong. There are two implementation phases: the Experimental Implementation Phase and the Full Implementation Phase. The Experimental Implementation Phase aims at accumulating experience from trial teaching and administrative arrangement. Program materials are revised and refined during this phase. The Full Implementation Phase aims at executing the programs in full force. Several lines of evidence support the effectiveness of the Tier 1 Program, including the evaluation findings based on randomized group trials [48, 49], subjective outcome evaluation [50], qualitative findings based on focus group interviews with program implementers and students [51], interim evaluation [52], analyses of the weekly diaries of students [53], and case studies [54]. The evaluation findings based on different evaluation strategies indicate that the Project P.A.T.H.S. promotes the development of its participants. Process evaluation has also been carried out in both the Experimental and Full Implementation Phases for different grades [55, 56]. The evaluation results generally indicate that the quality of implementation and program adherence are high. Program adherence is the objective estimation of the adoption percentage from the manualized plan for real service delivery. A variety of process factors exist. A review of the literature indicates that the following program attributes can affect the quality and success of the positive youth development program implementation [31, 42, 57–59]. Student Interest. A successful program usually elicits the interest of the students. Active Involvement of Students. The more involved the students are, the higher the possibility that the program can achieve its outcomes. Classroom Management. The program implementer can manage student discipline during student activities. Students obey the requirements set by the program implementer and are attentive. Interactive Delivery Method. Interactive delivery is better than didactic delivery for positive youth development programs. Strategies to Enhance the Motivation of Students. The use of various learning strategies can enhance the engagement of the students and result in positive learning outcomes. Positive Feedback. The use of praise and encouragement throughout the lessons by the program implementers can promote the engagement of the students. Familiarity of Implementers with the Students. All other things being equal, a high degree of familiarity with the students is positively related to student learning outcome. Reflective Learning. The program implementer should engage the students in reflection and deeper learning. This can lead to growth and meaningful changes among the students. Program Goal Attainment. The achievement of program goals constitutes program success. Time Management. Efficient time management ensures that the majority of the program materials are carried out with high program adherence. Familiarity of Program Implementers with the Implementation Materials. Familiarity with the material ensures that the messages are conveyed effectively to the students. These eleven attributes can form a checklist for evaluating the implementation process. On the other hand, program quality is the subjective appraisal of the program implementation process by the observer. It can be reflected from the implementation atmosphere and the interaction between program implementers and students. Program success refers to the extent of unit objective attainment and the subjective evaluation of the response of the students to the program. By conducting secondary data analyses on all datasets collected over the past 4 years regarding process evaluation, the current study has two purposes: (1) to understand the program implementation quality across different cohorts in terms of program adherence, process factors, program quality, and success and (2) to explore factors contributing to the overall quality and success of the Tier 1 Program. Correspondingly, there are two research questions: (1) what is the overall implementation quality of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong? and (2) how are program adherence and other indicators of process evaluation related to the implementation quality and success of the Tier 1 Program? 2. Method 2.1. Participants and Procedure From 2005 to 2009, the total number of schools that participated in the Project P.A.T.H.S. was 244. Among them, 46.27% of the respondent schools adopted the full program (i.e., 20 h program involving 40 units), whereas 53.73% of the respondent schools adopted the core program (i.e., 10 h program involving 20 units). A total of 62 schools were randomly selected to participate in the study of process evaluation (23 schools for Secondary 1, 21 for Secondary 2, and 18 for Secondary 3); 25.80% of the participating schools adopted the core program, while the remaining 74.2% adopted the full program. Around 65% of schools incorporated the program into their formal curriculum (e.g., Liberal Studies, Life Education, and Religious Studies), 27.42% used the class teacher's period to implement the program, and less than 8% used other modes. The average number of students in the participating schools was 33.91. The characteristics of the schools that joined this process evaluation study can be seen in Table 1. Process evaluation was carried out in each participating school using systematic observations of actual classroom program delivery. For each school that joined the process evaluation, one to two program units were evaluated by two independent observers who are project colleagues with master's degrees. A total of 97 units were observed for this study. During the observation, observers sat at the back of the classroom and evaluated the method by which the units were actually implemented through completing several evaluation forms. 2.2. Instruments Program Adherence Observers were requested to rate program adherence in terms of percentage (i.e., the correspondence between actual program delivery and stipulated program materials). Aggregating all data across studies, Pearson correlation analyses showed that the ratings of program adherence were highly reliable across raters (r = 0.84,   P < 0.001), suggesting that the assessment of program adherence was consistent. Implementation Process Checklist (IPC) Observers were requested to report their observations using a 7-point Likert scale ranging from 1 (extremely negative) to 7 (extremely positive) on the items. Aggregating the data across studies, the internal consistency of the scale, as shown by Cronbach's alpha, was 0.93. The interrater reliability of IPC, as shown by Pearson correlation, was 0.72 (P < 0.001). Process Outcomes Two items were used to evaluate the process outcome: implementation quality and implementation success. Observers were requested to indicate their observations using a 7-point Likert scale ranging from 1 (poor) to 7 (excellent). A higher score represents better implementation quality or success. The interrater reliability for implementation quality, as shown by Pearson correlation, was 0.74 (P < 0.001), whereas that for implementation success was 0.64 (P < 0.001). In short, the aggregated data across studies suggest that the observations on process outcome were consistent across raters. 3. Results As the interrater reliabilities of the scores across all units were high, the ratings of each item by the two observers were averaged to form a combined indicator. Table 2 shows the descriptive profile of the evaluative indicators for process evaluation. The overall program adherence to the established manual ranged from 13.00 to 100.00%, with an average overall adherence of 85.14%. For the 7-point items, a stringent score of 5.0 or more was used as the cut-off point to indicate high ratings. The mean scores for implementation quality and success were 5.32 (SD = 0.86) and 5.34 (SD = 0.77), suggesting a high level of implementation. The mean scores of the 11 process evaluation items ranged from 4.96 to 5.62. Classroom management (M = 5.62) and familiarity with students (M = 5.43) had the highest scores, whereas reflective learning (M = 4.96) had the lowest score. Apart from reflective learning, all scores were on the high side. Table 3 shows the intercorrelations among program adherence, implementation process, implementation quality, and implementation success. All variables were significantly correlated with each other. The correlations between quality and success (r = 0.93) and between process and quality (r = 0.81) were high, whereas the correlations between process and adherence (r = 0.40) and between quality and adherence (r = 0.49) were relatively low. Multiple regression analyses were further performed, in which program adherence and implementation process were entered as predictors, and implementation quality and implementation success as two separate dependent variables. Table 4 shows the results for the prediction of implementation quality. Both implementation process and program adherence significantly predicted program quality with a large proportion of variance in the dependent variable being explained (R 2 = 0.68). The effect size of the variance explained (Cohen f 2), as calculated by (R 2/1 − R 2), is 2.13, which is large. Table 5 shows the results of the prediction of implementation success. Similarly, implementation process and program adherence significantly predicted program success (R 2 = 0.67). The effect size is 2.03, which is also large. 4. Discussion This study attempts to integrate the process evaluation findings based on multiple studies via secondary data analyses. There are several unique features of this study. First, a large number of teaching units and schools was evaluated. Second, two observers who were independent raters conducted the assessment. Third, a structured and reliable measure of program implementation was used. Fourth, inter-rater reliability analyses showed that the observations were basically reliable. Finally, this is the first large-scale process evaluation of positive youth development programs in the Chinese context. Despite the discrepancy in the ratings of program adherence on different units, the overall degree of adherence to the program is on the high side. This observation is generally consistent with previous findings generated from separate process evaluation studies conducted by observers [55, 56] and subjective outcome evaluations reported by the program implementers [54]. Most program content is well designed for implementation. This can be attributed to the fact that all program materials have gone through trial teaching. They have already been revised and refined according to prior teaching experience. Thus, program implementers may not have great difficulty in following the teaching plans. These findings dispute the common myth that curriculum-based positive youth development programs cannot be used easily and require major adaptations or modifications. The findings on program adherence are very encouraging because program adherence is generally low in the international context. In a meta-analysis of evaluation studies of primary and early secondary prevention programs published between 1980 and 1994, Dane and Schneider [2] showed that only 39 out of 162 evaluation studies documented procedures of fidelity. Domitrovich and Greenberg [60] also reported that among the 34 effective prevention programs under review, only 21% examined whether the effective intervention was related to outcomes. O'Connor et al. [61] suggested that there are certain risky adaptations for program adherence, such as reducing the number or length of sessions, lowering the level of participant engagement, eliminating key messages, removing topics, and changing the theoretical approach. Obviously, program adherence coupled with the effective use of the self, and good interaction between implementers and students, can be more difficult than expected. This requires intensive training and personal reflexivity on the part of the social worker. The present study found that different aspects of the program delivery were perceived to be positive, highlighting the fact that the Tier 1 Program of the Project P.A.T.H.S. was well received by both program implementers and students. Moreover, the implementation was regarded as successful by the observers, although relatively low average ratings were reported on time management and reflective learning. These findings are similar to those based on the Experimental Implementation Phase [55, 56]. There are two possible explanations for the low ratings. First, due to the usual didactic teaching style in Hong Kong, students are not accustomed to reflecting on their everyday life practice in classroom settings. Hence, the students cannot easily shift their learning modes from one-way knowledge dissemination to reflective learning. Second, the overpacking of the curriculum may have prevented the students from carrying out reflections on their learning. Overpacking could have also contributed to the unsatisfactory rating of time management. The current study also found that program adherence and implementation process are closely associated with implementation quality and success. For positive youth programs, an interactive program delivery is the key milestone for program quality and success [57]. This explains the high correlations among these factors. Furthermore, implementation process and program adherence were found to predict implementation quality or success. Theoretically, both process and content are critical to program quality and success. All these findings suggested that the need for modifying the units in the implementation process was not high. Again, these findings could be used to challenge the common myth that curricula-based positive youth development programs cannot be easily used in reality and major modifications must be made for different adolescent populations. This demystification provides an evidence-based justification for following the manuals in an authentic manner. Findings of the present study have two implications. The first implication is on the conceptual level. When we are concerned about program implementation regardless of external environment (i.e., macrolevel implication and dosage), we can focus on three variables: program adherence, implementation process, and context. These variables are all related to implementation quality or success. The present findings provide conceptual insights for understanding program quality or success. The second implication is on a practical level. The process variables covered in the study can actually be used in other social work or health science contexts, especially in educational and developmental groups. All these measures are important for positive youth programs and should be brought to the fore in the training. Youth workers, social workers, and teachers should be aware that implementation process is critical for classroom-based psychosocial intervention programs. This study has several limitations. First, only 62 randomly selected schools participated in this study. Although the number of schools can be regarded as respectable, inclusion of more schools with diverse backgrounds would be helpful. Second, process evaluation with reference to macro-level implication, dosage issues [62], and school characteristics can help program developers to understand the quality of the program implementation process further. Third, the observation may have a confounding effect. Students may be more cooperative when there are visitors or outside observers because the students do not want to ruin the reputation of their schools. As Chinese students, they may also want to “give face” to the program implementers [63] and intentionally perform better in front of the raters. Fourth, consistent with the intrinsic problem of all observation studies where time sampling is involved, one needs to be conscious of the degree of generalizability of the present findings to other temporal and spatial contexts. Despite these limitations, the current process evaluation findings suggest that the quality of implementation of the Tier 1 Program of the Project P.A.T.H.S. is generally high. Acknowledgments The preparation for this paper and the Project P.A.T.H.S. were financially supported by the Hong Kong Jockey Club Charities Trust. Table 1 Descriptive profile of participating schools from 2005 to 2009. S1 S2 S3 2005/2006 (EIP) 2006/2007 (FIP) 2008/2009 (FIP) 2006/2007 (EIP) 2007/2008 (FIP) 2008/2009 (FIP) 2007/2008 (EIP) 2008/2009 (FIP) Total schools that joined P.A.T.H.S. 52 207 197 49 196 198 48 167  (i) 10 h program 23 95 104 27 113 110 29 104  (ii) 20 h program 29 112 93 22 83 88 19 63 Total schools that joined this study 6 14 3 4 14 3 4 14  (i) 10 h program 1 5 0 0 5 0 0 5  (ii) 20 h program 5 9 3 4 9 3 4 9 Background characteristics of participating schools Location (district) Hong Kong and Islands 1 2 1 1 2 1 1 4 Kowloon 0 7 1 2 2 1 1 4 N.T. 5 5 1 1 10 1 2 6 Finance mode Aided 6 11 3 3 10 3 4 13 Direct subsidy scheme 0 2 0 1 1 0 0 0 Government 0 1 0 0 3 0 0 1 Sex composition Coed 6 11 3 4 13 3 4 11 Unisex 0 3 0 0 1 0 0 3 Religious background Christian/Catholic 2 7 2 2 7 1 0 8 Buddhism/Taoism 0 3 0 0 2 0 2 3 Islam 0 0 1 1 0 0 0 0 Nil 4 4 0 1 5 2 2 3 Integration into school curriculum Formal curriculum (e.g., Liberal Studies, Religious Studies, Life Education) 3 12 2 3 7 2 3 8 Class teachers' period 1 2 1 1 7 1 1 4 Others 2 0 0 0 0 0 0 2 Average no. of students in the class 35.2 36.7 30 34.3 34.5 29.7 35.8 35.1 Program implementers Social worker 1 2 1 0 5 0 3 2 Teacher 1 6 1 1 7 2 0 6 Social worker + teacher 4 6 1 3 2 1 1 6 Total no. of units observed 12 22 3 7 21 5 7 20 Note. S1: Secondary 1 level; S2: Secondary 2 level; S3: Secondary 3 level; EIP: Experimental Implementation Phase; FIP: Full Implementation Phase. Table 2 Descriptive statistics of evaluation Items. Evaluation items Min Max M SD Interest 3.0 7.0 5.35 0.84 Involve 4.0 7.0 5.58 0.78 Class 3.0 7.0 5.62 0.82 Interact 3.0 7.0 5.27 0.84 Motivation 3.0 7.0 5.19 0.82 Feedback 3.0 7.0 5.03 0.89 FStudents 3.0 7.0 5.43 1.00 Reflect 3.0 7.0 4.96 0.91 Goal 2.0 7.0 5.35 0.87 Time 3.0 7.0 5.01 0.96 FMaterials 3.0 7.0 5.52 0.80 Adhere 13.00% 100.00% 85.14% 15.50% Quality 2.0 7.0 5.32 0.86 Success 3.0 7.0 5.34 0.77 Note. Interest: student interest; Involve: active involvement of students; Class: classroom management; Interact: interactive delivery method; Motivation: strategies to enhance the motivation of students; Feedback: positive feedback; FStudents: familiarity of implementers with students; Reflect: reflective learning; Goal: program goal attainment; Time: time management; FMaterials: familiarity of program implementers with the program materials; Adhere: program adherence; Quality: implementation quality; Success: implementation success. Table 3 Intercorrelations among program adherence, implementation process, implementation quality, and implementation success. Measure 1 2 3 4 (1) Implementation process — 0.45*** 0.81** 0.79*** (2) Program adherence 0.49*** 0.51*** (3) Implementation quality 0.93*** (4) Implementation success — Note. Bonferroni correction was used to evaluate the significance of the correlations. ***P < 0.001. **P < 0.005. Table 4 Regression table of implementation quality. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22701710PONE-D-12-0461410.1371/journal.pone.0038770Research ArticleBiologyMolecular cell biologySignal transductionSignaling CascadesTyrosine Kinase Signaling CascadeSignaling in cellular processesCell movement signalingGTPase signalingMitogenic signalingSignaling in Selected DisciplinesOncogenic SignalingSignaling PathwaysMedicineOncologyBasic Cancer ResearchEGFR Inhibition in Glioma Cells Modulates Rho Signaling to Inhibit Cell Motility and Invasion and Cooperates with Temozolomide to Reduce Cell Growth Erlotinib Alters Rho/ROCK and Glioma Cell MotilityRamis Guillem 1 Thomàs-Moyà Elena 1 Fernández de Mattos Silvia 1 2 Rodríguez José 1 ¤ Villalonga Priam 1 2 * 1 Cancer Cell Biology Group, Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Illes Balears, Spain 2 Departament de Biologia Fonamental, Universitat de les Illes Balears, Illes Balears, Spain Canoll Peter EditorColumbia University, United States of America* E-mail: [email protected] and designed the experiments: GR SF JR PV. Performed the experiments: GR ETM PV. Analyzed the data: GR ETM SF JR PV. Wrote the paper: PV. ¤ Current address: Hospital Severo Ochoa, Leganés, Madrid, Spain 2012 6 6 2012 7 6 e3877010 2 2012 13 5 2012 Ramis et al.2012This 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.Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G1. Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27kip1 levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion. ==== Body Introduction Malignant gliomas constitute the most common primary brain tumours in adults and rank among the most devastating and aggressive types of human cancer due to their dismal prognosis. Key biological features of these tumours are the ability of tumour cells to invade healthy brain tissue and their enhanced resistance to radio and chemotherapy-induced apoptosis [1]. Such characteristics have dramatic clinical consequences, since they critically challenge the success of therapeutic intervention. A number of genetic alterations are responsible for the malignancy of these tumours, often involving mutations leading to the hyperactivation of receptor tyrosine kinases. Among these, the epidermal growth factor (EGF) receptor (EGFR) is commonly overexpressed and amplified in gliomas, and contributes to uncontrolled proliferation and survival of glioma cells [2]. The EGFR is also frequently mutated in these tumours, leading to the expression of a truncated receptor termed EGFRvIII which lacks its extracellular domain and is constitutively active [3], [4]. Enhanced activation of the EGFR tyrosine kinase domain leads to the activation of intracellular signaling pathways such as the Raf/MEK/ERK and the PI3K/Akt pathways, which are ultimately responsible for the malignant phenotype of glioma cells. Accordingly, small molecule inhibitors of EGFR such as erlotinib (Tarceva) and gefitinib (Iressa) have been shown to attenuate glioma cell proliferation in vitro [5]–[7], although their clinical activity as single agents remains controversial due to the contradictory data obtained in clinical trials (reviewed in [8]). Similarly, opposing results have been reported regarding the predictive value of different biomarkers such as EGFR and EGFRvIII expression on EGFR inhibitor responsiveness. For instance, it has been described that sensitivity to erlotinib correlates with high EGFR expression levels but not with EGFRvIII expression [9] and, oppositely, that EGFRvIII expression correlates with sensitivity to erlotinib, whereas EGFR expression does not [6]. In contrast, it is well-established that sensitivity to EGFR inhibition correlates with low PI3K/Akt activity, since hyperactivation of the PI3K/Akt pathway through phosphatase and tensin homolog (PTEN) mutation renders glioma cells resistant to EGFR inhibition and, conversely, inhibition of PI3K signaling using different compounds has been shown to sensitize glioma cells to EGFR inhibitors [6], [9]–[11]. Altogether, despite the research efforts focused on understanding the molecular basis of sensitivity to EGFR inhibitors, the prevailing view is that an in-depth analysis of the molecular responses elicited by these compounds in glioma cells is still required. Here we have aimed to further characterize the cellular effects of EGFR inhibition with erlotinib in a panel of human glioma cell lines. Erlotinib treatment reduces cell growth and inhibits multicellular tumour spheroid formation. These effects are accompanied by alterations in signaling pathways and cell cycle regulators. Interestingly, EGFR inhibition can cooperate in a schedule-dependent manner with low doses of temozolomide (TMZ) to reduce glioma cell growth. We also show that EGFR inhibition induces a dramatic alteration in cell morphology through the modulation of Rho/ROCK signaling that leads to the inhibition of glioma cell motility and invasion. Importantly, EGFR inhibition is similarly effective in the reduction of proliferation, motility and invasion in cells expressing wild-type, mutant or amplified EGFR. Materials and Methods Cell culture and drug treatments LN229, U87MG, HS683, T98G, U251 and U373 cells were a gift from Joan Seoane (Institut de Recerca Hospital Universitari Vall d'Hebron, Barcelona). The U87MG derivative cell line U87ΔEGFR was a gift from Isabel Martínez-Lacaci (Hospital Universitario Virgen de la Arrixaca, Murcia). SKMG-3 cells were a gift of Hans Skovgaard (Rigshospitalet, Oslo). All cell lines were subconfluently grown and passaged, routinely tested for mycoplasma contamination and subjected to frequent morphological tests and growth curve analysis as quality-control assessments. Cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% foetal calf serum (Biological Industries, Israel) in a humidified incubator at 37°C with 5% CO2. Erlotinib (Roche, Basel, Switzerland), temozolomide (Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MA), C3 (Cytoskeleton, Denver, CO) and H-1152 (Calbiochem, Darmstadt, Germany) were added directly to the media at the indicated concentration and cells were harvested or analyzed at the time points indicated in the figure legends. Measurement of cell proliferation and viability For cellular growth assays, 2.5×104 cells were plated in 6-well plates and cell growth was assessed at the indicated time points counting cells after trypsinizing and incubating them with trypan blue solution (Sigma-Aldrich, St. Louis, MO). For cellular viability assays, 5×103 cells were plated in clear bottom 96-well plates, treated as indicated and processed using the CellTiter-Glo Luminiscent Assay Kit (Promega, Madison, WI) to measure cellular viability, following the manufacturer's instructions. Luminiscence was detected using a multiwell scanning spectrophotometer (Plate Chameleon, Hidex, Finland). Multicellular tumour spheroid formation assays To monitor the formation of multicellular tumour spheroids in culture, 4×105 U87MG cells were plated in 6-well plates and treated as indicated in the figure legends. After 4–6 days cells were stained with 0.5% (w/v) crystal violet in 70% ethanol and the number of multicellular tumour spheroids from representative fields (>10) counted under a light microscope. Flow Cytometry Cell cycle profile was measured by flow cytometry using propidium iodide. Briefly, trypsinized cells were collected by centrifugation, washed in PBS and fixed for 30 min at 4°C in 70% ethanol. After washing twice with PBS, DNA was stained with 50 µg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO) in the presence of 50 µg/ml RNase A (Sigma-Aldrich, St. Louis, MO). Stained cells were then processed using a Beckman Coulter EPICS XL Cytometer (Beckman Coulter, Fullerton, CA) and analyzed with the WinMDI software. Clonogenic assays For clonogenic assays, cells grown in 6-well plates were treated as indicated, trypsinized and plated at low density (3×103 cells per 60-mm plate) in fresh media. After 7–10 days cells were stained with 0.5% (w/v) crystal violet in 70% ethanol and the number of colonies counted. Actin Staining Cells grown on coverslips were fixed in 3.7% (v/v) paraformaldehyde for 20 min and permeabilized in 0.1% (v/v) Triton X-100 for 5 min. Actin filaments were visualized by incubating the fixed cells for 45 min at 37°C with TRITC-phalloidin (Sigma-Aldrich, St. Louis, MO, 1∶500). Stained cells were analyzed on a Leica TCS SPE confocal microscope (Leica Microsystems, Wetzlar, Germany). Measurement of Rho GTPase activity The capacity of Rho-GTP and Rac-GTP to bind to GST-Rhotekin and GST-PBD (p21-activated-kinase-binding-domain) respectively was used in order to analyze the amount of active GTPases [12] with Rho and Rac activation assay kits (Cytoskeleton, Denver, CO), according to the manufacturer's instructions. Briefly, cells (5–10×106) were lysed, cleared (10,000×g) and incubated for 45 min at 4°C with glutathione sepharose-4B beads coupled with GST-Rhotekin or GST-PBD for Rho-GTP or Rac-GTP pulldowns, respectively. Beads were washed 4 times in Lysis Buffer. Bound proteins were solubilized by the addition of 35 µl of Laemmli loading buffer and separated on 12.5% SDS-polyacrilamide gels. The amount of Rho or Rac in the bound fraction was detected by western blotting. Motility and invasion assays For 2D motility (wound-healing) assays, 4×105 U87MG cells were plated in 6-well plates, wounded thrice with a sterile tip and 3 representative images were collected. After 16 h, images of the same regions were collected and the ratio of cell motility in each experimental condition quantified. For 3D invasion assays, 2×104 U87MG cells were seeded on matrigel-coated transwells (BD Biosciences, San Diego, CA) containing DMEM supplemented with 0.5% FCS and placed in 24-well plates containing DMEM supplemented with 10% FCS to create a growth factor gradient. 24 h later, the matrigel layer was removed and cells were stained with 0.5% (w/v) crystal violet in 70% ethanol. Invading cells from 6 representative fields were counted under a light optic microscope. Gel electrophoresis and immunoblotting Cells were harvested in a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% (v/v) Triton X-100 plus protease and phosphatase inhibitors. Protein content was measured by the Bradford procedure [13]. Cell lysates were electrophoresed in SDS-polyacrylamide gels. After electrophoresis the proteins were transferred to Immobilon-P strips (Millipore, Billerica, MA) for 2 h at 60 V. The sheets were preincubated in TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl), 0.05% Tween 20 and 5% defatted milk powder for 1 h at room temperature and then incubated for 1 h at room temperature in TBS, 0.05% Tween 20, 1% BSA and 0.5% defatted milk powder containing the appropriate antibodies: Akt (sc-1618, 1∶1000), p27kip1 (sc-525, 1∶1000), cdk4 (sc-260, 1∶1000) and p21cip1 (sc-471, 1∶1000) from Santa Cruz (Santa Cruz, CA), anti-cyclin D1 (MS-210P, Neomarkers, Fremont, CA 1∶1000), pRB (14001A, BD-Pharmingen, San Diego, CA, 1∶500), Rho (ARH03, Cytoskeleton, Denver, CO, 1∶500), Rac (05–389, Millipore, Billerica, MA, 1∶1000), anti-β-tubulin (T0198, Sigma-Aldrich, St. Louis, MO, 1∶4000) and EGFR (#2232, 1∶1000), p-S473-Akt (#9271, 1∶500), p-ERK (#9101, 1∶1000) and ERK (#9102, 1∶1000) from Cell Signaling (Beverly, MA). After washing in TBS, 0.05% Tween 20, the sheets were incubated with a peroxidase-coupled secondary antibody (Dako, Glostrup, Denmark, 1/2000 dilution,) for 1 h at room temperature. After incubation, the sheets were washed twice in TBS, 0.05% Tween 20 and once in TBS. The peroxidase reaction was visualized by the enhanced chemiluminiscence detection system (Millipore, Billerica, MA). Statistical analysis The statistical significance of differences was assessed by Student's t test using GraphPad Prism (GraphPad Software Inc. La Jolla, CA). Statistically significant differences are indicated by ***p<0.001, **p<0.01 and *p<0.05. Results Erlotinib inhibits glioma cell proliferation and prevents multicellular tumour spheroid formation In order to characterize the cellular effects of EGFR inhibition in glioma cells, we treated a panel of 6 human glioma cell lines (LN229, U87MG, HS683, T98G, U251, U373) with erlotinib. Erlotinib reduced cell proliferation in all cell lines tested (Figures 1A, 1B). Growth curve experiments upon long-term erlotinib treatment indicated that erlotinib decreased total cell number (Figure 1B), but did not affect cellular viability as indicated by trypan blue staining (data not shown). Dose-response experiments confirmed that 10 µM erlotinib exerted an inhibitory effect on glioma cell growth ranging from 30% (U373 cells) to 80% inhibition (LN229 cells) (Figures 1C, 1D). Since U87MG cells spontaneously form multicellular tumour spheroids in culture [14], we also investigated whether erlotinib could prevent multicellular tumour spheroid formation. Whereas control U87MG cells formed high numbers of large and dense multicellular tumour spheroids, erlotinib-treated cells were largely resistant to spheroid formation (Figures 1E). These observations confirm that EGFR inhibition with erlotinib severely reduces glioma cell proliferation. 10.1371/journal.pone.0038770.g001Figure 1 Erlotinib inhibits glioma cell proliferation. (A) Representative phase-contrast micrographs of glioma cell lines left untreated (control) or treated for 48 h with 10 µM erlotinib (erlotinib). (B) Glioma cell lines were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. Data from a representative experiment out of three repetitions is shown, representing the total number of viable cells in untreated and erlotinib-treated conditions at the indicated time-points. (C) Glioma cell lines were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (D) Glioma cell lines were treated for 72 h with 10 µM erlotinib. Sensitivity to erlotinib of each cell line is expressed as the mean ± SD percentage of growth inhibitory activity from three independent experiments, each conducted in duplicate. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05) (E) Representative phase-contrast micrographs of U87MG cells left for 4–6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: **P<0.01). Erlotinib induces G1 phase arrest in glioma cells In order to characterize the cell cycle arrest induced by erlotinib treatment in glioma cells, we performed flow cytometric analysis in a panel of control and erlotinib-treated glioma cell lines. Erlotinib treatment led to a significant accumulation of cells in G1 in all tested cell lines (Figure 2A), showing a sensitivity to erlotinib in correlation with our previous results (Figure 2B). Interestingly, erlotinib did not induce cell death as indicated by the absence of a detectable sub-G1 population (Figures 2A and 2B), in line with our previous data. Altogether, our results indicate that erlotinib inhibits glioma cell proliferation primarily by inhibiting S-phase entry. 10.1371/journal.pone.0038770.g002Figure 2 Erlotinib prevents multicellular tumour spheroid formation and induces G1 arrest in glioma cells. (A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in Materials and Methods. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line. EGFR inhibition cooperates with temozolomide to inhibit glioma cell growth The current therapy for glioma patients involves the use of the alkylating agent temozolomide (TMZ) in combination with radiotherapy. We therefore investigated whether erlotinib could potentiate the antiproliferative effects of TMZ in glioma cells. For this purpose we used different experimental strategies. First, we performed clonogenic assays upon TMZ treatment of control or erlotinib-treated cells to assess if EGFR inhibition could potentiate TMZ-induced genotoxicity. LN229, U251 and HS683 cells pre-treated for 24 h with erlotinib recovered some of their clonogenic ability when re-plated in the absence of erlotinib (Figure 3A). In contrast, a short exposure to TMZ (3 h) dramatically compromised their clonogenic capacity (Figure 3A). Interestingly, erlotinib pre-treatment protected cells from TMZ-induced genotoxicity (Figure 3A). To extend these observations, we next monitored cell proliferation in MTT-based assays. In order to test whether erlotinib could cooperate with TMZ we used sub-optimal doses of both erlotinib (1 µM) and TMZ (25 µM). As expected, neither erlotinib nor TMZ at sub-optimal doses were able to significantly reduce cellular growth (Figure 3B). However, when used in combination, erlotinib was able to cooperate with TMZ to reduce cell proliferation in both U87MG and U251 cells (Figure 3B). In order to validate these results, we performed multicellular tumour spheroid formation assays using U87MG cells. To this end, U87MG cells were treated with sub-optimal doses of erlotinib (1 µM) and TMZ (25 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to control cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figures 3C). In sharp contrast, the combination of sub-optimal doses of erlotinib and TMZ dramatically reduced spheroid formation, similarly to the standard erlotinib treatment (Figures 3C). These results suggest that the combination of low doses of erlotinib and TMZ can cooperate to reduce glioma cell proliferation. 10.1371/journal.pone.0038770.g003Figure 3 EGFR inhibition cooperates with temozolomide to inhibit glioma cell growth. (A) LN229, U251 and HS683 cells were left untreated or were treated for 24 h with erlotinib and subsequently exposed to vehicle or TMZ for 3 h, plated and after 7–10 days the remaining colonies were stained and counted as indicated in Materials and Methods. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the number of clones relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's t-test: *P<0.05 and **P<0.01, respectively). (B) U251 and U87MG cells were plated in 96-well plates, left untreated or treated as indicated for 48 h and cell viability monitored as described in Materials and Methods. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's t-test: **P<0.01). (C) Representative phase-contrast micrographs of U87MG cells treated as indicated and left for 4–6 days to allow formation of MCTS. The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between combined treatment and either treatment alone are statistically significant (Student's t-test: ***P<0.001). Erlotinib inhibits ERK and Akt signaling promoting cyclin D1 downregulation and increasing p27kip1 levels We next investigated the molecular mechanisms responsible for the cell cycle arrest induced by EGFR inhibition, monitoring alterations on relevant signaling intermediates and cell cycle regulatory proteins in glioma cells (Figure 4A). For this purpose we performed a time-course experiment upon erlotinib treatment in LN229 and T98G cells. Erlotinib induced a rapid inhibition of ERK phosphorylation and an inhibition of Akt phosphorylation that was apparent upon longer term treatment (Figure 4B). Erlotinib also induced a significant downregulation of cyclin D1 and similarly reduced the levels of p21cip1 (Figure 4B). In contrast, Erlotinib increased the levels of the cyclin-dependent kinase inhibitor p27kip1 (Figure 4B). In agreement with these observations, erlotinib inhibited pRb phosphorylation (Figure 4B). We next investigated whether the observed molecular events correlated with the sensitivity to erlotinib in different glioma cell lines. To this end, we compared the aforementioned molecular alterations in three representative glioma cell lines (LN229, T98G and U373) with high, medium and low sensitivity to erlotinib. Erlotinib clearly inhibited both ERK and Akt phosphorylation in LN229 cells, which also showed a marked downregulation of cyclin D1 and a strong increase in p27kip1 levels (Figure 4C). However, p27kip1 levels did not increase in T98G cells, which show slightly lower sensitivity to erlotinib (Figure 4C). The behaviour of U373 cells, the least sensitive cell line, was different to that of LN229 and T98G cells. In U373 cells, erlotinib did not alter cyclin D1 levels nor Akt or ERK phosphorylation, although it induced p27kip1 upregulation (Figure 4C). Interestingly, the sensitivity to erlotinib could not be correlated with the expression levels of EGFR (Figure 4A). Taken together, our results suggest that the inhibition of signaling pathways converging on cell cycle regulatory elements mediate the antiproliferative effects of erlotinib in glioma cells. 10.1371/journal.pone.0038770.g004Figure 4 EGFR inhibition alters the expression levels of key cell cycle regulators. (A) The indicated human glioma cell lines were harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (B) LN229 and T98G cells were treated with 10 µM erlotinib for the indicated time, harvested and the expression levels of the indicated proteins were analyzed by western blotting with specific antibodies. (C) As in B, but LN229, T98G and U373 cells were treated as indicated. EGFR inhibition leads to actin cytoskeleton reorganization and morphological changes in glioma cells perturbing Rho GTPase signaling Our initial observations suggested that EGFR inhibition induced morphological changes in glioma cells (Figure 1A). We used U87MG cells to further investigate this alteration, since in these cells erlotinib exerted a robust effect on cell morphology. Untreated U87MG cells were relatively small, morphologically heterogeneous and loosely attached, with many cells eventually rounding up (Figure 5A). In contrast, erlotinib-treated U87MG cells were significantly larger, more homogeneous, firmly attached and well spread (Figure 5A). These results suggested that EGFR inhibition induced a profound cytoskeletal rearrangement. We thus evaluated the organization of the actin cytoskeleton in control and erlotinib-treated U87MG cells. F-actin staining indicated that polymerized actin was mostly located within the cell periphery in untreated U87MG cells, which were largely devoid of actin stress fibres (Figure 5B). Interestingly, erlotinib increased actomyosin contractility, as indicated by the assembly of actin stress fibres (Figure 5B). Since the dynamic regulation of the actin cytoskeleton is controlled by Rho GTPases [15], we monitored the activity of both Rho and Rac upon EGFR inhibition. Basal Rho activity is low in U87MG cells, but erlotinib induced a rapid and sustained activation of Rho, in agreement with the observed increase in actin stress fibres (Figure 5C). In sharp contrast, erlotinib dramatically reduced Rac activity in U87MG cells (Figure 5C). These results indicate that EGFR inhibition promotes the reorganization of the actin cytoskeleton through the modulation of Rho GTPase signaling. 10.1371/journal.pone.0038770.g005Figure 5 EGFR inhibition leads to actin cytoskeleton reorganization through Rho GTPase modulation. (A) Representative phase-contrast micrographs of U87MG cells left untreated (control) or treated for 24 h with 10 µM erlotinib (erlotinib). (B) U87MG cells grown on coverslips were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib), fixed and stained with TRITC-labelled phalloidin. Bar, 5 µm. (C) U87MG cells were treated as indicated, harvested and RhoA and Rac1 activation were analyzed by GST-Rhotekin and GST-PBD pulldown, respectively, followed by western blotting with anti-RhoA and anti-Rac1 antibodies (upper panel). An aliquot of each lysate was also loaded in another gel to analyze total RhoA and total Rac1 levels (bottom panel). The graphs represent the quantified mean ± SD Rho/Rac activation values (Rho-GTP/Total Rho and Rac-GTP/Total Rac), relative to untreated cells, from three independent experiments. Erlotinib inhibits glioma cell motility and invasion Actin cytoskeleton reorganization in response to Rho GTPase signaling plays a crucial role in the regulation of cell motility [16]. We therefore investigated whether EGFR inhibition could also modulate glioma cell motility. For this purpose, we performed wound-healing assays in untreated and erlotinib-treated U87MG cells. Control U87MG cells were highly motile and migrated very efficiently in wound-healing assays (Figure 6A). Interestingly, erlotinib clearly inhibited cell motility in these assays (Figure 6A and 6B). Cell motility inhibition in response to erlotinib was also clearly observed in both T98G and LN229 cells (Figure 6A and 6B). Since increased motility and invasiveness are hallmarks of malignant glioma cells, we also tested whether EGFR inhibition could reduce glioma cell invasion in a 3D context. To this end, we monitored cell invasion using matrigel-coated transwells. In agreement with our previous data, whilst untreated glioma cells were highly invasive, cell invasion was strongly inhibited in erlotinib-treated cells (Figure 6C). 10.1371/journal.pone.0038770.g006Figure 6 EGFR inhibition reduces glioma cell motility and invasion. (A) Representative phase-contrast micrographs of U87MG cells left untreated or treated with 10 µM erlotinib as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in Materials and Methods. (B) Representation of the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of U87MG cell motility relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05 and **P<0.01, respectively). (C) U87MG cells were seeded onto Matrigel-coated transwells in the absence (−) or presence (+) or 10 µM erlotinib to perform invasion assays as described in Materials and Methods. The graph represents the mean ± SD rate of invasion from three independent experiments performed in duplicate, expressed as the percentage of invasion relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05 and ***P<0.001, respectively). Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity Our previous observations suggest that the increase in Rho activity and actomyosin contractility are responsible for the reduction in cell motility observed in response to EGFR inhibition. In order to confirm this hypothesis, we treated cells with erlotinib and tested whether Rho/ROCK inhibition could restore cell morphology and motility to similar conditions as those observed in untreated cells. As opposed to control cells, erlotinib-treated cells showed their distinctive morphology, characterized by the presence of larger, more homogeneous and firmly attached cells (Figure 7A). Interestingly, treatment of U87MG cells with either a Rho inhibitor (the C3 exoenzyme) or a small-molecule ROCK inhibitor (H-1152) together with erlotinib resulted in a very similar cellular morphology to that of control cells (Figure 7A). Accordingly, whereas erlotinib induced the formation of thick and robust stress fibres, co-treatment with either C3 or H-1152 completely prevented the formation of actin stress fibres (Figure 7B). We finally investigated whether Rho/ROCK inhibition could restore cell motility in erlotinib-treated glioma cells. To this end, we performed wound healing assays using U87MG cells in the presence of erlotinib alone or together with C3 or H-1152. In agreement with our previous data, EGFR inhibition led to a marked decrease in the rate of cell motility (Figure 7C). In sharp contrast, inhibition of either Rho or ROCK prevented erlotinib-induced reduction in cell motility, restoring the motility rate to that of untreated cells (Figure 7C). Rho/ROCK inhibitors also restored cell motility in erlotinib-treated T98G and LN229 cells (data not shown). Taken together, these results confirm that EGFR inhibitors alter glioma cell morphology and motility through the activation of the Rho/ROCK signaling pathway. 10.1371/journal.pone.0038770.g007Figure 7 Erlotinib-induced effects on cell morphology and motility require Rho/ROCK activity. (A) Representative phase-contrast micrographs of U87MG cells left untreated (control) or treated for 24 h with 10 µM erlotinib alone or in the presence of 0,5 µg/ml C3 or 0,5 µM H-1152. (B) U87MG cells grown on coverslips were left untreated (control) or were treated for 24 h with 10 µM erlotinib alone or in the presence of 0,5 µg/ml C3 or 0,5 µM H-1152, fixed and stained with TRITC-labelled phalloidin. Bar, 10 µm. (C) Representative phase-contrast micrographs of U87MG cells left untreated or treated as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in Materials and Methods. The graph represents the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of U87MG cell motility relative to untreated cells. The differences in motility between cells treated alone with erlotinib or together with C3 or H-1152 are statistically significant (Student's t-test: *P<0.05 and ***P<0.001, respectively). EGFR inhibition is effective in glioma cells with amplified or mutant EGFR EGFR is frequently activated through mutation or amplification in malignant gliomas, although commonly-used glioma cell lines lack amplified or mutant EGFR. We therefore investigated whether EGFR inhibition was equally effective in the context of amplified or mutant EGFR. For this purpose we took advantage of the U87MG derivative cell line U87ΔEGFR, stably expressing the truncated and constitutively active EGFR mutant EGFRvIII [6], [10], and the SKMG-3 cell line that maintains endogenous EGFR amplification and expresses high levels of wild-type EGFR [17]. Dose-response and growth curve experiments showed that both cell lines were sensitive to EGFR inhibition, similarly to the previously-characterized standard glioma cell lines (Figure 8A and 8B). In agreement with our previous data, EGFR inhibition also prevented multicellular tumour spheroid formation in U87ΔEGFR cells (Figure 8C). We next used multicellular tumour formation assays to investigate cooperation with TMZ. To this end, U87ΔEGFR cells were treated with sub-optimal doses of erlotinib (1–5 µM) and TMZ (25–50 µM), alone or in combination, and the formation of multicellular tumour spheroids was assessed. Similarly to untreated cells, cells treated with sub-optimal doses of erlotinib or TMZ alone gave rise to a high number of spheroids (Figure 8D). In contrast, the diverse combinations of sub-optimal doses of erlotinib and TMZ clearly reduced spheroid formation, similar to the standard erlotinib treatment (Figure 8D). We also investigated whether EGFR inhibition could reduce cell motility and invasion in the context of activated EGFR using wound-healing and transwell invasion assays, respectively. EGFR inhibition strongly reduced cell motility in both U87ΔEGFR and SKMG-3 cells (Figures 8E and 8F) and this inhibitory effect on cell motility was significantly reverted by Rho/ROCK inhibitors C3 and H-1152 (data not shown). Cell invasion within a 3D matrix was also strongly inhibited in the presence of erlotinib in both cell lines (Figure 8G). Taken together, these results confirm that EGFR inhibition can effectively reduce glioma cell proliferation, motility and invasion in cells with enforced EGFR activation. 10.1371/journal.pone.0038770.g008Figure 8 EGFR inhibition is effective in glioma cells with amplified or mutant EGFR. (A) SKMG-3 and U87ΔEGFR cells were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (B) SKMG-3 and U87ΔEGFR cells were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the fold increase in cell growth in untreated and erlotinib-treated conditions at the indicated time-points. (C) Representative phase-contrast micrographs of U87ΔEGFR cells left for 6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: ***P<0.001). (D) U87ΔEGFR cells were left untreated or treated as indicated and grown for 6 days to allow formation of multicellular tumour spheroids (MCTS). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between combined treatments and either treatment alone are statistically significant (Student's t-test: *P<0.05). (E) Representative phase-contrast micrographs of U87ΔEGFR (left panel) and SKMG-3 (right panel) cells left untreated or treated with 10 µM erlotinib as indicated, before (upper panel) and after (lower panel) performing wound healing assays as described in Materials and Methods. (F) Representation of the mean ± SD rate of motility, from three independent experiments performed in sextuplicate, expressed as the percentage of cell motility in each of the indicated conditions relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05 and **P<0.01, respectively). (G) U87ΔEGFR and SKMG-3 cells were seeded onto Matrigel-coated transwells in the absence or presence of 10 µM erlotinib to perform invasion assays as described in Materials and Methods. The graph represents the mean ± SD rate of invasion from three independent experiments performed in duplicate, expressed as the percentage of invasion relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: ***P<0.001). Discussion In this report we have investigated the cellular effects of EGFR inhibition with erlotinib in a panel of human glioma cell lines. We consistently observed that erlotinib inhibited cell growth in all tested cell lines, leading to the accumulation of treated cells in G1. A combination of dose-response and time-course growth assays indicated that 10 µM erlotinib inhibited glioma cell proliferation within a 30–80% range. Sensitivity to EGFR inhibition in cellular growth assays was strongly correlated with its ability to induce G1 arrest, and was most apparent in LN229 and U87MG cells. A similar pattern of sensitivity to the EGFR inhibitor AG1478 has been recently reported [7]. We did not detect cellular death upon EGFR inhibition neither in cell viability nor in flow cytometry assays, in correlation with previous reports showing that EGFR inhibition mainly exerts cytostatic effects in glioma cells [6]. In agreement with its ability to inhibit glioma cell proliferation in culture, EGFR inhibition also suppressed the formation of multicellular tumour spheroids in U87MG cells. EGFR inhibition was equally effective in the context of amplified or mutated EGFR, since both SKMG-3 cells (with EGFR amplification and overexpression) [17] and U87ΔEGFR (stably expressing the truncated and constitutively active EGFRvIII) [6], [10] were sensitive to erlotinib. Accordingly, EGFR inhibition also suppressed the formation of multicellular tumour spheroids in U87ΔEGFR cells. Since the alkylating agent TMZ is routinely used in chemotherapy to treat gliomas, we tested whether EGFR inhibition could cooperate with TMZ to prevent growth of glioma cells. We first used clonogenic assays to test whether inhibition of EGFR signaling sensitized glioma cells to TMZ-induced cytotoxicity. However, erlotinib pre-treatment had a protective effect against TMZ in this setting, probably related to accumulation of cells in G1 induced by erlotinib and the consequent attenuation of the DNA damage elicited by TMZ, a hypothesis that was recently suggested [18]. Indeed, these results are in agreement with the observation that the efficacy of TMZ in glioma cells is cell cycle dependent, since E2F expression in glioma cells increases TMZ sensitivity, whereas p21cip1 expression reduces it [19]. We therefore assessed whether concomitant treatment with erlotinib and TMZ had any cooperative effects, although clonogenic assays could not be used for this purpose since the effects of TMZ are very robust and do not follow a linear dose-dependent pattern in these assays (data not shown). We thus performed MTT-based cell viability assays using low, sub-optimal, concentrations of both erlotinib and TMZ. In these conditions, concomitant treatment exerted a cooperative effect in both U251 and U87MG cells. Interestingly, concomitant treatment with sub-optimal concentrations of erlotinib and TMZ was able to synergistically suppress multicellular tumour spheroid formation in both parental U87MG cells and their oncogenic, EGFRvIII-expressing, derivative U87ΔEGFR. Taken together, these data suggest that EGFR inhibitors can cooperate with TMZ for the treatment of gliomas, even at sub-optimal doses. However, our results also point out that drug scheduling is likely to influence the outcome of such treatments, and consequently should be taken into account in the design of clinical trials. In agreement with this, different scheduling regimens might be partially responsible for the conflicting data obtained in clinical trials with erlotinib in combination with TMZ in glioma patients. For instance, a phase I/II trial in which patients received treatment with erlotinib prior to its combination with TMZ and radiotherapy (RT) showed no benefit when compared to the standard historical treatment [20], whereas another clinical trial assessing a similar combination in which patients where administered erlotinib and TMZ continuosly from the beginning reported a better survival than historical controls [21]. Similarly, recent work in lung cancer cells has also highlighted the importance of drug scheduling when combining TKIs and conventional chemotherapeutic agents [22], [23]. Noteworthy, the timing of administration in combinations of erlotinib or other TKIs leading to G1 arrest together with radiotherapy in glioma patients should also be carefully considered, since cellular radiosensitivity has also been shown to be cell cycle dependent [24]. Our results do not support the notion that EGFR expression levels predict the response to EGFR inhibitors, in agreement with previous reports [5] and in contradiction with others [25]. Interestingly, it has been recently reported that small EGFR inhibitors such as AG1478, erlotinib, gefitinib and lapatinib, as opposed to the EGFR monoclonal antibody cetuximab, also reduce erbB3 and erbB4 phosphorylation, and this inhibition correlates with their cellular activity in glioma cells [7]. These observations raise the possibility that EGFR inhibitors might partially mediate their antitumoral effects through other members of the erbB family. Whatever the case, the cellular effects of EGFR inhibitors have been clearly associated with their ability to inhibit PI3K/Akt signaling and, accordingly, inhibitors of this pathway have been shown to potentiate the effects of EGFR inhibitors in sensitive cells and restore sensitivity in some resistant cell lines [10], [11], [26]. Not surprisingly, the most sensitive cell line in our panel, LN229, is the only one expressing wild type PTEN. Interestingly, our data indicate that PI3K/Akt inhibition is only apparent after several hours following EGFR inhibition, whilst ERK inhibition is an early event. This suggests that ERK inhibition is a direct consequence of EGFR receptor inhibition, probably through the inhibition of Ras/Raf signaling, whereas PI3K/Akt inhibition requires the suppression of further cellular mediators, probably inhibiting their expression. For instance, long term EGFR inhibition could be required to inhibit the production of growth factors responsible for sustaining PI3K/Akt activity in the presence of EGFR inhibitors. Accordingly, it has been reported that combinatorial treatment with several receptor tyrosine kinase (RTK) inhibitors might be required to fully inhibit PI3K signaling in glioma cells [27]. The rapid and sustained inhibition of ERK phosphorylation observed in response to erlotinib could be used as a biomarker of appropriate drug delivery in vivo (i.e. to test whether the drug has reached the target), as opposed to a biomarker of drug efficacy, which fits clearly better with the monitoring of Akt phosphorylation, in agreement with the reported correlation between EGFR inhibitor sensitivity and Akt inhibition found in glioma cell lines [25] and glioma tumour-initiating cells (TICs) [28]. The sensitivity to erlotinib in different cell lines also correlated with changes in the expression of essential cell cycle modulators. A similar pattern of molecular changes has been reported in response to the EGFR inhibitor AG1478 [7]. EGFR inhibition induced cyclin D1 downregulation, which has been shown to be regulated at different levels downstream of both the Ras/Raf/MEK/ERK and the PI3K/Akt pathways [29]–[31]. In parallel, EGFR inhibitors increased p27kip1 levels, which can also be regulated downstream of the PI3K/Akt pathway, for instance by FOXO-dependent transcription [32]. These changes in cyclin D1 and p27kip1 levels are likely to be responsible for the inhibition of G1 cyclin-dependent kinases regulating pocket protein phosphorylation and E2F release. Consequently, EGFR inhibition induced pRb hypophosphorylation in glioma cells, in correlation with the observed cell cycle arrest in G1. We also observed that EGFR inhibition in glioma cells induced dramatic morphological changes suggestive of actin cytoskeleton rearrangements. EGFR inhibition promoted the formation of robust actin stress fibres resulting in an increase in cellular spreading and attachment. Interestingly, such dramatic effects on cell morphology might be responsible, at least partially, of the strong inhibition of spheroid formation from U87MG monolayers elicited by erlotinib. Since these cytoskeletal alterations are under the control of Rho family GTPases [15], [33], we investigated whether EGFR inhibitors could regulate Rho and Rac activity in glioma cells. Interestingly, erlotinib treatment inhibited Rac and concomitantly increased Rho activity levels. These results are in agreement with several studies reporting such an inverse functional crosstalk between Rho and Rac GTPases [34]. For example, Ras inhibition with S-trans, trans-farnesyl thiosalicylic acid (FTS) in glioma cells also reduces Rac activity whilst increasing Rho activation [35]. Indeed, EGFR inhibition might lead to Rho activation as a consequence of Rac inhibition, which is probably due to the inhibition in PI3K/Akt signaling. Interestingly, Rac expression has been associated with resistance to erlotinib in glioma cells [36], and the combination of EGFR inhibitors with statins, which perturb Rho GTPase membrane localization and function [37], synergize to inhibit glioma cell growth irrespective of EGFRvIII and PTEN status [38]. Crucially, the increase in Rho-dependent actomyosin contractility resulted in a significant inhibition in glioma cell motility. This is probably associated with an increased rigidness of erlotinib-treated cells, as a consequence of the Rho-mediated assembly of actin stress fibres, which also leads to focal adhesion formation and an increase in cell attachment [16]. Noteworthy, these effects are likely to be cell-type dependent, since the balance of active Rho and Rac dictates different outcomes in different cell types. Importantly, these effects on glioma cell morphology and motility were causally related to the increase in Rho/ROCK activity, since inhibition of either Rho or ROCK alone was sufficient to restore the organization of the actin cytoskeleton and the rate of cell motility to control conditions. Moreover, invasion of glioma cells within a three-dimensional matrix was also compromised in the presence of EGFR inhibitors. This reduction in glioma cell motility and invasion was also observed in cell lines with amplified or mutant EGFR. These results are relevant considering that tumour cell invasion is a biological feature of particular importance for the clinical outcome of gliomas [39]. Our data thus support the use of EGFR inhibitors to reduce the infiltration of glioma cells. For example, EGFR inhibitors could be useful to minimize local invasion prior to surgery. Alternatively, EGFR inhibition could be used in combination with radiotherapy, since irradiation has been shown to increase glioma invasiveness under some circumstances [40], and this increase has been associated with Rho GTPase activity [41]. It would also be interesting to assess the combinatorial effects of different RTK inhibitors on glioma cell morphology, motility and invasion, since such combinations have already shown more effectiveness reducing glioma cell proliferation [27]. In summary, we have shown that EGFR inhibition in glioma cells perturbs intracellular signaling networks including Rho family GTPases and the ERK and Akt pathways, reducing glioma cell proliferation, motility and invasion. Our data also indicate that EGFR inhibition can cooperate with the alkylating agent TMZ in a schedule-dependent manner to reduce glioma cell growth. The implications of these findings could help to improve the design and interpretation of future clinical trials with EGFR inhibitors in glioma patients. We are grateful to Joan Seoane (Institut de Recerca Hospital Vall d'Hebron, Barcelona), Hans Skovgaard (Rigshospitalet, Oslo) and Isabel Martínez-Lacaci (Hospital Universitario Virgen de la Arrixaca, Murcia) for the gift of human glioma cell lines, Francisco Vega (King's College, London) for helpful advice on invasion assays and Cati Crespí (Hospital Son Dureta, Palma) for technical assistance with flow cytometry analysis. We are indebted to tenor Josep Bros, who performed a charity concert to raise funds for Cancer Research granted by Junta de Balears-AECC. We are also grateful to Roche and the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (NCI) for providing erlotinib and TMZ, respectively. Competing Interests: The authors have read the journal's policy and have the following conflicts: Their laboratory has received research funding from Roche-Farma (Spain). However, Roche-Farma had no role whatsoever in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Funding: This work was supported by funding from the “Direcció General de R+D+I, Govern de les Illes Balears” (PV), “Junta de Balears-AECC ” (PV and ET-M) and Roche- España (JR and PV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Maher EA Furnari FB Bachoo RM Rowitch DH Louis DN 2001 Malignant glioma: genetics and biology of a grave matter. 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==== Front ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2270137010.1100/2012/728613Research ArticlePhysiochemical and Phytochemical Properties of Wax Apple (Syzygium samarangense [Blume] Merrill & L. M. Perry var. Jambu Madu) as Affected by Growth Regulator Application Moneruzzaman Khandaker Mohammad 1 *Nasrulhaq Boyce Amru 1 *Osman Normaniza 1 Sharif Hossain ABM 2 1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia2Program of Biotechnolgy, Department of Biology, Faculty of Sciecne, University of Hail, Hail-2404, Saudi Arabia*Mohammad Moneruzzaman Khandaker: [email protected] and *Amru Nasrulhaq Boyce: [email protected] Editors: R. L. Jarret and J. R. Qasem 2012 4 6 2012 2012 7286131 11 2011 5 1 2012 Copyright © 2012 Mohammad Moneruzzaman Khandaker et al.2012This 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.This study represents the first paper of the effects of growth regulators on the physiochemical and phytochemical properties of the wax apple fruit, a widely cultivated fruit tree in southeast Asia. Net photosynthesis, sucrose phosphate synthase (SPS) activity, peel color, fruit firmness, juice content, pH value, total soluble solids (TSSs), and the sugar acid ratio were all significantly increased in growth regulators (PGRs) treated fruits. The application of gibberellin (GA3), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy acetic acid (2,4-D) significantly reduced titratable acidity and increased total sugar and carbohydrate content compared to the control. The 50 mg/L GA3, 10 mg/L NAA, and 5 mg/L 2,4-D treatments produced the greatest increases in phenol and flavonoid content; vitamin C content was also higher for these treatments. PGR treatment significantly affected chlorophyll, anthocyanin, and carotene content and produced higher phenylalanine ammonia lyase (PAL) and antioxidant activity levels. There was a positive correlation between peel color and TSS and antioxidant activity and both phenol and flavonoid content and PAL activity and anthocyanin formation. A taste panel assessment was also performed, and the highest scores were given to fruits that had been treated with GA3 or auxin. The study showed that application of 50 mg/L GA3, 10 mg/L NAA, and 5 mg/L 2,4-D once a week from bud development to fruit maturation increased the physiochemical and phytochemical properties of wax apple fruits. ==== Body 1. Introduction The wax apple, or jambu air madu, as it is known in Malaysia, is a nonclimacteric tropical fruit in the Myrtaceae family and is botanically identified as Syzygium samarangense [1]. Wax apple is widely cultivated throughout Malaysia, mainly in smallholdings ranging from 1 to 5 ha, with a total hectare estimated at 1,500 ha in 2005 [2]. It is also grown throughout the southeast Asian countries, such as Thailand, Indonesia, and Taiwan as well as other tropical countries. In Malaysia, there are three species which bear edible fruits, namely, the water apple (Syzygium aqueum), Malay apple (Syzygium malaccense), and wax apple or jambu air (Syzygium samarangense). S. samarangense is the most popular of the three in southeast Asia, and the trees are cultivated in home gardens, often planted along driveways and paths. Fruit production is nonseasonal and the peak periods are in February to April and October to December. It has become an increasingly popular fruit in the tropical region where it can fetch a price of up to 3USD per kilogram and has the potential to bring great benefit to local farmers and the country's economy. The pear-shaped fruits are usually pink, light red, or red but may be greenish-white or cream-colored, and are generally crisp, often juicy, refreshing, with a subtly sweet taste and aromatic flavor. Wax apple fruits are eaten raw with salt or cooked as a sauce. Almost all of the fruit is edible. The fruit pulp is a rich source of phenolics, flavonoids and several antioxidant compounds and as a result is believed to have great potential benefits for human health. In addition to its use as food, it has also been used in traditional medicine for a variety of illnesses and conditions. The fruit can be used to treat high blood pressure and several inflammatory conditions, including sore throat, and can also be used as an antimicrobial, antiscorbutic, carminative, diuretic, and astringent. It is important for fruit growers to have information on the differences in fruit quality among the available fruit varieties and the changes occurring in fruit quality parameters over time. PGRs enhance the rapid changes in physiological and biochemical characters and improve crop productivity. GA3 has been found to increase fruit firmness, color, yield, and soluble solid content [3]. NAA has been shown to significantly increase fruit yield, total soluble solids (TSSs), total sugar content, fruit color in Bing cherry, and vitamin C in guava fruits [4]. Synthetic auxin increases total antioxidant capacity and nutritional quality in transgenic Silcora seedless grape [5]. It was also reported that 2,4-D increased total sugar content and enhanced the activities of antioxidant enzymes [6]. The application of 2,4-D, GA3, and NAA significantly reduced acidity percentage and increased vitamin C content of citrus fruits [7]. It has been reported that GA3 significantly promotes the secondary metabolites, which affect the biosynthesis of flavonoids [8], hormonal regulation of anthocyanin formation, and enhancement of phenylalanine ammonia lyase (PAL) [9]. Currently, there is no available literature describing the effects of growth regulators on wax apple quality. This study investigated the effects of gibberellin and synthetic auxin on fruit quality and on the physiochemical and phytochemical properties of wax apple under field conditions. It is proposed that the application of PGRs can affect or promote the physiochemical and phytochemical quality of the wax apple fruit. 2. Materials and Methods 2.1. Experimental Site The experiments were performed in orchards located at Malaysian Agricultural Research and Development Institute (MARDI), Klang (2°30 N, 112°30 E), and at a commercial farm in Banting (1°28 N, 111°20 E), Malaysia, both at an elevation of approximately 45 m above sea level. The area under study has a hot and humid tropical climate. The soil in both orchards is peat, with a mean pH of approximately 4.6. The experiments were conducted between 2008 and 2011. The first season (December 2008–April 2009) of experiments was performed at MARDI, Jalan Kebun, Klang, and the second (May 2010–October 2010) and third (December 2010–May 2011) seasons' experiments were conducted at the farm in Banting. 2.2. Treatment Application and Fruit Harvesting Twelve-year-old wax apple plants were selected for the study. The trees were planted in a 4.2 m × 4.2 m hexagonal pattern and received the same horticultural management. Total-seventy of two trees were used for 1st season for GA3, NAA, and 2,4-D. Three hundred sixty uniform branches (five branches per tree), with approximately the same length, diameter, and number of leaves, were selected for sample branches for the GA3, NAA, and 2,4-D experiments. Similarly, same numbers of trees and uniform branches were selected for the second and third seasons. Different trees from two experimental sites were utilized for the different treatments in different years to avoid additive effects of growth regulators. Each experiment consisted of four treatments, including the control, in six replicates. The leaves, flowers, and young small fruits of selected uniform branches were sprayed with 20, 50, and 100 mg/L GA3, 5, 10, and 20 mg/L NAA and 2,4-D and water (the control) once each week from the beginning of flower opening until fruit maturation. A total of seven spraying times were carried out; two times before anthesis and five times after anthesis, and 250 mL hormone solution was used per treatment (30 branches). It takes ten weeks from bud development to fruit ripening, and all the fruits were harvested eight weeks after anthesis. Immediately after harvest, all fruits were aggregated, sub-sampled and kept in a 4°C refrigerator until completion of the analysis. 2.3. Measurement of Physiological, Biochemical and Phytochemical Parameters 2.3.1. Peel Color, Pulp Firmness, Juice and pH The peel color of the fruits was measured using a Minolta colorimeter (CR-300, Konica, Japan). Parameters such as “L” (lightness), “a” (greenness to redness) and “b” (blueness to yellowness) were determined at three different spots around the top, middle and end of the fruits. Sample averages were calculated and the color was expressed in L*, a*, b* Hunter parameter, using the following formula (L* × a*)/b*. Fruit firmness was determined with a digital hand-held penetrometer (Model KM-1, Fujiwara, Japan). The fruit juice of each harvested fruit was extracted and weighed, and the average juice weight was calculated separately for each treatment. The pH of the wax apple juice was recorded using a pH meter (Hanna pH 211, Italy). 2.3.2. Titratable Acidity and Sugar Acid Ratio The fruit juice (10 mL) was titrated with 0.1 M NaOH, and the results are expressed in terms of percentage citric acid. The percentage of citric acid was calculated using the formula of Bhattarai and Gautam [10], and the sugar acid ratio of the wax apple juice is given as the ratio of TSS/TA: (1) TA(%)=Nb×Vb×Ea×df×100Vs, where N b is normality of the base, V b is volume of the base, E a is mill equivalent weight of citric acid, V s is volume of sample, and df: dilution factor. 2.3.3. Determination of Soluble Carbohydrates and Total Sugar The total soluble solids (TSSs) value was determined at 25°C with an Atago 8469 hand refractometer (Atago Co. LTD., Tokyo, Japan) and expressed as °Brix. Glucose, fructose, and sucrose were evaluated at 25°C with the HI 96811 digital refractometer (Hanna instruments) and expressed as percentages. Total soluble sugar was determined using the phenol-sulfuric method [11]. 2.3.4. Determination of Vit-C, Polyphenolic Compounds, and Pigment Concentration Total ascorbic acid (vit-C) content was determined using the method modified by Hashimoto and Yamafuji [12]. The total phenolic contents (TPC) of wax apple fruits were determined with the Folin-Ciocalteu assay, as described by Singleton and Rossi [13]. Total flavonoid content (TFC) was determined with the aluminum chloride colorimetric assay, using catechin as a standard [14]. The chlorophyll and carotene contents of the leaves and fruit were determined using the methods described by Hendry and Price [15]. The total anthocyanin and carotenoid contents of the hydrophilic extracts were measured using the pH-differential method with cyanidin-3-glucoside used as a standard, as described by Rodríguez-Saona et al. [16]. 2.3.5. 2,2-Diphenyl-1-picryhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Free-Radical-Scavenging Assays The DPPH free-radical scavenging activity was determined as described in Yang et al. [17]. The ABTS free-radical-scavenging activity was determined as described in Re et al. [18]. 2.3.6. PAL Enzyme Activity and Taste Panel Evaluation PAL activity in the crude enzyme extracts was assayed using the method described by Zucker [19] and expressed as nmol cinnamic acid yield. To evaluate the overall sensory characteristics of the wax apple fruits from various treatments, a taste panel was performed with twelve panelists. All panelists had been previously trained with the specific sensory evaluation test. They evaluated the randomly offered fruits on a scale from 0 to 100 (low-high scores for each evaluated variable) based on the following criteria: taste, flavor, color, firmness, acidity, sweetness, mouth aroma, and taste remaining after swallowing. Net Photosynthesis, Enzyme Extraction, and SPS Assay Treated wax apple leaf samples (0.5 g) were homogenized in 2 mL of ice-cold 50 mM MOPS-KOH buffer (pH 7.3), 5 mM MgCl2, 1 mM ethylene diamine tetra acetic acid (EDTA), 16 mM mercaptoethanol, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM benzamidine, 1 mgL−1 leupeptin, and 2 mM phenylmethyl-sulfonyl fluoride. The crude extract (0.8 mL) was desalted by centrifugal gel filtration using a 4 mL Sephadex-G25 (Pharmacia) column equilibrated with 50 mM MOPS-KOH (pH 7.3), 5 mM MgCl2, 1 mM EDTA, 16 mM mercaptoethanol, and 10% (v/v) glycerol. The desalted crude extract was used for enzyme analysis, and protein content was determined by Bradford method [20] using bovine serum albumin as standard. SPS activity was assayed under V max⁡ condition as described by Huber et al. [21]. The photosynthesis rate (Pn) was measured using an Li-6400XT portable photosynthesis system (Li-COR Biosciences, Lincoln, NE, USA). Measurements were made immediately after treatment application at 9.00 am to 1.00 pm, and fifty four leaves were selected from GA3 and NAA treatments and control branches. Before measuring the photosynthetic parameter, the cuvette chamber conditions were set to provide photosynthetic photon flux density of 400, 800, 1200, and 2000 μmol m−2 s−1 and cuvette block temperature was maintained at 24°C, and the concentration of CO2 was set at 350 μmol mol−1 with a flow rate of 500 mL s−1. 2.4. Statistical Analysis The experimental design was a randomized complete block design (RCBD) with six replications. The data obtained from three successive seasons were pooled and analysed using MSTAT-C statistical software. A one-way ANOVA was applied to evaluate significant differences in the studied parameters in the different treatments. The least significant difference (Fisher's protected LSD) was calculated following a significance F-test (at P = 0.05). 3. Results 3.1. Peel Color Development Fruit color is considered to be one of the important external factors in determining fruit quality, as the appearance of fruit greatly influences consumer's preferences. Table 1 shows that fruit color development was greatly enhanced by the PGRs treatments. In the “4th” week of observation, fruits treated with 50 mg/L GA3 showed a*, b* value 99, followed by fruits treated with 100 and 20 mg/L GA3, whereas control fruits had only a*, b* value 30. The difference between the treatments and control was statistically significant (Table 1). For NAA treatments, fruits treated with 10 mg/L showed a*, b* value 80. In contrast, control fruits had only a*, b* value 30, and the differences between the treatments and the control were statistically significant. The application of 2,4-D also had a significant effect on peel color development of wax apple fruits (Table 1). 3.2. Pulp Firmness, Juice Content, and pH of Fruit Juice The pulp firmness of wax apple fruits treated with different growth regulators is presented in Table 1. The pulp firmness of wax apple fruits was significantly affected by PGR application. Our results showed that pulp firmness increased with growth regulator application. The highest pulp firmness was recorded in fruits treated with 10 mg/L NAA, 50 mg/L GA3, and 10 mg/L 2,4-D,with measured values of 8.0, 7.5, and 7 (N), respectively. The pulp firmness of untreated fruit was 6.5 (N). The results show that the highest amount of juice (81 mL/100 g) was obtained in 50 mg/L GA3-treated fruits. The next-highest quantities were obtained in fruits treated with 20 and 100 mg/L, with juice percentages of 80 and 78 mL/100 g, respectively. The lowest juice percentage of 69 mL/100 g was obtained in the control. The differences between the treatments and control were statistically significant. Synthetic auxin (NAA or 2,4-D) treatments also produced significant effects on juice content in wax apple fruits. As shown in Table 1, the pH of the fruit juice was affected by growth regulator application, and the pH values of the juice fell within the range of 4.9–5.29. The differences in pH were statistically significant between the GA3 treatments and control. The results show that the highest pH (5.29) was recorded for the 50 mg/L GA3 treatment, while the lowest pH value (4.9) was recorded in the control. For the NAA, and 2,4-D treatments, the highest pH values (5.15 and 5.12) were recorded for the 10 mg/L 2,4-D and NAA treatments, respectively. The control pH value was the lowest (4.9), and the differences in pH value were statistically significant between the treatments and control. 3.3. Titratable Acidity (TA) and Sugar Acid Ratio The results for the TA experiments are shown in Table 1. Our results clearly indicate that titratable acidity was significantly affected by growth regulator application. The lowest amount of TA (0.70%) was observed with the 100 mg/L GA3 treatment, followed by the TA amounts for the 50 and 20 mg/L GA3 treatments. The highest amount of titratable acidity (0.78%) was observed in the control. Similarly, significant changes in titratable acidity of wax apple fruits were recorded with NAA and 2,4-D application, and the lowest amount (0.71%) was recorded with the 20 mg/L NAA treatment. As shown in Table 1, the sweetness index (sugar acid ratio) of fruits was significantly enhanced by the GA3, NAA and 2,4-D treatments. The 50 mg/L GA3 treatment increased the sugar acid ratio by 87%, followed by the 10 mg/L NAA and 10 mg/L 2,4-D treatments with increases of 71% and 60%, respectively, relative to the control. 3.4. TSS, Soluble Carbohydrates, and Total Sugar TSS is an important quality factor attribute for many fresh fruits during ripening. The solids include acids and the soluble sugars sucrose, glucose, and fructose. As shown in Table 2, the highest TSS value of 11.5 (°Brix) was observed in 50 mg/L GA3-treated fruit, followed by fruit treated with 100 and 20 mg/L GA3 treatments, with TSS values of 10.1 and 9.07, respectively. The minimum TSS in the control samples was 6.70 (°Brix). NAA and 2,4-D treatments also produced higher TSS contents than the control, and the differences were statistically significant (Table 2). Carbohydrates, namely glucose, fructose, and inverted sugar, were evaluated in this study. The carbohydrate content of wax apple fruits was significantly elevated by GA3 and NAA application. The highest glucose, fructose and inverted sugar contents were observed in the fruits treated with 50 mg/L and 5 mg/L NAA. The control samples contained the lowest amounts of glucose, fructose, and inverted sugar. Nonsignificant changes in carbohydrate content were observed with the 2,4-D treatments (Table 2). After performing the above experiments, we determined the sugar contents of the wax apple fruits. For the GA3 treatments, statistically significant differences between the treatments and control were observed. The results for GA3 are shown in Table 2, and the highest total sugar content of 6.5 g/100 g was recorded for the 50 mg/L GA3-treated fruits, followed by the 100 and 20 mg/L GA3-treated fruits, with sugar contents of 5.82 and 5.57 g/100 g, respectively. In contrast, the control fruits showed the lowest sugar content of 3.65 g/100 g. Significant changes in total sugar content were also observed with NAA and 2,4-D application (Table 2). 3.5. Vit-C Content In this study, growth regulator application significantly affected the vit-C content of wax apple fruits (Table 3). With GA3 application, the highest vit-C content (6.6 mg/100 g) was recorded in 50 mg/L treated fruits, followed by fruits treated with 20 and 100 mg/L GA3, with vit-C contents of 5.9 and 5.8 mg/100 g, respectively. The lowest amount of vit-C (5.1 mg/100 g) was recorded in control fruit. Similarly, significant increases in vit-C content were observed in wax apple fruits treated with NAA and 2,4-D. The highest vit-C contents for auxin application were observed in the 5 mg/L NAA and 2,4-D treatments, with 6 and 5.6 mg/100 g, respectively. 3.6. Total Phenol and Flavonoid Content As shown in Table 3 the TPC and TFC contents were clearly increased following growth regulator application. For GA3 treatment, the highest phenol and flavonoid contents (535 mg GAE/100 g and 45 mg CE/100 g, resp.) were recorded with the 50 mg/L GA3 treatment, followed by contents recorded in the 20 and 100 mg/L treatments. The control had the lowest levels of phenols and flavonoids (311 mg GAE/100 g and 18.5 mg CE/100 g, resp.). The differences between the treatments and control were statistically significant. Significant changes in the phenol and flavonoid contents of wax apple fruits were also observed with NAA and 2,4-D treatments. The effect of 2,4-D treatment on phenol and flavonoid content was highest with lower concentrations of 2,4-D (Table 3). Higher concentrations of 2,4-D showed a negative effect on the phytonutrient content of wax apple fruits. 3.7. Degradation of Chlorophyll Chlorophyll content of ripening wax apple skin was also observed in this study. The results show that GA3, NAA, and 2,4-D application significantly reduced the chlorophyll content in fruits. The highest chlorophyll content (0.63 mg/L) was observed in the control, followed by amounts recorded in the 20 and 100 mg/L GA3 treatments. The lowest amount (0.24 mg/L) was recorded in 50 mg/L GA3-treated fruits (Table 3). These results indicate that growth regulator application stimulates the pigmentation of wax apple fruits under field conditions. For NAA and 2,4-D application, nonsignificant changes in chlorophyll content were observed. 3.8. Anthocyanin and Carotenoid Biosynthesis As shown in Table 3, the application of various growth regulators had significant effects on the anthocyanin and carotene contents in wax apple fruits. For GA3 treatment, the highest amount of anthocyanin (46.0 mg/100 g) was observed with the 50 mg/L GA3 treatment, followed by the 20 and 100 mg/L treatments, with values of 40.0 and 36.0 mg, respectively. The control fruits had the lowest anthocyanin content (24.3 mg/100 g). NAA and 2,4-D treatments also produced significant changes in anthocyanin content (Table 3). The results show that the 50 mg/L GA3 treatment nearly doubled the carotenoid content in wax apple fruits. For NAA treatment, the highest carotenoid content (10.5 μg/g) was observed with the 10 mg/L NAA treatment, followed by the 5 and 20 mg/L NAA treatments, with carotene contents of 9.83 and 8.90 μg/g, respectively. The carotenoid content was 5.97 μg/g in control fruits (Table 3). Similar to the GA3 and NAA treatments, 2,4-D treatments also yielded higher carotenoids in the fruits. 3.9. Antioxidant Content The DPPH and ABTS radical scavenging activity measured in wax apple fruit extracts was affected by different growth regulators, as shown in Figure 1. Our results showed that the IC50 of the DPPH and ABTS radical scavenging activity increased with PGR application. The results showed that the DPPH and ABTS radical scavenging activity increased up to 70% and 54% in fruit extracts from the 50 mg/L GA3 treatment, while activity in the control was only 50% (Figures 1(a) and 1(a1)). For NAA treatments (Figures 1(b) and 1(b1)), the highest antioxidant capacity, determined using both the DPPH and ABTS assays, was observed in 10 mg/L NAA-treated fruits, followed by the 5 and 20 mg/L NAA-treated fruits. Control fruits showed the least antioxidant capacity. As with the 2,4-D treatments, 24% and 30% more DPPH and ABTS radical scavenging activity was recorded for extracts of 5 mg/L 2,4-D-treated fruits (Figures 1(c) and 1(c1)). Overall, GA3, NAA, and 2,4-D application increased the antioxidant capacity of wax apple fruits (Figure 1). 3.10. PAL Enzyme Activity of Fruits Our results show that GA3 treatment had a significant effect on PAL activity of the treated fruits (Figure 2(a)). PAL activity, as measured by cinnamic acid yield, was highest (15.67 nmol-cinnamic acid min−1 mg protein−1) for the 50 mg/L GA3 treatment, followed by the 100 and 20 mg/L GA3 treatments, with PAL activity values of 10.59 and 9.39 nmol-cinnamic acid min-1 mg protein−1, respectively. The lowest PAL activity (8.15 nmol-cinnamic acid min−1 mg protein−1) was recorded in the control. The synthetic auxins NAA and 2,4-D also produced significant differences in PAL activity (Figures 2(b) and 2(c)). For NAA, the highest PAL activity was recorded in 10 mg/L NAA-treated fruits, followed by fruits treated with 20 and 5 mg/L NAA. Control fruits produced the lowest amount of cinnamic acid (Figure 2(b)). Significant changes were similarly observed in 2,4-D-treated fruits (Figure 2(c)). 3.11. Correlations between Peel Color and TSS and between Phenols and Antioxidant Activity, PAL Activity, and Anthocyanin Formation Figure 3 shows the relationship between peel color and TSS and between phenol content and antioxidant capacity, determined using the DPPH and ABTS assays, in the wax apple fruits analyzed. A high degree of correlation (R 2 = 0.97 for 50 mg/L GA3, R 2 = 0.95 for 10 mg/L NAA, and R 2 = 0.93 for 5 mg/L 2,4-D) was observed for the peel color and TSS content of treated fruits. Similarly, a high degree of correlation (R 2 = 0.86 for 50 mg/GA3, R 2 = 0.92 for 10 mg/L NAA, and R 2 = 0.74 for 5 mg/L 2,4-D was observed for total phenols and antioxidant capacity (Figure 3). We also got positive correlation between PAL activity and anthocyanin formation in PGR-treated fruits (Figures 2(d), 2(e), and 2(f)). 3.12. Taste Panel Results on Overall Fruit Quality Attributes A spider chart was constructed to diagram the overall fruit quality evaluation of the panelists and shows the evaluation grades for the various attributes tested in the panel (Figure 4). The results show that fruits from the 50 mg/L GA3 treatment were designated as having the best taste, mouth aroma, and highest sweetness and were also classified among those having the lowest acidity, best flavor, and best aftertaste. On the other hand, fruit from the 10 mg/L NAA and 5 mg/L 2,4-D treatments exhibited intermediate values for most of the attributes tested. The control fruits had the highest acidity and exhibited low values for aftertaste, appearance, and sweetness. 3.13. Net Photosynthesis and SPS Activity To measure the activity level of photosynthetic carbon metabolism, we determined the photosynthetic activity in terms of μmol CO2 fixed m−2 s−1. GA3 and NAA treatments increased the leaf photosynthesis activity considerably; this effect was statistically significant in the observations of the 2010-2011 season. The activities were 1.64-, 2.42-, and 2.57-fold higher than the control at 350 ppm CO2 and light intensities of 400, 800, and 2,000 μmol m−2 s−1, respectively, in the leaves treated with 50 mg/L GA3 (Figure 2(a)). Leaf photosynthesis was highest with the 50 mg/L treatment, followed by the 100 and 20 mg/L GA3 treatments, in that order, whereas the control leaves evidenced the least photosynthesis. For NAA treatments, 10 mg/L treated plant showed highest photosynthetic activity compared to others and control. Sucrose phosphate synthase activity of treated leaves also increased significantly for GA3 and NAA treatments (Figures 2(b) and 2(d)) 4. Discussion The colors, or pigments, in fruits and vegetables reflect the presence of certain biologically active phytochemical compounds and antioxidants that have been reported to promote good health. Positive values of a* and b*, as observed in this work, are attributed to carotenoids or anthocyanins present in the skin. Our results for peel color development are in agreement with those of Basak et al. [3] that application of GA3 increases the color of fruits. Raphael et al. [22] also observed that synthetic auxin enhanced fruit color development in Bing cherry fruit. The increase observed, as a result of growth regulator applications, is possibly due to an increase in the activity of enzymes responsible for color development. In our study, growth regulators had positive effects on the firmness of wax apple fruits. These findings are in agreement with those of Choi et al. [23] that GA3 increased fruit firmness at harvest and decreased the rate of fruit softening. Similar findings were reported by Iqbal et al. [4], who showed that application of synthetic auxin significantly increased pulp firmness in loquat fruit. The amount fruit juice produced is normally related to fruit size and genetic characteristics of a particular fruit. It has also been documented in previous studies that growth regulators can have a significant effect on fruit size and as a consequence the amount of fruit juice. In this study GA3 treatment was found to have a positive effect on the juice content of wax apple fruits. These results are in agreement with those of Wang et al. [24] that the application of gibberellic acid at flowering and preharvest significantly increased the juice percentage in various citrus species. Synthetic auxin increases absolute juice content in citrus fruits, through simultaneous increases in fruit size and juice content from pulp [7]. We obtained similar results with NAA and 2,4-D in wax apple fruits. Data for fruit juice pH were in agreement with the findings of Thakur et al. [25] that the acidity of tomato fruits was reduced when the plant was sprayed with GA3 and 2,4-D. Thakur et al. [25] also reported, however, that ascorbic acid content increased with higher concentrations of 2,4-D. In the present study, growth regulator application significantly reduced the titratable acidity content in wax apple fruits. The reduction in titratable acidity observed, with the application of PGRs, can probably be attributed to the conversion of the organic acids to sugar during fruit ripening. Thakur et al. [25] similarly reported that titratable acidity was significantly reduced with GA3 and auxin application. GA3 application had a greater effect on reducing acidity compared to NAA. Our results for acidity percentage were in agreement with those of Xiao et al. [7] that application of PGRs significantly reduced acidity percentage. It is also important to note that acidity increased with increasing concentrations of 2,4-D (Table 1), indicating that 2,4-D should be applied at low concentrations for quality improvement. Wahdan et al. [26] reported that GA3 treatments significantly increased the TSS, sugar acid ratio, and total sugar content of mango, whereby the ratio of sugar and acid determines the taste, flavor, and acceptability of fruit. We also observed significant changes in the sugar acid ratio due to growth regulator application in wax apple fruits. In fact, the sugar acid ratio may be also the key factor affecting the quality of wax apple under tropical climates. In our study, GA3 and auxin treatments significantly increased the TSS (°Brix) content of wax apple. These results are in agreement with those of Basak et al. [3] that auxin and gibberellins significantly increased the TSS contents of the citrus fruits tested. Carbohydrates, such as glucose, fructose, and sucrose, play a central role in metabolism and regulate many developmental and physiological processes in plants. We observed that gibberellin and auxin treatments significantly altered carbohydrate concentration in wax apple fruits. The results are in agreement with Wang et al. [24], who reported that application of 2,4-D, GA3, and some other growth regulators increased the sugar content in various mandarin and sweet orange cultivars. Synthetic auxin application during anthesis was found to increase the amount of sugar content in tomato [4]. Accordingly, in this study, also PGRs treatments produced significant effects on total sugar content of wax apple. PGRs treatments may influence the source-sink balance in a plant and as a result increase the accumulation rate of carbohydrate content in wax apple fruit. Vit-C content in fruits varies among crop species and is affected by environmental factors, time of fruit harvesting, plant vigor, the age of the plant, and the use of growth regulators. In this study, we observed that GA3 treatments had a significant effect on vit-C content in wax apple. With respect to vit-C content, GA3 treatments were more beneficial compared to 2,4-D and NAA treatments, as vit-C content decreased with increasing concentrations of synthetic auxin (2,4-D or NAA). Our results were consistent with those of Xiao et al. [7] that preharvest application of growth regulators increased vit-C content of the citrus fruits. From our findings, GA3 treatments clearly had a significant effect on the total flavonoid and phenolic content of wax apple fruits. Our results also showed that phenolic content positively correlated with antioxidant activity in GA3-treated fruits. These results are in agreement with the findings of Pourmorad et al. [27] that the extracts of M. officinalis containing the highest amounts of flavonoid and phenolic compounds exhibited the greatest antioxidant activity. In our study, synthetic auxin also had a significant effect on the total phenolic content of wax apple fruits. This finding correlates with that of Elisa et al. [5] in that auxins increased the total polyphenolic content, as well as the nutritional content in grape. Flavonoids are ubiquitous plant secondary metabolites and play a vital role in their physiology by producing the red and purple anthocyanin pigments. The present study indicates that the flavonoid content of wax apple is significantly affected by PGRs and is consistent with the findings of Klessig and Malamy [8] that GA3 promote synthesis of flavonoids, as increased anthocyanin synthesis, promoted by GA3, was found to promote levels of flavonoid-specific mRNAs. Elisa et al. [5] also reported that the flavonoid content of grape was significantly affected by auxin application. It is believed that the applied growth regulators elevated the level and activity of chalcone synthase, an enzyme responsible for the synthesis of pigments and thus stimulated flavonoid synthesis in treated fruits. In this study, it was observed that chlorophyll loss gradually occurred with PGRs application. Perez et al. [28] reported similar findings that the plant growth regulator methyl jasmonate promoted chlorophyll degradation in the skin of Golden Delicious apples. Our results indicate that growth regulators had positive effects on anthocyanin and carotenoids content in wax apple fruits. These results are in agreement with the findings of Roussos et al. [29] that anthocyanin content in strawberry fruit increased significantly when the plants were treated with GA3. These observations suggest that GA3 could also play a role in the accumulation of pigments in fruits. Our results for anthocyanin content with auxin treatment were in agreement with the findings of Teresa et al. [9] that growth regulators enhanced the accumulation of anthocyanin content in strawberry fruits. Our results showed that fruit treated with PGRs exhibited higher antioxidant capacity than control fruits. These findings are consistent with the results of Klessig and Malamy [8] that GA3 and auxin significantly promoted biosynthesis of secondary metabolites in fruit with the highest antioxidant activity. PAL is one of the key enzymes controlling anthocyanin biosynthesis from phenylalanine. In our study, growth regulators had significant effects on PAL activity in wax apple fruits. This increased PAL activity probably contributed to the enhanced red color development observed in the growth regulator treated fruits. These results were consistent with those of Teresa et al. [9] that GA3 and auxin increased PAL activity in strawberry plants. Our results for peel color and TSS of treated fruits were in agreement with the results of Moneruzzaman et al. [30] that fruit color positively correlated with soluble solids in tomato. In this study, a positive correlation was observed between antioxidant activity and total phenolic and flavonoid content also PAL activity with anthocyanin synthesis. This correlation indicates that phenolic and flavonoid compounds could be the primary factors governing antioxidant activity in the wax apple fruit samples, in agreement with previous findings that many phenolic compounds in plants are good sources of natural antioxidants [31]. Many researchers have identified positive relationships between biochemical analysis data and taste panel results, including a correlation between the sweetness of fruits and their TSS [32]. The panelists graded 50 mg/L GA3-treated fruits as the sweetest, followed by the 10 mg/L and 5 mg/L 2,4-D-treated fruits. These results were in agreement with the highest TSS and sweetness index values having been observed in these treatments. Furthermore, the panelists classified fruits from these treatments as having the lowest acidity, greatest color, and best taste, again in agreement with the high sweetness index found for these fruits. As is well known, during and after photosynthesis, sugars, namely sucrose, are exported from the source leaves to other plant parts. Sucrose is synthesised in the cytosol from triose phosphates made in the Calvin cycle and exported from the chloroplasts, where it is converted into fructose 6-phospate which combines with UDP glucose to form sucrose phosphate, catalysed by sucrose phosphate synthase. It has been shown in our study that GA3 and NAA treatments increased the net photosynthesis and SPS activity of wax apple plants (Figure 5). Hubbard et al. [33] also found positive relationships between SPS activity and sucrose accumulation in melon. PGRs treatments may also increase the invertage activity, the increase invertage activity suggests for sucrose synthesis and vice versa due to increased photosynthetic product in treated leaves. The increased SPS activity could raise not only sucrose level but also glucose and fructose level in leaves and fruits of wax apple. It is suggested from this study that PGRs treated fruits accumulated the high percentage of sugar, polyphenolic compound, and antioxidant substances in fruits, thus increase its taste, flavour, as well as quality. 5. Conclusions From the present study, we concluded that the PGRs (GA3, NAA, and 2,4-D) can improve the physiochemical and phytochemical status of wax apple fruits. Nevertheless, it must be emphasized that the positive effects of PGRs on the quality of wax apple are dependent on types, dose, and environmental conditions. 50 mg/L GA3 treatment produced greater increases in physiochemical and phytochemical nutrition than the 100 and 20 mg/L GA3 treatments. For NAA, the 10 mg/L NAA treatment had the greatest effect on physiochemical and phytochemical nutrition. For 2,4-D, another synthetic auxin, the most promising results were obtained with a low concentration of 5 mg/L, and treatments in excess of 10 mg/L actually produced adverse effects on physiochemical and phytochemical properties. Our results also showed that the treated fruits with the highest antioxidant content were also ranked most favorably for fruit quality attributes and taste. From experiments performed under field conditions, we concluded that the 50 mg/L GA3, 10 mg/L NAA, and 5 mg/L 2,4-D treatments show particular promise for enhancing the physiochemical and phytochemical quality of wax apple fruits. Acknowledgment This research was supported by a Grant from the University of Malaya, Kuala Lumpur, Malaysia (Project no. RG002/09BIO). Figure 1 Changes in antioxidant activity: (a) and (a1) GA3 treated, (b) and (b1) NAA treated, (c) and (c1) 2,4-D treated wax apple fruits using the DPPH and ABTS assays. Vertical bars represent the LSD at the 5% level. Figure 2 Effects of treatments of GA3 (a) and (d), NAA (b) and (e) and 2,4-D (c) and (f) on PAL activity and correlation between PAL and anthocyanin synthesis in wax apple fruits. Data are means of six replicates ± SE. Different letters represent the significance at the 5% level of LSD test. Figure 3 Correlation between peel colour and TSS and total phenols and antioxidant capacity in the GA3, NAA, and 2,4-D treated fruits. Figure 4 Taste panel scoring of fruits from the GA3, NAA and 2,4-D treatments, based on the examined quality attributes. Figure 5 Effect of GA3 treatment on (a) SPS activity, (b) net photosynthesis and NAA treatment on (c) SPS activity, and (d) net photosynthesis of wax apple leaves. Data are means of six replicates ± SE. Different letters represent the significance at the 5% level of LSD test. Table 1 Effect of GA3, NAA and 2,4-D treatments on physicochemical properties of wax apple fruits. Treatment (mg/L) Peel color L*a*/b* Pulp firmness (N) Fruit juice (mL/100 g) PH TSS (°Brix) Titratable acidity (%) Control 30 ± 3.2b 6.5 ± 0.45b 69 ± 0.66b 4.90 ± 0.10b 6.70 ± 0.3b 0.78 ± 0.04a GA3 20 85 ± 2.6a 7.2 ± 0.38a 80 ± 2.30a 5.29 ± 0.12a 9.07 ± 0.3a 0.72 ± 0.03b GA3 50 99 ± 3.0a 7.5 ± 0.40a 81 ± 2.08a 5.17 ± 0.25a 11.5 ± 0.6a 0.71 ± 0.04b GA3 100 90 ± 2.8a 6.9 ± 0.26a 78 ± 1.15a 5.15 ± 0.20a 10.1 ± 0.5a 0.70 ± 0.03b LSD (5%) 7.283 1.250 9.42 0.32 1.665 0.085 Control 30 ± 3.2b 6.5 ± 0.45b 69 ± 0.66b 4.90 ± 0.10c 6.70 ± 0.3c 0.78 ± 0.04a NAA 5 74 ± 3.6a 7.8 ± 0.43a 79 ± 2.92a 4.95 ± 0.16b 9.76 ± 0.9b 0.73 ± 0.02b NAA 10 80 ± 2.5a   8.0 ± 0.29a 82 ± 2.20a 5.12 ± 0.29a 10.7 ± 0.9a 0.72 ± 0.03b NAA 20 76 ± 4.0a 7.5 ± 0.37a 80 ± 1.66a 5.09 ± 0.20b 9.70 ± 0.9b 0.71 ± 0.04b LSD (5%) 5.47 1.43 9.27 0.35 0.836 0.0823 Control 30 ± 3.2b 6.5 ± 0.45b 69 ± 0.66b 4.90 ± 0.10b 6.70 ± 0.3b 0.78 ± 0.04b 2,4-D 5 79 ± 3.0a 6.9 ± 0.35a 78 ± 1.76a 4.99 ± 0.14a 8.70 ± 0.8b 0.73 ± 0.04b 2,4-D 10 83 ± 5.0a 7.0 ± 0.42a 75 ± 2.60a 5.15 ± 0.17a 8.90 ± 0.9b 0.74 ± 0.02b 2,4-D 20 72 ± 3.0a 6.7 ± 0.31a 74 ± 1.54a 4.85 ± 0.12b 7.26 ± 0.7b 0.74 ± 0.03b LSD (5%) 5.57 1.33 9.53 0.38 1.582 0.093 Means (±SE) within the same column followed by the same letter do not differ significantly according to the LSD test at α′ = 0.05. Table 2 Effects of growth regulators on TSS/acidity ratio, soluble carbohydrates, and total sugar content of wax apple fruits. Treatment (mg/L) TSS/acidity ratio Glucose (%) Fructose (%) Inverted sugar (%) Total sugar (mg/100 g) Control 8.58 ± 1.3b 5.60 ± 0.29b 5.73 ± 0.14c 6.25 ± 0.7c 3.65 ± 0.47b GA3 20 12.6 ± 1.5a 7.00 ± 0.64a 7.08 ± 0.17b 7.37 ± 0.4b 5.57 ± 0.25a GA3 50 16.1 ± 1.7a 8.00 ± 0.20a 8.37 ± 0.15a 8.12 ± 0.4a 6.56 ± 0.23a GA3 100 14.4 ± 1.5a 7.60 ± 0.26a 8.18 ± 0.25a 7.93 ± 0.5a 5.82 ± 0.32a LSD (5%) 2.548 1.065 1.081 1.319 0.526 Control 8.58 ± 1.3b 5.60 ± 0.29c 5.73 ± 0.14b 6.25 ± 0.70c 3.65 ± 0.47b NAA 5 12.8 ± 2.0a 8.00 ± 0.23a 8.02 ± 0.15a 8.26 ± 0.60a 5.84 ± 0.20a NAA 10 14.7 ± 2.1a 7.20 ± 0.32b 7.62 ± 0.10a 6.96 ± 0.43b 6.35 ± 0.17a NAA 20 13.6 ± 1.7a 6.70 ± 0.18b 7.10 ± 0.27a 6.90 ± 0.45b 6.09 ± 0.23a LSD (5%) 2.473 0.767 0.990 1.072 0.581 Control 8.58 ± 1.3b 5.60 ± 0.29a 5.73 ± 0.14b 6.25 ± 0.70a 3.65 ± 0.47b 2,4-D 5 12.8 ± 1.8a 6.25 ± 0.30a 6.30 ± 0.25a 6.50 ± 0.45a 5.83 ± 0.30a 2,4-D 10 13.7 ± 1.9a 6.16 ± 0.29a 6.26 ± 0.30a 6.40 ± 0.65a 6.20 ± 0.32a 2,4-D 20 10.1 ± 2.0a 6.00 ± 0.34a 5.80 ± 0.19a 6.17 ± 0.53a 4.81 ± 0.27b LSD (5%) 2.872 0.653 0.782 0.890 0.672 Means (±SE) within the same column followed by the same letter do not differ significantly according to the LSD test at α′ = 0.05. Table 3 Effect of growth regulators on phytochemical properties and pigmentation of wax apple fruits. Treatment (mg/L) Ascorbic acid (mg/100 g) Phenol mg GAE/100 g Flavonoids (mg CE/100 g) Chlorophyll (mg/L) Anthocyanin (mg/100 g) Carotenoid (μg/g) Control 5.1 ± 0.07c 311 ± 21.6c 18.5 ± 0.5b 0.63 ± 0.06a 24.3 ± 2.07c 5.97 ± 0.24b GA3 20 5.9 ± 0.06b 589 ± 51.4a 40.0 ± 2.0a 0.44 ± 0.02b 40.2 ± 3.13b 10.5 ± 0.36a GA3 50 6.6 ± 0.18a 535 ± 32.4b 45.0 ± 1.2a 0.24 ± 0.04c 46.0 ± 3.70a 11.3 ± 0.20a GA3 100 5.8 ± 0.19b 552 ± 99.5b 37.9 ± 1.7a 0.26 ± 0.02b 36.0 ± 2.83b 10.3 ± 0.12a   LSD (5%) 1.469 40.92 2.545 0.093 3.18 0.648 Control 5.1 ± 0.07c 311 ± 21.6c 18.5 ± 0.5b 0.63 ± 0.06a 24.3 ± 2.07c 5.97 ± 0.  24b NAA 5 6.0 ± 0.10a 581 ± 25.3a 27.40 ± 1.8a 0.36 ± 0.03c 40.2 ± 0.25a 9.83 ± 0.40a NAA 10 5.8 ± 0.14a 537 ± 40.7a 28.87 ± 1.8a 0.46 ± 0.05b 39.0 ± 1.34a 10.5 ± 0.32a NAA 20 5.5 ± 0.16b 423 ± 55.6b 27.38 ± 1.2a 0.48 ± 0.04b 35.3 ± 1.46b 8.90 ± 0.25a LSD (5%) 1.49 37.31 2.53 0.0987 5.440 0.625 Control 5.1 ± 0.07c 311 ± 21.6c 18.5 ± 0.5b 0.63 ± 0.06a 24.3 ± 2.07c 5.97 ± 0.  24b 2,4-D 5 5.6 ± 0.12a 525 ± 4.83a 27.31 ± 2.1a 0.44 ± 0.03b 36.0 ± 0.23b 9.00 ± 0.39a 2,4-D 10 5.4 ± 0.16a 415 ± 2.94b 25.57 ± 2.5a 0.48 ± 0.05b 35.0 ± 0.17a 7.80 ± 0.28a 2,4-D 20 5.1 ± 0.13b 303 ± 3.92c 22.83 ± 1.6a 0.45 ± 0.03b 28.0 ± 0.11b 7.50 ± 0.35a LSD (5%) 1.52 41.54 3.934 5.47 5.574 0.643 Means (±SE) within the same column followed by the same letter do not differ significantly according to the LSD test at α′ = 0.05. ==== Refs 1 Morton J Java apple Fruits of Warm Climates 1987 381 382 2 Shü ZH Meon Z Tirtawinata R Thanarut C Wax apple production in selected tropical asian countries Acta Horticulturae 2008 773 161 164 3 Basak A Rozpara E Grzyb Z Use of bioregulators to reduce sweet cherry tree growth and to improve fruit quality Acta Horticulturae 1998 468 719 723 4 Iqbal M Khan MQ Jalal UD Rehman K Munir M Effect of foliar application of NAA on fruit drop, yield and physico-chemical characteristics of guava (Psidium guajava L.) Red flesh cultivar Journal of Agricultural Research 2009 47 3 259 269 5 Elisa C Lucia L Oriana S Tiziana P Angelo S Mezzetti B Auxin synthesis-encoding transgene enhances grape fecundity Plant Physiology 2007 143 4 1689 1694 17337528 6 Baogang W Jianhui W Hao L Reduced chilling injury in mango fruit by 2, 4-dichlorophenoxyacetic acid and the antioxidant response Postharvest Biology and Technology 2008 48 2 172 181 7 Xiao JX Peng S Hua HP Jiang LH Effects of calcium nitrate and IAA on calcium concentration and quality of Satsuma mandarin fruit Journal of Fruit Science 2005 22 211 215 8 Klessig DF Malamy J The salicylic acid signal in plants Plant Molecular Biology 1994 26 5 1439 1458 7858199 9 Teresa M Esperanza M Maria A Cabrejas M Francisco JLA Effects of gibberellic acid (GA3 ) on strawberry PAL (phenylalanine ammonia-lyase) and TAL (tyrosine ammonia-lyase) enzyme activities Journal of the Science of Food and Agriculture 1998 77 2 230 234 10 Bhattarai DR Gautam DM Effect of harvesting method and calcium on post harvest physiology of tomato Nepal Agriculture Research Journal 2006 7 37 41 11 Dubois MK Gils JK Hanniton PA Robes SF Use of phenol reagent for the determination of total sugar Analytical Chemistry 1956 28 350 354 12 Hashimoto S Yamafuji K The determination of diketo-L-gulonic acid, dehydro-L22 ascorbic acid, and 1-ascorbic acid in the same tissue extract by 2, 4-dinitrophenol hydrazine 23 method The Journal of Biological Chemistry 2006 174 201 208 13 Singleton VL Rossi JA Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents American Journal of Enology and Viticulture 1965 16 3 144 158 14 Zhishen JT Mengcheng T Jianming W The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals Food Chemistry 1999 64 4 555 559 15 Hendry GAF Price AH Hendry GAF Grime JP Stress indicators: chlorophylls and carotenoids Methods in Comparative Plant Ecology 1993 London, UK Chapman & Hall 148 152 16 Rodríguez-Saona LE Glusti MM Wrolstad RE Color and pigment stability of red radish and red-fleshed potato anthocyanins in juice model systems Journal of Food Science 1999 64 3 451 456 17 Yang B Zhao M Shi J Yang N Jiang Y Effect of ultrasonic treatment on the recovery and DPPH radical scavenging activity of polysaccharides from longan fruit pericarp Food Chemistry 2008 106 2 685 690 18 Re R Pellegrini N Proteggente A Pannala A Yang M Rice-Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radical Biology and Medicine 1999 26 9-10 1231 1237 10381194 19 Zucker M Induction of phenylalanine deaminase by light and its relation chlorogenic acid synthesis in potato tuber tissue Plant Physiology 1965 40 779 784 16656157 20 Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Analytical Biochemistry 1976 72 1-2 248 254 942051 21 Huber SC Nielsen TH Huber JLA Pharr DM Variation among species in light activation of sucrose-phosphate synthase Plant and Cell Physiology 1989 30 2 277 285 22 Raphael AS Moshe F Steve A Ruth BA Effect of synthetic auxins on fruit development of “Bing” cherry (Prunus avium L.) Scientia Horticulturae 2007 114 4 275 280 23 Choi C Wiersma P Toivonen P Kappel F Fruit growth, firmness and cell wall hydrolytic enzyme activity during development of sweet cherry fruit treated with gibberellic acid (GA3 ) Journal of Horticultural Science and Biotechnology 2002 77 5 615 621 24 Wang CF You Y Chen F Lu XS Wang J Wang JS Adjusting effect of brassinolide and GA3 on the orange growth Acta Agron 2004 26 759 762 25 Thakur BR Singh RK Nelson P Quality attributes of processed tomato products: a review Food Reviews International 1996 12 3 375 401 26 Wahdan MT Habib SE Bassal MA Qaoud EM Effect of some chemicals on growth, fruiting, yield and fruit quality of “Succary Abiad” mango cv The Journal of American Science 2011 7 651 658 27 Pourmorad FS Hosseinimehr J Shahabimajd N Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants African Journal of Biotechnology 2006 5 11 1142 1145 28 Perez AGS Sanz C Richardson G Olias JM Methyl jasmonate vapor promotes beta-carotene synthesis and chlorophyll degradation in golden delicious apple peel Journal of Plant Growth Regulation 1993 12 3 p. 163 29 Roussos PA Denaxa NK Damvakaris T Strawberry fruit quality attributes after application of plant growth stimulating compounds Scientia Horticulturae 2009 119 2 138 146 30 Moneruzzaman KM Hossain ABMS Sani W Saifuddin M Alenazi M Effect of harvesting and storage conditions on the post harvest quality of tomato (Lycopersicon esculentum Mill) cv. Roma VF Australian Journal of Crop Science 2009 3 2 113 121 31 Stratil P Klejdus B Kubáň V Determination of phenolic compounds and their antioxidant activity in fruits and cereals Talanta 2007 71 4 1741 1751 19071517 32 Azodanlou R Darbellay C Luisier JL Villettaz JC Amado R Quality assessment of strawberries (Fragaria species) Journal of Agricultural and Food Chemistry 2003 51 3 715 721 12537447 33 Hubbard NL Huber SC Pharr DM Sucrose phosphate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon (Cucumis melo L.) fruit Plant Physiology 1989 91 1527 1534 16667212
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==== Front PLoS Negl Trop DisPLoS Negl Trop DisplosplosntdsPLoS Neglected Tropical Diseases1935-27271935-2735Public Library of Science San Francisco, USA 22720109PNTD-D-11-0041010.1371/journal.pntd.0001696Research ArticleMedicineCardiovascularDrugs and DevicesInfectious DiseasesOral Administration of GW788388, an Inhibitor of Transforming Growth Factor Beta Signaling, Prevents Heart Fibrosis in Chagas Disease GW788388 for Chagas Diseasede Oliveira Fabiane L. 1 2 Araújo-Jorge Tania C. 3 de Souza Elen M. 2 de Oliveira Gabriel M. 2 Degrave Wim M. 1 Feige Jean-Jacques 4 5 6 Bailly Sabine 4 5 6 * Waghabi Mariana C. 1 1 Laboratório de Genômica Funcional e Bioinformática, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil 2 Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil 3 Laboratório de Inovações em Terapias, Ensino e Bioprodutos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil 4 INSERM, Unité 1036, Biology of Cancer and Infection, Grenoble, France 5 UJF-Grenoble 1, Biology of Cancer and Infection, Grenoble, France 6 CEA, DSV/iRTSV, Biology of Cancer and Infection, Grenoble, France Rodrigues Mauricio Martins EditorFederal University of São Paulo, Brazil* E-mail: [email protected] and designed the experiments: TCAJ EMdS WMD JJF SB MCW. Performed the experiments: FLdO GMdO MCW. Analyzed the data: FLdO GMdO JJF SB MCW. Contributed reagents/materials/analysis tools: GMdO. Wrote the paper: TCAJ JJF SB MCW. 6 2012 12 6 2012 6 6 e16966 5 2011 19 3 2012 de Oliveira et al.2012This 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 Chagas disease induced by Trypanosoma cruzi (T. cruzi) infection is a major cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. Transforming Growth Factor beta (TGFß) has been involved in several regulatory steps of T. cruzi invasion and in host tissue fibrosis. GW788388 is a new TGFß type I and type II receptor kinase inhibitor that can be orally administered. In the present work, we studied its effects in vivo during the acute phase of experimental Chagas disease. Methodology/Principal Findings Male Swiss mice were infected intraperitoneally with 104 trypomastigotes of T. cruzi (Y strain) and evaluated clinically. We found that this compound given once 3 days post infection (dpi) significantly decreased parasitemia, increased survival, improved cardiac electrical conduction as measured by PR interval in electrocardiography, and restored connexin43 expression. We could further show that cardiac fibrosis development, evaluated by collagen type I and fibronectin expression, could be inhibited by this compound. Interestingly, we further demonstrated that administration of GW788388 at the end of the acute phase (20 dpi) still significantly increased survival and decreased cardiac fibrosis (evaluated by Masson's trichrome staining and collagen type I expression), in a stage when parasite growth is no more central to this event. Conclusion/Significance This work confirms that inhibition of TGFß signaling pathway can be considered as a potential alternative strategy for the treatment of the symptomatic cardiomyopathy found in the acute and chronic phases of Chagas disease. Author Summary Cardiac damage and dysfunction are prominent features in patients with chronic Chagas disease, which is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi) and affects 10–12 million individuals in South and Central America. Our group previously reported that transforming growth factor beta (TGFß) is implicated in several regulatory aspects of T. cruzi invasion and growth and in host tissue fibrosis. In the present work, we evaluated the therapeutic action of an oral inhibitor of TGFß signaling (GW788388) administered during the acute phase of experimental Chagas disease. GW788388 treatment significantly reduced mortality and decreased parasitemia. Electrocardiography showed that GW788388 treatment was effective in protecting the cardiac conduction system, preserving gap junction plaque distribution and avoiding the development of cardiac fibrosis. Inhibition of TGFß signaling in vivo appears to potently decrease T. cruzi infection and to prevent heart damage in a preclinical mouse model. This suggests that this class of molecules may represent a new therapeutic tool for acute and chronic Chagas disease that warrants further pre-clinical exploration. Administration of TGFß inhibitors during chronic infection in mouse models should be further evaluated, and future clinical trials should be envisaged. ==== Body Introduction Chagas disease, caused by the intracellular kinetoplastid parasite Trypanosoma cruzi, is a widely spread distributed debilitating human illness, affecting 10–12 million people in Central and South America. It is a major cause of mortality and morbidity, killing 15,000 persons each year [1], [2]. Chagas disease presents an acute phase of infection that is characterized by mild clinical symptoms (fever and malaise) and high parasitemia, but is often unmarked. Due to a potent specific immune response which control parasitemia, patients usually attain the indeterminate stage of the infection, with low-level of parasite persistence that can last from 10 to 40 years. About one in three infected individuals develops the symptomatic chronic stage of infection, which is characterized mainly by myocardiopathy or/and intestinal megasyndrome. A century has passed since the discovery of Chagas disease and the development of an efficient drug is still a challenge. As other neglected diseases, it has not received much attention of the pharmaceutical industry and present available therapies are insufficient [3]. Nifurtimox and benznidazole, the only two trypanocide drugs available, have toxic side effects, are not effective for all parasite strains and the effect in human chronic phase is still under clinical trial [4]. Moreover, no therapeutic approach targeting Chagas disease heart fibrosis is presently available. Transforming Growth Factor ß1 (TGFß1) is the prototypic member of a family of polypeptide growth and differentiation factors that play a great variety of biological roles in such diverse processes as inflammation, fibrosis, immune suppression, cell proliferation, cell differentiation, and cell death [5], [6]. TGFß is also involved in many direct and indirect interactions between infectious agents and their hosts [7]. Several studies have demonstrated that TGFß plays a major role in the establishment and pathogenesis of T. cruzi infection (reviewed in [8]). Moreover, significantly higher circulating levels of TGFß1 have been observed in patients with Chagas disease cardiomyopathy [9] and in a culture system of cardiomyocytes infected by T. cruzi [10]. In order to establish its biological functions, TGFß must be activated into a mature form mainly by proteases, allowing its interaction with a specific transmembrane receptor called TGFß receptor-II (TßRII), which phosphorylates and stimulates the serine/threonine kinase activity of TßRI, also called activin receptor-like kinase 5 (ALK5). Upon activation, ALK5 phosphorylates the cytoplasmic signaling proteins Smad-2 and -3, which then associate with Smad-4, translocate into the nucleus as a multiprotein complex, and stimulate the transcription of TGFß-responsive genes, thereby inducing specific biological responses. We have recently described that the ALK5 inhibitor, 4-(5-benzo[1,3]dioxol-5-yl-4- pyridin-2-yl-1H-imidazol-2-yl)-benzamide (SB431542) reduces the infection of cardiomyocytes by T. cruzi in vitro [11] and we could further show that it also inhibited T. cruzi infection in vivo and prevented heart damage in a mouse model [12]. This work therefore clearly demonstrated that blocking the TGFß signaling pathway could be a new therapeutical approach in the treatment of Chagas disease heart pathology. However the limitation of this compound was the preclusion to oral administration and some toxic effects. To reinforce the prove of concept, the aim of the present work was therefore to test, in the same parasite-mouse model of experimental Chagas disease, another inhibitor of the TGFß signaling pathway, 4-(4-[3-(Pyridin-2-yl)-1H-pyrazol-4-yl] pyridin-2-yl)-N-(tetrahydro-2Hpyran-4-yl) benzamide (GW788388) which can be orally administered and that has an improved pharmacokinetic profile [13], [14]. We found that GW788388 added 3-day post infection (dpi) decreased parasitemia, increased survival, prevented heart damage, and decreased heart fibrosis. Very importantly, we also demonstrated here for the first time that when added after the end of the intense parasite growth and consequent metabolic shock phase at 20 dpi, GW788388 could still decrease mortality and heart fibrosis. Methods Parasites Bloodstream trypomastigotes of the Y strain were used and harvested by heart puncture from T. cruzi-infected Swiss mice at the parasitemia peak, as described previously [15]. Ethics statement Mice were housed for at least one week before parasite infection at the Animal Experimentation Section at the Laboratory of Innovations in Therapies, Education and Bioproducts-IOC/FIOCRUZ under environmental factors and sanitation according to “Guide for the Care and Use of Laboratory Animals”. Animal studies adhered to the International guidelines (National Research Council. 1996, National Academy press, Washington, DC). This project was approved by the FIOCRUZ Committee of Ethics in Research (protocol number 028/09). In vivo infection Male Swiss mice (age 6–8 weeks, weight 18–20 g) were obtained from the animal facilities of CECAL (FIOCRUZ, Rio de Janeiro, Brazil). Infection was performed by intraperitoneal (IP) injection of 104 bloodstream trypomastigotes. Age-matched non-infected mice were maintained under identical conditions. Experimental groups The animals were divided into the following groups: non-infected (NI), infected and untreated (Y DMSO), infected and treated with 3 mg/kg GW783388 (Y GW788388). Ten mice from each group were used for analysis at each different dpi and 5 independent experiments were performed. Drug and treatment The compound GW783388 (GlaxoSmithkline, France) or vehicle dilution buffer (4% DMSO, 96% [0.5% Hydroxypropylmethylcellulose (HPMC), 5% Tween 20, 20% HCl 1 M in NaH2PO4 0.1 M]) was used for oral administration. Mice received GW788388 at 3 mg/kg at 3 dpi or 20 dpi by gavage in a single administration (0.2 mL). The control group received vehicle buffer using the same schedule. Survival rates and parasitemia Parasitemia was individually checked by direct microscopic counting of parasites in 5 µL of blood, as previously described [15]. Mortality was checked daily until 30 dpi and expressed as percentage of survival. Biochemistry Blood was collected from the tip of mice tails of all experimental groups at 15 dpi and immediately analyzed for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea levels with Reflotron Plus (Roche), according to the manufacturer recommendations. ALT and AST activities were used to evaluate hepatic dysfunction and the results were expressed as enzyme concentration (mg/dL). ALT and AST belong to the group of transaminase that catalyses the conversion of amino acids into corresponding α-ceto acids and vice-versa by transference of amine groups. Urea was measured to evaluate renal function and the results were expressed in concentration (mg/dL). Histopathology Fixed tissue was dehydrated and embedded in paraffin. Sections (3 µm) stained by routine haematoxylin-eosin (HE) were analyzed by light microscopy. The number of amastigote nests and of inflammatory infiltrates (more than 10 mononuclear cells), were determined in 30 microscopic fields/slide (total magnification, 400×). The mean number of amastigotes or inflammatory infiltrates per field was obtained at 15 dpi from at least three infected mice, with three sections per mouse per group. The sections were observed using a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) coupled with Axiovision image acquisition system (Zeiss). The area (%) of inflammatory infiltrates was evaluated using NIH ImageJ software in at least 10 images per group. Histological assessment of cardiac fibrosis Heart fibrosis was studied by (a) Masson's trichrome staining at 15, 20 and 24 dpi as previously described [16], (b) immunohistochemical staining of specific extracellular matrix proteins (collagen type I and fibronectin, see below), and (c) Western blot analysis of collagen type I and fibronectin protein levels (see below). For collagen type I and fibronectin immunostainings, fixed tissue slides were obtained as described above and heart fibrosis was studied by collagen type I and fibronectin immunostainings at 15 dpi. Briefly, after blocking with 3% BSA for 1 hour, the following primary antibodies were applied overnight to the sections: rabbit polyclonal anti-collagen type I (Novotec, France, kindly provided by Dr. Daniella Areas Mendes-da-Cruz, IOC/Fiocruz) and rabbit polyclonal anti-fibronectin (Sigma Aldrich, USA). The secondary antibody was Alexa 594 goat anti-rabbit IgG (Invitrogen). Negative control sections were incubated with non-immune rabbit serum and the secondary antibody alone and indicated no cross reactivity (data not shown). Host-cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The sections were observed using a Zeiss Axioplan microscope (Zeiss) coupled with Axiovision image acquisition systems (Zeiss). Subsequent automated analysis of the captured images (6–10 mice per group) was carried out using an ImageJ macrobased algorithm that identifies, separates and quantifies the light blue areas stained with Masson's trichrome representing fibrosis. Cx43 plaque quantification Cx43 staining on fixed tissue was performed as described above. Briefly, heart sections were blocked with 3% BSA for 1 hour, and incubated overnight with rabbit polyclonal anti-connexin 43 (Sigma Aldrich, USA). The secondary antibody was Alexa 488 goat anti-rabbit IgG (Invitrogen). Host-cell nuclei were stained with DAPI. The images were obtained using a Zeiss Axioplan microscope at final magnification of ×400. At this magnification, a typical test area included approximately 34.600 µm2 of tissue area. The images obtained in each studied case were analyzed using the NIH software ImageJ to determine the mean number and the length of Cx43 plaques by quantification of 3-field images/animal. Immunoblot analysis Left ventricular heart proteins from each group (NI, Y DMSO and Y GW788388) were extracted from 100 mg tissue/mL phosphate-buffered saline, to which 0.4 mol/L sodium chloride, 0.05% Tween 20, and protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride and 1/100 protease inhibitors cocktail (Sigma)) were added. The samples were sonicated twice and centrifuged for 10 min at 3000 g, and the supernatant was kept frozen at −70°C. Proteins in the lysates (20 µg/lane) were separated by SDS/PAGE and analyzed by immunoblotting with specific primary antibodies (rabbit Anti-fibronectin and rabbit Anti-connexin-43 from Sigma Aldrich and rabbit Anti-collagen type I from Novotec, France). To confirm equal protein loading, the same membranes were stripped and reprobed with a monoclonal antibody against GAPDH (Pierce). Electrocardiography (ECG) ECG recording and analysis were performed in the three groups, as reported (30). Briefly, mice were fixed in the supine position, and transducers were carefully placed on the skin in accordance to chosen preferential derivation (lead II). Traces were recorded using a digital system (Power Lab 2/20) connected to a bio-amplifier in 2 mV for 1 s (PanLab Instruments). Filters were standardized between 0.1 and 100 Hz and traces were analyzed using the Scope software for Windows V3.6.10 (PanLab Instruments). ECG parameters were evaluated in the acute phase at 15 dpi, using the following standard criteria: (i) the heart rate, monitored by beats/minute (bpm), and (ii) the variation at P wave and PR, QRS and QT intervals, all measured in milliseconds (ms). Statistical analysis Differences were considered statistically significant when p<0.05 (*) or p<0.01 (**), as determined by GraphPadPrism 4.0 software (Graph- Pad Software Inc., San Diego, CA, USA). The Kaplan- Meier test was used to analyze the significances of the survival rates while all the other analyses were performed using the non-parametric Mann–Whitney test. Results The aim of the present work was to evaluate whether the compound GW788388, which is an ATP-competitive inhibitor of the kinase activity of ALK5, could have a beneficial effect in vivo in an experimental model of mouse acute infection by T. cruzi and whether it could protect infected mice from parasite-induced alterations of cardiac functions and fibrosis when administrated early (3 dpi) and late (20 dpi). Oral administration of GW788388 at 3 dpi reduced parasitemia and heart damage and increased mice survival rates in T. cruzi-infected mice In the first set of experiments, the inhibitor GW788388 was orally administered to male Swiss mice infected with 104 bloodstream trypomastigotes of the Y strain (day 0), at the 3rd dpi. We first performed a dose-response study by administering different doses of GW788388 (0.3, 3, 6 and 15 mg/kg) and analyzed parasitemia and survival rate. The results showed a dose-dependent inhibition of parasitemia at 8 dpi from 0.3 to 15 mg/kg of GW788388 (Methods S1 and Fig. S1A). On the other hand, the survival rate was increased with 3 or 6 mg/kg of GW788388 but unaltered at 0.3 and 15 mg/kg, suggesting some toxicity of the drug at this largest dose (Fig. S1B). For the subsequent studies, the dose of 3 mg/kg was chosen since it was the lowest GW788388 concentration that significantly affected parasitemia without worsening mortality. The choice for 3 mg/kg GW788388 administration was further reinforced by the assays performed by Gellibert and collaborators [13], who showed in a model of kidney fibrosis that doses as low as 3 mg/kg/mice of GW788388 significantly inhibited collagen type I mRNA levels. The control group received the vehicle buffer in which GW788388 was diluted (4% DMSO, 96% [0.5% Hydroxypropylmethylcellulose (HPMC), 5% Tween 20, 20% HCl 1 M in NaH2PO4 0.1 M]) and could be considered as the placebo group. The responses of DMSO-treated infected mice were not significantly different from those of untreated infected mice, excluding any sham or placebo effect (data not shown). In our model of acute infection, as previously described [12], parasitemia peaked at 8 dpi (Fig. 1A). We found that GW788388 administration at 3 dpi significantly reduced the blood parasitemia peak (Fig. 1A). Further, as previously described with the compound SB421543 [11], we could demonstrate that in vitro administration of GW788388 on cardiomyocytes impaired T. cruzi replication in host cells (Fig. S2) supporting the decreased parasitemia peak found in vivo. On the other hand, no effect of GW788388 on trypomastigote forms of T. cruzi viability could be observed after direct incubation of the drug with the parasites (unpublished result). We also showed that GW788388 administration significantly increased survival rates at 30 dpi (65% in the treated-group versus 34% in the untreated group, Fig. 1B). The infection induced a loss of body weight at 14 dpi [12], which was not modified by the administration of GW788388 (data not shown). To investigate whether GW788388 treatment would also affect myocardial parasitism and infiltration of inflammatory cells, we analyzed mouse infected heart sections collected at 15 dpi using histochemical techniques. Non-infected animals showed no inflammatory infiltration in the myocardium (data not shown). Myocardial sections from the T. cruzi-infected sham-treated group (Y DMSO) had many amastigote nests (Fig. 1C, open arrows) and large inflammatory foci (Fig. 1E, filled arrows) that were frequently associated with fibrotic areas. GW788388 treatment significantly decreased the number of amastigote nests (Fig. 1D and 1G). GW788388 administration also significantly decreased the area invaded by inflammatory infiltrates (Fig. 1F and 1H). A more detailed count of the number of cells per inflammatory foci showed that GW788388 treatment more particularly decreased the number of large inflammatory foci within the myocardium (larger than 20 or 50 cells per inflammatory infiltrates) (Table 1). 10.1371/journal.pntd.0001696.g001Figure 1 GW788388 administration at 3 dpi decreased parasitemia and heart inflammatory infiltrates and increased survival rates. Male Swiss mice were injected IP with 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) or DMSO was administered by gavage at 3 dpi. (A) Parasitemia was measured by direct counting of parasites in blood. (B) Percent survival was monitored during the experiment until 30 dpi. (C–F) At 15 dpi, mice were sacrificed and heart sections stained with hematoxylin-eosin were analyzed by light microscopy. Numerous amastigote nests (C, open arrows) and large inflammatory infiltrates (E, filled arrows) were observed in untreated T. cruzi-infected mice. GW788388 administration decreased the number of amastigote nests (D, open arrow) and of inflammatory infiltrates (F). (G and H) The mean number of amastigote nests in 30 fields (G) and the area (%) of inflammatory infiltrates (more than 10 mononuclear cells) (H) are shown. Values for the infected group treated with GW788388 that were significantly different from the value for the DMSO infected group are indicated (**p<0.01 and * p<0.05). n = 10 mice/group in 4 independent experiments. 10.1371/journal.pntd.0001696.t001Table 1 Effect of GW788388 on the number of inflammatory infiltrates in the heart at 15 dpi. Number of cells per Inflammatory Infiltrates Y DMSO Y GW788388 Fold decrease P value Total 168±45 94±29 1.78 0.008a >50 29±18 4±2 6.07 0.004a 21–50 66±19 24±10 2.75 0.002a 10–20 73±28 65±18 1.12 0.68 T. cruzi infected mice were treated with 3 mg/kg GW788388 at 3 dpi and the number of inflammatory infiltrates in the heart was counted at 15 dpi. a Significant differences (p<0.01) between the values for infected non treated (Y DMSO) and treated (Y GW788388) groups of mice. GW788388 controlled liver alteration caused by acute experimental T. cruzi infection T. cruzi infection induces a strong hepatitis during the acute phase of Chagas disease [17]. We therefore analyzed several parameters of the liver in sham-treated versus GW788388-treated mice. Analysis of liver sections at 15 dpi revealed the presence of large inflammatory infiltrates in DMSO-treated animals (Fig. 2A, arrow). GW788388 administration significantly decreased the number of these infiltrates (Fig. 2B and C). We also measured two circulating markers of hepatic function which are induced by T. cruzi infection: AST (aspartate aminotransferase) and ALT (alanine aminotransferase). We found that GW788388 administration significantly decreased the serum levels of AST and ALT (Fig. 2D and E). We also measured urea, which reflects the renal functional status. Urea level was significantly increased at 15 dpi in DMSO-treated animals while GW788388 administration significantly reduced it (Fig. 2F). 10.1371/journal.pntd.0001696.g002Figure 2 GW788388 administration at 3 dpi decreased liver and renal alterations. Male Swiss mice were injected IP with or without (NI) 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) or DMSO was administered by gavage at 3 dpi. (A and B) At 15 dpi mice were sacrificed, and liver sections stained with hematoxylin-eosin were analyzed by light microscopy. Large inflammatory infiltrates (A, white arrow) were observed in untreated T. cruzi-infected mice. (C) The mean number of inflammatory infiltrates (more than 10 mononuclear cells) in 30 fields is shown. (D and E) The serum levels of AST and ALT, two markers of hepatic lesion, were measured at 15 dpi. (F) The serum urea levels were measured to evaluate renal function. Each symbol shows the value for one mouse. The short black bars show the mean value for each group. Values that were significantly different from studied groups are indicated by asterisks (**p<0.01 and * p<0.05). n = 3 mice/group in 3 independent experiments. GW788388 prevented heart damage from T. cruzi infection We next analyzed electrocardiograms (ECG) of the different groups of mice at 15 dpi. As expected, analysis of the ECG demonstrated an atrial ventricular block with PR interval higher than 40 ms, leading to sinus bradycardia in sham-treated T. cruzi-infected animals as compared to the non-infected control group (495.8 and 774.2 bpm, respectively, Figure 3 and Table 2). GW788388 administration significantly limited the bpm decrease at 15 dpi, with a mean heart rate of 554.3 (Fig. 3 and Table 2). The other parameters analyzed demonstrated that infected mice had higher QT, PR and QRS intervals compared to non-infected mice (Table 2), and that GW788388 administration (3 mg/kg) also significantly decreased the QT intervals to 25.3 ms as compared to 29.6 in the infected DMSO-treated group (Table 2). A possible cause of this worsening in heart electrical conduction after the infection could be the direct effect of TGFß in heart cells. It has been already proposed that elevated TGFß levels during T. cruzi infection disorganize gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia in Chagas disease [12]. To test this hypothesis, we measured connexin 43 (Cx43) expression in the different groups of mice. Heart sections from at least three mice per group at 15 dpi were immunostained for Cx43. We observed by confocal microscopy that non-infected hearts presented a dense structure of gap junction plaques (Fig. 4A, green staining). A drastic change in Cx43 expression was observed in the infected hearts of vehicle-treated mice, with an important decrease in Cx43 expression and a disruption of gap junction plaques (Fig. 4B). We found that GW788388 treatment reduced Cx43 disassembly and prevented the dissolution of gap junctions, preserving organized plaque distribution (Fig. 4C). The mean number of Cx43 plaques and their mean length were significantly lower in the heart of infected mice at 15 dpi as compared to the non-infected group (Fig. 4D and E). GW788388 treatment protected infected-mice from this loss as the decrease in the mean number of plaques was only reduced by 30% versus 45% in non-treated mice (Fig. 4D) and the mean length was similar to the non-infected mice (Fig. 4E). Immunoblotting analysis of Cx43 expression from heart ventricles confirmed these data (Fig. 4F and G). 10.1371/journal.pntd.0001696.g003Figure 3 GW788388 administration at 3 dpi restored Electrocardiographic parameters. Male Swiss mice were injected IP with or without (NI) 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) (Y GW788388) or DMSO (Y DMSO) was administered by gavage at 3 dpi. Representative electrocardiographic tracings of the three groups at 15 dpi are shown. n = 6 mice/group in 3 independent experiments. 10.1371/journal.pntd.0001696.g004Figure 4 GW788388 administration at 3 dpi inhibited connexin 43 disruption. Male Swiss mice were injected IP with or without (NI) 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) (Y GW788388) or DMSO (Y DMSO) was administered by gavage at 3 dpi. (A–C) At 15 dpi, mice were sacrificed and heart sections stained with anti-Cx43 antibody (green, Cx43 plaques are indicated by white arrows) and DAPI (nuclei, colored in blue). Quantitative analysis of the number of Cx43 plaques (D) and length (E), on images from each group studied (NI, Y DMSO and Y GW788388). Values are expressed as the mean±SD (F) 20 µg of total proteins from hearts at 15 dpi were resolved in 12% SDS/PAGE and immunoblotted with anti-Cx43 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. (G) Densitometric histograms of the normalized levels of Cx43 as related to GAPDH are shown. Values that were significantly different from studied groups are indicated by asterisks (* p<0.05). n = 3 mice/group in 3 independent experiments. 10.1371/journal.pntd.0001696.t002Table 2 Effect of GW788388 on electrocardiograph parameters at 15 dpi. ECG parameters (Mean ± SD) Non-infected Y + DMSO Y + GW788388 Heart rate (bpm) 774.2±30.6 495.8±79.2a 554.3±44.5c PR intervals (ms) 28.6±3.1 50.4±8.2a 45.1±8.4 QRS intervals (ms) 8.6±1.4 10.6±2.7b 9.7±2.0 QT intervals (ms) 22.8±8.9 29.6±5.5b 25.3±6.4c Frequency of AVB1 0/10 (0%) 15/18 (83%) 5/18 (28%) Frequency of AVB2 0/10 (0%) 13/18 (72%) 6/18 (33%) ECG parameters were evaluated in the acute phase at 15 dpi, using the following standard criteria: (i) the heart rate was monitored by beats/minute (bpm), and (ii) the variation at P wave and PR, QRS and QT intervals, all measured in milliseconds (ms). The incidence of AVB1, atrioventricular block type 1 and AVB2, atrioventricular block type 2 are stated in absolute numbers and in percentage. Significant differences between the values for non-infected and infected groups of mice: a (p<0.01) and. b (p<0.05). c Significant differences (p<0.05) between the values for infected non treated (Y DMSO) and treated (Y GW788388) groups of mice. GW788388 prevented heart fibrosis development in T. cruzi-infected mice One of the best established biological function of TGFß is the stimulation of extracellular matrix (ECM) protein deposition. Therefore, we checked whether GW788388 treatment would affect heart fibrosis that occurs in response to T. cruzi infection. Left ventricular heart tissues were obtained from each group and the deposition of ECM proteins was studied by immunostaining for collagen type I and fibronectin at 15 dpi. We observed an interstitial fibrous heart with high levels of both collagen type I and fibronectin deposition, as observed in red on Figure 5A and C, respectively. Interestingly, we could show that oral administration of GW788388 significantly reduced collagen type I and fibronectin levels (Fig. 5B and D, respectively). These data were confirmed by immunoblotting analysis of collagen type I and fibronectin expression from heart ventricles (Fig. 5E, F and G). 10.1371/journal.pntd.0001696.g005Figure 5 GW788388 administration at 3 dpi decreased heart fibrosis. Male Swiss mice were injected IP with or without (NI) 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) (Y GW788388) or DMSO (Y DMSO) was administered by gavage at 3 dpi. At 15 dpi, mice were sacrificed and heart sections stained with anti-collagen type I antibody (A and B, open arrow) or anti-fibronectin antibody (C and D, white arrow) colored in red and DAPI (nuclei, colored in blue). (E) 20 µg of total proteins from hearts at 15 dpi were resolved in 12% SDS/PAGE and immunoblotted with anti-fibronectin or anti-collagen type I antibodies and reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. (F and G) Densitometric histograms of the normalized levels of fibronectin or collagen type I as related to GAPDH are shown. Values that were significantly different from studied groups are indicated by asterisks (**p<0.01 and * p<0.05). n = 3 mice/group in 3 independent experiments. We found that GW788388-treatment decreased the phosphorylation level of Smad2 in infected hearts, demonstrating that GW788388-treatment was related to TGFß dependent signaling in vivo (data not shown). Oral administration of GW788388 at 20 dpi also increased mice survival rates and reduced heart fibrosis in T. cruzi infected mice Because most of the beneficial effects that we observed here with the TGFß inhibitor (GW788388) might be due to the resulting decreased parasitemia due to the inhibitory effect of TGFß signaling inhibitors in host cell invasion and intracellular proliferation [11], [12], we next studied the effect of GW788388 oral administration after the parasitemia peak. We chose to add GW788388 at 20 dpi as by this time, only 18% of infected mice survived and 30% of them died at 24 dpi. Interestingly, we found that GW788388 administration at 20 dpi completely protected these mice (n = 12) from death until 24 dpi (Fig. 6A, inset). In the inset, 100 represents the percentage of survival rate calculated from 20 dpi. GW788388 administration still decreased the number of inflammatory infiltrates within the myocardium (Table 3). To verify if GW788388 treatment presented an effect in the reversion of installed fibrosis, we performed Masson's trichrome staining on heart cross-sections of infected untreated mice at 15 dpi (Fig. 6B), 20 dpi (Fig. 6C) and 24 dpi (Fig. 6D), and of infected GW788388-treated mice at 24 dpi (Fig. 6E). We observed a progressive increase in collagen deposition visualized as light blue staining, which followed fibrosis progression (from 15 to 24 dpi, Table 4). At 20 dpi, which corresponded to the day of GW788388 administration, we observed a fibrotic pattern on the heart of infected mice frequently associated to inflammatory infiltrates (Fig. 6C). Interestingly, four days after GW788388 administration (i.e. 24 dpi) we observed a decrease in collagen deposition (Fig. 6E) as compared to the untreated group (Fig. 6D, Table 4). Immunoblotting assays were performed to compare the expression levels of collagen type I between each group. We observed a significant increase in collagen type I expression in the DMSO infected group as compared to the non-infected group (Fig. 6F and G, 9 fold increase), while GW788388 administration to infected mice significantly decreased the expression levels of collagen type I (Fig. 6F and G). 10.1371/journal.pntd.0001696.g006Figure 6 GW788388 administration at 20 dpi protected T. cruzi-infected mice from death and decreased heart fibrosis. Male Swiss mice were injected IP with or without (NI) 104 bloodstream trypomastigotes. Then GW788388 (3 mg/kg) (Y GW788388) or DMSO (Y DMSO) was administered by gavage at 20 dpi. (A) Percent survival was monitored during the experiment until 24 dpi (Inset shows a blow-up of the survival curve from 20 to 24 dpi (100 represents the percentage of survival calculated from 20 dpi)). (B, C, D, E) Untreated infected mice were sacrificed at 15 dpi (B), 20 dpi (C), 24 dpi (D) and GW788388-treated mice at 20 dpi were sacrificed at 24 dpi (E). Heart sections were stained for collagen deposition by Masson's trichrome (light blue staining, open arrows). (F) 20 µg of total proteins from hearts at 24 dpi were resolved in 12% SDS/PAGE and immunoblotted with anti-collagen type I antibody and reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. (G) Densitometric histograms of the normalized levels of collagen type I as related to GAPDH are shown. Values that were significantly different from studied groups are indicated by asterisks (**p<0.01 and * p<0.05). n = 4 mice/group in 3 independent experiments. 10.1371/journal.pntd.0001696.t003Table 3 Effect of GW788388 on the number of inflammatory infiltrates in the heart at 24 dpi. Number of cells per Inflammatory Infiltrates Y DMSO Y GW788388 Fold decrease P value 10–20 40±4 27±5 1.5 0.02a 21–50 14±6 9±4 1.5 0.3 >50 11±7 2±1 4.3 0.1 Total 66±18 39±5 1.7 0.1 T. cruzi infected mice were treated with 3 mg/kg GW788388 at 20 dpi and the number of inflammatory infiltrates in the heart was counted at 24 dpi. a Significant differences (p<0.05) between the values for infected non treated (Y DMSO) and treated (Y GW788388) groups of mice. 10.1371/journal.pntd.0001696.t004Table 4 Effect of GW788388 on the fibrosis scores of the heart at 20 dpi. dpi 15 DMSO dpi 20 DMSO dpi 24 DMSO dpi 24 GW788388 Number of mice 7 8 10 6 % area stained for Masson' Trichrome 25.7 33.8 55.8 4.8 23.9 18.8 35.7 6.2 22.6 14.4 30.0 3.1 5.2 53.8 7.7 2.9 56.1 55.9 8.2 2.9 5.1 13.2 13.7 8.3 nd 39.4 54.0 16.1 54.0 20.2 9.2 Mean ± SD 23.1±18.6 30.7±17.6 28.9±20.0 4.7±2.2** T. cruzi infected mice were treated with 3 mg/kg GW788388 at 20 dpi and fibrosis was quantified before (15 and 20 dpi) and after (24 dpi) treatment. Percent of Masson's Trichrome stained area (light blue areas) were quantified using NIH ImageJ Software in the microscopic images of heart sections. **: Significant differences (p<0.01) between the values for Y DMSO and Y GW788388 groups of mice observed at 24 dpi. nd: not detected. Discussion We have recently demonstrated that in vivo inhibition of the TGFß signaling pathway can decrease infection and prevent heart damage [12], suggesting that this new class of therapeutic agents should be considered in association with trypanocidal compounds for the potential treatment of Chagas disease cardiomyopathy. In the present work, we demonstrated that a more potent inhibitor of the TGFß signaling pathway, GW788388, which can be orally administered, significantly decreased parasitemia, increased survival and restored cardiac function as measured by ECG heart frequency (increase in bmp) and atrial conduction (decrease in QT interval). When administered at 3 dpi, we observed that GW788388 treatment reduced parasitemia and its subsequent deleterious effects. Whether the protective effect of GW788388 results only from this sole anti-infectious effect remains to be established. However, the short half-life of GW788388 in vivo (plasma T1/2 = 1.3 hours; [13]) makes it unlikely that it is mediated by long-term effects on e.g. fibrosis or cardiac rhythm. In contrast, administration of GW788388 at 20 dpi to mice that survived the metabolic distress syndrome clearly resulted in improved survival, which correlated with decreased cardiac fibrosis and has probably no causal relationship with the anti-infectious effect of the drug. Given the recent availability of reliable mouse models for chronic chagasic cardiomyopathy [18], the present proof that orally administrated GW788388 is feasible and efficient in the acute phase will offer in the near future the possibility of testing TGFß inhibitors in the chronic phase in pre-clinical assays. Taken together, these data further support that blocking TGFß signaling could represent a potential new therapeutic approach for Chagas disease heart fibrosis treatment. It is now well established that the involvement of the TGFß signaling pathway plays an important role in the development of Chagas disease [8]. TGFß has been shown to be involved during parasite-host cell invasion, proliferation and differentiation [19]–[22]. Moreover, significantly higher circulating levels of TGFß1 have been observed in patients with Chagas disease cardiomyopathy [9], [16]. These data incited us to test the possibility of treating the development of Chagas disease by blocking the TGFß signaling pathway. Here, we show that oral administration of GW788388 kinase signaling inhibitor prevents parasitemia, mortality, and heart fibrosis to acutely T. cruzi-infected mice in comparison to untreated-infected experimental group of animals. In lack of demonstration of GW788388 direct killing effect upon T. cruzi, we postulate the protein kinase inhibitor used may induce intracellular parasite latency [23], [24], such as that involved with the Plasmodium sporozoites cell cycle inhibition of initiation factor-2alpha (elF2alpha) kinase (IK2); its down-regulation by removal of PO4 from elF2alpha-P gives rise to the latency [25], [26]. In this regard, ongoing investigations in chronically T. cruzi-infected mouse model will determine whether GW788388 beneficial effects can be explained by the drug-induced parasite latency and long lasting cryptic infections. Several approaches have been developed to abrogate TGFß signaling. Antibodies directed against TGFß have been administered in diabetic rodents and this was shown to efficiently prevent glomerulosclerosis and renal insufficiency [27]. Antisense TGFß oligonucleotides were found to reduce kidney weight in diabetic mice [28]. Recently, a soluble fusion protein of TßRII was reported to reduce albuminuria in a chemically induced model of diabetic nephropathy in rats [29]. And finally, inhibitors of the kinase activity of the TßRI (ALK5) have been developed. These inhibitors interact with the ALK5 ATP-binding site, thereby preventing TGFß intracellular pathways [30]. The first ALK5 inhibitor described, SB431542, is an ATP-competitive kinase inhibitor [31]. SB431542 significantly reduced procollagen1alpha (I) in rat kidneys in a model of induced nephritis. It was also described that SB431542 triggers antitumor activity in vivo [32]. Our work also demonstrated that SB431542 reduced mortality, decreased parasitemia and prevented heart damage as observed by histological and ECG analysis during the acute phase of experimental Chagas disease [12]. However, the limitations of SB431542 were the need of intraperitoneal injection and the in vivo toxic effects that have been demonstrated. Recently, GW788388 was developed as an alternative to SB431542 with better in vivo exposure. GW788388 is orally active and has a good pharmacokinetic profile [13], [14], [30]. GW788388 administration reduced liver and renal fibrotic response in a model of chemically induced fibrosis in rats and in the db/db mouse model of spontaneous diabetic nephropathy [13], [14]. Treatment with GW788388 also showed efficacy for preventing the fibrotic response in a skin fibrosis model [33] and attenuated cardiac dysfunction following myocardial infarction [34]. These data prompted us to test this compound during the acute phase of experimental Chagas disease. We found that oral administration of GW788388 at 3 dpi significantly reduced peripheral parasitemia and lowered parasite load in hearts of infected mice observed 15 dpi. This effect was achieved with lower administration doses (3 mg/kg) than the one we previously used for SB431542 (10 mg/kg) [12], and with a single oral administration. More importantly, oral administration of GW788388 also significantly improved mice survival (70% in GW788388-treated mice against 30% in non-treated infected mice at 30 dpi). This is probably due to the combined impairment of the second wave of T. cruzi parasitemia due the decrease of parasite burden and of the early inflammatory cytokines secretion balance. Infection with T. cruzi in the acute phase is followed by a strong mononuclear cell inflammation on target tissues such as heart and liver, which could cause tissue disruption, necrosis followed by fibrotic deposition and abnormalities in electrical impulse conduction. Our data showed less inflammation on both heart and liver tissues and, moreover, less mononuclear cells by inflammatory focus. An improved ECG profile was also observed after GW788388 administration, characterized mainly by the absence of sinus node dysfunctions and reduced sinus bradycardia. PR intervals larger than 40 ms suggested slower transmission of the electrical impulses and atrioventricular block (AVB), which is characteristic of acute T. cruzi infection [35]. We observed an improvement of the QT intervals following GW788388 administration, which represent the wave of ventricular recuperation and this could be related to the decrease of sudden death [36] and to the progression to a pathological chronic phase [35]. Heart failure and sudden death are the most common causes of death in patients with chronic cardiac Chagas disease [37] and altered ECG parameters correlates with increasing myocardial scar and decreasing myocardial function in these patients [38]. This results from disorganized gap junctions that could contribute to abnormal impulse conduction and arrhythmia that characterize severe cardiopathy in Chagas disease and heart fibrosis [10]. Gap junction Cx43 molecules are responsible for electrical impulse conduction in the heart [39] and are affected by TGFß [10], [40]. We observed that GW788388 treatment preserved a correct Cx43 plaque pattern in the heart and blocked the down-regulation of Cx43 expression commonly observed following T. cruzi infection. GW788388 treatment therefore favored a regular and correct electrical impulse transmission. TGFß is also a key factor in the generation of tissue fibrosis [41] and has been correlated to development of Chagas disease symptoms in cardiac chronic phase [8]. Our data showed that administration of GW788388 to T. cruzi-infected mice significantly prevented the increase of fibronectin and collagen type I, two important components involved in heart fibrosis. These data are consistent with previous studies showing that GW788388 reduced fibrosis markers in the kidney following chemically induced nephropathy [14], [42]. In the human acute phase of Chagas disease, symptoms are frequently mild and not noticed and it is therefore difficult to propose correct treatments with trypanocidal drugs. Therefore, in the present study, we also treated mice with GW788388 at the end of the acute phase, when there are scarce circulating parasites. Interestingly, we found that oral administration of GW788388 at 20 dpi completely protected mice from death (100% survival). Analysis of cardiac fibrosis by Masson's trichrome staining on heart cross-sections of T. cruzi-infected mice showed a strong increase of fibrosis from 15 dpi to 24 dpi (Fig. 5, Table 4). Interestingly, we found that mice treated with GW788388, in a single dose scheme at 20 dpi, reversed heart fibrosis observed four days later (24 dpi) as compared to untreated infected mice. The level of collagen type I was also restored in GW788388 treated mice versus untreated mice. Taken together these data demonstrated that blocking TGFß signaling could decrease an installed heart fibrosis. This important finding encourages further pre-clinical assays targeting fibrotic lesions that are always involved in the severity of the clinical picture observed in the chronic cardiac disease. The development of an efficient drug for Chagas disease is still a challenge and trypanocidal drugs such as nifurtimox and benznidazole are still the only drugs employed for specific Chagas disease treatment, although the observation of serious side effects. Treatment strategy approaching the reversion of fibrosis has been demonstrated here at the end of the acute phase of experimental Chagas disease. Still, further studies on a chronic experimental model are necessary previously to clinical assays. The inhibition of TGFß signaling pathway and its biological functions could then be considered as an alternative strategy for the treatment of the symptomatic cardiomyopathy found in the acute and chronic phases of Chagas disease, in synergy with current administered drugs, enabling lower dosages and avoiding toxic effects. Supporting Information Figure S1 Dose-dependent analysis of the effect of oral administration of GW788388 at 3 dpi on parasitemia and survival rates. Male Swiss mice were injected ip. with 104 bloodstream trypomastigotes. Then GW788388 (0.3–15 mg/kg) or vehicle (DMSO) was administered once by gavage at 3 dpi. (A) Parasitemia was measured by direct counting of parasites in blood. (B) Percent survival was monitored until 30 dpi. (EPS) Click here for additional data file. Figure S2 Effects of in vitro GW788388 administration to cardiomyocytes on T. cruzi invasion and replication. Cardiomyocytes were infected with trypomastigotes of the Y strain in a parasite∶host cell proportion of 10∶1 and 24 h post- infection, cultures were treated or not with GW788388 (10 µM). Cells were fixed 48 h (A and B) and 96 h (C and D) post-infection and stained with Giemsa. Magnification: 400×. Quantification of the percentage of cardiomyocytes containing parasites (E) and the number of parasites per infected cell (F) were determined by counting 400 cells/slide on two distinct coverslips at 48 and 96 h post-infection. (TIFF) Click here for additional data file. Methods S1 GW 788388 dose-response effect in vivo. (DOCX) Click here for additional data file. The authors would like to thank Marcos Meuser and Wanderson da Silva Batista for their technical help on animal care and Dr E. Tillet (U1036, University Joseph Fourier, Grenoble) for her help with the confocal microscope. We would like to thank Dr. A-C de Gouville (Department of Medicinal Chemistry and Biology, GlaxoSmithKline, 25–27 Avenue du Québec, 91951 Les Ulis, France) for providing us with the compound GW788388. The authors have declared that no competing interests exist. This work was supported by an INSERM-FIOCRUZ collaborative research program, by grants from PAPES/FIOCRUZ, IOC, Conselho Nacional de Desenvolvimento Científico e Tecnlógico-CNPq, and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho-FAPERJ to the Brazilian laboratories, and by recurrent funding from INSERM, University Joseph Fourier and CEA to the French laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Jannin J Villa L 2007 An overview of Chagas disease treatment. Mem Inst Oswaldo Cruz 102 95 97 17906803 2 Rassi A Jr Rassi A Little WC 2000 Chagas' heart disease. 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PLoS Negl Trop Dis. 2012 Jun 12; 6(6):e1696
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22745752PONE-D-11-2565110.1371/journal.pone.0039426Research ArticleBiologyBiochemistryBiotechnologyGenomicsMicrobiologyProteomicsRadiobiologyToxicologyChemistryMedicinal ChemistryRadiochemistryMedicineOncologyRadiologyToxicologyDMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFκB Transactivation through NIK/IKK in Human Glioma Cells Bisbenzimidazole a RadiomodulatorKaur Navrinder 1 Ranjan Atul 2 Tiwari Vinod 2 Aneja Ritu 3 Tandon Vibha 2 * 1 Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India 2 Department of Chemistry, University of Delhi, Delhi, India 3 Department of Biology, Georgia State University, Atlanta, Georgia, United States of America Deutsch Eric EditorInstitut Gustave Roussy, France* E-mail: [email protected] and designed the experiments: V. Tandon NK AR V. Tiwari. Performed the experiments: V. Tandon NK AR V. Tiwari. Analyzed the data: V. Tandon NK AR RA V. Tiwari. Contributed reagents/materials/analysis tools: V. Tandon RA. Wrote the paper: V. Tandon NK AR V. Tiwari. 2012 22 6 2012 7 6 e394267 12 2011 21 5 2012 Kaur et al.2012This 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 Ionizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage. Methods and Results Treatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2′-(3, 4-dimethoxyphenyl)-5′-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFκB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFκB inducing kinase mediated phosphorylation of IKKα and IKKβ both alone and in the presence of ionizing radiation (IR). The TNF-α induced NFκB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFκB activation at 10, 25 and 50 µM DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 µM +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h. Conclusions Our studies show that NIK/IKK mediated NFκB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFκB signaling. This subsequently leads to the radioprotective effect exhibited by DMA. ==== Body Introduction Exposure to IR, as well as other stresses, triggers several complex signaling pathways, including DNA damage recognition and repair, induction of cell cycle checkpoints, senescence and/or apoptosis [1], [2]. Damage to other cellular components, such as the cell membrane, mitochondria, endoplasmic reticulum, and non-DNA constituents of chromatin, may also initiate or modify stress signaling in response to IR. Some of the important pathways responding to radiation include the ATM/P53pathway, MAPK cascades and NFκB activation, as well as signaling events initiated at the cell membrane and within the cytoplasm [3]. The signalling loop concept exists as activation of membrane and cytoplasmic kinases in response to DNA damage inflicted by ionizing radiation. A complex of nuclear factor-κB (NFκB), essential modulator and ataxia telangiectasia- mutated kinase activated by genotoxic agents is sent to cytoplasm, prompting nuclear translocation of the active transcription factor NFκB. In parallel, linear signalling pathways are initiated in the cytoplasm, mostly by reactive oxygen species (ROS), resulting in NFκB activation and nuclear translocation. The choice of NFκB activation pathway and the extent of activation of various pathways may be influenced by the relative degree of damage inflicted by genotoxic agents in the nuclear and cytoplasmic compartments. The ultimate pattern of cellular response is determined by availability, abundance and localization of the proteins participating in the signal transduction. The fate of damaged cells depends on the balance between pro- and antiapoptotic signals. In this decisive life or death choice, the transcription factor NFκB has emerged as a prosurvival actor in most cell types [4], [5]. Early microarray analysis of radiation exposed human cells revealed several novel radiation-induced genes, including FRA1 and ATF3, which encode important transcription factors and require functional p53 for radiation responsiveness [6]. Signaling through MAP kinase pathways can also contribute to the molecular response to radiation exposure. It has been shown that ERK 1/2 pathway has been involved in the upregulation of EGR-1 [7] and VEGF [8] expression in the presence of ionizing radiation mediated by activator protein 1(AP-1) which may lead to further neovascularization and proliferation of glioblastoma cells. The complexity of signaling that occurs following exposure to IR allows flexibility in determining the ultimate fate of damaged cell or tissue. Competition between p53 and NFκB has been shown to sway the balance between apoptosis and survival of irradiated cell. The NFκB-inducing kinase (NIK) is a mitogen-activated protein kinase kinase kinase that potently induces NFκB. Although the upstream receptor remains uncertain, downstream NIK signaling leads to marked activation of the IκB kinases (IKKs) [9], which phosphorylate IκBα, leading to its rapid ubiquitination and degradation by the 26S proteasome. Inactive NFκB/IκB may continuously shuttle between the cytoplasm and nucleus; however, degradation of IκBα allows DNA binding by the liberated NFκB complex and promotes target gene expression [10]–[12]. NFκB plays a key role in cytokine and growth factor signaling, serving to regulate the expression of panoply of genes that mediate inflammatory, anti-apoptotic, and proliferation or differentiation signaling [13], [14]. Hoechst 33342 had been shown to protect genomic DNA against radiation-induced damage. It was reported that the radioprotective activity may be improved by the addition of electron-donating substituents to the ligand [15]–[17]. On the other hand, inhibition of topoisomerase as well as chromatin structure dependent DNA repair by Hoechst 33342 lead to a higher level of residual DNA damage, resulting in a higher level of cytogenetic damage, cell cycle perturbations, and genomic instability, thereby enhancing cytotoxicity [18]. Therefore, development of DNA binding ligands that afford a radioprotective effect without causing mutagenic and cytotoxic effects can play a significant role in biological radioprotection. Here we report the radioprotective effects of two minimally cytotoxic synthetic disubstitutedbenzimidazoles-DMA (Fig. 1A) and TBZ (5-{4-methylpiperazin-1-yl}-2-[2′{2′′-(4-hydroxy-3-methoxyphenyl)-5′′-benzimidazolyl}-5′-benzimidazolyl]. Their radiomodifying effects were investigated on BMG1, a human glioma cell line exposed to Co-60 gamma irradiation by determining cell survival and cell proliferation compared with that of the parent compound, Hoechst 33342. Results from radiation induced growth inhibition showed that DMA & TBZ afforded 84% and 100% radioprotection respectively in human brain glioma cells (BMG1 cells) [19]. Both ligands quench free radicals in isolated free radical system suggesting their dual mode of action against radiation induced damage to DNA. Our data indicate a 2-fold protection by DMA [20], which acts through altering DNA structure and free radical quencher both, as compared to Hoechst, which had shown free radical quenching only. The cell survival assay on four cell lines; human epithelial cancer cells (Hela), human breast cancer cell line (MCF7), human glioma cell line (U87), Human embryonic kidney cell line (HEK) was done previously in our laboratory [21]. 10.1371/journal.pone.0039426.g001Figure 1 Effect of DMA on survival of HDF, MCF10A and U87 cell lines. (A) Structure of the DNA minor groove binding ligand DMA –5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxyphenyl)-5′-benzimidazolyl] benzimidazole.(B) Effect of varying concentrations of DMA on metabolic viability studied by MTT assay in exponential growing HDF,MCF10A and U87 cells at 24 h, (C) At 48 h (D) At 72 h.Values are mean (±SD) of three independent experiments.Statistical significance by T-test p<0.05. Herein, we sought to gain insights on the enhanced radioprotective efficacy and noncytotoxicity of DMA (Fig. 1A). Microarray hybridization and protein expression analysis by 2D PAGE revealed large number of genes that are regulated in response to DMA and/or ionizing radiation. Real time quantitation of the identified proteins and headers confirmed differential regulation. On the basis of our results we propose the involvement of NFκB inducing kinase as the regulator of gene expression modulation in response to DMA and/or ionizing radiation. Results DMA Exerts Reduced Cytotoxicity In our earlier publications, we have already established that DMA is a radioprotector and it protects the human brain glioma cells (BMG1) from radiation and it is less cytotoxic to several cancer as well as transformed cell lines [19]. In view of above, it became imperative to evaluate the cytotoxicity of DMA in normal cells. We examined the cytotoxicity of DMA in primary human dermal fibroblasts (HDF) and near normal mammary epithelial cells (MCF10A) over a wide range of concentrations (0.1–150 µM) up to 72 h post DMA treatment. Interestingly, the IC50 of DMA in HDF and MCF10A could not be determined up to 72 h suggesting that DMA is less cytotoxic to normal cells in comparison to cancerous cells and can be developed as safe therapeutic agent. In addition to it we have measured the toxicity of DMA in U87 cell line also as it possess wild type p53 status and have been taken up as a system for studying irradiation and cytoprotective effects previously.The half maximal inhibitory concentration (IC50) of DMA was found to be 100 µM at 24 h in U87 cells (Fig. 1B, C, D). Differential Gene Expression in Response to DMA and Radiation Treatment in U87, Human Glioma Cells PANTHER gene expression tool was used to classify the differentially expressed genes affected by the three different treatment conditions in U87 human glioma cells, when compared to non-treated control, into specific pathways (Table 1). A total of 30 pathways were found to be differentially expressed in U87 cells treated with DMA, radiation and DMA+radiation (P<0.005). In DMA treated U87 cells, higher percentage of genes modulated were from the General transcription by RNA polymerase I (45%), General transcription regulation (8.82%), Cell cycle (13.636%), Transcription regulation by bZIP transcription factor (9.43%), Insulin/IGF pathway-MAP kinase cascade (14.28%), Insulin/IGF pathway-protein kinase B signaling cascade (13.48%) pathways as compared to radiation alone. On the contrary, higher percentages of genes modulated in the radiation treated U87 cells were from oxidative stress response (11.66%), p53 pathway (10.619%), p53 pathway feedback loops 2 (11.538%) as compared to DMA or DMA+ radiation with respect to control (Untreated cells). 10.1371/journal.pone.0039426.t001Table 1 List of pathways and number of genes in each pathway identified in U87 cells in three different treatment conditions DMA (50 µM), radiation (8.5 Gy) and DMA+ radiation (50 µM +8.5 Gy) after 4 h by Microarray hybridization studies. Pathways DMA Radiation DMA+Radiation VEGF signaling pathway 14 6 10 Oxidative stress response 5.5 13 7.4 FAS signaling pathway 14.6 17.4 9 Glutamatereceptor group II pathway 14 8.8 8.8 PDGF signaling pathway 13.8 10.5 13.5 Cadherin signaling pathway 13 12 11 Nicotinic acetylcholine receptor signaling 14.2 10 15 IGF pathway-protein kinase B signaling 14.2 10 10 IGF pathway-MAP kinase cascade 15 12.3 9 Ras pathway 14.8 4 8 Ubiquitin proteosome pathway 15 15 15 p53 pathway feedback loops 2 10 12 6 bZIP transcription factor 10 4.6 4.6 Interleukin signaling pathway 11.2 7.2 8 TGF-beta signaling pathway 14.8 9 8 p53 pathway 5.4 10.8 7.5 PI3 kinase pathway 10.6 6 6.4 Salvage pyrimidine ribonucleotides 22 14.4 7.5 Cell cycle 14 5 12.2 Wntsignaling pathway 10.6 10.6 11 Apoptosis signaling pathway 5 11 8 Integrin signaling pathway 10.6 6 6 General transcription regulation 9 5 6.4 Notch signaling pathway 7 4.6 4.6 Cytokine signaling pathway 10.4 5.7 6 Synaptic vesicle trafficking 21.4 18 15.8 DNA replication 28 14.6 14.6 FGF signaling pathway 6.2 3 2 General transcription 45 15 35 From these pathways, a number of representative differentially expressed genes identified are given in Table 2. Two important NFκB signaling related genes - CREB3L4 and TRERF, were induced in DMA treated cells. Other related transcription factors- HIF1A, ASCC3 and CREBBP were found to be upregulated in response to DMA treatment. MAP3K14 (NFκB inducing kinase) was upregulated in DMA and DMA + radiation treated cells. 10.1371/journal.pone.0039426.t002Table 2 List of genes differentially regulated in U87 cells 4 h after treatment with DMA (50 µM), radiation (8.5 Gy) and DMA + radiation (50 µM+8.5 Gy). Accessionnumber Genesymbol Gene Name Function DMA Radiation DMA+Radiation NM_130898 CREB3L4 cAMP responsive element binding protein3-like 4 regulation of transcription,DNA-dependent 4.03 1.55 1.65 AF297872 TRERF1 transcriptional regulating factor 1 Transcription regulation 4.9 0.54 6.45 NM_005902 ASCC3 Activating signal cointegrator 1 Complexsubunit 3 Transcription regulation 5.290 0.86 1.68 AC004760 CREBBP CREB binding protein mRNA transcriptionregulation 2.18 0.18 1.92 BC012527 HIF1A hypoxia-inducible factor 1, alpha subunit Transcription activation 0.981 0.909 5.075 AC008105 HIF!A Mitogen activated protein kinasekinasekinase 14 Positive regulation ofNFkB cascade 4.236 0.456 1.375 NM_004045 ATOX1 ATX1 antioxidant protein 1 homolog (yeast) response to oxidative stress 0.137 0.82 0.11 NM_012111 AHSA1 AHA1, activator of heat shock 90 kDa proteinATPase homolog 1(yeast) response to stress 0.146 1.5 0.17 NM_005742 PDIA6 Protein disulfide isomerase family A, member 6 protein folding 0.15 0.84 0.15 BC015484 CALB2 calbindin 2, 29 kDa (calretinin) calcium ion binding 0.131 1.07 0.12 NM_007236 CHP calcium binding protein P22 calcium ion binding 0.52 1.05 0.086 NM_002155 HSPA6 heat shock 70 kDa protein 7 (HSP70B) unfolded protein response 0.441 0.91 0.493 NM_006597 HSPA8 heat shock 70 kDa protein 8 unfolded protein response 0.327 0.74 0.474 BC024034 HSPA9 heat shock 70 kDa protein 9 (mortalin) unfolded protein response 0.395 0.93 0.59 NM_005347 HSPA5 heat shock 70 kDa protein 5(glucose-regulated protein 78 kDa unfolded protein response 0.06 0.77 0.118 NM_002157 HSPE1 heat shock 10 kDa protein 1 (chaperonin 10) unfolded protein response 1.07 3.74 1.03 NM_145046 CALR3 calreticulin 3 unfolded protein binding 1.28 5.09 1.04 BT019740 PRDX1 peroxiredoxin 1 peroxiredoxin 1 0.97 1.22 0.002 BT020007 PRDX3 peroxiredoxin 3 redox regulation 0.66 0.70 0.169 NM_012473 TXN2 thioredoxin 2 response to hypoxia 1.49 1.32 0.067 NG_005218 HSP90AA3P heat shock 90 kDa protein 1, alpha-like 1 Chaperone 1.43 1.74 0.071 NM_006993 NPM3 nucleophosmin/nucleoplasmin, 3 nucleic acid binding 0.20 1.27 0.105 BC009623 NPM1 nucleophosmin (nucleolarphosphoproteinB23, numatrin) nucleic acid binding 0.27 1.55 0.16 NM_006472 TXNIP thioredoxin interacting protein response to oxidative stress 0.18 1.29 0.202 NM_001540 HSPB1 heat shock 27 kDa protein-like2 pseudogene; heat shock 27 kDa protein unfolded protein response 0.44 1.66 0.464 NM_022121 PERP PERP, TP53 apoptosis effector induction of apoptosis 0.28 3.87 0.244 BC022307 ATM similar to Serine-protein kinase ATM (Ataxia telangiectasia mutated) DNA Repair 0.69 0.84 0.194 NM_001924 GADD45A growth arrest and DNA-damage-inducible, alpha DNA Repair 0.329 1.24 0.64 NM_015675 GADD45B growth arrest and DNA-damage-inducible, beta DNA Repair 0.113 0.64 0.378 NM_006705 GADD45G growth arrest and DNA-damage-inducible,gamma DNA Repair 0.177 0.94 0.206 NM_007233 TP53BP1 TP53 activated protein 1 Response to DNA damage 0.119 0.783 0.51 NM_005657 TP53AP1 TP53 binding protein 1 Response to DNA damage 0.14 2.41 1.13 NM_006034 TP53I11 TP53 inducible protein 11 Response to stress 0.203 1.06 0.69 NM_002578 PAK3 p21 (CDKN1A)-activated kinase 3 protein serine/threoninekinase activity 0.154 1.16 0.17 NM_020168 PAK6 p21 (CDKN1A)-activated kinase 6 protein serine/threoninekinase activity 0.22 1.22 0.21 NM_004964 HDAC1 histone deacetylase 1 regulation of transcription,DNA-dependent 0.216 0.69 0.35 (D = 50 µM DMA treated cells, R = 8.5 Gy irradiated cells, D+R = 50 µM DMA+8.5 Gy treated cells; Fold change values in red indicate upregulation in comparison with control untreated cells (2 fold and above); Fold change values in green indicate downregulation in comparison with untreated control (0.5 fold and. 2D PAGE Reveals Modulation of Protein Expression in Response to DMA and Radiation Treatment A total of 19 proteins were identified and observed as differentially regulated (upregulated or downregulated) in response to DMA, radiation and DMA+ radiation with respect to control (untreated cells) by 2D PAGE and ESI-MS/MS analysis (Fig. 2 and Table S1, S2). 10.1371/journal.pone.0039426.g002Figure 2 One representative image of 2D PAGE of the proteome of U87, cells in response to DMA, radiation and DMA+radiation treatment. Protein spots showing differential expression in the DMA, radiation and DMA+ radiation at 4 h time point, an average of three replicates (data derived from replicate wells) are annotated with arrows and numbers. The numbers given in this figure denote the 19 differentially expressed proteins identified by 2D PAGE and ESI-MS/MS analysis. The serial numbers 1–19 in table S1 denote the protein spots numbered in Figure 2. The left hand side spots are of protein marker. These differentially expressed proteins belonged to four different categories- chaperones and folding catalysts (HSP70, CALR, TRX, PRX2, PDIA3); structural proteins (UBCEP80, H2B1, ACTG, TPM4, VIM, FABPE); Single stranded nucleic acid binding proteins (NPM1,PCBP1); Glycolytic pathway enzymes (PGM, ENO1,TPI, PARK7,NACA). Since most of these proteins are interacting partners, a pathway construction was attempted by online available sources (KEGG, NCBI). This pathway converged onto three mitogen activated protein kinases–MAP3K3, MAP3K7 & and MAP3K14 (Fig.3). 10.1371/journal.pone.0039426.g003Figure 3 Pathway construction based on the proteins identified from 2D PAGE and ESI-MS/MS Analysis . Genes/proteins identified from proteomic data analyses were categorized into pathways based on NCBI, OMIM, KEGG and Gene Cards. Colour boxes represent the proteins identified by ESI-MS/MS- chaperones and folding catalysts (HSP70, CALR, TRX, PRX2, PDIA3); structural proteins (UBCEP80, H2B1, ACTG, TPM4, VIM, FABPE); Single stranded nucleic acid binding proteins NPM1,PCBP1); Glycolytic pathway enzymes (PGM, ENO1,TPI); PARK7,NACA. Blue boxes indicate interacting partners not identified by ES-MS/MS but are common between the identified proteins as interacting partners. Red arrows indicate that the proteins identified belonging to common categories and possessing common interacting partners converge onto MAPK signal transduction pathway. Relative Quantitation of Gene Expression of 2D PAGE Identified Proteins by Real Time PCR Out of the total genes identified from 2D page, a total of 5 genes of MAP kinase pathway were quantitated by RT PCR. Out of the five MAP kinase pathway genes, YWHAZ and MAP3K14 (NIK) were found upregulated in response to DMA and DMA + radiation treated cells. However, no effect was observed in response to irradiation only. In the case of MAP3K3, MAP3K7 and MAP3K10, no significant differential expressions were observed in all the treatment conditions. Similarly, NFκB (RelA subunit) was observed to be differentially regulated in DMA and DMA+ radiation treated cells in comparison to radiation alone (Fig. 4). 10.1371/journal.pone.0039426.g004Figure 4 Relative quantitation of gene expression for MAPK pathway genes in U87 cells. U87 cells were treated with 50 µM DMA and/or 8.5 Gy ionizing radiation. RNA samples were prepared 4 h after treatments. YWHAZ (14-3-3 zeta), MAP3K3 (Mitogen activated protein kinase kinasekinase 3), MAP3K7 (Mitogen activated protein kinase kinasekinase 7), MAP3K14 (Mitogen activated protein kinase kinase 14), MAPK10 (Mitogen activated protein kinase 10) and NFκB (Nuclear Factor Kappa B/Rel A subunit) were quantitated by Real Time PCRusing RNA samples from U87 glioma cell line. Values represent Mean±S.D.(*indicates statistical significance by T-test, *p<0.05). DMA Potentiates NFκB- Inducing Kinase Mediated Activation of IKKα and IKKβ The phosphorylation of both IKKα and IKKβ were not observed in control and radiation treated U87 cells, transfected with control siRNA through western blot experiments (Fig.5). However, in response to DMA and DMA + radiation, phosphorylated IKKα was observed. Phosphorylated IKKβ was also observed albeit to a weaker extent (Fig. 6). Over expression of NFκB- inducing kinase by transfecting U87 cells with p3XFLAG CMV10 NIK plasmid potentiated the activation of IKKα and IKKβ in DMA treated cells (Fig. 7). Minimal level of IKKα phosphorylation was observed in control cells, with no phosphorylation of IKKβ being detected. In radiation treated cells, activation of both IKKα and IKKβ was not observed. In response to DMA and DMA+radiation, phosphorylated form of both IKKα and IKKβ was detected. In all the samples, detection of IκBα was abrogated concurrent with observation of phosphorylated form of either IKKα or IKKβ (Fig.7). Transfection of U87 cells with siRNA-NIK and subsequent knock down of NFκB- inducing kinase expression gave a different response (Fig.6). In all the treated samples, phosphorylated form of both IKKα and IKKβ was not observed. However, total IKKα, IKKβ and IκBα were detected at all the time points. There was no change observed in total protein levels of both IKKα and IKKβ, following DMA and TNF-α stimulation. 10.1371/journal.pone.0039426.g005Figure 5 Effect of transfection with control-siRNA on the phosphorylation of IKKα and IKKβ. U87, human glioma cells were transiently transfected with control-siRNA. Cells were induced with 10 ng/ml TNF-α for 0,5,10,20,40,60 min and treated with DMA (50 µM) and/or irradiated at 8.5 Gy. Phosphorylation of IKKα, IKKβ was observed in DMA treated cells, alone or in combination with IR. 10.1371/journal.pone.0039426.g006Figure 6 Effect of transfection with siRNA-NIK on the phosphorylation of IKKα and IKKβ. U87, human glioma cells were transiently transfected with siRNA-NIK. Phosphorylation of IKKα, IKKβ was absent concurrent with equivalent expression of IκBα in response to both DMA and radiation treatment. 10.1371/journal.pone.0039426.g007Figure 7 Effect of NFκB inducing kinase overexpression on the phosphorylation of IKKα and IKKβ. U87, human glioma cells were transiently transfected with p3XFLAG CMV10 NIK. Phosphorylation of IKKα, IKKβ was advanced in NIK overexpressing cells as compared to siRNA transfected cells in response to DMA treatment, alone or in combination with IR. siRNA -NIK Knock down Abrogates DMA-induced Radioprotection in Transfected Cells In vitro cell proliferation assay was used to study radioprotective ability of DMA in siRNA-NIK transfected and control siRNA transfected cells with or without radiation [22]. As shown in Fig. 8, the percentage of radioprotection by DMA in control siRNA transfected cells with respect to drug only control at 24, 48 and 72 h was 51%,30% and 16% respectively while in siRNA-NIK transfected cells the percentage of radioprotection by DMA was 15%, 18% and 10% at 24, 48 and 72 h respectively. 10.1371/journal.pone.0039426.g008Figure 8 Radioprotection afforded DMA studied by proliferation kinetics assay in Control siRNA and siRNA-NIK transfectedU87 cells. (A) U87 cells (Control siRNA transfected) were treated with 50 µM DMA and 8.5 Gy Radiation. Post treatment cells were seeded in 6 well plates seeded at 8000 cells/cm2, and their proliferation kinetics was studied at 24 h intervals following trypsinization and counting total cells per well using a hemocytometer. (B) U87 Cells post siRNA-NIK oligonucleotide transfection were treated with 50 µM DMA and 8.5 Gy Radiation. Post treatment cells were seeded in 6 well plates seeded at 8000 cells/cm2, and their proliferation kinetics was studied at 24 h intervals following trypsinization and counting total cells per well using a hemocytometer. Values are mean (±SD) of three independent experiments.Statistical significance by T-test p<0.05. DMA does not Perturb Cell Cycle To further study the effect of DMA on cell cycle, we examined the cell cycle in control siRNA transfected as well as siRNA-NIK transfected U87 cells. We have observed normal cell cycle in control siRNA treated cells, whereas cells did show a strong G2 check point after irradiation alone. There was clear G2 phase accumulation (18.9%) at 6 h after exposure to 8.5 Gy, which was increased till 18 h (34.5%), came near to normal at 24 h (22.3%) (Fig. 9E, G). There was no strong/significant G2 check point observed after DMA (50 µM) treatment in control siRNA treated cells, but in DMA + radiation treated cells we have observed a significant G2-M (43.5%) arrest than the irradiated cells (34.5%) at 18 h(Fig. 9E, G). The delay in cell cycle progression is probably exploited by the cells to perform DNA repair, and as a consequence to reduce the adverse effects of radiation, such as cell killing and induction of mutations. The cell cycle analysis of siRNA-NIK transfected cells also showed strong G2 check point after radiation treatment (11.9% at 0 h, 13.3% at 3 h, 18.6% at 6 h, 30.3% at 12 h, 43.6% at 18 h and 29.7% at 24 h), whereas the G2 check point response in DMA + radiation treated cells was almost similar to radiation only treated group reassuring that the activity of DMA is diminished in NIK abrogated cells (Fig 9. F, H). 10.1371/journal.pone.0039426.g009Figure 9 DMA attenuates radiation induced apoptosis in U87 through NIK. (A) Apoptosis analysis of control siRNA transfected U87 cells at 3 h, 6 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation (8.5 Gy). Percentage of apoptosis is defined by % of cells that are Annexin V+ and Annexin V +PI+. (B) Apoptosis analysis of siRNA-NIK transfected U87 cells at 3 h, 6 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation (8.5 Gy). Percentage of apoptosis is defined by % of cells that are Annexin V+ and Annexin V +PI+. (C) Percentage of Annexin V+ and Annexin V +PI+.positive cells in control siRNA transfected U87 cells at, 3 h,6 h, and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation(8.5 Gy). (Data Not shown for 6 h and 12 h for only DMA and only radiation treatment condition). (D) Percentage of ofAnnexin V+ and Annexin V +PI+.positive cellssiRNA-NIK transfected U87 cells at 3 h, 6 h, and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation (8.5 Gy).(Data Not shown for 6 h and 12 h for only DMA and only radiation treatment condition). (E) Time course graphs showing the progression of cell cycle of control siRNA transfected U87 cells at 0, 3, 6, 12, 18 and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation(8.5 Gy). (F) Time course graphs showing the progression of cell cycle of siRNA-NIK transfected U87 cells at 0, 3, 6, 12, 18 and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation(8.5 Gy). (G) Cell cycle progression of control siRNA transfected U87 cells showing % G2-M fraction at 0, 3, 6, 12, 18 and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation(8.5 Gy). (H) Cell cycle progression of siRNA-NIK transfected U87 cells showing % G2-M fraction at 0, 3, 6, 12, 18 and 24 h in different treatment condition i.e. 50 µM DMA, radiation (8.5 Gy) and DMA (50 µM) + Radiation(8.5 Gy). The Annexin V assay showed lot of early and late apoptotic events in radiation treated U87 cells upto 24 h. The percentage apoptosis kept increasing in radiation treated cells till 24 h in a significant manner. In DMA + radiation treated cells the total (35% at 3 h, 30% at 6 h and 18% at 24 h) of early and late apoptotic cells were observed less in comparison to radiation treated cells only (45% at 3 h, 6 h and 24 h data not shown) (Fig 9. A, C). But in NIK knocked down cells the apoptotic events were almost similar in radiation (60% at 3 h, 6 h and 24 h data not shown) as well as DMA + radiation treated cells (58% at 3 h, 52% at 6 h, and 52% at 24 h), suggesting that NIK plays a role in the radioprotection activity of DMA. (Fig 9. B, D). Recently, there is a report where role of NIK was implicated in cell survival, supporting our observation [23]. DMA Affects NFκB Activation in a dose Dependent Manner TNF-α induced NFκB dependent reporter gene transcription assay showed no activation of NFκB in control and radiation treated cells. To confirm that the activation of NFκB was specifically caused by DMA, a series of concentration of DMA (0, 10, 25, 50 µM) was used in cells co-transfected with pNFκB-Luc (0.2 µg/well) and pRenilla (0.02 µg/well) and induced with 20 ng/ml TNF-α (Fig. 10). The results showed that NFκB activation was promoted in a dose-dependent manner by DMA. From 10 to 50 µM, a concentration dependent increase in induction of NFκB activation was observed in response to DMA. Higher induction of NFκB activation was observed in 50 µM DMA treated cells exhibiting a fold change of 3.62. However, NFκB activation was abrogated in U87 cells following irradiation. In DMA + radiation treated cells, activation of NFκB was again dose dependent with higher activation occurring in 50 µM + radiation treated cells i.e.; 5.77 fold as compared to control cells. 10.1371/journal.pone.0039426.g010Figure 10 Luciferase assays for NFκB activation in U87 cells. Cells were co-transfected with pNFκB-Luc and pRenilla. After 24 h, cells were irradiated (8.5 Gy) with or without DMA treatment (50 µM) and stimulated with TNF-α (20 ng/ml) for 4 h prior to cell lysis. Luciferase activity was determined to quantify NFκB transcription activity. Relative Luciferase activity (RLA) for NFκB activation was increased in DMA treated cells compared with irradiated or control cells. The triplicate samples were used and the data was presented as mean±SD from three independent experiments. (*indicates statistical significance by T-test, *p<0.00001). Identification of NFκB Inducing Kinase as the Candidate Gene From microarray hybridization and data analysis (Table 2), three important NFκB signaling related genes - CREB3L4, ASCC3 and TRERF were induced in DMA treated cells. It has been reported that CREB3L4 is a bZIP transcription factor that localizes to the membrane of endoplasmic reticulum and plays a role in cell homeostasis. It also has transcriptional activation activity from NFκB regulatory elements [24]. Another related transcription factor-ASCC3 was found to be upregulated in response to DMA treatment. ASCC3 has been reported to exist as a steady-state complex associated with three polypeptides, P200, P100, and P50, which stimulates transactivation by serum response factor (SRF), activating protein 1 (AP-1), and Nuclear factor kappaB (NF-kappaB) through direct binding to SRF, c-Jun, p50, and p65; and relieves the transrepression between nuclear receptors and either AP-1 or NF-kappaB. TRERF is another important transcription factor that has been observed to interact with CREBBP and EP300 resulting in a synergistic transcriptional activation of CYP11A1. Expression of HIF1A was found to be increased in DMA + radiation treated cells. It was reported that activation of HIF1A requires recruitment of transcriptional coactivators such as CREBBP and EP300.It also binds to core DNA sequence 5′-[AG] CGTG-3′ within the hypoxia response element (HRE) of target gene promoters. Since CREBBP is a coactivator of P300/CREB, increase in its expression would oppose the proapoptotic function of p53. Expression of HIF1A [25] was increased in DMA + radiation treated cells. Hence, we observed upregulation of NFκB inducers and effectors from microarray and real time PCR results. The key signaling molecule observed as upregulated was NFκB inducing kinase, which subsequently leads to mobilization of NFκB from cytosol to the nucleus thereby activating the nuclear targets and hence the transactivation of downstream genes. Thus, activation of NFκB inducing kinase in DMA treated cells perhaps may explain counteraction of radiation- induced damage. Discussion On the basis of microarray hybridization and data analysis of differentially regulated genes following pathways were observed to be differentially regulated on DMA and ionizing radiation treatment in U87 cells – Apoptosis pathway, Chaperones and folding catalysts, Transcription regulator, oxidative stress response cell cycle, signal transduction and p53 pathway. Two prominent NFκB signaling related genes - CREB3L4 and TRERF were induced in DMA treated cells. We observed expression of a number of NFκB signaling related genes (MAP3K14, CREBBP, CREB3L4, TRERF, ASCC3 and HIF1A) to be regulated in response to DMA from Microarray hybridization and data analysis. Thus, the concerted action of CREB3L4, TRERF, ASCC3 and HIF1A induces CREB/p300 and NFκB results in opposition of proapoptotic response and increase in transcriptional activation. Regulation of MAP3K14, CREB3L4, ASSC3, HIF1A points towards the activation of the NFκB mediated signaling pathway. Marked increase in NFκB activation in DMA treated cells indicates promotion of cell survival and proliferation. Simultaneously, downregulation of HSP70, stress responsive gene as observed in cells treated with DMA could further lead to reduction in inhibition of the NFκB inducing kinase, since HSP70 blocks NFκB activation via inhibition of both I kappa B kinase (IκK) activation and subsequent degradation of I kappa B alpha (IκBα) [26], [27]. Induction of NFκB inducing kinase subsequently leads to mobilization of NFκB from cytosol to the nucleus thereby activating the nuclear targets and hence the transactivation of downstream genes. This as an effect could result in the suppression of misfolded protein response, apoptosis and regulation of gene expression to ensure enhanced cell survival (Fig. 11). 10.1371/journal.pone.0039426.g011Figure 11 Proposed hypothesis for the mechanism of action of DMA. From gene expression analysis, important inducers of NFκB-CREB3L4, TRERF, ASCC3, HIF1A and MAP3K14 were observed to be activated. Moreover, HSP70 was found to be downregulated which inhibits NFκB activation at the level of I kappa B Kinase. Suppression of chaperones, folding catalysts, stress responsive genes in DMA treated cells further adds onto cellular proliferation. Thus, in cells treated with DNA minor groove binding ligand DMA, induction of NFκB inducing kinase and related transcription factors converges onto the propagation of the pro-survival signal. In addition following p53 pathway genes were downregulated in DMA treated cells- PERP,ATM,GADD45A,GADD45B,GADD45G,TP53BP1,TP53AP1,TP53I11,PAK3,PAK6,HDAC1. In cells treated with DMA alone, we observed simultaneous activation of NFκB and downregulation of p53 responsive genes (Table 2). p53 is generally a proapoptotic transcription factor, while NFκB promotes resistance to programmed cell death. The NFκB had an ability to suppress p53 transactivation and thus inhibitthe transcriptionally dependent induction of apoptosis by p53.Sequestration of p300 and CBP competitively; is likely to be understood as an increasingly common mechanism regulating inducible gene expression. It has been shown that p53 and RelA (NFκB) can bind p300 and CBP competitively, the answer may lie in the utilization of different p300/CBP complexes, being used by distinct subsets of DNA-binding proteins, thus functionally separating them and limiting cross talk to certain regulatory families of transcription factors [28], [29].Hence, the activity of NFκB may directly oppose the proapoptotic function of p53 activation through competition for the p300/CREB-binding protein transcriptional coactivator complexes. Thus, treatment with DMA leads to activation of NFκB inducers and effectors and provides comparatively enhanced radioprotective efficacy. In cells treated with DMA alone, we observed simultaneous activation of NFκB and CCNB2, CASP8, THBS1 and CCNG2 of p53 pathway were downregulated. Similarly BAX, CDK4, CCNG1, SESN1were found to be upregulated in DMA+ radiation treated U87 cells with no change in radiation alone. In our earlier work, we had observed that DMA modulates radiation damage to DNA through free radical quenching, which was supported by observing regulation of a number of genes in apoptotic pathway in U87 cell line. TNFRSR1A, CASP8, PIK3R5, BCL1, CAPN1 were found to be downregulated in DMA treated cells. Since these genes are the central components of apoptotic response their downregulation in response to DMA further implicates the absence of DNA damage. Thus, treatment with DMA leads to activation of NFκB inducers and effectors and provides comparatively enhanced radioprotective efficacy. In summary, suppression of expression of stress activated genes, absence of activation of DNA damage responsive genes, and over expression of signal transduction genes as well as transcription regulation factors may be possible reasons behind the radioprotective ability of this molecule as observed by attenuated DNA damage at the cellular level. A total of 19 proteins were identified as differentially expressed and categorized into four pathways using KEGG-chaperones and folding catalysts, structural proteins, single stranded nucleic acids binding proteins and metabolic enzymes on the basis of 2D PAGE analysis of U87 cells. Protein expression of five chaperones and folding catalysts was observed to be altered- HSP70, CALR, TRX, PRDX, and PDIA. A number of structural proteins were also identified as differentially expressed- UBCEP80, H2B1, ACTG, TPM4, VIM and FABPE. Two single stranded nucleic acids binding proteins-NPM and PCBP1 were found to be upregulated in radiation treated cells, however, in cells treated with DMA only, were found to be normally expressed. Enzymes belonging to the glycolytic pathway found to be differentially regulated were- PGAMB, ENO1 and TPI. Pathway construction using online available resources, on the basis of identified proteins and their interacting partners led onto the identification of the headers (MAP3K14, MAP3K7 and MAP3K3) that could be involved in modulation of radiation response by DMA in U87 cells (Fig. 3). RT PCR was performed to verify changes in expression of 21 genes. 19 genes were chosen from 2D PAGE results and two more i.e.; MAP3K14, header of the pathway constructed by us on the basis of 2D PAGE results, and NFκB a downstream gene of MAP3K14 were also included for real time study. Relative quantitation of above genes(data not shown for all genes) suggested modulation of a number of genes - HSP70, TXN, PDI, CALR, PRDX, YWHAZ, NPM1, PCBP1, MAP3K14, NFκB, H2B1. RT PCR experiments validated modulation of YWHAZ, MAP3K14, NFκB in MAP kinase pathway genes in all treatment conditions (Fig. 4). YWHAZ belongs to the family of 14-3-3 proteins that are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress [30]. Upregulation of this gene in response to DMA, both alone and in combination with IR shows regulation of cell cycle machinery by DMA. NFκB is known as a proinflammatory transcription factor and is now confirmed as an antiapoptotic and prosurvival force in the most cell types [31]–[33]. This gene was upregulated in DMA and DMA + radiation treated cells, thus implicating the activation of pro-survival genes in response to DMA. MAP3K14, an upstream kinase was upregulated in DMA and DMA+ radiation treated cells. It has been reported that MAP3K14 binds to Traf2 and stimulates NFκB activity [34]–[36]. Amifostine, along with other thiol-containing drugs, such as N-acetylcysteine (NAC), mesna, and oltipraz, which have been approved for clinical use as radioprotector, are capable of participating in intracellular reductive/oxidative processes that have broad clinical implications. These include effects on the activation of redox-sensitive transcription factors, alteration of gene expression, and modification of protein activities. Amifostine, NAC and oltipraz activate the redox-sensitive transcription factor NFκB in the absence of oxidative stress–inducing agents [37]–[41]. DNA-binding motifs for NFκB were found in the promoter regions of more than 150 genes, many of them involved in inflammatory processes and the survival response [14]. NFκB is an inducible transcription factor that plays an essential role in the expression of a number of gene families that include cytokines and their receptors, cell adhesion molecules, growth factors, and antioxidant genes [42]. A number of reports have described the activation of NFκB by reducing agents, when present at mM concentrations, such as NAC, dithiothreitol, 2-mercaptoethanol 2-ME, oltipraz, and amifostine [38]–[40], [43]. It has been reported that exposure of glioma cells to mM concentrations of WR-1065 significantly protected these cells from the cytotoxic and mutagenic effects of ionizing radiation [44], [45]. The Putrescine, spermidine, and spermine have each been reported to participate in the activation of NFκB and to enhance its binding to nuclear response elements (NREs) [46]. The underlying mechanism of action is not attributed to changes in oxidative stress, but rather to the ability of these positively charged molecules to activate NFκB and affect structural/conformational changes in DNA that facilitate an enhanced binding of NFκB to its NRE sequences. On the basis of above results, we identified NFκB inducing kinase as the candidate gene affected by DMA. Western blots results suggested that DMA affects NIK mediated NFκB activation. In cells overexpressing NIK, DMA promoted activation and phosphorylation of IKKα and IKKβ, although extent of phosphorylation of IKKβ was weaker than IKKα. Activation of IKKα and IKKβ in DMA treated samples, alone or in combination with radiation, was concurrent with degradation of IκBα. As compared to cells treated with control-siRNA, activation was advanced in time in cells over expressing NIK. On the contrary, cells in which the NIK expression was knocked down by siRNA-NIK, no activation and phosphorylation of IKKα and IKKβ was observed in response to DMA, alone or in combination with IR. Luciferase reporter assay confirmed dose dependent induction of NFκB activation. Biochemically the apoptotic process is first recognized by alterations of surface lipid composition, in that, phosphotidylserine, which is normally on the inner leaflet of plasma membrane, translocates to the outer leaflet, which can be easily measured by fluorescent labeled Annexin V binding assay. An impermeable dye propidium iodide, cannot enter the cells unless the cells are under late apoptosis, when membrane permeability is compromised. This can be quantified in large population of cells by flow cytometry. The control siRNA (untreated) cell culture contained very few apoptotic cells (6.5%) (Data not shown), which were assigned as the background cell death due to stress during normal cell culture. We found that in DMA (50 uM ) treated U87 transfected with control siRNA, resulted in 9% early apoptotic cells within 24 h, but radition (8.5 Gy ) treated cells showed 45% early apoptotic cells within 3 h which were reduced to 35% at 3 h, 30% at 6 h and 18% at 24 h in DMA + radiation treated cells. The U87 cells transfected with siRNA-NIK, showed 60% apoptotic cells after irradiation (8.5 Gy) at 3 h time point, which were consistant to 58% at 3 h, 52% at 6 h and 52% at 24 h in DMA + radiation treated siRNA-NIK transfected cells. The Annexin V assay showed that DMA causes a very few apoptotic events in U87 cells, hence DMA can be proposed as a safe molecule. This result is in concurrence of our earlier results in which DMA was observed less cytotoxic in comparison to parent molecule Hoechst 33342, a free radical quencher, no significant DNA damage was observed through comet assay [20]. We observed higher RQ value of NFκB in DMA treated cells as compared to DMA+radiation treated cells in RT-PCR. However, in luciferase reporter assay higher activation of NFκB was observed in DMA+radiation treated cells as compared to DMA treated cells in luciferase reporter assay. From a RT-PCR experiment only the transcript level is measured, but luciferase reporter assay takes into account promoter binding dependent activation and/or post transcriptional modification or transactivation. Hence, reporter assay is a more direct study of the NFκB protein level in response to DMA and/or radiation treated cells. Therefore, ultimately we observed higher activation of NFκB in DMA + radiation treated cells. We observed absence of IKKα and IKKβ activation and phosphorylation as well as subsequent NFκB activation in irradiated cells. Reactive oxygen species (ROS) increase first promotes NFκB activation by triggering NIK activation, which usually leads to prosurvivaltranscriptional events. However, as ROS become overwhelming, the nuclear environment is shifted from reductive to oxidative, which inhibits activation of NFκB and other transcriptional factors by preventing them from binding to DNA. As a consequence, the prosurvival transcription activity is abolished [47]. This increased ROS then leads to shift in survival/death control. We examined the effect of NIK inhibition by siRNA on the proliferation of U87 cells in DMA treated cells in the presence and absence of radiation. We observed a significant decrease of 36% in Radiation protection in siRNA- NIK treated cells as compared to control siRNA treated U87 cells at 24 h. The complete absence of radioprotection was not observed as we had already established that DMA, has a dual mode of action against radiation [20], it also acts as free radical quencher. The parent analogues Hoechst 33258 & 33342 were well identified as free radical quencher, generated directly or indirectly by IR [18], [48]. These results are in the agreement with our microarray, real time and western blot results suggesting that the radioprotective effect of DMA is NIK mediated. Collectively, our data suggests that, one of the primary targets of DMA is NIK. We may conclude that radioprotective effect of DMA is due to NIK mediated NFκB activation leading irradiated cells to antiapoptotic and prosurvival pathway, one of the plausible mechanisms of action of DMA as a radioprotector. Amifostine is converted into active form in normal cells by alkaline phosphatase which is inactive in an acidic environment of tumors making it a selective drug for normal cells. The major side effects of amifostine are transient hypotension, flushing, somnolence, metallic taste and transient hypocalcaemia. Also amifostine effect will be reduced in tumor cells due to hypoxia state [49]. On the contrary, our molecule DMA has dual mode of action i.e.;free radicals quenching and binding to DNA which makes it a better molecule [19]. Our cytotoxicity data suggest that DMA is more toxic to cancerous cells then normal cells. Abnormal vasculature of cancerous cells can be one of the possible modes of differential uptake of DMA in cells. Thus our correlation of NIK and DMA in U87 cells can be further elucidated for clinical importance in normal cell lines. NIK/IKK mediated NFκB activation is at a pivotal role for promoting pro-survival in cells hence the clinical relevance of this study; as further work in normal cells and animal model experiments can lead to development of a novel radioprotector with enhanced survival and radioprotection with less cytotoxicity which is a major drawback among most other well known radioprotectors. It was suggested that coactivation of prosurvival ATM/ERK/NFκB pathway offer an effective therapeutic approach for enhancing radiation tolerance in human skin cells [50] . Several groups have tested the effects of NFκB inhibitors on cellular radiosenstivity and shown that the NFκB activity is necessary for enhancing cell survival under high dose radiation. In addition, it was also observed that IKKβdependent NFκB activation provides radioprotection to the intestinal epithelial cells [51]. Similarly our results suggests that, DMA activates the NIK/IKKα/IKKβ mediated NFκB activation providing protection to cells from higher γ –radiation doses. Materials and Methods Cell Culture The human glioma cell line U87 was obtained from the National Centre for Cell Science (Pune, India). MCFA10A was obtained from American Type Culture Collection (ATCC) USA and HDF cells were obtained from Dermatology department at Emory University School of Medicine, Atlanta, GA, USA. These cells lines were maintained according to the ATCC recommendations at 37°C, 5% CO2, in DMEM supplemented with 10% heat inactivated FCS, 50 units/ml penicillin, 50 µg/ml streptomycin. Cells were cultured, grown until ∼ 80% confluent, trypsinized and seeded in 90 mm culture dishes for 24 h before experiments. We have used U87 cells for the present study due to number of reasons. Earlier, we have showed in BMG1 cell line (Glioma cells) that DMA is a noncytotoxic radioprotector. We measured the radio sensitivity of U87cells with a standard clonogenic assay [52] and found U87 cells were medium sensitive to radiation and survival of these cells, after radiation is higher than other cells. U87 cell lines possess wild type p53 status and have been taken up as a system for studying irradiation and cytoprotective effects previously [53]–[56]. Amifostine, a radioprotector which is in clinical trial and lot of previous work was done and validated with U87 cells. DMA and Exposure Conditions DMA was synthesized in our laboratory as described previously [19]. IC50 for DMA was determined at 100 µM in U87 cells. Radioprotection survival assay was performed using increasing dose of DMA from 0–100 µM concentration at four different radiation doses- 2, 5, 8.5 and 10 Gy respectively (data not shown). The radiation dose (8.5 Gy) and drug dose (50 µM) was determined by clonogenecity assay at increasing ligand concentration as well as increasing radiation dose. From the clonogenecity assay it was observed that, 50 µM at 8.5 Gy provided best radioprotection. At higher radiation dose most of the cells died in DMA untreated cells (control cells). Hence, in this study all the experiments were performed at 50 µM DMA with or without 8.5 Gyradiation. Stock of DMA was prepared in methanol and further dilution to 1 mM was done using DMEM. This solution was then filtered & sterilized and U87 cells were subjected to DMA treatment at a final concentration of 50 µM for 1 h. Four treatment conditions were studied- control (untreated), ligand treated (50 µM DMA), radiation treated (8.5 Gy radiation) and ligand + radiation treated (50 µM DMA+8.5 Gy radiation). Irradiation Procedure 1 h subsequent to DMA treatment, cells were exposed to 8.5 Gy γ- irradiation (1.3 Gy/min dose rate) using Co-60 source (Institute of Nuclear Medicine and Allied Sciences, Delhi, India). Following irradiation, cells were incubated for 4 h in a 5% CO2 humidified incubator. Trypsin was used to detach the cells. Cells were centrifuged, washed twice in 1X PBS (pH 7.4) and processed for further sample preparation. Metabolic Viability Assay Exponentially growing U87, HDF and MCF10A cells were plated at cell densities 3000 cells per well in 96-well tissue culture plates. After 24, 48 and 72 h cells were treated with increasing concentrations of DMA i.e.; 0.1–150 µM. Cytotoxicity was measured by MTT assay according to the manufacturer’s instructions (Promega, Madison, WI, USA). The experiments were performed in triplicate and repeated thrice. The percentage survival was calculated as percent of 570 nm OD of DMA treated cells to that of DMA untreated control cells with 630 nm OD as reference. Percentage survival  =  (Calculated difference between measurement and reference OD of DMA treated cell/Calculated difference between measurement and reference OD of DMA untreated cell) X 100. Microarray Hybridization and Analysis Total RNA was extracted from the cell lines using Tri Reagent (Ambion), digested by DNase (RNase free, MBI Fermentas) and purified using Qiagen™ RNeasyminikit. RNA concentration and purity was determined using NanoDrop spectrophotometer (Thermo Fischer, MA, USA). RNA quality was evaluated by Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Palo Alto, CA), and RNA integrity number (RIN) was found9.0. The 5.0 µg of total RNA was used for amplification using the Express Art® mRNA amplification kit. Amplified aRNA (aminoallyl antisense RNA) was then labelled with Cy3™ post-labelling reactive dye pack (GE Healthcare, United Kingdom) at RT. 20 µg of the labelled aRNA in 200 µl of Ocimum’sHyb buffer was denatured at 95°C for 3 min and 100 µl was used for hybridization performed at 50°C for 2 h on Tecan HS 4800 automated Hyb station. Human 40 KA and 40 KB OciChip™ (OcimumBiosolutions, Hyderabad) consisting of 20160 and 19968 spots respectively were printed on Corning® epoxy coated glass slides using Omnigrid (Gene Machines). Oligos were printed using spotting buffer A at 50% humidity and 22°C. The RNA samples were then hybridized and processed according to OciChip™ 40K Array manual specifications. For each treatment condition, three independent experiments were performed. Hybridized Chips were scanned using Affymetrix 428™ array scanner at three different PMT gains setting- 40, 50, 60. Data were collected and stored as Tiff images and further analyzed using Imagene software. The mean background intensity was subtracted from the mean target intensity to produce the signal intensities. Initial dataset that consisted of 40,320 probes was filtered to exclude quality control (QC) probes and empty spots. The data was transformed using log-transformation and median absolute deviation was performed. After normalization, genes in the treatment groups with at least two-fold change in expression were considered as up-regulated or downregulated in comparison to non treatedcells (control). To determine significant proportions of differentially expressed genes within functional groups, the hypergeometric probability P was calculated. P<0.005 was considered significant. The gene expression values generated were analyzed using online available tool- PANTHER (www.pantherdb.org).All microarray data are MIAME compliant and the raw data has been deposited in Gene Expression Omnibus (GEO) with accession number GSE30043. The individual accession number of genes is mentioned in the table 2.The links for access to microarray data submitted to the GEO are as mentioned below: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30043(U87 cell line). Protein Extraction, 2D PAGE and Image Analysis Protein samples were prepared from cells harvested from DMA and radiation treated cells as described elsewhere [57]. Cells were lysed in lysis buffer containing 8 M urea, 0.5% (w/v) CHAPS, 50 mMTris, 0.2% (w/v) Bio-Lyte 4/7, 50 mM DTT, 1 mM PMSF, 1 mM EDTA, 10% (v/v) Glycerol and 1–2 µg/µl protease inhibitor cocktail (Bio-Rad, Hercules, CA), vortexed at RT for 10 min, incubated on ice for 15 min and centrifuged in a microcentrifuge at 12,000 rpm for 10 min. The protein concentration was determined using the Bradford method with bovine serum albumin as a standard. The supernatant was combined with 200 µl of rehydration Buffer (Bio-Rad) containing 8 M urea, 2% CHAPS, 50 mM DTT, and 0.2% (w/v) Bio-Lyte 4/7 ampholytes, before isoelectric focusing. First dimensional separation of proteins (200 µg of protein in 200 µl) was performed on Ready Strip IPG strips (pH 4/7, 11 cm; Bio-Rad) for a total of 30,000 V-hours. The second dimensional separation was performed on a 12% acrylamide gel following DTT reduction and IAA alkylation. The gels were subsequently stained with Coomassie Brilliant Blue R250 and scanned using a Gel Documentation System (Alpha Imager) at 300 dpi and saved as a gray scale tiff file. Images were analyzed using Bio-Rad’s PD Quest software. The protein pattern differences between the control, radiation treated and DMA treated samples were elucidated by analyzing images from each sample using Bio-Rad’s PD Quest software. For each treatment sample, three independent experiments were performed. The software assigns a standard spot parameter number based on the quality and type of spots. Using the Gaussian Model during Test, quality and quantity values were assigned to spots based on total quantity in valid spots following normalization. In-Gel Trypsin Digestion and ESI-MS/MS Differentially regulated protein spots were excised from gels and destained using 50 mM Ammonium Bicarbonate and 50% Acetonitrile. Spots were subsequently dried using 100% Acetonitrile, speed dried under vacuum and digested with 20 ng/µl trypsin (Promega Trypsin Gold, Mass Grade) in 25 mM Ammonium Bicarbonate overnight at 37°C. Peptides were extracted from gel pieces using 60% Acetonitrile, 0.5% Formic Acid. Samples were purified using a C18 microbed column (Millipore ZipTip). Samples were then analyzed by ElectroSprayIonization tandem mass spectrometry (ESI-MS/MS; Applied Biosystems Q-Star XL) at a voltage of 1200 V. Sample spectra were acquired using TOF MS and tandem mass spectral data as well as using an IDA (information dependent acquisition) feature in the Analyst QS software. The IDA experiments selected multiply charged precursors (charged state from 2 to 5) for fragmentation with an intensity threshold of 10 counts per second. Precursor ions were excluded for 60 seconds using a window of 6 atomic mass units and collision energy settings were automatically determined by IDA based on m/z values. Database Queries and Protein Identifications The acquired tandem mass spectral data were queried against protein database using the MASCOT search engine (version 1.8, Matrix Science Ltd., U.K.) with a mass tolerance of 100 ppm and one trypsin miscleavage. Carbamidomethylation of cysteine was set as variable amino acid modifications. For a given sequence, the EST analyzer searched NCBInr protein database to identify a homologous protein (the best hit of BLASTX search), then used the homologous protein to annotate the query sequence. Proteins with MOWSE scores equal to the accepted significant threshold (determined at 95% confidence level as calculated by MASCOT) were reported in this study. Pathway Construction Online resources (NCBI, Gene Cards, OMIM and KEGG) were used to construct a pathway for proteins identified from 2D PAGE and their interacting partners. This pathway was utilized to identify the candidate genes. Real Time RT- PCR The transcript levels of genes identified from 2D PAGE were analyzed for their expression by Real-Time Polymerase Chain Reaction (RT-PCR) using gene specific primers obtained from Sigma Aldrich (Table 3). RNA was isolated using TriReagent (Ambion), digested with DNase I (MBI Fermentas) and purified using Qiagen™ RNeasy mini kit. RNA samples were quantified using NanoDrop Spectrophotometer. The RIN was found to be 8.0.1 µg of RNA sample was converted into cDNA, and transcript levels were quantitated using Sybr Green I (Power Sybr Green I, Applied Biosystems) and Roche’s light cycler 480 instrument (LC480) according to manufacturer’s guidelines. β Actin gene was used as a internal control. Each experiment for samples was performed three times independently. The calculations and data analysis were done using the efficiency calibrated mathematical model for the relative expression ratio in real time PCR [58]. 10.1371/journal.pone.0039426.t003Table 3 Sequences of primers used for quantitation of gene expression. S. No. Gene Primer sequences (FP- forward primer; RP-reverse primer) Tm (°C) 1. MAP3K3 FP-GAACTCCCCCTCACTGTTGARP-GAAGCAGGCACCTCTCTGTC 60.060.1 2. MAP3K7 FP-CAAACAACTCCAAAGGCTCCRP-CTCCTCCTCCTCCTCGTCTT 59.759.9 3. MAP3K14 FP-CCCTTCTCTCACAGCTCCATRP-ATGGAGGACAAGCAGACTGG 59.460.2 4. MAPK10 FP-TTCACATCCAATGTTGGTTCARP-CAGGCCCATCTCAGATCTTC 59.859.7 5. YWHAZ FP-GAAGCATTGGGGATCAAGAARP-ACAAAAGACGGAAGGTGCTG 60.060.2 6. NFκB FP-CCACCTTTACGAGGACCTATTCCRP- CAGCACTCGCTCTCCATGAA 60.160.3 Expression Vectors The construct for NIK was obtained by cloning the requisite sequence into p3XFLAG CMV10 generously provided by Dr. P. C. Rath, School of Life Sciences, Jawaharlal Nehru University, Delhi. The construct of the NFκB-driven luciferase reporter gene and corresponding control construct for Renilla luciferase were kindly provided by Dr. Balram Ghosh, Institute of Genomics and Integrative Biology, Delhi. Transfection with Expression Vectors The day before transfection, 6 well plates were seeded with 2×105 cells/well in serum containing antibiotic free medium. The cells were incubated at 37°C in a 5% CO2 incubator overnight. For each well, plasmid was prepared in required volume of serum free medium. Transfectin™ Lipid Reagent (Bio-Rad, Hercules, CA) was prepared in serum-free medium. The DNA and Transfectin solutions were mixed together and incubated for 20 min at room temperature. DNA-Transfectin complexes were added directly to cells in serum-containing medium and swirled gently. Cells were incubated at 37°C in a 5% CO2 incubator. Additional media was added 4–6 h after addition of complexes. After 48 h, cells were harvested and assayed for protein expression. Cloning and Expression of NFκB Inducing Kinase RNA was extracted from HEK cell line and cDNA was synthesized. The cDNA template of NFκB inducing kinase was amplified by PCR with sense primer NIK FP (5′-TCTAGAATGGCAGTGATGGAAATG-3′) and antisense primer NIK RP (5′-GGATCCTTAGGGCCTGTTCTCC-3′) using Phusion DNA polymerase (Finnzymes, Finland) and cloned into p3XFLAG CMV10. Subsequently, p3XFLAG CMV10 NIK plasmid was purified using EndoFree Plasmid Maxi Kit (QIAGEN™) according to manufacturer’s guidelines and dissolved in TE buffer (pH 7 to 8), before using for transfection. The transient transfection of the constructed plasmid was done by Transfectin Lipid Reagent (Bio-rad) according to the manufacturer’s guidelines. Knockdown of NIK using siRNA In a 6 well tissue culture plate, human glioma (U87) cells were plated (2×105 cells/well) in antibiotic free media supplemented with FBS. After 24 h, cells were transfected with siRNA-NIK (sc-36065, Santa Cruz Biotechnology Inc.) and control-siRNA (sc-37007, Santa Cruz Biotechnology Inc.) according to the manufacturer’s instructions. Knockdown of NIK was observed at 50 pM concentration of siRNA-NIK (Figure S1). Immunoblot Analysis Human glioma (U87) cells were transfected with p3XFLAG CMV10- NIK, siRNA-NIK and control-siRNA using Transfectin (Lipid Reagent) according to manufacturer’s guidelines. For the experiment following transfections, cells were treated with 50 µM DMA for 1 h. Subsequently, cells were induced with TNF-α (10 ng/ml) for 0,5,10,20,40,60 min and irradiated at 8.5 Gy.Trypsin was used to detach the cells and the cells were collected, centrifuged at 2000 rpm for 2 min and resuspended in RIPA buffer (50mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% sodium deoxycholate, 10% sodium orthovanadate and cocktail of protease inhibitors) on ice for 30 min. Following centrifugation at 25,000 g for 30 min, the supernatant was collected as total cell lysate and protein concentration was determined using Bradford’s Assay. Samples were prepared in SDS gel loading buffer (625 mM Tris pH 6.8, 10% SDS, 25% glycerol, 100 mM DTT and 0.015% bromophenol blue). Western blotting was performed using standard protocols with antibodies against human IKKα, IKKβ, IκBα, phospho-IKKα/IKKβ (180/181) (Santa Cruz Biotechnology Inc.) and horseradish peroxidase-conjugated mouse anti-human immunoglobulin (secondary antibody, Abcam). Detection was done by an enhanced chemiluminescence (ECL) system (Pierce). Proliferation Kinetics U87 Cells post siRNA oligonucleotide transfections were treated with 50 µM DMA and 8.5 Gyradiation. Post treatment cells were seeded in 6 well plates at 8000 cells per well, and their proliferation kinetics was studied at 24 h intervals following trypsinization and counting total cells per well using a hemocytometer. Same experiment was done with control siRNA-transfected U87 cells. Percentage (%) radioprotection of cells was calculated by following formula. % growth of Radiation treated cells  =  (Number of cells in Radiation treated cells/Number of cells in only DMA treated cells) X 100. % growth of DMA+Radiation treated cells  =  (Number of cells in DMA+ Radiation treated cells/Number of cells in only DMA treated cells) X 100. % Radioprotection  =  % growth of DMA+Radiation treated cells - % growth of Radiation treated cells. Cell-cycle Analysis U87 cells (2×105 cells/plate) in 60 mm plates, transfected with control siRNA and siRNA-NIK, were cultured and DMA, Radiation and DMA + Radiation treatment were given as described earlier. After irradiation, cells were collected after the incubation period of 0,3,6,12,18 and 24 h. After incubation cells were collected, washed twice with ice-cold PBS, and fixed in 70% ethanol. Cell pellets were stored at 4oC for 24 h. Cells pellets were washed twice with ice-cold PBS and stained with 0.5 mL of RNase (2 mg/mL) and 0.5 mL of propidium iodide (0.1% in 0.6% Triton X-100 in PBS) for 30 minutes in dark. Samples were then analysed on a FACSCalibur flow-cytometer (Becton Dickinson). Annexin-V Staining U87 cells (2×105 cells/plate) in 60 mm plates, transfected with control siRNA and siRNA-NIK, were cultured and DMA, Radiation and DMA +Radiation treatment were given as described earlier. After irradiation, cells were incubated for 3, 6, 12, 18 and 24 h (data not shown for 12 and 18h). Cells were collected by mild trypsinization and were pooled together with detached cells of respective group. Samples were prepared according to the manufacturer’s instructions (BD Pharmingen™ Annexin V: FITC Apoptosis Detection Kit I, Catalog Number 556547, USA) and the samples were subjected to flow cytometry analysis (Becton Dickinson). Luciferase Reporter Assay for NFκB Activity The effect of DMA on TNF-α induced NFκB dependent reporter gene transcription was measured. Briefly, human glioma (U87) cells (2×104 cells/well) were plated in 96 well plates. Next day cells were following serum starvation, cells were co-transfected with pNFκB-Luc (0.2 µg/well )and pRenilla (0.02 µg/well) using Transfectin™ Lipid Reagent (Bio-Rad, Hercules, CA) and β-galactosidase reporter vector (used as an internal control) according to the manufacturer’s instructions. pNFκB-Luc contains the firefly luciferase (luc) gene from Photinuspyralis. This vector also contains multiple copies of the NFκB consensus sequence fused to a TATA-like promoter (P TAL) region from the Herpes Simplex Virus thymidine kinase (HSV-TK) promoter. After endogenous NFκB proteins bind to the kappa (κ) enhancer element (GGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCCGGGAATTTCC), transcription is induced and the reporter gene is activated. Twenty four hours after transfection, U87 cells were treated with DMA (0,10,25,50 µM) for 1 h prior to irradiation at 8.5 Gy. Following irradiation, induction with TNF-α (20 ng/ml) was done for 4 h to induce NFκB driven luciferase expression. Subsequently, cells were assayed for reporter activity using Dual™ Luciferase Reporter Assay System (Promega) according to the manufacturer’s guidelines.The relative luciferase activity was calculated by normalizing results with the β –galactosidase expression. Statistical Analysis Data are expressed as means ±SD. Statistical significance among groups was determined using the Student t test and the differences were considered significant at P<0.05. Supporting Information Figure S1 Knock down of NFκB inducing kinase gene expression using siRNA against NFκB inducing kinase. (A) Lanes 1-5 - RT-PCR with siRNA treated cells with NFkB inducing kinase primers (control, 20 pM siRNA-NIK,50 pM siRNA-NIK,80 pM siRNA-NIK, NTC); Lanes 6-10- RT-PCR with siRNA treated cells with ACTB primers (control, 20 pM siRNA-NIK,50 pM siRNA-NIK,80 pM siRNA-NIK, NTC); M (DNA ladder). (B) Lanes 1-5 - RT-PCR with control siRNA treated cells with NFkB inducing kinase primers (control, 20 pM siRNA-NIK,50 pM siRNA-NIK,80 pM siRNA-NIK, NTC) Lanes 6-10- RT-PCR with siRNA treated cells with ACTB primers (control, 20 pM siRNA-NIK,50 pM siRNA-NIK,80 pM siRNA-NIK, NTC); M (DNA ladder). (TIF) Click here for additional data file. Table S1 Differentially regulated proteins identified by 2D PAGE and ESI-MS/MS analysis. (DOCX) Click here for additional data file. Table S2 MS/MS analysis of reported proteins. (DOCX) Click here for additional data file. The authors thank Dr. B.S. Dwarakanath, Scientist G for providing the Radiation facility at Institute of Nuclear Medicine & Allied Sciences, DRDO, Delhi, India. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported in part by the Department of Science & Technology, Delhi, India, and the Department of Biotechnology Delhi, India. PhD Fellowship was from Council of Scientific & Industrial Research, Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Jackson SP 2002 Sensing and repairing DNA double-strand breaks. Carcinogenesis23 687 696 2 Iliakis G Wang Y Guan J Wang H 2003 DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 22 5834 5847 12947390 3 Amundson SA Bittner M Fornace AJ Jr 2003 Functional genomics as a window on radiation stress signaling. 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PLoS One. 2012 Jun 22; 7(6):e39426
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22761868PONE-D-12-1210810.1371/journal.pone.0039684Research ArticleBiologyMolecular cell biologySignal transductionSignaling cascadesTGF-beta signaling cascadeSignaling in Selected DisciplinesOncogenic SignalingMembrane Receptor SignalingMedicineGastroenterology and HepatologyOncologyCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerBasic Cancer ResearchCancer TreatmentConcomitant Targeting of EGF Receptor, TGF-beta and Src Points to a Novel Therapeutic Approach in Pancreatic Cancer EGFR and TGF-beta Targeting in Pancreatic CancerDeharvengt Sophie 1 Marmarelis Melina 1 ¤ Korc Murray 2 * 1 Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire, United States of America 2 Departments of Medicine, and Biochemistry and Molecular Biology, Indiana University School of Medicine, the Melvin and Bren Simon Cancer Center, and the Pancreatic Cancer Signature Center, Indianapolis, Indiana, United States of America Munshi Hidayatullah G. EditorNorthwestern University, United States of America* E-mail: [email protected] and designed the experiments: MK. Performed the experiments: SD MM. Analyzed the data: SD MM. Contributed reagents/materials/analysis tools: SD MM MK. Wrote the paper: SD MK. ¤ Current address: Medical Student, Harvard Medical School, Boston, Massachusetts, United States of America 2012 27 6 2012 7 6 e3968424 4 2012 29 5 2012 Deharvengt et al.2012This 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.To test the hypothesis that concomitant targeting of the epidermal growth factor receptor (EGFR) and transforming growth factor-beta (TGF-β) may offer a novel therapeutic approach in pancreatic cancer, EGFR silencing by RNA interference (shEGFR) was combined with TGF-β sequestration by soluble TGF-β receptor II (sTβRII). Effects on colony formation in 3-dimensional culture, tumor formation in nude mice, and downstream signaling were monitored. In both ASPC-1 and T3M4 cells, either shEGFR or sTβRII significantly inhibited colony formation. However, in ASPC-1 cells, combining shEGFR with sTβRII reduced colony formation more efficiently than either approach alone, whereas in T3M4 cells, shEGFR-mediated inhibition of colony formation was reversed by sTβRII. Similarly, in vivo growth of ASPC-1-derived tumors was attenuated by either shEGFR or sTβRII, and was markedly suppressed by both vectors. By contrast, T3M4-derived tumors either failed to form or were very small when EGFR alone was silenced, and these effects were reversed by sTβRII due to increased cancer cell proliferation. The combination of shEGFR and sTβRII decreased phospho-HER2, phospho-HER3, phoshpo-ERK and phospho-src (Tyr416) levels in ASPC-1 cells but increased their levels in T3M4 cells. Moreover, inhibition of both EGFR and HER2 by lapatinib or of src by SSKI-606, PP2, or dasatinib, blocked the sTβRII-mediated antagonism of colony formation in T3M4 cells. Together, these observations suggest that concomitantly targeting EGFR, TGF-β, and src may constitute a novel therapeutic approach in PDAC that prevents deleterious cross-talk between EGFR family members and TGF-β-dependent pathways. ==== Body Introduction Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States, with a 5-year survival rate of 6% [1]. These dismal statistics are due, in part, to the advanced stage of the cancer at presentation, a low rate of resectability, multiple molecular alterations that promote pancreatic cancer cell growth and survival, marked chemoresistance, and intense desmoplasia which attenuates drug penetration [2]–[5]. PDAC is associated with a high frequency of mutations in the K-ras oncogene (95%), and the p16 (85%), p53 (75%) and SMAD4 (55%) tumor suppressor genes [4]. Moreover, when p16 gene is not mutated, it is epigenetically silenced [6]. There is also elevated expression of the epidermal growth factor (EGF) receptor (EGFR), other tyrosine kinase receptors and their ligands, and transforming growth factor beta (TGF-β) isoforms [7]. EGFR mediates cell-autonomous mitogenic and motogenic signaling cascades by activating diverse pathways, including mitogen activated protein kinase (MAPK), p38 MAPK, and jun kinase (JNK), whereas TGF-β activates Smad-dependent and -independent signaling and is believed to exert paracrine effects on cells within the tumor mircroenvironment in PDAC [8]–[10]. Excessive EGFR activation and dysfunctional signaling by TGF-β receptor (TβR)-dependent pathways, as observed in PDAC, generates multiple aberrant autocrine and paracrine interactions between the cancer cells and the tumor microenvironment that contribute to tumor desmoplasia and that may intersect with one or another of the dozen signaling cascades that are implicated in the majority of PDACs [5], [11]. Disappointingly, targeting EGFR only slightly prolongs the survival of patients with PDAC, and only when given in conjunction with gemcitabine [12], whereas anti-TGF-β therapies for PDAC are currently being developed and tested in pre-clinical studies [13]–[15]. We recently established a 3-dimensional culture system in which cells are embedded in Matrigel consisting of 3% collagen IV/laminin-enriched gelatinous medium and placed over a solidified layer of soft agar [16]. We determined that concomitant treatment with TGF-β1 and EGF enhanced growth in this 3-D model system to a greater extent than either growth factor alone, and conferred increased chemoresistance to cytotoxic compounds [16]. Moreover, pharmacological inhibition of TβRI with SB431542 or EGFR with erlotinib enhanced the efficacy of gemcitabine and cisplatin in human pancreatic cancer cells and in primary cell cultures established from pancreata of genetically-engineered mouse models of PDAC [16], underscoring the usefulness of this 3-D culture system for testing the efficacy of therapeutic agents. In view of the importance of EGFR and TGF-β in PDAC, we sought to test the hypothesis that targeting both pathways may exert beneficial growth-suppressive effects that are greater than suppressing either pathway alone. Because small molecule inhibitors that target EGFR and TβRI may exert non-specific effects and/or may target closely related kinases, we used a more specific approach consisting of a silencing strategy to suppress EGFR expression and a soluble TβRII strategy to sequester TGF-β ligands. We now report that simultaneous suppression of both pathways attenuated colony formation of ASPC-1 human pancreatic cancer cells grown in 3-D culture and tumor growth in vivo, but targeting TGF-β reversed the growth-inhibitory effects exerted by EGFR silencing in T3M4 human pancreatic cancer cells, and this reversal occurred in conjunction with src activation as reflected by increased src phosphorylation on tyrosine 419. Results Effects of EGFR Knockdown and sTβRII Expression on Colony Formation Human pancreatic cancer cell lines express transforming growth factor alpha (TGF-α) and other growth factors that activate EGFR [17]–[19], as well as all three TGF-βs [20]. To determine whether abrogating EGFR and TGF-β signaling modulated the growth of such cell lines, ASPC-1 and T3M4 cells were co-infected at an m.o.i. of 10 for each virus with shRNA-lentivirus targeting Luciferase (shLuc-LV with pWPT-GFP), EGFR (shEGFR-LV with pWPT-GFP), sTβRII (hLuc-LV with pWPT-sTβRII), or both EGFR and sTβRII (shEGFR-LV with pWPT-sTβRII). shEGFR-LV efficiently suppressed EGFR levels, whereas pWPT-sTβRII expression was associated with the presence of abundant levels of sTβRII protein in the medium in all four cell lines (Fig. 1A). 10.1371/journal.pone.0039684.g001Figure 1 EGFR knockdown and sTβRII expression modulate colony formation in pancreatic cancer cells. (A) ASPC-1 and T3M4 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates and conditioned media were then subjected to immunoblotting with anti-EGFR and anti-HA-tag antibodies, respectively, the latter serving to confirm sTβRII release by the cancer cells. An anti-ERK antibody served to assess lane loading. (B) The consequences of EGFR silencing with shEGFR and TGF-β sequestration with sTβRII were assessed by monitoring colony formation in 3-D culture (B). Data are the means ± SE of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, when compared with respective controls. The consequences of EGFR silencing and TGF-β sequestration were assessed next by monitoring colony formation in a 3-D culture assay in which Matrigel provides an acellular scaffold and soft agar supports anchorage-independent growth [16]. We chose to use this 3-D model system since we have previously shown that concomitant treatment with TGF-β1 and EGF in this model enhanced growth to a greater extent than either growth factor alone [16], thereby recapitulating TGF-β‘s tumor promoting effects previously demonstrated in xenograft and orthotopic mouse models of PDAC [13]–[14]. Colony formation with ASPC-1 cells infected with pWPT-sTβRII or shEGFR-LV was decreased by 21% (p<0.05) and 33% (p<0.01), respectively, whereas infection with both shEGFR-LV and pWPT-sTβRII resulted in a 56% (p<0.01) decrease in colony number by comparison with shLuc-expressing ASPC-1 cells (Fig. 1B). By contrast, after infection with shEGFR-LV, colony formation by T3M4 cells was decreased by 45% (p<0.05), whereas pWPT-sTβRII attenuated colony formation in T3M4 cells by 27% (p<0.05). However, pWPT-sTβRII completely reversed the inhibitory actions of shEGFR-LV on colony formation (Fig. 1B). Thus, ASPC-1 cells exhibited synergistic inhibitory effects on colony formation when infected with both shEGFR-LV and pWPT-sTβRII, whereas in T3M4 cells there was paradoxical reversal by pWPT-sTβRII of the inhibitory actions of shEGFR-LV. To determine whether other pancreatic cancer cell lined that behaves like T3M4 cells, we next performed the colony forming assay detailed in COLO-357 and PANC-1 pancreatic cancer cells (Fig. S1). COLO-357 cells were only growth inhibited in response to concomitant EGFR knockdown and sTβRII expression. By contrast PANC-1 cells were growth inhibited by EGFR knockdown, but exhibited a reversal of this growth inhibitory effect in the presence of sTβRII (Fig. S1). In Vivo Effects of EGFR Knockdown and sTβRII Expression We next examined the consequences of EGFR silencing and sTβRII expression in a subcutaneous nude mouse tumor model, to determine whether the paradoxical reversal of EGFR silencing observed in the 3-D in vitro model also occurred in vivo. Compared with tumors generated by ASPC-1 cells infected with shLuc-LV, tumor volumes on day 24 were decreased by 36% (p<0.05) with shEGFR-LV, 38% (p<0.05) with pWPT-sTβRII, and 85% (p<0.01) with both vectors (Fig. 2A). Moreover, 2 of 8 mice injected with pWPT-sTβRII-expressing ASPC-1 cells were tumor-free. Dramatically, 4 of 8 mice injected with ASPC-1 cells expressing both pWPT-sTβRII and shEGFR-LV were tumor-free on day 24, and the remaining 4 tumors only became visible 21 days following injection of the cancer cells. In the case of T3M4-derived tumors, experiments were terminated on day 16 due to rapid tumor growth in two of the four groups. At this time point, tumor volume was decreased by 37% (p<0.05) for cells infected with pWPT-sTβRII and by 97% (p<0.01) for shEGFR-LV-infected cells (Fig. 2B). By contrast, T3M4 cells infected with both pWPT-sTβRII and shEGFR-LV formed large tumors, each of which exhibited areas of necrosis (Fig. 2B). 10.1371/journal.pone.0039684.g002Figure 2 Targeting EGFR and TGF-β pathways exerts different effects on the formation and growth of tumors formed by ASPC-1 and T3M4 cells. ASPC-1 (A) and T3M4 (B) cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), sTβRII, or both EGFR-LV and sTβRII, and injected subcutaneously (one injection per mouse) into the flank region of nude mice. Tumor volumes were monitored for the indicated number of days. Values are the means ± SEM of 8 mice per group, indicated in the denominator to the right of each curve. The number of tumors that formed in each group is indicated in the numerator. *p<0.05, and **p<0.01, when compared with respective controls. Tumors arising from either ASPC-1 or T3M4 cells exhibited abundant Ki-67 immunoreactivity and foci of CD-31-positive endothelial cells (Fig. 3A). In ASPC-1-derived tumors, expression of pWPT-sTβRII did not alter proliferation, whereas expression of shEGFR-LV was associated with a 60% (p<0.05) decrease in both Ki-67 and CD31 immunoreactivity, and expression of both vectors caused a further decrease in Ki-67 (72%, p<0.01) and CD31 (76%, p<0.01) immunoreactivity (Fig. 3A). In T3M4 cells, expression of pWPT-sTβRII was associated with decreased proliferation (40%, p<0.01) and angiogenesis (77%, p<0.01), expression of shEGFR-LV did not significantly alter proliferation but markedly decreased CD31 immunoreactivity (71%, p<0.01), whereas expression of both vectors markedly increased cancer cell proliferation (196%, p<0.01) in spite of a persistent decrease (85%, p<0.01) in CD31 immunoreactivity (Fig. 3A). 10.1371/journal.pone.0039684.g003Figure 3 Effects of targeting EGFR and TGF-β pathways on proliferation, angiogenesis and apoptosis. A. The ASPC-1- and T3M4-derived tumors described in figure 2 were immunostained for Ki67 to assess proliferation and CD31 to assess angiogenesis. B. The same tumors were scored for TUNEL-poisitive cells and cleaved PARP immunoreactivity to assess apoptosis. Data are the means ± SEM of triplicate determinations from three independent experiments. *p<0.05, and **p<0.01, when compared with respective controls. In view of the presence of regions of necrosis in T3M4 tumors expressing both pWPT-sTβRII and shEGFR-LV, it was important to avoid spurious results that may occur in areas about to undergo necrosis. Therefore, both the TUNEL assay and cleaved PARP immunostaining were performed next to assess apoptosis, both methods yielding generally concordant results (Fig. 3B). Thus, pWPT-sTβRII did not significantly alter the percentage of cells undergoing apoptosis in either ASPC-1 or T3M4-drived tumors, whereas shEGFR-LV expression was associated with a marked increase in apoptosis in ASPC-1 cells (p<0.01), but not in T3M4 cells. Moreover, in ASPC-1-derived tumors, pWPT-sTβRII did not alter shEGFR-LV-associated apoptosis, but in T3M4-derived tumors it was associated with enhanced apoptosis (Fig. 3B). Effects of EGFR Knockdown and sTβRII Expression on Phosphorylation State of EGFR Family Members EGFR, HER2 and HER3 have all been implicated in the pathobiology of PDAC [7], [21]–[23]. Since EGFR forms heterodimers with HER2 and HER3, it was important to determine whether its silencing could modulate signaling by these EGFR family members. Therefore, ASPC-1 and T3M4 cell lysates were subjected to immunoblotting to assess the levels of phospho-HER2, and phospho-HER3 (Fig. 4A). Densitometric analysis of data from three experiments showed that pWPT-sTβRII expression in ASPC-1 cells induced a 17% and 20% decrease in phospho-HER2 and phospho-HER3 levels, respectively (p<0.05), whereas EGFR knockdown induced a 61% decrease in phospho-HER2 levels (p<0.01) and a 30% decrease in phospho-HER3 (p<0.01) levels. ASPC-1 cells expressing both shEGFR-LV and pWPT-sTβRII exhibited a similar decrease in phospho-HER2 levels (52%, p<0.01), but a more pronounced decrease in phospho-HER3 levels (56%, p<0.01). By contrast, in T3M4 cells, pWPT-sTβRII did not alter phospho-HER2 or phospho-HER3 levels, whereas EGFR knockdown was associated with increased levels of both phospho-receptors (Fig. 4A). In three experiments, there was a 60% increase in phospho-HER2 and phospho-HER3 levels in T3M4 cells following EGFR knockdown, and 100% and 80% increases in phospho-HER2 and phospho-HER3 levels, respectively, in cells expressing both vectors. 10.1371/journal.pone.0039684.g004Figure 4 Effects of EGFR knockdown and sTβRII expression on receptor phosphorylation and downstream signaling. (A) Effects on receptor phosphorylation. ASPC-1 and T3M4 cells were infected as indicated with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII. Cell lysates were subjected to immunoblotting with antibodies directed against the indicated receptors and phospho-receptors. (B) Cells were infected as indicated in A, and cell lysates were subjected to immunoblotting with antibodies directed against the indicated proteins and phospho-proteins. Each panel shows data from a representative of at least two independent experiments. In both panels A and B, immnoblotting with an anti-ERK antibody confirmed equivalent lane loading, but not all ERK blots are shown. To determine whether HER2 and HER3 phosphorylation was also modulated in vivo in T3M4 cells, tumors derived from these cells were evaluated by immunohistochemsitry (Fig. S2). Moderate phospho-HER2 immunoreactivity was evident in tumors from cells infected with shLuc, and shEGFR-LV, which was decreased in tumors infected with pWPT-sTβRII, but increased in tumors expressing both vectors. By contrast, phospho-HER3 immunoreactivity was low in tumors from shLuc-infected T3M4 cells, slightly increased in pWPT-sTβRII-expressing tumors, moderately increased in shEGFR-LV-expressing tumors, and markedly increased in tumors expressing both vectors (Fig. S2). Thus, both HER2 and HER3 are aberrantly activated in vivo in T3M4 cells when both EGFR and TGF-β pathways have been targeted. Effects of EGFR Knockdown and sTβRII Expression on Downstream Signaling ERK, src, and AKT are mitogenic and pro-survival signaling modules that are downstream of EGFR family members and that contribute to PDAC progression [12], [24]. Therefore, ASPC-1 and T3M4 cell lysates were subjected to immunoblotting to assess the effects of EGFR knockdown and sTβRII expression on these pathways (Fig. 4B). In ASPC-1 cells, shEGFR-LV, pWPT-sTβRII, and their combination was associated with attenuated phospho-ERK levels, but only the combination decreased phospho-AKT levels whereas none of these transfection conditions induced the de-phosphorylation of Src(Tyr527), which would be reflective of src activation (Fig. 4B). By contrast, in T3M4 cells, shEGFR-LV alone or in combination with pWPT-sTβRII resulted in increased phospho-ERK and decreased phospho-src(Tyr527) levels, without any alterations in phospho-AKT levels (Fig. 4B). To confirm that the combination of shEGFR-LV and pWPT-sTβRII activated src in T3M4 cells, lysates were also subjected to a phospho-kinase antibody array. EGFR silencing led to inhibition of the phosphorylation of src(Tyr419), Fyn, Hck, Lyn, Yes and Fgr, which was especially pronounced with respect to src (Fig. 5). By contrast, expression of sTβRII inhibited the phosphorylation of Lyn Yes, and Fgr, without altering src, Fyn or Hck phosphorylation (Fig. 5). However, the inhibitory effects of shEGFR-LV on all 6 kinases were completely reversed by sTβRII (Fig. 5), indicating that expression of sTβRII reactivated src family kinases. 10.1371/journal.pone.0039684.g005Figure 5 Effects of targeting EGFR and TGF-β pathways on phosphorylation status of src family members. T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), and/or WPT-sTβRII (sTβRII) as indicated. Cell lysates were then analyzed with a phospho-kinase antibody array to assess the phosphorylation status of the indicated src family members. Results were quantified as described in Methods. Data are the means ± SEM of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 when compared with control. Effects of HER2 Silencing and src Inhibition on Colony Formation in T3M4 Cells We next sought to assess the role of HER2 in mediating the deleterious effects of simultaneous targeting EGFR and TGF-β. As expected, shEGFR-LV markedly suppressed EGFR levels in T3M4 cells, shHER2-LV markedly suppressed HER2 levels, whereas infection with both vectors silenced the expression of both EGFR and HER2 (Fig. S3). Moreover, T3M4 cells expressing either shEGFR-LV or shHER2-LV exhibited a significant decrease in colony numbers in the 3-D assay (Fig. 6A). In the case of shEGFR-LV, but not shHER2-LV or shEGFR-LV together with shHER2-LV, this effect was reversed by pWPT-sTβRII (Fig. 6A). Thus, concomitant infection with shEGFR-LV and shHER2-LV markedly inhibited colony growth (66%, p<0.01). Similarly, lapatinib (1 µM), a dual tyrosine kinase inhibitor that interrupts HER2 and EGFR signaling pathways, reduced colony number by 47% (p<0.01) and prevented the reversal observed following co-infection with shEGFR-LV and pWPT-sTβRII (Fig. 6). 10.1371/journal.pone.0039684.g006Figure 6 Effects of HER2 silencing, lapatinib, and src inhibition on sTβRII-mediated reversal of growth suppression. A. HER2 silencing and inhibition. T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), shHER2-LV (shHER2), or both shEGFR and shHER2, in the absence or presence of WPT-sTβRII (sTβRII) or 1 µM lapatinip. Colony formation was monitored in 3-D culture. Data are the means ± SEM of triplicate determinations from three independent experiments. *p<0.05, and **p<0.01, when compared with control. B. Effects of c-Src inhibition. T3M4 cells were incubated in the absence or presence of the src kinase inhibitors SKI-606 (1 µM), PP2 (1 µM) and dasatinib (100 nM), and effects on colony formation in 3-D culture were determined. Data are the means ± SE of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, when compared with respective controls. In view of the up-regulation of phospho-src (Tyr419) and the dephosphorylation of Src(Tyr527) by the combination of shEGFR-LV and pWPT-sTβRII in T3M4 cells, we sought to determine whether the deleterious effects of this combination might be mediated by activated src. Therefore, the effects of three distinct src inhibitors on colony formation in 3-D culture were examined next. Only dasatinib (100 nM) significantly inhibited the growth of T3M4 cells infected with shLuc-LV (Fig. 6B). However, SKI-606 (1 µM), PP2 (1 µM), and dasatinib (100 nM) completely blocked the pWPT-sTβRII-mediated reversal of shEGFR-LV-induced growth inhibition (Fig. 6B), indicating that this effect was dependent on src kinase activity. Discussion Members of the EGF family, including TGF-α, heparin-binding EGF-like growth factor (HB-EGF), betacellulin, and amphiregulin, are expressed at high levels in PDAC and act on the cancer cells in PDAC and on the adjoining stromal elements [7]. EGFR activation by these ligands initiates multiple signaling cascades, such as Ras/Raf/MAPK and Rac/JNK/MAPK-p38 [24]. EGFR heterodimerization with other members of the EGFR family leads to the activation of other signaling pathways that include Src, Raf1, B-Raf, Crk, and Nck, which further promote tumor progression and biological aggressiveness [25]. EGFR cross talk with multiple pathways is enhanced by the high frequency of Kras and Smad4 mutations, and by the abundance of TGF-β which alters the extracellular matrix in a manner that promotes cancer cell growth, induces aberrant epithelial-mesenchymal interactions, enhances angiogenesis, and promotes metastasis [4]–[7], [10], [13]–[14], [26]–[27]. Moreover, TGF-β synergizes with EGF in promoting proliferation in 3-D culture [16]. Together, these observations suggest that aberrant EGFR and TGF-β-dependent signaling pathways are pivotal in promoting pancreatic cancer progression and may represent crucial therapeutic targets in PDAC. In the present study we demonstrated that lentiviral-based silencing of EGFR efficiently attenuated its pro-mitogenic actions in 3 of 4 pancreatic cancer cell lines, and that lentiviral-based sequestration of TGF-β also attenuated proliferation in 3-D culture in the same three cell lines. However, in ASPC-1 and COLO-357 cells, concomitantly silencing EGFR and sequestering TGF-β resulted in enhanced growth suppression, whereas in T3M4 and PANC-1 cells there was nearly complete reversal of the growth-suppressive effects of EGFR down-regulation. Under standard tissue culture conditions, ASPC-1 and T3M4 cells are resistant to TGF-β-mediated growth inhibition, whereas COLO-357 and PANC-1 cells are growth-inhibited by TGF-β [19], [28]. Thus, the observed paradoxical reversal cannot be attributed to differences in the growth-inhibitory responsiveness of the cancer cells. Instead, in T3M4 cells, this reversal is due, in part, to the up-regulation of phospho-HER2 and phospho-HER3 elicited by EGFR downregulation and enhanced in the presence of sTβRII. In agreement with this conclusion, the growth-inhibitory effects induced by silencing HER2 or both EGFR and HER2 were not reversed by sTβRII. Similarly, lapatinib, which inhibits both EGFR and HER2 kinase activities, also inhibited the growth of T3M4 cells and this effect was resistant to sTβRII-mediated reversal. ERK can be activated by multiple upstream signals, and increased HER2/3 phosphorylation in T3M4 cells was associated in the present study with increased ERK phosphorylation, indicating that HER2/3 downstream signaling was also being activated. Several lines of evidence suggest that src activation mediated by TGF-β sequestration is also crucial for the reversal phenomenon. First, src inhibition by EGFR silencing was completely reversed by TGF-β sequestration. Second, EGFR Signaling is known to activate src [29], and src activation is known to induce the release of the precursors of EGF-like ligands [6] and attenuate EGF internalization [29], [30]. These mechanisms may promote EGFR heterodimerization with HER2 and HER3, thereby further enhancing mitogenic signaling. Third, sTβRII increased the levels of src phosphorylation on tyrosine residue 419 in T3M4, and phosphorylation at this site correlates with increased src activity. Moreover, sTβRII did not alter the phosphorylation of Src(Tyr527) in ASPC-1 cells, but decreased its phosphorylation in T3M4 cells in the absence and presence of shEGFR, confirming that src was being activated in T3M4 cells by sTβRII. Fourth, three src kinase inhibitors, SKI-606, PP2, and dasatinib, blocked the TβRII-mediated reversal of growth inhibition. We have previously determined that addition of purified sTβRII protein to the medium of these cells also sequesters TGF-β and blocks TGF-β actions in vitro (unpublished observations). TGF-β binds to type II TGF-β receptor (TβRII) homodimer, which then forms a heterotetrameric complex with the TβRI homodimer, leading to the activation of TβRI serine-threonine kinase activity [9]. This activation initiates a signaling cascade that includes the phosphorylation of receptor-regulated Smads (R-Smads), Smad2 and Smad3, at their C-terminal SSXS motif, their subsequent oligomerization with the common mediator Smad4, and translocation of the complex to the nucleus where regulation of gene transcription is then effected [9], [31]. TβRII can also be phosphorylated on tyrosine residue 284 leading to the activation of alternate pathways such as p38 MAPK [32]. While src activation often occurs downstream of tyrosine kinase receptors, TGF-β may also increase src activity, but in a transient manner [33]. However, TGF-β also acts to induce src degradation [34]. It is possible, therefore, that TGF-β sequestration in T3M4 cells may prevent cancer cell-derived TGF-β from inducing src degradation and/or inactivation. To assess the biological relevance of these in vitro findings, we used a subcutaneous nude mouse model which allows for reproducible assessment of the in vivo biological relevance of signaling pathways that are altered in vitro. Thus, with respect to ASPC-1 cells, either EGFR down-regulation or TGF-β sequestration resulted in significant (36 to 38%) decreases in tumor volume, with a further decrease to 85% when both approaches were combined. Impressively, tumors failed to form in 2 of 8 mice injected with ASPC-1 cells expressing pWPT-sTβRII, and in 4 of 8 mice expressing both pWPT-sTβRII and shEGFR-LV. Moreover, there was a marked delay in the appearance of the 4 tumors that arose from cells expressing both pWPT-sTβRII and shEGFR-LV, all of which exhibited greatly decreased proliferation and angiogenesis, and increased apoptosis. These findings support strategies for targeting TGF-β in PDAC [13], [14], [35], and are consistent with the observation that there is a strong EGFR in situ hybridization signal in the tumor vasculature in PDAC in humans [36] and with proposed roles of EGFR in tumor angiogenesis. While targeting TGF-β by ligand sequestration or by TβRI kinase inhibition attenuates pancreatic tumor growth and metastasis in mouse models [13]–[15], our findings indicate that, in certain instances, targeting both EGFR and TGF-β-dependent pathways can exert synergistic inhibitory effects on PDAC proliferation and angiogenesis. In T3M4-derived tumors, TGF-β sequestration resulted in a 37% decrease in tumor volume and decreased proliferation and angiogenesis, whereas EGFR down-regulation resulted either in the failure to form tumors or in the formation of exceedingly small tumors and markedly attenuated angiogenesis. Thus, T3M4 cells are highly dependent on EGFR for tumor initiation, progression and angiogenesis in vivo, and this exquisite dependence on EGFR is consistent with EGFR-mediated mitogenesis as well as with its role in angiogenesis-dependent oncogene addiction [37], [38]. These dramatic effects were reversed by sTβRII which restored proliferation but did not alter angiogenesis or apoptosis, resulting in large tumors that exhibited foci of necrosis. Thus, while the in vitro and in vivo growth inhibitory actions of EGFR silencing were reversed by TGF-β sequestration, the paracrine anti-angiogenic effects of EGFR silencing and effects on apoptosis persisted, underscoring the pro-mitogenic effects of src activation. Moreover, it has been recently demonstrated that angiogenesis is important in a Kras-driven genetically engineered mouse model of PDAC [39] and that variant 161R form of interlukin-17F (IL-17F), which is a natural antagonist of the anti-angiogenic effects of wild-type 161H IL-17F, is associated with a worse prognosis in PDAC [40], providing indirect evidence that angiogenesis may play an important role in its metastatic spread. In view of these observations, the current findings suggest that targeting EGFR and TGF-β may be important for normalizing tumor angiogenesis in the primary tumor and suppressing angiogenesis in metastatic lesions in PDAC. ASPC-1 and T3M4 cells harbor mutated KRAS and p53 genes, and express high EGFR levels [5], [41]. These cells also produce high levels of TGF-β, TGF-α and amphiregulin [19], [42], which are auto-inducible, TGF-β-inducible, and pro-angiogenic. Moreover, ASPC-1 cells harbor a mutated SMAD4 gene [43], whereas T3M4 cells are wild type for Smad4 [44]. As such, ASPC-1 and T3M4 cells exhibit alterations that are highly representative of the spectrum of typical molecular alterations seen in PDAC. In spite of the presence of oncogenic Kras in ASPC-1 cells, the concomitant targeting of EGFR and TGF-β provided an effective therapeutic strategy in these cells, suggesting that targeting two key upstream events in PDAC may overcome therapeutic resistance engendered by oncogenic Kras in some pancreatic cancer cells. However, as evidenced in T3M4 cells, targeting both EGFR and TGF-β can also lead to deleterious effects as a consequence of HER2/3 and src activation. Inasmuch as src may be an important mediator of cross-talk between EGFR family members and several growth-modulating pathways such as Met, Notch-1 and furin [45]–[47], our findings suggest that concomitantly targeting the activation of cell-surface receptors such as EGFR, HER2, and TβRI and the intracellular src kinase may represent a novel strategy for suppressing pancreatic cancer growth in the presence of oncogenic Kras. It has been recently demonstrated that most cases of PDAC develop slowly over approximately two decades before acquiring the capacity to metastasize [48], [49]. Moreover, targeting TGF-β in an orthotopic murine model of PDAC markedly suppresses metastasis [14]. Together with the current findings, these observations also raise the possibility that combinatorial targeted therapy aimed at EGFR, TGF-β, and src may constitute a novel approach in PDAC that interferes with multiple signalling components downstream of EGFR and TβR, attenuating disease progression while preventing potentially deleterious cross-talk between these pathways. Moreover, targeting these pathways may attenuate PDAC desmoplasia [5], thereby potentially allowing for improved drug delivery within the tumor mass. In theory, therefore, delivery of lentiviral vectors into the pancreatic tumor mass via endoscopic ultrasonography administered prior to the presence of metastatic disease in conjunction with the systemic administration of a small molecule src inhibitor could prove to be an effective approach in PDAC. Antibodies or small molecule inhibitors that target both EGFR and TGF-β pathways given together with a src inhibitor could also be used even when metastatic disease is present, perhaps followed by the addition of chemotherapeutic agents such as gemcitabine. It will now be important to conduct additional pre-clinical testing of these approaches and to delineate specific biomarkers to indicate which subgroups of PDAC patients would be responsive to this form of combinatorial therapy. Material and Methods Cell Culture ASPC-1 and PANC-1 human pancreatic cancer cells were obtained from ATCC (Manassas, VA), whereas T3M4 and COLO-357 human pancreatic cancer cells were a gift from R. Metzger (Duke University). Both T3M4 and COLO-357 cells were originally isolated from PDAC metastases [50]–[51]. ASPC-1 and T3M4 cells were grown in RPMI (Mediatech Inc., Herndon, VA). COLO-357 and PANC-1 cells were grown in DMEM. Media were supplemented with 10% fetal bovine serum (FBS) from Omega Scientific Inc. (Tarzana, CA), and 100 U/ml penicillin and 100 µg/ml streptomycin. Vector Construction The soluble type II TGF-β receptor construct (pWPT-sTβRII) encodes a fusion protein consisting of the extra cellular domain (amino acid residues 1-477) of TβRII fused with an Ig Fc tail and an HA-tag. The construct encoding the tagged fusion protein was subcloned into XhoI sites from a lentivirus plasmid pWPT-GFP (Addgene, Cambridge, MA), replacing the GFP gene. The recombinant pWPT-sTβRII and pWPT-GFP plasmids were propagated in E. coli top ten competent cells (Invitrogen, Carlsbad, CA). Authenticity was confirmed by sequencing, and sTβRII expression was assessed by immunoblotting for HA (Cell Signaling, Danvers, MA). To prepare the shRNA targeting EGFR, a pool of siRNA sequences directed against EGFR (Dharmacon, Lafayette, CO) were transfected into ASPC-1 cells using Jet PEI (Qbiogene, Solon, OH) according to the manufacturer’s protocol. The siRNA pool efficiently silenced EGFR protein expression, and each sequence was then tested to select the most efficient siRNA sequences for designing the oligonucleotides for the shRNA targeting EGFR. The same procedure was used to target human EGFR 2 (HER2) and luciferase (negative control). Oligonucleotides were annealed and cloned into pLentiLox 3.7 (pll3.7) (Addgene, Cambridge, MA), yielding highly efficient lentiviral vectors carrying the shRNA targeting EGFR (shEGFR-LV), HER2 (shHER2-LV) or Luciferase (shLuc-LV). Virus stocks were prepared by co-transfecting pll3.7 with three packaging plasmids (pMDLg/pRRE, CMV-VSVG and RSV-Rev) into 293T cells [52]. Viral supernatants were harvested 36–48 hours later, filtered and centrifuged (90 min at 25,000 X g). Viral titers were determined by fluorescence-activated analysis (FACS) analysis and all cells were infected at a multiplicity of infection (m.o.i.) of 10. Colony Formation in 3-Dimensional Matrigel Assays A 3-dimensional (3-D) cell culture system was used to assess colony formation, as reported previously [16]. Briefly, cells (2,000 per well) were suspended in 3% growth factor reduced (GFR) Matrigel (BD Biosciences, San Jose, CA), dissolved in 0.2 ml of medium containing 5% FBS, and plated on top of solidified 0.2 ml of 1% noble agar in the same medium, using 48-well culture plates. Medium (0.2 ml) containing 3% GFR Matrigel and 5% FBS was added every 3 days, in the absence or presence of lapatinib (1 µM), SKI-606 (1 µM), PP2 (1 µM) and dasatinib (0.1 µM). After 2 weeks, colonies were stained by incubating for 4 hours with of 3-4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO) and counted. Immunoblotting Immunoblotting was performed as reported previously [13], using PVDF membranes (Perkin Elmer, Boston, MA). Membranes were incubated overnight with the following primary antibodies: anti-EGFR (15F8) (#4405), anti-phospho-EGFR (Tyr845) (#2231), anti-phospho-HER2 (Tyr1221/1222) (#2243), anti-phospho-HER3 (Tyr1289) (#4791), anti-HA-Tag (#2367), and anti-phospho-src(Tyr527) all form Cell Signaling Technology (Danvers, MA; 1∶500 to 1∶1000 dilution); and anti-HER2 (#06-562) and anti-HER3 (#05-390) from Upstate Biotechnology, Lake Placid, NY). The membranes were washed, incubated for 30 minutes with secondary horseradish peroxidase-conjugated antibody (Biorad, Hercules, CA), and bound antibodies were visualized using enhanced chemiluminescence (Pierce, Rockford, IL). Membranes were stripped and blotted with a 1∶10,000 dilution of rabbit anti-ERK antibody (Santa-Cruz Biotechnology, Santa Cruz, CA). Tumorigenicity Assay To assess effects on tumorigenicity, 1 x106 ASPC-1 cells and 0.5 x106 T3M4 cells expressing shLuc-LV, shEGFR-LV, pWPT-sTβRII, or both shEGFR-LV and pWPT-sTβRII, were injected subcutaneously into the flank region of 6–8 week-old, female, athymic nude mice (Harlan, Indianapolis, IN). Fewer T3M4 cells were used because these cells form rapidly growing tumors. Studies with mice were approved by Dartmouth Medical School and Indiana University School of Medicine Institutional Animal Care and Use Committees. Mice were monitored twice weekly and sacrificed 8–15 weeks after injection when tumor diameter reached a maximally allowable 15 mm. Tumor volumes were calculated as π/4 × width × height × length of the tumor [13]. Immunohistochemistry and TUNEL Assay Following rapid tumor removal, tissues were cryo-embedded in cryo-OCT compound (Fisher Scientific, Pittsburgh, PA). All immunohistochemistry experiments were done as described previously [53] using an anti-Ki-67 antibody (Abcam, Cambridge, MA; 1∶50 dilution) to assess proliferation, anti-CD31 to detect endothelial cells (PharMingen, San Jose, CA) and anti-cleaved PARP (#9141) (Cell Signaling Technology, Danvers, MA) to assess apoptosis. Phospho-HER2 and phospho-HER3 immunoreactivity was determined using the respective anti-phospho antibodies described above. Quantitative morphometry (10 areas/slide) was performed as reported previously [53], using an Olympus DP70 camera (100 X magnification), and quantified with the Image-Pro plus program (Version 4.51, Media cybernetics, L.P., Silver Spring, MD). Apoptotic cells were also detected by measuring DNA fragmentation using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method (In Situ Cell Death Detection Kit, POD, Roche Applied Science, Indianapolis, IN), according to the manufacturer’s protocol. Sections were incubated with peroxidase-conjugated anti-digoxigenin antibody for 30 min at 23°C to detect digoxigenin-dUTP labelling, and for 5 min in a solution of 0.05% 3,3′-diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA) and 0.01% H2O2. In all immunostaining and TUNEL assays, three randomly selected tumors per group were analyzed. Phospho-kinase Array T3M4 cells were analyzed in a panel of phosphorylation profiles of kinases (Human Phospho-Kinase Array, ARY003; R&D Systems, Minneapolis, MN). A cocktail of biotinylated detection antibodies, streptavidin–horseradish peroxidase and chemiluminescent detection reagents were used to detect the phosphorylated protein. The relative expression of specific phosphorylated proteins was determined following quantification of scanned images by Image-Pro plus program. Statistical Analysis Data were analysed using either ANOVA or the Kruskall and Wallis tests for mean comparisons, using the Dunn-Benferroni test for multiple comparisons. p<0.05 was taken as the level of significance. Supporting Information Figure S1 COLO-357 and PANC-1 human pancreatic cancer cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), WPT-sTβRII (sTβRII), or both shEGFR and sTβRII, and the consequences of EGFR silencing with shEGFR and TGF-β sequestration with sTβRII were assessed by monitoring colony formation in 3-D culture. Data are the means ± SE of triplicate determinations from three independent experiments. *p<0.05, **p<0.01, when compared with respective controls. (EPS) Click here for additional data file. Figure S2 T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), sTβRII, or both EGFR-LV and sTβRII, and injected subcutaneously into the flank region of nude mice. Tumor immunoreactivity for phospho-HER2 and phospho-HER3 was determined 16 days later using the indicated anti-phospho antibodies. Scale bars, 50 µm. (TIFF) Click here for additional data file. Figure S3 T3M4 cells were infected with shLuc-LV (shLuc), shEGFR-LV (shEGFR), shHER2-LV (shHER2) or a combination of shEGFR and shHER2, in the absence or presence of WPT-sTβRII (sTβRII). Immunoblotting was then carried out with antibodies directed against the indicated proteins. Data shown are from a representative of three experiments. (EPS) Click here for additional data file. The authors thank Dr. Jesse Gore for helpful discussions in the course of this work. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by National Cancer Institute (NCI) grant CA-R37-075059 (M.K.). 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==== Front Int J BiomaterInt J BiomaterIJBMInternational Journal of Biomaterials1687-87871687-8795Hindawi Publishing Corporation 10.1155/2012/380845Research ArticleFree Radical Production in Immune Cell Systems Induced by Ti, Ti6Al4V and SS Assessed by Chemiluminescence Probe Pholasin Assay P. Cachinho Sandra C. *Pu Fanrong Clinical Engineering, University of Liverpool, Liverpool L69 3GA, UK*Sandra C. P. Cachinho: [email protected] Editor: Mohamed Rahaman 2012 19 6 2012 2012 38084512 3 2012 13 5 2012 Copyright © 2012 S. C. P. Cachinho and F. Pu.2012This 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 oxidative burst of human blood cells in the presence of different metal materials was investigated using chemiluminescence assay. Commercial pure titanium (Ti), titanium alloy (Ti6Al4V), and stainless steel 316L (SS) in particulate form with <20 μm in size were used. The effect of particulate materials opsonisation on the upregulation of the respiratory burst production by blood cells was also assessed. The largest chemiluminescence response was achieved after simultaneous injection of the stimulants fMLP+PMA. Moreover, Ti and SS induced a greater inflammatory reaction compared to Ti6Al4V, since the respiratory burst mounted was higher for both materials after opsonisation treatment. These results suggest that in vitro chemiluminescence response and respiratory burst measurements proved to be composition and treatment dependent. ==== Body 1. Introduction Implanted biomaterials, introduced into the human body, interact initially with blood inducing a nonspecific host defence responses which are critical to their integration and susceptibility to infection [1–4]. Generally, when infection occurs, it tends to be persistent and intractable with antibiotics and the removal of the implant to clear the infection is often required [2, 5]. The body's response to a foreign body is characterised by an initial infiltration of polymorphonuclear leukocytes (PMNs), where neutrophils and monocytes are the first inflammatory cells to be recruited to an implanted surface exposed to blood. These blood cells can subsequently release cytokines and inflammatory mediators, which recruit additional cells—macrophages, fibroblasts, lymphocytes, and endothelial cells—that proliferate and mediate a chronic response [4, 6–8]. Adhesion, chemotaxis, aggregation, and activation in response to the inflammatory stimuli are processes in which cells may go through upon material contact [4, 9]. After adhesion to biomaterial surfaces, different leukocyte reactions such as phagocytosis, respiratory burst (generation of reactive oxygen species, ROS), and protease release may also occur, resulting in the deterioration of the implanted material and injury to peripheral tissue [10, 11]. The production of highly reactive oxygen intermediates (“oxygen burst” or “respiratory burst”) by PMNs, such as superoxide anion (O2 −), hydrogen peroxide (H2O2), and hydroxyl radical (OH•), may be a major source of damage to inflamed tissues and implanted materials degradation [12–14]. These oxidative metabolites are potent bactericides and constitute an important part of the oxygen-dependent bactericidal killing mechanism of the PMNs [12, 15, 16]. The oxidative response is mounted mainly against bacteria to destroy them, but the oxygen radicals are secreted during inflammatory reactions, too; moreover, they have been recently suggested to be mediators of cytokine activity on target cells, mainly for tumour necrosis factor (TNF) and interleukin-8 (IL-8) [16, 17]. The adhesion of neutrophils and other leukocytes to implanted surfaces and their activation, with the subsequent release of potential biomaterial and tissue-damaging agents, are important phenomenon in the host response to biomaterials, and whether implanted biomaterials increase the risk for infection is an important issue to be addressed. The NADPH oxidase-mediated respiratory burst is a result of neutrophil activation, which is triggered following occupation of specific receptors on neutrophil surfaces, binding to certain soluble inflammatory molecules or to invading microorganisms. In vitro the burst may also be initiated by soluble phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) [15, 18, 19]. In this work, the effect of three commercial particulate metal materials on leukocyte adhesion and their oxidative response was investigated. Whole blood was used to investigate leukocytes oxidative burst activity in contact to the particulate materials. The aim of this part of the research was to evaluate the potential of particulate materials with different compositions, to directly stimulate nonspecific activation of human inflammatory cells. The respiratory burst activity of cells from whole human fresh blood in the presence of materials was studied using in vitro chemiluminescence assay where fMLP and PMA were used as reference activators. Stimulated cell function in vitro has been largely studied using isolated peripheral blood mononuclear cells (PBMC); however, in this work, whole blood assay was preferred because of the better approximation to the in vivo situation. 2. Materials and Methods 2.1. Materials Spherical pure titanium (Ti), titanium alloy (Ti6Al4V), and stainless steel 316L (SS) in particulate form with <20 μm in size were purchased from Alfa Aesar (UK). Prior to use in cell culture, material particles were sterilised in 70% ethanol followed by washing in PBS and media. For cell culture particles were weighed, from this the number of particles was calculated; a ratio of 1 : 1 particle per cell was used. 2.2. Whole Blood Sample Preparation Fresh blood was collected from healthy adult volunteers by venous puncture and gently mixed with heparin (20 mL human whole blood gently mixed with 40 μL heparin); informed consent was obtained from each individual enrolled in this study. For chemiluminescence experiments, 20 μL of whole blood was used in each well. 2.3. Chemiluminescence Assay For the determination of the respiratory burst resulting from cells and biomaterials interaction, pholasin-dependent chemiluminescence was used. Free radicals and other reactive oxygen species produced by activated cells interact with Pholasin, and the luminescent response can be measured. Pholasin is a photoprotein that emits light after excitation with reactive oxygen species. 20 μL of whole blood was diluted in 2 mL of blood dilution buffer. Duplicate wells were prepared for each material with 90 μL of reconstitution and assay buffer, 20 μL adjuvant-K and 50 μL of Pholasin (all reagents from Abel Cell Activation Test Kits with Pholasin, Knight Scientific Limited, UK) and 20 μL of blood suspension. Two wells without materials were prepared to verify the activation effect of the materials on the cells. Opsonised particles were also used to determine the effect of complement on cells activation. The plate was then placed in the microplate reader FLUOstar OPTIMA (BMG Labtech, UK) previously set to 37°C and automatically shaken for 3 sec at 300 rpm before measurements were started. The microplate reader was controlled by the computer using the OPTIMA program. The program was set such that the luminometer measured light emission from each well every 20 sec. The light output from each well was measured for 40 min. At that time point, 20 μL of each stimulant, formyl-methionyl-leucyl-phenylalanine (fMLP, 12 μmol/L) and phorbol-myristate-acetate (PMA, 8 μmol/L), were injected into each well and the light emitted measured for another 40 min. Luminescence values were plotted as the amount of light emitted versus time. Data were analysed using OPTIMA MARS-data analysis software. 2.4. Graphical Representation and Statistical Analysis Experiments were repeated for four volunteers, and the means and standard deviations were calculated; this will be expressed as mean ± 1 standard deviation. Statistical significances were evaluated by one-way analysis of variance (ANOVA) and by post hoc Tukey test using Statistical Comparisons Sciences (SPSS) version 16.0 for Windows software. Differences were considered to be statistically significant at P < 0.05. All the results are represented in graphical form using Microsoft Excel. 3. Results 3.1. Chemiluminescence The results for the oxidative burst analysis of whole blood cells by continuous measurement of the cells chemiluminescence response to Ti, Ti6Al4V and SS particles are shown in Figures 1, 2, and 3, respectively. Each figure shows representative curves of the chemiluminescence response of the cellular interaction with the surface of the particle for a period of 80 min. In the measurement of the oxidative burst activity of blood cells, PMA and fMLP activators were used in combination to maximally stimulate cells. The stimulants were added to the reaction mixture after the blood cells were allowed to interact with the surfaces for 40 min. A general trend can be observed in all figures where maximum ROS production was reached between 2700 sec and 3100 sec and after (fMLP+PMA) stimulation. The chemiluminescence response elicited a slight decrease after stimulation and remained constant during the rest of the assay. Low chemiluminescence response was elicited by the particulate materials in the absence of stimulants. The largest chemiluminescence response was determined for SS, with or without opsonisation (2008 ± 206 and 6077 ± 3008 at 3060 sec for SS and SSops, resp.) (Figure 3), while on the other particulate metals, the response was smaller (1283 ± 379, 3397 ± 901, 485 ± 140, and 1395 ± 372 at 3060 sec for Ti, Tiops, Ti6Al4V, and Ti6Al4Vops, resp.) (Figures 1 and 2). The chemiluminescence response, after (fMLP+PMA) stimulation, was more sustained when blood cells were in contact with Tiops and SSops particulates, indicative of a more prolonged production of oxygen radicals by cells on these materials. In order to compare ROS production between materials, two specific time points were chosen: one before stimulant injection and the other after stimulant injection at 1800 and 3200 sec, respectively. The results are shown in Figures 4 and 5. Figure 4 shows the chemiluminescence response of whole blood cells on particulate surfaces prior to the addition of stimulants, while Figure 5 shows the chemiluminescence response subsequent to fMLP+PMA addition. With the exception of opsonised SS (5967 ± 2689), after stimulant injection, the highest production of ROS was detected for whole blood cells (negative control) without particulate materials contact (3705 ± 161) when compared with cells in contact with metal particles (≤3402) (Figure 5). Statistical differences (P < 0.05) were observed between the negative control and whole blood cells cultured in the presence of particulate Ti and particulate Ti6Al4V. Particulate materials downregulated the chemiluminescence response and a decrease in the respiratory burst activity was demonstrated. The lowest ROS production can be observed for all situations before stimulant injection (268 ≤ ROS ≤ 940) (Figure 4) when compared to the response after cell stimulation (468 ≤ ROS ≤ 5967) (Figure 5). At 1800 sec, similar chemiluminescence levels were detected between Ti and Tiops (853 ± 311 and 847 ± 452, resp.) and Ti6Al4V and Ti6Al4Vops (239 ± 39 and 268 ± 16, resp.), while opsonised SS showed a lower chemiluminescence signal compared to the nonopsonised SS (514 ± 47 and 898 ± 217, resp.) indicating a decrease in the respiratory burst activity (Figure 4). No statistical differences were demonstrated between opsonised and nonopsonised particulate materials, indicative that opsonisation did not affect the production of ROS; however, significant differences (P < 0.05) were noted between Ti and Ti6Al4V, Ti6Al4V and SS, and Tiops and Ti6Al4Vops, demonstrating that the respiratory burst activity was material dependent. The respiratory burst activity induced by the presence of particulate Ti6Al4V, opsonised (268 ± 16) and nonopsonised (239 ± 39), was statistically significant when compared to the negative control (whole blood cells: 940 ± 296) and the positive control (whole blood cells+zymops: 819 ± 41), showing lower ROS production. Ti6Al4V particles downregulated the respiratory burst, and no further increase in ROS production was induced by the presence of opsonised particles. No statistical differences were recorded for the other particulate materials composition compared to the negative and positive controls. 3200 sec after stimulation with PMA+fMLP, the respiratory burst activity was upregulated for all experimental conditions with maximum ROS production observed when whole blood cells were in contact with opsonised SS (5967 ± 2689) (Figure 5). An increase in the ROS production was induced when whole blood cells were in contact with opsonised particulates compared to the amount produced when cells were in contact with nonopsonised ones (from 1290 ± 347, 468 ± 105, and 2000 ± 158 to 3402 ± 746, 1459 ± 454, and 5967 ± 2689 for Ti, Tiops, Ti6Al4V, Ti6Al4Vops, SS, and SSops, resp.), indicating that opsonisation increased the production of oxygen radicals by whole blood cells. Statistical differences, P < 0.05, were recorded between Ti and Tiops and between SS and SSops, showing the effect of opsonisation on stimulation of a respiratory burst. No statistical differences were observed between nonopsonised particulate metals. The highest production of ROS observed for opsonised SS was statistically different compared to the other particulate materials, either opsonised or nonopsonised; providing further evidence of material composition effect (Figure 5). Significant differences (P < 0.05) were also recorded between opsonised SS and the positive control, whole blood cells cultured with opsonised zymosan, (2481 ± 336) supporting the evidence mentioned above. No differences were observed between the positive control and the other opsonised particulate materials. 4. Discussion The measurement of chemiluminescence serves as an excellent parameter for the quantitative evaluation of nonspecific leukocytes activation, and it has been widely used as a sensitive and accurate method for the assessment of ROS production [20, 21]. In this study, a chemiluminescence assay was used to evaluate the potential of immune cells to become activated after direct contact with particulate materials of different compositions, opsonised and nonopsonised. The light resulting from the interaction of free radicals, produced by activated cells, with the photoprotein Pholasin was detected and plotted as a function of time. The oxidative burst depends on the activity of NADPH oxidase, a transmembrane electron transport chain that reduces oxygen to superoxide. When PMNs are stimulated by soluble stimulants or phagocytose materials, the multicomponent system of NADPH oxidase is rapidly assembled and activated. [22–24]. There are differences in the final species of released ROS that depends on the type of stimulant and the pathway of activation. The positive references stimulants used in this work to activate the respiratory burst were the receptor stimulant fMLP and the phorbol ester PMA which act through different pathways. fMLP, a formylated oligopeptide, is structurally similar to bacteria-derived peptides. It is a ligand that binds to specific fMLP receptors on the cell surface and acts by means of a mechanism that is dependent on both intra- and extracellular calcium and on a process that includes phospholipase and protease activation. PMA is a chemical that lacks specific cell surface receptors, and so it bypasses this step and activates the specific enzymes involved in the signal transduction sequences (i.e., protein kinase C). In this study, the injection of Pholasin into each well did not show any effect on the light detected, showing that whole blood cell-particulate material interaction did not cause any effect on the respiratory burst; this situation, which happened before PMA+fMLP injection, might indicate that materials downregulate the production of ROS or that cells might have been activated at some earlier point in the assay. The production of ROS was also low in unstimulated whole blood cells without particulate materials contact. Unstimulated whole blood cells were expected to generate no measurable or very low amounts of ROS since the NADPH oxidase system should have been inactive at that stage. After simultaneous injection of the stimulants, fMLP+PMA, a peak for the light emitted was observed due to an increase in the production of ROS. This was found in stimulated whole blood cells with and without particulate materials contact. The response after the injection of stimulants was higher in all situations. This was indicative that whole blood cells were not activated by any of the materials tested in this work prior to the addition of PMA+fMLP, since a lower luminescent signal was demonstrated due to the lower production of ROS. In vitro evidence [25–27] indicated that macrophages and neutrophils on biomaterial surfaces have a low chemiluminescence response and, in addition, a low capacity to mount an oxidative response when stimulated by a soluble stimulus. It has been also shown that the human PMN function and the respiratory burst in vitro are influenced by cell adhesion to a surface. Increased adherence and spreading of PMNs to materials too large to phagocytose can lead to increased activation of oxygen metabolism [1, 28]. Moreover, the protein adsorption to a surface influences the in vitro cell attachment and function [25]. Opsonised metal particles induced higher levels of light emitted when compared to the nonopsonised ones. The greatest difference was found for whole blood cells in contact with opsonised stainless steel where the highest light emitted was recorded immediately after fMLP+PMA injection. Furthermore, the data indicated that Ti and SS could induce a greater inflammatory reaction compared to Ti6Al4V, since the respiratory burst mounted was higher for both after opsonisation treatment. If large amounts of H2O2 and O2 − are produced in vivo when a biomaterial is inserted into the human body, host tissues may be damaged. Titanium has been suggested as a nonreactive material used in vivo due to its passive oxide layer; however, the results from this study indicated that cells in contact with opsonised particulate titanium may be stimulated when challenged with fMLP+PMA. The same result was found by Eriksson et al. [9] where PMNs on titanium did not produce ROS spontaneously but were able to elicit a response when exposed to opsonised zymosan. Cells adherent to titanium were not active but have the ability to respond to a microorganism when challenged with the correct stimuli. In another work by Nygren et al. [29], titanium and stainless steel sheets implanted in vivo were analysed by chemiluminescence and the ability of the surface-adhering leukocytes to mount a respiratory burst after stimulation with PMA or zymosan was measured. The results from their study showed higher ROS production for stainless steel when challenged with opsonised zymosan when compared with titanium or PMA challenge. This suggests that titanium might not be as inert as previously described and when implanted in vivo might complex with serum proteins and induce the production of ROS. From this study, the production of ROS was found to be lower when whole blood cells were in contact with nonopsonised particulate metals compared to the opsonised ones. Comparison of the degree of inhibition of stimulated chemiluminescence, resulting from exposure of whole blood cells to various particulate metals, can be summarised as follows: (Ti6Al4V < Ti < SS) < (Ti6Al4Vops < Tiops < SSops), indicating that opsonisation upregulates the production of ROS by immune cells when in contact with those particulate materials; the same effect can be observed even after PMA+fMLP stimulation, resulting in higher respiratory burst activity. Some research [28, 30, 31] has shown in vitro that PMNs adhering to protein-coated substrates do not produce ROS unless exposed to a stimulus, which is in accordance with the results from this study. Opsonisation did not affect the activation state of cells until PMA+fMLP was injected and the respiratory burst was induced. It has been also published [32–34] that particles coated with serum constituents might make those surfaces less reactive. Serum proteins, for example, albumin, fibrinogen, and hemoglobulin, bind to and change the nature of particulate powder surfaces such that they lose the potential to stimulate cells. It has been suggested [28] that serum provides a mixed protein coating for biomaterials possibly creating a biocompatible interface between materials and cells. Terkeltaub et al. [35] found that the presence of serum in hydroxyapatite (HA) particles incubated with cells or the pretreatment of HA crystals with serum resulted in a marked suppression of neutrophil activation to HA crystals and the superoxide release was reduced. The same results were found by Remes and Williams [33] where serum proteins bound to CaHPO4 powders reduced the stimulation of neutrophils. This inhibitory effect was observed in this study where opsonised particles, metals and zymosan, did not affect the respiratory burst activity and the production of ROS was not affected, until challenged with fMLP+PMA. This low peak luminescence was a feature of all opsonised zymosan samples used in this study and was reproducible across different donors. Clearly, long-exposure times before stimulant addition did not affect the response. Besides evidence [24, 36] that zymosan, a yeast particle which is opsonised and commonly used stimulus for chemiluminescence studies, is particularly useful for stimulating neutrophils phagocytosis which results in a chemiluminescent response that can be 10–20 times higher than unstimulated values, some in vitro studies showed a reduced capacity of human cells to mount an oxidative response when the cells were challenged with opsonised zymosan [37]. Ginis and Tauber [30] found that nonopsonised zymosan did not stimulate O2 − generation by neutrophils, but rigorous O2 − response could be induced in cells adherent to fibronectin. The type of protein bound to a surface might explain the ability for surfaces to stimulate differing respiratory burst effects from cells in direct contact. Surface contact alters neutrophils behavior, and the oxidative burst can be reduced compared to control cells. Since macrophages and neutrophils are the major cells presented in blood, that contribute to the respiratory burst, the lower ROS production in the presence of opsonised zymosan, before stimulants addition, might be explained on the experimental evidence mentioned above. Macrophages are far less potent than neutrophils and eosinophils at producing ROS, and they might need time to acquire an activated phenotype to upregulate their bactericidal capability [38]. 5. Conclusions The chemiluminescence assay was demonstrated to be a useful technique to understand the interaction of immune cells and implanted materials in terms of reactive oxygen species production, known to be harmful to the tissues and potentially damaging to implanted medical devices. In this study, the stimulatory effects on whole blood cells caused by a series of particulate metals were assessed using the chemiluminescence method based on the photoprotein Pholasin. The results indicated that the exposure of whole blood cells to particulate metal materials could generate a respiratory burst when challenged with fMLP+PMA. ROS production was upregulated in the presence of opsonised particulate metals, where the highest production was found for opsonised stainless steel followed by opsonised titanium; suggesting these materials have the potential to stimulate an inflammatory response directly and consequently activation is directed by protein modification or by complement activation. Acknowledgment This work was supported by Marie Curie Funds (MEST-CT-2005-021089). List of Abbreviations Ti:Pure titanium Ti6Al4V:Titanium alloy SS:Stainless Steel 316L fMLP:N-Formyl-methionyl-leucyl-phenylalanine PMA:Phorbol myristate acetate PMNs:Polymorphonuclear leukocytes ROS:Reactive oxygen species PBMC:Peripheral blood mononuclear cells Ops:Opsonisation. Figure 1 Chemiluminescence response of whole blood cells to Ti, opsonised and nonopsonised, and opsonised zymosan (positive control). Reaction mixture included whole blood cells without particulate materials (negative control) and in the presence of Ti, Tiops, and Zymops particles. Cells were stimulated with fMLP+PMA at 2400 s. Figure 2 Chemiluminescence response of whole blood cells to Ti6Al4V, opsonised and nonopsonised, and opsonised zymosan (positive control). Reaction mixture included whole blood cells without particulate materials (negative control) and in the presence of Ti6Al4V, Ti6Al4Vops, and Zymops particles. Cells were stimulated with fMLP+PMA at 2400 s. Figure 3 Chemiluminescence response of whole blood cells to SS, opsonised and nonopsonised, and opsonised zymosan (positive control). Reaction mixture included whole blood cells without particulate materials (negative control) and in the presence of SS, SSops, and Zymops particles. Cells were stimulated with fMLP+PMA at 2400 s. Figure 4 ROS production during whole blood cell interactions to opsonised and nonopsonised particulate materials before fMLP+PMA injection and at 1800 sec of chemiluminescence measurement (reaction mixture included whole blood cells without particulate materials, whole blood cells in the presence of Ti, Ti6Al4V, SS, Tiops, Ti6Al4Vops, SSops, and Zymops particles) (∗, +, ♦ P < 0.05). Figure 5 ROS production during whole blood cell interactions to opsonised and nonopsonised particles materials after fMLP+PMA injection and at 3200 sec of chemiluminescence measurement (reaction mixture included whole blood cells without particulate materials, whole blood cells in the presence of Ti, Ti6Al4V, SS, Tiops, Ti6Al4Vops, SSops, and Zymops particles) (∗, +, ♦ P < 0.05). ==== Refs 1 Lim F Cooper SL Chemiluminescent oxidative products generated by in vitro leukocyte- material interactions Journal of Materials Science 1996 7 2 69 76 2-s2.0-0030085527 2 Kaplan SS Heine RP Simmons RL Defensins impair phagocytic killing by neutrophils in biomaterial- related infection Infection and Immunity 1999 67 4 1640 1645 2-s2.0-0033037234 10084997 3 Giridhar G Gristina AG Myrvik QN Altered oxidative responses and antibacterial activity of adult rabbit alveolar macrophages exposed to poly(methyl methacrylate) Biomaterials 1993 14 8 609 614 2-s2.0-0027626401 8399955 4 Anderson JM Mechanisms of inflammation and infection with implanted devices Cardiovascular Pathology 1993 2 33S 41S 5 Greco RS Body parts: in vivo veritas Journal of Biomedical Materials Research 1997 34 4 409 410 2-s2.0-0031569171 9054524 6 Chen FS Scher DM Clancy RM Vera-Yu A Cesare PED In vitro and in vivo activation of polymorphonuclear leukocytes in response to particulate debris Journal of Biomedical Materials Research 1999 48 904 912 10556858 7 Renò F Lombardi F Cannas M UHMWPE oxidation increases granulocytes activation: a role in tissue response after prosthesis implant Biomaterials 2003 24 17 2895 2900 2-s2.0-0038755157 12742728 8 Dokka S Toledo D Wang L Free radical-mediated transgene inactivation of macrophages by endotoxin American Journal of Physiology 2000 279 5 L878 L883 2-s2.0-0033697730 11053023 9 Eriksson C Lausmaa J Nygren H Interactions between human whole blood and modified TiO2 -surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation Biomaterials 2001 22 14 1987 1996 2-s2.0-0034991893 11426876 10 Nimeri G Öhman L Elwing H Wetterö J Bengtsson T The influence of plasma proteins and platelets on oxygen radical production and F-actin distribution in neutrophils adhering to polymer surfaces Biomaterials 2002 23 8 1785 1795 2-s2.0-0037089692 11950049 11 Hyslop PA Hinshaw DB Scraufstatter IU Cochrane CG Kunz S Vosbeck K Hydrogen peroxide as a potent bacteriostatic antibiotic: implications for host defense Free Radical Biology and Medicine 1995 19 1 31 37 2-s2.0-0029074049 7635356 12 Nathan CF Neutrophil activation on biological surfaces. 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Evidence for a local granulocyte defect Journal of Clinical Investigation 1984 73 4 1191 1200 2-s2.0-0021236861 6323536 28 Kaplan SS Basford RE Jeong MH Simmons RL Mechanisms of biomaterial-induced superoxide release by neutrophils Journal of Biomedical Materials Research 1994 28 3 377 386 2-s2.0-0028401582 8077253 29 Nygren H Hrustic E Karlsson C Öster L Respiratory burst response of peritoneal leukocytes adhering to titanium and stainless steel Journal of Biomedical Materials Research 2001 57 238 247 11484187 30 Ginis I Tauber AI Activation mechanisms of adherent human neutrophils Blood 1990 76 6 1233 1239 2-s2.0-0025088402 2400811 31 Yan SR Novak MJ Diverse effects of neutrophil integrin occupation on respiratory burst activation Cellular Immunology 1999 195 2 119 126 2-s2.0-0345493897 10448011 32 Rae T The haemolytic action of particulate metals (Cd, Cr, Co, Fe, Mo, Ni, Ta, Ti, Zn, Co-Cr alloy) Journal of Pathology 1978 125 2 81 89 2-s2.0-0018097153 722392 33 Remes A Williams DF Stimulation of a neutrophil respiratory burst by calcium hydrogen phosphate powder Clinical Materials 1992 9 2 71 76 2-s2.0-0026504129 34 Lindfors NC Klockars M Immunoglobulin enhances the bioactive-glass-induced chemiluminescence response of human polymorphonuclear leukocytes Journal of Biomedical Materials Research 2001 55 613 617 11288090 35 Terkeltaub RA Santoro DA Mandel G Mandel N Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals Arthritis and Rheumatism 1988 31 9 1081 1089 2-s2.0-0023733775 2844196 36 Hosker HSR Kelly C Corris PA Assessment of phagocytic function using chemiluminescence Blood Reviews 1989 3 2 88 93 2-s2.0-0024318335 2673449 37 Nakagawara A Nathan CF Cohn ZA Hydrogen peroxide metabolism in human monocytes during differentiation in vitro Journal of Clinical Investigation 1981 68 5 1243 1252 2-s2.0-0019866087 6271809 38 Forman HJ Torres M Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling American Journal of Respiratory and Critical Care Medicine 2002 166 12 S4 S8 2-s2.0-0037115158 12471082
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Int J Biomater. 2012 Jun 19; 2012:380845
==== Front Front Plant SciFront Plant SciFront. Plant Sci.Frontiers in Plant Science1664-462XFrontiers Research Foundation 2278326910.3389/fpls.2012.00149Plant ScienceOriginal ResearchInfluence of ATP-Binding Cassette Transporters in Root Exudation of Phytoalexins, Signals, and in Disease Resistance Badri Dayakar V. 1Chaparro Jacqueline M. 1Manter Daniel K. 2Martinoia Enrico 3Vivanco Jorge M. 1*1Center for Rhizosphere Biology, Colorado State UniversityFort Collins, CO, USA2Soil-Plant-Nutrient Research Unit, United States Department of Agriculture-Agricultural Research ServiceFort Collins, CO, USA3Zurich-Basel Plant Science Center, Institute of Plant Biology, Molecular Plant Physiology, University of ZurichZurich, SwitzerlandEdited by: William David Nes, Texas Tech University, USA Reviewed by: Dorothea Tholl, Virginia Polytechnic Institute and State University, USA; Huazhong Shi, Texas Tech University, USA *Correspondence: Jorge M. Vivanco, Center for Rhizosphere Biology, Colorado State University, 1173 Campus Delivery, Fort Collins, CO 80523-1173, USA. e-mail: [email protected] article was submitted to Frontiers in Plant Metabolism and Chemodiversity, a specialty of Frontiers in Plant Science. 05 7 2012 2012 3 14902 3 2012 16 6 2012 Copyright © 2012 Badri, Chaparro, Manter, Martinoia and Vivanco.2012This 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.The roots of plants secrete compounds as a way to exchange information with organisms living in the soil. Here, we report the involvement of seven root-expressed ATP-binding cassette (ABC) transporters corresponding to both full and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3, Atnap5, and Atath10) in root exudation processes using Arabidopsis thaliana. Root exuded phytochemicals were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), and it was determined that some of the root exudates from the corresponding ABC transporter mutants were significantly different compared to the wild type. For example, Atabcg37 and Atabcc5 secreted higher levels of the phytoalexin camalexin, and Atabcg36 secreted higher levels of organic acids, specifically salicylic acid (SA). Furthermore, we analyzed the root tissue metabolites of these seven ABC transporter mutants and found that the levels of SA, quercetin, and kaempferol glucosides were higher in Atabcg36, which was correlated with higher expression levels of defense genes in the root tissues compared with the wild type. We did not observe significant changes in the root exudates of the half-size transporters except for Atabcf1 that showed lower levels of few organic acids. In summary, full-size transporters are involved in root secretion of phytochemicals. ABC transportersphytoalexinroot exudatesdisease resistancesalicylic aciddefense proteins ==== Body Introduction ATP-binding cassette (ABC) transporters are one of the largest protein families in bacteria, fungi, plants, and animals, including both membrane bound and soluble proteins (Henikoff et al., 1997; Yazaki, 2005; Rea, 2007; Verrier et al., 2008; Yazaki et al., 2009). In prokaryotes, most members of this family are involved in high-affinity uptake of small molecules like maltose or histidine (Higgins, 1992). In humans, ABC transporters play a key role in multidrug resistance of cancer cells, by detoxifying chemotherapeutic drugs; and in genetic diseases such as cystic fibrosis (Gottesman and Pastan, 1993; Borst and Elferink, 2002). In plants, ABC transporters are responsible for the transportation of a multitude of compounds (Kang et al., 2010). They are involved in the excretion (extracellular over the plasma membrane or intracellular into the vacuoles of plants) of potentially toxic compounds, lipid translocation, transport of steroids and their derivatives, conferring heavy metal tolerance, and transporting phytohormones (Yazaki, 2005; Rea, 2007; Yazaki et al., 2009; Kang et al., 2010). Recent reports have shown that the Arabidopsis ABC transporters AtPDR8, AtPDR12, and NpPDR1 are involved in plant defense responses against Pseudomonas syringae pv. Lycopersici (DC3000), Peronospora parasitica, Phytophthora infestans, Golovinomyces orontii, and Plectosphaerella cucumerina (Lipka et al., 2005; Kobae et al., 2006; Stein et al., 2006). It has also been demonstrated that AtPDR8 and AtPDR9 are involved in regulation of auxin homeostasis and plant development by directional transport of the auxin precursor indole-3-butyric acid (IBA; Strader and Bartel, 2009; Ruzicka et al., 2010). In addition, it was demonstrated that AtPDR8 was required for the extracellular accumulation of the glucan polymer callose, in response to treatment with a microbe-associated molecular pattern (MAMP) derived from bacterial flagellin (Clay et al., 2009). Recent literature has also reported that some ABC transporters are involved in the secretion of compounds by roots (Badri et al., 2008, 2009). Roots secrete phytochemicals into the soil that mediate complex interactions with soil-borne organisms (Badri et al., 2009). Recently, it has been reported that root-specific ABC transporters of Arabidopsis are involved in exudation of phytochemicals (Badri et al., 2008), and that mutants of a specific transporter (Atabcg30) when grown in natural soils of Arabidopsis promote a complete overhaul of the microbial community found in association with wild-type Arabidopsis roots (Badri et al., 2009). Specifically, the Atabcg30 mutant promoted the growth of beneficial bacteria such as plant growth promoting rhizobacteria (PGPRs), nitrogen fixers, and bacteria involved in heavy metal remediation. Metabolomic profiling revealed an increase in phenolic compounds and a decline in sugars in the root exudates of the Atabcg30 mutant compared to the wild type and other ABC transporter mutants. Therefore, it was hypothesized that the modified ratios of sugars to phenolics in the root exudates of the mutant promote the growth of specific soil microbes at the expense of others. The Arabidopsis genome encodes 129 ABC transporter proteins that are sub-divided into 13 subfamilies (Sanchez-Fernandez et al., 2001; Martinoia et al., 2002; Garcia et al., 2004; Rea, 2007); exceeding the numbers reported in yeast (Decottignies and Goffeau, 1997) and in humans (Dean et al., 2001). Interestingly, 25 of these transporters are highly expressed in roots, and the root exudate profiles of seven of those mutants have been determined (Badri et al., 2008). Based on previous studies highlighting the dramatic changes that one mutation in an ABC transporter can have on the overall diversity of compounds secreted by roots, we report here the effect of seven additional root-expressed ABC transporter mutants, corresponding to both full-size and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3, Atnap5, and Atath10), in terms of root exudates secretion. Materials and Methods Plant material and growth conditions Arabidopsis seeds were surface-sterilized with laundry bleach for 1 min followed by four rinses in sterile distilled water and plated on Murashige and Skoog (MS; Murashige and Skoog, 1962) salts supplemented with 3% sucrose and 0.7% bactoagar in Petri plates. Petri plates were incubated in a growth chamber (Percival Scientific) at 25°C, with a photoperiod of 16 h light/8 h dark for germination. For vertical plate assays, mutant and wild-type seeds were plated on MS agar plates supplemented with 0.5 and 1% sucrose and incubated in a growth chamber with a photoperiod of 8 h light/16 h dark and readings were taken after 14 days. The list of ABC transporter T-DNA KO mutants used in this study is provided in Table A1 in Appendix. Screening of T-DNA mutant homozygous lines T-DNA mutants for the seven ABC transporter genes used in this study were obtained from the Arabidopsis Biological Resources Center (ABRC) and from the mutant collection of Dr. Enrico Martinoia. The T-DNA insertion and homozygote nature of the mutants were verified by PCR analysis with a gene-specific primer and left border primer, and further confirmed by sequencing. The complete knockout of the gene product was confirmed by RT-PCR. Briefly, total RNA was isolated from root tissue using a Trizol reagent and subsequently cDNA was prepared using Superscript Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. PCR was performed using gene-specific primers. The primers used for homozygote screening and gene expression are listed on Tables A2 and A3 in Appendix. Root exudate collection and phytochemical extraction To collect root exudates, 7-day-old seedlings were transferred to six-well culture plates (Fischer Co.) each containing 5 ml of liquid MS (MS basal salts supplemented with 1% sucrose), incubated on an orbital shaker at 90 rpm and illuminated under cool white fluorescent light (45 μmol m−2 s−1) with a photoperiod of 16 h light/8 h dark at 25°C ± 2. According to the methods of Badri et al. (2008, 2009), when plants were 18 days old they were washed with sterile water to remove the surface adhering exudates and transferred to new six-well plates containing fresh 5 ml MS liquid media and incubated on an orbital shaker at 90 rpm and illuminated under cool white fluorescent light (45 μmol m−2 s−1) with a photoperiod of 16 h light/8 h dark at 25°C ± 2. The exudates were collected 3 days after transfer (plants were 21 days old). For each replicate analysis, we collected 60 ml exudates from 12 individually grown Arabidopsis plants. The collected root exudates were filtered using nylon filters of pore size 0.45 μm (Millipore) to remove root sheathing and root-border-like cells. After filtration, the exudates were freeze-dried (Labconco), dissolved in 5 ml double distilled water and the pH was adjusted to 3.0 with 1 N HCl and partitioned two times with equal volume of ethyl acetate (Fisher Scientific). The ethyl acetate fractions were pooled and dried under nitrogen gas. The dried concentrate was again dissolved in 100 μl absolute methanol (Fisher Scientific) and analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Root tissue metabolites were extracted by grinding 50 mg of freeze-dried root tissue in 5 ml of 80% methanol and centrifuging for 20 min at 8000 rpm to collect the supernatant before drying under N2 gas. The dried concentrate was again dissolved in 1 ml methanol and analyzed by HPLC-MS. The experiment was repeated twice and each experiment had three replicates. High-performance liquid chromatography and mass spectrometry analyses The phytochemicals extracted from root exudates were chromatographed by gradient elution on a 150 mm × 4.6 mm reverse phase, C18 column (Dionex). The chromatographic system (Dionex Co., Sunnyvale, CA, USA) consisted of two P680 pumps connected to an AS1-100 automated sample injector and detected at 280 nm with a UV-visible detector. Mass determination of the peaks was analyzed by an MSQ-MS detector system (Thermo Electron Co., Waltham, MA, USA). A gradient was applied for all separations with a flow rate of 0.7 ml min−1. The gradient was as follows: 0–3 min, 90% (v/v) water and 10% (v/v) methanol; 3–43 min, 10–90% (v/v) methanol, 90–10% (v/v) water; 43–51 min, 90% (v/v) methanol and 10% (v/v) water. Gas chromatography and mass spectrometry analyses Root exudates for gas chromatography-mass spectrometry (GC-MS) analyses were collected similarly as for the HPLC-MS analyses except that the 18-day-old plants were washed in sterile water and transferred into new six-well plates containing 5 ml sterile water instead of MS liquid media with 1% sucrose. This procedure was adopted to prevent the interference of exogenously supplemented salts and sucrose present in the MS liquid media for the subsequent GC-MS analyses. After 3 days’ continuous secretion, the root exudates were collected, freeze-dried, and processed following the standard methoximation and trimethylsilylation derivative procedure (Broeckling et al., 2005). One microliter of each sample was injected onto an Agilent 6890 GC coupled to a 5973 MS at a split ratio of 1:1. The initial oven temperature was 80°C and was held for 2 min, ramped at 5°C min−1 to a final temperature of 315°C and then held for 12 min. Separation was achieved using a 60 m DB-5MS (J & W Scientific – 0.25 mm ID and 0.25 μm film thickness) at a flow rate of 1.0 ml min−1. Data transformation and statistical analysis Data from HPLC-MS analysis of root exudates and root tissue metabolite profiles were analyzed by calculating the retention times in minutes and peak area for each sample. Data detected at wavelengths of 254, 280, and 310 nm were analyzed but only data from 280 nm are shown here because the results were similar in all three wavelengths. The most prominent 25 peaks that were consistently present in all replicates of a given mutant were considered for further multivariate statistical analysis. For GC-MS analysis, peak detection and deconvolution was performed with automated mass spectral deconvolution and identification system (AMDIS; Halket et al., 1999) for several samples of each treatment and peak lists were compiled in a metabolomics ion-based data extraction algorithm (MET-IDEA; Broeckling et al., 2006). MET-IDEA was then used to extract quantitative peak area values for polar and non-polar data. Redundant peaks were removed from the data set, peak area values were scaled to mean zero and standard deviation 1.0 and the resulting data matrix analyzed for further multivariate analyses. Compound identifications were performed by matches with the mass spectral library developed by Dr. Lloyd Sumner laboratory, Samuel Roberts Noble Foundation. Relative Sorenson distances were calculated between GC-MS or LC-MS profiles and visualized by Bray–Curtis ordination (endpoint selection: Bray–Curtis original, Axis Projection and Residual Distances: Euclidean). Statistical differences between mutants were determined by using the non-parametric multi-response permutation procedure (MRPP) assuming a completely randomized design with one factor (i.e., mutant). All analyses were conducted in PC-ORD (McCune and Grace, 2002). Identification of candidate peaks showing quantitative and qualitative differences in ABC transporter mutant root exudates and root tissue metabolites by HPLC-MS In an effort to identify the missing compounds in the ABC transporter mutants compared to the wild type, we identified compound 4 in the root exudates with retention time 40.0 and positive ESIMS201 as camalexin by running standard camalexin that was obtained as a gift from Dr. Eugene Nester, University of Washington. Similarly, we identified four additional compounds from the root tissue metabolites profiles: peak retention time 24.3 and positive ESIMS: m/z 741 is kaempeferol-3-O-rutinoside-7-O-α-l-rhamnopyranoside (C33H40O19); peak retention time 27.1 and positive ESIMS: m/z 611 is quercetin 3-O-glucoside-7-O-rhamnoside (C27H30O16); peak retention time 29.0 and positive ESIMS: m/z 595 is quercetin 3,7-di-O-α-l-rhamnopyranoside (C27H31O15); and peak retention time 31.8 and positive ESIMS: m/z 579 is kaempferol 3,7-α-l-dirhamnoside (C27H29O14) by running authentic standard compounds isolated from Arabidopsis root tissues and characterized by proton nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LCMS; low resolution), and finally by LCMS/MS (high resolution; Badri et al., 2008). Total salicylic acid (SA) extractions were performed on root tissues of 18-day-old plants as described by Bowling et al. (1994) and measured by GC-MS. RNA isolation and semi-quantitative reverse transcriptase polymerase chain reaction Total RNA was isolated from frozen root tissues collected from different mutants and wild type by using the RNAeasy midi kit (Qiagen Inc.) and quantified by Nanodrop ND-1000 Spectrophotometer. RNA integrity was checked on a formamide denaturing agarose gel. Two micrograms of purified total RNA from root tissues were reverse-transcribed using Superscript III RT and poly (T) primer at 42°C for 1 h (Invitrogen) according to the manufacturer’s instructions. The reaction product was diluted to a concentration of 50 ng μl−1 and 1 μl used for each polymerase chain reaction (PCR) reaction. The reaction mix (20 μl) contained 0.4 μmol of each gene-specific primer, 200 μmol of dNTPs, 1× reaction buffer, and one unit of Taq DNA polymerase (Takara). PCR included 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min in an Applied Biosystems thermal cycler (GeneAmp PCR system 2700). Actin primers were used as a control to determine the uniformity of the concentration of cDNA. The gene-specific primers used for RT-PCR assays are listed in Table A4 in Appendix. Results Phenotypic characterization of ABC transporter mutants Organ-specific expression of the seven ABC transporters was determined by RT-PCR in root, leaf, and flower tissues of the wild-type Col-0. Among the seven ABC transporters, only AtABCF1 was expressed equally in roots, leaves, and flowers. In contrast, the other six ABC transporters (AtABCG36, AtABCG37, AtABCC5, AtABCF3, AtNAP5, and AtATH10) showed higher levels of expression in the roots compared to leaves and flowers (Figure 1A). Further, we determined the T-DNA insertions for the ABC transporter mutant’s Atabcf1, Atabcf3, Atnap5, and Atath10 were found on the first, fifth, third, and second exons, respectively, in their genes (data not shown). RT-PCR assays on root tissues from all seven mutant homozygous lines verified the complete knockout of the corresponding gene, except Atabcf3, which had a faint band because the primers designed to amplify the fragment are upstream of the T-DNA insertion sequence (Figure 1B). Root phenotype analysis showed that the Atath10 mutant has a significant increase in the number of lateral roots compared with other mutants and the wild type (Figure 1C), when grown under MS media supplemented with 0.5% sucrose as a carbon source. In contrast, there were no significant differences observed in primary root length compared with the other mutants and the wild type (Figure 1D). Besides this difference, there were no observable differences in terms of shoot morphology, bolting, and flowering time between the mutants and the wild type under the conditions tested. We did not perform the root phenotype assays for mutant’s Atabcc5, Atabcg36, and Atabcg37 because these were already described in the literature. Mutants Atabcg36 and Atabcg37 did not show any phenotypic differences when compared with wild type (Ito and Gray, 2006; Strader and Bartel, 2009), but Atabcc5 exhibits decreased root growth and increased lateral root formation (Gaedeke et al., 2001). Figure 1 Phenotypic characterization of ABC transporter T-DNA KO mutants used in this study. (A) Organ-specific expression of ABC transporters in wild type roots, leaves, and flowers. (B) RT-PCR assays of the ABC transporter mutants used in this study. (C) Characterization of root phenotype by growing wild type and mutants in MS agar medium supplemented with 0.5 and 1% sucrose. Number of lateral roots was measured when the seedlings were 14 days old. (D) Characterization of root phenotype by growing wild type and mutants in MS agar medium supplemented with 0.5 and 1% sucrose. Root length was measured when the seedlings were 14 days old. cDNA was prepared from respective tissues. Total RNA and RT-PCR was performed using gene-specific primers. The lower panel is the actin control for the cDNA of wild type root, leaf, and flower. R, root; L, leaf; F, flower. The results represent experiments repeated two times with three replicates each. Analysis of secondary metabolites in the root exudates of ABC transporter mutants by HPLC-MS We examined the role of these ABC transporters in the root secretion of phytochemicals by examining the root exudation profiles of the mutants by HPLC-MS and comparing those to the wild-type profiles (see Materials and Methods). Comparison of the root exudates’ profiles by MRPP analysis showed that the mutants Atabcg37, Atabcc5, Atabcf3, Atnap5, and Atath10 were significantly different from the wild type (Figure 2; Tables A5 and A6 in Appendix). In addition, a careful peak-by-peak analysis allowed determining four non-polar phytochemicals missing in the root exudates of the various mutants compared to that of the wild type. (Table 1; Figure A1 in Appendix). Compound 1, with a retention time of 13.8 min and a molecular mass of 277, was absent only in the exudates of Atabcg37. Similarly, compound 2 with retention time of 35.58 and molecular mass of 176 was only absent in the exudates of Atabcc5. In contrast, compound 3 with retention time of 39.3 and molecular mass of 224 was present only in Atabcg37 and absent in the other mutants and wild type. Compound 4 with a retention time 40.0 and a molecular mass of 200 was identified as camalexin and was present only in the exudates of Atabcc5 and Atabcg37, but absent in the other mutants and wild type. Table 1 Retention times and molecular masses of the compounds missing in the ABC transporter mutants compared with wild type after 3 days’ secretion (21-day-old plants) of root exudate profiles analyzed by HPLC-MS. Retention time (min), molecular mass Col-0 Atath10 Atabcg36 At abcf3 Atabcf1 Atnap5 Atabcc5 Atabcg37 13.8, 277 + + + + + + + − 35.58, 176 + + + + + + − + 39.3, 224 − − − − − − − + 40.0, 200 − − − − − − + + +, indicate presence of compound; −, indicate the absence of compound. Figure 2 Bray–Curtis ordination analyses with Sorenson distance of the root exudates of wild type and ABC transporter mutants constructed from HPLC-MS data obtained from root exudates profiles. Analysis of primary and secondary metabolites in the root exudates of ABC transporter mutants by GC-MS We also analyzed the primary and secondary metabolites present in the root exudates profiles of the mutants by GC-MS and compared the results with wild type’s root exudate profiles. Comparison of the GC-MS profiles of the mutants and wild type by MRPP revealed that all of the mutants were significantly different from the wild type (Figure 3; Tables A5 and A6 in Appendix). Further quantitative analysis of the identified compounds showed that these mutants exuded varying levels of organic acids and sugars compared to the wild type. Mutant Atabcc5 showed significantly lower levels of organic acids (oxalic acid, succinic acid, fumaric acid, malic acid, trihydroxybutyric acid, p-hydroxy benzoic acid, etc.). In contrast, Atabcg36 exudates contained significantly higher levels of organic acids (oxalic acid, benzoic acid, succinic acid, fumaric acid, nicotinic acid, SA, vanillic acid, p-hydroxy benzoic acid, etc.) compared to the wild type and the other mutants (Figure A2 in Appendix). We did not observe any significant differences in the levels of sugars between the mutants and wild type except that Atabcc5 had significantly decreased levels of ribose and d-galactose in the root exudates (Figure A3 in Appendix). In addition, there were significant differences in the “unknown” compounds between mutants and wild type. Figure 3 Bray–Curtis ordination analyses with Sorenson distance of the root exudates of wild type and ABC transporter mutants constructed from GC-MS data obtained from root exudates profiles. Root tissue metabolites profiles of ABC transporter mutants by HPLC-MS analysis The root tissue metabolite profiles were also analyzed in all seven mutants at the same time point (21 days old) as those used for the root exudates collection. The LC-MS profiles utilized only the retention times and peak areas of the 17 most prominent peaks present in all of the samples. Based on the MRPP analysis, we found that the profiles of all the mutants, except Atabcc5, were significantly different from those of wild type and the other mutants (Figure 4; Tables A5 and A6 in Appendix). Among those 17 peaks, four peaks were identified based on the retention time (Rt), positive-ion molecular mass (M+), and UV absorbance by running the authentic standard compounds as follows: kaempeferol-3-O-rutinoside-7-O-α-l-rhamnopyranoside (Rt = 24.3, M+ = 741), quercetin 3-O-glucoside-7-O-rhamnoside (Rt = 27.1, M+ = 611), quercetin 3,7-di-O-α-l-rhamnopyranoside (Rt = 29.0, M+ = 595), and kaempferol 3,7-α-l-dirhamnoside (RT = 31.8, M+ = 579). We further analyzed the profiles with peak-by-peak analysis and found both quantitative and qualitative differences in the mutants compared with wild type. For example, the compounds with retention time 25.58, 49.31, and 51.24 were absent in Atabcg37 but those compounds were present in wild type and other mutants (Table 2). Similarly, we observed that several other compounds differed quantitatively in the mutants compared with wild type and some are worth noting here (Table 2). For example, the concentrations of kaempeferol-3-O-rutinoside-7-O-α-l-rhamnopyranoside (Rt = 24.4 and M+ = 741), quercetin 3-O-glucoside-7-O-rhamnoside (Rt = 27.1 and M+ = 611), and quercetin 3,7-di-O-α-l-rhamnopyranoside (Rt = 29.0 and M+ = 595) were significantly higher in Atabcg36 compared with the wild type. Similarly, kaempeferol-3-O-rutinoside-7-O-α-l-rhamnopyranoside concentration was also higher in Atath10 compared with the wild type. Interestingly, we did not observe camalexin in the root tissue metabolites profiles of wild type and the other transporter mutants used in this study but we observed camalexin only in the root exudates profiles of the mutants Atabcg37 and Atabcc5. Table 2 Retention times of the compounds of the root tissue profiles of 21-day-old wild type and ABC transporter mutants analyzed by HPLC-MS. RT Col-0 Atabcf1 Atabcf3 Atath10 Atabcc5 Atnap5 Atabcg36 Atabcg37 10.42 132.3 (8.9) 145.5 (4.9) 105.1 (6.7) 59.5 (5.2) 42.8 (2.9) 94.3 (7.6) 135.7 (15.2) 44.6 (2.7) 14.91 4.9 (0.2) 10.3 (0.5) 10.3 (0.1) 12.4 (0.7) 5.7 (0.1) 6.4 (0.5) 11.6 (0.3) 7.5 (0.7) 19.0 11.0 (1.3) 18.4 (0.7) 25.3 (0.7) 30.8 (2.6) 33.5 (1.3) 20.5 (1.9) 28.8 (0.3) 18.5 (1.5) 20.36 16.9 (1.1) 23.5 (0.8) 35.8 (0.8) 36.6 (2.7) 31.7 (0.8) 28.5 (3.0) 40.5 (0.9) 20.1 (1.2) 23.0 6.5 (1.7) 9.7 (0.5) 3.6 (0.5) 8.4 (2.0) 3.1 (0.8) 10.5 (0.7) 17.5 (1.3) 2.5 (0.3) 24.33 20.7 (1.7) 28.1 (0.7) 25.9 (1.4) 34.3 (0.7) 23.3 (0.6) 24.2 (1.8) 32.7 (0.8) 33.4 (2.6) 25.58 26.1 (2.9) 27.8 (0.7) 16.4 (1.3) 21.4 (1.5) 21.6 (1.3) 16.8 (1.2) 22.6 (0.8) 0 (0) 27.11 18.1 (1.5) 23.1 (0.5) 28.5 (0.7) 35.4 (2.6) 11.0 (1.0) 30.2 (2.3) 48.1 (1.5) 29.5 (1.7) 29.0 29.1 (2.6) 39.7 (1.0) 39.8 (1.3) 49.1 (3.1) 28.1 (1.1) 41.1 (2.3) 57.9 (1.2) 37.0 (2.6) 30.59 17.3 (1.5) 22.1 (0.4) 14.7 (0.7) 19.3 (1.2) 10.8 (0.3) 15.3 (0.9) 18.1 (0.6) 12.4 (0.8) 31.38 18.8 (4.6) 37.7 (1.5) 10.0 (0.4) 37.7 (0.7) 36.1 (1.1) 12.2 (0.7) 17.9 (1.9) 10.9 (0.7) 34.86 17.3 (2.0) 27.5 (1.9) 17.2 (1.7) 33.1 (0.6) 16.7 (0.6) 16.5 (1.4) 26.7 (1.2) 5.3 (0.5) 36.6 2.4 (0.0) 3.9 (0.5) 2.9 (0.2) 4.55 (0.7) 1.3 (0.0) 1.5 (0.0) 1.7 (0.0) 2.8 (0.3) 37.32 48.0 (4.6) 74.1 (3.7) 59.3 (2.9) 70.1 (0.5) 33.5 (1.5) 65.1 (4.8) 59.2 (1.5) 32.6 (2.0) 40.49 7.6 (1.3) 11.2 (1.8) 6.5 (1.2) 15.6 (0.9) 4.8 (0.7) 14.2 (1.1) 7.9 (0.8) 9.7 (1.6) 49.31 6.7 (0.8) 10.4 (1.3) 7.8 (0.7) 12.5 (0.9) 4.7 (0.4) 9.4 (1.2) 10.4 (0.8) 0 (0) 51.24 10.2 (1.4) 15.9 (2.3) 8.1 (0.7) 14.2 (1.8) 6.5 (0.6) 8.4 (0.9) 13.6 (1.1) 0 (0) Values represented are peak areas of the corresponding compounds in the wild type and mutants. The values in bold are statistically significant when compared to the wild type at p ≤ 0.05, n = 8. Values represented in parentheses are SE values. Figure 4 Bray–Curtis ordination analyses with Sorenson distance of the root tissue metabolites of wild type and ABC transporter mutants constructed from HPLC-MS data obtained from root tissue metabolites profiles. Further characterization of Atabcg36 in defense response Based on our primary and secondary metabolites’ analyses, Atabcg36 mutants (both Atabcg36-1 and Atabcg36-2) secreted higher concentrations of organic acids such as SA, vanillic acid, oxalic acid, nicotinic acid, and other unknown compounds in the root exudates. Previous published information has involved Atabcg36 in non-host resistance and heavy metal tolerance (Kobae et al., 2006; Stein et al., 2006; Kim et al., 2007). Taking this information together, we hypothesized that higher levels of SA secretion by roots indirectly indicates the increase in the concentration of SA in tissues that contributes to the disease resistance response exhibited by Atabcg36. Therefore, we measured the relative SA concentrations in the root tissues of wild type and Atabcg36 mutants (Atabcg36-1 and Atabcg36-2) and found that there was a significant increase in SA concentration in the mutant plants of Atabcg36 compared to wild type (Figure 5). The increase in concentration of SA in Atabcg36 is probably due to a pleiotropic effect in the gene mutation. The observed variability in the SA concentrations between the mutant lines is unexplainable and warrants further studies in order to dissect the specific pleiotropic effects. We further investigated the basal resistance level in Atabcg36 by determining the expression of defense genes in the root tissues compared with that of the wild type under aseptically grown conditions. We found that Atabcg36 showed higher levels of expression of defense genes’ (PR1, PR2, PR5, and PDF1.1) in the root tissues compared with the wild type (Figure 6A). We did not observe the expression of PR1, PR5, and PDF1.1 in wild type roots. It was reported that mutations in certain transporters could modify the otherwise normal expression of other transporters in Arabidopsis (Badri et al., 2009). Thus, we analyzed the expression of other ABC transporters that could be highly expressed in roots (Badri et al., 2008) of Atabcg36 to take over the function of ABCG36. We checked the expression of 15 ABC transporters which includes both full-size and half-molecule transporters. Among those, only AtABCB1 (full-size transporter) expression was up-regulated in Atabcg36 compared to the wild type (Figure 6B). However, further evaluation is required to corroborate these gene expression patterns by real-time quantitative PCR analysis. Figure 5 Total salicylic acid concentrations of the root tissues of wild type and Atabcg36 mutant lines (Atabcg36-1 and Atabcg36-2). *Indicates the values are significant at p-value below 0.05 compared to wild type. Values represented are the mean of three biological replicates. Figure 6 Gene expression analyses of wild type (Wt) and Atabcg36 by RT-PCR assays. (A) RT-PCR assay of defense genes expressions in wild type and Atabcg36 root tissues. (B) RT-PCR assay of ABC transporters gene expressions in wild type and Atabcg36 root tissues. *Indicates the gene expression of AtABCB1 is higher in Atabcg36 compared to wild type. The results represent experiments repeated two times with three replicates each. Discussion The ABC proteins encompass a large protein family ubiquitous in all organisms, which includes both membrane bound and soluble proteins (Higgins, 1992; Yazaki et al., 2009). These proteins are classified as primary transporters which are directly energized by ATP hydrolysis to translocate solutes across cellular membranes (Higgins, 1992), a process that is independent of membrane potential and proton gradients across the membrane. ABC proteins are involved in various biochemical and physiological functions in plants, few are involved in defense mechanisms against biotic stresses (Yazaki et al., 2009) and others are involved in basic functions necessary for plant life such as hormone transport or phytol accumulation (Geisler and Murphy, 2006; Nagy et al., 2009). A recent report demonstrated the influence of some full-size ABC transporters in root exudation processes (Badri et al., 2008) by employing a mutant approach. In an expansion of that study, here we presented a detail characterization of a distinct set ABC transporters highly expressed in root cells (Badri et al., 2008) and their role in root exudation. ABC transporters are involved in root secretion of phytochemicals Based on our metabolomic analyses (HPLC-MS and GC-MS) the root exudates profiles of the mutants Atabcg36, Atabcg37, Atabcc5, Atabcf3, and Atath10 were significantly different from those of the wild type (Figures 2 and 3). Qualitative analyses of HPLC-MS root exudate profiles revealed that one compound (Rt = 13.8 and M+ = 277) was completely absent in Atabcg37 compared with the wild type and the other mutants used in this study. Similarly, another compound (Rt = 35.58 and M+ = 176) was absent in Atabcc5 compared with the other mutants and the wild type (Table 1). These results reinforce the previous notion describing that one transporter could transport a particular type of compound (Badri et al., 2008). Also, the differences observed in the secretion of phytochemicals by different ABC transporters could be attributed to their localization in the cell. For example, AtABCG36 and AtABCG37 are localized in the plasma membrane and AtABCC5 is localized in the vacuolar membranes (Dunkley et al., 2006; Knoller and Murphy, 2011). Previously, ABCG36 was reported to be involved in resistance to heavy metal (cadmium) and auxinic herbicides (Ito and Gray, 2006; Kim et al., 2007). It has also been reported that ABCG36 and ABCG37 are involved in regulation of auxin homeostasis and plant development by directional transport of the auxin precursor IBA (Strader and Bartel, 2009; Ruzicka et al., 2010). On the other hand, ABCC5 is involved in root development and stomata movement by transporting inositol hexakis phosphate (Gaedeke et al., 2001; Nagy et al., 2009). Based on the earlier published results and the results of the present study, it is assumed that one ABC protein might be involved in different kinds of functions depending upon their expression in particular plant organs. In contrast, we did not observe any qualitative differences in the root exudates profiles of secondary metabolites in the half-size transporters mutants (AtATH10, AtABCF1, AtABCF3, and AtNAP5) compared with the wild type. Therefore, these half-size transporters might not be involved in exporting secondary metabolites from the root cells. This is probably true, because these half-size transporters are thought to be soluble since they lack any detectable transmembrane domain and probably function in processes other than transport, as is the case for their yeast and human orthologs, which participate in ribosome recycling and translational control (Vazquez de Aldana et al., 1995; Tyzack et al., 2000; Braz et al., 2004; Dong et al., 2004; Pisarev et al., 2010). However, the phenotype differences observed in the root exudates profiles of these half-size transporter mutants need further confirmation by examining other allelic mutants. Interestingly, we found one compound (Rt = 40.0 and M+ = 201), identified as camalexin, which is present significantly only in the root exudates of Atabcc5 and Atabcg37 (Table 1; Figure A4 in Appendix). However, we observed the camalexin mass trace in other mutant lines and wild type but they were relatively very low and close to the threshold levels (background noise; Figure A4 in Appendix). Camalexin is a phytoalexin produced by the plant in response to pathogen infection and MAMPs but absent in plants without stress (Consonni et al., 2010; Millet et al., 2010). The secretion of camalexin in Atabcc5 and Atabcg37 is probably due to the pleiotropic effect of these mutations resulting in an elevated expression of genes involved in indolic metabolite biosynthesis, as reported in the case of Arabidopsis MLO2, 6, and 12 mutants (Consonni et al., 2010). We also analyzed the root tissue metabolite profiles of all the mutants by HPLC-MS analyses and did not find camalexin in the root tissues of any of the mutants or the wild type, which indicates that this compound might be toxic to the cells and extruded outside the cells by other means of transportation (Rogers et al., 1996; Glawischnig, 2007). The genetics and biochemistry of the biosynthesis and secretion of camalexin in Atabcc5 and Atabcg37 root exudates warrant further experimentation. Besides the absence of camalexin in the root tissue metabolite profiles of all mutants and wild type, we observed significant quantitative differences in other compounds between the wild type and the mutants used in this study (Table 2). Among those compounds, the concentrations of kaempeferol-3-O-rutinoside-7-O-α-l-rhamnopyranoside, quercetin 3-O-glucoside-7-O-rhamnoside, and quercetin 3, 7-di-O-α-l-rhamnopyranoside were significantly higher in the root tissues of Atabcg36. Circumstantial evidence indicates that flavonoids function in plant defense against bacterial and viral pathogens; however specific molecule interactions have yet to be identified (Shirley, 1996; Iriti and Faoro, 2009). Specifically, glycosides of the flavonols, kaempferol, and quercetin have been shown to induce the vir genes of Agrobacterium tumefaciens (Zerback et al., 1989). In addition, Atabcg36 root exudates showed higher concentrations of organic acids like SA, vanillic acid, p-hydroxybenzoic acid, nicotinic acid, etc. in our GC-MS analyses (Figure A2 in Appendix). We found higher concentrations of SA in the root tissues compared to wild type, which partly explains Atabcg36 role in non-host resistance (Kobae et al., 2006; Stein et al., 2006). Basal defense level is higher in Atabcg36 Previous reports have shown that the Arabidopsis ABC transporters AtABCG36, AtABCG40, and NpPDR1 are involved in plant defense responses (Lipka et al., 2005; Kobae et al., 2006; Stein et al., 2006). For example, Stein et al. (2006) demonstrated that SA genes are hyperinduced in Atpen3/Atabcg36 after pathogen inoculation. In addition, it was demonstrated that AtABCG36 was required for the extracellular accumulation of the glucan polymer callose, mediated by the glucan synthase like enzyme PMR4/GSL5, in response to treatment with a MAMP derived from bacterial flagellin (Clay et al., 2009). However, microbes can trigger callose formation in Arabidopsis to restrict further pathogen growth via the plant hormone SA-dependent pathway (DebRoy et al., 2004). In the present study, we found higher concentrations of SA in the root exudates, and SA, kaempeferol, and quercetin glucosides in the root tissues of Atabcg36 which suggested that the basal defense levels of Atabcg36 are higher than the wild type. It is worth noting here that the biosynthesis of these three compounds (SA, kaempferol, and quercetin) is derived from phenylalanine involved in the phenylpropanoid pathway, though SA can also be synthesized from the isochorismate pathway (Iriti and Faoro, 2009). Our results showed that the basal expression level of defense genes PR1, PR2, PR5, and PDF1.1 are higher in Atabcg36 compared to the wild type (Figure 6A). Previously, it was reported that the expression of PR proteins is higher in the leaves of Atabcg36 grown under non-sterile conditions without challenging with pathogen inoculation, but might be induced by beneficial microbes present in the soil (Kobae et al., 2006). In addition, AtABCG36 expression was also up-regulated upon pathogen inoculation (Kobae et al., 2006). In the present study, we showed that the basal defense proteins (PR proteins) expression is higher in the roots of Atabcg36 grown under sterile conditions without challenging the plant with any pathogen or beneficial microbe. This higher expression of PR proteins is likely induced by the over-production of SA in the roots of Atabcg36 plants. It is worth noting that callose formation in Arabidopsis in response to MAMPs is initiated by SA-dependent and SA-independent pathways (DebRoy et al., 2004; Bednarek et al., 2009). Further mechanistic studies are needed to dissect the specific pathways modified by the AtABCG36 mutation. In addition, Atabcg36 will serve as a good model system to study the regulatory networks associated with elevated basal defense response in the host compared with those induced upon pathogen attack. Overall this study draws the following conclusions: (1) Full-size ABC transporters that are highly expressed in the root cells play a role in the root secretion of secondary metabolites. (2) Basal defense gene expression levels are higher in the Atabcg36 mutant due to overproduction of SA. (3) Mutations of ABC transporters like ABCC5 and ABCG37 induce the secretion of the phytoalexin camalexin. (4) This study opens a new dimension for the role of ABC transporters in defense response and plant-microbe interaction in addition to their function in the transportation of substances across cellular membranes. This study also provides clues for developing strategies for disease resistance. Appendix Figure A1 Root exudates profile of 21-day-old wild type (Col-0) and ABC transporter mutant plants analyzed by HPLC-MS at wavelength 280 nm. Arrows indicate the peaks present or absent in respective mutants. The numbers indicate the positive ESIMS of the peaks. The chromatogram represent experiments repeated two times with three replicates each. Figure A2 Graphs illustrating the representative phenolic compounds that show different levels in the ABC transporter mutants compared to wild type analyzed by GC-MS. *Indicates the values are statistically significant (p < 0.05) compared with wild-type (Col-0) line (n = 8). Figure A3 Graphs illustrating the representative sugars that show different levels in the ABC transporter mutants compared to wild type analyzed by GC-MS. *Indicates the values are statistically significant (p < 0.05) compared with wild-type (Col-0) line (n = 8). Figure A4 Mass trace of compound camalexin (positive ESIMS 201) in the root exudates of wild type (Wt) and ABC transporter mutants used in this study overlayed with authentic camalexin. Table A1 List of ABC transporters and their T-DNA KO mutants used in this study. AGI code New systemic name Mutant name Reference At1g59870 AtABCG36 Salk_000578 (Atabcg36-1) Kobae et al. (2006) Salk_142256 (Atabcg36-2) Kobae et al. (2006) At3g53480 AtABCG37 pdr9-1 Kim et al. (2007) At1g04120 AtABCC5 Mrp5-1 Knoller and Murphy (2011) At5g60790 AtABCF1 Salk_129048 In this study At1g64550 AtABCF3 Salk_055035 In this study At1g71330 AtNAP5 Salk_047823 In this study At4g01660 AtATH10 Salk_105250 In this study Table A2 List of primers used in this study for screening homozygous lines of ABC transporters T-DNA KO mutant lines. AGI code new systemic name Mutant name Primer pairsa At5g60790 AtABCF1 Salk_129048 F: 5′-TGCTCGTCAAGCACAGAGTAA-3′ R: 5′-CTGAGTCACCTTTCCAACGAA-3′ At1g64550 AtABCF3 Salk_055035 F: 5′-CCGGTTTTGAACTGAGAATTTG-3′ R: 5′-CAAAAGAACATGCAACGATTTC-3′ At1g71330 AtNAP5 Salk_047823 F: 5′-CCAACTCAGAAGTTTTTGGC-3′ R: 5′-CTTGACTTTCTTGTTTCCCGTC-3′ At4g01660 AtATH10 Salk_105250 F: 5′-GCTCGTTTGCAATACATCAGAG-3′ R: 5′-TGTTGAATCAGGTACGGTAACG-3′ aF indicates left gene-specific primer and R indicates right gene-specific primer. Table A3 List of primers used in this study for screening the expression of ABC transporter genes in T-DNA KO lines. AGI Code New systemic name Primer pairsa At1g59870 AtABCG36 F: 5′-AGAGCAGCGGCTATTGTGATGA-3′ R: 5′-TGGCGTAGACGATGAGTGAGAT-3′ At3g53480 AtABCG37 F: 5′-TGAGGAGAGGTATAACAGGAGGTC-3′ R: 5′-GAGAGATTCAAAGAACGAGAGAGG-3′ At1g04120 AtABCC5 F: 5′-CCTGCTCTTGGGTTTCTCTGCT-3′ R: 5′-ATGGTGGGCAGACACTAAGGCC-3′ At5g60790 AtABCF1 F: 5′-CTGCGATTGGTAGGTTTGGT-3′ R: 5′-CTCTCAAGTGTGTGGCTTTA-3′ At1g64550 AtABCF3 F: 5′-AAAGCATGGATTGGTGAAGC-3′ R: 5′-CAATTCGCATTCTCCATCCT-3′ At1g71330 AtNAP5 F: 5′-TACATATTGCACGCGCTCTCTACC-3′ R: 5′-TTCACAGGAGCTTGCACATCCTCA-3′ At4g01660 AtATH10 F: 5′-ACGGTAACGGAAGCATCATCTC-3′ R: 5′-GTTGTTGGTTCGGAAAGCATAG-3′ aF indicates forward and R indicates reverse. Table A4 List of primers used in this study for screening the expression of genes for RT-PCR assays. AGI Code Gene Primer pairsa At2g14610 AtPR1 F: 5′-GTAGGTGCTCTTGTTCTTCCC-3′ R: 5′-CACATAATTCCCACGAGGATC-3′ At3g57260 AtPR2 F: 5′-CTACAGAGATGGTGTCA-3′ R: 5′-AGCTGAAGTAAGGGTAG-3′ At1g75040 AtPR5 F: 5′-CACATTCTCTTCCTCGTGTTC-3′ R: 5′-tAGTTAGCTCCGGTACAAGTG-3′ At5g44420 AtPDF1.2 F: 5′-CACCCTTATCTTCGCTGCTC-3′ R: 5′-TGTTTGGCTCCTTCAAGGTT-3′ At1g71330 AtNAP5 F: 5′-TACATATTGCACGCGCTCTCTACC-3′ R: 5′-TTCACAGGAGCTTGCACATCCTCA-3′ At3g47730 AtATH1 F: 5′-TCAAGTGGGTTTCCCTATGC-3′ R: 5′-CCCGGTATCCCGATAAATCT-3′ At3g47780 AtATH6 F: 5′-GGGAAACTTGAGAGGGGAAG-3′ R: 5′-CTTCAGCTCTTTTGGGTTGC-3′ At4g01660 AtATH10 F: 5′-ACGGTAACGGAAGCATCATCTC-3′ R: 5′-GTTGTTGGTTCGGAAAGCATAG-3′ At5g39040 AtTAP2 F: 5′-ATGGAAGCGAGAATGGTTTG-3′ R: 5′-CTACTAGCGCCTGCAGCTTT′-3′ At5g60790 AtABCF1 F: 5′-CTGCGATTGGTAGGTTTGGT-3′ R: 5′-CTCTCAAGTGTGTGGCTTTA-3′ At1g64550 AtABCF3 F: 5′-AAAGCATGGATTGGTGAAGC-3′ R: 5′-CAATTCGCATTCTCCATCCT-3′ At2g34660 AtABCC2 F: 5′-CCGCAGAAATCCTCTTGGTCTTGATG-3′ R: 5′-CCTTGTAAGTGGTGTGAGTCATCTTTGG-3′ At1g04120 AtABCC5 F: 5′-CCTGCTCTTGGGTTTCTCTGCT-3′ R: 5′-ATGGTGGGCAGACACTAAGGCC-3′ At2g36380 AtABCG34 F: 5′-AGATGTTGACGTCACGAATCTTGCT-3′ R: 5′-GTTGCCCTGCGTGAAAAGAATTG-3′ At1g15210 AtABCG35 F: 5′-GGACATACACGCTTCCCACT-3′ R: 5′-AAGCACACTTGTTCCCAACC-3′ At3g53480 AtABCG37 F: 5′-TGAGGAGAGGTATATCAGGAGGTC-3′ R: 5′-GAGAGATTCAAAGAACGAGAGAGG-3′ At2g36910 AtABCB1 F: 5′-AGACCTGGAAGCGGCACATGC-3′ R: 5′-TAGAGTCGCGGCTTGTATGAT-3′ At2g47000 AtABCB4 F: 5′-TTCATCAGTGGTCTGCAACAG-3′ R: 5′-TGAAGCTGAACTAACGAAGCA-3′ aF indicates forward and R indicates reverse. Table A5 Multi-response permutation procedure (MRPP) analysis of the root exudates and root tissue profiles of the wild type and mutants analyzed by GC-MS and HPLC-MS methods. Statistic GC-MS exudates HPLC-MS exudates HPLC-MS tissues Test statistic (T) −12.044 −12.562 −11.834 Observed delta 0.194 0.192 0.095 Expected delta 0.256 0.248 0.192 Variance of delta <0.001 <0.001 <0.001 Skewness of delta −0.440 −0.481 −0.513 p-Value <0.001 <0.001 <0.001 Table A6 Pairwise comparisons of the root exudates and root tissue profiles of the wild type and mutants analyzed by GC-MS and HPLC-MS methods. Comparison p-Values Mutant 1 Mutant 2 GC-MS exudates HPLC-MS exudates HPLC-MS tissues Col-0 Atabcc5 0.001 0.001 0.084 Col-0 Atabcf1 0.001 0.052 0.006 Col-0 Atabcf3 0.002 0.002 0.006 Col-0 Atabcg37 0.003 0.013 0.006 Col-0 Atabcg36 0.002 0.066 0.007 Col-0 Atnap5 0.002 <0.001 0.010 Col-0 Atath10 0.002 <0.001 0.009 Atabcc5 Atabcf1 0.001 0.058 0.008 Atabcc5 Atabcf3 0.001 0.040 0.006 Atabcc5 Atabcg37 0.002 <0.001 0.006 Atabcc5 Atabcg36 0.001 <0.001 0.010 Atabcc5 Atnap5 0.002 <0.001 0.007 Atabcc5 Atath10 0.004 <0.001 0.009 Atabcf1 Atabcf3 0.003 0.011 0.020 Atabcf1 Atabcg37 0.002 0.025 0.007 Atabcf1 Atabcg37 0.001 0.095 0.141 Atabcf1 Atnap5 0.008 <0.001 0.068 Atabcf1 Atath10 0.082 <0.001 0.012 Atabcf3 Atabcg37 0.145 0.009 0.006 Atabcf3 Atabcg36 0.006 0.001 0.007 Atabcf3 Atnap5 0.003 <0.001 0.007 Atabcf3 Atath10 0.021 <0.001 0.010 Atabcg37 Atabcg36 0.010 0.132 0.006 Atabcg37 Atnap5 0.003 <0.001 0.006 Atabcg37 Atath10 0.020 <0.001 0.010 Atabcg36 Atnap5 0.030 <0.001 0.088 Atabcg36 Atath10 0.162 <0.001 0.009 Atnap5 Atath10 0.322 0.466 0.009 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. 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==== Front BMC Res NotesBMC Res NotesBMC Research Notes1756-0500BioMed Central 1756-0500-5-1182236136610.1186/1756-0500-5-118Research ArticleAssociation of body weight and physical activity with blood pressure in a rural population in the Dikgale village of Limpopo Province in South Africa Mkhonto Seth S [email protected] Demetre [email protected] Musawenkosi LH [email protected] Population Health, Health Systems and Innovation, Human Sciences Research Council, 134 Pretorius Street, Pretoria, 0002, South Africa2 Department of Medical Science, University of the Limpopo, Turfloop Campus, Fauna Park Polokwane, 0787, South Africa3 HIV/AIDS, STIs and TB, Human Sciences Research Council, 750 Francois Road, Durban, 4001, South Africa2012 23 2 2012 5 118 118 13 9 2011 23 2 2012 Copyright ©2012 Mkhonto et al; BioMed Central Ltd.2012Mkhonto et al; 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 Africa is faced with an increasing burden of hypertension attributed mainly to physical inactivity and obesity. Paucity of population based evidence in the African continent hinders the implementation effective preventive and control strategies. The aim of this study was to determine the association of body weight and physical activity with blood pressure in a rural black population in the Limpopo Province of South Africa. Methods A convenient sample of 532 subjects (396 women and 136 men) between the ages 20-95 years participated in the study. Standard anthropometric measurements, blood pressure, and physical activity were recorded by trained field workers. Results Anthropometric measurements showed that a high percentage of women were significantly (p < 0.001) overweight and obese than men. Hypertension was significantly high among women (38.1%) compared to men (27.9%). In the univariate analysis mean body mass index (BMI), waist circumference (WC), hip circumference (HC) and waist hip ratio (WHR) showed a significant positive association (p ≤ 0.05) with systolic and diastolic BP in women, and only WHR was statistically significant in men. The odds of being hypertensive also increased with BMI, WC and WHR in both women and men, including HC in women. No relationship was found between physical activity and high blood pressure. In the multivariate analysis only increase in HC and WHR was consistently associated with increase in SBP in women and WHR with hypertension in men. Conclusions The study findings indicate that women in this black South African rural population are overweight and obese than men and are at higher risk of hypertension as determined by selected anthropometric parameters. ==== Body Background High blood pressure (BP) or hypertension is a major public health problem worldwide, the disease is estimated to cause 7.1 million premature deaths globally [1]. Previously, the burden of disease was thought to be highest in developed countries. In fact, until recently, high blood pressure was thought to be rare in Africa. However, there is mounting evidence that developing countries are faced with an increasing burden of both hypertension and cardiovascular disease [2,3]. A number of studies have reported that hypertension is common in both rural and urban African populations due to lifestyle changes [4-7]. Hypertension has been associated with physical inactivity and obesity [8]. However, in most African countries including there is limited evidence base useful for designing and implementing effective preventive and control strategies [3] In South Africa population based studies have also reported higher blood pressure among urbanized black Africans females compared to other race groups [9-12]. Even though there is an indication that the prevalence of hypertension is increasing in rural areas few studies have been conducted in these settings in the country [13]. In 1995 a demographic surveillance survey was established in the Digkale village in the Limpopo Province of South Africa and conducted one of the first measurements of physical activity and anthropometric parameters in a rural black population in the country [14]. Therefore the aim of this study was to investigate the association of body weight and physical activity with blood pressure using the Dikgale Demographic Surveillance System (DDSS). Methods Study area The Dikgale Demographic Surveillance System (DDSS) site is located in the Central Region in Mankweng district, about 40-50 km northeast of Polokwane, the capital city of Limpopo Province (Figure 1). All villages in Dikgale consist of communal grazing land some distance away from residential area. Settlements in Dikgale are a mixture of traditional mud huts, conventional brick houses and shacks with an estimated total population of 7900 people. Few households have water taps in the yards, but most of them fetch water from taps situated at strategic points in the villages. The area is impoverished with high unemployment and a large segment population working as migrant workers, farm labourers and domestic workers. The villages have poor infrastructure and most households have pit latrines with no organized waste disposal and roads are not tarred [14]. Figure 1 Maps of South Africa and Limpopo showing the Dikgale District (shaded in black), the insert is the Dikgale Demographic Surveillence System site (shaded in blue) from Alberts et al. [14]. Study participants Prior to field work Chiefs or Induna's in Dikgale were visited to explain the rationale behind the study and after the visit the Induna's informed the people in the villages. Initially, a random sample of 1000 subjects was generated from the DDSS relational database and distributed to trained fieldworkers. However, fieldworkers reported difficulty in contacting the subjects during house-to-house visits. Because of time and financial constraints, it was decided that fieldworkers would recruit participants house to house, at common meeting places, and through general mouth-to-mouth promotion of the survey. Therefore a total of 830 subjects were conveniently recruited between December 2005 and December 2007. Signed informed consent was obtained from all willing participants. Ethical approval was obtained from the Ethics Committee of the University of Limpopo (Turfloop Campus). Out of 830 participants we excluded 298 who were below 20 years of age and therefore, only 532 participants (396 women and 136 men) between 20 and 95 years of age were included in the analysis. Pregnant women or participants with renal disease were also excluded. Ten field workers were trained by research supervisor to take blood, measure physical activity and do anthropometric measurements in accordance with the standard procedures of the International Society for the Advancement of Kinanthropometry [15]. Instruments and measurements Anthropometry All anthropometric measurements were taken twice and the average was recorded. Before the main survey, we conducted a small pilot study on 100 randomly selected students of different ages from the University of Limpopo to determine standard error of measurement and coefficients of variation between different observers. Once we were satisfied with the reliability of the measurements the main study began. Body weight was measured using an electronic scale (Fazzini, EB6371B, China) to the nearest 0.1 kg with the participants wearing light clothing and barefoot. Height was measured with the participants barefoot to the nearest 0.1 cm using a stadiometer and with their heads in the Frankfort plane. Body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Waist circumference (WC) midway between the lower rib margin and the iliac crest was measured with the steel anthropometric tape to the nearest 0.1 cm extended around the waist parallel to the ground. The hip circumference (HC) was measured at the maximal circumference of the buttock with a steel tape to the nearest 0.1 cm. Circumferences were measured with the cross-hand technique, with the tape at right angles to the body and the readings were done on the right hand side. The waist to hip ratio (WHR) was calculated as WC divided by HC. Only one field worker in each village took the measurements to ensure uniformity and to avoid interstater variation. Physical activity Physical activity was measured using a pedometer (New Lifestyles Inc., Kansas City, MO, USA) to calculate the average number of steps per day [16]. The device was mounted on the belt which the participants put around their waist on the right hand side except when bathing or sleeping. The participants were not visited or followed up they were instructed to wear the pedometer over nine consecutive days after which the data were downloaded. Blood pressure Blood pressure was monitored using the Omron electronic blood pressure equipment (Omron M5-I, R5-I and HEM-907) [17]. The blood pressure of the participants was measured three times with at least 2-3 min between successive measurements. All measurements were taken in a quiet room after the participants had been sitting in a chair for 5 min. Hypertension (high blood pressure) was defined as systolic (SBP) ≥ 140 mm Hg and diastolic blood pressure (DBP) ≥ 90 mm Hg [18]. Statistical analysis For statistical analysis the BMI (kg/m2) was classified into four categories: under-weight: < 18.5, normal weight: 18.5-24.9, over-weight: 25.0-29.9 and obese: ≥ 30.0 [12]. Physical activity measured by average step per day was categorized into five groups: sedentary: ≤ 5000, low active: 5000-7499, somewhat active: 7500-9999, active: 10000-12499 and very active: ≥ 12500 [19]. Descriptive statistics was used to define the characteristics of study participants. Differences between male and female participants were assessed using Wilcoxon-Mann-Whitney test for continuous variables and Chi-square test was used for categorical variables. Univariate linear regression analysis was used to assess the association of anthropometric parameters and physical activity to SBP and DBP. Binary logistic regression was used to assess the association of anthropometric parameters and physical activity to hypertension (normal or hypertensive) by estimating the odds ratio with 95% confidence interval (CI). Only variables significant in the univariate models for SBP, DBP and hypertension were fitted into multivariate models for each primary outcome. Variables were considered significant at a p-value ≤ 0.05. The analysis was done using STATA version 10 (STATA Corporation, College Station, Texas, USA). Results Characteristics of study participants Anthropometric measurements showed that a high percentage of women were significantly (p < 0.001) overweight and obese compared to men. There was no statistically significant difference in SBP between women and men. However, women had significantly higher DBP than men. DBP among women varied between 48 and 157 mm Hg and between 55 and 115 mm Hg among men. Hypertension was significantly high among women (38.1%) compared to men (27.9). There was no significant difference in physical activity between women and men (Table 1). Table 1 Descriptive characteristics of female and male participants (n = 532) Characteristics Female (n = 396) Male (n = 136) All (n = 532) Continuous variables Mean (SD) Mean (SD) Mean (SD) p-value± Age(years) 47.7 (18.7) 40.9 (19.2) 45.9 (19.0) < 0.001 Height(cm) 158.9 (7.6) 167.8 (8.5) 161.2 (8.7) < 0.001 Weight(cm) 68.6 (15.9) 64.9 (13.6) 67.6 (15.5) < 0.018 Body mass index (kg/m2) 27.2 (6.2) 23.1 (4.8) 26.1 (6.1) < 0.001 Waist circumference(cm) 86.4 (14.3) 77.6 (12.4) 84.2 (14.4) < 0.001 Hip circumference (cm) 104 (14.3) 92.6 (11.2) 101.3 (14.5) < 0.001 Waist-to-hip ratio 0.83 (0.08) 0.84(0.08) 0.83 (0.08) 0.330 Systolic blood pressure(mm Hg) 123 (24) 123 (20) 123 (23) 0.907 Diastolic blood pressure(mm Hg) 81 (14) 77 (11) 80 (13) 0.001 Physical activity (average steps/day) 11466 (5204) 12048 (4936) 11615 (5139) 0.173 Categorical variables N (%) N (%) N (%) BMI categories (kg/m2) Underweight (< 18.5) 16 (4.0) 24 (17.7) 40 (7.5) < 0.001 Normal weight (18.5-24.9) 148 (37.4) 75 (55.2) 223 (41.9) Overweight (25.0-29.9) 115 (29.0) 24 (17.7) 139 (26.1) Obese (≥ 30) 117 (29.6) 13 (9.6) 130 (24.4) Physical activity (average steps/day) Sedentary (< 4999) 40 (10.1) 13 (9.6) 53 (10.0) 0.205 Low active (5 000-7 499) 38 (9.6) 7 (5.2) 45 (8.5) Moderate active (7 500-9 999) 76 (25.0) 27 (19.7) 103 (19.4) Active (10 000-12 499) 99 (25.0) 32 (23.5) 131 (24.6) Very active (≥ 12 500) 143 (36.1) 57 (41.9) 200 (37.6) Hypertension (mm Hg)* Normotensive 249 (62.90) 98 (72.1) 347 (65.2) 0.053 Hypertensive 147 (37.1) 38 (27.9) 185 (34.8) Differences between women and men significant at p ≤ 0.001, SD-Standard deviation, *systolic blood pressure ≥ 140 mm Hg and diastolic blood pressure ≥ 90 mm Hg Univariate analysis Mean age showed a statistically significant positive association with SBP and DBP in both women and men (Tables 2 and 3). However, age was highly correlated with all other independent variables and was therefore excluded in subsequent analysis. Mean BMI showed a significant positive association with both SBP and DBP only in women, however, no significant association was found with BMI categories in both women and men. Both SBP and DBP were significantly and positively associated with WC, HC and WHR in women, and only WHR showed a significant positive association with both SBP and DBP in men. The association between categorical variables for physical activity (average steps per day) and blood pressure (SBP and DBP) were not plausible in both women and men. The risk of hypertension increased with BMI, WC and WHR in both women and men (Table 4). Increase in HC was only significantly associated with increased risk of hypertension in women. No statistically significant association was found between hypertension and categorical variables of BMI and physical activity in both women and men. Table 2 Univariate association of anthropometric parameters and physical activity with diastolic blood pressure (mm Hg) in women and men Variables Women (n = 396) Men (n = 136) β 95% CI p-value± β 95% CI p-value± Age (years) 0.654 0.546 0.763 0.000 0.354 0.186 0.522 0.001 Body mass index (kg/m2) 0.567 0.188 0.945 0.003 0.187 -0.530 0.905 0.606 Waist circumference (cm) 0.401 0.241 0.561 0.000 0.242 -0.032 0.515 0.082 Hip circumference (cm) 0.234 0.071 0.398 0.005 -0.039 -0.346 0.267 0.800 Waist-Hip-Ratio 70.121 40.898 99.344 0.000 59.885 21.145 98.625 0.003 Average steps/day 0.000 0.000 0.001 0.747 0.001 0.000 0.002 0.011 BMI categories(kg/m2) Underweight (< 18.5) ** ** ** ** ** ** ** Normal weight (18.5-24.9) -17.590 -29.710 -5.469 0.005 0.127 -9.276 9.529 0.979 Overweight (25.0-29.9) -13.230 -25.519 -0.941 0.035 1.375 -10.199 12.949 0.815 Obese (≥ 30) -8.402 -20.679 3.874 0.179 1.756 -12.050 15.563 0.802 Physical activity (average steps/day) Sedentary (< 4999) ** ** ** ** ** ** ** Low active (5 000-7 499) 16.755 6.247 27.263 0.002 1.385 -17.241 20.010 0.883 Moderate active (7 500-9 999) 7.545 -1.517 16.606 0.102 2.570 -10.842 15.982 0.705 Active (10 000-12 499) 9.973 1.282 18.664 0.025 0.822 -12.245 13.889 0.901 Active (≥12 500) 8.349 0.052 16.646 0.049 7.771 -4.440 19.982 0.210 **First category taken as the reference group, β Regression coefficients, CI Confident interval, ±significant at ≤ 0.05 Table 3 Univariate association of anthropometric parameters and physical activity with systolic blood pressure (mm Hg) in women and men Variables Women (n = 396) Men (n = 136) β 95% CI p-value± B 95% CI p-value± Age (years) 0.263 0.195 0.331 0.000 0.190 0.095 0.284 0.001 Body mass index (kg/m2) 0.554 0.340 0.768 0.000 0.225 -0.176 0.626 0.269 Waist circumference (cm) 0.302 0.212 0.392 0.000 0.119 -0.035 0.272 0.128 Hip circumference (cm) 0.216 0.123 0.309 0.000 -0.073 -0.245 0.098 0.399 Waist-Hip-Ratio 40.122 23.222 57.022 0.000 40.538 19.173 61.902 0.000 Average steps/day 0.000 0.000 0.000 0.453 0.000 0.000 0.001 0.119 BMI categories(kg/m2) Underweight (< 18.5) ** ** ** ** ** ** ** Normal weight (18.5-24.9) -9.667 -16.540 -2.795 0.006 -1.310 -6.539 3.919 0.621 Overweight (25.0-29.9) -8.220 -15.188 -1.252 0.021 0.625 -5.812 7.062 0.848 Obese (≥ 30) -1.145 -8.415 5.506 0.681 3.455 4.223 11.133 0.375 Physical activity (average steps/day) Sedentary (< 4999) ** ** ** ** ** ** ** Low active (5 000-7 499) 10.021 3.959 16.083 0.001 4.0.659 -5.733 15.052 0.377 Moderate active (7 500-9 999) 3.442 -1.785 8.670 0.196 1.083 -6.401 8.566 0.775 Active (10 000-12 499) 5.711 0.697 10.725 0.026 -2.269 -9.560 5.022 0.539 Active (≥ 12 500) 4.313 -0.473 9.100 0.077 2.652 -4.161 9.465 0.443 **First category taken as the reference group, β Regression coefficients, CI Confident interval, ±significant at ≤ 0.05 Table 4 Univariate association of anthropometric parameters and physical activity with hypertension (mm Hg) between women and men Variables Women (n = 396) Men (n = 136) Odds Ratio 95% CI p-value± Odds Ratio 95% CI p-value± Age (years) 1.047 1.034 1.060 0.000 1.035 1.014 1.056 0.001 Body mass index (kg/m2) 1.048 1.013 1.084 0.006 1.092 1.010 1.181 0.026 Waist circumference (cm) 1.038 1.022 1.054 0.000 1.045 1.012 1.079 0.007 Hip circumference (cm) 1.020 1.005 1.035 0.009 1.018 0.984 1.054 0.296 Waist-Hip-Ratio 971.916 55.559 17002.150 0.000 2488.920 14.248 434771.3 0.003 Average steps/day 1.000 1.000 1.000 0.882 1.000 1.000 1.000 0.650 Age Quartile categories(years) BMI categories(kg/m2) Underweight (< 18.5) ** ** ** ** ** ** ** Normal weight (18.5-24.9) 0.480 0.170 1.356 0.166 0.500 0.180 1.387 0.183 Overweight (25.0-29.9) 0.456 0.158 1.310 0.145 0.824 0.242 2.797 0.756 Obese (≥ 30) 0.887 0.312 2.523 0.822 3.200 0.787 13.017 0.104 Physical activity (average steps/day) Sedentary (< 4999) ** ** ** ** ** ** ** Low active (5 000-7 499) 2.333 0.922 5.904 0.074 1.333 0.165 10.743 0.787 Moderate active (7 500-9 999) 0.891 0.384 2.069 0.788 1.667 0.365 7.607 0.510 Active (10 000-12 499) 1.517 0.690 3.333 0.300 0.933 0.200 4.347 0.930 Active (≥ 12 500) 1.502 0.706 3.196 0.291 1.417 0.346 5.800 0.628 **First category taken as the reference group, CI Confident interval Multivariate analysis Categorical variables for BMI and physical activity were excluded due to lack of meaningful results in the univariate analysis. Only HC (β = 1.68, CI = 0.27-3.11, p = 0.020) and WHR (β = 252.70, CI = 69.06-436.34, p = 0.007) showed a significant positive association with SBP in women. The odds of being hypertensive increased significantly with WHR (OR = 597.04; CI = 0.97-0.36E06, p = 0.051) only in men. All the other selected variables showed no statistically significant association with SBP, DBP and hypertension in the final multivariate models. Discussion This study examined the association of anthropometric parameters, physical activity to blood pressure in women and men in a black South African rural population. Anthropometric measurements (BMI, WC, HC and WHR) showed that women were overweight and obese than men. While there was no difference in mean SBP between men and women, mean DBP differed significantly by sex and was higher in women (mean = 81 mm Hg) compared to men (mean = 77 mm Hg). Furthermore, hypertension was significantly high among women (38.1%) than men (27.9%). The 2002 South African Demographic Health Survey (SADHS) also found a had high prevalence of overweight and obesity among black women and this was associated with increased risk of hypertension [12-14]. In the current study univariate analysis identified central (BMI) and abdominal (BMI, WC, HC and WHR) measures of obesity as significant determinants of elevated SBP and DBP in women, and only WHR was significant in men. Hypertension also increased with increasing BMI, WC, and WHR in both women and men. HC showed a positive relationship with hypertension only in women. This is biologically plausible because in women most fat is distributed in the hips and in man around the waist [20]. However, no relationship was found between BMI categories and BP in both women and men. A study across three different populations in Africa found that although in general there was an increase in SBP and DBP with increasing BMI the risk of hypertension was not continuously distributed at all levels of BMI [21]. They found that there were BMI groups with an increased risk of hypertension and the cut of points varied markedly between men and women depending on ethnicity, biological, behavioural and environmental factors, including diet and nutrition, which have been implicated as determinants of high BP in different population groups [22,23]. Furthermore, in this study physical activity showed no clear association with blood pressure and hypertension. Marti et al. [24] and Manjoo et al. [25] also found no association between SBP and physical activity as measured by daily steps. Other studies in South Africa showed that average steps per day could not be used to define the intensity physical activity [26,27]. This can be attributed to the fact that physical activity levels are difficult to standardize and measure across populations in different countries [21,27,28]. In the final multivariate analysis only increase in HC and WHR was consistently associated with increase in SBP in women and WHR with hypertension in men. This is consistent with other studies which found WHR to be a strong independent indicator of hypertension than BMI for both sexes in some population groups [29-32]. It has been suggested that an increased WHR may reflect both a relative abundance of abdominal fat (increased WC) and a relative lack of gluteal muscle (decreased hip circumference) [33]. In addition, WHR not only shows body fat distribution but also reflects most of the lifestyle-related factors of an individual [34]. The current study may be limited by potential risk factors or confounders which were not accounted for in the analysis such as dietary intake, substance abuse (alcohol and smoking) and other life style behavioural risk factors. The small sample size especially for men given the fact that the subsample of individuals used in the analysis was purposefully recruited makes generalization of the results difficult. Nevertheless, the findings of this study indicate that overweight and obese people especially women are more at risk of hypertension in this rural black population. Conclusion Based on the finding of this study it is possible therefore as postulated by Grimm [35] that modernization of rural villages such as Dikgale has significantly changed lifestyle with consequent increase in obesity and hypertension. This highlights the importance of population based survey to monitor high blood pressure for effective prevention and control. Abbreviations BMI: Body mass index; BP: Blood pressure; DBP: Diastolic blood pressure; DDSS: Dikgale Demographic Surveillance System; HC: Hip circumference; SBP: Systolic blood pressure; SADHS: South African Demographic Health Survey; WC: Waist circumference; WHR: Waist hip ratio. Competing interests The authors declare that they have no competing interests. Authors' contributions SSM was involved in data collection, performed initial data analysis and drafted the manuscript. MMLH assisted with statistical analysis, interpretation of data and revised the manuscript. DL made significant intellectual input, provided direction and helped revise the final manuscript. All authors read and approved the final manuscript. Acknowledgements We would like to thank Prof. Marianne Alberts and Dr. Ian Cook of University of the Limpopo, Turfloop Campus, Polokwane in South Africa for providing the data and for commenting on the early versions of the manuscript. We are also grateful to Solomon Choma of University of the Limpopo, Turfloop Campus, Polokwane in South Africa. ==== Refs WHO World Health Report: Reducing risks, Promoting Healthy Life 2002 Geneva: World Health Organization WHO Diet, Nutrition and the Prevention of Chronic Diseases, Report of a Joint WHO/FAO Expert Consultation 2003 WHO Technical Report Series No. 916. Geneva: World Health Organization Kearney PM Whelton M Reynolds K Muntner P Welton PK Global burden of hypertension: analysis of worldwide data Lancet 2005 365 217 223 15652604 Seedat YK Hypertension in developing nations in sub-Saharan Africa J Hum Hypertens 2000 14 739 747 10.1038/sj.jhh.1001059 11095164 Sobngwi E Mbanya JC Unwin NC Kengne AP Fezeu L Minkoulou EM Aspray TJ Alberti KGMM Physical activity and its relationship with obesity, hypertension and diabetes in urban and rural Cameroon Int J Obes Relat Metab Disord 2002 26 1009 1016 12080456 Cooper RS Amoah AG Mensah GA High blood pressure: the foundation for epidemic cardiovascular disease in African populations Ethn Dis 2003 13 S48 S52 13677414 Tesfaye F Byass P Wall S Population based prevalence of high blood pressure among adults in Addis Ababa: uncovering a silent epidemic BMC Cardiovasc Disord 2009 9 39 10.1186/1471-2261-9-39 19698178 Froberg K Andersen LB Mini Review: physical activity and fitness and its relations to cardiovascular disease risk factors in children Int J Obes 2005 29 S34 S39 Seedat YK Race, environment and blood pressure: the South African experience J Hypertens 1983 1 7 12 6681027 Steyn K Gaziano TA Bradshaw D Laubscher R Fourie J Hypertension in South African adults: results from the Demographic and Health Survey, 1998 J Hypertens 2001 19 1717 1725 10.1097/00004872-200110000-00004 11593090 Department of Health South Africa Demographic and Health Survey 1998. Full report 2002 Department of Health, Pretoria, Republic of South Africa Puoane T Steyn K Bradshaw D Laubscher R Fourie J Lambert V Mbananga N Obesity in South Africa: The South African Demographic and Health Survey Obes Res 2002 10 1038 1048 10.1038/oby.2002.141 12376585 Mollentze WF Moore AJ Joubert G Steyn K Oosthuizen GM Weich DJ Coronary heart disease risk factors in a rural and urban Orange Free State black population S Afr Med J 1995 85 90 96 7597541 Alberts M Burger S Tollman SM The Dikgale field site S Afr Med J 1999 89 851 852 10488360 Norton K Olds T Anthropometrica 1996 Sydney: University of New South Wales Press 120 267 Crouter SE Schneider PL Bassett DR Spring-levered versus piezo-electric pedometer accuracy in overweight and obese adults Med Sci Sports Exerc 2005 37 10 1673 1679 10.1249/01.mss.0000181677.36658.a8 16260966 Omboni S Riva I Giglio A Caldara G Groppelli A Parati G Validation of the Omron M5-I, R5-I and HEM-907 automated blood pressure monitors in elderly individuals according to the International Protocol of the European Society of Hypertension Blood Press Monit 2007 2 233 242 17625396 Chalmers J MacMahons S Mancia G Whitworth J Hansson L Neal B Rodgers A Mhurchu CN Clark T World Health Organization International Society of management of hypertension J Hypertens 1999 17 151 183 10067786 Tudor-Locke C Bassett DR How many steps/day are enough? Preliminary pedometer indices for public health Sports Med 2004 34 1 8 10.2165/00007256-200434010-00001 14715035 Chan DC Watts GF Barrett PHR Burke V Waist circumference, waist-to-hip ratio and body mass index as predictors of adipose tissue compartments in men Q J Med 2003 96 441 447 10.1093/qjmed/hcg069 Tesfaye F Nawi NG Van Minh H Byass P Berhane Y Bonita R Wall S Association between body mass index and blood pressure across three populations in Africa and Asia J Hyperten 2007 21 28 37 Treloar C Porteous J Hassan F Kasniyah N Lakshmanudu M Sama M Sja'bani M Heller RF The cross cultural study Health Policy 1999 5 279 286 Sobngwi E Mbanya JC Unwin NC Porcher R Kengne AP Fezeu L Minkoulou EM Caroline Tournoux C Gautier JF Aspray TJ Alberti K Exposure over the life course to an urban environment and its relation with obesity, diabetes, and hypertension in rural and urban Cameroon Int J Epidemiol 2004 33 769 776 10.1093/ije/dyh044 15166209 Marti B Tuomilehto J Salonen JT Puska P Nissinen A Relationship between leisure-time physical activity and risk factors for coronary heart disease in middle-aged Finnish women Acta Med Scand 1987 222 223 230 3425377 Manjoo P Joseph L Pilote L Dasgupta K Sex differences in step count-blood pressure association: A preliminary study in Type 2 Diabetes PLoS One 2010 5 11 1 6 Cook I Alberts M Brits JS Choma S Mkhonto SS Descriptive epidemiology of ambulatory activity in rural, black South Africans Med Sci Sports Exerc 2010 43 1261 1268 20019642 Malhotra R Hoyo C Ostbye T Hughes G Schwartz D Tsolekile L Zulu J Puoane T Determinants of obesity in an urban township of South Africa S Afr J Clin Nutr 2008 21 315 320 Hu G Tuomilehto J Silverntoinem K Barengo N Jousilahti P Joint effects of physical activity, body mass index, waist circumference and waist-to-hip ratio with the risk of cardiovascular disease among middle-aged Finnish men and women Eur Heart J 2004 25 2212 2219 10.1016/j.ehj.2004.10.020 15589638 Welborn TA Satvinders D Bennet SA Waisthip-ratio is the dominant risk factor predicting cardiovascular death in Australia Med J Australia 2003 179 580 585 14636121 Sayeed MA Mahtab H Latif ZA Khanam PA Ahsan KA Banu A Azad Khan AK Waist-to-height ratio is a better obesity index than body mass index and waist-to-hip ratio for predicting diabetes, hypertension and lipidemia Bangladesh Med Res Counc Bull 2003 29 1 10 14674615 Yalcin BM Sahin EM Yalcin E Which anthropometric measurements is more closely related to elevated blood pressure? Fam Pract 2005 22 541 547 10.1093/fampra/cmi043 15964872 Sanya AO Ogwumike OO Ige AP Ayanniyi OA Relationship of Waist Hip Ratio and Body Mass Index with Blood Pressure of Individuals Afr J Physiother Med Rehabil Sci 2009 1 7 11 Seidell J Han T Feskens E Lean M Narrow hips and broad waist circumference independently contribute to increased risk of non-insulin dependent diabetes mellitus J Intern Med 1997 242 401 406 10.1046/j.1365-2796.1997.00235.x 9408070 Han TS Bijan FC Lean MEJ Seidell JC Separate associations of waist and hip circumference with lifestyle factors Int J Epidemiol 1998 27 422 430 10.1093/ije/27.3.422 9698130 Grimm JJ Interaction of physical activity and diet: implications for insulin-glucose dynamics Public Health Nutr 1999 2 363 368 10610074
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==== Front PLoS Negl Trop DisPLoS Negl Trop DisplosplosntdsPLoS Neglected Tropical Diseases1935-27271935-2735Public Library of Science San Francisco, USA 22802977PNTD-D-12-0024210.1371/journal.pntd.0001719Research ArticleBiologyImmunologyImmune ResponseModel OrganismsAnimal ModelsMouseZoologyParasitologyKinetics of Antibody Response in BALB/c and C57BL/6 Mice Bitten by Phlebotomus papatasi Mice Antibody Response to P. papatasi BitesVlkova Michaela 1 * Rohousova Iva 1 Hostomska Jitka 1 Pohankova Lucia 1 Zidkova Lenka 1 Drahota Jan 1 Valenzuela Jesus G. 2 Volf Petr 1 1 Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic 2 Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Rockville, Maryland, United States of America Louzir Hechmi EditorInstitut Pasteur de Tunis, Tunisia* E-mail: [email protected] and designed the experiments: IR JH PV. Performed the experiments: MV JH LP LZ JD. Analyzed the data: MV IR JH LP PV. Contributed reagents/materials/analysis tools: MV IR JGV PV. Wrote the paper: MV IR PV. 7 2012 10 7 2012 6 7 e171927 2 2012 20 5 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.2012This 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.Background Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins. Methodology/Principal Findings Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera. Conclusions Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi–mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission. Author Summary Leishmania major is the causative agent of zoonotic cutaneous leishmaniasis and Phlebotomus papatasi serve as the major vector. In endemic foci, rodents are the natural reservoirs of this disease. Thus, we studied anti-P. papatasi saliva antibody response in BALB/c and C57BL/6 mice that are commonly used as model organisms sensitive and resistant to cutaneous leishmaniasis, respectively. We followed the kinetics and persistence of specific antibody response in both mice strains and we characterized the main P. papatasi salivary antigens. We demonstrated that sand fly bites elicit production of specific IgG that reflect the intensity of sand fly exposure. In endemic areas, this could provide useful information about the effectiveness of anti-vector control programs. We also examined the reaction of mice sera with four P. papatasi recombinant proteins. Our data indicate that a combination of these proteins could be used instead of crude salivary gland homogenate for the monitoring of anti-sand fly saliva antibodies in natural hosts in endemic foci. ==== Body Introduction Sand flies (Diptera: Phlebotominae) serve as vectors of leishmaniasis, a neglected disease with symptoms ranging from non-lethal cutaneous to life-threatening visceral form. The causative agents of the disease are protozoan parasites of the genus Leishmania which are transmitted to the hosts by the bites of infected sand fly females. The percentage of infected flies in foci of leishmaniasis fluctuates and humans and animals are more frequently exposed to the bites of uninfected sand flies. Repeated exposure to sand fly saliva elicits anti-saliva antibodies that could be used as a marker of exposure to sand fly bites [1]–[5]. Moreover, the antibodies are sand fly species-specific. Therefore they can be utilized to differentiate between exposure to vector and non-vector species [1], [4], [6]–[9]. In several epidemiological studies, anti-sand fly saliva antibodies were already employed as a reliable tool to monitor exposure to sand fly bites, to evaluate the effectiveness of vector control programs, and in some instances to estimate the risk of Leishmania transmission [1], [4], [5], [10]–[14]. In endemic areas sand fly population fluctuate seasonally [15], which may influence host anti-saliva antibody response. However, very little is known about the kinetics and persistence of anti-saliva antibodies in sera of hosts bitten by blood-feeding insects. Few data on antibody kinetics are available from mice, chicken and guinea pigs experimentally exposed to bites of Triatoma infestans [16]–[18], from humans bitten by mosquitoes [19]–[22] as well as from humans [4], [23] and dogs [3], [5], [24] bitten by sand flies. Screening for antibodies is, however, unsuitable for broader use in epidemiological studies until recombinant proteins could be employed instead of the crude salivary gland homogenate, which requires maintenance of sand fly colonies and laboratory dissections of insects. So far, only recombinant salivary proteins from Lutzomyia longipalpis have been tested for reactivity with sera of naturally bitten humans, dogs, and foxes [8], [9]. We studied mice antibody response to P. papatasi, the main vector of Leishmania major, and compared long-term kinetics and persistence of anti-saliva antibodies in BALB/c and C57BL/6 mice that are widely used as model organisms sensitive or resistant to L. major infection, respectively. Furthermore, we characterized and compared main P. papatasi salivary antigens recognized by sera of experimentally bitten BALB/c and C57BL/6 mice. The reactivity of mice sera was also tested with the four P. papatasi recombinant proteins; two Yellow-related proteins (PpSP44/AF335492 and PpSP42/AF335491) and two D7-related proteins (PpSP30/AF335489 and PpSP28/AF335488). Methods Ethical statement BALB/c and C57BL/6 mice were maintained and handled in the animal facility of Charles University in Prague in accordance with institutional guidelines and Czech legislation (Act No. 246/1992 coll. on Protection of Animals against Cruelty in present statutes at large), which complies with all relevant European Union and international guidelines for experimental animals. The experiments were approved by the Committee on the Ethics of Animal Experiments of the Charles University in Prague (Permit Number: 24773/2008-10001) and were performed under the Certificate of Competency (Registration Number: CZU 934/05; CZU 307/09) in accordance with the Examination Order approved by Central Commission for Animal Welfare of the Czech Republic. Sand flies and salivary gland dissection A colony of Phlebotomus papatasi (originating from Turkey) was reared under standard conditions as described in [25]. Salivary glands were dissected from 4–6-day-old female sand flies, placed into 20 mM Tris buffer with 150 mM NaCl and stored at −20°C. Experimental exposure Twelve mice of BALB/c or C57BL/6 strains (6 weeks old) were divided into experimental and control groups of six mice each. Mice in the experimentally bitten groups were exposed individually to 30 Phlebotomus papatasi females (22±0.6 (standard error) blood-fed females per mouse per exposure on BALB/C mice; 26±0.7 (standard error) blood-fed females per mouse per exposure on C57BL/6 mice), once a week in a total of 5 exposures (weeks 1–5). Mice in the control groups remained without any exposure to sand flies. Animals in both groups were anaesthetized (ketamin 150 mg/kg and xylazin 15 mg/kg body weight, intraperitoneally). Blood samples were taken weekly from the tail vein of each mouse one day before exposure to sand flies from week 0 (pre-immune serum) to week 12 and than every other week till the end of the experiment (week 28 for BALB/c mice; week 27 for C57BL/6 mice). In total, mice were followed for 29 and 28 weeks, respectively. Two independent experiments were done for each mice strain. To test the presence of memory cells, BALB/c mice were additionally exposed to P. papatasi bites (21±0.5 (standard error) blood-fed females per mouse) in the week 27. Preparation of recombinant proteins Genes coding for P. papatasi salivary gland secreted proteins PpSP28 (AF335488), PpSP30 (AF335489), PpSP42 (AF335491) and PpSP44 (AF335492) were amplified from VR2001-TOPO vector [26] by PCR. Two specific restriction sites (Nde I and Bam HI) were incorporated into the PCR primers: PpSP28Fw (CATATGAAGTACCCTAGGAATGCCGAT), PpSP28Rev (GGATCCGTACGTTCTTGCGGATTGGTCATC), PpSP30Fw (CATATGCGATTTCCTAGGAATGGAGAC), PpSP30Rev (GGATCCGTATTTCCAAGATTCAATATCAAG), PpSP42Fw (CATATGAAAAGAGATGATGTTGGA), PpSP42Rev (GGATCCCCCTTGACACTTTTCTCC), PpSP44Fw (CATATGAAAAGAGACGATGTTGAA), and PpSP44Rev (GGATCCTTTAGGTTTTCTCACTTC). Afterwards, PCR products were ligated into E. coli pGEM-T Easy Vector (Promega) using TA cloning and the ligation products were transformed into E. coli competent cells TOP10 (Invitrogen). Vectors were replicated in bacteria and after that, genes restricted by Nde I and Bam HI enzymes and restricted E. coli pET-42 Expression Vectors (Novagen) were ligated. Ligation products were transformed into E. coli competent cells TOP10 (Invitrogen) again. Plasmids were isolated from the bacteria, and transformed into E. coli BL21 (DE3) gold (Agilent) for expression. E. coli lysates were prepared under denaturing conditions and His-tagged proteins were purified by FLPC on a Ni-NTA Superflow column with The QUIaxpressionist kit (Quiagen) according to manufacturers manual. Detection of anti-P. papatasi saliva antibodies Anti-P. papatasi saliva IgG antibodies and IgG subclasses were measured in sera of BALB/c and C57BL/6 mice using indirect ELISA. Microtiter plate wells were coated with P. papatasi salivary gland homogenate (SGH) made by three freeze-thaw cycles (about 60 ng of protein per well). To block free binding sites, washed wells were incubated with 6% low fat dry milk diluted in 20 mM phosphate-buffered saline with 0.05% Tween 20. Mice sera were diluted 1∶200 in 2% low fat dry milk and incubated for 90 min at 37°C for specific IgG or overnight at 4°C for IgG subclasses. Secondary antibodies (goat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3; Serotec) conjugated with horseradish peroxidase (HRP) were diluted and incubated at 37°C as described in Table S1. Orthophenylendiamine and H2O2 in McIlwein phosphate-citrate buffer (pH 5.5) were used as substrate solution. Absorbance was measured at 492 nm using an Infinite M200 microplate reader (Tecan). The cut-off value was determined as two standard errors of the mean of the absorbance of pre-immune serum. The intensity of booster effect was measured by increased levels of specific antibodies in sera of bitten mice after the last sand fly exposure (comparing week 24 and 28). Anti-P. papatasi IgE were measured in sera of BALB/c mice as described above with the following modifications. Microtiter plate wells were coated with P. papatasi SGH (about 300 ng of protein per well). To block the free binding sites, washed wells were incubated with 6% fetal calf serum. Mouse sera were diluted 1∶100 in 2% fetal calf serum. Secondary antibody (rat anti-mouse IgE; BD PharMingen) was diluted and incubated as listed in Table S1. Western blot analysis Phlebotomus papatasi SGH (about 10 µg of protein per well) was separated on 10% SDS-PAGE gel under non-reducing conditions using the Mini-Protean III apparatus (BioRad). Salivary proteins were blotted onto a nitrocellulose membrane by Semi-Phor equipment (Hoefer Scientific Instruments) and cut into strips. The strips were then blocked with 5% low fat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBS-Tw) and subsequently incubated with mice sera (week 28 for BALB/c mice; week 5 for C57BL/6 mice) diluted 1∶200 for 1 hour. In the next step the strips were incubated for 1 hour with peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2b (Serotec) diluted in TBS-Tw as follows: IgG and IgG1 1∶5000; IgG2b 1∶2000 for BALB/c mice sera and IgG, IgG1 1∶2000 for C57BL/6 mice sera. The chromogenic reaction was developed using a solution containing diaminobenzidine and H2O2. Similar protocol was used for western blot analysis with P. papatasi recombinant proteins PpSP28, PpSP30, PpSP42, and PpSP44. Briefly, recombinant proteins were loaded on the 10% SDS-PAGE gel (3 µg protein per well) and separated under reducing conditions. BALB/c mice sera (week 28) were diluted 1∶50 and secondary antibody (goat anti-mouse IgG from Serotec) was diluted 1∶1000 in TBS-Tw. Mass spectrometry The proteins from the P. papatasi salivary glands used for mass spectrometric analysis were run on the same gel as salivary glands used for western blot analysis. Proteins were visualized by Coomassie Blue G-250 staining (Bio-Rad). The individual bands were cut and incubated with 10 mM dithiothreitol (DTT) and then treated with 55 mM iodoacetamid. Washed and dried bands were digested with trypsin (5 ng, Promega). Alpha-cyano-4-hydroxycinnamic acid was used as a matrix. Samples were measured using a 4800 Plus MALDI TOF/TOF analyzer (AB SCIEX). A peak list from MS spectra was generated by 4000 Series Explorer V 3.5.3 (AB SCIEX) without smoothing. Peaks with local signal to noise ratio greater than 5 were picked and searched by local Mascot v. 2.1 (Matrix Science) against a database of putative salivary protein sequences derived from GenBank. Database search criteria were as follows – enzyme: trypsin, taxonomy: Phlebotomus, fixed modification: carbamidomethylation, variable modification: methionine oxidation, peptide mass tolerance: 80 ppm, one missed cleavage allowed. Only hits that scored as significant (p<0.05) are included. Statistical analysis The data obtained by ELISA were subjected to GLM ANOVA and Tukey-Kramer Multiple Comparison procedure to analyze differences in kinetics of anti-P. papatasi saliva antibody response between experimentally bitten and control mice at all sampling points. The non-parametric Wilcoxon rank sum test for differences in medians was used for evaluation of booster effect, the comparison of antibody level between week 24 and 28. For correlation tests we used the non-parametric Spearman rank correlation matrix. For all tests statistical significance was regarded as a p-value less than 0.05. All statistical analyses were performed using NCSS 6.0.21 software. Results Kinetics of anti-P. papatasi saliva antibody response in BALB/c mice To investigate the kinetics and persistence of anti-P. papatasi saliva antibody response, experimentally bitten and control mice were followed for 29 weeks. Mice exposed five times to bites of sand flies at one-week interval had significantly increased levels of specific IgG, IgG1, and IgG2b as compared to control group (Figure 1A, C, E). In contrast, specific IgG2a, IgG3, and IgE levels in sera of bitten mice were comparable to non-exposed controls with some differences only at the last data points (Figure S1). No anti-saliva antibodies were detected in any pre-immune sera tested. 10.1371/journal.pntd.0001719.g001Figure 1 Anti-sand fly saliva antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi. BALB/c mice (A, C, E) and C57BL/6 mice (B, D, F) were divided into control (squares) and experimentally bitten groups (circles). Mice in the latter group were exposed to sand fly bites (arrows) in weeks 1–5 and additionally in the week 27 (only BALB/c mice). Levels of specific IgG (A, B); IgG1 (C, D); and IgG2b (E, F) were measured by ELISA at all sampling points. Full circles represent significant difference between control and bitten mice (p<0.05). Data are presented as the means ± standard errors of the means. Two independent studies were done. OD = optical density. In bitten mice, anti-P. papatasi saliva IgG and IgG1 levels increased significantly (p<0.05) after the fourth exposure (Figure 1A, C). IgG2b levels differed between experimental and control group from week 9 onward, with the exception of weeks 10 and 11 (Figure 1E). Anti-saliva IgG increased steadily till the end of the study, while specific IgG2b increased slowly until week 22 followed by a slight decrease at week 24. Anti-saliva IgG1 increased steadily and peaked at week 7 and persisted on this level until the end of the study. To test the presence of putative memory cells to P. papatasi salivary proteins, BALB/c mice were additionally exposed to sand flies 22 weeks after the last exposure (week 27). One week after the booster (at week 28) anti-P. papatasi saliva antibodies increased significantly in IgG by 43%, in IgG1 by 80% and in IgG2b by 79% (Figure 1A, C, E). Positive correlation was found between the number of blood-fed sand fly females during the individual immunization weeks (sum of the blood-fed females from the relevant week and the weeks before) and the corresponding levels of anti-P. papatasi IgG (r = 0.62, p<0.0001), IgG1 (r = 0.74, p<0.0001), and IgG2b (r = 0.29, p<0.05) (Figure 2A, C, E). Furthermore, positive correlation was detected between the total amount of blood-fed females and the levels of specific IgG (r = 0.72, p<0.0001) and IgG1 (r = 0.8, p<0.0001) after the fifth sand fly exposure (week 5). 10.1371/journal.pntd.0001719.g002Figure 2 Correlation between the intensity of sand fly exposure and the anti-Phlebotomus papatasi saliva antibodies. The correlation between the number of blood-fed sand fly females and the levels of anti-saliva antibodies in experimentally bitten BALB/c (A, C, E) and C57BL/6 (B, D) mice was performed using Spearman Rank Correlation Matrix. Positive correlation was detected in specific IgG (A); IgG1 (C); and IgG2b (E) in BALB/c mice and in specific IgG (B); and IgG1 (D) in C57BL/6 mice. OD = optical density. Kinetics of anti-P. papatasi saliva antibody response in C57BL/6 mice Experimentally bitten and control mice of C57BL/6 strain were followed in experiments lasting 28 weeks. Five exposures at one-week interval significantly increased levels of specific IgG and IgG1 in bitten mice (Figure 1B, D). In contrast, specific IgG2b, IgG2c, and IgG3 levels of bitten mice were comparable to controls. No anti-saliva antibodies were detected in any pre-immune sera tested. Similarly to BALB/c mice, anti-P. papatasi IgG and IgG1 levels differed significantly between experimentally bitten and control C57BL/6 mice from week 4 onward (Figure 1B, D). Anti-saliva IgG gradually increased until week 8 and then with a slight fluctuation of antibody levels decreased until the end of the study. Specific IgG1 developed with similar kinetics to IgG, however, it peaked earlier (at week 6) and then slowly decreased till the end of the study. Anti-saliva IgG2b, IgG2c, and IgG3 antibodies did not differ between the exposed and control group throughout the study (Figure 1F; Figure S1B, D) with the exception of week 21 for IgG3 subclass (Figure S1D). We also detected a positive correlation between the number of blood-fed sand fly females during the individual immunization weeks (sum of the blood-fed females from the relevant week and the weeks before) and the corresponding levels of anti-P. papatasi IgG (r = 0.80, p<0.0001) and IgG1 (r = 0.86, p<0.0001) (Figure 2B, D). Moreover, positive correlation was detected between the total amount of blood-fed females and the levels of specific IgG (r = 0.85, p<0.0001), IgG1 (r = 0.86, p<0.0001), and IgG2c (r = 0.5, p<0.05) after the fifth sand fly exposure (week 5). Identification and characterization of P. papatasi salivary antigens Phlebotomus papatasi salivary antigens were studied using sera of experimentally bitten BALB/c and C57BL/6 mice. Only the antibody classes and subclasses shown to be produced in high titers by ELISA were tested in a western blot; specific anti-P. papatasi IgG and IgG1 in both mice strains and additionally specific IgG2b in BALB/c mice. BALB/c mice sera recognized up to 10 protein bands with approximate molecular weights of 70, 65, 51, 49, 47, 35, 31, 30, 23, and 15 kDa, the last three being the most intensively recognized by all BALB/c sera in all IgG subclasses tested. Sera of C57BL/6 mice reacted additionally with the 53 kDa protein but did not recognize the 49 and 47 kDa protein bands. The most intensive reaction in all C57BL/6 mice was detected with the 65, 53, and 30 kDa protein bands in IgG as well as in IgG1 (Figure 3). Comparison of two mice strains therefore revealed an interesting difference in recognition of four protein bands of 53, 51, 49, and 47 kDa. No reaction was detected with any pre-immune mice sera tested (Figure 3). 10.1371/journal.pntd.0001719.g003Figure 3 Anti-sand fly saliva antibody response in BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi. (A) Total protein profile, Coomassie blue-stained SDS-PAGE gel with P. papatasi salivary gland homogenate. (B) Western blot of P. papatasi salivary proteins recognized by IgG, IgG1, or IgG2b from sera of P. papatasi-bitten BALB/c (week 28) and C57BL/6 mice (week 5). Pre-immune sera of BALB/c and C57BL/6 mice were used as negative controls (Neg). In BALB/c mice, the 51 kDa protein was recognized only by one out of 5 sera tested in IgG and IgG1, while in C57BL/6 mice, this protein band was recognized by all mice sera tested in IgG1 and by two out of five sera tested in IgG. Anti-P. papatasi IgG2b antibodies reacted consistently with the 65, 35, 31, 30, 23, and 15 kDa proteins (Figure 3). In C57BL/6 mice, 70, 65, 53, 31, and 30 kDa proteins were recognized by all mice sera tested (IgG as well as IgG1), while the 51, 35, 23, and 15 kDa antigens were recognized by some sera only (Figure 3). Specific IgG1 of C57BL/6 mice predominantly recognized the 65, 53, 51, 31, and 30 kDa antigens (Figure 3). Mass spectrometry analysis identified the salivary proteins with the same mobility in the SDS-PAGE as the proteins recognized by the sera of experimentally bitten mice as the Yellow-related proteins (GenBank acc. no. AF335492 and AF335491), apyrase (AF261768), D7-related proteins (AF335489; AF335488), antigen-5 protein (DQ205724), and proteins of the SP15 protein family (AY628879, AY628880; AF335486; AF335485) (Table 1). 10.1371/journal.pntd.0001719.t001Table 1 Phlebotomus papatasi salivary proteins recognized by sera of bitten mice. MW NCBI acc.number Best match to NR protein database (kDa) Sequence name E-value Protein family 70 N.D. N.D. N.D. N.D. 65 N.D. N.D. N.D. N.D. 53 N.D. N.D. N.D. N.D. 51 AF335492 44 kDa yellow-related salivary protein (PpSP44) 0.00E+00 yellow-related 49 N.D. N.D. N.D. N.D. 47 AF335491 42 kDa yellow-related salivary protein (PpSP42) 0.00E+00 yellow-related 35 AF261768 salivary apyrase (PpSP36) 0.00E+00 apyrase 31 AF335490 32 kDa salivary protein (PpSP32) 1.10E−05 PpSP32-like 30 AF335489 30 kDa D7-related salivary protein (PpSP30) 4.50E−12 D7-related 30 DQ205724 29 kDa antigen 5-related salivary protein 7.10E−05 antigen 5-related 23 AF335488 28 kDa D7-related salivary protein (PpSP28) 0.00E+00 D7-related 15 AY628879 SP-15 protein 1.80E−05 PpSP15-like 15 AY628880 SP-15 protein 3.50E−07 PpSP15-like 15 AF335486 14 kDa salivary protein (PpSP14) 0.00E+00 PpSP15-like 15 AF335485 12 kDa salivary protein (PpSP12) 0.00E+00 PpSP15-like N.D. – not determined. Reactivity of anti-saliva antibodies with the P. papatasi recombinant proteins The reactivity of PpSP44 (yellow related protein), PpSP42 (yellow related protein), PpSP30 (D7 related protein), and PpSP28 (D7 related protein) recombinant proteins was studied using sera from BALB/c mice exposed to P. papatasi bites and positive for anti-P. papatasi IgG antibodies. Sera of control mice did not recognize any of the recombinant proteins tested. The most intensive reaction was detected with the PpSP30, although, this protein was not recognized by all sera tested (4 out of 5). Three out of five mice sera reacted with the PpSP42 and PpSP44 recombinant proteins and very weak reaction was detected with the PpSP28 recombinant protein in two out of five mice sera (Figure 4). 10.1371/journal.pntd.0001719.g004Figure 4 Reactivity of anti-Phlebotomus papatasi saliva IgG with P. papatasi recombinant proteins. Sera from BALB/c mice experimentally bitten by P. papatasi (Bitten mice) that were positive for anti-P. papatasi IgG (OD>cut-off: 0.19) were tested by Western blot analysis against (A) P. papatasi salivary gland homogenate, (B) a combination of bacterially-expressed PpSP42 and PpSP28 (Yellow-related protein AF335491 and D7-related protein AF335488, respectively), and (C) a combination of bacterially-expressed PpSP44 and PpSP30 (Yellow-related protein AF335492 and D7-related protein AF335489, respectively). The SDS-PAGE protein profiles of the P. papatasi salivary gland homogenate as well as the recombinant proteins were blotted and stained by Amido Black. Pre-immune sera of BALB/c mice were used as the controls (Neg). Discussion This study describes in detail long-term kinetics and persistence of anti-P. papatasi saliva antibodies in sand fly-exposed BALB/c and C57BL/6 mice strains that are widely used as model organisms sensitive or resistant to Leishmania infection, respectively (e.g. [27], [28]). Four IgG subtypes have been described in mice: IgG1, IgG2a, IgG2b, and IgG3. Additionally, certain strains such as C57BL/6 produce the IgG2c subclass instead of IgG2a [29]. The nomenclature of murine IgG subtypes does not correlate with the subtypes of human or canine IgG. The most abundant subclass is IgG1; it binds to Fc-receptors of mast cells and basophils, and it mediates the immediate hypersensitivity reactions. Both IgG1 and IgG2a activate the complement cascade via the alternative pathway, whereas IgG2b employs the classical pathway of complement activation [30]. Moreover, production of IgG1 is the marker of Th2 profile of immune response in mice, while IgG2a predicts Th1 type of immune response in these animals [31]. We showed that repeated exposure to sand fly bites elicits increased levels of anti-saliva IgG and IgG1 in both BALB/c and C57BL/6 strains, and additionally IgG2b in BALB/c mice. In comparison, higher levels of specific IgG were detected in BALB/c mice. This finding complies well with the fact that BALB/c mice mostly respond to repeating antigens by Th2 humoral immune response while C57BL/6 mice produce mainly Th1 cellular response [30]. It seems that P. papatasi saliva elicits mainly production of specific IgG1 subclass, which suggests the polarization to the Th2 type of immune response in bitten mice regardless of the strain. The production of anti-sand fly saliva IgG1 was previously described in BALB/c mice repeatedly bitten by Lutzomyia longipalpis, but they did not observe any production of neither IgG2a nor IgG2b [32]. As the composition of sand fly saliva varies in different sand fly species [33] and the sand fly saliva compounds elicit different profile of specific antibody response [34], this could be the feasible explanation for the production of different antibody subclasses in mice bitten by different sand fly species. To our knowledge, there are no data available about the anti-sand fly saliva antibody subclasses elicited by sand fly feeding in the C57BL/6 mice. In Swiss Webster mice immunization by P. ariasi saliva produced also predominantly IgG1 antibodies [34]. Production of specific IgG2b in BALB/c mice compared to the absence of this antibody subclass in C57BL/6 mice may be the result of different cytokine responses in both mice strains against sand fly saliva. The switch to IgG2b subclass is initialized by production of TGF-β [35], a suppressive cytokine that blocks the activation of lymphocytes and monocytes derived phagocytes. This could positively contribute to the susceptibility of BALB/c mice to Leishmania parasites. Importantly, positive correlation was found in both mice strains between the intensity of sand fly exposure and the levels of specific antibodies in aforementioned subclasses. Our results correspond well to previously published data showing that the antibody response in dogs [3], [5] as well as in humans [4] reflected the intensity and the time-course of sand fly exposure. We found that sand fly exposure did not affect the production of IgG2a and IgG3 in BALB/c mice, and IgG2b, IgG2c, and IgG3 in C57BL/6 mice. Neither did the levels of specific IgE differ significantly between non-exposed and exposed groups of mice, and the IgE kinetics showed high variation during the study. Similarly, high fluctuation in specific IgE response was detected in humans [11], [23] and dogs [3] bitten by Lutzomyia longipalpis in the field as well as under laboratory conditions. While some of the individuals and animals presented high levels of specific IgE, others did not mount specific IgE response at all [3], [11], [23]. To mimic the situation commonly occurring in endemic foci of leishmaniases, where sand fly-free periods last up to 6 months [15], BALB/c mice were exposed to P. papatasi bites again 23 weeks after the last sand fly exposure. This single sand fly exposure elicited statistically significant increase of anti-P. papatasi IgG, IgG1, IgG2b which suggests the persistence of memory cells generated during the previous round of exposures. This could be related to the “previous sand fly season” in the field. Furthermore, in both mice strains, the differences between non-exposed and exposed groups of mice in production of specific IgG1 and IgG2b were detectable from week four or nine, respectively, until the end of the study. Similarly, the levels of specific IgG, IgG1, and IgG2 in sera of dogs exposed to L. longipalpis or P. perniciosus bites differed significantly from pre-immune sera for more than 14 weeks after the last sand fly exposure [3], [5]. In individuals repeatedly bitten by P. argentipes, elevated levels of specific antibodies persisted after the 30-day sand fly-free period, although anti-saliva antibodies significantly decreased throughout this time [4]. Thus, regardless the host-sand fly combination, anti-sand fly saliva antibodies can persist in sera of repeatedly bitten hosts until the next sand fly season. We also characterized the reactivity of mice sera with P. papatasi salivary proteins as well as with selected recombinant proteins. Mice sera of BALB/c and C57BL/6 strains reacted with up to eleven P. papatasi antigenic protein bands. The 30 kDa protein band recognized by both mice strains was identified by mass spectrometry as a mixture of a D7-related (AF335489) and an antigen 5-related (DQ205724) protein. The other proteins which were intensively recognized either by BALB/c (47, 23, and 15 kDa proteins) or by C57BL/6 mice (65, 53, and 51 kDa proteins) were determined as members of the Yellow-related protein family (51 kDa - AF335492, 47 kDa - AF335491), D7-related protein family (23 kDa – AF335488), and SP-15 protein family (15 kDa – AY628879, AY628880, AF335486, AF335485). The 70, 65, 53, and 49 kDa bands were not identified by mass spectrometry. Our results correspond to previously published data, where the human and BALB/c mice IgG antibodies recognized preferentially the P. papatasi 30 kDa protein band [1], [14]. To our knowledge, the only study describing the reactivity of specific IgG subclasses with P. papatasi antigens was performed on humans [14]. In accordance with our results, the 30 kDa D7-related protein was also found to be the most immunogenic antigen in all human antibody subclasses tested [14]. Taken together, our data complies well with previously published studies, where Yellow-related proteins, D7-related proteins, as well as SP-15 proteins from P. papatasi saliva were identified as potent antigens for mice and humans [1], [14]. Sera of BALB/c mice experimentally bitten by P. papatasi were tested also with four bacterially expressed recombinant proteins belonging to two salivary protein families: Yellow-related proteins (PpSP44/AF335492 and PpSP42/AF335491) and D7-related proteins (PpSP30/AF335489 and PpSP28/AF335488). Within the salivary gland homogenate, sera reacted with proteins identified as PpSP42, PpSP30, and PpSP28 proteins, but no reaction was detected with PpSP44. In contrast, PpSP30 and PpSP44 recombinant proteins were strongly recognized and PpSP42 gave a weak reaction. Reaction of anti-saliva IgG with recombinant proteins may, however, differ between mouse strains. For example, the C57BL/6 mice reacted predominantly with PpSP42 and PpSP28 recombinant proteins (data not shown). Although none of the recombinant proteins were recognized by all sera. Each mouse serum tested reacted with at least one of the recombinant proteins. Our data suggest that recombinant proteins could be used as markers of sand fly exposure instead of crude salivary gland homogenates, ideally as a mixture of several different proteins to cope with various host species and individual reactivity of each serum sample. In sand flies this concept has been demonstrated using Lutzomyia longipalpis recombinant proteins; the reactivity of anti-L. longipalpis seropositive human sera with the salivary gland sonicate was comparable to the reaction with the combination of the two L. longipalpis recombinant Yellow-related proteins (LJM11/AY445935 and LJM17/AF132518) [8]. In conclusion, we detected a significant increase of specific IgG and IgG1 in exposed mice of both strains, and of IgG2b in exposed BALB/c mice. The other IgG subclasses were comparable to controls. Specific IgG response was shown to reflect the intensity of sand fly exposure and furthermore, anti-P. papatasi saliva antibody response persisted in mice for more than 5 months. Thus, in endemic areas the antibodies could persist till the following sand fly season. The 30 kDa band recognized by sera of experimentally bitten BALB/c as well as C57BL/6 mice was identified as a mixture of D7-related and antigen 5-related proteins. Moreover, the reactivity of mice sera with PpSP44, PpSP42, PpSP30, and PpSP28 recombinant proteins suggested that their combination could substitute the salivary gland homogenate. Taken together, the kinetics, persistence and the individual variability of anti-sand fly saliva antibody response are important aspects to consider in further experiments, where anti-saliva antibodies are used as the markers of sand fly exposure. Supporting Information Figure S1 Anti-sand fly saliva antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi . BALB/c mice and C57BL/6 mice were divided into control (squares) and experimentally bitten groups (circles). Mice in the latter group were exposed to sand fly bites (arrows) (30 P. papatasi females per week) in weeks 1–5 and additionally in the week 27 (only BALB/c mice). Anti-P. papatasi saliva antibodies - IgG2a (A), IgG3 (C), and IgE (E) in BALB/c mice and IgG2c (B) and IgG3 (D) in C57BL/6 mice - were measured using ELISA as described in Methods. Data are presented as the means ± standard errors of the means. Two independent studies were done. OD = optical density. (TIF) Click here for additional data file. Table S1 Dilution and incubation time of secondary antibodies. (DOC) Click here for additional data file. We would like to thank Dr. Helena Kulikova and Stepanka Hlavova for excellent technical and administrative assistance. The authors have declared that no competing interests exist. This project was funded by EU grant 2011-261504 EDENEXT and the paper is catalogued by the EDENEXT Steering Committee as EDENEXT026. The research was supported by Ministry of Education of the Czech Republic (MSM 0021620828, LC 06009), by Czech Science Foundation (206/09/0777; 206/09/H026; 206/09/0822) and by Charles University (GAUK – 13009/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Rohousova I Ozensoy S Ozbel Y Volf P 2005 Detection of species-specific antibody response of humans and mice bitten by sand flies. 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==== Front Front Endocrinol (Lausanne)Front Endocrinol (Lausanne)Front. Endocrin.Frontiers in Endocrinology1664-2392Frontiers Research Foundation 2279895810.3389/fendo.2012.00087EndocrinologyMini Review ArticleColonic flora, Probiotics, Obesity and Diabetes Marik Paul E. *Division of Pulmonary and Critical Care Medicine, Department of Medicine, Eastern Virginia Medical School,Norfolk, VA, USAEdited by: Tsuguhito Ota, Kanazawa University, Japan Reviewed by: Undurti Narasimha Das, UND Life Sciences, USA Constance Boyer, Centre National de l’Interprofession Laitière, France *Correspondence: Paul E. Marik, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 410, Norfolk, VA 23507, USA. e-mail: [email protected] article was submitted to Frontiers in Diabetes, a specialty of Frontiers in Endocrinology. 11 7 2012 2012 3 8724 5 2012 25 6 2012 Copyright © Marik.2012This 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.Obesity results from alterations in the body’s regulation of energy intake, expenditure, and storage. Animal and human data demonstrate that phylogenic changes occur in the microbiota composition in obese individuals. Furthermore, evidence from animal models suggest that the alterations of the gut microbiota with obesity results in increased energy extraction and lipid deposition, altered release of entero-hormones, increased intestinal permeability and metabolic endotoxemia. Treatment with pre- and probiotics may reverse many of metabolic effects linked with the altered microbiota in obese patients. The gut microbiota is, therefore, a potential nutritional and pharmacological target for the management of obesity and obesity-related disorders. probioticsobesity and diabetes ==== Body Mammals, including humans, have, throughout their evolution, been associated with complex microbial communities, which inhabit the surfaces and alimentary tract of their host and which outnumber the human somatic cells by a factor of 10. The human intestinal microbiota is estimated to be composed of 1013 to 1014 microorganisms whose collective genome, the microbiome, contains at least 100-fold more genes than the complete human genome (Eckburg et al., 2005; Gill et al., 2006). The gut of a neonate is sterile. However, at birth, the gut is immediately colonized by maternal and environmental bacteria and the complexity of the resulting gut microbiota increases until weaning to solid foods. Although incompletely understood the gut microbiota is implicated in a variety of host functions involving intestinal development and function, including epithelial turnover, immune modulation, gastrointestinal motility, and drug metabolism. The gut microbiota also has important metabolic functions, breaking down dietary toxins and carcinogens, synthesizing micronutrients, fermenting indigestible food substances, assisting in the absorption of certain electrolytes and trace minerals, and affecting the growth and differentiation of enterocytes and colonocytes through the production of short-chain fatty acids (SCFA; Macfarlane and Macfarlane, 1997; Zoetendal et al., 2001; Ouwehand et al., 2002; Stappenbeck et al., 2002). Finally, the normal gut microbiota helps prevent luminal colonization by pathogenic bacteria, such as Escherichia coli, Clostridia, Salmonella, and Shigella species. The composition of the adult intestinal microbiota has been intensively studied, by means of culture-based methodologies and,more recently, through culture independent technologies based on the amplification and direct sequencing of small subunit ribosomal DNA sequences. Among the novel investigative methods that are used to study the microbial ecology of complex bacterial communities, the so-called metagenomics approach is considered to represent the method that produces results of “gold standard” quality (Eckburg et al., 2005; Gill et al., 2006). Metagenomics is the study of microbial communities through sequence-based, compositional and/or functional analyses of all the combined microbial genomes contained within an environmental sample. With respect to its application to human intestinal microbiota, metagenomic studies of mucosal as well as fecal samples revealed the presence of representatives of the phylogenetic groups Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Verrucomicrobia, and Actinobacteria (Eckburg et al., 2005; Gill et al., 2006; See Table 1). With a relative abundance of 25 and 65%, respectively, the two dominant phyla Bacteroidetes and Firmicutes represent together up to 90% of the total microbiota, whereas Actinobacteria, Proteobacteria, and Fusobacteria are the subdominant phyla with a relative abundance of about 5, 8, and 1%, respectively (Candela et al., 2010). At least 1800 genera and 16,000 phylotypes at the species level have been so far identified, and an even greater diversity at the species level has been predicted. The bacterial phyla are unevenly distributed along the length of the gastrointestinal tract. Metagenomic analyses of jejunum samples reveal a distinctive community composition which includes an abundance of bacteria belonging to the Streptococcus genus, whereas Bacteroidetes and Firmicutes genera are the predominant microbial groups identified in the distal ileum, ascending colon and rectum. Table 1 Major Bacteria and Archaea phyla and genera found in the human gut microbiota. Reproduced with permission from DiBaise et al. (2008). Phyla Representative genera Bacteria Firmicutes Ruminococcus Clostridium Peptostreptococcus Lactobacillus Enterococcus Bacteroidetes Bacteroides Proteobacteria Desulfovibrio Escherichia Helicobacter Verrucomicrobia Actinobacteria Bifidobacterium Cyanobacteria Synergistes Archaea Euryarchaeota Methanobrevibacter The composition of flora differs between individuals, with each person having unique strains that generally remain constant over time. Genetics, diet, immune status, infections, gastrointestinal disorders as well as antibiotics and other drugs interact to determine an individual’s unique microbiome. The effect of genotype on the composition of the human microbiota was demonstrated in a study involving monozygotic twins (Zoetendal et al., 2001). According to this report, there were greater similarities between the gut microbiota of monozygotic twins than between monozygotic twins and their unrelated marital partners. The importance of the initial colonizing microbial community on the eventual microbial composition of the gut is evident from animal studies. The composition of the mouse gut microbiota is significantly affected by maternal transmission (Ley et al., 2005). Diet is also a pivotal variable in influencing the composition of the intestinal microbiota. Studies on chemically well-defined diet components have proven a clear correlation between diet and the presence of specific bacterial groups. It has been shown that a diet rich in inulin and related fibers promote an increase in bifidobacteria, whereas the intake of dietary sulfate favors several genera of sulfate-reducing bacteria over methanogenic Archaea. Diet affects not only the microbiota composition, but more significantly the metabolic activities of the microorganisms. Obesity results from alterations in energy balance, that is, how the body regulates energy intake, expenditure, and storage. Because starvation poses a greater danger to an organism than overabundance, our biological systems are geared to better protect against weight loss than weight gain (i.e., a thrifty genotype). Considerable effort has been made to improve the availability and stability of the food supply, resulting in an abundance of inexpensive, palatable, and energy-dense foods. Consequently, humans adapted for a situation of insufficiency are now confronted with the easy availability of such foods. Recent evidence suggests that the gut microbiota affect nutrient acquisition and energy regulation and that obese and lean people have differences in their gut microbial landscape. These findings raise the possibility that the gut microbiota may have a role in the development of obesity (See Figure 1; DiBaise et al., 2008; Cani and Delzenne, 2009). FIGURE 1 Proposed mechanism whereby alteration of the bowel flora with a high-fat low-fiber diet alters the microbiota leading to metabolic endotoxemia with increased lipid storage and decreased insulin sensitivity. Adapted with permission from Cani and Delzenne (2009). In an elegant series of experiments, Backhed et al. (2004) found that young conventionally reared mice have a 40% higher body fat content than germ-free mice (gnotobiotic mice) even though they consumed less food than their germ-free counterparts. The distal gut microbiota from the normal mice were then transplanted into the gnotobiotic mice (a process known as conventionalization), resulting in a 60% increase in body fat within 2 weeks without any increase in food consumption or obvious differences in energy expenditure. The authors proposed that the gut microbiota promotes intestinal monosaccharides absorption, energy extraction from non-digestible food components via SCFA production through fermentation, de novo hepatic lipogenesis and adipocyte fatty acid storage. The gut microbiota influence the expression of host genes expressed in the intestine that control fatty acid absorption, oxidation, and storage. One such target is angiopoietin-related protein 4 (ANGPTL4), a potent lipoprotein lipase inhibitor. ANGPTL4 inhibits the uptake of fatty acids from circulating triglyceride-rich lipoproteins in white adipose and muscle tissues. Backhed et al. (2004) demonstrated that colonizing germ-free mice with gut microbiota leads to a drop in the intestinal expression of ANGPTL4. Several other proteins/systems (such as the endocannabinoid system and the tight junction protein zona occludin-1) are influenced by gut colonization and are changed upon dietary modulation of gut microbiota composition, which are implicated in the control of inflammation, gut barrier function, gut motility, nutrient oxidation, and storage (Delzenne and Cani, 2011). Recent data suggests that SCFAs act not only as an energy substrate for the host, but also as a signaling molecule. SCFAs act as ligands for at least two G-protein-coupled receptors, GPR41 and GPR43 (Le Poul et al., 2003). Samuel et al. (2008) have demonstrated that GPR41-/- mice colonized with a model of fermentative microbial community did not gain fat mass at the same extent as the wild-type littermates. On the basis that obesity and insulin resistance is associated with low-grade chronic systemic inflammation, Cani et al. (2007a) postulated another mechanism linking the intestinal microbiota to the development of obesity. They hypothesized that bacterial lipopolysaccharide (LPS) derived from Gram-negative bacteria residing in the gut microbiota acts as a triggering factor linking inflammation to a high-fat diet-induced metabolic syndrome. In a series of experiments in mice fed a high-fat diet they showed that a high-fat diet increases endotoxemia, favors an increase in the Gram-negative to Gram-positive colonizing bacteria and that chronic endotoxemia induces obesity, insulin resistance, and diabetes (See Figure 1; Cani et al., 2007a,b, 2009b). These authors demonstrated that modulation of the intestinal microbiota by using prebiotics in obese mice acts favorably on the intestinal barrier, lowering the high-fat diet-induced LPS endotoxemia and systemic and liver inflammation. Modulation of the gut flora with prebiotics has been demonstrated to increase glucagon-like peptide-2 (GLP-2) production in the colon. Increased GLP-2 production is associated with higher expression of ZO-1 which improves mucosal barrier function leading to a decrease of plasma LPS. To assess the relative abundance of various types of gut bacteria in obese and lean mice, Ley et al. (2005) analyzed bacterial 16S rRNA gene sequences from the cecal microbiota of genetically obese (ob/ob) mice, their lean ob/+ and +/+ siblings, and their ob/+ mothers, all fed the same polysaccharide-rich diet. They found that the ob/ob mice had 50% fewer Bacteroidetes and correspondingly more Firmicutes than their lean littermates, a finding unrelated to differences in food consumption. Cani et al. (2007a,b) demonstrated that induced obesity in rodents markedly reduced the number of Bifidobacterium as well as Bacteroides-related bacteria. Ley et al. (2006) compared the distal gut microbiota of obese and lean human subjects and found that obese people had fewer Bacteroides and more Firmicutes than did lean control subjects. Interestingly, the authors observed that after weight loss (following a fat restricted or a carbohydrate restricted low-calorie diet), the ratio of Bacteroidetes to Firmicutes approached a lean type profile after 52 weeks of diet-induced weight loss (Ley et al., 2006). Collado et al. (2008) observed significant differences in gut microbiota composition according to the body weight during pregnancy. Interestingly, they found significantly higher numbers of Bacteroides group and of S. aureus in the overweight state compared with normal-weight women, and they established a positive correlation between the number of Bacteroides on the one hand, and the weight and BMI (before and over pregnancy), on the other hand. The Bifidobacterium group was present in higher numbers in normal weight than in overweight women and also in women with lower weight gain over pregnancy. In addition, these researchers demonstrated that overweight mothers give birth to neonates that have a decreased number of Bifidobacteria (Collado et al., 2010). The infants’ fecal microbial composition was related to the weight and weight gain of their mothers during pregnancy. This finding may be related to the effect of colonization at birth and/or inheritable obesogenic microbiota. A recent paper has shown for the first time in humans that differences in the gut microbiota may precede overweight development. Kalliomaki et al. (2008) have shown that Bifidobacterium spp. number was higher in children who exhibited a normal weight at 7 years than in children becoming overweight. They observed that the Staphylococcus aureus was lower in children who maintain a normal weight than in children becoming overweight several years later. The authors proposed that S. aureus may act as a trigger of low-grade inflammation contributing to the development of obesity. Gastric-bypass surgery has been shown to be very effective in reversing insulin resistance (Mingrone et al., 2012; Schauer et al., 2012). This effect has been observed to occur early after surgery and before the patients have had a significant loss of weight. It has been postulated that the surgically altered anatomy and flow of nutrients results in alteration of the microbiome which may explain this finding (Cani and Delzenne, 2011). Li et al. (2011) performed bacterial profiling to determine the effect of Roux-en-Y gastric bypass in a non-obese rat model. At 2 weeks post-surgery they noted substantial shifts of the main gut phyla toward higher concentrations of Proteobacteria (52-fold). Lower concentrations of Firmicutes (4.5-fold) and Bacteroidetes (twofold) in comparison with sham-operated rats were also found. EFFECT OF PREBIOTICS, PROBIOTICS, AND SYNBIOTICS ON WEIGHT LOSS AND INSULIN RESISTANCE The best non-surgical strategy for reversing obesity in the population may be to promote small but long-term changes in diet and physical activity that take advantage of our biological systems for regulating energy balance and preventing positive energy balance. Although clearly no substitute for proper diet and exercise, manipulation of the gut microbiota may represent a novel approach for treating obesity, one that has few adverse effects. A probiotic is defined as “a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora of the host and by that exert beneficial health effects in this host” (Schrezenmeir and de Vrese, 2001). The term prebiotic was introduced by Gibson and Roberfroid (1995), who defined prebiotics as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon.” The term synbiotic is used when a product contains both probiotics and prebiotics. PREBIOTICS Prebiotic agents are non-digestible oligosaccharides that act as “fertilizers” of the colonic microbiota, enhancing the growth of beneficial commensal organisms (e.g., Bifidobacterium and Lactobacillus species; Schrezenmeir and de Vrese, 2001). Fructo-oligosaccharides are prebiotic agents that are fermented by a number of colonic bacteria to modulate the growth of beneficial bacteria (Roberfroid, 2002). The number of Bifidobacteria has been shown to increase in the presence of inulin-type fructans with prebiotic properties. This increase occurs within a few days, but rapidly disappears upon withdrawal of the prebiotic compounds (after 1 week; Gibson and Roberfroid, 1995). The extent of increase in the number of Bifidobacteria is also dependent on their initial number in the gut. Breast-milk contains oligosaccharides with prebiotic properties that contribute to the increase in the number of Bifidobacteria after birth (Bode, 2009; Chichlowski et al., 2011). Dietary fructans, which are present in various fruits and vegetables and added to food products, are used as an energy substrate by bacteria, including Bifidobacterium spp., that express β-fructofuranosidase, which promotes their development in the gut. A remarkable increase has been observed in the number of Bifidobacterium spp. in mice with diet-induced or genetically determined obesity that were supplemented with inulin-type fructans (Cani et al., 2007b). Interestingly, the number of Bifidobacteria was inversely correlated with the development of fat mass, glucose intolerance, and LPS level (Cani et al., 2007b). Inulin-type fructans increased the number of endocrine L cells in the jejunum and in the colon of rodents, and promoted the production and release of the active forms of GLP-1 and GLP-2 in the portal vein (Cani et al., 2004). GLP-1 participates in prebiotic-driven decreases in appetite, fat mass and hepatic insulin resistance, whereas GLP-2, as mentioned before, contributes to the reduced permeability of the intestinal wall and endotoxemia that are associated with obesity (Cani et al., 2006, 2009b). Interestingly, a 2-week treatment with inulin-type fructans (16 g per day) in healthy volunteers increased the postprandial release of gut peptides (namely GLP-1 and gastric inhibitory peptide; Cani et al., 2009a). Cani and colleagues demonstrated that in rats fed a standard or high-fat diet, the addition of oligofructose to the diet reduced energy intake and consumption and protected against weight gain and fat-mass development, effects shown to be mediated by the modulation of endogenous gut peptides involved in appetite and weight regulation (Cani et al., 2004, 2005; Delzenne et al., 2005). The authors also reported that oligofructose increased the gut bifidobacterial content of high-fat diet-fed mice and that endotoxemia significantly and negatively correlated with Bifidobacterium species (Cani et al., 2007b). Parnell and Reimer (2009) randomized 48 otherwise healthy adults with a BMI > 25 kg/m2 to receive 21 g oligofructose or a placebo (maltodextrin) for 12 weeks. There was a reduction in body weight of 1.03 ± 0.43 kg with oligofructose supplementation, whereas the control group experienced an increase in body weight of 0.45 ± 0.31 kg over 12 weeks (p = 0.01). A lower area under the curve (AUC) for ghrelin (p = 0.004) and a higher AUC for peptide YY (PYY) with oligofructose (p = 0.03) coincided with a reduction in self-reported caloric intake (p > 0.05). Glucose decreased in the oligofructose group and increased in the control group between initial and final tests (p > 0.05). PROBIOTICS AND SYNBIOTICS Probiotics have generated considerable interest in recent years because studies investigating their use in a variety of clinical conditions have yielded encouraging results (Floch and Montrose, 2005). Probiotics have been demonstrated to be beneficial in necrotizing enterocolitis, in adults and children with infective diarrhea and for the prevention and treatment of antibiotic associated diarrhea (Floch and Montrose, 2005; Hempel et al., 2012). Probiotics belonging to a number of genera including Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, and Enterococcus (Enterococcus faecium SF68) have been used in these studies. The genus Lactobacillus comprises more than 90 species, the most commonly used include L. acidophilus, L. rhamnosus, L. casei, L. bulgaricus, L. plantarum, and L. reuteri. Most of the clinical trials reported to date have used blends of various probiotic genera, primarily Lactobacillus spp. in combination with other probiotics. The study of probiotics is complicated by the fact that the efficacy of these agents may be strain specific and the results from one probiotic (or combination) cannot be extrapolated to another. Furthermore, the results of clinical trials may depend on the potency (concentration) and the measures used to ensure the “bio-availability” of the bacteria. While probiotics are regarded as safe, few prebiotics have received the FDA GRAS (Generally Recognized as Safe) approval. Probiotics should be used with caution in critically ill and immunocompromised patients. In rare cases, probiotics have been associated with serious adverse effects in these patients including fungemia (Saccharomyces boulardii) and bacterial sepsis (Riquelme et al., 2003; Land et al., 2005). While clinical data supports the use of prebiotics and experimental data supports the use of probiotics, few studies have investigated the role of probiotics in patients with diabetes, insulin resistance, and obesity. Luoto et al. (2010) randomized 159 women to receive Lactobacillus rhamnosus GG or placebo 4 weeks before expected delivery with the intervention extending for 6 months postnatally. Anthropometric measurements of the children were followed for 10 years. The perinatal probiotic intervention appeared to moderate the initial phase of excessive weight gain (up to 48 months), especially among children who later became overweight. Kadooka et al. (2010) conducted a multicenter, double-blind, randomized, placebo-controlled intervention trial in which subjects (n = 87) with an increased BMI (24.2–30.7 kg/m2) and abdominal visceral fat area (81.2–178.5 cm2) were randomly assigned to receive either fermented milk containing Lactobacillus gasseri SBT2055 (active FM; n = 43) or fermented milk without LG2055 (control FM; n = 44) for 12 weeks. Abdominal fat area was determined by computed tomography. In the active FM group, abdominal visceral and subcutaneous fat areas significantly (p > 0.01) decreased from baseline by an average of 4.6 and 3.3% respectively. Body weight and other measures also decreased significantly while, none of these parameters changed significantly in the control group. Probiotics and synbiotics have been demonstrated to decrease intestinal permeability and endotoxemia in patients with liver disease and may serve as a model for reducing “metabolic endotoxemia” (Malaguarnera et al., 2010). In patients with minimal hepatic encephalopathy, Liu et al. (2004) demonstrated that treatment with a synbiotic significantly increased the fecal content of non-urease-producing Lactobacillus species at the expense of other bacterial species. Such modulation of the gut flora was associated with a significant reduction in blood ammonia levels, reversal of encephalopathy and a reduction in endotoxemia. CONCLUSION Experimental and clinical data demonstrate that obesity is associated with changes in the intestinal microbiota. Furthermore, provocative data suggest that manipulation of the microbiome using prebiotics, probiotics, and synbiotics may reduce insulin resistance and fat accumulation. While probiotics are regarded as safe additional studies are required before such therapy can be widely recommended. 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Front Endocrinol (Lausanne). 2012 Jul 11; 3:87
==== Front Crit CareCrit CareCritical Care1364-85351466-609XBioMed Central 1364-8535-16-R322235654710.1186/1364-8535-16-R32ResearchFlavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis Bitto Alessandra [email protected] Letteria [email protected] Antonio [email protected] Natasha [email protected] Mariagrazia [email protected] Francesco S [email protected] Francesco [email protected] Domenica [email protected] Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology, University of Messina, via C. Valeria Gazzi, Messina, 98125, Italy2 Department of Neurosciences, Psychiatry and Anaesthesiology, University of Messina, via C. Valeria Gazzi, Messina, 98125, Italy2012 22 2 2012 16 1 R32 R32 24 10 2011 25 1 2012 22 2 2012 Copyright ©2012 Squadrito et al.; licensee BioMed Central Ltd.2012Squadrito 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 Cecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis. Methods C57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies. Results COX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver. Conclusions Flavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition. ==== Body Introduction Cecal ligation and puncture (CLP) is an experimental model of shock that reproduces all the pathologic sequelae of sepsis that occur in intensive care patients [1]. Despite a massive effort invested in developing potential therapies, to date, severe sepsis is still a common, frequently fatal, and expensive pathologic condition [2]. Sepsis is a systemic inflammatory response to infection triggered by Gram-positive and/or -negative organisms, which can proliferate and/or release endotoxin and exotoxins that stimulate monocytes, macrophages, endothelial cells, and neutrophils to an overproduction and release of major inflammatory mediators, followed by tissue injury which in turn, culminates in multiple-system organ failure (MOF). The first step of this signaling pathway involves Toll-like receptors (TLRs) binding to the bacterial cell-wall components, which induces nuclear factor (NF)-κB/IκB system activation that exerts transcriptional regulation on proinflammatory genes and encodes various adhesion molecules, cytokines, and other proinflammatory mediators. In addition, TLRs activate MAP kinases, including extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), and p38 [3,4]. Recently, it was demonstrated that β-arrestins, adaptor proteins involved in G protein-coupled receptor (GPCR) desensitization, are also implicated in regulation of TLR signaling and proinflammatory gene expression. Furthermore, it has been suggested that β-arrestin 1 and 2 differentially regulate TLR4 signaling pathways and, in particular, β-arrestin 2 negatively regulates the inflammatory response in CLP-induced mortality [5,6]. During septic shock, eicosanoids, proinflammatory cytokines, such as TNF-α, IL-1β, IL-3, IL-6, IL-8, and antiinflammatory mediators, such as IL-2, IL-4, and IL-10, are dramatically increased to block the infection and tissue damage [7-10]. Severe sepsis associated with hypotension, acute respiratory distress syndrome (ARDS), hepatic failure, disseminated intravascular coagulation, and organ dysfunction, is characterized by a poor prognosis; these changes are well documented first in the lung and then in the liver [11]. In addition, COX-2 and 5-LOX products derived from arachidonic acid, such as prostaglandins and leukotrienes, are responsible for the microvasculature failure, and are implicated as pathogenic mediators in endotoxemia [12]. Pharmacologic agents that modulate eicosanoid metabolism have been used to block the inflammatory response of various diseases, including septic shock [9,13-15]. Ito and co-workers [14] have shown that mice pretreated with a 5-LOX inhibitor have a reduced TNF-α production and attenuated liver injury during endotoxemia. Moreover, early survival improvement was found in endotoxin-challenged mice, but not in CLP mice, treated with a selective COX-2 inhibitor [15]. Conversely, it has been demonstrated that anti-TNF antibody and IL-1 receptor antagonist, as well as COX or LOX pathways single inhibitors, failed to protect patients with septicemia and septic shock. Altogether, these results support the effort to find a novel strategy; the development of dual inhibitors of COX-2 and 5-LOX pathways may represent new insights into the treatment of sepsis, thanks to the anti-inflammatory efficacy and the lower incidence of gastric toxicity [16]. Recently, we demonstrated that flavocoxid, which contains both the naturally occurring flavonoids baicalin and catechin isolated from Scutellaria baicalensis (S. baicalensis) and Acacia catechu (A. catechu), respectively, acts as a dual inhibitor of COX-2 and 5-LOX and blunts the proinflammatory phenotype in LPS-stimulated macrophages [17]; in addition, it holds immunomodulatory and antiinflammatory activities in experimental models in vivo, as shown in a murine model of Duchenne muscular dystrophy [18] and in rats subjected to acute caerulein-induced pancreatitis [19]. To date, a dual inhibitor of both COX-2 and 5-LOX pathways has not been investigated in CLP-induced lethal sepsis in mice. Therefore, the aim of our study was to evaluate the potential therapeutic effect of flavocoxid, in a murine cecal-ligation puncture model of polymicrobial sepsis-induced mortality. Materials and methods Animals, experimental procedure, and treatments All procedures complied with the standards for the care and use of animal subjects, as stated in the Guide for the Care and Use of Laboratory Animals, and were approved by the Committee on Animal Health and Care of Messina University. The 5-week-old male C57BL/6J mice (Charles River, Calco, LC, Italy), used for this study, had free access to a standard diet and tap water. They were maintained on a 12-hour light/dark cycle at 21°C. Cecal ligation and puncture (CLP) was performed in C57BL/6J mice as previously described [6]. Specifically, mice were anesthetized with ether, and a midline incision was made below the diaphragm to expose the cecum. The cecum was ligated at the colon juncture with a 4-0 silk ligature suture without interrupting intestinal continuity. The cecum was punctured once with a 22-gauge needle. The cecum was returned to the abdomen, and the incision was closed in layers with a 4-0 silk ligature suture. After the procedure, the animals were fluid-resuscitated with sterile saline (1 ml) injected subcutaneously (sc). Sham controls were subjected to the same procedures as were those with CLP without ligation and puncture of the cecum. Shams were treated with flavocoxid or vehicle. CLP animals were randomized to receive flavocoxid with and without gentamicin (3.2 mg/kg, sc), or baicalin (16 mg/kg, ip), or catechin (4 mg/kg, ip), or its vehicle (0.9% NaCl solution). On the basis of previous data, 20 mg/kg was chosen as the optimal dose of flavocoxid for these experiments [19]. The doses of baicalin and catechin reflected the ratio (4:1) of these two molecules in the formulation [17-19]. Flavocoxid intraperitoneal (ip) or oral treatment was started 1 hour after the CLP procedure and repeated every 12 hours. The same protocol was used for baicalin and catechin treatment. In a first experiment, mice were subjected to CLP (n = 15 per group) or sham operation (n = 10 per group), and survival was monitored every 24 hours for a total of 120 hours. In a second experiment, six mice per group were subjected to sham operation or CLP, and NF-κB, MAP kinases, TNF-α, IL-6, IL-10, and β-arrestin 2 expression was determined in the lung and liver after 18 hours. In the same animals, a specimen from lung and liver was also collected for MPO activity and histologic analysis, and blood was drawn for measuring serum cytokines, eicosanoids, and lipoxin A4 levels. Isolation of cytoplasmic and nuclear proteins In brief, lung and liver samples were homogenized in 1 ml lysis buffer (25 mM Tris/HCl, pH 7.4, 1.0 mM ethylene glycol tetraacetic acid, 1.0 mM ethylenediamine tetraacetic acid, 0.5 mM phenylmethyl sulfonylfluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 100 mM Na3VO4). The homogenate was subjected to centrifugation at 15,000 g for 15 minutes. The supernatant was collected and stored at -80°C. The concentration of total proteins was determined by using a Bio-Rad protein-assay kit (Milan, Italy). Determination of β-arrestin 2, NF-κB, TNF-α, p-ERK 1/2, p-JNK, COX-2, 5-LOX, IL-6, and IL-10 with Western blot analysis β-Arrestin 2, NF-κB, TNF-α, p-ERK 1/2, p-JNK, COX-2, 5-LOX, IL-6, and IL-10 expression was evaluated with Western blot, according to the method described previously [17]. Primary antibodies for NF-κB, TNF-α, IL-6, IL-10, COX-2, 5-LOX, and β-arrestin 2 were purchased from Abcam (Cambridge, UK), whereas primary antibodies for p-ERK 1/2 and p-JNK were purchased from Biovision (Mountain View, CA, USA). Secondary antibodies, peroxidase conjugated, were obtained from Pierce (Rockford, IL, USA). The protein signals were quantified by scanning densitometry by using a bio-image analysis system (Bio-Profil Celbio, Milan, Italy) and were expressed as integrated intensity in comparison with those of control normal animals measured with the same batch [17]. TNF-α, IL-6, IL-10, and lipoxin A4 production TNF-α, IL-6, and IL-10 were measured by using enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA). Lipoxin A4 production was analyzed with the ELISA kit (Oxford Biomedical Research, Oxford, MS, USA) and by following the manufacturer's instructions. LTB4 and PGE2 production Blood was collected, stored at -80°C, and analyzed for LTB4 by using a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA). Samples were run in duplicate, and the absorbance was read at 450 nm. The intensity of the color was proportional to the concentration of LTB4 in the sample. PGE2 production was assayed without purification by using the Cayman EIA kit (Cayman, Ann Arbor, MI, USA). Samples were run in duplicate, and the absorbance was read at 412 nm and was directly proportional to the content of PGE2 in samples. Myeloperoxidase activity MPO activity was determined in the lung and liver as an index of neutrophil accumulation, as previously described [6]. MPO activity of tissue was expressed in units per 100 mg of tissue. Histologic evaluation For light microscopy, lung and liver tissues were rapidly removed and fixed in 10% buffered formalin. Subsequently, they were embedded in paraffin, cut, and stained with hematoxylin and eosin (H&E). Assessment of tissue changes was carried out by an experienced pathologist who was blinded to the treatments. The histologic study of liver sections was based on the following parameters: infiltration of inflammatory cells, steatosis, necrosis, and ballooning degeneration. The parameters considered for scoring lung damage were infiltration of inflammatory cells, vascular congestion, and interstitial edema. All parameters were evaluated by the following score scale of values: 0, absent; 1, mild; 2, moderate; and 3, severe. Drugs Flavocoxid (Limbrel), baicalin, and catechin were a kind gift of Primus Pharmaceuticals, Inc. (Scottsdale, AZ, USA). Gentamicin sulfate was purchased from Sigma Aldrich (St. Louis, MO, USA). All the compounds were dissolved in 0.9% NaCl solution. Statistical analysis Data are expressed as the mean ± SD. All other data were analyzed with ANOVA followed by post hoc evaluation (Bonferroni test) by using GraphPad Prism Software, (San Diego, CA, USA). For the histologic results, statistical analysis was performed by using the Kruskal-Wallis test followed by the Mann-Whitney U test. Survival rates were analyzed with the Kaplan-Meier method, followed by the Log Rank test. A value of P < 0.05 was selected as the criterion for statistical significance. Results Effects of flavocoxid on CLP-induced mortality For mortality studies, C57BL/6 mice subjected to CLP or sham operation were treated with flavocoxid (20 mg/kg/ip) or vehicle, 1 hour after CLP. All the CLP animals were fluid resuscitated with sterile 0.9% NaCl saline solution (1 ml/mouse). Flavocoxid treatment was repeated every 12 hours. Mouse survival was monitored for up to 120 hours. CLP-induced sepsis in mice produced severe mortality (P < 0.01) with respect to sham animals. Flavocoxid treatment did not affect survival in sham animals, but significantly reduced mortality (P < 0.01) in CLP mice (Figure 1). Flavocoxid, administered orally at the same dosage, produced an overlapping effect on the survival rate (Figure 1). Furthermore, the combination of flavocoxid with gentamicin (administered subcutaneously at the dose of 3.2 mg/day throughout the experiment) caused a greater improvement in the survival rate (Figure 1). Neither baicalin (16 mg/kg/ip) nor catechin (4 mg/kg/ip), administered in the same percentage as in the 20 mg/kg/ip of flavocoxid, significantly modified survival (Figure 1). Figure 1 Effect of flavocoxid on survival in mice with cecal ligation and puncture (CLP). C57BL/6J mice were subjected to CLP (n = 15 per group) or sham operation (n = 10 per group). All the CLP animals were fluid resuscitated with sterile NaCl 0.9% saline solution (1 ml/mouse). Animals were treated with flavocoxid (20 mg/kg, ip) with and without gentamicin (3.2 mg/kg, sc), or baicalin (16 mg/kg, ip), or cathechin (4 mg/kg, ip), or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Mouse survival was monitored every 24 hours for a total of 120 hours, and expressed as percentage of mice survived at each time point. *P < 0.01 compared with sham + vehicle or flavocoxid; #P < 0.01 compared with CLP + vehicle; §P < 0.01 compared with CLP + vehicle. Effects of flavocoxid on TNF-α, IL-6, and IL-10 expression Inflammatory cytokines have a crucial role in CLP-induced sepsis and mortality. Therefore, we studied the effects of flavocoxid on CLP-induced TNF-α, IL-6, and IL-10 expression in lung and liver of mice. Basal protein levels of TNF-α, IL-6, and IL-10 were measured in lung (Figure 2a-c) and liver (Figure 2d-f) of sham mice. CLP mice showed a significant increase in these cytokines (p < 0.005) compared with sham (Figure 2a-f). Flavocoxid did not change protein expression in sham animals, but markedly inhibited the expression of TNF-α and IL-6 in CLP animals (P < 0.005); moreover, IL-10 protein expression was further enhanced after treatment in both lung (Figure 2a-c) and liver (Figure 2d-f) tissues of septic animals. Figure 2 Western blot analysis of TNF-α, IL-6, and IL-10 in the lung (a through c) and liver (d through f) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Flavocoxid reduces COX-2 and 5-LOX CLP-induced expression, serum cytokines, LTB4 and PGE2, and increases lipoxin A4 To verify in vivo the protective effect of flavocoxid, we first evaluated COX-2 and 5-LOX expression in lung and liver of both CLP and sham mice. Sham mice showed a low expression of COX-2 and 5-LOX in both lung and liver (Figure 3a-d), and no differences were found between sham animals given either flavocoxid or vehicle. In contrast, the expression of both enzymes was increased in lung and liver of CLP mice, with respect to sham mice. Flavocoxid treatment significantly reduced COX-2 and 5-LOX expression either in lung and in liver of CLP mice (Figure 3a-d). Figure 3 Western blot analysis of COX-2 and 5-LOX in the lung (a and b) and liver (c and d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Circulating levels of TNF-α, IL-6, and IL-10 were also measured 18 hours after surgery. Serum cytokines were significantly augmented in the bloodstream of CLP mice (Figure 4a-c). Flavocoxid treatment markedly blunted the serum concentration of TNF-α (Figure 4a) and IL-6 (Figure 4b), but increased serum IL-10 (Figure 4c). Figure 4 Serum TNF-α (a), IL-6 (b), and IL-10 (c) levels obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Serum PGE2 and LTB4 measurements were also determined in CLP and sham mice, 18 hours after surgery. Figure 5a and 5b shows a significant increase of both PGE2 and LTB4 in serum of CLP mice. As a consequence of COX-2 and 5-LOX inhibition by flavocoxid treatment, PGE2 and LTB4 serum levels were significantly reduced in CLP mice (P < 0.005) (Figure 5a and 5b). Lipoxin A4 was also enhanced in the serum of CLP mice 18 hours after surgery (Figure 5c). Flavocoxid administration caused a greater increase in the circulating antiinflammatory lipid mediator, thus suggesting that this antiinflammatory mechanism may concur with the protective effect of flavocoxid (Figure 5c). Figure 5 Serum PGE2 (a), LTB4 (b), and lipoxin A4 (c) levels obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Flavocoxid regulates CLP-induced expression of NF-κB, p-ERK, p-JNK, and β-arrestin 2 NF-κB is a transcription factor that is triggered by TLR activation and causes the nuclear activation of inflammatory genes involved in sepsis. Nuclear NF-κB was markedly expressed in the lung (Figure 6a) and in the liver (Figure 6c) of CLP mice, 18 hours after surgery. Flavocoxid treatment blunted the increased nuclear expression of NF-κB in both the lung (Figure 6a) and the liver (Figure 6c). To determine whether β-arrestin 2 plays a key role in modulating the TLR signaling pathway in CLP-induced inflammation, β-arrestin 2 protein expression in lung and liver of sham and CLP mice was studied. Basal levels of β-arrestin 2 were measured in lung and liver of sham animals (Figure 6b and 6d). As shown in Figure 6b and 6d, β-arrestin 2 expression was significantly reduced in lung and liver of mice subjected to CLP (P < 0.005) compared with sham-operated animals. Treatment with flavocoxid preserved β-arrestin 2 expression in both lung and liver of CLP mice (Figure 6b and 6d), but it did not affect basal values of β-arrestin 2 in sham animals. Figure 6 Western blot analysis of NF-κB and β-arrestin 2 in lung (a, b) and liver (c, d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, and repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Furthermore, to better characterize the mechanism of action of flavocoxid, p-ERK 1/2 and p-JNK were evaluated. A low expression of either p-ERK (Figure 7a and 7c) or p-JNK (Figure 7b and 7d) was determined in both lung and liver of sham mice. No significant differences were found in MAPKs expression in sham animals administered flavocoxid. CLP mice showed a significant increase in the expression of MAPKs with respect to sham animals (Figure 7a to 7d). In contrast, flavocoxid treatment significantly reduced (P < 0.005) p-ERK and p-JNK expression in lung and liver of CLP animals (Figure 7a to 7d), suggesting that β-arrestin 2 regulates sepsis-induced MAPK activity in vivo. Figure 7 Western blot analysis of p-ERK 1/2 and p-JNK in lung (a, b) and liver (c, d) of CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Effects of flavocoxid on histologic changes and myeloperoxidase activity Sham animals treated with either vehicle or flavocoxid showed a normal architecture of lung and liver (Figure 8a and 8d; Tables 1 and 2). CLP induced damage in lung and liver, characterized by microscopic alterations, hemorrhagic areas, and marked leukocyte infiltration (Figure 8b and 8e; Tables 1 and 2). In CLP mice, lung histologic examination showed ample inflammatory infiltrate, interstitial edema, and marked vascular congestion (Figure 8b and Table 1). The histologic picture showed that liver damage was characterized by a high degree of inflammatory infiltrate, steatosis, necrosis, ballooning degeneration, as well as morphologic alterations of the nuclei (Figure 8e and Table 2). In contrast, treatment with flavocoxid significantly reduced the histopathologic alterations in the lung (Figure 8c) and liver (Figure 8f) of CLP mice. Lung histology revealed a reduced inflammatory infiltrate, vascular congestion, and interstitial edema (Figure 8c and Table 1). After flavocoxid treatment, liver inflammatory infiltrate and steatosis were less evident, necrosis was absent, and normal cellular architecture was restored (Figure 8f and Table 2). Figure 8 Histology of treated and untreated mice. (a) Normal histology of lung obtained from sham mice. (b) Representative cecal ligation and puncture (CLP)-induced lung damage. (c) Representative lung of CLP mice treated with flavocoxid (20 mg/kg, ip, administered 1 hour after surgery, repeated every 12 hours). (d) Normal histology of liver obtained from sham mice. (e) Representative CLP-induced liver damage. (f) Representative liver of CLP mice treated with flavocoxid (20 mg/kg, ip, administered 1 hour after surgery, repeated every 12 hours). Sham and CLP mice were killed 18 hours after surgery. Histopathology was performed on lung and liver. Original magnification, ×20. Table 1 Flavocoxid effects on lung histologic damage in CLP mice Parameter Sham Sham + flavocoxid CLP CLP + flavocoxid Inflammatory infiltrate 0 0 2.27 ± 0.59 1.15 ± 0.57a Vascular congestion 0 0 2.12 ± 0.20 1.25 ± 0.27a Interstitial edema 0 0 2.33 ± 0.41 1.25 ± 0.52a Histologic grading as reported in Methods; six mice per group treated with flavocoxid (20 mg/kg, ip).aP < 0.05 versus CLP. Table 2 Flavocoxid effects on liver histologic damage in CLP mice Parameter Sham Sham + flavocoxid CLP CLP + flavocoxid Inflammatory infiltrate 0 0 2.52 ± 0.52 1.55 ± 0.46a Steatosis 0 0 2.33 ± 0.41 1.47 ± 0.26a Necrosis 0 0 2.77 ± 0.23 0b Ballooning degeneration 0 0 2.17 ± 0.26 0.21 ± 0.12b Histologic grading as reported in Methods; six mice per group treated with flavocoxid (20 mg/kg, ip). aP < 0.05 versus CLP; bP < 0.001 versus CLP. To confirm the antiinflammatory effects of flavocoxid, we evaluated myeloperoxidase (MPO) activity, a marker of neutrophil infiltration in both the lung (Figure 9a) and the liver (Figure 9b) of CLP mice 18 hours after surgery. MPO content was markedly increased in the lung (Figure 9a) and in the liver (Figure 9b) of septic mice. The dual inhibitor of COX-2 and 5-LOX markedly reduced leukocyte infiltration in the lung (Figure 9a) and in the liver (Figure 9b) of CLP mice. Figure 9 Myeloperoxidase (MPO) activity in the lung (a) and liver (b) obtained from CLP and sham mice. Animals were treated with flavocoxid (20 mg/kg, ip) or vehicle (NaCl 0.9%) 1 hour after surgery, repeated every 12 hours. Bars represent the mean ± SD of six animals. *P < 0.005 compared with sham + vehicle or flavocoxid; #P < 0.005 compared with CLP + vehicle. Discussion Flavonoids have been used in traditional remedies for the treatment of a variety of diseases, including inflammatory conditions [20-22]. Flavocoxid, marketed as Limbrel, is an FDA-regulated prescription, medical food, for the clinical dietary management of OA in United States, which contains the flavonoids, baicalin and catechin [23]. We previously showed that flavocoxid reduced the expression of both COX-2 and 5-LOX enzymes and inhibited the activation of the transcription factor NF-κB in LPS-stimulated macrophages in vitro [17]. Therefore, we evaluated the effects of flavocoxid in an in vivo murine model of polymicrobial sepsis, which better reproduces human sepsis. Li et al. [9] demonstrated that NS-398, a selective inhibitor of COX-2, ameliorates cytokine imbalance and decreases liver injury in septic rats. Additionally, Reddy et al. [15] showed that treatment with a COX-2 inhibitor improves early survival in murine endotoxemia but not in CLP mice. Our study demonstrates for the first time that a dual COX-2 and 5-LOX inhibitor exerts beneficial effects in a murine CLP model of abdominal sepsis. In the present study, flavocoxid significantly improved survival in CLP mice; this protective effect was strongly associated with reduced expression and production of proinflammatory cytokines TNF-α and IL-6, as well as with increased antiinflammatory IL-10 protein level. Recently, different studies have shown that β-arrestin 2, a member of the arrestin family originally known for its prominent role in desensitization process of G protein-coupled receptors, plays an important function in the signal-transduction pathway of TLR4 [5,24,25]. We found that β-arrestin 2 expression was significantly reduced in lung and liver of CLP mice compared with sham mice, and, in parallel experiments, CLP-induced TNF-α, IL-6, and IL-10 expression was significantly higher in control mice. Our findings, therefore, are in agreement with those of other studies, which indicate β-arrestin 2 as a negative regulator of the CLP-induced inflammatory response [5,6,26,27]. It was demonstrated that β-arrestin 2 downregulates inflammatory responses in polymorphonuclear leukocytes [26] as well as in splenocytes after CLP induction [6]. An altered cytokine production was also found by Wang et al. [27] in bone marrow-derived macrophages from β-arrestin 2-deficient mice, in response to LPS. All these data suggest that β-arrestin 2 is an essential negative regulator of sepsis-induced inflammation via TLR signaling and may represent an innovative target for antisepsis drugs. However, conflicting results were obtained by Porter et al. [28] in regulating inflammatory cytokines production in beta arrestins knockout (KO) mice during endotoxic shock; the observed differences might be explained by the use of a different shock model. Flavocoxid, given curatively, restored β-arrestin 2 to basal levels and blunted CLP-induced increase in TNF-α and IL-6, but not IL-10 production in both lung and liver tissues. In addition, flavocoxid reduced serum levels of PGE2 and LTB4 as a consequence of a downregulation of COX-2 and 5-LOX expression in lung and liver of mice subjected to CLP, implicating the same mechanism of action of flavocoxid as reported in our previous studies [17-19,23]. These findings are further supported by histologic examinations: flavocoxid treatment restored organ damage specifically in lung and liver that are profoundly involved in the pathophysiologic changes that occur during sepsis. Interestingly, besides acting as a dual inhibitor of COX-2 and 5-LOX, flavocoxid might stimulate β-arrestin through some yet-unrecognized mechanism. It has been suggested that β-arrestin 2 acts as scaffold for ERK and JNK cascades, thus leading to modulation of transcriptional events [24,25]. In our experimental model, CLP-induced MAPKs expression was significantly increased in both lung and liver tissues compared with that in sham mice. Reduced p-ERK1/2 and p-JNK expression was found after flavocoxid treatment of CLP mice, thus suggesting that the compound may upregulate β arrestin 2 through a MAPKs-dependent mechanism. All these data, taken together, confirm that β-arrestin 2 plays a critical role in a clinically relevant model of sepsis, as already shown by Fan et al. [6], and support the hypothesis that β-arrestin 2 improved expression is protective in a model of abdominal sepsis. However, additional mechanism(s) underlying flavocoxid beneficial effects in experimental sepsis might be taken into account. NF-κB is an early signal that activates an inflammatory cascade leading to the expression of cell-adhesion molecules and cytokines and finally culminating in the deleterious accumulation of leukocytes in peripheral organs such as lung and liver [29]. Because of this, CLP animals showed an increased nuclear NF-κB expression and an enhanced content of MPO, a marker of neutrophil accumulation, in both lung and liver. Flavocoxid administration succeeded in reducing these steps of the inflammatory cascade, thus suggesting that the dual inhibitor of COX-2 and 5-LOX may work as an NF-κB inhibitor. Furthermore, lipoxin A4, another lipid mediator that is endogenously derived from arachidonic acid metabolism, has been shown to possess marked antiinflammatory and pro-resolving effects [30]. Our results suggest that flavocoxid treatment augmented the levels of lipoxin A4: therefore, this effect may concur in the overall protective activity of this natural antiinflammatory drug. Finally, we established for the first time that a natural derived dual inhibitor of COX-2 and 5-LOX activity is able to improve survival and restore the histologic architecture of lung and liver of mice subjected to polymicrobial sepsis. However, further studies will be necessary to characterize better whether other mediators contribute in the mechanism of action of flavocoxid in this model of sepsis. Conclusions This is the first report demonstrating that flavocoxid, a dual COX-2 and 5-LOX inhibitor of natural origin, exerts an antiinflammatory response and protects mice from sepsis in a murine model of CLP. Therefore, on the basis of efficacy and safety profile [17-19,23,31], flavocoxid represents a novel antiinflammatory therapy and a potential approach for future clinical trials in patients with sepsis. Key messages • Despite a massive effort invested in developing potential therapies, to date, severe sepsis is still a common, frequently fatal, and expensive pathologic condition. • Because of the enhanced antiinflammatory efficacy and the lower incidence of gastric toxicity, the development of dual inhibitors of COX-2 and 5-LOX pathways may represent a new strategy to fight severe sepsis. • Flavocoxid, which contains both the naturally occurring flavonoids baicalin and catechin isolated from S. baicalensis and A. catechu, respectively, acts as a dual inhibitor of COX-2 and 5-LOX and blunts the proinflammatory phenotype in LPS-stimulated macrophages. • Cecal ligation and puncture (CLP) is an experimental model of shock that reproduces all the pathologic sequelae of sepsis that occur in intensive care patients. β-Arrestin 2 is an essential negative regulator of sepsis-induced inflammation via TLR signaling and may represent an innovative target for antisepsis drugs. Flavocoxid reduced the inflammatory cascade associated with sepsis, enhanced β-arrestin 2, and improved the survival rate. • By inhibiting both COX-2 and 5-LOX expression and stimulating the negative regulator β-arrestin 2 through an MAPKs-dependent mechanism, flavocoxid may represent a potential new approach to the treatment of sepsis. Abbreviations 5-LOX: 5-lipooxygenase; ARDS: acute respiratory distress syndrome; CLP: cecal ligation and puncture; COX-2: cyclooxygenase 2; ELISA: enzyme-linked immunosorbent assay; FDA: Food and Drug Administration; GPCR: G-protein-coupled receptor; H&E: hematoxylin and eosin; IL: interleukin; iNOS: inducible nitric oxide synthase; ip: intraperitoneal; KO: knockout; LPS: lipopolysaccharide; LTB4: leukotriene B4; MAPK: mitogen-activated protein kinase; MOF: multiple-system organ failure; MPO: myeloperoxidase; NF-κB: nuclear factor κB; OA: osteoarthritis; p-ERK: phospho-extracellular-regulated kinase; p-JNK: phospho-Jun N-terminal kinase; PGE2: prostaglandin E2; TLRs: Toll-like receptors; TNF-α: tumor necrosis factor α; sc: subcutaneous. Competing interests The authors declare that they have no competing interests. Authors' contributions AB carried out the in vivo studies and drafted the manuscript, LM carried out the in vitro studies and drafted the manuscript, AD participated in the design of the study, NI and MR carried out ELISA and biomolecular measurements, FSV drafted the manuscript, FS participated in the design of the study, and DA designed studies, analyzed data, and drafted the manuscript. All authors read and approved the final manuscript. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22815821PONE-D-12-0767110.1371/journal.pone.0040789Research ArticleBiologyImmunologyImmune SystemCytokinesImmunityInnate ImmunityImmunologic SubspecialtiesPulmonary ImmunologyImmune CellsImmune ResponseModel OrganismsAnimal ModelsMouseMedicinePulmonologyEnvironmental and Occupational Lung DiseasesInterstitial Lung DiseasesInnate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice LPS Exacerbates Pulmonary Fibrosis in MiceBrass David M. 1 * Spencer Jennifer C. 1 Li Zhuowei 2 Potts-Kant Erin 2 Reilly Sarah M. 3 Dunkel Mary K. 3 Latoche Joseph D. 3 Auten Richard L. 1 Hollingsworth John W. 2 4 Fattman Cheryl L. 3 1 Neonatology Division, Department of Pediatrics, Neonatal Perinatal Research Institute, Duke University Medical Center, Durham, North Carolina, United States of America 2 Pulmonary Allergy and Critical Care Division, Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America 3 Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, United States of America 4 Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America Ryffel Bernhard EditorFrench National Centre for Scientific Research, France* E-mail: [email protected] and designed the experiments: DMB RLA JWH CLF. Performed the experiments: DMB JCS ZL EP-K SMR MKD JDL CLF. Analyzed the data: DMB JCS CLF. Contributed reagents/materials/analysis tools: DMB RLA JWH CLF. Wrote the paper: DMB. 2012 16 7 2012 7 7 e4078915 3 2012 13 6 2012 Brass et al.2012This 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.Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress. ==== Body Introduction The factors that contribute to the pathogenesis and progression of fibrotic lung disease remain poorly understood. Fibrotic lung disease may be caused by inhalation of organic or inorganic substances, by medications or infection; may be associated with clinical conditions such as connective tissue disease; or may be idiopathic in nature. Rates of incidence and prevalence of fibrotic lung diseases are difficult to estimate, although a recent study shows that incidence can range between 19.4 and 34.3 per 100,000 [1]. In particular, exposure to crystalline silica in occupational settings has historically been estimated to be in the millions annually in the US alone [2] and thus continues to be a significant occupational risk factor. We use crystalline silica instillation in mice as a clinically relevant model of human disease because silica instillation leads to lung injury that is consistent and uniformly similar with pulmonary lesions observed in patients with occupational silicosis. Broadly, fibrotic lung diseases may be characterized as slowly progressing, rapidly progressing or punctuated by episodes of disease acceleration known as acute exacerbations [3]. It is increasingly recognized that a significant portion of the population with interstitial lung diseases that develop lung fibrosis suffer from episodes of accelerated disease progression for unknown reasons [4], [5], [6]. Acute exacerbations of pulmonary fibrosis occur in approximately 14% of patients with idiopathic pulmonary fibrosis each year [7]. The differences between slowly and rapidly progressing fibrotic lung disease are becoming somewhat more clear [8] although the causes for these differences remain obscure. Similarly, the causes of acute exacerbations of lung fibrosis remain to be determined. First described in 1993 [9], the definition of an acute exacerbation has come to include 1) increased dyspnea within 1 month; 2) hypoxemia; 3) new pulmonary infiltrates that are visible by radiography; and 4) absence of infection or evidence of a cardiac event [10]. The idiopathic and apparently stochastic nature of acute exacerbations renders them very difficult to study. Nevertheless, some attempts have been made to identify mechanisms that could contribute to such exacerbation events. For example, viral infections have frequently been observed in cases of lung fibrosis [11], and viral accelerations of lung fibrosis have been successfully modeled in mice and are characterized by Th1 cytokine and fibrogenic growth factor expression that is consistent with activation of innate immunity [12], [13]. Viral particles are recognized by Pathogen Associated Molecular Pattern (PAMP) receptors in the Toll-Like Receptor (TLR) family and are able to activate an innate immune response in the lung. We have previously shown that innate immune stimulation of the lung with the canonical activator of innate immunity, lipopolysaccharide (LPS), can increase expression of genes commonly associated with lung fibrosis and can induce fibroproliferative airway remodeling [14]. Therefore, our overall hypothesis is that environmental exposures that activate innate immunity contribute to acceleration of or exacerbation of stable pulmonary fibrosis. To address this hypothesis, we first exposed mice to crystalline silica in the lung and 28 days later, exposed these mice to LPS by aerosol inhalation. Our observations suggest that exposure to LPS, or other common environmental exposures that activate innate immunity, could contribute to acceleration of, or acute exacerbations of fibrotic lung disease in patients with pulmonary fibrosis. Materials and Methods Animals and Exposures Eight week old male C57BL/6 male mice (Jackson Laboratory, Bar Harbor, ME) were given a single oropharyngeal aspiration of 0.2 g/kg crystalline silica (Min-U-Sil 5 [15], [16]; a generous gift from Dr. Andy Ghio, US Environmental Protection Agency) in 60 µl of sterile 0.9% saline or an equivalent volume of 0.9% saline alone at day 0. On day 28, groups of mice (n = 6/group) were then either exposed to an aerosol of LPS as previously described [17] at a target concentration of 0.5 µg/m3 or to an aerosol of sterile 0.9% saline for 2.5 hours per day for five consecutive days under barrier conditions. Groups of mice were then sacrificed immediately after the end of the last LPS exposure or were allowed to recover for 21 days before sacrifice. Treatment groups are referred to by exposures in the order administered. Saline-Saline first received the vehicle control for silica via oropharyngeal aspiration followed 28 days later by the aerosolized saline control for LPS; Saline-LPS first received the vehicle control for silica via oropharyngeal aspiration followed 28 days later by aerosolized LPS; Silica-Saline first received 0.2 g/kg silica via oropharyngeal aspiration followed 28 days later by the aerosolized saline control for LPS, and; Silica-LPS first received 0.2 g/kg silica via oropharyngeal aspiration followed 28 days later by aerosolized LPS. Mice treated with N-acetylcysteine (NAC) were provided with drinking water supplemented with 2 mg/ml NAC as described previously [18] for the period beginning three days prior to the start of LPS inhalation and lasting until phenotyping (n = 8/group). All animal procedures were approved by the Duke University Institutional Animal Care and Use Committee. Hydroxyproline Analysis Whole lungs were lyophilized and then acid hydrolyzed in sealed, oxygen-purged glass ampules containing 2 ml of 6 N HCl at 110°C for 24 h. Samples were resuspended in 1.5 ml of phosphate buffered saline and incubate at 60°C for 1 h. Samples were centrifuged at 13,000 rpm for 10 min. and the hydroxyproline analysis was performed on the resulting supernatant using chloramine-T spectrophotometric absorbance as previously described [19]. Lung Histology Lung sections were stained with Masson’s trichrome to visualize collagen. Images were captured at either 5× or 20× magnification, and the color balance for each image was adjusted equally using Adobe Photoshop. Analysis of Bronchoalveolar Lavage Fluid (BALF) Mice were euthanized by CO2 inhalation, and lungs were lavaged three times to capacity with 0.9% saline through PE-90 tubing at a pressure of 20 cmH2O. The collected lavage fluid was processed and stored for further analysis as previously described [20]. Total MCP-1, MIP-2 and KC concentrations in the BALF were determined using Duo-Set ELISA kits (R&D Systems; Minneapolis MN) according to the manufacturer’s instructions. Protein carbonyl content of the BALF was determined using the Cell BioLabs, Inc (San Diego, CA) OxiSelectTM protein carbonyl ELISA kit according to the manufacturer’s instructions. Statistical Analyses All data are expressed as means ± SE. Differences between groups and between variables were analyzed by ANOVA. Probability values of P<0.05 (two-tailed) were considered statistically significant. Results Innate Immune Stimulation Significantly Increases Total Lung Hydroxyproline Content in Mice Previously Exposed to Crystalline Silica To address whether innate immune stimulation would contribute to pre-existing lung fibrosis, we first exposed 8 week old C57BL/6 mice to either crystalline silica or vehicle alone via oropharyngeal aspiration. This was followed 28 days later by exposure of these groups of mice to either aerosolized LPS or sterile saline. We observed that immediately and 21 days after the aerosol exposure (Figure 1) there was no difference in total lung hydroxyproline between Saline-Saline (closed square) and Saline-LPS groups (open square), indicating that LPS exposure by itself did not increase total lung hydroxyproline in these mice. Both immediately and 21 days after the aerosol exposure (Figure 1) we observed that exposure to crystalline silica by itself (Silica-Saline group, closed triangle) significantly increased total lung hydroxyproline content when compared with the Saline-Saline group. Importantly, we observed that mice exposed to both silica and inhaled LPS (Silica-LPS, open triangle) had significantly more total lung hydroxyproline than control mice exposed to saline alone (Saline-LPS), or mice exposed to silica alone (Silica-Saline). These observations taken together demonstrate that activation of the innate immune system in the lung increases total lung hydroxyproline in mice with pre-existing lung fibrosis. 10.1371/journal.pone.0040789.g001Figure 1 Hydroxyproline content in whole lung tissue. Total hydroxyproline per lung immediately after and 21 days after the end of inhalation challenge. Data are presented as Mean + SEM and are representative of two such experiments. n = 6/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). The significant increases in hydroxyproline associated with LPS inhalation challenge consequent to silica instillation may be appreciated histologically as shown in Figure 2 (immediately after) and Figure 3 (21 days after). We observed that immediately after either saline or LPS inhalation challenges (Saline-Saline and Saline-LPS groups; Figures 2A & 2C) there were no significant histological differences between groups. However, in the Silica-Saline group, immediately after the end of saline inhalation challenge we observed prominent areas of inflammation and loosely organized fibrotic tissue (Figure 2B). Importantly, in the Silica-LPS group immediately after the end of the LPS inhalation challenge, we observed a greater overall degree of fibrosis and the presence of enlarged macrophages with diffusely transparent cytoplasm (Inset in Figure 2D) consistent with pulmonary alveolar proteinosis [21]. While these macrophages were observed in the Silica-Saline group, their presence was more prominent in the Silica-LPS group. 10.1371/journal.pone.0040789.g002Figure 2 Histology immediately after the end of inhalation challenge. (A) Saline-Saline; (B) Silica-Saline; (C) Saline-LPS; and (D) Silica-LPS exposed mice immediately after the end of either saline or LPS inhalation challenge. n = 6/group. Images were taken in anatomically comparable regions of the lung at 5× (main images) or 20× (Inset in panel D) magnification. Bar = 100 microns. 10.1371/journal.pone.0040789.g003Figure 3 Histology 21 days after the end of inhalation challenge. (A) Saline-Saline; (B) Silica-Saline; (C) Saline-LPS; and (D) Silica-LPS exposed mice immediately after the end of either saline or LPS inhalation challenge. n = 6/group. Images were taken in anatomically comparable regions of the lung at 5× (main images) or 20× (Inset in panel D) magnification. Bar = 100 microns. Similarly, at 21 days after the end of inhalation challenge there were no apparent differences between the Saline-Saline and Saline-LPS groups (Figures 3A & 3C). As with the mice sacrificed immediately after the end of inhalation challenge, we observed prominent areas of inflammation and consolidated fibrotic tissue in mice in the Silica-Saline group (Figure 3B). In the histological sections from the Silica-LPS group mice sacrificed 21 days after the end of inhalation challenge, we observed notable areas of condensed fibrotic tissue with prominent collagen staining (Figure 3D). We also observed the persistent presence of enlarged macrophages with diffusely transparent cytoplasm (Inset in Figure 3D) in this group of mice. Innate Immune Stimulation in Mice Previously Exposed to Crystalline Silica Causes a Significantly Increased Inflammatory Response As we and others have previously observed [17], [22], exposure to inhaled LPS alone (Saline-LPS, open square) causes an immediate significant increase in total lung inflammatory cells when compared to mice exposed to aerosolized saline (Saline-Saline, closed square) (Figure 4A). Consistent with previous observations, we show here that the majority of the inflammatory cells recruited to the lung in response to inhaled LPS are polymorphonuclear leukocytes (PMNs) (Figure 4C), although there are also significant increases in total lung macrophages (Figure 4B) and lymphocytes (Figure 4D). We also observed that, consistent with previous reports [23], silica exposure alone (Silica-Saline, closed triangle) significantly increased total lung inflammatory cells (Figure 4A), of which equal numbers are PMNs (Figure 4C) and macrophages (Figure 4B) while total lung lymphocytes are also increased (Figure 4D). Notably, prior exposure to silica significantly increases the immediate inflammatory response to inhaled LPS (Silica-LPS, open triangle; Figure 4A) with the majority of the additional cells recruited also being PMNs (Figure 4C; also compare Figure 4B Silica-Saline and Silica-LPS showing no increase in total lung macrophages resulting from the combined exposure). At 21 days after LPS exposure the immediate cellular inflammatory response to inhaled LPS alone (Saline-LPS) resolves completely (Figure 4A showing total cells and 4C showing total PMNs). Consistent with previous reports there is still a significant inflammatory response to silica exposure alone (Silica-Saline) that is comprised of macrophages, PMNs and lymphocytes (Figure 4A–D). Importantly, in mice exposed to both silica and LPS (Silica-LPS) the effects of the combined exposure cause a significant and persistent increase in total cellular inflammation in the lung (Figure 4A). The total number of macrophages in this group is also significantly increased when compared to total number of macrophages recruited by silica exposure alone (Silica-Saline). Consistent with the cellular response we also observed that the pro-inflammatory cytokines MCP-1, MIP-2 and KC, that are known to recruit macrophages (MCP-1) and neutrophils (MIP-2 and KC) to sites of ongoing inflammation, are similarly increased in whole lung lavage fluid immediately after the end of the aerosol exposure (Figure 5). Interestingly, IL-1β which we have previously shown to be increased by LPS inhalation alone [17] is not significantly increased by combined exposure to silica and LPS at this time point (Figure 5D). Notably, at 21 days after the aerosol exposure total MCP-1 in whole lung lavage fluid remains significantly increased in the Silica-LPS group when compared to the Silica-Saline group at this time point (Figure 5A). There is no difference between these groups in total MIP-2 or IL-1β at this time point. These observations taken together show that innate immune stimulation causes a significant and persistent increase in whole lung inflammation in mice with pre-existing silica-induced lung fibrosis. 10.1371/journal.pone.0040789.g004Figure 4 Whole lung lavage immediately after and 21 days after the end of inhalation challenge. (A) Total cells×105; (B) total macrophages×105; (C) total neutrophils×105; and total lymphocytes×105. Data are presented as Mean + SEM and are representative of two such experiments. n = 6/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). 10.1371/journal.pone.0040789.g005Figure 5 Whole lung cytokines immediately after and 21 days after the end of inhalation challenge. Total (A) MCP-1; (B) MIP-2; (C) KC; and (D) IL-1β in whole lung lavage. Data are presented as Mean + SEM and are representative of two such experiments. n = 6/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). To investigate the role of oxidative damage resulting from exposure to silica and to LPS we measured the concentration of protein carbonyls in whole lung lavage fluid [24], [25]. We observed that by themselves neither prior silica exposure (Silica-Saline) nor LPS exposure (Saline-LPS) caused an increase in protein carbonyl concentration in whole lung lavage fluid. However, combined exposure to both silica and LPS (Silica-LPS) resulted in a significant increase in protein carbonyl concentration in whole lung lavage fluid immediately after LPS exposure (Figure 6). By 21 days after the LPS exposure there was no difference in the concentration of protein carbonyls in whole lung lavage fluid between any of the groups (Figure 6). These observations taken together suggest that oxidative stress resulting from exposure to inhaled LPS in the context of pre-existing silica-induced lung fibrosis could be contributing to the increased collagen deposition seen both immediately after and 21 days after LPS exposure in Silica-LPS mice. 10.1371/journal.pone.0040789.g006Figure 6 Protein carbonyls in whole lung lavage protein. Concentration of protein carbonyls in whole lung lavage protein immediately after and 21 days after the end of inhalation challenge is shown. Data are presented as Mean + SEM and are representative of two such experiments. n = 6/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). Blocking Oxidative Stress with N-acetylcysteine during LPS Inhalation Attenuates Inflammation but not Fibrosis To investigate the role of oxidative stress resulting from exposure to silica and to LPS we exposed groups of mice to silica and then to either inhaled saline or LPS as before. Groups of mice were given either normal drinking water or drinking water supplemented with 2 mg/ml of N-acetylcysteine (NAC), which has previously been shown to block oxidative stress and to protect from oxidative stress-induced airspace enlargement [18], prior to and during LPS inhalation challenge. We observed that blocking oxidative stress with NAC failed to protect from the enhanced fibrosis caused by LPS inhalation challenge in silica-exposed mice (Figure 7). However, NAC treatment prior to and during LPS inhalation significantly reduced the total inflammatory response (Figure 8A) by attenuating the recruitment of PMNs (Figure 8C) while the total numbers of macrophages and lymphocytes was unchanged. NAC treatment significantly reduced the macrophage chemoattractant chemokine MCP-1 (Figure 9A), but not the neutrophil-specific chemoattractant chemokines MIP-2 (Figure 9B) or KC (Figure 9C). Interestingly, NAC treatment significantly reduced the amount of IL-1β (Figure 9D) present in whole lung lavage fluid. 10.1371/journal.pone.0040789.g007Figure 7 Hydroxyproline content in whole lung tissue in mice treated with drinking water alone or drinking water supplemented with 2 mg/ml NAC during LPS exposure. Data are presented as Mean + SEM. n = 8/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge). 10.1371/journal.pone.0040789.g008Figure 8 Whole lung lavage immediately after the end of inhalation challenge in mice treated with drinking water alone or drinking water supplemented with 2 mg/ml NAC during LPS exposure. (A) Total cells×105; (B) total macrophages×105; (C) total neutrophils×105; and (D) total lymphocytes×105. Data are presented as Mean + SEM. n = 8/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); ∧ p<0.05 vs. regular drinking water (significant difference due to NAC treatment alone); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). 10.1371/journal.pone.0040789.g009Figure 9 Whole lung cytokines immediately after the end of inhalation challenge in mice treated with drinking water alone or drinking water supplemented with 2 mg/ml NAC during LPS exposure. Total (A) MCP-1; (B) MIP-2; (C) KC; and (D) IL-1β in whole lung lavage. Data are presented as Mean + SEM. n = 8/group. * p<0.05 LPS vs. Saline (significant difference due to inhalation challenge); # p<0.05 Silica vs. Saline (significant difference due to silica instillation or combined Silica and LPS). Discussion We have shown that innate immune stimulation with LPS in the context of pre-existing silica-induced lung fibrosis either accelerates or exacerbates the existing disease. This is accompanied by significant increases in cellular inflammation at both time points examined and concomitant increases in pro-inflammatory cytokine production. Furthermore, this is accompanied by evidence that only combined exposure to both silica and LPS leads to an increase in evidence of oxidative damage to proteins in whole lung lavage fluid. However, blocking oxidative stress during the LPS exposures failed to attenuate the observed LPS-induced increases in hydroxyproline. These observations taken together provide evidence that exposure of the lung to LPS, which is ubiquitous in the environment, could lead to exacerbations or acceleration of disease progression in patients with stable pulmonary fibrosis. They further suggest that anti-oxidant therapy in vulnerable patients could reduce inflammation associated with such exacerbations but that this treatment would likely have limited effect on loss of normal lung architecture. The pathogenesis of pulmonary fibrosis remains poorly understood [26]. This is due in large part to the obvious challenge presented by the study of human end stage disease that may not reveal the mechanistic early events that lead to later development of fibrosis. An additional challenge is the lack of similarity of common disease models with human lung fibrosis. Silica is a biologically relevant fibrogenic environmental dust and the resulting pathology in mice has many features in common with human silicosis. The silica model gives us the opportunity to study both early and late stage events in development of experimental lung fibrosis and to use this model to understand how common environmental exposures may accelerate or exacerbate existing disease in human patients. Our data show that activation of innate immunity by LPS in mice previously exposed to crystalline silica enhances many previously observed features of human silicosis including inflammation, evidence of oxidative stress-induced lung damage, collagen deposition and the presence of enlarged foamy macrophages that are consistent with pulmonary alveolar proteinosis [21], [27]. While we observed enlarged foamy macrophages most prominently in the Silica-LPS groups both immediately after and 21 days after the end of the aerosol challenge it is unclear what the role of these cells may be in the disease process. Our observations taken together support our previously published observations [14] and other studies [28], [29] showing that LPS inhalation by itself induces many genes that are also induced in experimental fibrosis. Importantly, the current observations showing that blocking oxidative stress with NAC blocks inflammation but not fibrosis exacerbation supports the notion that inflammation and fibrosis are at least partially independent processes [30], [31], [32], [33], [34]. Interestingly, the role of inflammation in development and progression of lung fibrosis remains controversial. For example, a recent study has demonstrated that nucleotide-binding domain and leucine-rich repeat containing (NLR) protein family member 3 (Nalp3)-dependent pro-inflammatory mediators are critical to development of lung fibrosis in the silica model [35] and these multi-protein complexes contribute significantly to development of inflammation. Interestingly, recent studies have demonstrated that innate immune activation may play a central role in the response to crystalline silica [36] and that macrophages in particular may drive the complete response to silica in the lung through the inflammasome [35], [36], [37]. In general, inflammasomes sense both exogenous and endogenous danger signals through intracellular NOD-like receptors (NLRs) [38] that activate caspase-1 which in turn promotes cleavage and activation of the pro-inflammatory cytokine IL-1β [38], [39]. Previous studies have shown the importance of IL-1β in the response to crystalline silica exposure. Silica-treated macrophages secrete IL-1β, and IL-1β-deficient mice have an altered inflammatory response to silica [40], [41]. In addition, a polymorphism in the gene encoding the endogenous receptor antagonist for the IL-1 receptor (IL-1Ra) has been associated with increased susceptibility to silicosis in a human population [42]. Silica exposure activates the inflammasome [43] and Nlrp3 deficient mice are protected from the effects of silica in the lung [35] suggesting that the inflammasome is critical to a complete in vivo response to silica. LPS has also been shown to activate the inflammasome, and mice deficient in Caspase-1, a key component of the inflammasome, have a primary defect in the ability to produce and secrete IL-1β in response to LPS [44]. We therefore hypothesized that LPS exposure would significantly enhance IL-1β protein concentrations in whole lung lavage fluid over the concentrations induced by silica exposure and that this could be causally linked to the observed increases in fibrosis. However, our observations demonstrate clearly that LPS inhalation challenge failed to enhance IL-1β concentrations in whole lung lavage over silica alone. Blocking oxidative stress with NAC fails to protect from the enhanced fibrosis seen in mice exposed to both silica and inhaled LPS. NAC treatment significantly attenuated inflammation and the concentration of IL-1β in whole lung lavage fluid in mice exposed to both silica and LPS while having no effect on hydroxyproline content of the lung. These observations are consistent with previous reports showing that inhibitors of reactive oxygen species (ROS) inhibit IL-1β secretion [35], [45]. LPS inhalation exposure, by itself, can increase ECM gene expression [14] and therefore, exacerbation or acceleration of fibrosis observed in Silica-LPS mice may be a result of direct innate immune stimulation of ECM gene expression rather than being a by-product of enhanced inflammation. In summary, we have demonstrated that inhalation of the TLR4 ligand LPS, exacerbates or accelerates silica-induced fibrotic lung disease. This was associated with increases in total lung inflammation and in cytokines and chemokines that are chemoattractants for macrophages and neutrophils. Additionally we have shown that only mice exposed to both silica and LPS have significant increases in evidence of oxidative damage in the lung but that blocking oxidative stress with NAC fails to attenuate the enhanced hydroxyproline deposition observed in mice exposed to both silica and LPS. These data suggest that inhalation of environmental LPS and possibly other TLR ligands may contribute to exacerbation or acceleration of pre-existing disease in patients with pulmonary fibrosis in a manner that is independent of inflammasome activation or inflammation. Competing Interests: The authors have declared that no competing interests exist. Funding: Funding was provided by the March of Dimes, ES016126, ES016000, AI 081672. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Kornum JB Christensen S Grijota M Pedersen L Wogelius P 2008 The incidence of interstitial lung disease 1995–2005: a Danish nationwide population-based study. BMC Pulm Med 8 24 18983653 2 Peters JM 1986 Silicosis. NIOSH 219 241 3 Kim DS Collard HR King TE Jr 2006 Classification and natural history of the idiopathic interstitial pneumonias. Proc Am Thorac Soc 3 285 292 16738191 4 Papanikolaou IC Drakopanagiotakis F Polychronopoulos VS 2010 Acute exacerbations of interstitial lung diseases. Curr Opin Pulm Med 2010 13 5 Park IN Kim DS Shim TS Lim CM Lee SD 2007 Acute exacerbation of interstitial pneumonia other than idiopathic pulmonary fibrosis. Chest 132: 214–220 Epub 2007 Mar 2030 6 Selman M Carrillo G Estrada A Mejia M Becerril C 2007 Accelerated variant of idiopathic pulmonary fibrosis: clinical behavior and gene expression pattern. 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PLoS One. 2012 Jul 16; 7(7):e40789
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22829863PONE-D-11-1499810.1371/journal.pone.0027571Research ArticleBiologyBiotechnologyEnvironmental BiotechnologyEcologyConservation ScienceEarth SciencesEnvironmental SciencesEnvironmental EngineeringGeomorphologyErosionLandform DynamicsEngineeringEnvironmental EngineeringHazardous WastesSolid Waste ManagementImprovement of Landfill Leachate Biodegradability with Ultrasonic Process Improvement of Landfill Leachate BiodegradabilityMahvi Amir Hossein 1 2 3 * Roodbari Ali Akbar 1 Nabizadeh Nodehi Ramin 1 Nasseri Simin 1 Dehghani Mohammad Hadil 1 Alimohammadi Mahmood 1 1 School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran 3 Center for Solid Waste Research, Institute for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran Magar Vanesa EditorPlymouth University, United Kingdom* E-mail: [email protected] and designed the experiments: AHM RNN. Performed the experiments: AAR MA. Analyzed the data: AHM AAR SN MHD. Wrote the paper: AHM AAR. 2012 19 7 2012 7 7 e2757131 7 2011 19 10 2011 Mahvi et al.2012This 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.Landfills leachates are known to contain recalcitrant and/or non-biodegradable organic substances and biological processes are not efficient in these cases. A promising alternative to complete oxidation of biorecalcitrant leachate is the use of ultrasonic process as pre-treatment to convert initially biorecalcitrant compounds to more readily biodegradable intermediates. The objectives of this study are to investigate the effect of ultrasonic process on biodegradability improvement. After the optimization by factorial design, the ultrasonic were applied in the treatment of raw leachates using a batch wise mode. For this, different scenarios were tested with regard to power intensities of 70 and 110 W, frequencies of 30, 45 and 60 KHz, reaction times of 30, 60, 90 and 120 minutes and pH of 3, 7 and 10. For determining the effects of catalysts on sonication efficiencies, 5 mg/l of TiO2 and ZnO have been also used. Results showed that when applied as relatively brief pre-treatment systems, the sonocatalysis processes induce several modifications of the matrix, which results in significant enhancement of its biodegradability. For this reason, the integrated chemical–biological systems proposed here represent a suitable solution for the treatment of landfill leachate samples. ==== Body Introduction The generation of leachate remains an inevitable consequence of the practice of waste disposal in sanitary landfills [1]. Leachate from mature landfills contains less biodegradable carbon due to loss from the landfill via methane gas production and is typically characterized by high ammonium (NH4 +) content, low biodegradability (low BOD5/COD ratio) and high fraction of refractory and large organic molecules such as humic and fulvic acids [2]. Usually young landfill leachates contain low organic compound concentrations and are treated more easily as compared to the old one [3]. Biodegradable organic compounds and ammonia are leachate constituents that pose the most significant environmental threats [4], [5]. Biological treatment of leachate is often the most cost-effective alternative when compared to other treatment options [6]. Nevertheless, mature leachate effluents are known to contain recalcitrant and/or non-biodegradable organic substances and biological processes are not efficient in these cases [7], [8]. Studies have demonstrated that the major fraction of dissolved organic carbon (DOC) in biologically pre-treated landfill leachates consists of humic substances, mainly in humic and fulvic acids. Traditionally, the degradation of organic compounds and the removal of nitrogen can be achieved by advanced oxidation processes (AOP) [9], [10]. AOP have been used to enhance the biotreatability of wastewaters containing various organic compounds that are non-biodegradable and/or toxic to common microorganisms [11], [12]. Ultrasonic process is one of AOP and involve the generation of the hydroxyl radical (•OH) and pyrolysis phenomenon, which has a very high oxidation potential and is able to oxidize almost all organic pollutants and volatile matter such as NH3. Although these processes are very effective in completing mineralization of pollutants, if they are applied as the only treatment process, they will be expensive. A promising alternative to complete oxidation of biorecalcitrant leachate is the use of ultrasonic process as pre-treatment to convert initially biorecalcitrant compounds to more readily biodegradable intermediates, followed by biological oxidation of these intermediates to biomass and water. The major pollutants contained in leachate are biodegradable/non-biodegradable organic material, ammonia and inorganic salts, with anthropogenic organic chemicals, such as phthalates and other endocrine disrupting compounds becoming an increasing concern [13], [14]. Because of the variation in leachate composition and the wide range of pollutants contained in leachate, it is difficult to predict a treatment technique that will be effective for leachate. Usually combinations of physical, chemical and biological methods are used for effective treatment of landfill leachate, since it is difficult to obtain satisfactory results by using any of those methods alone. Sedimentation, air stripping, adsorption, membrane filtration are the major physical methods used for leachate treatment [15], [16]. These methods are usually used in combination with chemical and biological methods. Coagulation–flocculation [17], [18], chemical precipitation [19], [20], chemical–electrochemical oxidations [21], [22] are the major chemical methods used for the landfill leachate treatment. Biological treatment methods used for the leachate treatment can be classified as aerobic, anaerobic and anoxic processes which are widely used for the removal of biodegradable compounds. Physicochemical methods are used along with the biological methods mainly to remove non-biodegradable compounds from the leachate [23], [24], [25]. As a result, parameters have been developed to characterize leachate and predict its treatment efficiency. The ratio of biochemical oxygen demand (BOD) to chemical oxygen demand (COD) (BOD/COD) is a common classification approach. Leachate is classified as stabilized, intermediate, or fresh given BOD/COD values of <0.1, 0.1–0.5, and >0.5, respectively [26], [27], [28]. The BOD/COD ratio indicates that biological processes are appropriate for treatment of fresh leachate because of a higher fraction of biodegradable organic material, while physical–chemical processes are more appropriate for treatment of stabilized leachate because of the high fraction of non-biodegradable organic material. The objectives of this study are to investigate the effect of ultrasonic process on leachate biodegradability improvement. Results and Discussion Characterization of the Raw Landfill Leachate Main chemical characteristics of raw leachate summarized in Table 1. With biodegradability ratio (BOD5/COD) lower than 0.35 and a pH higher than 8, the samples can be considered as moderately stabilized leachates [29], normally classified as refractory to conventional biodegradation processes. In most cases, intensive and sophisticated physicochemical processes are necessaries for the treatment of aged leachates. 10.1371/journal.pone.0027571.t001Table 1 Chemical characteristics of the studied landfill leachates. Parameters Values Parameters Values COD 5691±83 pH 7.9–8.1 Calcium 10.61±0.2 Magnesium 8.65 BOD5 1738±36 NH3-N 726±25 TOC 1536±20 TS 1420±29 Alkalinity as CaCO3 3650±123 Values (except pH) in mg/l. Effect of Sonocatalyst on Biodegradability of Leachate The results indicated that sonocatalyst process can improve leachate biodegradability (BOD5/COD ratio). BOD5/COD ratio for raw leachate was 0.35 but it reached to 0.786 (with TiO2) and 0.783 (with ZnO) after sonication. Independent Samples T-test showed there is significant difference between BOD5/COD ratio of raw leachate and pretreated leachate with sonocatalysis process. (pvalue = 0.000 for both TiO2 and ZnO). The results indicated that the system operates with great efficiency (BOD5/COD ratio = 0.786) in pH of 10, power of 110 watt, frequency of 60 KHz and TiO2 concentration of 5 mg/l. Effect of Ultrasound Power Input on Biodegradability Improvement Figure 1 shows the effect of power input on leachate biodegradability for TiO2 and ZnO. As shown in this Figure, the power input clearly improves biodegradability. Independent Samples T-test showed there is significant difference between BOD5/COD ratio of raw leachate and pretreated leachate at different powers. (Pvalue = 0.000 for both TiO2 and ZnO). 10.1371/journal.pone.0027571.g001Figure 1 BOD/COD ratio input for TiO2 and ZnO at different powers (Frequency = 30 KHz, concentration = 5 mg/l, pH = 3). According to sonochemistry theory, when the ultrasound intensity reaches or exceeds the cavity threshold, bubbles will be formed easily and the cavities collapse violently. Increasing the ultrasonic power will increase the energy of cavitation, lowering the threshold limit of cavitation, and enhancing the quantity of the cavitation bubbles [30]. In other words, at higher intensities, the concentration of hydroxyl radicals and mass transfer are higher which lead to more degradation of organic materials [31] and also more biodegradable intermediate compounds. The efficiency of ratio improvement then increases with the increase of ultrasonic intensity. Effect of Exposure Time on Biodegradability Improvement Results indicated that the exposure time improve biodegradability somehow. Figure 2 shows the effect of exposure time on leachate biodegradability for TiO2 and ZnO. As shown in this Figure, the exposure time improve biodegradability somehow. One-Way ANOVA test showed there is no significant difference between BOD5/COD ratio of raw leachate and pretreated leachate with sonocatalysis process at different exposure times. (Pvalue = 0.467 for TiO2 and 0.398 for ZnO). 10.1371/journal.pone.0027571.g002Figure 2 BOD/COD ratio for TiO2 and ZnO at different exposure times (Frequency = 30 KHz, concentration = 5 mg/l, pH = 3). Effect of Frequency on Biodegradability Improvement Figure 3 shows the effect of frequency on leachate biodegradability for TiO2 and ZnO. As shown in this Figure, the frequency improves biodegradability. One-Way ANOVA test showed there is significant difference between BOD5/COD ratio of raw leachate and pretreated leachate with sonocatalysis process at different frequency. (pvalue = 0.000 for both TiO2 and ZnO). Results of Tukey statistical test also showed that there are significant difference between frequencies of 30 and 60 KHz (Pvalue = 0.000) and 45 and 60 KHz (Pvalue = 0.000). 10.1371/journal.pone.0027571.g003Figure 3 BOD/COD ratio for TiO2 and ZnO at different frequencies (power = 70watt, concentration = 5 mg/l, pH = 3). Effect of pH on Biodegradability Improvement Figure 4 shows the effect of pH on leachate biodegradability for TiO2 and ZnO. As shown in this Figure, pH improves biodegradability somehow. One-Way ANOVA test showed there is no significant difference between BOD5/COD ratio of raw leachate and pretreated leachate with sonocatalysis process at different pHS. (pvalue = 0.503 for TiO2 and0.170 for ZnO). 10.1371/journal.pone.0027571.g004Figure 4 BOD/COD ratio for TiO2 and ZnO at different pH (power = 70watt, concentration = 5 mg/l, frequency = 30 KHz. Effect of Type of Catalysts on Biodegradability Improvement by Ultrasound Figure 5 shows the effect of types of catalysts on leachate biodegradability (BOD5/COD ratio). Results showed that effects of two catalysts on leachate biodegradability was similar but Independent Samples T-test indicated that the there is no significant difference between BOD5/COD ratio of raw leachate and pretreated leachate with TiO2 and ZnO. (Pvalue = 0.287). 10.1371/journal.pone.0027571.g005Figure 5 BOD5/COD ratio for TiO2 and ZnO (power = 70watt, concentration = 5 mg/l, frequency = 30 KHz). Concurrent Effect of Power Input and Frequency on Biodegradability Improvement by Ultrasound As mentioned above, power and frequency were effective parameters (statistically significant) on biodegradability improvement by sonocatalysis process. Univeriate statistic test showed that estimates of effect size were 88.6% for power and 74.9% for frequency but concurrent effect was declining (44.4%). Biodegradability Changes During Ultrasonic Decomposition Initially, the biodegradability of the leachates was evaluated through the evolution of the BOD/COD ratio. For untreated samples, this parameter attains values of about 0.35 while ultrasonic treatment of 120 min permit its enhancement up to values near 0.786, which represent substantial biodegradability according to the current literature [32], [33]. This result indicates that the ultrasonic process can break down or rearrange molecular structures of organic matter and convert the non-biodegradable organics to more biodegradable forms. This is a fact of remarkable importance in the case of the application of chemical–biological integrated system to wastewater treatment [33]–[34]. In general, it is admitted that ultrasonic process can transform organic recalcitrant compounds into easily biodegradable products, improving the efficiency and reducing the cost of further biological steps. In a second phase, raw and pre-treated leachate was submitted to a biological degradation process using a sequential batch reactor. The evolution of COD during the biological treatment (figure 6) confirms the low biodegradability of raw leachates, which achieve a maximal COD removal of about 30% at 72 h treatment times. On the other hand, the COD of pre-treated leachates fades progressively attaining COD removal higher than 90% at the end of the 72-h cycle. Additionally, the use of ultrasonically pre-treated samples favored the preservation of physical characteristics of the biological sludge, which could be corroborated by the measurement of traditional physical parameters and microscopic observation [35]. 10.1371/journal.pone.0027571.g006Figure 6 Evolution of COD during biological treatment of the leachates (pretreated sample sonicated with power of 110 watt, frequency of 60 KHz, pH of 7 and 5 mg/l of TiO2 for 120 minutes). Landfill leachates contain some macromolecular organic substances that are resistant to biological degradation. With very low biodegradability ratios (BOD/COD), usually lower than 0.35, these complex matrixes show a recognized resistance toward conventional activated sludge systems [36]. When applied as relatively brief pre-treatment systems, the sonocatalysis processes induce several modifications of the matrix, which results in significant enhancement of its biodegradability. For this reason, the integrated chemical–biological systems proposed here represent a suitable solution for the treatment of landfill leachate samples with an efficient remediation of the relevant parameters (COD, TOC). Materials and Methods Materials Samples of landfill leachate were obtained from a municipal landfill site (over 10 years old) located in Shahrood (Semnan, Iran). All leachate samples were collected from leachate lift stations or storage tanks, stored at 3°C, and tested within 2 d of collecting the samples. Characteristics of the leachate samples were COD = 5830 mg/l, BOD5 = 3940 mg/l, NH3-N = 730 mg/l and pH of 8. At the experiments, 5 mg/l of TiO2 and ZnO were also been used as catalysts. The ammonia- nitrogen concentrations were analyzed with C203 8 parameter test meter (Hanna electronics co., Ltd.). The pH was measured by a Benchtop pH Meters (Cole-Parmer Co., Ltd).The pH meter was calibrated before each use with pH 3, 7 and 10 buffer solutions. BOD and COD measurements were determined following Standard Methods 5210 and 5220, respectively. Reagents and standard chemicals were purchased from Hach Co., except the BOD buffer solution, which was prepared according to Standard Method 5210. BOD check standards were performed with each batch of BOD measurements [27]. The results were considered good when the value of the BOD check standard fell within the range of 198±30.5 mg/l. The average ± standard deviation of the BOD check standards for the entire duration of the project was 169±29 mg/l, which demonstrates good results given the inherent variability in BOD measurements. COD check standards were also performed with each batch of COD measurements. A COD standard solution of 1000 mg/l was diluted to 200 and 500 mg/l to ensure the accuracy of COD measurements. The relative difference for calibration check standards (RDcal) is defined as the absolute difference of the check standard concentration and the known concentration all divided by the known concentration. The RDcal for COD was <10% for the entire duration of the project [28]. Experimental Set-up As shown in Figure 7, for the laboratory experiments a cylindrical shape Plexiglas reactor with total volume of 1 L was prepared. The solution in the reactor was mixed with a magnetic stirrer, while sufficient aeration was provided by a compressor connected to a porous stone located in the bottom of the reactor. The compressor was used to ensure completely mixed condition in the reactor. The ultrasonic source was a Model UGMA-5000 ultrasound generator with three 30, 45 and 60 kHz transducers having a titanium probe with 20 mm diameter. The power input could be adjusted continuously from 60 to 120 W. A leachate sample of 1000 ml was sonicated in a covered cylindrical glass vessel. Aeration was supplied by a Model SALWAT air compressor. Samples of activated sludge inoculums were collected directly from the aeration tank of the Shahrood municipal wastewater treatment plant. The sludge was continuously aerated using aeration pumps. Ferrous sulphate (FeSO4·7H2O), sulphuric acid and hydrogen peroxide (Merck, 30 wt. %) were of analytical grade. 10.1371/journal.pone.0027571.g007Figure 7 Schematic diagram of the reactor. Procedure After the optimization by factorial design, the ultrasonic were applied in the treatment of raw leachates using a batch wise mode. At first, the raw leachate sample was filtered by filter paper (0.45) to remove any suspended solid impurity [28]. Then the sample was adjusted to the required pH with H2SO4 or NaOH. Then different scenarios were tested with regard to power intensities of 70 and 110 W, frequencies of 30, 45 and 60 KHz, reaction times of 30, 60, 90 and 120 minutes and pH of 3, 7 and 10. For determining the effects of catalysts on sonication efficiencies, 5 mg/l of TiO2 and ZnO have been also used. To evaluate the effect of ultrasonic on the biodegradability of raw and sonochemically pre-treated leachates, BOD5 and COD of both samples were measured. Biological Procedure The activated sludge system was applied in cylindrical aeration glass-vessels (30 cm of internal diameter and 60 cm of height). The system was aerated by using air pumps placed at the bottom of the reactors. The initial volume of the culture was 150 mL, which was completed to 300 mL with substrates (leachate, pre-treated leachate or glucose) at the beginning of each cycle. The pH was controlled by a probe and adjusted at 7.0 by using H2SO4 or NaOH. The oxygen concentration was monitored by using an O2 probe, located at the top of the reactor. All the experiments were carried out in duplicate and at room temperature (20–25°C) by periods of 72 h. For COD determinations, samples (10 ml each) were taken every 12 h, after they had been centrifuged and filtered through a 0.45 µ Millipore filter [28]. Ethic Statement No specific permits were required for the described field studies. No specific permissions were required for these locations/activities. Location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. Authors would like to thank from research affair of Tehran University of medical sciences. Competing Interests: The authors have declared that no competing interests exist. Funding: The authors have no funding or support to report. ==== Refs References 1 Hamidi AA Jyy LT AbuAhmed MH Muhammad U Nordin AM 2011 leachate treatment by swim-bed bio fringe technology. 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==== Front Eur J PsychotraumatolEur J PsychotraumatolEJPTEuropean Journal of Psychotraumatology2000-81982000-8066Co-Action Publishing 1766010.3402/ejpt.v3i0.17660Short CommunicationCombining biofeedback and Narrative Exposure Therapy for persistent pain and PTSD in refugees: a pilot study Morina Naser 1*Maier Thomas 2Bryant Richard 3Knaevelsrud Christine 4Wittmann Lutz 1Rufer Michael 1Schnyder Ulrich 1Müller Julia 11 Department of Psychiatry and Psychotherapy, University Hospital Zurich, Switzerland2 Psychiatric Services of the Canton St. Gallen-North, Switzerland3 School of Psychology, University of New South Wales, Sydney, Australia4 Department of Psychology, Free University Berlin, Germany* Naser Morina, Department of Psychiatry and Psychotherapy, University Hospital Zurich, Culmannstrasse 8, CH-8091 Zürich, Switzerland. Email: [email protected] 6 2012 2012 3 10.3402/ejpt.v3i0.1766028 2 2012 03 5 2012 29 5 2012 © 2012 Naser Morina et al.2012This 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.Objective Many traumatised refugees suffer from both persistent pain and posttraumatic stress disorder (PTSD). To date, no specific guidelines exist for treatment of this group of patients. This paper presents data on a pilot treatment study conducted with 15 traumatised refugees with persistent pain and PTSD. Methods Participants received 10 sessions of pain-focused treatment with biofeedback (BF) followed by 10 sessions of Narrative Exposure Therapy (NET). Structured interviews and standardised questionnaires were used to assess symptoms of pain intensity, pain disability, PTSD and quality of life directly before and after treatment and at 3 months follow-up. Results Following the combined intervention, participants showed a significant reduction in both pain and PTSD symptoms, as well as improved quality of life. Additionally, biofeedback increased motivation for subsequent trauma-focused therapy, which in turn was related to larger PTSD treatment gains. Conclusion This pilot study provides initial evidence that combining BF and NET is safe, acceptable, and feasible in patients with co-morbid persistent pain and PTSD. Refugeespersistent painPTSDtreatmentbiofeedbackNarrative Exposure TherapyFor the abstract or full text in other languages, please see Supplementary files under Reading Tools online This article has been retracted. Please find Retraction note at http://dx.doi.org/10.3402/ejpt.v4i0.21913 ==== Body There are an estimated 15.4 million refugees worldwide (United Nations High Commissioner for Refugees [UNHCR], 2011). Due to sequential and longstanding interpersonal trauma such as torture, often experienced in the context of war, refugees are at special risk for suffering from a more complex pattern of trauma-spectrum disorders and appear less responsive to treatment, compared to other victims of traumatisation (Robjant & Fazel, 2010). Research indicates that refugee populations typically report high rates of psychopathology (Steel et al., 2009); with PTSD being especially prominent (Schubert & Punamäki, 2011). Furthermore, these mental health problems persist many years after their first occurrence (Schubert & Punamäki, 2011), often compounded by difficulties with emotion tolerance (Momartin, Silove, Manicavasagar, & Steel, 2003; Zarowsky, 2004) and post-migration living difficulties (Steel et al., 2011). A critical feature that frequently complicates the treatment of mental health in refugees is the concurrent problem of persistent pain (Carinci, Mehta, & Christo, 2010). It is a particularly frequent co-morbid condition of PTSD (Johnson & Thompson, 2008). This specific co-morbidity is especially common in traumatised refugees, asylum seekers and torture survivors (Hinton & Lewis-Fernández, 2011; Silove, Steel, McGorry, & Mohan, 1998). Co-morbidity of both disorders generally results in more severe functional disturbance and reduced quality of life, and has been reported in up to 76% of treatment-seeking traumatised refugees (Dahl, Dahl, Sandvik, & Hauff, 2006; De Jong et al., 2001). Current models postulate that PTSD and pain are mutually maintaining, because PTSD symptoms can heighten arousal, which leads to increased muscle tension and muscular pain, which can subsequently function as reminders of traumatic experiences, which in turn elicit further PTSD re-experiencing reactions (Carty, O'Donnell, Evans, Kazantzis, & Creamer, 2011; Liedl et al., 2010; Sharp & Harvey, 2001). Despite the observed interplay between pain and PTSD, no studies have been published on treating these co-morbid conditions in this population. Accordingly, there is a need for proof-of-concept studies to determine appropriate treatments to manage the co-morbidity of persistent pain and PTSD. One proven means of alleviating pain-related distress is a biofeedback-based cognitive behavioural intervention (BF; Morley, Eccleston, & Williams, 1999). BF is a treatment technique by which people learn how to change psychophysiological parameters to improve their health by using signals from their own bodies. Some limited evidence exists demonstrating beneficial effects of BF on posttraumatic pain (Müller et al., 2009; Tatrow, Blanchard, & Silverman, 2003), but the effects are less pronounced than for mono-morbid persistent pain patients. Additionally, it appears to be insufficient as a stand-alone treatment for PTSD (Foa, Keane, Friedman, & Cohen, 2008). A pilot study with traumatised refugees suffering from co-morbid pain and PTSD revealed BF to be well-accepted and exert strong effects on patients’ coping with pain (Müller et al., 2009). Amongst different PTSD psychotherapies for refugees (Heide, Mooren, Kleijn, Jongh, & Kleber, 2011; Nickerson, Bryant, Silove, & Steel, 2011), Narrative Exposure Therapy (NET; Schauer, Neuner, & Elbert, 2005) is a well-established therapy with strong treatment effects for the reduction of PTSD symptom severity (Neuner, Schauer, Klaschik, Karunakara, & Elbert, 2004; Nickerson et al., 2011; Robjant & Fazel, 2010). NET is a short-term trauma-focused therapy that focuses on imaginary exposure to memories of the traumatic event and the reorganisation of these memories into a coherent chronological narrative. So far, only one study has demonstrated any efficacy of NET in refugees and/or asylum seekers in a Western setting (Neuner et al., 2010). Taking into account that many refugees suffer from co-morbid PTSD and persistent pain, combining biofeedback for pain with NET may provide additive effects relative to either intervention alone. The use of biofeedback is further indicated for refugees, because many refugees come from cultures in which distress is expressed somatically (Hinton, Pich, Chhean, Safren, & Pollack, 2006) and somatically-based interventions may carry a certain level of face validity. Generally, the first step in evaluating a treatment is to determine its feasibility, acceptance, and safety within a proof-of-concept study, prior to more rigorous testing through controlled trials. To this end, we evaluated the preliminary effects of a combined, pain-focused, 10-session BF program followed by 10-session trauma-focused NET protocol for traumatised refugees. This was an uncontrolled pilot study to examine the feasibility, acceptance, and safety of the combined intervention. Methods Participants The study was conducted between March 2008 and October 2009, with approval from the Ethics Commission of the Canton of Zurich. Of all the patients who had been referred to the Outpatient Unit for Victims of Torture and War in the Department of Psychiatry and Psychotherapy (University Hospital of Zurich) over this time, 18 individuals were preselected based on their referral letter indicating co-morbid persistent pain and PTSD, and asked if they would be willing to participate in the study. All these 18 patients committed to the study and fulfilled inclusion criteria, according to our psychiatric and neuropathic examination. The inclusion criteria were: experience of torture/war; current DSM-IV diagnoses of persistent pain (excluding neuropathic pain) and PTSD; and either no psychoactive medication or a stable dose (i.e., participants on medication were required to maintain their dosage for the duration of the trial). Exclusion criteria were: additional pain-/trauma-focused psychotherapy over the course of the study; current psychotic or substance-related disorders; a history of any organic mental disorder; prominent current homicidal or suicidal ideations; severe dissociative symptoms; and any perceived risk of deportation within the next 12 months. Fifteen participants completed the treatment program.. The majority of the participants were male (60%, n=9). Eight participants originated from Turkey (of which six were of Kurdish descent), three from Bosnia, and one each from Sri Lanka (Singhalese), Iraq, Syria and Vietnam. Participants had an average age of 43.1 years (SD=6.9) and an average of 9.7 years of schooling (SD=4.1), and had lived in Switzerland for an average of 11.0 years (SD=5.8). No patient who was asked to participate had to be excluded due to insecure visa status. Assessments were conducted at four time points: T1 = pre-BF, T2 = post-BF/pre-NET, T3 = post-NET, and T4 = 3 months of follow-up. The assessment and treatment protocols were translated by professional interpreters. Patients completed 10 sessions of manualised BF, as described elsewhere (Liedl et al., 2011; Müller et al., 2009) followed by 10 sessions of Narrative Exposure Therapy (Schauer et al., 2005). In accordance with the study protocol, psychological assessment and treatment were provided by four postgraduate clinical psychologists who had received special training in the assessment as well as in the application of the manual. All of them participated in weekly supervision sessions dealing with questions regarding assessment and treatment. Neurological assessments were performed by psychiatrists (at least resident level). Instruments The M.I.N.I. Plus version (Ackenheil, Stotz-Ingenlath, Dietz-Bauer, & Vossen, 1999) is a standardised interview that assesses major Axis I psychiatric disorders including somatoform pain disorders according to DSM-IV; this only was used to determine if individuals satisfied inclusion/exclusion criteria. Pain intensity was measured using the self-reported Verbal Rating Scale (item 7 of the SF-36; Ware & Sherbourne, 1992), which assesses current pain levels on a six-point rating scale (1 = no current pain to 6 = extreme current pain). The self-reported Pain Disability Index (Tait, Chibnall, & Krause, 1990) assesses the impact of pain on a person's ability to participate in essential life activities (ranging from 0 = no disability to 10 = worst disability). Only those four of seven items relevant to refugees (family/home responsibilities, sexual behaviour, self-care, and life-support activities) were administered. PTSD symptoms were assessed using the Clinician-Administered PTSD Scale (CAPS; Blake et al., 1990). The CAPS is a structured clinical interview that assesses the 17 DSM-IV PTSD criteria. Quality of life was assessed with the WHO self-reported EUROHIS-QOL which has been widely used and validated internationally in many different cultures and languages (Nossikov & Gudex, 2003); it is comprised of eight items that index personal satisfaction with various life domains on five-point rating scales. Motivation for trauma-focused therapy was assessed via six items administered at T1 and T2 that employed a 100 mm Visual Analogue Scale ranging from 0 = not at all to 100 = completely; (“How big is your motivation for psychotherapy, which focuses on the processing of the trauma you have experienced?”). Cronbach's alpha (α=.69) was acceptable. Data analysis Data were analysed using SPSS 19.0 and were performed on completers only. Time courses were performed using the GLM for repeated measures and paired t-tests. Because of the small sample size, missing data were imputed (data missing at each time point: T1 = 1.6%; T2 = 6.7%; T3 = 13.3%; T4 = 26.6%) to maximise the use of information. As data were missing at random (Tsikriktsis, 2005), five complete data sets were imputed applying an EM algorithm (PRELIS) and an average of the imputations was constructed. To determine changes in treatment effects, Cohen's d effect size was calculated (Cohen, 1988). Regarding motivation for trauma-focused therapy, subgroups were created to represent highly (n=7) and poorly (n=8) motivated patients using a median split. Results Generally, the procedure was accepted very well; no patient demonstrated any deterioration. Of 18 patients included in the study, 15 completed all treatment sessions, while two had to be excluded due to life crises unrelated to the treatment protocol (death of a best friend and severe maternal illness) and one dropped out after the third session of BF treatment. After completion of the BF intervention, there were no significant reductions in pain intensity (d=0.45; t(14) = 1.47, p=.164) or PTSD symptoms (d=0.1; t(14) = 1.89, p=.79). Motivation for trauma-focused therapy increased significantly from pre to post BF with Cohen's d=–1.01 and t(14) = –3.50, p=.004; T1: M=5.10, SD=2.37; T2: M=7.44, SD=2.24. As expected, effect sizes indicated that additional NET had a strong effect on posttraumatic stress symptoms, and moderate effects on pain intensity. Table 1 presents the mean scores at each assessment time for each dependent variable, as well as effect sizes for pre-post-BF, pre-post-NET, post-NET, and 3-month follow-up, and GLM time courses T1–T4 for repeated measures. Table 1 Means, standard deviations, treatment effect sizes and time course (N=15 for all time points) Means (standard deviations) Effect sizes (Cohen's d) Symptom time course Variables T1a T2b T3c T4d T1–T2 T2–T3 T3–T4 Time contrasts (F) GLM VRS 4.80 (.94) 4.40 (.83) 3.87 (.92) 3.35 (1.12) 0.45 0.61 0.44 12.82*** (3, 42) PDI 23.40 (9.46) 22.33 (8.31) 20.82 (10.69) 15.39 (9.50) 0.11 0.15 0.53 4.39** (3, 42) CAPS 83.19 (22.51) 78.47 (22.76) 47.33 (23.87) 48.95 (26.70) 0.1 1.43 −0.01 22.01*** (1.3, 18.39) QoL 17.88 (4.08) 18.62 (5.79) 19.51 (5.34) 21.47 (6.20) −0.15 −0.15 −0.37 5.54** (3, 42) Note: VRS, Verbal Rating Scale (range 1–6); PDI, Pain Disability Index (range 0–40); CAPS, Clinician-Administered PTSD Scale (0–136); QoL, Eurohis Quality of Life (8–40). a T1 = pre-biofeedback (BF) b T2 = post-BF/pre-NET c T3 = post-NET d T4 = 3 months follow-up. ** p<.01 *** p<.001. Figure 1 illustrates the time courses of the outcome variables. As anticipated, a steady reduction in pain intensity and pain disability and an increase in quality of life were observed over time. At follow-up, medium effects were found relative to post-treatment for pain intensity, pain disability, and quality of life, but not for PTSD. Fig. 1 Changes over time of observed variables at four time points (time course). Note: VRS, Verbal Rating Scale; PDI, Pain Disability Index; CAPS, Clinician-Administered PTSD Scale; QoL, Eurohis Quality of Life. Brackets indicating standard deviations. In order to examine the impact of BF on NET, participants were divided into groups representing high and poor gains in motivation for trauma-focused therapy during BF, using a median split. Patients with strong BF-related gains in motivation exhibited larger decreases in PTSD symptoms during NET than subjects without pronounced gains in motivation (t(13) = 3.17, p=.007). Discussion The aim of this pilot study was to assess the potential feasibility, acceptance, and safety of combined BF and NET for co-morbid persistent pain and PTSD in refugee trauma survivors. Regarding the following findings, the combined intervention seemed to be feasible, well accepted, tolerated and safe. None of the patients declined to participate; there were only three drop-outs, none of them related to the intervention; and no patient experienced any exacerbations in pain or PTSD symptoms or any form of personal crisis during the intervention or follow-up period. All outcomes improved significantly after the combined intervention. Improvements in posttraumatic stress symptoms persisted at 3 months of follow-up, and improvements were even more marked for all other measures. Finally, we noted increased motivation for trauma-focused therapy after BF. In terms of pain response, there was a moderate pre to post decrease in pain intensity. Following NET, there was a moderate reduction in pain intensity, a small reduction in pain disability, and slightly improved quality of life. Conversely, by 3 months of follow-up, there were marked improvements in terms of pain and quality of life. Interestingly, there was no reduction in pain intensity following BF; however, there was noted pain reduction after NET and at follow-up. This pattern concurs with models that PTSD symptoms themselves cause pain to persist, and that reducing PTSD intensity through NET yields a subsequent decrease in pain (Liedl et al., 2010; Sharp & Harvey, 2001). Alternatively, it is possible that the effects of BF were not immediately apparent, and that they only were observed following the additive NET intervention. Given that somatisation is quite frequent among refugees (Aragona et al., 2011; Schweitzer, Melville, Steel, & Lacherez, 2006), a physiologically-oriented approach such as BF may provide refugees with an intervention of strong face validity, because it concurs with their somatic experience of distress, and addresses the significant pain that they experience. In terms of reducing PTSD, there were marked reductions following NET and these were maintained at follow-up (effect size of 1.45), which is in accordance with previous studies on NET for refugees (Ertl, Pfeiffer, Schauer, Elbert, & Neuner, 2011; Neuner et al., 2004). The question arises concerning the role of BF in preparing refugees for NET. BF may have enabled patients to benefit from exposure treatment, because enhanced pain management may prevent exacerbation of pain during exposure (Wald, Taylor, & Fedoroff, 2004). Furthermore, BF may play a role in establishing the therapeutic relationship that provides stabilisation for refugees, who frequently experience distrust as a result of interpersonal traumatic experiences (Nickerson et al., 2011). It is possible that BF also contributes a sense of mastery or self-efficacy to refugees, which results in greater distress tolerance and an enhanced capacity to manage traumatic memories. In this sense, this finding also might be understood in the context of recent evidence that treating complex PTSD can be optimised by training patients in skills that enhance emotion regulation prior to commencing exposure therapy (Cloitre et al., 2010). To determine the relative contributions of BF and NET to reductions in pain and PTSD in refugees, it will be essential to conduct randomised controlled trials that allocate refugees to (a) BF, (b) NET, or (c) combined BF and NET. The pattern of findings in this pilot study may be interpreted in terms of the key role of NET; one could conclude that BF was unnecessary because symptom reduction was not observed after this intervention. Additionally, it will be necessary to examine if BF is more efficacious than other forms of preparatory skills training that have been found to be very effective in other trauma populations prior to NET, such as the emotion regulation training incorporated within Cloitre's STAIR program (Cloitre et al., 2010). One limitation of the current study is that it was an uncontrolled study with a very small sample. Furthermore, we used the CAPS without blinding the assessors; we therefore do not know if their ratings may have been influenced by their assumptions on the efficacy of either intervention. Additionally, as no clear classification or treatment guidelines exist for DESNOS to date, we decided to diagnose persistent pain and PTSD as two distinct co-morbid concepts in our study. However, we are aware that these two disorders are highly interrelated and might be better captured in a single diagnosis, as was suggested for DESNOS (American Psychiatric Association, 1994). Despite these limitations, the present study provides initial evidence that combining BF and NET is safe, acceptable, and feasible in patients with co-morbid persistent pain and PTSD. Furthermore, biofeedback increased motivation for subsequent trauma-focused therapy, which in turn was related to larger PTSD treatment gains. Given the prevalence of refugees with these co-morbid conditions and the documented impairment these conditions cause (Dahl et al., 2006; De Jong et al., 2001), this first step offers one avenue for controlled trials to test the relative efficacies of these treatment strategies. Acknowledgements The study was supported by the Gottfried and Julia Bangerter-Rhyner Foundation, the EMDO Foundation, the Hartmann Müller Foundation, and the Department of Psychiatry and Psychotherapy, University Hospital Zurich/Switzerland. We want to thank all the participating patients, assessors and therapists: Claudine Pfister, Klaudia Perret, Jacqueline Kamm, Yildiz Ünver, Lukas Jann, Angela Georgiev-Kill and Sonja Schmidt. Last, but not least, many thanks to all interpreters, particularly for their excellent translations. Conflict of interest and funding There is no conflict of interest in the present study for any of the authors. ==== Refs References Ackenheil M Stotz-Ingenlath G Dietz-Bauer R Vossen A Mini international neuropsychiatric interview (German Version 5.0.0, DSM-IV) 1999 München Psychiatrische Universitätsklinik American Psychiatric Association Diagnostic and statistical manual of mental disorders (DSM-IV) 1994 4th ed. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22911823PONE-D-12-1108810.1371/journal.pone.0041610Research ArticleBiologyGeneticsGene expressionDNA transcriptionGenetic MutationMutation TypesHuman GeneticsGenetic Association StudiesCancer GeneticsMedicineOncologyCancer Risk FactorsGenetic Causes of CancerCancers and NeoplasmsBone and Soft Tissue SarcomasOsteosarcomaLysyl Oxidase Polymorphisms and Susceptibility to Osteosarcoma Lysyl Oxidase Polymorphisms and OsteosarcomaLiu Yang 1 Lv Bitao 1 He Zhimin 2 Zhou Yujia 3 Han Carrie 4 Shi Guodong 1 Gao Rui 1 Wang Ce 1 Yang Lili 1 Song Haihan 5 * Yuan Wen 1 * 1 Department of Spine Surgery, Changzheng Hospital, Second Military Medical University, Shanghai, China 2 Department of Orthopedic Surgery, Fengxian Branch of the Shanghai No.6 People’s Hospital, Shanghai, China 3 School of Occupational Therapy, Western University, London, Ontario, Canada 4 Department of Immunology, University of Toronto, Toronto, Ontario, Canada 5 Emergency Center, Shanghai East Hospital, Tongji University, Shanghai, China Toland Amanda Ewart EditorOhio State University Medical Center, United States of America* E-mail: [email protected] (WY); [email protected] (HS)Conceived and designed the experiments: YL BL HS WY. Performed the experiments: YL BL ZH GS RG CW LY. Analyzed the data: YL BL ZH YZ CH HS WY. Contributed reagents/materials/analysis tools: YL HS WY. Wrote the paper: YL BL YZ CH HS WY. 2012 23 7 2012 7 7 e4161018 4 2012 22 6 2012 Liu et al.2012This 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.Despite the knowledge of many genetic alterations present in osteosarcoma, the complexity of this disease precludes placing its biology into a simple conceptual framework. Lysyl oxidase (LOX) catalyzes the cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue. In the current study, we performed genomic sequencing on all seven exons -including the intron-exon splice sites, and the putative promoter region of LOX gene - followed by luciferase reporter assay to analyze the function of newly identified polymorphisms. Associations between LOX polymorphisms and osteosarcoma were then evaluated. Our sequencing data revealed three polymorphisms (−22G/C, 225C/G, and 473G/A) in the exons and promoter region of LOX. The −22G/C polymorphism lies in the downstream core promoter element (DPE) region and caused a decrease in promoter activity of LOX. The prevalence of the −22C allele and 473A allele were significantly increased in osteosarcoma patients compared to controls (odds ratio [OR] = 3.88, 95% confidence interval [CI]  = 1.94−7.78, p = 4.18×10−5, and OR = 1.38, 95%CI = 1.07−1.78, p = 0.013; p 0.0167 was considered significant after Bonferroni correction). Analyzing haplotype showed that the frequency of CCG haplotype (−22, 225, 473) was significantly higher in osteosarcoma cases than in healthy controls after Bonferroni correction (p = 4.46×10−4). These results indicate that the −22G/C polymorphism may affect the expression of LOX, and that −22G/C and 473G/A polymorphisms may be new risk factors for osteosarcoma. These findings reveal a potential new pathway by which genetic polymorphisms may affect human diseases. ==== Body Introduction Osteosarcoma is the most common pediatric bone malignancy in the world [1]. Fifty years ago, surgery was the only available treatment, and survival was abysmal at less than 20% [2]. Treatment of patients with osteosarcoma was transformed in the 1980s and 1990s with advances in chemotherapy and orthopedic surgical techniques, leading to long-term survival rates that now approach 70% [2], [3]. Unfortunately, further increases in survival have not occurred in the last decade despite numerous trials targeting increased intensities of chemotherapy treatments and use of new chemotherapeutic agents [3]. Therefore a new focus on the natural history and biology of osteosarcoma may be necessary to improve our therapeutic approaches. Type I collagen is the principal constituent of extracellular bone matrix and is a crucial determinant for mechanical properties of bone tissue [4], [5]. Posttranslational collagen modifications result in the formation of a mature functional matrix, which is essential for subsequent matrix mineralization [6]–[9]. Lysyl oxidase (protein-lysine 6-oxidase, LOX) is a copper-dependent enzyme that initiates cross-linking of collagen and elastin by catalyzing oxidative deamination of ε-amino groups of lysine and hydroxylysine residues [6]. In bone tissue, pyridinolines and deoxypyridinolines are the primary cross-links of mature type I collagen that provide mechanical integrity, rigidity, and strength [10], [11]. Diminished LOX enzyme activity results in an increased risk of bone deformities and fractures [12], [13]. Findings also show that the LOX protein is down-regulated in ras-transformed cells, many tumor cell lines [14], [15] as well as in human cancers [16]–[19], suggesting an additional function of LOX. Meanwhile, spontaneous reversion of cancers and induced phenotypic reversions are accompanied by increased LOX expression [20]. Its function as a tumor suppressor has been directly shown by transfection using anti-sense LOX, which triggers transformation of normal rat fibroblasts and reversion of stable phenotypic revertants of ras-transformed NIH3T3. This tumor suppressing role of LOX is recently attributed to the 18-kDa propeptide [15]–[17], which is processed from the secreted 50-kDa proenzyme by the procollagen C-proteinase bone morphogenic protein-1 [21]. Human genetic polymorphisms can play critical roles in various diseases. For example, the CC chemokine receptor 5 (CCR5) delta 32 polymorphism can greatly protect people against HIV infection [22], whereas the CTLA-4 49A/G SNP can increase the risk of developing lung cancer [23]. Polymorphisms can also affect bone tumors. It has been reported that the CD86+1057G/A SNP is correlated with increased risk of osteosarcoma [24]. Despite the importance of LOX, studies on LOX gene polymorphisms still remain superficial. Only one functional polymorphism, 473G/A (rs1800449), has been identified for its effect on different diseases such as breast cancer and coronary artery disease [25], [26]. In the current study, we screened all seven exons, including the intron-exon splice sites, and the putative promoter region of LOX gene and detected three polymorphisms in the Chinese population. We further investigated the function of −22G/C polymorphism and evaluated the association between these three polymorphisms and susceptibility to osteosarcoma. Materials and Methods Ethics Statement The study was approved by the Review Boards of Changzheng Hospital and the Shanghai No.6 People’s Hospital. Written informed consent was obtained from each participant or guardians on the behalf of the children participants involved in the study. Study Population The study population consisted of 326 osteosarcoma patients (10–67 years of age) and 433 healthy controls (12–70 years of age) recruited from Changzheng Hospital and the Shanghai No.6 People’s Hospital from May 2005 to July 2011 (Table 1). The diagnosis of osteosarcoma was established by histological examination in all cases. In the same period, 433 subjects who underwent regular physical examinations at the same hospital were recruited as controls. Relatives of study participants were excluded from this study. Those with a history of familial cancer syndromes were also excluded from this study. All the control subjects were matched with patient population in terms of age, sex, and residence area (urban or rural). All subjects were of Han Chinese ethnicity and were unrelated to each other. Each study participant provided a peripheral blood sample. 10.1371/journal.pone.0041610.t001Table 1 General characteristics of the subjects. Characteristics Osteosarcoma Control P value (n = 326) (%) (n = 433) (%) Age 0.828 ≤20 232 (71.2) 305 (70.4) >20 94 (28.8) 128 (29.6) Gender 0.884 Male 188 (57.7) 252 (58.2) Female 138 (42.3) 181 (41.8) Tumor location Long tubular bones 253 (77.6) Axial skeleton 73 (22.8) Metastasis With 98 (30.1) Without 228 (69.9) Screening Polymorphisms Genomic DNA was extracted from peripheral blood lymphocytes using a commercially available kit according to the manufacturer’s instructions (Blood genomic DNA miniprep kit; Axygen Biosciences). Direct sequencing of all 7 exons, including the exon-intron junctions and the putative promoter region, in 30 control subjects and 15 osteosarcoma patients was performed on an ABI 3100 automated sequencer with BigDye (Applied Biosystems) chemistry. PCR products were prepared using the genomic primer pairs and standard PCR conditions as shown in Table 2. The PCR primers were also used as sequencing primers. 10.1371/journal.pone.0041610.t002Table 2 Primers used in this study. Location Forward primers Reverse primers Temperature Promoter 5′-AGCATGCGATGTTTTACTGACTTT-3′ 5′-GCCATGGGGCGACGCCAAAATA-3′ 54°C 5′-TGCGTTGGGAAATGTGTCTGGTTA-3′ 5′-GCAAAGTTACACAAGCCGTTCTGG-3′ 59°C 5′-GGAAGGAGGGCAGGGACGGGAGA-3′ 5′-GAGGCGAGCGGAGCACGGGTATC-3 63°C Exon 1 5′-TTGCACGTTTCCAATCGCATTACG-3′ 5′-CGGCGGCGCTGAGGCTGGTA-3′ 63°C 5′-GCTTCGCCTGGACCGTGCTCCTG-3′ 5′-CAGCCACGTCGAGAAGCCACATA-3′ 61°C 5′-GGCGGCCGGGCCAGAACG-3′ 5′-GGGCGGCCAGCGGTGACTCC-3′ 60°C 5′-ACAACGGGCAGGTGTTCAG-3′ 5′-GGGAGGGATCGGATCTGCGAGG-3′ 58°C Exon 2 5′-CCGGGCGTCCCCAGTTCT-3′ 5′-CCAGCTCTTGTCCCACTTCCTAAC-3′ 61°C Exon 3 5′-GTTGGGAAAGGAGGATTGTCACTA-3′ 5′-AGCAATTTTCTCCCTTCAGGTAAG-3′ 51°C Exon 4 5′-TTGTGAAGCCATGGGGGAAGTCAT-3′ 5′-TTTAATGCTAACTAACGGTAGATG-3′ 51°C Exon 5 5′-TGGAGGTGCTATAAGGCTGAGTAA-3′ 5′-GCATATTTTCCCCTGAAGTTCTTT-3′ 50°C Exon 6 5′-ATCCCTTCCCTGCTTGATTTAGC-3′ 5′-CCATTTTCTGCCTTTGGTCTGCT-3′ 53°C Exon 7 5′-TGCTTAGGTGGAGGGAAACTGTTG-3′ 5′-TTCTCAGCACCAGATGTGTCCATA-3′ 51°C 5′-CATGTTCCTTTTGAAATTGTAGTG-3′ 5′-CCTCAAGAAATATCAGACAGGAT-3′ 50°C Polymorphisms Genotyping After identification of the three polymorphisms by direct sequencing, the 473G/A polymorphism was detected in the rest of the subjects via a polymerase chain reaction (PCR)–restriction fragment length polymorphism method. The PCR primers were designed based on the GenBank reference sequence and Primer 5.0. The primers were as follows: forward primer 5′-CTCACAGTACCAGCCTCAGCG-3′ and reverse primer 5′-CCAGGTCTGGGCCTTTCATA-3′. The PCRs were performed in a total volume of 35 µL reaction mixture containing 100 ng genomic DNA, 12.5pmol of each primer, 0.1 mM of each dNTP, 1xPCR buffer, 1.0 mmol/L MgCl2, and 1.5 unit of Taq DNA polymerase (Fermentas). Reactions were conducted using the thermal cycler (Biometra) under the following conditions: an initial incubation at 95°C for 10 min, 30 cycles of 95°C for 30s, 57°C for 30s, 72°C for 30s, and a final extension at 72°C for 7 min. The efficiency of the PCR was confirmed by gel electrophoresis on a 1.5% agarose gel. After DNA amplification, the PCR products were digested overnight at 37°C with 10U of the specific restriction endonuclease PstI (Fermentas), which cut allele A. The digestion products were then resolved and separated on a 2% agarose gel stained with ethidium bromide for visualization under ultraviolet light. After electrophoresis, homozygous A alleles were represented by DNA bands at 291 and 114 bp in length. An uncut fragment of 405 bp indicated the homozygous G allele while the heterozygous genotype was displayed as a combination of 405, 291, and 114 bp fragments. The detection of 225C/G polymorphism was performed by similar methods. In brief, the polymorphism was first identified by direct sequencing and was then followed by PCR and enzyme restriction method using SacII (Fermentas). The detection of −22G/C polymorphism in the rest of the subjects was performed by direct sequencing using the primer 5′ -TTGACTGGGGAAGGGTCTGA- 3′. Cell Culture MG-63 cells were purchased from American Type Culture Collection. It is a human osteosarcoma cell line that offers a fibroblast-like morphology. The MG-63 cells were grown in aMEM (a-minimum essential medium; Biochrom) containing 5% fetal calf serum (FCS; Biochrom), supplemented with 4.5 g/l L-glucose, 50 ug/ml ascorbic acid (Sigma) and 10 ug/ml gentamycin (Sigma). Jurkat cells (CD4+ T lymphocyte cell line) and U937 cells (monocytic cell line) were cultured in RPMI-1640 medium supplemented with 10% FCS. Construction of Reporter Plasmids and Cell Transfection Since the −22G/C polymorphism lies in the downstream core promoter element (DPE) region of the LOX gene, we hypothesized that the polymorphism may affect the transcription of the LOX gene. Sixty-five nucleotides of the 5′ upstream region of the LOX gene covering the Initiator (Inr) and DPE regions were obtained by PCR using human genomic DNA. The forward primer was 5′ -GTCAGAGCTCAGCCCCGTTTTTATTTTCTG- 3′ and contained a SacI site. The reverse primer was 5′ -CCCGGGATCACGCCTTTTGCCAGATTGA- 3′, which corresponded to the −20 to +2 positions in the LOX gene sequence, referencing the first nucleotide preceding the ATG codon as position −1. This primer contained an XmaI site in the 5′ end. The PCR product carrying wild type −22G or mutation −22C was subsequently cloned into the SacI/XmaI sites of the promoterless and enhancerless firefly luciferase reporter vector pGL3-Basic. Similarly, we made a shorter PCR product (42bps), which only contained the DPE with or without the −22G/C polymorphism (Fig. 1). Along with pRL-CMV, which is an internal control for monitoring transfection efficiency, each resulting recombinant construct was transiently cotransfected into MG-63 cells by FuGENE 6 (Roche). Cells were harvested 48 hours after transfection and luciferase activities were measured according to the manufacturer’s instructions (Dual-Luciferase Reporter Assay System, Promega) with a Luminometer Centro LB960 (Berthold, Bad Wildbad, Germany). Relative luciferase expression (fold increase) was calculated with the following equation: fold increase = (firefly luciferase activity of upstream region of LOX gene construct/Renilla luciferase activity)/(firefly luciferase activity of promoterless vector pGL3-Basic/Renilla luiferase activity). 10.1371/journal.pone.0041610.g001Figure 1 (A) Fragment containing 65-bp upstream non-coding region of the lysyl oxidase (LOX) gene. The −22G/C polymorphism is underlined. Core promoters Initiator (Inr) and downstream core promoter element (DPE) are labeled with boxes. (B) Schematic representation of LOX promoter-reporter chimeras with or without Inr, DPE and −22G/C polymorphisms. (C, D) Luciferase activity mediated by the upstream non-coding region of the wild-type and −22G/C polymorphism in MG-63 cells (C) and Jurkat cells (D). Data represent three independent experiments with similar results. Data shown are the mean ± S.D. of three experiments, each determined with triplicate dishes. P values for differences in fold increase are shown. Statistical Analysis The SPSS statistical software package ver.13.0 (SPSS Inc., Chicago, USA) was used for statistical analysis. Demographic data between the study groups were compared by the chi-square test and by the Student t-test. The polymorphisms were tested for deviation from Hardy-Weinberg equilibrium (HWE) by comparing the observed and expected genotype frequencies using the chi-square test. For single nucleotide polymorphism (SNP) analysis, genotype and allele frequencies of LOX were compared between groups using the chi-square test; odds ratio (OR) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression. The linkage disequilibrium (LD) between these polymorphisms and the haplotypes of LOX were conducted using the SHEsis software from the website http://analysis.bio-x.cn/(Bio-X Inc.,Shanghai, China). Promoter activity data were expressed as mean ± S.D. of at least three independent experiments. For the purpose of correcting for multiple testing, Bonferroni correction was applied. Consequently, differences were considered significant when the P value was less than 0.0167. Results Polymorphisms in LOX Gene Genomic DNA sequencing analysis was performed on all 7 exons, all exon/intron splice sites, and 850 bp upstream including the predicted promoter region of LOX, in 30 control subjects and 15 osteosarcoma patients. Three polymorphisms (−22G/C, 225C/G, and 473G/A) were detected in both cases and controls (Table 3). No SNP was found exclusively in osteosarcoma patients. The −22G/C polymorphism was a novel SNP. It lies 22 nucleotides upstream from the open frame ATG codon of the LOX gene, and changes the first nucleotide of DPE from GGTCA to CGTCA (Fig. 1a). The frequency of the polymorphic −22C allele is 1.3% in the Chinese population (Table 3). The other two SNPs detected, 225C/G (rs2278226) and 473G/A (rs1800449), were previously reported polymorphisms. The 225C/G SNP lies in exon 3 and is a silent polymorphism with both alleles coding for alanine. The 473G/A polymorphism is located in exon 1 and results in a conservative amino acid substitution from arginine to glutamine. We also observed several mutations in the LOX gene. However, frequencies of these mutations were too low to be considered as polymorphisms (frequency of minor allele <0.5%), and they were not investigated further (Data not shown). 10.1371/journal.pone.0041610.t003Table 3 LOX polymorphisms in osteosarcoma patients and controls. Polymorphisms Osteosarcoma Control subjects OR (95%CI) P value (n = 326) (%) (n = 433) (%) −22 G/C Genotype GG 306 (93.9) 425 (98.1) Referent GC 9 (2.8) 5 (1.2) 2.50 (0.83−7.54) 0.093 CC 11 (3.3) 3 (0.7) 5.09 (1.41−18.41) 0.006 * Allele G 621 (95.2) 855 (98.7) Referent C 31 (4.8) 11 (1.3) 3.88 (1.94−7.78) 4.18×10−5 * 225 C/G Genotype CC 315 (96.6) 416 (96.1) Referent CG 11 (3.4) 17 (3.9) 0.85 (0.39−1.85) 0.690 Allele C 641 (98.3) 849 (98.0) Referent G 11 (1.7) 17 (2.0) 0.86 (0.40−1.84) 0.693 473 G/A Genotype GG 209 (64.1) 301 (69.5) Referent GA 86 (26.4) 112 (25.9) 1.11 (0.79−1.54) 0.552 AA 31 (9.5) 20 (4.6) 2.23 (1.24−4.02) 0.006 * Allele G 504 (77.3) 714 (82.4) Referent A 148 (22.7) 152 (17.6) 1.38 (1.07−1.78) 0.013 * Haplotypes (−22, 225, 473) GCG 472 (72.4) 690 (79.7) Referent GCA 138 (21.2) 147 (17.0) 1.37 (1.06−1.78) 0.017 GGG 8 (1.2) 14 (1.6) 0.84 (0.35−2.01) 0.687 CCG 24 (3.7) 9 (1.0) 3.90 (1.80−8.46) 4.46×10 −4 * * p<0.0167 was considered significant after Bonferroni correction. LOX, lysyl oxidase; OR, odds ratio; CI, confidence interval. The −22G/C SNP and LOX Promoter Activity Sequence analysis revealed that the LOX promoter region lacked the canonical TATA box and CAAT box (Data not shown). Instead, it contained 5′-TTATTTT- 3′ from −55 to −49 upstream from the open frame ATG codon of the LOX gene, which is identical to the Initiator (Inr) element consensus sequence YYAN(T/A)YY (Y is pyrimidine; N is any nucleotide) [24], [25]. Furthermore, a sequence 5’ -GGTCA- 3’ was located at the region from −22 to −18 upstream of ATG; this is consistent with the consensus sequence of the DPE, (A/G)G(A/T)(C/T)(G/A/C) [25]. Thus, a potential Inr core promoter element combined with the DPE was mapped in the LOX promoter region (Fig. 1A). Since the −22G/C SNP changed the DPE of LOX from GGTCA to CGTCA (Fig. 1A), we hypothesized that the polymorphism could affect the activity of DPE. Therefore, we constructed a reporter plasmid containing the Inr and DPE with the −22G/C polymorphism and compared its luciferase activity with that of the wild-type version (Fig. 1B). As shown in Fig. 1C, the reporter activity of the wild type containing Inr and DPE was approximately 7.2-fold higher than that of the pGL3-Basic plasmid in MG-63 cells. The −22C reduced the reporter activity to about 4-fold. We also generated shorter versions of the wild type as well as mutant constructs carrying the DPE only (Fig. 1B). Without the Inr, the reporter activity of the wild type was approximately 5.6-fold higher than that of the pGL3-Basic plasmid, and the −22C reduced the reporter activity to about 2-fold in MG-63 cells (Fig. 1C). In order to determine whether the function of −22C/G SNP is cell-specific, we performed the same experiments in an additional two different cell lines - Jurkat cells (CD4+ T lymphocyte cell line) and U937 cells (monocytic cell line). In the Jurkat cells (Fig. 1D), the −22C decreased the promoter activity of longer construct from 5.1-fold to 3.1-fold and from 4.0-fold to 1.8-fold for the shorter version. The −22C/G SNP showed similar effects in U937 cells (Data not shown). These results suggest that the Inr and the DPE do not work as one combined unit initiating the LOX gene transcription because deletion of the Inr only partially inhibited the reporter gene expression. Furthermore, the −22G/C SNP could decrease the promoter activity by affecting the DPE, and this effect has no cell specificity. The LOX Polymorphisms and Susceptibility to Osteosarcoma We further analyzed the association between the three LOX polymorphisms and susceptibility to osteosarcoma in the Chinese population. A total number of 326 osteosarcoma cases and 433 controls were recruited for the present study. All subjects were ethnic Chinese. Demographic and other selected characteristics of the cases and controls are presented (Table 1). Cases and controls did not show statistically significant differences with regard to age and sex (Table 1). The LOX polymorphisms (−22G/C, 225C/G, and 473G/A) in osteosarcoma patients and healthy controls are summarized in Table 3. All SNPs genotyped were in HWE (P>0.05). The prevalence of the −22CC genotype and −22C allele were significantly increased in osteosarcoma patients compared to controls (OR = 5.09, 95% CI = 1.41–18.41, p = 0.006; and OR = 3.88, 95%CI = 1.94–7.78, p = 4.18x10−5; p<0.0167 was considered significant after Bonferroni correction). Similarly, the frequencies of the 473AA genotype and A allele were significantly higher in osteosarcoma cases (OR = 2.23, 95% CI = 1.24−4.02, p = 0.006; and OR = 31.38, 95%CI = 1.07–1.78, p = 0.013; p<0.0167 was considered significant after Bonferroni correction). Analysis of linkage disequilibrium (LD) between polymorphic sites revealed that there was no significant LD among the polymorphisms. Further, we analyzed the haplotypes constructed by these three polymorphisms. The four most common haplotypes are shown (Table 3). The frequency of the CCG (−22, 225, 473) haplotype was significantly higher in osteosarcoma cases than in controls after Bonferroni correction (p = 4.46×10−4). These data suggest that LOX −22G/C and 473G/A SNPs are associated with increased susceptibility to osteosarcoma in the Chinese population. We further evaluated the association of LOX−22G/C, 225C/G, and 473G/A polymorphisms with different clinical-pathological factors in the osteosarcoma patients. The 326 osteosarcoma patients were divided into two subsets based on age, gender, tumor location, or metastasis (Table 4), and SNPs were compared between these two subsets. The frequencies of the 473AA genotype and 473A allele were higher in osteosarcoma patients with metastasis than those without metastasis (OR = 2.44, 95%CI = 1.13–5.26, p = 0.020; OR = 1.52, 95%CI = 1.04–2.24, p = 0.032). However, the differences did not reach statistical significance after Bonferroni correction. 10.1371/journal.pone.0041610.t004Table 4 Stratification analysis of LOX polymorphisms in osteosarcoma patients. Frequencies Age (%) ≤20/>20 (232)/(94) OR 95%CI P Gender (%) Male/Female (188)/(138) OR 95%CI P −22 G/C GG 216 (93.1) 90 (95.8) Referent 177 (94.1) 129 (93.5) Referent GC 7 (3.0 ) 2 (2.1) 1.46 (0.60−7.16) 0.640 5 (2.7) 4 (2.9) 0.91 (0.24−3.46) 0.891 CC 9 (3.9) 2 (2.1) 1.88 (0.40−8.85) 0.420 6 (3.2) 5 (3.6) 0.87 (0.26−2.93) 0.828 G 439 (94.6) 182 (96.8) Referent 359 (95.5) 262 (94.9) Referent C 25 (5.4) 6 (3.2) 1.73 (0.70−4.28) 0.233 17 (4.5) 14 (5.1) 0.89 (0.43−1.83) 0.744 225 C/G CC 223 (96.1) 92 (97.9) Referent 182 (96.8) 133 (96.4) Referent CG 9 (3.9) 2 (2.1) 1.86 (0.39−8.76) 0.428 6 (3.2) 5 (3.6) 0.88 (0.26−2.94) 0.831 C 455 (98.1) 186 (98.9) Referent 370 (98.4) 271 (98.2) Referent G 9 (1.9) 2 (1.1) 1.84 (0.39−8.60) 0.432 6 (1.6) 5 (1.8) 0.88 (0.27−2.91) 0.833 473 G/A GG 150 (64.7) 59 (62.8) Referent 123 (65.4) 86 (62.3) Referent GA 62 (26.7) 24 (25.5) 1.02 (0.58−1.78) 0.955 49 (26.1) 37 (26.8) 0.93 (0.58−1.54) 0.767 AA 20 (8.6) 11 (11.7) 0.72 (0.32−1.58) 0.407 16 (8.5) 15 (10.9) 0.75 (0.35−1.59) 0.446 G 362 (78.0) 142 (75.5) Referent 295 (78.5) 209 (75.7) Referent A 102 (22.0) 46 (24.5) 0.87 (0.58−1.30) 0.493 81 (21.5) 67 (24.3) 0.86 (0.59−1.24) 0.411 Frequencies Location (%) L/A (253)/(73) OR 95%CI P Metastasis (%) Yes/No .(98)/(228) OR 95%CI P −22 G/C GG 237 (93.7) 69 (94.6) Referent 90 (91.8) 216 (94.7) Referent GC 7 (2.8) 2 (2.7) 1.02 (0.21−5.02) 0.982 4 (4.1) 7 (3.1) 1.37 (0.39−4.80) 0.620 CC 9 (3.5) 2 (2.7) 1.31 (0.28−6.21) 0.733 4 (4.1) 5 (2.2) 1.92 (0.50−7.32) 0.331 G 281 (91.8) 140 (95.9) Referent 184 (93.9) 439 (96.3) Referent C 25 (8.2) 6 (4.1) 2.08 (0.83−5.18) 0.110 12 (6.1) 17 (3.7) 1.68 (0.79−3.40) 0.174 225 C/G CC 245 (96.8) 70 (95.9) Referent 93 (94.9) 222 (97.4) Referent CG 8 (3.2) 3 (4.1) 0.76 (0.20−2.95) 0.693 5 (5.1) 6 (2.6) 1.99 (0.59−6.68) 0.257 C 498 (98.4) 143 (97.9) Referent 191 (97.4) 450 (98.7) Referent G 8 (1.6) 3 (2.1) 0.77 (0.20−2.93) 0.695 5 (2.6) 6 (1.3) 1.96 (0.59−6.51) 0.262 473 G/A GG 165 (65.2) 44 (60.3) Referent 58 (59.2) 151 (66.2) Referent GA 65 (25.7) 21 (28.8) 0.83 (0.46−1.50) 0.526 25 (25.5) 61 (26.8) 1.07 (0.61−1.86) 0.819 AA 23 (9.1) 8 (10.9) 0.77 (0.32−1.83) 0.549 15 (15.3) 16 (7.0) 2.44 (1.13−5.26) 0.020 G 395 (78.1) 109 (74.7) Referent 141 (71.9) 363 (79.6) Referent A 111 (21.9) 37 (25.3) 0.83 (0.54−1.27) 0.387 55 (28.1) 93 (20.4) 1.52 (1.04−2.24) 0.032 p<0.0167 was considered significant after Bonferroni correction. LOX, lysyl oxidase; OR, odds ratio; CI, confidence interval. L: long tubular bones. A: axial skeleton. Discussion The core promoter is an important, yet often overlooked, component in the regulation of transcription by RNA polymerase II. It includes the TATA box, the TFIIB recognition element (BRE), the Inr and the DPE. The DPE is most commonly found in TATA-less promoters, although some promoters contain both DPE and TATA motifs [27], [28]. Our study showed that human LOX promoter region lacks the canonical TATA and CAAT boxes. Instead, it contains 5′ -TTATTTT- 3′ from −55 to −49 upstream from the open frame ATG codon of the LOX gene, which is identical to the Inr element consensus sequence YYAN(T/A)YY (Y is pyrimidine; N is any nucleotide) [27], [28]. Furthermore, a sequence of 5’ -GGTCA- 3’ was located in the region −22 to −18 nucleotides upstream of ATG, consistent with the consensus sequence of the DPE (A/G)G(A/T)(C/T)(G/A/C) [28]. While it is generally believed that the DPE functions in coordination with the Inr [25], our results suggest that the Inr and the DPE do not work as one combined unit initiating the LOX gene transcription because deletion of the Inr only partially inhibited the reporter gene expression (Fig. 1). These data are consistent with the research about the Inr and the DPE in rat LOX gene [29]. Although a previous study has shown that promoter activity can be affected by mutagenesis of DPE [29], effects of DPE change on human diseases have never been reported. To our knowledge, this is the first report demonstrating that a SNP causes functional change of the DPE and is subsequently associated with human disease. LOX catalyzes cross-linking of elastin and collagen, which is essential for the structural integrity and function of bone tissue [12], [13]. A study by Pischon et al has shown that LOX gene deficiency can affect osteoblastic phenotype [30]. In addition, LOX can act as a tumor suppressor. To date, a decrease in LOX mRNA and/or protein has been observed in basal and squamous cell, bronchogenic, colon, esophageal, gastric, head and neck squamous cell, pancreatic, and prostatic carcinomas, as well as osteosarcoma [31]. It has been reported that suramin may influence proliferation and differentiation of osteosarcoma cells by up-regulating LOX mRNA expression [32]. These studies indicate that changes in LOX may be correlated with the development of osteosarcoma. The LOX G473A polymorphism has shown to be associated with a higher risk of breast cancer and coronary artery disease [25], [26]. While the mechanism remains to be fully elucidated, it is possible that the G473A polymorphism, which causes an Arg158Gln substitution in a highly conserved region within LOX-PP, may reduce the ability of LOX-PP to suppress Ras signaling [26]. Our data showed that the 473G/A SNP is correlated with increased susceptibility to osteosarcoma (Table 3), which may also be due to the decreased activation of the LOX-PP pathway caused by the SNP. In addition, 473AA genotype and A allele revealed higher prevalence in metastatic osteosarcoma cases compared to normal patients (p = 0.02 and p = 0.32) (Table 4). Although values were not statistically significant after Bonferroni correction, these data demonstrate a trend that 473G/A polymorphism might be associated with metastatic osteosarcoma patients. Further studies are necessary to confirm this. As for the −22G/C SNP, the reporter activity of DPE carrying the −22G/C was about 2.6-fold lower than that of the DPE wild-type, but about 2-fold higher than the reporter activity of the negative control (p<0.01 in MG-63 cells; p<0.05 in Jurkat cells) (Fig. 1C, D). These data indicate that the −22G/C polymorphism partially affects the function of DPE. This SNP was also associated with an increased risk of osteosarcoma (Table 3). The mechanism is not yet clear. It may be because the −22G/C SNP reduces the LOX mRNA level, which results in a diminished amount of LOX-PP; or because the decreased LOX by the SNP causes bone deformities. According to the International HapMap Project, LOX 473G/A SNP lies in European, Asian, Sub-Saharan African, and African American populations. The LOX −22G/C polymorphism is a novel SNP. Studies in different populations and diseases may be helpful for further understanding this polymorphism. In conclusion, our results demonstrated that the LOX gene −22G/C in the DPE could decrease promoter activity. Furthermore, through the association of −22G/C and 473G/A SNPs with increased susceptibility to osteosarcoma in the Chinese population, this study revealed a novel pathway by which genetic polymorphisms may affect human diseases. Competing Interests: The authors have declared that no competing interests exist. Funding: This work was supported by National Natural Science Foundation of China (Grant No. 81171677). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Hameed M Dorfman H 2011 Primary malignant bone tumors–recent developments. Semin Diagn Pathol 28 86 101 21675380 2 Subbiah V Kurzrock R 2011 Phase 1 clinical trials for sarcomas: the cutting edge. Curr Opin Oncol 23 352 60 21519259 3 Posthuma DeBoer J Witlox MA Kaspers GJ van Royen BJ 2011 Molecular alterations as target for therapy in metastatic osteosarcoma: a review of literature. Clin Exp Metastasis 28 493 503 21461590 4 Boskey AL 1996 Matrix protein and mineralization: an overview. Connect Tissue Res 35 357 363 9084675 5 Fratzl P Gupta HS Paschalis EP Roschger P 2004 Structure and mechanical quality of the collagen-mineral nano-composite in bone. 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Inflammopharmacology 19 117 129 21107914 32 Buchinger B Spitzer S Karlic H Klaushofer K Varga F 2008 Lysyl oxidase (LOX) mRNA expression and genes of the differentiated osteoblastic phenotype are upregulated in human osteosarcoma cells by suramin. Cancer Lett 265 45 54 18374478
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PLoS One. 2012 Jul 23; 7(7):e41610
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22844422PONE-D-11-2515210.1371/journal.pone.0040992Research ArticleBiologyBiochemistryLipidsLipid MetabolismMetabolismLipid MetabolismProteinsLipoproteinsMolecular Cell BiologyCellular TypesEpithelial CellsMedicineGastroenterology and HepatologySmall IntestineNutritionModulatory Role of PYY in Transport and Metabolism of Cholesterol in Intestinal Epithelial Cells PYY and Intestinal Lipid TransportGrenier Emilie 1 2 Garofalo Carole 1 Delvin Edgard 1 3 Levy Emile 1 2 * 1 Research Centre, Centre Hospitalier Universitaire (CHU) Ste-Justine, Montreal, Quebec, Canada 2 Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada 3 Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada Blachier François EditorNational Institute of Agronomic Research, France* E-mail: [email protected] and designed the experiments: EG CG ED EL. Performed the experiments: EG CG. Analyzed the data: EG ED EL. Contributed reagents/materials/analysis tools: CG. Wrote the paper: EL. 2012 23 7 2012 7 7 e4099215 12 2011 19 6 2012 Grenier et al.2012This 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 Gastrointestinal peptides are involved in modulating appetite. Other biological functions attributed to them include the regulation of lipid homeostasis. However, data concerning PYY remain fragmentary. The objectives of the study were: (i) To determine the effect of PYY on intestinal transport and synthesis of cholesterol, the biogenesis of apolipoproteins (apos) and assembly of lipoproteins and (ii) To analyze whether the effects of PYY are similar according to whether cells are exposed to PYY on apical or basolateral surface. Methodology/Principal Findings Caco-2/15 cells were incubated with PYY (1–36) administered either to the apical or basolateral medium, at concentrations of 50 or 200 nM for 24 hours. De novo synthesis of cholesterol, cholesterol uptake, and assembly of lipoproteins were evaluated through the incorporation of [14C]-acetate, [14C]-cholesterol, and [14C]-oleate, respectively. Biogenesis of apos (A-I, A-IV, E, B-48 and B-100) was examined by the incorporation of [35S]-methionine. The influence of PYY on protein and mRNA levels of many key mediators of lipid metabolism was analyzed by Western blot and PCR, respectively. Our results show that PYY influenced cholesterol metabolism in Caco-2/15 cells depending on the site of PYY delivery. Apical addition of PYY significantly lowered the incorporation of [14C]-cholesterol likely via the reduction of NPC1L1, stimulated intracellular cholesterol synthesis probably through an increase in SREBP-2 expression, whereas it concomitantly increased apo A-I synthesis and decreased LDL secretion. In contrast, basolateral PYY reduced the production of chylomicrons (CM) as well as the biogenesis of apos B-48 and B-100, while lowering the expression of the transcription factors RXRα and PPAR(α,β). Conclusions/Significance PYY is capable of influencing cholesterol homeostasis in intestinal Caco-2/15 cells depending on the site delivery. Apical PYY was able to decrease cholesterol uptake via NPC1L1 downregulation, whereas basolateral PYY diminished CM output through the biogenesis decline of apos B-48 and B-100. ==== Body Introduction Peptide YY (PYY), a 36 amino acid straight chain polypeptide, is produced by epithelial entero-endocrine L cells throughout the gut, but its concentrations increase distally, reaching higher levels in the colon and rectum [1], [2]. The predominant form of PYY is released into the circulation as PYY(3–36), with the Nterminal truncated by the enzymatic action of dipeptidyl peptidase (DPP-IV) [3], [4]. Peptide YY(3–36) is secreted in proportion to the amount of calories in the meal, with serum concentrations increasing within 15 min of a meal, peaking at about 60 min and sustaining for up to 6 h [5]. The secretion is mediated by neural reflex and by direct contact of nutrients; however, fat intake promotes higher secretion of PYY(3–36) than carbohydrates and proteins [6]. Although PYY is localized in mucosa, it may act as an endocrine modulator of distant target tissues, particularly within the gastrointestinal tract. In fact, PYY exhibits various actions on this tissue, including the delay of both gastric emptying and mouth to caecum transit time [7], the inhibition of jejunal pressure wave activity [8], the reduction of pentagastrin-stimulated acid secretion [6], the cephalic phase related to negative pancreatic exocrine secretion in man [9]–[11], and actin arrangement and expression of cytoskeletal proteins in intestinal epithelial cells, which interact with organizing intracellular structure, such as cellular-extracellular matrix [12]. This regulation by PYY enhances the efficiency of nutrient digestion and absorption, and ensures efficient utilization of ingested food. Furthermore, PYY can initiate an “ileal brake” when the rate of triglyceride (TG) hydrolysis exceeds the rate of fatty acid (FA) absorption excess [10], [13], [14]. In addition, PYY interacts with FAs to induce differentiation in mucosal cells [15], [16]. As reported above, PYY modulates important gastrointestinal functions with important effects on food intake and energy expenditure, which influences the delivery of nutrients and gut hormones to the circulation. There is also evidence that PYY is closely involved in insulin secretion and glucose homeostasis [17]. Furthermore, peripheral administration of PYY has been shown to increase blood pressure in healthy humans [18], which suggests that this gut hormone may also influence cardiovascular diseases in addition to modulating appetite and metabolism. Accordingly, recent studies have reported an inverse correlation between fasting PYY and total cholesterol [19], as well as low- and high-density lipoprotein cholesterol levels [20], which indicates that the PYY gut hormone may be involved in the modulation of cholesterol metabolism. Intriguingly, the role of PYY in intestinal cholesterol transport has not been investigated. Therefore, the major aim of this study is to determine whether PYY is able to modulate intestinal cholesterol synthesis, apolipoprotein (apo) biogenesis and lipoprotein assembly and secretion. These issues were tackled using the Caco-2/15 cell line that spontaneously differentiates into polarized mature enterocytes under standard culture conditions, and lends itself to the in vitro study of human gut in view of its efficient intestinal transport processes [21]. With Caco-2/15 cells granting access to both apical and basolateral sides of the bipolar intestinal epithelium when cultured on Transwell filters, we were also able to underline the importance of the regulatory site of intestinal cholesterol transport by PYY since this gastrointestinal factor was found to be secreted in the two compartments [22]–[25]. Materials and Methods 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) [26]–[28]. Caco-2/15 cells were grown at 37°C with 5% CO2 in MEM (GIBCO-BRL, Grand Island, NY) containing 1% penicillin-streptomycin and 1% MEM nonessential amino acids (GIBCO BRL) and supplemented with 10% decomplemented fetal bovine serum (FBS) (Flow, McLean, VA) as described previously [29]. Caco-2/15 cells (passages 30–40) were maintained in T-75-cm2 flasks (Corning Glass Works, Corning, NY). Cultures were split (1∶6) when they reached 70–90% confluence, by use of 0.05% trypsin-0.5 mM EDTA (GIBCO-BRL). For individual experiments, cells were plated at a density of 1×106 cells/well on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores (Costar, Cambridge, MA), in MEM (as described above) supplemented with 5% FBS. The inserts were placed into six-well culture plates, permitting separate access to the apical and basolateral compartments of the monolayers. Cells were cultured for 14 days at which the Caco-2/15 cells are highly differentiated into polarized mature enterocytes and appropriate for lipid metabolism [29], [30]. The medium was refreshed every second day. PYY (1–36) Human PYY (1–36) (Sigma) was added to cells in apical or basolateral compartments at different concentrations (50 nM or 200 nM) for 24 h. Isolation of Apical or Basolateral Membranes from Differentiated Caco-2/15 14 days after reaching 100% confluence until fully differentiated, the brush border fractions were prepared by a modification of the method of Schmitz et al. [31]. Briefly, the culture medium was removed, and the cells were rinsed twice with Phosphate Buffer Saline (PBS, GIBCO-BRL). The cells were homogenized in 50 mM mannitol-HCL solution, pH 7.5, supplemented with 0.15 mg/ml leupeptin, 100 U/ml trasylol, 1×10−13 mg/ml pepstatin, and 1 mM PMSF. After the addition of CaCl2 to a final concentration of 10 mM, the homogenate was centrifuged for 15 min at 5000×g and 4°C to provide a pellet containing basolateral membrane. The supernatant was subsequently centrifuged first for 20 min at 30000×g and 4°C and then for 30 min at 30000×g and 4°C. The final pellet containing the apical membranes was frozen. Cholesterol Absorption by Caco-2/15 Cells Cholesterol uptake by the cells was assessed following its solubilisation in albumin as described previously [32], [33]. The differentiated cells were incubated at 37°C for 24 h in MEM containing 50 nM or 200 nM of PYY, in apical or basolateral compartments, as well as cholesterol solution added to the apical medium. At the end of the treatment, cells were washed twice with cold PBS (sufficient to complete removal of labelled cholesterol bound on cell membranes as established in our laboratory by various methods such as cyclodextrin), scrapped in 1 ml lysis buffer (5 mM Tris, 15 mM NaCl, EDTA 5 mM, 0.1% SDS, 1% Triton×100, 0.5% sodium deoxycholate) and homogenized by sonication followed by a 5 min at 13800×g centrifugation to remove cell debris. An aliquot of 0.1 ml was placed in a scintillation vial with Ready Safe counting fluid (Beckman, Fullerton, CA). Radioactivity was measured by scintillation counting (LS 5000 TD, Beckman). Cell protein was quantified by the Bradford method (BioRad). Lipid Carrier Blood (20 mL) was collected by venipuncture 2–3 h after the ingestion of a high fat meal (50 g/1,73 m2) from two human healthy volunteers. This procedure was approved by the Institutional Ethic Committee. After a 1000×g centrifugation to pellet red blood cells, postprandial plasma was supplemented with 1 mM of aprotinin and 0.1% of sodium azide and was mixed with basolateral media to serve as a carrier for the isolation of labeled chylomicron (CM) as described previously [29]. De novo Lipogenesis Caco-2/15 cells were serum-starved after 14 days of differentiation on Transwell filter inserts. After 18 h-incubation in serum-free medium, cells were cultured in the presence of 5 µCi of sodium-[14C]-acetate (specific activity of 50–62 mCi/mmol; GE Healthcare, Piscataway, NJ) for 24 h. Cells and media were collected, supplemented with a mixture of anti-proteases and lipids were extracted overnight in chloroform-methanol (2∶1, vol:vol). Lipids recovered were separated on TLC plates, and bands corresponding to free cholesterol and cholesteryl ester were scraped off the plates, mixed with scintillation fluid and counted for the amount of radioactivity incorporated as described previously [34]. Isolation of Lipoproteins Radiolabeled [14C]-oleic acid (sp act, 53 mCi/mmol, Amersham, Oakville, ON, Canada) was added to unlabeled oleic acid and then solubilised in FA-free bovine serum albumin (BSA) [BSA/oleic acid, 1∶5 (mol:mol)]. The final oleic acid concentration was 0,7 mM (0,45 µCi)/well. Cells were first washed with PBS, and the [14C]-oleic acid-containing medium was added to the apical compartment. PYY (1–36) was added to the apical and basolateral chamber in serum-free MEM. At the end of a 24 h incubation period, cells were washed and then scraped with a rubber policeman in a PBS solution containing antiproteases (phenylmethylsulfonyl fluoride, pepstatin, EDTA, aminocaproic acid, sodium azide and trasylol, all at a final concentration of 1 mM). The medium was first mixed with a plasma lipid carrier (4∶1, vol/vol) to efficiently isolate de novo lipoproteins synthesized. The lipoproteins were then isolated from basolateral media by serial ultracentrifugation using a TL-100 ultracentrifuge (Beckman-Coulter, Fullerton, CA) according to the method described previously [35]. Briefly, CM were first isolated after an ultracentrifugation (20000 rpm for 20 min). Very-low density lipoprotein (VLDL; 1.006 g/mL) and low density lipoprotein (LDL; 1.063 g/mL) were subsequently separated by centrifugation at 100000 rpm for 2.5 h with a tabletop ultracentrifuge 100.4 rotor at 4°C. The High density lipoprotein (HDL) fraction was obtained by adjusting the LDL infranatant to density at 1.21 g/mL and centrifuging for 6 h at 100000 rpm. Each lipoprotein fraction was exhaustively dialyzed against 0.15 M NaCl and 0,001 M EDTA, pH 7.0 at 4°C for 24 h. De novo Apolipoproteins Synthesis and Secretion The effect of PYY (1–36) on newly synthesized apos (A-I, A-IV, B-48, B-100 and E) was assessed as described previously [29]. To induce apo synthesis, cells were incubated apically with unlabeled oleic acid bound to albumin in serum-free medium, 18 h before [35S]-methionine incubation. Thereafter, cells as well as the outer chambers were rinsed twice with PBS. The apical compartment was replaced with 1.5 ml of methionine-free medium containing the unlabeled substrate (oleic acid) and 100 µCi/ml [35S]-methionine (50 mCi/mmol, Amersham Life Sciences). During this time, PYY (1–36) (Sigma) was added to the apical or basolateral chamber. Following basolateral medium removal, cells were scraped off the inserts in the cell lysis buffer. The cell lysates and media were supplemented with an antiprotease cocktail and analysed for apo synthesis and secretion, respectively, using antibodies with high specificity, which was tested by assessing cross reactivity, the recognition of a specific apo among a cocktail of various proteins, and the use of control conditions (omission of antibodies or exclusive treatment with pre-immune rabbit sera). Immunoprecipitation of Apolipoproteins Immunoprecipitation of apos in cell lysates and basolateral media was performed in the presence of excess polyclonal antibodies to human apos (A-I, A-IV, E, with a dilution of 1/1000; Santa Cruz, CA) and apo B (with a dilution of 1/1000; Boehringer, Mannheim) overnight at 4°C. Samples were then washed with lysis buffer. They were subsequently centrifuged and resuspended in sample buffer (1.2% SDS, 12% glycerol, 60 mM Tris, pH 7.3, 1.2% β-mercaptoethanol, and 0.003% bromophenol blue) and analyzed by a linear 4–15% polyacrylamide gradient preceded by a 3% stacking gel. Radioactive molecular weight standards (Amersham Life Sciences) were run in the same conditions. Gels were sectioned into 2-mm slices and counted after an overnight incubation with 1 ml of Beckman tissue solubilizer (0.5 N quaternary ammonium hydroxide in toluene) and 10 ml of liquid scintillation fluid (Ready Organic, Beckman). Results for each apo studied were expressed as percent disintegrations per minute per milligram protein to assess the specific effect of PYY (1–36) on apo synthesis. Western Blot To assess the presence of Niemann-Pick C1 like 1 (NPC1L1), Scavenger receptor-class B type I (SR-BI), CD36, ATP binding cassette subfamily (ABCG5, ABCG8), LDL-receptor (LDL-R), HMG-CoA reductase (HMG-CoA-R), Acyl-Coenzyme A:cholesterol acyltransferase (ACAT)-2, and microsomal triglyceride transfer protein (MTP), Caco-2/15 cells were homogenized and prepared for Western blotting. The Bradford assay (Bio-Rad) was used to determine protein concentration. Proteins were denatured in sample buffer containing SDS and β-mercaptoethanol, separated on a 7.5% SDS-PAGE gel, in the presence of a cocktail of a high-range rainbow molecular weight markers (myosin, 220, 000 Da; phosphorylase b, 97,000 Da; BSA, 66,000 Da; ovalbumin, 45,000 Da; carbonic anhydrase 30,000 Da; trypsin inhibitor, 20,100 Da; Lysozyme, 14,300 Da from GE Healthcare) and blotted onto nitrocellulose membranes. Purified apos also served as markers for apo mobility. Nonspecific binding sites of the membranes were blocked with 5% defatted milk proteins. Reactions took place by the addition of primary antibodies directed against targeted proteins: NPC1L1 (1/3000; Novus), SR-BI (1/50000; Novus), CD-36 (1/5000; Santa Cruz), ABCG5 (1/1000; Santa Cruz), ABCG8 (1/500; Novus), LDL-R (1/20000; Fitzgerald), HMG CoA-R (1/10000; Upstate), phosphorylated (P)-HMG CoA-R (1/10000; Millipore), ACAT-2 (1/5000; Cayman), and MTP (1/3000; Provided by Dr. David Gordon, Bristol-Myers-Squibb). Reaction was revealed with species-specific horseradish peroxidise-conjugated secondary antibody (1/20000; Roche Diagnostic, Mannheim) for 1 h at room temperature. β-actin (with Ab dilution of 1/5000) was used as an internal control to confirm equal loading protein on SDS-PAGE. Blots were developed with the chemiluminescent substrate Luminol (Roche) and proteins were quantified by use of a Hewlett-Packard scanner equipped with a transparency adaptor and UN-SCAN-IT (Silk Scientific) software. RT-PCR PCR experiments for the various genes [(liver X receptors (LXRs); retinoid X receptors (RXRs); peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element binding protein-2 (SREBP-2) as well GAPDH (as a housekeeping gene)] were performed by using the mastercycler gradient (Eppendorf). Approximately 30–40 cycles of amplification were used at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. Amplicons were visualized on standard ethidium bromide-stained agarose gels. Importantly, we have established the experimental conditions relative to RT-PCR, which correspond to the linear portion of the exponential phase for every gene expression. Statistical Analysis All values were expressed as the mean ± SD. The data were evaluated by ANOVA where appropriate, and the differences between the means were assessed using the Dunnett’s post test. A P value <0.05 was considered statistically significant. Results PYY and Cell Integrity Before starting the evaluation of PYY effects on lipid homeostasis, it was mandatory to determine that this gastrointestinal peptide does not disturb cell viability and monolayer formation. As assessed by trypan blue exclusion, cell viability was not affected (data not shown). Moreover, measurement of transepithelial resistance, alkaline phosphatase, and sucrase activity did not disclose any significant perturbation in mucosal barrier function and cell differentiation (data not shown). Therefore, it could be concluded that PYY does not exert any cytotoxic effects on Caco-2/15 cells. Since various investigators have reported the regulation of PYY output by lipids, it was important to assess that the cellular model Caco-2/15 also responds to these stimuli. Indeed, PYY was positively stimulated by fatty acids as shown in Figure 1. However, the addition of fatty acids to the basolateral medium resulted in a higher PYY output than fatty acids administered to the apical compartment. 10.1371/journal.pone.0040992.g001Figure 1 Influence of oleic acid on PYY secretion in the apical and basolateral media. Oleic acid at the concentrations of 0.7 mM and 1.4 mM was added either to the apical or basolateral medium. Following the 2-h incubation period, both apical and basolateral media were assessed for their PYY content. Values are expressed as means ± SD for n = 3 separate experiments in each group. CTR, Control; Baso, basolateral; OA, Oleic Acid. Localization of PYY Receptor in Caco-2/15 Cell Line When seeded on porous filters (Transwell), Caco-2/15 cells allow access to both sides of the bipolar intestinal epithelium. Therefore, it is possible to explore the role of PYY in the regulation of lipoprotein secretion as a function of PYY site delivery: apical and basolateral compartments corresponding to intestinal lumen or serosal circulation, respectively. However, it was important to first show whether NPY1R receptor is localized in the apical and the basolateral membrane. Therefore, both membranes were isolated as described in our previous studies [36]–[38]. Careful scrutiny was carried out to assess the purity of the brush-border and basolateral membranes and to exclude the contamination by other organelle membranes. First, brush-border membrane (BBM) purity was determined by assessing the enrichment of specific brush-border markers, such as sucrase/isomaltase. Western blot analysis showed that the protein mass of this enzyme was increased by a factor of 55–62.5 in isolated intestinal BBM in comparison with homogenates of intestinal mucosa or Caco-2/15 cells (data not shown). Second, the typical microvillus carcino-embryonic antigen was enriched 7.0- to 8.2-fold in BBM (data not shown). Third, we excluded contamination by the endoplasmic reticulum and the Golgi by measuring the activity of glucose 6- phosphatase and galactosyltransferase, respectively, which were undetectable in BBM. Forth, Na+/K+-ATPase, a classical basolateral protein, was detectable only in basolateral membranes and not found in BBM (data not shown). Following purification and purity assessment, Western blot analysis, using a specific antibody, indicates the presence of Y1 receptor in both the apical and basolateral membranes (Figure 2). However, the receptor protein mass was preponderant in the basolateral membrane. Given the distribution of the receptor on both sides of the Caco-2/15 cells, we decided to investigate its actions by adding PYY separately to apical and basolateral compartments. 10.1371/journal.pone.0040992.g002Figure 2 Detection of Y1 receptor (NPY1R) in Caco-2/15 cells in culture. Following differentiation, Caco-2/15 cells were homogenized and apical and basolateral membranes were isolated. Aliquots were fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. The blots were then incubated with the polyclonal antibody overnight at 4°C. Immunocomplexes were revealed by means of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin and an enhanced chemiluminescence kit. Y1 receptor mass was quantitated by use of an HP Scanjet scanner equipped with a transparency adapter and software. Values are expressed as means ± SD for n = 3 separate experiments in each group. Effect of PYY on Apical Cholesterol Uptake Following pre-incubation (24 h) of Caco-2/15 cells with medium containing 50 nM and 200 nM of PYY, we determined how the addition of PYY to either the apical or basolateral compartment modifies cholesterol uptake. As illustrated in Figure 3, only the addition of PYY to the apical compartment was able to lower the capacity of Caco-2/15 cells to incorporate cholesterol. 10.1371/journal.pone.0040992.g003Figure 3 Effects of the administration of PYY (1–36) to the apical or basolateral medium on cholesterol uptake in Caco-2/15 cells. Differentiated Caco-2/15 cells were cultured for 24 h in MEM containing 50 nM or 200 nM of PYY in their apical or basolateral medium, in the presence of 100 µM cholesterol in apical (with 250 000 dpm [14C]-cholesterol). Data are reported as % of control values representing 100%. Values represent the mean ± SD for n = 3 separate experiments in each group. * P<0.05 vs. controls, ** P<0.01 vs. controls. Influence of PYY on Cholesterol Protein Transporters The decrease in cholesterol uptake exhibited by Caco-2/15 cells exposed apically to PYY may be due to differences in the expression of cholesterol transporters. To test this hypothesis, the protein expression of cholesterol transporters (NPC1L1, SR-BI and CD36) was examined by Western blot. As shown in Figure 4, the apical addition of PYY at the two concentrations (50 and 200 nM) resulted in a significant decrease in the protein expression of NPC1L1 without any changes in SR-BI and CD36. Besides, no alterations in all these cholesterol transporters were noted when PYY was delivered to the basolateral medium. Since ABCG5 and ABCG8 restrain cholesterol absorption in the lumen of the intestine by excreting absorbed cholesterol, it was mandatory to assess their protein expression in response to PYY administration. Figure 5 shows no alterations in the protein mass of ABCG5 and ABCG8 following PYY inclusion either in the apical or basolateral compartments. 10.1371/journal.pone.0040992.g004Figure 4 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of transporters mediating cholesterol absorption. Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of Figure2. Western blot was used to analyze the protein expression of NPC1L1 (A), SR-BI (B) and CD36 (C). Values are means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. *P<0.01 vs. controls. 10.1371/journal.pone.0040992.g005Figure 5 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of ABCG5 and ABCG8. Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of Figure 2. Western blot was used to analyse the protein expression of ABCG5 (A) and ABCG8 (B). Data represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. Effects of PYY on De novo Cholesterol Synthesis Experiments were performed to examine the regulatory role of PYY on newly intracellular cholesterol synthesis. Caco-2/15 cells were, therefore, incubated with [14C]-acetate for 24 h and the two free and esterified forms of cholesterol were analyzed by TLC. Apical and basolateral administration of PYY at the largest concentration slightly enhanced the synthesis of free and esterified cholesterol (Figure 6). Interestingly, the delivery of PYY to the basolateral compartment was more effective in raising cholesterol formation. 10.1371/journal.pone.0040992.g006Figure 6 Effects of the administration of PYY (1–36) to the apical or basolateral medium on cholesterol synthesis. After 24 h incubation with [14C]-acetate, cells were homogenized. Lipids were extracted in chloroform/methanol and separated by TLC. The free cholesterol (FC) and cholesteryl ester (CE) bands were scraped off the plate and counted. Data represent means ± SD for n = 3 separate experiments in each group. *P<0.05 vs. controls, **P<0.01 vs. controls. PYY and the Key Regulatory Proteins of Cholesterol Metabolism Next, we determined the impact of PYY on the regulatory sterol enzymes: HMG-CoA-R, the rate-limiting step in cholesterol synthesis, and ACAT-2, an integral protein present in the rough endoplasmic reticulum that catalyzes the formation of cholesteryl ester from free cholesterol. HMG-CoA-R protein expression increases with only the addition of 200 nM PYY to the basolateral medium (Figure 7A). Moreover, the same concentration of PYY at the same delivery site displayed a trend of decrease in HMG-CoA-R phosphorylation, which reflects an activation of the enzyme (Figure 7B). Confirmatory data were obtained when the P-HMG-CoA reductase/HMG-CoA reductase ratio was calculated (Figure 7C). As to ACAT-2, while the apical addition of PYY remained without effect on its protein mass, a significant decrease was noted with in the protein expression with the highest PYY concentration administered in the basolateral compartment (Figure 8). Finally, we tested the effects of LDLR and could not find a significant impact of PYY administration to the apical and basolateral media (Figure 9). 10.1371/journal.pone.0040992.g007Figure 7 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of HMG-CoA-R (A) and phosphorylated HMG-CoA-R (B). Caco-2/15 cells were cultured for 24 h in MEM as described in Figure 2. Western blot was used to analyze the protein expression and phosphorylation of HMG-CoA-R. Values are means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. *P<0.05 vs. controls, ** P<0.01 vs. controls. 10.1371/journal.pone.0040992.g008Figure 8 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of ACAT-2. Caco-2/15 cell line was cultured for 24 h in MEM described in Figure 2. Western blot was used to analyze the protein expression of ACAT-2. Values are means ± SD for n = 3 separate experiments in each group and are reported as percent. *P<0.01 vs. controls. 10.1371/journal.pone.0040992.g009Figure 9 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of LDL-receptor (LDL-R). Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of Figure 2. Western blot was used to analyse the protein expression of LDL-R. Data represented means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. PYY and Lipoprotein Production The ability of Caco-2/15 cells to assemble and secrete lipoproteins was assessed as a function of PYY concentrations. Figure 10 shows little effect of PYY on the production of lipoproteins. Nevertheless, a small decrease in CM output was noted with the addition of 200 nM to the basolateral medium (Figure 10A). The same trend was observed in LDL with the administration of 200 nM to the apical medium (Figure 10C). However, no alterations were noted in VLDL (Figure 10B) and HDL (Figure 10D). 10.1371/journal.pone.0040992.g010Figure 10 Effects of the administration of PYY (1–36) to the apical or basolateral medium on lipoproteins output. Differentiated Caco-2/15 cells were cultured for 24 h in MEM containing 50 nM or 200 nM of PYY in their apical or basolateral medium, in the presence of [14C]-oleic acid for 24 h. Thereafter, the media were ultracentrifuged to isolate lipoproteins at their specific densities. Radioactivity incorporated into each fractions was further determined. Data were analyzed as dpm/mg of total protein but were reported as percent difference relative to control. Data represent means ± SD for n = 3 separate experiments in each group. * P<0.05 vs. controls. CM; Chylomicrons (A), VLDL; Very-low density lipoprotein (B), LDL; low density lipoprotein (C) and HDL; high density lipoprotein (D). PYY and Apolipoprotein Biosynthesis In attempt to explore whether PYY influences the synthesis and secretion of apos, the gastrointestinal peptide was added either to the apical or basolateral compartment in combination of [35S]-methionine as a precursor of peptide elongation. Only the biogenesis of apo A-I was increased with the apical addition of 50 nM PYY. On the other hand, the addition of PYY to the basolateral compartment was more effective in reducing the synthesis of apos B-48 and B-100 (Figure 11). 10.1371/journal.pone.0040992.g011Figure 11 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the synthesis of apolipoproteins (apo). Epithelial cells at 14 days post-confluence were incubated with [35S]-methionine in the presence of PYY (1–36) (50 or 200 nM) and unlabeled oleic acid for 24 h to stimulate apo biogenesis. At the end of the labelling period, cells were washed, homogenized, and centrifuged. Supernatants from the cell homogenates were then reacted with excess antibodies for 18 h at 4°C to precipitate specific apos. Immune complexes were washed and analyzed by linear 4–15% SDS-PAGE. After electrophoresis, gels were sliced and counted for radioactivity. Data represent means ± SD for n = 3 separate experiments in each group. Values are reported as % of control values representing 100%. *P<0.05 vs. controls. PYY and Microsomal Trigyceride Transfer Protein MTP has been identified as a crucial protein for intracellular apo B lipoprotein assembly. We have, therefore, determined the influence of PYY on its protein expression. No significant changes were noticed in the protein mass assessed by Western blot between controls and cells treated with PYY(1–36). However, there was a divergence between apical and basolateral compartments (Figure 12). 10.1371/journal.pone.0040992.g012Figure 12 Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of MTP. Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of Figure 2. Western blot was used to analyse the protein expression of MTP. Data represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. * P<0.05 vs. 50 nM or 200 nM in apical side. PYY and Transcription Factors To approach the mechanisms triggered by PYY, we assessed the gene expression of several factors that affect the transcription of a variety of genes associated with lipid and cholesterol metabolism, including RXR(α, β), liver LXR(α, β), PPAR(α, β, γ), and SREBP-2. Data on Figures 13 and 14 illustrate how PYY at the concentrations of 50 and 200 nM impacted on the expression of the different nuclear and transcription factors in Caco-2/15 cells. It did not cause any significant variation on the mRNA levels of RXRβ (Figure 13B), LXRα (Figure 13C), LXRβ (Figure 13D), and PPARγ (Figure 14C), whereas it produced a significant reduction in gene expression of RXRα (Figure 13A), PPARα (Figure 14A) and PPARβ (Figure 14B) in Caco-2/15 cells incubated with 200 nM PYY in basolateral medium. Finally, when we explored the effect of PYY on SREBP-2 gene expression, we detected a significant enhancement upon exposure to 50 nM or 200 nM PYY in apical compartment (Figure 14D). 10.1371/journal.pone.0040992.g013Figure 13 Effects of the administration of PYY (1–36) to the apical or basolateral medium on RXR and LXR gene expression in Caco-2/15 cells. PCR analysis was performed on Caco-2/15 cell line at 14 days post-confluence to analyze mRNA of RXRα (A), RXRβ (B), LXRα (C), LXRβ (D). Values represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%. *P<0.05 vs. controls, **P<0.01 vs. controls. 10.1371/journal.pone.0040992.g014Figure 14 Effects of the administration of PYY (1–36) to the apical or basolateral medium on PPAR and SREBP-2 gene expression in Caco-2/15 cells. PCR analysis was performed on Caco-2/15 cell line at 14 days post-confluence to analyze mRNA of PPARα (A), PPARβ (B), PPARγ (C), SREBP-2 (D). Values represent means ± SD for n = 3 separate experiments in each group and are reported as percent difference relative to control. * P<0.05 vs. controls, **P<0.01 vs. controls. Discussion PYY exerts anorexigenic effects via satiety signalling actions in the brain and the periphery. It is known that PYY regulates intestinal motility [39], [40] and prolonged gastric emptying [41]. Surprisingly, little information is available, to our knowledge, on the role of PYY in intestinal cholesterol uptake, synthesis and transport by lipoproteins in the small intestine. For the first time, our work has attempted to unravel the role of PYY in cellular cholesterol metabolism. Our data indicate that PYY could decrease cholesterol uptake by reducing the protein mass of NPC1L1, an essential protein for dietary cholesterol absorption. PYY also elicited cholesterogenesis by translational regulation of HMG-CoA-R. Small negative effects of PYY were noted on CM and LDL along with alterations of certain types of apo. Finally, PYY was able to regulate specific transcription factors implicated in the regulation of lipid and cholesterol metabolism. Importantly, the effects of PYY were noted to depend on two factors in intestinal cells: the PYY site of administration (apical vs. basolateral) and PYY concentrations (50 vs. 200 nM). 10.1371/journal.pone.0040992.g015Figure 15 Integrative scheme documenting the asymmetrical regulation of lipid transport by apical and basolateral PYY stimuli using differentiated, polarized Caco-2/15 cells. Influences of apical and basolateral domains of the enterocyte, which correspond to the intestinal lumen and the serosal circulation, respectively, are actually limited. Indeed, there are only few studies dealing with the stimuli, including PYY, originating from apical and basolateral compartments. Some investigators have also detected distinct effects on various targets depending on the stimulus location. For example, addition of transforming growth factor beta (TGF-β) to the basolateral medium resulted in phosphorylation of the intracellular protein signalling Smad2, whereas no phosphorylation was observed when TGF-β was added to the apical chamber [42]. According to the authors, there are elements of communication between epithelial and mesenchymal in polarized epithelia, which allow vectorial signalling. Furthermore, intestinal epithelial cells were capable of organizing their response to inflammatory signals and producing inflammatory mediators in a bidirectional, vectorial fashion [43]. Finally, we have recently shown that there is a discrete regulation of SR-BI from stimuli, originating from apical and basolateral media, such as n−3 and n−6 fatty acids, fibrate, cholesterol, 7-ketocholesterol, methyl β-cyclodextrin, lipopolysaccharide, tumor necrosis factor-α, interferon γ, insulin, growth hormone and epidermal growth factor [44]. It is the first time that vectorial response in link with PYY location has been treated. Interestingly, we have added new studies evaluating how FAs regulate the secretion of PYY from the apical and basolateral media (Figure 1). Our experiments showed that oleic acid at the concentration of (1.4 mM), administred to the basolateral medium, upregulated PYY output from the basolateral pole. This increased output was higher than that caused by the stimulation of apical PYY secretion, by oleic acid at the same concentration. Interestingly, these observations are in line with data from Figure 2 illustrating the preponderance of receptor protein mass in the basolateral membrane. To evaluate the involvement of PYY in cholesterol homeostasis, 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 [21], [29], [30], [32]–[35], [38] and from other groups have shown that Caco-2/15 monolayers are fully appropriate for the study of lipid/lipoprotein homeostasis. Importantly, when seeded on porous filters (Transwell), Caco-2/15 cells allow access to both sides of the bipolar intestinal epithelium. Therefore, we were able in the present work to explore the regulation of cholesterol metabolism as a function of PYY site delivery: apical and basolateral compartments corresponding to intestinal lumen or serosal circulation, respectively. As a prerequisite to our study, it was necessary for us to demonstrate the presence of PYY receptor in Caco-2/15 cells and its distribution on both sites (apical and basolateral membrane), thus favouring binding of the ligand (PYY) and its internalization [45]. Importantly, L cells in the intestinal epithelium secrete PYY (1–36) into the lumen of the intestine across the apical membrane and the blood circulation via the basolateral membrane [22]–[24]. The presence of PYY in the apical and basolateral membranes may be dependent on the type of nutrient ingested and may serve to modulate the diverse physiologic roles of PYY. Moreover, the concentrations of PYY are variable in the circulation depending on the physiological states: they are low during fasting and significantly increase after meal consumption, reaching a plateau following 2 h post-prandially. In the current research, we used human PYY(1–36) instead of PYY(3–36), since previous reports have shown the stimulation of apo A-IV by PYY(1–36) in Caco-2/15 cells [46]. According to the authors of this paper, only Y1 receptor mediated this stimulatory effect following binding with the PYY (1–36) ligand given that the other Y2 and Y5 receptors for PYY (3–36) seemed not operational in view of their undetectable mRNAs in Caco-2/15 cells. In these studies, the concentrations of PYY reached 200 nM in order to obtain the stimulation of Apo A-IV. Additionally, the PYY (1–36) was repeatedly used in studies associated with lipid metabolism in the intestine [12], [15], [47]. Finally, in terms of cell differentiation, maintenance of the intestinal epithelium and the regulation of absorption in the intestine, investigators have largely turned to the PYY (1–36), which has a high affinity for the receptor NPY1R [48]. Accordingly, the presence of the receptor NPY1R has already been noted in previous studies using intestinal Caco-2/15 cells. Taking into account the secretion of PYY from both apical and basolateral compartments of the enterocytes [22], [49], it appeared important for us to determine the status of this receptor both in apical and basolateral sides. In the present work, PYY was used in the 50–200 nM range, since numerous investigators reported plasma concentrations of PYY in the range of pM to nM [50]. But these concentrations could considerably be augmented following exercise and meal tests [51], [52]. Additionally, levels of PYY varied according to the intestinal region and reached values of ∼1000 pmol/g colonic or rectal tissue [53], [54]. Therefore, given these collective data, we do not think that PYY was employed in high pharmacological doses in our studies. NPC1L1 has been identified at the surface of plasma membrane as a critical protein in the exogenous cholesterol absorption [55]. It transports cholesterol from the lumen into enterocytes where intestinal ACAT-2 then converts cholesterol to CE that is packed into CM. SR-BI and CD36 are also located on the brush border membrane and can contribute to the absorption of cholesterol. Our experiments showed an inhibition of cholesterol uptake with the addition of PYY to the apical medium, which was confirmed by NPC1L1 downregulation, but SR-BI and CD36 remained unaffected. To approach the underlying mechanisms, we analyzed the gene expression of specific transcription factors involved in cholesterol homeostasis. We first examined PPARs that represent a subgroup of the nuclear receptor superfamily, including PPAR(α, β, γ) heterodimerizing with the RXR and, upon ligand binding, become transcriptionally active and control a series of genes of lipid and energy metabolism. Even if previous studies showed that specific activation of PPARα and PPARβ decreases cholesterol absorption via an inhibitory effect on NPC1L1 expression in the proximal small intestine [56], , no significant differences were noted, in our study, in the gene expression of apical PPARα and PPARγ in contrast with a substantial decrease in PPARβ mRNA. These findings are not consistent with an implication of PPARs in PYY-mediated NPC1L1 downregulation. We then turned to LXRs that act as oxysterol sensors of intracellular cholesterol homeostasis [58]. LXRs form obligate heterodimers with RXRs, which activate their target genes by binding to specific response elements (LXREs). Recently, it has been revealed that LXRs seem to govern dietary cholesterol fate in the enterocyte by downregulating apical absorption through NPC1L1 [59]. Our data displaying no changes in LXRα,β and decreased RXRα are not lining up with a possible involvement of these transcription factors in PYY-mediated NPC1L1 downregulation. Finally, the upregulation of SREBP-2 gene expression in response of PYY could not explain the reduction in cholesterol transport and in NPC1L1 protein abundance. An important direction to follow in next studies in order to detect the mechanisms of action of PYY will consist in investigating the formation of NPC1L1–flotillins-positive cholesterol-enriched membrane microdomains. In fact, NPC1L1 performs the task of apical cholesterol uptake by collaborating with flotillins to form cholesterol-enriched membrane microdomains. Internalization of these membrane microdomains brings a large amount of cholesterol into the cells which might be a mechanism accounting for high efficiency of cholesterol absorption [60]. In our studies, HMG-CoA R expression was found to be 3-fold upregulated, but the cholesterol synthesized was only increased 1.2 fold in response to the PPY. In fact, HMG-CoAR can be regulated through alterations of its quantity and/or of its catalytic efficiency. In particular, mechanisms implicated in the regulation of HMG-CoAR include the manipulation of enzyme quantity through transcriptional [61], [62] and post-transcriptional processes [63], [64] and enzyme degradation [65], [66], as well as the alteration of enzyme catalytic activity by membrane composition and fluidity [67], by thiols [68], by microtubules [69], or by cytosolic lipid inhibitors and their binding proteins [70]. It is, therefore, possible that despite the increase in HMG-CoA-R mass, PYY may elicit another regulatory mechanism, e.g. the reversible inactivation and reactivation of HMG-CoA-R via its covalent phosphorylation and dephosphorylation. Interestingly, the addition of PYY to the basolateral medium resulted in a significant decrease in the biogenesis of apo B-48, an essential protein in the assembly of TG-rich lipoprotein. A slight, but significant, decrease was also observed in CM output. It is generally accepted that the intracellular mechanism of TG-rich lipoprotein assembly requires apo B synthesis and association. An early step in this process is the cotranslational lipidation of apo B that is transiently bound to the endoplasmic reticulum membrane where it is folded. The addition of lipids stabilizes apo B-48 and prevents its proteolytic degradation via the ubiquitin-dependent proteasomal pathway [71], [72]. It is therefore reasonable to suggest that the defective synthesis of CM in Caco-2/15 cells, supplemented in their basolateral medium with PYY, is due to limited apo B-48 protection from misfolding and degradation despite MTP protein expression enhancement, which does not reflect MTP activity or alternatively could not compensate for the marked apo B-48 proteolytic degradation by the proteasome. Finally, although the decrease of CM appears modest despite the statistical significance, it is reasonable to propose that PYY can affect the intestinal transport of lipids given the significant length of the intestine and especially the perpetual postprandial state characterizing nowadays the Western population, which is actually subjected to abundant and frequent feeding. This study shows interesting results in relation with the influence of PYY on intestinal cholesterol metabolism. Previous reports documented that binding of PYY to its receptors resulted in inhibition of electrolyte secretion [49], [73] via inhibition of cAMP production [74] suggesting that PYY was likely to exert its effect through an inhibition of adenylyl cyclase. At this time, we have no evidence as to the role of cAMP in PYY-mediated regulation of cholesterol metabolism. However, the present data point out that PYY in the apical compartment can essentially decrease cholesterol uptake through the downregulation of NPC1L1, while basolateral PYY can reduce the output of CMs via a decrease in apo B-48 biosynthesis. More in vivo and in vitro studies are absolutely required to determine the specific endocrine and paracrine mechanisms triggered by PYY for the regulation of the transport of various nutrients. Although the PYY receptor is more concentrated on the basolateral membrane vs. the apical membrane, only the administration of PYY apically was able to decrease cholesterol uptake. This intriguing finding may be explained by distinct intracellular signalling pathways or discrete regional regulation at the two intestinal poles, triggered by the two PYY receptors located on basolateral and apical membranes. In previous studies, fasting PYY correlated negatively with plasma total cholesterol, LDL-cholesterol and HDL-cholesterol in response to bariatric surgery in severely obese patients [19] and overfeeding in normal weight subjects [20]. Our data indicate for the first time that PYY may act on the intestine to down-regulate cholesterol absorption, which may influence cardiovascular risk factors. Although most early studies so far have been concerned by the concept that factors secreted from the gut endocrine system participate in the regulation of gastrointestinal functions, growing evidence underlines their functions in metabolic pathways and in the pathophysiology of various metabolic diseases, including obesity, insulin resistance and diabetes, which lead to cardiovascular diseases. Indeed, a great number of gastrointestinal peptides, including PYY affect numerous organs while modulating energy storage, lipolysis, body weight, appetite, satiety, β-cell preservation and glucose metabolism [75]. Recently, it has been shown that the gut plays a major role in glucose homeostasis via the regulation of both insulin secretion and sensitivity [76]–[78], while bariatric surgery likely influences several GI pathways in complementary ways to improve glucose control and diabetes. Accordingly, the selective activation of the neuropeptide Y receptor by PYY (3–36) suppresses appetite, reduces acute food intake, causes body weight loss and provides a promising approach to obesity management. Therefore, the control of receptor Y by PYY becomes an attractive mechanism for the therapeutic management of obesity and its associated morbidities such as insulin resistance and diabetic dyslipidemia. Limited studies are available on the effects of PYY on cholesterol metabolism, but indirect evidence points to this important issue. As an example, two obese women groups differed from lean controls by showing lower plasma PYY (3–36) both under basal conditions and after meal, and this was accompanied by higher postprandial blood glucose and insulin levels, as well as total cholesterol and LDL-cholesterol [79]. Furthermore, with the sustained rise in GLP-1 and PYY levels, there was a decline in glucose, insulin, total cholesterol, LDL-cholesterol, and triglyceride levels [80]. In another investigation, long-term exercise training displayed beneficial effects for overweight adolescents with respect to the increase in PYY, decrease in TG and lowering of total cholesterol and LDL-cholesterol although the changes of the cholesterol variables did not reach statistical significance [81]. In another study, overfeeding significantly raised fasting PYY, which was negatively correlated with the changes of total cholesterol, HDL and LDL while being positively associated with HDL cholesterol [20]. Altogether, the information obtained from these research groups indicates an indirect link between PYY and lipid (cholesterol and triglyceride) metabolism. As to the relationship between PYY and cardiovascular diseases, various investigators reported that the beneficial effects of dietary feeding (e.g. diminution of postprandial glycemia, lipidemia and insulinemia along with the reduction of cardiovascular disease risks) could be due to its actions on the levels of PYY among many peptides [82]. In addition, Hanusch-Enserer et al. concluded that, in restrictive bariatric surgery, PYY correlates with major cardiovascular risk factor and surrogate parameters of insulin secretion [19]. Finally, according to Zwirska-Korczala et al., down-regulation of PYY secretion may lead to progression of endothelial dysfunction and may promote acceleration of atherosclerosis [51]. Nevertheless, additional studies are needed to scrutinize this important aspect and to evaluate cause-effect evidence of PYY and cardiovascular risk factors. However, it is important to note that, following binding with PYY, Y1 receptors rapidly internalize through clathrin-coated pits and recycle back to the plasma membrane [83], [84]. It remains unknown whether these internalized receptors enter a recycling pathway leading to relocalization at the cell surface. In summary, our data suggest that PYY may exert an impact on intracellular lipid metabolism depending on the route of administration as well illustrated in Figure 15. Although it is known that, following binding with PYY, Y1 receptors rapidly internalize through clathrin-coated pits and recycle back to the plasma membrane, no information is available to indicate whether internalized PYY may be transferred from one membrane to another, which may influence the effects of PYY on one specific pole of the cell. Further investigations are required to explore this exciting issue. For now, our efforts at least highlighted a segregation in the function of PYY in the two cell poles: (i) at the apical compartment, it decreased LDL secretion and lowered cholesterol uptake via the down-regulation of NPC1L1 transporter, while it enhanced certain types of apos and cholesterogenesis; and (ii) at the basolateral compartment, it disclosed ability to augment cholesterol synthesis and to lower chylomicron output through the lowering of apos and transcriptional factors. More work is necessary to further establish the role and mechanisms action of PYY in lipid transport in the enterocytes. The authors thank Mrs Shohraya Spahis for her technical assistance. Competing Interests: The authors have declared that no competing interests exist. Funding: The current work was supported by research grants from the JA deSève Research Chair in nutrition (EL), Natural Sciences and Engineering Research Council of Canada (EL), Diabète Québec (ED), and the scholarship award from the “Fonds de la Recherche en Santé du Québec” (EG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Arantes RM Nogueira AM 1997 Distribution of enteroglucagon- and peptide YY-immunoreactive cells in the intestinal mucosa of germ-free and conventional mice. Cell Tissue Res 290 61 69 9377643 2 Bottcher G Sjolund K Ekblad E Hakanson R Schwartz TW 1984 Coexistence of peptide YY and glicentin immunoreactivity in endocrine cells of the gut. Regul Pept 8 261 266 6548568 3 Neary NM Small CJ Druce MR Park AJ Ellis SM 2005 Peptide YY3-36 and glucagon-like peptide-17-36 inhibit food intake additively. 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PLoS One. 2012 Jul 23; 7(7):e40992
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22848667PONE-D-12-1153510.1371/journal.pone.0041940Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyCytokinesImmunologyImmunityAdaptive ImmunityHumoral ImmunityInnate ImmunityMedicineClinical ImmunologyImmunityAdaptive ImmunityHumoral ImmunityInnate ImmunityIL-21 Regulates the Differentiation of a Human γδ T Cell Subset Equipped with B Cell Helper Activity IL-21 Sustains Vγ9Vδ2 TFH CellsCaccamo Nadia 1 Todaro Matilde 2 La Manna Marco P. 1 Sireci Guido 1 Stassi Giorgio 2 Dieli Francesco 1 * 1 Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Università degli Studi di Palermo, Palermo, Italy 2 Dipartimento di Discipline Chirurgiche ed Oncologiche, Università degli Studi di Palermo, Palermo, Italy Ryffel Bernhard EditorFrench National Centre for Scientific Research, France* E-mail: [email protected] and designed the experiments: FD NC G. Stassi. Performed the experiments: MPL G. Sireci MT. Analyzed the data: FD G. Stassi. Wrote the paper: FD G. Stassi. 2012 25 7 2012 7 7 e4194013 4 2012 27 6 2012 Caccamo et al.2012This 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.Vγ9Vδ2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and display pleiotropic features. Here we report that coculture of Vγ9Vδ2 cells with phosphoantigen and IL-21 leads to selective expression of the transcription repressor Bcl-6 and polarization toward a lymphocyte subset displaying features of follicular B-helper T (TFH) cells. TFH-like Vγ9Vδ2 cells have a predominant central memory (CD27+CD45RA−) phenotype and express ICOS, CD40L and CXCR5. Upon antigen activation, they secrete IL-4, IL-10 and CXCL13, and provide B-cell help for antibody production in vitro. Our findings delineate a subset of human Vγ9Vδ2 lymphocytes, which, upon interaction with IL-21-producing CD4 TFH cells and B cells in secondary lymphoid organs, is implicated in the production of high affinity antibodies against microbial pathogens. ==== Body Introduction Development of efficient humoral immune response results in the production of a high-affinity antibodies that are essential for the clearance of many infectious pathogens. Generation of these protective antibodies takes place in specialized structures in secondary lymphoid organs, known as germinal centers (GCs), and requires the combination of diverse events such as isotype-switch, somatic hypermutation and affinity maturation, which occur upon interaction between activated B lymphocytes and CD4 follicular helper T (TFH) lymphocytes [1], [2]. TFH cells are defined by follicular localization and high expression of specific markers [3]–[7]: CXCR5 that drives TFH cells to migrate into the B cell follicles; the inhibitory receptor PD-1 and the costimulatory molecule ICOS, which interact with their corresponding ligands on B lymphocytes; the signature cytokine IL-21, which predominantly acts as a paracrine factor for GC B lymphocytes, but has only limited autocrine function as regulator of TFH lineage fate. The transcriptional repressor Bcl-6 is a crucial intrinsic regulator of TFH lineage commitment, but the differentiation pathway from naive CD4 to TFH cells is the subject of intense studies. Despite CD4 TFH cells, other T cell subsets including CD8 T cells, NKT cells and γδ T cells are capable to provide B cell help and as such contribute to the outcome of antibody response [8]–[10]. Early studies in αβ T cell–deficient mice demonstrated a nonredundant role for γδ T cells in the generation of antimicrobial antibodies [11], [12] and autoantibodies [13]–[15]. However, the finding that γδ T cell-deficient mice do not show marked defects in IgM and IgG production, suggested that γδ T cells may have a modulatory, rather then a primary function in the control of humoral immunity. Antibody production was also increased in in vitro cultures of human γδ T cells with B cells [16], [17], but the amount of secreted antibody was low and the mechanisms underlying the observed B-cell help were not examined. More recent studies have shown that human γδ T cells are found in secondary lymphoid tissues [18], [19], where they are scattered throughout the T zone and clustered within follicles [20], express costimulatory molecules after TCR-triggering and provide B-cell help in vitro, suggesting their participation in humoral immunity [20]. The majority of human peripheral blood γδ T cells, express a TCR consisting of the Vγ9 and the Vδ2 chains (here and thereafter referred to as Vγ9Vδ2 cells) and recognize nonpeptidic phosphorylated metabolites of isoprenoid biosynthesis produced by microorganisms and stressed cells [21]–[23]. Upon activation, Vγ9Vδ2 cells can be skewed toward distinct effector functions depending on polarizing cytokines, in analogy to CD4 helper T cells [24]–[26]. Accordingly, under appropriate culture conditions, Vγ9Vδ2 cells divert from the typical Th1-like phenotype and polarize to Th2 [26], [27], Th17 [28], [29] and Treg cells [30]. Such a broad plasticity emphasizes the capacity of Vγ9Vδ2 cells to influence the nature of immune response to different challenges. We and others have shown that antigen-stimulated Vγ9Vδ2 cells acquire TFH-associated features (ICOS, CD40L and CXCR5 surface expression, IL-21R mRNA expression, IL-4 and IL-10 secretion) and provide B-cell help for antibody production [30], [25], [31]; moreover, a recent study by Bansal et al. [32], reported that Vγ9Vδ2 T cells stimulated with the phosphoantigen HMB-PP in the presence of IL-21, express markers associated with TFH cells and support antibody production by B cells, clearly pointing to IL-21 as the key cytokine for differentiation of this TFH-like Vγ9Vδ2 cell subset. Yet no data are available on the relative role of antigen and cytokines for the regulation of lineage-specifying factors required for the differentiation of TFH Vγ9Vδ2 cells. We show here that in human Vγ9Vδ2 cells, Bcl-6 expression and polarization towards TFH cells are efficiently induced by coordinated TCR triggering and IL-21. Moreover, we provide detailed phenotypic and functional analysis of TFH-like Vγ9Vδ2 cells, and in agreement with the study of Bansal et al. [32], suggest that the interaction between Vγ9Vδ2 TFH cells, CD4 TFH cells and B cells in reactive secondary lymphoid tissues may profoundly impact on the production of high affinity antibodies against microbial pathogens. Methods Subjects Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy volounteers by density gradient centrifugation using Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). PBMC and mononuclear cells were also isolated from fresh tonsils of patients undergoing tonsillectomy. According to Italian rules (art. 13, DLgs n. 196/03), this study did not require authorisation by the local ethical committee. The study was performed in accordance to the principles of the Helsinki declaration and all individuals gave written informed consent to participate. Cell Purification and Culture Peripheral blood CD14+ monocytes and Vγ9Vδ2 cells were isolated by positive selection with CD14- and Vδ2-specific microbeads, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). Dendritic cells (DCs) were obtained from sorted CD14+ monocytes after culture for 5–6 days in the presence of 25 ng/ml GM-CSF and 1000 U/ml IL-4 (both from Euroclone, Milan, Italy). Subsets of Vγ9Vδ2 cells were isolated to over 99% purity of total Vγ9Vδ2 T cells, after staining with phycoerythrin (PE)-conjugated anti-CD27 (1A4, BD Biosciences, San Josè, CA) and allophycocyanin (APC)-conjugated anti-CD45RA (M-T271, BD Biosciences) monoclonal antibodies (mAbs), followed by cell sorting with a FACSAria (BD Biosciences). Sorted Vγ9Vδ2 T cell subsets were labeled with CFSE (Molecular Probes, Eugene, OR) and were cultured in RPMI-1640 medium (Euroclone, Milan, Italy) supplemented with 2 mM L-glutamine, 20 nM Hepes buffer, 10 µg/ml gentamycin, 100 U/ml penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO) and 10% pooled human AB+ serum (kindly provided by the Blood Bank of the University Hospital, Palermo), and 5×104 cells were cultured in U-bottom 96-well plates, with an equal number of irradiated (30 Gy from a cesium source) DCs and isopentenyl pyrophosphate (IPP; Sigma-Aldrich; 10−5 M final concentration). After 48 hrs, recombinant human cytokines were added to cultures: IL-2 (Novartis Pharma; 50 IU/ml final concentration), or recombinant IL-15 (10 ng final concentration, R&D Systems), or recombinant IL-21 (100 ng/ml final concentration, eBioscience through Prodotti Gianni, Mlan, Italy). Every three days, half of the medium was removed and replaced with fresh medium containing the recombinant cytokine. The cells were harvested, following 9–12 days of culture. FACS Staining and Sorting The following conjugated antibodies were used in different combinations: anti-Vδ2 (B6, BD Biosciences), anti-Vγ9 (B3, BD Biosciences), anti-CD27 (1A4, BD Biosciences), anti-CD45RA (M-T271, BD Biosciences), anti-CD25 (M-A251, BD Pharmingen), anti-CCR7 (a gift of Dr. M. Lipp, Max-Delbruch-Center for Molecular Medicine, Berlin, Germany), anti-HLA-DR monomorphic (a gift of Prof. V. Horejsi, Institute of Molecular Genetics, Academy of Science of the Czech Republic, Prague), anti-ICOS (a generous gift of Dr. R.A. Kroczek), anti-CD40L (TRAP1, BD Biosciences), anti-CCR3 (61828.111, R&D Systems), anti-CCR4 (1G1, BD Biosciences), anti-CCR5 (2D7, BD Bioscences), anti-CCR6 (11A9, BD Biosciences), anti-CXCR3 (1C6/CXCR3, BD Biosciences), anti-CXCR5 (a gift of Dr. R. Keoczek, Molecular Immunology, Robert Koch Institute, Berlin, Germany) and isotype control mAbs. Vγ9Vδ2 cells were incubated in U-bottom 96-well plates with labelled mAbs in PBS containing 1% FCS, for 30 min at 4°C according to manufacturers’ recommendations, washed, and analyzed by flow cytometry on an FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). Viable cells were gated by forward and side scatter, and the analysis was performed on 100,000 acquired events for each sample. Proliferation, Cytokine Analysis and Chemotaxis Assay Proliferation of primed Vγ9Vδ2 cells was assessed 72 hours after stimulation of cells (105/ml) with IPP (10−5 M final concentration) and irradiated DCs and measured by CFSE dilution. The cytokine-producing capacity of primed Vγ9Vδ2 cells was assessed by stimulation of cells (105/ml) for 24 hrs with IPP (10−5 M final concentration) and irradiated DCs. Cytokines (TNF-α, IFN-γ, IL-2, IL-4, IL-10 and IL-17) and chemokine (CXCL13) in culture supernatants were measured by ELISA, according to the manufacturer’s instructions (R&D Systems). Intracellular staining for IFN-γ, IL-4, IL-10 and IL-17 was done on Vγ9Vδ2 cells stimulated for 6 hrs with IPP (10−5 M final concentration) in the presence of GolgiStop (BD Biosciences) for the final 4 hrs of culture. Cells were fixed and made permeable with BD Cytofix/Cytoperm Plus (BD Biosciences) according to the manufacturer’s instructions. Cells were incubated with fluorescein isothiocyanate (FITC)-labeled anti-IFN-γ mAb (B27, BD Biosciences), PE-labelled anti-IL-4 mAb (8D4-8, BD Biosciences), PE labeled anti-IL-10 mAb (JES5-16E3, BD Biosciences) and APC-labeled anti-IL-17 mAb (eBIO64-DEC17, eBioscience), or isotype-control mAbs. Cells were washed and data were acquired on a FACSCalibur or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star). The chemotactic ability of IL-21-primed Vγ9Vδ2 cells was assayed using a double-chamber system with 3-µm pores (Transwell, Costar), according to [31]. Briefly, 105 Vγ9Vδ2 cells were added to the upper chamber and recombinant human CXCL13 (R&D Systems, 3 µM final concentration) to the lower chamber and incubated at 37°C for 2 h in a 5% CO2 humidified incubator. In some experiments, anti-CXCR5 or isotype control mAbs were added to the lower chamber during the test. Assays were performed in triplicate. Afterward, the membrane was removed, washed on the upper side with PBS, fixed, and stained. Migrated cells were counted microscopically at x1000 magnification in five randomly selected fields per well. Percentage migration was calculated by measuring the counts recovered from the lower chamber and comparing them to the total input counts. Results represent the mean ± SD of three independent experiments. Antibody Production in vitro Vγ9Vδ2 T cell help in antibody production was studied as follows. IL-21-primed Vγ9Vδ2 T cells were co-cultured with sorted tonsillar B cells in 96-well plates at 105 cells/well each of T and B cells in the presence or absence of IPP, in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (Euroclone), 2 mM L-glutamine, 20 nM HEPES and 100 U/ml penicillin/streptomycin. 10 days later IgM, IgG, and IgA levels in the culture supernatants were determined by ELISA. Real-time Quantitative RT-PCR Total RNA was extracted with the ABI PRISM 6100 Nucleic Acid PrepStation (Perkin-Elmer Applied Biosystems) according to the manufacturer’s instructions. Random hexamers and an MMLV Reverse Transcriptase kit (Stratagene, La Jolla, CA) were used for cDNA synthesis. Transcripts were quantified by real-time quantitative PCR on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems) with Applied Biosystems predesigned TaqMan Gene Expression Assays and reagents according to the manufacturer’ s instructions. The following probes were used (identified by Applied Biosystems assay identification number): RORC, Hs01076112_m1; TBX21, Hs00203436_m1; BCL6, Hs00277037_m1; GATA3, Hs00231122_m1; IL21R, Hs00222310_m1; IFNG, HS00174143_m1; IL-4, HS00174122_m1; IL-10, HS00174086_m1; IL-13, HS00174379_m1; IL-21, Hs00222327_m1. For each sample, mRNA abundance was normalized to the amount of 18S rRNA. Statistics A standard two-tailed t-test or a t-test with Welch’s correction was used for statistical analysis. P values of <0.05 were considered significant. Results Factors Inducing the Differentiation of Vγ9Vδ2 TFH Cells It has been previously shown that antigen-stimulated Vγ9Vδ2 cells acquire TFH-associated features (ICOS, CD40L and CXCR5 surface expression, IL-4 and IL-10 secretion and B-cell help for antibody production) [20], [25], [31], including expression of IL-21R mRNA [25], yet no data are available on the antigen and cytokine requirements for the differentiation of this subset of TFH Vγ9Vδ2 cells. To identify conditions that permit the polarization of human TFH Vγ9Vδ2 cells, we stimulated highly purified subsets of naive (Tnaive, CD45RA+CD27+), central memory (TCM, CD45RA−CD27+), effector memory (TEM, CD45RA−CD27−) and terminally-differentiated effector memory (TEMRA, CD45RA+CD27−) Vγ9Vδ2 cells with irradiated autologous DCs and antigen (IPP), together with different cytokines (see Materials and Methods for details), and analyzed their surface marker expression. Primarily, we used ICOS expression as a signature of TFH Vγ9Vδ2 cells because (a) expression of high levels of ICOS is a defining feature of CD4 TFH cells [33], (b) ICOS is not expressed by resting Vγ9Vδ2 cells and (c) the ability of CD4 [34] and Vγ9Vδ2 [31] TFH cells to help B cells is mediated by ICOS. In the absence of exogenous cytokines, only a small percentage (2% or less) of antigen-primed Vγ9Vδ2 cells expressed ICOS. Addition of IL-2 or IL-15 did not enhance ICOS expression ( Figure 1A ), but addition to cultures of IL-21 strongly induced expression of ICOS on the majority of Vγ9Vδ2 T cells ( Figure 1A ). Vγ9Vδ2 cells with a Tnaive and a TCM phenotype were the only subsets that can be polarized to ICOS expression upon culture with antigen and IL-21, while TEM and TEMRA Vγ9Vδ2 cells failed to express any of the tested TFH surface markers under similar cytokine priming conditions ( Figure 1B ). Therefore, in subsequent in vitro culture experiments we used sorted CD27+ Vγ9Vδ2 T cells (which contains both Tnaive and a TCM cells) as a starting population. 10.1371/journal.pone.0041940.g001Figure 1 Surface phenotype of Vγ9Vδ2 T cells differentiated by antigen and IL-21. Vγ9Vδ2 T cells were primed for 12 days with an equal number of irradiated DCs and IPP, in the presence of IL-21. Cells were surface stained for several different markers. (A) shows CD27 and ICOS expression on Vγ9Vδ2 T cells that had been cultured with antigen alone (Nil) or with antigen but in the presence of IL-2, IL-15 or IL-21. Data are representative of seven independent experiments, each carried out in triplicate. (B) FACS analysis to determine the percentage of ICOS+ Vγ9Vδ2 T cells among highly purified TNaive,TCM, TEM and TEMRA subsets Vγ9Vδ2 T cells from eight different donors, primed with antigen and IL-21. Each symbol represents a single donor, small horizontal bars indicate the mean. In (C), expression of activation and costimulatory molecules, and chemokine receptors is shown upon gating on Vγ9Vδ2 T cells. The vertical line in each panel indicates the negative cut-off as determined by staining with isotype-control mAbs. (D) Time-course of ICOS expression on Vγ9Vδ2 T cells that had been cultured with medium alone (filled squares), with IL-21 alone (filled circles) or with antigen but in the absence (open circles) or presence (open squares) of IL-21. Data are representative of five independent experiments each carried out in triplicate. The vast majority of Vγ9Vδ2 T cells differentiated in the presence of antigen and IL-21 had a predominant central memory phenotype as they did not express CD45RA, but expressed CD27. Moreover, they expressed the activation markers CD25 and HLA-DR, and the costimulatory molecules CD40L and ICOS. They also expressed low levels CXCR5, but they did not express CXCR3, CCR3, CCR5 and CCR6 ( Figure 1C ). Time-course experiments showed, that ICOS expression by Vγ9Vδ2 T cells differentiated in the presence of antigen and IL-21 was evident after 2 days of culture, reached a peak at day 4–6 and declined by day 8 onwards ( Figure 1D ). The Relative Role of Antigen and IL-21 in the Regulation of Lineage-specifying Factors Bcl-6 was recently identified as a master regulator of TFH differentiation [35]–[37]. We therefore measured the expression of mRNA encoding human Bcl-6 (BCL6) in Vγ9Vδ2 TFH cells. Culture of sorted Vγ9Vδ2 T cells with antigen and IL-21 induced high expression of BCL6, while expression of RORC (RORγt), TBX21 (T-bet) and GATA3 was induced only slightly or not at all ( Figure 2A ). Addition of IL-1β, IL-2, IL-6, IL-12, IL-15 or TGFβ, either alone, or in combination with IL-21 (data not shown), to cultures of Vγ9Vδ2 T cells and antigen did not induce BCL6 expression ( Figure 2B ). 10.1371/journal.pone.0041940.g002Figure 2 Antigen and IL-21 differently regulate expression of lineage-specifying transcription factors in Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were cultured with an equal number of irradiated DCs and IPP, in the presence of IL-21. (A) RT-PCR of the expression of RORC, TBX21, GATA3 and BCL6 in cells primed with antigen in the absence (white columns) or presence (black columns) of IL-21. (B) RT-PCR of the expression of BCL6 on Vγ9Vδ2 T cells that had been cultured with antigen in the presence of different cytokines. (C) and (D), RT-PCR of the expression of IL21R (C) and BCL6 (D), in Vγ9Vδ2 T cells primed for various times (horizontal axes) with medium (filled circles), with IL-21 alone (open circles) or with antigen but in the absence (filled squares) or presence (open squares) of IL-21. Data represent the mean values ± SD of five separate experiments, each carried out with cells from three different donors. To investigate early events in the differentiation of Vγ9Vδ2 TFH cells and the relative role of antigen and the polarizing cytokine IL-21, we assessed the kinetics of expression of mRNA encoding for IL-21R and BCL6. Data are shown in Figure 2C and 2D , respectively. Resting, unstimulated Vγ9Vδ2 T cells do not constitutively express both IL21R and BCL6. Vγ9Vδ2 TCR stimulation by antigen induced expression of IL21R mRNA, as early as 6 hrs after stimulation. Expression of IL21R mRNA peaked on day 2–3 and consistently decreased on day 6. Antigen stimulation alone was not sufficient to induce detectable BCL6, indicating that upregulation of lineage-specifying transcription factors requires combination of antigen and IL-21. Accordingly, BCL6 was significantly induced by antigen in the presence of IL-21, which peaked on days 3–6 and decreased by day 9 onwards. These results indicate that the coordinated combination of TCR triggering by antigen and the presence of IL-21, induces sustained expression of BCL6 in human Vγ9Vδ2 T cells, which is consistent with their ability to promote differentiation and polarization towards TFH cells. Proliferation Potential and Cytokine Production of Vγ9Vδ2 TFH Cells In the following set of experiments, we assessed the proliferation potential of purified Vγ9Vδ2 TFH. To this end, we generated in vitro Vγ9Vδ2 TFH cells with antigen and IL-21, as previously described. Vγ9Vδ2 TFH cells were sorted, labelled with CFSE and stimulated again in vitro with IPP in the presence of irradiated DCs. As shown in Figure 3A , proliferation of Vγ9Vδ2 TFH cells was very low in the presence of DCs, but without antigen. As expected, upon stimulation with antigen and DCs, or with immobilized anti-CD3 mAb, Vγ9Vδ2 TFH cells, showed significant proliferation, indicating that they have an high proliferative potential to TCR ligands, and suggesting that they are at an early stage of memory cell differentiation. 10.1371/journal.pone.0041940.g003Figure 3 Proliferation and cytokine production of Vγ9Vδ2 TFH cells. Vγ9Vδ2 TFH cells were obtained upon culture with antigen and IL-21, as previously described. Vγ9Vδ2 TFH cells were sorted, labelled with CFSE and re-stimulated in vitro with IPP in the presence of irradiated DCs. (A) Proliferation was measured by CFSE dilution, after 72 hrs stimulation. Cytokine levels were assessed by ELISA (B) or intracellular FACS analysis (C), 24 and 6 hrs after stimulation, respectively. Numbers in the quadrant of dot plots in Figure 3C indicate the percentages of positive cells. (D) RT-PCR of the expression of IL4, IL10, IL13, IL17, IL21 and IFNG in Vγ9Vδ2 TFH cells stimulated in vitro with IPP in the presence of irradiated DCs for 6 hrs. The Figure shows one out of five independent experiments. We then studied the pattern of cytokine production in Vγ9Vδ2 TFH cells, after a 24 hrs stimulation period with antigen and DCs in vitro. As shown in Figure 3B , Vγ9Vδ2 TFH cells produced very few, if any, amounts of cytokines upon stimulation with DCs, but in the absence of antigen. However, Vγ9Vδ2 TFH cells that had been stimulated by antigen and DCs, produced IL-2, IL-4 and, to a lower extent, IL-10, but not IFN-γ, TNF-α or IL-17. Moreover, and differently than CD4 TFH cells, Vγ9Vδ2 T cells cultured with antigen and IL-21 did not produce IL-21, in agreement with previously published results [25]. This pattern of cytokine production was confirmed by flow cytometry studies in Vγ9Vδ2 TFH cells 6 hrs after in vitro culture with antigen and DCs: as expected from the ELISA data, antigen-stimulated Vγ9Vδ2 T cells expressed IL-4 and IL-10, but not IFN-γ or IL-17 ( Figure 3C ). Given that Vγ9Vδ2 TFH cells are capable of producing the Th2 cytokines, IL-4 and IL-10 in the spite of very low, if any, GATA-3 expression, we analysed whether this pattern involved other Th2 cytokines. In agreement with the ELISA and flow cytometry data, antigen-stimulated Vγ9Vδ2 TFH cells expressed IL-4 and IL-10 mRNA, but not IL-13 mRNA ( Figure 3D ). Moreover, and as expected, antigen-stimulated Vγ9Vδ2 TFH cells did not express both IL-13, IFNG and IL-21 mRNA. Thus Vγ9Vδ2 TFH cells are characterized by the distinctive pattern of IL-4 and IL-10 expression in the absence of significant GATA-3 and IL-13 expression. Finally, and differently than CD4 TFH cells, Vγ9Vδ2 TFH cells neither express nor produce IL-21. Chemokine Production and Migratory Properties of Vγ9Vδ2 TFH Cells It has been previously demonstrated that Vγ9Vδ2 T cells stimulated with HMB-PP in the presence of IL-21, but not of IL-2 or IL-4, express CXCL13 mRNA, and the secretion of CXCL13 by PBMC stimulated with antigen and IL-21 depends on the presence of Vγ9Vδ2 T cells [25]. Data reported in Figure 4A confirm that Vγ9Vδ2 TFH cells stimulated by IPP and DCs, secrete CXCL13 into the supernatant. 10.1371/journal.pone.0041940.g004Figure 4 Production of CXCL13 and migration to CXCL13 Vγ9Vδ2 TFH cells. Vγ9Vδ2 TFH cells were obtained upon culture with antigen and IL-21, as previously described. In (A), Vγ9Vδ2 TFH cells were sorted and re-stimulated in vitro with IPP in the presence of irradiated DCs. Supernatants were collected after 24 hrs stimulation and CXCL13 levels assessed by ELISA. In (B) Vγ9Vδ2 TFH cells were tested for in vitro migration to CXCL13 (3 µM, final concentration) in the absence or presence of anti-CXCR5 or isotype-matched control mAbs (15 µg/ml, final concentration). Data are representative of three independent experiments. *p<0.02 when compared with groups consisting of Vγ9Vδ2 TFH cells migrating to CXCL13 in the absence of any Ab or in the presence of isotype-matched control mAb. The finding that IL-21-primed Vγ9Vδ2 TFH cells express low levels of CXCR5 led us to explore if the expressed CXCR5 is functional, by assessing migration in response to the CXCR5 ligand, CXCL13 in a 2 hrs assay. IL-21-primed Vγ9Vδ2 TFH cells migrated readily in response to CXCL13 ( Figure 4B ), but migration was significantly inhibited by an anti-CXCR5 mAb added to cultures ( Figure 4B ), indicating that the expressed CXCR5 receptor is functional. Vγ9Vδ2 TFH Cells Help B Cells for Antibody Production As Vγ9Vδ2 TFH cells express costimulatory molecules and produce IL-4 and IL-10 upon antigen stimulation, we tested whether or not these cells were able to support B cells to secrete immunoglobulins. To this end, we generated in vitro Vγ9Vδ2 TFH cells with IPP and IL-21, as previously described. Vγ9Vδ2 TFH cells were sorted, and cultured with CD19 B cells isolated from the tonsil of the same donor, in the presence or absence of antigen. As shown in Figure 5 , B cells produced comparable low amounts of IgA, IgG and IgM when cultured for 10 days without Vγ9Vδ2 T cells, but co-culture of B cells with Vγ9Vδ2 TFH cells and IPP resulted in an 15-fold increase in the production of IgG, a 10-fold increase in the production of IgA and a 5-fold increase in the production of IgM. Of note, Vγ9Vδ2 TFH cells failed to cause significant increase of antibody production in co-cultures with B cells carried out in the absence of antigen. 10.1371/journal.pone.0041940.g005Figure 5 Vγ9Vδ2 TFH cells help B cells for antibody production. Vγ9Vδ2 T cells were cultured with an equal number of irradiated DCs and IPP, in the presence of IL-21. At the end of culture, Vγ9Vδ2 TFH cells were sorted, and cultured with CD19 B cells isolated from the tonsil of the same donor, in the presence or absence of IPP. Ten days later, total IgG, IgA and IgM levels in culture supernatants were assessed by ELISA. *p<0.001 and **p<0.02 when compared with the group consisting of B cells cultured with Vγ9Vδ2 TFH cells but in the absence of antigen. One out of five independent experiments is shown. The B cell helper activity of Vγ9Vδ2 TFH cells in in vitro co-cultures was strictly dependent on their provision of both costimulatory molecules and cytokines. In fact, blocking of CD40L or ICOS caused a drastic reduction of both IgG and IgA production ( Figure 6 ). Similarly, addition to co-cultures of antibodies neutralizing IL-4 and IL-10 caused reduction of IgG production, while a modest, not significant decrease of IgA production was only observed upon neutralization of IL-10, but not of IL-4 ( Figure 6 ). 10.1371/journal.pone.0041940.g006Figure 6 Vγ9Vδ2 TFH cell helper activity requires costimulatory molecules and cytokines. Vγ9Vδ2 T cells were cultured with an equal number of irradiated DCs and IPP, in the presence of IL-21. At the end of culture, Vγ9Vδ2 TFH cells were sorted and cultured with CD19 B cells isolated from the tonsil of the same donor, in the presence of IPP and mAbs to costimulatory molecules or cytokines (see Materials and Methods). Ten days later, total IgG and IgA levels in culture supernatants were assessed by ELISA. *p<0.005 and **p<0.02 when compared with the group consisting of B cells cultured with Vγ9Vδ2 TFH cells and antigen. One out of five independent experiments is shown. These data therefore suggest that antigen stimulation in the presence of IL-21 induces a population of Vγ9Vδ2 TFH cells which supplies B cells with costimulatory signals and cytokines required for immunoglobulin production. Discussion Vγ9Vδ2 T cells display in vitro a certain degree of plasticity in their function, that is reminiscent of conventional CD4 T cells. In analogy with CD4 T cells, where a plethora of specialized subsets affect the host’s response, Vγ9Vδ2 T cells may readily and rapidly assume distinct Th1-, Th2-, Th17- and Treg-like effector functions, [24]–[30] suggesting that they profoundly influence cell-mediated immune responses. Comparatively, little is known about their role in antibody-mediated immune responses. We [31] and others [20], [25] previously identified a unique subset of peripheral blood and tonsil Vγ9Vδ2 cells with TFH-like properties, which upon antigen stimulation express ICOS, CD40L, CXCR5, and IL-21R, secrete IL-4 and IL-10 and provide B-cell help for antibody production in vitro, but the cytokine requirements for differentiation of this TFH-like Vγ9Vδ2 cell subset have not been examined yet. Here we show that in human Vγ9Vδ2 T cells, Bcl-6 expression and polarization towards a TFH-like phenotype is efficiently induced by coordinated antigen stimulation of the specific TCR and IL-21. The in vitro differentiated Vγ9Vδ2 TFH cells exhibit a TCM phenotype, illustrated by the expression of CD27 in the absence of CD45RA. Vγ9Vδ2 TFH cells distinctively express both activation (CD25 and HLA-DR) and costimulatory (CD40L and ICOS) molecules and also express, although at low levels, CXCR5, a chemokine receptor that has been identified as a marker of TFH cells [1], [2], but they do not express any other tested chemokine receptor (CXCR3, CCR3, CCR4, CCR5, and CCR6). Conversely, Th1-like Vγ9Vδ2 T cells express CXCR3 and CCR5 [19], and Th17-like Vγ9Vδ2 T cells express CCR6 [29]. Expression of CXCR5 on human Vγ9Vδ2 TFH cells is a matter of debate. Brandes and colleagues [20] did not detect CXCR5 expression on both peripheral blood and tonsillar Vγ9Vδ2 T cells, while other studies [31], [38] found this receptor being expressed by a subset of Vγ9Vδ2 TFH. Moreover, Forster et al. [39] found that in healthy individuals, 2% of peripheral blood γδ T cells, but ∼23% of tonsillar γδ T cells express CXCR5 and this percentage consistently increased in HIV-infected individuals. While we have no obvious explanation for the discrepancy in CXCR5 expression on peripheral blood Vγ9Vδ2 T cells between these studies, in mice, CXCR5 expression during primary responses depends on sequential signaling by CD28 and OX40, suggesting the requirement for APCs [40], [41]. Hence, the presence or absence of DCs in the in vitro cultures might influence the outcome of CXCR5 expression. Thus, Vγ9Vδ2 TFH cells differentiated in vitro with antigen and IL-21 can clearly express CXCR5, providing a molecular explanation for their clustering in germinal centres [20], [25]. Our present data also show that IL-21 plays a role in stimulating expression of the CXCR5 ligand, the chemokine CXCL13, by Vγ9Vδ2 TFH cells. Overall, our results indicate that IL-21 drives Vγ9Vδ2 T cells to assume a TFH-like phenotype, thus evoking the crucial effect of IL-21 in the generation of CD4 TFH cells [42]–[44]. Similar results have been published very recently by Bansal at al. [32], who have reported that Vγ9Vδ2 T cells stimulated with the phosphoantigen HMB-PP in the presence of IL-21, express markers associated with TFH cells and support antibody production by B cells. Although these findings suggest that γδ T cells follow a similar differentiation pathway as conventional CD4 TFH cells in their IL-21 requirement, expression of other important TFH cell markers including PD-1, SAP, BTLA and CD57 is necessary to precisely define the Vγ9Vδ2 TFH cell population. The determination of the co-expression of these markers is also important as this would resolve the proportion of the TFH cell subset within the total Vγ9Vδ2 T cells derived from the in vitro culture. This is important for three reasons: (1) expression of two activations markers, CD25 and HLA-DR, rises the question of the heterogeneity of the in vitro activated Vγ9Vδ2 T cells; (2) it is not clear whether or not all Vγ9Vδ2 T cells can be biased towards a TFH phenotype by IL-21, or are rather specific subsets of Vγ9Vδ2 T cells pre-programmed to become TFH-like cells and expand rapidly under the right conditions, as suggested by the heterogeneity of TFH-like Vγ9Vδ2 T cells in peripheral blood and tonsils [31]; (3) ICOS is not exclusively associated with TFH functions [45]. In our experiments, only Tnaive and a TCM subsets of Vγ9Vδ2 T cells acquire some TFH features when stimulated with IPP and IL-21 in the presence of irradiated DCs: since these subsets have the highest proliferative potential amongst Vγ9Vδ2 T cells [19], high ICOS expression after 12 days of culture should be the hallmark of their proliferation, rather than differentiation to a TFH subset. Thus, differential ICOS expression by Tnaive/TCM and/or TFH subsets might also explain the observed trend with peak expression on day 4–6, and again on day 12. IL-21 is the main cytokine shown to induce CD4 TFH cells, but other cytokines have also been shown to induce TFH cells, and these include IL-6, and IL-12. The requirements for the generation of conventional CD4 TFH cells seem to be different for human and for mouse. While surprisingly in humans IL-12 also induces IL-21 production in a STAT-4-dependent manner [46], [47], in mouse IL-6 signaling also induces IL-21-secreting CD4 TFH cells [44], [48]. Although we have not formally determined the signaling pathway that operates in Vγ9Vδ2 TFH cells, data reported in Figure 2B clearly show that addition of IL-1β, IL-2, IL-6, IL-12, IL-15 or TGFβ, either alone, or in combination with IL-21, to cultures of Vγ9Vδ2 T cells and antigen did not induce or even enhance BCL6 expression. The acquisition of TFH-associated markers by Vγ9Vδ2 T cells and their dependence on IL-21 was initially suggested by microarray studies [25]. IL-21 turned out to have a similar capacity as the related cytokine IL-2 to support and sustain antigen-induced Vγ9Vδ2 T cell proliferation, yet without promoting the supposedly signatory cytokines IFN-γ and TNF-α [49], thus highlighting a much greater plasticity of Vγ9Vδ2 cell responses than previously appreciated [25]. While IL-21 may potentiate the cytolytic function of Vγ9Vδ2 T cells when combined with IL-2 [50], previous findings [25] and results here reported demonstrate that IL-21 on its own specifically co-stimulates expression of the chemokine receptor CXCR5, that enables TFH cells to migrate into the B cell follicles, and also the CXCR5 ligand, CXCL13 that attracts further CXCR5+ cells, such as naive B cells and early activated CD4 T cells. As CXCR5 and CXCL13 are uniquely expressed in B cell follicles but mostly absent from extrafollicular areas, including the T zones of lymph nodes, spleen and Peyer’s patches, this implicates a role for IL-21-stimulated Vγ9Vδ2 T cells in orchestrating immune cell trafficking to the GCs. Differently than CD4 TFH cells, Vγ9Vδ2 TFH cells generated by culture with antigen and IL-21 do not produce IL-21, in agreement with previously published results [25]. On the other hand, the in vitro differentiated Vγ9Vδ2 TFH cells have a Th2-type pattern of cytokine production upon short-term antigen stimulation in vitro, as they secrete IL-2, IL-4, and IL-10, but not IL-17, IFN-γ and TNF-α. This finding clearly contrasts with the cytokine production pattern of the Th1-like TEM subsets of Vγ9Vδ2 T cells, which preferentially secrete IFN-γ and TNF-α [19]. The finding of a population of Vγ9Vδ2 T cells that secretes IL-4 and IL-10 is not new, and expands previous results demonstrating IL-4 production by resting [27], [51] and Vγ9Vδ2 clones [26], [52], most of which express CD27 (M. Bonneville and E. Scotet, unpublished observations). Moreover, and accordingly, we previously found that secretion of IL-4 and IL-10 was confined to the CD27+ subset of CXCR5+ Vγ9Vδ2 T cells [31]. However, Vγ9Vδ2 TFH cells lack expression of GATA-3, and IL-13 mRNAs, both signatures of Th2 cells. The dissociated expression of IL-4 and IL-13/GATA-3 was unexpected but confirms a very recent paper in a mouse model of helminth infection [53], showing that IL-4, but not IL-13, was made by TFH cells. In contrast, Th2 cells produced both cytokines. IL-13 production by Th2 cells was associated with large amounts of cellular transcription factor GATA-3, which was necessary for sustaining IL-13-producing. Conversely, TFH cells produced only IL-4 and did not express GATA-3. Altogether, the results in mice and the data here reported in human Vγ9Vδ2 TFH cells, indicate previously unappreciated regulation of these duplicated cytokines, as suggested by the differences between IL-4- and IL-13-expression in dependence on and expression of GATA-3. It is likely that high levels of Bcl-6 expression in TFH cells restrict GATA-3 to levels insufficient to activate IL13. Although Bcl-6 is a direct transcriptional repressor for many genes, it might suppress GATA-3 at a post-transcriptional level [37]. Because Bcl-6 overexpression can induce a TFH phenotype [35], it is possible to speculate that decay of Bcl-6 or expression of Blimp-1 in T cells [36] is a prerequisite for relieving repression of the genetic programs, such as extended cytokine expression, necessary for the completion of Th2 differentiation in the periphery. Production of Th2-type cytokines together with expression of CD40L and ICOS strongly suggests that IL-21-stimulated Vγ9Vδ2 T cells are engaged in B cell activation and help for antibody production. Accordingly, we observed enhanced production of IgM, IgG and IgA when tonsillar B cells were coculturing with IL-21-stimulated Vγ9Vδ2 TFH cells in the presence of Ag, thus fully identifying this cell population as a classical helper cells. Moreover, Ig production was consistently inhibited by blocking CD40-CD40L and ICOS-ICOSL interactions, or by neutralization of IL-4 or IL-10. In theory one may argue that, due to the preactivation status of tonsillar B cells, Vγ9Vδ2 TFH cells may only be active on already activated B cells and hence during secondary antibody responses. While we have no evidence to support or exclude such a possibility, our previous findings that circulating CXCR5+ Vγ9Vδ2 T cells are also able to help circulating naive B cells for antibody production [31], strongly suggests that Vγ9Vδ2 TFH cells may play an important regulatory role in all aspects of humoral immunity. γδ T cells have been reported to support antibody production in immunised and infected mice [11]–[14]. Of note, GCs are present in TCRαβ−/− mice and develop in SCID mice upon adoptive transfer of γδ T cells and B cells, demonstrating that γδ T cells are sufficient to orchestrate follicular responses [11], [15], [54]. In humans, γδ T cells can be found in secondary lymphoid tissues [17], [18], where they are scattered throughout the T zone and clustered within GCs [18]–[20]. Contribution of Vγ9Vδ2 TFH cells to antibody-mediated immune responses may occur early during microbial infections, before full development of acquired responses mediated by CD4 T cells. In humans, Vγ9Vδ2 TCM cells are resident in the paracortical areas of lymph nodes, where they may become stimulated by antigen and express IL-21R: these, pre-activated cells may thus encounter IL-21 produced by CD4 T cells and as a consequence express a distinct set of molecules associated with providing B cell help. The interaction between with Vγ9Vδ2 TFH cells, IL-21 producing CD4 T cells and B cells in reactive secondary lymphoid tissues is likely to impact on the production of high affinity antibodies against microbial pathogens. In humans, the vast majority of Vγ9Vδ2 T cells directly recognize nonpeptide ligands without presentation by MHC molecules. Because αβ and γδ T cells recognize different types of antigens, the presence of a subset of each of these populations capable of inducing immunoglobulin secretion would provide a mechanism whereby humoral immune responses could be elicited against a diverse array of antigens irrespective of the type of responding T cell. Thus, the presence of Vγ9Vδ2 T cells in germinal centers would broaden the repertoire of antibodies produced by the B cell response. We thank Martin Lipp, Richard Kroczek and Vaclav Horejsi for providing us with reagents and Matthias Eberl, Marc Bonneville and Emmanuel Scotet for sharing unpublished data. Competing Interests: The authors have declared that no competing interests exist. The corresponding author, Francesco Dieli, is an Academic Editor of PLoS ONE. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Funding: This work has been supported by grants from the European Commission within the 6th Framework Programme, TB-VAC contract no. LSHP-CT-2003-503367 (The text represents the authors’ views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information), the Italian Ministry for Instruction, University and Research (contract no. 2008L57JXW to FD), the Italian Ministry of Health (Progetto ricerca finalizzata 2007 “Stem cells in different pathological conditions innovative therapeutical approches” to FD) and the University of Palermo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Refs References 1 Breitfeld D Ohl L Kremmer E Ellwart J Sallusto F 2000 Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J Exp Med 192 1545 1552 11104797 2 Schaerli P Willimann K Lang AB Lipp M Loetscher P 2000 CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. 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PLoS One. 2012 Jul 25; 7(7):e41940
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22848619PONE-D-11-0892710.1371/journal.pone.0041820Research ArticleMedicineClinical Research DesignRetrospective StudiesGastroenterology and HepatologyLiver DiseasesCirrhosisOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaSurgeryGastrointestinal SurgeryTransplant SurgerySalvage Liver Transplantation for Patients with Recurrent Hepatocellular Carcinoma after Curative Resection Salvage Liver Transplantation for Recurrent HCCWu LinWei 1 Hu AnBin 1 Tam Ngalei 1 Zhang JianWei 2 Lin Min 2 Guo ZhiYong 1 * He XiaoShun 1 * 1 Organ Transplant Center of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 2 Zhongshan Medical School of Sun Yat-sen University, Guangzhou, China Mandell Mercedes Susan Editor University of Colorado, United States of America * E-mail: [email protected] (XH); [email protected] (ZG)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: LW ZG XH. Performed the experiments: LW AH NT JZ ML. Analyzed the data: AH NT JZ. Contributed reagents/materials/analysis tools: LW ZG XH. Wrote the paper: LW NT ZG. 2012 26 7 2012 7 7 e418209 2 2012 26 6 2012 © 2012 Wu et al2012Wu 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.Objective To summarize the experience with salvage liver transplantation (SLT) for patients with recurrent hepatocellular carcinoma (HCC) after primary hepatic resection in a single center. Methods A total of 376 adult patients with HCC underwent orthotopic liver transplantation (OLT) at Organ Transplantation Center, the First Affiliated Hospital of Sun Yat-sen University, between 2004 and 2008. Among these patients, 36 underwent SLT after primary liver curative resection due to intrahepatic recurrence. During the same period, one hundred and forty-seven patients with HCC within Milan criteria underwent primary OLT (PLTW group), the intra-operative and post-operative parameters were compared between these two groups. Furthermore, we compared tumor recurrence and patient survival of patients with SLT to 156 patients with HCC beyond Milan criteria (PLTB group). Cox Hazard regression was made to identify the risk factors for tumor recurrence. Results The median interval between initial liver resection and SLT was 35 months (1–63 months). The intraoperative blood loss (P<0.05) and transfusion volume (P<0.05) were larger in the SLT group than in the PLTW group. The operation time was longer in the SLT group (P<0.05). The post-operative complications incidence, tumor recurrence rate, patients' survival rate, and tumor-free survival rate were comparable between these two groups (all P>0.05). When compared to those patients with HCC beyond Milan criteria undergoing primary OLT, patients undergoing SLT achieved a better survival and a lower tumor recurrence. Cox Proportional Hazards model showed that vascular invasion, including macrovascular and microvascular invasion, as well as AFP level >400 IU/L were risk factors for tumor recurrence after LT. Conclusions In comparison with primary OLT, although SLT is associated with increased operation difficulties, it provides a good option for patients with HCC recurrence after curative resection. This study was supported by the National High Technology Research and Development Program of China (863 Program) (2012AA021008), the Key Clinical Project from the Ministry of Health (2010159), the National Natural Science Foundation of China (30972951, 81102244, 81102245, and 81170448), the Special Fund for science research by Ministry of Health (201002004), the Research Fund for the Doctoral Program of Higher Education of China by Ministry of Education (20100171110063 and 20110171120077), the Science and Technology Planning Key Clinical Project of Guangdong Province (2011A030400005), Science and Technology Planning Project of Guangdong Province (2011B0318000099), Medical Scientific Research Foundation of Guangdong Province (B2011072) and Project by Division of Medical Service Management of Ministry of Health (2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction In China mainland, the incidence of hepatocellular carcinoma (HCC) is increasing, and it is the third leading cause of cancer mortality [1]. Orthotopic liver transplantation (OLT) remains the most effective treatment for patients with small HCC. However, due to organ shortage, economic or conceptional constraints, only a small population of such patients could receive a transplant. Hepatic resection (HR) remains a more reasonable choice for the vast majority of such patients although they might face a higher risk of tumor recurrence or liver function deterioration. However, for those patients with HCC recurrence after hepatic resection, 80% might be rescued by means of liver transplant [2], [3]. The concept of liver transplant performed after HCC recurrence post HR, namely salvage liver transplantation (SLT) has been introduced by Pietro E. Majno [2] in 2000. Herein we retrospectively reviewed and compared the patient survival and cancer recurrence rates between patients undergoing SLT and primary OLT in our single center between 2004 and 2008. Methods Baseline characteristics of patients From January 2004 to December 2008, 376 adult patients with HCC received OLT at our center, including 296 males and 80 females (mean age, 50.8 years; age range, 18–75 years). The patients who had tumor thrombosis of the main trunk of portal vein or hepatic vein (vena cava) (n = 19), and those who had ABO incompatible match (n = 8), had split grafts (n = 6) and living donors (n = 4) were excluded from this study. Thirty-six patients received SLT after radical HR due to intra-hepatic recurrence. All the 36 patients were included in the SLT group. One hundred and forty-seven patients who received primary OLT for HCC within Milan criteria were classified into PLTW (primary OLT for HCC within Milan criteria) group. The demographics and clinical data of the patients in the two groups are shown in Table 1. There was no significant difference in pre-operative baseline characteristics between the two groups. We also compared the tumor recurrence and patients survival of SLT group to 156 patients undergoing primary OLT for HCC beyond Milan criteria (PLTB group), and the risk factors for tumor recurrence was identified by Cox regression analysis. 10.1371/journal.pone.0041820.t001Table 1 Demographics of the patients. SLT (n = 36) PLTW (n = 147) PLTB (n = 156) P value Age (yr) Mean ± SD 49.4±17.1 47.6±15.8 51.1±15.6 0.160 Median 48 (20–65) 49 (18–63) 46(22–68) Gender(M/F) 27/9 106/31 118/38 0.775 HBV infection 35 (97.2) 144 (97.9) 149(95.5) 0.479 HCV infection 2 (5.5) 9 (6.1) 11(7.1) 0.921 ALT (IU/L) 110.5±90.2 100.4±84.8 122.7±88.6 0.085 Sodium (mmol/L) 137.9±7.3 139.4±6.8 141.1±9.8 0.059 Albumin (mg/L) 36.9±17.9 38.8±20.9 34.9±21.5 0.269 Creatinine (µmol/L) 95.5±93.2 86.8±95.3 105.1±89.8 0.229 Bilirubin(µmol/L) 48.3±39.6 57.2±46.5 60.5±37.3 0.282 INR 1.59±0.48 1.60±0.59 1.75±0.62 0.065 MELD score 18.6±6.7 17.9±7.1 19.4±4.8 0.103 Liver cirrhosis 36(100%) 131(89.1%) 104(66.7%) <0.001 Tumor demographics Nodules numbers solitary 21 86 40 <0.001 ≤3 13 61 56 0.580 >3 2 0 60 <0.001 Largest tumor size(mm) 48 50 130 0.465 AFP levels (IU/L) <400 12 44 10 <0.001 ≥400 24 103 146 <0.001 Macro vascular invasion 0 0 37 <0.001 Microvascular invasion 2 14 55 <0.001 Pathologic characteristics Poor differentiated 8 30 68 <0.001 Well differentiated 28 117 88 <0.001 Pre-transplant treatment radiofrequency ablation 9 34 25 0.224 ethanol injection 6 25 30 0.860 TACE 3 14 18 0.777 Abbreviation: SLT, salvage liver transplantation; PLTW, primary liver transplantation for HCC within Milan criteria; PLTB, primary liver transplantation for HCC beyond Milan criteria; TACE, transcatheter arterial chemoembolization. All the participants received whole liver graft from deceased donors. Different transplant techniques were performed, including 16 cases of classic OLT, 303 cases of modified piggyback technique introduced by YM Wu [4] and 20 cases of classic piggyback. The immunosuppressive regimens used included double regimen (steroid and Tacrolimus or Cyclosporine (CsA)) and triple regimen (steroid, Tacrolimus or CsA, and Mycophenolate mofetil (MMF)). MMF was used in patients with a low serum Tacrolimus concentration even under a relatively high dose intake, and in those patients with hyperglycemia or renal impairment during the early period post-transplantation. When MMF was given, Tacrolimus was maintained in a lower tough level. Liver function and blood concentration of immunosuppressants were monitored according to the protocols in our center. Interleukin-2 receptor monoclonal antibody (Basiliximab) induction was adopted in patients with high-risk factors, such patients with age >60-year old, hypoalbuminemia, hepatorenal syndrome and a model for end-stage liver disease (MELD) score over 30. The interleukin-2 monoclonal antibody was administered intraoperatively and on day 4 post-transplantation, respectively. A dose of 500 mg steroid was administered during operation. When induction therapy was used, Tacrolimus or CsA was used from day 4 post-transplantation, and then was adjusted according to plasma concentration. Prior to the study, the protocol, which was in accordance with the ethical guidelines of the 1975 Helsinki Declaration, was approved by the institutional ethics committee of the First Affiliated Hospital of Sun Yat-sen University. Written, informed consent was obtained from all subjects. Statistical analysis All statistical analysis was performed using the Statistical Package for Social Science 13.0 (SPSS, version 13.0; Chicago, IL). Continuous variables were tested for normal distribution and expressed as mean ± standard deviation (SD) or median (range) as appropriate. Categorical variables were compared by Pearson chi-squared test and continuous variables were compared by Student's t-test. Univariate survival curves were estimated using the Kaplan-Meier method and compared by the log-rank test. Risk factors for tumor recurrence were evaluated by a Cox Proportional Hazards model. For all analyses, P values<0.05 were considered statistically significant. Results Implementation of SLT Thirty-six patients, including 29 males and 7 females, received SLT after radical HR due to intrahepatic recurrence. Sixteen patients received primary right hemihepatectomy, 10 left hemihepatectomy and the others irregular hepatic segmentectomy pre-transplantation. Before HR, 22 patients had single tumor nodule, 12 had 2 or 3 nodules, and only 2 had >3 tumor nodules. In all patients, the tumors were located within a liver lobe without macrovascular invasion. The post-operative pathologic studies showed poorly differentiated HCC in 24 cases and well differentiated HCC in the other 16 cases. The median interval between initial HR and SLT was 35 months (1–63 months). Among the 36 patients, 15 had received radiofrequency ablation or ethanol injection pre-transplantation, 3 patients received transcatheter arterial chemoembolization (TACE). Operative parameters and postoperative complications The operative parameters and post-operative complications between the two groups of patients were described in Table 2. The intraoperative blood loss (1560±670 ml versus 1180±910 ml, P<0.05) and transfusion volume (1060±780 ml versus 820±910 ml, P<0.05) were larger in the SLT group than in PLTW group. The operation time was longer in the SLT versus PLTW group (340±77 min versus 302±81 min, P<0.05). These indicated that SLT would increase the operation difficulties. However, the postoperative recovery and intraoperative complications between the two groups were not significantly different (P>0.05). 10.1371/journal.pone.0041820.t002Table 2 Operative parameters and postoperative complications. SLT (n = 36) PLTW (n = 147) t value or χ 2 value P value Donor liver cold ischemic time (h) 8.1±2.5 7.4±2.8 1.37 0.127 Anhepatic time (min) 39.6±5.2 38.3±6.9 1.06 0.268 Operative time (min) 340±77 302±81 2.54 0.024* Application of arterial jump grafts 3 2 5.29 0.021 Roux-n-Y 4 3 6.47 0.011 Intraoperative bleeding volume (ml) 1560±670 1180±910 2.35 0.028* Intraoperative transfusion volume (ml) 1060±780 820±910 2.02 0.043* Postoperative ICU time (h) 34±12 29±16 1.76 0.078 Primary graft nonfunction 0 0 – – Delayed graft function 1 4 <0.001 0.985 Intra-abdominal bleeding 0 4 1.001 0.317 Infections 2 14 0.571 0.450 Renal failure 1 3 0.073 0.786 Acute rejection 2 15 0.742 0.389 Biliary complications 2 14 0.571 0.450 Vascular complications 0 3 0.747 0.387 Recurrence of hepatitis 0 2 0.495 0.482 Abbreviation: SLT, salvage liver transplantation; PLTW, primary liver transplantation for HCC within Milan criteria; ICU, intensive care unit. Patient survival There were 11 deaths in the SLT group, including 1 perioperative death due to severe infection. In PLTW group, there were 36 deaths, including 3 perioperative deaths (1 died from renal failure, 1 from severe infection and 1 from hepatic artery thrombosis). Survival curves generated by the Kaplan-Meier method are shown in Figure 1A. Log-Rank test showed that the patient survival was not significantly different between the two groups (χ 2 = 0.926, P = 0.336). 10.1371/journal.pone.0041820.g001Figure 1 (A) shows the cumulative survival rate between SLT group and PLTW group, a Log-Rank test showed P = 0.336. (B) shows the cumulative survival rate between SLT group and PLTB group, a Log-Rank test showed P = 0.041. Abbreviation: SLT salvage liver transplantation, PLTW primary liver transplantation within Milan criteria, PLTB primary liver transplantation beyond Milan criteria. And in those patients undergoing primary OLT for HCC beyond Milan criteria, there were 75 deaths including 7 perioperative deaths (3 from severe infection, 2 from renal failure, 1 from GVHD and 1 from biliary ischemia). Compared with this group of patients, patients in SLT group achieved longer survival (P<0.05) (Figure 1B). Tumor recurrence and risk factors By December 2010, all the patients were follow-up 25–83 months. The median follow-up time of SLT group was 61 months. Five patients of the SLT group experienced recurrences during follow-up, with the mean recurrence time of 28.2±15.1 months. Two patients had metastases in the lung, and the other 3 in the liver graft, bone and brain, respectively. While in the PLTW group, the median follow-up time was 62 months, 15 patients experienced recurrences in 30.4±11.8 months, which were also found in lungs (8 cases), liver grafts (5 cases) and bone (2 cases). The recurrence time (P>0.05) and recurrence rate (13.9% versus 10.2%, χ 2 = 0.403, P>0.05) were comparable between the two groups. The tumor recurrence profiles of the patients in the two groups are shown in Table 3. The cumulative recurrence rate curves are shown in Figure 2A. 10.1371/journal.pone.0041820.g002Figure 2 (A) shows the cumulative recurrence rate between SLT group and PLTW group, a Log-Rank test showed P = 0.525. (B) shows the cumulative recurrence rate between SLT group and PLTB group, a Log-Rank test showed P = 0.006. Abbreviation: SLT salvage liver transplantation, PLTW primary liver transplantation within Milan criteria, PLTB primary liver transplantation beyond Milan criteria. 10.1371/journal.pone.0041820.t003Table 3 Survival and tumor recurrence rate. Group Cases Follow-up (month) Recurrence Overall survival rate (%) Tumor-free survival rate (%) Rate time (month) 1 yr 3 yrs 5 yrs 1 yrs 3 yrs 5 yrs SLT 36 58.7±20.7 5/36 28.2±15.1 97.2 80.6 69.4 97.1 87.9 74.2 PLTW 147 64.2±18.1 15/147 30.4±11.8 98.0 86.4 75.5 97.9 89.9 80.3 PLTB 156 57.2±33.1 63/156 22.5±14.9 96.2 64.7 48.7 88.5 53.2 33.6 P value 0.065 <0.001 <0.001 0.657 <0.001 <0.001 0.002 <0.001 <0.001 Abbreviation: SLT, salvage liver transplantation; PLTW, primary liver transplantation for HCC within Milan criteria; PLTB, primary liver transplantation for HCC beyond Milan criteria. In 156 patients who received primary OLT for HCC beyond Milan criteria, 63 (40.3%) patients experienced recurrence in 16.5±10.9 months. The recurrence rate was higher in this group of patients compared with SLT group (χ 2 = 8.977, P = 0.003). The cumulative recurrence rate curves are shown in Figure 2B. A Cox Proportional Hazards model was made to evaluate the risks factors for tumor recurrence in all of the patients here, the result showed that vascular invasion, including macrovascular and microvascular invasion, as well as AFP level >400 IU/L were risk factors for tumor recurrence after LT, as showed in Table 4. 10.1371/journal.pone.0041820.t004Table 4 Risk factors for Overall survival of liver transplantation by Multivariate Analysis. Items n Mean±SD (months) Univariate analysis Multivariate analysis Age (yr) 0.192 <47 169 63.21±2.23 ≥47 170 59.23±3.41 Gender (M/F) 0.241 Male 251 57.34±2.16 Female 78 59.54±3.31 HBV infeciont 0.105 Present 328 58.94±2.16 Absent 11 62.54±5.31 HCV infection 0.143 Present 22 59.24±2.16 Absent 317 61.54±3.31 ALT 0.267 <135 169 63.52±2.34 ≥135 170 58.12±3.64 MELD score 0.228 <18 169 66.21±4.43 ≥18 170 62.03±5.22 Liver cirrhosis 0.312 Present 271 55.34±2.16 Absent 68 59.54±3.31 Nodules numbers 0.109 ≤3 227 65.52±1.54 >3 62 59.12±2.75 AFP levels 0.001 0.031a <400 66 73.66±2.09 ≥400 273 62.09±1.64 Macrovascular invasion <0.001 <0.001b Present 37 40.28±4.16 Absent 302 67.22±2.35 Microvascular invasion <0.001 0.009c Present 71 46.56±3.46 Absent 268 69.31±3.45 Pathologic characteristics 0.092 Poor differentiated 106 62.34±1.46 Well differentiated 233 59.86±3.17 Pre-transplant treatment 0.185 Present 164 62.54±3.46 Absent 175 59.91±4.32 a Relative risk:1.91, 95% CI: 1.04–3.02. b Relative risk:3.33, 95% CI: 1.77–6.24. c Relative risk:2.93, 95% CI: 1.31–6.11. Cox Proportional Hazards model showed that vascular invasion, including macrovascular and microvascular invasion, as well as AFP level >400 IU/L were risk factors for tumor recurrence after OLT. Discussion HCC is one of the most common malignant tumors in China, with the incidence rate up to 80 per million populations. More than 80% patients had simultaneous liver cirrhosis, which leads to an actual rate of resection less than 30%, while the 5 year recurrence rate after resection reached above 70% [3], [5], [6]. Theoretically, OLT was the most effective treatment for HCC patients, which not only achieves radical tumor resection, but also deals with the frequently concurrent end stage liver diseases. In China mainland, nearly half of liver transplant recipients were patients with HCC [7]. However, considering the severe organ shortage, high cost and perioperative risk of this procedure, as well as constraint in concept, HR remains to be the mainstay treatment for patients with resectable early HCC. Although OLT can yield a higher tumor-free survival than HR in early HCC patients with Child A stage liver function, the 5-year survival rate had no significant difference between the two procedures [8]. The reasons might be as follows: (1) the perioperative mortality of liver transplant is higher than that of HR; (2) the survival rate of liver transplant recipients declines because of rejection, recurrence of hepatitis, side effects of immunosuppressants and transplant-related complications; (3) re-excision, radiofrequency ablation and OLT can be applied for recurrence after HR, which prolong patient survival time; (4) due to organ shortage, 15%∼33% of patients lost the opportunities of transplantation because of tumor progression [7], [9], [10]. However, SLT remained a life-saving treatment for patients with intrahepatic recurrence or liver function deterioration after primary HR [2], [10], [11]. In some cases, surgical resection can be taken as an initial treatment so as to control tumor progression when patients are on the waiting list. Transplantation can be implemented as soon as donor liver is available [12]. Previous studies have documented that 80% of patients suffered from intrahepatic recurrence after HR, and about 52% of the recurrent patients are still transplantable [13], [14], [15]. Whether the patients can receive SLT depends on the tumor factors, age of patients, the time waiting for a donor liver, and the patients' willingness. The surgical difficulty in SLT increases due to peritoneal adhesions caused by previous upper abdominal surgery. The adhesions usually lie between the cut surface and the omentum and/or the intestine. In addition, some recipients may have vigorous portal collaterals, which might lead to massive intraoperative bleeding. The dissection of the liver hilum might be another technical difficulty in SLT, especially when hilar dissection has been done extensively during previous hepatectomy. Several such cases were included in our study, we used the iliac artery from the same donor as a bridge and the celiac trunk of the graft was anastomosed to the recipients' abdominal aorta via the bridge. A Roux-en-Y hepaticojejunostomy was utilized for biliary reconstruction. Although some studies have shown that SLT does not increase the difficulty of surgery [13], [16], [17], the SLT group did have longer operative time, more introperative blood loss and transfusion volume in our study. The interval between initial LR and SLT may be a factor related to the operative difficulty since the severity of adhesion is associated with this interval. However, the increased difficulty neither increases the post-operative complications, nor negatively affects the short and long term prognosis. There was one patient received SLT just 1 month after resection, the later was performed in another hospital. This patient had liver cirrhosis and a tumor with 5 cm in diameter locating at the right lobe, while two susceptive small nodules in S4 was also noted, which was regarded as cirrhotic nodules by the doctors. During the first operation, the cirrhosis was more severe than what had been expected before the operation, right lobectomy was performed. Importantly, a PET-CT scanning one month after the resection revealed that the two nodules in S4 were HCC nodules. Therefore, LT was suggested immediately after the PET-CT scanning. This patient seems more likely had remnant cancer, and he had tumor recurrence about 24 months after transplantation. Nowadays, the treatment for liver cancer develops rapidly. There are a variety of interventional therapy that can be chosen for recurrence, including radiofrequency ablation, ethanol injection, and TACE. Besides, some studies [14] have shown that SLT has no obvious advantage over the treatment strategies mentioned above. However, most studies still support that transplantation is the most effective treatment for HCC. In this study, we observe that the Milan criteria are still eligible for the 36 patients at the time of recurrence. And the 5-year overall survival rate and disease-free survival rate are not lower than those undergoing primary OLT. In conclusion, in comparison with primary OLT, although SLT would increase the operation difficulties, it provides a good option for patients with HCC recurrence after curative resection. Identification of recurrent patients who would gain favorable outcomes from SLT will help decision-making among multiple choices of treatment. ==== Refs References 1 Venook AP , Papandreou C , Furuse J , de Guevara LL (2010 ) The incidence and Epidemiology of Hepatocellular Carcinoma: A global and Regional perspective . The Oncologist 15 : 5 –13 . 2 Majno PE , Sarasin FP , Mentha G , Hadenque A (2000 ) Primary Liver Resection and Salvage Transplantation or Primary Liver Transplantation in Patients With Single, Small Hepatocellular Carcinoma and Preserved Liver Function: An Outcome-Oriented Decision Analysis . Hepatology 31 : 899 –906 .10733546 3 Fong Y , Sun RL , Jarnagin W , Blumqart LH (1999 ) An analysis of 412 cases of hepatocellular carcinoma at a Western center . Ann Surg 229 : 190 –800 . 4 Wu YM , Voigt M , Rayhill S , Katz D , Chenhsu RY , et al (2001 ) Suprahepatic venacavaplasty (cavaplasty) with retrohepatic vava extension in liver transplantation: experience with first 115 cases . Transplantation 72 : 1389 –1394 .11685109 5 Poon RT , Fan ST , Lo CM , Ng IO , Liu CL , et al (2001 ) Improving survival results after resection of hepatocellular carcinoma: a prospective study of 377 patients over 10 years . Ann Surg 234 : 63 –70 .11420484 6 Ercolani G , Grazi GL , Ravaioli M , Del Gaudio M , Gardini A , et al (2003 ) Liver resection for hepatocellular carcinoma on cirrhosis: univariate and multivariate analysis of risk factors for intrahepatic recurrence . Ann Surg 237 : 536 –543 .12677151 7 Mazzaferro V , Chun YS , Poon RT , Schwartz ME , Yao FY , et al (2008 ) Liver Transplantation for Hepatocellular Carcinoma . Ann Surg Oncol 15 : 1001 –1007 .18236119 8 Jonas S , Bechstein WO , Steinmuller T , Herrmann M , Radke C , et al (2001 ) Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis . Hepatology 33 : 1080 –1086 .11343235 9 Facciuto ME , Koneru B , Rocca JP , Wolf DC , Kim-Schluger L , et al (2008 ) Surgical treatment of hepatocellular carcinoma beyond Milan criteria. Results of liver resection salvage transplantation, and primary liver transplantation . Ann Surg Oncol 15 : 1383 .18320284 10 Sala M , Fuster J , Llovet JM , Navasa M , Sole M , et al (2004 ) High pathological risk of recurrence after surgical resection for hepatocellular carcinoma: An indication for salvage liver transplantation . Liver Transplantation 10 : 1294 –1300 .15376311 11 Adam R , Azoulay D , Castaing D , Eshkenazy R , Pascal G , et al (2003 ) Liver resection as a bridge to transplantation for hepatocellular carcinoma on cirrhosis: a reasonable strategy? Ann Surg 238 : 508 .14530722 12 Kim BW , Park YK , Kim YB , Wang HJ , Kim MW , et al (2008 ) Salvage liver transplantation for recurrent hepatocellular carcinoma after liver resection: feasibility of the Milan criteria and operative risk . Transplant Proc 40 : 3558 –3561 .19100437 13 Hu RH , Ho MC , Wu YM , Yu SC , Lee PH , et al (2005 ) Feasibility of salvage liver transplantation for patients with recurrent hepatocellular carcinoma . Clin Transplant 19 : 175 –180 .15740552 14 Poon RT , Fan ST , Wong J (2000 ) Risk factors, prevention and management of postoperative recurrence after resection of hepatocellular carcinoma . Ann Surg 232 : 10 –24 .10862190 15 Poon RT , Fan ST , Lo CM , Liu CL , Wong J (2002 ) Long-term survival and pattern of recurrence after resection of small hepatocellular carcinoma in patients with preserved liver function: implication for a strategy of salvage transplantation . Ann Surg 235 : 373 –382 .11882759 16 Cucchetti A , Vitale A , Gaudio MD , Ravaioli M , Ercolani G , et al (2010 ) Harm and benefits of primary liver resection and salvage transplantation for hepatocellular carcinoma . American Journal of Transplantation 10 : 619 –627 .20121741 17 Eguchi S , Hidaka M , Tomonage T , Miyazaki K , Inoluma T , et al (2009 ) Actual therapeutic efficacy of pre-transplant treatment on hepatocellular carcinoma and its impact on survival after salvage living donor liver transplantation . J Gastroenterol 44 : 624 –629 .19381752
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PLoS One. 2012 Jul 26; 7(7):e41820
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22859931PONE-D-12-0985510.1371/journal.pone.0040148Research ArticleBiologyMolecular cell biologySignal transductionSignaling cascadesAkt signaling cascadeMAPK signaling cascadesSignaling in Cellular ProcessesMedicineDiagnostic MedicinePathologyGeneral PathologyBiomarkersNephrologyBladder and Ureteric DisordersOncologyCancers and NeoplasmsGenitourinary Tract TumorsBladder CancerBladder Carcinoma in SituBasic Cancer ResearchUrologyBladder and Ureteric DisordersBladder Cancer and Urothelial Neoplasias of the Urinary TractProline-Rich Tyrosine Kinase 2 (Pyk2) Regulates IGF-I-Induced Cell Motility and Invasion of Urothelial Carcinoma Cells Pyk2 and IGF-IR in Bladder CancerGenua Marco 1 Xu Shi-Qiong 1 Buraschi Simone 2 Peiper Stephen C. 2 Gomella Leonard G. 1 Belfiore Antonino 3 Iozzo Renato V. 2 Morrione Andrea 1 * 1 Endocrine Mechanisms and Hormone Action Program, Department of Urology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America 2 Cancer Cell Biology and Signaling Program, Department of Pathology, Anatomy and Cell Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America 3 Endocrinology, Department of Health, University of Catanzaro, Catanzaro, Italy Andre Frederic EditorAix-Marseille University, France* E-mail: [email protected] and designed the experiments: MG RVI AB AM. Performed the experiments: MG SQX SB. Analyzed the data: MG AB RVI AM. Contributed reagents/materials/analysis tools: SCP LGG AB RVI AM. Wrote the paper: RVI AM. 2012 28 6 2012 7 6 e401484 4 2012 1 6 2012 Genua et al.2012This 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 insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. We have recently demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues and promotes motility and invasion of urothelial carcinoma cells. These effects require IGF-I-induced Akt- and MAPK-dependent activation of paxillin. The latter co-localizes with focal adhesion kinases (FAK) at dynamic focal adhesions and is critical for promoting motility of urothelial cancer cells. FAK and its homolog Proline-rich tyrosine kinase 2 (Pyk2) modulate paxillin activation; however, their role in regulating IGF-IR-dependent signaling and motility in bladder cancer has not been established. In this study we demonstrate that FAK was not required for IGF-IR-dependent signaling and motility of invasive urothelial carcinoma cells. On the contrary, Pyk2, which was strongly activated by IGF-I, was critical for IGF-IR-dependent motility and invasion and regulated IGF-I-dependent activation of the Akt and MAPK pathways. Using immunofluorescence and AQUA analysis we further discovered that Pyk2 was overexpressed in bladder cancer tissues as compared to normal tissue controls. Significantly, in urothelial carcinoma tissues there was increased Pyk2 localization in the nuclei as compared to normal tissue controls. These results provide the first evidence of a specific Pyk2 activity in regulating IGF-IR-dependent motility and invasion of bladder cancer cells suggesting that Pyk2 and the IGF-IR may play a critical role in the invasive phenotype in urothelial neoplasia. In addition, Pyk2 and the IGF-IR may serve as novel biomarkers with diagnostic and prognostic significance in bladder cancer. ==== Body Introduction Bladder cancer is a major epidemiological problem, whose incidence continues to rise. The most recent cancer statistic has estimated 73,510 new cases and 14,880 estimated deaths in the United States for 2012 [1]. The majority of bladder tumors (∼70%) are low-grade noninvasive papillary tumors that do not penetrate the epithelial basement membrane (Ta stage). The remainder comprise tumors that have penetrated the basement membrane but not invaded the muscle layer of the bladder wall (T1 stage) and muscle-invasive tumors (T2, T3 and T4 stages) [2], [3], [4]. The prognosis for low-grade tumors is generally good, but about 10%–15% of these patients will later develop invasive disease. For invasive tumors the prognosis is much less favorable, with only 50% survival at 5 years. Invasive tumors frequently progress to life-threatening metastases, which is associated with a 5 year survival rate of 6% [3], [4]. Thus, understanding the mechanisms that regulate bladder tumor invasion is critical to predict and treat this devastating condition in bladder cancer patients. It is well established that the insulin-like growth factor receptor I (IGF-IR) plays a critical role in cell growth both in vitro [5] and in vivo [6]. Mice with targeted ablation of the IGF-IR gene have severe growth retardation, being only 45% the size of wild-type littermates [7], [8]. Studies performed in mouse embryo fibroblasts derived from the IGF-IR-deficient mice (R- cells) have really underscored the essential role of the IGF-IR in transformation [9]. R-cells are indeed refractory to transformation induced by several tumorigenic agents (viral oncogenes such as Ras and SV40 large T Ag, as well as over-expressed PDGFR and EGFR, and various chemical agents) but are transformed upon IGF-IR re-expression [10], [11]. Experiments on tumor cell lines and epidemiological studies have confirmed that activation of the IGF-IR is involved in the development of many common neoplastic diseases, including carcinomas of lung, prostate, pancreas, liver, colon and breast [10], [12], [13]. The transforming capability of the IGF-IR most likely depends on its ability to protect cancer cells from apoptosis [11], [12], [14], [15], [16]. We have recently demonstrated that the IGF-IR is upregulated in invasive and high-grade bladder cancer tumor tissues compared to low-grade and normal tissue controls and promotes motility and invasion of urothelial cancer cells [17], [18]. Significantly, IGF-IR activation did not induce cell proliferation of bladder cancer cells, indicating that the IGF-IR acts as a “scatter factor” for urothelial carcinoma-derived cells and may regulate the transition to the invasive stage of bladder cancer [17]. We also showed that IGF-IR-dependent cell motility and invasion required the activation of the Akt and MAPK pathway [17], [18] and Akt- and ERK-dependent activation of paxillin, which upon IGF-I-stimulation colocalized with focal adhesion kinase (FAK) in dynamic adhesions at the leading edge of migrating urothelial cancer cells and was critical for IGF-I-induced motility of these cells [17]. Here we show that while FAK was not required for IGF-IR-dependent signaling and motility of invasive urothelial carcinoma cells, the FAK-related Pyk2 [19], [20] was strongly activated by IGF-I in urothelial carcinoma cells, was critical for IGF-IR-dependent motility and invasion and regulated IGF-I-dependent activation of the Akt and MAPK pathways. We also discovered that Pyk2 is overexpressed in bladder cancer tissues compared to normal tissue controls and that there is a striking increase in Pyk2 translocation to the nuclei of these malignant cells. Collectively, these results provide novel information toward a better understanding of the mechanisms that regulate tumor progression in bladder cancer and suggest that Pyk2 and the IGF-IR may be critical for the transition to the invasive phenotype. In addition, these studies could potentially contribute to the identification of novel targets for therapeutic intervention in bladder tumors. Results FAK Activity in the Regulation of IGF-I-induced Migration, Invasion and Signaling We recently discovered that paxillin plays an important role in regulating IGF-IR-dependent motility of urothelial carcinomas [17]. It is well established that FAK regulates paxillin activation [21] and the assembly/disassembly of focal adhesions (adhesion turnover) at the cell front of migrating cells [22]. However, it is not yet established whether FAK or its homolog Pyk2 [19], [20], which is also expressed by urothelial cancer cells, may play a role in regulating IGF-I-induced motility of bladder cancer cells. Thus, we first employed small interfering RNA (siRNA) strategies to transiently deplete endogenous FAK in 5637 invasive urothelial carcinoma cells and then assessed FAK function in the regulation of IGF-I-induced motility and invasion. We reached a very significant depletion of endogenous FAK with the anti-FAK siRNA (Figure 1A). The oligos were specific for FAK insofar as there was no effect on Pyk2 expression levels (Figure 1A). Notably, FAK depletion did not induce a statistically-significant decrease in IGF-I-mediated migratory response in 5637 cells compared to either parental or scrambled oligos-transfected cells (Figure 1B). However, the invasive ability of 5637 cells was reduced, although at levels barely below statistical significance (P = 0.046) as compared to control oligos-transfected cells (Figure 1C). In addition, using immunoblot analysis with phospho-specific antibodies, we discovered that FAK depletion did not affect IGF-I-mediated activation of the MAPK or Akt pathways (Figure 1D), which are both necessary for IGF-IR-dependent motility and invasion of urothelial cancer cells [17], [18]. 10.1371/journal.pone.0040148.g001Figure 1 FAK is not important for IGF-I-mediated motility and signaling of invasive urothelial cancer cells. (A) 5637 cells were transfected with the FAK siGenome pool or control oligos. After 72 hours FAK and Pyk2 expression was detected by immunoblot with specific antibodies. Blot is representative of three independent experiments with an average FAK depletion level of 93.3±3.5 (arbitrary units) as assessed by densitometric analysis (B and C) Migration and invasion assays of 5637 cells were performed as described in Materials and Methods and assessed after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. *P = 0.046. (D) FAK-depleted 5637 cells were tested for Akt and MAPK activation after 10 minutes of IGF-I stimulation using a mix of phospho-specific antibodies (PathScan Cocktail I). eIF4E monitors protein loads. Blot is representative of three independent experiments. Collectively, these results do not support a critical role for FAK in regulating IGF-IR-dependent motility and invasive capability of urothelial cancer cells. Pyk2 is Critical for IGF-I-induced Motility, Invasion and Signaling It is known that Pyk2 can promote both distinct and overlapping signaling events with FAK [23], [24]. As we could not establish a major role for FAK in IGF-I-evoked motility of urothelial cancer cells, we considered the alternate hypothesis that Pyk2 is a predominant intracellular kinase that could mediate the downstream signaling pathway triggered by activation of the IGF-IR in urothelial cancer cells. First, we discovered that IGF-I stimulation of 5637 cells induced a prolonged Pyk2 activation, which was sustained for up to 2 hours, as determined by immunoblot with anti-Phospho-Pyk2 antibody (Figure 2A). Second, we performed transient transfection assays in 5637 cells and determined that overexpression of wild type Pyk2 significantly increased IGF-I-induced migration, which was inhibited by the expression of a kinase-dead dominant negative Pyk2 (**P<0.01, compared to V-transfected cells) (Figure 2B). Proper expression of Flag-tagged wild-type or kinase-dead Pyk2 proteins was determined by immunoblot with anti-Flag antibodies (Figure 2C). 10.1371/journal.pone.0040148.g002Figure 2 IGF-I-activated Pyk2 is critical for IGF-IR-dependent motility of invasive urothelial cancer cells. (A) Serum-starved 5637 cells were stimulated with 50 ng/ml of IGF-I for the indicated time points. Pyk2 phosphorylation was detected by immunoblot using anti-phospho-Pyk2 (Tyr402) antibodies, while total Pyk2 protein level was assessed using anti-Pyk2 polyclonal antibodies. Blot is representative of two independent experiments. (B) Migration of 5637 cells transiently transfected with either Flag-tagged wild type (PYK2 WT) or a dominant negative (KD PYK2) Pyk2 proteins was assessed after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. ** P<0.01. (C) Expression levels of transiently transfected Pyk2 proteins were assessed by immunoblot with anti-flag M2 antibodies. Blot is representative of two independent experiments. Next, to confirm Pyk2 function, we depleted endogenous Pyk2 in 5637 cells by siRNA approaches. Pyk2 depletion (Figure 3A) severely inhibited IGF-I-induced tumor cell migration (Figure 3B) and invasion through Matrigel™ (Figure 3C). Interestingly, Pyk2 depletion slightly upregulated FAK levels (Figure 3A), although FAK was unable to compensate for Pyk2 loss. In addition, Pyk2 knockdown in 5637 cells affected IGF-IR-downstream signaling and inhibited IGF-I-dependent activation of Akt and ERK1/2 and downstream effectors S6K and p90RSK (Figure 3D). 10.1371/journal.pone.0040148.g003Figure 3 Pyk2 is critical for IGF-IR-induced motility, invasion and signaling of invasive urothelial cancer cells. (A) 5637 cells were transfected with the Pyk2 siGenome pool or control. After 72 hours Pyk2 and FAK expression was detected by immunoblot with specific antibodies. Blot is representative of three independent experiments with an average Pyk2 depletion level of 92.6±3 (arbitrary units) as assessed by densitometric analysis (B and C) Migration and invasion of 5637 cells were assessed as described in Materials and Methods after 16 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. *P<0.05; **P<0.01. (D) Pyk2-depleted 5637 cells were tested for the activation of the Akt and MAPK pathways after 10 min of IGF-I stimulation using a mix of phospho-specific antibodies (PathScan Cocktail I). eIF4E monitors protein loads. Blot is representative of three independent experiments. To corroborate our results on Pyk2 function, we transiently depleted by siRNA approaches endogenous Pyk2 in T24 cells, another IGF-I-responsive invasive urothelial cancer cell line [17], [18]. We achieved a significant reduction in Pyk2 levels (Figure S1A) with a concurrent reduction in the ability of T24 cells to migrate (Figure S1B) and invade (Figure S1C) in response to IGF-I stimulation (*P<0.05, compared to either mock transfected or control oligo-transfected cells). In addition, Pyk2 ablation in T24 cells was associated with reduced IGF-I-dependent activation of ERK1/2 and S6K, while Akt and p90RSK activation was not affected (Figure S1D). Collectively, our findings reveal an essential role for Pyk2 in the IGF-IR functional regulation of tumor cell motility and invasion, key properties of the aggressive cancer phenotype. Pyk2 colocalizes with the IGF-IR and Complexes with IRS-1/2 and Grb2 in Urothelial Cancer Cells To determine whether Pyk2 may interact with the IGF-IR in 5637 cells, we initially performed co-immunoprecipitation assays but we were unable to detect an interaction between endogenous IGF-IR and Pyk2 proteins. Thus, we used confocal microscopy analysis to determine whether Pyk2 may colocalize with the IGF-IR in 5637 urothelial cancer cells. While in serum-starved 5637 cells Pyk2 did not colocalize with the IGF-IR (Figure 4A), 30 minutes of IGF-I stimulation induced significant colocalization of endogenous Pyk2 and IGF-IR proteins (Figure 4A) suggesting that Pyk2 may be recruited to the IGF-IR upon ligand stimulation. 10.1371/journal.pone.0040148.g004Figure 4 Pyk2 colocalizes with the IGF-IR and complexes with IRS-2 and Grb2 after IGF-I stimulation of urothelial cancer cells. (A) 5637 cells were serum-starved over night and then treated with 50 ng/ml of IGF-I for 30 minutes. After fixation, cells were labeled with a monoclonal anti-IGF-IR (green) and a polyclonal anti-Pyk2 (red) and imaged by confocal microscopy. The pictures of merged fields show colocalization (yellow) of IGF-IR and Pyk2 in the IGF-I treated cells (arrows) but not in unstimulated control cells. The distinct co-localization of Pyk2 and IGF-IR is detectable in the Z stacks (yellow staining, bottom panel). Pictures are representative of at least 10 independent fields from two independent experiments. An average of 300 cells was examined for each condition. Bar: 10 µm. (B) 5637 and(C) T24 bladder cancer cells were serum-starved for 24 hours and then stimulated with 50 ng/ml of IGF-I for 30 minutes. Two mg of cell lysates were immunoprecipitated with anti-Pyk2 polyclonal antibodies. IRS-1, IRS-2, Grb2 and Pyk2 levels were assessed by immunoblot with specific polyclonal antibodies. Blots are representatives of three independent experiments. Next, to investigate the mechanisms by which Pyk2 regulates IGF-IR downstream signaling, we performed co-immunoprecipitation assays in both 5637 and T24 cells. The main goal of these studies was to determine whether Pyk2 would complex with the docking proteins IRS-1 and/or IRS-2 or Grb2 adaptors, known to regulate IGF-IR-dependent activation of the Akt and MAPK pathways, respectively [25], [26], [27]. In 5637 cells, IRS-1 was detectable in complex with Pyk2 in unstimulated cells but uncoupled from Pyk2 after 30 minutes of IGF-I stimulation (Figure 4B). In contrast, IRS-2 binding to Pyk2 was barely detectable in serum-starved 5637 cells but strongly increased after IGF-I stimulation (Figure 4B). Grb2 recruitment to Pyk2 was detectable in unstimulated 5637 cells but it was strongly enhanced after ligand stimulation (Figure 4B). The same results were recapitulated in T24 cells with only some differences in the levels of IRS-1, IRS-2 and Grb2 detectable in Pyk2 co-immunoprecipitates (Figure 4C). These qualitative differences may be likely due to differences in the relative abundance of these proteins in 5637 and T24 cells as in fact 5637 cells express higher level of Pyk2 proteins compared to T4 (data not shown). In addition, the interaction between Pyk2, IRS-1 and IRS-2 could be indirect and mediated by additional associated proteins, which may differ between 5637 and T24 cells. These results indicate that Pyk2, by recruiting IRS-2 and Grb2, could play a critical role in regulating IGF-IR-dependent activation of downstream signaling pathways required for motility and invasion of urothelial cancer cells. Pyk2 is Overexpressed in Bladder Cancer Tissues We have recently shown that the IGF-IR is overexpressed in invasive bladder cancer tissues compared to normal tissue controls [17] and IGF-IR levels increase with bladder cancer progression [18]. Thus, we determined the expression of Pyk2 in a well annotated bladder cancer tissue microarray using immunofluorescence and AQUA analysis (Automated Quantitative Analysis) [28]. Pyk2 expression significantly increased in various bladder cancer tissues types (Figure 5A and B) as compared to normal tissue controls. In addition, the AQUA analysis for Pyk2 expression in various cellular compartments revealed that there was a significantly higher level of Pyk2 expression in the nuclei of urothelial cancer tissue cells when compared to cells in normal tissues (*P = 0.012, Figure 5C). 10.1371/journal.pone.0040148.g005Figure 5 Pyk2 is up-regulated in bladder cancer tissues. (A) Pyk2 expression on a bladder cancer tissue microarray was determined by immunofluorescence and AQUA analysis using the AQUA PM-2000 system (HistoRx, Inc). Automated quantification and statistics on the different types of bladder cancer tumor tissues (B) and in the cytoplasmic and nuclear fractions of urothelial carcinoma cells (C) was calculated by AQUA Software. (B) *P<0.05. **P<0.01 compared to normal tissue controls. (C) *P = 0.012 compared to non-neoplastic nuclear fraction. Nuclear Pyk2 staining is better visualized at higher magnification of selected field of normal and urothelial carcinoma tissues (Figure S2). Collectively, our results have identified a novel protein in the IGF-IR pathway that may be critical for bladder cancer. They also provide the first evidence that Pyk2 may translocate into the nuclei of bladder cancer cells. In addition, Pyk2 may serve in conjunction with the IGF-IR as a novel diagnostic and possibly prognostic biomarker for bladder cancer. Discussion The molecular mechanisms that determine malignant transformation of urothelial cells in the bladder are still very poorly characterized. In addition, there is an urgent need to identify proteins that may play a key role in driving the progression to the invasive and possibly metastatic phenotype in bladder neoplasia [2], [3], [4]. We have recently established that activation of the IGF-IR does not evoke in vitro cell proliferation but promotes motility and invasion of urothelial cancer cells [17], [18]. These results support the hypothesis that the IGF-IR may not be so critical for bladder cancer initiation, but may play a prominent role during progression to the invasive and possibly metastatic stage of bladder cancer. Based on our previous observation that upon IGF-I-stimulation FAK localizes with paxillin at dynamic adhesion sites of migrating cells [17], we investigated whether FAK, or its homolog Pyk2, would modulate IGF-IR action in urothelial cancer cells. We demonstrate that: (i) Depletion of endogenous FAK protein by siRNA strategies does not affect IGF-I-dependent motility and signaling of 5637 urothelial cancer cells. (ii) The FAK homolog Pyk2 is activated upon IGF-I stimulation of 5637 cells. (iii) Transient expression of wild type Pyk2 enhances IGF-I-induced migration, which is severely inhibited instead by the expression of a dominant-negative kinase-dead Pyk2 mutant. (iv) Pyk2 depletion by siRNA approaches inhibits IGF-I-dependent migration and invasive ability of 5637 and T24 cells and affects IGF-IR downstream signaling. (v) Upon IGF-I stimulation Pyk2 complex with IRS-2 and Grb2 in 5637 and T24 urothelial cancer cells. (vi) Pyk2 is overexpressed in various bladder cancer tissue types compared to normal tissue controls. (vii) Pyk2 expression increases in the nuclei of urothelial cancer tissue cells compared to normal tissue cells. FAK and Pyk2 are related tyrosine-kinases involved in the dynamic regulation of the actin cytoskeleton, a process critical for cell motility, mitosis and tumor progression [24], [29]. FAK and Pyk2 share a conserved molecular architecture and exhibit an overall 45% sequence identity with the greatest sequence identity (60%) in the kinase domain [24], [29]. FAK is ubiquitously expressed while Pyk2 expression has a more limited tissue distribution with the highest Pyk2 expression levels detected in cells of the central nervous system and in hematopoietic lineage [29]. In addition, FAK and Pyk2 differs for their intracellular distribution, with FAK prevalently expressed at focal adhesions while Pyk2 expression is more distributed throughout the cell and sometimes enriched in perinuclear regions [29]. We have recently shown that IGF-I stimulation of invasive urothelial cells induces paxillin phosphorylation at Tyrosine 31 [17], a process mediated by FAK in other cellular models [21]. We further showed that paxillin localizes with FAK at the leading edge of migrating cells [17]. Because in other tumor models FAK is required for PI3K- and Ras-dependent tumorigenesis [30] and the integrins/FAK complex activates Ras signaling to MAPK [31], [32] a plausible mechanism by which IGF-I promotes migration and invasion of bladder cancer cells would be by activating FAK and the signaling cascade leading to Akt, MAPK and paxillin activation. Surprisingly, FAK depletion in 5637 cells had no effect in modulating both IGF-I-induced migration and IGF-IR-dependent activation of the Akt and MAPK pathways. The modest inhibitory effect on invasion detected in FAK-depleted 5637 cells in the absence of MAPK and Akt inhibition suggest that additional MAPK- and Akt-independent pathways may partially contribute to FAK-dependent invasive signaling in these cells. However, we discovered that altering Pyk2 expression by transient overexpression of either wild type or dominant-negative Pyk2 proteins, or by siRNA-mediated Pyk2 depletion, had a major effect on IGF-I-induced motility and invasive ability of 5637 and T24 urothelial cancer cells. These functional assays were further corroborated by biochemical assays showing a significant inhibition of IGF-IR-activation of downstream signaling when intracellular Pyk2 levels were reduced. Thus, these results suggest that Pyk2 may have a more prevalent role than FAK in regulating IGF-IR-dependent biological responses in invasive urothelial cancer cells. As Pyk2 depletion severely inhibits IGF-I-induced signaling, it could be argued that the effects of Pyk suppression on migration are a consequence of reduced proliferation/survival. However, we have previously shown that in both 5637 and T24 urothelial cancer cells the ability of IGF-I to induce motility (migration and invasion) is totally independent from the IGF-IR ability to sustain proliferation/survival, as in fact IGF-I does not enhance cell growth in these cells, which proliferate in the absence of serum [17]. To investigate the mechanisms by which Pyk2 may regulate IGF-I-dependent biological responses in urothelial cells, we initially assessed by confocal microscopy whether upon IGF-I-stimulation Pyk2 colocalized with paxillin in focal adhesions. However, in both serum-starved and IGF-I-stimulated 5637 and T24 cells we could not detect any colocalization of Pyk2 and paxillin, and Pyk2 staining was more diffuse throughout the cytoplasm and not enriched in focal adhesions (not shown). In addition, we performed co-immunoprepitation experiments in which we failed to detect a Pyk2/paxillin complex (not shown). These results strongly indicate that Pyk2 action in regulating IGF-I-dependent motility of urothelial cancer cells can be separated from paxillin function at focal adhesions. Ligand-dependent recruitment of IRS-1/2 and Grb2 proteins to the IGF-IR is a critical step in the activation of the Akt and MAPK pathways in various IGF-IR-dependent biological responses [25], [33], [34], [35], [36]. Interestingly, IGF-I stimulation of 5637 and T24 urothelial cancer cells evoked the formation of a complex containing Pyk2, IRS-2 and Grb2 suggesting that in urothelial cancer cells Pyk2 may work as a critical signaling hub downstream of the IGF-IR. Whether Pyk2 binds directly to the IGF-IR and mediates the recruitment of IRS-2 and Grb2 to the receptor has not been demonstrated. So far we have not being able to detect an interaction between the IGF-IR and Pyk2 by co-immunoprecipitation experiments in 5637 cells but this negative result could be likely attributed to the relative low level of endogenous proteins. On the other hand, this result could also indicate that the IGF-IR and Pyk2 may interact indirectly in a complex with other signaling molecules of the IGF-IR system, such as IRS-1 and IRS-2. However, we have demonstrated by confocal microscopy that the IGF-IR and Pyk2 colocalize in ligand-dependent fashion suggesting that Pyk2 upon IGF-I stimulation may complex with the IGF-IR and facilitate the recruitment of signaling molecules to the receptor. Recent experiments in vascular smooth muscle cells have demonstrated that upon IGF-I stimulation Pyk2 mediates the recruitment of Grb2 to the signaling SHP-1/SHP2/Src complex thus promoting MAPK activation and cell proliferation [37]. However, whether a similar mechanism may be conserved in urothelial cancer cells remains to be elucidated. Our recent data have demonstrated that the IGF-IR is overexpressed in invasive bladder cancer tissues compared to normal tissue controls [17] and IGF-IR levels increase with bladder cancer progression [18]. The AQUA analysis we performed shows that Pyk2 expression is significantly upregulated in various bladder cancer tissue subtypes compared to normal controls but we could not detect a statistically significant difference in Pyk2 expression levels associated with different stages of urothelial carcinoma. A study with a larger sampling representing different stages of urothelial carcinoma is required to clearly establish whether Pyk2 may work as a prognostic marker for bladder cancer progression. Interestingly, in urothelial carcinoma cells the AQUA analysis revealed a statistically significant increase in the fraction of Pyk2 detected in the nucleus compared to cells in normal controls. Pyk2 localization in the nucleus has been previously demonstrated [38], [39] but the function of Pyk2 in the nucleus has not been characterized. Our results provide the first evidence of increased levels of nuclear Pyk2 in bladder cancer cells thereby suggesting the novel hypothesis that in bladder cancer cells IGF-I-activated Pyk2 may act not only in the cytoplasm but also translocate into the nucleus, where it might work as a transcription factor. Significantly, IRS-1 and IRS-2 proteins have been shown to translocate to the nucleus in several cancer cell models [40], [41], [42], [43], where they regulate gene expression [43], [44]. In addition IRS-1 level in the nucleus predicts tamoxifen response in patients with early breast cancer [45], Thus, our results suggest the attractive hypothesis that IRS-1 or IRS-2 proteins may play a role in regulating Pyk2 translocation and/or interact with Pyk2 in the nucleus. Experiments are currently under way to determine whether Pyk2 nuclear translocation is detectable in various urothelial cancer cell lines and is mediated by IGF-I. Future experiments will also determine IRS-1 or IRS-2 action in regulating Pyk2 nuclear translocation and function. In conclusion, we have identified Pyk2 as a novel critical regulator of IGF-IR-dependent motility and invasion of urothelial cancer cells. These studies will greatly contribute to the identification of novel targets for therapeutic intervention in bladder tumors. In addition IGF-IR and Pyk2 may work as novel biological markers for bladder cancer progression. Materials and Methods Cells and Materials Urothelial carcinoma-derived human 5637 and T24 cells were obtained from ATCC (Manassas, VA, USA. 5637 and T24 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS). Serum-free medium (SFM) is DMEM supplemented with 0.1% bovine serum albumin and 50 µg/mL of transferrin (Sigma-Aldrich, St Louis, MO, USA). Recombinant IGF-I was purchased from Calbiochem (San Diego, CA, USA). siRNA-mediated Gene Silencing To silence FAK or Pyk2 we used RNA interference by using small-interfering RNA (siRNA). 5637 and T24 cells were transfected with vehicle (DEPC-treated water), control siRNA (scrambled), or siRNA specific oligos (200–400 pmol/L) using the TransIT-siTKO reagent (Mirus Bio LLC, Madison, WI, USA). Both scrambled and anti-FAK or anti-PYK2 siRNA oligos were from Thermo Scientific Dharmacon (siGenome Smartpool siRNA) (Lafayette, CO, USA). Cells were analyzed for motility and signaling 72 hours post-transfection. siRNA efficiency was detected by immunobloting using anti-FAK (#3285) and anti-Pyk2 (#3090) polyclonal antibodies (both from Cell Signaling Technology, Beverly, MA, USA). ß-actin was detected using anti-ß-actin polyclonal antibody (Sigma-Aldrich). Densitometric analysis was performed using the ImageJ program (rsbweb.nih.gov/ij/). Transient Transfection Assays 5637 cells were transiently transfected using the TransIT®-Prostate Transfection Kit (Mirus BIO LLC) with the expression plasmid pShCMV.3X FLAG expressing either wild type or kinase-dead (K457A) Pyk2 mutant protein. Forty-eight hours post transfection, cells were serum-starved for additional 24 hours and then stimulated or not with 50 ng/mL of IGF-I. Migration was determined after 18 hours of incubation with the ligand, as stated below. In parallel, cells were lysed with cold RIPA buffer and the expression of the transfected plasmids was detected by western blot analyses using an anti-FLAG antibody (Santa Cruz Biotechnologies, Inc.). Migration and Invasion Assays 5637 or T24 cells were plated in duplicate at a density of 3×104 cells/35-mm2 plates in serum-supplemented medium. After 24 hours, cells were transferred to SFM or SFM supplemented with 50 ng/mL of IGF-I. Migration or invasion experiments were carried on for 4 hours or 16 hours, depending on the cell line used (T24 or 5637, respectively). Migration experiments were performed using HTS FluoroBloks™ inserts (BD, San Jose, CA, USA) as previously described [17], [18], [46], [47]. Membranes were mounted on a slide and migrated cells were counted and photographed with a Zeiss Axiovert 200 M cell live microscope at the Kimmel Cancer Center Bioimaging Facility. Cell invasion through a 3D-extracellular matrix was assessed using BD Matrigel™-coated Invasion Chambers (BD Biocoat) [17], [18], [47]. After 24 hours filters were washed, fixed, and stained with Coomassie Brilliant Blue. Cells that had invaded to the lower surface of the filter were counted under the microscope. Analyses of Protein/Protein Interactions 5637 or T24 cells were serum-starved for 24 hours and then stimulated with IGF-I (50 ng/mL) for 30 minutes. Cells were lysed in cold RIPA buffer without sodium deoxycholate. The insoluble material was separated by centrifugation and the supernatants were incubated at 4°C under rotation for 18 hours with anti-Pyk2 polyclonal antibody (Sigma-Aldrich). At the end of the incubation, immunocomplexes were separated by adding 30 µL of mix protein A/G-Sepharose for additional 30 minutes. The resolved proteins were reduced in 40 µL of Laemmli buffer and subjected to SDS-PAGE. IRS-1 and IRS-2 interactions were determined by immunoblot using Anti-IRS-1 and Anti-IRS-2 polyclonal antibodies from Millipore (Burlington, MA, USA). The anti-Grb2 monoclonal antibody is from BD Biosciences. Blots are representative of three independent experiments. Detection of Activated Signaling Pathways 5637 or T24 cells were serum-starved for 24 hours and then stimulated with IGF-I (50 ng/mL) for 5, 10, 30 and 120 minutes. Pyk2 phosphorylation was detected by immunoblot using anti-phospho-Pyk2 (Tyr-402) antibodies (Cell Signaling Technology). The activation of p90RSK, Akt, ERK1/2 and S6 Ribosomal Protein was analyzed by western immunoblot using the PathScan Multiplex Western Cocktail I (Cell Signaling Technology). ElF4E protein is used as control to monitor the loading of the samples. Confocal Microscopy 5637 cells were plated onto 4-well chamber slides (BD Bioscences) and serum-starved over night prior to treatment with 50 ng/ml of IGF-I for 10, 30 and 60 minutes. Cells were then washed with 1X PBS and fixed with 4% PFA for 30 minutes at room temperature. Subsequently, slides were subjected to immunofluorescence and confocal analysis as previously described [18], [46], [48], [49], [50]. Primary antibodies were anti-IGF-IR monoclonal (Calbiochem) and anti-Pyk2 polyclonal antibodies (Santa Cruz Biotechnologies). Secondary antibodies were goat anti-mouse IgG Alexa Fluor® 488 and goat anti-rabbit IgG Alexa Fluor® 594 antibodies (Invitrogen). Confocal analysis was performed on a Zeiss LSM810 microscope. The filters were set to 488 and 594 nm for dual channel imaging. All the images were then analyzed using Image J and Adobe Photoshop CS3 (Adobe Systems, San Jose, CA) software. Pyk2 Expression in Bladder Cancer Tissues Pyk2 expression levels in bladder cancer tissue were determined by AQUA analysis (Automated Quantitative Analysis) [28] on an Accumax bladder cancer tissue microarray (TMA #A215), composed by 4 non neoplastic spots and 45 different bladder cancer tissues (n = 33 urothelial carcinoma, n = 5 adenocarcinoma, n = 4 squamous carcinoma and n = 3 urothelial carcinoma in situ, two spots for each case). Detailed information regarding the TMA used is available on Accumax website. The antibodies used for immunofluorescence were rabbit pan-cytokeratin antibody (Cy2 conjugated, DAKO), Pyk2 antibody (Rabbit monoclonal YE353, Abcam) and DAPI. Pyk2 antibody was conjugated with Cy5 since it is outside the auto-fluorescence spectrum of tissue. Nuclear and cytoplasmic mask were automatically defined by AQUA Software, and applied to quantify Pyk2 expression on TMA. The analysis was performed at the Kimmel Cancer Center Translational Core Facility using an AQUA PM-2000 system (HistoRx, Inc). Automated quantification and statistics was calculated by AQUA Software. *P<0.05. **P<0.01 compared to normal. Statistical Analysis Experiments were carried out in triplicate and repeated at least three times. Results are expressed as mean ± SD. All statistical analyses were carried out with PRISM GraphPad Software, v.5. Results were compared using the two-sided Student’s t test. Differences were considered statistically significant at P<0.05. Supporting Information Figure S1 Pyk2 is critical for IGF-IR-induced motility, invasion and signaling of invasive urothelial cancer cells. (A) T24 cells were transfected with the Pyk2 siGenome pool or control. After 72 hours Pyk2 and FAK expression was detected by immunoblot with specific antibodies. Blot is representative of three independent experiments with an average Pyk2 depletion level of 93.4±3.5 (arbitrary units) as assessed by densitometric analysis (B and C) Migration and invasion of T24 cells were assessed as described in Materials and Methods after 4 hours of IGF-I stimulation. Values are expressed as fold change over SFM and represent mean ± SD. *P<0.05. (D) Pyk2-depleted T24 cells were tested for the activation of the Akt and MAPK pathways after 10 min of IGF-I stimulation using a mix of phosphor-specific antibodies (Pathscan cocktail I). eIF4E monitors protein loads. Blot is representative of three independent experiments. (TIF) Click here for additional data file. Figure S2 Pyk2 is up-regulated in urothelial carcinoma. Pyk2 expression on a bladder cancer tissue microarray was determined by immunofluorescence and AQUA analysis using the AQUA PM-2000 system (HistoRx, Inc). Higher magnification images from the same normal and urothelial carcinoma tissue samples shown in Figure 5 were acquired using a LEICA DM5500B microscope equipped with Leica Application Suite, Advanced Fluorescence 1.8 software (Leica Mycrosystem, Inc.) using a 63X Objective. Pictures are representative of at least 10 independent fields. Bar ∼10 µm. (TIF) Click here for additional data file. We thank Dr. Joseph C. Loftus, Mayo Clinic Arizona, for generously providing wild-type and Pyk2 mutated constructs. Competing Interests: The authors have declared that no competing interests exist. Funding: This work has been supported by the Benjamin Perkins Bladder Cancer Fund, the Martin Greitzer Fund, AIRC grant IG-10625/2011, AIRC project Calabria 2011 and Fondazione Cassa di Risparmio di Calabria e Lucania (to A.B.) and National Institutes of Health Grants RO1 DK068419 (A.M.) and RO1 CA39481 and RO1 CA047282 (R.V.I.). 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PLoS One. 2012 Jun 28; 7(6):e40148
==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, USA 22860118 PONE-D-12-07041 10.1371/journal.pone.0042361 Research Article Biology Biochemistry Glycobiology Glycoproteins Proteins Cytoskeletal Proteins Ion Channels Transmembrane Transport Proteins Biomacromolecule-Ligand Interactions Chemical Biology Chemistry Chemical Biology Medicine Infectious Diseases Neglected Tropical Diseases Leishmaniasis Chemistry Infectious Diseases Biochemistry Biology and Life Sciences Cell Biology Cellular Types Animal Cells Blood Cells Red Blood Cells Biology and Life Sciences Biochemistry Proteins Cytoskeletal Proteins Spectrins Medicine and Health Sciences Tropical Diseases Neglected Tropical Diseases Leishmaniasis Medicine and Health Sciences Parasitic Diseases Protozoan Infections Leishmaniasis Medicine and Health Sciences Infectious Diseases Zoonoses Leishmaniasis Biology and Life Sciences Biochemistry Metabolism Metabolic Processes Proteolysis Biology and Life Sciences Biochemistry Proteins Proteolysis Research and Analysis Methods Spectrum Analysis Techniques Spectrophotometry Cytophotometry Flow Cytometry Biology and Life Sciences Biochemistry Lipids Lipid Peroxidation Biology and Life Sciences Cell Biology Cellular Types Animal Cells Blood Cells White Blood Cells Macrophages Biology and Life Sciences Cell Biology Cellular Types Animal Cells Immune Cells White Blood Cells Macrophages Biology and Life Sciences Immunology Immune Cells White Blood Cells Macrophages Medicine and Health Sciences Immunology Immune Cells White Blood Cells Macrophages Medicine and Health Sciences Hematology Anemia Sialoglycosylation of RBC in Visceral Leishmaniasis Leads to Enhanced Oxidative Stress, Calpain-Induced Fragmentation of Spectrin and Hemolysis Spectrin Proteolysis and Hemolysis of RBC Samanta Sajal 1 Ghoshal Angana 1 ¤ Bhattacharya Kaushik 1 Saha Bibhuti 2 Walden Peter 3 Mandal Chitra 1 * 1 Cancer Biology and Inflammatory Disorder Division; CSIR-Indian Institute of Chemical Biology, Kolkata, India 2 Department of Tropical Medicine, School of Tropical Medicine, Kolkata, India 3 Department of Dermatology, Charité-Universitätsmedizin Berlin, Humboldt University Berlin, Germany Datta Kaustubh Editor University of Nebraska Medical Center, United States of America * E-mail: [email protected] Competing Interests: The authors have declared that no competing interests exist. ¤ Current address: Department of Zoology, Triveni Devi Bhalotia College, Burdwan, India Conceived and designed the experiments: SS AG KB CM. Performed the experiments: SS AG KB. Analyzed the data: SS AG KB PW CM. Contributed reagents/materials/analysis tools: CM BS. Wrote the paper: SS CM. 2012 31 7 2012 24 10 2019 7 7 e423616 3 2012 4 7 2012 Samanta et al https://creativecommons.org/licenses/by/4.0/ Except for Figure 1, 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. Visceral leishmaniasis (VL) caused by the intracellular parasite Leishmania donovani accounts for an estimated 12 million cases of human infection. It is almost always associated with anemia, which severely complicates the disease course. However, the pathological processes leading to anemia in VL have thus far not been adequately characterized to date. In studying the glycosylation patterns of peripheral blood cells we found that the red blood cells (RBC) of VL patients (RBCVL) express eight 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) that are not detected in the RBC of healthy individuals (RBCN). At the same time, the patients had high titers of anti-9-O-AcSGP IgG antibodies in their sera. These two conditions appear to be linked and related to the anemic state of the patients, as exposure of RBCVL but not RBCN to anti-9-O-AcSGPs antibodies purified from patient sera triggered a series of responses. These included calcium influx via the P/Q-type but not L-type channels, activation of calpain I, proteolysis of spectrin, enhanced oxidative stress, lipid peroxidation, externalization of phosphatidyl serine with enhanced erythrophagocytosis, enhanced membrane fragility and, finally, hemolysis. Taken together, this study suggests that the enhanced hemolysis is linked to an impairment of membrane integrity in RBCVL which is mediated by ligand-specific interaction of surface 9-O-AcSGPs. This affords a potential explanation for the structural and functional features of RBCVL which are involved in the hemolysis related to the anemia which develops in VL patients. This work received financial support from the CSIR-IICB, Department of Biotechnology (GAP235) (http://dbtindia.nic.in/index.asp), Indian Council of Medical Research (GAP266) (http://www.icmr.nic.in/), New Delhi, J.C. Bose Fellowship, Department of Science and Technology (P90807) (http://www.dst.gov.in/), Govt. of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body pmcIntroduction Leishmania donovani, the causative organism of visceral leishmaniasis (VL), is an obligatory intracellular parasite that resides and proliferates within the hostile environment of host macrophages [1]. Approximately 12 million humans suffer from VL with an incidence of 0.5 million cases per year and increasing prevalence on the Indian subcontinent [1]. The clinical spectrum of VL ranges from asymptomatic infection to mortality, if untreated. VL is usually associated with severe anemia which severely complicates the clinical courses and adds to the patients' suffering [2]. In general, the average life span of the erythrocytes of patients with VL (RBCVL) is significantly reduced [3]. Accordingly the hemoglobin content in blood of these patients is lower than in normal healthy individuals. Despite its profound impact on the patients' fate and chances for recovery, little is known about the pathological processes contributing to the hemolysis and anemia. Sialic acids (SA) are 9-carbon acidic sugars that constitute a family of monosaccharide and terminal components of glycoproteins and glycolipids that play very crucial roles in intercellular communication and defense against different pathogens under various pathological conditions [4]–[7]. Earlier we have demonstrated exclusive presence of eight VL-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) on RBCVL not detected on erythrocytes of healthy individuals [8]–[11]. Also the white blood cells of VL patients display different derivatives of sialic acids [12]–[21]. Additionally, high titers of anti-9-O-AcSGP IgG were found in the patients' sera [18], [22] whereas only a fraction of the polyclonal IgG2 purified from normal human serum shows specificity for 9-O-AcSA [23]–[24]. The structural integrity of mammalian erythrocyte is supported by a complex network of different cytoskeleton proteins which comprises of five to seven spectrin subunits linked to actin filaments [25]. Structural and biochemical changes of RBC that cause alterations in the cytoskeleton proteins may lead to degradation of the cell. Modifications like glycation and oxidation of spectrin have been documented in diabetes mellitus and associated to erythrocyte membrane changes [26]–[27]. We recently could demonstrate glycosylation and proteolytic cleavage of spectrin in RBC of VL patients [11]. With the present study we addressed the possible contribution of 9-O-AcSGPs associated with RBCVL and of anti-9-O-AcSGP IgGVL antibodies in VL. We report that anti-9-O-AcSGP IgG purified from serum of VL patients trigger a series of pathological events in RBCVL including (1) altered membrane properties as indicated by increased osmotic fragility, hydrophobicity, morphological changes like vesiculation, cell shrinkage, (2) influx of extracellular calcium ion (Ca2+) into the cell through P/Q-type channel and elevation of intracellular Ca2+ level, (3) activation of calpain I caused by elevated cytosolic Ca2+ accompanied by enhanced fragmentation of human erythrocytic α-spectrin to a 60 kDa 9-O-AcSGP (SGP-60) fragment, (4) enhanced production of reactive oxygen species (ROS) and lipid peroxidation, and (5) externalization of phosphatidyl serine (PS) and erythrophagocytosis of sensitized RBCVL. This study thus suggests a mechanism of hemolysis in VL patients that may relate to their anemic state. Materials and Methods Clinical samples The study involved clinically confirmed VL patients (Table 1, n = 40; 20 males, 20 females; median age: 30 years) admitted to the School of Tropical Medicine, Kolkata. The diagnosis of VL was based on WHO recommended microscopic demonstration of Leishmania sp. amastigotes in splenic aspirates [28]. Blood was sent to the Indian Institute of Chemical Biology where it was processed immediately and the diagnosis validated by two in-house techniques in which the increased presence of linkage-specific 9-O-AcSGPs on erythrocytes was quantified by an erythrocyte binding assay [9] and anti-9-O-AcSGP antibodies in serum or plasma were detected by ELISA [18], [29]. The serum was also checked for the level of parasite-specific antibodies by ELISA with parasite lysates as coating antigen [22]. The hematological parameters of the patients were indicative of anemia but no other blood cell disorder. Controls included normal healthy individuals from endemic (n = 20) and non-endemic areas (n = 20) of the median age 28 for age matched study. The Institutional Human Ethical Committee had approved the study and samples were taken with the consent of donors, patients. 10.1371/journal.pone.0042361.t001 Table 1 Clinical and laboratory features of patients with visceral leishmaniasis (VL). Parameters PatientVL Normal Number Male (n = 20), Female (n = 20) endemic area (n = 20), non-endemic area (n = 20) Age (yr) 30±2.25 28±4.32 Height (m) 1.55±0.72 1.65±0.52 Weight (kg) 38.50±4.25 60±5.25 Body Mass Index (BMI)a 16±0.55 22±1.25 Duration of illness (month) 4.02±0.25 Not applicable RBC count 0.92–2.5×106/µl 4.5–6.2×106/µl Leukocyte count (/mm3) 3.48–3.55×103 5–11×103 Hemoglobin concn. (g/dl) 5.32±0.32 11.5±2.2 Mean cell hemoglobin concn. (g/dl) 30–31 g/dl 30–36 g/dl Hematocrit (%) 35 41–53 Reticulocyte count (%) 4.25–5.05 0.5–2.5 Spleen size (cm) 10.72±1.06 Not palpable Splenic aspirate scoreb 4.05±0.17 Negative Bilirubin (mg/dl) 1.5–2.5 0.9–1 Albumin (g/dl) 3.0–3.5 3.5–5.5 Aminotransferase Normal Normal Alkalinephosphatase Normal Normal RBC-ELISAc 0.9–1.12 0.2–0.3 BSM-ELISAd 0.77–1.1 0.16–0.23 Parasite-ELISAe 1.1–1.7 0.22–0.32 a BMI is weight in kilograms divided by height square in meter, normal BMI = 18.5–24.9. b Splenic aspirate score is taken to be 4 when the number of parasites per microscopic field ranges from 1–10. c RBC-ELISA refers to the antigen-ELISA as described elsewhere [9]. The presence of 9-O-AcSGPs on RBCVL was determined by exploiting the binding specificity of a lectin, Achatinin-H, which has a restricted specificity towards 9-O-acetylated sialic acids [62] and therefore used as coating antigen. Briefly, Achatinin-H was immobilized in a 96-well plate and allowed to bind with RBC in 4°C for overnight. After washing, bound RBC was lysed by double distilled water. The extent of binding was determined by using a chromogenic substrate 2,7-diamino fluorine dihydrochloride and measuring absorbance values at 620 nm. d Anti-9-O-AcSGP antibody was detected by using BSM as coating antigen as described elsewhere [22]. e Parasite specific antibody was detected by using parasite lysate as coating antigen as described elsewhere [8]. Anti-9-O-AcSGP IgG and sensitization of erythrocytes Antibodies (IgG) specific for 9-O-AcSGPs were affinity purified from pooled sera from three patients (anti-O-AcSGP IgGVL) and normal healthy (NHS) individuals (anti-O-AcSGP IgGNHS), respectively, as described by Pal et al. [24]. Briefly, the succession of purification steps was 33% ammonium sulfate fractionation, removal of galactose-binding antibodies by passage through asialo-BSM Sepharose 4B, purification of anti-O-AcSGP by BSM (bovine submandibular mucin) Sepharose 4B affinity chromatograpgy, and isolation of IgG by Protein G affinity chromatography. The yield of anti-9-O-AcSGP IgG purified from 12 ml VL sera was 588±25 µg (49 µg/ml serum) and from 20 ml normal control sera 240±19 µg (12 µg/ml serum). For sensitization with anti-O-AcSGP IgG, RBC (1×107) were suspended in Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 32 mM HEPES, 5 mM glucose, 5 mM CaCl2, pH 7.4) and incubated with the antibodies at 37°C for 30 min. As positive controls, RBC were incubated with calcium ionophore (A23187, 1.0 µM, Sigma) in Ringer solution. Osmofragility of erythrocytes RBC (1×107) were incubated in NaCl concentrations from 0.1 to 0.9 g % as indicated for 1 h at 37°C and extent of hemolysis measured spectrophotometrically at 412 nm. Likewise, erythrocytes in Ringer solution were incubated with anti-O-AcSGP IgG antibodies (6 µg/ml) or A23187 (1 µM) or buffer only for 30 min at 37°C. After centrifugation at for 3 min 4000 rpm, the extent of hemolysis in supernatant was determined spectrophotometrically at 412 nm. Hemolysis in distilled water was taken as 100% lysis. Percent hemolysis (%) was calculated as OD412 nm at the given condition/OD412 nm with 100% lysis×100 [20]. Hydrophobicity measurements Hydrophobicity was detected before and after sensitization of RBC (1×107) with anti-9-O-AcSGP IgGVL antibodies (6 µg/ml) in phosphate buffered saline (PBS) at 37°C for 30 min. Sensitized erythrocytes were washed with PBS, suspended in PBS and loaded with ANS in PBS (5 µl, 1 mM) for 1 h at 37°C. The binding of ANS to hydrophobic sites on erythrocyte membrane was measured with a spectrofluorimeter (Perkin-Elmer, LS55, Exmax = 365 nm) as described elsewhere [20], [30]. Fluorescence emission spectra were recorded from 400 to 800 nm with excitation and emission band passes of 5 nm. Scanning electron microscopy (SEM) The morphology of RBC before and after sensitization with anti-9-O-AcSGP IgGVL antibodies (6 µg/ml) at 37°C for 30 min was done by SEM. Cells were fixed overnight with 2.5% glutaraldehyde in PBS followed by an overnight incubation with osmium tetroxide (1%), dehydration in an ethanol series, carbon dioxide by the critical point method, sputter coating with gold and examined with a SEM (Vegaii Lsu, Tescan, Czech Republic) [31]. Micrographs were taken at magnification of 18,000 and about 200 erythrocytes were counted to calculate the percentage of deformed cells. Measurement of reactive oxygen species RBC were incubated with 2′,7′-dichlorofluorescein diacetate (H2DCF-DA,100 µM) in PBS for 30 min at 37°C and sensitized with anti-9-O-AcSGP IgG. ROS generation was detected and quantified by measuring the fluorescence intensity at λex, = 485 nm and λem = 538 nm. As controls, ROS generation was determined after treatment of the RBC with N-acetyl cysteine (10 mM), a scavenger of ROS. The results are expressed as fold increase in comparison to untreated erythrocytes [32]. Lipid peroxidation Lipid peroxidation of erythrocyte membrane was measured by the thiobarbituric acid (TBA) method in which malondialdehyde (MDA), a product of peroxidation reaction of polyunsaturated fatty acids and thioberbituric acid-reactive species (TBA-RS) is used as indicator [33]. In brief, RBC membrane proteins were precipitated in twice volume of trichloro acetic acid (10%) for 30 min at 37°C and centrifuged at 1000× g for 10 min at 4°C. The cleared supernatant was incubated with thiobarbituric acid (0.67% in 7.1% sodium sulphate) at 100°C for 25 min in a water bath. After centrifugation at 1000× g for 10 min the absorbance of the pink color reaction product of MDA with TBA was measured at 535 nm. Calibration standards were generated with 1,1,3,3-tetramethoxypropane. Results are expressed as fold increase in comparison with unsensitized RBC. Phosphatidyl serine (PS) externalization RBC (1×106) in annexin V-binding buffer (10 mM HEPES, pH 7.4; 140 mM NaCl, 2.5 mM CaCl2) were incubated with FITC-labeled annexin V in the dark for 15 min at room temperature and analyzed by flow cytometry. A23187 (1.0 µM) treated and unsensitized RBC served as positive and negative controls, respectively. Erythrophagocytosis assay RBC (2×106) with or without sensitization with 6.0 µg/ml anti-O-AcSGP IgG for 30 min at 37°C, were layered over macrophages adhered on cover slips and incubated at 37°C for 1 h. Non-adherent erythrocytes were removed by gentle washing with PBS and cell surface-bound erythrocytes lysed by treatment with Tris-NH4Cl (140 mM NH4Cl, 17 mM Tris-HCl, pH 7.6) for 5 min. The slides were stained with diaminobenzidine for erythrocytes and counterstained with Giemsa stain for macrophages. Phagocytosis was calculated as the percentage of macrophages that had ingested one or more erythrocytes [34]. Measurement of cytoplasmic Ca2+ Erythrocytes (2×107) were washed in Ringer solution, loaded with Fluo-3/AM (2 µM, Calbiochem, Germany) in Ringer solution for 10 min at 37°C in dark under gentle shaking and washed twice with the same buffer [35]. Fluo 3-loaded erythrocytes were sensitization with buffer only or varying concentrations of anti-9-O-AcSGP IgG (0–40 µg/ml) in Ringer solution for 30 min at 37°C, washed twice and resuspended in same buffer and analyzed by spectrofluorimetry (λex 506 nm, λem 530 nm) and flow cytometry. Time dependent increase of intracellular calcium ion was also carried out by incubating RBC with anti-9-O-AcSGP IgG (2.5 µg/ml) for different time point at 37°C followed by flow cytometry. Calibration was done at the end of each experiment. To determine the Ca2+ channel type, Fluo 3-loaded RBCVL (2×107) in Ringer solution containing Ca2+ (5 mM) were preincubated without or with the indicated doses of ω-agatoxin TK, a spider venom peptide (P/Q-type Ca2+ channel blocker, Calbiochem) or nifedipine (L-type Ca2+ channel blocker, Sigma) for 20 min at 37°C, washed, sensitized with anti-9-O-AcSGP IgG (2.5 µg/ml) as above and analyzed by flow cytometry [35]. The Ca2+ concentrations were calculated as [Ca2+] = Kd [(F−Fmin)/(Fmax−F)] where Kd is the dissociation constant of the Ca2+ Fluo-3 complex (864 nM at 37°C), F fluorescence of anti-9-O-AcSGP IgG-treated RBC, Fmax the maximal fluorescence intensity with A23187 treated, Fmin corresponds to minimum fluorescence intensity with A23187 and EGTA [36]. Electrophoresis and Western Blotting RBC (2×107) were incubated or not with anti-9-O-AcSGP IgGVL (10 µg/ml) for 60 min at 37°C in Ringer solution, washed and lysed by sonication on ice. Cell lysates were centrifuged to remove debris, the proteins separated in SDS-PAGE (10% or 7.5%) and blotted onto nitrocellulose. The Western blots were developed separately with horse reddish-labeled anti-calpain I antibodies (1∶1000, Cell Signaling, USA) and rabbit anti-spectrin antibodies (1∶1000, Sigma, USA). Positive and negative controls were RBC treated with A23187 (1 µM) in Ringer solution for 30 min at 37°C in the absence or presence of EGTA (25 mM), respectively. For confirmation of specificity, RBC were preincubated (15 min, 37°C) or coincubated during sesitization with the calpain inhibitor I N-acetyl-leucyl-leucyl-norleucinal (ALLN; 200 µM, Sigma) in Ringer solution before sensitization or A23187 treatment. The blots were developed with diaminobenzidine and peroxide, scanned densitometrically and analyzed with the Quantity One software (BIO-RAD, USA). Calpain I assays Spectrin was purified separately from RBC as described by Ungewickell et al. [37] and confirmed by SDS-PAGE and Western blot analysis as above. Spectrin (3.0 µg) was digested with the indicated doses of active calpain I (Sigma) for 60 min at 24°C in reaction buffer (50 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM NaN3, 1 mM CaCl2, 1 mM DTT), the reactions stopped with EGTA (15 mM final concentration) [38] and the reaction product analyzed by SDS-PAGE. For specificity control calpain I (10 µg/ml) was pre-incubated with ALLN (150 µM) for 15 min on ice prior to addition of reaction buffer containing purified spectrin. Gels were stained by Coomassie brilliant blue and band densities were compared by densitometric analysis. Statistical analysis Results are reported as mean ± SD. All statistical analyses were done using Excel software (Microsoft Co.). The one or two-tailed t test for significance was performed, P<0.05 was considered significant. Results Alterations in the membrane characteristics of RBC in VL To test the stability of RBCVL in comparison to RBCN, the cells were incubated in NaCl solutions with at concentrations ranging from 0 to isoosmotic 0.9 g%, and the degree of hemolysis was determined spectrometrically. The osmofragility thus determined is an indicator of cell stability and alterations in membrane properties. RBCVL were osmotically more fragile than RBCN, as indicated by an approximately 3-fold enhancement of lysis at 0.5 g% NaCl (Fig. 1A). 10.1371/journal.pone.0042361.g001 Figure 1 Altered membrane properties and reduced stability of RBCVL after sensitization with anti-9-O-AcSGP IgGVL antibodies. A. The osmotic fragility of RBCVL (open square) compared to RBCN (filled square) was measured by degree of hemolysis with increasing osmotic stress by incubation in NaCl (0–0.9%) determined by spectrophotometry. Data are represented as % hemolysis, are means of triplicates ± SD and representative of three independent experiments. B. Sensitization of RBCVL for hemolysis with anti-9-O-AcSGP IgGVL antibodies. RBCVL (open bars) and RBCN (filled bars) were incubated for 30 min at 37°C with increasing concentration (0.5 µg/ml–12.0 µg/ml) of anti-9-O-AcSGP IgGVL and anti-9-O-AcSGP IgGNHS, respectively. The extent of hemolysis was then determined by spectrophotometry. The Spectrophotometric readings of RBCVL or RBCN in buffer under similar condition were taken as base values and subtracted from the experimental values. Data are expressed as means ± SD of three independent experiments. C. Kinetics of the hemolysis of RBCVL and RBCN after sensitization with anti-9-O-AcSGP IgGVL antibodies. RBCVL (open squares) and RBCN (filled squares) were incubated with equal (6.0 µg/ml) amounts of anti-9-O-AcSGP IgGVL and anti-9-O-AcSGP IgGNHS, respectively, for different time points (0–120 min). Base value was routinely subtracted from each experimental value as in Fig. 1B. Data are means ± SD of three independent experiments. D. Hemolysis of RBCN (filled bars) and RBCVL (open bars) after sensitization of RBCVL with anti-9-O-AcSGP IgGNHS or anti-9-O-AcSGP IgGVL (6.0 µg/ml), respectively, or incubation with the calcium ionophore A23187 (1 µM) in Ringer solution for 30 min. Data are means ± SD of three independent experiments. E. Enhanced membrane hydrophobicity of RBCVL after sensitization with anti-9-O-AcSGP IgGVL. The hydrophobicity was determined spectrofluorimetrically using ANS as indicator. RBCVL were incubated with anti-9-O-AcSGP IgGVL antibodies or PBS only for 30 min at 37°C. As controls, RBCN were incubated with anti-9-O-AcSGP IgGNHS. The cells were washed with PBS, loaded with ANS and incubated for 1 h at 37°C. The fluorescence emission spectra were recorded from 400 to 850 nm with excitation at 365 nm. The graphs are representatives of the results obtained from three independent experiments. F. Morphological changes of RBCVL after sensitization with anti-9-O-AcSGP IgGVL antibodies. RBCN and RBCVL were not sensitized or sensitized with anti-9-O-ACSGP IgGNHS or anti-9-O-AcSGP IgGVL antibodies, respectively, (6.0 µg/ml) for 30 min at 37°C, and then processed for SEM as described in Materials and Methods. Representative images from three independent experiments are shown. G. Decrease of RBCVL cell size after sensitization detected by low-angle scattering light flow cytometry (forward scatter, FCS). FCS was measured of RBCN (upper left panel) and of RBCVL without sensitization (upper right panel) or with sensitization with anti-9-O-AcSGP IgGVL (2.5 µg/ml) (lower left panel) or treatment with A23187 (1.0 µM) (lower right panel). Figure 1F is excluded from this article's CC-BY license. See the accompanying retraction notice for more information. Both RBCVL and RBCN were sensitized for 30 min, at 37°C with increasing concentrations of anti-9-O-AcSGP IgGVL and anti-9-O-AcSGP IgGNHS, respectively. Sensitization of RBCVL with 6.0 or 10.0 µg/ml of anti-9-O-AcSGP IgGVL resulted in a high percentage of lysis, i.e. 50.25±2.0% and 61±3.0%, respectively (Fig. 1B). An increase in the lysis of sensitized RBCVL with 6.0 µg/ml of anti-9-O-AcSGP IgGVL was observed over time compared to sensitized RBCN (Fig. 1C). Sensitization of RBCVL using 6.0 µg/ml of anti-9-O-AcSGP IgGVL for 30 min at 37°C resulted in a 4.5 fold higher lysis than un-sensitized RBCVL i.e. 63±4.0% vs. 14±2.0% (Fig. 1D). Sensitized RBCVL displayed a 5.7-fold higher lysis compared to sensitized RBCN. A23187 in the presence of Ca2+ induced the maximum possible hemolysis of the erythrocytes. RBCVL exhibited higher membrane hydrophobicity than RBCN, as indicated by increased fluorescence due to enhanced ANS binding. Sensitized RBCVL demonstrated a further increase in ANS binding and enhanced hydrophobicity compared to un-sensitized erythrocytes (Fig. 1E). A blue shift of the emission maxima from 544 to 500 nm in sensitized RBCVL suggested that sensitization increased the membrane hydrophobicity resulting in a greater number of accessible sites for the binding of ANS. Unsensitized RBCVL exhibited a higher hydrophobicity than sensitized RBCN (emission maxima at 560 nm). Unsensitized RBCN displayed only a negligible reading. SEM revealed sensitized RBCVL to have a greater number of ultra structural morphological changes compared to un-sensitized RBCVL, suggesting a stressed condition in these erythrocytes (Fig. 1F). The presence of shrunken RBCVL, as reflected by morphometric analyses indicating membrane alterations due to a sensitization of the 9-O-AcSGPs. The sensitized RBCN did not exhibit any noteworthy alterations of the membrane, retaining a normal discoid shape. Altered cell morphology in RBCVL For further demonstration of changes in cell size, we used flow cytometry to monitor the forward light scattering of RBCVL before and after sensitization. The forward scattering data (FSC) showed there were only 27% un-sensitized RBCVL in M1 as compared to 73% cells in M2 (Fig. 1G). In contrast, sensitized RBCVL exhibited a higher percentage (59%) of cells in M1. RBCN exhibited only 17% cells in M1. A considerably enhanced (80%) percentage of cells were observed in the A23187-treated RBCVL. Sensitized RBCVL exhibit enhanced ROS generation, lipid peroxidation and externalization of PS The generation of ROS is usually linked with membrane damage, specifically lipid-peroxidation, which may lead to membrane alterations such as PS externalization which are associated with cell death. As mature RBC is devoid of intracellular components such as nuclei and mitochondria, alterations may also result in these cells in a stressed condition. To address the question whether anti-9-O-AcSGP IgGVL is capable of inducing stress, we measured ROS generation, lipid peroxidation and PS-externalization in sensitized RBCVL. 5.7 and 3-fold higher levels of ROS and TBA-RS were observed in sensitized RBCVL compared to un-sensitized erythrocytes (Fig. 2A–B). RBCN displayed negligible ROS generation and insignificant lipid peroxidation. Sensitized RBCVL also showed enhanced annexin-V binding (32±5%) compared to un-sensitized (0.2±0.01%) erythrocytes, indicating a rapid externalization of PS, whereas sensitized and unsensitized RBCN demonstrated only a negligible percentage of annexin-V positive cells (Fig. 2C–D). A23187 treated RBCVL exhibited an increase in annexin-V positivity and served as a positive control. 10.1371/journal.pone.0042361.g002 Figure 2 Enhanced ROS, lipid peroxidation and externalization of phosphatidyl serine (PS) in RBCVL after sensitization with anti-9-O-AcSGP IgGVL. A. Induction of ROS in sensitized RBCVL. Prior to sensitization with anti-9-O-AcSGP IgGVL or anti-9-O-AcSGP IgGNHS RBCVL and RBCN were incubated with the hydroperoxide indicator H2DCF-DA in PBS for 30 min at 37°C. Then the antibodies were added, the cell incubated as before and ROS generation determined by fluorimetry. As controls, ROS generation was determined after prior treatment of the RBC with N-acetyl cysteine, a quencher of hydroperoxides. The results are expressed as means ± S.D. (n = 5) of fold increases in comparison to the fluorescence levels detected with untreated erythrocytes. B. Increased lipid peroxidation in sensitized RBCVL. Lipid peroxidation of erythrocyte membrane was detected and quantified with thiobarbituric acid (TBA) and the TBA-reactive species (TBA-RS) measured with a spectrophotometer at 532 nm. The results are mean ± S.D of fold increase in comparison with unsensitized RBC of five independent measurments. C–D. Enhanced externalization of phosphatidylserine on sensitized RBCVL. Sensitized or unsensitized erythrocytes were incubated in annexin-V binding buffer with FITC-annexin-V and analyzed by flow cytometry. A23187-treated RBC in the presence of Ca2+ served as positive control. The results are shown as representative histogram of three independent experiments (C) and as comparative flow-cytometric analysis (D). Sensitized RBCVL exhibit increased erythrophagocytosis The exposure of PS on the outer leaflet of the plasma membrane is one of the signals that induce macrophages to bind and ingest apoptotic cells [39]. Sensitization of 9-O-AcSGPs on RBCVL by anti-O-AcSGP IgGVL antibodies resulted in an increase in phagocytosis of RBCVL compared to unsensitized, as evidenced by the increase in the percentage of positive macrophages that ingested one or more erythrocytes, from 3.0±1.0 to 52.0±5.0 (Table 2). The extent of phagocytosis may be dependent on the externalization of PS, as suggested by the good correlation (r = 0.92) with annexin-V positivity. In contrast, RBCN demonstrated negligible uptake by macrophages under identical conditions. 10.1371/journal.pone.0042361.t002 Table 2 Erythrophagocytosis Assay. RBCN RBCVL a Group bPositive macrophages (%) uptaking diseased erythrocytes RBC 2±1 3±1 Sensitized RBC 3±2 52±5 a RBCVL and RBCN (2×106), without or with sensitization by anti-O-AcSGP IgGVL and anti-O-AcSGP IgGNHS antibodies and processed for erythrophagocytosis assay as described in Materials and Methods. Results represent the mean ± S.D. of five separate determinations. b Positive macrophages (%) are the percentage of macrophages that ingested one or more erythrocytes and was used as the index of phagocytosis. Results represent the mean ± S.D. of five separate determinations. Intracellular accumulation of Ca2+ in sensitized RBCVL Spectrofluorimetric data suggested that the cytosolic Ca2+ ion content of un-sensitized RBCVL is slightly higher than RBCN, suggesting a stressed condition in VL (Fig. 3A). However, the sensitization of 9-O-AcSGPs on Fluo-3-loaded RBCVL showed a significant increase in fluorescence with increasing concentrations of anti-9-O-AcSGP IgGVL antibodies, indicating an enhanced Ca2+ influx as compared to the un-sensitized erythrocytes (Fig. 3B). 10.1371/journal.pone.0042361.g003 Figure 3 Calcium influx into RBC upon sensitization with anti-9-O-AcSGP IgGVL antibodies. For all experiments, erythrocytes were washed and loaded with the Ca2+-indicator fluorochrome Fluo-3/AM in Ringer solution. A23187-treated Fluo-3/AM-loaded RBCVL served as positive controls (black line) and A23187-treated cells in the presence of EGTA as negative controls (gray background). A. Fluo-3/AM loaded RBCVL and RBCN (2×107) were left unsensitized or sensitized with varying concentrations of anti-9-O-AcSGP IgGVL or anti-9-O-AcSGP IgGNHS, respectively, (0–40 µg/ml) in Ringer solution for 30 min at 37°C. After washing the cells twice with same solution the fluorescence intensities of the Ca2+-Fluo-3 complexes was determined by spectrofluorimetry and the levels of intracellular calcium calculated as described in Materials and Methods. B. Cells treated as in A with 0.5 µg/ml (red line), 1.0 µg/ml (green line) and 2.5 µg/ml (pink line) anti-9-O-AcSGP IgGVL were analyzed by flow cytometry to determine the fraction of responding RBC. C. Similarly, RBCN were analyzed without and with sensitization with 5 µg/ml anti-9-O-AcSGP IgGNHS (blue line). D–E. Time dependent increase of intracellular Ca2+ in RBCVL after sensitization with anti-9-O-AcSGP IgGVL (2.5 µg/ml) for 5 min (red line), 10 min (green line), 20 min (pink line) and 30 min (blue line) at 37°C. Cells were washed and analyzed by flow cytometry. D. Histogram representation; E. Mean fluorescence intensities calculated from D. F–G. Inhibition of the Ca2+ influx into RBC sensitized with anti-9-O-AcSGP IgGVL. Prior to sensitization with anti-9-O-AcSGP-IgGVL (2.5 µg/ml), the cells were incubated without (green line) or with the P/Q-type channel blocker ω-agatoxin TK (1 µM, pink line, F), or without (green line) or with the L-type channel blocker nifedipine (10 µM, blue line, G) and analyzed by flow cytometry. Sensitized Fluo-3-loaded RBCVL also displayed enhanced fluorescence with an increasing concentration of anti-9-O-AcSGP IgGVL antibodies as compared to un-sensitized erythrocytes, as determined by flow cytometry (Fig. 3C). It is noteworthy that not all of the cells responded equally to stimulation at the lower doses of the antibody (1.0 µg/ml) compared to 2.5 µg/ml, at which almost all of the cells showed higher fluorescence. In contrast, Fluo-3-loaded sensitized RBCN displayed only a minimal increase in fluorescence, even at higher doses, as compared to un-sensitized RBCN, suggesting the absence of 9-O-AcSGPs (Fig. 3B, 3D). However, a lack of signaling for other, undetermined reasons cannot be ruled out. A time dependent increase in intracellular Ca2+ was observed in sensitized RBCVL (Fig. 3E–F). Almost all of the cells exhibited higher Fluo-3 fluorescence after 30 min of stimulation. As expected, Fluo-3-loaded RBCVL and RBCN incubated with A23187 exhibited maximum fluorescence and were used as positive control, which effect was decreased in the presence of EGTA, confirming the assay specificity. P/Q-type channel mediated Influx of Ca2+ ions In order to understand the Ca2+ influx pathway of sensitized RBCVL, we used different concentrations (0.01 µM, 0.05 µM, 0.1 µM, 0.5 µM, 1.0 µM and 2.0 µM) of ω-agatoxin TK, a P/Q-type Ca2+-ion channel blocker (Fig. 3G). At a concentration of 1 µM, ω-agatoxin TK strongly inhibited the influx of Ca2+ ions by reducing the MFI from 210 arbitrary units to the background value of 43 arbitrary units, suggesting the involvement of P/Q-type calcium channels in the Ca2+ ion influx in sensitized RBCVL. In contrast, nifedipine, an L-type channel blocker, even at a 10 µM concentration, did not inhibit the influx of Ca2+ (Fig. 3H). No inhibition could be detected at up to a 50 µM concentration of nifedipine. Enhanced cytoplasmic Ca2+ activated calpain I in sensitized RBCVL Increased intracellular Ca2+ activates the Ca2+-dependent protease calpain I [40]. Therefore, we investigated the involvement of enhanced Ca2+ in activating calpain I in sensitized/unsensitized RBCVL (Fig. 4A). Sensitized RBCVL displayed an approximate 2-fold increase of the 75 kDa active form of calpain I as a result of Ca2+-dependent autoproteolysis of the inactive membrane localized 80 kDa native form, indicating that an increased cytosolic Ca2+ level was sufficient for protease activation. It also suggested that the activation of calpain I was dependent on Ca2+ through sensitized 9-O-AcSGPs. A23187-treated RBCVL or RBCN produced an intense 75 kDa band. Unsensitized RBCVL inherently contain a less intense 75 kDa active form of calpain I which was completely absent in RBCN. Sensitization of RBCN with A23187 in the presence of EGTA resulted in only the native 80 kDa form. 10.1371/journal.pone.0042361.g004 Figure 4 Activation of calpain I in RBCVL or RBCN. A. Cells (2×107) were incubated with or without anti-9-O-AcSGP IgGVL, anti-9-O-AcSGP IgGNHS (10 µg/ml) or the Ca2+ ionophore A23187 in the absence or presence of EGTA (25 mM) for 60 min at 37°C in Ringer solution, washed with same buffer and lysed by sonication on ice. After removal of cell debris by centrifugation the lysates were separated by SDS-PAGE, and calpain I was detected by Western blot analysis with an anti-calpain-I antibody. B. Enhanced degradation of α-spectrin in sensitized RBCVL. RBCVL and RBCN were suspended separately in Ringer solution and sensitized with anti-9-O-AcSGP IgGVL, anti-9-O-AcSGP IgGNHS or A23187 in the absence or presence of EGTA for 30 min at 37°C. The cells were then lysed as before and the lysates subjected to SDS-PAGE and Western blot analysis with a spectrin-specific antibody. C. Involvement of calpain I in the degradation of spectrin in RBCVL. RBCVL and RBCN were pre-incubated with the calpain I inhibitor ALLN in Ringer solution for 30 min at 37°C before sensitization or co-incubated together with anti-9-O-AcSGP IgGVL or A23187 as positive controls. The cells were processed as before and spectrin detected by Western blot after SDS-PAGE of the cellulaproteins. D. Dependence of the hemolysis of RBCN on the activation of calpain I. RBCN were incubated without or with A23187 and with A23187 plus EGTA or ALLN for 1 h at 37°C, and the degree of hemolysis determined spetrophotometrically as in Figure 1. The results are shown as representative bar graphs of three independent experiments. Activated calpain I induced proteolysis of spectrinVL in sensitized RBCVL Active calpain I cleaves cytoskeleton proteins such as spectrin [40]. The enhancement of the Ca2+ influxes upon sensitization suggests an underlying relationship of the 9-O-AcSGPs on RBCVL and the elevated level of calcium. A four-fold more intense band of the SGP-60 kDa protein and reduced intensity of the α-spectrin band in sensitized RBCVL suggests an enhanced fragmentation of spectrinVL compared to un-sensitized RBCVL (Fig. 4B). EGTA reduced the proteolysis of spectrinVL to SGP-60, indicating a role for calcium-dependant proteolysis in the fragmentation of spectrin. In support of this notion, A23187/Ca2+-treated RBCVL/RBCN exhibited an intense SGP-60 band and complete loss of the α-spectrin band, and effect which was reversed by the addition of EGTA (Fig. 4B). Similar treatment of RBCVL in the presence of ALLN did not exhibit any enhancement of the SGP-60 band (Fig. 4C). The SGP-60 band was absent in sensitized RBCN in the presence of ALLN (Fig. 4C). Activated calpain I induced hemolysis in RBCN The activation of calpain I caused a degradation of spectrin in RBC. Does this degradation lead to RBC hemolysis? We checked the percentage of hemolysis in A23187-treated RBCN (Fig. 4D). Approximately 85% of the RBC hemolysis was observed after the activation of calpain I by the A23187 treatment, suggesting a positive correlation of spectrin degradation with cells hemolysis. Calpain I-specific inhibitor (ALLN)-treated RBC or the chelation of cytosolic Ca2+ by EGTA reduced the percentage of hemolysis to 12–14%, confirming the assay specificity. RBCN exhibited only 8.4% hemolysis. Activated calpain I cleaved purified spectrin Purified spectrinVL showed bands of 280, 246 and 60 kDa (Fig. 5A). SGP-60 was not present in spectrinN. Purified spectrinVL digested with active calpain I displayed a similar enhancement of fragmented spectrinVL, as evidenced by the increased presence of the SGP-60 band and reduced intensity of α-spectrin compared to undigested spectrinVL, suggesting the presence of active calpain in the patient's erythrocytes may be responsible for such proteolysis (Fig. 5B). Similar treatment in the presence of ALLN exhibited a reduced intensity of the SGP-60 band, thus showing the specificity of the reaction (Fig. 5C). SpectrinN digested with active calpain I also displayed an increase in the intensity of SGP-60 and corresponding decrease in α-spectrin in a calpain I dose-dependent manner (Fig. 5B). The SGP-60 band was absent in both undigested spectrinN and in the case of treatment with active calpain I in the presence of ALLN (Fig. 5C). 10.1371/journal.pone.0042361.g005 Figure 5 Degradation of purified α-spectrin by activated calpain I. A. Characterization of purified spectrins. SpectrinVL and spectrinN were purified from RBCVL and RBCN as described in Materials and Methods, and analyzed on SDS-PAGE (7.5%). The purified spectrins were Western blotted and detected with polyclonal rabbit anti-spectrin antibodies. B. Proteolysis of purified spectrinVL and spectrinN by active calpain I. SpectrinN and SpectrinVL (3.0 µg) were digested with different doses of active calpain I as indicated in reaction buffer, the reaction was stopped with EGTA and the products analyzed by SDS-PAGE. C. Inhibition of proteolysis by the calpain I inhibitor ALLN. Calpain I was preincubated with ALLN for 15 min on ice prior to addition of reaction buffer containing purified spectrinVL or spectrinN and processed as before. Discussion Studies reported by our group initially demonstrated the presence of 9-O-AcSGPs and anti-9-O-AcSGP antibodies in VL [22], [29]. Earlier we have reported the presence of only two 9-O-AcSGPs of molecular weight 36 kDa and 144 kDa on PBMCN respectively [41]–[43]. In contrast, several other distinct VL–associated newly induced 9-O-AcSGPs (19, 56, 65 kDa) were demonstrated on PBMCVL [21], [41], [44]. Interestingly, almost 40% of the membrane proteins present on the RBCVL were 9-O-acetylated, that were totally absent on RBCN which further signified their link with disease pathogenesis [8]–[11]. However, little progress has been made determining the extent of the contribution of 9-O-AcSGPs to the enhanced hemolysis of RBC in VL in the active disease state. Accordingly, our aim was to investigate the specific role of 9-O-AcSGPs in RBCVL hemolysis and their ligand-specific interaction with anti-9-O-AcSGP IgGVL. The major achievement of the study was to demonstrate the involvement of VL-associated 9-O-AcSGPs in triggering the altered cellular and membrane biochemical characteristics leading to the phagocytosis of these altered erythrocytes. Sensitization of 9-O-AcSGP using anti-O-AcSGP IgGVL antibodies led to an alteration of the membrane characteristics, as evidenced by enhanced osmotic fragility and hydrophobicity, suggesting a mechanism for the membrane damage which developed in RBCVL in contrast with RBCN. Moreover, profound ultrastructural changes in morphology from the normal discoid shape and oxidative stress induced in the sensitized RBCVL indicate definite alterations in their membranes, suggesting a key role for 9-O-AcSGPs. Similar alterations in erythrocyte membrane organization have been documented in Fanconi's anemia [45] and acute childhood lymphoblastic leukemia [33]. The physiological concentration of anti-9-O-AcSGPs antibodies in normal serum against two 9-O-AcSGPs (36 kDa and 144 kDa) present on PBMCN is only 11–13 µg/ml [13], [18], [22], [46]. However, the total anti-9-O-AcSGPs (54 µg/ml) in VL-serum are developed against several 9-O-AcSGPs (112, 107, 103, 57, 51, and 48 kDa) newly induced both on PBMCVL and RBCVL. Therefore, it may be envisages that the enhanced anti-9-O-AcSGP antibody found in the VL serum is definitely different from the antibody present in normal human serum. Hence, affinity purified anti-9-O-AcSGPVL antibody used to sensitize 9-O-AcSGPs on RBCVL certainly has a distinct identity, specific and active. Accordingly, even a lower concentration (6 µg/ml) of anti-9-O-AcSGP IgGVL antibody used for sensitization is capable of inducing 5.7-fold higher degree of RBCVL hemolysis compared to sensitized RBCN. This observation signifies that even lower doses of the anti-9-O-AcSGP IgGVL antibody through the ligand-specific interaction could play an important role in hemolysis of RBCVL leading to anemia. In contrast, no significant increase in the hemolysis (%) of RBCN was observed even at higher concentration of the anti-9-O-AcSGP IgGN antibody because of the negligible presence of 9-O-AcSGPs on normal cells. This further indicated that this change in red cell morphology due to this specific ligand-mediated interaction was VL-associated. ROS are linked to cell death signaling in a variety of cell types. Loss of membrane PS asymmetry has been reported in human erythrocytes, sickle cell disease, thalassemia and diabetes [39], [47]–[48]. Increased oxidative stress in erythrocytes of Leishmania-infected hamsters has been reported [49]. A 6-fold higher ROS generation and a 4-fold increased lipid peroxidation in sensitized RBCVL suggested that the signaling through 9-O-AcSGPs was indeed VL-associated. PS exposure on the outer leaflet of the cell membrane serves as a signal for the removal of apoptotic cells from the circulation [50]. Sensitized RBCVL demonstrated display an enhanced externalization of PS, an event which is reported to be correlated with membrane damage in other diseases [46], [47]–[48], [50]–[51]. A 17-fold higher erythrophagocytosis of sensitized RBCVL was demonstrated which indicated their efficient removal from the circulation, suggesting a probable cause for the anemia- associated VL patients. Sensitization of RBCVL with anti-9-O-AcSGP IgGVL resulted in an increase in the cytosolic Ca2+ level. However, the uptake of Ca2+ was not equal in all of the cells, suggesting that the 9-O-AcSGP content of the cells is also not equal, with possibly a few cells having a higher number of 9-O-AcSGPs stimulated earlier at a lower dose of antibody and earlier time point. The increased cytosolic Ca2+ causes the activation of calpain I, which in turn meditated an enhanced proteolysis of spectrinVL. Hence, the possible mechanism of a destabilization of RBC by damaging cytoskeleton proteins which in turn leads to hemolysis has been established in VL, with a cell-specific role for 9-O-AcSGPs. Calcium must be taken up from the extracellular compartment into the inside of the cell, as erythrocytes are devoid of any Ca2+ storage organelles such as the endoplasmic reticulum and mitochondria. We performed a channel-inhibition experiment in order to characterize the specific type of channel utilized in the course of ion influx. Inhibition of Ca2+ influx by ω-agatoxin TK confirmed the involvement of the P/Q-type channel. In contrast, even at higher doses, an L-type channel blocker (nifedipine) was unable to block the influx of Ca2+ during the sensitization process, clearly showing that this influx was not through the L-type channel. The sensitization of RBCVL caused activation of the Ca2+ channel and along with an enhanced influx of the Ca2+ ion, which may have further caused the activation of the Na+/K+ ion channel [52]. Activation of the Na+/K+ ion channel opens up the Gardos channel followed by an efflux of water from the cells, as reflected by the shift of a significant population of sensitized cells towards a lower FSC. The RBCVL cell size was typically lower than RBCN, possibly due to the higher level of intracellular Ca2+ in the VL condition. Spectrin, a cytoskeleton membrane protein which is crucial for the maintenance of the structural integrity of the cell, is thought to be a target of calpain-mediated proteolysis [53]. Supporting this idea, we observed increased cytosolic Ca2+ activated calpain I in sensitized RBCVL, as evidenced by the appearance of the active form mediating the proteolysis of spectrinVL. Calpain is activated by autoproteolysis and calpain I relocates from the cytoplasm to the inner surface of the plasma membrane, where it may cause damage to the cytoskeleton structure [40]. It degrades cytoskeletal proteins in neuronal cells during cerebral malaria, traumatic/post-traumatic neurodegeneration [54]–[57], aneurysmal subarachnoid hemorrhage [58] and spinal cord ischemia [59]. Calcium and phenylhydrazine-induced proteolysis of spectrin in rat and human erythrocytes has been documented [60]–[61]. The enhanced presence of SGP-60 in sensitized RBCVL which indicates the enhanced degradation of spectrin may be due to Ca2+-mediated proteolysis of cytoskeleton proteins destabilizing RBCVL. However, SGP-60 is already present in unsensitized RBCVL and its lack in RBCN suggested an association of degraded spectrin with the active disease state. The identification of SGP-60 as a fragment of erythrocytic α-I spectrin was confirmed by sequencing [11]. The fragmentation of purified spectrinVL/spectrinN by active calpain I displayed a similar pattern of spectrin-proteolysis as in RBCVL, suggesting an enhanced activation of calpain in RBCVL in vivo. Erythrocytes may have lost their membrane integrity as a consequence of spectrin degradation, as reflected in the enhanced hemolysis of the A23187-sensitized RBC. A higher degree of RBCN hemolysis may occur through the activation of calpain I, as chelating the cellular Ca2+ by EGTA suppressed the hemolysis. This was corroborated when a specific inhibitor of calpain I which blocks spectrin proteolysis (ALLN) was used, resulting in a decrease in the degree of hemolysis. Taken together, the evidence suggests that 9-O-acetylated sialoglycoproteins have an important role in Ca2+ influx, activating calpain-I, which in turn cleaves spectrin, causing destabilization of RBCVL and ultimately their removal through phagocytosis by macrophages. Hence the study findings have yielded important insight into the pathophysiological role of 9-O-AcSGPs on RBCVL, including potential cell-biological mechanisms which result in anemia (Fig. S1). Supporting Information Figure S1 Overview of the proposed mechanism. A hypothetical model has been shown in describing the role of 9-O-AcSGPs for the hemolysis of erythrocytes in VL. The model highlights the possible events inside the RBCVL including activation of calpain I followed by spectrin degradation and phosphatidyl serine exposure after sensitization of 9-O-AcSGPs by anti-9-O-AcSGP IgGVL. (TIF) SS, AG and KB are Senior Research Fellows of the Council of Scientific and Industrial Research (CSIR) Govt. of India. We are thankful to Mr. A. Mallick, Dr. R. Bhadra, Mr. B. Das, IICB, Dr. R.B. Bhar and Mr. Pallab Dasgupta Instrumentation Science, Jadavpur for providing facility of SEM and their cooperation in this experiment. 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Neurochem Int 48 : 108–113.16236382 56 Brophy GM , Pineda JA , Papa L , Lewis SB , Valadka AB , et al . (2009) alphaII-Spectrin breakdown product cerebrospinal fluid exposure metrics suggest differences in cellular injury mechanisms after severe traumatic brain injury. J Neurotrauma 26 : 471–479.19206997 57 Deng Y , Thompson BM , Gao X , Hall ED (2007) Temporal relationship of peroxynitrite-induced oxidative damage, calpain-mediated cytoskeletal degradation and neurodegeneration after traumatic brain injury. Exp Neurol 205 : 154–165.17349624 58 Lewis SB , Velat GJ , Miralia L , Papa L , Aikman JM , et al . (2007) Alpha-II spectrin breakdown products in aneurysmal subarachnoid hemorrhage: a novel biomarker of proteolytic injury. J Neurosurg 107 : 792–796.17937225 59 Lee JC , Hwang IK , Yoo KY , Kim DS , Kim WK , et al . (2006) Degradation of spectrin via calpains in the ventral horn after transient spinal cord ischemia in rabbits. 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PLoS One. 2012 Jul 31; 7(7):e42361
==== Front Ital J PediatrItal J PediatrItalian Journal of Pediatrics1824-7288BioMed Central 1824-7288-38-342282399310.1186/1824-7288-38-34ReviewInfantile colic, facts and fiction Kheir Abdelmoneim E M [email protected] Department of Paediatrics, Faculty of Medicine, University of Khartoum, Khartoum, Sudan2012 23 7 2012 38 34 34 16 1 2012 23 7 2012 Copyright ©2012 Kheir; licensee BioMed Central Ltd.2012Kheir; 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.Infantile colic is one of the major challenges of parenthood. It is one of the common reasons parents seek medical advice during their child’s first 3 months of life. It is defined as paroxysms of crying lasting more than 3 hours a day, occurring more than 3 days in any week for 3 weeks in a healthy baby aged 2 weeks to 4 months. Colic is a poorly understood phenomenon affecting up to 30% of babies, underlying organic causes of excessive crying account for less than 5%. Laboratory tests and radiological examinations are unnecessary if the infant is gaining weight normally and has a normal physical examination. Treatment is limited and drug treatment has no role in management. Probiotics are now emerging as promising agents in the treatment of infantile colic. Alternative medicine (Herbal tea, fennel, glucose and massage therapy) have not proved to be consistently helpful and some might even be dangerous. In conclusion infantile colic is a common cause of maternal distress and family disturbance, the cornerstone of management remains reassurance of parents regarding the benign and self-limiting nature of the illness. There is a critical need for more evidence based treatment protocols. ColicCryingInfantBaby ==== Body Introduction and definition It is a behavioural syndrome characterized by excessive paroxysmal crying, that is most likely to occur in the evenings without any identifiable cause. There are so many definitions but the most widely used one is based on the amount of crying by Wessel et al. which states that it is paroxysms of excessive crying in an otherwise healthy baby lasting more than 3 hours per day, occurring > 3 days in any week for 3 weeks, aged 2 weeks to 4 months [1]. Rule of three. Colic is one of the common reasons parents seek medical advice for their baby in his first 3–4 months of life. Those most affected by colic are the parents. Sleepless nights and the inability to console a newly arrived baby cause a great deal of stress, especially among first-time parents. Mothers of infants with colic were found to be more concerned about their infants’ temperament and even to feel rejection compared with mothers of infants without colic [2,3]. Colicky babies may be at an increased risk of abuse at the hands of exhausted and frustrated parents. Additionally, the parent may not properly bond with the child because of feelings of inadequacy and anger, leading to developing behavioral problems as the child grows [4]. The term colic derives from the Greek word kolikos or kolon, it is quite characteristic, a baby who has colic often cries about the same time every day, usually in the late afternoon or evening. Colic episodes may last from a few minutes to three hours or more on any given day. The crying usually begins suddenly and for no clear reason. The baby may have a bowel movement or pass gas near the end of the colic episode. Colic crying is intense and often high pitched. The face may flush, and he or she is extremely difficult — if not impossible — to comfort. Posture changes like Curled up legs, clenched fists and tensed abdominal muscles are common during colic episodes [5,6]. Epidemiology Internationally Colic affects 10-30% of infants worldwide. This condition is encountered in male and female neonates and infants with equal frequency. The colic syndrome is commonly observed in neonates and infants aged 2 weeks to 4 months. The incidence of colic in breastfed and bottle-fed infants is similar with no difference [7]. Increased susceptibility to recurrent abdominal pain, allergic disorders and certain psychological disorders may be seen in some babies with colic in their childhood. Illingworth found no association between the mother’s age, parity, or pregnancy history and colic [8]. Aetiology The cause of infantile colic remains unclear. Underlying organic causes of excessive crying must be considered during the evaluation. Organic causes account for less than 5 percent of infants presenting with excessive crying. These includes CNS causes like infantile migraine and subdural haematoma, GIT causes include constipation ,Cow’s milk protein intolerance, Gastro esophageal reflux ,Lactose intolerance, Intussusception, rectal fissure, strangulated inguinal hernia. Infections like Meningitis, Otitis media, Urinary tract infection and viral illness can also mimic colic. Trauma has to be excluded in a colicky baby namely child abuse, corneal abrasions, Foreign body in the eye, fractured bone and hair tourniquet syndrome [9,10]. Gastrointestinal, psychosocial, and neurodevelopmental disorders have been suggested as the cause of colic. Gastrointestinal disorders have been implicated in colic because of the infant’s leg position and grimacing during a crying spell. Excessive crying or increased gas production from colon function can result in intraluminal gas formation and aerophagia. This mechanism does not appear to be the cause of colic, however, because radiographic images taken during a crying episode have shown a normal gastric outline [11]. Gut hormones such as motilin also may play a causative role in colic. Motilin is thought to cause hyperperistalsis, leading to abdominal pain and colic [12]. Although studies have addressed possible psychosocial causes of colic, no evidence has been found in support of this mechanism. Even when colicky infants are cared for by trained occupational therapists, they cry twice as long as infants without colic. The hypothesis that colic is an early manifestation of a difficult temperament is not supported by prospective longitudinal studies [13,14]. Studies have suggested that colic may lie at the upper end of the normal distribution of crying in infants. The fact that most infants outgrow colic by four months of age lends support to a neurodevelopmental cause of colic [9]. New epidemiologic evidence suggests that exposure to cigarette smoke and its metabolites may be linked to infantile colic. Moreover, studies of the gastrointestinal system provide corroborating evidence that smoking is linked to increased plasma and intestinal motilin levels and higher-than-average intestinal motilin levels are linked to elevated risks of infantile colic [15]. Work up Colic is a common condition in early infancy that causes a great deal of concern. Parents and caregivers should be encouraged to document crying and fussing spells for review by the physician. A period of wellness followed by specific periods of crying is reassuring. Diagnosis is by exclusion. Further workup should be considered in infants who have frequent regurgitation of more than 28 g (1 oz), apneic or cyanotic episodes, fever, respiratory difficulties, poor weight gain, or abnormal findings on neurologic examination. Serial examination of the infant during times of the day when the infant is less fussy may be necessary [16-18]. Laboratory tests and radiographic examinations usually are unnecessary if the child is gaining weight normally and has a normal physical examination [19]. If the patient’s stools are excessively watery, testing them for excess reducing substances (Clinitest) may be worthwhile. If results are positive, this may be an indication of an underlying GI problem, such as acquired (post infectious) lactose intolerance. Stool may be tested for occult blood to rule out cow’s milk allergy. Mothers often believe that their baby’s crying may be related to milk formula or disease which can be managed by changing the infant’s formula from cow’s milk to a soya protein or casein hydrolysate formula. When a formula was changed, mothers more frequently believed that the cause of the problem was intrinsic to the child (P<0.001) and that their infant had had a “disease or illness” (P<0.001). When formula changes occurred, 26% of mothers believed that their infants were allergic to cow milk. These beliefs may affect a mother’s perceptions of her child’s vulnerability [20]. Treatment The mainstay of colic management is an acknowledgment by the physician of the difficulties the parents are facing and an inquiry into the well-being of the parents [21]. The single most effective step remains reassurance of parents regarding the benign and self-limiting nature of the illness as most of the babies improve by the age of 3 to 4 month. Drug treatment generally has no place in the management of colic, unless the history and investigations reveal gastro esophageal reflux. Simethicone, a safe, over-the-counter drug for decreasing intraluminal gas, it is a non absorbable medication that changes the surface tension of gas bubbles, allowing them to coalesce and disperse and releasing the gas for easier expulsion. It has been promoted as an agent to decrease colicky episodes. A randomized, placebo-controlled, multicenter trial concluded that treatment with this agent produces results similar to those of placebo. The perceived improvement may be a placebo effect. Two other RCTs found no benefit for treatment with simethicone [22]. Dicyclomine hydrochloride is an anticholinergic drug that has been proven in clinical trials to be effective in the treatment of colic. However, because of serious, although rare, adverse effects (eg, apnea, breathing difficulty, seizures, syncope), its use cannot be recommended [23]. Cimetropium bromide, which is widely used in Italy to treat infantile colic, showed a decrease in duration of crying crises in the treated group compared with placebo. The major side effect was sleepiness; there were no reports of life-threatening events [24]. Dietary changes like eliminating cow’s milk proteins is indicated only in cases of suspected intolerance to the protein (eg, positive family history, eczema, onset after the first month of life, association with other GI symptoms such as vomiting or diarrhea). In infants with suspected allergy to cow’s milk protein (formula fed) a protein hydrolysate formula is indicated. Extensively hydrolyzed protein formulas may reduce colic, while partially hydrolyzed formulas should not be used in infants with colic due to cow’s milk protein allergy [25]. Use of soy-based formula is not recommended because many infants allergic to cow’s milk protein may also develop intolerance to soy protein. if the mother is breastfeeding, a period of elimination of allergenic foods (e.g. dairy, nuts, soy, citrus, etc.) from her diet in order to observe changes in the baby’s condition. If the crying is related to a cow’s milk allergy benefits are usually seen within 2–7 days [26-28]. Hill et al. suggested that a period of dietary modification with a low allergen diet and appropriate nutritional support should be considered in healthy infants with colic .He also found that exclusion of allergenic foods from the maternal diet was associated with a reduction in distressed behavior among breastfed infants with colic presenting in the first 6 weeks of life [29,30]. In some patients, exercise-induced anaphylactic reactions (EIAN) occur only when a particular food is eaten before exercise. Food-exercise combined challenge may be useful in identifying foods that favor exercise-induced anaphylactic reactions in children with multiple food-dependent EIAn [31,32]. Probiotics may have a role in treatment of infantile colic. Lactobacillus reuteri endogenous to the human GI tract was found to relieve colic symptoms in breastfed infants within one week of treatment. In a more recent study, 50 exclusively breastfed colicky infants were randomly assigned to receive either L reuteri or placebo daily for 21 days. A 50% reduction in crying time from baseline was noted in the L reuteri group compared with the placebo group on days 7. The study concluded that L reuteri at a dose of 108 colony-forming units per day improved symptoms of infantile colic and was well tolerated and safe. Further studies are needed before this can be recommended as a routine therapy for colic in infants [33,34]. Recently as revised by Critch there is insufficient evidence to recommend for or against the use of probiotics or prebiotics in the management of colic [35]. Alternative medicine Oral hypertonic glucose and sterile water were compared for treatment of colic in infants in a randomized trial. In the group receiving glucose, 30% had significantly less colic than the placebo group [36]. Herbal teas containing mixtures of chamomile, vervain, licorice, fennel, and lemon balm, used up to three times a day (150 mL per dose) have been shown to decrease crying in colicky infants. Given the multiplicity of herbal products, the lack of standardization of strength and dosage, and potential interference with normal feeding, parents should be cautioned about their use [37,38]. Spinal manipulation is a traditional form of treatment practiced by chiropractors, osteopaths, physiotherapists and other healthcare providers mostly (but not exclusively) to treat musculoskeletal problems. Spinal manipulation can be described as ‘the use of hands applied to the patient incorporating the use of instructions and maneuvers to achieve maximal painless movement and exposure of the musculoskeletal system. Evidence for the efficacy of spinal manipulation in treating infantile colic is inconclusive. Physicians should be cautious about recommending spinal manipulations in infants [39,40]. Rhythmic calming techniques are effective in calming colicky babies which forms the core of the 5 Ss approach. 1. Swaddling, safe swaddling carefully avoiding overheating, covering the head, using bulky or loose blankets, and allowing the hips to be flexed [41,42]. 2. Side or stomach (holding a baby on the back is the only safe position for sleep, but it is the worst position for calming a fussy baby); 3. Shhh sound (making a strong shush sound near the baby's ear [43,44]. 4. Swinging the baby with tiny jiggly movements (no more than 1" back and forth) always supporting the head and neck [44,45] 5. Sucking (Letting the baby suckle on the breast, your clean finger or a pacifier) Numerous studies mentioned above have shown that when key components of the “5 S’s” (e.g. swaddling, shushing, swinging) are used all night they can improve sleep or reduce crying; and, when the “5 S’s” are done correctly and in combination, they offer significant potential to promptly reducing infant crying and promote sleep. Remind parents about the importance of feeding a hungry baby, changing wet diapers, and comforting a baby who is cold and crying as a result of these factors. Soothing music accompanied with parental attention (including eye contact, talking, touching, rocking, walking, and playing) may be effective in some infants and is never harmful. Encourage parents to discuss their feelings and concerns with each other to obtain support. Emphasize the responsibility of the whole family in the care of a baby with colic. Conclusion Infantile colic is a common cause of maternal distress and family disturbance, the cause remains unclear, the cornerstone of management remains reassurance of parents regarding the benign and self-limiting nature of the illness as most of the babies grow out of it by the age of 3–4 months. Investigations are rarely required and drug treatment is usually ineffective. Consistent follow up and a sympathetic physician forms the basis of management in patients with colic. There is a critical need for more evidence based treatment protocols. 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yes
Ital J Pediatr. 2012 Jul 23; 38:34
==== Front PLoS One PLoS One plos PLOS ONE 1932-6203 Public Library of Science San Francisco, USA 22879938 PONE-D-12-01886 10.1371/journal.pone.0042310 Research Article Biology Molecular Cell Biology Cell Growth Medicine Oncology Basic Cancer Research Tumor Physiology Cancer Treatment Antiangiogenesis Therapy Cancers and Neoplasms Hematologic Cancers and Related Disorders Leukemias Chronic Myeloid Leukemia Oncology Agents Cell Biology Oncology Biology and Life Sciences Cell Biology Cellular Structures and Organelles Vesicles Exosomes Biology and Life Sciences Cell Biology Cellular Types Animal Cells Epithelial Cells Endothelial Cells Biology and Life Sciences Anatomy Biological Tissue Epithelium Epithelial Cells Endothelial Cells Medicine and Health Sciences Anatomy Biological Tissue Epithelium Epithelial Cells Endothelial Cells Biology and Life Sciences Physiology Cardiovascular Physiology Angiogenesis Medicine and Health Sciences Physiology Cardiovascular Physiology Angiogenesis Biology and Life Sciences Developmental Biology Angiogenesis Biology and life sciences Genetics Gene expression Gene regulation Small interfering RNAs Biology and life sciences Biochemistry Nucleic acids RNA Non-coding RNA Small interfering RNAs Medicine and Health Sciences Oncology Cancer Treatment Medicine and Health Sciences Oncology Cancers and Neoplasms Hematologic Cancers and Related Disorders Leukemias Medicine and Health Sciences Hematology Hematologic Cancers and Related Disorders Leukemias Biology and Life Sciences Biochemistry Proteins Post-Translational Modification Phosphorylation Biology and Life Sciences Physiology Immune Physiology Cytokines Medicine and Health Sciences Physiology Immune Physiology Cytokines Biology and Life Sciences Immunology Immune System Innate Immune System Cytokines Medicine and Health Sciences Immunology Immune System Innate Immune System Cytokines Biology and Life Sciences Developmental Biology Molecular Development Cytokines Carboxyamidotriazole-Orotate Inhibits the Growth of Imatinib-Resistant Chronic Myeloid Leukaemia Cells and Modulates Exosomes-Stimulated Angiogenesis CTO Modulates Exosome-Stimulated Angiogenesis Corrado Chiara 1 Flugy Anna Maria 1 Taverna Simona 1 Raimondo Stefania 1 Guggino Giuliana 1 Karmali Rashida 2 De Leo Giacomo 1 ¶ Alessandro Riccardo 1 * ¶ 1 Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi, Sezione di Biologia e Genetica, Università di Palermo, Italy 2 Tactical Therapeutics Inc., New York, New York, United States of America Loges Sonja Editor University Hospital Hamburg-Eppendorf, Germany * E-mail: [email protected] Competing Interests: RA received a grant research from Tactical Therapeutics that owns the anticancer drug (CTO) studied in the research. However, this does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Conceived and designed the experiments: CC ST AF GDL RA. Performed the experiments: CC AF ST SR GG. Analyzed the data: CC AF ST SR GG RA. Contributed reagents/materials/analysis tools: RK. Wrote the paper: CC AF ST RA. ¶ These authors also contributed equally to this work. 2012 3 8 2012 16 10 2019 7 8 e4231020 1 2012 5 7 2012 Corrado et al https://creativecommons.org/licenses/by/4.0/ Except for Figure 3a, 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. The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients. This work was supported by Tactical Therapeutics Inc, New York, United States of America; University of Palermo (International Cooperation) to RA; ex 60% Ministero per l’Università e per la Ricerca Scientifica e Tecnologica to RA, AF and to GDL. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body pmcIntroduction Chronic myeloid leukaemia is characterized by the Philadelphia (Ph) chromosome encoding the chimeric Bcr–Abl oncoprotein with a constitutive tyrosine kinase activity that drives disease pathogenesis by stimulating a number of downstream signalling cascades [1], [2]. Whereas CML can be effectively treated, during chronic phase (CP), with tyrosine kinase inhibitors (TKIs) such as imatinib [3], the acquisition of imatinib resistance, mainly due to point mutations, causes disease progression (blast crisis) that can be fatal within months. To circumvent resistance, more potent TKIs, such as nilotinib and dasatinib, have been recently approved [4]. However, these compounds do not have therapeutic activity against all imatinib-resistant mutants of Bcr-Abl and therefore, a long-term tolerability problem has emerged [5]. Combination strategies of imatinib with drugs that target downstream signalling molecules have shown some success in the treatment of the imatinib-resistant cells in in vitro settings and in mouse models but have not been studied in clinical trials yet [6], [7]. Therefore, there is an urgent need for new anticancer agents and combinations that can improve responses and survival rates for CML. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable small molecule that was recently shown to decrease in vitro cell viability and to augment apoptosis in three different imatinib-resistant CML cell lines through the down-regulation of Bcr-Abl protein, inhibition of tyrosine phosphorylation of Bcr-Abl, STAT5, CrkL, as well as inhibition of ERK1/2 phosphorylation [8], [9]. CTO shows a reduced toxicity, increased oral bioavailability and achieves higher plasma concentrations and stronger efficacy when compared to the parental compound [10]. We have recently demonstrated that LAMA84 CML cells release exosomes and that the addition of those microvesicles to HUVEC affects several steps of in vitro angiogenesis including motility, cytokine production, cell adhesion, and cell signalling as well as in vivo angiogenesis in nude mice [11]. A number of studies have recently described exosomes as new players in modulating the tumor microenvironment, promoting angiogenesis and tumor development [12]; furthermore, neovascularization is known to exert an important role in the progression of chronic myeloid leukaemia and may represent a valid alternative target for therapy. Taking these data into account, the aims of our study were (i) to test if CTO is able to inhibit in vivo the growth of imatinib-resistant CML cells and (ii) to investigate the ability of CTO to affect tumor microenvironment by modulating exosome-stimulated angiogenesis in vitro and in vivo. Our results indicate that administration of CTO to a CML xenograft model in NOD/SCID mice may increase survival and that CTO reduces in a dose- and time-dependent fashion exosomes-stimulated angiogenic process. Further work is necessary to demonstrate in a clinical setting, the possible use of CTO as an alternative therapeutical option in the treatment of imatinib-resistant forms of chronic myelogenous leukaemia. Materials and Methods Ethic Statement All animal experiments were conducted in full compliance with Universita’ di Palermo and Italian Legislation for Animal Care and Dipartimento di Biopatologia e Biotecnologie Mediche e Forensi (DiBiMef) review board has approved this study. Cell Culture and Reagents Imatinib resistant LAMA84 and K562 cells (LAMA84R and K562R) were kindly provided by Dr. P. Vigneri, Università di Catania [13]. Cells were cultured in RPMI 1640 medium (Euroclone, UK) supplemented with 10% fetal bovine serum (Euroclone, UK), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Euroclone, UK) and 1 µM of imatinib to maintain the resistance. HUVEC were obtained from Lonza and grown in Endothelial Growth Medium (EGM) according to supplier’s information (Clonetics, Verviers, Belgium). Imatinib mesylate (Selleck chemicals, Houston, TX, USA) was prepared as a 1 mM stock solution in sterile phosphate-buffered saline (PBS); CAI orotate (CTO) from Tactical Therapeutics Inc, New York, USA was solubilized in DMSO at 0.1 M for in vitro assay. For in vivo assay CTO was prepared at 50 mg/ml in 80% PEG-100, sonicated, aliquoted and kept at −20°C; imatinib was dissolved in PBS at a concentration of 2 mg/ml. All other reagents were purchased from Sigma (St. Louis, MO), if not cited otherwise. Proliferation Assay (MTT Assay) Methyl-thiazol-tetrazolium (MTT) assay was done as previously described [8], cells were plated in triplicate or quadruplicate at 1.5×104 per well and exposed to escalating doses of CTO for up to 4 days. Means and standard deviations generated from 3 to 4 independent experiments are reported as the percentage of growth. Cell proliferation curves were derived from these data by using Microsoft Excel software. Western Blot Total protein cell lysates or exosome lysates were obtained and analyzed by SDS-PAGE followed by Western blotting as previously described [8]. Antibodies used in the experiments were: c-Abl, phospho-Abl, phospho-CrkL, Erk 1/2, phospho Erk 1/2, Hsc70, β-actin (all from Cell Signalling Technology, MA, USA); anti-CrkL, VCAM1-FITC, ICAM1-FITC and anti-CD63 (all from Santa Cruz Biotechnology, CA, USA). CML Mouse Xenograft Male NOD/SCID mice four-to-five week old were purchased from Charles River (Charles River Laboratories International, Inc, MA, USA) and acclimated for a week prior to experimentation. Mice received filtered water and sterilized diet ad libitum. Animals were observed daily and clinical signs were noted. Each mouse was inoculated subcutaneously (sc) in the right flank with viable single cells (1×107) suspended in 0.2 ml of PBS. The day of injection was considered as Day 0. On Day 7, when tumors were palpable, mice were randomly assigned to groups of ten and were treated with imatinib administered intraperitoneally (i.p) (50 mg/Kg, three days a week for two rounds) or with its vehicle (PBS) in combination either with CTO 342 mg/kg (Q1D×5 for two rounds) or with CTO 513 mg/kg (Q1D×5 for two rounds) or with their vehicle (80% PEG-100). All mice received both p.o. and i.p. doses of the vehicle to control for morbidity associated with the treatment. Tumor xenografts were measured and the mice were weighed three times a week starting on Day 7. Tumor volume was determined by calliper by using the following formula: L×W2/2 =  mm3 where L and W are the longest and shortest perpendicular measurements in millimeters, respectively. The same formula was used to calculate tumor weights assuming that 1 mm3 = 1 mg. Due to Università di Palermo rules and Italian legislation, animals were euthanized when sc tumor xenografts reached 4000 mg in weight. Exosome Isolation and Characterization Exosomes produced by LAMA84R cells during a 24 h culture period, were isolated from conditioned culture medium by different centrifugations as described previously [11]. Exosome protein content was determined by the Bradford assay (Pierce, Rockford, IL, USA). The activity of acetylcholinesterase, an exosome marker protein, was determined as described by Savina et al [14]. Briefly a total of 10 µg of exosomes or 10 µg of total cell lysate in 100 µl of PBS were resuspended in a solution of 1.25 mM acetylthiocoline and 0.1 mM 5,5′-dithiobis (2-nitrobenzoic acid) in a final volume of 1 ml. The incubation was carried out in cuvettes at 37°C and the change in absorbance at 412 nm was followed at different time points (from 0 to 180 min). Isolated exosomes were observed with a scanning electron microscope. They were fixed with 2% glutaraldehyde in PBS for 10 min, attached onto stubs, coated with gold in a sputterer (Sputter Coater 150A, Edwards, UK) and observed using a field emission scanning electron microscope (FEGESEM QUANTA 200 FEI) at a working voltage of 30 kV. RNA Interference Small interfering RNAs (siRNA) targeting IL8 or scramble siRNA were purchased from Dharmacon (ON-TARGET plus SMART pool, Human, Dharmacon inc. CO, USA) and used to transfect HUVEC by employing oligofectamine (Invitrogen, UK), according to the suggested protocol. Briefly, 85% confluent cells were incubated with a mix of oligofectamine- Opti-MEM containing 75 pmol of siRNA or equal volume of PBS (CN). After 24 h, the conditioned medium was collected for ELISA assay and cells were lysed for RNA extraction. Knockdown efficiency was determined by Real-time PCR and ELISA quantitation of IL8 release. RNA Extraction and Real-time PCR HUVEC were grown to confluence in 12-well plates, pre-treated or not with CTO (5 and 10 µM) for 24 h or 48 h and incubated for 6 h with exosomes (20 and 50 µg/ml). To investigate efficacy of IL8 silencing, HUVEC were grown to confluence in 6-well plates, transfected with siRNAs, incubated for 6 h with 50 µg/ml of exosomes and then lysed to extract RNA. For all experiments, IL8, VCAM1, ICAM1 transcript levels were measured by reverse transcription (RT) and TaqMan real-time quantitative polymerase chain reaction (RQ-PCR) and were analyzed as previously described [11]. The following primers were used: IL8 Hs00174103 m1, VCAM1 Hs00174239 m1, ICAM1 Hs00277001 m1 and GAPDH Hs99999905 m1 (Applied Biosystems, Foster City, CA, USA) used as internal controls. Flow Cytometry Expression of HUVEC cell surface VCAM1 and ICAM1 was determined by flow cytometry analysis. HUVEC were pre-treated or not with 10 µM CTO for 24 h and incubated over night with 50 µg/ml of LAMA84R-exosomes in a low serum medium (EGM:RPMI, 1∶9). 5×105 cells were washed in PBS and incubated with 0.5 µg anti VCAM1-FITC or ICAM1-FITC (Santa Cruz Biotechnology, CA, USA) for 15 min at 4°C according to manufacturer’s recommendations. Isotype-matched irrelevant antibodies were used as a negative control. Viable cells were gated by forward and side scatter and analysis was performed on 100,000 acquired events for each sample. Samples were analyzed on a FACS Calibur with the use of the CellQuest software (BD Biosciences, NJ, USA). Elisa HUVEC conditioned medium (CM) was collected from cells pre-treated or not for 24 h with 10 µM CTO and then stimulated for 6 h with indicated treatments; HUVEC CM was also collected from cells after 24 h of transfection with siRNA (scramble or IL8) and 6 h of incubation with 50 µg/ml of exosomes; CM aliquots were centrifuged to remove cellular debris and afterwards IL8 protein concentrations were quantified using an ELISA kit (R&D Systems, MN, USA), according to manufacturer’s protocol. IL8 was also measured directly in LAMA84R exosomes. Migration Assay Migration assays were performed in Transwell chemotaxis chambers assay (NeuroProbe, Cabin John, MD, USA) [8]. Briefly, HUVEC (2×106/ ml) were suspended in serum-free RPMI 1640 medium supplemented with 0.1% BSA with or without CTO (1, 5, 10 µM), in transwell chemotaxis chamber equipped with 8 µm pore filters and exposed to chemoattractants with exosomes (20–50 µg/ml), 10 ng/ml of recombinant IL8 or neutralizing antibodies anti IL8 (5 µg/ml) (R&D system, MN, USA) as indicated. To evaluate the migration ability of HUVEC transfected with siRNA, endothelial cells were suspended in RPMI 1640 medium supplemented with 0.1% BSA with or without 10 µM CTO, were exposed to chemoattractants with 50 µg/ml of exosomes and were processed as above. Filters were removed after 6 h, were fixed in methanol and were stained with Diff-Quick (Medion Diagnostics GmbH, Dudingen, Switzerland). Each test group was tested in three independent experiments; the number of migrating cells in five high-power fields per well were counted at 400X magnification. Adhesion Assay Adhesion assays were performed as previously described [11]. Briefly, HUVEC were pre-treated or not with 5–10 µM of CTO for 24 h and HUVEC monolayer was incubated for 6 h with indicated conditions, as described in the results. After treatment, cells were washed with PBS and CML cells were added for 2 h at 37°C. Adherent cells were stained with hematoxylin/eosin, each test group was assayed in triplicate; five high power (400X) fields were counted for each condition. Tube Formation of HUVEC on Matrigel Matrigel was used to test the effects of exosomes on in vitro vascular tube formation as described [11], [15]. 50 µg/ml of exosomes were added to HUVEC plated on Matrigel in low serum medium and 10 µM CTO. For HUVEC transfected with siRNA the same experiment were performed after 24 h of transfection. Cells were incubated for 6 h and then evaluated by phase-contrast microscopy and photographed. The length of the cables was measured manually with the IMAGE-J software (http://rsbweb.nih.gov/ij/) [16]. Matrigel Plug Assay Groups of six NOD/SCID mice (4 weeks) (Charles River) were injected subcutaneously with 400 µL of Matrigel (BD Biosciences Pharmingen, San Diego, CA, USA) as follows: respectively, animals in group 1 and 2 were injected with matrigel plus PBS (PBS) or plus 100 µg LAMA84R-derived exosomes (Exo), and were treated with 80% PEG-100 and PBS. Animals in group 3-4-5 were injected with matrigel plus 100 µg LAMA84R-derived exosomes and were treated either with CTO 513 mg/Kg/inj and imatinib vehicle (group 3, Exo + CTO) or with CTO 513 mg/Kg/inj and imatinib 50 mg/kg (group 4, Exo + IM + CTO) or with CTO vehicle and imatinib 50 mg/kg (group 5, Exo + IM). Animals in group 6 and 7 were injected with matrigel plus 50 ng of recombinant IL8 and were respectively treated either with vehicles of CTO and imatinib (Rec IL8) or with CTO 513 mg/Kg/inj and imatinib vehicle (Rec IL8+ CTO), The degree of vascularization was evaluated by determination of hemoglobin content using the Drabkin method (Drabkin’s reagent kit) [17]. Statistics Data were expressed as means ± SEMs of the indicated number of experiments. Statistical analysis was performed by using a paired samples t test. Differences were considered to be significant when P values were smaller than 0.05. Results Effects of CTO on Growth of Human CML Cells and Bcr-Abl Mediated Tyrosine Phosphorylation MTT assays were performed to determine the antiproliferative effects of CTO on LAMA84R and on K562R cells. Data presented in figure 1 (panel a) show results of 4 days treatment. CTO inhibits cell growth of LAMA84R and K562R in the low micromolar range in a dose dependent fashion (p<0.001). The results herein show a 50% growth reduction of the CML lines with 5 µM CTO at 96 h time point. In order to correlate the antiproliferative effects of CTO on CML cells with the Bcr-Abl activity, cells were incubated with increasing concentrations of CTO, were harvested and were subjected to immunoblotting with antibodies against phosphorylated Bcr-Abl and CrkL. As shown in figure 1 (panel b), a dose-dependent inhibition of both total and phosphorylated Bcr-Abl levels was observed after 72 and 96 h of drug exposure. Consistent with this conclusion, CTO inhibits the phosphorylation of a selected target of Bcr-Abl kinase; tyrosine phosphorylation of CrkL was reduced by 5 µM CTO treatment. 10.1371/journal.pone.0042310.g001 Figure 1 CTO inhibits cell proliferation of LAMA84R and K562R cell lines. (a) Cell growth was measured by MTT assay after 24, 48, 72, 96 h of treatment with increasing doses of CTO (0.1-1-5-10 µM). The values were plotted as a percentage of the control (cells treated with DMSO). Each point represents the mean ± SD for three independent experiments. *p≤0.001. (b) CTO treatment decreases Bcr–Abl expression, inhibits Bcr–Abl tyrosine phosphorylation and its downstream substrate CrkL on LAMA84R and K562R cell lines. These cell lines were treated with increasing doses of CTO (0.1–5 µM) or DMSO (CN) for 72 h and 96 h; afterwards protein lysates were subjected to western blot analysis as described in Material and Methods using anti-phospho-Abl, anti-cAbl, anti-phospho-CrkL and anti-CrkL antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading. Effects of CTO on Tumor Xenograft Growth On the basis of the in vitro growth and Bcr-Abl signalling inhibitory effects of CTO, we further examined the antineoplastic effect of CTO on LAMA84R using a xenograft CML tumor model. Otherwise CTO–treated mice seemed healthy and did not exhibit any signs of distress during the drug treatment. For these analyses, mice receiving either CTO alone or in combination with imatinib were treated, as described in Material and Methods, until day 26 of treatment regimen. Animals were then maintained until tumor weight reached 4000 mg. Tumor weight curve analysis (figure 2) showed that mice bearing LAMA84R tumor- reached on average the 4000 mg weight after 26 d in the following subsequent groups: CTO 342 mg/Kg plus imatinib; CTO 342 mg/Kg plus PBS and imatinib plus PEG. By contrast, CTO 513 mg/Kg group resulted in a slight longer period of time to reach the 4000 mg weight (33 d) compared with the control group (PBS plus PEG). The combination of CTO 513 mg/Kg plus imatinib slowed tumor growth to a greater extent than the control group (P<0.01), attaining the experimental end point after 40 d. 10.1371/journal.pone.0042310.g002 Figure 2 Antitumor activity of CTO on human CML xenografts. LAMA84R cells were injected subcutaneously in NOD/SCID mice as described. After palpable tumor formation, mice were treated as described in Material and Methods. Comparison of the median tumor weight was used as index of the antitumor efficacy of the compounds. Exosome Vesiscles Released by LAMA84R Cells LAMA84R cells release exosomes into the culture medium during a 24 h period as demonstrated by their characteristic shape and diameter (70 nm ±10) observed with scanning electron microscope (figure 3a) and by the presence of Hsc70 and CD63 proteins (figure 3b). Furthermore, acetylcholinesterase activity, a characteristic enzyme localized in exosomes, was found associated with the exosome fraction (Figure 3c). 10.1371/journal.pone.0042310.g003 Figure 3 LAMA84R exosomes characterization. (a) Exosomes released by LAMA84R cells observed with a scanning electron microscope. (b) Detection of Hsc70 and CD63 in 30 µg of cell lysate (lane 1) and 30 µg of exosomes lysate (lane 2). (c) Acetylcholinesterase assay. The activity of acetylcholinesterase, an exosome-specific protein marker, was determined in 10 µg either of total cell lysate (Cells) or of Exosomes (Exo); exosome-deprived conditioned medium (CM-Exo) and exosome-deprived Fbs (Fbs–Exo) were used as negative controls. Figure 3a is excluded from this article's CC-BY license. See accompanying retraction notice for more information. CTO Inhibits the Exosome-stimulated Increase of Cell-cell Adhesion Molecules and IL8 Expression in HUVEC To determine the potential effects of CTO on exosome-mediated induction of genes associated with angiogenesis, we evaluated by TaqMan PCR analysis the mRNA levels of cell-cell adhesion molecules and cytokines after adding the drug to exosome-stimulated HUVEC. Figure 4 panel I shows that LAMA84R-derived exosomes added to HUVEC monolayer caused, compared to control, a dose-dependent increase of VCAM1 (a), ICAM1 (b) and IL8 (c) mRNA expression. Increase of mRNA production was statistically significant and reached approximately a 24-, 10-, 60-fold induction respectively after 6 h stimulation of the endothelial monolayer with 50 µg/ml of exosomes. Treatment of endothelial cells with exosomes together with increasing doses of CTO caused a dose-dependent inhibition of VCAM1, ICAM1 and IL8 mRNA levels. Figure 4 panel II shows that a comparable effect to the one of LAMA84R exosomes on the mRNA induction was observed when endothelial cells (EC) were incubated with 10 ng/ml of recombinant IL8; adding 10 µM CTO or neutralizing anti-IL8 antibody revert the increase in VCAM1 (a), ICAM1 (b) and IL8 (c) mRNA expression. 10.1371/journal.pone.0042310.g004 Figure 4 Effects of CTO on cell adhesion molecules and cytokines mRNA expression. (I) CTO reverts the effects of CML exosome treatment on VCAM1, ICAM1 and IL8 mRNA expression in HUVEC cells. VCAM1 (a), ICAM1(b) and IL8 (c) mRNA expression increased in a dose dependent manner after adding exosomes (Exo) to endothelial cell monolayer. CTO (1-5-10 µM) reverts these effects in a time- and dose dependent manner. (II) VCAM1, ICAM1 and IL8 mRNA expression in HUVEC treated for 6 h either with low serum medium (CN), or with 50 µg/ml exosomes (Exo), or with 10 ng/ml of recombinant IL8 (Rec IL8) with or without CTO 10 µM, or with 50 µg/ml exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8). Values are representative for three independent experiments. *p≤0.05; **p≤0.01. FACS analysis confirmed that incubation of HUVEC with LAMA84R exosomes resulted in detection of VCAM1 (figure 5a) and ICAM1 (figure 5b) on the surface of HUVEC which was blunted by treatment of cells with 10 µM CTO. ELISA assay demonstrated the increasing release of IL8 into HUVEC conditioned medium after treatment with CML exosomes (figure 5c). Small amount of IL8 was also found in LAMA84R exosomes (41 pg/50 µg exosomes). Transfection of HUVEC with IL8 siRNA caused, as expected, a striking reduction in both IL8 mRNA levels and cytokine release in conditioned medium compared to EC transfected with scramble siRNA (figure S1 and b). 10.1371/journal.pone.0042310.g005 Figure 5 Effects of CTO on cell adhesion molecules and cytokines production. CTO inhibits the exosomes-stimulated increase of VCAM1, ICAM1 expression and IL8 secretion on HUVEC cells. Representative overlay histograms showing an increase of surface expression of VCAM1 (a) and ICAM1 (b) on HUVEC treated with 50 µg/ml of LAMA84R exosomes (blue line) compared to HUVEC treated with 50 µg/ml of LAMA84R exosomes plus 10 µM CTO (red line) or untreated HUVEC, as control (green line). (c) ELISA for IL8 release by HUVEC. Aliquots of conditioned medium (CM) from cells were collected after 6 h of stimulation either with 50 µg/ml of exosomes (CM HUVEC + Exo) or with 50 µg/ml of exosomes plus 10 µM CTO (CM HUVEC + CTO + Exo); low serum medium (CM HUVEC) or low serum medium plus 10 µM CTO (CM HUVEC + CTO) were used as negative controls. The amount of IL8 in 50 µg/ml of exosomes was also quantified. CTO Inhibits the Adhesion of CML Cells to HUVEC Monolayer While leukaemia progresses, cancer cells adhere to endothelial cells in order to infiltrate and colonize extramedullary sites. Figure 6a shows that the increase of adhesion of LAMA84R cells (arrows) to HUVEC monolayer was inhibited by pre-treatment of EC with 10 µM CTO. Accordingly, figure 6b shows that pre-treatment of HUVEC with 50 µg/ml of LAMA84R exosomes induces an 80 fold increase of CML cells adhesion to EC; CTO inhibits CML cells adhesion in a dose dependent manner. Figure 6c shows the inhibitory effects of both a neutralizing anti-IL8 antibody and CTO on IL8-stimulated adhesion of CML cells to EC. 10.1371/journal.pone.0042310.g006 Figure 6 CTO inhibits the adhesion of LAMA84R cells to exosome-treated HUVEC monolayer. (a) Phase contrast micrographs showing the adhesion of LAMA84R cells (arrows) on HUVEC monolayer treated with 50 µg/ml of exosomes (Exo) and with 50 µg/ml of exosomes after pre-treatment of 24 h with 10 µM CTO. (b) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo) and exosomes plus increasing doses of CTO (1–10 µM); (c) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo), 50 µg/ml of exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8), 10 ng/ml of recombinant IL8 (Rec IL8), 10 ng/ml of recombinant IL8 plus 10 µg/ml of a neutralizing anti-IL8 antibody and 10 ng/ml of recombinant IL8 with increasing doses of CTO (1–10 µM). Values are representative for three independent experiments. *p≤0.05; **p≤0.01. Effects of CTO on Exosomes-stimulated Migration of Endothelial Cells We examined the effect of CTO treatment on exosome-stimulated EC motility by Boyden chamber assay. Our results showed that adding a range of concentrations of CTO (1–10µM) to the upper well of the chamber caused, after 6 h, a dose-dependent inhibition of LAMA84R exosome-stimulated endothelial cell migration (figure 7a). Figure 7b shows the inhibitory effects of a neutralizing anti-IL8 antibody and CTO on IL8-stimulated motility of EC cells. Furthermore, we used EC transfected with IL8 siRNA to demonstrate the role of IL8 in exosome-stimulated migration. Our results show that exosome-induced HUVEC migration decreased when IL8 was silenced compared to cells transfected with scramble siRNA control (Figure S2a). 10.1371/journal.pone.0042310.g007 Figure 7 CTO inhibits the effects of LAMA84R exosomes on HUVEC migration. (a) Effects on migration of CTO-treated endothelial cells using 50 µg/ml of exosomes as chemoattractant. (b) 50 µg/ml of exosomes (Exo) with or without 10 µg/ml of neutralizing anti-IL8 antibody (N Ab IL8), or 10 ng/ml of recombinant IL8 (Rec IL8) with or without neutralizing anti-IL8 antibody were added as chemoattractants to the bottom wells., Motility of endothelial cells with or without increasing doses of CTO (1–10 µM) was evaluated as described in Material and Methods. *p≤0.05; **p≤0.01. CTO Treatment Inhibits in vitro and in vivo Exosome-mediated Angiogenesis We evaluated the properties of CTO to inhibit exosome-stimulated angiogenesis by using in vitro and in vivo angiogenesis models. Angiogenesis is a very complex process involving several kinds of cells; tube formation of endothelial cells is one of the key steps of angiogenesis. Therefore, firstly we showed that, compared to cells maintained in low serum medium, LAMA84R exosomes stimulate in vitro tube formation as tested by Matrigel assay to a similar extent of addition of recombinant IL8 used as positive control (figure 8a). Addition of a neutralizing anti-IL8 antibody or 10 µM CTO, inhibited exosome-induced tube formation (figure 8a). These results were confirmed by the measurement of the length of tubular connections that showed a more than threefold increase in cellular projections interconnecting HUVEC after treatment with LAMA84R exosomes, or recombinant IL8 compared to control. Adding CTO or neutralizing anti-IL8 antibody caused a dramatic inhibition of exosome-mediated effects on tube formation (figure 8b). LAMA84R exosomes do not induce the tube formation on IL8-silenced endothelial cells (Figure S2b). 10.1371/journal.pone.0042310.g008 Figure 8 in vitro inhibition of exosome-stimulated angiogenesis by CTO. (a) Phase contrast micrographs showing the effects of LAMA84R exosomes and CTO treatment on endothelial network formation (matrigel assay). Few cables are observed when HUVEC are plated in low serum medium (CN). Addition to HUVEC cells of 10 ng/ml of recombinant IL8 (Rec IL8) or 50 µg/ml of LAMA84R exosomes (Exo) induces the formation of capillary-like structures. No tube formation is observed when HUVEC are plated in the presence of 50 µg/ml of exosomes plus neutralizing anti-IL8 antibody (Exo + N Ab IL8). CTO inhibits the effects of recombinant IL8 (10 µM CTO + Rec IL8) or exosomes (Exo +10 µM CTO) on tube formation by HUVEC on matrigel. (b) Histograms showing the quantitative analysis of the cables length by Image J software. Then we evaluated in vivo antiangiogenic effect of CTO on a mouse Matrigel plug model. An initial sign of the levels of activity of CTO as inhibitor of the exosome-stimulated angiogenesis was visually assessed because of the color difference in the vascularized plugs when compared with the controls. The reddish color of LAMA84R-exosomes (Exo) or recombinant IL8 (Rec IL8) containing plugs in the vehicle control-treated mice reflected the development of a dense neovascularization. In contrast, the pale color of the plugs removed from CTO–treated mice indicated inhibition of exosome-stimulated vascularization over a 4 weeks period (figure 9a). Drabkin’s assay was used to measure haemoglobin content in the plugs as a marker of vascularity (figure 9b). Next we examined whether LAMA84R exosomes stimulate phosphorylation of signalling proteins, particularly Akt and Erk 1/2, which are the principal mediators of cell proliferation, survival, and chemotaxis in endothelial cells and if CTO was able to modulate these pathways. Figure 10 shows that over night addition of 50 µg/ml LAMA84R exosomes or of 10 ng/ml IL8 to endothelial cells trigger the phosphorylation of both the signalling molecules; this result is consistent and extends our previous findings [11] suggesting that microvesicles are able to interact directly with target cells and act as a signalling molecule. The enhanced Erk 1/2 and Akt phosphorylation was reduced by treatment of endothelial monolayer with 10 µM CTO (figure 10). 10.1371/journal.pone.0042310.g009 Figure 9 CTO treatment inhibits exosome-stimulated angiogenesis in vivo. NOD/SCID mice treated with CTO show a decreased exosome-stimulated angiogenesis in matrigel plug assay. (a) Matrigel plugs implanted subcutaneously in mice and containing: PBS as negative control,or 100 µg of LAMA84R exosomes (Exo) or 50 ng of recombinant IL8 (Rec IL8). Mice were treated with vehicles or drugs (CTO and/or imatinib) as described in Material and Methods and plugs were removed after 4 weeks. (b) Evaluation of haemoglobin concentration in the matrigel plugs by Drabkin assay. *p≤0.05; **p≤0.01, ***p≤0.001. 10.1371/journal.pone.0042310.g010 Figure 10 CTO inhibits Akt and Erk 1/2 phosphorylation in exosomes-stimulated HUVEC. HUVEC were incubated with 50 µg/ml of exosomes with or without 10 µM CTO or with 10 ng/ml of IL8 with or without 10 µM CTO for 1 h (pErk 1/2 and Erk 1/2) or over night (pAkt and Akt). After the treatments, protein lysates were subjected to western blot analysis as described in Material and Methods using anti-phospho-Akt, anti- Akt, anti-phospho-Erk, anti-Erk antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading. Discussion In the present study we tested the anti-tumor effects of carboxyamidotriazole-orotate in vitro and in xenograft model of imatinib-resistant human CML, furthermore we evaluated the ability of CTO to inhibit CML exosome-stimulated angiogenesis both in vitro and in vivo models. Imatinib treatment has been remarkably successful in reducing the tumor burden and suppressing the progression of patients in the chronic phase of CML, however, a lower efficacy has been observed during the accelerated phase of the disease, mainly due to the evolution of clinical resistance. Second generation inhibitors such as dasatinib or nilotinib, do not show activity against all point mutations responsible for imatinib resistance. Among patients with CML who developed imatinib resistance, Bcr-Abl mutations was reported just in 31% to 42% of cases, suggesting mechanisms of disease progression that are Bcr-Abl-independent [18]. New approaches reling on the use of compounds targeting either pathways downstream of Bcr-Abl activation or events that contribute or modulate leukaemia progression are necessary. We have previously demonstrated that CAI, an inhibitor of calcium-mediated signal transduction [19], decreases cell viability and induces apoptosis of imatinib-resistant CML cells, reducing both total and phosphorylated Bcr-Abl [8]; furthermore, we showed that CAI activity against imatinib-resistant CML cells was due to the ability to increase intracellular reactive oxygen species [9]. Although CAI has been tested in phase II clinical trial for treatment of solid tumours, its use in clinical settings has been hampered by the limited solubility, toxicity, or evident clinical benefits [20]. CTO is the orotate salt form of carboxyamidotriazole showing a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound [10]. One of the initial findings of our study was that CTO caused in both K562R and LAMA84R cells, inhibition of proliferation concomitant to Bcr-Abl down-regulation, dephosphorylation and decrease in tyrosine phosphorylation of CrkL, more rapidly than the parental compound. Furthermore, it showed to be more active than CAI on a molar basis [8]. Rapid reduction of Bcr-Abl protein coupled with kinase inactivation, as seen with CTO, can be particularly advantageous because of the multiple Bcr-Abl domains that mediate protein interactions triggering different signalling pathways responsible for cell proliferation, adhesion, and inhibition of apoptosis [21]. The inhibitory effects of CTO against CML cells in culture, is mirrored by its activity against CML xenografts in NOD/SCID model. Tumor growth retardation in mice treated with CTO 513 mg/kg plus imatinib was evident suggesting that CTO was acting in this model as an antileukaemic agent. Recently, there are increasing data showing that angiogenesis plays an important role in the development and progression of chronic myeloid leukaemia [22], [23]. The bone marrow of patients with CML exhibit marked neovascularization and increased number of endothelial cells [24]; the cross-talk between tumor cells and endothelial cells leads to enhanced tumor growth, metastasis and altered response to anti-cancer therapy [25]. Recently, a number of studies have recently described exosomes as new players in modulating tumor microenvironment, promoting angiogenesis and tumor progression [12]. Our group and other collaborators have shown that exosomes released by imatinib-sensitive LAMA84 [11] and K562 CML cells [26] have a potential to influence in vitro and in vivo angiogenesis by affecting directly endothelial cells properties. One of the findings of the present study was the confirmation, by morphological and biochemical analysis, that LAMA84R CML cells secrete exosomes and that these vesiscles are able to modulate angiogenesis in vitro and in vivo. These findings drove us to investigate if CTO could target both tumor cells and the tumor microenvironment. Therefore, we focused on the inhibitory effects of CTO on in vitro selected functional steps of angiogenesis as well as on in vivo angiogenesis in NOD/SCID mice. Our in vitro studies with HUVEC demonstrated that CTO inhibits exosome stimulated motility, cytokines and cell-adhesion molecules (ICAM1 and VCAM1) expression of endothelial cells; moreover CTO inhibits exosomes activated signalling pathways and capillary-like structure formation. The matrigel plug assay that mimics physiological neo-angiogenesis, was used as in vivo model; our study showed that CTO drastically decreased exosome-stimulated angiogenesis. To investigate on the possible molecular mechanisms of the CTO-mediated antiangiogenic effect, we examined whether CTO inhibited the activation of intracellular signalling pathways involved in endothelial cell activation. Treatment of the EC with CTO blocked significantly the exosome-induced phosphorylation of signalling proteins, particularly Akt and Erk 1/2, which are the principal mediators of cell proliferation, survival, and chemotaxis in endothelial cells [27]. Kinase-dependent and kinase-independent mechanisms are known to contribute to the abnormal adhesion and migration of CML progenitors, thus the effect of CTO on both endothelial cells and leukemic cells may concomitantly inhibit adhesion of leukaemia cells to vascular endothelium and conditions that favour leukostasis and tissue infiltration. IL8 is a member of the CXC family of chemokines, a potent proangiogenic factor [28], and its plasma levels are found significantly higher in patients affected by chronic myelogenous leukaemia [29]. Interestingly we showed, through the use of IL8 neutralizing antibodies and short interfering RNAs, that IL8 was in part responsible for the effects of LAMA84R exosomes on EC activation; furthermore, treatment of EC with CTO inhibited the IL8-stimulated angiogenic phenotype. It is conceivable to hypothesize that IL8 secreted by EC stimulated with CML exosomes, may modulate both myeloid malignant cells and endothelial cells, thus generating a paracrine machinery between hematopoietic malignant cells and newly generated endothelium. In this tumor microenvironment, CTO could inhibit the angiogenic process through blocking the exosome-mediated crosstalk, thus causing the interruption of a reciprocal stimulatory loop between leukemic and endothelial cells. Other groups have pointed their attention on the close relationship between exosome production and tumor microenvironment modulation; Hood and collaborators demonstrated that exosomes released by melanoma cells modulate both angiogenic and immunological cytokine signalling, thus serving as paracrine nanocarriers that might prepare distal sites for the arrest of metastatic cells [30]. In this context, the inhibition of either exosomes shedding or modulation of their function has been proposed as worthwhile approach to cancer therapy. Al-Nedawi et al. showed that the treatment of A431 tumor xenografts with Diannexin, which inhibits the uptake of the A431 (human squamous cell carcinoma cell line)-derived microvesicles into endothelial cells, led to a reduction of tumor growth rate and microvascular density [31]. As far as we are aware, this is the first study that demonstrates the inhibitory effect of an anticancer drug on angiogenesis stimulated by exosomes released from drug-resistant cancer cells; collectively, our findings generate a rationale for investigating clinical efficacy of molecules such as CTO that are endowed with antitumor and antiangiogenic properties. Supporting Information Figure S1 IL8 siRNA inhibits IL8 mRNA expression and cytokine release from HUVEC. IL8 mRNA expression levels (a) or IL8 protein release in conditioned medium (b) were evaluated in HUVEC transfected either with oligofectamine (CN), or with scramble siRNA or with IL8 siRNA. HUVEC transfected were treated or not for 6 h with 50 µg of LAMA84R exosomes (Exo). (TIF) Figure S2 IL8 siRNA inhibits the effects of LAMA84R exosomes on migration and tube formation capabilities of HUVEC. (a) Addition of exosomes to the bottom wells of Boyden chamber increases the migration of either HUVEC and HUVEC transfected with scramble siRNA while concomitant CTO treatment reverts this effect. On the contrary exosomes have not significative effects on migration of IL8-silenced HUVEC.(b) Phase contrast micrographs showing the effects of LAMA84R exosomes and CTO treatment on endothelial network formation after silencing of HUVEC with IL8 siRNA (matrigel assay). Exosomes (Exo) induce formation of capillary-like structures on HUVEC transfected with scramble siRNA compared to control cells (siRNA scramble). No tube formation is observed when exosomes stimulated EC were silenced for IL 8 mRNA expression with short interfering RNAs. (TIF) ==== Refs References 1 Rowley J (1973) A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. 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PLoS One. 2012 Aug 3; 7(8):e42310
==== Front Cancer Cell IntCancer Cell IntCancer Cell International1475-2867BioMed Central 1475-2867-12-212264264210.1186/1475-2867-12-21Primary ResearchThe stem cell factor antibody enhances the chemotherapeutic effect of adriamycin on chemoresistant breast cancer cells Jelly Neil D [email protected] Issam I [email protected] Jennifer [email protected] Oleg [email protected] Mohamed [email protected] University of Lincoln, Brayford Pool, Lincoln, LN6 7TS, UK2 Research & Development, Lincoln County Hospital, Greetwell Road, Lincoln, LN2 5QY, UK3 Queens Medical Centre, University of Nottingham, Derby Road Nottingham, Nottingham, NG7 2UH, UK2012 29 5 2012 12 21 21 12 12 2011 29 5 2012 Copyright ©2012 Jelly et al.; licensee BioMed Central Ltd.2012Jelly 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 outcome of chemotherapy in breast cancer is strongly influenced by multidrug resistance (MDR). Several surrogate markers of chemoresistance have been identified including - CD24 (cluster differentiation 24) expression, stem cell growth factor (SCF), B-cell lymphocyte protein 2 (Bcl-2) and annexin V. The present study aimed to examine the expression of CD24 in the sensitive breast cancer cell line MCF-7 (Michigan Foudation-7) and MCF-7/adriamycin resistant (MCF-7/AdrRes) cells, and, if minimal effective doses of the anthracycline drug adriamycin (0.579 μM and 88.2 μM) would be enhanced by the antibody to SCF (anti-SCF). Methods CD24 expression was analysed by flow cytometry. Both Bcl-2 and annexin V protein expression were quantitatively assessed by the enzyme-linked immunosorbent assay (ELISA). Results In MCF-7/AdrRes cells the expression of CD24 was significantly higher compared to MCF-7 cells, 86.6% and 16.3% (p < 0.001), respectively. Bcl-2 expression was significantly increased in the presence of adriamycin and SCF (p < 0.038) and decreased in the presence of adriamycin and anti-SCF. When adriamycin, anti-SCF and SCF were combined or when adriamycin was used alone the decrease in Bcl-2 expression was insignificantly altered. In the presence of both adriamycin and SCF the expression of annexin V was decreased. However, it was significantly increased in the presence of adriamycin and anti-SCF (p < 0.042), as well as adriamycin, anti-SCF and SCF combined. In MCF-7 cells the effect of adriamycin alone or with either SCF, anti-SCF or anti-SCF or SCF combined, did not significantly alter the expression of Bcl-2. However, in the presence of both adriamycin and SCF the expression of annexin V was decreased, but was significantly increased in the presence of adriamycin and anti-SCF (p < 0.001), adriamycin, anti-SCF and SCF combined and adriamycin alone. Our results demonstrate that anti-SCF with low dose of adriamycin reduces Bcl-2 expression in MCF-7/AdrRes cells and increases annexin V expression in both MCF7/AdrRes and MCF-7 cells. Conclusion Adding anti-SCF to the chemotherapeutic regime of adriamycin may strongly enhance its chemotherapeutic effect in the treatment of patients with breast cancer. ==== Body Introduction Breast cancer is the most common form of cancer and the principal cause of death from cancer among women worldwide [1] Neoadjuvant chemotherapy (NAC) is frequently used to treat breast cancer patients particularly those with locally advanced disease in order to downstage and downgrade the disease [2] However, a complete pathological response is only observed in 30% of patients, whilst 70% of patients show an incomplete or no pathological response [3-7] Despite advances in understanding the molecular basis of breast cancer the poor responses to chemotherapeutic agents are not well defined. Several factors are attributed to drug resistance including - drug efflux, cancer stem cells (CSCs), cytokine overexpression and resistance to drug-induced apoptosis [8,9]. The ability to predict the response to NAC may result in a more cost-effective therapy. Therefore, targeting therapy to these potential responders would also avoid significant and unnecessary morbidity in nonresponders [3]. Adriamycin is an important drug component in NAC regimens however; breast cancer cells often become resistant to its effects. Critical apoptotic pathways, which are initiated by adriamycin and other cytotoxic drugs, are altered by several mechanisms resulting in chemoresistance. The ability to evade programmed cell death is a phenotypic characteristic of most tumours [10]. Negative regulators of apoptosis are amongst the most frequently studied particularly the proto-oncogene Bcl-2. Both B-cell lymphocytes and CSCs are characterised by extracellular protein expression of CD24, which may have an important role in both tumour growth and resistance. Nonetheless, it is thought that cancer stem cells (CSCs) are involved in carcinogenesis, local invasion and metastasis which play a key role to both radiotherapy and chemotherapy resistance [9]. Also, SCF may be co-expressed with Bcl-2 however their relationship requires further definition. Recently, an antibody to SCF (anti-SCF) significantly enhanced the cytotoxic effects of chemotherapy in human resistant haematological cancer [11]. However, it is not known whether anti-SCF enhances cytotoxicity in solid cancer e.g. breast cancer. On developing new molecular therapeutics understanding pharmacodynamic endpoints is critical. One of the characteristics of apoptosis is the externalization of phosphatidylserine (PS). It is documented that Annexin V is able to bind with high specificity to PS [12]. Therefore, the aim of this study was to evaluate the expression of CD24, and the ability of anti-SCF to enhance adriamycin by examining their combined effects on both Bcl-2 and annexin V expression in MCF-7 and MCF-7/AdrRes breast cancer cells. Materials and methods Cell culture of MCF-7 and MCF-7/AdrRes cell lines The MCF-7 and MCF-7/AdrRes human breast adenocarcinoma cell lines were a kind gift from Queens Medical Centre, University of Nottingham, UK [3,9]. The MTT 3(4, 5 Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to establish minimal effective dose concentrations of adriamycin. In MCF-7/AdrRes cells the IC50 concentration for adriamycin was 88.2 μM versus 0.579 μM in MCF-7 cells. The cells were grown in tissue culture medium (TCM) consisting of RPMI-1640 (with L-glutamine) (Sigma Aldrich, UK), supplemented with 10% foetal calf serum (FCS) (Sigma Aldrich, UK), and 100 units/ml of penicillin/and 0.1- mg/ml of streptomycin (Sigma Aldrich, UK), and placed in an incubator. The incubator was set at 0.05 CO2 and humidified at 37°(C) throughout the study. All cells used subsequently in this study were between 15–20 passages. 2.2 Immunophenotyping for CD24 Expression Using Flow Cytometry Cultured cells were resuspended (0.5 × 106 cells) in ice cold Phosphate Buffered Saline (PBS) and 10% FCS. Cells (0.5 × 106) were then incubated for 30 minutes (mins) at 4°C with primary CD24 fluorescein isothiocyanate (FITC) murine monoclonal antibody (MoA) (Cambridge Biosciences, Cambridge, UK), which was prepared by diluting in 3% bovine serum albumin (BSA) and PBS (BSA/PBS) to a final concentration of 1 in 20. Cells were washed in PBS centrifuged at 300 g for 5 mins and resuspended in 500μls of ice cold PBS with 10% FCS for analysis by the Epics xL flow cytometer (Beckman Coulter, High Wycombe, UK) which was equipped with a standard argon laser for 488 nm excitation and with 525 nm band pass (FL1), 575 nm band pass (FL2), 620 nm band pass (FL3) and 675 nm band pass (FL4) filters. Treatment of MCF-7 and MCF-7/AdrRes cells MCF-7 and MCF-7/AdrRes cell lines were cultured (5 × 103 cells perwell) and were treated with adriamycin (Sigma Aldrich, UK) using 0.579 μM and 88.2 μM, respectively, to be effective in accordance with IC50 concentrations, 0.4 μg/ml anti-SCF (PeproTech EC LTD, UK), and/or 100 units/ml of SCF (0.4 μg/ml of anti-SCF fully inhibits the biological activity of SCF as previously established by our colleagues) [11]. The final concentration of 100 μl/well of treatment was incubated with the cells using the following parameters: (1) adriamycin with SCF; (2) adriamycin with anti-SCF; (3) adriamycin with both SCF and anti-SCF; (4) adriamycin alone. Cells were treated without adriamycin using the same parameters. Five replicates of each treated culture (2.5 × 104 cells) were set and incubated for 48hs. Analysis of bcl-2 using the quantitative ELISA assay The human Bcl-2 ELISA kit (Bender Med SystemsTM) was used to quantitatively analyse Bcl-2 protein, as described previously [11]. Cells were washed twice with PBS and centrifuged at 500 g for 5 mins. The cell pellets were resuspended at 5 × 106/ml of lysis buffer for 60 mins at room temperature (RT). Cells were re-centrifuged and resuspended in sample diluent (serum matrix, buffered solution) 1:5. Each sample of cell lysate and lyophilized standards of serially diluted Bcl-2 (100μls) were duplicated and added with FITC-labelled biotin-conjugate (anti-Bcl-2 murine MoA) diluted 1:100 in the assay buffer (PBS, with 1% Tween 20 and 10% BSA), and incubated in a 96-microwell plate coated with murine MoA of human Bcl-2 for 2hs at RT. Using wash buffer (PBS containing 1% Tween 20) the microwell strips were thoroughly washed. Streptavidin-horseradish peroxidase (HRP) - conjugate was then added to all wells at a dilution of 1:100 in the assay buffer and incubated for 1 h at RT. Microwell strips were thoroughly washed again and then TMB substrate solution (1 × tetramethylbenzidine added to 1 × 0.02% - buffered hydrogen peroxide) was added for 10 mins and incubated at RT. The change in colour from the substrate reaction was monitored and was terminated by adding stop solution (1 M phosphoric acid). The absorbance of each microwell was then read at 450 nm using an ELISA plate reader (BMG Lab Technologies, Germany). Analysis of annexin V using the quantitative ELISA assay The human annexin V ELISA kit (Bender Med SystemsTM) was used to quantitatively analyse annexin V protein, as previously reported [11]. Cells were washed twice with PBS and centrifuged at 500 g for 5 mins. The cell pellets were re suspended in sample diluent (serum matrix, buffered solution) (1:2). Each sample and lyophilized standards of serially diluted annexin V (100μls) were duplicated and added with FITC-labelled biotin-conjugate (anti-annexin V murine MoA) diluted 1:100 in the assay buffer (PBS, with 1% Tween 20 and 10% BSA). Samples were then incubated in a 96-microwell plate coated with murine MoA of human annexin V for 2hs at RT. Using wash buffer, (PBS containing 1% Tween 20) the microwell strips were washed thoroughly. Then at a dilution of 1:200 in the assay buffer, streptavidin-horseradish peroxidase (HRP) - conjugate was added to all wells and incubated for 1 h at RT. Microwell strips were again washed thoroughly with wash buffer with TMB substrate solution (1 × tetramethylbenzidine added to 1 × 0.02% - buffered hydrogen peroxide) then being added for 10 mins at RT. The change in colour from the substrate reaction was monitored and was terminated by adding stop solution (1 M phosphoric acid). The absorbance of each microwell was read at 450 nm using the ELISA plate reader (BMG Lab Technologies, Germany). Statistical analysis The mean ± standard error of all readings underwent statistical analysis, whenever appropriate. Independent two-sample, two-tailed tests were used to analyse the difference in mean values of CD24 expression. Multi-factorial (three way) Analysis of Variance (ANOVA) was used to analyse the two response variables of Bcl-2 and annexin V. Statistical significance is recorded when the p-value was less than 0.05 (P < 0.05). Results CD24 expression CD24 expression was measured by flow cytometry in MCF-7/AdrRes and MCF-7 cell lines (Figures 1 and 2). Levels of CD24 expression ranged from 86.6 ± 3.0% in resistant cells, compared with 16.3 ± 1.9% in wild type cells which was highly significant (p < 0.001). Figure 1 Expression of CD24 in MCF-7/AdrRes cells. Cells (104) were analysed by flow cytometry using CD24 monoclonal antibody (FITC). The histogram shows high level expression for CD24 in cells (positives - 87%). Both the plots of forward scatter (FSC) versus side scatter (SSC) were used to gate cells (not shown). To show positivity of CD24 an (FL1) plot was used (shown). For the Epics xL flow cytometer λ excitation = 488 nm and λ emission = 525 nm. Figure 2 Expression of CD24 in MCF-7 cells. Cells (104) were analysed by flow cytometry using CD24 monoclonal antibody (FITC). The histogram shows low level expression for CD24 in cells (positives - 16%). Both the plots of forward scatter (FSC) versus side scatter (SSC) were used to gate cells (not shown). To show positivity of CD24 an (FL1) plot was used (shown). For the Epics xL flow cytometer λ excitation = 488 nm and λ emission = 525 nm. Effect of adriamycin/anti-SCF on bcl-2 expression in MCF-7/AdrRes and MCF-7 cells The therapeutic effects of adriamycin, anti-SCF and SCF on Bcl-2 expression were examined by ELISA in MCF-7 and MCF-7/AdrRes cells, after being treated simultaneously for 48 hrs. The combination of adriamycin and SCF significantly increased Bcl-2 expression in MCF-7/AdrRes cells (p < 0.038), with a decrease in Bcl-2 expression observed after treatment with adriamycin and anti-SCF, adriamycin and both SCF and anti-SCF combined and adriamycin alone (Figure 3). In MCF-7 cells there was no significant difference in Bcl-2 expression observed after treatment with either adriamycin and SCF or adriamycin and anti-SCF, unlike in MCF-7/AdrRes cells. Similarly, no significant difference in Bcl-2 expression was observed in cells after treatment with adriamycin and both anti-SCF and SCF combined, or adriamycin (Figure 4). Figure 3 Effect of Adriamycin/Anti-SCF on Bcl-2 Expression in MCF-7/AdrRes Cells. Cell lysates were used to determine the expression of Bcl-2 using ELISA after treatment with the drug adriamycin (D) + SCF, D + anti-SCF, D + SCF + anti-SCF and Drug alone. For each individual experiment data are represented as the mean ± SE of duplicate determinations of 1.25 × 104 cells. ANOVA showed a significant* difference (p < 0.05) between treatments (D) ± SCF and D ± anti-SCF, D ± SCF ± anti-SCF and Drug alone. Figure 4 Effect of Adriamycin/Anti-SCF on Bcl-2 Expression in MCF-7 Cells. Cells lysates were used to determine the expression of Bcl-2 using ELISA after treatment with the drug adriamycin (D) + SCF, D + anti-SCF, D + SCF + anti-SCF and Drug alone. For each individual experiment data are represented as the mean ± SE of duplicate determinations of 1.25 × 104 cells. ANOVA showed no significant difference (p > 0.05) between treatments (D) ± SCF, D ± anti-SCF, D ± SCF ± anti-SCF and Drug alone. Effect of adriamycin/anti-SCF on annexin V expression in MCF-7/AdrRes and MCF-7 cells To determine the therapeutic effects of adriamycin, anti-SCF and SCF on annexin V expression, MCF-7 and MCF-7/AdrRes cells were also treated simultaneously for 48 hrs before being examined by ELISA. The expression of annexin V was decreased in MCF-7/AdrRes cells after treatment with adriamycin and SCF, but significantly increased after treatment with adriamycin and anti-SCF (p < 0.042), adriamycin and both SCF and anti-SCF combined and adriamycin alone (Figure 5). There was also a decrease in annexin V expression in MCF-7 cells after treatment with adriamycin and SCF, with a significant increase observed after treatment with adriamycin and anti-SCF (p < 0.001), adriamycin and both anti-SCF and SCF combined and adriamycin alone (Figure 6). Figure 5 Effect of Adriamycin/Anti-SCF on Annexin V Expression in MCF7/AdrRes Cells. Cells were used to determine the expression of annexin V using ELISA after treatment with the drug adriamycin (D) + SCF, D + anti-SCF, D + SCF + anti-SCF and Drug alone. For each individual experiment data are represented as the mean ± SE of duplicate determinations of 1.25 × 104 cells. ANOVA showed a significant* difference (p < 0.05) between treatments D ± anti-SCF, D ± SCF ± antiSCF, Drug alone and (D) ± SCF. Figure 6 Effect of Adriamycin/Anti-SCF on Annexin V Expression in MCF-7 Cells. Cells were used to determine the expression of annexin V using ELISA after treatment with the drug adriamycin (D) + SCF, D + anti-SCF, D + SCF + anti-SCF and Drug alone. For each individual experiment data are represented as the mean ± SE of duplicate determinations of 1.25 × 104 cells. ANOVA showed a significant* difference (p > 0.05) between treatments D ± anti-SCF, D ± SCF ± antiSCF and Drug alone and (D) ± SCF. Discussion Neoadjuvant chemotherapy is used as a multimodality treatment for breast cancer patients with large and locally advanced disease. One important component of the drug combinations used are anthracyclines (adriamycin), which are given in association with other chemotherapeutic agents such as Taxanes [13]. However, this treatment is compromised by the presence of multidrug resistance (MDR) resulting in treatment failure and subsequent increased morbidity and mortality. Several covariate mechanisms are responsible for multi-drug resistance in breast cancer cells including increased drug efflux (P-glycoprotein-P-gp), CSCs (CD24), drug detoxification (glutathione S-transferase) deregulated apoptosis (Bcl-2 overexpression) and overexpression of cytokines (SCF)[8]. The anti-apoptotic expression of Bcl-2 is well documented and its affects on tumourigenic activity [14]. CD24 is a phenotypic surface marker for granulocytes, Bcl’s and CSCs, which are implicated in resistance to both chemotherapy and radiotherapy. Subsequently, some of these progenitor cells differentiate into new mature tumour cells with a chemoresistant phenotype [15]. CSCs were originally described in haematologic malignancies, but this emerging concept is now also applied to solid tumours. Consequently, there is an increasing need to develop assays which determine the pharmacodynamic effects of both existing and new cancer therapeutics [10]. Annexin V, is a 35 kilodalton (kDa) calcium-dependent protein which binds with high affinity (K = 10-9 M) to phosphatidylserine (PS) residues that are displayed on the outer surface of apoptotic cells [16]. This is not only an alternative non-invasive technique in order to quantify apoptosis and determine pharmacodynamic endpoints after treatment with anti-cancer therapies, but may also be of potential benefit when developing new molecular therapies. The administration of high-dose chemotherapy facilitated by autologous progenitor cell support such as SCF, the haematopoietic growth factor, is being more frequently applied to the treatment of cancer [17]. In patients with breast and ovarian cancer, lymphoma and multiple myeloma and in conjunction with G-CSF (Granulocyte Colony Stimulating Factor) SCF is used in clinical practice to mobilise CD34+ cells into peripheral blood [18]. In the past SCF expression has been demonstrated in malignant melanomas, pancreatic cancer, glioma cells, gastrointestinal stromal tumours (GIST) and colon cancer [19-23]. Therefore, in preventing chemotherapy induced haematological depression in cancer patients the clinical relevance of SCF and other growth factors or cytokines which may possibly influence tumour proliferation and survival at the level of cancer stem cells, opens questions. Another major concern about this treatment is that SCF may induce protection against chemotherapy in tumour cells that also express the SCF receptor (c-Kit), which has been observed in both haematological and solid cancers. It has been suggested that the maintenance and normal growth of mammary epithelial tissue is influenced by the ckit/SCF pathway with their progressive loss occuring adjacent to malignant transformation [24]. However, one alternative suggestion is that SCF modulates tumour growth and angiogenesis via the involvement of mast cells [25]. Consequently, further examination of the effects of SCF on breast cancer growth and progression is required. Additionally, the activity of anti-SCF may have a multiple role in antagonising the negative growth of breast cancer cells by affecting those pathways involved in CSC and Bcl-2 mediated chemoresistance. The difference in CD24 expression in MCF-7/AdrRes and MCF-7 cells was demonstrated in this study, and also the ability of anti-SCF to enhance the effects of adriamycin chemotherapy. To the best of our knowledge it is the only study to analyse the synergistic effects of adriamycin and anti-SCF in MCF-7/AdrRes and MCF-7 cells, and their combined effects on Bcl-2-related resistance and annexin V-related cytotoxicity. Initially, the IC50 minimal dose concentrations obtained from in vitro cytotoxicity assays showed a 1.52 × 102 fold higher resistance to adriamycin in MCF-7/AdrRes cells, compared with MCF-7/WT cells (88.2 μM and 0.579 μM), respectively. In MCF-7/AdrRes cells there was a significant increase in the positivity of CD24 expression compared with a markedly low level in MCF-7 cells. Recently, there have been high levels of CD24 observed in mouse xenografts derived from both CD44+/CD24-/low and CD44+/CD24hi breast cancer cells suggesting important role for CD24 in tumour growth, whilst CD44+/CD24-/low breast cancer cells were not associated with increased tumourigenicity [26]. Further to this, ovarian tumour specimens of a patient showed a sub-population enriched for ovarian CSCs defined by CD24 phenotype. It was observed that the CD24+ sub-population remained quiescent and more chemoresistant compared with the CD24-/low fraction as well as having stem cell-like characteristics such as specific capacity for self-renewal and differentiation. Additionally, CD24 + cells were able to form tumour xenografts in nude mice, whereas equal numbers of CD24- cells did not [27]. Finally, CD24+ cells had lower E-cadherin mRNA levels in comparison to CD24- cells, whilst the mRNA levels of certain stemness genes (Nestin, β-catenin, Bmi-I, Oct4, Oct3/4, Notch1 and Notch4) were more highly expressed. Interestingly, both adriamycin and SCF combined significantly increased Bcl-2 expression in MCF-7/AdrRes cells, however in MCF-7/WT cells there was no effect. In addition to its cell cycle inhibitory function which markedly increases the cell cycle withdrawal into the G0 quiescent phase Bcl-2 also has the ability to enhance cell survival [28]. This also protects cells from the effects of chemotherapy with increased expression of CD24 being a further contributory factor, modulating not only Bcl-2 expression, but also the presence of CSC populations. The decrease in Bcl-2 expression observed after treatment with both adriamycin and anti-SCF has reduced Bcl-2 related resistance. Subsequently, there were higher annexin V levels observed in MCF-7/AdrRes (and MCF-7/WT cells) after treatment with adriamycin and anti-SCF indicating increased apoptosis, but these were decreased after treatment with adriamycin and SCF. This result differs with our previous study in MCF-7/paclitaxel resistant cells (unpublished data, Jelly et al., 2008). The mechanistic action of each drug combined with anti-SCF may subsequently not only affect Bcl-2 expression, but also the pathways involved in apoptosis leading to annexin V expression, therefore explaining these differences. Other studies have also reported that Bcl-2 may have distinct biological properties depending on the anticancer agent used when affecting antineoplastic sensitivity [29]. The major advantage of using anti-SCF is that it specifically inhibits SCF and not other key cytokines unlike other generic tyrosine kinase inhibitors such as imatinib, which block several cytokine receptors including c-Kit [30]. In addition there is also the potential when administering this as a combined therapy with adriamycin, to slow the proliferation of normal CD34+ bone marrow cells protecting them from chemotherapy-induced myelosuppression. Also, it may be possible to shorten post-chemotherapy neutropenia. However, normal CD34+ bone marrow cells may become susceptible to cytotoxicity when treating with chemotherapy if anti-SCF reduces Bcl-2 expression [11]. If this combined therapy increases apoptosis in these normal cells of the bone marrow then any therapeutic advantage gained may be lost and therefore further evaluative studies are warranted. In this study it has been demonstrated that Bcl-2 expression is reduced in MCF-7/AdrRes cells after treatment with adriamycin and anti-SCF with annexin V being increased in both MCF-7/AdrRes and MCF-7/WT cells. This possible advantage as well as their cytotoxic effects on normal CD34 + bone marrow cells requires further investigative study. Both adriamycin and anti-SCF combined may potentially improve response to treatment in chemoresistant breast cancer and also improve long term clinical outcome. Competing interests There are no competing interests financial or non-financial (political, personal, religious, ideological, academic, intellectual, commercial or any other) to declare in relation to this manuscript. Authors’ contribution NJ was jointly responsible for the concept, design and completion of all laboratory studies, IH assisted in the laboratory studies. JE and OE participated in the coordination of the study and helped to draft its manuscript. MES was jointly responsible for the concept, design, coordination of the study and also helped to draft its manuscript. All authors read and approved the final manuscript. Authors’ information I. Mr Neil Jelly (NJ) is a PhD student and Lecturer at University of Lincoln. II. Dr Issam Hussain (IH) is a Lecturer at University of Lincoln III. Dr Jennifer Eremin (JE) is a Senior Consultant in Medical Oncology IV. Professor Oleg Eremin (OE) is a Special Professor at the Department of Surgery at Queens Medical Centre, University of Nottingham and Director of Research & Development, Lincoln Hospital V. Director of the study. Mr. Mohamed El-Sheemy (MES) is a Senior Lecturer at the University of Lincoln & Breast Surgeon at Lincoln Hospital. Director of Clinical Research Laboratory at United Lincolnshire Hospitals NHS Trust Funding Grant from the collaborative Joint Research Fund between University of Lincoln and United Lincolnshire Hospitals NHS Trust. Acknowledgements We pleased to acknowledge the considerable help we received from the laboratory technicians Ms Angela `Murtagh and Ms Beverley Shepherd for their technical help. Also, we appreciate the help received in statistical analysis from Mr John Flynn, Consultant Statistician, University of Lincoln. ==== Refs Washbrook E Risk factors and epidemiology of breast cancer Wom Health Med 2006 3 8 14 10.1383/wohm.2006.3.1.8 Atalay C Gurhan ID Irkkan C Gundaz U Multidrug Resistance in Locally Advanced Breast Cancer Tumour Biol 2006 27 309 318 10.1159/000096086 17033200 Chuthapisith S Layfield R Kerr ID Hughes C Eremin O Proteomic profiling of MCF-7 breast cancer cells with chemoresistance to different types of anti-cancer drugs Int J Oncol 2006 30 1545 1551 17487377 Bear HD Anderson S Brown A Smith R Mamounas EP Fisher B Margolese R Theoret H Soran A Wickerham DL Wolmark N National Surgical Adjuvant Breast and Bowel Project Protocol B-27: The effect on tumour response of adding sequential preoperative docetaxel to preoperative doxorubicin and cyclophosphamide: preliminary results from National Surgical Adjuvant Breast and Bowel Project Protocol B-27 J Clin 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Expression of stem cell factor (SCF), a KIT ligand, in gastrointestinal stromal tumours (Gist’s): A potential marker for tumour proliferation Pathol Res Pract 2008 204 799 807 10.1016/j.prp.2008.05.002 18602222 Attoub S Rivat C Rodrigues S Van Bocxlaer S Bedin M Bruyneel E The c-kit Tyrosine Kinase Inhibitor STI571 for Colorectal Cancer Therapy Cancer Res 2002 62 4879 4883 12208734 Ulivi P Zoli W Medri L Amadori D Saragoni L Barbanti F Calistri D Silvestrini R c-kit and SCF expression in normal and tumor breast tissue Breast Canc Res Treat 2004 83 33 42 10.1023/B:BREA.0000010694.35023.9e Zhang W Stoica G Tasca SI Kelly KA Meininger CJ Modulation of Tumor Angiogenesis by Stem Cell Factor Cancer Res 2000 60 6757 11118063 Rappa G Anzanello F Lorico A CD24 expression and breast cancer Stem cell phenotype J Clin Oncol 2009 27 15 s 10.1200/JCO.2008.21.7695 Gao Q Choi Y-P Kang S Youn JH Cho N-H CD24+ cells from hierarchically organized ovarian cancer are enriched in cancer stem cells Oncogene 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==== Front Pan Afr Med JPan Afr Med JPAMJThe Pan African Medical Journal1937-8688The African Field Epidemiology Network PAMJ-12-42ResearchDental caries and oral health practices among 12 year old children in Nairobi West and Mathira West Districts, Kenya Gathecha Gladwell 1&Makokha Anselimo 2Wanzala Peter 3Omolo Jared 1Smith Perry 41 Field Epidemiology and Laboratory Training Programme, Ministry of Public Health and Sanitation2 Jomo Kenyatta University of Agriculture and Technology3 Kenya Medical Research Institute4 State University of New York at Albany, USA, School of Public Health& Corresponding author: Gladwell Gathecha, Field Epidemiology and Laboratory Training Programme and Ministry of Public Health and Sanitation P.O. Box 27236-00100, Nairobi, Kenya22 6 2012 2012 12 4206 9 2011 18 6 2012 © Gladwell Gathecha et al.2012The 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.Background Dental caries is a common disease in children which causes pain with resultant effect on various physiological and social functions. The main objective of the study was to determine the association between dental caries and oral health knowledge and practice among children in Nairobi West and Mathira West Districts. Methods A cross-sectional study was conducted among 639 children aged 12 years attending public primary schools in Nairobi West and Mathira West districts between August 2009-February 2010. A questionnaire was used to determine the level of knowledge and practices employed. Oral screening was performed using World Health Organisation (WHO) recommended methods. Dental caries was measured using the Decayed, Missing, Filled Teeth (DMFT) index. Results Nairobi West District had significantly higher caries prevalence of 37.5% than Mathira West District (24.0%). The DMFT in Nairobi West District was 0.76±1.2, while in Mathira West District it was 0.36±0.7. On multivariate analysis high consumption of soda was found to be a significant risk factor for dental caries in Nairobi West District(Odds Ratio (OR) = 3.0). In Mathira West District having an illiterate mother was a significant risk factor for dental caries (OR = 4.3). Conclusion Countrywide intensive oral health promotion should be carried out especially in urban areas, to reduce the higher prevalence of dental caries. The school health policy should be used to promote oral health by provision of oral health instructions and highlighting harmful dietary practices. Preventive practices such as regular dental checkups should be advocated and promoted in schools. Dental carieschildrenknowledgehealthpracticeattitudeKenya ==== Body Background Dental caries is a chronic infectious disease that causes demineralization of teeth. The interaction of four factors allows this to happen: a susceptible tooth surface, specific bacteria in dental plaque (e.g, Streptococcus mutans, lactobacillus), time and a diet rich in fermentable carbohydrates, particularly refined sugars. The impact of dental caries includes oral pain which may affect speech, eating, sleeping, swallowing and breathing. The altered appearance it causes can lead to low self esteem and undermine social acceptance [1]. The World Health Organization (WHO) recognizes dental caries as a pandemic and reports that the prevalence of dental caries among school aged children is 60% to 90% [2]. In several industrialized countries the prevalence and severity of dental caries have declined substantially because of preventive oral health care programmes and changes in living conditions and lifestyles [3]. In developing countries, especially sub-Saharan Africa, the prevalence varies according to country population group and socio-economic status [4]. To date, no national oral survey has been carried out to determine the prevalence of dental caries in Kenya. A study done in 1992 found the prevalence of dental caries to be 40% to 50% among children aged 13-15 [5] in Nairobi, while in 2006 Kassim et. al found the prevalence among adults living in a rural arid region, to be 43% [6]. Basic oral health education is taught in Kenyan primary schools. Children are instructed to avoid sugary foods because they cause dental caries. They are also taught that they should brush their teeth three times a day. The two messages are important but do not cover the entire aspects of dental caries and its prevention. Another source of dental education is dental clinics, but access to these facilities is limited due to several barriers including poverty. A visit to a public dental clinic will require one to purchase a card, the cost of which is prohibitive to many people. Government facilities, of which 80% are situated in the urban areas, are also understaffed and are unable to provide dental education to visiting clients as demonstrated by the dentist- population ratio of 1:60,000 [7]. Dental caries has been shown to affect a significant number of children in our country, but there is limited recent information about dental caries prevalence and oral health habits among children in Kenya. This study describes the dental caries experience among 12 year old children in two Kenyan districts and relates it to their oral health habits. Methods Study design The study employed a cross-sectional design determine the relationship between dental caries and oral health practices among 12 year old children attending public primary schools in Nairobi West and Mathira West districts, Kenya. Setting Nairobi West District is located in Nairobi Province, the capital city of Kenya, and is predominantly an urban area. It is a cosmopolitan district. Drinking water is supplied by pipes to homes by the Nairobi Water Company. Mathira West District is located in Central Province and is predominantly a rural area. The main economic activity is farming, and the main sources of drinking water are boreholes, rivers and streams. Study population The study population consisted of 12 year old children attending public primary schools. Children aged 12 years were used as the study population as this is an index age recommended by WHO [8]. Children who assented to the study and whose parents/guardians gave written informed consent were included in the study. Time frame The Study was conducted between August 2009 and February 2010. Sampling A two stage sampling technique was used. Stratified sampling was used to sample the primary schools. A total of twelve schools were included, six out of 54 schools from Nairobi West District and six out of 36 from Mathira West District. In both Nairobi West and Mathira West Districts, two schools were randomly selected from each of the three divisions in each District. To select the children, simple random sampling was used. A register was obtained containing all the 12 year olds. A unique number was assigned to each child, and then a list of random numbers was generated using the computer via Microsoft Excel software. A total of sixty children were selected from each school, and the refusal rate ranged from 0%-15% in the two districts. Data collection An oral interviewer-administered pre-tested questionnaire was used to collect data from the children. Consumption of cariogenic foods was classified into three categories: high consumption (consumes at least three times a week), low consumption (consumes two times a week or less) and never consumes. Clinical examination Dental caries status was determined by oral screening using the WHO caries diagnostic criteria: Decayed Missing Filled Teeth (DMFT) index [8]. Dean′s index was used to record the prevalence of dental fluorosis [9]. Results were reported as either sound (score 0) or very mildly to severely fluorosed (score 1-4). Instruments used during the screening included dental mirrors and tongue depressors. The children were examined while seated in an upright chair using natural day light. All examinations were done in the classrooms independently by two examiners. A calibration exercise was conducted to assess the consistency of the two examiners, which revealed agreement between the two examiners for all but 8% of the children. Differences between the two examiners were discussed and a consensus reached. Fluoride content in water One water sample was collected from each of the six schools in Nairobi West District for analysis of their fluoride content. In Mathira West District six water samples were collected from nearby rivers, water tank and boreholes. The fluoride analysis was done at Kenya Water Institute Laboratory. Fluoride concentration was determined using photometric analysis [10]. Ethical considerations Approval to conduct the study was given by The Jomo Kenyatta University of Agriculture and Technology, National Council of Science and Technology and the Nairobi City Council. Written informed consent was obtained from the children's guardian/parents and assent was sought from the children. All information was treated confidentially. The children were subsequently given oral health education, advice and referral instructions if indicated. Data management and analysis The collected data were entered, cleaned and stored using Epi info version 3.3.2. Measures of statistical significance were done using the T-test, Chi-square and Wilcoxon test. P- value of less than 0.05 was considered significant. Logistic regression was done separately for each district using the forward method by including variables that had p-values of less than 0.25. Results Study Population A total of 639 children participated in the study. Of these, 347 (54%) were from Nairobi West District while 292 (46%) were from Mathira West District. Females were 57% and 52% in Nairobi West and Mathira West, respectively. The mothers of the children in Nairobi West had a higher level of education (75% with secondary level and above) than mothers in Mathira West (56%). Two percent and 3% of mothers in Nairobi West and Mathira West Districts, respectively, had no formal education. Dental caries status The prevalence of dental caries was significantly higher in Nairobi West District (37.5%) than in Mathira West District (24.0%) (OR= 1.9, CI = 1.3-2.7). The major portion of caries experience for both districts was decayed teeth. The DMFT index was 0.76 in Nairobi West and 0.36 in Mathira West. None of the children in Mathira West District had their teeth filled (Table 1). The indices for decayed teeth, missing teeth and DMFT were significantly higher (p < 0.05) in Nairobi West than Mathira West. Table 1 Caries experience among 12 year old children in two Kenyan districts, 2009-2010 Nairobi West (347 Children) Mathira West (292 Children) Number of teeth Index* Number of teeth Index* Decayed Teeth 233 0.67** 102 0.35 Missing Teeth 19 0.05** 3 0.01 Filled Teeth 13 0.02 0 0 DMFT 265 0.76** 105 0.36 DMFT: Decayed, missing and filled teeth * Index was calculated as the number of affected teeth divided by the total number of children ** p< 0.05 Sixty-one percent and 45% of the children in Nairobi West and Mathira West districts, respectively, brushed their teeth two or more times per day as indicated in Table 2. Chewing sticks were reported to have been used by 14%, and of these, 1.2% and 30.0% were from Nairobi West and Mathira West districts, respectively. In all, 77.0% of the children admitted to brushing each of their teeth carefully. Twenty-nine percent of children in Nairobi West District had received instructions on tooth brushing, while in Mathira West District 40% had received instructions. The proportion of those who had ever visited the dentist was more than twice for Nairobi West District children (38.0%) in comparison to Mathira West District (17.5%). Table 2 Oral health practices among 12 year old children in two Kenyan districts, 2009–2010 Nairobi West n = 347 Mathira West n = 292 Total n = 639 Frequency of tooth brushing  Less often than daily 12 (3.5%) 10 (3.0%) 22 (3.0%)  Once /day 122 (35.2%) 152 (52.0%) 274 (43.0%)  Two or more/day 213 (61.4%) 130 (45.0%) 343 (54.0%) Tooth cleaning aids  Toothbrush and toothpaste 322 (96.0%) 192 (66.0%) 514 (80.0%)  Chewing stick* 4(1.2%) 88 (30.0%) 92 (14.0%)  Others** 21 (2.8%) 12 (4.0%) 33 (6.0%) Reported brushing every tooth very carefully 320 (93.0%) 168 (58.0%) 488 (77.0%) Reported receiving instructions on tooth brushing 100 (29.0%) 116 (40.0%) 216 (34.0%) Reported visiting a dentist at least once 132 (38.0%) 51 (17.5%) 183 (28.6%) Of those visiting a dentist, reasons for dental visits  “For a checkup” 22 (17.0%) 6 (12.0%) 28 (15.0%)  “ My gums were bleeding” 8 (6.0%) 3 (6.0%) 11 (6.0%)  “Teeth were growing badly” 14 (11.0%) 3 (6.0%) 18 (10.0%)  “My teeth were loose” 6 (4.0%) 2 (4.0%) 8 (4.0%)  “My tooth was aching” 82 (62.0%) 37 (72.0%) 118 (65.0%) Reported visiting a dentist within the past 12 months 70 (20.0%) 34 (12.0%) 104 (16.0%) * Piece of stick cut from a tree that is flayed out at the end for brushing teeth ** Includes salty water, charcoal and limestone In Mathira West District, having a mother with no formal education was a significant risk factor for dental caries (OR = 4.3) as illustrated by Table 3. In both districts children who had visited a dentist in the past 12 months were more likely to have dental caries than those who had never visited a dentist. In Nairobi West district children who ate cakes/biscuits had significantly more caries than those who never ate cakes/biscuits. The risk of dental caries was significantly higher among children who drank sodas at a higher frequency compared to those who did not drink (OR = 2.2) in Nairobi West District. There was no significant difference in the distribution of dental caries by sex, frequency of tooth brushing and presence of dental flourosis (Table 3). Table 3 Dental caries in relation to sociodemographic characteristics, oral health habits and consumption of cariogenic foods among children in two Kenyan districts, 2009–2010 Nairobi West Mathira West n Children with Caries OR (95%CI) n Children with Caries OR (95% CI) Sex-Female 199 78 (39) 1.2 (0.8–1.9) 152 38 (25) 1.1 (0.6–1.9) Mothers Education No formal education 8 3 (38) 0.9 (0.2–3.9) 9 5 (56) 4.3(1.1–6.7)* Primary 49 16 (33) 0.7 (0.4–1.4) 99 21 (21) 0.9 (0.5–1.7) Secondary and above 259 103 (40) ref 163 37 (23) ref Dental fluorosis present 28 11 (40) 1.1 (0.5–2.4) 3 0 (0) 0 Frequency of tooth brushing Less often than daily 12 4 (33) 1.1 (0.3–3.1) 10 2 (20) 1.2 (0.1–4.3) Once /day 122 51 (31) 1.3 (0.8–2.0) 152 39 (20) 1.2 (0.6–2.0) Twice or more/day 213 75 (35) ref 130 29 (22) ref Reported receiving instructions on tooth brushing 100 37 (37) 0.9 (0.6–1.6) 116 21 (18) 0.6 (0.3–1.0) Reported visiting a dentist at least once 132 64 (49) 2.1 (1.4–3.3)* 51 17 (33) 1.7 (0.9–3.4) Reported visiting a dentist within the past 12 months 70 40 (57) 2.7 (1.6–4.8)* 34 13 (38) 2.1 (1.0–4.6)* Consumption of cakes/Biscuits-High 148 63 (42) 2.3 (1.2–4.6)* 92 23 (25) 0. 6 (0.3–1.6) Low 136 52 (38) 2.0 (1.0–3.9)* 161 39 (21) 0.5 (0.2–1.5) Never 63 13 (24) ref 26 (33) ref Consumption of soda-High 117 55 (47) 2.2 (1.1–4.5)* 30 3 (23) 0.9 (0.2–3.6) Low 177 60 (34) 1.3 (0.7–2.5) 203 48 (24) 0.9 (0.5–1.7) Never 55 15(28) ref 76 19(25) ref ref = reference group OR = Odds Ratio CI = Confidence Interval * p < 0.05 The mean Fluoride water content for Nairobi West District was 0.59 mg/L (range 0.35-0-0.85), while for Mathira West District it was 0.77mg/L (range 0.4-1.05). The difference between the fluoride content in the two regions was not significant. Logistic regression analysis revealed that high consumption of soda was a significant risk factor for dental caries in Nairobi West District (OR = 3.2, CI = 1.3-8.0). In Mathira West District having an illiterate mother was a significant risk factor for dental caries (OR = 4.3, CI = 1.1-1.6). Discussion The prevalence of dental caries was found to be 37.5% (DMFT 0.76) in Nairobi West and 24.0% (DMFT 0.36) in Mathira West. These results indicate a decline in dental caries as compared to previous Kenyan studies that found a prevalence of 50% in 11-13 year olds [5] and 64% in 3-5 year olds [11]. The decrease may be attributed to increased oral health awareness and number of available dental professionals. The prevalence of dental caries and the DMFT were significantly higher in Nairobi West District than in Mathira West District. Living in urban areas has implications for lifestyle, including dietary pattern and has been shown to be associated with an increased prevalence of dental caries [12]. The prevalence of dental caries found here is slightly lower than other East African countries which have recorded a prevalence of 41% in urban areas and 29% in rural areas in Uganda [13], and 41.5% among urban children in Tanzania [14]. Our results were quite similar, however, to the findings in a study done in Burkina Faso where the urban area prevalence was 33.8%, while the rural area prevalence was 21.2% [15]. The decayed teeth component of the DMFT index formed the major component in both districts: 0.67 in Nairobi West District and 0.35 in Mathira West District. These high numbers of untreated teeth may be a result of a low perception of the need for treatment and the low priority placed on oral health care compared with other needs [16, 17]. None of the children in Mathira West District had any of their teeth filled, compared with 13 fillings in children in Nairobi West District. This can be explained by the low number of dental clinics, both private and public, and lack of resources in the few clinics that are available in Mathira West District. A study done in South Africa also indicated that none of the 12 year olds in the rural population had their teeth filled [17]. Children who had illiterate mothers had higher caries prevalence than children whose mothers had secondary and above level of education in Mathira West District. Similar findings have been reported in Uganda [18]. Mothers with no formal education may lack access to literature on caries prevention and oral health in general. Sixty-one percent of children in Nairobi West District and 45% of children in Mathira West District brushed their teeth at least twice per day. These figures are higher than what has been reported in Sudan, where 30% of twelve year old children brushed their teeth at least twice a day [19]. Sixty-two percent of the children in Nairobi West District and 82% in Mathira West District had never visited a dentist compared to 76% in Tanzania [14], 60% in India [16], and 34% in Thailand [20]. Although an earlier study done in Sudan [19] showed that children who had ever visited a dentist had lower caries prevalence than those who had never visited a dentist, this was not found in our study. It was aslo found that children who had reported visiting a dentist at least once had significantly higher caries prevalence than those who had not in Nairobi West District. The same results applied to those who reported visiting a dentist within the past 12 months though the association was slightly more for both districts. This finding is in agreement with other studies [16, 18, 20] and may be explained by the fact that children tended to visit the dentist when there was already a problem instead of going for routine checkups as confirmed by the reasons they gave for visiting the dentist; only 15% had gone for a checkup. Reasons for visiting were curative rather than preventive. In Nairobi West District, children who ate cakes/biscuits and children who had a high consumption of sodas had significantly higher dental caries prevalence than those who did not. A study done in Mexico revealed that drinking of sodas particularly between meals was significantly associated with dental caries [21]. This study had several potential limitations. First, the study involved school children who are twelve years old, thereby missing the small percentage of children not attending school. The children in this study may therefore not have been representative of the general population of the target children. Secondly, there was not way to verify the information reported on the questionnaire and so its validity could not be assessed. Therefore, there could have been information bias, including over-reporting of socially accepted behavior such as tooth brushing and under-reporting of less accepted behavior such as consumption of cariogenic foods. Nevertheless the self-reported information clearly shows deficits of healthy oral hygiene behavior, and the dental examinations documented a high degree of inadequate dental care. Conclusion A high prevalence of dental caries and poor dental hygiene practices was observed in 12 year old children in Nairobi West and Mathira West Districts, Kenya. It is recommended that countrywide intensive oral health promotion should be carried out especially in urban areas, to reduce the high prevalence of dental caries. The school health policy should be used to promote oral health by provision of oral health instructions and education on harmful dietary practices. Preventive practices such as regular dental checkups should be advocated and promoted in schools. Acknowledgments We would like to thank the children, their parents and teachers of the twelve schools we visited for their support and co-operation. We are grateful to the Field Epidemiology and Laboratory Training Programme Division in the Ministry of Public Health and Sanitation for financial support. Competing interests Authors declared they have no conflict of interest. Authors’ contributions Gladwell Gathecha: Principal investigator of the study and main author of the manuscript. Anselimo Makokha: Provided technical assistance in conducting the study. Peter Wanzala: Provided technical assistance in conducting the study Jared Omolo: Provided technical assistance in conducting the study. Perry Smith: Co-author of the manuscript. Provided technical assistance in writing the manuscript. ==== Refs References 1 Weir E Dental caries: a nation divided CMAJ. 2002 167 1035 12403746 2 Petersen PE Bourgeois D Ogawa H Estupinan-Day S Ndiaye C The Global burden of oral disease and risks to oral health Bull World Health Organ. 2005 9 83 9 661 9 16211157 3 Petterson GH Brathall D The caries decline: A review of reviews Eur J Oral Sci. 1996 104 436 443 8930595 4 Cleaton-Jones P Fatti P Dental caries trend in Africa Community Dent Oral Epidemiol. 1999 10 27 5 316 20 10503791 5 Ng'ang'a PM Valderhaug J Dental caries in primary school children in Nairobi, Kenya Acta Odontol Scand. 1992 50 269 72 1441930 6 Kassim BA Noor MA Chindia ML Oral Health status among Kenyans in a rural arid setting: dental caries experience and knowledge on its causes East Afr Med J. 2006 83 2 100 5 16708882 7 Kaimenyi JT Oral health in Kenya Int Dent J. 2004 12 54 6 Suppl 1 378 82 15631100 8 World Health Organization Oral health surveys, basic methods 1987 3rd ed Geneva 9 Rozier RG Epidemiologic indices for measuring the clinical manifestations of dental fluorosis: overview and critique Adv Dent Res. 1994 8 1 39 55 7993559 10 Nollet LML Handbook of Water Analysis 2007 Florida Boca Raton 11 Ngatia EM Imungi JK Muita JW Nganga PM Dietary patterns and dental caries in nursery school children in Nairobi, Kenya East Afr Med J. 2001 78 12 673 7 12199451 12 Ismail AI Tanzer JM Dingle JL Current trends of sugar consumption in developing societies Community Dent Oral Epidemiol. 1997 12 25 6 438 43 9429817 13 Wandera M Twa-Twa J Baseline survey of oral health of primary and secondary schools in Uganda Afr Health Sci. 2003 4 3 1 19 22 12789084 14 Mwakatobe AJ Mumghamba EG Oral health behavior and prevalence of dental caries among 12-year-old school-children in Dar-es-Salaam, Tanzania Tanzania Dental Journal. 2007 14 1 7 15 Varenne B Petersen PE Ouattara S Oral health status of children and adults in urban and rural areas of Burkina Faso, Africa Int Dent J. 2004 54 83 89 15119798 16 David J Wang NJ Åstrom AN Kuriakose S Dental caries and associated factors in 12-year-old schoolchildren in Thiruvananthapuram, Kerala, India Int J Paediatr Dent. 2005 11 15 6 420 8 16238652 17 Bajomo AS Rudolph MJ Ogunbodede EO Dental caries in six, 12 and 15 year old Venda children in South Africa East African Medical Journal. 2004 81 236 243 15508337 18 Kiwanuka SN Astrom N Trovik TA Dental caries experience and its relationship to social and behavioural factors among 3-5-year-old children in Uganda Int J Paediatr Dent. 2004 9 14 5 336 46 15330999 19 Nurelhuda NM Trovik TA Ali RW Ahmed MF Oral health status of 12-year-old school children in Khartoum state,the Sudan; a school-based survey BMC Oral Health. 2009 9 15 34 19527502 20 Petersen PE Hoerup N Poomvise N Prommajan J Watanapa A Oral health status and oral health behavior of urban and rural schoolchildren in Southern Thailand Int Dent J. 2001 51 95 102 11569670 21 Cook SL Martinez-mie EA Dean JA Weddel JA Sanders BJ Eggertsson H Dental caries experience and association to risk indicators of remote rural populations Int J Paediatr Dent. 2008 7 18 4 275 83 18284473
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Pan Afr Med J. 2012 Jun 22; 12:42
==== Front Case Rep RheumatolCase Rep RheumatolCRIM.RHEUMATOLOGYCase Reports in Rheumatology2090-68892090-6897Hindawi Publishing Corporation 2293744110.1155/2011/210795Case ReportChronic Recurrent Multifocal Osteomyelitis with Concomitant Features of Juvenile Idiopathic Arthritis Tsitsami Elena 1 *Dermentzoglou Vasiliki 2 Moschovi Mary 3 Chrousos George P. 4 1Pediatric Rheumatology Unit, 1st Department of Pediatrics, Children's Hospital Aghia Sophia, University of Athens, 115 27 Athens, Greece2Department of Radiology, Children's Hospital Aghia Sophia, 115 27 Athens, Greece3Hematology Oncology Unit, 1st Department of Pediatrics, Children's Hospital Aghia Sophia, University of Athens, 115 27 Athens, Greece41st Department of Pediatrics, Children's Hospital Aghia Sophia, University of Athens, 115 27 Athens, Greece*Elena Tsitsami: [email protected] Editor: B. Bannwarth 2011 29 12 2011 2011 2107957 11 2011 13 12 2011 Copyright © 2011 Elena Tsitsami et al.2011This 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.We report a case of a 13-year-old girl with chronic recurrent multifocal osteomyelitis (CRMO) who developed severe arthritis in four different joints within the first year from the onset of the disease. Her multiple vertebrae lesions showed significant amelioration after a 2-month treatment with prednisolone. In parallel, the initial severe symmetrical arthritis of both knees showing overt synovitis and joint effusion, in the absence of lesions in the metaphyses of the femur or the tibia, responded remarkably well in intra-articular triamcinolone hexacetonide injections. However, upon discontinuation of prednisolone, the patient developed severe arthritis of her right ankle and the proximal interphalangeal joint of her right middle finger. Thus, prednisolone was reinitiated combined with methotrexate, and the patient went into remission, which persists one year after prednisolone tapering. The appearance of arthritis in both knees in the absence of bone lesions and the emergence of severe arthritis of the ankle after remission of spinal bone lesions suggest that CRMO and juvenile idiopathic arthritis may coexist and be causally related. ==== Body 1. Introduction Chronic recurrent multifocal osteomyelitis (CRMO) or CRMO syndrome (OMIM no. 259680) is a rare inflammatory bone disease presenting usually early in life (∼10 years of age) although adults with CRMO have been described. It shares many features with and is, therefore, regarded to be the pediatric subset of SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome considered together to constitute the unique disease spectrum of chronic nonbacterial osteomyelitis (CNO). Accordingly, CRMO syndrome is characterized by multiple foci of nonbacterial osteomyelitis appearing radiologically as a mixture of osteolytic/sclerotic lesions. It presents as bone pains with or without fever, has an unpredictable course alternating between exacerbations and spontaneous remission, and is frequently associated with other inflammatory and/or autoimmune conditions [1, 2]. The underlying defect and the pathogenesis of CRMO are largely unknown. Based on the finding that spontaneous recessive mutations in Pstpip2 (a murine gene encoding a protein homologous to the PAPA-pyogenic arthritis, pyoderma gangrenosum, and acne-syndrome protein PSTPIP1) cause, in the cmo mouse model, an autoinflammatory bone disease most closely resembling CRMO [3, 4], the later is considered as an autoinflammatory disorder [5]. Despite the fact that the human PSTPIP2 gene is encoded within a genomic interval found to be associated with sporadic CRMO by transmission disequilibrium testing [6], no PSTPIP2 mutations have been as yet identified in CRMO syndrome. As a result of its unknown etiology, diagnostic tests specific for CRMO do not exist, patients seem to be underdiagnosed, and its treatment is still empiric and not always successful. The histologic findings in bone biopsies are nonspecific, showing inflammatory changes with granulocytic infiltration. The situation is further confused due to the various classifications under which the syndrome appears in the literature. Beyond its already mentioned relation with SAPHO syndrome and based on the recurrent purulent manifestations involving joints, eye, skin, and bones, CRMO is classified amongst pyogenic autoinflammatory disorders, along with PAPA and Majeed syndromes [7]. On the other hand, several clinicians, looking at clinical, radiological, and histological findings, assume a common pathway for the different acute and chronic diseases associated with osteitis [8, 9]. In this context, the diagnostic criteria for nonbacterial osteomyelitis (acute and chronic) proposed by Jansson et al. [2], based on the analysis of a series of 89 patients with sterile bone inflammation (39 with CRMO without arthritis at first presentation), are expected to better discriminate CRMO patients among those with similar clinical and radiologic presentation. This case report describes a 13-year-old girl with CRMO who, early after her initial presentation, developed polyarthritis that is incompatible with the as yet described physical history of the syndrome. This interesting fact brings not easily answerable questions about a possible coexistence of CRMO with juvenile idiopathic arthritis (JIA) as well as about an underlying autoinflammatory process able occasionally to give rise in a wider spectrum of symptoms, including polyarthritis. 2. Case Presentation In June 2010, a previously healthy, 13-year-old girl initially presented to a local hospital with a three-month history of dorsal spine pain. At the time of presentation, physical examination revealed nothing remarkable, except arthritis of the left knee. No cutaneous involvement was noted. Laboratory test results, including bone marrow aspiration smear, were normal except for elevated ESR (33 mm/h) and CRP (9 mg/L), and a titer of antinuclear antibodies of 1 : 160. Despite magnetic resonance imaging (MRI) showing characteristic lesions (Figure 1), the diagnosis of CRMO was not considered, and the patient was hospitalized on several occasions for diagnostic investigation. During the subsequent 6 months, the initial symptoms of the patient were worsening, and she therefore underwent an arthroscopy of the left knee and an open vertebral biopsy. Neither synovium nor vertebral biopsies provided any conclusive histopathologic features except a nonspecific chronic inflammatory process, while their standard cultures failed to identify any causal microorganism. All extensive investigation of infectious agents was negative. Based on a working diagnosis of bacterial osteomyelitis and septic arthritis, antibiotics, including amoxicillin/clavulanate potassium, ceftriaxone, and cefuroxime, were unsuccessfully administered, and the patient, in December 2010, was referred to our Pediatric Rheumatology Unit. At that time, the patient was in bad general condition. She was febrile (39.5°C) and unable to remain standing for more than a few minutes. Physical examination showed severe muscular atrophy in both of the legs, slight dorsal scoliosis with convexity to the right and exacerbated arthritis of the left knee. The remainder of the examination was normal. There were no cutaneous lesions or lymphadenopathy and there was not family history of a chronic inflammatory disorder (psoriasis, inflammatory bowel disease, inflammatory arthritis severe acne, or any other). Ultrasound examination and MRI demonstrated synovitis and joint effusion in both knees, most prominent in the left (Figure 2). MRI of the knees did not detect bone lesions in the distal femur or the proximal tibia (Figures 2(a), 2(c)). Bone scan showed increased uptake mainly in both knees, in thoracic vertebrae (T5, T6), in the left hip, and in the manubrium and body of the sternum (Figure 3). A new MRI of the spine showed again several vertebrae lesions of the cervical, thoracic, and lumbar spine with no evidence of deterioration or improvement. The lesions did not cross intervertebral discs as can occur in infection. The ESR was 68 mm/h, CRP was 134 mg/L, leukocytes were 7.410/μL and anti-TPO antibodies were 90 IU/mL. HLA-B27 was absent. Despite the inconclusive information provided by the previous histopathologic evaluation of the vertebral and synovial biopsies, as neither universal polymerase chain reaction to amplify eubacterial and mycobacterial genes, nor immunohistochemical examination were performed, further invasive diagnostic approach was avoided due to the bad general condition of the patient. Taking into account the failure of antibiotic treatments as well as the fact that the case was fulfilling the criteria of Jansson et al. [2], the diagnosis of CRMO was assumed. Treatment with indomethacin (2 mg/kg/day) was started, while intra-articular triamcinolone hexacetonide injections (40 mg) were performed in both knees. After 24 hours, the patient became afebrile and able to stand up and started walking. Concomitantly, however, she started complaining of headache that was attributed to indomethacin. Therefore, indomethacin was discontinued and prednisolone (1 mg/kg/day) was initiated. The patient responded remarkably well and, over the following weeks, she returned to her normal physical activity. The prednisolone was gradually tapered and discontinued two months after the initiation. At that time, that is, one year after the appearance of the disease, the patient was reevaluated. She was in good general condition, the spinal MRI showed significant amelioration of bone lesions most of which had completely disappeared (Figure 4), and the US examination of both knees did not reveal findings of arthritis. However, she was complaining of pain and edema in the proximal phalanx of her right middle finger as well as in her right ankle. Therefore, prednisolone was reinitiated combined with methotrexate, and the patient went into remission, which persisted after prednisolone tapering and discontinuation. 3. Discussion The diagnosis of CRMO is hampered by the lack of a specific diagnostic test and is essentially one of exclusions. Infective osteomyelitis, malignancy, and Langerhans' cell histiocytosis are the main differential diagnoses [10]. JIA cannot be excluded from the differential diagnosis [11], since in a recent report by Beck et al. [12], at the time of diagnosis, arthritis has been noted in as many as 38%, morning stiffness in 16.2%, functional impairment of legs in 43.2%, and asymmetry of extremities/thorax in 37.8% out of a series of 37 children patients. This high incidence of arthritis is not astonishing, bearing in mind that it affects always joints adjacent to the bone lesions as well as that metaphyses or metaphyseal equivalents are the most common sites of disease, accounting for approximately 75% of all lesions [13]. The arthritis of CRMO seems to be accompanied by mild synovitis and/or joint effusion although, to the best of our knowledge, exact data about their degree are lacking from the literature. Due to the presence of the characteristic for CRMO multiple bone lesions, there was no difficulty to differentiate the case reported herein from JIA. However, the presence of severe symmetrical arthritis of the knees in the absence of lesions in the metaphyses of the femur or the tibia as well as the emergence of arthritis of the right ankle and the right proximal interphalangeal joint after the remission of spinal lesions is quite interesting. Furthermore, the severe muscular atrophy in both of the legs with which the patient presented implies that arthritis of the knees was initiated long before the appearance of CRMO lesions and was possibly not perceived or overridden by the dorsal spine pain. A suspicion of possible coincidence of the two diseases is therefore arisen, that necessitates a closer followup of the patient. Nevertheless, from the pathophysiological point of view and assuming an autoinflammatory nature of both disorders, this would be a simply rhetoric question, since a common autoinflammatory process could occasionally give rise in a brooder than the usual spectrum of CRMO symptoms, including polyarthritis. It is also worth mentioning that, as in the case of our patient, improvement of CRMO arthritis by intra-articular corticosteroid injections has been also previously attempted [14]. Finally, the appearance of arthritis very early in the disease course of our patient deterred us from the major differential diagnosis of Langerhans' cell histiocytosis. Histiocytic oligoarthritis is a very rare finding of the later condition [15]. Even though JIA does not represent the major differential diagnosis of CRMO, the emergence of arthritis is not uncommon during the protracted course of the syndrome [16]. Moreover, evolution into enthesitis-related arthritis or spondyloarthropathy has been documented in children and young adults [16–18] underlining the need for long-term followup. However, the management of the reported patient before her referral to our unit indicates that a high index of suspicion is needed to diagnose CRMO syndrome, in order for misdiagnoses of infection and neoplasm, and unnecessary aggressive surgical and antibiotic therapy to be avoided. Figure 1 MRI of the spine at the disease presentation. Multiple vertebrae lesions are demonstrated in the cervical, thoracic, and lumbar spine, which exhibit increased signal intensity on sagittal T2-weighted images (a), (b), and (c). The appearance of the intervertebral discs is normal. Figure 2 MRI and US of the left knee at the disease presentation. T2-weighted sagittal image (a) and US (b) demonstrated synovial thickening with increased vascularity (b) and joint effusion. The signal intensity of the distal femur and proximal tibia is normal (a), (c). Figure 3 Bone scan at the disease presentation. Since the radiological investigation for bone lesions in this area proved negative, the increased uptake in both knees (a) was attributed to synovitis. Increased uptake was also demonstrated in the sternum (manubrium and body synchondrosis) (b), in thoracic vertebrae (c), and in the left hip (d). Figure 4 MRI of the spine one year from the disease presentation. ==== Refs 1 El-Shanti HI Ferguson PJ Chronic recurrent multifocal osteomyelitis: a concise review and genetic update Clinical Orthopaedics and Related Research 2007 462 11 19 2 Jansson A Renner ED Ramser J Classification of non-bacterial osteitis: retrospective study of clinical, immunological and genetic aspects in 89 patients Rheumatology 2007 46 1 154 160 16782988 3 Byrd L Grossmann M Potter M Shen-Ong GLC Chronic multifocal osteomyelitis, a new recessive mutation on chromosome 18 of the mouse Genomics 1991 11 4 794 798 1686018 4 Ferguson PJ Bing X Vasef MA A missense mutation in pstpip2 is associated with the murine autoinflammatory disorder chronic multifocal osteomyelitis Bone 2006 38 1 41 47 16122996 5 Masters SL Simon A Aksentijevich I Kastner DL Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease Annual Review of Immunology 2009 27 621 668 6 Golla A Jansson A Ramser J Chronic recurrent multifocal osteomyelitis (CRMO): evidence for a susceptibility gene located on chromosome 18q21.3–18q22 European Journal of Human Genetics 2002 10 3 217 221 11973628 7 Rigante D Autoinflammatory syndromes behind the scenes of recurrent fevers in children Medical Science Monitor 2009 15 8 RA179 RA187 19644432 8 Baltensperger M Grätz K Bruder E Lebeda R Makek M Eyrich G Is primary chronic osteomyelitis a uniform disease? Proposal of a classification based on a retrospective analysis of patients treated in the past 30 years Journal of Cranio-Maxillofacial Surgery 2004 32 1 43 50 14729050 9 Earwaker JWS Cotten A SAPHO: syndrome or concept? Imaging findings Skeletal Radiology 2003 32 6 311 327 12719925 10 Girschick HJ Zimmer C Klaus G Darge K Dick A Morbach H Chronic recurrent multifocal osteomyelitis: what is it and how should it be treated? Nature Clinical Practice Rheumatology 2007 3 12 733 738 11 Robertson LP Hickling P Chronic recurrent multifocal osteomyelitis is a differential diagnosis of juvenile idiopathic arthritis Annals of the Rheumatic Diseases 2001 60 9 828 831 11502607 12 Beck C Morbach H Beer M Chronic non-bacterial osteomyelitis in childhood: prospective follow-up during the first year of anti-inflammatory treatment Arthritis Research &amp; Therapy 2010 12 2, article R74 13 Mandell GA Contreras SJ Conard K Harcke HT Maas KW Bone scintigraphy in the detection of chronic recurrent multifocal osteomyelitis Journal of Nuclear Medicine 1998 39 10 1778 1783 9776287 14 Eleftheriou D Gerschman T Sebire N Woo P Pilkington CA Brogan PA Biologic therapy in refractory chronic non-bacterial osteomyelitis of childhood Rheumatology 2010 49 8 1505 1512 20430869 15 Aouba A Larousserie F Le Guern V Martin A Guillevin L Spumous histiocytic oligoarthritis coexisting with systemic Langerhans’ cell histiocytosis: case report and literature review Joint Bone Spine 2009 76 6 701 704 19640768 16 Girschick HJ Raab P Surbaum S Chronic non-bacterial osteomyelitis in children Annals of the Rheumatic Diseases 2005 64 2 279 285 15647436 17 Vittecoq O Said LA Michot C Evolution of chronic recurrent multifocal osteitis toward spondylarthropathy over the long term Arthritis and Rheumatism 2000 43 1 109 119 10643706 18 Job-Deslandre C Krebs S Kahan A Chronic recurrent multifocal osteomyelitis: five-year outcomes in 14 pediatric cases Joint Bone Spine 2001 68 3 245 251 11394625
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Case Rep Rheumatol. 2011 Dec 29; 2011:210795
==== Front J NanobiotechnologyJ NanobiotechnologyJournal of Nanobiotechnology1477-3155BioMed Central 1477-3155-10-312281765810.1186/1477-3155-10-31ReviewProspects and applications of nanobiotechnology: a medical perspective Fakruddin Md [email protected] Zakir [email protected] Hafsa [email protected] Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka, Bangladesh2 Department of Microbiology, Primeasia University, Dhaka, Bangladesh2012 20 7 2012 10 31 31 3 6 2012 20 7 2012 Copyright ©2012 Fakruddin et al.; licensee BioMed Central Ltd.2012Fakruddin 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 Nanobiotechnology is the application of nanotechnology in biological fields. Nanotechnology is a multidisciplinary field that currently recruits approach, technology and facility available in conventional as well as advanced avenues of engineering, physics, chemistry and biology. Method A comprehensive review of the literature on the principles, limitations, challenges, improvements and applications of nanotechnology in medical science was performed. Results Nanobiotechnology has multitude of potentials for advancing medical science thereby improving health care practices around the world. Many novel nanoparticles and nanodevices are expected to be used, with an enormous positive impact on human health. While true clinical applications of nanotechnology are still practically inexistent, a significant number of promising medical projects are in an advanced experimental stage. Implementation of nanotechnology in medicine and physiology means that mechanisms and devices are so technically designed that they can interact with sub-cellular (i.e. molecular) levels of the body with a high degree of specificity. Thus therapeutic efficacy can be achieved to maximum with minimal side effects by means of the targeted cell or tissue-specific clinical intervention. Conclusion More detailed research and careful clinical trials are still required to introduce diverse components of nanobiotechnology in random clinical applications with success. Ethical and moral concerns also need to be addressed in parallel with the new developments. NanobiotechnologyApplicationsMedicalProspects ==== Body Introduction Nanotechnology is a novel scientific approach that involves materials and equipments capable of manipulating physical as well as chemical properties of a substance at molecular levels. On the other hand, biotechnology uses the knowledge and techniques of biology to manipulate molecular, genetic and cellular processes to develop products and services and is used in diverse fields from medicine to agriculture. Nanobiotechnology is considered to be the unique fusion of biotechnology and nanotechnology by which classical micro-technology can be merged to a molecular biological approach in real. Through this methodology, atomic or molecular grade machines can be made by mimicking or incorporating biological systems, or by building tiny tools to study or modulate diverse properties of a biological system on molecular basis. Nanobiotechnology may, therefore, ease many avenues of life sciences by integrating cutting-edge applications of information technology & nanotechnology into contemporary biological issues. This technology has potential to remove obvious boundaries between biology, physics and chemistry to some extent, and shape up our current ideas and understanding. For this reason, many new challenges and directions may also arise in education, research & diagnostics in parallel by the extensive use of nanobiotechnology with the passage of time. Nanobiotechnology at a glance Biotechnology and nanotechnology are two of the 21st century’s most promising technologies. Nanotechnology (sometimes referred to as nanotech) is defined as the design, development and application of materials & devices whose least functional make up is on a nanometer scale [1,2]. Generally, nanotechnology deals with developing materials, devices, or other structures possessing at least one dimension sized from 1 to 100 nanometers. Meanwhile, Biotechnology deals with metabolic and other physiological processes of biological subjects including microorganisms. Association of these two technologies, i.e. nanobiotechnology can play a vital role in developing and implementing many useful tools in the study of life. Nanotechnology is very diverse, ranging from extensions of conventional device physics to completely new approaches based upon molecular self-assembly, from developing new materials with dimensions on the nanoscale to investigating whether we can directly control matters on/in the atomic scale/level. This idea entails the application of fields of science as diverse as surface science, organic chemistry, molecular biology, semiconductor physics, microfabrication, etc. Advantages of nanobiotechnology The pathophysiological conditions and anatomical changes of diseased or inflamed tissues can potentially trigger a great deal of scopes for the development of various targeted nanotechnological products. This development is like to be advantageous in the following ways: 1. Drug targeting can be achieved by taking advantage of the distinct pathophysiological features of diseased tissues [3]; 2. Various nanoproducts can be accumulated at higher concentrations than normal drugs [4]; 3. increased vascular permeability coupled with an impaired lymphatic drainage in tumors improve the effect of the nanosystems in the tumors or inflamed tissues through better transmission and retention [5,6]. 4. Nanosystems have capacity of selective localization in inflammed tissues [7]. 5. Nanoparticles can be effectively used to deliver/transport relevant drugs to the brain overcoming the presence of blood–brain barrier (meninges) [8,9]. 6. Drug loading onto nanoparticles modifies cell and tissue distribution and leads to a more selective delivery of biologically active compounds to enhance drug efficacy and reduces drug toxicity [10,11]. Applications of nanobiotechnology in medical and clinical fields A number of clinical applications of nanobiotechnology, such as disease diagnosis, target-specific drug delivery, and molecular imaging are being laboriously investigated at present. Some new promising products are also undergoing clinical trials [12,13]. Such advanced applications of this approach to biological systems will undoubtedly transform the foundations of diagnosis, treatment, and prevention of disease in future. Some of these applications are discussed below. (a) Diagnostic applicationsCurrent diagnostic methods for most diseases depend on the manifestation of visible symptoms before medical professionals can recognize that the patient suffers from a specific illness. But by the time those symptoms have appeared, treatment may have a decreased chance of being effective. Therefore the earlier a disease can be detected, the better the chance for a cure is. Optimally, diseases should be diagnosed and cured before symptoms even manifest themselves. Nucleic acid diagnostics will play a crucial role in that process, as they allow the detection of pathogens and diseases/diseased cells at such an early symptomless stage of disease progression that effective treatment is more feasible. Current technology, such as- polymerase chain reaction (PCR) leads toward such tests and devices, but nanotechnology is expanding the options currently available, which will result in greater sensitivity and far better efficiency and economy. 1. Detection:Many currently used/conventional clinical tests reveal the presence of a molecule or a disease causing organism by detecting the binding of a specific antibody to the disease-related target. Traditionally, such tests are performed by conjugating the antibodies with inorganic/organic dyes and visualizing the signals within the samples through fluorescence microscopy or electronic microscopy. However, dyes often limit the specificity and practicality of the detection methods. Nanobiotechnology offers a solution by using semiconductor nanocrystals (also referred to as "quantum dots"). These minuscule probes can withstand significantly more cycles of excitations and light emissions than typical organic molecules, which more readily decompose [14]. 2. Individual target probesDespite the advantages of magnetic detections, optical and colorimetric detections will continue to be chosen by the medical community. Nanosphere (Northbrook, Illinois) is one of the companies that developed techniques that allow/allowing doctors to optically detect the genetic compositions of biological specimens. Nano gold particles studded with short segments of DNA form the basis of the easy-to-read test for the presence of any given genetic sequence. If the sequence of interest in the samples, it binds to complementary DNA tentacles on multiple nanospheres and forms a dense web of visible gold balls. This technology allows/facilitates the detection of pathogenic organisms and has shown promising results in the detection of anthrax, giving much higher sensitivity than tests that are currently being used [15]. 3. Protein chipsProteins play the central role in establishing the biological phenotype of organisms in healthy and diseased states and are more indicative of functionality. Hence, proteomics is important in disease diagnostics and pharmaceutics, where drugs can be developed to alter signaling pathways. Protein chips can be treated with chemical groups, or small modular protein components, that can specifically bind to proteins containing a certain structural or biochemical motif [16]. Two companies currently operating in this application space are Agilent, Inc. and NanoInk, Inc. Agilent uses a non-contact ink-jet technology to produce microarrays by printing oligos and whole cDNAs onto glass slides at the nanoscale. NanoInk uses dip-pen nanolithography (DPN) technology to assemble structure on a nanoscale of measurement [17]. 4. Sparse cell detectionSparse cells are both rare and physiologically distinct from their surrounding cells in normal physiological conditions (e.g. cancer cells, lymphocytes, fetal cells and HIV-infected T cells). They are significant in the detection and diagnosis of various genetic defects. However, it is a challenge to identify and subsequently isolate these sparse cells. Nanobiotechnology presents new opportunities for advancement in this area. Scientists developed nanosystems capable of effectively sorting sparse cells from blood and other tissues. This technology takes advantage of/exploits the unique properties of sparse cells manifested in differences in deformation, surface charges and affinity for specific receptors and/or ligands. For example, by inserting electrodes into microchannels, cells can be precisely sorted based on surface charge. They can also be sorted by using biocompatible surfaces with precise nanopores. The nano-biotechnology center at Cornell University (NBTC) is currently using these technologies to develop powerful diagnostic tools for the isolation and diagnosis of various diseases [18]. 5. Nanotechnology as a tool in imagingIntracellular imaging can be made possible through labelling of target molecules with quantum dots (QDs) or synthetic chomophores, such as fluorescent proteins that will facilitate direct investigation of intracellular signalling complex by optical techniques, i. e. confocal fluorescence microscopy or correlation imaging [19,20]. (b) Therapeutic applications:Nanotechnology can provide new formulations of drugs with less side effects and routes for drug delivery. 1. Drug Delivery:Nanoparticles as therapeutics can be delivered to targeted sites, including locations that cannot be easily reached by standard drugs. For instance, if a therapeutic can be chemically attached to a nanoparticle, it can then be guided to the site of the disease or infection by radio or magnetic signals. These nanodrugs can also be designed to "release" only at times when specific molecules are present or when external triggers (such as infrared heat) are provided. At the same time, harmful side effects from potent medications can be avoided by reducing the effective dosage needed to treat the patient. By encapsulating drugs in nanosized materials (such as organic dendrimers, hollow polymer capsules, and nanoshells), release can be controlled much more precisely than ever before. Drugs are designed to carry a therapeutic payload (radiation, chemotherapy or gene therapy) as well as for imaging applications [21]. Many agents, which cannot be administered orally due to their poor bioavailability, will now have scope of use in therapy with the help of nanotechnology [22,23]. Nano-formulations offer protection for agents vulnerable to degradation or denaturation when exposed to extreme pH, and also prolong half-life of a drug by expanding retention of the formulation through bioadhesion [24,25]. Another broad application of nanotechnology is the delivery of antigens for vaccination [26,27]. Recent advances in encapsulation and development of suitable animal models have demonstrated that microparticles and nanoparticles are capable of enhancing immunization [28]. 2. Gene deliveryCurrent gene therapy systems suffer from the inherent difficulties of effective pharmaceutical processing and development, and the chance of reversion of an engineered mutant to the wild type. Potential immunogenicity of viral vectors involved in gene delivery is also problematic [29,30]. To address this issue, nanotechnological tools in human gene therapy have been tested and nanoparticle-based nonviral vectors (usully 50-500 nm in size) in transportation of plasmid DNA described. Therefore, successful introduction of less immunogenic nanosize gene carriers as a substitution of the disputed viral vectors seems beneficial in repairing or replacing impaired genes in human [31]. 3. LiposomesA liposome being composed of a lipid bilayer can be used in gene therapy due to its ability to pass through lipid bilayers and cell membranes of the target. Recent use of several groups of liposomes in a local delivery has been found to be convincingly effective [32,33]. Liposomes can also help achieve targeted therapy. Zhang et al demonstrated widespread reporter expression in the brains of rhesus monkeys by linking nanoparticle (such as polyethylene glycol) treated liposomes to a monoclonal antibody for human insulin reporter [34]. These successful trials reflect the future of targeted therapy and the importance of nanometer-sized constructs for the advancement of molecular medicine. 4. SurfacesIn nature, there are a multitude of examples of the complicated interactions between molecules and surfaces. For example, the interactions between blood cells and the brain or between fungal pathogens and infection sites rely on complex interplays between cells and surface characteristics. Nanofabrication unravels the complexity of these interactions by modifying surface characteristics with nanoscale resolutions, which can lead to hybrid biological systems. This hybrid material can be used to screen drugs, as sensors, or as medical devices and implants. Nanosystems, owned by the Irish drug company Elan, developed a polymer coating capable of changing the surface of drugs that have poor water solubility [35]. 5. Biomolecular EngineeringThe expense and time involved in traditional biomolecule designing limit the availability of bioactive molecules. Nanoscale assembly and synthesis techniques provide an alternative to traditional methods. Improvements can be achieved due to the ability to carry out chemical and biological reactions on solid substrates, rather than through the traditional solution based processes. The use of solid substrate usually means less waste and the ability to manipulate the biomolecule far more precisely. EngeneOS (Waltham, Massachusetts) pioneered the field of biomolecular engineering. The company developed the engineered genomic operating systems that create programmable biomolecular machines employing natural and artificial building blocks. These biomolecule machines have broad range of commercial applications-as biosensors, in chemical synthesis and processing, as bioelectronic devices and materials, in nanotechnology, in functional genomics and in drug discovery. 6. BiopharmaceuticalsNanobiotechnology can develop drugs for diseases that conventional pharmaceuticals cannot target. The pharmaceutical industry traditionally focuses on developing drugs to treat a defined universe of about five hundred confirmed disease targets. But approximately 70 to 80 percent of the new candidates for drug development fail, and these failures are often discovered late in the development process, with the loss of millions of dollars in R&D investments. Nanoscale techniques for drug development will be a boon to small companies, which cannot employ hundreds of organic chemists to synthesize and test thousands of compounds. Nanobiotechnology brings the ability to physically manipulate targets, molecules and atoms on solid substrates by tethering them to biomembranes and controlling where and when chemical reactions take place, in a fast process that requires few materials (reagents and solutions). This advance will reduce drug discovery costs, will provide a large diversity of compounds, and will facilitate the development of highly specific drugs. Potentia Pharmaceuticals (Louisville, Kentucky) is an early-stage company that is attempting to streamline the drug development process with the use of nanotechnologies (Harvard Business School 2001). 7. Nanotechnology in cardiac therapyNanotechnology is currently offering promising tools for applications in modern cardiovascular science to explore existing frontiers at the cellular level and treat challenging cardiovascular diseases more effectively. These tools can be applied in diagnosis, imaging and tissue engineering [36]. Miniaturized nanoscale sensors like quantum dots (QDs), nanocrystals, and nanobarcodes are capable of sensing and monitoring complex immune signals in response to cardiac or inflammatory events [20]. Nanotechnology can also help detect and describe clinically-significant specific mechanisms implicated in cardiac disorders. In addition, it is useful in designing atomic-scale machines that can be incorporated into biological systems at the molecular level. Introduction of these newly designed nanomachines may positively change many ideas and hypotheses in the treatment of critical cardiovascular diseases. Nanotechnology could also have great impact in tackling issues like unstable plaques and clarification of valves. Thus, this approach could be a real milestone of success in achieving localized and sustained arterial and cardiac drug therapy for the management of cardiovascular diseases [37]. 8. Nanotechnology in dental careNanotechnology will have future medical applications in the field of dentistry. The role of nanodentistry by means of the use of nanomaterials [38,39], biotechnology [40,41], and nanorobotics will ensure better oral health. Millions of people currently receiving poor dental care will benefit from such remarkable breakthrough in the science of dental health [42,43]. Moreover, nanodental techniques in major tooth repair may also evolve. Reconstructive dental nanorobots could be used in selective and precise occlusion of specific tubules within minutes, and this will facilitate quick and permanent recovery. The advantage of nanodentistry in natural tooth maintenance could also be significant [44]. Covalently-bonded artificial materials like sapphire may replace upper enamel layer to boost the appearance and durability of teeth [43]. 9. Nanotechnology in orthopedic applicationsNanomaterials sized between 1 and 100 nm have role to play as new and functional constituents of bones being also made up of nanosized organic and mineral phases [45,46]. Nanomaterials, nanopolymers, carbon nanofibers, nanotubes, and ceramic nanocomposites may help with more efficient deposition of calcium-containing minerals on implants. Based on these evidences and observations, nanostructure materials represent a unique realm of research and development that may improve the attachment of an implant to the surrounding bone matters by enhancing bone cell interactions, and this will indeed aid in improving orthopedic implant efficacy while drastically minimizing patient-compliance problems. Future prospects of nanobiotechnology There is much debate on the future implications of nanobiotechnology. It could create and suggest implementation of a choice of various new materials and devices potentially useful in the field of medicine, electronics, biomaterials and energy production. Nevertheless, this approach raises many of the same issues as any new technology, including problems with toxicity and environmental impact of nanomaterials [47] and their potential effects on global economics, as well as speculation about various doomsday scenarios. These concerns have accounted for a debate among advocacy groups and governments on whether special statutory regulation of nanobiotechnology is warranted. Despite the existence of some disputes, this technology renders immense hope for the future. It may lead to innovations by playing a prominent role in various biomedical applications ranging from drug delivery and gene therapy to molecular imaging, biomarkers and biosensors. One of these applications being the prime research objective of the present time would be target-specific drug therapy and methods for early diagnosis and treatment of diseases [2]. Two types of medical applications are already emerging, both in clinical diagnosis and in R&D. Imaging applications, such as quantum dot technology are already being licensed and applications for monitoring cellular activities in tissue are coming soon. The second major type of application involves the development of highly specific and sensitive means of detecting nucleic acids and proteins [48]. By 2015 to 2020, we will see that products being tested in academic and government laboratories will be creeping into commercialization. Sparse cell isolation and molecular filtration applications should, by then, make it to market. Some of the drug delivery systems should be commercialized or in advanced clinical trials. For example - drug delivery systems have been developed by NanoSystems or by American Pharmaceutical Partners, which is testing the encapsulation of Taxol, a cancer drug in a nanopolymer called paclitaxel. Most medical devices and therapeutics are a decade or more away from market. Therefore, drug target manipulation as well as device implantation requires a complex technical infrastructure like nanotechnology as well as complex regulatory management [49]. Continuous advancements in nanomedicine have opened up its opportunities for application in a variety of medical disciplines. Its future application as diagnostic and regenerative medicine is currently being investigated. In diagnosis, detection of diseased cells would be faster, possibly at the point of a single sick cell, while allowing diseased cells to be cured at once before they spread into and affect other parts of the body. Also, individuals suffering from major traumatic injuries or impaired organ functions could benefit from the use of nanomedicine. Challenges for nanobiotechnology No single person can provide the answers to challenges that nanotechnology brings, nor can any single group or intellectual discipline. The five main challenges are to develop instruments to assess exposure to engineered nano-materials in the air and water. It is fairly understood that exposure of humans and animals to the environment potentially contaminated with nano-materials may need to be monitored for any adverse consequence. The challenge becomes increasingly difficult in more complex matrices like food. The second challenge would be to develop applicable methods to detect and determine the toxicity of engineered nano-materials within next 5 to 15 years. Then again, proposing models for predicting effects of these nano-materials on human health and the environment would be an inevitable issue. The next challenge would be to develop reverse systems to evaluate precise impact of engineered nano-materials on health and the environment over the entire life span that speaks to the life cycle issue. The fifth being more of a grand challenge would be to develop the tools to properly assess risk to human health and to the environment. Commercialization challenges of nanobiotechnology include uncertainty of effectiveness of innovation, scalability, funding, scarce resources, patience etc. A broad majority of company recognizes a great potential in nanotechnology for the development of new products and the improvement of existing products. A new potentially disruptive technology like nanotechnology raises fundamental questions about the need for new regulations. Authorities around the world should evaluate possible risks and an appropriate regulatory response to the extensive use of this advanced technology. Potential hazards of nanoparticles Nanoparticles, as a result of their extreme microscopic dimension, which gives unique advantage, have potential hazards similar to particulate matters [50]. These particles have the potential to cause varied pathologies of respiratory, cardiovascular and gastrointestinal system [51]. Intra-tracheal instillation of carbon nanotube particles in mice has shown that carbon nanotubes have the potential to cause varied lung pathologies like epitheloid granuloma, interstitial inflammation, peribronchial inflammation and necrosis of lung. The toxicity produced by carbon nanotube was found to be greater than that produced by carbon black and quartz [52]. It has been shown that nanomaterials can enter the human body through several ports. Accidental or involuntary contact during production or use is most likely to occur via the lungs, from which a rapid translocation is possible to other vital organs through the bloodstream. On the cellular level, an ability to act as a gene vector has been demonstrated for nanoparticles [53]. Nanoparticles can enter the central nervous system either directly through axons of olfactory pathway or through systemic circulation through the olfactory bulb [54]. Studies done on monkeys and rats have shown accumulation of carbon and manganese nanoparticles in the olfactory bulb through the olfactory pathway [55]. This shows that nanoparticle-mediated delivery can, in future, provide a means of alternate route, circumventing the blood brain barrier. However, this can also result in the inflammatory reactions/responses in the brain, which needs to be evaluated. Radomski et al[56] have observed the pro-aggregatory effects of nanotubes on platelets in in vitro studies and acceleration of vascular thrombosis in rat. It was also observed that fullerenes do not have the property of inducing platelet aggregation. Thus, for designing nanoparticle-based drug delivery systems, fullerenes may be a safer approach as compared to nanotubes [57]. The toxicity of nanoparticles can also be extrapolated to gastrointestinal system, resulting in inflammatory bowel diseases. The toxicity of nanoparticles may be related to its ability to induce release of pro-inflammatory mediators resulting in inflammatory response and organ damage. If ingested, the nanoparticles can reach the circulation and reach different organs and systems and possibly result in toxicity [58]. These have been studied in vitro and in animal models and the effect on human system is difficult to extrapolate from such studies. Their use in humans requires further research and much needed caution. Conclusion Nanobiotechnology is still in its early stages. The multidisciplinary field of nanobiotechnology is bringing the science of the almost incomprehensibly small device closer and closer to reality. The effects of these developments will at some point be so vast that they will probably affect virtually all fields of science and technology. Nanobiotechnology offers a wide range of uses in medicine. Innovations such as drug delivery systems are only the beginnings of the start of something new. Many diseases that do not have cures today may be cured by nanotechnology in the future. Although the expectations from nanobiotechnology in medicine are high and the potential benefits are endlessly enlisted, the safety of nanomedicine is not yet fully defined. Use of nanotechnology in medical therapeutics needs adequate evaluation of its risk and safety factors. Scientists who are against the use of nanotechnology also agree that advancement in nanotechnology should continue because this field promises great benefits, but testing should be carried out to ensure the safety of the people. It is possible that nanomedicine in future would play a crucial/unparallel role in treatment of human diseases and also in enhancement of normal human physiology. If everything runs smoothly, nanobiotechnology will, one day, become an inevitable part of our everyday life and will help save many lives. Competing interests The authors declare that they have no competing interests. Authors' contributions MF planned the study and outlined it, prepared final manuscript. ZH performed literature review and helped in preparing manuscript. HA also performed literature review and helped in preparing manuscript. All authors read and approved the final manuscript. 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J Nanobiotechnology. 2012 Jul 20; 10:31
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22912868PONE-D-12-1340710.1371/journal.pone.0043406Research ArticleBiologyAnatomy and PhysiologyMusculoskeletal SystemHand StrengthMuscleMusculoskeletal AnatomySkinSkin PhysiologyMedicineAnatomy and PhysiologyMusculoskeletal SystemHand StrengthMuscleSkinSurgeryDermatologic and Cosmetic SurgeryReconstructive SurgeryLate Complications of Clinical Clostridium Histolyticum Collagenase Use in Dupuytren's Disease Clostridium Histolyticum Collagenase ComplicationsRozen Warren M. 1 2 * Edirisinghe Yasith 1 2 Crock John 1 2 1 Department of Plastic and Reconstructive Surgery, Dandenong Hospital, Southern Health, Dandenong, Victoria, Australia 2 Department of Surgery, Faculty of Medicine, Monash University, Clayton, Victoria, Australia Awad Hani A. Editor University of Rochester, United States of America * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: WMR YE JC. Performed the experiments: WMR YE JC. Analyzed the data: WMR YE JC. Contributed reagents/materials/analysis tools: WMR YE JC. Wrote the paper: WMR YE JC. Surgical planning and execution: WMR YE JC. 2012 17 8 2012 7 8 e434068 5 2012 23 7 2012 © 2012 Rozen et al2012Rozen 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.Introduction While Dupuytren's disease can cause disabling contractures requiring open surgery, a less-invasive option using Clostridium Histolyticum collagenase (CHC) via percutaneous injection was recently reported. A recent prospective, randomized trial demonstrated few complications during 90 days follow-up, however did not assess any longer term follow-up for these patients. Long-term outcomes in this setting have not been adequately reported, and the current manuscript aims to identify late complications from the clinical use of percutaneous CHC. Methods The current manuscript reports an extended 12-month follow-up for a cohort of twelve of patients enrolled in the original prospective, randomized trial, treated at a single institution. An analysis of complications requiring surgical intervention was undertaken. Results Two of twelve patients reported debilitating pain and triggering requiring surgical intervention. Extensive deep-tissue scarring and adhesions were identified, providing the first visual and qualitative analysis of the pathologic effects of CHC. Conclusion Late complications from CHC use can and have occurred, outside the follow-up period of the initial phase III trials. Longer term follow-up of such patients is thus essential, and further investigation and characterization of the late effects of CHC use is warranted. The authors have no support or funding to report. ==== Body Introduction Dupuyten's disease is a fibroblastic proliferative disorder in which normal fascial bands of the palmar fascia become diseased cords, which shorten progressively leading to soft-tissue and joint contractures. With limited conservative measures available, the only definitive management option for disabling Dupuyten's disease has been surgical intervention, ranging from percutaneous cordotomy to radical dermo-fasciectomy. A new drug containing Clostridium Histolyticum collagenase (CHC) has been reported to be capable of lysing Dupuytren's cords and is being marketed as a product that can be used to treat Dupuyten's disease in an office-based setting by simply injecting the product subcutaneously into the affected cords (Xiaflex, Auxillium Pharmaceuticals) [1]–[4]. This product has entered clinical use, largely based on phase III trials with the ‘Collagenase Option for Reduction of Dupuytren's (CORD1 and CORD2)’ studies reporting high efficacy and low complication rates. One cohort of these patients were tested in a single centre in Melbourne Australia, and in this group, longer follow-up outside of the study period was able to be assessed. While a low complication rate was reported within initial trials, in which a 90 day follow-up period was included in the study protocols, we were able to achieve longer term follow-up (12 months) for 12 patients, and were able to identify some significant complications which were not previously recognised. Methods Ethics Statement Inclusion of patients in this study was approved by the institutional ethical committee of Southern Health and the National Health and Medical Research Council of Australia guidelines. The study complies with the Declaration of Helsinki, 1995. The subjects gave full written informed consent, and patient anonymity has been preserved. This institutional review board specifically approved this study. A cross-sectional study was undertaken of a selected group of patients from the cohort of participants included in the phase III trials of CHC use as described in the CORD1 and CORD2 studies, comprising prospective, randomized, double-blind, placebo-controlled, multicentre trials [1]. The cohort of patients comprised 12 patients who had all been treated at a single institution. CORD1 and CORD2 trial protocols for recruitment, consent, randomization, treatment and initial follow-up had been adhered to, and as such, the patients included were patients with Dupuyten's disease who had fixed-flexion contractures of the metacarpophalangeal joint or proximal interphalangeal joint of 20 degrees or more in one finger, were of good health, and were able to appropriately consent. Exclusion criteria included bleeding disorders or anticoagulant use, and previous treatment of the primary joint. Written informed consent was provided in all cases. The primary intervention for each of the 12 patients had been the administration of up to three CHC injections at 1-month intervals, together with early mobilisation. CHC dosing comprised 0.58 mg per injection reconstituted in 0.39 ml of sterile saline for metacarpophalangeal joints or 0.31 ml of sterile diluent (a combination of sterile saline and calcium chloride) for proximal interphalangeal joints. After reconstitution, 0.25 ml was injected into a cord affecting an metacarpophalangeal joint and 0.20 ml injected into a cord affecting a proximal interphalangeal joint. The injections were applied subcutaneously, directly into the affected cords. While initial reporting of the CORD studies reported 90 day outcomes, the current study reports the 12 month follow-up of the cohort described. All early-term follow-up results were as reported in the original trial, with the reported adverse effects in this cohort being pain/discomfort during the action of the CHC in dividing the Dupuytren's cords, insignificant bruising and pain around the injection sites. Furthermore, all twelve patients achieved a full range of flexion and extension, as per the end criteria of the CORD1 and CORD2 trials. However, the primary endpoint of this study was the incidence of complications requiring surgical intervention, presenting outside the initial follow-up period. The findings of surgical exploration following CHC use has not been described, and this study provides a visual and qualitative analysis of the pathologic effects of CHC use. Results Within the extended follow-up period of the current study, between 3 and 12 months post-injection, 10 of the 12 patients did not report any further complications. Two patients however presented with significant disability from painful triggering of the injected finger. Both patients were male, with no significant past or present comorbidities or confounding factors. For both patients, their symptoms were managed conservatively throughout the study period, with monthly medical assessment, and conservative therapy comprising mobilisation, night splinting and scar management. At each of the monthly follow-up measurements, these 2 patients recorded full range of motion in flexion and extension, despite the clinically evident persistence of pain and stiff fingers. By 12 months, it was evident that these patients had no further functional recovery and with a clinical diagnosis of significant scarring/adhesions, operative intervention was undertaken. Similar clinical findings were experienced with both patients. Each patient underwent surgical exploration for both diagnosis and management, with the aim of confirming the pathology, determining the anatomical structures involved and managing the pathology through fasciectomy, tenolysis and soft tissue reconstruction as required. This intervention also served as a means to assess the anatomy and changes caused by CHC injection, an assessment not previously reported, and to photographically record these effects. Figures 1, 2, 3 demonstrate the photographic sequence of dissection from superficial to deep, with findings consistent between both cases. Superficial dissection demonstrated the presence of an ongoing pretendinous band of Dupuyten's disease, albeit attenuated, confirming the incomplete action of CHC on the disease (Figure 1). Deeper dissection, to a plane immediately deep to the superficial palmar fascia, revealed dense adhesions around the flexor tendon and a thickened A1 pulley distally. The adhesions extended laterally around the flexor tendons to involve the digital neurovascular bundles circumferentially, outside a plane involved by Dupuyten's disease (Figure 2). Deeper dissection still identified further dense adhesions and flexor tendon scarring, limiting tendon glide. This scar tissue was within the tendon sheath, surrounding the tendons themselves, however both flexor digitorum superficialis and flexor digitorum profundus tendons were intact. Both patients underwent neurolysis, tenolysis, adhesiolysis, fasciectomy and closure. Histopathologic assessment of the excised tissue confirmed dense scar tissue. Longer term follow-up, 18 months after CHC injection and 6 months after revision surgery, demonstrated a return to full range of motion and no need for subsequent intervention. 10.1371/journal.pone.0043406.g001Figure 1 Superficial dissection revealing residual attenuated pretendinous Dupuytren's cord. 10.1371/journal.pone.0043406.g002Figure 2 Deeper dissection through dense peritendinous adhesions and scarring. 10.1371/journal.pone.0043406.g003Figure 3 Further dissection revealing dense scarring of the flexor tendons with adhesions limiting tendon glide. Discussion Dupuytren's disease has been an extensively investigated disease process throughout the 19th and 20th centuries, with even the earliest reference to Dupuyten's disease in 1614 by Felix Plater highlighting the debility caused by a condition causing flexion in the little fingers, which he postulated to be caused by flexor tendon shortening [5]. In 1777, Sir Henry Cline demonstrated the causal relationship of palmar fascia to the condition from a series of cadaveric dissections, followed by Baron Guillaume Dupuytren in 1831 comprehensively describing the differential diagnosis, suspected pathology, clinical course, and management of the disease. Since that time, the etiology, pathophysiology and differing management strategies have been extensively studied by various research groups [6]. These have highlighted the more common associations with the disease: men are more likely to have Dupuyten's disease than women, with 9 of every 10 patients being male; the incidence increases with age; the disease is common in Scandinavia, Great Britain, Ireland, Australia, and North America, while it is uncommon in southern Europe and South America, and rare in Africa and China. Other studies have shown associations with diabetes, alcoholism and hypercholesterolemia, and twin and genetic studies conducted thus far show a strong genetic influence, with current evidence supporting an autosomal dominant inheritance pattern with variable penetrance [7], [8]. With limited conservative measures available, the mainstay of management options for disabling Dupuyten's disease has been surgery, with McGrouther et al in 2006 publishing a comprehensive algorithm for the surgical management of Dupuyten's disease [9]. This strategy involves clear identification of symptoms on presentation and close follow-up for a year, with guidelines recommending operative intervention if clear progression of the condition or clear deterioration of function are observed. Operative intervention can vary depending on the degree of involvement of the disease, and may involve percutaneous aponeurotomy, limited fasciectomy or dermofasciectomy for more extensive disease. The introduction of CHC boasted a revolutionary therapy in the treatment of Dupuyten's disease, suggesting a minimally invasive means to releasing palmar fascial contractures with good efficacy and a safe side-effect profile [1]–[4]. The compound, isolated from the culture medium of Clostridium Histolyticum, comprises a fixed-ratio mixture of a clostridial type-I collagenase (termed by the manufacturer as AUX-I) and a clostridial type-II collagenase (AUX-II). The type-I collagenase is a 114-kDa single polypeptide chain containing approximately 1000 amino acids of known sequence. The type-II collagenase is 113 kDa in weight and is also approximately 1000 amino acids long. These enzymes differ from each other in terms of domain structure, substrate affinity, catalytic efficiency, and preferred cleavage site on the collagen molecule. According to the pre-clinical studies conducted by the manufacturer, these class-I and class-II collagenases work synergistically rather than independently [3]. The form and pharmacokinetics of the compound is straightforward, with in-vivo and in-vitro studies suggesting that the compound can lyse Dupuytren's cords while leaving the adjacent neurovascular structures unaffected [10]. CHCs are metalloprotease enzymes of the matrixin subfamily, which function via lysis of the three-dimensional structure of collagen molecules. The CHC molecule has demonstrated in-vivo and in-vitro catalytic activity against all collagen types, except for collagen type IV. Dupuytren's cords show an abundance of type 1 collagen as well as unusually large amounts of collagen type III, which is otherwise not commonly seen within palmar fascia. The basement membrane of the neurovascular structures mostly contains type IV collagen, and is therefore not affected even in the case of direct injection [10]. Non-clinical studies failed to show significant degradation of blood vessels, nerves and epithelia following local injection, which may be related to its poor activity against type IV collagen [3]. Pre-clinical animal studies did not reveal any detectable systemic effects following intravenous injection of collagenase, and this has been supported by clinical trials that have not shown any detectable levels of collagenase systemically after its subcutaneous injection [11]. Consequently, clinical drug interaction studies have not been conducted. Of note, the tetracycline family of antibiotics has been shown to inhibit matrix metalloproteinase-mediated collagen degradation in published in-vitro experiments, which suggests that concomitant use of tetracyclines may inhibit collagenase action [12]. Other complications have included reports of local inflammatory reactions at the injection site as a result of vascular leakage and neutrophil chemotaxis [10], [11]. The CORD1 trial reported the clear success of CHC injections for treating Dupuyten's disease. In this trial, 3 successive injections spaced 30 days apart, followed by aggressive early mobilization and night splinting, was able to achieve post-injection extension to 5 degrees or less in 64% of the cases, while the placebo arm achieved this extension in 6.8% of the cases. This benefit was associated with a relatively low side-effect profile, with reports of local immune/inflammatory reactions such as pruritis, injection site pain, lymphadenopathy, ecchymosis, erythema, and blistering. Two cases of flexor tendon rupture were reported, with the cause for these 2 cases identified as suboptimal injection technique. With a corrected technique, the extension of the study reported 3 flexor tendon ruptures out of 2600 injections [1]. The combined analysis of outcomes and complications from the CORD1 and CORD2 studies suggested that anatomically, the CHC technique employed resulted in effects on the diseased palmar fascia and some action on the flexor tendon. The current study highlighted several key outcomes. Firstly, tendon scarring and adhesions causing painful triggering of the affected flexor tendon is a noteworthy late complication that is worthy of assessment and consideration in the consent process, and may require revisionary surgery. Secondly, it is clear that the action of CHC is more anatomically extensive than previously considered. Post-injection adhesions were evident lateral to the flexor tendons, within the flexor sheath (at the A1 pulley) and circumferentially around the neurovascular structures of the digits. In addition to the presenting complaints described, the longer term impact on digital function or on surgical outcomes in recurrent disease, is yet to be established. Combining the flexor tendon ruptures reported in the CORD1 study and the extensive adhesions presented in this study, alteration of CHC dosing or drug concentration, injection technique or image-guidance during the injection, are all techniques worth investigating as a means to improved outcomes. Conclusions While the use of CHC can achieve excellent results in most patients, complications can occur outside of early follow-up, such as occurred in initial phase III trials. Late complications include flexion pain and triggering, and may require operative intervention as late as 12 months post-injection. With operative findings of deep tissue scarring and tendon adhesions following CHC use, the likely mechanism for these complications is through a broad anatomical action of CHC on palmar and digital anatomy, and these effects warrant further investigation and characterization. ==== Refs References 1 Hurst LC , Badalamente MA , Hentz VR , Hotchkiss RN , Kaplan FT , et al (2009 ) Injectable collagenase clostridium histolyticum for Dupuytren's contracture . N Eng J Med 361 (10) : 968 –979 . 2 Amadio PC (2009 ) What's new in hand surgery . J Bone Joint Surg (Am) 91 : 496 –502 .19182010 3 Desai SS , Hentz VR (2010 ) Collagenase clostridium histolyticum for Dupuytren's contracture . Expert Opin Biol Therapy 10(9) : 1395 –1404 . 4 Badalamente MA , Hurst LC (2007 ) Efficacy and safety of injectable mixed collagenase subtypes in the treatment of Dupuytren's contracture . J Hand Surg (Am) 32 : 767 –774 .17606053 5 Saar JD , Grothaus PC (2000 ) Dupuytren's Disease: An Overview . Plas Reconstr Surg 106(1) : 125 –134 . 6 Rayan GM (2007 ) Dupuytren's Disease: Anatomy, Pathology, Presentation and Treatment . J Bone Joint Surg (Am) 89 (1) : 190 –198 . 7 Townley WA , Baker R , Grobbelaar AO (2006 ) Dupuytren's contracture unfolded . BMJ 332 : 397 –400 .16484265 8 Hindocha S , McGrouther DA (2009 ) Epidemiological Evaluation of Dupuytren's Disease – Incidence and Prevalence Rates in Relation to Etiology . Hands 4 : 256 –269 . 9 Bayat A , McGrouther DA (2006 ) Management of Dupuytren's disease – Clear advise for an elusive condition . Ann Roy Coll Surg Engl 88 : 3 –8 . 10 Rydevik B , Ehira T , Linder L , Olmarker K , Romanus M , et al (1989 ) Microvascular response to locally injected collagenase . Scand J Plast Reconstr Surg 23 : 17 –21 . 11 Badalamente MA , Hurst LC , Hentz VR (2002 ) Collagen as a clinical target: nonoperative treatment of Dupuytren's disease . J Hand Surg (Am) 27 : 788 –798 .12239666 12 Suomalainen K , Sorsa T , Golub L , Ramamurthy N , Lee HM , et al (1992 ) Specificity of the anticollagenase action of tetracyclines; relevance to their anti-inflammatory potential . Antimicr Agents Chemotherapy 36 : 227 –229 .
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PLoS One. 2012 Aug 17; 7(8):e43406
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22912865PONE-D-12-1357410.1371/journal.pone.0043383Research ArticleBiologyDevelopmental BiologyStem CellsMesenchymal Stem CellsCell DifferentiationMolecular cell biologyCellular TypesStem CellsMesenchymal Stem CellsSignal transductionSignaling cascadesERK signaling cascadeMAPK signaling cascadesP38 and ERK1/2 MAPKs Act in Opposition to Regulate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells P38 and ERK1/2 on BMP9-Induced OsteogenesisZhao Yingze Song Tao Wang Wenjuan Wang Jin He Juanwen Wu Ningning Tang Min He Baicheng Luo Jinyong * Key Laboratory of Diagnostic Medicine designated by the Chinese Ministry of Education, Chongqing Medical University, Chongqing, 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: YZ JL. Performed the experiments: YZ TS WW JW JH NW MT BH. Analyzed the data: MT BH. Contributed reagents/materials/analysis tools: JH NW. Wrote the paper: JL. 2012 17 8 2012 7 8 e433835 5 2012 23 7 2012 © 2012 Zhao et al2012Zhao 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.Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-indcued osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The in vitro effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation in vivo. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs. This work was supported in part by research grants from the Natural Science Foundation of China (#31071304 and #30800658 to Dr. Luo), and the Natural Science Foundation Project of Chongqing Science and Technology Commission (#2009BB5060 to Dr. Luo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Mesenchymal progenitor cells (MPCs) are non-hematopoietic stem cells capable of differentiating into osteoblastic, chondrogenic, myogenic, or adipogenic lineages [1], [2], [3], [4], [5]. Although recent studies have demonstrated that MPCs are also able to differentiate into other lineages, including neuronal and cardiomyogenic lineages [6], [7], [8]. Bone morphogenetic proteins (BMPs), members of transforming growth factors beta (TGFβ) superfamily, are known to play important roles in stem cell biology and perform pivotal functions in the areas of embryogenesis, multiple growth and differentiation processes [9], [10], [11], [12]. Genetic disruptions of BMPs have led to various skeletal and extra-skeletal abnormalities during development [9], [13]. To date, more than twenty BMPs have been identified. Several forms of recombinant BMPs, most notably BMP2 and BMP7, have been shown to promote osteogenesis and are now used as adjunctive therapy in the clinical setting [14], [15], [16], [17], [18]. However, it remains unclear whether BMP2 and BMP7 are in fact the most potent BMPs in inducing osteogenic differentiation and bone formation. Recent studies have implied us that BMP9, one of the least studied BMPs, is probably a more potent inducer in promoting osteogenic differentiation of MPCs in vitro and in vivo [11], [19], [20], [21]. Furthermore, a distinct set of downstream target genes that may play a role in regulating BMP9-induced osteogenic differentiation of MPCs were identified by microarray [11], [20]. Our recent study revealed that TGFβ type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in MPCs [22]. We also elucidated that TGFβ type II receptors BMPRII and ActRII are the functional receptors necessary to transmit osteoinductive signaling of BMP9 [23]. Despite these valuable discoveries, BMP9 remains as the least characterized BMPs, and the signaling mechanism through which BMP9 regulates osteogenic differentiation of MPCs is still largely unknown and warrants extensive studies. It has been well established that BMPs fulfill their signaling activity through activation of transcription factors Smads. In this case, BMPs signal transduction begins with binding to heterodimeric complex of two transmembrane serine/threonine kinase receptors, BMPs type I and type II receptors [9], [10], [22], [23]. These activated receptors transmit signals by phosphorylating the transcription factors Smads (Smad1, 5, and/or 8) [9], [10], [22], [23], [24], [25], [26], which in turn form a heterodimeric complex with Smad4 in the nucleus and modulate context-dependent gene expression in collaboration with other co-activators. However, growing evidences have implied that BMPs also utilize diverse intracellular signaling molecules in addition to Smads to regulate a wide array of cellular functions [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45]. These are collectively called the non-Smads (non-canonical) pathway of BMPs signaling. Mitogen activated protein kinases (MAPKs), such as p38 and extracellular signal-regulated kinase (ERK1/2), are key branch of non-Smads BMPs pathway [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]. MAPKs are a group of well-described serine/threonine-specific protein kinases generally expressed in all cell types [46], [47], [48]. At least four subfamilies of mammalian MAPKs have been identified, including ERK1/2, ERK5 (also known as MAPK7 or BMK1), the Jun amino-terminal kinases (JNKs) and p38 MAPKs. Each class of MAPKs is activated by a distinct kinase cascade in which a MEKK phosphorylates and stimulates a downstream dual-specificity MEK, which in turn phosphorylates threonine/tyrosine residues on MAPKs. Phosphorylation of these threonine/tyrosine residues on MAPKs results in a conformational change that evokes MAPKs activity [49]. MAPKs are essential components of signal transduction machinery and occupy a central position in regulation of gene expression, mitosis, metabolism, survival, motility, apoptosis, proliferation and differentiation [47], [50], [51], [52]. However, the different MAPKs members are activated in response to different extracellular stimuli and have different downstream targets, and thus play distinct roles in cellular responses. Although it has been demonstrated that BMP9 plays a critical role in the processes by which MPCs undergo commitment to the osteoblastic lineage, little is known about the downstream signaling pathway(s) involved. In our previous studies, we have found that BMP9 is capable of simulating the expression of Smad6 and Smad7 (early targets of BMPs-Smads signaling) [22], [23]. Moreover, we further demonstrated that BMP9 is able to increase Smad1/5/8 transcriptional activity and enhance translocation of Smad1/5/8 to the nucleus in MPCs [22], [23], [24]. In another study by Bergeron et al., BMP9 was found to fulfill osteoinductive effects on MC3T3-E1 preosteoblastic cells by phosphorylating downstream Smads transcriptional factors [25]. These reports intensively validate that BMP9 can induce osteogensis and bone formation through activation of the Smads signaling pathway. However, the non-Smads pathways in BMP9-induced osteogenesis are largely unknown and need to be intensively explored. It has been well demonstrated that MAPKs play a critical role in transmitting intracellular signaling of osteoinductive BMPs, such as BMP2 and BMP4, in MPCs [34], [35], [36], [37], [38], [39], [40], [41], [42]. However, whether MAPKs are also relevant to BMP9-induced osteogenic differentiation of MPCs are currently unclear. In the present study, we try to probe the possible involvement and exact function of two classic MAPKs subfamilies, p38 and ERK1/2 in regulating BMP9-indcued osteogenic differentiation of MPCs. Our results show that p38 and ERK1/2 MAPKs are both activated by BMP9 treatment in MPCs. Inhibition of p38 by specific inhibitor SB203580 or RNAi dramatically reduces BMP9-indcued osteogenic differentiation and Smads signaling in vitro, decreases BMP9-induced bone formation in vivo. Conversely, blocking of ERK1/2 by specific inhibitor PD98059 or RNAi enhances BMP9-induced osteogenic differentiation and Smads signaling, and increases BMP9-induced bone formation. Taken together, these results strongly suggest that BMP9 can simultaneously stimulate phosphorylation/activation of p38 and ERK1/2 MAPKs. Also, it’s important to note that p38 and ERK1/2 MAPKs are probably to act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs. Materials and Methods Cell Culture and Chemicals C3H10T1/2, C2C12, HEK293 and HCT116 cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA)and maintained in complete DMEM medium supplemented with 10% fetal calf serum (FCS, Hyclone) and antibiotics. MAPKs inhibitors PD98059 and SB203580 were obtained from Santa Cruz Biotechnology (California, USA). Inhibitors were dissolved in DMSO and aliquots were stored in −80°C. Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (Saint Louis, USA). Isolation of Mouse Embryo Fibroblasts (MEFs) MEFs were isolated from post coitus day 13.5 mice, as previously described [22], [23]. Briefly, each embryo was dissected into 10 ml sterile PBS, voided of its internal organs, and sheared through an 18-gauge syringe in the presence of 1 ml 0.25% trypsin and 1 mM EDTA. After 15 min incubation with gentle shaking at 37°C, DMEM with 10% FCS was added to inactivate trypsin. The cells were plated on 100 mm dishes and incubated for 24 hr at 37°C. Adherent cells were used as MEFs. Aliquots were kept in a liquid nitrogen tank. All MEFs used in this study were less than five passages. Construction of Recombinant Adenoviruses Recombinant adenoviruses expressing BMP9 (Ad-BMP9) were generated previously using the AdEasy system, as demonstrated [21], [22], [23], [24]. Recombinant adenoviruses expressing small interference RNA (siRNA) targeted p38 (AdR-si-p38), ERK1/2 (AdR-si-ERK1/2) were kindly provided by Dr. Tong-chuan He of University of Chicago Medical Center. Adenoviruses expressing only GFP (Ad-GFP) and RFP (Ad-RFP) were used as controls. Preparation of Conditioned Medium BMP9 conditioned media (BMP9-CM) were prepared as described [22], [23]. Briefly, subconfluent HCT116 cells (in 75-cm2 flaks) were infected with an optimal titer of Ad-BMP9. At 24 hrs after infection, the culture medium was changed to serum-free DMEM. Conditioned medium was collected at 48 hrs after infection and used immediately. Western Blotting Analysis Briefly, cells were collected and lysed in RAPI buffer. Cleared total cell lysate was denatured by boiling and loaded onto a 10% gradient SDS–PAGE. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. Membrane was blocked with Super-Block Blocking Buffer, and probed with the primary antibody, followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected by using SuperSignal West Pico Chemiluminescent Substrate kit. Primary antibodies were obtained from Santa Cruz, as follows: anti-phosphor-p38, anti-p38, anti-phosphor-ERK1/2, anti-ERK1/2, anti-phosphor-Smad1/5/8, anti-Smad1/5/8, anti-Runx2 and anti-β-actin. Alkaline Phosphatase (ALP) Assays ALP activity was assessed by a modified Great Escape SEAP Chemiluminescence assay (BD Clontech, Mountain View, CA), as described previously [19], [20], [21], [22], [23]. Each assay condition was performed in triplicate and the results were repeated in at least three independent experiments. ALP activity was normalized by total cellular protein concentrations among the samples. Matrix Mineralization Assay Cultured cells were seeded in 24-well cell culture plates and infected with Ad-BMP9 followed by treated with PD98059 or SB203580, or infected with AdR-si-p38 (or AdR-si-ERK1/2) followed by treat with BMP9-CM. Cells were cultured in the presence of ascorbic acid (50 µg/ml) and β-glycerophosphate (10 mM). At 21 days after cultured, mineralized matrix nodules were stained for calcium precipitation by means of Alizarin Red S staining, as described previously [21], [22], [23]. Briefly, cells were fixed with 0.05% (v/v) glutaraldehyde at room temperature for 10 min. After being washed with distilled water, fixed cells were incubated with 0.4% Alizarin Red S (Sigma-Aldrich) for 5 min, followed by extensive washing with distilled water. The staining of calcium mineral deposits was recorded under bright field microscopy. Immunocytochemical Stain Cultured cells were infected with Ad-BMP9 or Ad-GFP followed by treated with PD98059 and SB203580, or infected with AdR-si-p38 (or AdR-si-ERK1/2) and followed by BMP9-CM treatment. At the indicated time points, cells were fixed with 4% formalin and washed with PBS. The fixed cells were permeabilized with 0.25% Triton X-100 and blocked with 10% goat serum, followed by incubation with an anti-osteocalcin antibody (Santa Cruz Biotechnology) overnight. After washing, cells were incubated with biotin-labeled secondary antibody for 30 min, followed by incubating cells with streptavidin-HRP conjugate for 20 min at room temperature. The presence of the expected protein was visualized by DAB staining and examined under a microscope. Stains without the primary antibody, or with control IgG, were used as negative controls. Immunofluorescence Staining Cultured cells were treated with PD98059 and SB203580 followed by stimulated with BMP9-CM. At the indicated time points, cells were fixed with 4% formalin and washed with PBS. The fixed cells were permeabilized with 0.25% TriTon X-100, followed by incubation with a polyclone anti-Smad1/5/8 antibody (Santa Cruz Biotechnology) overnight. After washing, cells were incubated with FITC fluorescein conjugated secondary antibody for 30 min. Fluorescence signal was recorded under a fluorescence microscope. RNA Isolation and Quantitative Real-time RT-PCR (qPCR) Total RNA was extracted from cells with Trizol reagents (Invitrogen), and was used to generate cDNA by hexamer (Takara) and MMLV reverse transcriptase (Promega, CA, USA). PCR primers (Table S1) were designed by using the Primer3 program. SYBR Green-based qPCR analysis was carried out to amplify the genes of interest using RG-3000 Real-Time DNA Detection System (Corbett Research, Australia). Triplicate reactions were carried out for each sample. The cycling program was as: 94°C for 2 min for 1 cycle and 30 cycles at 92°C for 20 s, 57°C for 30 s, and 72°C for 20 s, followed by a plate read at 78°C for each cycle. All samples were run in triplicate and normalized by the endogenous expression level of β-actin. Subcutaneous MPCs Implantation All animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) of Chongqing Medical University. The ectopic bone formation by MPCs implantation was conducted as described [22], [23,23]. C3H10T1/2 cells were co-infected with Ad-BMP9 and Ad-si-p38 (or Ad-si-ERK1/2). Cells were collected at 80% confluence and for subcutaneous injection (5×106/injection) into the flanks of athymic nude mice (6 animals per group, 4–6-week old, male, Harlan Sprague Dawley). At 5 weeks after implantation, animals were killed, and the implantation sites were retrieved for histological stains, as follows: Retrieved tissues were decalcified, fixed in 10% formalin overnight, and embedded in paraffin. Serial sections of the embedded specimens were stained with hematoxylin and eosin (H&E) stain. Masson’s Trichrome stain was carried out as described [22], [23]. Deparaffinized and rehydrated sections were stained with 1% Alcian Blue (pH 2.5), as described previously [22], [23]. Mouse Calvarial Organ Culture Calvariae was isolated from 4 days old mouse pulps, and culture in media containing BMP9 and SB203580 (or PD98059) for 4 days. Then, the calvariae was placed into fresh media and return to incubator for another 3 day until day 7. The hematoxylin and eosin staining (H&E staining) was conducted to measure the total area of original bone remaining. In H&E staining, the eosin Y differentially stains the original bone darker whereas the new bone matrix that is formed appears as a lighter color [53]. The width of new bone was determined by Image Pro Plus. Results BMP9 Stimulates Phosphorylation/activation of p38 and ERK1/2 MAPKs in MPCs First of all, we sought to determine if p38 and ERK1/2 MAPKs can be activated by BMP9 in MPCs. Proliferating C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, then phosphorylation of Smad1/5/8, p38 and ERK1/2 were assessed by western blotting. Consistent with the results from pervious reports [22], [23], [24], [25], BMP9 was found to effectively activate Smads signaling, leading to an increased level of phosphorylated Smad1/5/8 (Fig. 1A). What is most noteworthy, however, is that exposure of C3H10T1/2 cells to BMP9 effectively increased the levels of phosphorylated p38 and ERK1/2, without altering the total amounts of these proteins. Similar results were also obtained in MEFs (Fig. 1B) and C2C12 cells (Fig. 1C). Furthermore, we also tested the effects of BMP9-conditioned medium (BMP9-CM) on activation of p38 and ERK1/2 in C3H10T1/2 cells. As illustrated in Fig. 1D, an increased level of phosphorylated p38 was first observed at 5 min, with its summit at 30 min post BM9-CM treatment. Similarly, the level of phosphorylated ERK1/2 was also increased, and peaked at 30 min. Collectively, these results strongly suggest that BMP9 can effectively promote activation of p38 and ERK1/2 MAPKs in MPCs. However, the exact functional roles of p38 and ERK1/2 in BMP9-induced osteogenic differentiation of MPCs need to be precise investigated. 10.1371/journal.pone.0043383.g001Figure 1 BMP9 induces phosphorylation/activation of p38 and ERK1/2 in MPCs. (A). Western blotting analysis of BMP9-induced phosphorylation of Samd1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of Smad1/5/8, p38 and ERK1/2 was analyzed by western blotting. β-actin was used to demonstrate equal loading of all samples. (B) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (C) Western blotting analysis of BMP9-induced phosphorylation of p38 and ERK1/2 in C2C12 cells. C2C12 cell were infected with Ad-BMP9 or Ad-GFP (MOI = 5), at 24 hrs, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed by western blotting. (D) Western blotting analysis of BMP9 conditioned medium (BMP9-CM)-induced phosphorylation of p38 and ERK1/2 in C3H10T1/2 cells. C3H10T1/2 cells were treated with BMP9-CM, total amount and phosphorylated forms of p38 and ERK1/2 was analyzed at indicated time points by western blotting. BMP9-induced Early Osteogenic Differentiation of MPCs is Dramatically Blocked by the p38 Inhibitor, SB203580, but is Enhanced by the ERK1/2 Inhibitor, PD98059 To inspect the detail roles of p38 and ERK1/2 in BMP9-induced osteogenic differentiation of MPCs. C3H10T1/2 cells were exposed to BMP9 in the presence of SB203580 and PD98059, which are specific inhibitor of p38 and ERK1/2 respectively. Then, ALP activity was determined using chemiluminescence quantitative assay. By treating C3H10T1/2 cells with varying concentrations of SB203580 (0, 2, 5 and 10 µM), we found that SB203580 was able to significantly inhibit BMP9-induced ALP activity in a dose-dependent manner (Fig. 2A). Conversely, PD98059 (0, 10, 25 and 50 µM) treatment was found to remarkably enhance BMP9-induced ALP activity mostly in a dose-dependent manner (Fig. 2B). We also found similar effects of SB203580 and PD98059 on BMP9-induced ALP activity in MEFs (Fig. 2C) and C2C12 MPCs (Fig. 2D). These results strongly suggest us that p38 and EKR1/2 may exhibit opposing roles in regulating BMP9-induced early osteogenic differentiation of MPCs. 10.1371/journal.pone.0043383.g002Figure 2 Opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity of MPCs. (A) Inhibition of BMP9-induced ALP activity by p38 inhibitor SB203580 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 2, 5 and 10 µM) of SB203580. ALP activity was quantitative measured at day 7. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (vs. control groups); “*”, p<0.05 (vs. control groups). (B) Enhancement of BMP9-induced ALP activity by ERK1/2 inhibitor PD98059 in C3H10T1/2 cells. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with varying concentrations (0, 10, 25 and 50 µM) of PD98059. ALP activity was quantitative measured at 7 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01; “*”, p<0.05 (vs. control groups). (C) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in MEFs. MEFs were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 5 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (vs. control groups). (D) Opposing effects of SB203580 and PD98059 on BMP9-induced ALP activity in C2C12 cells. C2C12 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), followed by treatment with fixed concentrations of SB203580 (25 µM) or PD98059 (10 µM). ALP activity was quantitative measured at 3 days. Each assay condition was carried out in triplicate in at least two independent batches. “**”, p<0.01 (vs. control groups). As p38 and ERK1/2 are involved in regulating both cell proliferation and differentiation [52], [54], we thought that the opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity of MPCs may partly correlate with the change of cell proliferation. Therefore, C3H10T1/2 cells were treated with BMP9 in the presence of SB203580 and PD98059 respectively, then ALP activity and cell proliferation was detected at selected time points. As illustrated in Fig. 3A and Fig. 3B, inhibition of ALP activity with SB203580 was accompanied by a lower level of cell proliferation, whereas PD98059 treatment led to a marked increase both in ALP activity and cell proliferation. Together, these findings indicate that the opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity are partly in line with the change of cell proliferation. 10.1371/journal.pone.0043383.g003Figure 3 Opposing effects of p38 and ERK1/2 on BMP9-induced ALP activity is correlated with the change of cell proliferation. (A) Effect SB203580 and PD98059 on BMP9-induced ALP activity in C3H10T1/2 cells. Subconfluent C3H10T1/2 cells were infected with Ad-BMP9 (MOI = 5) or Ad-GFP (MOI = 5), followed by treatment with SB203580 (25 µM) or PD98059 (10 µM), ALP activity was quantitative measured at the indicated time points. Each assay condition was carried out in triplicate in at least two independent batches. (B) Effect of SB203580 and PD98059 on BMP9-induced cell proliferation in C3H10T1/2 cells. Subconfluent C3H10T1/2 cells were infected with Ad-BMP9 (MOI = 5) or Ad-GFP (MOI = 5), followed by treatment with SB203580 (25 µM) or PD98059 (10 µM), cell proliferation was monitored by MTT assay at the indicated time points. Each assay condition was carried out in triplicate in at least two independent batches. Opposing Effects of p38 and ERK1/2 on BMP9-induced Late Osteogenic Differentiation of MPCs Although ALP is a well-established early osteogenic marker, it is hardly an accurate predictor of the late stage of osteogenic differentiation and bone formation [11], [22], [23]. Thus, we sought to determine if p38 and ERK1/2 have any opposing effects on BMP9-induced late osteogenic markers, such as matrix mineralization and osteocalcin (OC) expression. We infected C3H10T1/2 cells and MEFs with Ad-BMP9 or Ad-GFP and then treated with SB203580 (10 mM) or PD98059 (25 mM). At 21 days post BMP9 treatment, Alizarin Red S staining was conducted to judge the effects of p38 and ERK1/2 on BMP9-induced matrix mineralization. Interestingly, SB203580 treatment deceased BMP9-induced matrix mineralization, on the contrary, PD98059 treatment increased matrix mineralization in C3H10T1/2 cells and MEFs (Fig. 4A). Next, we infected C3H10T1/2 cells with Ad-BMP9 in the presence of SB203580 or PD98059. At 9 days and 11 days post infection, total RNA was isolated from the cells for qPCR analysis. Likewise, we found that SB203580 resulted in a significant decrease in BMP9-induced OC expression. However, treatment by PD98059 led to a dramatically increase in BMP9-indcued OC expression at gene level (Fig. 4B). Moreover, we utilized immunocytochemical stain and further confirmed that BMP9-induced OC expression was inhibited by SB203580, and yet was enhanced by PD98059 at protein level (Fig. 4C). Taken together, the above results intensively suggest us that p38 and ERK1/2 may play important but opposing roles in regulating both early and late stages of BMP9-induced osteogenic differentiation of MPCs. 10.1371/journal.pone.0043383.g004Figure 4 Opposing effects of p38 and ERK1/2 on BMP9-induced late osteogenic differentiation and Runx2 activation in MPCs. (A) Opposing effects of p38 and ERK1/2 on BMP9-induced matrix mineralization of MPCs. C3H10T1/2 cells and MEFs were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 21, cells were subjected to Alizarin Red S staining. Magnification, ×200. (B) qPCR analysis of SB203580 and PD98059 on BMP9-induced osteocalcin expression at gene level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 9 and day 11, osteocalcin expression was detected by qPCR. All samples were normalized using endogenous levels of β-actin. “**”, p<0.01 (vs. control groups). (C) Immunocytochemical staining analysis of SB203580 and PD98059 on BMP9-induced osteocalcin expression at protein level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP (MOI = 5), and then treated with SB203580 (25 mM) or PD98059 (10 mM). At day 12, cells were fixed and subjected to immunocytochemical staining analysis. Magnification, ×200. (D) Opposing effects of p38 and ERK1/2 on BMP9-induced Runx2 transcriptional activity. C3H10T1/2 cells were transfected with OC promoter containing Runx2-responsive element reporter, p6xOSE-Luc. Next, cells were infected with Ad-BMP9 in the presence of SB203580 (25 mM) or PD98059 (10 mM). At 36 hrs post infection, cells were lyzed for luciferase activity assay. Each assay condition was carried out in triplicate in at least two independent batches. Luciferase activity was normalized by total cellular protein concentrations among the samples. “**”, p<0.01 (vs. control groups). (E) qPCR analysis of SB203580 and PD98059 on BMP9-induced Runx2 expression at gene level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At 24 and 48 hrs post treatment, Runx2 gene expression was detected by qPCR. Each assay condition was carried out in triplicate. All samples were normalized by endogenous level of β-actin. “**”, p<0.01 (vs. control groups). (F) Western blotting analysis of SB203580 and PD98059 on BMP9-induced Runx2 expression at protein level. C3H10T1/2 cells were infected with Ad-BMP9 or Ad-GFP, and then treated with SB203580 (25 mM) or PD98059 (10 mM). At 48 hrs post treatment, Runx2 protein expression was detected by western blotting. β-actin was used to demonstrate equal loading of all samples. Opposing Effects of p38 and ERK1/2 on BMP9-induced Runx2 Activation in MPCs Runt-related transcription factor 2 (Runx2), also known as core-binding factor subunit alpha-1 (CBFα-1), is a key transcription factor associated with osteogenesis [55], [56]. Runx2 can directly stimulate expression of most of the well-established osteogenic markers, including osteocalcin (OC) [55]. It has been reported in our previous studies that Runx2 is a target of BMP9 [11], [20]. Therefore, we asked whether BMP9-induced activation of Runx2 was also influenced by p38 and ERK1/2 in an opposing model. Using a commonly used Runx2-regulated OC promoter reporter (p6xOSE-luc), we found that SB203580 treatment was able to inhibit BMP9-induced luciferase activity of p6xOSE-luc reporter, which contains Runx2-responsive elements and reflects Runx2 transcriptional activity. Conversely, PD98059 treatment increased BMP9-induced p6xOSE-luc reporter activity (Fig. 4D). These findings imply us that the effects of p38 and ERK1/2 on BMP9-induced Runx2 transcriptional activity are possibly opposing. Next, we examined the Runx2 expression at gene and protein level using qPCR and western blotting. As shown in Fig. 4E and Fig. 4F, BMP9-induced Runx2 expression was reduced by SB203580 treatment, and yet enhanced by PD98059 treatment at both gene and protein level. These results further confirm that p38 and ERK1/2 can exhibit opposing effects on BMP9-induced activation of Runx2 in an opposing manner. Token together, these above data reveal that p38 and ERK1/2 MAPKs are highly probably to play opposing roles in regulating BMP9-induced osteogenic differentiation of MPCs. Block of p38 and ERK1/2 Activity Leads to Opposing Effects on BMP9-induced Activation of Smads Signaling We next sought to explore the possible mechanism behind the opposing effects of p38 and ERK1/2 on BMP9-induced osteogenic differentiation of MPCs. Smad1, 5, and 8 (Smad1/5/8) are classic mediators for BMPs osteoinductive signaling. It has been reported that candidate phosphorylated sites for MAPKs are existed in Smad1/5/8 proteins [57]. Moreover, MAPKs activation by BMPs has shown to influence Smads signaling in different cell models [9], [10], [26], [39]. Fig. 1A has confirmed that BMP9 was able to simultaneously induce activation of Smad1/5/8, p38 and ERK1/2. Therefore, we asked whether BMP9-activated Smads signaling was also changed in the presence of SB203580 or PD98059, respectively. Firstly, we examined the effects of SB203580 and PD98059 on phosphorylation of Smad1/5/8, the activated mediator of BMPs signaling. Upon BMP9 stimulation, phosphorylated Smad1/5/8 was increased and was effectively inhibited by SB203580. However, BMP9-induced phosphorylation of Smad1/5/8 was further enhanced by PD98059 (Fig. 5A). As BMP9-induced Smad1/5/8 phosphorylation was changed, we therefore postulated that the change of Smad1/5/8 phosphorylation ought to result in alternated nuclear translocation of these proteins. Using immunofluorescence stain, we found that SB203580 interestingly disrupted translocation of Smad1/5/8 to the nucleus. Conversely, PD98059 treatment enhanced BMP9-induced nuclear translocation of Smad1/5/8 (Fig. 5B). Lastly, we examined the ability of SB203580 and PD98059 on BMP9-induced transcriptional activity of Smad1/5/8. Using the BMPs responsive Smads reporter, p12xSBE-luc [22], [23], we found that SB203580 was able to neutralize BMP9-induced transcriptional activity of Smad1/5/8, however, PD98059 was capable of augmenting BMP9-induced Smad1/5/8 transcriptional activity (Fig. 5C). To sum up, inhibition of p38 and ERK1/2 activity led to opposing effects on BMP9-induced phosphorylation of Smad1/5/8, as well as its translocation to the nucleus, and subsequently transcriptional activity of Smad1/5/8. These above results imply us that p38 and ERK1/2 are likely to play opposing roles in regulating BMP9-induced osteogenic differentiation of MPCs partly through influence on Smads signaling cascade. 10.1371/journal.pone.0043383.g005Figure 5 Inhibition of p38 and ERK1/2 activity leads to opposing effects on BMP9-indcued classical Smads signaling in MPCs. (A) Western blotting analysis of SB203580 and PD98059 on BMP9-induced Smad1/5/8 phosphorylation. Subconfluent C3H10T1/2 cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 30 mins post BMP9-CM treatment, total amount and phosphorylated forms of Smad1/5/8 was analyzed by western blotting. (B) Immunofluorescence staining analysis of SB203580 and PD98059 on BMP9-induced nuclear translocation of Smad 1/5/8. Subconfluent C3H10T1/2 cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 2 hrs post BMP9-CM treatment, nuclear translocation of Smad 1/5/8 by Immunofluenrescence staining. Magnification, ×200 (C) Luciferase reporter analysis of SB203580 and PD98059 on BMP9-induced Smad1/5/8 transcriptional activity. C3H10T1/2 cells were transfected with p12xSBE-luc. Next, cells were pretreated with SB203580 (25 mM) or PD98059 (10 mM), and then treated with BMP9-CM. At 36 hrs, cells were lyzed for luciferase activity assay. Luciferase activity was normalized by total cellular protein concentrations among the samples. “**”, p<0.01 (vs. control groups). Gene Silence of p38 and ERK1/2 Leads to Opposing Effects on BMP9-induced Osteogenic Differentiation of MPCs Using specific inhibitors for p38 and ERK1/2 respectively, we found that p38 and ERK1/2 may exhibit opposing effects on BMP9-induced early and late osteogenic differentiation of MPCs through influence on classic Smads signaling cascade. To confirm that the effects of inhibitors were truly due to p38 and ERK1/2 inhibition and not a nonspecific drug effect, we employed adenoviruses expressing small interference RNA (siRNA) targeted p38 and ERK1/2 to infect C3H10T1/2 cells. Western blotting was carried out to assess the inhibitory efficiency of these siRNA on expressions of p38 and ERK1/2, respectively (Fig. 6A). Then, the effects of p38 and ERK1/2 knockdown on BMP9-induced osteogenic differentiation of MPCs were assessed. Similar to the results observed with SB203580, p38 knockdown was shown to inhibit BMP9-indcued ALP activity, as well as matrix mineralization and OC protein expression. In contrast, consistent with the data obtained from PD98059, ERK1/2 knockdown was found to enhance BMP9-induced ALP activity, matrix mineralization and OC protein expression (Fig. 6B, Fig. 6C, Fig. 6D). These results suggest that gene silence of p38 and ERK1/2 lead to opposing effects on BMP9-induced osteogenic differentiation of MPCs. 10.1371/journal.pone.0043383.g006Figure 6 Knockdown of p38 and ERK1/2 leads to opposing effects on BMP9-indcued osteogenic differentiation of MPCs. (A) Effective knockdown of p38 and ERK1/2 expression by RNAi. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2. At 24 hrs, total amount forms of p38 and ERK1/2 were analyzed by western blotting. NC, negative control. (B) Effect of p38 and ERK1/2 knockdown on BMP9-induced ALP activity. Subconfluent C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. ALP activity was quantitatively assessed at the indicated time points. “*”, p<0.05 (vs. control groups). (C) Effect of p38 and ERK1/2 knockdown on BMP9-induced matrix mineralization. C3H10T1/2 cells were Infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At 21 days, cells were subjected to Alizarin Red S staining. Magnification, ×200. (D) Effect of p38 and ERK1/2 knockdown on BMP9-induced OC protein expression. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At day 12, cells were fixed and subjected to Immunocytochemical stain. Magnification, ×200. (E) Effect of p38 and ERK1/2 knockdown on BMP9-induced Smad1/5/8 phosphorylation. C3H10T1/2 cells were infected with AdR-si-p38 or AdR-si-ERK1/2, and then treated with BMP9-CM. At 30 min post BMP9-CM treatment, total amount and phosphorylated forms of Smad1/5/8 was analyzed by western blotting. Gene Silence of p38 and ERK1/2 Results in Opposing Effects on BMP9-induced Activation of Smads Pathway We next sought to determine the influences of p38 and ERK1/2 knockdown on BMP9-induced Smad1/5/8 activity. Interestingly, BMP9-induced phosphorylation of Smad1/5/8 were accordingly decreased along with p38 RNAi, and yet increased along with ERK1/2 RNAi (Fig. 6E). Together, these results further confirm that p38 and ERK1/2 are likely to influence BMP9-induced Smads signaling cascade in a converse manner, through which to exert opposing effects on BMP9-induced osteogenic differentiation of MPCs. Opposing Effects of p38 and ERK1/2 on BMP9-induced New Bone Formation in Calvarial Organ Culture The above in vitro data demonstrate that p38 and ERK1/2 may act in opposition to regulate osteoinductive activity of BMP9. To further investigate the regulatory roles of p38 and ERK1/2 on BMP9-induced bone formation, we conducted the calvarial organ culture experiments. Using calvariae of 4 days mouse pups, we found that treatment of BMP9 significantly stimulates new bone formation (in H&E staining, appeared as lighter color) over 7 days period [Fig. 7A and Fig. 7B]. It is noteworthy that inhibition of p38 activity by SB203580 led to a decrease in new bone formation compared with the BMP9 group, however, PD98059 treatment resulted in an increase in new bone formation (Fig. 7A and Fig. 7B). These results obtained from organ culture experiments suggest that p38 and ERK1/2 may act opposition to regulate BMP9-evokeed new bone formation. 10.1371/journal.pone.0043383.g007Figure 7 Opposing effects of p38 and ERK1/2 on BMP9-induced new bone formation in calvarial organ culture. (A) Mouse calvariae was infected with Ad-BMP9, and then treated with SB203580 (25 µM) and PD98059 (10 µM). At 4 days post infection, calvariae was placed into fresh media and return to incubator for another 3 day until day 7. The amount of original bone remaining and new bone was assessed by H&E staining. Magnification, ×600. (B) measurement of new bone width using Image Pro Plus. “*”, p<0.05 (vs. BMP9). Gene Silence of p38 and ERK1/2 Results in Opposing Effects on BMP9-induced Ectopic Bone Formation in Subcutaneous MPCs Implantation in vivo Lastly, we sought to confirm these above findings in vivo via MPCs implantation experiments. C3H10T1/2 cells were shown to be effectively co-infected with Ad-BMP9 and/or Ad-RFP, AdR-si-p38, AdR-si-ERK1/2 (Fig. 8A). The infected cells were collected and injected subcutaneously into athymic mice. At 5 weeks, the animals were euthanized, and the bony masses were retrieved (Fig. 8B). It seems that p38 knockdown did not affect the BMP9-transduced cells formed bony masses (Fig. 8C). However, ERK1/2 knockdown increased BMP9-transduced cells formed bony masses, which were noticeably bigger than those formed by the cells transduced by control groups (Fig. 8C). On histological evaluation, p38 gene silence inhibited BMP9-induced osteogenic differentiation and osteoblast maturation of C3H10T1/2 cells in vivo, with thinner trabeculae, more chondrocytes and a significant number of undifferentiated MPCs in H&E staining (Fig. 8D). Trichrome stain indicated that, while the BMP9 control samples exhibit abundantly fully ossified matrix, samples retrieved from p38 knockdown group display only focal ossification and a significant amount of cartilage matrix, implying immature osteogenesis (Fig. 8D). The presence of cartilage matrix in the samples co-infected with Ad-BMP9 and AdR-si-p38 was further confirmed by Alcian Blue stain (Fig. 8D). However, although ERK1/2 knockdown did not seem to affect BMP9-induced bone maturation, it was able to increase the quantity of bone formation, with more chicker trabeculae in H&E stain (Fig. 8D). Thus, in agreement with our in vitro studies, these in vivo results further substantiate the findings about the opposing roles of p38 and ERK1/2 in regulating BMP9-induced osteogenic differentiation of MPCs. 10.1371/journal.pone.0043383.g008Figure 8 Knockdown of p38 and ERK1/2 leads to opposing effects on BMP9-indcued ectopic bone formation. (A) Co-infection efficiency of Ad-BMP9 and AdR-si-p38 (or AdR-si-ERK1/2). C3H10T1/2 cells were co-infected with Ad-BMP9 and AdR-si-p38 (or AdR-si-ERK1/2). At 24 hrs post co-infection, the co-infection efficiency was determined under a fluorescence microscope. (B) Macrographic images of ectopic bone mass. (C) Total volume of retrieved samples. “**”, p<0.01 (vs. control groups) (D) Histological stains of retrieved samples. Retrieved tissues were decalcified, fixed in 10% formalin overnight, and embedded in paraffin. Serial sections of the embedded specimens were stained with H&E, Masson’s Trichrome stain and Alcian Blue stain. BM, Bone Matrix; Ch, Chondrocyte; UMPCs, Undifferentiated mesenchymal progenitor cells; CM, Cartilage Matrix. Magnification, ×150. Discussion BMP9 (also known as growth differentiation factor 2, or GDF2) was originally isolated from fetal mouse liver cDNA libraries and is a potent stimulant of hepatocyte proliferation [58]. Other roles of BMP9 include inducing the cholinergic phenotype of embryonic basal forebrain cholinergic neurons [59], regulating glucose and lipid metabolism in liver [60], and maintaining homeostasis of iron metabolism [61]. BMP9 is also a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures [62]. In previous studies, BMP9 has been proved to be most highly capable of inducing osteogenic differentiation of MPCs [11], [19], [20], [21]. Yet BMP9 remains as one of the least studied BMPs, and little is known about detail molecular mechanism underlying the BMP9-induced osteogenic differentiation of MPCs. Therefore, we are particularly interested in illuminating downstream signaling pathway(s) involved in BMP9 osteoinductive activity. In this report, we investigate the detail roles of p38 and ERK1/2 MAPKs in BMP9-induced osteogenic differentiation of MPCs. We find that BMP9 simultaneously stimulates phosphorylation/activation of p38 and ERK1/2 in the osteogenic differentiation process of MPCs. BMP9-induced early and late osteogenic differentiation is decreased by p38 inhibitor SB203580, yet enhanced by ERK1/2 inhibitor PD98059. SB203580 is shown to inhibit BMP9-induced Runx2 activation, and to disrupt BMP9-activated Smads signaling. On the contrary, PD98059 treatment promotes BMP9-induced Runx2 activation and enhances BMP9-evokeed Smads signaling. The effects of inhibitors were reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. We find that p38 and ERK1/2 act in opposition to regulate BMP9-induced new bone formation of cultured mouse calvarial organ. MPCs implantation studies also reveal that knockdown of p38 significantly decreases trabecular bone and osteoid matrix formation. However, ERK1/2 gene silence led to robust of trabecular bone. Together, our results strongly suggest that p38 and ERK1/2 are likely to play opposing regulatory roles in BMP9-induced osteogenic differentiation of MPCs partly through affecting Smads signaling cascade. The physiological functions of MAPKs (mainly p38, ERK1/2 and JNKs) in osteogenic differentiation and bone formation have been deeply investigated both in vitro and in vivo [41], [42], [63], [64], [65], [66], [67], [68], [69], [70]. However, the obtained results from in vitro studies are controversial, with some studies suggesting a stimulatory role of MAPKs in osteogenic differentiation and bone formation [63], [64], [65], [66], and others proposing that MAPKs is inhibitory [41], [42], [67], [68]. The conflicting results of in vitro studies emphasize the need to explore the role of the MAPKs in osteogenic differentiation and bone formation by in vivo assay. Ge et al., showed that ERK1/2 activation was capable of stimulating osteoblast differentiation and fetal skeletal development through a pathway involving Runx2 in mice [69]. Using a transgenic mice model, Greenblatt et al., evidenced that p38 functions to phosphorylate osteogenic master gene Runx2 and promotes skeletogenesis and bone homeostasis [70]. These in vivo studies affirmatively supported that p38 and ERK1/2 MAPKs are play positive roles in regulating osteogenic differentiation and bone formation. Although in vitro and in vivo studies about the precise roles of MAPKs in skeletal development didn’t lead to complete unanimity, it is well established that MAPKs plays a functional role in osteogenesis and bone metabolism. Several growth factors have shown to trigger MAPKs in different cell models [27], [28], [29], [30], [37], [71], [72], [73], [74]. IGF-1 promoted phosphorylation of p38, ERK1/2 and JNKs, through which to induce expression of osterix in human mesenchymal stem cells [37]. In MC3T3-E1 preosteoblastic cells, FGF-2 stimulated Runx2 phosphorylation and osteocalcin expression via activation of MAPKs [71]. Also, In PA-JEB or PA-JEB/β4 keratinocytes, p38 and ERK1/2 phosphorylation was increased after EGF stimulation during mitotic cell rounding [72]. HGF promoted activation of ERK1/2 and p38, and subsequently induced proliferation of lung adenocarcinoma cell line H441 [73]. In addition, MAPKs can also be activated by BMPs stimulation [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], which represents an important mechanism for non-Smads pathway(s) of BMPs signaling. The activation of MAPKs by BMPs could lead to various effects depending on the cell context. For example, BMP4 induced hepatocellular differentiation of rat hepatic progenitor cell was mediated by increase in activation of ERK1/2 [27]. BMP2-activated p38 was found to participate in BMP2-induced commitment of C3H10T1/2 MPCs to the adipocyte lineage [28]. BMP2 increased ALP activity and matrix mineralization through phosphorylation of p38α in human dental pulp cells (HDPCs) [29]. Moreover, BMPs-induced activation of MAPKs was also found to be essential for other physiological and pathological process, such as chondrogenesis, retina regeneration, tumor angiogenesis and cancer cells metastasis [30], [31], [32], [33]. Although BMPs could regulate tooth, kidney, skin, hair, muscle, haematopoietic and neuronal development, and maintain the iron metabolism and vascular homeostasis, the most intensive characterized function of BMPs is to induce osteogenic differentiation and bone generation. It has been described by various studies that MAPKs can activated by BMPs [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]. However, the exact effects of MAPKs on BMPs-induced osteogenesis are diverse and disputable, depending on the extracellular stimuli, the type of MAPKs and context of specific cells. By now, the studies about BMPs-activated MAPKs on BMPs-induced osteogensis were mainly focus on BMP2, and theses studies did not reach on consensus. For example, BMP2 inducement increased ERK1/2 activity in C3H10T1/2 MPCs, whereas nonfunctional ERK1/2 partially eliminated BMP2-induced ALP activity [36]. Celil el al., reported that BMP2 mediates osterix expression via activation of p38 and JNKs in human mesenchymal stem cells [37]. A study by Guicheux el al., also showed that p38 and JNKs were activated in response to BMP2 treatment in MC3T3-E1 preosteoblastic cells and primary cultured osteoblastic cells, and evidenced that these MAPKs have positive roles in regulating BMP2-induced osteogenic differentiation [38]. A study using C2C12 cells indicated that BMP2 activates ERK1/2 and p38 to promoting osteoblastic differentiation [39]. These above studies agree on the notion that MAPKs (p38, ERK1/2 and JNKs) play positive roles in BMP2-induced osteogenic differentiation. However, other studies using C2C12 and MC3T3-E1 cells obtain opposite results, showing that MAPKs (p38 and JNKs) have a negative role in BMP2-indcued osteogenic differentiation [40], [41], [42]. All these experiments did not ascertain the exact roles of MAPKs in BMPs-induced osteogenesis because of the controversial results. However, it is convincingly supported that MAPKs play essential roles in regulating osteogenic activity of BMPs, with positive or negative effects. In the current study, we examine the ability of BMP9 to activate p38 and ERK1/2 MAPKs, and the contribution of each MAPKs to regulate BMP9-induced osteogenic differentiation of MPCs. BMP9 was able to activate ERK1/2 and p38 MAPKs in MPCs. Importantly, inhibition of p38 and ERK1/2 activity led to opposing effects on BMP9-induced osteogenic differentiation of MPCs both in vitro and in vivo. Therefore, we conclude that p38 and ERK1/2 exert opposing influences on BMP9-induce osteogenic differentiation of MPCs. The notion that ERK1/2 and p38 act in opposition has been reported in various studies. FGF2 was able to simultaneously increase phsphorylation of ERK1/2 and p38, blocking of p38 activity promotes process extension whereas inhibition of ERK1/2 activity leads to reduce of process extension [74]. Oncogenic transformation by H-Ras was found to involve down-regulation of tropomyosin, which in turn depended on the simultaneously activation of ERK1/2 and inactivation of p38 MAP kinase [75]. Dipyrithione (2, 2′-dithiobispyridine-1, 1′-dioxide, PTS2), a pyrithione derivate, was shown to activate p38 and ERK1/2, suppression of p38 by SB203580 reduced the PTS2-induced apoptosis of the HeLa cells, whereas inhibition of ERK1/2 with PD98059 increased apoptosis [76]. P38 and ERK1/2 have also been reported to mediate BMP4-induced osteogensis of muscle-derived stem cells in an opposing manner [77]. Thus, the balance of ERK1/2 and p38 activities may be a key regulatory signal for many biological and pathophysiological responses, including BMP9-induced osteogenic differentiation of MPCs. In conclusion, we find that BMP9 are shown to activate p38 and ERK1/2 in the induction of the osteogenic differentiation of MPCs, implying that p38 and ERK1/2 are essential regulatory components in BMP9 osteoinductive activity. Notably, using specific inhibitor and siRNA for p38 and ERK1/2 respectively, we find that p38 and ERK1/2 may act opposition to regulate BMP9-indcued early and late osteogenic differentiation of MPCs in vitro. Calvarial organ culture and subcutaneous MPCs implantation studies confirm the opposing effects of p38 and ERK1/2 on BMP9-indcued osteogenesis and bone formation in vivo. Furthermore, we explore the possible molecular mechanism involved, and find that p38 and ERK1/2 are likely to fulfill opposing regulatory effects on BMP9-indcued osteogenic differentiation of MPCs via exert influence on Smads signaling cascades. This knowledge will provide insights into the molecular mechanisms by which BMP9 induces osteogenic differentiation of MPCs. 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PLoS One. 2012 Aug 17; 7(8):e43383
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22912826PONE-D-12-0370110.1371/journal.pone.0043204Research ArticleBiologyBiochemistryNucleic acidsRNARNA interferenceBiophysicsNucleic acidsRNARNA interferenceGeneticsGene expressionRNA interferenceMolecular cell biologyGene expressionRNA interferenceMedicineGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BGastrointestinal CancersOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaBasic Cancer ResearchPhysicsBiophysicsNucleic acidsRNARNA interferenceThe Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation MiR-338-3p on HBx Deletion Mutants (HBx-d382)Fu Xiaoyu 1 Tan Deming 1 * Hou Zhouhua 1 Hu Zhiliang 2 Liu Guozhen 1 Ouyang Yi 1 Liu Fei 1 1 Department of Infectious Disease, Xiangya Hospital, Central South University, Changsha, Hunan, China 2 The Second Hospital of Nanjing, Nanjing, Jiangsu, China Bartosch Birke Editor Inserm, U1052, UMR 5286, France * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: XF DT Z. Hou. Performed the experiments: XF Z. Hou Z. Hu YO FL. Analyzed the data: XF DT GL. Wrote the paper: XF DT GL. 2012 17 8 2012 7 8 e432042 2 2012 18 7 2012 © 2012 Fu et al2012Fu 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.Objective Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation. Methods We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony. Results HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition. Conclusion miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx. This project was supported by grants from the National Natural Science Foundationof China (No 30872228). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Among the four open reading frames (ORFs) in the genome of hepatitis B virus (HBV), the HBV X gene (HBx) correlates the most to liver cancer development like hepatocellular carcinoma (HCC). The HBx protein is a multifunctional regulator that is essential for viral replication and plays an important role in regulating gene transcription, participating in cell signaling, and controlling cell proliferation and apoptosis [1]–[2]. However, there is controversy surrounding the direct causal effect of HBx on HCC development [3]–[5]. The integration of the HBx gene into the host genome in hepatocarcinoma tissues, and the gene mutants in HBx that arise due to this integration process, have been reported in many studies. The study conducted by Minenura M et al. [6] revealed a correlative relationship between HBx gene point mutations (at codon 130 [AAG → ATG] and 131 [GTC → ATC]) and liver cancer. Another report by Yeh et al. [7] found that HBx-A31 (containing a mutation at codon 31) was detected more frequently in patients with HCC. Constructs based on either naturally occurring or artificially designed carboxyl-terminal HBx gene deletion mutants were successfully cloned and encoded a carboxy-terminal truncated HBx protein. These HBx mutants have very different functions than wild-type HBx protein [8]–[9], since the mutants exhibited the ability to promote cell proliferation and to reduce the response to apoptotic stimuli [9]–[10] that play important roles in HCC development. However, the mechanism underlying HBx deletion mutant-induced malignant transformation of liver cells is still unclear. MicroRNAs (miRNAs) are a class of endogenous, noncoding small RNAs that regulate gene expression at the post-transcriptional level through binding the 3′-UTR region of the target gene mRNA. Several recent studies have shown that dysregulation of miRNA expression is associated with a variety of tumors, and that these miRNAs may play a tumor suppressor or oncogenic role [11]–[12]. Important to our present study, miRNAs have been reported to be closely related to the incidence and development of liver cancer [13]–[15]. However, only a few studies have evaluated the specific roles of either the HBx deletion mutants or miRNAs, or their possible interaction, in the incidence and development of HBV-related liver cancer. Our previous studies found that liver cancer tissues from chronically HBV-infected human patients contained an elevated incidence of a particular HBx deletion mutation, HBx-d382 (named because of a deletion mutation between nt 382–400) [16]. We speculated that this relationship was related to the higher incidence of HBx mutations that occur following HBV DNA integration, which often happens during chronic HBV infection that also correlates with the development of hepatocarcinoma. Upon further investigation by miRNA microarray analysis, we found that stable expression of HBX or HBx-d382 in LO2 cells decreased miRNA expression, including miR-338-3p, as compared to control LO2/pcDNA cells; interestingly, the LO2/HBx-d382 cells exhibited this phenotype in a more pronounced way than LO2/HBx cells. Additional analysis by real-time PCR confirmed this result (unpublished data). Furthermore, the miRNA target prediction based on bioinformatic analysis showed that CyclinD1 is a likely miR-338-3p target. To better understand the influence of the down-regulated miR-338-3p expression in cells expressing the HBx-d382 mutant and to determine whether there are any changes in miR-338-3p-mediated CyclinD1 expression, we used the stably transfected LO2/HBx and LO2/HBx-d382 (also used in our previously published experiments) as our experimental model. The results of our study suggest that the miR-338-3p-mediated CyclinD1 expression changes downstream of HBx mutant gene function may help to understand the mechanisms underlying the HBx-d382 mutant-induced hepatocarcinogenesis from a new perspective. Materials and Methods Cells Culture and Establishment of the Stably Transfected Cell Lines All cells were cultured in RPMI-1640 medium (GIBCO, USA) containing 10% fetal bovine serum (FBS) (GIBCO, USA) and 100 units/mL penicillin plus 100 µg/mL streptomycin at 37°C with 5% CO2. The control plasmid pcDNA3.0, recombinant plasmid pcDNA3.0/HBx-d382 (a mutant of the HBx gene with deletion from 382–400 bp), and pcDNA/HBx were previously established in our lab [16]. The HBx genetic fragments used here originate from the liver cell line HepG2.215. We established stably engineered LO2 cells transfected with the wild type HBx and HBx-d382 mutant plasmids; the hepatocyte cell line LO2 (obtained from Chinese Academy of Science, Cell Biology of Shanghai Institute) was transfected with LipofectamineTM2000 (Invitrogen, California, USA) according to the manufacturer’s instructions and was selected with G418 (Geneticin, GIBCO, USA). Empty pcDNA3.0 vector plasmid was used as a control. The stable transfection of pcDNA3.0/HBx-d382 (termed LO2/HBx-d382), pcDNA/HBx (termed LO2/HBx), or the empty vector (termed LO2/pcDNA3.0) was confirmed by RT-PCR for gene expression and western blotting for protein expression. 10.1371/journal.pone.0043204.g001Figure 1 HBx-d382 enhances the non-anchored growing ability of transfected hepatocytes. (A–B) The identification of stable HBx transfection in LO2 cells. (A) The HBx gene was identified by RT-PCR. (B) Western blotting showed the expression of HBx in LO2 cells. 1: LO2; 2: LO2/pcDNA3.0; 3: LO2/HBx-d382; 4: LO2/HBx; M: marker. (C) Soft agar colony formation assay of transfected LO2 cells. The rate of colony formation in HBx-expressing cells was significantly higher than in control LO2 and LO2/pcDNA3.0 cells (*P<0.01). While within the groups of LO2/HBx-d382 LO2/HBx, the former was higher than the latter (#P<0.01). Cell Transfection HBx-expressing cells were transfected with miRNA mimics or miRNA inhibitor (GeneCopoeia, USA) using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM, according to manufacturer’s instructions. The normal recommended controls for the miRNA-mimics and inhibitor (GeneCopoeia, USA) were used in the experiments. Changes in gene expression were detected 48 hr after transfection. miR-338-3p miRNA target verification was performed in LO2/HBx cells, which were co-transfected with miR-338-3p mimics and the dual luciferase reporter plasmid. miRNA-NC miRNA was used as the control for miR-338-3p. Inhibition of HBx Expression Small interfering RNA (siRNA) sequences specifically targeting HBx were selected accordingly [17] and synthesised by GenePharma (Shanghai, China). Approximately 50 nM HBx siRNA or control siRNA was transfected into LO2/HBx and LO2/HBx-d382 cells by Lipofectamine as described above. 10.1371/journal.pone.0043204.g002Figure 2 HBx, especially HBx-d382, enhances CyclinD1 expression. (A) CyclinD1 protein level of the engineered LO2 cells. (B) The histogram of CyclinD1 mRNA and protein level in LO2/HBx and LO2/HBx-d382 cells, compared with LO2/pcDNA3.0 cells (*P<0.001,**P = 0.017,***P = 0.029). RT-PCR Analysis Total RNA from the engineered LO2 cells was extracted by Trizol (Invitrogen, USA). The PCR primers used for HBx detection were as follows: for HBx, 5′-AAGGTACCATGGCTGCTAGGCTGTGCT-3′ (forward) and 5′-CTGGGCCCTTAGGCAGAGGTGAAAAAGTTG-3′ (reverse), leading to a 462 bp amplified product; for the β-actin control, 5′-CTCCATCCTGGCCTCGCTGT-3′ (forward) and 5′-GCTGTCACCTTCACCGTTCC-3′ (fragment), leading to a 242 bp amplified product. Soft Agar Colony Formation Assay The assay was conducted according to previously published methods [18], with slight modifications. Briefly, 5×103 transfected LO2 cells were first thoroughly mixed with 2 mL RPMI Medium 1640 containing 3 g/L agar and 10% FBS. This mixture was then added onto solid agar (RPMI Medium 1640 medium containing 5 g/L agar and 10% FBS) in a 6-well plate and incubated for 2 weeks. Finally, the derived clones from each group within a randomly selected area were selected and counted under a microscope at 50× magnification. Quantitative Real-time PCR (qRT-PCR) Analysis miR-338-3p expression in normal hepatocytes (LO2 and QSG7701 cells) and HBx-expressing cells after knocking down HBx expression was measured with SYBR qRT-PCR. CyclinD1 expression before and after miR-338-3p mimic or inhibitor introduction into HBx-expressing LO2 cells was measured with SYBR qRT-PCR. Total RNA was extracted with Trizol (Invitrogen) according to the manufacturer’s instructions. miR-388-3p cDNA was synthesized from 2 µg of total RNA with an All-in-one™ miRNA First-Strand cDNA Synthesis (GeneCopoeia) Kit using the supplied poly-A primer. Real-time PCR was performed in a 20 µL reaction mix including 2 µL of 5× diluted reverse transcription product, 2 µL miRNA specific primer, 10 µL SYBR 2× All-in-one qPCR Mix, 0.4 µL 50× ROX Reference dye, and 3.6 µL double distilled water. The cycling conditions for amplification on the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) were 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for 32 sec. The data were normalized against the U6 snRNA. CyclinD1 expression was analyzed with THUNDERBIRD SYBR qPCR Mix (ToYoBo, Japan). cDNA was synthesized with the RevertAid™ First Strand cDNA Synthesis Kit (MBI Fermentas, Canada) in a total volume of 20 µL. The primer sequences used were as follows: for CyclinD1, 5′-AGGAACAGAAGTGCGAGGAGG-3′ (forward) and 5′-GGATGGAGTTGTCGGTGTAGATG-3′ (reverse); for GAPDH, 5′-CGGATTTGGTCGTATTGGGC-3′ (forward) and 5′-CCTGGAAGATGGTGATGG. GATT-3′ (reverse). qRT-PCR was performed on an Applied Biosystems 7500 RT-PCR System. The cycling conditions for amplification were as follows: 95°C for 1 min, 40 cycles of 95°C for 15 sec, and 58°C for 35 sec. The data were normalized against GAPDH. Each sample was analyzed in triplicate, and the fluorescence signal was measured at each extension step. The relative expression was determined using the 2−ΔΔCT method, where the normalized CT (ΔCT) value was calculated by subtracting the CT of a control gene (U6 snRNA for miR-338-3p and GAPDH for CyclinD1) from the CT of the gene of interest. 10.1371/journal.pone.0043204.g003Figure 3 miR-338-3p inhibits cell proliferation, especially in LO2/HBx-d382 cells. (A) A representative cell cycle profile for miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells comparing to their control RNA transfection. (B) Average of G1 phase populations in HBx-expressed LO2 cells after transfected miR-338-3p mimics or inhibitor (*P<0.001). (C) Average of S phase populations after transfected miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells (*P<0.001). While miR-338-3p overexpresses or suppresses, miR-338-3p induce cell cycle arrest and anti-miR-338-3p increases cell growth, especially in LO2/HBx-d382 cells. Values in (B) and (C) are means ±SD of three separate experiments. 10.1371/journal.pone.0043204.g004Figure 4 miR-338-3p inhibits cell proliferation by Edu assay. (A–B) Representative profiles of Edu cell proliferation after transfection with miR-338-3p mimics or inhibitor in LO2/HBx-d382 and LO2/HBx cells compared to their negative control transfection (magnification 100×). (C) Rate of Edu positive cells in S phase. Gain of miR-338-3p inhibits the cellular DNA replication in HBx-expressed LO2 cells and control cells, whereas loss of miR-338-3p expression demonstrates an adverse result, especially in LO2/HBx-d382 cells. (*P<0.001; **P<0.01; ***P<0.05). 10.1371/journal.pone.0043204.g005Figure 5 miR-338-3p influence the colony formation of transfected LO2 cells. (A–B) Gain of miR-338-3p decreases the rate of colony formation of LO2/HBx-d382 and LO2/HBx cells, whereas loss of miR-338-3p enhances the non-anchored growing ability in HBx-expressing cells (*P<0.001, **P<0.01). (C) The non-anchored growing ability of control LO2/pcDNA3.0 cells are also inhibited by miR-338-3p, although the significant difference is lower than that in HBx-expressing cells (***P = 0.032,#P = 0.025). Western Blot Analysis HBx and CyclinD1 proteins were measured by western blot. Total protein was extracted from transfected cells using the RIPA lysis buffer (Beyotime, China) according to the manufacturer’s instructions. For western blot analysis, equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). Blots were blocked using with 5% skim milk, followed by incubation with antibodies specific for rabbit anti-HBx (Abcam, England, 1∶1500 dilution) or mouse anti-CyclinD1 (Santa Cruz, USA, 1∶100 dilution), and mouse anti-β-actin (Abcam, 1∶3000 dilution). Blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch, USA, 1∶5000 dilution) and visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences, USA). Gene Sequence Analysis and Primer Design Through in silico miRNA target prediction, we found two binding sites for miR-338-3p in the 3′ untranslated regions (3′-UTR) of CyclinD1. Two gene fragments corresponding to the two binding sites in CyclinD1-3′-UTR were cloned into a vector using the restriction enzymes XhoI and NotI. Using the NM_053056 (gene = “CCND1”) gene sequence, primers were designed to amplify binding site locations in the 3′-UTR region. Mutation primers mutating the specific binding sequence for miR-338-3p in CyclinD1 were also designed, named Mut (mutating both binding sites), Mut-1 (mutating nt 907–913), or Mut-2 (mutated nt 2397–2403). The PCR products were cloned into a pmirGLO Vector (Promega, Madison, WI, USA) that was designed to quantitatively evaluate miRNA binding and function by measuring luciferase activity after inserting miRNA target sites downstream or 3′ of the luciferase gene. In this vector, Renilla luciferase was used as the primary reporter, while firefly luciferase acts as a control reporter for normalization and selection. PCR products were 1573 bp in size. The primers used for cloning were as follows: Wt-Forward: 5′- CCGCTCGAGTC CTATTTTTGTAGTGACCTGTTTATG-3′, Wt-Reverse: 5′- GAATGCGGCCGCGC TA CGCCCCCGATCAGATGAAG-3′; Mutant-Forward: 5′-CCGCTCGAGTCC TAT TTTTGTAGTGACCTGTTTATGAGTTCCAGATTTTCTACCCAACGGCCC-3′, Mutant-Reverse: 5′-GAATGCGGCCGCGCTACGCCCCCGATCAGATGAAGT. GCTCTGGAACACAGGCGCAGGGAAGAGAAG-3′; Mutant1-Forward: 5′-CCGC TCGAGTCCTATTTTTGTAGTGACCTGTTTATGAGTTCCAGATTTTCTACCCAACGGCCC-3′, Mutant1-Reverse: 5′-GAATGCGGCCGCGCTACGCCCCCGATCAGAT GAAG-3′; Mutant2-Forward: 5′-CCGCTCGAGTCCTATTTTTGTAGTGACCTGT TTATG-3′, Mutant2-Reverse: 5′-GAATGCGGCCGCGCTACGCCCCCGATCAGATG AAGTGCTCTGGAACACAGGCGCAGGGAAGAGAAG-3′. 10.1371/journal.pone.0043204.g006Figure 6 CyclinD1 is a direct target of and is regulated by miR-338-3p, and the effect of miR-338-3p on CyclinD1 is mainly dependent on the CyclinD1-3′-UTR region (nt 2397–2403). (A–B) Successfully constructed plasmids of CyclinD1-3′-UTR. (A) Amplification of a DNA fragment containing CyclinD1-3′-UTR region. PCR amplification was performed using specific primers for the types of WT-, Mut-, Mut1-, and Mut2-Cyclin D1-3′-UTR, respectively, and genomic DNA isolated from the genome of LO2/HBx cells was used as PCR template. An approximately 1573 bp band PCR product was detected by agarose gel electrophoresis. (B) Identification of recombinant plasmids. The fragment digested with XhoI and NotI from the recombinant plasmids: pCyclinD1-3′-UTR-WT, pCyclinD1-3′-UTR-Mut, pCyclinD1-3′-UTR-Mut1, and pCyclinD1-3′-UTR-Mut2 were used as a template for confirmation PCR. The PCR product was about 1573 bp, which was finally confirmed by DNA sequencing. miR-338-3p targets CyclinD1. (C) Dual luciferase assay of LO2/HBx cells cotransfected with the Renilla luciferase constructs containing the CyclinD1 WT or Mut 3′-UTR and miR-338-3p mimics or negative RNA (*P = 0.404,**P<0.001). (D) The major site of CyclinD1 targeted by miR-338-3p was at position 2397–2403 nt. Effects of miR-338-3p and the negative control on the reporter constructs containing CyclinD1-3′-UTR Mut1 and Mut2 were determined 48 hours after transfection. Renilla luciferase values normalized to Firefly luciferase are presented. (**P<0.001,***P = 0.04). (E–G) CyclinD1 protein expression after transfection of miR-338-3p mimics, inhibitor, or negative control (*P<0.001,**P<0.01). The fold change before and after transfection is more pronounced in LO2/HBx-d382 cells than that in LO2/HBx and control cells. Data are shown as mean ±SD from at least 3 independent experiments. (H) qRT-PCR with the 2−ΔΔCT method to evaluate the CyclinD1 mRNA expression normalized to GAPDH in LO2/HBx-d382, LO2/HBx, and control cells transfected with miR-338-3p mimics or inhibitor or their respective controls(★P = 0.164,★★ P = 0.438, #P = 0.220,##P = 0.101,* P = 0.254,** P = 0.417). Amplification and Identification of CyclinD1-3′-UTR Constructs Amplification of wild-type and mutant plasmids was carried out using the same conditions. Briefly, 6 µL of 5× PrimeSTAR buffer (TaKaRa, Japan), 2 µL of 2.5 mM dNTPmix, 1 µL each of upstream and downstream primer (10 mM), 0.3 µL of PrimeSTAR HS DNA polymerase (2.5 U/µL), 1 µL of DNA template, and sterile water up to a total of 30 µL per reaction were mixed together. Touchdown PCR was used for amplification as follows: hot-start at 95°C for 5 min, followed by 10 cycles of denaturation at 98°C for 10 sec, annealing starting from 65°C and decreasing 1°C in each cycle, and extension at 72°C for 1 min 45 sec; for the remaining 18 cycles, the annealing temperature was kept at 55°C. A final extension step at 72°C for 7 min followed by decreasing the temperature to 4°C completed the PCR reaction. To verify the PCR products, 2 µL of DNA sample was loaded on 1% agarose for gel electrophoresis. PCR products were retrieved from the gel and double digested with XhoI and NotI. Meanwhile, double digestion of pmirGLO vectors with XhoI and NotI had been performed. The digested fragments from the vector and PCR products were purified and used to clone recombinant plasmids, including wt/Mut-CyclinD1-3′-UTR, CyclinD1-3′UTR-Mut1, and CyclinD1-3′UTR-Mut2. To identify the recombinant plasmids, the inserts were double digested with XhoI and NotI. Identification of miR-338-3p Target by Dual-luciferase Reporter Assay The reporter assay was performed according to Promega's instructions. Briefly, growth medium was removed from cultured cells, fresh medium (100 µL/well) was added, and luciferase substrate (100 µL/well) was prepared. The plate was gently rocked at room temperature for 10 min, and firefly luciferase activity (hLuc) was then measured using a microplate luminometer (Veritas™ microplate fluorescence reader; YuanPingHao Bio, Beijing, China). Next, 100 µL of Stop & Glo reagent was dispensed, incubated for 10 min by gentle rocking, and Renilla luciferase activity (hRluc) was measured. The luciferase counts were then normalized to hLuc counts to obtain final reporter activity. Each sample was measured in triplicate. EdU Assay Cell proliferation was measured by 5-ethynyl-2′-deoxyuridine (EdU) assay using an EdU assay kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, LO2/HBx-d382 and LO2/HBx cells were cultured in triplicate at 5×103 cells per well in 96-well plates and were transfected with 50 nM of miR-338-3p mimics, miR-338-3p inhibitor, or their respective control RNA for 48 h. The cells were then exposed to 50 µM of EdU for additional 4 h at 37°C. The cells were then fixed with 4% formaldehyde for 15 min at room temperature and treated with 0.5% Triton X-100 for 20 min at room temperature for permeabilization. After 3× washes with PBS, the cells were treated with 100 µL of 1× ApolloR reaction cocktail for 30 min. Subsequently, the DNA contents of each well of cells were stained with 100 µL of Hoechst 33342 (5 µg/mL) for 30 min and visualized under a fluorescent microscope (Olympus, Japan). Cell Cycle Assay Cell cycle analysis was determined by flow cytometry (BD, UA). Briefly, LO2/HBx-d382 and LO2/HBx cells at 1×106 cells per well were cultured in 6-well plates and transfected with 50 nM of miR-338-3p mimics, miR-338-3p inhibitor, or their respective control RNA for 48 h. The cells were then harvested and fixed in 70% ice-cold ethanol for 24 h, followed by propidium iodide (PI) staining. The different cell cycle phases were analyzed using a FACS Calibur instrument. Statistical Analysis The data from soft agar colony formation and CyclinD1 expression in the engineered LO2 cells was performed with Fisher’s Least Significant Difference (LSD)-t test. The data from miR-338-3p expression in normal hepatocytes was performed with Fisher’s Least Significant Difference (LSD)-t test. All the other data was analyzed by unpaired two-tailed t-test. All data was expressed as means and standard deviation from at least 3 independent experiments. All p values were obtained with SPSS 16.0 software package, and a p<0.05 was considered statistically significant. Results HBx-d382 Enhances the Non-anchored Growing Ability of Transfected Hepatocytes and Promotes CyclinD1 Expression There is an established correlation between the presence of HBx deletion mutations in liver cells and hepatocarcinogenesis. To study whether and how the HBx deletion mutation affected the growth and proliferation of hepatocytes, we observed the isolated effect of the HBx-d382 HBx deletion mutant, found previously in our studies to be prevalent in HCC patient tumors, on hepatocyte cell proliferation as compared to the wild-type HBx gene. To do this, we established a non-tumorigenic human hepatocyte cell line, LO2, expressing the mutated HBx-d382 gene as well as a control LO2 cell line expressing the wild-type HBx. We first used RT-PCR to identify the HBx gene in the cDNA of the engineered LO2 cells. β-actin was used as a loading control (Fig. 1A). The data showed that the HBx gene had been successfully introduced into the host genome in LO2 cells. Western blotting showed that HBx protein expression could be detected in LO2 cells (Fig. 1B). These data suggest that stably HBx-transfected LO2 cells were successfully established. We used these engineered cells to test whether HBx-d382 had an effect on cell proliferation. Soft agar colony formation analysis revealed that the rate of colony formation in LO2/HBx-d382 and LO2/HBx groups was significantly higher than that of the control LO2 group and LO2/pcDNA3.0 groups after culturing for 2 weeks (p<0.01); there was no significant difference in colony formation between the control LO2 and LO2/pcDNA3.0 groups (Fig. 1C). Within the LO2/HBx-d382 and LO2/HBx groups, the former was higher than the latter (p<0.01). This result indicated that HBx and HBx-d382 led to cell cycle dysregulation, with the HBx-d382 mutant leading to the most pronounced proliferation effect. As the cyclin proteins are known to regulate cell proliferation and cell cycle, we tested whether CyclinD1 was differentially expressed in the engineered LO2 cells. PCR and western blot results showed that CyclinD1 mRNA and protein levels, respectively, were up-regulated in cells transfected with HBx and HBx-d382, when compared with those transfected with pcDNA3.0 (Fig. 2); consistent with the data in Figure 1, the effect of HBx-d382 on CyclinD1 expression was more prominent than wild-type HBx. This result indicated that HBx and the HBx-d382 deletion mutant increased CyclinD1 expression in hepatocytes. Relative Expression of miR-338-3p in Normal Hepatocytes is Higher than that in HBx-expressing Cells Our unpublished studies showed that miR-338-3p was down-regulated in HBx-expressing LO2 cells compared with control LO2/pcDNA3.0 cells by microarray and real-time PCR; however, whether miR-338-3p expression was altered in normal hepatocytes compared to HBx-expressing cells was unknown. To address this, real-time PCR evaluation using the 2−ΔΔCT method showed that the relative expression of miR-338-3p in normal hepatocytes is higher than that in HBx-expressing cells (Fig. S1). Moreover, according to the results from Figure 2, we conclude that CyclinD1 expression inversely correlates with miR-338-3p expression in HBx-expressing LO2 cells. miR-338-3p Inhibits Cell Proliferation, Especially in LO2/HBx-d382 Cells We next wanted to determine whether the HBx-regulated miRNA miR-338-3p that we identified in our miRNA microarray could regulate the HBx-mediated increased proliferation we observed in Figure 1. To examine the functional role of miR-338-3p, we altered cellular miR-338-3p expression by transfecting the HBx- and HBx-d382–expressing LO2 cells with miR-338-3p mimics or an miR-338-3p inhibitor compared with LO2/pcDNA3.0 cells. Cell cycle and EdU incorporation assays were used to determine cell proliferation. Compared to their respective negative controls (NC), the HBx-expressing LO2 cells transfected with the miR-338-3p mimics displayed significantly higher frequency of cells at the G1 phase and a lower frequency of cells at S phase (Fig. 3A–C, Fig. S2, p<0.001). In contrast, cells transfected with miR-338-3p inhibitor showed a significant decrease of cells at G1 phase and an increase of cells at S phase (Fig. 3A–C, Fig. S2, p<0.001). This indicated that miR-338-3p inhibited cell cycle progression from the G1 phase to the S phase. Next, we used the EdU incorporation assay, which is a more sensitive and specific method [19]–[20], to evaluate the effects of miR-338-3p on cell proliferation. We found that the number of cells incorporating EdU in the miR-338-3p mimic-treated group was significantly reduced as compared to the negative control. In contrast, the number of cells incorporating EdU was significantly increased in the miR-338-3p inhibitor-treated group as compared with the negative control. Interestingly, this effect is especially significant in LO2/HBx-d382 cells (Fig. 4A–C). In addition to regulating cell cycle transition between the G1/S phase, this result indicated that miR-338-3p also inhibited cell proliferation, especially in LO2/HBx-d382 cells. Finally, the effects of miR-338-3p mimics/inhibitors on the colony-forming assay demonstrated that the miR-338-3p inhibited the non-anchored growing ability of transfected LO2 cells, especially in HBx-expressing cells (Fig. 5). CyclinD1 is a Direct Target of miR-338-3p, and the Effect of miR-338-3p on CyclinD1 is Mainly Dependent on the CyclinD1-3′-UTR Region (nt 2397–2403) As predicted by several in silico methods for target-gene prediction, including TargetScan [21], CyclinD1 was identified as one of the candidate genes regulated by miR-338-3p. Two putative binding sites, nt 907–913 and nt 2397–2403, are located in the CyclinD1-3′-UTR. To determine which putative binding site is used by miR-338-3p, or whether both are used, we mutated each of these putative sites alone (Mut1-CyclinD1-3′-UTR [mutated nt 907–913] and Mut2-CyclinD1-3′-UTR [mutated nt 2397–2403nt]) as well as both of these sites together (Mut-CyclinD1-3′-UTR) in the 3′-UTR region of the CyclinD1 gene. WT-CyclinD1-3′-UTR was used as a control. PCR amplification was performed using specific primers for the mutated binding sites. The amplified PCR products and genomic DNA isolated from the genome of LO2/HBx cells was used as a PCR template. As shown in Figure 6A, the different CyclinD1-3′-UTR gene-derived PCR products were found around the predicted 1573 bp band size. The plasmids were then extracted and subjected to a double digestion with XhoI and NotI enzymes that generated one product: a band at approximately 1573 bp (Fig. 6B) that corresponds to the CyclinD1-3′-UTR gene for all plasmids. Finally, the recombinant plasmid gene sequences were further verified by DNA sequencing. To validate whether miR-338-3p can directly regulate CyclinD1 through either or both of the putative binding sites in the 3′-UTR region of CyclinD1, the WT-, Mut-, Mut1-, and Mut2-CyclinD1-3′-UTR plasmids were cloned into the 3′-UTR of the Renilla luciferase gene and were co-transfected with miR-338-3p mimics or negative control RNA in LO2/HBx cells. The luciferase enzyme activity levels were then measured to determine the miR-338-3p effects on luciferase translation upon binding to the WT or mutated 3′-UTR regions from CyclinD1. Compared with the negative control group, Renilla luciferase activity was significantly decreased (i.e. the ratio of hRluc to hLuc deceased) in the group co-transfected with miR-338-3p and pCyclinD1-3′-UTR-WT (Fig. 6C, p<0.001), as predicted. However, luciferase activity in the group co-transfected with miR-338-3p and pCyclinD1-3′-UTR-Mut did not exhibit a significant difference from the NC group (p = 0.404), indicating that miR-338-3p could no longer bind. To determine if one or both of the binding sites were necessary for the functional effect of miR-338-3p, we compared the luciferase activity between pCyclinD1-3′-UTR-Mut1 and pCyclinD1-3′-UTR-Mut2 groups with the negative control group. The CyclinD1-3′-UTR-Mut1 maintained the significant difference (p<0.001) noted in the pCyclinD1-3′-UTR-WT in response to miR-338-3p as compared with the negative control group, indicating that miR-338-3p was independent of this binding site. In contrast, the luciferase activity in pCyclinD1-3′-UTR-Mut2 group was less affected by miR-338-3p (Fig. 6D, p = 0.04), indicating that this binding site is important for miR-338-3p binding and function. These data indicated that miR-338-3p has effect on both binding sites in CyclinD1-3′-UTR region, but that the main binding site is the second site (nt 2397–2403). miR-338-3p Represses CyclinD1 Protein Expression To directly test the validity of the putative target, HBx-expressing LO2 cells were transfected with miR-338-3p mimics or miR-338-3p inhibitor as well their respective negative controls. The mRNA and protein levels of CyclinD1 were measured by qRT-PCR and Western blotting, respectively. Relative to the control, miR-338-3p overexpression downregulated endogenous CyclinD1 protein, while miR-338-3p inhibition upregulated the CyclinD1 protein; consistent with our previous data, these changes in CyclinD1 are more prominent in the LO2/HBx-d382 cells (Fig. 6E–G). In contrast, no change of CyclinD1 mRNA level was noted (Fig. 6H), indicating post-transcriptional regulation of CyclinD1 by miR-338-3p. Changes in HBx Expression Affect miR-338-3p and CyclinD1 Expression in Transfected LO2 Cells Since the miR-338-3p and CyclinD1 expression was significantly changed in HBx-expressing LO2 cells, we wondered whether the reduction of HBx could also lead to differential miR-338-3p and CyclinD1 expression. To address this question, we transfected HBx siRNA or control RNA into LO2/HBx and LO2/HBx-d382 cells to inhibit HBx expression. We measured the miR-338-3p expression by qRT-PCR using the 2−ΔΔCT method 48 h after transfection and found that there was a significant increase of miR-338-3p expression after knocking-down HBx compared to the negative controls (Fig. S3), while the western blot showed that CyclinD1 protein levels were significantly down-regulated after HBx reduction (Fig. S3), Our findings revealed that high HBx expression up-regulated CyclinD1 and down-regulated miR-338-3p, while HBx reduction leads to down-regulated CyclinD1 and up-regulated miR-338-3p expression,indicating that HBx is necessary for cyclinD1 upregulation. Our data introduces a new mechanism suggesting that HBx up-regulated CyclinD1 through down-regulating miR-338-3p. Discussion Although the integration of HBV DNA into the host genome is not required for viral replication, it is a common phenomenon that HBV genes integrate with host genomic DNA in many liver tissues chronically infected with HBV. This promotes HBV persistent infection [22]–[23] by providing protection of the viral genome from immunological surveillance and, most importantly, promoting the development of genomic DNA instability that contributes to malignant transformation of liver cells [24]–[25]. In addition to effecting these changes on the infected host liver cells, the process of HBV DNA integration can induce mutations in the HBV DNA itself. Deletion mutations are frequently found in the integrated HBV DNA, especially in the X gene (including the HBx gene explored here), S gene, and enhancer regions, resulting in the expression of truncated proteins encoded by the mutated genes, such as the truncated HBx protein that can then lead to dysregulation of host cellular genes [9], [26]. For the HBx gene, deletions in the C-terminal region of the gene are common after HBV DNA integration. In the context of hepatocarcinogenesis, there is an intriguing correlation between the occurrence of this particular mutation and transformed liver cancer cells, as the level of C-terminal truncated HBx protein is higher in transformed tumor tissues as compared to distal non-tumor tissues in HBV-infected HCC patients [10], [26]. Supporting this finding, Iavarone et al. [9] found that a truncated HBx protein detected in HCC tissues derived from an HBx gene deletion that led to a reading frame shift mutation and the emergence of a new STOP codon. Currently, the mechanisms underlying how HBx induces hepatocarcinogenesis are not fully clear. Wang et al. [27] reported that HBx-mediated down-regulation of let-7a expression inhibited cell proliferation; this was the first study to report an interaction between HBx and miRNA. However, the role of miRNA in the relationship between the HBx deletion mutation and hepatocarcinogeness is largely unexplored. To better understand the mechanism underlying the effect of miRNAs on HBx deletion-mutation–related HCC, we performed the study described here, and our results provide new ideas for the diagnosis and treatment of liver cancer. Our previous studies found that the HBx-d382 mutant was detected in several HCC patients [16], and the ability of HBx-d382 to induce tumor formation was confirmed in a nude mice model [28]. miRNA microarray analysis and confirmatory real-time PCR data showed that HBx-d382 had a stronger ability to down-regulate miR-338-3p expression than wild-type HBx in transfected LO2 hepatocyte cells (unpublished data). To understand what genes targeted by miR-338-3p might function to promote HBx deletion-mutation related liver cancer, we used TargetScan to identify the potential miR-338-3p target gene and found that CyclinD1 was a likely candidate. Furthermore, HBx-d382 was able to promote proliferation and anchor-independent growth of LO2 cells as well as enhance CyclinD1 gene expression. Edu incorporation and cell cycle analysis by flow cytometry to measure cell proliferation demonstrated that ectopic expression of miR-338-3p mimics in both LO2/HBx and LO2/HBx-d382 cells inhibited cell proliferation, whereas repression of endogenous miR-338-3p expression enhanced cell proliferation. Furthermore, the forced expression or down-regulation of miR-338-3p reduced or enhanced, respectively, the protein expression of CyclinD1 in both LO2/HBx and LO2/HBx-d382 cells. In the above experiments, the phenotypes were more profound in LO2/HBx-d382 than in LO2/HBx cells. The dual luciferase reporter assay confirmed that CyclinD1 is a direct target gene of miR-338-3p, and that the main binding site of miR-338-3p in CyclinD1-3′-UTR region is located between nt 2397–2403 (the second predicted binding site). Finally, HBx reduction enhanced miR-338-3p expression and reduced CyclinD1 expression in HBx-expressing cells, supporting that HBx is necessary for cyclinD1 upregulation through miR-338-3p. The miR-338 gene is located on chromosome 17q25 and produces two mature forms (miR-338-3p and miR-338-5p). Our data showed that miR-338-3p expression is down-regulated in inducing tumor formation HBx-expressing cells compared with normal hepatocyte cell line LO2 and QSG7701, similar to the results found in another HCC cell line HepG2.2.15 cells [29]. Moreover, another study reported that down-regulated miR-338 expression, and miR-338-3p in particular, was frequently found in HCC tissues [30], which peripherally supports our data that miR-338-3p is associated with HCC formation. The miR-338 gene is located within the intron 8 region of the apoptosis -associated tyrosine kinase (AATK) gene [31]. Interestingly, the survivin gene, a new inhibitor of apoptosis protein family, is also located on chromosome 17q25 [32]. Overexpression of survivin gene was found in a wide variety of tumor and transformed cells, including liver cancer [32]–[34] and cells in chronic HBV infected livers that are deemed close to transformation to tumor cells [32], [35], suggesting that miR-338-3p may play an important role in liver cancer formation due to its location in the fragile site locus associated with liver cancer tumorigenesis. Currently, there are few studies on miR-338-3p, although it was reported that COXIV and SMO could be regulated by miR-338-3p [36]–[37]. SMO, a transmembrane protein, has a switch function in Sonic hedgehog (Hh) signaling pathway; the classic Hh signaling pathway has been associated with tumorigenesis in the literature [38]–[39], further implicating a close correlation between miR-338-3p and tumor development. The focus of our study is on CyclinD1, a novel target of miR-338-3p. G1/S-specific CyclinD1 is a protein in the highly conserved Cyclin family and functions at the transition between the G1 and S cell cycle phases where it is a key signaling protein for G1 cell proliferation [40]. CyclinD1 overexpression leads to a remarkable reduction in the lag time between the G1/S phase cell cycle transition as well as promotion of the cell cycle progression rate, eventually leading to uncontrolled cell proliferation and tumor formation. In contrast, CyclinD1 inhibition in KM20 cells by effective siRNA treatment leads to cell cycle arrest and cell growth inhibition [41]. Therefore, CyclinD1 can be considered as an oncogene [42]–[43]. Previous studies have suggested a link between HBx and CyclinD1, where CyclinD1 is shown to be up-regulated by HBx; in these studies, the process is independent of extracellular mitotic signals [44] or is mediated by the NF-κB2(p52)/BCL-3 complex [45]. However, our findings have revealed a new process to explain how HBx can up-regulate CyclinD1, and that mechanism is by down-regulating miR-338-3p. Therefore, HBx, especially the HBx-deletion mutant HBx-d382, is involved in regulating CyclinD1 expression through an HBx-mediated miR-338-3p down-regulation mechanism, suggesting that miR-338-3p plays a tumor suppressor role in the development of HBx- and HBx-d382 deletion-mutant–mediated HCC. The effects of HBx on cell cycle progression are potentially complicated. Most studies found that HBx stimulated cell cycle progression that accelerated transit through checkpoint controls at G0/G1 [44], [46]–[47], which is consistent with our studies. However, several other studies show that HBx actually inhibited cell proliferation by using Hepa1–6 (mouse HCC), HepG2 (human HCC) [48], or hepatocytes isolated from HBx-expressing mice [49]. One possible reason for the contradictory results may be the use of different cell lines with different differentiation statuses and different genetic backgrounds. Another possibility is that the HCC tumor cells may modify the biological activity of HBx, which is something we tried to address in our present study using normal hepatocytes. Yet another possibility is that p53 levels may influence HBx function, as p53 is known interact with HBx, and the normal physiological level of p53 displays anti-apoptotic activity, whereas a high level of p53 displays an adverse result [50]–[51]. The recent studies on HBV-related HCC found that genetic heterogeneity of the HBx gene is prevalent in liver cancer tissue. For example, wild-type HBx and mutant HBx can coexist in the same tumor [10]. The major mechanism underlying the creation of HBx deletion mutants is loss of C-terminal fragments of different sizes; this is a dominant phenotype that accounts for almost 100% of the HBx present in the integrated HBV DNA [8], [10], [52]. The HBx protein functional domains are divided into the regulatory region and the trans-activation region, where the regulatory region is located near the N-terminal end (1∼50 aa) and the trans-activation region is located near the carboxyl terminus (51∼154 aa) end. The 1∼20 aa in the regulatory region is responsible for inhibiting the activity of the trans-activation region [53]. Therefore, a point mutation or deletion mutation occurring at almost any region in the HBx gene could affect its trans-activation function. Since the deleted region of the HBx-d382 (nt 382–400 deletion) mutant is located near the C-terminal trans-activation region and is also close to where the trans-activation domain associates with mitochondria in HBV-infected cells (the first l32∼139 amino acids), the deleted region is necessary for HBx trans-activation activity as well as its known association with mitochondria. Therefore, the HBx-d382 mutant has a close correlation with the process of HBx gene replication and transcription, which are involved in the viral-host interaction, as well as the apoptosis machinery located in the mitochodria. While the loss of C-terminal expression in HBx-d382 mutant attenuates the effect of wild-type HBx on cell proliferation, the expressed truncated HBx protein has gained the ability to promote the malignant transformation of cells. In addition to the role described here, the deleted region in the HBx-d382 mutant is also known to be involved in the p53-binding region of the HBx gene (102∼136 aa). Direct binding of HBx to human tumor suppressor p53 has been reported to inhibit p53-stimulated transcription; additionally, the p53 and HBx interaction also reduced the HBx-mediated trans-activation [54]. Therefore, loss of the p53 binding site in HBx mutants can affect the tumor-suppression ability of p53 and further promote tumor formation. Taken together, these results collectively suggest that multiple HBx gene mutations found in HCC, including HBx-d382, may promote the development of HBx-mediated liver cancer. In conclusion, our study revealed that the HBx-d382 mutant has an inhibitory effect on miR-338-3p expression that is associated with HBx-d382-mediated promotion of liver cell proliferation. The finding that HBx-d382 attenuated the miR-338-3p-mediated inhibition of CyclinD1 expression may provide insight into HBx-mediated tumorigenesis and a new direction for further investigation into HBx function and therapeutic development. Supporting Information Figure S1 miR-338-3p expression in normal hepatocytes of LO2 and QSG7701 cells compared with HBx-expressing cells by real-time PCR. (A) Relative expression of miR-338-3p in engineered LO2 cells. 1: LO2; 2: LO2/pcDNA3.0; 3: LO2/HBx; 4: LO2/HBx-d382. (B) miR-338-3p expression in QSG7701 cells transfected with HBx using real-time PCR. 1: QSG7701; 2: QSG/pcDNA; 3: QSG/HBx; 4: QSG/HBx-d382 (*p>0.05, **p<0.001). (TIF) Click here for additional data file. Figure S2 Cell cycle analysis of control LO2/pcDNA3.0 cells. Histograms of average of G1/S phase populations in LO2/pcDNA3.0 cells after transfection with miR-338-3p mimics or inhibitor (*P<0.001, **P<0.01). (TIF) Click here for additional data file. Figure S3 miR-338-3p and CyclinD1 expression after knocking-down HBx in HBx-expressing cells. (A–B) HBx mRNA and protein expression levels after transfection with HBx siRNA compared with control RNA in LO2/HBx and LO2/HBx-d382 cells (*P<0.001). (C) Relative expression of miR-338-8p using the 2−ΔΔCT method of qRT-PCR after altering HBx in LO2/HBx and LO2/HBx-d382 cells (*P = 0.013,**P = 0.008). (D) CyclinD1 levels in HBx-expressing cells after transfection of the HBx siRNA or negative control (***P<0.001). Data are shown as mean ±SD of 3 separate experiments. (TIF) Click here for additional data file. ==== Refs References 1 Murakami S (2001 ) Hepatitis B virus X protein: a multifunctional viral regulator . J Gastroenterol 36 : 651 –660 .11686474 2 Venard V , Corsaro D , Kajzer C , Brnowicki JP , Le Fau A (2000 ) Hepatitis B virus X gene variability in French-born patients with chronic hepatitis and hepatocellular carcinoma . 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PLoS One. 2012 Aug 17; 7(8):e43204
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22952780PONE-D-12-1184110.1371/journal.pone.0043834Research ArticleBiologyBiochemistryLipidsLipid MetabolismMetabolismLipid MetabolismPopulation BiologyEpidemiologyEpidemiological MethodsMedicineClinical Research DesignEpidemiologyEndocrinologyDiabetic EndocrinologyDiabetes Mellitus Type 2EpidemiologyCardiovascular Disease EpidemiologyClinical EpidemiologyEpidemiological MethodsDevelopment and Evaluation of a Simple and Effective Prediction Approach for Identifying Those at High Risk of Dyslipidemia in Rural Adult Residents Neural Network to Predict DyslipidemiaWang Chong-Jian 1 Li Yu-Qian 2 Wang Ling 1 Li Lin-Lin 1 Guo Yi-Rui 1 Zhang Ling-Yun 3 Zhang Mei-Xi 1 Bie Rong-Hai 1 * 1 Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China 2 Department of Clinical Pharmacology, School of Pharmaceutical Science, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China 3 Department of Endocrinology, Military Hospital of Henan Province, Zhengzhou, Henan, People’s Republic of China Malaga German Editor Universidad Peruana Cayetano Heredia, Peru * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: RHB CJW. Performed the experiments: YQL LW LLL. Analyzed the data: LLL YRG. Contributed reagents/materials/analysis tools: YQL YRG LYZ MXZ. Wrote the paper: CJW LW. 2012 28 8 2012 7 8 e4383425 4 2012 30 7 2012 © 2012 Wang et al2012Wang 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 Dyslipidemia is an extremely prevalent but preventable risk factor for cardiovascular disease. However, many dyslipidemia patients remain undetected in resource limited settings. The study was performed to develop and evaluate a simple and effective prediction approach without biochemical parameters to identify those at high risk of dyslipidemia in rural adult population. Methods Demographic, dietary and lifestyle, and anthropometric data were collected by a cross-sectional survey from 8,914 participants living in rural areas aged 35–78 years. There were 6,686 participants randomly selected into a training group for constructing the artificial neural network (ANN) and logistic regression (LR) prediction models. The remaining 2,228 participants were assigned to a validation group for performance comparisons of ANN and LR models. The predictors of dyslipidemia risk were identified from the training group using multivariate logistic regression analysis. Predictive performance was evaluated by receiver operating characteristic (ROC) curve. Results Some risk factors were significantly associated with dyslipidemia, including age, gender, educational level, smoking, high-fat diet, vegetable and fruit intake, family history, physical activity, and central obesity. For the ANN model, the sensitivity, specificity, positive and negative likelihood ratio, positive and negative predictive values were 90.41%, 76.66%, 3.87, 0.13, 76.33%, and 90.58%, respectively, while LR model were only 57.37%, 70.91%, 1.97, 0.60, 62.09%, and 66.73%, respectively. The area under the ROC cure (AUC) value of the ANN model was 0.86±0.01, showing more accurate overall performance than traditional LR model (AUC = 0.68±0.01, P<0.001). Conclusion The ANN model is a simple and effective prediction approach to identify those at high risk of dyslipidemia, and it can be used to screen undiagnosed dyslipidemia patients in rural adult population. Further work is planned to confirm these results by incorporating multi-center and longer follow-up data. This research was supported by the National High Technology Research and Development Program of China (Grant No: 2006BAI01A01), China Postdoctoral Science Foundation (Grant No: 201104375), and Medical Scientific Research Foundation of Health Department of Henan Province (Grant No: 201004042). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Dyslipidemia is a widely recognized risk factor for cardiovascular diseases, a leading cause of death in both developed and developing countries [1], [2]. The World Health Organization (WHO) estimates that dyslipidemia is associated with more than half of the global cases of ischemic heart disease and more than four million deaths per year [3]. Evidence demonstrates that dyslipidemia can be prevented and controlled, which is cost-beneficial and with effective prevention programs can decrease the incidence and mortality of cardiovascular diseases [4], [5]. Estimating an individual’s risk across a range of presumed risk factors is fundamental to prevent dyslipidemia [6]. Due to its complex and multifactorial nature, the prevention of dyslipidemia must refer to multiple risk factors. Evidence showed that some predictors are associated with the occurrence of dyslipidemia [7]. Factors related to diet, lifestyle and family history might be associated with an increased risk of dyslipidemia [8], [9]. However, it is difficult to evaluate an individual’s risk of dyslipidemia when many predictors exist concomitantly. A model that integrates related factors and predicts the risk of dyslipidemia would be helpful to promote health education and counseling, and enable further development of computerized medical decision support systems for aiding healthcare practitioners to assess the risks of their patients quickly, inexpensively, and noninvasively [9], [10]. Logistic regression (LR) is often used to identify significant factors that correlate with disease, and has commonly been used to develop models for clinical diagnosis and treatment [11]. Artificial neural network (ANN) is a computer modeling technique based on the observed behaviors of biological neurons [12]. This is a non-parametric pattern recognition method that can recognize hidden patterns between independent and dependent variables [13]. Although ANN has been used in oncology, diabetes, hypertension, and other diseases [14]–[21], none have developed and evaluated the feasibility of the ANN model for predicting the risk of dyslipidemia in rural adults. Thus, it is unclear whether the ANN model is reliable and effective to identify those at high risk of dyslipidemia. Therefore, the purpose of this study was to develop and deliver an ANN model without biochemical parameters to identify those at high risk of dyslipidemia in rural adult population, and evaluate the predictive performance of the ANN model in comparison with the traditional LR model. Participants and Methods Study Population This study was a cross-sectional survey, and subjects were selected randomly from eligible candidates in the residential registration record from the rural areas in Henan Province, China. The eligibility of the candidate was defined as those who were stable residents for at least 10 years in the study areas aged 35–78 years, and were free from the following conditions: 1) severe psychological disorders, physical disabilities, cancer, Alzheimer’s disease, or dementia, within 6 months; or 2) currently diagnosed with tuberculosis, acquired immune deficiency syndrome (AIDS), and other infectious diseases. Ultimately, a total of 8,914 residents who met the criteria enrolled in the study. Informed consent was obtained from all participants. The procedure of the study was approved by the Zhengzhou University Medical Ethics Committee, and written informed consent was obtained for all participants. Data Collection and Measurements A home interview was conducted by physicians or public health workers from the local Centers for Disease Control and Prevention and the community hospital. All investigators and staff successfully completed a training program that oriented them both to the aims of the study and to the specific tools and methodologies used. At the training sessions, interviewers were given detailed instructions on administration of the study questionnaire. Demographic data Education level was classified into five categories: no education, primary school, middle school, high school, college and above. Marital status was categorized as unmarried, married/cohabitation, and divorced/widowed. Occupation was categorized as farmers, laborers, professionals and employers/managers. Individual annual income was calculated by total household income divided by the number of family members. Positive family history was defined as the participant’s parents or siblings having a history of dyslipidemia at or before the baseline examination. Dietary and lifestyle behaviors Three-day dietary intake data were collected from each subject using a 24-hour diet recall and a 2-day diet record. The daily intake of energy, nutrients, food and food groups for each subject were calculated using China Food Composition Table [22]. According to the Chinese Dietary Guidelines [23], vegetable and fruit intake was defined as consuming an average of more than 500 g per day, and fat intake is defined as consuming an average of more than 25 g per day. Current smoking status included smoker and non-smoker. Participants who currently smoked and had smoked at least 100 cigarettes during their lifetime were classified as current smokers if they answered affirmatively to the following questions: “Do you smoke cigarettes now?” and “Have you smoked at least 100 cigarettes during your lifetime?” Physical activity level was classified as low, moderate, or high according to the International Physical Activity Questionnaire (IPAQ) scoring protocol [24]. Anthropometric data Body weight and height were measured twice in light indoor clothing without shoes to the nearest 0.1 kg and 0.1 cm, respectively. Waist circumference (WC) was measured twice at the mid-point between the lowest rib and the iliac crest to the nearest 0.1 cm, after inhalation and exhalation. Central obesity based on WC (Male: WC ≥90 cm; Female: WC ≥80 cm) was defined according to WHO criteria for the Asia-Pacific region population [25]. Laboratory measurement An overnight fasting blood specimen was collected in a vacuum tube containing EDTA for measurement of lipid profile. Blood specimens were centrifuged at 4°C and 3,000 rpm for 10 minutes, and the plasma was transferred and stored at −20°C for biochemical analyses. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured enzymatically on an automatic biochemical analyzer (Hitachi 7080, Toky, Japan) with reagents purchased from Wako Pure Chemical Industries (Osaka, Japan). Definition of Dyslipidemia According to the China Adult Dyslipidemia Prevention Guide (2007 Edition) criteria [26], subjects were considered normal if their TC was less than 6.22 mmol/L, TG was less than 2.26 mmol/L, and HDL-C was greater than 1.04 mmol/L at the time of examination. The subjects were considered having dyslipidemia if one of their TC, TG or HDL-C were greater than 6.22 mmol/L, 2.26 mmol/L, or less than 1.04 mmol/L, respectively. Training and Validation Data Sets Of 8,914 participants who met inclusion criteria, 75% of subjects (N1 = 6,686) were randomly selected to provide the training group for constructing ANN and LR prediction models. The remaining 25% of participants (N2 = 2,228) were assigned to the validation group for performance comparisons of ANN and LR models. The proportion of dyslipidemia between the training group and validation group was similar to the surveyed population data, and there was no statistically significant difference by χ2 test for gender and age between the training and validation datasets (Table 1). 10.1371/journal.pone.0043834.t001Table 1 Comparison of baseline characteristics between the training and validation groups. Variable Participants (N = 8,914) P value Training group (N1 = 6,686) Validation group (N2 = 2,228) Age (years), mean (±sd) 53.89 (10.89) 52.56 (11.02) 0.9906 Gender (women), n (%) 3,712 (55.52) 1,276 (57.27) 0.1491 Occupation, n (%) Farmers 5,238 (78.34) 1,750(78.56) 0.9610 Laborers 586 (8.76) 191(8.57) Employers/managers 862 (12.89) 287(12.88) Education, n (%) 0.2317 No education 1,177 (17.60) 369 (16.56) Primary school 2,384 (35.66) 768 (34.47) Middle school 2,484 (37.15) 887 (39.81) High school 579 (8.66) 181 (8.12) College and above 62 (0.93) 23 (1.03) Marital status, n (%) Unmarried 34 (0.51) 9 (0.40) 0.8261 Married/cohabitation 6,126 (91.62) 2,043 (91.70) Divorced/widowed 526 (7.87) 176 (7.90) Physical activity, n (%) Low 1,913(28.61) 619(27.78) 0.4386 Moderate 1,579(23.62) 555(24.91) High 3,194(47.77) 1,054(47.31) Individual income (annual), mean (±sd) 2,075(1199) 2,037(1226) 0.1009 Waist circumference (cm), mean (±sd) 82.61(10.36) 82.46(10.15) 0.5222 TC (mmol/L), mean (±sd) 4.56 (0.96) 4.58 (0.95) 0.9492 TG (mmol/L), mean (±sd) 1.90 (0.53) 1.87 (0.65) 0.1052 H-DLC (mmol/L), mean (±sd) 1.11 (0.25) 1.17 (0.27) 0.1464 L-DLC (mmol/L), mean (±sd) 2.59 (0.79) 2.62 (0.77) 0.4164 Dyslipidemia, n (%) 3,115 (46.59) 1,011(45.38) 0.3200 Modeling Tools Logistic regression The LR model generates the coefficients for the following formula used in logit transformation for predicting the probability of an event among patients with certain characteristics of interest: Logit (P) = β0+β1 χ 1+β2 χ2+…+ βi χi [9]. The formula P = 1/(1+e-logit(P)) used for calculating the probability of dyslipidemia in this study, where 1 =  dyslipidemia and 0 =  non-dyslipidemia. A stepwise algorithm was used to construct the multivariate LR model. At each step, independent variables not yet included in the equation were tested for possible inclusion. The variable with the strongest significant contribution to improving the model was included. Variables already included in the logistic regression equation were tested for exclusion on the basis of a likelihood ratio test. The analysis ended when no further variables for inclusion or exclusion were available [27]. Furthermore, LR was used to estimate the coefficients (β) of these variables, from which the probability of dyslipidemia was estimated. Artificial neural network The ANN is a nonlinear statistical data modeling tool that can be used to model complex relationships between inputs and outputs or to find patterns in data. The processing elements or nodes are arranged in “input,” “hidden,” and “output” layers, each layer containing one or more nodes. The input layer consists of the data thought to be of value in predicting the outputs of the model [28], [29]. Each data point is represented by a node in the input layer. The output layer estimates the probability of the outcome as determined by the model. Each layer comprises one or more processing elements all interconnected in a way that each node in the hidden layer is connected to each node in the input and output layers [28], [29]. Each connection carries a “weight” or value that determines the relevance of a particular input for the resulting output [28], [29]. The ANN makes predictions based on the strength of connections between the neurons in the input, hidden, and output layers [28], [29]. The results of the output layer in ANN model represent the probability of a characteristic of interest (dyslipidemia). Figure 1 presents a flow chart describing the basic ANN design. A procedural description of the algorithm used for ANN in this study is available from the author and listed in Text S1. A more detailed description of ANN could be provided by Zou and Dumont [30], [31]. 10.1371/journal.pone.0043834.g001Figure 1 Framework of artificial neural network model for predicting an individual’s risk of dyslipidemia. The input layer contained 9 neurons. In the hidden layers, the numbers of neuron were 21. The output layer had only one neuron representing the probability of dyslipidemia. Abbreviations: XA, age; XG, gender; XEL, educational level; XS, smoking; XHFD, high-fat diet; XVFI, vegetable and fruit intake; XFH, family history of dyslipidemia; XPA, physical activity; XWC, waist circumference. Statistical Analysis Analysis was performed in three stages. Firstly, a set of indicators contributing to the prediction of dyslipidemia was identified by univariate and multivariate logistic regression analysis based on the training group. Secondly, The ANN and LR models were performed with the probability of having dyslipidemia as the dependent variable and the risk factors as the independent variables. In general, the analysis structure of the neural network included three layers: input layer to accept information, hidden layer to process information, and output layer to calculate responses. In this study, the ANN model used the same input variables as the LR model. Thirdly, predictive performance was assessed by using receiver operating characteristic (ROC) curve analysis. The LR model and ROC curve analysis were constructed using SAS 9.1 (SAS Institute, USA). The ANN model was performed with MATLAB 7.1 (MathWorks Institute, USA). All reported P-values were two-sided, and P-values less than 0.05 were considered statistically significant. Results Characteristics of the Participants Table 1 shows the characteristics of the 8,914 subjects in the training and validation groups. The mean age (±sd) was 53.89±10.89 and 52.56±11.02 years in these two groups, respectively, while the number of females were 3,712 (55.52%) and 1,276 (57.27%), respectively. The prevalence rates of dyslipidemia were 46.59% and 45.38% in the training and validation groups, respectively. The relevant variables did not significantly differ between training and validation groups (P>0.05), confirming the reliability of random subject selection. Predictors of Dyslipidemia Risk Table 2 shows the predictors of dyslipidemia risk identified from LR analysis based on the training group (N1 = 6,686). With the construction of a multivariable model (LR analysis, the independent predictors were included when P<0.05, and were eliminated when P>0.1). Factors significantly associated with dyslipidemia were age (odds ratio OR = 1.042), higher-educational level (OR = 1.362), smoking (OR = 1.165), more high-fat diet (OR = 1.403), positive family history of dyslipidemia (OR = 1.876), and central obesity (Male: WC ≥90 cm; Female: WC ≥80 cm) (OR = 2.327). There was also an inverse relationship for male gender (OR = 0.758), more vegetable and fruit intake (OR = 0.844), and more physical activity (OR = 0.924). 10.1371/journal.pone.0043834.t002Table 2 Multivariate logistic regression analysis on risk factors of dyslipidemia in the training group. Variable β S.E. Wald P-value OR (95%CI) Age, XA 0.0410 0.0029 203.5210 0.0001 1.042 (1.036–1.048) Male, XG −0.2774 0.0800 12.0404 0.0005 0.758 (0.648–0.886) Higher-educational level, XEL 0.3092 0.0609 25.7535 0.0001 1.362 (1.209–1.535) Smoking, XS 0.1529 0.0454 11.3361 0.0008 1.165 (1.066–1.274) More high-fat diet, XHFD 0.3385 0.1063 10.1416 0.0014 1.403 (1.139–1.728) More vegetable and fruit intake, XVFI −0.1701 0.0548 9.6278 0.0019 0.844 (0.758–0.939) Positive family history, XFH 0.6290 0.0671 87.8728 0.0001 1.876 (1.645–2.139) More physical activity, XPA −0.0786 0.0321 5.9851 0.0144 0.924 (0.868–0.984) Central obesity, Xwc 0.8444 0.0561 226.3747 0.0001 2.327 (2.084–2.597) Abbreviations: XA, age; XG, gender; XEL, educational level; XS, smoking; XHFD, high-fat diet; XVFI, vegetable and fruit intake; XFH, family history of dyslipidemia; XPA, physical activity; XWC, waist circumference. Prediction Models The logit probability of having dyslipidemia was described by the following LR model: −2.7155+0.0410 XA − 0.2774 XG +0.092 XEL +0.1529 XS +0.3385 XHFD - 0.1701 XVFI +0.6290 XFH - 0.0786 XPA +0.8444 XWC. According to the predictors of dyslipidemia risk from the LR analysis, the ANN model was built using the training group data. The predictors used as the model input were XA, XG, XEL, XS, XHFD, XVFI, XFH, XPA, and XWC. The probability of whether an individual had dyslipidemia was the output variable. The analysis structure of the neural network included three layers: input, hidden and output layers. Figure 1 shows the input layer with 9 neurons, 21 hidden layer neurons and one output layer neuron, corresponding to the forecast variable (that is the probability of having dyslipidemia). Comparison of Predictive Performance The ANN and LR models could successfully distinguish an individual’s risk of having dyslipidemia. We compared the individual’s predicted risk from the two models with the actual status using the validation group (N2 = 2,228). The ANN model detected 911 dyslipidemia patients from the 1011 actual dyslipidemia patients, whereas the LR model only detected 582 dyslipidemia subjects. Figure 2 summarizes the ROC curve obtained from the LR and ANN models. For the ANN model, the sensitivity, specificity, positive likelihood ratio (+ LR), Negative likelihood ratio (− LR), positive predictive value (PPV), and negative predictive value (NPV) were 90.41%, 76.66%, 3.87, 0.13, 76.33%, and 90.58%, respectively, while the corresponding numbers were only 57.37%, 70.91%, 1.97, 0.60, 62.09%, and 66.73% in the LR model, respectively (Table 3). The AUC value of the ANN model was significantly higher (AUC = 0.86±0.01, 95% CI: 0.85–0.88) than that of the LR model (AUC = 0.68±0.01, 95% CI: 0.66–0.70) (P<0.001). 10.1371/journal.pone.0043834.g002Figure 2 ROC curves of ANN and LR prediction models in the validation group. Areas under ROC curves were 0.86 and 0.68 for ANN and LR models, respectively. Area under ROC curve obtained by ANN was superior to that obtained by LR. Abbreviation: ANN, artificial neural network; LR, logistic regression; ROC, receiver operating characteristic. Discussion To our knowledge, this is the first study to develop and evaluate the reliability and effectiveness of the ANN model for predicting dyslipidemia risk of rural adults in comparison with the LR model. Our findings showed that the ANN model had superior predictive performance, and the sensitivity, specificity, PPV, NPV, and AUC value of the ANN model were significantly higher than that of the traditional LR model. The ANN model can be used to identify undiagnosed dyslipidemia patients in rural adult population. 10.1371/journal.pone.0043834.t003Table 3 Performance comparison of ANN and LR models for predicting dyslipidemia in the validation group. Variable ANN model LR model Sensitivity (%, 95% CI) 90.41 (88.40–92.22) 57.37 (54,31–60.38) Specificity (%, 95% CI) 76.66 (74.18–79.03) 70.91 (68.29–73.50) + LR (95% CI) 3.87 (3.42–4.40) 1.97 (1.71–2.28) − LR (95% CI) 0.13 (0.10–0.16) 0.60 (0.54–0.67) PPV (%, 95% CI) 76.33 (74.47–78.93) 62.09 (59.33–66.00) NPV (%, 95% CI) 90.58 (86.24–94.26) 66.73 (61.70–70.53) AUC (95% CI) 0.86 (0.85–0.88) 0.682 (0.66–0.70) Abbreviation: ANN, artificial neural network; LR, logistic regression; CI, confidence intervals; AUC, areas under ROC curve; + LR, positive likelihood ratio; − LR, Negative likelihood ratio; PPV, positive predictive value; NPV, negative predictive value. Because of the rapidly increasing prevalence of dyslipidemia, detecting people with undiagnosed dyslipidemia is very important in both public health and clinical practice [32], [33]. Lipid profile measurement is a standard method for identifying and diagnosing dyslipidemia [34], [35], but it is not available in resource limited settings, especially in some rural areas of developing countries. For example, the costs of TC, TG, HDL-C, and LDL-C measurement are more than ¥30 in China, which is probably the income of three days to a Chinese peasant. Therefore, an effective and inexpensive identifying approach has been sought which could be used to screen undiagnosed dyslipidemia in resource limited countries and areas. In this study, we used a general epidemiology survey database without biochemical parameters to develop and evaluate a prediction model to distinguish patients with dyslipidemia, which is not only inexpensive, but also quick and noninvasive. Model sensitivity and specificity are important when testing whether a model can accurately recognize positive and negative outcomes [9]. The ideal model has both high sensitivity and high specificity [36]. In this study, the results of the predictive performance showed that the ANN model could be used to accurately screen undiagnosed dyslipidemia patients because it had sufficient sensitivity (90.41%) and specificity (76.66%) compared to the standard LR model (57.37% and 70.91%) for identifying true positive or negative patients. Since AUC provides a superior performance index in addition to superior accuracy, it was often used to evaluate the predictive accuracy of classifiers [37]. The AUC of a classifier can be defined as the probability of the classifier ranking a randomly chosen positive example higher than a randomly chosen negative example, and higher AUC values can be interpreted as higher predictive accuracy [37], [38]. For ANN model, the AUC value (AUC = 0.86±0.01) was superior to that of the LR model (AUC = 0.68±0.01) in terms of predictive accuracy. The above comparisons confirm that the sensitivity, specificity, and AUC for the prediction model constructed using ANN were significantly higher than that of the traditional LR model. That is, the ANN model outperforms the traditional LR model for predicting individual’s risk of dyslipidemia in rural adult residents. The availability of the predictors is also very important when evaluating whether a model is feasible to identify positive and negative outcomes. This study examined the feasibility of the predictors of dyslipidemia in rural adult population. The findings based on the training dataset revealed nine common parameters significantly associated with dyslipidemia, including age, gender, educational level, smoking, high-fat diet, vegetable and fruit intake, family history, physical activity, and central obesity. These predictors without biochemical parameters were readily accessible in the rural population through routinely collected data in general practice or from general survey. In addition, the ANN predictive model can be easily navigated using a simple questionnaire through computerized medical decision support systems, in which the path depends on simple yes or no questions. The final result will help rural healthcare practitioners to quickly determine the individual’s risk of dyslipidemia. The ANN predictive model using demographic, lifestyle and anthropometric data provides a feasible approach to screen undiagnosed dyslipidemia patients in rural adult population. Although this is the first study to develop and evaluate the feasibility of the ANN model for predicting individual’s risk of dyslipidemia in rural adult residents, study limitations should be noted. Firstly, evaluations of the different models were based on a cross-sectional survey without a longer follow-up period. Secondly, the samples were limited geographically and ethnically, consisting of a rural community of individuals aged 35–78 years old. Thirdly, the relevant variables were measured, such as smoking, physical activity, waist circumference, fat, vegetable and fruit consumption, on only a single occasion. Finally, the prediction model was based on a sample without use of data from multiple regions. Despite these limitations, the results are based on a large population-based study combining multiple risk factors, and the prediction approach was reliable and effective to screen undiagnosed dyslipidemia patients in rural adult residents. Conclusion Our findings demonstrate that the ANN model had superior predictive performance compared with the traditional LR model to quickly identify those at high risk of dyslipidemia, and it can be used to screen undiagnosed dyslipidemia patients in rural adult population using general survey data or routinely collected data in general practice. This will help rural healthcare practitioners to evaluate the risks of their patients quickly, inexpensively, and noninvasively. Meanwhile, further work is planned to assess the utility of incorporating multiple rural locations and longer follow-up data. Supporting Information Text S1 This file contains the procedure of the artificial neural network model in the Matlab environment. (DOCX) Click here for additional data file. The authors thank all of the participants, coordinators, and administrators for their support and help during the research. The authors also wish to thank MSc. Shuihong Zhou for her help in constructing the ANN model. In addition, the authors would like to thank Mr. Mark Dickson (Doctoral Candidate) and Dr. Luo ZC for their critical reading of the manuscript. ==== Refs References 1 Barter P , Gotto AM , LaRosa JC , Maroni J , Szarek M , et al (2007 ) HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events . 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PLoS One. 2012 Aug 28; 7(8):e43834
==== Front BMC MedBMC MedBMC Medicine1741-7015BioMed Central 1741-7015-10-402252419710.1186/1741-7015-10-40Research ArticleMitochondrial aldehyde dehydrogenase (ALDH2) protects against streptozotocin-induced diabetic cardiomyopathy: role of GSK3β and mitochondrial function Zhang Yingmei [email protected] Sara A [email protected] Nan [email protected] Jacalyn R [email protected] Haichang [email protected] Jun [email protected] Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032, China2 Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, WY 82071, USA2012 23 4 2012 10 40 40 18 1 2012 23 4 2012 Copyright ©2012 Zhang et al; licensee BioMed Central Ltd.2012Zhang 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 Mitochondrial aldehyde dehydrogenase (ALDH2) displays some promise in the protection against cardiovascular diseases although its role in diabetes has not been elucidated. Methods This study was designed to evaluate the impact of ALDH2 on streptozotocin-induced diabetic cardiomyopathy. Friendly virus B(FVB) and ALDH2 transgenic mice were treated with streptozotocin (intraperitoneal injection of 200 mg/kg) to induce diabetes. Results Echocardiographic evaluation revealed reduced fractional shortening, increased end-systolic and -diastolic diameter, and decreased wall thickness in streptozotocin-treated FVB mice. Streptozotocin led to a reduced respiratory exchange ratio; myocardial apoptosis and mitochondrial damage; cardiomyocyte contractile and intracellular Ca2+ defects, including depressed peak shortening and maximal velocity of shortening and relengthening; prolonged duration of shortening and relengthening; and dampened intracellular Ca2+ rise and clearance. Western blot analysis revealed disrupted phosphorylation of Akt, glycogen synthase kinase-3β and Foxo3a (but not mammalian target of rapamycin), elevated PTEN phosphorylation and downregulated expression of mitochondrial proteins, peroxisome proliferator-activated receptor γ coactivator 1α and UCP-2. Intriguingly, ALDH2 attenuated or ablated streptozotocin-induced echocardiographic, mitochondrial, apoptotic and myocardial contractile and intracellular Ca2+ anomalies as well as changes in the phosphorylation of Akt, glycogen synthase kinase-3β, Foxo3a and phosphatase and tensin homologue on chromosome ten, despite persistent hyperglycemia and a low respiratory exchange ratio. In vitro data revealed that the ALDH2 activator Alda-1 and glycogen synthase kinase-3β inhibition protected against high glucose-induced mitochondrial and mechanical anomalies, the effect of which was cancelled by mitochondrial uncoupling. Conclusions In summary, our data revealed that ALDH2 acted against diabetes-induced cardiac contractile and intracellular Ca2+ dysregulation, possibly through regulation of apoptosis, glycogen synthase kinase-3β activation and mitochondrial function independent of the global metabolic profile. ALDH2cardiac contractiondiabetesGSK3βmitochondrial function ==== Body Background The mitochondrial isoform of aldehyde dehydrogenase (ALDH2) has been shown to play a pivotal role in the metabolism of acetaldehyde and other toxic aldehydes [1-4]. Ample evidence from our laboratory as well as others has revealed a rather singular role for ALDH2 in cardioprotection against ischemic injury, arrhythmias and alcoholism [2,3,5-7]. However, the role of ALDH2 in myopathic anomalies associated with metabolic disorders, including diabetes mellitus, has not been elucidated. The prevalence of diabetes and associated heart diseases has been steadily increasing, particularly in Asian countries, with approximately 50% of populations carrying one copy of the mutant ALDH2 gene [4,8-11]. A plethora of studies have depicted significant contribution from genetic variants, such as peroxisome proliferator-activated receptors (PPARs), in the predisposition of diabetes [12]; however, very few have examined the role of ALDH2 in the onset and progression of diabetes and its complications. Recent evidence revealed that ALDH2 polymorphism is closely associated with an increased risk of diabetes [11] while experimental findings showed reduced ALDH2 expression and activity associated with oxidative stress and cardiac dysfunction in diabetes [13]. These observations are somewhat consistent with the notion that inactive ALDH2 promotes hyperglycemia [9], while genotypes of ALDH2 can modify diabetes risk irrespective of alcohol intake [14]. To this end, this study was designed using a unique murine model to examine the impact of ALDH2 overexpression in the pathogenesis of diabetic cardiomyopathy and the underlying cellular mechanism(s) involved. Recent evidence from our group has revealed a pivotal role for the essential survival factor Akt and its downstream signaling molecules, including glycogen synthase kinase-3β (GSK3β), PPAR (mTOR) and the forkhead transcriptional factor in ALDH2, in cardioprotection against alcoholism and ischemia-reperfusion [3,7,15]. To better elucidate the interplay between these signaling cascades and mitochondrial function in diabetes and/or ALDH2-induced cardiac responses, we scrutinized apoptosis and mitochondrial integrity, including mitochondrial membrane potential, as well as cell signaling of Akt, GSK3β, mTOR and Foxo3a in wild-type FVB and ALDH2 transgenic mice with or without the induction of experimental diabetes. Given that Akt signaling is under the negative control of phosphatase and tensin homologue on chromosome ten (PTEN) in a wide variety of disease conditions, including myocardial hypertrophy, heart failure and preconditioning [16], we monitored pan protein expression and phosphorylation of PTEN. To evaluate if ALDH2 affects myocardial morphometric and functional anomalies in diabetes through any potential effect secondary to global metabolic alterations, we scrutinized whole body metabolism, including the respiratory exchange ratio (RER), and total physical activity as well as plasma levels of free fatty acid, insulin and glucose (fasting and postprandial) in control and diabetic mice. Methods Experimental animals, experimental diabetes and ALDH2 activity All animal procedures were approved by our Institutional Animal Care and Use Committee at the University of Wyoming (Laramie, WY, USA). Production of ALDH2 transgenic mice using the chicken β-actin promoter was as described previously [5]. All mice were housed in a temperature-controlled room under a 12 hour light-12 hour dark circadian cycle with access to water and food ad libitum. Five-month-old male FVB (used as wild-type) and ALDH2 transgenic mice received intraperitoneal injections of streptozotocin (STZ, 200 mg/kg). All mice were maintained for four weeks with free access to standard laboratory chow and tap water before their blood glucose levels were monitored. Mice with fasting blood glucose levels > 13 mM were deemed diabetic [17]. ALDH2 activity was measured in 33 mM sodium pyrophosphate containing 0.8 mM NAD+, 15 μM propionaldehyde and 0.1 mL protein extract. Propionaldehyde, the substrate of ALDH2, was oxidized in propionic acid, while NAD+ was reduced to NADH to estimate ALDH2 activity. NADH was determined by spectrophotometric absorbance at 340 nm. ALDH2 activity was expressed as nanomoles NADH per minute per milligram of protein [18]. Serum free fatty acid, plasma insulin and blood glucose levels Plasma free fatty acids were measured three hours after mice were denied access to food using a Free Fatty Acid Assay Kit (Cayman Chemical, MI, USA). In brief, mouse blood samples were centrifuged at 2000 × g for 15 minutes at 4°C. An excitation wavelength of 530 nm and an emission wavelength of 590 nm were used to detect the quantity of free fatty acids. Plasma insulin levels were measured using a mouse insulin ELISA kit from Diagnostic System Laboratory (Webster, TX, USA). Fasting (overnight) and postprandial (two hours after re-feeding following the overnight fasting) blood glucose levels were determined using a glucometer (Accu-ChekII, model 792, Boehringer Mannheim Diagnostics, Indianapolis, IN, USA) [17]. Metabolic measurement Indirect calorimetry and total physical activity were measured in light (10 a.m.) and dark (10 p.m.) phases using the Comprehensive Laboratory Animal Monitoring System (Oxymax/CLAMS; Columbus Instruments, Columbus, OH, USA). Volume of oxygen intake (VO2), volume of Carbon Dioxide exhaled (VCO2), the RER (VCO2/VO2) and physical activity were measured. All the parameters were measured every 10 minutes for six hours during daytime and six hours during nighttime. Result recorded in the first and last half hour was not be used. For simplicity, only the RER is presented without displaying original data from VO2 and VCO2 [19]. Echocardiographic assessment Cardiac geometry and function were evaluated in anesthetized mice using a two-dimensional guided M-mode echocardiography (Sonos 5500; Phillips Medical System, Andover, MA, USA) equipped with a 15-6 MHz linear transducer. Left ventricular (LV) wall thickness and diastolic and systolic dimensions were recorded from the M-mode images. Fractional shortening was calculated from end-diastolic diameter (EDD) and end-systolic diameter (ESD) using the equation: (EDD-ESD)/EDD×0.01 Estimated echocardiographic LV mass was calculated as: [(LVEDD+septal wall thickness+posterior wall thickness)3-LVEDD3]×1.055 where 1.055 (in mg/mm3) represents the density of the myocardium. Heart rate was calculated from 20 consecutive cardiac cycles [20]. Isolation of murine cardiomyocytes After intraperitoneal administration of a sedative (ketamine 80 mg/kg and xylazine 12 mg/kg), the hearts were removed and digested for 20 minutes with Liberase Blendzyme 4 (Hoffmann-La Roche Inc., Indianapolis, IN, USA). Cardiomyocyte yield was approximately 75% and was not affected by STZ treatment or ALDH2 overexpression. Only rod-shaped myocytes with clear edges were selected for the mechanical study [21]. For the in vitro study, cardiomyocytes from control FVB mice were exposed to high extracellular glucose (25.5 mM) [22] in the absence or presence of the ALDH2 activator Alda-1 (20 μM), the mitochondrial uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP, 1 μM) or the GSK3β inhibitor SB216763 (10 μM) [23,24] for 12 hours before an assessment of their mechanical and biochemical properties. Cell shortening and relengthening The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, MA, USA) [5]. In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70, Olympus Corporation, Tokyo, Japan) and superfused (approximately 1 mL/min at 25°C) with a buffer containing 131 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), at pH 7.4. The cells were field-stimulated with a supra-threshold voltage at a frequency of 0.5 Hz for 3 ms using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The studied myocyte was displayed on the computer monitor using an IonOptix MyoCam camera. IonOptix SoftEdge software was used to capture changes in cell length. Cell shortening and relengthening were assessed using the following indices: peak shortening (PS), the peak ventricular contractility; time-to-PS (TPS; contraction duration) and time-to-90% relengthening (TR90), the cardiomyocyte relaxation duration; and maximal velocities of shortening (+dL/dt) and relengthening (-dL/dt), the maximal velocities of ventricular pressure rise and fall. Intracellular Ca2+ transient measurement Myocytes were loaded with fura-2-acetoxymethyl ester (0.5 μM) for 10 minutes and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Cardiomyocytes were placed on an Olympus IX-70 inverted microscope and imaged through a Fluor × 40 oil objective. Cells were exposed to light emitted by a 75 W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 seconds then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca2+ concentration were inferred from the ratio of fura-2 fluorescence intensity (FFI) at the two wavelengths (360 and 380 nm). Fluorescence decay time was measured as an indication of the intracellular Ca2+ clearing rate. Both single- and bi-exponential curve fit programs were applied to calculate the intracellular Ca2+ decay constant [5]. Histological examination After anesthesia, hearts were excised and immediately placed in 10% neutral-buffered formalin at room temperature for 24 hours after a brief rinse with PBS. The specimens were embedded in paraffin, cut into 5 μm sections and stained with H & E as well as fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin. Heart sections were stained with H & E for gross morphology analysis. Thereafter, the slides were washed three times with PBS, mounted with aqueous mounting media and cover-slipped. Cardiomyocyte cross-sectional areas were calculated on a digital microscope (× 400) using the Image J (version1.34S) software [5,20]. Caspase-3 assay Tissue homogenates were centrifuged (10,000 g at 4°C, 10 minutes) and pellets were lysed in an ice-cold cell lysis buffer. The assay was carried out in a 96-well plate with each well containing 30 μL cell lysate, 70 μL of assay buffer (50 mM HEPES, 0.1% 3-([3-cholamidopropyl]-dimethyllammonio)-1-propanesulfonate (CHAPS), 100 mM NaCl, 10 mM dithiothreitol and 1 mM ethylenediaminetetraacetic acid) and 20 μL of caspase-3 colorimetric substrate Ac-DEVD-pNA. The 96-well plate was incubated at 37°C for one hour, during which time the caspase in the sample was allowed to cleave the chromophore pNA from the substrate molecule. Caspase-3 activity was expressed as picomoles of pNA released per microgram of protein per minute [5]. Aconitase activity Mitochondrial aconitase, an iron-sulfur enzyme located in the citric acid cycle, is readily damaged by oxidative stress via removal of an iron from the [4Fe-4S] cluster. Mitochondrial fractions prepared from whole heart homogenate were resuspended in 0.2 mM sodium citrate. An aconitase activity assay (Aconitase Activity Assay Kit, Aconitase-340 Assay; OxisResearch, Portland, OR, USA) was performed according to the manufacturer instructions with minor modifications. Briefly, the mitochondrial sample (50 μL) was mixed in a 96-well plate with 50 μL trisodium citrate (substrate) in Tris-HCl pH 7.4, 50 μL isocitrate dehydrogenase (enzyme) in Tris-HCl, and 50 μL NADP in Tris-HCl. After incubating for 15 minutes at 37°C, the absorbance was dynamically recorded at 340 nm every minute for five minutes with a spectrophotometer. During the assay, citrate is isomerized by aconitase into isocitrate and eventually α-ketoglutarate. The Aconitase-340 Assay measures NADPH formation, a product of the oxidation of isocitrate to α-ketoglutarate. Tris-HCl buffer (pH 7.4) was served as the blank [25]. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining of myonuclei positive for DNA strand breaks was determined using a fluorescence detection kit (Roche, Indianapolis, IN, USA) and fluorescence microscopy. Paraffin-embedded sections (5 μm) were incubated with Proteinase K solution for 30 minutes. TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein-dUTP was added to the sections in 50 μL drops and incubated for 60 minutes at 37°C in a humidified chamber in the dark. The sections were rinsed three times in PBS for five minutes each. Following embedding, sections were visualized with an Olympus BX-51 microscope equipped with an Olympus MaguaFire SP digital camera. DNase I and label solution were used as positive and negative controls. To determine the percentage of apoptotic cells, micrographs of TUNEL-positive and 4'-6-dia! midino-2-phenylindole-stained nuclei were captured using an Olympus fluorescence microscope and counted using the ImageJ software (ImageJ version 1.43r; National Institutes of Health) followed by manual exclusion of the false-positive staining from 10 random fields at 400 × magnification. At least 100 cells were counted in each field [26]. Measurement of mitochondrial membrane potential Murine cardiomyocytes were suspended in HEPES saline buffer and the mitochondrial membrane potential (ΔΨm) was detected as previously described [27]. Briefly, after incubation with JC-1 (5 μM) for 10 minutes at 37°C, cells were rinsed twice by sedimentation using the HEPES saline buffer free of JC-1 before being examined under a confocal laser scanning microscope (Leica TCS SP2, Leica Microsystems Inc. Buffalo Grove, IL, USA) at an excitation wavelength of 490 nm. The emission of fluorescence was recorded at 530 nm (monomer form of JC-1, green) and at 590 nm (aggregate form of JC-1, red). Results in fluorescence intensity were expressed as the 590 nm to 530 nm emission ratio. The mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (10 μM) was used as a positive control for the mitochondrial membrane potential measurement. Western blot analysis The myocardial protein was prepared as previously described [27]. The antibodies used for western blotting included anti-PGC-1α, anti-UCP-2 (EMD Millipore Billerica, MA, USA), anti-ALDH2 (gift from Dr. Henry Weiner, Purdue University, West Lafayette, IN, USA), anti-GSK3β, anti-phosphorylated(p)-GSK3β (Ser9), anti-Akt, anti-pAkt (Thr308), anti-Foxo3a, anti-pFoxo3a (Thr32), anti-mTOR, anti-pmTOR (Ser2448), anti-PTEN, anti-pPTEN (Ser380), anti-sarcoendoplasmic reticulum Ca2+-ATPase (SERCA2a; Affinity Bioreagents Inc., Golden, CO, USA), anti-Na+-Ca2+ exchanger (1:1000; Sigma, St. Louis, MO, USA), anti-phospholamban (1:500; Abcam, Cambridge, MA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; loading control). Antibodies for GSK3β, pGSK3β, Akt, pAkt, Foxo3a, pFoxo3a, mTOR, pmTOR, PTEN and pPTEN were purchased from Cell Signaling Technology (Beverly, MA, USA) while antibody for PGC-1α was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) unless otherwise indicated. The membranes were incubated with horseradish peroxidase-coupled secondary antibodies. After immunoblotting, films were scanned and detected with a Bio-Rad Calibrated Densitometer Hercules, CA, USA) Data analysis Data are presented as the mean ± standard error of the mean (SEM). Statistical comparison was performed by a one-way analysis of variance (ANOVA), with a two-way ANOVA for RER and physical activity studies, followed by Tukey's post hoc test. Significance was set as P < 0.05. Results General features, mitochondrial and metabolic properties of normal and diabetic mice STZ treatment significantly reduced body but not organ weights in FVB and ALDH2 mice. Although STZ treatment did not affect the size of the kidney (organ-to-body weight ratio), it overtly increased heart and liver sizes in FVB but not ALDH2 mice. ALDH2 transgene did not affect the body and organ weights or organ sizes. As expected, blood glucose (fasting and postprandial) and free fatty acid levels were significantly elevated whereas plasma insulin levels were severely decreased in STZ-challenged mice compared with the non-diabetic FVB mice. ALDH2 did not affect the levels of fasting or postprandial blood glucose, serum free fatty acids or plasma insulin in either normal or diabetic groups (Table 1). Furthermore, experimental diabetes decreased both protein level and enzymatic activity of ALDH2, the effect of which was masked by ALDH2 overexpression. STZ elicited overt apoptosis (as evidenced by caspase-3 activity) and mitochondrial damage (reduced aconitase activity). Although ALDH2 enzymatic overexpression itself had little effect on apoptosis and mitochondrial function, it significantly attenuated or nullified diabetes-induced apoptosis and mitochondrial damage (Figure 1). Table 1 Biometric parameters of control or diabetic FVB and ALDH2 mice Parameter FVB FVB-STZ ALDH2 ALDH2-STZ Body weight (g) 30.3 ± 1.3 24.2 ± 0.7a 29.5 ± 1.1 26.1 ± 0.8a Heart weight (mg) 165 ± 9 171 ± 8 163 ± 6 159 ± 5 Heart/body weight ratio (mg/g) 5.45 ± 0.19 7.23 ± 0.43a 5.66 ± 0.26 6.16 ± 0.18b Liver weight (g) 1.54 ± 0.07 1.44 ± 0.07 1.52 ± 0.05 1.46 ± 0.06 Liver/body weight (mg/g) 51.2 ± 1.4 59.4 ± 2.3a 51.9 ± 1.9 54.9 ± 1.9 Kidney weight (g) 0.45 ± 0.03 0.39 ± 0.02 0.42 ± 0.02 0.40 ± 0.03 Kidney/body weight (mg/g) 14.8 ± 0.4 16.1 ± 0.8 14.4 ± 0.4 15.4 ± 0.8 Plasma insulin (ng/mL) 0.27 ± 0.03 0.05 ± 0.01a 0.28 ± 0.03 0.06 ± 0.02a Fasting blood glucose (mM) 5.50 ± 0.26 19.7 ± 1.5a 5.43 ± 0.18 18.5 ± 1.7a Postprandial blood glucose (mM) 9.94 ± 0.67 25.3 ± 1.5a 10.57 ± 0.54 26.5 ± 1.8a Plasma free fatty acids (mM) 1.00 ± 0.11 1.30 ± 0.04a 1.00 ± 0.15 1.25 ± 0.08a Mean ± SEM, n = 21 to 22 mice per group. aP < 0.05 versus FVB group; bP < 0.05 versus FVB-STZ group. Figure 1 Influence of streptozotocin and ALDH2 on expression and activity of ALDH2, apoptosis and mitochondrial function. (A) ALDH2 expression; (B) ALDH2 enzymatic activity measured using spectrophotometry; (C) caspase-3 activity; (D) mitochondrial aconitase activity. Inset: Representative gel blots of ALDH2 and GAPDH (loading control) using specific antibodies. Mean ± SEM, n = 5 to 7 per group. *P < 0.05 versus FVB group; #P <0.05 versus FVB-STZ group. To evaluate the impact of ALDH2 overexpression on metabolic indices, calorimetric parameters were obtained in control and diabetic FVB and ALDH2 mice using the six-chamber Oxymax system (Columbus Instruments, Columbus, OH, USA) as previously described [19]. Diabetic FVB and ALDH2 mice had a significantly lower RER (VCO2/VO2), especially during the dark cycle, compared with control FVB and ALDH2 mice. This diabetes-associated change in RER was not due to altered physical activity. There was little difference in physical activity among the four mouse groups tested (Figure 2). These results indicate that STZ-induced diabetic mice oxidized a lower proportion of carbohydrate compared with control mice, reflecting a higher fractional reliance on lipid rather than glucose as the main energy source. ALDH2 overexpression did not alter the energy source globally in control or diabetic mice. Figure 2 Oxygen consumption and total activity during day and night time in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Respiratory exchange ratio (RER) during day time; (B) RER at night; (C) total activity during day time; (D) total activity at night. Mean ± SEM, n = 4 to 5 mice per group. *P < 0.05 versus FVB group for both STZ groups. Echocardiographic properties of normal and diabetic mice Heart rate and absolute LV mass (calculated using echocardiography) were comparable among all mouse groups, regardless of diabetic or ALDH2 state. However, diabetes increased the normalized LV mass value in the FVB but not ALDH2 group, consistent with its effect on gross heart weights. Experimental diabetes significantly increased EDD and ESD and decreased wall thickness, the effect of which was mitigated by ALDH2. Interestingly, STZ treatment significantly depressed fractional shortening in FVB mice. ALDH2 mitigated the diabetes-induced decrease in fractional shortening without eliciting any effect itself (Figure 3). Figure 3 Echocardiographic properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Representative echocardiographic images; (B) heart rate; (C) wall thickness; (D) left ventricular (LV) end-diastolic diameter; (E) LV end-systolic diameter; (F) LV mass; (G) LV mass normalized to body weight; (H) fractional shortening. Mean ± SEM, n = 12 to 13 mice per group. *P <0.05 versus FVB group; #P <0.05 versus FVB-STZ group. Cardiomyocyte contractile and intracellular Ca2+ properties Neither experimental diabetes nor ALDH2 overexpression affected resting cell length, as depicted in Figure 4. Experimental diabetes significantly reduced PS and maximal velocity of shortening and relengthening (± dL/dt) as well as prolonged TPS and TR90 in FVB cardiomyocytes, reminiscent of our earlier findings [28,29]. Importantly, ALDH2 abolished diabetes-induced mechanical abnormalities without eliciting any notable effect by itself. To explore the potential mechanism of action involved in the ALDH2-elicited beneficial effects against experimental diabetes, fura-2 fluorescence microscopy was employed to monitor the intracellular Ca2+ homeostasis. Data presented in Figure 5 reveal a significantly depressed intracellular Ca2+ rise in response to electrical stimulus (ΔFFI) and reduced intracellular Ca2+ decay rate (single or bi-exponential curve fit) along with unchanged baseline intracellular Ca2+ in cardiomyocytes from STZ-treated mouse hearts. ALDH2 overexpression negated STZ-induced prolongation in intracellular Ca2+ decay and depression in ΔFFI with little effect on baseline FFI. Last but not least, the ALDH2 transgene itself did not affect the intracellular Ca2+ indices tested. Figure 4 Cardiomyocyte contractile properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Resting cell length; (B) peak shortening (PS), normalized to cell length; (C) maximal velocity of shortening (+ dL/dt); (D) maximal velocity of relengthening (- dL/dt); (E) time-to PS (TPS); (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 101 to 102 cells from four mice per group. *P <0.05 versus FVB group; #P <0.05 versus FVB-STZ group. Figure 5 Cardiomyocyte intracellular Ca2+ handling properties in FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A) Resting fura-2 fluorescence intensity (FFI); (B) electrically-stimulated rise in FFI (ΔFFI); (C) intracellular Ca2+ decay rate (single exponential); (D) intracellular Ca2+ decay rate (bi-exponential). Mean ± SEM, n = 76 to 77 cells from four mice per group. *P <0.05 versus FVB group; #P <0.05 versus FVB-STZ group. Effect of ALDH2 on diabetes-induced change in myocardial histology To assess the impact of ALDH2 overexpression on myocardial histology after STZ treatment, heart gross morphology and the cardiomyocyte cross-sectional area were examined. Low magnification transverse heart sections indicated reduced LV wall thickness and enlarged chamber size in mice with experimental diabetes, with lesser alteration in ALDH2 transgenic mice. Findings from FITC-conjugated wheat germ staining sections revealed increased cardiomyocyte area after the induction of experimental diabetes, consistent with increased normalized LV mass, EDD and ESD in FVB-STZ mice. The experimental diabetes-induced change in cardiomyocyte size was effectively ablated by ALDH2 overexpression while the ALDH2 transgene itself did not affect cardiomyocyte size (Figure 6). Figure 6 Histological analyses in hearts from FVB and ALDH2 transgenic mice treated with or without streptozotocin. (A-D) Representative photomicrographs from gross morphological view of transverse myocardial sections (scale bar = 1 mm). (E-H) Representative fluorescein isothiocyanate-conjugated wheat germ agglutinin staining depicting cardiomyocyte size (× 200; scale bar = 100 μm). (I) Quantitative cardiomyocyte cross-sectional (transverse) area from 60 cells from three mice per group. Mean ± SEM, *P < 0.05 versus FVB; #P < 0.05 versus FVB-STZ group. Effects of ALDH2 on diabetes-induced apoptosis and mitochondrial damage To further examine the mechanism(s) of action behind ALDH2-elicited protection against STZ-induced cardiac mechanical dysfunction, the myocardium and cardiomyocytes from normal or diabetic FVB and ALDH2 mice were examined for myocardial apoptosis, using TUNEL staining, and mitochondrial integrity, using JC-1 fluorescence microscopy. Results shown in Figure 7 (panels A-I) indicate that the TUNEL-positive cells were more abundant in STZ-treated FVB mice, the effect of which was significantly attenuated by ALDH2 overexpression. ALDH2 itself did not affect myocardial apoptosis. Our fluorescence data displayed in Figure 7 (panels J and K) revealed loss of mitochondrial membrane potential in cardiomyocytes from STZ-treated FVB mice, the effect of which was reconciled by ALDH2 overexpression. ALDH2 itself did not affect the mitochondrial membrane potential. These findings indicate a corroborative role of apoptosis and mitochondrial function in ALDH2-offered cardioprotection against experimental diabetes. Figure 7 Effect of streptozotocin and ALDH2 on myocardial apoptosis and mitochondrial membrane potential using TUNEL staining and JC-1 fluorescence. TUNEL-positive nuclei were visualized with fluorescein (green) in panels (A) FVB, (C) FVB-STZ, (E) ALDH2 and (G) ALDH2-STZ. All nuclei were stained with 4'-6-diamidino-2-phenylindole (blue) in panels (B) FVB, (D) FVB-STZ, (F) ALDH2 and (H) ALDH2-STZ. Original magnification × 400. (I) Quantified data. (J) Quantitative analysis of cardiomyocyte mitochondrial membrane potential using JC-1 ratio in FVB and ALDH2 mice treated with or without STZ (10 μM carbonyl cyanide m-chlorophenylhydrazone was used as a positive control). (K) Representative JC-1 fluorochrome images depicting mitochondrial membrane potential in cardiomyocytes. Mean ± SEM, n = 12 and 7 fields from three mice per group for panel I and J, respectively. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. Expression of UCP-2, PGC1α, SERCA2a, Na+-Ca2+ exchanger and phospholamban To explore the possible mechanism behind ALDH2 and diabetes-induced responses on cardiac function, particularly on mitochondrial function and intracellular Ca2+ homeostasis, western blot analysis was performed to assess the levels of the key mitochondrial proteins UCP-2 and PGC1α as well as the essential intracellular Ca2+ regulatory proteins SERCA2a, Na+-Ca2+ exchanger and phospholamban. Our data shown in Figure 8 depict that diabetes significantly downregulated the expression of UCP-2, PGC1α, SERCA2a and Na+-Ca2+ exchanger while upregulating the level of phospholamban in FVB mice. Although the ALDH2 transgene itself did not alter the expression of UCP-2, PGC1α, SERCA2a, Na+-Ca2+ exchanger or phospholamban, it nullified STZ-induced changes in all five. Figure 8 Western blot analysis of the mitochondrial proteins UCP-2 and PGC1α as well as the Ca2+ regulatory proteins SERCA2a, Na+-Ca2+ exchanger and phospholamban in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) Representative gel blots of UCP-2, PGC1α, SERCA2a, Na+-Ca2+ exchanger, phospholamban and GAPDH (loading control) using specific antibodies; (B) UCP-2; (C) PGC1α; (D) SERCA2a; (E) Na+-Ca2+ exchanger; (F) phospholamban. All proteins were normalized to the loading control GAPDH. Mean ± SEM, n = 5 to 6 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. Western blot analysis for Akt, GSK3β, Foxo3a, mTOR and PTEN signaling To examine possible signaling mechanism(s) involved in the ALDH2-offered protection against diabetes-induced myocardial anomalies, the expression and phosphorylation of post-insulin receptor signaling, including Akt and the Akt downstream signaling molecules GSK3β, Foxo3a and mTOR, were evaluated. Western blot findings revealed that diabetes overtly dampened phosphorylation of Akt, GSK3β and Foxo3a without affecting that of mTOR, the effect of which was mitigated by ALDH2 overexpression. Neither diabetes nor ALDH2 affected the pan protein expression of Akt, Foxo3a or mTOR (Figure 9). To explore the possible mechanisms responsible for ALDH2- and experimental diabetes-elicited changes in Akt phosphorylation, levels of pan and pPTEN, a negative regulator of Akt signaling, were examined in control and diabetic FVB and ALDH2 mice. Our data shown in Figure 10 revealed that STZ treatment significantly increased phosphorylation of PTEN (both absolute and normalized value) without affecting pan protein expression of PTEN, the effect of which was mitigated by ALDH2 overexpression. The ALDH2 transgene itself did not affect pan or phosphorylated levels of PTEN. Figure 9 Phosphorylation of Akt, GSK3β, mTOR and Foxo3a in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) pAkt-to-Akt ratio; (B) pGSK3β-to-GSK3β ratio; (C) pmTOR-to-mTOR ratio; (D) pFoxo3a-to-Foxo3a ratio. Insets: representative gel blots of pan and phosphorylated Akt, GSK3β, mTOR and Foxo3a (GAPDH as loading control) using specific antibodies. Mean ± SEM, n = 6 to 7 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. Figure 10 Total and phosphorylated PTEN in myocardium from FVB and ALDH2 mice treated with or without streptozotocin. (A) Representative gel blots of pan and phosphorylated PTEN and GAPDH (used as loading control) using specific antibodies; (B) pan PTEN expression; (C) pPTEN levels; (D) pPTEN-to-PTEN ratio. Mean ± SEM, n = 6 mice per group. *P < 0.05 versus FVB group; #P < 0.05 versus FVB-STZ group. Influence of ALDH2 activation, mitochondrial uncoupling and GSK3β inhibition on high glucose-induced cardiomyocyte mitochondrial and contractile responses To further examine the causal relationships between ALDH2-induced mechanical and mitochondrial responses in diabetes, cardiomyocytes from control FVB mice were exposed to high glucose (25.5 mM) in the absence or presence of the ALDH2 activator Alda-1 (20 μM), the mitochondrial uncoupler FCCP (1 μM) or the GSK3β inhibitor SB216763 (10 μM) for 12 hours [6,9,30] prior to an assessment of mechanical and biochemical properties. Figure 11 depicts that high glucose significantly dampened mitochondrial function (shown as loss of aconitase activity) and cardiomyocyte contractile function (shown as reduced PS, ± dL/dt and prolonged TR90), the effect of which was abolished by Alda-1 and SB216763, without any additive effect between the two. Interestingly, FCCP abolished Alda-1-induced beneficial mitochondrial and mechanical effects. There was little effect on mitochondrial integrity and mechanical function by the pharmacological inhibitors themselves (data not shown for FCCP). Figure 11 Influence of the ALDH2 agonist Alda-1 on high glucose-induced responses of aconitase activity and cardiomyocyte contractile properties. Cardiomyocytes from control FVB mice were exposed to high glucose (HG; 30 mM) in the absence or presence of Alda-1 (20 μM), the mitochondrial uncoupler FCCP (1 μM) or the GSK3β inhibitor SB216763 (10 μM) for 12 hours prior to assessment of mitochondrial and mechanical properties. (A) Aconitase activity; (B) peak shortening (PS; normalized to cell length); (C) maximal velocity of shortening (+ dL/dt); (D) maximal velocity of relengthening (- dL/dt); (E) time-to PS (TPS); (F) time-to-90% relengthening (TR90). Mean ± SEM, n = 5 isolations (panel A) or 72 to 73 cells per group (Panel B-F). *P < 0.05 versus control group; #P < 0.05 versus HG group. Discussion Earlier findings from our group indicated that ALDH2 may rescue against ischemic and alcoholic injuries to the heart [3,7,15]. Data from this study provides, for the first time, compelling evidence that ALDH2 protects against diabetes-induced myocardial remodeling and contractile defect through lessened apoptosis, preserved mitochondrial function and post-insulin receptor signaling, including phosphorylation of Akt, GSK3β and Foxo3a transcriptional factor. These data favor a likely role of the activation of Akt and GSK3β as well as inactivation (phosphorylation) of Foxo3a in ALDH2-elicited preservation of mitochondrial and mechanical function in diabetes. Our data further reveal that ALDH2 may preserve Akt activation in diabetes through ablation of diabetes-induced mitochondrial injury and/or increasing the phosphorylation of PTEN, a negative regulator of Akt [16]. An analysis of global metabolism indicated that ALDH2 failed to alter diabetes-induced changes in plasma levels of glucose (fasting and postprandial), insulin and serum free fatty acids, the RER or total physical activity, excluding the possibility of a potential cardiac protective effect secondary to any ALDH2-elicited global metabolic benefits. Taken together, these findings should lead to a better understanding of the role of ALDH2 in myocardial anomalies in diabetes. Reduced contractility and prolonged duration of systole as well as diastole are hallmarks of diabetic cardiomyopathy [17,28,29]. Findings from our present study revealed reduced fractional shortening; enlarged EDD and ESD; decreased wall thickness, PS and ± dL/dt; and prolonged TPS and TR90 in whole hearts and isolated cardiomyocytes in diabetic mice. These findings are similar to our previous findings [17,28,29]. Several mechanisms may be postulated for diabetes-related abnormalities such as impaired intracellular Ca2+ homeostasis and oxidative stress [28,29]. In our study, the impaired intracellular Ca2+ handling (reduced intracellular Ca2+ clearance and intracellular Ca2+ rise (ΔFFI)) may likely underscore the prolonged duration of contraction and relaxation and the reduced PS, maximal velocity of shortening and relengthening and fractional shortening observed in STZ-induced diabetic mouse hearts. The fact that the ALDH2 transgene reconciled STZ-induced intracellular Ca2+ mishandling favors a possible role of intracellular Ca2+ homeostasis in diabetes-induced myocardial dysfunction and ALDH2-offered protection, somewhat reminiscent of the beneficial role of mitochondrial protection against diabetes or obesity-induced myocardial dysfunction [31,32]. Our findings revealed a loss of mitochondrial membrane potential and overt apoptosis (demonstrated by caspase-3 and TUNEL) along with downregulated levels of PGC1α and UCP-2 in STZ-induced diabetic hearts, suggesting a corroborative role of mitochondrial dysfunction and apoptosis in diabetic cardiomyopathy, as reported previously [31]. In addition, our observations that the ALDH2 transgene restored downregulated expression of SERCA2a and Na+-Ca2+ exchanger as well as upregulated phospholamban in diabetes also support a role of intracellular Ca2+ homeostasis in diabetes-induced cardiac contractile dysfunction and ALDH2-offered protection. Perhaps our most significant finding is that ALDH2 overexpression reconciled diabetes-induced cardiac remodeling (represented by cardiomyocyte cross-sectional area, changes in LV wall thickness, ESD and EDD) and contractile dysfunction in association with preserved myocyte survival and mitochondrial integrity. These beneficial effects of ALDH2 in cardiac geometry and function, cell survival and mitochondrial integrity were seen despite the persistent hyperglycemic and hyperlipidemic environments in STZ-induced experimental diabetes, thus excluding a possible secondary effect for ALDH2-induced protection against diabetic cardiomyopathy. This is further supported by the fact that ALDH2 failed to alter global metabolism (RER and physical activity) in diabetes. In our study, STZ failed to elicit any hypertrophic effect as evidenced by absolute heart weight and LV mass, although it enhanced cardiomyocyte size and heart-to-body weight ratio, and normalized LV mass. These effects were likely due to an STZ-induced loss in body weight. Interestingly, ALDH2 overexpression attenuated diabetes-induced changes in cardiomyocyte, heart and LV sizes, possibly due to the antagonism of ALDH2 against diabetes-induced cardiac apoptosis and mitochondrial damage. Both apoptosis and mitochondrial damage are known to regulate cardiac remodeling in diabetes and obesity [31,33]. Our observation of preserved levels of the mitochondrial proteins PGC1α and UCP-2 as well as of aconitase activity and mitochondrial membrane potential in ALDH2 mice after STZ treatment strongly supported a role of mitochondrial function in ALDH2-offered cardioprotection. The therapeutic role of the mitochondrial protein ALDH2 in diabetes is consistent with the fact that the protein level and enzymatic activity of ALDH2 are both reduced in experimental diabetes [13] (also seen in our study) while inactive ALDH2 promotes hyperglycemia and enhances the risk of diabetes [14]. It is noteworthy that the reduction in the ALDH2 expression and activity is relatively minor although such subtle loss of ALDH2 may be sufficient to trigger overt changes in mitochondrial integrity and cell survival. Although it is beyond the scope of our current study, the main substrate for ALDH2 detoxification, aldehydes, serve as the main source for oxidative stress and pathological changes in disease condition. Even with a moderately reduced ALDH2 level, sublethal levels of aldehydes may accumulate and interact with functional signaling systems to impose oxidative damage and associated gene alterations in response to the stress challenge [34]. The notion that ALDH2 protects against diabetic cardiomyopathy through preservation of mitochondrial integrity was further substantiated by our in vitro findings. Our results revealed that the ALDH2 activator Alda-1 effectively rescued against high glucose-induced mitochondrial and mechanical dysfunctions, and this effect was nullified by the mitochondrial uncoupling compound FCCP. These data convincingly support the permissive role of mitochondria in ALDH2-offered cardioprotection against hyperglycemia-induced anomalies. Data from our study showed dampened phosphorylation of the post-insulin receptor signaling Akt, GSK3β and Foxo3a in STZ-treated diabetic hearts, in line with mitochondrial injury in diabetes and observations from our earlier studies [28,35]. These signaling molecules play an essential role in the maintenance of cardiac survival, structure and function. Akt, GSK3β and mTOR are essential post-insulin receptor signaling molecules, which may be compromised after mitochondrial injury and contribute to apoptosis and cardiac dysfunction in pathological conditions [10,14,36]. Our data revealed that diabetes dampened phosphorylation of Akt and its downstream signaling molecules Foxo3a and GSK3β (although not mTOR), the effect of which was nullified by ALDH2 transgene. The decrease in the phosphorylation of Foxo3a and GSK3β is expected to result from dampened Akt phosphorylation. The reduced phosphorylation of Foxo3a appears to coincide with overt mitochondrial injury (as evidenced by mitochondrial membrane potential and levels of PGC1α, UCP-2 and aconitase) and apoptosis (demonstrated by caspase-3 and TUNEL staining) after STZ treatment, as reported previously by our group using the same diabetic model [35]. GSK3β, a serine/threonine kinase downstream of Akt that is inactivated by oxidative stress through the phosphorylation of Ser9, serves as a negative regulator of cardiac hypertrophy and mitochondrial function through mitochondrial permeation pore opening [24,25,37]. Data from our study revealed that ALDH2 abrogated the diabetes-induced decrease in GSK3β phosphorylation, aconitase activity and levels of PGC1α and UCP-2, favoring a possible role of GSK3β signaling and mitochondrial protection in ALDH2-offered cardioprotection. This is supported by the finding that GSK3β inhibition using SB216763 and mitochondrial uncoupling using FCCP respectively ablated high glucose and Alda-1-induced mitochondrial and mechanical changes. Mitochondrial injury is known to compromise insulin signaling at both insulin receptor and post-receptor levels [30]. A recent report from our group revealed that protection of mitochondrial integrity using cardiac-specific overexpression of insulin-like growth factor 1 effectively alleviates high fat diet intake-induced loss of insulin sensitivity, oxidative stress and contractile dysfunction in the heart [38], supporting the pivotal role of mitochondria in the maintenance of cardiac insulin signaling. Nonetheless, our data also depicted elevated phosphorylation of the Akt negative regulator PTEN in experimental diabetes, the effect of which was mitigated by ALDH2. This finding favors a possible role for PTEN in ALDH2 overexpression-rescued Akt activation in experimental diabetes. These observations suggest that ALDH2 offers cardioprotection against experimental diabetes, possibly through suppressed PTEN phosphorylation and subsequently preserved Akt-GSK3β phosphorylation, leading to protected mitochondrial integrity. Conclusion In summary, findings from our present study reveal a role of ALDH2 in the protection against diabetic cardiomyopathy, possibly via an Akt-GSK3β-mediated preservation of cell survival and mitochondrial integrity. These data indicate not only a role of ALDH2 in the prevalence of diabetic cardiomyopathy but also some therapeutic promise for ALDH2 in the management of diabetic complications. As the important cardioprotective aspects of ALDH2 begin to be unveiled, clinical implications of ALDH2, in particular ALDH2 polymorphism, still remain to be explored. Further studies should focus on a better elucidation of the link between the ALDH2 gene and cardiovascular risk in diabetic populations. Abbreviations + dL/dt: maximal velocity of shortening; - dL/dt: maximal velocity of relengthening; ALDH2: mitochondrial aldehyde dehydrogenase; ANOVA: analysis of variance; EDD: end-diastolic diameter; ELISA: enzyme-linked immunosorbent assay; ESD: end-systolic diameter; FCCP: carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; FFI: fura-2 fluorescence intensity; FITC: fluorescein isothiocyanate; GADPH: glyceraldehyde 3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase-3β; H & E: hematoxylin and eosin stain; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LV: left ventricular; mTOR: mammalian target of rapamycin; p: phosphorylated; PBS: phosphate-buffered saline; PPAR: peroxisome proliferator-activated receptor; PTEN: phosphatase and tensin homologue on chromosome ten; PS: peak shortening; RER: respiratory exchange ratio; SEM: standard error of the mean; SERCA2a: sarcoendoplasmic reticulum Ca2+-ATPase; STZ: streptozotocin; TPS: time-to peak shortening; TR90: time-to-90% relengthening; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. Competing interests The authors declare that they have no competing interests. Authors' contributions YZ, SAB, NH and JRM participated in data collection; YZ, HW, JR designed the study, secured the research funding and wrote the manuscript. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1741-7015/10/40/prepub Acknowledgements This work was supported by NIAAA 1R01 AA013412 and NCRR 5P20RR016474 to JR. ==== Refs Budas GR Disatnik MH Mochly-Rosen D Aldehyde dehydrogenase 2 in cardiac protection: a new therapeutic target? 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22984472PONE-D-12-1533610.1371/journal.pone.0044174Research ArticleBiologyModel OrganismsAnimal ModelsRatMolecular cell biologyCellular TypesEpithelial CellsNucleic acidsRNARNA synthesisCell DeathCell GrowthCellular Stress ResponsesGene ExpressionToxicologyToxic AgentsMedicineDiagnostic MedicinePathologyGeneral PathologyBiomarkersKidney Injury Molecule-1 Is Up-Regulated in Renal Epithelial Cells in Response to Oxalate In Vitro and in Renal Tissues in Response to Hyperoxaluria In Vivo KIM-1 as a Marker of HyperoxaluriaKhandrika Lakshmipathi 1 Koul Sweaty 1 Meacham Randall B. 1 Koul Hari K. 1 2 3 * 1 Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology- Department of Surgery, School of Medicine, University of Colorado at Denver, Aurora, Colorado, United States of America 2 University of Colorado Comprehensive Cancer Center, University of Colorado at Denver, Anschutz Medical Campus, Aurora, Colorado, United States of America 3 Denver Veterans Administration Medical Center, Denver, Colorado, United States of America Dahiya Rajvir Editor UCSF/VA Medical Center, United States of America * E-mail: [email protected] Interests: Senior author Dr. Koul is an Academic Editor with PLOS One. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials. Conceived and designed the experiments: HK SK LK. Performed the experiments: LK SK. Analyzed the data: HK RBM SK LK. Contributed reagents/materials/analysis tools: SK HK LK. Wrote the paper: HK LK SK. Designed experiments: HK. Conducted all experiments with animal model: LK. Conducted all experiments with cell cultures: SK. Analyzed data: HK SK LK RBM. Wrote the manuscript: HK SK LK. Supervised experiments: HK. 2012 12 9 2012 7 9 e4417423 5 2012 30 7 2012 © 2012 Khandrika et al2012Khandrika 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.Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic in vitro and in vivo models of hyper-oxaluria. Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria. This work was supported in part by a research grant to HKK from National Institutes of Health (NIH-RO1-DK-54084) and the Department of Surgery Academic Enrichment Funds (HKK).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Hyper-oxaluria, either as a result of inherited metabolic disorders or many other intrinsic and extrinsic factors, is one of the major risk factors for developing kidney stones [1], [2]. Studies with animal models as well as tissue culture model systems, have demonstrated injury to the epithelial cells of the kidney in presence of oxalate and or calcium oxalate crystals. Persistently high concentrations of Oxalate in the body can lead to end stage renal failure, severe systemic oxalosis and can finally result in premature death of most of the patients [3]. Current diagnostic methods depend on the estimation of concentrations of Oxalate and Glycolate in the urine, however, the accuracy of utilizing these measures is misleadingly low, especially in end stage renal failure [4]. Monitoring tubular injury in these conditions may offer a more reliable measure of severity of the disease. In 1994, we were the first group to note that oxalate renal cell interactions involved alterations in gene expression [5]. Over the past two decades, studies [5]–[12] have demonstrated that oxalate interactions with renal epithelial cells result in a program of events, including changes in gene expression and cell dysfunction, consistent with cellular stress. Oxalate a metabolic end product is freely filtered at glomerulous and undergoes bi-directional transport in the renal tubules. We demonstrated for the first time that oxalate exposure to the renal cells results in plethora of changes, including changes in gene expression, re-initiation of DNA synthesis, and cell death [5]. Over the years others and we have shown that exposure to Oxalate results in activation of many different pathways, gene expression changes and initiation of DNA synthesis in the epithelial cells [6]–[16]. Studies in our laboratory have focused on changes in signal transduction pathways mediated by JNK and p38 MAPK upon Oxalate exposure [7], [17]. Even though extensive studies have been attempted to understand cellular changes occurring as a result of hyperoxaluria, early detection of Oxalate exposure still remains a challenge. The need for a reliable and easily detectable early marker for tubular injury as a result of hyper-oxaluria has not been answered yet. Several recent studies have suggested the usefulness of Kidney Injury Molecule -1 (KIM-1) as an early indicator for renal injury resulting from chemical toxicity or ischemic injury [18], [19]. KIM-1 was first identified as a putative cell adhesion molecule that can recognize and induce phagocytosis of dead cells in the tubular lumen of the kidney [20]. Expression of KIM-1 is up-regulated during tubular injury, but most importantly, the stable ectodomain is cleaved from the membrane anchor and can be detected in the urine. This phenomenon has been observed in rodent and human models of nephrotoxicity including in Renal Cell Carcinoma [21]. KIM-1 has been suggested to be an ideal biomarker in many chemical and pathological nephrotoxicity models due to its robust and marked expression in the injured state. Moreover, there is a total lack of its expression in healthy kidneys [22]. In the present study, we determined the effect of hyper-oxaluria on the expression status and shedding of KIM-1. We used both in vitro and in vivo methods that are well documented as models for experimental hyperoxaluria for these studies. For in vitro studies, treating cells in tissue culture with different concentrations of Sodium Oxalate or COM-Crystals has been established as an efficient model system to test the effects of hyper-oxaluria [6]–[16]. In this study, HK-2 cells [23], a line of immortalized human proximal tubular epithelial cells, were exposed to oxalate for different time points. Administration of Ethylene Glycol either alone or in combination with Ammonium Chloride or Vitamin D as an efficient model in generating experimental hyper-oxaluria in rats [24]–[26]. Boeve et al [25]. identified nephrotoxic effects of Ammonium Chloride independent of Ethylene Glycol and though higher doses of ethylene Glycol may be toxic to rodents, 0.75% Ethylene Glycol has been successfully used for experimental hyper-oxaluria studies without deleterious effects [26]. In this study, Male Wistar rats, with an average weight about 150 gm, were supplied drinking water with 0.75% Ethylene Glycol over 4 week duration to induce hyper-oxaluria. Results from both the in vivo and in vitro studies indicate that hyperoxalria increases the expression of KIM-1 mRNA and protein in renal cells and tissues. Moreover, the ectodomain of KIM-1 was also identified in the urine collected from hyper-oxaluric rats by Western blot analysis. This study, thus, suggests that KIM-1 may be a potential urinary biomarker for oxalate mediated renal injury. Results KIM-1 mRNA and protein levels are increased in HK-2 cells upon Oxalate exposure Oxalate exposure has been shown to induce cellular injury in epithelial cells and one of the markers for tubular injury is the over-expression and shedding of Kidney Injury Molecule (KIM-1) [20]. To study the effect of Oxalate exposure on KIM-1 expression, we incubated HK-2 cells in the presence of oxalate for various time points and looked at the mRNA and protein levels of KIM-1. Results presented in Figure 1a suggest that there is a significant increase in the mRNA levels of KIM-1 in as little as 30 min after HK-2 cells are exposed to Sodium Oxalate. This increase in the mRNA levels is sustained over a period of more than 4 hours of Oxalate exposure. Western blot analysis indicated that the KIM-1 protein levels are also elevated 2 hours post Oxalate exposure (Figure 1b). The increase is highly significant as the control cells do not have any detectable KIM-1 protein. These results suggest that oxalate exposure triggers transcriptional up-regulation of KIM-1 gene expression in renal epithelial cells and this increase occurs very early in response to oxalate exposure. 10.1371/journal.pone.0044174.g001Figure 1 Human Kidney Epithelial Cells (HK-2) in culture, exposed to Sodium Oxalate showed increased KIM-1 mRNA and protein levels. (a) Oxalate was added to serum starved HK-2 cells and incubated for various time points. KIM-1 mRNA levels were estimated by Relative quantitative PCR and (Top Panel) representative gel image is shown. (Lower Panel) Quantification of mRNA levels was performed by band density analysis and represented as a relative intensity compared to control. Each data point represents mean +/− S.D. of two independent PCRs (* indicates p<0.05 compared to control, n = 2). (b) Total protein was extracted as in materials and methods. Western blot analysis was used to detect KIM-1 protein levels in the cells. Representative blots from two individual experiments are shown (Top Panel). Densitometric analysis was used to quantify the KIM-1 protein levels. Each data point represents mean +/− S.D. of two independent gels (*indicates p<0.05 compared to control, n = 2). Hyperoxaluria increased Kidney Injury Molecule (KIM-1) expression in rats Since we observed increased KIM-1 expression upon Oxalate exposure in vitro using proximal tubular epithelial cells, we extended the study to an in vivo model for hyper-oxaluria. Wistar rats were given 0.75% Ethylene Glycol in drinking water to develop hyper-oxaluria. Immuno-histochemical staining using the antibody that binds to the C-terminal region of KIM-1 suggested that the expression of the protein is significantly higher in hyper-oxaluric rat kidney in as little as 1 week (Figure 2). Regardless of the duration of hyper-oxaluric exposure, the levels of KIM-1 protein are higher compared to the control rats. 10.1371/journal.pone.0044174.g002Figure 2 Immunohistochemical analysis of KIM-1 expression in kidney tissue of hyper-oxaluric rats. (Left Panel) KIM-1 protein expression in kidney tissue was detected using immunohistochemistry. (Right Panel) protein expression was quantified based on color density from five different 1 cm2 regions of the section. Each data point represents mean +/− S.D. of two independent sections (* indicates p<0.05 compared to control, n = 2). RT-PCR analysis to determine the mRNA levels of KIM-1 also revealed significantly higher mRNA levels in rats exposed to Ethylene Glycol for as little as 1 week compared to control (Figure 3a). Densitometry analysis also suggested that the relative amounts of KIM-1 mRNA remain elevated in the treatment group at least up to three weeks, suggesting that there is sustained damage to the kidneys. To confirm that the increased mRNA levels lead to increased protein amounts, we performed western blot analysis using kidney tissue collected from control and hyper-oxaluric rats. Results presented in Figure 3b show increased levels of KIM-1 total protein in kidney tissue collected from hyper-oxaluric rats. Moreover, KIM-1 protein levels are consistently higher in hyper-oxaluric rats compared to control as long as 3 weeks beginning from the first week of Ethylene Glycol treatment. Taken together, these results suggest increased expression of KIM-1 mRNA and protein in renal tissues of hyper-oxaluric rats. 10.1371/journal.pone.0044174.g003Figure 3 Hyper-oxaluric rats have increased mRNA and protein levels of KIM-1 in renal tissue. (a) KIM-1 mRNA levels were detected by relative quantitative RT-PCR in kidney tissue of control and three individual hyper-oxaluric rats. Representative gel images from three individual PCRs are shown (Top Panel). Quantitative representation of relative KIM-1 mRNA expression across the hyper-oxaluric group compared to control is shown in the Bottom panel. Each data point represents mean +/− S.D. of three experiments (*indicates p<0.05 compared to control, n = 3). (b) Representative gel images from three individual western blots are shown (Upper Panel). Quantitative representation of the relative amounts of KIM-1 protein in the kidney tissue is shown in the lower panel. Each data point represents mean +/− S.D. of three blots (*indicates p<0.05 compared to control, n = 3). Increased KIM-1 expression is associated with deposition of COM-Crystals in the renal tubules of hyper-oxaluric rats Kidneys were dissected out of control and hyper-oxaluric rats after each week and processed for histology, to determine tubular injury. Hematoxylin and Eosin staining showed increased luminal volume suggesting injury leading to tubular dilation. These changes were observed in as little as 2 weeks after the rats were given 0.75% EG in their drinking water (data not shown). Kidney tissue dissected out of rats fed EG for 3 weeks showed evidence of crystal deposition in the lumens of the tubules. Bi-refringent crystals were observed under polarized light (Figure 4a) and there were no significant differences in crystal deposition in the cortical and papillary region. Moreover, the tubular epithelium in the vicinity of the deposited crystals showed evidence of structural damage. 10.1371/journal.pone.0044174.g004Figure 4 Hyper-oxaluric rats showed crystal deposits of Calcium Oxalate in renal tubules after 3 weeks. (a) Hematoxylin and Eosin stained paraffin embedded 5 um sections were photographed under polarized light to detect crystal deposits in the tubules. Representative Images captured using a 10× objective magnification are shown. (b) Von Kossa's staining method was used to identify Calcium deposition in tubules of hyper-oxaluric rat kidneys. Digital photo-micrographs were taken with a 10× magnification. Representative images are shown. To determine the nature of the chemical composition of the crystals, tissue sections were stained with Von Kossa's staining for Calcium. Tissue sections collected from rats after 3 week of EG diet, showed positive von Kossa's staining, suggesting that the observed crystal deposits were primarily composed of Calcium Oxalate (Figure 4b). Tissue collected from control rats did not show any staining further suggesting that the observed staining in the tissue from hyper-oxaluric rats was from Calcium oxalate crystal deposition. Hyper-oxaluria results in shedding of the ectodomain of KIM-1 in rats Tubular injury in rodents has been shown to cause shedding of the ectodomain of KIM-1 protein, which can be detected in the urine. Urine was collected both from control and hyper-oxaluric rats for a period of 24 hours and western blot analysis was used to identify KIM-1 in the urine. Hyper-oxaluric rat urine showed shed KIM-1 protein in the urine, where as the control rats did not have any protein in the urine (Figure 5a). This suggests that hyper-oxaluria not only increases the expression of KIM-1 but also causes protein cleavage and shedding in the urine. Shedding of the ectodomain of KIM-1 protein was observed in conjunction with increased urinary Oxalate excretion (Figure 5b). This increase in Oxalate excretion was significantly higher in as little as 1 week after drinking water with added Ethylene Glycol. 10.1371/journal.pone.0044174.g005Figure 5 Hyper-oxaluria results in increased shedding of KIM-1 ectodomain in rat urine. (a) Western blot to detect KIM-1 protein shed in the urine of two individual hyper-oxaluric rats. Representative gel images from three individual western blots are shown (Upper Panel). Quantitative representation of the relative amounts of KIM-1 protein in urine, averaged across the treatment groups, is shown in the lower panel. Each data point represents mean +/− S.D. of three blots (* indicates p<0.05 compared to control, n = 3). (b) Urine Volume (ml) and Creatinine excretion (mg) values (c) Oxalate (mg) and (d) Calcium (mg) over a period of 24 hours. Each data point represents mean +/− S.D. of individual animals in each group (* indicates p<0.05 compared to control). Our observations also did not suggest any changes in the water or feed intake of the treatment group compared to the control rats. Results presented in Figure 5c, showed that there were no appreciable differences in the volume of urine or in the creatinine clearance by the EG fed rats compared to the control group. In contrast, there was a significant reduction in Calcium excretion (Figure 5d). Moreover, there was a gradual reduction in the amount of total calcium excretion up to 3 weeks. Taken together, these results suggest that hyper-oxaluria increased the expression and shedding of the ectodomain of KIM-1 protein in rats. Discussion Hyper-oxaluria either as a result of increased Oxalate intake or metabolic disorders afflicts different age groups and is a primary source of discomfort. Persistent and prolonged exposure of the kidneys and urinary tract to higher Oxalate loads leads to increased renal injury and is usually considered to be an initiator for developing kidney stones [12]. Instances of uncontrolled hyper-oxaluria leading to multi organ oxalosis and renal failure are well documented [23] Recent studies have identified Kidney Injury Molecule-1 (KIM-1) over-expression and shedding upon nephrotoxicity. This molecule is known to be highly up-regulated in both human and rodent kidney during nephrotoxicity [18]. The present study was designed to determine the status of KIM-1 upon hyper-oxaluric exposure using both in vivo and in vitro models of hyper-oxaluria. In the present study, exposure of HK-2 cells to increased Oxalate concentrations over-expresses KIM-1 mRNA and protein. KIM-1 mRNA levels are significantly increased in as little as 30 min after exposure to oxalate (Figure 1a). Control HK-2 cells do not have KIM-1 protein but increased protein levels are observed in HK-2 cell lysates after exposure to oxalate (Figure 1b). This suggests that KIM-1 mRNA is expressed even in normal epithelial cells but protein expression is found only upon Oxalate exposure. Moreover, stable protein expression is detected in a very short time upon Oxalate exposure. For developing an in vivo model, Wistar rats were given drinking water with 0.75% Ethylene Glycol for a period of 3 weeks. Rat models of hyper-oxaluria were based on Ethylene Glycol in various concentrations ranging from 0.75% to 2.5% in drinking water [28] In addition to Ethylene Glycol; many studies have utilized Ammonium Chloride as an additive for generating hyper-oxaluria in rats. Though, these results in crystal deposition in a shorter time period [29], the nephrotoxic effects cannot be ignored. Our results indicated that rats fed with 0.75% Ethylene Glycol alone have a significant increase in the amount of urinary Oxalate and developed signs of hyper-oxaluria as early as 1 week, without any discomfort or perceptible weight or appetite loss. Our observed results are consistent with other studies wherein, only Ethylene Glycol was used to induce hyper-oxaluria [26]–[29]. Immuno-histochemical staining using antibodies against the C-terminal region of KIM-1 showed up-regulation of KIM-1 in tissue sections collected from hyper-oxaluric rats compared to control (Figure 2). The increase in KIM-1 protein was observed in as little as 1 week after Ethylene Glycol treatment. Even though crystal deposition was not observed after week 1 of Ethylene Glycol diet, KIM-1 mRNA levels were higher suggesting that tubular injury occurs earlier than crystal deposition (Figure 3a). Though, the Creatinine clearance in the rats on Ethylene Glycol diet was comparable to the control animals up to 3 weeks, the up-regulation of KIM-1 mRNA and increased shedding of KIM-1 protein (Figure 5a) in the urine of hyper-oxaluric rats suggests that this may be a better marker for associating tubular injury to hyper-oxaluria. This may be particularly useful as previous studies have not been able to identified tubular damage associated with this experimental model based on creatinine clearance data. KIM-1 has been shown to be localized to proximal tubular cells and may play an important role in the phagocytosis of apoptotic and dead cells [30]. Recent studies have also identified its potential role in removal of dead cells and therefore, contribute to tissue repair [31]. Though the functional significance of KIM-1 shedding is not known, Mitogen Activated Protein Kinases, especially ERK and p38 MAPK, have been shown to play an important role in this process [32]. Previous studies in our laboratory have identified activation of p38 MAPK in kidney epithelial cells exposed to Oxalate and COM-Crystals [7], [8], [17]. Based on these observations, it is tempting to speculate that signaling by p38 MAPK pathway may also play an important role in KIM-1 overexpression and shedding during hyperoxaluria mediated tubular injury, however, additional studies are need to support such conclusions. We observed a reduction in the Urinary Oxalate on week 3 in the hyper-oxaluric rats compared to the amount of urinary oxalate on weeks 1 and 2. This may be a reflection of the observed deposition on birefringent crystals in the lumen of the tubules (Figure 4a). In addition to increased urinary Oxalate, there was also a large reduction in the amount of calcium excreted in the urine after 2 weeks. This further suggested that the deposited crystals in the renal tubules may be composed of Calcium Oxalate. Positive staining results obtained by Von Kossa's staining further confirmed that the observed crystalline deposits were composed of Calcium (Figure 4b). In summary, our study shows that KIM-1 is over-expressed in human kidney epithelial cells upon exposure to higher oxalate concentrations. Since, increased KIM-1 expression is observed over a sustained period of time after hyper-oxaluric exposure, over-expression and shedding of KIM-1 may be potentially used as an early marker in hyper-oxaluria mediated tubular injury. Materials and Methods Chemicals and reagents All chemicals used in this study were procured from Sigma (Sigma-Aldrich, St. Louis, MO). Microcentrifuge concentrators Microcon YM-10 for concentrating rat urine were procured from Millipore Corporation (Billerica, MA). Animals and Treatments Male Wistar rats (Harlan Laboratories, Indianapolis, IN) 3 to 5 week old with an average weight of 150 gm were according to the Center for Laboratory Animal Care (CLAC) guidelines at the University of Colorado Vivarium. Randomized groups were given either control water or water with 0.75% Ethylene Glycol. For metabolic studies, the rats were housed in metabolic cages for a period of 24 hours for measuring water intake and collecting urine for chemical analysis. The rats were acclimatized for a period of 24 hours to ensure continuity. Urine was collected for a period of 24 h at the end of each week and tissue samples from sacrificed animals was asceptically collected and processed. For histological studies, part of the tissue was fixed in formalin and embedded in paraffin wax; or stored in RNAlater solution(Qiagen, Valencia, CA) for extracting RNA. Cells and Cell Culture Human Kidney Epithelial Cells, HK-2 were procured from ATCC and maintained in DMEM medium supplemented with 10% Fetal Bovine Serum and antibiotics. Before Oxalate treatments, cells were serum starved for 16 to 20 hours. Media components were procured form Invitrogen Corporation (Carlsbad, CA) as described previously [9]. Urine Composition analysis Urine analysis for concentrations of different metabolites was performed with the assistance of University of Colorado Hospital Clinical laboratory using standard procedures. Oxalate concentration in the urine was estimated using a AS10 ion chromatography column [33]. Immuno-histochemical analysis Kidney tissue dissected from rats was fixed in 10% Formaldehyde and embedded in Paraffin wax. 5 uM thin sections were used for histochemical staining; sections were stained for assessing fine structures by Hematoxylin and Eosin [34]. Immuno-histochemical staining for expression of KIM-1 was performed as described [35]. Briefly, the tissue sections were heated in Citrate buffer (10 mM sodium citrate and 1 mM EDTA, pH 6.0) for 15 minutes in a microwave oven and blocked with 10% donkey serum. Biotin conjugated secondary antibodies were used to detect primary antibody binding and then stained with streptavidin conjugated HRP antibodies using Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Antibody binding was detected using diaminobenzidine color reaction (Sigma-Aldrich, St. Louis, MO). Detection of Calcium Oxalate crystal deposits was by VonKossa's staining and was done by the Pathology Laboratory of the University of Colorado Denver using standard procedures [36]. RNA isolation and Reverse Transcriptase PCR RNA was isolated from tissue stored in RNAlater using the RNEasy Kit according to manufacturer's recommendations (Qiagen, Valencia, CA). 1 ug total RNA was used to synthesize cDNA using iScript cDNA synthesis Kit (Bio-Rad Laboratories, Hercules, CA). PCR was performed with gene specific primers using Platinum Taq Polymerase (Invitrogen, Carlsbad, CA) and separating the products on a 1% agarose gel. Primers were procured from Integrated DNA Technologies (Coralville, IA) and primer sequences for rat and human Kidney Injury Molecule (KIM-1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are described in Table 1. 10.1371/journal.pone.0044174.t001Table 1 Sequences of primers used in RT-PCR. Primer Sequence KIM-1 (Rat) forward 5′-GGT CAC CCT GTC ACA ATT CC-3′ KIM-1 (Rat) reverse 5′-CTC GGC AAC AAT ACA GAC CA-3′ KIM-1 (Human) forward 5′-CTG CAG GGA GCA ATA AGG AG-3′ KIM-1 (Human) reverse 5′-ACC CAA AAG AGC AAG AAG CA -3′ GAPDH forward 5′-ACC ACA GTC CAT GCC ATC AC-3′ GAPDH reverse 5′- TCC ACC ACC CTG TTG CTG TA-3′ Protein isolation and Western blot analysis Kidney tissue from rats and HK-2 cells were washed with Ice cold PBS and then lysed immediately with boiling hot 2X SDS gel loading buffer with 2- Mercaptoethanol. Cellular proteins were resolved using SDS-Polyacrylamide gels and then transferred onto Polyvinylidene difluoride membrane (Millipore Corporation). Two different antibodies that represent the N- terminal region and the C-terminal region of KIM-1 protein were procured from Abcam Inc. (Cambridge, MA), while GAPDH antibody was procured from Cell Signaling Technology Inc. Quantification of the band intensities was performed using the densitometry analysis function of Quantity One 1-D gel analysis software (Bio-Rad Laboratories, Hercules, CA). Statistical Analysis Unless otherwise mentioned, two dimensional two sample equal variance student's T-test was used for statistical analysis. A p<0.05 was considered significant. We thank Prof. Ross Holmes, Wake Forest University-School of Medicine, Winston-Salem, North Carolina for his assistance in determining in Urinary Oxalate concentrations and the Department of Pathology, University of Colorado for processing tissue sections and assistance in VonKossa's staining. ==== Refs References 1 Monico CG , Persson M , Ford GC , Rumsby G , Milliner DS (2002 ) Potential mechanisms of marked hyperoxaluria not due to primary hyperoxaluria I or II . Kidney Int 62 : 392 –400 .12110000 2 McConnell N , Campbell S , Gillanders I , Rolton H , Danesh B (2002 ) Risk factors for developing renal stones in inflammatory bowel disease . BJU Int 89 : 835 –841 .12010224 3 Hoppe B , Beck BB , Milliner DS (2009 ) The primary hyperoxalurias . Kidney Int 75 : 1264 –1271 .19225556 4 Williams EL , Acquaviva C , Amoroso A , Chevalier F , Coulter-Mackie M , et al (2009 ) Primary hyperoxaluria type 1: update and additional mutation analysis of the AGXT gene . 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PLoS One. 2012 Sep 12; 7(9):e44174
==== Front Rehabil Res PractRehabil Res PractRERPRehabilitation Research and Practice2090-28672090-2875Hindawi Publishing Corporation 10.1155/2012/479046Review ArticleTherapeutic Management of the Hallux Rigidus Aggarwal Anoop 1 *Kumar Suraj 2 Kumar Ratnesh 3 1Department of Physiotherapy, Institute for Physically Handicapped (Ministry of Social Justice and Empowerment), Pt. DDUIPH, New Delhi 110002, India2Department of Physiotherapy, NIOH, Kolkata 700090, India3NIOH, Bon Hooghly, Kolkata 700090, India*Anoop Aggarwal: [email protected] Editor: Jari P. A. Arokoski 2012 5 9 2012 2012 47904615 4 2012 24 7 2012 25 7 2012 Copyright © 2012 Anoop Aggarwal et al.2012This 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. Background. Hallux rigidus is a chronic, disabling condition of foot characterized by reduced great toe extension. The manual therapy approaches are described theoretically however their practical published evidence has not been analyzed well. Objective. Aim of the present paper was to systematically review the literature available for therapeutic management of the hallux rigidus by identifying and evaluating the randomized controlled trials (RCTs) and non-RCTs. Methods. To view the hallux rigidus and its rehabilitation, a webbased published literature search of Pubmed, Ovid Medline, Science direct, Cochrane Database, PEDro database, CINAHL was conducted for last 35 years in August 2010 using 4 specific keywords “hallux rigidus, physical therapy, chiropractic, and manual therapy” typed in exactly same manner in the search column of the databases. Result. the review finds that there is acute need of the quality studies and RCTs for the manual therapy, chiropractic, or physiotherapeutic management of the hallux rigidus. Conclusion. Review conclude that conservative programs for hallux rigidus consists of comprehensive intervention program that includes great toe mobilization, toe flexor strengthening, sesamoid bones mobilization and long MTP joint. The clinician should put an emphasis on the mobilization program with proper follow up along with comparative studies for rehabilitation of hallux rigidus. ==== Body 1. Introduction Hallux rigidus is a common condition of foot is characterized by reduced motion at the first meta-tarsophalangeal joint; particularly the extension range is reduced [1]. This is the manifestation of the osteoarthritis (OA) of first metatarsophalangeal (MTP) joint of great toe [2]. In early stage, it is also known as hallux limitus. Chief complaints of HL include inflammation, edema, pain, and reduced flexibility [3]. The condition may be accompanied with the stiffness and pain in the big toe, and may be the result of the acute or chronic injury to the MTP joint [4, 5]. This is a common problem among the patients suffering from the rheumatoid arthritis [4] and/or other chronic pathological conditions affecting the foot joints, however the incidence of this condition is not well documented. The reported incidence of the hallux rigidus vary from the 2% [1] to 50% [6]. 1.1. Biomechanical Fault Biomechanical abnormality of the first ray of the foot is considered to play an important role in the causation of various foot disorders. The decreased mobility of the first ray was found to be an associated factor in the patients with hallux rigidus [7]. During normal weight bearing phase (stance phase) of the gait, the planter aponeurosis gets stretched and this causes extension of the first MTP joint with the motion of the hallux [8]. This is important to maintain the Windlass effect for maintaining stability of the first MTP joint [9]. Also during toe-off phase of the gait the first metatarsal head bears more than 50% body weight [9]. During hallux rigidus, the restriction of the great toe extension ROM at MTP joint occurs along with commonly formed osteophyte at the dorsal aspect of the joint. These changes cause pain, and therefore to avoid pain and MTP extension the client attempts to put less weight on medial aspect, therefore he does internal rotation of tibia and bears more weight on the lateral MTP joints [9]. Due to the limited range of mot ion (ROM) the axis of movement gets shifted to more planter aspect to cause the jamming of MTP joint while attempting extension of great toe [10, 11]. This leads to an increased pressure on the plantar aspect of first ray [1]. During gait observation, the patient may shift weight laterally or rotate the hip joint externally to clear the first MTP from floor and to bear body weight on the little fingers [12, 13]. 1.2. Classification According to the severity of the involvement, the foot surgeons most commonly use the classification (Table 1) suggested by Drago et al. [14]. 1.3. Surgical Interventions Failure of the conservative treatment may warrant surgeries which may be in form of cheilectomy (debridement of joint), arthrodesis, proximal phalanx resection [1]. Cheilectomy is good choice only for patients with grade I and Grade II hallux rigidus, however, later on another operation may be required if the degenerative changes in the joints progress [9]. Arthrodesis is an option for moderate to severe joint involvement or the patients with active lifestyle [16]. Patients with grade-4 hallux rigidus or grade-3 hallux rigidus with less than 50% of the metatarsal head cartilage remaining at the time of surgery should be treated with arthrodesis [17]. Keller procedure is a resection arthroplasty advised in severely damaged first MTP or the older patients with less functional demands as it provides early symptomatic relief and needs minimal postoperative rehabilitation [9]. During this procedure the medial eminence and one third of proximal phalanx is removed due to Keller procedure the excessive shortening of great toe occurs, with impaired push-off strength and increased risk of metatarsalgia and the weak Windlass mechanism. The joint replacement surgeries for MTP joints are poorly satisfactory as complex bone grafting is needed leading to higher risk of nonunion and malunion. Beside this, the motion of first MTP joint is also reduced putting overloading on the interphalangeal joints [18]. Despite of the numerous options available for the surgery of this painful condition, a large number of patients having hallux rigidus have preference towards the conservative options of the management. In a long term followup (mean 14.4 years followup) study by Smith et al. [19] it was reported that the sufferers of this condition had learnt to satisfactorily live with this condition by making certain adaptations such as using shoes having ample toe-box, using stiff sole shoe, and appropriate shoe modifications. This study also had reported that though the radiologically the first MTP joint had deteriorated very significantly, yet the patients did not developed any further functional restriction due to this damage of the joint [20]. Similarly in another retrospective analysis [23] including 72% patients with hallux limitus, about 55% patients reported that they were successfully treated with conservative measures, including change of footwear, foot orthoses, and corticosteroid injection. These clients did not require any form of surgical intervention. 1.4. Physiotherapeutic Interventions The conservative management consists of the anti-inflammatory medicines to deal with synovitis, the weight offloading orthosis, and the rest [1]. Shoe modifications attempt to offload the first MTP joint during push off phase of the gait cycle. Therefore, medial arch support with metatarsal supports is advised. Orthotic intervention attempts to allow modifications which decrease pressure on the painful structures [24]. Besidess the nonsteroidal anti-inflammatory drugs and steroid injections are advised in the nonresponding cases [23]. In a randomized controlled trial by Mathew et al. [3], it was found that the occurrence of hallux limitus can be significantly reduced by using the dynamic splinting for first MTJ extension (60 minutes, three times per day). They found that dynamic splinting had effectively reduced the contracture of post-operative hallux and the patients gained a mean 250% improvement in Active range of motion (AROM) [3]. The development of hallux rigidus to certain extent can be expected only in cases if metatarsals are in a rectus pattern or if the angle of the metatarsus adductus is less than 10 degrees. If angle of metatarsus adductus is more than 10 degrees, than the greater transverse plane position of metatarsals allows the development of hallux abductovalgus [20]. Due to the chronic and disabling nature of the hallux rigidus, the management is very important to avoid the loss of function. However the physical therapy interventions for the management of the hallux rigidus are not much commonly available, moreover the quality of the available evidences has not been analyzed till date. Therefore, the purpose of this. Present review is to analyze the quality assessment of the available literature for the therapeutic management of hallux rigidus (Table 2). 2. Materials and Methods 2.1. Criteria for Considering Studies for This Paper 2.1.1. Types of Studies Objective of this study was to review the quality of the literature available for the physical therapy or manual therapy or chiropractic management of the hallux rigidus condition. Due to the absence of the RCTs on this topic the non-RCTs were included for this paper. Also the inclusion criteria and exclusion criteria were considered while selecting the study for consideration. The inclusion criteria that were followed while considering the study wereas follows. The published studies involving therapeutic intervention on the clients diagnosed with hallux rigidus. The intervention-based studies conducted on human beings of either gender and of any age group. Studies published in English language. Either case study, clinical or controlled trials. The studiyd which had used some form of manual therapy. Therapeutic intervention, or electrotherapy or exercise therapy or chiropractic approach. The comparative trials could be included if at least one intervention group contained some form of physical therapy intervention. The studies were excluded if the following applies. They did not involve the therapeutic interventions in at least one of its group. If the management was done was using surgical interventions or the medical interventions. Articles published in language other than English. If it was a review or simply descriptive study without any intervention. Studies including the non-touch techniques of management. Only those studies were included which met the inclusion criteria. 2.1.2. Types of Participants Trials were considered if the therapeutic intervention was done on patients presenting with the hallux rigidus and its associated complaints such as pain over the great toe joint line of foot; pain and tenderness over the MTP (Meta tarso-phalangeal) joint of foot; restricted great toe extension. 2.1.3. Types of Interventions Trials were considered in which at least one group of patient received the therapeutically (nonsurgical or nonpharmacological) intervention (s). The possible interventions included manipulation, mobilizations, stretching exercises, electrotherapeutic interventions, massage, shoe modifications, active exercises, and strengthening exercises. 2.2. Outcome Measure Outcome measures commonly used in patients of the hallux rigidus include: pain VAS (visual) analogue scale; (NRS) numerical rating scale), the great toe extension range of motion, functional score (LEFI-lower extremity functional index), the strength of FHL (flexor hallucis longus) muscle. 2.3. Procedure 2.3.1. Literature Search During 2012 the computerized literature searches were performed (between July 20th, 2010 and July 8th 2012) searching for the clinical or controlled trials and reviews of therapeutic interventions of hallux rigidus using the following databases between year 1975 and year 2012: Pub med, Ovid Medline, Science direct, the Cochrane Database, PEDro database (physiotherapy evidence database), CINAHL (cumulative index of nursing and allied health literature). The searched terms used were: “hallux rigidus, physical therapy, chiropractic, manual therapy.” During database searches the searches were limited only to clinical or controlled trials, case studies. 2.3.2. Study Selection Both reviewers (Aggarwal and Kumar) were involved in this review process. In the beginning session, they discussed the design, idea, and search strategy for the procedure. Databases were searched by the principal reviewer (Aggarwal) who downloaded the authors, title, abstracts. The possible studies which seemed to meet inclusion criteria were obtained in form of full text articles. The inclusion and exclusion criteria were then applied to the selected studies. 2.4. Data Analysis 2.4.1. Data Extraction The full text articles were then independently scrutinized by both reviewers. Any conflict regarding the suitability of the study was resolved by discussion between authors. For each study, the following details were documented: inclusion criteria, exclusion criteria, design, randomization, dropouts, blinding, details of interventions, followup and results. 2.4.2. Analysis and Quality Assessment For assessing the quality of the studies for this systematic review PEDro (physical therapy evidence database) quality score method was used to judge the quality of study on the scale of maximum 10 points each point related to specific criteria: as shown in Table 5. 3. Results 3.1. Selection of the Studies Fourteen trials were found using Science-direct database. These were screened and two trials were identified [5, 21] they involved therapeutic interventions and met inclusion criteria of this paper. The remaining nine studies were descriptive noninterventional studies, therefore rejected (Figure 1). Two studies were found in Pubmed Central Database. Out of these, the one study [21] was already selected and included from Science direct database. The other study was a literature review and thus did not meet the inclusion criteria and hence rejected. Beside this, one other study [22] was found in pubmed related articles and fulfilled necessary inclusion criteria of this review, therefore it was included for review. The Ovid, Medline, Cochrane Database, PEDro database, and CINAHL database did not returned any study on initial hit. Therefore no study was included from these databases. Thus only three studies (two from Science direct, one from Pub med related study) met the inclusion criteria and were selected for the quality assessment. 3.2. Methodological Quality Table 3 gives the details of the methodological assessment of the included studies. The scores assigned by both reviewers were same for each study, indicating the reliable process. Table 4 gives the details of the included studies. The quality of the study was evaluated using the PEDro scale for the RCT quality assessment. Since during search of the literature for this paper, we could found only the case studies (not RCTs) and few points of the PEDro were not applicable for the case study evaluation, therefore, if these studies were to be assessed by using full version of PEDro scale, their scoring would be obviously abysmal. To avoid this biasing against the scoring of case studies, a modified version was also used for the quality assessment of the case study. In this modified form those criteria (Random allocation; concealed allocation; baseline comparability; intergroup comparison; post estimate variability) were removed which do not apply to the case studies. Therefore, in this modified version the case studies could be evaluated using five criteria only (blinded assessor; blinded therapist; blinded subjects; adequate followup; intention to treat analysis). The methodological quality of all three studies was poor. None of them scored 50% or more on the quality score of PEDro on either original or in modified form version. Hence, none of them could be considered to be valid for the judgment. The most noticeable weakness of all three studies was the single subject intervention and the absence of the comparison with a control patient. The interventions were not clearly defined in terms of number of repetitions, duration, intensity, and the interval between the treatment sessions. This was the common limitation of all three studies (Table 4). Quality analysis was done according to the levels that rate the scientific evidence [25, 26]. Though these scoring system of scientific evidence—are used to judge quality of RCTs studies, but in this present paper this scoring system was used to describe non- RCTs studies due to lack of published RCTs on this topic [27]. Since there was no RCT available for the consideration, the level 4 scientific evidence (absence of RCT or the lack of clear evidence) was found in this paper. 4. Discussion Based on these results of included studies, it can be speculated that therapeutic interventions could be helpful for the management of patients with hallux rigidus, however, lack of adequate number of studies, their unsatisfactory methodology and ill-defined literatures does not strongly support this. There is limited evidence for the physiotherapeutic and manipulative therapy or exercise therapy for the management of the hallux rigidus conditions. The main reason for this limitation is that the previous studies differ from each other significantly in terms of the assessment parameters, the severity of the condition, the dosage of the interventions, and the nature of the interventions. The previous studies have not followed any specific classification system to classify the patients according to severity of the joint involvement. The quality scores of the included studies were below the acceptable levels. These scores were: 2/5 [5]; 1/5 [21]; 0/5 [22]. If all “n/r”— (not reported) scores were considered to be “yes” then the respective scores of the included studies would become 3/5, 3/5 and 2/5, respectively. Thus, in that case the scores for two studies would become more than the 50% of the total score of modified version (maximum score 5) which would then become more acceptable (more than 50% of total score) to strengthen the weak evidence that therapeutic interventions are helpful to manage the hallux rigidus. The RCTs provide stronger evidence while compared to the non-RCTs studies [28]. Due to the lack of RCT the non-RCT case studies were considered for the current paper. The PEDro scale and its modified version were used to evaluate the quality of the included studies. The original version of PEDro is applicable for the RCTs, therefore all quest ions were not suitable for evaluating the non-RCTs, and they were removed in the modified version. As per our best knowledge, this present study is the first paper to evaluate the therapeutic interventions in hallux rigidus. Therefore, the results of this paper cannot be compared with previously available studies and reviews. The major issues while conducting this paper included the lack of RCTs, low methodological quality of the found studies, the non availability of studies with adequate sample size, and the poor reporting of the trials. The study of Brant Ingham et al. [5] was a case study involving a professional player having big toe pain and grade I hallux rigidus. The management strategies included the graded mobilization of the first MTP (metatarsophalangeal) joint, cryotherapy, sesamoid bone mobilization. The methodlogy of the study was considered to be inferior and it did not mention the method of the treatment, exact protocol, the number of repetitions and sets on the intervened protocol. Right after the single session of intervention, they mentioned the significant improvement in the first MTP extension range, significant reduction in pain (NRS— numerical rating score dropped by about 67%) and the about 20% improvement of the lower extremity functional index score (LEFI). These improved effects lasted for about 5 months. They continued the same treatment for up to 17 sessions, however, no additional improvements were observed. The duration and repetitions of the stretching could be extremely important to determine the result of stretching, however this was not adequately explained in their case study. This emphasizes that the exact protocol what they used n the first visit, could be extremely helpful for the management of hallux rigidus, however in absence of the clear protocol explanation in their study, this vital information could not be made available for the use of clinicians. The manual methods that have been identified to be used in the hallux rigidus management include long axis traction of MTP joint [29], sesamoid joint mobilizations [11], flexor hallucis strengthening [11]. The manipulation of the Ist MTP by long axis traction is considered to be effective for management of the hallux rigidus (HR). The possible reduction in the pain upon manipulation of MTP joint could be related to improve first MTP joint bio mechanics [11]. The release of the capsular contracture and the associated tendon on plantar aspect could also result in the control of pain [30]. The joint mobilization is also helpful to improve the range of mot ion of the MTP joint [29]. The sesamoid joint mobilization of the first MTP joint is believed to improve the bio mechanics of this joint and to improve the pulley mechanism of flexor hallucis longus (FHL) tendon [11, 31]. The included case study of [21] recruited a professional tennis player having pain of the big toe for over 4 years. The diagnostic X-ray was not used however provisionally the symptoms and signs were considered as grade 2 hallux rigidus. They reported that during examination and assessment of the patient during preintervention phase the limited dorsiflexion of ipsilateral ankle; restricted inter-metatarsal joint movements, loss of axial mobility of toes, loss of subtalar eversion was found. The intervention involved the chiropractic adjustment of the MTP joint only, and the manual thrust to improve the mortise separation, forefoot eversion, first ray plantarflexion, and axial elongation of the digits. However, it must be noted that the quality of the presentation of case was not up to mark, as the dosimetry, number of repetitions, number of sets, and the grade of mobilization were not clearly mentioned. The volume of the manual treatment given, the number of repetitions and sets could be extremely vital for establishing the influence of them on the joint functioning. The limitation of their case study was that the effect of the interventions was not discussed properly and adequately in the discussion section. The study by Manral [22] considered a student having sudden onset pain on medial aspect of mid and forefoot while playing basketball. The protocol of the study was very vague as it did not consider the influence of manual therapy alone and the client was recommended to take the dietary supplements too. In such case, it is impossible to segregate the confounding influence of the manual therapy from that of the dietary supplements. The protocol just mentions the use of CMT (chiropractic manual therapy) without clarifying the method, dose, number of repetitions and sets, and the joints which were given manual therapy, and so forth. Additionally, the client also had radio logically established diagnosis of talonavicular and cuneionavicular joint, which were not given any treatment, not their confounding effect on the trial outcome was discussed. They found the reduction of pain intensity by about 80% (from intensity level 5 the pain reduced to level 1) after the first session of intervention; however the improvement was not properly discussed. They did not evaluate goniometric readings, yet the improvement in the range of the first MTP movement was completely restored within three sessions. The lack of the clarity in the protocol and the failure to frame a proper discussion preclude the understanding of the clinical or academic relevance of their study. 5. Conclusion The abysmal quality of the found literature makes it very evident that the good quality studies are majorly lacking for the physiotherapeutic management of hallux rigidus. The effect of the manual therapy must be examined on the patients grouped according to the grading of their joint involvement. The difference of the effect of manual therapy among males and females must be examined. There is an acute need of developing a standard protocol based on the manual therapy to standardize the practice of managing hallux rigidus. Future studies must also examine how many number of sessions are required to alleviate the patient's pain and associated disability. Since there was no RCT available for the consideration, the level 4 scientific evidence (absence of RCT or the lack of clear evidence) was found in this paper. The poor evidence for therapeutic interventions is due to the drastic variations in the methodology adopted by the researcher for classifying the degree and severity of joint involvement and the assessment methodology. Due to wide prevalence of the hallux rigidus, and the poor long term results of the surgical interventions, it is highly vital that the conservative means of the management must be adopted. This further needs to follow a uniform system of classification of the patients according to the radiological and functional severity of the joint mechanism. Acknowledgment The authors are thankful to Ms. Ruchika Madan (M. P. T. Orthopaedic medicine), Ms. Manju Vats (Physiotherapist, Pt. DUIPH), and Ms. Swati Sethi for their assistance during various phases of the formulating the search strategy and during formatting of the paper. This study has no conflict of interests. Figure 1 Flowchart indicating the method of selection of the studies. Table 1 Classification of the Hallux rigidus according to severity [14, 15]. Grade Clinical findings Radiological findings I Pain at end of passive ROM Functional limitation without radiographic degradation of articular cartilage Metatarsus primus elevatus, plantar subluxation of proximal phalanx no radiographic evidence of DJD II Limited passive ROM Dorsal spurring, subchondral eburnation, sclerosis, periarticular lipping, flattening of firstmetatarsal head, possible development of osteochondral defects III Grade II plus joint crepitation and pain with full ROM established joint destruction Subchondral bone cyst, severe flattening of joint, severe spurring, asymmetrical joint space loss, articular cartilage loss IV Grade III plus less than 10° first MTPJ ROM, possible total ankylosis Obliteration of joint space, intra-articular loose bodies Table 2 Various options available for the physiotherapeutic management of hallux limitus. Orthotic interventions (i) weight offloading orthotics (ii) offload the first MTP joint during push off phase of the gait cycle (iii) medial arch support (iv) dynamic splinting for first MTJ extension Manual methods (i) long axis traction of MTP joint (ii) sesamoid joint mobilizations (iii) flexor halluces strengthening (iv) manipulation of the first MTP (v) Stretching of the capsular contracture and the associated tendon on plantar aspect improves the pulley mechanism of the FHL (flexor hallucis longus) tendon (vi) Intrinsic foot flexor exercise Table 3 PEDro quality scoring of the studies included in this systematic review. Study, year Random allocation Concealed allocation Baseline Applicability Between group comparison Point estimate variability Blinded assessor Blinded subjects Blinded therapist Adequate follow up Intention to treat analysis Total PEDro score Modified PEDro score for case study Brantingham et al., 2007 [5] n/a n/a n/a n/a n/a No Yes no Yes n/r 2/10 2/5 Brantingham and Wood, 2002 [21] n/a n/a n/a n/a n/a No n/r no yes n/r 1/10 1/5 Manral, 2004 [22] n/a n/a n/a n/a n/a No n/r No No n/r 0/10 0/5 Keys: n/a: not applicable; n/r: not reported; ffi: the criteria which were used for the modified PEDro scoring of the included case studies. Table 4 Summary of therapeutic clinical trials included in this systematic review. Study, year Design PEDro score Modified PEDro score Number of patients Participant characteristics Complaints of patients Outcome measures Interventions Number of sessions Followup Result Brantingham et al., 2007 [5] Single subject, case study 2/10 2/5 1 31-year-old male, golfer by profession Post traumatic big toe pain since 7 month Grade I hallux rigidus on X-ray NRS rating for pain score LEFI ROM of big toe extension Graded axial elongation of MTP joint Ultrasonic therapy graded mobilization of MTP joint Heel raises great toe mobility and flexibility Foot flexor strengthening exercises 1 7 sessions distributed over 10 months 5 months Chiropractic interventions are helpful to reduce pain and management of hallux rigidus. RCT studies are needed to clear the interventions. Brantingham and Wood, 2002 [21] Single subject case study 1/10 1/5 1 36-year-old male professional tennis player Insidious onset of pain. Within six month pain increased to disabling limits; VAS score of pain was 10 MRI showed 80% of the foot extensor tendons had rubbed away due to spur on MTP dorsum and were repaired surgically X-ray was not taken though the clinical diagnosis of Grade 2 Hallux rigidus was made Pain on VAS scale Pain during playing tennis Plantar flexion strength of big toe 1st session: the 1st MTP axial elongation with grade 4 mobilization at slow oscillation for 10–15 times Sessions 2–4: Same as first session (i) Plus the HVLA thrust of MTP 3–5 times (ii) subtalar eversion thrust (iii) First ray planterflexion thrust (iv) MTP dorsiflexion thrust (v) axial elongation After 6 session the HEP consisted of gentle dorsiflexor ROM exercises cursive writing with toes Total 4 treatment sessions over 2 weeks 10 months                           Manral, 2004 [22] Single Subject case study 0/10 0/5 1   Borg pain scale score 5/10 Great toe extension ROM Borg pain scale, visualized ROM, Morton's test Chiropractic manipulative therapy, nutritional supplement, home advise of passive stretching great toe 3–5 repetition thrice a day Total 7 sessions over 8 weeks Not clearly mentioned Quality RCTs are needed to evaluate the effect of used interventions on hallux rigidus Keys: NRS: numerical rating score; LEFI: lower extremity functional index; ROM: range of motion; HEP: home exercise programs; RCT: randomized controlled trial; MTP: metarso-phalangeal joint. Table 5 PEDro scale criteria to assign scores to the RCT studies. Random allocation 1 point Concealed allocation 1 point Baseline similarity 1 point Blinding of participant 1 point Blinding of assessor 1 point Blinding of therapist 1 point Adequate followup (more than 85% of randomized patient report back on follow up) 1 point Between group comparison (not merely within group comparison) 1 point ==== Refs 1 Shereff MJ Baumhauer JF Hallux rigidus and osteoarthrosis of the first metatarsophalangeal joint Journal of Bone and Joint Surgery A 1998 80 6 898 908 2-s2.0-0031808809 2 Harisboure A Joveniaux P Madi K Dehoux E The Valenti technique in the treatment of hallux rigidus Orthopaedics and Traumatology 2009 95 3 202 209 2-s2.0-66149083682 19394916 3 Mathew MJ Stanley K Stephen VP Willis FB dynamic splinting for postoperative hallux limitus-a randomized, controlled trial Journal of American Podiatric Medical Association 2011 101 4 285 288 4 Shrader JA Siegel KL Nonoperative management of functional hallux limitus in a patient with rheumatoid arthritis Physical Therapy 2003 83 9 831 843 12940769 5 Brantingham JW Chang MN Gendreau DF Price JL The effect of chiropractic adjusting, exercises and modalities on a 32-year-old professional male golfer with hallux rigidus Clinical Chiropractic 2007 10 2 91 96 2-s2.0-34247577158 6 Clanton TO Ford JJ Turf toe injury Clinical Sports Medicine 1999 23 477 484 7 Greisberg J Sperber L Prince DE First ray mobility increase in patients with metatarsalgia Foot and Ankle International 2012 33 1 44 49 2-s2.0-78649889730 22381235 8 Hicks JH The mechanics of the foot. 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The plantar aponeurosis and the arch Journal of Anatomy 1954 88 1 25 31 2-s2.0-77049185349 13129168 9 Beertema W Draijer WF van Os JJ Pilot P A retrospective analysis of surgical treatment in patients with symptomatic hallux rigidus: long-term follow-up Journal of Foot and Ankle Surgery 2006 45 4 244 251 2-s2.0-33745503440 16818152 10 Lichniak JE Hallux limitus in the athlete Clinics in Podiatric Medicine and Surgery 1997 14 3 407 426 2-s2.0-0030788545 9257031 11 Shamus J Shamus E Gugel RN Brucker BS Skaruppa C The effect of sesamoid mobilization, flexor hallucis strengthening, and gait training on reducing pain and restoring function in individuals with hallux limitus: a clinical trial Journal of Orthopaedic and Sports Physical Therapy 2004 34 7 368 376 2-s2.0-3142777692 15296364 12 Mann RA Clanton TO Ha llux rigid us: treatment by cheilectomy Bone and Joint Surgery A 1988 70 400 406 13 Zammit GV Menz HB Munteanu SE Landorf KB Plantar pressure distribution in older people with osteoarthritis of the first metatarsophalangeal joint (Hallux limitus/rigidus) Journal of Orthopaedic Research 2008 26 12 1665 1669 2-s2.0-56749170801 18634037 14 Drago JJ Oloff L Jacobs AM A comprehensive review of hallux limitus Journal of Foot Surgery 1984 23 3 213 220 2-s2.0-0021191514 6376607 15 Lawrence MO Patel GJ A retrospective analysis of joint salvage procedures for grades III and IV hallux rigidus The Journal of Foot and Ankle Surgery 2008 47 3 230 236 2-s2.0-43049133682 18455670 16 Taylor DT Sage RA Pinzur MS Arthrodesis of the first metatarso-phalangeal joint The American Journal of Orthopedics 33 285 288 17 Coughlin MJ Shurnas PS Hallux rigidus The Journal of Bone and Joint Surgery 2004 86 2 119 130 2-s2.0-6044252523 15466753 18 Keiserman LS Sammarco VJ Sammarco GJ Surgical treatment of the hallux rigidus Foot and Ankle Clinics 2005 10 1 75 96 2-s2.0-17044438618 15831259 19 Smith RW Katchis SD Ayson LC Outcomes in hallux rigidus patients treated nonoperatively: a long-term follow-up study Foot and Ankle International 2000 21 11 906 913 2-s2.0-0033678697 11103761 20 Michaud TC Abnormal Motion during the Gait Cycle , Foot Orthotics and other forms of Conservative Foot Care 1993 1st edition Williams and Witkins 21 Brantingham JW Wood TG Hallux rigidus Journal of Chiropractic Medicine 2002 1 1 31 37 2-s2.0-33749619788 19674557 22 Manral DB Hallux rigidus: a case report of successful chiropractic management and review of the literature Journal of Chiropractic Medicine 2004 3 1 6 11 2-s2.0-33749632721 19674618 23 Grady JF Axe TM Zager EJ Sheldon LA A retrospective analysis of 772 patients with hallux limitus Journal of the American Podiatric Medical Association 2002 92 2 102 108 2-s2.0-0036480234 11847262 24 James MH Hunt GC Lerche FF Voi P Smith JW An external shoe modification for reducing metatarsal head pressure in people with metatarsalgia Journal of Prosthetics and Orthotics 2010 22 1 37 42 2-s2.0-75149167873 25 Jennifer JS Christopher JH Introduction to Evidence-Based Medicine, Levels of Evidence, and Systematic Reviews. Evidence-Based Otolaryngology 2008 New York, NY, USA Springer 26 Van-Tulder M Cherkin D Berman B Acupuncture for low back pain (CochraneReview), The Cochrane Library no. 2, Oxford Update Software, 2001 27 Reid SA Rivett DA Manual therapy treatment of cervicogenic dizziness: a systematic review Manual Therapy 2005 10 1 4 13 2-s2.0-12944299810 15681263 28 Mcpherson K Lord S Clinician guides to research part 2 New Zealand Journal of Physiotherapy 2000 28 2 20 28 29 Talarico LM Vito GR Goldstein L Perler AD Management of hallux limitus with distraction of the first metatarsophalangeal joint Journal of the American Podiatric Medical Association 2005 95 2 121 129 2-s2.0-17144402910 15778469 30 Shereff MJ Bejjani FJ Kummer FJ Kinematics of the first metatarsophalangeal joint Journal of Bone and Joint Surgery A 1986 68 3 392 398 2-s2.0-0022625831 31 Aper RL Saltzman CL Brown TD The effect of hallux sesamoid resection on the effective moment of the flexor hallucis brevis Foot and Ankle International 1994 15 9 462 470 2-s2.0-0028048289 7820237
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Rehabil Res Pract. 2012 Sep 5; 2012:479046
==== Front Reprod Biol EndocrinolReprod. Biol. EndocrinolReproductive Biology and Endocrinology : RB&E1477-7827BioMed Central 1477-7827-10-582290567810.1186/1477-7827-10-58ResearchThe proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways Du Xiao-Yu [email protected] Jian [email protected] Liang-Quan [email protected] Dan-Feng [email protected] Lei [email protected] Li-Xia [email protected] Xiao-Ling [email protected] Wei-Yun [email protected] Li-Ping [email protected] Yue-Hui [email protected] Medical Experimental Teaching Department, Nanchang University, Nanchang, 330006, China2 Department of Physiology Reproduction, Medical College of Nanchang University, Nanchang, 330006, China3 Department of Academic Journal, Nanchang University, Nanchang, 330006, China2012 20 8 2012 10 58 58 10 1 2012 13 8 2012 Copyright ©2012 Du et al.; licensee BioMed Central Ltd.2012Du 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 C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. Methods Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. Results The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c-src expression. Furthermore, our studies demonstrated that folliculogenesis onset was inhibited by Calphostin, PD98059 or LY294002 treatment,but none of them down-regulated c-src expression. In contrast, the expression levels of p-PKC, p-ERK1/2 and p-PI3K in the follicles were clearly decreased by c-src siRNA transfection. Correspondingly, both Calphostin and LY294002 treatment resulted in a decrease in the p-PKC level in follicles, but no change was observed in the PD98059 group. Finally, LY294002 treatment decreased the p-PI3K expression level in the follicles, but no changes were observed in the PD98059 and Calphostin groups. Conclusions C-src plays an important role in regulating primordial follicle activation and growth via the PI3K-PKC- ERK1/2 pathway. ==== Body Introduction Oocytes are surrounded by somatic cells in the ovaries of newborn mammals. In rats, during the first 3 days after birth, the primordial follicles are assembled and remain developmentally arrested thereafter until the primary follicles are formed later [1]. The primordial follicle growth signals the transition of the primordial follicle from quiescence to the next growth state—the primary follicle stage. As the process commences, the oocytes begin to grow and the granulosa cells around the oocyte become cubiform and proliferate rapidly. When the cubiform granulosa cells surrounding the growing oocytes reach more than one layer, the follicle become the secondary follicle [2]. This progress requires a coordinated interaction of events, such as cell cycle progression, apoptosis, and differentiation of pluripotent somatic cells into the granulosa cell lineage. Although the exact factors and mechanisms that regulate folliculogenesis initiation remain elusive, the accumulated evidence suggests that the early growth stage of follicle development is not dependent on the gonadotropins but is mainly controlled by a combination of local paracrine factors within the ovaries. Some factors, such as stem cell factor (SCF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), nerve growth factor (NGF), bone morphogenic protein (BMP), growth differentiation factor 9 (GDF-9) and insulin-like growth factor (IGF), promote the development of the primordial follicles. Other factors, such as AMH, E2 and P, inhibit primordial follicle development [3-6]. Although we still poorly understand at the molecular level how these factors regulate primordial follicle development, successful activation of follicle growth must involve genetic networks both in germ and somatic cells. In recent years, genetic factors have received increasing attention as determinants of primordial follicle onset [7-11]. In a recent study, we have showed that the mRNA of another Proto-oncogene, c-erbB2, is expressed in the primordial follicle, and ablation of c-erbB2 in neonatal rat ovaries results in excessive inhibition of primordial follicles [12], which demonstrates that c-erbB2 plays an important role in regulating primordial follicle onset. In addition to the evidence from our previous studies that c-src mRNA is expressed in mammalian ovaries, primordial follicle growth was retarded and the number of mature follicles was significantly reduced in c-src knock-out mice [13,14]. Based on this finding, it is tempting to speculate that c-src might play an important role in regulating primordial follicle onset as well. The proto-oncogene c-src, an evolutionarily conserved proto-oncogene and the first carcinoma gene to be discovered in cells by Bioshop et al. in 1976, is widely expressed in yeast, Drosophila and vertebrates, including humans. c-src participates in the regulation of cell growth, development, differentiation and other biological functions. Src protein was the first member of the Src protein family kinases (SFKs) to be identified, and it is a non-receptor tyrosine protein kinase. During the oocyte maturation process, phosphorylated SFKs and non-phosphorylated SFKs are concentrated in the nucleus and the cortical region of the oocytes before germinal vesicle breakdown (GVBD). Once GVBD occurs, the activated SFK is distributed throughout the oocytes [15-17]. These findings suggest that c-src plays an important role in oocyte maturation. However, whether c-src and Src protein are expressed during primordial follicle growth and what roles they play in this process have not been reported. A variety of signaling pathways, including the MAPK and PKC pathways, are involved in the activation of the growth of primordial follicles [18-21]. Signaling pathways, such as the PI3K and mTORC1 pathways, regulate the activation of primordial follicles and the early development of ovarian follicles [7,11]. It is possible that Src protein and the three intracellular signaling proteins (MAPK, PKC, PI3K) are inextricably linked. Both PKC isozymes and Fyn protein kinase exist in mammalian follicles, and PKC might induce the activation of eggs [22,23]. PP2, an inhibitor of Src protein, hindered the phosphorylation of PI3K and Akt [24-26]. In this study, we will explore the possible signaling roles of c-src in primordial follicle initiation in the context of other canonical signaling pathways. Methods Animals and reagents The animal use was approved by the Committee of Nanchang University for Animal Research. Two-day-old Sprague Dawley rats (weight approximately 4-6g) were used for all the experiments. The immunohistochemical kit was purchased from ZhongShan Co., Ltd. (Beijing, China). The monoclonal antibodies against Src protein, PD98059 (a MAPK inhibitor), Calphostin (a PKC inhibitor) and LY294002 (a PI3K inhibitor), β-actin and Lipofectamine2000 were purchased from Sigma (St. Louis MO). The EASY siRNA kit was purchased from Genechem Co., Ltd. (Shanghai, China) and the lentivirus-packaged siRNA was prepared by Genechem Co., Ltd. (Shanghai, China). Designation, construction and transfection of lentivirus c-src siRNA In brief, three c-src-targeting oligonucleotides (siRNA1, siRNA2, siRNA3, targeting to the c-src gene NM_031977) were designed, and another was used as the negative control (no siRNA). Transfection was performed according to the instructions provided with Lipofectamine2000. The ovaries were cultured in 24-well plates. After 36 h, they were transferred to serum-free culture solutions with 40 pmol/l of siRNA1, siRNA2 or siRNA3. After 12 h of transfection, the medium was replaced with fresh medium containing no siRNA, and the ovaries were cultured for an additional 8 days. Furthermore, the specimen with the interference effects were evaluated by RT-PCR and western blotting, and the siRNA that produced the most effective knockdown was synthesized and packed into a lentiviral vector (1.5 × 109IU/ml). The best interference effect for c-src siRNA was as follows: sense, 5’-CACUACAAGAUCCGGAAACtt-3’, antisense, 5’-GUUUC CGGAUCUUG UAGUGtt-3’. Culture of neonatal rat ovaries and experimental protocol Ovaries from postnatal Day 2 Sprague–Dawley rat pups were cultured as previously described [18]. For the in vitro studies, the ovaries were divided into three groups: c-src siRNA group (lentiviral c-src siRNA; c-src siRNA), negative control group (blank vector; lentivirus without siRNA), and blank control group (c-src-non-targeting oligonucleotides). The medium was replaced every 48 h with fresh medium containing no siRNA, and the ovaries were cultured for 8 days. To determine the upstream and downstream relationships between c-src, MAPK, PKC and PI3K, the ovaries were challenged with PD98059 (5 × 10-2 mmol/L), Calphostin (5 × 10-4 mmol/L) or LY294002 (5 × 10-2 mmol/L). After termination, the ovaries were processed for morphometric evaluation of follicular development by the detecting levels of mRNA, immunohistochemistry and western blotting analysis. Histological morphometric evaluation of folliculogenesis Ovaries from 2-day-old rats were collected fresh or cultured for 4 and 8 days (ovaries were cultured with/without inhibitors and lentiviral c-src siRNA), with 16 ovaries in each group. Fresh ovaries were fixed in Bouins solution for 1–2 h, embedded in paraffin, sectioned (3-5 × 10-3 mm) and stained with hematoxylin and eosin. The number of follicles at each developmental stage was counted in two serial sections from the largest cross-section through the center of the ovary. Typically, two ovaries were included in each treatment group as replicates, and 150–200 follicles were present in each ovary cross-section. The experiments were repeated three times (therefore, n = 6 for each treatment group). Primordial follicles are known to consist of one oocyte that is partially or completely encapsulated by flat squamous pregranulosa cells. Developing follicles contain successively more cuboidal granulosa cells in the layers around the oocyte. Immunohistochemistry to determine the localization of src protein Paraffin-embedded rat ovaries were sectioned to 3-5 × 10-3 mm and set in the oven at 60°C for half an hour. The tissue sections were deparaffinized, and the endogenous peroxidase activity was quenched with 3% H2O2 in methanol. Following rehydration, nonspecific binding was blocked with binding liquid, and the sections were then incubated for 2 h with monoclonal antibodies against the Src protein at 37°C. Following extensive washing with PBS, the tissue sections were incubated with a biotin-conjugated secondary antibody at 37°C for 20 min. After washing with PBS, the tissue sections were quenched with HRP-working liquid for 20 min at 37°C to detect and bind to the secondary antibodies. After treatment with DAB, the tissue sections were counterstained with hematoxylin. Following dehydration, hyaline, drying and finalizing, the sections were set under an inverted microscope for imaging. We used PBS instead of monoclonal antibodies toward Src protein as a negative control. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the levels of c-src and β-actin mRNA Expression of mRNA for c-src was assayed by RT-PCR. Ovaries from the same culture wells described above were pooled to prepare a single RNA sample. The ovaries that had been cultured with inhibitors and a reorganizing lentivirus were also assayed by RT-PCR. RNA was extracted using the Trizol reagent (Sigma, St. Louis, MO). Total RNA from each sample was reverse transcribed into cDNA using a standard oligo-dT RT protocol. cDNA samples were used as a template for polymerase chain reaction (PCR) analysis. The 2 × EasyTaq PCR Supermix kit (TRansGen Biotec) was used according to the manufacturer’s instructions. The c-src primers were as follows: forward sequence: 5’-CAT CCA AGC CTC AGA CCC A-3’, reverse: 5’-TGA CAC CAC GGC ATA CAG C-3’. The housekeeping reference gene β-actin primers were as follows: forward: 5’-ACA CTG TGC CCA TCT ACG AGG-3’, reverse: 5’-AGG GGC CGG ACG CGT CAT ACT- 3’. The samples were heated to 94°C for 4 min and then submitted to 35 cycles of 95°C for 45 sec, 55°C for 45 sec and 72°C for 1 min, and followed by an extension step at 72°C for 7 min. The fluorescent detection data for c-src mRNA were analyzed and normalized relative to the β-actin mRNA levels. The identities of the RT-PCR products were confirmed by direct sequencing (Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.). All experiments were repeated at least three times. Real-time PCR determination of the levels of c-src and β-actin mRNA To analyze c-src mRNA levels, total RNA was extracted from cultured rat ovaries and used as a template for cDNA synthesis using oligo (dT) primers and the SuperscriptIII kit (Invitrogen, CA). Total DNA was extracted from the rat ovaries to assess GFP DNA levels. Real-time quantitative PCR was performed using the ABI Prism 7500 detection system (PE Applied Biosystems, Foster City, CA) with the SYBR green DNA detection kit (Applera, NY). The expression levels of the house keeping gene encoding β-actin were also quantified using 50 ng of cDNA. The relative mRNA values were determined and used for normalization. All experiments were repeated at least three times. The PCR primers for c-src were as follows: forward primer: 5'- GGACAGTGGCGGATTCTACATC-3', reverse primer: 5'- AGCTGCTGCAGGCTGTTGA-3'. The reaction conditions were as follows: 95°C for 30 sec, 95°C for 5 sec, 60°C for 60 sec for a total of 45 cycles. The amplicon size was 57 bp. The PCR primers for β-actin were as follows: forward primer: 5'- TTCAACACCCCAGCCATGT-3', reverse primer: 5'- CAGAGGCATACAGGGACAACAC-3', and the amplicon size was 58 bp. The PCR primers for GFP were as follows: forward primer: 5'-TGCTTCAGCCGCTACCC-3', reverse primer: 5'-CTTGCCGTAGTTCCACTTGA-3'. The reaction conditions for GFP PCR were as follows: 95°C for 15 sec, 95°C for 5 sec, 60°C for 30 sec for a total of 45 cycles. Detection and quantification of src protein, p-ERK1/2, p-PKC and p-PI3K by Western blotting analysis Ovaries that had been cultured for 8 days were pooled to produce a single protein sample. The levels of Src, p-ERK1/2, p-PKC, p-PI3K or β-actin protein in ovaries that had been cultured with inhibitors and a reorganizing lentivirus were assayed by western blotting. Tissue protein extracts were electrophoretically separated under reduced conditions using NuPAGE 7.5–10% Bis-Tris gels (Invitrogen; Paisley, UK). Standard Mark (Invitrogen) was used as the molecular weight standard. The proteins were then electrotransferred to nitrocellulose membranes (BIORAD; Munich, Germany, 4°C, 230 mA, 1.5 h), and the immunoblots were subsequently blocked for 2 h at room temperature in TBST (TBS containing 0.1% Tween 20) containing 2.5% BSA. The membranes were incubated overnight at 4°C with antibodies against Src, p-ERK1/2, p-PKC, p-PI3K or β-actin (1:200). The β-actin bands were used as an internal control for equal loading. After rinsing with TBST, the membranes were incubated for 2 h at 37°C with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:500). Finally, the membranes were treated with ECL in a darkroom, exposed, developed and fixed and imaged. We analyzed the images with the Gel image analysis system. Fluorescence imaging After 8 days of culture with the lentivirus, the rat ovaries were removed and cut into 5-μm-thick serial sections. GFP fluorescence was observed using a fluorescence microscope (Leica, Germany). Statistics To evaluate follicle development, three ovaries from different rats were cultured for each treatment group, and the cultures were repeated at least twice. For immunohistochemistry, we used one ovary per group, and the cultures were repeated three times. For RNA and protein preparation, three ovaries per group were cultured and then pooled into one sample. The cultures were repeated three times. All data were presented as the means ± SEM and analyzed by ANOVA and Duncan’s new multiple range tests. p < 0.05 was considered significantly different. Results Src protein expressed in rat ovaries during primordial follicle culture The expression of c-src protein in the ovaries during primordial follicle growth was examined by immunohistochemistry analysis. Src protein was detected in the oocytes and granulosa cells of primordial follicles and primary follicles. During the development of the follicles, the expression of Src protein increased correspondingly (p < 0.01) (Figure 1). Figure 1 Expression of Src protein in rat ovaries during the activation and growth of primordial follicle were examined by immunohistochemistry analysis. Src protein expression in the oocyte and granulosa cell membrane of primordial and primary follicles (red arrows) is shown in A, B and C (which represent ovaries cultured in vitro for 0, 4 and 8 days, respectively). During the development of the follicles, the expression of Src protein increased corresponding. The negative controls are shown in D, E and F (which represent ovaries cultured in vitro for 0, 4 and 8 days, respectively). siRNA knockdown of c-src mRNA and protein suppresses primordial follicle development The specificity of the c-src siRNA effect was verified by examining the levels of c-src mRNA and protein in ovaries exposed to c-src siRNA. Compared with blank control and negative control, siRNA specifically and appreciably reduced the levels of c-src mRNA (Figure 2) and c-src protein (Figure 3) in the ovaries. When the ovaries were cultured with lentivirus packaging siRNA particles, the β-actin mRNA and protein were not affected by any treatment, suggesting the specificity of the c-src siRNA (Figure 2, Figure 3). Figure 2 The silencing efficiency of the lentivirus after transfection.A, c-src mRNA in ovaries 8 days after lentivirus transfection and a semiquantitative assay of the c-src mRNA levels 8 days after lentiviral transfection from six different replicates. B, Real-time PCR was used to analyze the relative c-src mRNA (c-src mRNA/β-actin mRNA). Blank control corresponds to the group that was transfected with c-src-non-targeting oligonucleotides, negative control corresponds to the group that was transfected with a blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with the lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). **P<0.01 compared with the negative control group. Figure 3 The silencing efficiency of the lentivirus after transfection.A, c-src protein in the ovaries 8 days after lentivirus transfection. B, Semiquantitative results of western blotting from three different replicates. Blank control corresponds to the group with c-src-non-targeting oligonucleotides, negative control corresponds to the group with blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). **P<0.01 compared with the negative control group. The delivery efficiency was measured by fluorescence imaging. Eight days after transfection of the rat ovaries with c-src siRNA, the GFP fluorescence of frozen ovarian sections was clearly observed under a fluorescence microscope. Furthermore, the GFP DNA level increased significantly, and its relative expression level was 5.07 times higher than that in the control group, and the c-src siRNAs lowered the GFP DNA relative expression level 1.96 times. These results demonstrated that the lentivirus was successfully delivered to the ovarian tissue by in vitro organ culture (Figure 4). Figure 4 The delivery efficiency of the lentivirus after transfection.A,The GFP fluorescence of frozen ovarian sections 8 days after lentivirus transfection. B, Semiquantitative assay of the relative level of GFP DNA 8 days after lentivirus transfection from six different replicates. Blank control corresponds to the group with c-src-non-targeting oligonucleotides, negative control corresponds to the group with blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). ** P<0.01 compared with blank control; Δ P<0.05 vs. negative control. To clarify whether c-src was involved in activation of the growth of primordial follicles, we transferred in vitro synthesized siRNAs into the newborn rats' ovaries to examine the effect of c-src on primordial follicle development. Neonatal rat ovaries cultured for 8 days in the blank control group contained approximately 35% primordial follicles, and the percentage was not altered when the ovaries were exposed to the negative control. c-src siRNA significantly retarded the development of primordial follicles, as approximately 64% of the primordial follicles remained in the siRNA-treated ovaries. Together with the evidence of constitutive Src distribution in the primordial follicles, the results of the effect of c-src inhibition by siRNAs on primordial follicle growth suggested that c-src might be essential for primordial follicle development (Figure 5). Figure 5 Effect ofc-srcsiRNA on the activation of primordial follicle growth.A, Ovaries were cultured for 8 days with the indicated treatments: blank control, negative control, 100 μmol/L siRNA targeting c-src. Ovarian sections were stained with hematoxylin and eosin to show the follicles at different developmental stages. Scale bar: 20 μm. HE staining X400 B, The number of follicles was counted in serial cross-sections. The percentage of the number of each category over the total number was plotted. The data are presented as the means ± SEM. Bars with different superscripts are statistically different (P<0.01). Blank control corresponds to the group with c-src-non-targeting oligonucleotides, the negative control corresponds to the group with blank vector (lentivirus without siRNA), and c-src siRNA corresponds to the group with lentivirus packaging c-src siRNA. The data are presented as the means ± SEM (n = 3). **P<0.01 compared with negative control group. The effect of c-src on primordial follicle development involving the MAPK, PKC and PI3K signaling pathway PD98059, Calphostin and LY294002 are widely used as MAPK, PKC and PI3K inhibitors, respectively. After culturing neonatal rat ovaries for 8 days, each of these compounds inhibited primordial follicle development significantly (Figure 6). These results suggest that the MAPK, PKC and PI3K signaling pathways are vital to primordial follicle development. We added PD98059, Calphostin and LY294002 to the culture solution and collected specimens to detect c-src mRNA levels by real time-PCR and Src protein abundance by western blotting. The development of primordial follicles was observed by hematoxylin/eosin (HE) staining. We found that, compared with the blank group, the c-src mRNA and Src protein levels in each inhibitor group were not reduced (P > 0.05) (Figure 7, Figure 8). Treatment of cultured primordial follicles with the c-src siRNA not only suppressed the levels of src protein, but also the phosphorylation of ERK1/2, PKC and PI3K (Figure 8), suggesting that the activation of MAPK, PKC and PI3K in primordial follicle is downstream of the c-src proto-oncogene. In addition, as shown in Figure 8, we found that p-ERK1/2 in the follicles decreased after PD98059, Calphostin and LY294002 treatment, suggesting that activation of MAPK in the follicles is downstream of PKC and PI3K. The levels of p-PKC in the follicles decreased after Calphostin and LY294002 treatment, but no change was observed after treatment with PD98059, suggesting that activation of PKC in the follicles is downstream of PI3K and upstream of MAPK. The levels of p-PI3K in the follicles decreased after LY294002 treatment, but no changes were detected in the PD98059 and Calphostin groups, suggesting that activation of PI3K in the follicles is upstream of MAPK and PKC. Therefore, we conclude that the effect c-src on primordial follicle development is involved in the MAPK, PKC and PI3K signaling pathways, and the direction of the cascade may be c-src → p-PI3K → p-PKC → p-ERK1/2 (Table 1 and Figure 9). Figure 6 Effect of PD98059, Calphostin and LY294002 on the activation of primordial follicle growth.A, Ovaries were cultured for 8 days with treatments: blank control, PD98059(5 × 10-2 mmol/L), Calphostin (5 × 10-4 mmol/L)and LY294002(5 × 10-2 mmol/L). Ovarian sections were stained with hematoxylin and eosin to show the follicles at different developmental stages. Scale bar: 20 μm. HE staining X400 B, The number of follicles was counted in serial cross-sections. ** P<0.01 vs. blank control. Figure 7 Effect of the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 on the expression ofc-srcmRNA.A, The expression of c-src mRNA (250 bp) in the ovaries by RT-PCR and semiquantitative analysis of the RT-PCR results. B, Real-time PCR was used to analyze the relative levels of c-src mRNA (c-src mRNA/β-actin mRNA). The data are presented as the means ± SEM (n = 3). Figure 8 The expression of Src, p-ERK1/2, p-PKC and p-PI3K protein afterc-srcsiRNA, PD98059, Calphostin and LY294002 treatment, respectively.A,The expression of Src, p-ERK1/2, p-PKC and p-PI3K protein in the ovaries detected by western blotting. B, The expression of Src protein according to a semiquantitative analysis of the western blot result. C, The expression p-ERK1/2 protein according to a semiquantitative analysis of the western blot result. D, The expression of p-PKC protein according to a semiquantitative analysis of the western blot result. E, The expressions of p-PI3K protein by semiquantitative analysis of the western blot result. Blank control corresponds to the group transfected with negative control siRNA. The data are presented as the means ± SEM (n = 3). ** P<0.01 vs. blank control group. Table 1 C-srcinvolved in p-PI3K, p-PKC and p-ERK1/2 signal cascades in primordial follicle development Treatment The relationship and the changes c-src→ p-PI3K→ p-PKC→ p-EPK1/2→ follicle growth c-src siRNA ↓ ↓ ↓ ↓ ↓ LY294002 - ↓ ↓ ↓ ↓ Calphostin - - ↓ ↓ ↓ PD98059 - - - ↓ ↓ ↓ stands for down regulation of expression; -symbols for no change. Figure 9 C-srcregulating primordial follicle activation and growth through PI3K → PKC → MAPK signaling pathway Discussion Location and analysis of src mRNA and protein in primordial follicles Our PCR results showed that c-src mRNA was present in SD rat primordial follicles, which was confirmed by sequence comparison with GeneBank. Our immunochemical results confirmed that Src protein mainly localized in the oocyte membrane and granulosa cell membrane in the primordial follicles of neonatal SD rats. Src protein increased during the development of primordial follicles in vitro. Previous studies have shown that the egg cortical region is rich in cytoskeletal structures such as actin, microtubules, microfilaments and so on [13]. The different subcellular localization of SFKs might be related to the regulation of intracellular activities, such as mitosis, the rearrangement of the cytoskeleton, and trans-membrane exchange of substances [17]. Src protein, which is also present in the granulosa cell cytoplasm, might be involved in other cytoskeletal activities, especially in terms of granulosa cell-oocyte interaction. However, the finding that the expression of Src protein increased with an increasing culture duration indicated that the activity of c-src increased with the development of primordial follicles. In this study, whether the activity of Src protein increased was unclear. Additionally, further examination of whether the crosstalk between the oocytes and the granulosa cells affects the dynamic localization of Src protein in primordial follicles is needed. The effect of c-src on primordial follicles and its mechanism of action Cultured ovarian tissue that was transfected with siRNA1 packaged into a lentiviral vector had decreased levels of c-src mRNA and Src protein compared with the control group. The ovarian HE staining demonstrated that the proportion of primordial follicles compared with total follicles increased in the transfected group compared with the control group, demonstrating the inhibitory effect of c-src siRNA1 on primordial follicle development. We conjectured that the activity of c-src may be essential for promoting the development of primordial follicles. c-src SH2 structural domain binds to Tyr527 at its C’ end (or terminal), and then Tyr57 is phosphorylated by the protein tyrosine kinase C-terminal Src kinase (CSK), which in turn causes the molecular structure to transform, preceding the inhibition of the activity center of the c-src kinase [27-29]. The dephosphorylation of Tyr572 can reactivate c-src. Once c-src kinase is released from its inhibited status, a key Tyr residue inside the PTK functional domain would be phosphorylated due to the activation of PTK. Inter-activation between CSK’s homolog domain SH2/SH3 with certain proteins causes self-phosphorylation of the Tyr residues in the catalytic region of the Src-family protein tyrosine kinases (SFKs), which in turn activates SFK [30]. Talmor-Cohen et al. [13] found that the fusion of rat oocytes and sperm triggers SFK activation, which induces calcium release via the PLC-IP3 pathway and the resumption meiosis. Meng et al. [17] found that transfection of c-src siRNA into mouse oocytes before GVBD could reduce the resumption of meiosis by more than 50% compared with controls, similar to the effect of a c-src inhibitor. SFKs are found in rat and mouse oocytes, and they have a role in regulating oocyte maturation [17]. The specific inhibitor of PP2 could decrease the rate of GVBD, which further implies that SFK is independent of mouse oocyte meiosis resumption [31]. SKF maintained its kinase activity by binding to the cell inner membrane through an N-terminal membrane-bound sequence that was covalently linked to myristic acid [32]. However, the execution of the SFK’s functions in oocytes might be related to cytoskeleton microfilaments and microtubules [17]. We conjectured that Src protein might be primarily synthesized in oocytes and granulosa cells and function as a regulator of the activation of primordial follicles via its N-terminal membrane-binding sequence and by mediating the cytoskeletal interactions between granulosa cells and oocytes. We also found that Src protein levels increased over time during culture, indicating that the activity of c-src might be elevated during the development of primordial follicles. Thus, we further presumed that endogenous factors inside primordial follicles, such as EGF, may bind to the SH2 structural domain of the Src protein. Alternatively, some factors (e.g., PI3K) produced during the development of the primordial follicle may bind to the SH3 structural domain of Src and caused SH3 to bind to the SH2 structural domain, or SH3 may directly bind to the SH2 structural domain of the Src protein. The exact mechanism of Src protein activation remains to be elucidated. Src protein in primordial follicle development and the PI3K, PKC and MAPK signaling pathways Many studies have shown that SFK can be activated by growth factors, integrin, superoxide and UV rays. Many growth factors, such as PDGF, FGF, EGF, are upstream activators of the Src protein, not only activating the Src protein but also resulting in ovum activation [30]. The activated SFKs promote the translocation of γ site-phosphates of adenosine triphosphate (ATP) to tyrosine residues in the corresponding target proteins, thus activating the target proteins to a further transmit the signal. Hindering the activity of Src kinase inhibits the activity of MAPK in granulosa cells [33]. The inhibition of mouse oocyte GVBD is due to the activation of PKCα, which may be involved in the regulation of chromatin condensation and nuclear envelope assembly as well as other cellular events during the process of oocyte maturation [15,22]. In mice, rats and other animal oocytes, it has been found that the inhibition of PKC activity in GV stage oocytes and blockade of GVBD also inhibit the activation of MAPK. After its activation, PKC may participate in other functions by inhibiting the upstream molecules of MAPK. However, MAPK is not necessary for GVBD to occur, and PKC may also regulate GVBD through cAMP [16,34]. The secretion of granulosa cells may be mediated by Ca2+ release or PKC activation, while SKF can activate PKC by producing DAG, causing a subsequent signaling cascade [13]. pten is the inhibitory regulating gene of PI3K. Reddy et al. found that the number of primordial follicles in the ovaries was greatly reduced in pten-knockout mouse, which led to premature activation of primordial follicles and premature depletion of the follicle reserve, similar to Foxo3a–knockout mice. Their findings indicate that pten plays an important role in the regulation of premature ovarian failure; meanwhile, the oocyte inhibits follicle activation [8,11]. Our results showed that the maturity of primordial follicles was not only dependent on the activation of Src protein, but also required the involvement of ERK1/2, PKC and PI3K signaling pathways in primordial follicles. ERK1/2, PKC and PI3K may positively regulate the maturation of primordial follicles. We speculated that Src protein, along with ERK1/2, PKC and PI3K, form a complex signaling network that regulates the development of primordial follicles. The putative signaling cascade is triggered by PI3K, which in turn activates PKC and ultimately causes the activation of MAPK activation. According to a new study [8], many genes, such as pten, Tsc1, Tsc2, Foxo3a, p27, are involved in the silencing and activation of primordial follicles. A better understanding of these signaling pathways and the genetic networks that orchestrate primordial follicle development will be helpful for uncovering the mechanisms underlying many disease conditions, such as female sterility, premature ovarian failure (POF), polycentric ovary disease syndrome (PCOS), and ovarian cancers. Conclusions C-src play an important role in regulating primordial follicle activation and growth through the PI3K-PKC-ERK1/2 signaling pathway. Competing interests The authors declare that they have no competing interests. Authors’ contributions DXY, HJ, TDF, XLQ and WL carried out all the experiments. ZYH, DXY, HJ, TDF, XLQ, WL, ZLP, PXL and CWY performed the statistical analysis and drafted the paper. ZYH and ZLP designed the study and amended the paper. All authors read and approved the final manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (No.30660053) and the Jiangxi Province Major Sciences and Technologies Supporting Program. We are also grateful to Dr. DaLei Zhang and Dr. ZhiSheng Zhong for their support of this work. ==== Refs Hirshfield AN Development of follicles in the mammalian ovary Int Rev Cytol 1991 124 43 101 2001918 McNatty KP Fidler AE Juengel JL Quirke LD Smith PR Heath DA Lundy T O'Connell A Tisdall DJ Growth and paracrine factors regulating follicular formation and cellular function Mol Cell Endocrinol 2000 163 11 20 10.1016/S0303-7207(99)00235-X 10963868 Parrott JA Skinner MK Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis Endocrinology 1999 140 4262 4271 10.1210/en.140.9.4262 10465300 Wang C Roy SK Expression of growth differentiation factor 9 in the oocytes is essential for the development of primordial follicles in the hamster ovary Endocrinology 2006 147 1725 1734 16384866 Skinner MK Regulation of primordial follicle assembly and development Human Reprod 2005 11 461 471 10.1093/humupd/dmi020 Kezele PR Skinner MK Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly Endocrinology 2003 144 3329 3337 10.1210/en.2002-0131 12865310 Liu K Rajareddy S Liu L Jagarlamudi K Boman K Selstam G Reddy P Control of mammalian oocyte growth and early follicular development by the oocyte PI3 kinase pathway: new roles for an old timer Dev Biol 2006 299 1 11 10.1016/j.ydbio.2006.07.038 16970938 Reddy P Liu L Adhikari D Jagarlamudi K Rajareddy S Shen Y Du C Tang W Hämäläinen T Peng SL Lan ZJ Cooney AJ Huhtaniemi I Liu K Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool Science 2008 319 611 613 10.1126/science.1152257 18239123 Reddy P Adhikari D Zheng W Liang S Hämäläinen T Tohonen V Ogawa W Noda T Volarevic S Huhtaniemi I Liu K PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles Hum Mol Genet 2009 18 2813 2824 10.1093/hmg/ddp217 19423553 Reddy P Zheng W Liu K Mechanisms maintaining the dormancy and survival of mammalian primordial follicles Trends Endocrinol Metab 2010 21 96 103 10.1016/j.tem.2009.10.001 19913438 Adhikari D Zheng W Shen Y Gorre N Hämäläinen T Cooney AJ Huhtaniemi I Lan ZJ Liu K Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles Hum Mol Genet 2010 19 397 410 10.1093/hmg/ddp483 19843540 Li-Ping Z Da-Lei Z Jian H Liang-Quan X Ai-Xia X Xiao-Yu D Dan-Feng T Yue-Hui Z Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways Reprod Biol Endocrinol 2010 8 66 74 10.1186/1477-7827-8-66 20565902 Talmor-Cohen A Tomashov-Matar R Eliyahu E Shapiro R Shalgi R Are Src family kinases involved in cell cycle resumption in rat eggs? 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==== Front Breast Cancer ResBreast Cancer ResBreast Cancer Research : BCR1465-54111465-542XBioMed Central bcr31332239464710.1186/bcr3133Research ArticleEffects of a pre-visit educational website on information recall and needs fulfilment in breast cancer genetic counselling, a randomized controlled trial Albada Akke [email protected] Dulmen Sandra [email protected] Jozien M [email protected] Margreet GEM [email protected] NIVEL (Netherlands Institute for Health Services Research), Otterstraat 118-124, Utrecht, 3500 BN, the Netherlands2 Department of Primary and Community Care, Radboud University Nijmegen Medical Centre, Geert Grooteplein-Noord 21, Nijmegen, 6525 EZ, the Netherlands3 Department of Psychology, Utrecht University, Heidelberglaan 1, Utrecht, 3584 CS, the Netherlands4 Department of Medical Genetics, University Medical Center Utrecht, Lundlaan 6, 3508 AB, the Netherlands2012 6 3 2012 14 2 R37 R37 27 5 2011 20 2 2012 6 3 2012 Copyright ©2012 Albada et al.; licensee BioMed Central Ltd.2012Albada 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 Pre-visit education which helps counselees to prepare for their first visit for breast cancer genetic counseling might enhance information recall and needs fulfilment. This study assessed the effects of a pre-visit website with tailored information and question prompt sheet (QPS), named E-info geneca. Methods A total of 197 counselees were randomized to receive usual care (UC) or UC plus E-info geneca. All counselees completed a pre- and post-visit questionnaire and visits were videotaped. We studied effects on counselees' information recall, knowledge about breast cancer and heredity, fulfillment of needs, risk perception alignment, anxiety and perceived personal control, using multilevel regression analyses. Results Intent-to-treat analysis showed that counselees in the intervention group (n = 103) had higher levels of recall of information from the consultation (β = .32; confidence interval (CI): .04 to .60; P = .02; d = .17) and post-visit knowledge of breast cancer and heredity (β = .30; CI: .03 to .57; P = .03) than counselees in the UC group (n = 94). Also, intervention group counselees reported better fulfilment of information needs (β = .31; CI: .03 to .60; P = .03). The effects of the intervention were strongest for those counselees who did not receive an indication for DNA testing. Their recall scores showed a larger increase (β = .95; CI: .32 to 1.59; P = .003; d = .30) and their anxiety levels dropped more in the intervention compared to the UC group (β = -.60; CI: -1.12 to -.09; P = .02). No intervention effects were found after the first visit on risk perception alignment or perceived personal control. Conclusions This study shows that pre-counseling education, using tailored information technology, leads to more effective first visits for breast cancer genetic counseling, in particular for counselees who received no indication for DNA testing and, therefore, had no indication for a second visit. Future study should focus on the effects of a pre-visit website on the outcomes after a complete series of visits. Trial registration Dutch Trial Register ISRCTN82643064. ==== Body Introduction Women who are diagnosed with breast cancer or have affected relatives often worry about their or their children's risk to (re)develop breast cancer. Therefore, they might be referred to genetic counseling. Breast cancer genetic counseling aims to educate individuals about their breast cancer risk to optimize risk management, increase personal control and decrease anxiety [1]. However, studies have consistently found that most counselees still have a marked overestimation of risks relative to counselors' evaluations after genetic counseling [2]. The aims of the first visit are mainly to educate the counselee about breast cancer and genetics, make a risk estimation and decide whether a DNA test is indicated. For counselees who are the first in their family to request breast cancer genetic counseling, an indication for a DNA test might be for themselves or an affected relative. These counselees are assigned with the transmission of information regarding this possible indication, breast cancer risks and preventive options to their relatives. Counselees are unlikely to transfer and act according to advice that they do not remember [3]. Their recall of information from the first visit is, therefore, especially important for the course of breast cancer genetic counseling and the risk management of themselves and their relatives. However, after the initial visit counselees show only small improvements in knowledge about breast cancer and genetics [4,5], risk perceptions, perceived personal control and anxiety [5] and their recall of information has not been studied yet. Generally, recall of information from medical visits is low. Kessels [6] reported that 40% to 80% of the information presented was forgotten immediately by patients. Studies in oncology found that patients recalled only half of the information provided in general oncology visits when they were cued with topics [7] and actively reproduced only 23% of the information from consultations about chemotherapy [3]. Cued recall within genetic counseling for diverse disorders was higher, namely three-quarters of the most important items [8]. These higher scores might be due to the percentages of patients who request genetic counseling at their own initiative because of a need for information. Alternatively, different ways to assess recall might result in higher estimates [3]. Unidirectional transfer of generic instead of tailored or counselee-specific information has long been the preferred method in cancer genetics, both in the consultation and in pre-visit information. However, counselee-specific information is expected to be more effective in reaching the aims of counseling by increasing the counselee's recall [9,10]. Tailoring information to individual needs is an effective way of reducing the amount of information and ensuring that only relevant information is provided [11]. According to the Elaboration Likelihood Model and confirmed by findings from neuropsychological research, increased personal relevance of information enhances central processing [12] and information retrieval [13,14]. Therefore, the use of counselee-specific rather than general recommendations should enhance recall [15]. Additionally, the provision of additional written information and spreading information over different time points might show increased retention [14,16,17]. The combination of pre-visit tailored information with more counselee-specific information provided during the visit, might thus result in increased information recall and knowledge. Additionally, improved recall of the risk information might increase the alignment of counselees' risk perception with the counselors' estimation. Previous studies of computer-based education prior to genetic counseling were limited to assessment of counselees' knowledge about breast cancer and heredity [18-20]. The current study assessed counselee's recall of the counselor's advice as discussed during the visit. Comparison with the consultation video determined whether this recall was correct. As opposed to general knowledge of hereditary breast cancer this recall of the counselor's explanation and advice determines whether counselees can transfer risk estimates correctly to relatives and can act upon the advice given [7]. Additionally, fulfillment of information needs evaluates whether the counselee's agenda was met. Studies that evaluated the impact of meeting counselees' individual needs found that [21] adjusting communication to these needs resulted in improved counselee's perceived personal control and anxiety [5]. This article describes an randomized controlled trial (RCT) focused on the effects of a pre-visit educational website, E-info geneca [22] to optimize the first visit. The website provided computer-tailored information concerning counselees' pre-visit needs, for example, the procedure and consequences of genetic counseling for counselees who were the first in their family to request breast cancer genetic counseling [23]. Additionally, the website offered a QPS to encourage counselees to formulate questions [24]. These questions were sent ahead to the counselor and were answered during the visit. Previous studies showed that after having accessed E-info geneca, counselees communicated more assertively by more often sharing their agenda, directing the flow of the visit and paraphrasing the counselor to check their understanding. As a result, counselors provided more counselee-specific information [25]. The current study evaluates whether the pre-visit educational website improved counselee outcomes after their first visit, that is, information recall, knowledge, fulfillment of needs, risk perception alignment, anxiety and perceived personal control. Methods Study design This study was conducted at the department of Medical Genetics of the University Medical Center (UMC) Utrecht. The study was approved by the institutional medical ethical committee and is registered in the Dutch Trial Register (ISRCTN82643064). The department of Medical Genetics enrolled counselees from February 2008 to April 2010. This department offers breast cancer genetic counseling according to the Dutch guidelines [26] and services are similar to those of the other eight family cancer clinics in the Netherlands. New counselees, 18 years old or older, who were the first in their family to seek breast cancer genetic counseling, were sent information about the study and an opt-out form. The opt-out form included a question about reasons for withdrawal. Counselees were ineligible if they lacked internet or email access or when they requested pre-symptomatic DNA testing in the presence of an identified BRCA1/2 gene mutation in a relative. All counselees who did not return the opt-out form were randomly assigned 1:1 to the usual care (UC) or intervention group (UC + website E-info geneca) by a secretary unaware of respondent's characteristics using sequentially numbered, sealed, opaque envelopes. UC comprised a brief standard pre-visit leaflet with information about the counseling procedure. Both UC and intervention group respondents received a login to access the web-based baseline questionnaire. Upon completion of the baseline questionnaire the intervention group respondents received a link to access the website E-info geneca. At the start of the consultation the counselor collected the informed consent form. The visits were videotaped. In the week after their visit, counselees again completed a web-based questionnaire. Because with 100 counselees in each group, there was an 80% power to detect effect sizes of d = .40 or higher, we aimed to include 200 counselees. Counselor characteristics Counselors were clinical geneticists, residents in clinical genetics, genetic counselors or genetic counselors in training. All will be referred to as 'counselor'. Their age, gender and number of years of experience with cancer genetic counseling was assessed at the start of their participation in the study. Counselee characteristics Age, having children, personal and family cancer history and educational attainments were assessed in the baseline counselee questionnaire. All but the latter were derived from the medical file if missing. Additionally, whether or not there was an indication for DNA testing for the counselee or an affected family member was collected from the medical files. Measures Counselees' recall of information from the visit was assessed in the post-visit questionnaire with seven questions for cued recall. Each question prompted a topic of the consultation, for example, limitations of DNA testing and involving family members, and these topics were based on the counselees' information needs questionnaire Quote-geneca [23]. Each question started with a multiple choice indication of whether the topic was discussed. Answer options were 1) no, not discussed, 2) yes it was discussed but I don't remember what was said, 3) yes, namely. With the latter, the counselee was invited to write down what she recalled about this topic [3]. Coders first assessed whether the topic was discussed in the visit based on the videotapes. Second, each item recalled was compared with the specific items mentioned by the counselor [3,7]. The percentage of accurate recall was calculated by dividing the sum of the accurately recalled items by the total number of items discussed [7]. Recall was coded by AA and a second coder recoded a random 10% of the visits. Coders were blinded to group allocation. Interrater reliability was assessed. Cohen's kappa for the recall per item averaged 0.70 (range 0.39 to 1.0). The intra class correlation (ICC) of the overall recall percentage was 0.89. The level of accurate knowledge about hereditary breast cancer was assessed using the validated Dutch hereditary breast cancer knowledge scale [5,27]. Respondents indicated whether each item was correct, incorrect, or whether they did not know. A knowledge score was computed as the mean number of correct answers, with higher scores indicating more accurate knowledge. Pre-visit, counselees' needs were assessed with the QUality Of counselling Through counselees' Eyes scale for cancer genetic counseling (QUOTE-geneca) [23]. At baseline, respondents indicated the importance (not important, fairly important, important, extremely important) of these needs. Post-visit, identical items were administered to measure fulfillment of these needs (inadequate, not really adequate, adequate, more than adequate) [28]. The QUOTE-geneca includes four generic needs, which refer to what a counselor should do during counseling (25 items) and four cancer genetic information needs, which refer to receiving explanations on hereditary cancer (19 items), as identified by principal component analysis with good internal consistency [23]. Risk perception alignment was defined as the degree of agreement of the counselee's risk perception with the counseled risk [2]. Counselees rated their perceived risk that they themselves would (re-) develop breast cancer in the future, that hereditary breast cancer runs in their family and that they themselves had inherited a breast cancer gene mutation, on visual analogue scales from 0 to 100%. Counselors rated their estimations of these risks for the counselee on identical scales after the visit. Additionally, we categorized this risk based on population or slightly increased (< 20%), moderate (17% to 30%) or high risk (> 30%) of developing breast cancer [29]. The perceived absolute risk of developing breast cancer was assessed on a 5-point scale from 1 'very low to 5 'very high'. Finally, the perceived relative risk was assessed, indicating whether the counselee perceives her breast cancer risk as lower, even or higher than the average risk for women of her age [30]. Pre- and post-visit anxiety was assessed with the validated Dutch state version of State-Trait Anxiety Inventory (STAI; 10 items) [28,31]. Scores range from 10 to 40, higher scores indicating greater anxiety. Pre- and post-visit perceived control was assessed using the validated Dutch version of the Perceived Personal Control questionnaire (PPC) [28,32]. Scores range from 0 to 2 with high scores indicating high perceived control. Analysis We analyzed respondents in the groups to which they were randomized regardless of whether they actually accessed E-info geneca or other protocol irregularities. The few missing values (seven cases) on the baseline measures of educational attainment were imputed with the median. Post-visit, the number of missing data on the outcome variables ranged from 18 for knowledge and recall, 20 for fulfillment, 22 for perceived personal control, 25 for anxiety and 43 for risk perception. All counselees were included in analyses through use of repeated measures analysis which makes use of available data of all cases (intention-to-treat analysis) [33]. Intervention and UC group counselees were compared on socio-demographics, cancer history and levels of pre-visit measures using Chi-square tests and t-tests. To account for the multilevel structure of measurements (level 1) nested within counselees (level 2) nested within counselors (level 3), random effects multilevel regression analyses were conducted. This method corrects for the number of consultations per counselor [34]. The percentage of variance explained at the counselor level ranged from zero for knowledge to 12% for anxiety. Analyses were controlled for baseline values, counselee disease status, education and receiving an indication for DNA testing. Additionally, analyses were controlled for background variables at the counselor level, namely whether the counselor was a clinical geneticist or a genetic counselor and whether or not the counselor was still in training. All analyses were conducted using Stata 10. Two-sided tests of significance were performed and results were considered statistically significant when P < .05. Furthermore, Cohen's effect sizes (d) were calculated [35]. Results Response Few counselees were ineligible because of lack of internet or email access (24 of 371; 6.5%). The response was 58.6%. Half of the decliners gave a reason (72 of 139; 50.4%). Most decliners preferred that the visit not be videotaped (48 of 72; 66.7%). There were no significant differences between participants and decliners in age (t = 1.62; P = .11), disease status (X2 = .05; P = .81), family history of cancer (X2 = .06; P = .82) and referral pathway (X2 = 87; P = .35). A flowchart of the study and reasons for non-response are shown in Figure 1. Figure 1 Flow diagram of study participants. Counselors All fourteen breast cancer genetic counselors of the department of Medical Genetics participated and recorded 4 to 29 consultations each. Six were genetic counselors, of whom three were in training (all women). Three were experienced (5 to 15 years) clinical geneticists (two men, one woman) and five were clinical geneticists in training (all women), see Table 1. Table 1 Characteristics of the genetic counsellors (N = 14). Mean SD (range) Age (years) 36.22 9.60 (26 - 53) N Genetic counselor 3 Genetic counselor in training 3 Clinical geneticists 3 Clinical geneticist in training (resident) 5 Male 2 Female 12 Experience in cancer genetic counseling < 1 year 8 1 to 5 years 1 > 5 years 3 5 to 10 years 0 > 10 years 2 Counselee characteristics As shown in Table 2 UC and intervention group respondents were similar with regard to all background characteristics except for being affected with breast cancer themselves (X2 = 5.10; P = .02). There was no significant association between having (had) breast cancer and baseline knowledge (T = .12; P = .91). One counselee was affected with ovarian cancer. There were no significant baseline differences in knowledge, information needs, risk perception, anxiety and perceived personal control between the study groups (Tables 3, 4 and 5). Table 2 Counselee characteristics (N = 197). UC group (n = 94) Intervention group (n = 103) Mean SD (range) Mean SD (range) Age (years) 41.3 11.5 (21 to 68) 41.5 11.3 (21 to 69) N % N % Children (having children) 64 68.1 71 68.9 Personal history of breast cancer (affected) 29 30.9 49 47.5 1st degree relatives affected with breast cancer 47 52.8 54 52.9 Educational attainment: University (MSc/BSc)/higher vocational education (BSc) 42 48.8 35 35.34 Middle vocational education 23 26.7 30 30.3 High school/Secondary education 18 20.9 33 33.3 < High school level 3 3.5 1 1.0 Referral pathway: GP 50 55.6 44 43.1 Specialist consultant UMC 18 24.4 32 31.4 Specialist consultant peripheral hospital 21 20.0 26 25.5 Indication for DNA-testinga 67 71.3 81 78.6 Breast cancer risk category: High (≥ 30% lifetime risk) 17 18.1 18 17.5 Moderate (20 to 30% lifetime risk) 34 36.2 38 36.9 Population (< 20% lifetime risk) 43 45.8 47 45.6 a Indication for testing the counselee or a relative as judged during the initial visit. Summations vary due to missing values. GP, general practitioner; UC, usual care; UMC, University Medical Center. Table 3 Counselees' level of accurate knowledge about breast cancer and heredity. Baseline (T0) Post-visit (T1) UC group (n = 94) Intervention group (n = 103) UC group (n = 94) Intervention group (n = 103) Scale Mean SD Mean SD Mean SD Mean SD P a Accurate knowledge score (0-7) 4.65 1.46 4.64 1.60 5.71 1.53 6.10 1.13 .03 True/false knowledge items: correct answer correct answer correct answer correct answer n % n % n % n % Early detection and treatment of bc lead to longer survival than late detection and treatment (True) 86 96.6 98 97.0 79 94.1 90 94.7 .74 All women who are carrier of an altered gene (mutation) for bc, will develop bc in the long term (False) 39 43.8 47 46.5 56 66.7 72 75.8 .048 A woman who has a sister with an altered gene (mutation) for bc, has a 50% change (1 in 2) to also carry the mutation herself (True) 31 35.2 34 33.7 52 61.9 70 73.7 .09 A woman who does not have an altered gene (mutation) for bc, can nevertheless develop bc (True) 70 78.7 77 77.8 78 92.9 89 93.7 .74 Physical examination is necessary only when you have complaints; at that point it is soon enough to prevent bc (False) 84 94.4 93 93.0 76 90.1 89 93.7 .56 If a father has an altered gene (mutation) for bc, then his children have 50% chance (1 in 2) of also having this mutation (True) 27 30.3 38 37.6 60 71.4 81 85.3 .02 If in a family, in which bc frequently occurs, no altered gene (mutation) for bc is found, then regular breast surveillance is no longer necessary (False) 77 86.5 82 81.2 79 94.1 88 92.6 .50 a Between group differences at T1. bc, breast cancer; UC, usual care. Table 4 Counselees' information needs at baseline and fulfilment of these needs concerning breast cancer genetic counselling post-visit. Baseline (T0) needs Post-visit (T1) fulfilment UC group (n = 94) Intervention group (n = 103) UC group (n = 94) Intervention group (n = 103) Scale (1-4) Factors (1-4) Mean SD Mean SD Mean SD Mean SD P a Cancer genetic information needs 3.24 .38 3.25 .39 3.02 .55 3.14 .53 .03 Determination and meaning of being a carrier of a cancer gene 3.32 .47 3.33 .49 3.03 .62 3.15 .58 .04 Emotional consequences for counselee and family 3.13 .55 3.08 .59 2.88 .69 2.97 .67 .26 Own risk of developing breast cancer 3.44 .47 3.46 .56 2.92 .74 2.99 .77 .36 Heredity of breast cancer 2.67 .53 2.80 .53 3.25 .59 3.44 .53 .008 Generic needs 3.25 .36 3.25 .37 3.21 .44 3.26 .43 .22 Procedural aspects of counseling 2.93 .52 2.82 .58 3.08 .46 3.14 .52 .23 Sensitive communication 2.72 .74 2.65 .79 3.46 .47 3.50 .43 .53 Emotional support 2.93 .53 2.90 .62 3.04 .55 3.06 .49 .46 Assessment of susceptibility 3.09 .54 3.1 .49 3.03 .62 3.12 .59 .17 a Between group differences at T1. UC, usual care. Table 5 Effects of E-info geneca on risk perception, anxiety and perceived personal control. Baseline (T0) Post-visit (T1) UC group (n = 94) Intervention group (n = 103) UC group (n = 94) Intervention group (n = 103) Range Mean (SD) Mean (SD) Mean (SD) Mean (SD) Ba P a da Anxiety (10-40) 10.0 - 40.0 19.94 (6.25) 19.73 (5.81) 18.54 (6.11) 17.91 (5.58) -.70 .24 .11 Perceived personal control (1-3) .22 -2.00 1.23 (.43) 1.15 (.41) 1.33 (.41) 1.34 (.44) -.03 .49 .00 Breast cancer risk perception alignment (mean difference between counselee and counselor's risk estimation) 1-100 26.69 (19.89) 27.83 (18.20) 18.00 (16.17) 16.81 (16.35) .07 .79 .07 No indication for DNA-testing (n = 49): Anxiety 10-34 20.52 (6.76) 20.00 (6.50) 19.15 (7.96) 16.65 (5.49) -.60 .02 .36 Perceived personal control (1-3) .22 -2.00 1.37 (.41) 1.19 (.46) 1.25 (.49) 1.19 (.59) .09 .83 .00 Breast cancer risk perception alignment (mean difference between counselee and counselor's risk estimation) 1-100 29.75 (20.87) 25.94 (15.69) 12.37 (20.27) 11.32 (17.59) -.08 .79 .06 a Between group difference at T1. UC, usual care. Information recall Less than half of all information items were recalled correctly (Table 6). The total number of recalled items was significantly higher in the intervention compared to the UC group (β = .32; CI: .04 to .60; P = .02; d = .17). The effect size of the intervention was larger for the counselees who did not receive an indication for DNA testing (β = .95; CI: .32 to 1.59; P = .003; 52.69(23.72) versus 59.94(25.09); d = .30). The educational level of the counselee was also a significant predictor (β = .59; CI: .32 to .87; P = .000). Additionally, counselees who were asked to return for a follow-up consultation recalled less than those who had only one consultation (β = -.46; CI: -.92 to -.01; P = .046). And finally, counselees tended to have more recall when they had seen a genetic counselor (nurse) instead of a clinical geneticist (β = .27; CI: .00 to .55; P = .047) and less recall when they had seen a counselor who was still in training (β = -.29; CI: -.57 to .00; P = .048). This was not related to the total number of information items discussed (geneticists M = 7.01 SD = 2.45; counselors M = 6.93 SD = 2.56). Table 6 Counselees' recall of information items concerning topics discussed in the consultation. UC group (n = 94) Intervention group (n = 103) N items discussed Recalled items % items recalled Discussed Recalled items % items recalled M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) P a P b Total 6.79 (2.43) 3.17 (2.07) 45.43% (21.98) 7.12 (2.55) 3.49 (2.28) 49.64% (24.45) .10 .02 Topics (items) N (%) ce topic discussed M (SD) N (%) ce topic discussed M (SD) P a P b Probability that hereditary breast cancer runs in the family 84 (100) .93 (.30) 91 (97) .90 (.45) .90 .85 Possibilities DNA-test 82 (97) .78 (.46) 88 (95) .80 (.45) .11 .78 Limitations DNA-test 77 (92) .66 (.58) 82 (89) .78 (.67) .50 .09 (the risk of hereditary bc can not be ruled out, testing only performed on affected individuals, unclassified variant) Possibilities for early detection of breast cancer 77 (93) 1.00 (.85) 88 (95) .89 (.98) .51 .76 (mammography, MRI, ultrasound, breast self examination, clinical breast examination of GP/surgeon) Possibility of risk reducing breast surgery 25 (30) .48 (.51) 43 (46) .68 (.52) .04 .15 Emotional consequences of genetic counselling 47 (57) .73 (.73) 60 (65) .80 (.67) .82 .85 (for the counselee and/or for relatives, for example being reminded of the illness period, feelings of guilt towards relatives, possibilities for support) Involving family members in the genetic counselling procedure 68 (82) 1.01 (.50) 82 (88) .95 (.44) .75 .58 (Asking for permission to request medical file, asking cooperation for DNA-test, informing about DNA-test) a Between group differences for items discussed; bBetween group differences for items recalled. bc, breast cancer; ce, counselees; GP, general practitioner; MRI, magnetic resonance imaging; UC, usual care. In almost all visits, the counselor provided information about the probability that hereditary breast cancer runs in the counselee's family and of the possibilities and limitations of DNA testing (Table 6). The possibility of risk reducing breast surgery was significantly more often discussed in intervention group visits (X2 = 4.81; P = .03). The probability that hereditary breast cancer runs in the family was recalled best and information about risk reducing breast surgery and the emotional consequences of genetic counseling had the lowest recall scores. Knowledge of breast cancer and heredity Intervention group counselees had relatively more post-visit knowledge of breast cancer and heredity than UC group counselees (β = .30; CI: .03 to .57; P = .03; d = .28). Additionally, counselees in whose family there was an indication for DNA testing had more post-visit knowledge (β = .72; CI: .31 to 1.15; P = .001). In the intervention group, knowledge especially concerning inheritance and penetrance of BRCA1/2 mutations was enhanced (Table 3). Needs fulfilment As shown in Table 4 the intervention group counselees had higher fulfillment scores concerning their cancer genetic information needs (β = .31; CI: .03 to .60; P = .03; d = .22). Intervention group counselees especially indicated higher fulfillment of their need for information about the determination and meaning of being a carrier of a mutation in one of the breast cancer genes (β = .29; CI: .01 to .56; P = .04) and about hereditary breast cancer than the UC group counselees (β = .42; CI: .12 to.72; P = .006). Counselees in both groups reported high fulfillment of generic needs (β = -.18; CI: .36 to .12; P = .22). Risk perception Intervention condition was not associated with the alignment of the counselee's perception of her breast cancer risk with the counselor's risk estimation (β = .07; P = .79). Table 5 shows the mean difference between counselee and counselor's risk estimation. Neither was there an intervention effect on the perceived relative risk or the verbal indication of the height of the risk of developing breast cancer. Most counselees (101; 67.3%) overestimated their risk of (re-)developing breast cancer post-visit. On average their estimation was 21.62 (SD = 17.42) percentage points higher than that of the counselor. A minority of 40 counselees (26.7%) underestimated their risk with a mean of 10.83 (SD = 8.08) percentage points. The alignment of counselees' risk perception with counselors' estimation did improve for counselees who did not receive an indication for DNA testing, regardless of group allocation. While at baseline 68.8% of counselees overestimated their breast cancer risk based on the risk categories, this was 32.7% post-visit. Most counselees (75; 48.7%) also overestimated the risk of hereditary cancer running in their family, (average overestimation of 25.68 percentage points (SD = 17.84)) and their risk of being a carrier of a BRCA1/2 mutation (92; 61.3%; average overestimation of 23.46 percentage points (SD = 18.66)). Other counselees underestimated these risks (69; 45.4%; mean 16.90 (SD = 13.70) and (52; 34.7%; mean 14.89 (SD = 12.69), respectively). The alignment of these risk perceptions with the counselors' estimations was not significantly associated with the intervention condition (β = .01; P = .91 and (β = .07; P = .57, respectively; not shown in Table). Anxiety For the whole group of counselees, post-visit anxiety was unrelated to the intervention condition (Table 5). Overall explained variance was 48% and 4% of the variance in the model was due to counselor variation. However, the group of counselees who did not receive an indication for DNA testing (n = 49) had significantly lower anxiety scores in the intervention compared to the UC group when controlled for baseline values (β = -.60; CI: -1.12 to -.09; P = .02). Perceived personal control Perceived personal control was not significantly related to the intervention condition (Table 5). Levels tended to be higher if there was an indication for DNA testing for the counselee or a family member (β = .34; CI: .01 to .69; P = .06). Discussion This study showed that a pre-visit educational website with QPS improves counselees' recall of information discussed in their first visit for breast cancer genetic counseling. As counselees' transfer of information from the visit to relatives has been described as a whisper game where the counselor's information fades out [36], our finding of less noise in counselees' recollection of information is very important. Additionally, counselees considered their need for information better addressed after the visit. Furthermore, we confirmed previous findings that pre-visit education improves breast cancer knowledge [18-20]. These findings are in line with the theoretical notion that provision of pre-visit information combined with increased adjustment of information to the individual increases recall. In 45-minute first consultations for breast cancer genetic counseling a large amount of information is transferred with the intention that the counselee will understand and remember this information so as to make decisions about DNA testing and to involve relatives [37,38]. This study is the first to assess how much counselees actually remember and found that less than half of the information was recalled. This percentage is similar to levels of recall in oncology outpatient settings [7]. The intervention effect on recall was strongest for those counselees who did not receive an indication for DNA testing and will therefore not have a second visit. Although the effect size was modest, other interventions have failed to produce significant differences [17] or produced small effect sizes [10]. Future endeavors should determine ways to further increase recall. Effects of the intervention on anxiety were only found for counselees who did not receive an indication for DNA testing. The lack of improvements for those who received an indication for DNA testing is understandable as the indication itself and waiting for the test results can be a source of distress [39]. For counselees who did not receive an indication, their counseling is limited to one visit and they are generally at population risk or slightly increased risk. A decrease in their anxiety is therefore appropriate. Apparently, when counselees had learned about breast cancer and heredity through the website they were better able to process the reassuring information in the visit. The computer-tailored information on the website about indications for hereditary breast cancer and about the need for an indication for DNA testing might have been helpful to prepare the counselee for the population risk estimate. This would result in lower cognitive dissonance during the visit, which contributes to enhanced processing of information [40]. Additionally, they might have better processed the reassuring message because of the slight increase of counselee-specific information in the first visit due to the QPS [25]. QPS studies have found mixed effects on anxiety [10,41]. Possibly, the QPS in the current study showed effects because the questions were sent ahead to the counselor and were endorsed by the counselor. The pre-visit web-based education had no effect on alignment of counselees' risk perceptions with the counselors' estimation. An explanation for this lack of effect might be the fact that the risk perception, as opposed to factual recall of information, seems to be determined to a large extent by personal experiences of loss [42], identification with affected family members and personality factors [43]. Counselees' perception of their risk of developing breast cancer may therefore be less open to change [44]. Additionally, there were no intervention effects on perceived personal control. Effects of web-based information alone might be limited to cognitive outcomes and might therefore not include perceived personal control [11,45]. In this study, effects on personal control were expected through provision of more counselee-specific information in the visit, but this improvement was small [25]. Most counselees only receive estimations for their personal risk in the final visit when the results of the DNA tests are discussed. Improvements in personal control might thus be more likely after this final visit as only after receiving advice for surveillance or preventive options can counselees take the required actions to control their risk [32]. Additionally, counselees who did not receive an indication for DNA testing for themselves did not gain increased control over decisions. Research on the effects after the final visit is needed. Analyses were performed with multilevel analysis to take differences in counselor styles into account. This study is the first to show the percentage of variance at the counselor level and this might provide insight into the extent to which differences in individual counseling styles affect genetic counseling outcomes. Although differences between counselors have not been studied before with appropriate methods, several authors have mentioned the need for this [37,44]. For example, geneticists and genetic counselors have received different training and might have different counseling styles resulting in better or worse outcomes. Our study detected a significant difference in the recall of counselees who were counseled by a genetic counselor compared to a clinical geneticist. However, the number of counselors (14) is too small to guarantee reliable effects for variables at the counselor level [46]. The variation in counselors' communication styles and its impact on outcomes should be the focus of future multi-center research allowing for inclusion of more counselors. The first visit for breast cancer genetic counseling has been described as an educational session in which large amounts of standard information are being transferred and the counselee's understanding is scarcely checked upon [37]. This finding has been reported not only for the Dutch situation [38], but also for the USA [47,48], UK [49] and Australia [47,50,51]. Therefore, we expect that the reported benefits of a pre-visit educational website are generalizable to other countries. However, countries have different health care systems due to which populations of breast cancer genetic counselees and their genetic knowledge might vary slightly [52]. Pre-visit educational websites should therefore be adapted to country-specific settings and populations of counselees. Limitations There are some limitations. First, counselors were not blinded to group allocation as they received counselees' question sheet (QPS) and their responses to these questions were part of the intervention. Second, the response rate is moderate, but relatively high for studies using video recordings of genetic counseling visits [5,38,53]. Importantly, there were no significant differences between responders and decliners and the results of the study are therefore representative for breast cancer genetic counseling counselees. Third, the knowledge scale showed a clear ceiling effect. On at least two of the items hardly any improvements from baseline were possible and this might have hampered the effect size. Fourth, the high percentage of missing values on counselees' risk perception was due to a technical error in the web-based questionnaire and is therefore unlikely to be related to counselee characteristics. Fifth, as we have studied the effects of a combined intervention, we can not distinguish between effects of the two components of this web-based intervention: tailored information and the QPS. Both the fact that counselees learned from reading information on the website and the endorsement of the QPS in the visit might have contributed to the reported effects. Sixth, this study lacked power to untangle gender differences in counselor communication. Conclusions When counselees had prepared with a pre-visit website, they remembered more information from their first consultation for breast cancer genetic counseling and their information needs were better addressed. If adopted in practice, pre-counseling education could lead to more effective first visits for breast cancer genetic counseling due to improved cognitive outcomes. Counselees who need to transfer information to their family might better succeed as a result of increased recall of what was discussed with the counselor. Abbreviations B: beta; BC: breast cancer; CI: confidence interval; d: Cohen's effect size; GP: general practitioner; ICC: intra class correlation; M: mean; MRI: magnetic resonance imaging; N: number; P: probability; PPC: perceived personal control; QPS: question prompt sheet; QUOTE: Quality of counselling Through counselees' eyes questionnaire; RCT: randomized controlled trial; SD: standard deviation; STAI: State-Trait Anxiety Inventory; UC: usual care; UMC: University Medical Center. Competing interests The authors declare that they have no competing interests. Authors' contributions AA carried out the acquisition of data, performed the statistical analyses and drafted the manuscript. SvD conceived of the study, participated in its design and coordination and helped to interpret the data and helped to draft the manuscript. JB participated in the design of the study, helped to interpret the data and helped to revise the manuscript. MA participated in the study design and coordination, helped to interpret the data and helped to revise the manuscript. All authors read and approved the final manuscript. Acknowledgements We want to thank all counselees who participated in this study. We also owe our gratitude to the clinical geneticists, genetic counselors and residents in clinical genetics of the department of Medical Genetics of the UMC Utrecht, in particular, Angela Schoemaker and Ivette Wieffer who arranged the logistics of the study. We are grateful to Anita Wallet, secretary of the department of Medical Genetics and Doortje Saya, secretary of Nivel, for organizing many practicalities of the study. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23029172PONE-D-12-1845810.1371/journal.pone.0045667Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyCytokinesModel OrganismsAnimal ModelsMouseMedicineClinical ImmunologyImmune SystemCytokinesImmunityInflammationAllergy and HypersensitivityImmune ResponseNutritionMicronutrient DeficienciesPulmonologyAsthmaIron Supplementation Decreases Severity of Allergic Inflammation in Murine Lung Iron Supplements Decrease Allergic InflammationHale Laura P. 1 3 * Kant Erin Potts 2 Greer Paula K. 1 Foster W. Michael 2 1 Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States of America 2 Department of Medicine/Division of Allergy, Pulmonary, and Critical Care Medicine, Duke University Medical Center, Durham, North Carolina, United States of America 3 Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina, United States of America Ryffel Bernhard Editor French National Centre for Scientific Research, France * E-mail: [email protected] Interests: Part of this study was supported by personal funds derived from payments to Dr. Hale for services that she renders that were diverted for use in the laboratory, rather than being taken as income. 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: LPH WMF. Performed the experiments: LPH EPK PKG. Analyzed the data: LPH EPK PKG WMF. Contributed reagents/materials/analysis tools: LPH EPK PKG WMF. Wrote the paper: LPH PKG. Edited and approved manuscript and figures: EPK WMF. 2012 20 9 2012 7 9 e4566725 6 2012 21 8 2012 © 2012 Hale et al2012Hale 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 incidence and severity of allergic asthma have increased over the last century, particularly in the United States and other developed countries. This time frame was characterized by marked environmental changes, including enhanced hygiene, decreased pathogen exposure, increased exposure to inhaled pollutants, and changes in diet. Although iron is well-known to participate in critical biologic processes such as oxygen transport, energy generation, and host defense, iron deficiency remains common in the United States and world-wide. The purpose of these studies was to determine how dietary iron supplementation affected the severity of allergic inflammation in the lungs, using a classic model of IgE-mediated allergy in mice. Results showed that mice fed an iron-supplemented diet had markedly decreased allergen-induced airway hyperreactivity, eosinophil infiltration, and production of pro-inflammatory cytokines, compared with control mice on an unsupplemented diet that generated mild iron deficiency but not anemia. In vitro, iron supplementation decreased mast cell granule content, IgE-triggered degranulation, and production of pro-inflammatory cytokines post-degranulation. Taken together, these studies show that iron supplementation can decrease the severity of allergic inflammation in the lung, potentially via multiple mechanisms that affect mast cell activity. Further studies are indicated to determine the potential of iron supplementation to modulate the clinical severity of allergic diseases in humans. This work was funded by NIH grants ES 016347 and AI 081672 to WMF and by additional funds paid out of LPH’s “discretionary account” rather than from existing grants. The funds in the discretionary account are derived from payments to LPH for services that she renders and are personal funds, diverted for use in the laboratory, rather than being taken as income. The National Institutes of Health had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Under normal conditions, the immune system responds to common ingested or inhaled antigens by inducing a state of specific non-responsiveness called tolerance. Allergy develops when antigen-presenting cells process these otherwise innocuous antigens and present them to T cells in a cytokine milieu that promotes B cell production of allergen-specific IgE. When allergen-specific IgE bound to mast cell Fc receptors is cross-linked by allergen, the mast cells release pre-formed granules containing histamine, TNF, IL-8 and other inflammatory mediators. Mast cell degranulation thus induces a cascade that results in infiltration by immune cells and and stimulates de novo production of additional inflammatory mediators. Inflammation may be localized to the site of exposure (e.g. respiratory or gastrointestinal tracts) or may become systemic, resulting in life-threatening episodes of anaphylaxis. Asthma is a chronic inflammatory disease of the airways associated with airway hyperresponsiveness that often occurs in allergic patients. Despite an intensive research effort to identify mechanisms central to the diathesis of inflammatory airway disease, control factors remain unclear [1]. The incidence of asthma is increasing and affects 300 million people worldwide, including 23 million in the US, where it leads to 5,000 deaths annually [2]. Current treatments that decrease the severity of airway obstruction include bronchodilators and corticosteroids, but these fail to address any underlying allergic reactions. Methods to decrease the severity of allergic reactions include IgE depletion using anti-IgE monoclonal antibodies, induction of IgG blocking antibodies by allergen immunotherapy, and blocking downstream effects of mast cell degranulation using antihistamines or corticosteroids. Controlled oral exposures to allergens have in some cases allowed subjects to develop tolerance, although the mechanisms involved are still under active investigation. However, many asthma patients go on to develop treatment-resistance and disease progression despite optimized treatments, [3], [4]. Identification of additional therapies that could prevent or decrease the severity of allergic reactions would provide a major improvement in clinical care of patients with allergen-triggered asthma. Iron status is well-known to affect the ability to oxygenate tissues and generate energy, but how iron affects immune and non-immune-triggered inflammatory processes is less clear. We recently identified iron deficiency as a trigger for increased mast cell activation that was associated with mast cell-dependent hair loss in IL-10-deficient (Il10 −/−) mouse pups [5]. The effects of iron status on mast cell reactivity in classic mast cell-mediated diseases such as allergy have not been reported previously. These studies were designed to test the hypothesis that iron supplementation could beneficially decrease the severity of allergic disease by decreasing mast cell activation, using a classic model of IgE-mediated allergic asthma in mice. Methods Ethics Statement All animal studies were approved (protocol numbers A151-09-05 and A190-09-07) by the Institutional Animal Care and Use Committee of Duke University, an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International. Suffering was minimized by providing anesthesia for all procedures (the Flexivent measurements) with the potential to cause pain and/or distress. Animal Studies Breeding pairs of C57BL/6 wild type (WT) and mast cell-deficient Kit W-sh/Kit W-sh (Sash) mice (strain name  =  B6.Cg-KitW-sh/HNihrJaeBsmJ; stock #005051) [6], [7] were obtained from Jackson Laboratories (Bar Harbor, ME). Experimental mice were placed on AIN-93G- based custom diets containing 8 or 200 ppm iron at 3–4 weeks of age. Four weeks later, mice were sensitized to ovalbumin (OVA) by intraperitoneal immunization with 20 µg OVA +2 mg alum on day 0 and 10 µg OVA +1 mg alum on day 7, then challenged by a 20 min exposure to aerosolized 1% OVA or saline on days 14, 15, 16. On day 17, mice were anesthetized and total airway resistance in response to nebulized methacholine was measured as described [8]. Only male mice were used, since males and females differ in both iron homeostasis [9] and susceptibility to asthma [10]. Erythropoiesis was determined by a complete blood count (CBC). Non-heme iron stores in liver and spleen were determined colorimetrically [11]. Serum iron was measured using a commercially available kit (Thermo Scientific, Middletown, VA). Hepcidin transcript levels were determined by real-time reverse transcriptase PCR of liver tissue [12] and expressed as 2−ΔΔCt, based on the threshold cycle (Ct) for hepcidin compared to β-actin, OVA-specific IgG was quantitated by antibody capture enzyme immunoassay on immobilized OVA [13]. OVA-specific IgE was measured by antibody capture on immobilized anti-IgE, followed by binding and quantitation of biotinylated OVA via a standard curve with known concentrations of anti-OVA IgE (clone PMP68; AbD Serotec, Raleigh, NC). Bronchoalveolar lavage (BAL) samples were obtained following euthanasia. Cytokines were quantitated by a Luminex bead-based multiplex fluorescent immunoassay (BioRad). Mucous (goblet cell) metaplasia was evaluated using periodic acid-Schiff (PAS)-stained lung sections. For analysis of Muc5AC transcripts, total RNA was extracted from ∼30 mg lung tissue. cDNA was synthesized and real-time PCR analyses were performed using SYBR green fluorescence and specific primers [14]. Relative levels of Muc5AC transcripts were expressed as 2−ΔΔCt versus β-actin. In vitro Studies of Mast Cell Activation Primary bone marrow-derived mast cells (BMMC) were derived as described [15] using 10% fetal bovine serum with an iron content of 193 µg/dL (79% transferrin saturation). For degranulation assays, sensitized BMMC were incubated with ± iron for 30 min prior to activation. RBL-2H3 mast cells (American Type Culture Collection; Manassas, VA) were cultured for 20 hrs in media containing 1 µg/ml IgE and 10% calf serum with low (25 µg/dL Fe; 5% transferrin saturation), medium (low supplemented to 642 µg/dL Fe; 83% transferrin saturation), or high iron content (medium serum supplemented to 1402 µg/dL Fe with 50 µM FeSO4). Degranulation was triggered by cross-linking with goat anti-mouse IgE antibody in serum-free Tyrode’s buffer. Percent degranulation was determined using a colorimetric substrate assay for β-hexoseaminidase as described [16]. Total granule content was determined following lysis with Triton X-100. Viability after degranulation was monitored using a tetrazolium-based colorimetric assay. Results Iron Supplementation Decreases the Severity of Allergic Asthma At 3–4 weeks of age, wild type (WT) C57BL/6 male mice were placed either on an unsupplemented diet containing 8 ppm iron or a matched diet supplemented to contain 200 ppm iron. The recommended daily amount of iron for mice is 35 ppm [17] and 200 ppm is a level commonly used for iron-supplemented rodent diets. Beginning at 8–9 weeks of age, all mice were sensitized to ovalbumin (OVA) then challenged x 3 with aerosolized OVA or saline and airway resistance was determined following methacholine challenge. OVA-challenged mice that consumed the unsupplemented (8 ppm iron) diet showed large methacholine-induced increases in airway resistance compared with saline-challenged mice (Figure 1A). These changes in airway resistance were markedly attenuated (∼30% reduction) in OVA-challenged mice on the iron-supplemented (200 ppm) diet (Figure 1A; p≤0.0005). Allergen triggering induced marked infiltration of inflammatory cells into bronchoalveolar spaces of unsupplemented mice (mean ± SEM = 202,408 total cells/ml), the majority of which were eosinophils. This cellular infiltration was also markedly attenuated by iron supplementation (Figure 1B). Specifically, the eosinophils present in bronchoalveolar spaces decreased from 192,679±12,608 eosinophils/ml BAL fluid in OVA-challenged unsupplemented mice (95% of total cells; n = 5 mice) to 93,224±13,770 eosinophils/ml in OVA-challenged iron-supplemented mice (85% of total cells; n = 5 mice), a decrease of ∼50%. Production of a variety of proinflammatory cytokines and chemokines within pulmonary tissue were also significantly decreased by iron supplementation. These included IL-1β, IL-3, IL-4, IL-5, Il-6, IL-9, IL-10, IL-12(p40), IL-13, IL-17, IFN-γ, KC, GM-CSF, and MIP1α (Figure 2A–C). 10.1371/journal.pone.0045667.g001Figure 1 Iron supplementation decreases severity of allergic asthma. WT and mast cell-deficient Sash mice on the indicated diets were sensitized to OVA then challenged with OVA or saline. A. Airway resistance (measured in units of cm H2O/ml/sec) in response to methacholine was significantly decreased following allergen challenge for mice that received iron supplementation (*indicates p≤0.0005). B. Iron supplementation decreased infiltration of eosinophils into bronchoalveolar spaces (decrease of 52%; *indicates p = 0.0007). 10.1371/journal.pone.0045667.g002Figure 2 Effects of iron supplementation on allergen-induced cytokine secretion into bronchoalveolar spaces. Cytokine and chemokines present in bronchoalveolar lavage (BAL) fluid obtained from OVA-sensitized mice 24 hrs following the 3d challenge with aerosolized antigen or saline were markedly increased in unsupplemented compared with iron-supplemented mice (*indicate p<0.01). The percentage increase ranged from 25–35% for IL-1β, IL-5, IL-6, and IL-13 to 170% for IL-17. As typically seen in allergic asthma [18], allergen challenge also induced mucous metaplasia, with increased numbers of goblet cells present in the large airways of OVA-challenged mice from both diet groups (Figure 3A–D). Mucus production increased following allergen challenge as measured by qPCR for Muc5AC mRNA, but was not affected by iron supplementation (Figure 3E). 10.1371/journal.pone.0045667.g003Figure 3 Iron supplementation does not affect mucus production. Periodic acid-Schiff (PAS)-stained formalin-fixed, paraffin-embedded lung sections show allergen-induced goblet cell differentiation in the large airways of mice on both 8 ppm (A  =  saline-challenged, B  =  OVA-challenged) and 200 ppm diets (C  =  saline-challenged, D  =  OVA-challenged). Expression of Muc5AC mRNA in the lungs was similar for both diet groups (E). Effect of the Unsupplemented Diet on Iron Status and Allergic Sensitization Total body iron content is reflected by iron present in red blood cells, serum, and stored within tissues. When dietary iron is limited, its absorption is enhanced by inhibition of the iron regulatory hormone hepcidin and additional iron is mobilized from tissue stores to maintain normal levels of serum iron and hematopoiesis. Once tissue iron stores are depleted, iron deficiency anemia will result if iron intake remains insufficient to meet daily iron needs. Tissue iron content is thus the most sensitive measure of body iron status. Hepcidin production was determined in allergen-sensitized but saline-challenged mice to avoid confounding due to effects of inflammation. At the time of allergen challenge, hemoglobin and hematocrit were slightly decreased for mice on the 8 ppm iron diet compared with the iron-supplemented diet (Table 1), but remained in the normal range. Tissue analysis revealed that these mice had markedly decreased levels of iron stored in liver and spleen. The low ratios of hepcidin:actin mRNA (Table 1) measured in unsupplemented mice are consistent with the expected ongoing release of stored and newly absorbed iron into the systemic circulation, based on their mild to moderate iron deficiency without anemia. Interestingly, serum iron levels were slightly elevated in unsupplemented mice, most likely reflecting extensive mobilization of body iron stores (Table 1). Thus, although the mice on the unsupplemented diet had deficient iron stores, their degree of iron deficiency was not sufficient to result in anemia. The higher levels of hepcidin transcripts seen in the mice on the iron-supplemented diet is consistent with their higher total body iron stores and therefore a decreased need for release of recently absorbed or stored iron. 10.1371/journal.pone.0045667.t001Table 1 Iron Status of Mice Studied.** UnsupplementedDiet (8 ppm iron) SupplementedDiet (200 ppm) RBC (M/µL) 10.2±0.3 10.1±0.3 Hgb (g/dL) 14.6±0.5 15.7±0.3* Hct (%) 46±2 50±1 MCV (fL) 44.7±1.9 49.7±0.9* Serum iron (µg/dL) 193±8 174±9* Liver iron (µg/g wet tissue) 16±2 88±3 Spleen iron (µg/g wet tissue) 45±9 270±19 Hepcidin:actin mRNA (liver) 0.05 (0.02–0.12) 1.82* (1.50–2.22) * indicates p≤0.05 for comparisons of unsupplemented vs. iron-supplemented diets. ** Data shown are mean ± SEM for 6 mice/diet group for red blood cells (RBC), hemoglobin (Hgb), hematocrit (Hct), and mean cellular volume (MCV). The numbers of mice studied for serum and tissue iron ranged from 10–25 per group. Hepcidin measurements are given as the geometric mean (upper - lower confidence interval) for 10 allergen-sensitized, saline-challenged mice/group. Similar levels of OVA-specific antibodies were present in the serum of mice on the unsupplemented diet (186±7 µg/ml OVA-specific IgG and 122±15 ng/ml OVA-specific IgE; n = 25) compared with mice on the iron-supplemented diet (173±7 µg/ml OVA-specific IgG and 123±20 ng/ml OVA-specific IgE; n = 12). Thus, under the conditions used for this study, iron supplementation did not affect asthma severity through effects on allergic sensitization. Although it has been reported that the OVA-alum model of asthma is not necessarily mast cell-dependent [19], this has been refuted by others [20]–[22]. We found that allergen-induced airway responses were minimal in mast cell-deficient KitW-sh/KitW-sh (Sash) mice that were sensitized and challenged with OVA (Figure 1A). This indicated that, under the experimental conditions used for this study, contributions of non-mast cells to the changes in airway resistance in this model were small. Thus, the differences in airway resistance measured primarily resulted from allergen-triggered activation of mast cells. Iron Supplementation Decreases Mast Cell Activation in vitro To directly assess the effects of iron supplementation on mast cells, IgE-sensitized primary bone marrow-derived murine mast cells (BMMC) were incubated in Tyrode’s buffer, with and without addition of 100 µM Fe2+ for 30 min prior to activation via IgE cross-linking. Iron supplementation had no effect on spontaneous degranulation, but decreased IgE-mediated degranulation by 30% (p = 0.01; Figure 4A). Following degranulation, BMMC were cultured for 18 hrs in their standard growth media and selected cytokines and chemokines were measured by multiplex fluorescent immunoassay. Production of TNF, MCP-1, and IL-6 by IgE-activated mast cells was markedly decreased by iron supplementation, with reductions of 94%, 29%, and 27%, respectively (Figure 4B). Baseline levels of IL-1β, IL-4, IL-10, IFN-γ, KC, and MIP-1α produced by BMMC were extremely low at this time point and were not affected by IgE-mediated degranulation or by iron supplementation. Additional studies using the RBL-2H3 rat mast cell line showed that iron supplementation also decreased mast cell granule content, measured both by total cellular content of the granule-associated enzyme β-hexoseaminidase content and by flow cytometric detection of cytoplasmic granularity (Figure 4C–E). 10.1371/journal.pone.0045667.g004Figure 4 Effects of iron supplementation on IgE-mediated mast cell degranulation, cytokine production, and total granule content. A. Supplementation with iron decreased degranulation of primary BMMC by 30% (*indicates p = 0.01). Data shown is the mean ± SEM of 4 experiments. B, C. In duplicate experiments, iron supplementation decreased mast cell production of TNF, MCP-1, and IL-6 by 94%, 29%, and 27%, respectively. An MTS assay confirmed continued viability of the BMMC (not shown). D, E. RBL-2H3 mast cells showed a dose-dependent reduction in total granule contents per cell with iron supplementation, as indicated by β-hexoseaminidase activity following lysis with Triton X-100 (panel D) and flow cytometric analysis of side scatter (panel E). Data shown represents the mean ± SEM of 3 − 4 independent experiments, each conducted in triplicate. *indicates p≤0.02. Discussion These studies show that dietary iron supplementation decreased the severity of allergic asthma in wild type mice, as measured by significant decreases in infiltration of eosinophils and secretion of proinflammatory cytokines and chemokines into bronchoalveolar spaces, confirming in part similar results recently reported by Maazi et al [23]. In addition, we made the novel observation using a direct measure of airflow resistance that supplementation attenuates the acute development of airway hyperresponsiveness in the common ovalbumin model of allergic asthma. In our studies, the benefits of iron supplementation were observed in mice with levels of iron deficiency that would be classified clinically as mild, since body iron stores were decreased but not serum iron levels or hematopoiesis. In vitro studies showed that iron supplementation affected mast cell activity via multiple mechanisms that included decreased mast cell granule content, decreased IgE-mediated degranulation, and decreased production of pro-inflammatory cytokines and chemokines following degranulation. Few studies regarding potential effects of iron on mast cell activity have been reported previously. Incubation of mast cells with media containing serum or the iron-containing protein transferrin was previously described to inhibit mast cell degranulation [24]. Conversely, exposure to iron chelators has been shown to activate primary human mast cells [25], [26], rat peritoneal mast cells [25], [27], and a human mast cell line [28]. Our in vitro studies confirm that iron supplementation also has direct inhibitory effects on mast cell activation. Our in vivo studies also clearly show that iron supplementation can decrease the severity of inflammation in an in vivo model of allergic asthma in mice with mild iron deficiency. It is tempting to speculate that these effects are directly due to effects of iron on mast cells, particularly in light of our data that mast cells are required for the differences in airway resistance seen in iron-supplemented vs. unsupplemented mice following allergen challenge (Figure 1A). However, these results do not rule out potential significant contributions by other cell types to decrease airway responses upon iron supplementation. Although iron supplementation was shown to decrease total mast cell granule content as well as IgE-triggered mast cell degranulation and production of pro-inflammatory cytokines and chemokines post-degranulation, studies that allow manipulation of iron levels within specific cell types, including mast cells, will likely be required to further define the cell types involved as well as the precise mechanisms by which iron regulates these processes. Iron supplementation reduced production of a number of pro-inflammatory cytokines and chemokines following respiratory exposure to aerosolized allergen (Figure 2). Each of these agents has previously been shown to contribute to the pathogenesis of asthma [29], [30], and demonstration of these important biologically relevant mechanisms for decreased airway hypperreactivity and inflammation adds strength to the study. In particular, IL-9 has been shown to be critical in the pathogenesis of bronchial hyperresponsiveness. Our finding that IL-17 production is also decreased by iron supplementation is also of particular interest, given recent data suggesting that IL-17-producing Th17 cells can also play a pathogenic role in allergic asthma [30]. In addition to effects on allergen-induced pathways of inflammation, it is also possible that iron may affect mast cell activity directly via its redox properties. Cho et al recently showed that stimulation of mast cells through FcεRI induced the production of intracellular reactive oxygen species that act as second messengers in a signal transduction pathway leading to degranulation and cytokine synthesis [31]. Iron is a well-known quencher of free radicals and can be readily oxidized or reduced under conditions present within cells. But at present, neither the source of the reactive oxygen species generated in response to Ag stimulation nor the pathway by which they are generated in Ag-stimulated mast cells is clearly understood. Iron homeostasis in mast cells is also poorly understood. Thus, additional studies will be required to determine the potential contribution of these pathways to asthma severity. Although mice with decreased iron stores had more severe inflammation in this model of allergic asthma, the iron-dependent changes in immune cell reactivity that we observed may potentially be beneficial to host defense. Mast cells are best known for their pathogenic role in allergy and asthma. However, their immediate release of pre-stored TNF upon degranulation stimulates a rapid influx of neutrophils that is critical for control of bacterial infections. Mice deficient in mast cells are thus highly susceptible to fatal infections with enteric bacteria [32], [33]. TNF released by mast cells is also critical for inducing lymph node changes that stimulate the development of antigen-specific immune responses [34]. Following degranulation, mast cells continue to secrete cytokines that further stimulate the inflammatory response. Inflammation induces the peptide hormone hepcidin, which decreases bioavailable iron to additionally limit bacterial growth [35]. Thus, based on current understanding of these pathways, enhanced mast cell activation due to decreased iron could be beneficial to enhance host antibacterial defenses, but enhanced mast cell activation is detrimental in allergy. The potential to decrease the severity of allergic reactions by normalizing iron status has major implications for public health, since correction of iron deficiency is simple, safe, and inexpensive and is medically desirable for a variety of reasons. Iron deficiency is the most common nutritional deficiency in humans world-wide [36]. The Centers for Disease Control and Prevention estimates that, in the United States, 7% of children 1–2 yrs of age, 12% of females from ages 12–49, and 2% of males from ages 16–69 have severe iron deficiency with or without anemia [9]. In a setting of iron sufficiency, the iron-regulatory hormone hepcidin maintains body iron homeostasis by decreasing dietary iron absorption and preventing release of stored iron into the bloodstream [37]. Since production of hepcidin is also up-regulated by inflammation [38], the inflammatory sequelae of allergic reactions would be predicted to exacerbate any dietary iron deficiency by further decreasing iron absorption and release. It is important to note that, although the unsupplemented mice in our study had significantly decreased iron stores, the large increases in airway resistance occurred while hematologic parameters (e.g. Hgb, Hct) remained within the normal range. Humans with a similar degree of iron deficiency would not typically be classified as being iron-deficient based on a complete blood count. Measurement of iron stores in humans currently requires expensive (e.g. magnetic resonance imaging [39]) or invasive testing (e.g. bone marrow biopsy). Identification of alternative blood-based biomarkers of iron status that can accurately predict mast cell reactivity will facilitate follow-up studies to determine the potential of iron supplementation to modulate the clinical severity of allergic diseases in humans. The authors thank Chau Trinh for excellent technical assistance and animal husbandry. ==== Refs References 1 Kim HY , DeKruyff RH , Umetsu DT (2010 ) The many paths to asthma: phenotype shaped by innate and adaptive immunity . Nat Immunol 11 : 577 –584 .20562844 2 Beasley R (2002 ) The burden of asthma with specific reference to the United States . J Allergy Clin Immunol 109 : S482 –S489 .11994720 3 Gaga M , Papageorgiou N , Yiourgioti G , Karydi P , Liapkou A , et al (2005 ) Risk factors and characteristics associated with severe and difficult to treat asthma phenotype: an analysis of the ENFUMOSA group of patients based on the ECRHS questionnaire . 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PLoS One. 2012 Sep 20; 7(9):e45667
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23029364PONE-D-12-1401210.1371/journal.pone.0046010Research ArticleBiologyMolecular cell biologyGene expressionProtein translationRNA interferenceCell DeathCell GrowthMedicineOncologyCancer TreatmentGene TherapyCancers and NeoplasmsNeurological TumorsGliomaGlioblastoma MultiformeOncology AgentsMicroRNA-330 Is an Oncogenic Factor in Glioblastoma Cells by Regulating SH3GL2 Gene MiR-330 Regulates SH3GL2 Gene in GlioblastomaQu Shengtao 1 Yao Yilong 1 Shang Chao 2 3 Xue Yixue 2 3 Ma Jun 2 3 Li Zhen 1 Liu Yunhui 1 * 1 Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of China 2 Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang, People’s Republic of China 3 Institute of Pathology and Pathophysiology, China Medical University, Shenyang, People’s Republic of China Ulasov Ilya Editor University of Chicago, United States of America * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YHL YXX CS STQ. Performed the experiments: STQ YLY JM ZL CS. Analyzed the data: STQ ZL CS YXX YHL. Contributed reagents/materials/analysis tools: CS YXX YHL. Wrote the paper: STQ YXX YHL. SPSS. 2012 21 9 2012 7 9 e4601015 5 2012 23 8 2012 © 2012 Qu et al2012Qu 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 have recently emerged as key regulators of cancers. This study was therefore conducted to investigate the role of miR-330 in biological behaviors of human glioblastoma U87 and U251 cell lines and its molecular mechanism. SH3GL2 gene was identified as the target of miR-330. MiR-330 overexpression was established by transfecting miR-330 precursor into U87 and U251 cells, and its effects on proliferation, migration, invasion, cell cycle and apoptosis were studied. Overexpression of miR-330 can enhance cellular proliferation, promote migration and invasion, activate cell cycle and also inhibit apoptosis in U87 and U251 cells. Collectively, these above-mentioned results suggest that miRNA-330 plays an oncogenic role in human glioblastoma by regulating SH3GL2 gene and might be a new therapeutic target of human glioblastoma. This work is supported by grants from Natural Science Foundation of China (Nos. 81171131, 81172197, 30973079, 81072056), the special fund for Scientific Research of Doctor-degree Subjects in Colleges and Universities, (Nos. 20092104110015, 20102104110009), Natural Science Foundation of Liaoning Province in China (No. 201102300), Science and Technology Plan Projects of Liaoning Province in China (No. 2011225020) and Shenyang Science and Technology Plan Projects (Nos. F-10-205-1-22, F-10-205-1-37). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction With a prevalence of approximately 60%, glioblastoma remains the most common malignant primary brain tumors in adult central nervous system. Despite aggressive surgery, combined radiation and chemotherapy, the median survival time is approximately 14 months [1]. Therefore, how to prolong the survival time of patients of glioblastoma is an urgent problem we are facing. The recent study of miRNAs brings us possibilities for the treatment of human glioblastoma [2], [3]. MicroRNAs (miRNAs) are now recognized as a class of small non-coding RNA molecules throughout the genomes of mammal [4]. They post-transcriptionally regulate protein expression by targeting the 3′-UTR of target mRNA which causes either degradation or repression of translation. Recently many miRNAs are found to play important roles in the development and maintenance of tumorigenesis. A large set of miRNAs are lower expressed or overexpressed in human tumors compared to normal tissues and miRNAs-mediating gene silencing promotes or inhibits tumor cell growth. Such regulators are usually regarded as the enhancers or suppressors of tumor progression. MiR-21 is overexpressed and has been identified as an antiapoptotic factor in human glioblastoma cells [5], [6]. Additionally, miR-128, miR-34a, miR-7 and many other miRNAs also act as tumor suppressors in human glioblastoma cells [7]–[9]. MiR-330 gene was firstly found by Weber in 2005, located at 19q13.32 [10] . Previous studies show that miR-330 was able to acts as tumor suppressor and induced apoptosis of prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation [11]. However, the function and molecular mechanism of miR-330 in determining the malignant phenotype of human glioblastoma are less elusive. SH3GL2 gene is a candidate tumor suppressor gene that particularly more highly expressed in central nervous system [12], [13]. Moreover, decreased expression of SH3GL2 is shown to be associated with tumorigenesis of laryngeal carcinoma [14]. Our previous study has shown that SH3GL2 gene is obviously less expressed in human glioblastoma which indicates the correlation of its expression with the incidence of glioblastoma [15]. Potter, et al also observed deletion of the locus in pilocytic astrocytomas suggesting a tumor suppressor role of SH3GL2 in brain tumors [16]. However, the underlying mechanism is still unclear. The recent prediction of miR-330 targeting SH3GL2 3′-UTR has led our further study on the mechanism of downregulation of SH3GL2 gene in glioblastoma. Here, for the first time, we uncovered a comprehensive analysis of the regulation of miR-330 and SH3GL2 expression in glioblastoma. We investigated the regulatory effects of miR-330 on SH3GL2 and explored the potential oncogenic mechanism of miR-330 in glioblastoma cells. Materials and Methods Human Tissue Samples All human normal brain and glioma tissue samples were obtained from the Department of Neurosurgery, Shengjing Hospital of China Medical University. This study procedure was approved by The Institutional Review Board at the hospital. All participants provided written informed consent. The tissue samples were obtained from those without necrosis and coagulation parts. For each sample, the major portion of tissue was frozen immediately in liquid nitrogen for molecular analysis, and the remaining tissue was fixed in paraformaldehyde for histological examination. All samples were histologically classified and graded according to WHO guidelines by tow experienced clinical pathologists. Reagents and Cell Culture Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) and fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Trizol and Lipofectamine™ 2000 transfection reagent were purchased from Invitrogen (Carlsbad, CA, USA). MicroRNAs and their negative control molecules were synthesized by Ambion (Austin, TX, USA). The siRNA targeting SH3GL2 gene and its negative control molecules were synthesized in vitro using the Ambion Silencer ™ siRNA Construction Kit. All other chemicals and reagents were purchased from Sigma-Aldrich (Shanghai, China) unless otherwise specified. Human glioblastoma cell lines U87 and U251 were obtained from the Chinese Academy of Medical Sciences and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin/ml and 100 ng of streptomycin/ml. HEK-293 cells were also from the Chinese Academy of Medical Sciences and cultured in DMEM medium of high glucose without penicillin and streptomycin. All cells were incubated in a 5% CO2 humidified incubator at 37°C. Vector Construction The 3′-UTR sequence of human SH3GL2 gene was amplified by PCR using the following primers: SH3GL2 forward primer, 5′-TCG AGG ATG TTA TGC TGG CTG-3′; SH3GL2 reverse primer, 5′-CTG CGG CCT GCA CTT GGG ATGT-3′. For its mutagenesis, the sequence complementary to the binding site of miR-330 in its 3′-UTR (TGC TTT G) was replaced by GAA GCC A using the overlap PCR method. The wild type and mutant type 3′-UTRs of SH3GL2 were cloned into pmirGLO Dual-Luciferase miRNA target expression vector using the Xho I and Sal I sites. These constructs were validated by sequencing. Cell Transfection Cells at 50–70% confluence were transfected using lipofectamine 2000 reagent (Invitrogen, CA, USA) 24 h after plating. Transfection complexes were prepared according to the manufacturer’s instructions and added directly to the cells to a final oligonucleotide concentration of 50 nmol/L. At 6 h after the transfection, the medium was replaced with fresh DMEM/F12 with 10% fetal bovine serum, and the cells were incubated for an additional 48 h or 72 h. Then the transfected cells were harvested for further study. Those transfected with miRNA precursors were divided into 5 groups: mock group with no miRNA precursor but PBS, pre-miR-con group with pre-miR negative control precursor, pre-miR-330 group with miR-330 precursor, anti-miR-con with anti-miR negative control precursor and anti-miR-330 group with miR-330 inhibitor precursor. Those transfected with siRNA were divided into 2 groups: siRNA control (siRNA-con) group and siRNA-SH3GL2 group. Bioinformatics Method and Luciferase Reporter Assay The common targets of miR-330 predicted by computer-aided algorithms were obtained from multiple target prediction programs: Targetscan and Miranda (http://www.targetscan.org/and http://www.microrna.org/). HEK-293 cells were seeded into a 24-well plate. After cultured overnight, cells were co-transfected with the wild-type or mutated SH3GL2 3′-UTR reporter plasmid, and transfected with pre-miR-330 or pre-miR-con precursors. Luciferase assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, WI, USA). RNA Extraction, Reverse Transcription PCR and Quantitative Real-time PCR Total RNA of each group was extracted from the cells using Trizol according to the manufacturer’s instruction. RT-PCR was conducted using an RT-PCR kit (TaKaRa, Dalian, China). The primers used were all synthesized by Sangon (Shanghai, China). To analyze miR-330 expression levels, the stem-loop RT-PCR assay was used to quantify the miRNAs expression levels. The RT-PCR primers were as following: miR-330 RT primer: 5′-GTC GTA TCC AGT GCG TGT CGT GGA GTC GGC AAT TGC ACT GGA TAC GAC TCT CTG C-3′. MiR-330 PCR primers are: forward: 5′-CGG CAA AGC ACA CGG CCT G-3′; reverse: 5′-TGC GTG TCG TGG AGT CGG C-3′. U6 RT primer is: 5′-TGG TGT CGT GGA GTC G-3′. U6 PCR primers are: forward: 5′-CTC GCT TCG GCA GCA CA-3′; reverse: 5′-AAC GCT TCA CGA ATT TGC GT-3′. qRT-PCR was performed using SYBR Premix Dimer Eraser (TaKaRa, Dalian, China) on a 7500HT system. The expression levels of miR-330 were normalized with reference to expression levels of U6, and fold changes were calculated by relative quantification (2−ΔΔCt). The primer sequences for SH3GL2 gene expression were as follows: β-actin forward: 5′-ATC ATG TTT GAG ACC TTC AAC A-3′, reverse: 5′-CAT CTC TTG CTC GAA GTC CA-3′; SH3GL2, forward: 5′-GGC CCT GTC ACT CCT GAG AT-3′, reverse: 5′-GGC ATC CAG GTT ATC GGG GA-3′; Amplification was performed over 35 cycles: 94°C/60 s (denaturation), 58°C/45 s (annealing) and 72°C/45 s (extension). The relative SH3GL2 mRNA levels were normalized to those of β-actin mRNA levels using Quality One analysis software (Bio-Rad, USA). Western Blot Analysis Total protein from transfected cells was extracted in RIPA buffer supplemented with protease inhibitors (100 mM Tris, pH 7.4, 150 mM NaCl, 5 mMEDTA, 1% Triton X-100, 1% deoxycholate acid, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM DTT, 2 mM leupeptin, 2 mM pepstatin). The samples were centrifuged (12,000 g, 4°C) for 20 min and the protein concentration was determined by the BCA method (Beyotime Institute of Biotechnology, Jiangsu, China). The proteins were separated using 12% SDS-PAGE and then electrophoretically transferred to a PVDF membrane (Millipore, USA). The membranes were blocked in blocking buffer (5% non-fat milk dissolved in Tris-buffered saline-Tween, TBS-T) overnight at 4°C. The blots were then incubated with mouse monoclonal anti-SH3GL2 antibody (diluted 1∶500, Santa Cruz Biotechnology, CA, USA) and mouse monoclonal anti-β-actin antibody (diluted 1∶4000, Santa Cruz Biotechnology). Protein bands were visualized by ECL (Santa Cruz Biotechnology, CA, USA) and detected by ECL Detection Systems (Thermo Scientific). The relative integrated density values (IDVs) were calculated based on β-actin protein as an internal control. Proliferation Assay U87 and U251 cells were seeded into 96-well plates at a density of 2000 cells/well with five replicate wells for each group, transfected and assayed 24, 48, 72, 96, 120 h after transfection. 20 µl of MTT (5 mg/ml) was added into each well and incubated for another 4 h, and then the supernatant was discarded, 150 µl of DMSO was added to each well to dissolve the precipitate. Optical density (OD) value was measured at the wavelength of 490 nm every 24 h for 5 consecutive days after treatment. The data were derived from three independent experiments. Cell Migration and Invasion Assay Migration of U87 and U251 cells was assayed using chamber (Costar, USA) with polycarbonic membrane (6.5 mm in diameter, 8 µm pore size). Cells were grown to about 70% confluence and transfected with the desired miRNAs. After 24 h, the cells were replaced with serum-free medium incubated for another 24 h. Then cells were trypsinized and collected. 5×104 cells in serum-free medium were added to the upper chamber. Then 600 µl medium with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37°C, and then non-migrating cells on the top of membrane were removed with cotton swabs. Cells that migrated to the bottom of the membrane were then fixed with methanol and stained with 20% Giemsa solution for 30 min at 37°C and washed twice with PBS. Then stained cells were subjected to a microscopic inspection counted within five randomly chosen fields and the average number was taken. For the cell invasion ability assay, the procedure was similar to migration assay, but the transwell membrane was coated with a 500 ng/µl Matrigel solution (BD, Franklin Lakes, NJ), and incubated for 4h at 37°C then collected the after-transfected cells added to the upper chamber for the further assay. Invasive cells were fixed, stained with 5% crystal violet and counted as previously described. Cell Cycle Assay To assess the effect of miR-330 on the cell cycle, the U87 and U251 cells were transfected with pre-miR-con and pre-miR-330. Briefly, 72 h after transfection 1×106 cells were washed with phosphate buffer saline (PBS), trypsinized and resuspended in ice-cold PBS. Cells were then gently pelleted by centrifugation (500 g for 5 min at 4°C), the supernatant was removed and the cells were fixed and permeabilized in 70% ethanol at −20°C. Fixed cells were washed with PBS and incubated in the dark for 30 min with a propidium iodide (PI) staining solution containing 50 µg/ml propidium iodide (PI) and 100 µg/ml RNase A in PBS. The cells were then analyzed for DNA content on a FACscan (BD FACSCalibur, USA) and G0/G1, S and G2/M fractions were determined. Apoptosis Assay The effect of miR-330 expression on cell apoptosis was assessed by Annexin V-FITC/PI staining. U87 and U251 cells were transfected with mock, pre-miR-330, anti-miR-330 and their negative control molecules for 72 h. Then, the cells were harvested and stained with Annexin V-FITC and PI according to the instruction of the manufacturer. Cell samples were analyzed on a FACsan and apoptotic fractions were determined. Statistical Analysis For all the experiments, data were obtained from three independent experiments. All results were expressed as the mean ± SD for each group. Data were analyzed by use of SPSS 13.0 software. When appropriate, two group comparisons were analyzed with a t-test and more than two groups comparisons were analyzed with one-way ANOVA. P<0.05 was considered significant and symbolized by an asterisk in the graphs. Results MiR-330 Expression was Increased in Glioblastoma To determine the levels of miR-330 in established glioblastoma cell lines, glioblastoma tissues and normal brain tissues, total RNAs were extracted from U87, U251 and U373 cells, glioblastoma tissues and normal brain tissues. The expression levels of miR-330 were analyzed using RT-PCR and quantitative real-time PCR. U6 RNA level was used as an internal control. As shown in Figure 1, the expression levels of miR-330 normalized to U6 were significantly up-regulated in five glioblastoma tissues and three glioblastoma cells when compared to the three normal brain tissues. This result indicates that miR-330 is up-regulated in glioblastoma. 10.1371/journal.pone.0046010.g001Figure 1 MiR-330 was overexpressed in human glioblastoma tissues and cells. Relative miR-330 expression levels were analyzed by qRT-PCR in normal brain tissues, glioblastoma tissues and established glioblastoma cell lines. U6 RNA level was used as an internal control. Asterisks indicate significant difference when compared to normal brain tissues (P<0.05). MiR-330 Directly Targeted the 3′-UTR of SH3GL2 Bioinformatics analyses available on the public miRNA databases were used (http://www.targetscan.org/and http://www.microrna.org/) to identify miRNAs that specifically target SH3GL2. MiR-330 was shown to have a putative binding site to the 3′-UTR region of SH3GL2 with 100% conserved sequence. A highly conservative miR-330 binding site at SH3GL2 3′-UTR 56–62 base position was predicted in many species, such as the H. Sapiens, the M. muculus and the R. Norvegius et al. The seed for miR-330 to SH3GL2 3′-UTR is shown in Figure 2A. The wild type of SH3GL2 3′-UTRs was cloned into pmirGLO Dual-Luciferase miRNA target expression vector. Overexpression of miR-330 decreased luciferase activity of this reporter to about 60% of the control level (Figure 2C), suggesting that miR-330 inhibits the 3′-UTR function of SH3GL2. To test whether miR-330 specifically inhibited SH3GL2 by its potential binding site of seed sequence, the mutated reporter at miR-330 binding site was constructed (Figure 2B). Forced expression of miR-330 did not affect the mutant SH3GL2 reporter activities (Figure 2D). These results indicate that SH3GL2 is a direct target of miR-330 with the specific binding site at the seed sequence. 10.1371/journal.pone.0046010.g002Figure 2 MiR-330 negatively regulated SH3GL2 through binding to the 3′-UTR of SH3GL2. (A) Schematic diagram of putative miR-330 binding site in the 3′-UTR of SH3GL2 in human. The seed sequence of miR-330 matches 3′-UTR of SH3GL2 (in bold). (B) The corresponding mutated nucleotides of the SH3GL2 3′-UTR was labeled in bold bellow. (C) Relative luciferase activities of SH3GL2 wild type 3′- UTR were obtained by co-transfection of PBS (mock), negative control miRNA or miR-330 precursors; and calculated as the ratio of firefly/renilla activities in the cells and normalized to those of the control. The results were presented as mean±SD from three independent experiments with each experiment in triplicate. Asterisk indicates significant difference when compared to control (P<0.05). (D) Relative luciferase activities of pmirGLO plasmid, SH3GL2 wild type and mutant type 3′- UTR were obtained by co-transfection of miR-330 precursor; and calculated as the ratio of firefly/renilla activities in the cells and normalized to those of the control. The results were presented as mean±SD from three independent experiments with each experiment in triplicate. Asterisk indicates significant difference when compared to control (P<0.05). MiR-330 Post-transcriptionally Inhibited SH3GL2 Expression The relationship between miR-330 level and SH3GL2 expression was analyzed in U87 and U251 cells. When cell lines were transfected with exogenous miR-330 and its inhibitors, the level of SH3GL2 protein was detected by western blotting. As shown in Figure 3B , there was a significant inverse correlation between miR-330 and SH3GL2 protein level in the pre-miR-330 group versus pre-miR-con group (P<0.05), while there was no obvious difference between mock group and pre-miR-con group. What’s more, the anti-miR-330 played the opposite effect of pre-miR-330 with an increasing level of SH3GL2 protein expression versus either pre-miR-330 or anti-miR-con group (P<0.05). Meanwhile, the SH3GL2 mRNA level after transfection in each group was also detected by RT-PCR. However, there was no statistical significance among the five groups (Figure 3A). As shown in Figure 3, miR-330 induced a significant decrease of SH3GL2 protein expression without influence on mRNA level. To further validate the effect of miR-330 on SH3GL2 gene, rescue experiment by siRNA on SH3GL2 in U87 cells was performed. As shown in Figure S1, there were decreases at both mRNA and protein level of SH3GL2 gene (P<0.05). All of these results indicate that miR-330 post-transcriptionally inhibits SH3GL2 expression and miR-330 is negatively correlated with SH3GL2 protein expression in glioblastoma cells. 10.1371/journal.pone.0046010.g003Figure 3 MiR-330 regulated SH3GL2 expression at the post-transcriptional level. (A) Overexpression of miR-330 or transfection of exogenous miR-330 inhibitor did not change the SH3GL2 mRNA level detected by RT-PCR. The effectiveness of miR-330 on SH3GL2 mRNA was analyzed by RT-PCR. The mRNA levels of the SH3GL2 were shown and normalized against that of β-actin. (B) Overexpression of miR-330 inhibited SH3GL2 expression at protein level. The expression level of SH3GL2 and β-actin in U87 and U251 cells transfected with exogenous miR-330 or its inhibitor were analyzed by Western blot 48 h after transfection. β-actin was used as an internal loading control (P<0.05). Accompanying graphs show densitometry analysis of SH3GL2 expression. Data are means of three independent experiments ± SD.* P<0.05 compared with control group. MiR-330 Promoted Cellular Proliferation of Glioblastoma To investigate whether miR-330 could influence glioblastoma cell proliferation, MTT assay was performed in U87 and U251 cells. The result demonstrated that U87 and U251 cells transfected with pre-miR-330 exhibited a significant increase of cellular viability compared with cells treated by pre-miR-con (P<0.05). While, there was no significant difference between mock group and pre-miR-con group in both cell lines. On the contrary, anti-miR-330 decreased cellular proliferation in both U87 and U251 cells compared with anti-miR-con group (Figure 4). In the rescue experiment, U87 cells transfected with siRNA-SH3GL2 showed higher cellular proliferation than that of siRNA-con group (Figure S2A). These results demonstrate that miR-330 can promote cellular proliferation in glioblastoma cells in an SH3GL2-dependent way. 10.1371/journal.pone.0046010.g004Figure 4 MiR-330 increased cell proliferation of U87 and U251 cells. MTT assay showed that U87 (A) and U251 (B) cells transfected with pre-miR-330 proliferated at a higher rate than the pre-miR-con group (P<0.05). Those transefected with anti-miR-330 group showed a lower proliferation rate than the anti-miR-con group (P<0.05).There was no obvious difference between mock group and the two control groups in the experiment. Values represent the mean ± SD of three independent experiments.* P<0.05 compared with control group. MiR-330 Increased Glioblastoma Cell Migration and Invasion To examine whether overexpression of miR-330 affected the migration and invasion capacity of glioblastoma cells, transwell assays were introduced. We observed that miR-330 overexpression enhanced FBS-induced migration ability of glioblastoma cells compared with pre-miR-con group (P<0.05) (Figures 5A and 5C). In parallel, a Matrigel invasion assay was also performed to study the effects of miR-330 on the invasion of glioblastoma cells. The results clearly revealed that miR-330 also increased FBS-induced invasion of U87 and U251 cells compared with pre-miR-con group (P<0.05) (Figures 5B and 5D). However, there was no significant difference between mock group and pre-miR-con group. We also found the opposite effects of anti-miR-330 exerted on both cell lines compared with anti-miR-con or pre-miR-330 group (P<0.05). While the U87 cell showed higher migration and invasion capacity in siRNA-SH3GL2 group than in siRNA-con group (P<0.05) (Figure S2B).These results above strongly suggest that miR-330 is an important factor involved in the migration and invasion of glioblastoma cells by regulating SH3GL2 gene. 10.1371/journal.pone.0046010.g005Figure 5 MiR-330 increased migration and invasion of U87 and U251 cells. U87 and U251 cells were transfected miRNA precursors and then subject to transwell migration and invasion assays. After 24 h and 48 h, migration and invasion cells were correspondingly counted after staining with Giemsa (A and B) or 5% crystal violet (C and D). Representative photographs of migration and invasion cells on the membrane and accompanying statistical plots were presented. There were obvious differences between pre-miR-330 group and pre-miR-con group; the anti-miR-330 group showed a contrary effect compared with mock, negative control and pre-miR-330 groups (P<0.05). However, there was no obvious difference between mock group and negative control groups. Values represent the mean ± SD from three independent experiments.* P<0.05 compared with control group. MiR-330 Accelerated Glioblastoma Cell Cycle The cell cycle distribution of the cells treated with miR-330 was examined using flow cytometry. The results showed that there were higher S, G2/M phase fractions in glioblastoma cells transfected with pre-miR-330 compared with the cells transfected with pre-miR-con (P<0.05) (Figure 6). In the rescue experiment, the cell cycle distribution between siRNA-SH3GL2 group and siRNA-con group showed a significant difference (P<0.05) (Figure S3A). These data suggest that overexpression of miR-330 cause acceleration of cell cycle through influencing SH3GL2 gene. 10.1371/journal.pone.0046010.g006Figure 6 MiR-330 promoted cell cycle progression of U87 and U251 cells. Cell cycle distribution of U87 and U251 cells transfected with pre-miR-con and pre-miR-330. The percentage of cells in the different cell cycle phases was plotted, and the results represent the mean ± SD. * P<0.05 compared with control group. MiR-330 Inhibited the Apoptosis of Glioblastoma To confirm that the overexpression of miR-330 was associated with apoptosis, we examined the apoptosis of the cells by flow cytometry 72 h after transfection. The results obviously demonstrated that there was a significant decrease of apoptosis in glioblastoma cells transfected with pre-miR-330 compared with cells transfected with pre-miR-con (P<0.05). While those transfected with anti-miR-330 showed an increase of apoptosis compared with anti-miR-con and pre-miR-330 groups (P<0.05) (Figure 7). In the rescue experiment, the siRNA-SH3GL2 group also showed a lower apoptotic proportion than the siRNA-con group (P<0.05) (Figure S3B). These data confirm that miR-330 plays an antiapoptotic role in glioblastoma cells by regulating SH3GL2 gene. 10.1371/journal.pone.0046010.g007Figure 7 MiR-330 inhibited apoptosis of U87 and U251 cells. Apoptosis of U87 and U251 cells were monitored by flow cytometry. Early apoptotic cells are Annexin V+/PI−, late apoptotic cells are Annexin V+/PI+, necrotic cells are Annexin V−/PI+ and healthy cells are Annexin V−/PI−. A representative experiment of three performed was shown. The percentage of apoptotic cells is indicated and the results represent the mean ± SD. * P<0.05 compared with control group. Discussion In this study, we firstly revealed the oncogenic role of miR-330 in human glioblastoma cells and its relationship with SH3GL2 gene. We found that miR-330 could influence the proliferation, migration, invasion, cell cycle and apoptosis of human glioblastoma by regulating SH3GL2 gene. SH3GL2, also termed as endophilin-1, is a multifunctional gene [17]. SH3GL2 mainly distributes in central nervous system, particularly enriched in the presynaptic ganglion [12]. Besides its endocytic functions, the non-endocytic functions of SH3GL2 may play a more important part in the malignant progression of tumor. Previous studies have shown that SH3GL2 gene is expressed less and functions as a tumor suppressor gene in many different tumor tissues. Gene chip demonstrated that the SH3GL2 gene expression was significantly decreased in laryngeal cancer. Clinical evidences also showed that there is a reduction of SH3GL2 expression in the samples from the follow-up patients [14]. Osterberg also proved that low expression of SH3GL2 was associated with increased chemotherapy resistance in ovarian cancer [18]. Sinha confirmed the decreased SH3GL2 gene expression in breast cancer [19]. In addition, it was reported that the SH3GL2 gene was involved in head and neck dysplastic lesions [20]. Our previous study found that the SH3GL2 expression was obviously lower in glioblastoma tissues compared with normal brain tissues [15]. These suggest that the SH3GL2 gene may also be a tumor suppressor gene and plays an important role in human glioblastoma. However, the underlying mechanism is still unclear. Recently, miRNAs have been rapidly developed as potential important molecular markers for cancer and other diseases [21]–[23]. They are found to regulate apoptosis, proliferation, differentiation, development, and metabolism in worm, fly, fish, mouse and human cells [24]. Many studies have shown that miRNAs function as oncogenes or tumor suppressor genes [25]–[27]. MiR-34a is repressed and promotes tumorigenesis in proneural malignant gliomas [28]. Inhibition of the highly expressed miR-221 and miR-222 impairs the growth of prostate carcinoma xenografts in mice [29]. To explore whether there were some microRNAs involved in the regulation of SH3GL2, we applied bioinformatics method (TargetScan and Miranda) to search for the possible microRNAs that target SH3GL2. We found that there was a binding site of miR-330 in the 3′-UTR of SH3GL2, suggesting SH3GL2 as a potential target of miR-330. Subsequently, luciferase reporter assay confirmed the prediction of miR-330′s targeting on the 3′-UTR of SH3GL2 gene. Meanwhile, we found a higher relative expression level in glioblastoma tissues and established glioblastoma cell lines than that of normal brain tissues. By transfection of exogenous miR-330, we detected a reduction of SH3GL2 protein expression in two glioblastoma cells. Glioblastoma cells transfected with anti-miR-330 showed higher expression of SH3GL2 protein level. Neither of these miRNA precursor transfections affected the SH3GL2 mRNA level. Meanwhile, we revealed that overexpression of miR-330 could increase the viability of glioblastoma cells. We also found that miR-330 obviously increased migration and invasion of glioblastoma cells. Besides, miR-330 could also influence the cell cycle and act as an antiapoptotic factor in both cell lines. What’ more, we found the opposite effects by transfecting anti-miR-330 precursor. To further confirm the biological behavior change of glioblastoma cells through miR-330′s regulation of SH3GL2, we also transfected the siRNA targeting on SH3GL2 and got similar results as miR-330. In line with the previous studies, these results indicated that miR-330 functioned as an oncogenic miRNA by negatively regulating the candidate tumor suppressor gene SH3GL2 in human glioblastoma cells. Since the close relationship between miR-330 and the SH3GL2 gene, we analyzed the potential mechanism of this process according to the previous studies. SH3GL2 can regulate intracellular transit and maturation of metalloprotease disintegrin by binding to its cytoplasmic tail, and then affects cell adhesion and growth factor signaling [30]. When binding to Cbl-interacting protein of 85 kDa (CIN85) and Cbl, SH3GL2 can also influence internalization, degradation and intracellular signaling of tyrosine kinase receptors for hepatocyte and epidermal growth factors [31]–[33]. At the same time, this complex mediates ligand-induced downregulation of c-Met and EGF receptors [31], [34]. Both c-Met and EGF receptors cause the development of cancers by influencing their differentiation, proliferation, migration, invasion, cell cycle progression and apoptosis, etc. Hence, SH3GL2 may play a key role in the progression of malignant tumors as a tumor suppressor gene. A recent study showed that miR-330 induced apoptosis in prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation [11]. Our study showed that exogenous overexpression of miR-330 caused some opposite biological behavior changes in glioblastoma cells compared with those in prostate cancer cells. However, it has been reported that even the same miRNA targets the same gene, which may show different biological effects in different tissues [35]. Hodzic reported that miR-330 post-transcriptionally regulated the expression of deoxycytidine kinase by targeting its 3′-UTR and decreased its sensitivity to gemcitabine in cancer cells [36]. Though, by targeting different gene and maybe through different pathways, miR-330 seems to exert the same effect in this report and our study to some extent. What is to be explored next is the molecular mechanism, downstream signal pathway of SH3GL2 mediates apoptosis and whether there is some crosstalk between other genes and signal pathways. Moreover, in vivo studies are to be performed to validate these findings. In conclusion, this is the first study to show that the tumor suppressor gene SH3GL2 is negatively regulated by miR-330 at the posttranscriptional level. We also showed that overexpression of miR-330 played an oncogenic role by inhibiting SH3GL2 and affected cell proliferation, migration, invasion and activation of cell cycle in U87 and U251 cells. MiR-330 is a potential novel oncogenic miRNA in glioblastoma and provides a new therapeutic target of human glioblastoma. Supporting Information Figure S1 Inhibition of SH3GL2 expression by siRNA in U87 cells. U87 cells were transfected with SH3GL2 siRNA (siRNA-SH3GL2) and its scramble control (siRNA-con) oligonucleotides. Both mRNA and protein levels lower expression of SH3GL2 were revealed compared with scramble control group. * P<0.05 compared with control group. (TIF) Click here for additional data file. Figure S2 Effect of SH3GL2 knocked-down on U87 cell proliferation, migration and invasion. (A) The proliferation of siRNA-con and siRNA-SH3GL2 transfected U87 cells was analyzed by MTT assay. The cell proliferation rate of siRNA-SH3GL2 group was much higher than that of siRNA-con group. (B) Transwell assay was performed to evaluate the migration and invasion capacity in U87 cells. The migration and invasion capacity of siRNA-SH3GL2 cells was larger than that of siRNA-con group. * P<0.05 compared with control group. (TIF) Click here for additional data file. Figure S3 Effect of SH3GL2 knocked-down on U87 cell cycle and apoptosis. The cell cycle distribution of siRNA-con and siRNA-SH3GL2 transfected cells was analyzed by flow cytometry. The percentage of cells in the different cell cycle phases was plotted, and the results represent the mean ± SD. * P<0.05 compared with control group. (B) Apoptosis of U87 cells were monitored by flow cytometry after transfected with siRNA to SH3GL2. Early apoptotic cells are Annexin V+/PI−, late apoptotic cells are Annexin V+/PI+, necrotic cells are Annexin V−/PI+ and healthy cells are Annexin V−/PI−. A representative experiment of three performed was shown. The percentage of apoptotic cells is indicated and the results represent the mean ± SD. * P<0.05 compared with control group. (TIF) Click here for additional data file. ==== Refs References 1 Ohgaki H (2009 ) Epidemiology of brain tumors . Methods Mol Biol 472 : 323 –342 .19107440 2 Li C , Feng Y , Coukos G , Zhang L (2009 ) Therapeutic microRNA strategies in human cancer . AAPS J 11 : 747 –757 .19876744 3 Zhong X , Coukos G , Zhang L (2012 ) miRNAs in human cancer . Methods Mol Biol 822 : 295 –306 .22144208 4 Stark A , Brennecke J , Bushati N , Russell RB , Cohen SM (2005 ) Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3′UTR evolution . 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==== Front BMC Complement Altern MedBMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central 1472-6882-12-1042281800010.1186/1472-6882-12-104Research ArticleIn vitro and in vivo anti-colon cancer effects of Garcinia mangostana xanthones extract Aisha Abdalrahim F A [email protected] Khalid M [email protected] Zhari [email protected] Amin Malik Shah Abdul [email protected] Department of Pharmacology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800, Pulau Penang, Malaysia2 The Chair of Cancer Targeting and Treatment, Biochemistry Department and King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, 11451, Saudi Arabia3 Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800, Pulau Penang, Malaysia4 Australian Institute for Nanotechnology and Bioengineering, University of Queensland, Queensland, 4072, Australia2012 20 7 2012 12 104 104 21 11 2011 20 7 2012 Copyright ©1900 Aisha et al.; licensee BioMed Central Ltd.1900Aisha 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 Xanthones are a group of oxygen-containing heterocyclic compounds with remarkable pharmacological effects such as anti-cancer, antioxidant, anti-inflammatory, and antimicrobial activities. Methods A xanthones extract (81% α-mangostin and 16% γ-mangostin), was prepared by crystallization of a toluene extract of G. mangostana fruit rinds and was analyzed by LC-MS. Anti-colon cancer effect was investigated on HCT 116 human colorectal carcinoma cells including cytotoxicity, apoptosis, anti-tumorigenicity, and effect on cell signalling pathways. The in vivo anti-colon cancer activity was also investigated on subcutaneous tumors established in nude mice. Results The extract showed potent cytotoxicity (median inhibitory concentration 6.5 ± 1.0 μg/ml), due to induction of the mitochondrial pathway of apoptosis. Three key steps in tumor metastasis including the cell migration, cell invasion and clonogenicity, were also inhibited. The extract and α-mangostin up-regulate the MAPK/ERK, c-Myc/Max, and p53 cell signalling pathways. The xanthones extract, when fed to nude mice, caused significant growth inhibition of the subcutaneous tumor of HCT 116 colorectal carcinoma cells. Conclusions Our data suggest new mechanisms of action of α-mangostin and the G. mangostana xanthones, and suggest the xanthones extract of as a potential anti-colon cancer candidate. ==== Body Background Garcinia mangostana L. or mangosteen is a tropical tree from the family Clusiaceae. The tree is cultivated for centuries in Southeast Asia rainforests, and can be found in many countries worldwide [1]. Pericarps of the fruit have been used in folk medicine for the treatment of many human illnesses such as skin and wound infections, and inflammatory diseases [2]. Mangosteen is also used as an ingredient in several commercial products including nutritional supplements, herbal cosmetics, and pharmaceutical products [1]. Mangosteen fruit rinds contain high concentration of xanthones. α-Mangostin (1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9 H-xanthen-9-one), and γ-mangostin (1,3,6,7-tetrahydroxy-2,8-bis(3-methylbut-2-enyl)xanthen-9-one) (Figure 1) are the main xanthones isolated from G. mangostana[3,4]. Figure 1 Chemical structure of α-mangostin and γ-mangostin, the main constituents of the G. mangostana xanthones extract. The G. mangostana xanthones are gaining more and more interest due to their remarkable pharmacological effects including analgesic [5], antioxidant [6], anti-inflammatory [7], anti-cancer [8-11] anti-allergy [12], antibacterial [13], anti-tuberculosis [14], antifungal [15], antiviral [16], cardioprotective [17], neuroprotective [18], and immunomodulation [19] effects. Colorectal cancer is the third in incidence after lung and breast cancers and accounts for almost 10% of total cases of cancer and almost 8% of total cancer deaths [20]. According to the World Health Organization (WHO), more than 70% of all cancer deaths occurred in countries with low and middle income, and deaths from cancer worldwide are projected to continue to rise to over 11 million in 2030 [21]. Hence, there is an increasing demand for cost-effective therapeutics and chemoprevention agents for the various types of cancer. Several studies have shown natural products, particularly medicinal plants as potential chemoprevention and anti-cancer candidates. Anti-cancer properties of G. mangostana extracts or pure xanthones have been extensively studied in vitro, however few reports of in vivo anti-cancer effects could be traced. Xanthone extracts from G. mangostana have been reported with chemoprevention effects against the chemically induced colon cancer [8], suppression of tumor growth and metastasis in a mouse model of mammary cancer [9], and a recent report showed the inhibition of prostate cancer growth by α-mangostin, the main constituent of the G. mangostana xanthones [22]. This study aims to investigate the in vitro anti-colon cancer properties of a G. mangostana xanthones extract (81% α-mangostin and 16% γ-mangostin) on HCT 116 human colorectal carcinoma. The in vitro anti-cancer effects include cytotoxicity, apoptosis, cell migration, cell invasion, and clonogenicity. The mechanism of action of the xanthones extract and α-mangostin on the transcription factor level of 10 signalling pathways involved in colon carcinogenesis was also investigated. The study also aims to investigate the in vivo anti-colon cancer effect on a pre-established subcutaneous tumor of HCT 116 cells in NCR nude mice. Methods Cell lines and reagents Human colorectal carcinoma cell line HCT 116; Catalogue number (CCL-247) and CCD-18Co normal colonic fibroblast; Catalogue number (CRL-1459) were purchased from the American Type Culture Collection (ATCC; Manassas, Virginia). RPMI 1640, Opti-MEM® and DMEM cell culture media, heat inactivated fetal bovine serum (HI-FBS), and phosphate buffered saline (PBS) without calcium and magnesium were purchased from Bio-Diagnostics (Petaling Jaya, Selangor, Malaysia). Cignal finder™ 10-pathway reporter-array system, and matrigel matrix (10 mg/ml) were purchased from SABiosciences (Frederick, Maryland). Wizard® SV genomic DNA purification system, caspases-3/7, -8 and −9 reagents, trans fast liposome, and dual luciferase reporter system were purchased from Promega (Petaling Jaya, Selangor, Malaysia). Cisplatin, Hoechst 33258, Rhodamine 123, agarose, ethidium bromide, penicillin/streptomycin (PS) solution, dimethylsulfoxide (DMSO), phenazine methosulfate (PMS), and 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide inner salt (XTT) were purchased from Sigma-Aldrich (Kuala Lumpur, Malaysia). The solvents were of analytical or HPLC grade and were obtained from Avantor Performance Materials (Petaling Jaya, Selangor, Malaysia). Plant material and extraction Ripened G. mangostana fruit was collected from a local fruit farm at Island of Penang, Malaysia. A voucher specimen (11155) was deposited at the Herbarium of School of Biological Sciences, USM. The fruit rind was chopped and dried at 45–50°C for 24 h. Toluene extract was prepared by maceration method at 1:5 plant: solvent ratio (wt/v), at 60°C for 48 h. The extract was filtered, concentrated at 60°C by rotavapor to about 150 ml, and crystallized at 2–8°C for 24 h. A yellow solid was formed, which was collected and dried at 50°C. Animals Athymic NCR nu/nu nude mice were obtained from Taconic Farms Inc. (Hudson, New York). Mice were housed in specific pathogen free (SPF) cages supplied with high efficiency particulate air (HEPA) filters. Free access to autoclaved food and water was provided and the autoclaved bedding was changed twice weekly. The procedures were approved by the USM Animal Ethics Committee with a reference number PPSG/07(A)/044/(2010) (59). Liquid chromatography-mass spectrometry (LC-MS) The xanthones extract was analyzed by a Dionex-Ultimate® 3000 Rapid Separation LC system (Dionex, Sunnyvale, California), connected with a Micro TOF-Q mass spectrometer (Bruker, Madison, Wisconsin). Chromatographic separation was performed using Nucleosil C18 column (5 μm, 4.6 × 250 mm) (Macherey-Nagel, Bethlehem, Pennsylvania), at 30°C, the mobile phase was consisting of 95% acetonitrile and 5% of 0.1% formic acid in water. The flow rate was set at 0.5 ml/min for 15 min, and spectral data were collected at 244 nm. Mass analysis was performed in the range 50–1000 m/z, under negative ion mode, and the nebulizer was set at 3.0 bar and heated to 150°C. The capillary voltage was set at 3000 V using a nitrogen dry gas at 8.0 L/min. The end plate offset was maintained at −500 V. Cell culture HCT 116 cells were maintained in RPMI 1640 medium supplemented with 10% HI-FBS and 1% PS, and the CCD-18Co cells were maintained in DMEM medium supplemented with 10% HI-FBS and 1% PS. Cells were cultured in a 5% CO2 in a humidified atmosphere at 37°C. Cell viability Cell viability was determined by the XTT test as described previously [23]. Briefly, cells were treated for 48 h, the culture medium was removed and replaced with a fresh one containing XTT and PMS at 100 μg/ml and 1 μg/ml, respectively. After incubation for 4 h, the optical density was measured at a wavelength of 450 nm, using a microplate reader (Thermo Fisher Scientific, Ratastie, Vantaa, Finland). The results are presented as a percentage inhibition to the negative control (0.5% DMSO) as the following: (1) Percentage inhibition=(1–ODSamples– ODBlankODVehicle– ODBlank)×100 The median inhibitory concentrations (IC50s) were calculated from the dose response curves (n = 3). Caspases-3/7, -8 and −9 HCT 116 cells were treated in a white 96-well plate for 90 min. Subsequently, the caspases activity was measured by caspase Glo 3/7, Glo 8 and Glo 9 as described previously [24]. Luminescence was measured by a microplate reader (Hidex, Mustionkatu, Turku, Finland), and the results are presented as a mean of relative light units (RLU) ± SD (n = 4). Mitochondrial membrane potential and chromatin condensation Rhodamine 123 and Hoechst 33258 were used as probes to study the effect on mitochondrial membrane potential and chromatin condensation [25,26]. Briefly, HCT 116 cells were treated with α-mangostin or the xanthones extract at different concentrations for 2 h. Subsequently, cells were fixed in 4% paraformaldehyde for 20 min, simultaneously stained with rhodamine 123 at 1 μg/ml and Hoechst 33258 at 10 μg/ml for 20 min, washed extensively with PBS, and examined immediately using IX71 inverted fluorescent microscopy (Olympus, Shinjuku, Tokyo, Japan). Cell morphology was evaluated by studying 5 randomly selected microscopic fields and the apoptotic index was calculated. DNA fragmentation HCT 116 (2 × 106) cells were treated for 48 h. Subsequently, the floating and attached cells were collected by centrifugation at 3000 rpm for 10 min, the total genomic DNA was extracted using Wizard® SV genomic DNA purification system, and analyzed by electrophoresis on 1.2% agarose gel stained with 0.5 μg/ml ethidium bromide. Anti-tumorigenicity Anti-tumorigenicity studies including clonogenicity, cell migration, and cell invasion were investigated on HCT 116 cells. Effect on the clonogenicity was evaluated by the colony formation assay as previously described [27]. Five hundred cells were seeded in 6-well plate in 2.5 ml of RPMI 1640 medium, and were incubated to allow attachment. Subsequent to 48 h treatment, the drug was removed and cells were incubated in a fresh medium for 12 days. Colonies were fixed in 4% paraformaldehyde, stained with 0.5% crystal violet, and counted under a stereomicroscope. The plating efficiency (PE) of untreated cells and the survival fraction (SF) of treated cells were then determined (n = 3). The effect on cell migration was studied by the wound healing assay as described previously [28]. Cell’s monolayer was scratched using a 200-μl micropipette tip, the detached cells were washed off, and the cells were treated in a medium containing 2% serum. The wounds were then photographed at zero time and incubated for 24 h. The distance of cell-free wounds was then measured using a Leica QWin image analysis software (Leica Microsystems Inc., Buffalo Grove, Illinois), and the percentage of wound closure was calculated relative to zero time. Effect on cell invasion was studied by a modification of the Boyden chamber assay using matrigel matrix [29]. Basically, 50 μl of matrigel (5 mg/ml) was loaded into 96-well plate and allowed to solidify for 45 min. Treated cells (5 × 103 in 150 μl RPMI medium) was added to each well and incubated for 48 h. Subsequently, cells were washed with PBS and the number of the invading cells was determined under inverted light microscopy. The results are presented as a percentage inhibition to untreated cells (n = 3). Effect on cell signalling pathways The assay was performed in 96-well plate format according to the manufacturer’s instructions. Briefly, HCT 116 cells were transfected by reverse transfection with DNA constructs of 10 signalling pathways, a positive control, and a negative control. After overnight incubation, cells were treated for 6 h in complete RPMI medium. Subsequently, the activity of Firefly and Renilla luciferases was measured using dual-luciferase assay. The results are displayed as relative luciferase units, generated by dividing the Firefly/Renilla ratio of transcription factor-responsive reporter transfections by the Firefly/Renilla ratio of negative control transfections (n = 3). The fold change in the transcription factor activity was then calculated by dividing the results of the treated cells by that of untreated cells. In Vivo anti-tumor activity Twenty four nude mice aged 6–8 weeks with average weight of 25 g were injected subcutaneously in right flank with 5 × 106 cells in 150 μl RPMI. After 7–10 days, animals with uniform tumor size were divided into 3 groups of 6 animals. Tumor size and body weight were recorded before starting the treatment and at 5-days intervals for 20 days. Animals were treated by mixing the extract with the animal food at 0.25% and 0.5% extract: food ratio (wt/wt). Tumor dimensions were measured by a calibre in 2 angles, length and width [30]. Tumor size was then calculated as described previously [30-32], by applying the formula (((W + L)/2) ^ 3) × 2, where W is the width and L is the length. Tumor size in tumors with more than a lobe was calculated by summation of the size of the individual lobes [30]. Cross sections of the tumors were then prepared, stained with Eosin/Hematoxylin, and were studied for presence of necrotic cells and for the number of intratumor blood vessels. Blood vessels were counted at 20× magnification in 25 microscopic fields per tumor, and the results are presented as average number of blood vessels per tumor ± SD. Statistical analysis The results are presented as mean ± SD. The differences between groups were compared by One-way ANOVA, and were considered significant at P < 0.05. Data analysis was carried out using SSPS 16.0 software. Results Phytochemical analysis The extract was obtained at 5% yield (wt/wt) relative to the dry plant material. LC-MS analysis indicates the presence of 5 compounds; α-mangostin, was 81%, γ-mangostin was 16%, and the other 3 compounds were 3%, the percentage of the compounds was calculated based on the peak area (Table 1). Table 1 Mass spectrometry of the G. mangostana xanthones extract Peak No Retention time (min) % Intensity Isotopic pattern [M-H]- (m/z) Molecular formula Compounds 1 7.4 ± 0.006 1.4 ± 0.1 413.1408 C23H26O7 Garcinone C       414.1443           415.1397     2 7.8 ± 0.001 15.6 ± 1.6 395.1308 C23H24O6 γ-mangostin       396.1338           397.1360     3 8.8 ± 0.013 1.2 ± 0.1 379.1370 C23H24O5 8-deoxygartanin       380.1381           381.1440     4 9.2 ± 0.001 80.8 ± 1.6 409.1452 C24H26O6 α-mangostin       410.1489           411.1526     5 13.5 ± 0.005 0.9 ± 0.03 423.1604 C25H28O6 β-mangostin       424.1631           425.1667     The mass was recorded in the negative ion mode (n = 4). Cytotoxicity The xanthones extract, α-mangostin, and γ-mangostin caused dose dependent killing of the colon cancer cells (Figure 2a), showing IC50s of 6.5 ± 1.0 μg/ml, 5.1 ± 0.2 μg/ml, and 7.2 ± 0.4 μg/ml, respectively. CCD-18Co normal cells, unlike HCT 116 cells, were 2 folds less sensitive showing IC50 of 11.1 ± 0.4 μg/ml (α-mangostin), and 13.0 ± 0.6 μg/ml (xanthones extract). Cisplatin, as a positive control, also showed dose dependent cytotoxicity on colon cancer cells giving IC50 of 6.1 ± 0.2 μg/ml. Figure 2 Cytotoxic and apoptotic effects of α-mangostin and the xanthones extract on HCT 116 cells. Dose response curves of the xanthones extract on HCT 116 and CCD-18Co cells in the concentration range 2.5 – 30 μg/ml (a). Effect on caspases-3/7 (b), and caspases-8 and −9 (c). Effect on DNA fragmentation (d): the negative control was 0.5% DMSO, the positive control was α-mangostin at 20 μg/ml, and the xanthones extract was applied at 10, 20, 30 and 40 μg/ml. (*) indicates P < 0.05. Effect on caspases-3/7,-8 and −9 α-Mangostin and the xanthones extract at 10 and 20 μg/ml, showed a rapid enhancement of the caspases-3/7 activity after a treatment for 90 min (Figure 2b). At a concentration of 5 μg/ml, a slight but not significant increase in the activity was achieved (P > 0.05). The treatment compounds also caused significant enhancement of the caspase-9 activity in HCT 116 cells, but not caspase-8 activity (Figure 2c). The increase in caspase-9 activity was almost 8-folds more than that of caspase-8. Effect on DNA fragmentation Analysis of the total genomic DNA by agarose gel electrophoresis revealed apparent DNA fragmentation in HCT 116 cells (Figure 2d). The results indicate that the effector caspases executed the apoptotic signal stimulated by the treatment compounds. Effect on mitochondrial membrane potential of HCT 116 cells The rhodamine staining showed a distinct morphology of the apoptotic cells, which were stained more brightly than the non-apoptotic cells (Figure 3a). The result indicates lower concentration of rhodamine 123 due to loss of mitochondrial membrane potential. The apoptotic index of α-mangostin-treated cells at 20 μg/ml was (55 ± 9)%, and that of the xanthones extract was (13.2 ± 2.4)%, (38 ± 4.5)%, (47 ± 4.5)%, and (68 ± 9)% at 7.5, 10, 15 and 20 μg/ml, respectively. Significant induction of apoptosis, compared to untreated cells (5.1 ± 2.3)%, was obtained at the last 3 concentrations (P = 0.0), whereas no significant effect was observed at a concentration of 7.5 μg/ml (P = 0.2). Figure 3 Effect the xanthones extracts on mitochondrial membrane potential and chromatin condensation. The mitochondrial membrane potential (a): negative control (1), α-mangostin at 20 μg/ml (2) and the xanthones extract at 20 μg/ml (3). Chromatin condensation (b): negative control (1), α-mangostin at 20 μg/ml (2) and the xanthones extract at 20 μg/ml (3). Effect on chromatin condensation and nuclear fragmentation α-Mangostin at 20 μg/ml, and the xanthones extract caused significant and dose dependent induction of chromatin condensation and nuclear fragmentation in HCT 116 cells after 2 h treatment. Staining with the DNA probe Hoechst 33258 produced a distinct nuclear morphology of the apoptotic cells, which were stained more brightly, with or without nuclear fragmentation, whereas the non-apoptotic cells showed uniformly stained nuclei at lower intensely (Figure 3b). The apoptotic index of α-mangostin-treated cells was (47 ± 5.5)%, and that of the extract was (4.4 ± 3)%, (37 ± 7)%, (39 ± 10)%, and (52 ± 9)% at 7.5, 10, 15 and 20 μg/ml, respectively. Compared with the vehicle alone (3.3 ± 3)%, significant induction of apoptosis was obtained at 10, 15 and 20 μg/ml (P = 0.0), whereas the treatment at 7.5 μg/ml did not show any apoptotic effect, (P = 0.99). Anti-tumorigenicity The compounds inhibited the clonogenicity of HCT 116 cells (Figure 4a). The PE was (54 ± 2)%, and the SF in cells treated with the xanthones extract was 0% at all concentrations. The SF in α-mangostin treated cells was 0% at 20, 15, 10 and 7.5 μg/ml, and (7.8 ± 0.3)% at 5 μg/ml. Figure 4 Anti-tumorigenicity effect of the xanthones extract on HCT 116 cells. Clonogenicity (a): negative control (1), α-mangostin at 5 μg/ml (2) and the xanthones extract at 5 μg/ml (3). Cell migration (b): wounds photographed at zero time (1), and the treated cells after 24 h; negative control (2), α-mangostin at 5 μg/ml (3) and the xanthones extract at 5 μg/ml (4). Matrigel invasion (c): untreated cells (1), α-mangostin at 6 μg/ml (2) xanthones extract at 6 μg/ml (3) and at 4.5 μg/ml (4). Cell migration was also inhibited in both treatments (Figure 4b). The percentage of wound closure in the untreated cells was (65 ± 4.3)%. α-Mangostin, at 5 μg/ml, reduced the percentage of wound closure to (41 ± 2.7)%, (P = 0.0). Likewise, the xanthones extract, at 3 and 5 μg/ml, reduced the wound closure percentage to (42 ± 4.2)% and (56 ± 3.4)%, (P < 0.05). The cell invasion of matrigel was also inhibited by α-mangostin at 6 μg/ml (78 ± 6)%, and by the xanthones extract at 6 μg/ml (78 ± 8)% and 4.5 μg/ml (57 ± 8)%. Besides reducing the number of matrigel-invading cells, the treatment compounds also caused morphological changes in the treated cells characterized by cytoplasmic shrinkage and contraction of cellular polypodia (Figure 4c). Effect on cell signalling pathways The transfected HCT 116 cells were treated at 2 concentrations 7.5 and 10 μg/ml for 6 h, and the results in the treated cells were compared to those treated with the vehicle alone (0.5% DMSO). The transcription factor activity of the 10 pathways is reduced by treating the cells with 10 μg/ml of the xanthones extract and α-mangostin. However, the treated cells showed apoptotic morphology, which indicates the downregulation of signalling pathways occurred as a consequence of apoptosis. Treatment at 7.5 μg/ml did not induce apoptotic changes in the treated cells, but resulted in differential effects on the signalling pathways. The fold changes in the transcription factor activity in cells treated at 7.5 μg/ml is displayed in Figure 5. The transcription factor activity of the MAPK/ERK pathway was increased by 71% in α-mangostin-treated cells and 97% in the xanthones extract-treated cells. Activity of the Myc/Max signalling pathway was also increased by 48% in α-mangostin and 60% in the xanthones extract-treated cells. In addition, the activity of the p53 signalling pathway was increased by 30% in α-mangostin-treated cells and 50% in the xanthones extract-treated cells. On the contrary, the activity of the NFKB pathway was inhibited by 30% in α-mangostin treatment and by 13% in the extract-treated cells. On other hand, the treatment compounds did not cause any significant changes in the Wnt, Notch, TGFB, cell cycle, hypoxia and MAPK/JNK signalling pathways. Figure 5 Effect of the xanthones extract and α-mangostin (7.5 μg/ml) on the transcription factor activity of 10 cell signalling pathways. The fold changes in the transcription factor activity were calculated by dividing the relative light units in the treated cells by that of the untreated cells. The fold change of (1) indicates no activity. In Vivo anti-colon cancer effect The in vivo anti-colon cancer effect of the xanthones extract was investigated on the HCT 116 subcutaneous tumor model established in NCR nu/nu nude mice. The results are presented as average tumor size ± SD (n = 6). The treatment with the α-mangostin extract caused apparent necrosis of the pre-established tumors in 2 animals (Figure 6a), and caused significant reduction in the tumor size compared to untreated group. Data analysis was performed by considering the tumor size on 5-days intervals and showed that significant reduction in tumor size was achieved after 15 days (0.5% wt/wt), and 20 days (0.25% wt/wt) of treatment, P < 0.05, (Figure 6b). Analysis of the tumor cross sections revealed apparent differences in the extent of necrotic regions between the treated versus untreated tumors (Figure 6c). The necrotic/apoptotic cells in treated tumors predominate over the viable tumor cells which appear as islands in the middle of necrotic cells. On the contrary, untreated tumors were more compact with more abundance of viable tumor cells. Figure 6 The subcutaneous tumors in NCR nude mice (a): Untreated group (1), and the treated group at 0.5% wt/wt of the xanthones extract (2). Analysis of tumor size (b): analysis of tumor size versus time (days) after treatment with the xanthones extract at 2 doses 0.5% and 0.25% wt/wt compared to the control group (untreated). (*) refers to significant difference between both treated groups (P < 0.05) and the control, and (#) refers to significant difference between the 0.5% group and the control in each corresponding interval. Cross sections of tumor tissues (c): untreated animals (1), 0.5% treated group (2) and the 0.25% treated group (3). The tissues were stained with Hematoxylin-eosin and the pictures were captured at 5× magnification. (N) refers to necrotic cells and (V) refers to viable tumor cells. The average number of intratumor blood vessels was 3.9 ± 0.6/microscopic field (0.5% wt/wt) and 4 ± 0.3/microscopic field (0.25% wt/wt), was significantly lower than that in the control group (7.8 ± 1.2), P = 0.0. Additionally, effect on the animal body weight was also investigated and the results are presented as average percentage of weight gain or loss. The data showed a slight, but not statistically significant weight loss in the treated groups −4.4 ± 10% (0.5% wt/wt) and −1.5 ± 2.4% (0.25% wt/wt), compared to 5.3 ± 6% (control group), P = 0.1 and 0.4, respectively. Discussion The xanthones extract of G. mangostana fruit rinds contains mainly α-mangostin and γ-mangostin. The HCT 116 cell line was selected as a model of human colorectal carcinoma [33], and CCD-18Co human normal fibroblast was selected as a control cell line. The cytotoxicity of the xanthones extract, α-mangostin and γ-mangostin was comparable to that of cisplatin, and the xanthones extract was almost 2 times more cytotoxic on the colon cancer cells than on the normal cells, which indicates higher selectivity towards the colon cancer cells. Apoptosis studies revealed enhancement of the executioner caspases-3/7, activation of the initiator caspase-9, induction of DNA fragmentation and chromatin condensation, and loss of mitochondrial membrane potential. These results indicate the role of the mitochondrial pathway of apoptosis in mediating cytotoxicity of the compounds. Our results are consistent with the previous results of other researchers [10,34], and provide further evidence on apoptotic effects of G. mangostana, and indicate the xanthones of this fruit as potential anti-cancer candidates. Sub-cytotoxic concentrations of α-mangostin and the xanthones extract inhibited 3 key steps in tumor metastasis including the cell migration, cell invasion and clonogenicity. These results, in combination the results of other researchers [9,35], indicate the potential anti-metastatic effect of the G. mangostana xanthones. In order to gain deeper insights into the mechanism of action, a cell-based reporter assay was used to study the effect of α-mangostin and the xanthones extract on the transcription factor activity of the Notch, Wnt/β-Catenin, TGFβ, p53, HIF, Myc, E2F, NFKB, MAPK/ERK (SRE), and MAPK/JNK (AP-1) signalling pathways. The compounds enhanced the transcription factor activity of the MAPK/ERK, Myc/Max, and p53/DNA damage signalling pathways. Previous research showed that the activated ERK pathway is associated with increased stability and activity of p53, and increased stability of c-Myc that in turn increases the proapoptotic effects of p53 tumor suppressor gene [36,37]. Recent studies showed that activation of the ERK pathway is implicated in inducing apoptosis, as a consequence of DNA damage caused by cisplatin [38], etoposide [39], doxorubicin, and ionizing and Ultraviolet irradiation [40]. Therefore, upregulation of the ERK pathway may provide a therapeutic target for different types of cancer [41-43], however further investigation is required to study the effect of the activated ERK pathway on the expression of the proapoptotic proteins such as p21 and Bax. α-Mangostin also caused inhibition of the NFKB pathway. The downregulation of this pathway is associated with increased sensitivity of chemoresistant cells [44], and hence α-mangostin may sensitize the colon cancer cells to the apoptotic effect of chemotherapeutics. Different mechanisms of action of mangostins have been reported including upregulation of the ERK ½ in DLD-1 colon cancer cells [8], inhibition of TCF/β-catenin transcriptional activity in colon cancer cells [11], and inhibition of the MAPK/ERK, MAPK/JNK and Akt signalling pathways in human chondrosarcoma cells [45]. These findings indicate that mangostins may work by different mechanisms in different tumor cells. Drug concentration and duration of treatment have significant effects on viability of cells, and hence these may have substantial effect on the activity of signalling pathways. The In vivo anti-colon cancer study revealed significant inhibition of the tumor growth. The Anti-tumor effect of the extract may be explained due to direct cytotoxicity on the tumor cells as evident by the presence of extensive necrosis in the subcutaneous tumors, or due to reducing the intratumor blood supply as evident by the significant reduction in the number of intratumor blood vessels, or due to combination of both mechanisms. Conclusions Taken together, our data suggest new mechanisms of action of α-mangostin and suggest the xanthones extract of G. mangostana as a potential anti-colon cancer candidate. Competing interests The authors declare no conflict of interest related to this work. Authors’ contributions AFA carried out the experiments, performed the statistical analysis, and drafted the manuscript. KM interpreted the results of cell signalling pathways and helped in editing the manuscript. ZI interpreted the LC-MS data. AMS participated in the design of the study and edited the 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/1472-6882/12/104/prepub Acknowledgements Abdalrahim F. A. Aisha would like to acknowledge Universiti Sains Malaysia (USM) for providing fellowship for the academic year 2010/2011. The Authors would like to thank Dr. Tan Mei Lan and Mr. Ahmad Ismail (IPHARM, USM) for providing and helping in fluorescent microscopy, Associate Prof. Dr. Gurjeet Kaur (INFORMM, USM) for helping in analysis of tumor cross sections. This work was financially supported by the USM-Research University Grant [1001/PFARMASI/81144], and was supported partially by the research chair funded by King Saud University on drug targeting and treatment of cancer using nanoparticles. ==== Refs Ji X Avula B Khan IA Quantitative and qualitative determination of six xanthones in Garcinia mangostana L. by LC-PDA and LC-ESI-MS J Pharm Biomed Anal 2007 43 1270 1276 17129697 Harborne JB Baxter H Moss GP Phytochemical dictionary: a handbook of bioactive compounds from plants 1999 CRC, Pedraza-Chaverri J Cardenas-Rodriguez N Orozco-Ibarra M Perez-Rojas JM Medicinal properties of mangosteen (Garcinia mangostana) Food Chem Toxicol 2008 46 3227 3239 18725264 Obolskiy D Pischel I Siriwatanametanon N Heinrich M Garcinia mangostana L.: a phytochemical and pharmacological review Phytother Res 2009 23 1047 1065 19172667 Cui J Hu W Cai Z Liu Y Li S Tao W Xiang H New medicinal properties of mangostins: analgesic activity and pharmacological characterization of active ingredients from the fruit hull of Garcinia mangostana L Pharmacol Biochem Behav 2010 95 166 172 20064550 Jung H Su B Keller W Mehta R Kinghorn A Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen) J Agric Food Chem 2006 54 2077 2082 16536578 Chen LG Yang LL Wang CC Anti-inflammatory activity of mangostins from Garcinia mangostana Food Chem Toxicol 2008 46 688 693 18029076 Akao Y Nakagawa Y Iinuma M Nozawa Y Anti-cancer effects of xanthones from pericarps of mangosteen Int J Mol Sci 2008 9 355 370 19325754 Doi H Shibata MA Shibata E MorimotoN J Akao Y Iinuma M Tanigawa N Otsuki Y Panaxanthone isolated from pericarp of Garcinia mangostana L. suppresses tumor growth and metastasis of a mouse model of mammary cancer Anticancer Res 2009 29 2485 2495 19596918 Matsumoto K Akao Y Kobayashi E Ohguchi K Ito T Tanaka T Iinuma M Nozawa Y Induction of apoptosis by xanthones from mangosteen in human leukemia cell lines J Nat Prod 2003 66 1124 1127 12932141 Yoo J-H Kang K Jho EH Chin Y-W Kim J Nho CW [alpha]- and [gamma]-Mangostin Inhibit the Proliferation of Colon Cancer Cells via [beta]-Catenin Gene Regulation in Wnt/cGMP Signalling Food Chem 2011 129 1559 1566 Nakatani K Atsumi M Arakawa T Oosawa K Shimura S Nakahata N Ohizumi Y Inhibitions of histamine release and prostaglandin E2 synthesis by mangosteen, a Thai medicinal plant Biol Pharm Bull 2002 25 1137 1141 12230104 Sakagami Y Iinuma M Piyasena KG Dharmaratne HR Antibacterial activity of alpha-mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics Phytomedicine 2005 12 203 208 15830842 Suksamrarn S Suwannapoch N Phakhodee W Thanuhiranlert J Ratananukul P Chimnoi N Suksamrarn A Antimycobacterial activity of prenylated xanthones from the fruits of Garcinia mangostana Chem Pharm Bull(Tokyo) 2003 51 857 859 12843596 Kaomongkolgit R Jamdee K Chaisomboon N Antifungal activity of alpha-mangostin against Candida albicans J Oral Sci 2009 51 401 406 19776506 Chen S Wan M Loh B Active constituents against HIV-1 protease from Garcinia mangostana Planta Med 1996 62 381 382 8792678 Devi Sampath P Vijayaraghavan K Cardioprotective effect of alpha-mangostin, a xanthone derivative from mangosteen on tissue defense system against isoproterenol-induced myocardial infarction in rats J Biochem Mol Toxicol 2007 21 336 339 17994576 Weecharangsan W Opanasopit P Sukma M Ngawhirunpat T Sotanaphun U Siripong P Antioxidative and neuroprotective activities of extracts from the fruit hull of mangosteen (Garcinia mangostana Linn.) 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23049906PONE-D-12-0966110.1371/journal.pone.0045965Research ArticleBiologyBiochemistryNucleic acidsRNARNA interferenceMolecular cell biologyGene expressionDNA transcriptionRNA interferenceCell AdhesionCell DeathCell DivisionCell GrowthMedicineOncologyBasic Cancer ResearchMetastasisTumor PhysiologyCancers and NeoplasmsBreast TumorsInvasive Ductal CarcinomaMiR-145 Regulates Epithelial to Mesenchymal Transition of Breast Cancer Cells by Targeting Oct4 MiR-145 Suppresses EMT in Breast CancerHu Jiajia 1 Guo Hua 2 Li Hongyan 2 Liu Yan 2 Liu Jingjing 3 Chen Liwei 2 Zhang Jin 3 Zhang Ning 1 2 * 1 Tianjin Medical University, Research Center of Basic Medical Sciences, Tianjin, China 2 Laboratory of Cancer Cell Biology,Cancer Institute and Hospital, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin, China 3 Cancer Institute and Hospital, 3rd Department of Breast Cancer, Tianjin, China Hotchin Neil A. Editor University of Birmingham, United Kingdom * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: NZ. Performed the experiments: JH HG HL. Analyzed the data: YL LC. Contributed reagents/materials/analysis tools: JL JZ. Wrote the paper: JH HG. 2012 26 9 2012 7 9 e4596531 3 2012 23 8 2012 © 2012 Hu et al2012Hu 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.MiR-145 could regulate tumor growth, apoptosis, migration, and invasion. In our present study, we investigated its role in epithelial-mesenchymal transition (EMT). Expression of miR-145 was decreased in breast tumor tissues at T3&4 stages in comparison with those at T1&2. Over-expression of miR-145 mimics enhanced protein levels of E-cadherin and dampened those of α-SMA and Fibronectin, indicative of its inhibitory role in EMT occurrence. Mechanistic studies showed that miR-145 mimics inhibited Oct4 expression and miR-145 inhibitor enhanced it. Over-expression of Oct4 reversed miR-145-regulated expression of EMT markers, suggesting that Oct4 mediated the inhibitory effects of miR-145. MiR-145 could inhibite the expression of Snail, ZEB1, and ZEB2, while over-expression of Oct4 rescued the effects. Furthermore, Oct-4 induced over-expression of transcription factor Snail, ZEB1 and ZEB2 was mediated by β-catenin. Expression of Slug and Twist were not altered by miR-145/Oct4. Taken together, our results have revealed a novel role of miR-145 on EMT. It inhibits EMT by blocking the expression of Oct4, and downstream transcriptional factors, Snail, ZEB1 and ZEB2. The founders of the National Scientific Foundation of China (NSFC #81072160) and 973 program grants (#2011CB933100 and #2010CB933900) had roles in providing financial support. The founders of Tianjin Science and Technology Higher Education Development fund (#20090138), The Key (Key grant) Project of Chinese Ministry of Education (#211010), Tianjin Health Bureau Technology Fund (#2010KZ73) had roles in study design. The founders of the Changjiang Scholars and Innovative Research Team in University in China (Grant IRT 1076), Tianjin Medical University Innovation Fund 146-200007 had roles in providing reagents and materials. ==== Body Introduction Metastasis, the major cause of mortality among cancer patients, is a multi-step process, including detachment of tumor cells from the primary sites, intravasation into circulation, migration along the circulation, extravasation to the secondary sites, and proliferation [1]. Epithelial-mesenchymal transition (EMT) plays a critical role during the initiation stage of metastasis. Immotile epithelial cells with the apical-basal polarity are converted to the motile, dispersed mesenchymal-like cells with spindle shape [2]. Consequently, tumor cells are detached from original sites and start to invade surrounding tissue. Enhanced motility of tumor cells is essential for the following steps of metastasis, such as invasion, intravasation and extravasation [3]. Thus, EMT is a pre-requisite step for cancer cell migration. Increasing reports have demonstrated that epigenetic dysregulation, as well as genomic instability, contributes to tumor metastasis. Abnormalities in DNA methylation or histone acetylation induce tumorigenesis and metastasis [4], [5]. MicroRNAs (miRNAs), a highly conserved group of small non-coding RNAs, regulate the expression of mRNA transcripts at post-transcriptional level [6]. Increasing evidences have proven that miRNAs take part in the regulation of many physiological and pathological processes, especially EMT and tumor metastasis [7], [8], [9], [10]. Gregory et al reported that miR-200 family and miR-205 mediated EMT through targeting ZEB1 and SIP1, which in turn regulated metastasis [11]. It has been documented that miR-21, miR-181a, miR-429, miR-137 and miR-661 were also involved in EMT [12], [13], [14], [15], [16]. Several reports have revealed that the expression level of miR-145 is decreased in various human cancers [17]. Early studies have shown that miR-145 plays an important role in suppressing tumor growth and promoting tumor apoptosis [18], [19], [20]. Recently, Xin et al pointed out that miR-145 and miR-143 could modulate cytoskeletal dynamics of smooth muscle cells in response to vascular injury [21]. Gotte et al and Sachdeva et al indicated that miR-145 suppressed breast cancer cell migration via inhibiting the expression of junctional adherin molecule A (JAMA), fascin and mucin1 [22]–[23]. Thus, it is clear that miR145 regulates the expression of proteins directly involved in cell migration. EMT is a key step before cancer cell invasion and migration. However, the role of miR-145 in EMT is still largely unknown. 10.1371/journal.pone.0045965.t001Table 1 Primer Sequences. Target Forward primer sequence Forward primer sequence Oct4 AAGCGATCAAGCAGCGAC GGAAAGGGACCGAGGAGTA Snail CCTCCCTGTCAGATGAGGAC CCAGGCTGAGGTATTCCTTG Slug GGGGAGAAGCCTTTTTCTTG TCCTCATGTTTGTGCAGGAG ZEB1 TTCAAACCCATAGTGGTTGCT TGGGAGATACCAAACCAACTG ZEB2 TTCCTGGGCTACGACCATAC TGTGCTCCATCAAGCAATTC Twist GGAGTCCGCAGTCTTACGAG TCTGGAGGACCTGGTAGAGG GAPDH ACCCAGAAGA CTGTGGATGG TCTAGACGGCAGGTCAGGTC In a search for negative regulators of cancer cell chemotaxis, we identified that miR-145 inhibited breast cancer cell chemotaxis. During a preliminary characterization, we found that over-expression of miR-145 reversed the expression of EMT markers in MDA-MB-231 cells, suggesting that miR-145 suppressed EMT. In this study, we investigated the molecular mechanism of miR-145-mediated EMT in cancer cells, revealing a signaling pathway involving transcription factor Oct4 and Snail/ZEB1/ZEB2. Furthermore, our results have demonstrated that miR-145-mediated EMT is required for cancer cell to acquire migration and invasion properties. Materials and Methods Ethics Statement This project entitled “MiR-145 regulates epithelial to mesenchymal transition of breast cancer cells by targeting Oct4” will analyze the expression of miR-145 in 41 fresh samples of human breast cancer specimens obtained from patients who underwent breast cancer surgery at the Cancer Hospital of Tianjin Medical University from January 2002 to December 2004. This project had the informed consents from all the patients. This study is consistent with the regulations of the Ministry of Health, ‘biomedical research involving human ethics review (tentative)” and the Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Subjects. 10.1371/journal.pone.0045965.g001Figure 1 MiR-145 inhibits cancer cell migration and EMT (A) Expression of miR-145 mimics inhibited EGF-induced chemotaxis in MDA-MB-231 cells. Statistical analysis was performed by one-way ANOVA.*, P<0.01 versus control. Error bars indicating SD. (B) Expression of miR-145 mimics inhibited cell migration in a wound-healing assay. Statistical analysis was also performed by one-way ANOVA. (Points, mean of three independent experiments; bars, SD.) (C) Cell migration and invasion were inhibited upon treatment with miR-145 mimics. Statistical analysis was assessed by the two-tailed Student’s t test. Columns, mean of three independent experiments; bars, SD. (D) Expression of miR-145 mimics enhanced the expression of E-cadherin, and suppressed the expression of α–smooth muscle actin and Fibronectin. A representive data of there separate experiments is shown. (E) Expression of miR-145 was examined in breast tumor tissues from 41 patients. Cell Culture MDA-MB-231, SK-BR-3, BT-549, ZR-75-30 and T47D cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA USA). All the cell lines were cultured at the normal conditions according to the protocol from ATCC. Reagents and Antibodies Micro-Boyden chambers for chemotaxis assay were obtained from Neuroprobe (Neuro Probe,Gaithersburg, MD USA ); miR-145 mimics, miR-145 inhibitor, miR-145 probe and U6 probe were all from Qiagen (QIAGEN, Hilden, Germany). Recombinant human epidermal growth factor (EGF) from R&D Systems (R&D Systems, Minneapolis, MN USA), Fibronectin from Sigma (Sigma, St Louis, MO USA), and Matrigel was from BD Biosciences (BD Biosciences, Franklin Lakes, NJ USA). Antibodies against Oct4, Fibronectin, Snail1, ZEB2, β-catenin and β-actin were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA USA); E-cadherin from BD Biosciences; α-smooth muscle actin (α-SMA) from Sigma and ZEB1 was from AbCam (AbCam, Cambridge, UK). HRP-conjugated goat anti- mouse IgG and HRP-conjugated goat anti-rabbit IgG were brought from Santa Cruz Biotechnology (Santa Cruz Biotechnology,CA USA).The pGL3-Control Luciferase Report vector, pRL Renilla Luciferase Report vector and Dual-luciferase reporter assay system were all brought from Promega (Promega, Madison, WI USA) and pcDNATM3.1/ZEO (+) plasmid was form Invitrogen (Invitrogen, Carlsbad, CA USA). 10.1371/journal.pone.0045965.g002Figure 2 MiR-145 directly targeted Oct4 in breast cancer cells. (A) Identification of miR-145 target site in Oct4 mRNA 3′UTR. (B) Treatment with miR-145 mimics did not alter the mRNA levels of Oct4. (C) Treatment with miR-145 mimics inhibited the protein levels of Oct4. (D) Treatment with miR-145 inhibitor enhanced the protein levels of Oct4. (E) Treatment with miR-145 mimics inhibited translational activity of a luciferase-expressing plasmid containing a 3′-UTR from Oct4. Statistical analysis was performed by t test. Values in B and E are shown as means±SD of three independent experiments. *, P<0.01, significance difference between two compared groups. Error bar indicates SD. (F) Western blot assay showed that knockdown of Oct-4 inhibited the expression of β-catenin while overexpression of Oct-4 enhanced it. miRNA Microarray Experiments For miRNA microarray experiments, total RNA samples were analyzed by CapitalBio (CapitalBio Corp, Beijing, China). Procedures were performed as described in detail on the website of CapitalBio (http://www.capitalbio.com). Image intensities were measured as a function of the median of foreground minus the background, as previously described [24]. Raw data were normalized and analyzed in GenePix Pro 4.0 software (Axon Instruments). Expression data were median-centered using the global median normalization function of the Bioconductor package (http://www.bioconductor.org). Statistical comparisons were performed with the SAM software [25]. 10.1371/journal.pone.0045965.g003Figure 3 Over-expression of Oct4 rescued the inhibitory effects of miR-145 on EMT. (A) Over-expression of Oct4 reversed miR145-elicited protein expression profiles of E-cadherin, N-cadherin, α–smooth muscle actin and Fibronectin. A Western blot representative of 3 independent experiments is shown. (B) Over-expression of Oct4 rescued miR-145-suppressed cancer cell migration and invasion. Statistical analysis was performed by t test. The data are shown as means±SD for triplicate measurement. *, P<0.01, significant difference; Bars, SD. 10.1371/journal.pone.0045965.g004Figure 4 Snail, ZEB1, and ZEB2 are the downstream molecules of miR-145/Oct4 in MDA-MB-231, BT-549, ZR-75-30 and T47D cells. (A) MiR-145/Oct4 did not alter the expression of Slug and Twist. (B), (C) and (D) Real-time PCR and Western blot assay showed that miR-145/Oct4 regulated the expression of Snail, ZEB1 and ZEB2 both in mRNA level and protein level. Statistical analysis was performed by t test. Columns, mean of three separate experiments; Error bars show the SD of three independent experiments measured in triplicate. *, P<0.01, significant difference between two compared groups. (E) Western blot assay showed that down-regulation of β-catenin inhibited Oct-4 induced expression of ZEB1, ZEB2 and Snail in MDA-MB-231, BT-549, ZR-75-30 and T47D cells. RNA Extraction, Reverse Transcription PCR and Real-time PCR Assay The materials consisted of 41 patients due to breast cancer who had not undergone chemotherapy or radiotherapy prior to surgery between 2002 and 2004. The samples of patients were obtained from the department of tissue library, Tianjin Cancer Institute and Hospital, Tianjin Medical University, China. Total RNA was collected by using Trizol (Invitrogen). Real-time PCR was carried out by using miScript SYBR Green PCR kit (Qiagen). For transcription factor detection, 2 µg of total RNA was used to perform reverse transcription by using TransScript First-strand cDNA Synthesis SuperMix kit. The real-time PCR was performed by using STBR Premix Ex Taq™ kit (TaKaRa Bio,Otsu, Japan). The primers of miR-145 and U6 are acquired from miScript Primer assays kit (Qiagen). The primers for the detection of transcription factors were list in Table 1. Chemotaxis, Wound-healing Assay and Invasion Assay Chemotaxis assay was performed as previous report [26]. The 10 µm filter membrane should be pretreated with 10 µg/ml of Fibronectin at 4°C overnight. For wound-healing assay, scrape wounds were created with a sterile pipette tip in a 6-well plate and the wound distances were measured at different time points (0, 3, 6, 9, 12, 24 hours) under the microscope. Matrigel-coated invasion inserts (BD Biosciences) with 8 µm pore membrane were used for invasion assays. 10.1371/journal.pone.0045965.g005Figure 5 MiR-145 inhibited EMT and cell migration. (A) MiR-145 regulated EMT through Oct4 and its downstream transcriptional factors, Snail, ZEB1, and ZEB2. (B)Treatment with miR-145 mimics in BT-549, ZR-75-30 and T47D cells inhibited the protein levels of Oct4. Treatment with miR-145 inhibited cell migration (C) and invasion (D) in another three breast cancer cell lines BT-549, ZR-75-30 and T47D. Statistical analysis was performed by t test. Values in B and C are means±SD of three independent experiments. *, P<0.01, significant difference between two compared groups. Immunoblotting Oct4 (1∶200), E-cadherin (1∶1000), α-SMA (1∶800), Vimentin (1∶5000), Fibronectin (1∶250), ZEB1 (1∶200), ZEB2 (1∶200), Snail (1∶500) and β-actin (1∶5000) antibodies were used for Western blot assay. Secondary horseradish peroxidase-conjugated goat anti- mouse or rabbit antibodies (Bio-Rad) were used at a 1∶5000 dilution and detected by the enhanced chemiluminescence reagent (Millipore, Billeria, MA USA). Luciferase Assay We cloned the sequence of human Oct4 3′UTR into pGL3-Control Luciferase reporter vector, then co-transfected pre-miR-145 or negative control sequence into MDA-MB-231 cells under the control of human pGL3-Control Luciferase reporter vector inserted with human Oct4 mRNA 3′UTR or without insert for luciferase assay. pRL renilla luciferase reporter vector was as internal control in each assay. At 48 h after transfection, cell lysates were collected and measured the luminometers both the firefly luciferase luminescence and the renilla luciferase luminescence in the same well of a 96-well format in the luminometer. Statistical Analysis Results were expressed as the means±SD. Statistical analysis of significance was calculated using one-way ANOVA. Results Expression of miR-145 Suppressed EMT Chemotaxis plays an essential role in metastasis [27]. In a search for microRNAs that negatively regulated chemotaxis and metastasis, eight pairs of breast cancer samples and matched adjacent normal tissues were screened by using miRNA gene chip V3.0 (from Capital Bio Corp.). As shown in Table 2, ten microRNAs showed significant decrease in cancer tissues, consistent with previous reports [28]. Among them, expression of miR-145 mimics blocked EGF-induced chemotaxis in MDA-MB-231 cells, a highly metastatic breast cancer cell (Fig. 1A). Wound-healing assay results further confirmed that miR-145 suppressed cancer cell migration (Fig. 1B). Furthermore, upon expression of miR145 mimics, migration and invasion, induced by NIH3T3 cell conditioned medium, were inhibited (Figure 1C). 10.1371/journal.pone.0045965.t002Table 2 miRNA differentially expressed between breast cancer tissues and adjacent normal tissues. miRNA Name Gene ID Fold change Location has-miR-199-5p 406976 0.436908 19p13.2 has-miR-497 574456 0.297475 17p13.1 has-miR-132 406921 0.408389 17p13.3 has-miR-145 406937 0.446519 5q32 has-miR-143 406935 0.590019 5q32 has-miR-10a 406902 0.755212 17q21.32 has-miR-10b 406903 0.545784 2q31.1 has-miR-7b 406884 0.702293 22q13.31 has-miR-7c 406885 0.626236 21q21.1 has-miR-12+ 406913 0.764364 9q34.3 Next, we tested the hypothesis that miR-145 inhibited cancer cell migration by blocking EMT. Over-expression of miR-145 mimics enhanced expression of E-cadherin and impaired expression of α-SMA and Fibronectin, suggesting that miR-145 suppressed EMT (Figure 1D). If miR-145 negatively regulated EMT, it would be expected to detect a decrease in miR-145 in highly malignant tumors. Indeed, analysis of 41 breast cancer tissues (14 samples at stage T1, 14 samples at stage T2, 13 samples at stage T3&4) revealed a loss of miR-145 expression in stage T3&4 cancer samples (Fig. 1E). Taken together, our results revealed that miR-145 inhibited cancer cell migration, probably due to its inhibition of EMT. A Transcription Factor, Oct4, was a Direct Target of miR-145 in Breast Cancer Cells Based on sequence analysis, we hypothesized that miR-145 inhibited Oct4 which in turn mediated EMT in breast cancer cells (Fig. 2A). Expression of miR-145 mimics inhibited Oct4 protein expression, but not mRNA levels, suggesting that miRNA-145 regulated Oct4 translation, not transcription level (Fig. 2B&2C). Treatment with a miR-145 inhibitor enhanced Oct4 protein levels (Fig. 2D). Upon expression of miR-145 mimics, luciferase activities were significantly impaired in cells transfected with a plasmid containing 3′UTR of Oct4 mRNA, indicating a direct interaction between miR145 and Oct4 mRNA in breast cancer cells (Fig. 2E). WNT/β-catenin regulates EMT [29]. In MDA-MB-231 cells, knockdown of Oct4 inhibited the expression of β-catenin while overexpressing Oct4 enhanced the expression of β-catenin (Figure 2F). Over-expression of Oct4 Rescued miR-145 Mediated Suppression of EMT In order to further investigate the role of Oct4 in miR-145- elicited EMT, an Oct4 over-expression vector was transfected into MDA-MB-231 cells treated with or without miR-145 mimics. Expression of miR-145 mimics inhibited Oct4 expression while over-expression of Oct4 reversed the inhibitory effects (Fig. 3A). Furthermore, over-expression of Oct4 reversed expression of E-cadherin, N-cadherin, α-SMA, and Fibronectin in cells treated with miR-145 mimics, suggesting that miR-145 suppressed EMT via Oct4 (Fig. 3B&3C). Consequently, over-expression of Oct4 also rescued the migration and invasion defects induced by miR-145 (Fig. 3D). Thus, our results suggest that Oct4 plays an important role in miR-145 regulated EMT of breast cancer cells. Expression of Snail, ZEB1 and ZEB2 was Regulated by miR-145/Oct4 Signaling Pathway Extensive studies have reported that a number of transcription factors, including Snail, Slug, Twist, ZEB1, and ZEB2, regulated EMT during tumorigenesis [30], [31]. Treatment with miR-145 mimics significantly decreased the expression of Snail, ZEB1, ZEB2, but not Slug or Twist, at both mRNA and protein levels in four breast cancer cell lines MDA-MB-231, BT-549, ZR-75-30 and T47D. Over-expression of Oct4 enhanced the mRNA and protein levels of Snail, ZEB1, and ZEB2 (Fig. 4, A, B, C, and D). Thus, our results suggest that Snail, ZEB1, and ZEB2, three regulators of EMT, are the downstream effectors of miR-145/Oct4 pathway. It has been reported that β-catenin regulates the expression of Snail, ZEB1 &2 [29], [32], [33]. Thus, we tested the hypothsis that over-expression of transcription factor Snail, ZEB1 and ZEB2 induced by Oct4 was mediated by β-catenin. Indeed, knockdown of β-catenin dampened Oct-4 induced expression of Snail, ZEB1 &2. Taken together, our results suggest that miR-145/Oct-4 regulate the expression of Snail, ZEB1 and ZEB2 through β-catenin in MDA-MB-231, BT-549, ZR-75-30 and T47D cells. MiR-145 Inhibited Cell Migration by Blocking Oct4-mediated EMT in Breast Cancer Cells Based on the above investigation of breast cancer tissues and MDA-MB-231 cells, a novel mechanism was proposed to explain the role of miR-145 in regulating cancer cell migration and metastasis (Fig. 5A). During the progress of tumorigenesis, a decrease in miR-145 expression promoted the expression of Oct4, which in turn stimulated the expression of Snail, ZEB1, and ZEB2, three key transcriptional factors, through β-catenin. Consequently, cancer cells switched from polarized epithelial-like cells to mobile mesenchymal-like cells, as indicated by the expression of EMT markers. To demonstrate that miR-145 played a general role, several additional breast cancer BT-549, ZR-75-30, and T47D cells were tested. Expression of miR-145 mimics inhibited Oct4 protein expression in these three cell lines (Fig. 5B). As shown in Fig 5 C and D over-expression of miR-145 inhibited migration and invasion of BT-549, ZR-75-30, and T47D cells, consistent with our hypothesis. Discussion Our study has indicated a critical role of miR-145 in EMT and revealed its molecular mechanism. Our results suggest that miR145 targets genes essential for EMT, a prerequisite step for migration and invasion during metastasis. MiR145 targets Oct4 in cancer cells, which in turn regulates the expression and function of β-catenin. Oct-4/β-catenin regulates the expression of Snail, ZEB1&2, three transcriptional factors in EMT. Consequently, tumor cells acquire properties of mesenchymal cells, and become invasive and migratory. The results from immunohistochemical analysis of 41 clinical samples showed that miR-145 expression correlated with aggravation of breast cancer, supporting a role of miR-145 in EMT. Apparently, miR-145 is just one of the regulators. Slug and Twist, another two important transcriptional factors in EMT, may be regulated by other mechanisms. Taken together, our results suggest miR-145/Oct4 plays a balanced regulatory role in EMT. The function of miR-145 appears to be cell type specific. Xu et al have demonstrated that miR-145 could repress pluripotency in human embryonic stem cell by direct targeting Oct4, Sox2 and Klf4 [34]. Takahashi et al demonstrated that co-transfection of Oct4, Sox2, Klf4, and c-Myc could reprogram human fibroblasts to generate induced pluripotent cells [35]. Li et al have shown that Oct4 and Sox2 could suppress the pro-EMT signals in mouse fibroblasts, thus promoting the MET occurrence [36]. Thus, in fibroblast cells, miR-145 appears to promote the EMT program. However, miR-145 clearly suppresses EMT and its inhibitory role in metastasis has been well-documented. So, we speculate that miR-145/Oct4 may play different roles in normal cells and tumor cells. The molecular mechanism behind such difference needs further investigation. In sum, we have identified miR-145 as one of the key blockers of EMT in cancer. It exerts its function by specifically inhibits the expression of Oct4/β-catenin. We identified Slug, ZEB1, and ZEB2 as the specific downstream effectors of Oct4 in cancer cells. Due to its multiple roles in tumor formation and metastasis, miR-145 may serve as an effective target for cancer diagnosis and therapy. We thank R.Xiang from the medical school of Nankai University, Tianjin city, China for the pcDNATM3.1/ZEO (+) plasmid donation. ==== Refs References 1 Gupta GP , Massague J (2006 ) Cancer metastasis: building a framework . Cell 127 : 679 –695 .17110329 2 Thiery JP , Acloque H , Huang RY , Nieto MA (2009 ) Epithelial-mesenchymal transitions in development and disease . Cell 139 : 871 –890 .19945376 3 Hanahan D , Weinberg RA (2011 ) Hallmarks of cancer: the next generation . 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PLoS One. 2012 Sep 26; 7(9):e45965
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23049889PONE-D-12-2055610.1371/journal.pone.0045903Research ArticleBiologyGeneticsPopulation GeneticsPopulation BiologyEpidemiologyInfectious Disease EpidemiologyPopulation GeneticsGenetic PolymorphismHaplotypesZoologyEntomologyMedicineEpidemiologyGenetic EpidemiologyInfectious DiseasesBacterial DiseasesBartonellosisBorrelia InfectionRickettsia ProwazekiiParasitic DiseasesPediculosisVectors and HostsLiceInfectious Disease ControlEvidence That Head and Body Lice on Homeless Persons Have the Same Genotype Human Head and Body Lice GenotypeVeracx Aurélie 1 Rivet Romain 1 McCoy Karen D. 2 Brouqui Philippe 1 Raoult Didier 1 * 1 URMITE UMR 6236, CNRS-IRD, Faculté de Médecine, Université Aix-Marseille, Marseille, France 2 MIVEGEC UMR 5290, CNRS-IRD-UM1-UM2, Centre IRD, Montpellier, France Li Wenjun Editor Duke University Medical Center, United States of America * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: PB DR. Performed the experiments: AV RR KDM. Analyzed the data: AV RR KDM PB DR. Wrote the paper: AV RR KDM PB DR. 2012 26 9 2012 7 9 e459033 7 2012 23 8 2012 © 2012 Veracx et al2012Veracx 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.Human head lice and body lice are morphologically and biologically similar but have distinct ecologies. They were shown to have almost the same basic genetic content (one gene is absent in head lice), but differentially express certain genes, presumably responsible for the vector competence. They are now believed to be ecotypes of the same species (Pediculus humanus) and based on mitochondrial studies, body lice have been included with head lice in one of three clades of human head lice (Clade A). Here, we tested whether head and body lice collected from the same host belong to the same population by examining highly polymorphic intergenic spacers. This study was performed on lice collected from five homeless persons living in the same shelter in which Clade A lice are prevalent. Lice were individually genotyped at four spacer loci. The genetic identity and diversity of lice from head and body populations were compared for each homeless person. Population genetic structure was tested between lice from the two body regions and between the lice from different host individuals. We found two pairs of head and body lice on the same homeless person with identical multi locus genotypes. No difference in genetic diversity was found between head and body louse populations and no evidence of significant structure between the louse populations was found, even after controlling for a possible effect of the host individual. More surprisingly, no structure was obvious between lice of different homeless persons. We believe that the head and body lice collected from our five subjects belong to the same population and are shared between people living in the same shelter. These findings confirm that head and body lice are two ecotypes of the same species and show the importance of implementing measures to prevent lice transmission between homeless people in shelters. The authors have no support or funding to report. ==== Body Introduction Human head lice (Pediculus humanus capitis) and body lice (Pediculus humanus humanus) are obligate parasites; head lice live on the scalp and lay their eggs at the base of hair shafts, and body lice live on the body surface and lay their eggs on clothing [1]. Head and body lice are considered to be sub-species and are generally thought to colonize their host in an independent manner [2]. However, in circumstances involving people heavily infested with lice, such as in homeless populations, head and body lice are often found on the same person. This finding raises the question of whether the lice can migrate between the different body areas. Although numerous studies have attempted to elucidate this issue, the species status of these two types of lice is still debated [3]. Body lice pose a serious public health problem as they are vectors of the pathogens Rickettsia prowazekii, Bartonella quintana and Borrelia recurrentis, which are responsible for epidemic typhus, trench fever and relapsing fever, respectively [4], [5]. A comparison of the humoral and cellular immune responses of head and body lice following bacterial challenge showed reduced cellular (phagocytic) activity in body lice which may explain the higher level of vector competence that has been found in this subspecies [6]. The epidemiological role of head lice in the transmission of human pathogens has not yet been demonstrated, but several studies have reported the presence of Bartonella quintana in head lice [7]–[10]. It is therefore important to better understand the dynamics of human lice populations to minimize their propagation and the transmission of their associated pathogens in at-risk populations. The first classifications of head and body lice were based on morphological characters. Some authors maintained that the morphological, behavioral and ecological differences between the two lice populations were not sufficient to recognize them as distinct species [1], [11]. Others, argued the reverse, that these differences required the recognition of these two groups as distinct taxonomic entities [12]–[15]. An analysis of primary endosymbionts indicated that these two types of lice are conspecific [16], but louse isoenzymes suggested that genetic differentiation may exist between the two forms [17]. After these phenotypic studies, numerous DNA-based molecular studies were performed, and again presented conflicting conclusions [3], [18], [19]. Currently, three deeply divergent clades (or phylotypes) of human lice with different geographic distributions are recognized: clades A, B and C. Phylotypes B and C contain only head lice, but phylotype A includes both head and body lice [20], [21]. Clade A lice have been further subdivided into subclusters of non-Sub-Saharan African lice (called A1) and Sub-Saharan African lice (called A2), as reported by two independent studies. The first study was based on the 18S rRNA gene sequence [22] and reported the divergence of head and body lice as being a recent event that occurred independently in each geographical group. The second study was based on the PM2 spacer [23] and could not show head and body louse divergence within each of the two clusters. Head and body lice were also shown to be genetically indistinguishable in a worldwide study based on four intergenic spacers [24] and in a very recent study based on the louse transcriptome [25]. Finally, based on a Bayesian coalescent model, ancestral migration events between head and body lice were shown to happen in both directions [26]. As multispacer typing was shown to be useful in addressing population-level questions [24], we used this genotyping method to determine if homeless people were infested by head and body lice of the same population. We examined the genetic population structure between lice from two body regions of five human subjects. However, one of the critical problems associated with this experimental design is that lice can migrate temporarily from one site to the other (with or without reproduction), making it difficult to determine their true origin (head or body). Consequently, to avoid any possible confusion regarding the origin of the tested lice, we collected eggs from the hair and clothing of homeless people from one shelter in Marseille, France. After hatching these eggs in the lab, we genotyped the first instar larvae and tested the genetic population structure of the lice from the two body areas. Methods Ethics statement In Marseille, there are an estimated 1500 homeless people, and 600 of them sleep in one of two available shelters (A and B) [27]. Because these individuals live in poor sanitary conditions, homeless persons are exposed to a number of health problems and belong to the social class with the most limited access to healthcare. To implement appropriate preventive and curative interventions, a snapshot investigation of the two shelters of Marseille has been performed each year since 2000 by a multidisciplinary team [28], [29]. The study protocol was reviewed and approved by the Institutional Review Board and Ethics Committee of Marseille No. 10.005 as it is in accordance with the French Bioethics law N° 2004-800 60 (06/08/2004). The study reported here was made based on samples collected in 2010. Homeless persons were informed of the purpose of the intervention and were asked if they would agree to participate by reading and signing an informed consent form . The document was divided into two parts: one for the patient with all information about the study and the other part including patient signature was kept by the investigators. The homeless persons were then interviewed and given a physical examination by a medical doctor. A nurse collected blood and other microbiological samples. One of the researchers (AV) was assigned to this team to meet the homeless and to collect head and body lice. When an individual had a body louse infestation, we provided clean clothes and kept the lice-infested undergarments and T-shirts in a sealed container to later harvest the eggs. In the cases of head louse infestations, the hair was cut and used to harvest the eggs. The infested homeless person was then invited to take a shower and was offered treatment with ivermectin [30]. The investigations consisted in a clinical exam that was offered to any homeless who presented even if he would not agree to participate to our study. Every homeless including participating and non-participating persons were offered the same services and a prescription was given if needed. Moreover, depending on the results of the samples analysis, the patient was taken in charge in the hospital if needed. All homeless in France are eligible for a social security cover (free healthcare for people on low incomes), this permit us to include all potential participants. Eggs incubation before hatching In the laboratory, the infested clothes were cut to separate the collar from the rest of the clothes. Eggs situated on ball caps (Figure 1A), collars (Figure 1B) or beards were not included in the analysis to avoid using lice located on the “borders” between head and body lice. The fabric and hair that contained eggs were put in labeled and separated boxes with holes, and grouped by the homeless person that they were isolated from. All of the eggswere incubated at 29 degrees Celsius with 70 to 80 percent relative humidity until hatching. Each day for 8 days, the newly hatched larvae were collected and stored at −20°C until further processing. Figure 2 shows a head louse (Figure 2A) and a body louse (Figure 2B) with their respective nits (empty egg shells) one day after hatching in the laboratory. 10.1371/journal.pone.0045903.g001Figure 1 Lice eggs attached to a homeless cap (A) and a homeless collar (B). 10.1371/journal.pone.0045903.g002Figure 2 Picture of a head louse (A) and a body louse (B) and their respective nits. Genotyping Total genomic DNA extraction, PCR and sequencing of the intergenic spacers S2, S5, PM1 and PM2 were performed as described previously [23]. As lice are diploid, cloning was necessary to identify the different allelic sequences; therefore, PCR products were cloned using a previously described protocol [23]. The resulting sequences were aligned with genotypes published in GenBank [23], [24] for identification. When less than 100% homology was obtained, the new genotype was recorded, a new number was assigned to it and it was published in GenBank (JX041640–JX041654). This was done according to a new set up of GenBank submissions providing the gene names that this is the intergenic spacer between: PHUM005704-PHUM006210 for intergenic spacer 2 , PHUM007351-PHUM002191 for intergenic spacer 5, PHUM007934-PHUM003340 for intergenic spacer PM1 and PHUM002215-PHUM002223 for intergenic spacer PM2 We also used high-throughput 454 sequencing of the amplicons using tagged libraries. Libraries were created by PCR using the same protocol as above and the same specific primers with the addition of the 454 adaptor and a Multiplex Identifier sequence (MID). The same 8 nucleotide barcode was used for all primer pairs (spacer S2, S5, and PM2). A total of 13 barcodes were designed using Barcrawl software [31]. We excluded barcodes with the same 5′ base as 3′ end of the upstream 454 adaptor, and we added a guanine to the 3′ end of the barcode to avoid the presence of the same 3′ barcode base at the 5′ end of the downstream primer. Barcodes that were converted to other barcodes by deletion were excluded. The numbers of 454 GS-FLX nucleotide flows to sequence the barcodes were as low as possible and were used between 5 and 9 flows (Supplementary Tables 1 and 2). The preparation of the 163 libraries was performed, as described in the Amplicon Library Preparation Method Manual from Roche. Additionally, 8 pools of 20 to 21 libraries were created to perform the clonal amplification, as described in the emPCR Method Manual from the Lib-A SV GS FLX Titanium Series from Roche. We worked with two Small Volume Emulsions of capture beads A and two Small Volume Emulsions of capture beads B per pool of libraries at a ratio of 1.8 copies of library per bead. The sequencing was performed in accordance with Roche using a GS FLX Titanium sequencing Kit XLR70 and the PicoTiter plate that was divided into 8 medium-sized regions. 10.1371/journal.pone.0045903.t001Table 1 Diversity estimates for each locus and population of lice from the bodies and heads of the sampled homeless persons. Locus Diversity Host S Host 33 Host F Host M Host D estimate Body Head Body Head Body Head Body Head Body Head (n = 17) (n = 2) (n = 6) (n = 9) (n = 5) (n = 4) (n = 5) (n = 3) (n = 5) (n = 9) S2 h 0.836 0.500 0.833 0.764 0.650 0.708 0.875 0.667 0.667 0.804 π 0.025 0.011 0.029 0.033 0.029 0.012 0.037 0.009 0.008 0.030 S5 h 0.669 0.000 0.667 0.743 0.375 0.833 0.675 1.000 0.667 0.518 π 0.024 0.000 0.024 0.026 0.006 0.024 0.024 0.033 0.028 0.009 PM2 h 0.836 1.000 1.000 0.722 0.700 1.000 1.000 0.833 0.875 0.736 π 0.011 0.010 0.009 0.005 0.006 0.007 0.012 0.007 0.004 0.005 Gene diversity (h) and nucleotide diversity (π) are based on Nei's (1987) estimates. n refers to the number of lice genotyped for each locus. 10.1371/journal.pone.0045903.t002Table 2 Summary of Hardy-Weinberg tests when louse populations are defined at the level of the body location of each homeless person (head or body), or when combined across body locations for each homeless person. Dataset Locus n Fis (SE) P-value Body location S2 10 0.2210 (0.1688) 0.1809 S5 9 0.5843 (0.1328) 0.0003 PM2 10 0.6782 (0.0862) <0.0001 Overall 29 0.4914 (0.0835) <0.0001 Homeless person S2 5 0.3306 (0.0609) 0.0032 S5 5 0.5891 (0.1047) <0.0001 PM2 5 0.6222 (0.1138) <0.0001 Overall 15 0.5140 (0.0620) <0.0001 P-values represent the combined value across populations (Fisher's procedure). n refers to the number of combined values. Fis (± standard error) refers the average unweighted value across populations and measures the deviation from panximia. For each region, barcodes were associated with only one DNA sample. We used mothur software [32] to trim the sequences and identify the barcodes using the following parameters: minlength = 100, bdiffs = 1, qwindowsize = 50, qwindowaverage = 25 (Supplementary Table 3). The trimmed sequences were mapped to the 3 reference genes using the program CLC Genomics Workbench. A probabilistic variant table was created for each mapped gene and every SNP (small nucleotide polymorphisms) and DIP (deletion and insertion polymorphisms) were verified and associated to extract the two alleles. 10.1371/journal.pone.0045903.t003Table 3 Analysis of molecular variance (AMOVA) of louse populations for each spacer locus. Locus Level df % variation Fixation index P-value S2 Among homeless persons 4 0.31 Fct = 0.0031 0.57283 Between body locations (homeless) 5 9.25 Fsc = 0.093 0.06647 Within body locations 114 90.45 Fst = 0.096 0.00782 S5 Among homeless persons 4 10.84 Fct = 0.11 0.14374 Between body locations (homeless) 5 11.30 Fsc = 0.13 0.19129 Within body locations 116 77.86 Fst = 0.22 0.00759 PM2 Among homeless persons 4 5.11 Fct = 0.051 0.12401 Between body locations (homeless) 5 4.48 Fsc = 0.047 0.58660 Within body locations 116 90.41 Fst = 0.096 0.08566 % variation indicates the amount of overall variation in the data explained at a given level of organization. The fixation indices refer to the amount of genetic structure attributed to each level. It should be noted that at the Within body locations level, the % variation refers the amount of variation found within populations, whereas Fst measures the structure among populations. % variation indicates the amount of overall variation in the data explained at a given level of organization. The 454 sequencing results were blasted against the results obtained from the PCR and cloning method. Differences in numbers of A or T in homopolymers were not taken into account. Population genetic structure The genotypic data were analyzed using tests based on both the allelic identity and the allele sequence. For the tests based on allelic identity, each unique sequence was assigned an allele number and the genetic distance among the sequences was considered equal. Using these data, we first tested to see if Hardy-Weinberg proportions (HW) were found within the populations. To determine the correct level of a population, we tested for HW by using two different combinations of the sampled lice. First, we broke the lice into the smallest possible biological unit by grouping all lice from a given body area on a given homeless person (body location data, n = 10 populations). Next, we considered all lice from the same homeless person as representing as single population, regardless of whether the lice were found on the head or body (homeless person data, n = 5 populations). If there was significant isolation between head and body lice, we expected to find higher deviations from HW in the latter case due to a Wahlund effect [33]. Deviations from the expected HW proportions for each population and locus were measured by Weir and Cockerham's estimator of Wright's Fis index and tested for significance using exact probability tests implemented in the software GENEPOP v4.1. Exact p-values were calculated using the Markov chain method, and tests across body locations, individuals and loci were combined using Fisher's procedure [34]. Gene diversity and nucleotide diversity was estimated for each locus and population using the body location dataset and the software F-STAT v 2.9.3 [35] and Arlequin v.3.5.1.3 [36], respectively. Differences in diversity among head and body lice were tested using paired t-tests for each locus. Tests across loci were combined using Fisher's procedure [34]. We used the sequence-based genotypic data to carry out an Analysis of Molecular Variance (AMOVA) that considers the allelic content of the genotypes and their frequencies to measure the population structure at different hierarchical levels of organization (i.e., within populations, among populations within groups, among groups) [37]. This analysis was carried out using the software Arlequin v.3.5.1.2 [36] and tests for the significance of the covariance components associated with each organizational level were performed using a non-parametric permutation procedure (20,000 permutations of the data where the type of permutation depends on the organizational level). This analysis also provided fixation index estimates for each level, ie, a measure of population structure [38]. Results Collections During our investigations, not all homeless people were willing to cooperate, either because the rooms of the shelters were cold and not comfortable enough to allow them to change clothing or because they preferred to have their meal and go directly to bed. Additionally, because of the regular head shaving of homeless diagnosed with head lice that had been previously offered, we had difficulty finding head lice on many of the individuals. The presence of head lice was most frequently noted on hair near the neck or above and behind the ears. For body lice, we noted that eggs and even motile forms were found much more often near seams, particularly in the armpit. During our investigations, we met 210 homeless people. Among them, 29 subjects had lice with 2 who had only head lice, 14 had only body lice and 13 had both. In addition to the head lice, we collected 163 body eggs (with 44 attached on the collar) and 727 mobile forms (larvae or adults). The head lice that we collected included 116 head eggs (with 10 attached to the beard) and 340 mobile forms. However, genotyping was only performed on the first instar larvae that were hatched from the eggs collected from homeless persons that had both head and body eggs. These criteria left us with 38 body lice larvae and 27 head lice larvae from 5 homeless people that were all sleeping at shelter A. Interestingly, we never found body lice eggs without larvae or adult body lice on the same body. In contrast, some homeless people had head lice eggs without larvae or adults found in hair (among our 5 studied homeless subjects, this is the case of homeless person S). Genotyping The PM1 spacer region was monomorphic (genotype 13) for all of the genotyped lice and was therefore not included in the analyses. Many of the collected lice were heterozygous as multiple sequences were overlaid in the chromatograms. For these individuals, cloning was needed to assess the genotypes. This was the case for almost all of the S2 sequences, many of the PM2 sequences and some of the S5 sequences. To ensure that all genotypes had been correctly assessed, the results obtained from the PCR and cloning method were compared with the results obtained from the high-throughput 454 sequencing of the same samples. In general, our results were congruent. However, it happened very often that the number of Ts or As found in homopolymers varied. Indeed, the polymerase can easily make mistakes at these positions, resulting in differences between the two sequencing methods and even between different clones or reads generated by the same sequencing method. Differences in homopolymer length were therefore not taken into consideration in the analyses. Moreover, the cloning method is long and fastidious when sequencing diploid organisms. The 454 sequencing method offers many advantages, including the production of hundreds of clones in one step. However, in some cases the reads obtained were not long enough to cover the studied region, so some adjustments to the protocol or to the chosen primers might prove useful. Overall, the sequencing run produced 285,002 reads with an average length of 484.5 nt and a median length of 507 nt. The total number of bases sequenced was 138,103,874, and the average quality score was 26.98. As shown in figure 3, we observed that the majority of genotypes, including the most common genotypes, were shared between head and body lice (in green). The most prevalent alleles in head and body lice were the same. For the PM2 spacer region, alleles 1, 38 and 33 were present in the majority of lice. In spacer S5, the more frequent alleles were 42 and 12. Finally, in spacer S2, the most frequent alleles were 48 and 68 (Figure 3). The raw data are provided in Supplementary Table 4. The concatenated genotypes of the S2, S5 and PM2 spacers that occurred at least twice in our sample are presented in Figure 4. We found two pairs of head and body lice on the same patient (homeless person 33) that had a unique multi-locus genotype, indicated with green arrows in Figure 4 (genotype 68, 42, 33 and genotype 68, 42, 48). This suggests that related individuals can be found on both regions of the body. 10.1371/journal.pone.0045903.g003Figure 3 Proportion of each allele among the head and body lice. The names (ID numbers) of the alleles are mentioned followed by the letter H for head lice and B for body lice. The alleles found in both the head and body lice are shown in green. The blue alleles were found only in the head lice, and the yellow alleles were found only in the body lice. 10.1371/journal.pone.0045903.g004Figure 4 Repartition of head and body lice haplotypes found at least twice in our samples. The haplotypes are concatenated alleles of the S2, S5 and PM2 spacers. A single color was assigned to each unique haplotype. Each block represents one louse, but may be separated into several colored units if the alleles at the three spacers could be combined to generate multiple haplotype possibilities. The green arrows indicate identical head and body lice haplotypes collected from the same homeless person. Genotypic data analyses Genetic diversity and nucleotide diversity were similar among the head and body lice populations sampled from the homeless individuals (Table 1; Fisher's combined test Chi2 h = 2.323, df = 6, P = 0.888; Chi2 π = 4.616, df = 6, P = 0.594). HW proportions were not found within lice populations. Indeed, the fixation index (Fis value) was positive (indicating a deficit in heterozygotes), and significant in almost all cases regardless of the spatial organization of the data (Table 2). Only the estimate for locus S2 was non-significant when populations were considered at the level of the body location, but this change is most likely caused by the high standard error of this estimate associated with the reduced population sizes of lice populations when divided into two groups for each homeless person. Indeed, restricting a louse population to only those lice found on the respective head or body zones of a given homeless person did not significantly reduce the overall heterozygote deficits present in the dataset (Table 2) and suggests that deficits at the homeless person level are not due to a Wahlund effect, i.e., an artifact of mixing different isolated populations. AMOVA analyses revealed no significant population structure between head and body lice after controlling for the sampled person at any of the three loci tested (Table 3). Indeed, the fixation index at this level of population organization was low (Fsc) and non-significant. Furthermore, no structure was evident among lice of different homeless persons (Fct). However, some population structure was evident among all populations (Table 3, Fst), but detailed pairwize comparisons could not reveal any interpretable pattern to this structure (data not shown). This structure may therefore be due different colonization histories and drift among some of the head and body populations. Discussion In people infested with both head and body lice, the original ecosystem of the lice can be dubious. Indeed, we found numerous eggs on collars, beards and ball caps that could have been from either the head or body (Figure 1). The method used to sample the two types of lice is therefore critical. For this reason, eggs collected from hair (head lice) and eggs collected from clothes (body lice) were kept separate and incubated until hatching, and molecular analyses were performed on the newly hatched larvae. All precautions to avoid DNA contamination were taken, and negative controls were used at each step of the study. Moreover, the use of two sequencing approaches strengthened our results because both techniques were concordant. Based on this data, and despite the elimination of individual lice in the potential overlap zones, our results strongly suggest a genetic mixing of lice from head and body populations. After genotyping lice, the genetic diversity and the nucleotide diversity was calculated for each spacer and used to compare head and body lice populations. This was not calculated for the spacer PM1 because it was monomorphic in the tested populations. The spacers S2, S5 and PM2 showed high heterogeneity in both the head and body lice (Table 1). However, these two groups of lice did not differ significantly in gene diversity or nucleotide diversity at the studied loci. This contrasts with a previous study that reported a higher nucleotide diversity in head lice compared to body lice [39]. This may be caused by a sampling bias as this previous study was based on 40 lice collected from across 12 different countries. We also found two pairs of head and body lice with identical genotypes collected from the same homeless person (homeless 33) (Figure 4) suggesting that related individuals move between body areas on the host. Strong heterozygote deficits were present in all populations, regardless of how a population was defined (all lice from a given homeless person, or only those lice from the respective head or body populations). This result is not surprising given that transmission ratio distortion, that is, a non-Mendelian inheritance pattern of alleles, is known to occur in P. humanus populations and may have caused the HW disequilibria found in this study [40]. However, regardless of the presence of this distorter, the deviation from HW equilibrium proportions at the level of homeless person was not reduced by dividing lice into the smallest possible population unit, that of the body zone within a host individual. Although it is possible to have a Wahlund effect due to mixing of different louse families within each body zone, no Wahlund effect seems to be related to mixing lice from head and body populations on single host. These results are further supported by those of the AMOVA analyses which showed little genetic variation attributable to between host and body lice populations and no significant structure between these populations. These results validate previous assumptions that the clade A lice may evolve and colonize both the hair and clothing niches [24], [25]. First, our results suggest that the lice collected from our five subjects belong to a single population and, thus, that lice are shared between people living in the same shelter. They further indicate that head and body lice likely move frequently from one part of the body to the other. These results support recent data comparing the transcriptional profiles of head and body lice [25]. Fourteen putative differentially transcribed genes were identified between head and body lice that could explain phenotypic differences [25]. The presence of two clades of lice living on some host individuals may help explain previous reports of independent head and body lice population [9], [19]. Indeed, a study on doubly infested persons in Ethiopia showed that all of the head lice were black and of clade C and all of the body lice were gray and of clade A [9]. Moreover, the only other study that reported independent head and body lice populations on individuals infested by the two forms was undertaken in Nepal where both Clade A and C lice are prevalent [19]. In the case of the clade A lice from our study, it seems that migration occurs between the two body zones and that it may increase in case of massive infestations. However, here we consider only lice that hatched from collected eggs. It remains to be shown whether these individuals could durably establish in the ecological niche where they were found. From our data, we can also not say whether migration is bidirectional between body zones (from both head to body and body to head) or whether one zone acts as a source for the other. However, a previous study showed that a single gene of an unknown function seems to be lost in all head lice [25]. This finding suggests that head lice may originate from body lice rather than the reverse. More complete phylogeographic studies are called for to test this hypothesis. Our failure to find population structure among homeless persons living in the same shelter may indicate that louse transmission frequently occurs in shelters. Prevention measures should therefore focus on avoiding the sharing of items such as mattresses, blankets and other personal belongings through which lice transmission is likely to occur from one homeless person to another. Supporting Information Table S1 Primer sequences. (XLS) Click here for additional data file. Table S2 Barcode sequences. (XLS) Click here for additional data file. Table S3 Recovery statistics after Mothur processing. (XLS) Click here for additional data file. Table S4 Results of the Multi-Spacer-Typing of homeless lice. (XLS) Click here for additional data file. ==== Refs References 1 Buxton PA (1946) The Louse. An Account of the Lice which Infest Man, their Medical Importance and Control. Edward Arnold & Co, London, England. 2 Durden LA (2002) Lice (Phthiraptera). In: Mullen G, Durden L, editors, Medical and Veterinary Entomology. San Diego, CA: Academic Press. pp. 45–65 3 Light JE , Toups MA , Reed DL (2008 ) What's in a name: the taxonomic status of human head and body lice . Mol Phylogenet Evol 47 . 4 Cutler SJ , Abdissa A , Trape JF (2009 ) New concepts for the old challenge of African relapsing fever borreliosis . 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Inf.Microbio.Frontiers in Cellular and Infection Microbiology2235-2988Frontiers Research Foundation 10.3389/fcimb.2012.00123MicrobiologyOpinion ArticleHuman Gut Microbiota: Dysbiosis and Manipulation Shen Dongqian 1*Liu Chuan 1Xu Ran 1Zhang Faxing 11Beijing Genomics Institute-ShenzhenShenzhen, ChinaEdited by: Lorenza Putignani, Children's Hospital and Research Institute Bambino Gesù, Italy Reviewed by: Lorenza Putignani, Children's Hospital and Research Institute Bambino Gesù, Italy *Correspondence: [email protected] 9 2012 2012 2 12330 8 2012 07 9 2012 Copyright © 2012 Shen, Liu, Xu and Zhang.2012This 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. ==== Body The adult human intestine is home to an almost inconceivable number of microorganisms. Their population is up to 100 trillion, nearly 10 times larger than the total number of our somatic and germ cells. All three domains, including bacteria, archaea, and eukarya, are contained in the adult human gastrointestinal (GI) tract and the bacteria achieve the highest cell densities and phylogenetic diversities (Whitman et al., 1998). Such gut microbiome can be viewed as a microbial organ placed within a host organ and the genomes of our affiliated microbial partners (the microbiome) may contain more than 100 times the number of genes in our genome. Once established, the indigenous microbiota provides many crucial functions to the host endows us with functional features that we have not had to evolve ourselves (MacDonald and Monteleone, 2005). These have been reviewed elsewhere (Ley et al., 2009; Neish, 2009) and include the contribution to digestion (such as the ability of microbes to break down host non-digestible polysaccharides) and its secondary benefits (the generation of SCFA), the metabolism of xenobiotics, the development of human immune system, and the colonization resistance. Generally, a healthy human state is a homeostasis between the microbiota and the host. Maladies such as Crohn's disease, chronic periodontitis, and bacterial vaginosis are characterized by a disruption of this homeostasis, a state known as dysbiosis (Tamboli et al., 2004). Meanwhile, the composition of the intestinal microbiota can undergo dynamic changes as a result of its interactions with diet, genotype/epigenetic composition, and immune-metabolic function (Kau et al., 2011). We envision a future in which new therapeutics and diagnostics enable the management of our microbiota to treat and prevent disease. Here, the relationship between gut microbiome and diseases and the effort in adjusting the gut microbiome will be discussed briefly. The Human Gut Microbiota and Diseases The human gut harbors diverse microbes that play a fundamental role in the health of their host. It differs from person to person depending on the unique species of bacteria accumulated over a lifetime. This means that every person's health is distinctly influenced by the specific byproducts created by their particular microbiota (Goodacre, 2007). Microbiome, considered as our “forgotten organ,” has been studied by some large-scale international projects such as HMP (Turnbaugh et al., 2007; Peterson et al., 2009) and MetaHIT (Qin et al., 2010). Based on an increasing number of studies on human microbiome, including the microbial community structure and function (Huttenhower et al., 2012), the researchers are shifting their concerns to the role of human microbiota in development of some acute or chronic diseases, especially GI Disorder. And the understanding that pathogenesis of some diseases is associated with result of complex interactions between microbiota and host was accepted more and more commonly. The most frequently reported disease that has been proved to associate with dramatic changes in the gut microbiota is Inflammatory Bowel Disease (IBD; Dicksved et al., 2008). MacFarlane et al. (2009) revealed significant reductions in Bifidobacterium populations in rectal biopsies from IBD patients. Zhang et al. (2007) have shown that bacterial diversity of Lactobacilli varied greatly between ulcerated tissue and non-ulcerated tissue in the same UC individuals. The number of mucosal adherent bacteria, such as invasive E. coli, Proteobacteria, Enterobacteriaceae are increased in IBD patients’ gut (Nagalingam and Lynch, 2012). Despite the involvement of microorganisms in inflammatory processes, antibiotic therapy was unsuccessful in IBD. However, recent studies demonstrated the use of probiotics, prebiotics, and synbiotics suggested the potential for controlling these diseases through manipulation of the composition of the gut microbiota, and direct interactions with the gut immune system (MacFarlane et al., 2009). Obesity and type 2 diabetes (T2D), the most prevalent metabolic diseases worldwide, are considered to be induced by impact of the microbiota on our metabolic health. Turnbaugh et al. (2009) observed that obesity is associated with phylum-level changes in the microbiota, reduced bacterial diversity, and altered representation of bacterial genes and metabolic pathways. The hypothesis is that the microbiota in obese individuals can harvest the more energy from food than the one in lean individuals. And another hypothesis is that gut microbiota can modulate plasma LPS levels which triggers chronic low-grade inflammation leading to obesity and diabetes (Cani et al., 2007). Another disease that related with obese tightly is non-alcoholic fatty liver. The intestinal microbiota may contribute to the development of non-alcoholic fatty liver disease through the complex and cooperative activities of two microbe-sensing protein families, namely nucleotide oligomerization domain receptors (NLRs) and Toll-like receptors (TLRs; Mukhopadhyay et al., 2011; Yeretssian, 2012), and through inflammasomes (Henao-Mejia et al., 2012) that shape metabolic pathway such as lipid accumulation. The gut microbiota has a considerable impact on the host's intestinal immunity and immune responses. Rheumatoid Arthritis (RA), a systemic autoimmune disease, is considered to be linked with gut microbiome. The antibodies to P. gingivalis have been reported to be more frequent in RA subjects compared with controls and that the titer of RA-related autoantibodies and C-reactive protein concentrations are also higher in individuals infected with P. gingivalis suggesting that this organism plays a role in disease risk and progression in RA (Mikuls et al., 2009). Furthermore, RA is closely related to periodontal disease. In a case-control study, serum antibodies against disease-producing periodontal bacteria were identified more frequently in subjects affected by RA and periodontitis than control subjects (Ogrendik et al., 2005; Moen et al., 2006). Commensal gut bacteria are essential to immune system development, and exposures disrupting the infant gut microbiota have been considered to be linked to asthma. The western diet has been found associated with increased risk of asthma for children (Nagel et al., 2010), and fast food consumption might counteract the protective effects of prolonged breastfeeding (Mai et al., 2009). Following birth, exclusive breastfeeding confers “beneficial” gut microbiota to infants, including increased colonization by Bifidobacteria and reduced prevalence and abundance of C. difficile compared to formula-fed infants (Penders et al., 2007; Fallani et al., 2010; Roger and McCartney, 2010). Infants who are not sufficiently exposed to Bifidobacteria in breast milk may have inappropriate immune responses to microbial exposures later in childhood, leading to atopic disorders including asthma. Beside of breast milk and other nutritional supplements, antibiotics affecting colonization of the intestinal bacteria by suppressing commensal bacteria, and causing the emergence of asthma-associated pathogens such as C. difficile are the next most commonly ingested substances by infants. The research shows that antibiotic use in the immediate period after birth can severely alter the composition and population of gut microbiota in infants (Fallani et al., 2010). Additional, the perinatal prevention from asthma via the intestinal microbiome is a relatively new perspective that has evolved long side modern technologies for the study of microbial communities (Azad and Kozyrskyj, 2012). The Pursue of Manipulate The Gut Microbiota The increasingly serious chronic health issues, ranging from obesity and diabetes to bowel disease and RA, are being demonstrated to be linked with perturbations in gut flora. Hence, it is feasible to treat these complex diseases through adjusting the gut microbiome. Modern medicine is struggling to seek methods of treating these multi-component diseases. The ancient medical philosophies and practices of Asia – particularly those of traditional Chinese medicine (TCM) – can offer an alternative approach. TCM's reliance on complex mixtures of compounds and its philosophy – complete system needs to be balanced – of treating the human body, match up well with the synergistic properties of the gut microbiome (Crow, 2011). In addition, most herbal medicines are orally administered, which will result in the unavoidable exposure of these medicines to the microorganisms in the gut. During this process, some of them are selectively metabolized into active or absorbable components by enzymes derived from intestinal microbiota. Then the therapeutic effects can be achieved. The Chinese microbiologist Zhao (2012) adopted a regimen involving Chinese yam and bitter melon – fermented prebiotic foods – that are believed to change the growth of bacteria in the digestive system. When he adopted the regimen by combing these prebiotics with diet composed of whole grains, he lost 20 kg in 2 years. Furthermore, his blood pressure, heart rate, and cholesterol level came down as well. The content of Faecalibacterium prausnitzii, a bacterium with anti-inflammatory properties, increased from an undetectable level to 14.5 percentage. of his total gut bacteria. The animal experiments showed that when rats were given a high fat diet (HFD) together with berberine, the major pharmacological component of the Chinese herb Coptis chinensis or Huanglian, they did not develop obesity or insulin resistance. What is more, the populations of known pathogens decreased while those of known beneficial bacteria increased in the gut. Other studies in mice also showed that the change from a low fat, plant polysaccharide diet to a western diet high in sugar, and fat would rapidly and profoundly reconfigure the composition of microbes in the gut. The gut microbiota in response to HFD feeding may allow the host to harvest more energy from food (Ley et al., 2005; Ley et al., 2006; Sanderson et al., 2006). Challenge and Prospect Though the development of human gut microbiome research burst in the last decade, we are still unenlightened in facing to the complex gut composition and its influence on human health. Several challenges remain to be overcome in a near future. Firstly, the list of the diseases that related to the gut microbiome is just growing and growing, and these diseases are usually complex in terms of both pathogenesis and complication, while sequencing and computational technologies would be a bottleneck in large-scale correlation analysis between the human microbiome and diseases. Secondly, despite a growing number of researches discovered the relationship between alterations in the gut microbiome and diseases, it remains to be established whether these are causes or effects. Further studies are required to distinguish disease-associated changes from a mass of interindividual variations that observed in the microbiome. Thirdly, time-series studies of individuals to monitor the status alter process from health to disease and back to the health is necessary for exploring the changeable human microbiome. High-resolution time-series studies provide a feasibility to discriminate between “normal” perturbations and pathologic states, and between organisms that are simply passing through a body habitat and are entrenched residents of an ecosystem (Eckburg et al., 2005; Palmer et al., 2007; Koenig et al., 2011). In some studies, (Huse et al., 2008; Dethlefsen and Relman, 2010) rapid decreases in alpha diversity and a characteristic shift in community composition were observed in association with antibiotic therapy, followed by a rapid post-antibiotic increase in diversity as the gut community returned to a state similar (but not identical) to the pre-treatment state. Furthermore, despite a large number of reports have showed the different gut microbiome between patient and healthy person, the definition of “the healthy gut microbiome” remains unclear. And the methodologies that can change the unhealthy gut microbiome to a healthy one are still in investigation. In terms of its application of human gut microbiome for human health development, we propose to monitor the microbiome when being healthy, and to establish a baseline indicating healthy, with more intensive monitoring when being sick and during treatment period. Such method demands the development of new diagnostic tools that are both accurate and sufficiently rapid to inform decisions regarding therapeutics. Such diagnostics are not yet feasible, but given recent advances in our ability to survey the human microbiome, this possibility is not far in the future, especially if we are able to identify particular components of the human microbiome that contribute disproportionately to the maintenance of human health. An adaptive management approach to clinical medicine provides a wonderful example of personalized medicine, with treatments tailored to individuals on the basis of diagnostic changes in an individual's microbiome, and continually adjusted through regular monitoring. Such an information-intensive approach, guided by ecological theory, has the potential to revolutionize the treatment of disease. ==== Refs References Azad M. B. Kozyrskyj A. L. (2012 ). Perinatal programming of asthma: the role of gut microbiota . Clin. Dev. 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Front Cell Infect Microbiol. 2012 Sep 27; 2:123
==== Front J Insect SciJ. Insect SciinscJournal of Insect Science1536-2442University of Wisconsin Library 2295809410.1673/031.011.17701ArticleMolecular Phylogeny and Identification of the Peach Fruit Fly, Bactrocera zonata, Established in Egypt Abd-El-Samie Emtithal M. 1aEl Fiky Zaki A. 2b*1 Entomology Department, Faculty of Science, Cairo University, Giza 12613, Egypt2 Genetics Department, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypta [email protected] [email protected]*Corresponding authorEditor: Craig Coates was Editor of this paper. 2011 31 12 2011 11 17710 6 2010 14 10 2011 © 20112011This 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.The genetic structure of the Egyptian peach fruit fly (Bactrocera zonata (Saunders) (Diptera: Tephritidae)) population was analyzed using total RNA from adult females. A portion of mitochondrial cytochrome oxidase I (COI), 369 bp was amplified using RT-PCR, and was sequenced and analyzed to clarify the phylogenetic relationship of B. zonata established in Egypt. The data suggested that the gene shared a similarity in sequence compared to Bactrocera COI gene found in GenBank. Molecular phylogenetic analyses were performed based on nucleotide sequences in order to examine the position of the Egyptian population among many other species of fruit flies. The results indicate that four accession numbers of B. zonata (three from New Zealand and one from India) are closely related, while the Egyptian B. zonata are close to the 71 accession numbers of Bactrocera include one B. zonata from New Zealand. These two B. zonata from Egypt and New Zealand showed a close relationship in neighbor—joining analysis using the seven accession numbers of B. zonata. In addition, a theoretical restriction map of the homology portion of the COI gene was constructed using 212 restriction enzymes obtained from the restriction enzyme database to identify the Egyptian and New Zealand B. zonata. Keywords mitochondrial cytochrome oxidase 1 gene (COI)restriction mapRT-PCR ==== Body Introduction Tephritid fruit flies in the genus Bactrocera (Diptera: Tephritidae) are distributed worldwide. The genus Bactrocera is a group of fruit flies containing more than 450 species (Drew and Hancock 2000; White 2000), and several Bactrocera species are serious pests of fruits and vegetables (Allwood et al. 1999). At least 28 Bactrocera subgenera have been denoted, and these are divided into four groups: Bactrocera, Melanodacus, Queenslandacus, and Zeugodacus (Drew 1989). The phylogenetic relationships among these Bactrocera species are poorly understood. Genetic markers and sequences from the mitochondrial genome in particular have proven informative in this respect (Shi et al. 2005; Xie et al. 2006). This is due to the availability of efficient PCR primers (Simon et al. 1994) and a wealth of comparative data (Jamnongluk et al. 2003b; Mun et al. 2003; Nardi et al. 2003; Reyes and Ochando 2004; Shi et al. 2005; Nardi et al. 2005; Boykin et al. 2006; Xie et al. 2006). Mitochondrial DNA (mtDNA) has been employed in phylogenetic relationships among tephritid fruit fly species, but the relationship among higher taxa could not be resolved (Han and McPheron 1997; Han 2000). Recently, by using 1.6 kb sequences of mtDNA, the more resolved phylogenetic relationship among higher taxa of the genus Bactrocera has been reported (Muraji and Nakahara 2001). The sequences of mtDNA contain the tRNAleu and flanking cytochrome oxidase I and II (COI and COII) of regions (1.3 Kb) provide some useful perspectives on Bactrocera species relationships (Nakahara and Muraji 2008). Cytochrome oxidase I (COI) sequences were shown to be appropriate for intraspecific analysis because of the observed high degree of polymorphism. Furthermore, COI sequences have been used in some studies to address similar problems on a comparable geographic range, and using the same marker might facilitate comparisons (B. depressa: Mun et al. 2003; B. dorsalis: Shi et al. 2005; Nakahara and Muraji 2010; Tetranychus urticae: Xie et al. 2006). Additionally, PCR-RFLP-based methods of Bactrocera species identification was considered based on nucleotide sequences of the mtDNA (Muraji and Nakahara 2002). COI sequences are at the base of the barcoding identification system (Hebert et al. 2003); a valuable tool for species identification and discovery that has been proposed as a powerful methodology in biosecurity and invasive species identification (Armstrong and Ball 2005). A case study on tephritid fruit flies (Armstrong and Ball 2005) reported high rates of success, but also mentioned some difficulties with the identification of few species (e.g., B. dorsalis, B. Cucurbitae, A. fraterculus), where the occurrence of cryptic species, inadequate sampling of all genetic subgroups, and high levels of geographic differentiation might complicate identification. The peach fruit fly, B. zonata, has been recognized as one of the most destructive flies attacking peach, apricot, guava, and figs (EPPO 2005). As this species is considered to be native to south and southeast Asia, it is thought to have been introduced to the Middle East, namely Saudi Arabia, Oman, and Egypt in recent years. Taher (1998) recorded this fly for the first time in Egypt, and it is now well— established, widespread, and well—adapted to local conditions (Hashem et al. 2001). Aedeagal length, body size, and number of pectin septa were used to distinguish between B. zonata found in Egypt with the sympatric species, B. dorsalis and B. correcta in Thailand (Iwahashi and Routhier 2001), and the study concluded that the aedeagal length can differentiate between these three species. A larger genetic distance was observed between populations of the peach fruit fly B. zonata collected from Thailand and Egypt than between many other pairs of distinctly different species (Nakahara and Muraji 2008). These populations were closely related with B. correcta in lineage clade (Muraji and Nakahara 2001; Nakahara and Muraji 2008), while B. correcta was close to B. dorsalis (Jamnongluk et al. 2003b). In this study, RT-PCR was performed to amplify a portion of the COI gene from B. zonata fruit flies established in Egypt. Comparative analysis of this sequence with Bactrocera COI genes found in the GenBank has been carried out to determine phylogenetic relationship. Moreover, a theoretical restriction map of COI fraction was performed to identify both the Egyptian and New Zealand B. zonata populations. Materials and Methods Fruit fly collection and handling The infested guava (Psidium guajava L.) fruits were collected from five locations (Abu Rawash, Badrashin, Ayyat, Imbaba, and El Saf) in Giza, Egypt during July 2008. Guavas were washed and placed in traps containing autoclaved sand. Fully—grown larvae of B. zonata that naturally jumped to the sand were allowed to pupate and rear to the adult stage in the laboratory at Cairo University, Giza, Egypt. Emerging adults were identified morphologically (E-B.z.) according to White and Hancock (1997). The identified female adults were rinsed in 70% ethanol, washed twice with double distilled water, dried using sterile tissue papers, and finally stored at -70 °C for RNA extraction. RNA isolation and RT-PCR analysis Total RNA was extracted from one female adult for each location using Gentra Purescript RNA Kit (www.qiagen.com). One µg of total RNA was reversely transcribed with RevertAid™ Minus Kit #K1631 (Thermo Fisher Scientific, www.thermoscientific.com) according to manufacturer instructions. PCR amplification was performed in 50 µL total volume with the following forward 5′ CATACGGATACAATGGTTAT 3′ and reverse 5′ TCGCGATCTGTCATATCCTG 3′ primers. PCR conditions were as follows: an initial denaturation step at 95 °C for four min, 40 cycles of 94 °C for 40 sec, 58 °C for 40 sec, and 72 °C for 40 sec, and a final extension step at 72 °C for 10 min, using Perkin Elmer Gene Amp 9600 (www.perkinelmer.com). PCR products were checked by electrophoresis using 1.5% agarose gel in 1× TAE buffer. The products were then purified using QIAQuick Gel Extraction Kit #28706 (QIAGEN, www.quiagen.com) following manufacturer instructions and sequenced by automated DNA sequencing reactions, which were performed using a sequencing ready reaction kit (Life Technologies, www.invitrogen.com) in conjunction with ABI-PRISM and ABI-PRISM big dye terminator cycler. DNA sequence and phylogenetic analyses A consensus sequence of COI fragments from one female of each location was constructed by using the SeqMan™ II (Windows 32 SeqMan 4.05) package (DNAStar, www.dnastar.com). The sequence obtained in this study was submitted to the GenBank nucleotide sequence databases (Accession number: GQ225768). This sequence was subjected to alignment with COI sequences of the GenBank, EMBL, DDBJ, and PDB sequence database using the program BioEdit version 7.0.0 (Hall 1999). The PAUP version 4.Ob10 package (Swofford 2005) was used to generate a phylogenetic tree using the neighbor—joining methods based on Saitou and Nei (1987). A total of 500 bootstrap replicates were used for analysis. Identification of B. zonata established in Egypt The restriction map of homology portion (57%) of COI of Egyptian B. zonata (accession number: GQ225768) was compared to three B. zonata (accession numbers: DQ116357, DQ116360, and DQ116361) (Armstrong and Ball 2005). The sequences were retrieved from NCBI as a GenBank file via their accession number by using NEBcutter program version 2.0 (Vincze et al. 2003). A restriction map was constructed using 212 restriction enzymes from a restriction enzyme database. Results Properties of DNA sequence After amplifying cDNA, a single fragment of approximately 390 bp nucleotide sequences of the COI gene from five B. zonata female adults was amplified. Sequencing results exhibited that the total nucleotide length obtained from each one contained 390 bases. Alignments of these five sequences revealed 100% similarity between them. The DNA sequence compositions are 99 (A), 70 (C), 79 (G), 118 (T), and 3 (N). The nucleotide frequencies were 0.2538 (A), 0.3025 (T), 0.1794(C), and 0.2025 (G). Phylogenetic analysis The topology of neighbor—joining tree and bootstrap support of the Egyptian B. zonata population (accession number: GQ225768) with 76 accession numbers of subgenus Bactrocera in the GenBank database represented a monophyletic group, bootstrap support < 50% (Figure 1). The three B. zonata fruit flies from New Zealand (accession numbers: DQ116357, DQ116360, DQ116361) and one from India (accession number: DQ838980) were clustered with each other showing bootstrap support < 50%, while B. zonata from New Zealand (accession number: DQ116359) was clustered with 72 fruit fly accession numbers of Bactrocera and showed bootstrap support 99%. The 72 accession numbers represented a monophyletic group with a 100% bootstrap support. Within this group, nine B. umbrosa fruit flies were closely related and formed a monophyletic lineage (100% bootstrap support). The Egyptian B. zonata population was found to cluster with 71 accession numbers of Bactrocera including B. zonata from New Zealand (accession number: DQ116358) (bootstrap support 100%); this accession number was found in a clade that consisted of B. dorsalis and B. papayae (bootstrap support < 50%). The seven accession numbers of the peach fruit fly B. zonata (Figure 1) were used to construct a phylogenetic tree of B. zonata (Figure 2). This tree represented a monophyletic group (bootstrap support < 50%) and the Egyptian B. zonata (accession number: GQ225768) showed a close relationship to B. zonata (accession number: DQ116358) from New Zealand (100% bootstrap support), while B. zonata (accession number: DQ116359) from New Zealand was closely related to the two previous accession numbers (90% bootstrap support). Two B. zonata fruit flies from New Zealand and India (accession numbers: DQ116357 and DQ838980, respectively) were closely related with each other (bootstrap support <50%) and to the three previous accession numbers (70% bootstrap support). The two B. zonata fruit flies from New Zealand (accession numbers: DQ116360 and DQ116361) were clustered with each other (bootstrap support < 50%). Identification of Egyptian B. zonata A theoretical restriction map patterns of homology portion of COI using NEBcutter software program showed recognition sites of 22, 15, and 14 restriction enzymes in B. zonata from Egypt and New Zealand accesion numbers GQ225768, DQ116357, and DQ116360/ DQ116361, respectively (Figure 3). The map showed the presence of 32 cut sites in GQ225768 and 23 cut sites in DQ116357, DQ116360, or DQ116361. The DQ116357 differed in restriction enzymes SetI and Sth132I cut sites, whereas the DQ116360 and DQ116361 had the same restriction enzyme map. The restriction enzymes CstMI, HpyAV, Tsp509I, and TspDTI had the same restriction cut sites in the four accesion numbers, and the Egyptian B. zonata (GQ225768) differed in all other enzymes. Discussion The adaptation to the environmental conditions produced by the host plants might play a role in speciation of tephritid fruit flies in the genus Bactrocera (Jamnongluk et al. 2003b). Total RNA of one B. zonata female for each location has been used to amplify a fragment of COI gene (390 bp). Alignment of these five sequences revealed 100% similarity between them. This similarity may be due to the fact that the five locations, which represent five districts at Giza governorate, have the same environmental conditions where the infested fruits were collected from the same host plant. Molecular analysis of the consensus sequence showed that the A+T content in Egyptian B. zonata population was 59%. These data are in agreement with the molecular analysis of Jamnongluk et al. (2003a) who reported that the A+T content of the 639 bp downstream segment of COI in species of the genus Bactrocera was slightly lower (63–68%) than those reported in other insects over the same segment; for example, 71% in L. migratoria (Flook et al. 1995), 69% in An. gambiae (Beard et al. 1993), and 70% in C. capitata (Spanos et al. 2000). The results clearly indicate that the four accession numbers of B. zonata (three from New Zealand and one from India) were closely related, while the Egyptian B. zonata (accession number: GQ225768) was close to the 71 accession numbers of Bactrocera, including other one B. zonata from New Zealand (accession number: DQ116358). The latter was found in a clade consisting of B. dorsalis and B. papaya, and the different Bactrocera species did not form a monophyletic lineage. This is in agreement with data obtained by Jamnongluk et al. (2003b), who reported that B. correcta was close to B. dorsalis when using COI. Muraji and Nakahara (2001, 2002) also reported the disagreement between morphological classification and molecular phylogeny. In reality, many fruit fly species such as B. dorsalis and B. carambolae are very capable invaders; however, it is difficult to distinguish between them since they have overlapping host and geographic ranges with B. verbascifoliae, which is not a recognized pest. Some morphologically indistinct regulated species such as B. philippinensis and B. papayae have different host and geographic ranges. This is important information for assessing the specific risk and pathway involved. For example, with the fruit flies, COI could not confidently discriminate some of the species within the B. dorsalis complex, for which an additional gene region may be appropriate (Armstrong and Ball 2005). Phylogenetic analysis of COI sequences suggests that tephritid fruit fly species that attack cucurbit plants (Asiadacus, Hemigymnodacus, and Zeugodacus) were more closely related to each other than to fruit fly species of the subgenus Bactrocera, which attack plants of numerous families (Jamnongluk et al. 2003b). They also suggested that adaptation to the environmental conditions produced by the host plants might play a role in the speciation of tephritid fruit flies in the genus Bactrocera. Moreover, The Queensland fruit fly B. tryoni and a sibling species B. neohumeralis are sympatric and produce viable and fertile hybrids (Pike et al. 2003). These two species could not be clearly discriminated in both neighbor—joining and maximum parsimony analyses (Nakahara and Muraji 2008). When comparing these results with other studies addressing similar problems on phylogenetic relationships, it is possible to observe different levels and patterns of genetic differentiation. It is worth mentioning that the oriental fruit fly B. dorsalis showed higher variability in the COI sequences (5.94% of variable sites, compared to 1.15% in the melon fly) with almost no sharing of haplotypes among populations and only weak signs of differentiation in the westernmost samples (Shi et al. 2005). On the other hand, the pumpkin fly B. depressa shows equally high levels of genetic differentiation (4.14%) of variable sites, but with strong differentiation between Japanese and Korean populations (Mun et al. 2003). Host plant differences and geographic isolation could have played an important role in species differentiation within seven closely related species of B. tau complex (Baimai et al. 2000) and 52 sibling species of B. dorsalis species complex (Drew and Hancock 1994). The phylogenetic analysis of the 77 accession numbers indicated that some species were placed within other species of Bactrocera, having weak bootstrap support even though the adults were morphologically distinct. Consequently, phylogenetic analysis of seven accession numbers of the peach fruit fly B. zonata from Figure 1 was used to indicate the relationship among these populations. This analysis showed a close relationship between B. zonata from Egypt and B. zonata (accession number: DQ116358) from New Zealand (100% bootstrap support). Moreover, the peach fruit fly B. zonata collected from Thailand and Egypt were closely related to B. correcta in the lineage clade by using rDNA (Muraji and Nakahara 2001; Nakahara and Muraji 2008). Accuracy of identification is also dependent on reliability of the simple sequence similarity approach. In this case, a portion of the COI of Egyptian B. zonata poplation was selected, which is similar in sequence with the three B. zonata populations from New Zealand, to construct the theoretical restriction map. This map, produced by 28 restriction enzymes, was used to identify the peach fruit fly B. zonata from Egypt and New Zealand. As a result, recognition sites of several restriction enzymes have been found, which could be used in PCR-RFLP (i.e., restriction enzyme SetI). The PCR-RFLP analysis was used to identify B. zonata fruit flies (i.e., AseI was expected to differ among 16 of 18 species). The remaining two species, B. dorsalis and B. philippinensis, were expected to be discriminated by analyses using DpnI and MseI (Muraji and Nakahara 2002). Also, the restriction enzymes DraI and SspI have been used to recognize 44 haplotypes of B. dorsalis complex (Nakahara and Muraji 2010). In addition, this information could promote the development of a realistic system of B. zonata diagnostics based on PCR-RFLP analysis (useful for practical purposes such as field research) and quarantine inspection. Conclusion The sequence analysis of the isolated COI gene showed 100% similarity between the five sequences of B. zonata collected from five locations having the same environmental conditions and the same host plant. The properly rooted tree might indicate that most B. zonata samples form a single lineage of uncertain relationship (polytomy) with the Egyptian B. zonata and most other Bactrocera lineages. To resolve the disagreement between morphological classification and molecular phylogeny of fruit fly species in the future, we suggest that combined sequences from more than one gene (i.e., COI, non-transcribed region between COI and tRNAleu, cytochrome B, 16S rDNA, ITS1, and ITS2) could be used to identify the same species collected from the same host. Figure 1. Neighbor—joining dendrogram of 77 fruit flies Bactrocera generated based on Saitou and Nei distances. Bootstrap confidence limits are shown adjacent the branches of clades supported in more than 50% of 500 replications. High quality figures are available online. Figure 2. Neighbor—joining dendrogram of seven peach fruit flies Bactrocera zonata generated based on Saitou and Nei distances. Bootstrap confidence limits are shown adjacent the branches of clades supported in more than 50% of 500 replications. High quality figures are available online. Figure 3. Homology portion theoretical restriction map of four Bactrocera zonata COI showing the recognition sites of 28 restriction enzymes. (A): DQ116360 (63–229bp) and DQ116361 (63–229bp), (B): DQ116357 (63–229bp) and (C): GQ 225768 (156–366bp). High quality figures are available online. Abbreviations: PCRpolymerase chain reaction; RTreverse transcription; RFLPrestriction fragment length polymorphism UA ==== Refs References Allwood AJ Chinajariyawong A Kritsaneepaiboon S Drew RAI Hamacek EL Hancock DL Hengsawad C Jipanin JC Jirasurat M Kong Krong C Leong CTS Vijaysegaran S. 1999 Host plan records for fruit flies (Diptera: Tephritidae) in Southeast Asia. Raffles Bulletin Zoology Supplement 7 1 92 Armstrong KF Ball SL. 2005 DNA barcodes for biosecurity: invasive species identification. Philosophical Transactions of the Royal Society of London B—Biological Sciences 360 1813 1823 Baimai V Phinchongsakuldit J Sumrandee C Tigvattananont S. 2000 Cytological evidence for a complex of species within the taxon Bactrocera tau (Diptera: Tephritidae) in Thailand. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23056300PONE-D-12-2082810.1371/journal.pone.0046410Research ArticleMedicineOncologyCancer Detection and DiagnosisEarly DetectionCancer Risk FactorsHormonal Causes of CancerPredisposing Conditions and SyndromesCancer TreatmentEndocrine TherapyCancers and NeoplasmsBreast TumorsDuctal Carcinoma in SituInvasive Ductal CarcinomaInvasive Lobular CarcinomaBasic Cancer ResearchGREB1 Functions as a Growth Promoter and Is Modulated by IL6/STAT3 in Breast Cancer GREB1 Functions as a Growth PromoterLiu Mingli 1 3 * Wang Guangdi 2 * Gomez-Fernandez Carmen R. 3 Guo Shanchun 1 * 1 Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, Georgia, United States of America 2 Department of Chemistry, Xavier University of Louisiana, New Orleans, Louisiana, United States of America 3 Department of Pathology, Department of Medicine, University of Miami School of Medicine, Miami, Florida, United States of America Deb Sumitra Editor Virginia Commonwealth University, United States of America * E-mail: [email protected] (ML); [email protected] (GW); [email protected] (SG)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ML SG. Performed the experiments: ML CRG. Analyzed the data: ML GW SG. Contributed reagents/materials/analysis tools: ML GW SG. Wrote the paper: ML GW SG. 2012 3 10 2012 24 7 2014 7 10 e4641010 7 2012 29 8 2012 © 2012 Liu et al2012Liu 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 Growth Regulation by Estrogen in Breast cancer (GREB1) was an estrogen receptor (ER) target gene, and GREB1 expression inversely correlated with HER2 status, possibly as a surrogate marker for ER status and a predictor for tamoxifen resistance in breast cancer patients. In the present study, we examine the function and regulation of GREB1 in breast cancer, with the goal to develop GREB1 as a biomarker in breast cancer with de novo and acquired tamoxifen resistance. Methods We overexpressed GREB1 using adenovirus containing the full length GREB1 cDNA (Ad-GREB1) in breast cancer cell lines. The soft agar assay was used as a measure of anchorage independent growth. The effects of GREB1 on cell proliferation in MCF-7 cells transduced with Ad-GREB1 were also measured by the me olic activity using AlamarBlue assay. We tested whether there was interaction between STAT3 and ER, which could repress GREB1 expression by immunoprecipitation assay. The effects of IL-6/JAK/STAT3 cascade activation on estrogen-induced GREB1 promoter activity were determined by luciferase assay and those on gene expression were measured by real time reverse transcription polymerase chain reaction (qRT-PCR). Results We found that the ability of breast cancer cells to grow in soft agar is enhanced following GREB1 transfection. In MCF-7 cells transduced with Ad-GREB1 or transfected with siRNA GREB1, the metabolic activity was increased or completely abolished, suggesting that GREB1 may function as a growth promoter in breast cancer. E2 treatment increased GREB1 promoter luciferase activity. IL-6 inhibited E2-induced GREB1 transcription activity and GREB1 mRNA expression. Constitutively expressing active STAT3 construct (STAT3-C) dramatically decreased GREB1 transcription. Conclusions These data indicate that overexpression of GREB1 promotes cell proliferation and increases the clonogenic ability in breast cancer cells. Moreover, Il6/STAT3 modulates estrogen-induced GREB1 transcriptional activity in breast cancer cells. This work was partly supported by a grant from the Woman’s Cancer Association of the University of Miami awarded to ML and RCMI grant 5G12RR026260 to GW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Current endocrine therapies for breast cancer patients target the estrogen receptor (ER) by reducing its ligand-induced activation, blocking its function and ultimately inducing ER degradation. Although these therapies are effective in many patients with ER-positive tumors, long-term follow up and clinical trials have demonstrated that up to 62% of breast cancers that are initially responsive to endocrine therapy eventually relapse, with the patients then requiring salvage surgery [1], [2], [3]. Therefore, identification of molecular markers that can predict responses to anti-estrogen therapy in ER positive breast cancer is critically needed. Tamoxifen treatment is the most frequently utilized therapy for patients with estrogen receptor α (ERα) positive breast cancer. Although many patients benefit from tamoxifen, one-third of ERα positive (ER+) breast cancers exhibit primary resistance to tamoxifen treatment (intrinsic, or de novo resistance) [4]. The remaining 70% of ER-positive breast cancers initially respond to the tamoxifen but have a great tendency to relapse and subsequently fail to respond to tamoxifen (acquired resistance) [5], [6]. Tamoxifen competes with estrogen for ER binding sites and functions as an antagonist of ER [6]. Binding of tamoxifen to ERα results in conformational changes of the receptor, thereby impairing the ability of ERα to recruit coactivators or corepressors to the tamoxifen-ER complex [6]. The interaction between tamoxifen and ER not only determines the antagonist effects of tamoxifen on the tissues, but also indicates possible mechanisms by which resistance might develop in breast cancer. A better understanding of the biological and molecular mechanisms underlying intrinsic and acquired tamoxifen resistance could provide novel strategies to circumvent resistance to tamoxifen, and aid in the optimal design of order and duration of endocrine reagents for further improvements in disease outcomes. Numerous predictive and prognostic factors for endocrine response have been evaluated in breast cancer. Established biomarkers such as ER and progesterone receptor (PgR) are important positive predictive markers for response to endocrine therapy in patients with breast cancer [7]. Total loss of PgR predicts relative resistance to the anti-estrogen tamoxifen, but may not indicate resistance to aromatase inhibitors [8]. New adjuvant setting studies also indicate that high levels of epidermal growth factor receptor 2 (HER2) is associated with tamoxifen resistance, but not resistance to aromatase inhibitors [8]. Ki67, a typical although modest prognostic factor, has recently been recognized as a more effective predictor of treatment efficacy for both endocrine and chemotherapy [9]. An estrogen targeted gene Zinc transporter LIV-1 (SLC39A6) was recently shown to be associated with Ki67 conferring resistance to tamoxifen and fulvestrant [9]. New clinical studies indicate NF-κB p50 activation as a potential prognostic marker capable of identifying a high-risk subset of primary ER-positive breast cancer destined for early relapse in spite of adjuvant tamoxifen therapy. The sensitivity to tamoxifen can be restored by interrupting activation of NF-κB [10], [11]. ErbB3 has also played an important role in the development of resistance to antiestrogens such as tamoxifen [12]. Since the inception and broad application of DNA microarray technology, numerous multigene expression profiling assays have been developed with the aim of identifying new prognostic biomarkers predicting response to endocrine therapy. Among them, the Oncotype DX assay and the MammaPrint profile are currently undergoing clinical evaluation to determine their efficacy [7]. Gene expression signatures suggest that a “proliferation cluster” including Ki-67, proliferating cell nuclear antigen (PCNA), some proliferation-related genes and cell cycle genes may be the strongest predictor for metastasis and relapse in tamoxifen treated ER-positive breast cancer patients, emphasizing the important roles of proliferation genes in prognosis [13], [14], [15]. Although biomarkers as described above appear to be of certain biological importance, only few such as ER, PgR and HER2 have proven clinically applicable. We have reported that GREB1 correlates with ERα expression in breast cancer cell lines and breast cancer tissues [16]. However, in several anti-estrogen resistant cell lines including BT-474, T47D and SUM44, which are ER-positive, GREB1 expression is either reduced or absent. It has been well documented that there is interplay between HER2 activity and loss of ER transcription [17], and anti-estrogen resistant tumors are characterized by elevated HER2 levels [18], [19], [20]. Consistent with these findings, we have reported that GREB1 expression inversely correlated with HER2 expression in ER-positive breast cancer patients. In other words, ER-positive, GREB1-negative patients have a significantly greater tendency to positively express HER2 protein compared to ER-positive, GREB1-positive patients [21]. Patients with GREB1 positive expression exhibit significant tamoxifen sensitivity and prolonged survival compared to the patients with GREB1 negative expression [22]. In addition, we also showed previously that not only GREB1 but also other ER-regulated genes such as IGFBP4, IRS-1 and BCL-2 mRNA expressions were increased as HER2 signaling was blocked, suggesting that HER2 may regulate GREB1 through ER pathways [21]. However very little is known about the function of GREB1 and the mechanism by which it is regulated by ER. Earlier reports indicated that signal transducer and activator of transcription (STAT3) acts downstream of HER2 [23], [24]. STAT3 is tyrosine phosphorylated through the interleukin-6 (IL-6)/glycoprotein 130/Janus kinase pathway in breast cancer [25]. STATs and ER can physically interact in vivo [26], [27]. In the present study, we investigated the function of GREB1 gene on cell proliferation and trasnfrormation and whether the HER2 downstream signaling molecule STAT3 regulates ER transcription resulting in negative or decreased expression of GREB1 in breast cancer cells. We believe that our study will provide a basis for development of GREB1 as a novel biomarker in combination with ER to better identify breast cancer patients who will benefit from tamoxifen therapy and those who will likely develop resistance to endocrine therapy. Results GREB1 is Induced by E2 in ER-positive Breast Cancer Cell Line GREB1 was detected in MCF-7 cells treated with E2 for 24 and 48 hrs, while no protein is detected in the ER-negative BT-549 cells or in MCF-7 cells grown in estrogen-free conditions in Western blotting assay (Figure 1A). Treatment with ICI 182,780 (ICI), an estrogen receptor antagonist, and silencing the GREB1 gene by siRNA led to the loss of GREB1 protein expression. As shown in Figure 1B, GREB1 protein expression is reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared to that observed in cells treated with estrogen alone. Figure 1C shows the loss of detectable GREB1 protein when GREB1 is knocked down by GREB1 siRNA (SiGREB1) at 48 hours compared to control (CSiRNA), CSiRNA has no effect on E2-induced GREB1 production. Corresponding densitometric analysis of the bands performed with the ImageQuant program (Bio-Rad) areshown below the Western blot. E2-induced GREB1mRNA levels were similarly analyzed as above, with GREB1 mRNA levels correlating well with GREB1 protein expressions. Upon E2 stimulation, GREB1 mRNA is notable as early as 24 hours and lasts up to 48 hours as presented in a time course study in Figure 1D. BT-549 cells express GREB1 mRNA as low as the control. ICI treatment (Figure 1E) and silencing the GREB1 gene (Figure 1F) both significantly reduced the GREB1 expression at the transcriptional level. 10.1371/journal.pone.0046410.g001Figure 1 GREB1 is induced by E2 in ER-positive breast cancer cell lines. A, Western blotting detects a single band of ∼216 kD in E2-deprived MCF-7 cells treated with estrogen for 24 and 48 hrs while no protein is detected in the ER-negative BT-549 cells or in MCF-7 cells grown in estrogen-free conditions. B, GREB1 protein expression was reduced in the MCF-7 cells treated with estrogen plus ICI 182,780 (ICI) compared to that observed in cells treated with estrogen alone. C, Figure C shows loss of detectable GREB1 protein when GREB1 is knocked down by GREB1 siRNA (SiGREB1) at 48 hours. Control siRNA (CSiRNA) has no effect on E2-induced GREB1 production. Corresponding densitometric analysis of the bands performed with the ImageQuant program (Bio-Rad) were shown below the Western blot. E2-induced GREB1mRNA levels were also analyzed, GREB1 mRNA levels were well correlated with GREB1 protein expressions. D, GREB1 mRNA is notable as early as 24 hours and lasts up to 48 hours as presented in a time course study. BT-549 cells express GREB1 mRNA as low as the control. E and F, ICI treatment (Figure 1E) and silencing the GREB1 gene (Figure 1F) significantly reduced the GREB1 expression at transcriptional level. Data are shown as mean ± SD. *P<0.05; **P<0.01. GREB1 mRNA Correlates with ER Status in Breast Cancer Patients, and Predicts Patient Survival and Responses to Tamoxifen Treatment GREB1 is the most sensitive ER-regulated gene in response to E2 stimulation in breast cancer patients [28]. ERα is the prototypic phenotypic marker used in prognosis of breast cancer and it directly controls GREB1 expression [29], [30]. Furthermore, GREB1 is tightly correlated with ERα expression in breast cancer cell lines and it is required for breast cancer cell growth [28], [31]. However, GREB1 as a cancer biomarker and the clinical significance of GREB1 protein expression in human breast cancer is underexplored. GREB1 function and regulation need to be fully investigated. To this end, we analyzed GREB1 expression in publicly available breast cancer microarray studies using the Oncomine database and gene microarray data analysis tool [32], [33]. Meta-analysis of microarray gene expression data sets related to human cancer genes revealed that GREB1 mRNA is highly expressed in breast carcinomas compared to normal breast tissues (T-test: 4.815; P-value: 2.1E-5) (Figure 2A) [34]. Using the same Oncomine research platform, microarray data obtained from 2321 patients of human breast cancer patients (1651 ER-positive, 670 ER-negative) through 15 studies were also evaluated for the relationship between GREB1 expressions and other clinical parameters. Meta-analysis from published database demonstrated that GREB1 expression is significantly increased in ER-positive cancer patients compared to ER-negative cancer patients (Figure 2B) [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. We further analyzed the effects of GREB1 mRNA on patients’ survival using the data from the North Central Cancer Treatment Group Trial, Tamoxifen (TAM) arm of 89-30-52 which recruited 225 patients (Phase III trial of adjuvant therapy with Tamoxifen alone or combined with Fluoxymesterone in postmenopausal women with resected estrogen receptor positive breast cancer) [48], [49], [50]. Although PgR expression alone has historical precedence as a clinical prognosticator of breast cancer patient in response to hormone therapy [8], [51], [52], the analysis showed that GREB1 mRNA predicts disease-free survival (DFS) in Tamoxifen-treated patients better than PgR or ER (Table 1). This assessment of clinical relevance was further corroborated in a patient survival analysis using an online database containing the expression of 22,277 genes and 20-year survival information of 1809 patients [53]. The database has recently been updated to include survival information of 2898 breast cancer patients (http://www.kmplot.com/analysis/). GREB1 downregulation was found to correlate strongly with poor relapse free survival (RFS) for all breast cancer patients followed for 20 years (Fig. 2C, hazardous ratio 0.66, p = 1.1×10−10). For patients with ER+ breast tumor, lower expression of GREB1 was also seen as significantly associated with decreased survival (Fig. 2D, hazardous ratio 0.75, p = 0.0029). As shown in Fig. 2E, for ER+ patients who have received endocrine therapy, reduced GREB1 expression predicts worse outcome in RFS (hazardous ratio 0.63, p = 0.041). 10.1371/journal.pone.0046410.t001Table 1 225 patients from the TAM arm of 89-30-52. Log-rank Test Results, when factor dichotomized at its median value Median (range) OS (n = 87 events) DFS (n = 97 events) TTBR (n = 43 events) GREB1 −3.00 (−9.99 to −0.14) P = 0.038 P = 0.023 P = 0.038 ER 0 (−8.59 to 2.31) P = 0.365 P = 0.434 P = 0.646 PR −4.21 (−10.85 to 0.81) P = 0.108 P = 0.140 P = 0.056 OS- death due to any cause. Disease-free survival (DFS). Disease-free survival was defined as the time from randomization to the documentation of the first adverse event where an adverse event is defined as local, region, or distant disease progression, the development of contralateral breast disease, or death from any cause without documentation of another adverse event. Time to breast cancer recurrence (TTBR). Time to breast cancer recurrence was defined as the time from randomization to the documentation of the first adverse breast event where an adverse breast event is defined as local, region, or distant disease progression. 10.1371/journal.pone.0046410.g002Figure 2 GREB1 mRNA correlates with ER status in breast cancer patients, and predicts responses in Tamoxifen-treated patients. Meta-analysis was performed using the Oncomine Research Platform- based microarray studies. A. Microarray analysis of 7 normal breast tissues (red bar) and 40 breast cancers (blue bar). The mean values of GREB1 mRNA expression are shown here. B. Microarray analysis of 2321 patients (1651 ER-positive, 670 ER-negative). The level of GREB1 expression is higher in ER-positive breast cancer patients, which are depicted by red-colored bars and ER-negative patients are represented by blue ones. Kaplan-Meier survival plots demonstrate the prognostic relevance of GREB1 expression on patient survival using various data cohorts. C. Reduced expression of GREB1 is predictive of lower relapse free survival (HR = 0.66, p = 1.1×10−10) for all patients; D. For ER+ breast cancer patients, lower GREB1 expression is correlated with decreased relapse free survival (HR = 0.75, p = 0.0029); E. Endocrine treated ER+ breast cancer patients have reduced relapse free survival if their tumors expressed lower GREB1 level (HR = 0.63, p = 0.041). GREB1 is Localized in the Nucleus in ER-positive Breast Cancer Cell Line and Breast Cancer Tissues The immunofluorescence staining was performed to determine the subcellular localization of GREB1 protein. E2-deprived MCF-7 cells were treated with 1 nM E2 for 24 h and then stained for GREB1 in red (Alexa Fluor® 555, Figure 3A) and nucleus in blue (DAPI, Figure 3B). The merged picture is given in Figure 3C, which demonstrates a clear nuclear localization of GREB1 (there are some specks of GREB1 proteins in the surrounding cytoplasm, but GREB1 appears mostly nuclear). Figure 3D to 3F show staining of negative control lacking primary antibody GREB1. The nuclear localization of GREB1 was further confirmed by introducing exogenous GREB1 into MCF-7 cells. We overexpressed GREB1 using adenovirus containing the full length GREB1 cDNA in estrogen-deprived MCF-7 breast cancer cells. E2-deprived MCF-7 cells were infected with Ad-CMV-null and Ad-GREB1 at a MOI of 10. After 72 h, the cells were stained for GREB1 in red (Alexa Fluor® 555, Figure 3G) or nucleus in blue (DAPI, Figure 3H), the merged image is shown in Figure 3I. Figure 3J to 3L show the staining of Ad-CMV-null as negative control. There was low background in the negative controls, however MCF-7 cells showed a significant efficiency of nuclear transfection of Ad-GREB1 compared with the transfection of empty vector (Fig. 3J to 3L). Immunohistochemistry staining (IHC) of ER-positive MCF-7 cells (Figure 3M, 3N) and ER-positive breast cancer tissues (Figure 3Q) confirmed the observation of GREB1 nuclear localization obtained from immunofluorescent staining described as above. ER-negative breast cancer cell line MDA-MB-231 and ER-negative breast cancer tissues expressed undetectable levels of GREB1 (Figure 3O, P and R). Therefore, both immunofluorescence and IHC staining results show that GREB1 is localized predominantly to the nucleus in both ER-positive breast cancer cell line and ER-postive primary breast cancer tissues. 10.1371/journal.pone.0046410.g003Figure 3 GREB1 is localized in the nucleus in ER-positive breast cancer cell line and breast cancer tissues. Subcellular localization of GREB1 in breast cancer cells was determined by immunofluorescent microscopy and immunohistochemical staining (IHC). A to C, E2-deprived MCF-7 cells were stimulated with E2 for 24 h and then stained for GREB1 in red (Alexa Fluor 555, A) and nucleus in blue (DAPI, B), and C gave the merged picture. GREB1 is almost exclusively localized in the nucleus. D to F showed staining of negative control lacking primary GREB1 antibody. The nuclear localization of GREB1 was further confirmed by introducing exogenous GREB1 into MCF-7 cells. GREB1 was stained in red in Figure 3G, nucleus was stained in blue in Figure 3H, the merged image was shown as Figure 3I. Figure 3J to 3L showed the staining of negative control. IHC staining showed that ER-positive MCF-7 cells (Figure 3M) and ER-positive breast cancer tissues (Figure 3Q) detected GREB1 expression (brown). ER-negative breast cancer cell line MDA-MB-231 (Figure 3O) and ER-negative breast cancer tissues expressed undetectable levels of GREB1 (Figure 3R). HE staining was also showed for ER-positive breast cancer (Figure 3N) and ER-negative breast cancer (Figure 3P). Overexpression of GREB1 Promotes Cell Proliferation and Increases the Clonogenic Ability of Breast Cancer Cells To investigate the biological functions of GREB1 in tumor formation and progression, we tested the effects of overexpression of GREB1 on breast cancer cell growth. We overexpressed GREB1 using adenovirus vector containing the full length GREB1 cDNA, which has been shown to significantly improve transfection efficiency. We checked the transfection efficiency using quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Briefly, MDA-MB-231, MDA-MB-453, MDA-MB-468 and 3-day estrogen depleted MCF-7 were infected with Ad-CMV-null and Ad-GREB1 at an MOI of 10, respectively. After 24 h, the cells were lysed and the expression of GREB1 mRNA in infected cells was analyzed by qPCR assay. Our results showed that the cells transduced with Ad-GREB1 express high GREB1 mRNA levels, whereas GREB1 was at non detectable level in all cells including estrogen deprived MCF-7 asynchronous cultures (Figure 4D, middle upper panel), MDA-MB-231 (Figure 4A, right upper panel), MDA-MB-453 (Figure 4B, right upper panel), and MDA-MB-468 (Figure 4C, right upper panel) cell cultures transduced with empty vector. Subsequently, the soft agar assay was used as a measure of anchorage independent growth, a defining characteristic of transformed cells. We found that the ability of MDA-MB-231 (Figure 4A, left panel), MDA-MB-453 (Figure 4B, left panel), MDA-MB-468 (Figure 4C, left panel), and MCF-7 (Figure 4D, middle lower panel) to grow in soft agar is substantially enhanced by 1.9-, 2.2-, 1.7- and 2.0- fold following GREB1 transfection. We next tested E2 deprived MCF-7 cells for the effects of GREB1 on cell proliferation. In parallel, cells were also treated with vehicle and estradiol (E2) as controls. On day 4, the mitogenic effects were measured using an AlamarBlue reduction assay. The growth rate in MCF-7 cells treated with E2 is 3.5 fold higher than in estrogen depleted, quiescent controls. Transfection with the SiGREB1 had marked effect on cells treated with E2, as cell proliferation was completely abolished (Figure 4D, right upper panel). In MCF-7 cells transduced with Ad-GREB1, the metabolic activity as measured by AlamarBlue was also increased by 1.5 fold compared to Ad-CMV-null transfected controls (Figure 4D, right lower panel), suggesting that GREB1 may function as a growth promoter in breast cancer and promote cell proliferation. It is noteworthy that the transfection efficiency varied among different breast cancer cell lines. For example, the transfection efficiency in E2-deprived MCF-7 cells (21- fold in Figure 4D, middle upper panel) is slightly lower than in ER-negative cells such as MDA-MB-231 (63-fold in Figure 4A, right upper panel), MDA-MB-453 (45-fold in Figure 4B, right upper panel) and MDA-MB-468 (40-fold in Figure 4C, right upper panel). 10.1371/journal.pone.0046410.g004Figure 4 Overexpression of GREB1 promotes cell proliferation and increases the clonogenic ability of breast cancer cells. MDA-MB-231, MDA-MB-453, MDA-MB-468 and estrogen depleted MCF-7 for 3 days were infected with Ad-CMV-null and Ad-GREB1 at an MOI of 10 respectively. The cells transduced with Ad-GREB1 expressed high GREB1 mRNA levels, whereas GREB1 was at extremely low detectable level in all cells including estrogen deprived MCF-7 asynchronous cultures (Figure 4D, middle upper panel), MDA-MB-231 (Figure 4A, right upper panel), MDA-MB-453 (Figure 4B, right upper panel), and MDA-MB-468 (Figure 4C, right upper panel) cell cultures transduced with empty vector. The soft agar assay was used as a measure of anchorage independent growth. The ability of MDA-MB-231 (Figure 4A, left panel), MDA-MB-453 (Figure 4B, left panel), MDA-MB-468 (Figure 4C, left panel), and MCF-7 (Figure 4D, middle lower panel) to grow in soft agar is substantially enhanced 1.9-, 2.2-, 1.7- and 2.0- fold following GREB1 transfection. We next tested E2 deprived MCF-7 cells for the effects of GREB1 on cell proliferation. The growth rate in MCF-7 cells treated with E2 is 3.5 fold higher than in estrogen depleted, quiescent controls. Transfection with the SiGREB1 had significant effect on cells treated with E2, as showed here that cell proliferation was completely abolished (Figure 4D, right upper panel). In MCF-7 cells transduced with Ad-GREB1, the metabolic activity as measured by AlamarBlue was also increased by 1.5 fold compared to Ad-CMV-null transfected controls (Figure 4D, right lower panel). The transfection efficiency in E2-deprived MCF-7 cells (21- fold in Figure 4D, middle upper panel) is a little lower than those of ER-negative cells such as MDA-MB-231 (63-fold in Figure 4A, right upper panel), MDA-MB-453 (45-fold in Figure 4B, right upper panel) and MDA-MB-468 (40-fold in Figure 4C, right upper panel). Figure 4E showed the little higher basic expression level of GREB1 in E2-deprived MCF-7 cells than other cells. Data are shown as mean ± SD. *P<0.05; **P<0.01. IL6/STAT3 Modulates Estrogen-induced GREB1 Transcriptional Activity in Breast Cancer Cells Previous reports indicated that STAT3 acts downstream of HER2 [23], [24]. STAT3 is tyrosine phosphorylated through the interleukin-6 (IL-6)/glycoprotein 130/Janus kinase pathway in breast cancer [25]. Interestingly, we found several STAT3 binding sites TT(N4)AA and TT(N5)AA located in the human GREB1 promoter region (http://www.cbrc.jp/research/db/TFSEARCH.html), which incidentally also contains three consensus EREs. STATs and ER can physically interact in vivo [26]. Based on this information, we hypothesize that STAT3 may physically connect with ER to repress GREB1 transcriptional activity or compete with ER for DNA binding sites within the multiple estrogen inducible enhancer regions of GREB1, resulting in decreased or non-detectable GREB1 expression. To test this hypothesis, we first assayed the effects of IL-6 on estrogen-induced GREB1 gene expression. Total RNA was extracted from estrogen-deprived MCF-7 cells for 3 days followed by exposure to E2 for 48 h. A region specific to GREB1a transcripts was amplified by real-time polymerase chain reaction (qPCR). The results show that E2 treatment increases GREB1 mRNA levels by approximately 43-fold after 48 h treatment (Figure 5A). ICI abolishes E2-induced GREB1 expression. As expected, IL-6 has an inhibitory effect on GREB1 expression, which inhibits GREB1 expression by approximately 44% (Figure 5A). IL-6 is known to activate many downstream signaling pathways, we thus asked whether the inhibitory effect of IL-6 on E2-induced GREB1 transcription is due to JAK/STAT3 cascade activation. Therefore, we designed the experiments to test what would happen to E2-mediated GREB1 induction if STAT3 gene was knocked down or kept constitutively active. Transfection with STAT3 siRNA (SiSTAT3) duplexes increased E2 induced expression of GREB1 (Figure 5B) whereas transfection with constitutively active STAT3 vector (STAT3-C) suppressed E2 induced expression of GREB1 (Figure 5C). We then asked how IL-6 affects transcriptional activation of GREB1. The construct was established by cloning a 1.7-kb fragment containing ERE1 and the GREB1a promoter region into pGL3 reporter plasmid, followed by inserting ERE2 and ERE3 upstream of ERE1. The DNA fragment contains all three EREs. MCF-7 cells were estrogen deprived for 3 days before transfection with 0.5 µg luciferase GREB1 promoter-reporter construct and 0.1 µg phRL-SV40 Renilla internal control (Promega). The following day, transfected cells were continued to befed with medium containing 1 nM E2 and 10 ng/ml IL-6 for a further 24 h before lysis and measurement of luciferase activity using the Dual Luciferase Assay kit (Promega). As shown in Figure 5D, E2 treatment increases luciferase activity by approximately 37-fold over the control. Estrogen receptor antagonist ICI 182,780 blocks the E2-mediated GREB1 induction, confirming that the increased GREB1 is due specifically to the E2 stimulation. Interestingly IL-6 inhibits E2-induced GREB1 transcriptional activity by approximately 38%. To determine the most efficient concentration for IL-6, hormone-starved MCF-7 cells were treated with various concentrations of IL-6 (0∼100 ng/ml) for 24 h. As shown in Figure 5E, inhibition of of E2-mediated GREB1 activity by IL-6 peaked at 10 ng/ml. In the subsequent time course experiments, cells were treated with 10 ng/ml IL-6 for 0, 10, 20, 30 min until 24 h, and the resulting inhibition of E2-induced GREB1 level was similarly analyzed. We observed that the effects on the reduction of GREB1 by IL-6 did not vary significantly from 1 h to 24 h, GREB1 transcriptional activity indicated by GREB1 luciferase activity was significantly diminished at each time point compared to E2 stimulation alone (*P<0.05, compared to E2 stimulation alone). Interestingly, pretreatment with IL-6 for less than half an hour significantly dampered the antagonistic function of IL-6 on suppression of E2-induced GREB1 (Figure 5F). To determine if the expression levels of STAT3 would have any effect on the transcriptional activity of GREB1, MCF-7 cells were cotransfected with a GREB1 luciferase reporter construct, a STAT3 constitutively expressing vector (STAT3-C), and a control vector, respectively. Some samples were treated with E2 as indicated before assaying for luciferase activity. As shown in Figure 5G, STAT3-C decreases GREB1 transcription by approximately 39%. 10.1371/journal.pone.0046410.g005Figure 5 IL6/STAT3 modulates estrogen-induced GREB1 transcriptional activity in breast cancer cells. E2 treatment increases GREB1 mRNA levels by approximately 43-fold after 48 h treatment (Figure 5A). ICI abolishes E2-induced GREB1 expression. IL-6 has an inhibitory effect on GREB1 expression, which inhibits GREB1 expression by approximately 44% (Figure 5A). Transfection with STAT3 siRNA (SiSTAT3) duplexes increased E2 induced expression of GREB1 (Figure 5B) whereas transfection with constitutively active STAT3 vector (STAT3-C) suppressed E2 induced expression of GREB1 (Figure 5C). Figure 5D to 5G, MCF-7 cells were estrogen deprived for 3 days before transfection with 0.5 µg luciferase GREB1 promoter-reporter construct and 0.1 ug phRL-SV40 Renilla internal control. The following day, transfected cells were refed with medium containing 1 nM E2 and 10 ng/ml IL-6 for a further 24 h before lysis and measurement of luciferase activity. As shown in Figure 5D, E2 treatment increases luciferase activity by approximately 37-fold over the control. Estrogen receptor antagonist ICI blocks the E2-mediated GREB1 induction. IL-6 inhibits E2-induced GREB1 transcriptional activity by approximately 38%. To determine the best efficient concentration for IL-6, hormone-starved MCF-7 cells were treated with various concentrations of IL-6 (0∼100 ng/ml) for 24 h. Figure 5E shows that the maximum reduction of E2-mediated GREB1 activity is seen at 10 ng/ml, the inhibitory effects are decreased regardless of whether the doses are further increased or decreased throughout the experiment. In time course experiments, cells were treated with 10 ng/ml IL-6 at indicated time,. Pretreatment with IL-6 for less than half an hour dampered the antagonist function of IL-6 on suppression of E2-induced GREB1 greatly (Figure 5F). MCF-7 cells were then cotransfected with a GREB1 luciferase reporter construct, a STAT3 constitutively expressing vector (STAT3-C) and control vector respectively. The samples were treated with E2 as indicated before assaying for luciferase activity. As shown in Figure 5G, STAT3-C decreases GREB1 transcription by approximately 39%. Data are shown as mean ± SD. *P<0.05; **P<0.01. STAT3 physically Interacts with ERα To confirm whether there is a physical interaction between STAT3 and ERα, we transfected 293T cells with FLAG-tagged STAT3-C alone or together with ERα. 48 h after transfection, cells were treated with 1 nM E2 for 3 h. Cell lysates were then immunoprecipitated with anti-ERα and immunoblotted with anti-FLAG (Figure 6A). Immuno-complex was probed with anti- ERα antibody as internal loading control. Norml rabbit IgG served as negative control. Cell lysates were also immunoprecipitated with anti-FLAG from two different commercially available sources (Millipore and Sigma respectively), followed by immunoblotted with anti-ERα as indicated in Figure 6B. Immuno-complex was probed with anti- FLAG antibody as internal loading control. The results from Figure 6 indicate that STAT3 indeed directly interacts with ERα. 10.1371/journal.pone.0046410.g006Figure 6 STAT3 physically interacts with ERα. To confirm whether there is a physical interaction between STAT3 and ERα, we transfected 293T cells with FLAG-tagged STAT3-C (2 µg) alone or together with ERα (2 µg). 48 h after transfection, cells were treated with 1 nM E2 for 3 h. Cell lysates were immunoprecipited with anti-ERα and immunoblotted with anti-FLAG as shown in Figure 6A. Figure 6B showed that cell lysates were first immunoprecipitated with anti-FLAG from two different commercially available resources (Millipore and Sigma) respectively followed by immunoblotted with anti-ERα. The results from Figure 6 indicate that STAT3 indeed directly interacts with ERα. Discussion Whether tamoxifen acts as an agonist or antagonist is intimately related to AF domain activation. AF-1 activity is regulated by phosphorylation and is ligand-independent; AF-2 is the ligand-binding domain (LBD). AF-1 and AF-2 act synergistically under most conditions, but each can also act independently. When tamoxifen binds to the ERα LBD, the changes in structural conformation prevent binding of co-activators, suppressing AF-2 promoted transcription. In this situation, tamoxifen acts as an antagonist. In genes where AF-2 function is redundant and transcription is driven by the AF-1 region, tamoxifen may act as an agonist. Multiple mechanisms have been proposed as responsible for tamoxifen resistance in breast cancer [6]. Among them, the alterations in ERα expression/function contribute greatly to resistance to tamoxifen. ER/PgR negative tumors do not respond to tamoxifen. In ER positive breast cancer patients, the levels of ERα expression reflect the possibility of benefit from endocrine therapy [54]. Transcriptional silencing of ERα by DNA methylation has been well documented in patients with recurrent breast cancer who have received tamoxifen therapy, whereas mutations within the open reading frame of ER in patients are not common although some mutations in the ER have been examined in resistant cell lines [6], [54]. The majority of ER-positive patients still expresses quite high levels of ERα at the time disease progresses and develops acquired tamoxifen resistance [17], [54]. Phosphorylation of ERα (Ser118) and sequential activation of its downstream pathway induced by tamoxifen has been reported to contribute to the development of endocrine therapy resistance and predict poor prognosis [54]. While ER expression status has important treatment and prognosis implication in breast cancer patients, ER alone is not perfectly correlated with hormonal response [55]. To determine the possibility of GREB1 functions as a surrogate marker for ER in clinical situation, we analyzed the GREB1 expression status in normal breast tissues, ER-positive and ER-negative breast cancer tissues using Oncomine public database and gene microarray data analysis tool [32], [33]. Our results show that GREB1 mRNA is highly expressed in breast carcinomas than normal breast tissues (T-test: 4.815; P-value: 2.1E-5), GREB1 mRNA is significantly overexpressed in ER-positive breast cancer patients compared to that of ER-negative patients (Figure 2) [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47]. Furthermore, analysis of GREB1 mRNA in 225 patients from the Tamoxifen (TAM) arm of 89-30-52 indicates that GREB1 can serve as an independent predictor of good disease-free survival in Tamoxifen-treated patients in ways even more reliable than ER or PgR, which itself has historical precedence as a clinical prognosticator in breast cancer patients in response to endocrine therapy (Table 1) [8], [51], [52]. This conclusion is further supported by a patient survival analysis to correlate GREB1 gene expression and relapse free survival for 2898 breast cancer patients (www.kmplot.com) where loss or reduced level of GREB1 is strongly predictive of worse disease outcome for all breast cancer patients in general, and for ER+, and ER+ endocrine treated patients in particular (Figure 2C to 2E). Based on these data, we believe that GREB1 protein may have a great potential to be a new biomarker not only for predicting ER and/or PgR status, but also for predicting Tomoxifen treatment response in breast cancer patients. The role of GREB1 in regulating hormone-related cancer including mammary carcinoma proliferation has been investigated for some time [28], [56]. However, no studies have systemically explored the role of GREB1 in cellular proliferation and cell growth [28], [56]. In the results presented here, we showed for the first time that increased levels of GREB1 have the growth advantage seen in breast tumors, with a 150% increase in MCF-7 cells expressing Ad-GREB1 than cells containing control vector at time points of 48 h after infection (Figure 4D, right lower panel). We also showed that RNAi directed against GREB1 significantly decreased the growth rate of MCF-7 cells (Figure 4D, right upper panel). We then tested whether adeno-GREB1-mediated increase in GREB1 expression could influence the ability of different breast cancer cell lines including MCF-7, MDA-MB-231, MDA-MB-453, and MDA-MB-468 to form colonies in soft agar. Adeno-directed increase in GREB1 resulted in a significant increase (about 170%–220%) in colony formation in all four breast cancer cell lines regardless of ER status (Figure. 4). These results clearly demonstrate that increased GREB1 levels enhance the clonogenic ability of breast cancer cells. In another word, elevation of GREB1 levels has substantial effects on the transformed phenotype of breast cancer cells. The tumorigenesis assays in which GREB1-expressing cells exhibit a greater capability to display a transformed phenotype are important since GREB1-mediated signaling pathways may be useful in inhibiting tumor formation in vivo. A slightly higher basic expression level of GREB1 in E2-deprived MCF-7 cells as shown in Figure 4E may cause difficulty in introducing exogenous GREB1 gene into these cells and may account for the higher transfection efficiency in MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells than E2-deprived MCF-7 cells. IL-6 is a multifunctional cytokine that has important roles in the immune system, hematopoiesis, and acute phase reactions [27]. In addition, IL-6 has been found to inhibit the growth of human breast cancer cells in vitro in the presence of E2 and modulate the ER and PgR [57]. In the present study, the breast cancer growth promoter, E2-induced GREB1 transcriptional activity was found to be significantly diminished by IL-6 compared to E2 stimulation alone (Figure 5F). Clinical studies showed that patients with STAT3 nuclear expression had a significantly improved 5-year survival, patients with phospho-STAT3 (Tyr705) nuclear expression had a significantly improved survival at both short-term (5-year ) and long-term (20-year) survival [58], and phosphor-STAT3 (Tyr705) is a marker for improved overall survival independent of other prognostic markers [58]. The glycoprotein 130 (gp130) receptor and gp130-associated JAKs are known mediators of STAT3 phosphorylation [25]. JAK2 was recently found to negatively regulate expression of endogenous ERα target genes, such as GREB1 and pS2 [59]. JAK2 mediated downregulation of ERα via the ubiquitin-proteasome pathway. This negative feedback modulation of ERα is physiologically essential to limit estrogen action in target tissues [59]. It is therefore not surprising for us to show here that IL-6 as well as its downstream pathway molecules STAT3 inhibit the transcriptional expression of E2-target gene GREB1. It is noteworthy in our observation that GREB1-expressing MDA-MB-231 and MDA-MB-468 cells still displayed enhanced ability to form colonies in soft agar even though these cells constitutively expressed tyrosine-phosphorylated STAT3 [25]. This indicates that endogenous phosphor-STAT3 is unable to completely offset the exogenous GREB1 function in these two cell lines. ER has been found to interact with some STAT proteins [26], [27]. Early research on the cross-talk between ER and STAT5a indicated that cross-talk between ER and STAT5a was through a direct physical association between the two proteins, and their C-termini were mainly responsible for this interaction [26]. We provided evidence here that the inhibitory function of STAT3 on GREB1 expression was also caused by a direct physical interaction between STAT3 and ER, and subsequently in the presence of E2, the low receptor level of ER leads to reduced transcriptional activity of ER-target genes such as GREB1. GREB1 is a primary E2 target gene and is strongly and sustainably induced by estrogen [28], [29], [30], [31]. We show that GREB1 expression is associated with ERα expression in breast cancer cell lines and breast cancer tissues. ERα directly controls GREB1 expression, and GREB1 is required for breast cancer cell growth. Clinically, like ER status, the loss or reduced expression of GREB1 is predictive of worse therapeutic outcome and decreased relapse free survival. Overall, our studies provide new insight into the biological function of GREB1 and its role in the pathogenesis of breast cancer. Materials and Methods Antibodies and Reagents Monoclonal antibody against GREB1 was obtained from ProMab Biotechnologies Inc (Richmond, CA). Antibody to β-actin was obtained from Sigma-Aldrich. All secondary antibodies used for Western blot were purchased from Calbiochem. GREB1 siRNA, STAT3 siRNA and control siRNA were purchased from Dharmacon (Lafayette, CO). The GREB1 promoter-luciferase construct was obtained as a generous gift from Joyce Slingerland (Department of Medicine, University of Miami, Miami, FL). Constitutive active STAT3 construct (STAT3-C) is kindly provided by Jacqueline F. Bromber (Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY). Microarray Analysis from Meta-analysis of Oncomine Database The expression of GREB1 transcript in breast normal and cancer tissues was obtained from meta-analysis of cancer gene microarray meta-analysis public database [32], [33]. Statistical analysis of differences was performed using Oncomine algorithms to account for the multiple comparisons among different studies similar to a meta-analysis as previously described [60]. Brest Cancer Cell Lines and Culture Conditions Breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453 and MDA-MB-468 were from American Type Culture Collection (ATCC) and were maintained and growth assays were performed as described previously [28], [61]. For defined estrogen culture experiments, cells were washed and grown in steroid depleted media (phenol red-free IMEM supplemented with 5% charcoal stripped calf bovine serum-Valley Biomedical Products, VA). SiRNA Transfection Small interfering RNA (siRNA) duplexes (total four pairs) of GREB1 and STAT3 were designed and purchased from Dharmacon (Lafayette, CO). A scrambled siRNA, with no homology to any known sequence was used as control. Hormone-depleted MCF-7 cells were transfected with 100 nM specific siRNA or control using Lipofectamine™ reagent (Invitrogen, Carlsbad, CA) in serum free OptiMEM-1 medium (Invitrogen) according to the manufacture’s instruction. After six hours of transfection, MCF-7 cells were split into two groups and grown in 10% CCS for another 24 h, then the cells were treated with 1 nM E2 or 0.01% ethanol; MDA-MB-231, MDA-MB-453, and MDA-MB-468 cells were grown in 10% FBS for 24 h. All studies were done in triplicates. Real-time RT-PCR Analysis Cell pellets were stored in Trizol reagent and homogenized in fresh Trizol. Total RNA were isolated from cells using an RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA were synthesized from the isolated RNA using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc). Reverse transcription was performed by using random hexamers at 25°C for 5 minutes, 42°C for 30 minutes, and 85°C for 5 minutes. Quantitative PCR were performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.) in a CFX96 Real-Time PCR System machine (Bio-Rad Laboratories, Inc). The data was analyzed using CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc). Primer sequences for the human GREB1 are: GREB1a-F: 5′-AAATCGAGGATGTGGAGTG-3′, GREB1a-R: 5′-TCTCACCAAGCAGGAGGA-3′. Luciferase Reporter Gene Assay MCF-7 cells were transfected using lipofectamine 2000 (Invitrogen) with 0.75 µg of GREB1 promoter-luciferase construct together with 100 µg of pRL-TK, a cytomegalovirus-Renilla vector to control transfection efficiency. The amount of total DNA transfected was equalized with the appropriate amounts of control vectors. After transfection at different indicated points, cells were harvested and lysed in reporter lysis buffer (Promega, Madison, WI). Luciferase activity was determined by using the Dual Luciferase Kit (Promega) and a luminometer (Turner Design, Sunnyvale, CA) according to the manufacturer’s recommendation. All luciferase results were normalized to Renilla activity from the co-transfected pRL-TK plasmid. The data for luciferase activity was expressed as fold induction with respect to control cells and was the mean ± standard error of triplicate samples. Immunoprecipitation and Western Blot Cells were lysed with lysis buffer (50 mmHEPES, 150 mmNaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Nonidet P-40, 100 mm NaF, 10 mm sodium pyrophosphate, 0.2 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 µg/ml aproptinin, and 10 µg/ml leupeptin). After centrifugation, protein lysates (50 µg) or the immunoprecipitates from cell lysates (500 µg) were separated on a 4–15% gradient gel (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane and immunoblotted with the appropriate antibodies. The membrane was blocked in 5% nonfat dry milk in TBST for 1 h at room temperature and incubated with mouse antibody to human GREB1 (ProMab Biotechnologies Inc) at a dilution of 1∶1000 in TBST +2.5% nonfat dry milk, followed by horseradish peroxidase-conjugated antimouse secondary antibody (Amersham) at a dilution of 1∶2,000. Immunoblots were reprobed with β-actin monoclonal antibody to confirm equal loading. The expression levels of GREB1 and β-actin detected by immunoblotting were quantitated using the ImageQuant program (Bio-Rad) for the integrated density of each band. Western blot assays were conducted in duplicate for each sample and the mean value was used for the calculation of protein expression levels. AlamarBlue Assay After trypsinization, the indicated cancer cell lines were counted and resuspended to a final concentration of 1×104 cells/ml. A 100 µl aliquot of the cell suspension were seeded per well in 96 well plates. The stock solution of AlamarBlue was aliquoted and stored and protected from light at −20°C. 20 µl of AlamarBlue was added to each well at 48 h and the 96 well plates were returned to the incubator for 6 h. Absorbance was monitored with excitation at 570 nm and emission at 600 nm using a CytoFluorTM 2300 plate reader and the software CytoFluorTM 2300 v. 3A1 (Millipore Co, Bedford, MA, USA). Anchorage-Independent Growth Assays MCF-7 cells were hormone starved for 3 days in PRF IMEM containing 10% CS-FCS (HyClone). MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were grown in regular media containing 10% FBS. At 2 days after transfection, cells (300 cells per well) transfected with indicated plasmids were mixed with tissue culture medium containing 0.7% agar to result in a final agar concentration of 0.35%. Then 1 ml samples of this cell suspension were immediately plated in six-well plates coated with 0.6% agar in tissue culture medium (2 ml per well) and cultured at 37°C with 5% CO2. After 2 weeks, tumor cell colonies measuring at least 150 µm were counted from three replicates per treatment under a dissecting microscope. Immunoflurescence Microscopy Cells grown in monolayer cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100, and blocked with 10% fetal calf serum prior to antibody staining. Staining by anti-GREB1 antibody (1∶100) was visualized with corresponding Alexa Fluor® 555-labeled secondary antibody. Cover slips were mounted onto slides with Vectashield mounting medium with DAPI (H-1200; Vector Laboratories Inc). Fluorescent images were collected by using a Zeiss LSM510 confocal microscope, and images were captured with LSM software, version 2.3. Immunohistochemistry Staining Breast tumor tissue microarrays (TMA) were provided by Tissue Array Networks (http://Tissue-Array.Net). Slides containing formalin-fixed, paraffin-embedded samples were deparaffinized, hydrated in water, and subjected to antigen retrieval in 10 mM citrate buffer, pH 6.0. Immunostaining was performed as described previously with some modifications [62]. Briefly, slides were probed with GREB1 antibody at a dilution of 1∶100 for 1 hour, then probed with secondary antibody for another one hour. The reaction products were finally visualized by immersing slides in 3, 3-diaminobenzidine tablet sets (Sigma Fast, Sigma) and counterstained with hematoxylin. The anti-ER antibody (clone 1D5, dilution 1∶100; Dako) used is FDA approved [63]. TMAs were reviewed and scored by two pathologists (C.R. G. and M.J.). Patient Survival Analysis An online database [Gyorffy et al 2010] was used to assess relevance of GREB1 expression to relapse free survival. The database was established using gene expression data and survival information of 1,809 patients (recently increased to 2898 patients) downloaded from Gene Expression Omnibus (GEO) (Affymetrix HGU133A and HGU133+2 microarrays). Briefly, GREB1 gene was entered into the database (http://kmplot.com/breast/) to obtain Kaplan-Meier survival plots where the number-at-risk is indicated below the main plot. Hazard ratio (and 95% confidence intervals) and logrank P were calculated and displayed on the webpage. Statistical Analysis Results were expressed as mean ± SEM of at least 2 independent experiments done in triplicate. Paired t-test or ANOVA tests were performed for data analysis, and significant difference was defined as p<0.05. 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23056300
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PLoS One. 2012 Oct 3; 7(10):e46410
==== Front BMC NeurosciBMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2202-13-502262139810.1186/1471-2202-13-50Research ArticleDose-dependent changes in neuroinflammatory and arachidonic acid cascade markers with synaptic marker loss in rat lipopolysaccharide infusion model of neuroinflammation Kellom Matthew [email protected] Mireille [email protected] Vasken L [email protected] Mei [email protected] Stanley I [email protected] Jagadeesh S [email protected] Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, 9000 Rockville Pike, Bldg. 9, 1S-126, Bethesda, MD, USA2012 23 5 2012 13 50 50 16 12 2011 8 5 2012 Copyright ©2012 Kellom et al.; licensee BioMed Central Ltd.2012Kellom 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 Neuroinflammation, caused by six days of intracerebroventricular infusion of bacterial lipopolysaccharide (LPS), stimulates rat brain arachidonic acid (AA) metabolism. The molecular changes associated with increased AA metabolism are not clear. We examined effects of a six-day infusion of a low-dose (0.5 ng/h) and a high-dose (250 ng/h) of LPS on neuroinflammatory, AA cascade, and pre- and post-synaptic markers in rat brain. We used artificial cerebrospinal fluid-infused brains as controls. Results Infusion of low- or high-dose LPS increased brain protein levels of TNFα, and iNOS, without significantly changing GFAP. High-dose LPS infusion upregulated brain protein and mRNA levels of AA cascade markers (cytosolic cPLA2-IVA, secretory sPLA2-V, cyclooxygenase-2 and 5-lipoxygenase), and of transcription factor NF-κB p50 DNA binding activity. Both LPS doses increased cPLA2 and p38 mitogen-activated protein kinase levels, while reducing protein levels of the pre-synaptic marker, synaptophysin. Post-synaptic markers drebrin and PSD95 protein levels were decreased with high- but not low-dose LPS. Conclusions Chronic LPS infusion has differential effects, depending on dose, on inflammatory, AA and synaptic markers in rat brain. Neuroinflammation associated with upregulated brain AA metabolism can lead to synaptic dysfunction. Arachidonic acidCytokineSynapseDrebrinLipopolysaccharideSynaptophysinPhospholipase A2NeuroinflammationNF-κB ==== Body Background Neuroinflammation has been reported in many progressive neurodegenerative and neuropsychiatric brain illnesses, such as Alzheimer’s disease, Parkinson’s disease, HIV-1 dementia and bipolar disorder [1-4]. Low-grade neuroinflammation can induce slow progressive cellular and tissue damage, whereas high-grade inflammation is associated with robust cytokine release. Despite its pathophysiologic significance, molecular mechanisms underlying effects of low-grade and high-grade neuroinflammation are not fully understood. Six or 30 days of intracerebroventricular (icv) low-dose bacterial lipopolysaccharide (LPS) (0.5-1.0 ng/h) infusion in rats produces behavioral deficits, induces amyloid deposits, and activates microglia and astrocytes [5,6]. Low-dose LPS infusion in rats increases arachidonic acid (AA, 20:4n-6) turnover in brain phospholipids, brain activities of AA-selective cytosolic Ca2+-dependent phospholipase A2 (cPLA2) type IVA and of secretory phospholipase A2 (sPLA2) type II, and concentrations of prostaglandin (PG) E2, PGD2 and thromboxane (TX)B2 metabolites [6-9]. However, net brain cyclooxygenase (COX) activity, COX-1 and COX-2 protein levels, and calcium-independent phospholipase A2 (iPLA2-VI) activity are unchanged with low-dose LPS infusion [6,8]. Depending on the infusion rate and duration, LPS infusion has been reported to activate rat brain microglia and astrocytes, increase expression of the transcription factor nuclear factor-kappa B (NF-κB) and inflammatory cytokines, stimulate microglial inducible nitric oxide synthase activity (iNOS) to produce nitric oxide, and increase brain glutamate [10,11]. A 6-day icv infusion of high-dose LPS (250 ng/h) has been shown to increase activated microglia in the rat thalamus [12], activities of cPLA2-IVA and sPLA2-IIA, and unesterified AA and PGE2 concentrations in rat brain [8]. The same high-dose LPS infusion for 28 days increased mRNA levels of interleukin-1 beta (IL-1β) and of tumor necrosis factor-alpha (TNFα), reduced pyramidal cells in layers II and III of the entorhinal cortex, attenuated long-term potentiation (LTP), and impaired spatial memory in adult rats [10,13]. Synaptic proteins such as synaptophysin, drebrin and post-synaptic density-95 (PSD-95) play important roles in synaptic plasticity. Drebrin is an actin-binding neuron-specific protein [14], abundant within dendritic spines at postsynaptic excitatory synapses [15]. Suppressing drebrin expression reduces spine density and results in the formation of thin immature dendritic spines [16]. Thus, the drebrin-actin complex plays a crucial role in the regulation of dendritic spine morphology. Synaptophysin is a 38-kD glycoprotein localized in presynaptic vesicle membranes. Functions of synaptophysin include docking, fusion, and endocytosis, otherwise known as membrane trafficking [17]. PSD-95 is a neuronal protein that associates with receptors and cytoskeletal elements at synapses, and is involved in regulating the number and size of dendritic spines and developing glutamatergic synapses [18]. Changes in these synaptic markers have been used to evaluate neuronal damage [19]. The impact of low- and high-dose LPS infusion on brain AA cascade, neuroinflammatory and synaptic markers has not been examined consistently. Therefore, we thought of interest to measure effects of the two doses of LPS on AA, neuroinflammatory and synaptic markers in rat brain after a six-day LPS infusion, compared to infusion of artificial cerebrospinal fluid (aCSF). Based on reported upregulated brain AA metabolism in the LPS-infused rat brain, we hypothesized that 6-day icv infusion of low- and high-dose LPS would increase expression of AA cascade and neuroinflammatory markers, and reduce pre- and post-synaptic markers such as synaptophysin and drebrin, in a dose-dependent manner. An abstract of some of this work has been presented [20]. Methods Animals The study was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 86–23), and was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Three-month-old male Fischer F344 rats (Taconic Farms, Rockville, MD) (n = 24) were housed in a facility with controlled temperature, humidity, and 12-hour light/dark cycle. Food (Rodent NIH-31 auto 18–4 diet, Zeigler Bros, Gardners, PA) and water were provided ad libitum. The diet contained (as % of total fatty acid) 20.1% saturated, 22.5% monounsaturated, 47.9% linoleic, 5.1% α-linolenic, 0.02% AA, 2.0% eicosapentaenoic, and 2.3% docosahexaenoic acid [21]. LPS infusion Low-dose (1 μg/ml infused at a rate of 0.5 ng/h) or high-dose (0.5 mg/ml infused at a rate of 250 ng/h) of LPS was infused icv in rats for 6 days as described previously [6-9]. The rationale for choosing the 6-day infusion period is based on a pilot study performed when measuring AA incorporation into brain at 2, 3, 4, 6, 8, and 10 days of low-dose LPS infusion. The study found no increase in total AA incorporation over control values until day 4 (10–15% increase) of infusion. Incorporation of AA reached a maximum at day 6 and remained elevated until day 10 [22]. We chose to infuse LPS into the fourth ventricle based on earlier studies that showed activation of microglial cells by such LPS infusion [23] and of unregulated brain AA cascade markers [7-9]. Briefly, the prefilled pump was placed in sterile 0.9% NaCl at 37°C overnight before surgery to start immediate pumping. aCSF (140 mmol/L NaCl, 3.0 mmol/L KCl, 2.5 mmol/L CaCl2, 1.0 mmol/L MgCl2, and 1.2 mmol/L NaPO4, pH 7.4) or LPS from Escherichia coli (Sigma, Saint Louis, MO; serotype 055:B5; source strain, CDC 1644–70; chemotype, rough type) at a low or high dose was infused into the fourth ventricle through the cannula via an osmotic pump (Alzet, Model 2002, Cupertino, CA). Postoperative care included triple antibiotic ointment applied to the wound; 5 ml of sterile 0.9% NaCl was injected subcutaneously to prevent dehydration during recovery. Following 6 days of LPS or aCSF infusion, a rat was anesthetized with an overdose of CO2, and decapitated. The brain was rapidly excised, frozen in 2-methylbutane at −50°C, and stored at −80°C until use. The whole brain was cut into two hemispheres. One half of the brain was used for preparing cytosolic and nuclear extracts and the other half was used for extracting total RNA. Protein homogenates were prepared from the cerebrum and cerebellum without including the brainstem. Preparation of cytosolic and nuclear fractions Cytosolic and nuclear extracts were prepared from control (aCSF), low- and high-dose LPS-infused rats, as previously described [24]. Briefly, brains were homogenized in 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM KCl, and a protease inhibitor cocktail (Roche, Indianapolis, IN), using a Teflon-glass homogenizer. After adding 0.5% tergitol type NP-40, five additional strokes of homogenization were performed. The suspension was incubated for 30 min on ice, and then centrifuged in a microcentrifuge (13,000 × g, 1 min, 4°C). The resulting supernatant was used as the cytosolic fraction. To the nuclear pellet, solution B (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.4 M NaCl) and a protease inhibitor cocktail (Roche) were added. The samples were mixed and placed on a small rotatory shaker for 30 min, then centrifuged at 13,000 × g for 3 min at 4°C. The supernatant containing the proteins from the nuclear extracts was transferred to a fresh tube. Protein concentrations of cytosolic fractions and nuclear extracts were determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA). Western blot analysis Proteins from the cytosolic and nuclear fractions (65 μg) were separated on 4-20% SDS-polyacrylamide gels (PAGE) (Bio-Rad) and then electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Cytosolic protein blots were incubated overnight in TBS containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for IL-1β (1:500), TNFα (1:500), glial fibrillary protein (GFAP) (1:1000), CD11b (1:1000), ionized calcium-binding adapter molecule 1Iba-1 (1:1000) (monoclonal), iNOS (1:1000), phosphorylated p38 mitogen-activated protein kinase (MAPK) (1:1000) (R&D Systems, Minneapolis, MN), cPLA2-IVA, sPLA2-IIA, sPLA2-V, iPLA2-VIA, COX-1 (1:1000), COX-2 (1:500), cytochrome P450 epoxygenase (CYP2B1), 5-, 12-, and 15-lipoxygenase (LOX) (1:1000), PSD-95 (1:1000), drebrin (1:1000) (Santa Cruz, Santa Cruz, CA), synaptophysin, (1:1000), and β-actin (1:10,000) (Sigma Aldrich, St. Louis, MO). Nuclear blots were incubated overnight in TBS containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for specificity protein 1 (SP-1) (1:500) and nuclear export factor (NXF) (1:500) (Abcam, Cambridge, MA). Cytosolic blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) and were visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ) on X-ray film (XAR-5, Kodak, Rochester, NY). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA) and were normalized to β-actin (Sigma) to correct for unequal loading. All experiments were carried out with 8 independent samples. Values are expressed as percent of control. Total RNA isolation and real time RT-PCR Total RNA was isolated from half-brains using an RNeasy lipid tissue mini kit (Qiagen, Valencia, CA). Briefly, tissue was homogenized in Qiagen lysis solution and total RNA was isolated by phenol-chloroform extraction. Complementary DNA was prepared from total RNA using a high-capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). mRNA levels (cPLA2-IVA, sPLA2-IIA and -V, iPLA2-VIA, COX-1, COX-2, 5-, 12-, 15-LOX, cytochrome P450 epoxygenase, drebrin, synaptophysin) were measured by quantitative RT-PCR, using an ABI PRISM 7000 sequence detection system (Applied Biosystems). Specific primers and probes for these markers, purchased from TaqManR gene expression assays (Applied Biosystems), consisting of a 20X mix of unlabeled PCR primers and Taqman minor groove binder (MGB) probe (FAM dye-labeled). The fold-change in gene expression was determined by the ΔΔCT method [25]. Data are expressed as the relative level of the target gene in the LPS-infused rat normalized to the endogenous control (β-globulin) and relative to the control (calibrator). All experiments were carried out in duplicate with 8 control and 8 brain samples from LPS-infused rats and data are expressed as relative expression. Transcription factor NF-κB p50 and NF-κB p65 activity Nuclear extracts were assayed for brain NF-κBp50 and NF-κBp65 activities according to the manufacturer’s instructions (Panomics, Freemont, CA). Briefly, 10 μg of nuclear extract was preincubated with biotin-labeled NF-κB p50/p65 for 60 min in a microfuge tube. The labeled oligonucleotide-nuclear protein complexes were transferred to immobilized streptavidin-coated 96-well plates. The bound oligonucleotide protein complex was detected by using a specific primary antibody directed against either NF-κB p50 or p65, followed by addition of HRP-conjugated secondary antibody. Color was developed by adding tetramethylbenzidine substrate, and optical densities were measured at 450 nm. Values (n = 8) are expressed as percent of control. Statistics Data are presented as mean ± SEM. When three groups were compared (low-dose LPS, high-dose LPS and aCSF control), statistical significance was determined using a one-way ANOVA with Newman-Keuls Multiple Comparison post-hoc test for multiple comparisons between the groups. Statistical significance was set at p ≤ 0.05. Results Body weight Body weight (grams) was significantly reduced by 9% and 19% in low- (264 ± 7; p < 0.05) and high- (235 ± 5; p < 0.001) dose LPS-infused rats, respectively, compared to control (290 ± 7), as previously reported [8]. Brain microglia, astrocyte markers and proinflammatory cytokines Six days of high-dose but not low-dose LPS infusion significantly elevated the brain protein level (89%) of the microglia/macrophage marker CD11b [26], compared to control (Figure 1A). Consistent with that, a significant increase in the protein level of microglia marker Iba-1 was found in high-dose but not low-dose LPS infused rats (Figure 1B). The astroglial marker GFAP protein level was unchanged in rats infused with either LPS dose compared to control (Figure 1C). Proinflammatory cytokine TNFα protein levels were significantly increased independently of the dose of LPS (332% and 340% with low- and high-dose LPS, respectively) (Figure 1D), whereas the IL-1β protein level was unaltered (Figure 1E). The protein level of iNOS, a major free radical-generating enzyme in activated microglia [27], was increased significantly by 180% and 155%, with the low- and high-LPS doses, respectively (Figure 1F). These significant protein changes in CD11b, TNFα and iNOS did not correspond to significant changes in their respective mRNA levels (data not shown). Further, there was no significant difference between low and high-dose LPS infused rats in any neuroinflammatory marker. Figure 1 Protein levels of (A) CD11b, (B) Iba-1 (C) iNOS, (D) TNFα, (E) IL-1β and (F) GFAP (with representative immunoblots) in control, low- and high-dose LPS rat brain. The protein level was determined in a homogenate of cerebrum and cerebellum, as in all subsequent figures (see methods). Data are ratios of optical densities of protein to β-actin, expressed as percent of control. Comparisons between LPS groups were made using one-way ANOVA, mean ± SEM (n = 8). *p < 0.05. Brain arachidonic acid cascade markers High-dose LPS significantly increased phosphorylated cPLA2-IVA (120%) as did low-dose LPS (60%) (p < 0.05) compared to control (Figure 2A). The protein and mRNA levels of cPLA2-IVA were significantly elevated by 178% and 2-fold, respectively, in rats infused with high-dose LPS (Figures 2B and 2C). High-dose LPS significantly increased phospho-cPLA2 and cPLA2 levels compared to low-dose LPS (Figures 2A and 2B). sPLA2-V protein levels were significantly increased by 34% and 24% in low- and high-dose LPS-infused rats respectively, compared to control (Figure 2D), but without a significant change in sPLA2-V mRNA (Figure 2E). sPLA2-IIA and iPLA2-VIA protein levels were unaltered. Figure 2 Mean (A) phosphorylated cPLA2-IVA, (B) cPLA2-IVA and (D) sPLA2-V protein levels (with representative immunoblots) in control, low- and high-dose LPS rat brain. Data are ratios of optical densities of protein to β-actin, expressed as percent of control and were compared using one-way ANOVA, mean ± SEM (n = 8). mRNA levels of (C) cPLA2-IVA and (E) sPLA2-V in control, low- and high-dose LPS in rat brain, measured using real time RT-PCR. Data are normalized to the endogenous control (β-globulin) and relative to control level (calibrator), using the ΔΔCT method. Comparisons between LPS groups were made using one-way ANOVA, mean ± SEM (n = 8), *p < 0.05, **p < 0.01. COX-2 protein (61%) and mRNA (5-fold) levels were significantly elevated in rats infused with high- but not low-dose LPS compared to controls (Figures 3A and 3B), whereas COX-1protein was unchanged compared to control (Figure 3C). High-dose LPS significantly increased both COX-2 protein and mRNA levels compared to low-dose LPS infused rats (Figures 3A and 3B). Protein (74%) and mRNA levels of 5-LOX (8-fold) were significantly increased only with the high-dose LPS infusion (Figures 3C and 3D). Protein levels of 12-LOX, 15-LOX and cytochrome P450 epoxygenase were unaltered by either LPS infusion (Figures 3F and 4A-B). Figure 3 Protein levels of (A) COX-2, (C) COX-1, (D) 5-LOX and (F) 12-LOX (with representative immunoblots) in control, low- and high-dose LPS rat brain. Data are ratios of optical densities of protein to β-actin, expressed as percent of control, and were compared using one-way ANOVA, mean ± SEM (n = 8). mRNA levels of (B) COX-2 and (E) 5-LOX in control, low- and high-dose LPS, measured using real time RT-PCR. Data normalized to the endogenous control (β-globulin) and relative to control level (calibrator), using the ΔΔCT method. Comparisons between LPS were made using one-way ANOVA, mean ± SEM (n = 8), *p < 0.05, **p < 0.01. Figure 4 Protein levels of (A) 15-LOX, and (B) p450 epoxygenase (with representative immunoblots) in control, low- and high-dose LPS rat brain. Data are ratios of optical densities of protein to β-actin, expressed as percent of control and were compared using one-way ANOVA, mean ± SEM (n = 8). Representative brain transcription factor binding activities (DNA-protein complex) of (C) NF-κB p50 and (D) NF-κB p65 in control, low- and high-dose LPS rat brains. DNA binding activity was measured in brain nuclear extracts as described in Materials and Methods. Mean ± SEM (n = 8), **p < 0.01. Protein levels of (E) phospho-p38 MAPK (with representative immunoblots) in control, low- and high-dose LPS rat brain. Data are ratios of optical densities of protein to β-actin, expressed as percent of control. Comparisons between LPS groups were made using one-way ANOVA, mean ± SEM (n = 8), *p < 0.05. NF-κB activity and phosphorylated p38 MAPK DNA-binding activity of NF-κBp50 was significantly increased (40%) in the high-dose LPS-infusion compared to control rats (p < 0.001) (Figure 4C), whereas NF-κBp65 DNA-binding activity was unaltered (Figure 4D). High-dose LPS significantly increased NF-κBp50 activity compared to low-dose LPS infused rats (Figures 2A and 2B). Active phosphorylated p38 MAPK was significantly increased by 108% and 109% with low- and high-dose LPS infusion, respectively, compared to control (Figure 4E). Synaptic markers Protein levels of the presynaptic marker synaptophysin were significantly decreased by high-dose (−35%) and low-dose (−16%) LPS, compared to control (p < 0.05) (Figure 5A). However, the mRNA level of synaptophysin was significantly reduced (0.7-fold) only by high-dose LPS (Figure 5B). Postsynaptic marker drebrin protein (−30%) and mRNA (−0.6-fold) levels were significantly reduced in high- but not low-dose LPS infused rats compared to control (Figures 5C and 5D). High-dose LPS significantly decreased protein and mRNA levels of synaptophysin and drebrin compared to low-dose LPS infused rats (Figures 5A and D). Further, another marker of post-synaptic marker PSD-95 was significantly decreased by high-dose LPS but not low-dose LPS (Figure 5E). The protein levels of NXF, a transcription factor of drebrin [28], and of SP-1, a constitutive transcription factor of synaptophysin [29], were unaltered (Figures 5F and G). Figure 5 Protein levels of (A) synaptophysin, (C) drebrin and (E) PSD-95 (with representative immunoblots) in control, low- and high-dose LPS rat brain. Data are ratios of optical densities of protein to β-actin, expressed as percent of control and were compared using one-way ANOVA, mean ± SEM (n = 8). mRNA levels of (B) synaptophysin and (D) drebrin in control, low- and high-dose LPS in rat brain, measured using real time RT-PCR. Data are normalized to the endogenous control (β-globulin) and relative to control level (calibrator), using the ΔΔCT method. Comparisons between LPS groups were made using one-way ANOVA, mean ± SEM (n = 8), *p < 0.05, ***p < 0.001. Discussion The present study demonstrates that a 6-day icv infusion of low- or high-dose LPS in unanesthetized rats, compared with aCSF infusion, significantly increased brain levels of AA cascade and neuroinflammatory markers in a dose-dependent manner (Table 1). These changes were associated with decreased levels of pre-and post-synaptic markers in a dose dependent manner as well. Similar changes have been demonstrated in postmortem brain tissue from patients with bipolar disorder, schizophrenia, AIDS and Alzheimer’s disease [4,30-35], suggesting that LPS infusion at different rates in rats are reasonable models for understanding interactions of brain AA metabolism, neuroinflammation and synaptic integrity in these progressive human brain diseases, and perhaps for designing treatments for them [8,9,36,37]. Table 1 Summary of effects compared to control, 6 days of LPS infusion on neuroinflammatory, AA cascade, and synaptic markers in rat brain   Low-dose LPS High-dose LPS Neuroinflammation TNFα (protein) ↑ ↑ CD11b (protein) No change ↑ Iba-1( Protein) No change ↑ iNOS (protein) ↑ ↑ AA cascade p-cPLA2-IVA (protein) ↑ ↑ cPLA2-IVA (protein, mRNA) No change ↑ sPLA2-V (protein) ↑ ↑ COX-2 (protein, mRNA) No change ↑ 5-LOX (protein, mRNA) No change ↑ Transcription regulators NF-κBp50 (activity) No change ↑ p38 MAPK (protein) ↑ ↑ Synaptic markers Synaptophysin (protein) ↓ ↓ (+ mRNA) Drebrin (protein, mRNA) ↓ ↓ (+ mRNA) PSD-95 (protein) No Change ↓ IL-1β, GFAP, iPLA2-VI, sPLA2-IIA, COX-1, 12- and 15-LOX, cytochrome P450 epoxygenase, mPGES, NF-κBp65, NFX, and SP-1 were unchanged. Previously, we reported that a 6-day icv infusion of either LPS dose increased cPLA2-IV activity, but that the low-dose infusion did not alter the cPLA2-IV protein level [6-8]. In the present study, although both LPS doses increased phosphorylated cPLA2-IVA and phosphorylated p38 MAPK (linked to activation and phosphorylation of cPLA2 and AA release [38]), only the high-dose induced mRNA and protein increases of cPLA2-IVA. These new data demonstrate that high-dose but not low-dose LPS infusion induced transcriptional level activation. Both doses also increased brain sPLA2 activity [6-8], which can be ascribed to upregulated protein levels of sPLA2-V [39,40] since sPLA2-IIA protein was unchanged. In contrast, Ca2+-independent docosahexaenoic acid (DHA)-selective iPLA2-VIA protein and mRNA levels were not significantly altered by either dose of LPS. This is consistent with evidence that neither LPS infusion dose changed the brain unesterified DHA concentration, DHA turnover in brain phospholipids or iPLA2-VI activity [6,8,22,41]. The brain PGE2 concentration was elevated in rats infused with both LPS doses, consistent with increased concentration of unesterified AA released by cPLA2-IVA and sPLA2-V [6-8]. Protein and mRNA levels of COX-2, the rate-limiting enzyme in PGE2 biosynthesis, were upregulated with only the higher dose of LPS, showing a dose-dependent effect. In line with a previous observation, low-dose LPS infusion did not alter the brain COX-2 protein level [6]. LOX and cytochrome P450 epoxygenase convert AA to 5-hydroxyeicosatetraenoic acid (5-HETE)/leukotrienes/lipoxins and epoxyeicosatrienoic acids, respectively. The high-dose LPS infusion increased 5-LOX protein and mRNA levels, without altering 12- or 15-LOX levels. Concentrations of 12-HETE and 15-HETE remain unchanged with both doses [8]. In this regard, LPS has been reported to induce expression of 5-LOX [42] and the 5-LOX-activating protein (FLAP) via NF-κB-mediated transcriptional mechanisms in mononuclear phagocytes, which is critical for leukotriene synthesis [43]. Another AA cascade marker, cytochrome P450 epoxygenase, remained unchanged after LPS infusion. Cell culture studies have shown that two major cytokines, IL-1β and TNFα, can induce transcription of cPLA2, sPLA2, COX-2, and iNOS genes through an NF-κB-mediated mechanism [44-46], as NF-κB binding sites are present on the promoter regions of these genes [46-49]. Our study suggesting that increases of cPLA2-IVA, COX-2, and iNOS in high-dose LPS-infused rats were due to elevated levels of TNFα following increased NF-κB p50 activity, was recently supported by in vitro studies [50]. The response was not observed in low-dose LPS infusion. NF-κB p50 also is known to regulate transcription of many proinflammatory genes [51,52]. Since p38 MAPK can activate NF-κB mediated cell signaling [53], the increases found in phosphorylated p38 MAPK (the active form) [54] may be involved in LPS-mediated NF-κB activation. These changes my induce TNFα and COX-2 [55,56]. NF-κB also can be activated by other cellular signal transduction factors, such as extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) [57,58]. Consistent with the increased TNFα level, a pro-inflammatory cytokine produced by microglia [59], high-dose but not low-dose LPS increased the microglia/macrophage marker CD11b, without altering the astroglial marker GFAP. Microglia but not astrocytes express the LPS CD14/toll-like receptor 4 (TLR4) [60], but we cannot rule out localized regional changes in brain GFAP. Indeed, we reported that low-dose LPS increased lectin-reactive microglia in the cerebral ventricular surround, pia mater, and glial membrane of the cortex, and produced morphological changes of GFAP-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles [6]. An increase of CD11b with NF-κB-induced activation of COX-2 by high-dose LPS infusion is consistent with the observation that CD11b plays a significant role in the optimal production of COX-2 via NF-κB [56]. Since CD11b integrins originally were identified as LPS receptors [61], our data suggest that high-dose but not low-dose LPS modifies the CD11b receptor. Further, the microglial specific marker [62], Iba-1 was significantly increased in high-dose compared to low-dose LPS infused rats, suggesting the presence of activated microglia in high-dose LPS infused rats. Increases in TNFα and iNOS levels and in AA cascade enzyme expression have been implicated in neuronal damage [63] and cognitive-behavioral impairments in rats infused with high doses of LPS [10]. The latter impairments might be due to reduced expression of the postsynaptic dendritic spine actin-regulatory protein drebrin, which is involved in spinogenesis and synaptogenesis [15]. LPS reduced the presynaptic vesicle marker synaptophysin in a dose-dependent manner. The molecular mechanisms by which neuroinflammation downregulate drebrin in rat brain are not clear. Several studies reported that inhibition of p38 MAPK activity prevented cytokine-induced loss of synaptophysin in rat primary cortical neuronal cultures and in an animal model of Alzheimer’s disease [64,65]. Similarly, drebrin loss was attributed to p38 MAPK activity in hippocampal cultures [66]. Apart from cytokines, PGE2 is known to activate p38 MAPK in rat primary astrocytes [67]. Both LPS doses increase the PGE2 concentration in rat brain [6-8] and may be involved in activating p38 MAPK. Increased p38 MAPK activity observed in this study may explain the loss of synaptophysin following low- and high-dose LPS infusion. Interestingly, the increased p38 MAPK may not be solely responsible for synaptic loss, because low-dose LPS increased p38 MAPK activity without changing the synaptic proteins. This suggests that other mechanisms are involved regulating the synaptic proteins. Further specific in vitro studies are required to understand synaptic regulation by LPS. Both LPS doses also did not change protein levels of NXF or SP-1, transcription factors for drebrin and synaptophysin, respectively [28,29]. The changes in the synaptic markers might be related to post-transcriptional regulation or changes of other transcription factors/kinases [28,29]. Consistent with loss of post-synaptic drebrin, the protein level of another postsynaptic marker PSD-95 was decreased in high-dose LPS infused rats compared to controls and low-dose infused rats. A limitation of current study is that one time point (6 days) was taken to study the effects LPS infusion based on the AA cascade metabolism. This choice was based on a prior study showing that AA incorporation in rat brain with low-dose LPS infusion did not increase until day 4 (10–15% increase) of infusion, reached a maximum at day 6 and remained elevated until day 10; no difference was evident at day 28 [22]. However, at earlier time points Toll receptors and other pathways are activated [68]. Future studies should aim at earlier and perhaps later time points. Conclusions We demonstrated that 6-day icv LPS infusion dose-dependently increased neuroinflammatory and AA cascade markers associated with an increase in phosphorylated p38 MAPK, and decreased synaptic markers (Table 1). Low-dose LPS increased TNFα, iNOS, sPLA2-V, cPLA2-IVA and p38 MAPK phosphorylation, and reduced presynaptic synaptophysin. High-dose LPS upregulated gene expression of AA cascade enzymes via NF-κB, CD11b, and downregulated postsynaptic drebrin. Targeting these disturbed pathways by specific anti-inflammatory drugs and/or cPLA2 inhibitors could lead to therapeutic treatments of neuronal damage and behavioral changes associated with neuroinflammation [7-9,69]. Abbreviations aCSF, Artificial CSF; cPLA2, Calcium-dependent cytosolic phospholipase A2; COX, Cyclooxygenase; GFAP, Glial fibrillary acidic protein; HETE, Hydroxyeicosatetraenoic acid; IL-1β, Interleukin-1β; iNOS, Inducible nitric oxide synthase; iPLA2, Calcium-independent phospholipase A2; LOX, Lipoxygenase; LPS, Lipopolysaccharide; MAPK, Mitogen-activated protein kinase; NF-kB, Nuclear factor-kappa B; NXF, Nuclear export factor; p, Phosphorylated; mPGES, Membrane PGE synthase; sPLA2, Secretory phospholipase A2; SP-1, Specificity protein 1; TNFα, Tumor necrosis factor α. Competing interests The authors declare that they have no competing interests. Authors’ contributions MK participated in the sacrifice of animals, carried out the molecular biology studies, performed the statistical analysis, and drafted the manuscript. MB wrote the animal protocol, participated in the rat surgery, prepared osmotic pumps, and revised the manuscript. VLK performed Western blot studies. MC performed the surgery. SIR participated in the design of the experimental work and corrected the final version of the manuscript. JSR participated in the design of the experimental work, performed the statistical analysis, drafted the manuscript and wrote its final version. All authors read and approved the final manuscript. Acknowledgements This research was supported entirely by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23071727PONE-D-12-1330310.1371/journal.pone.0047110Research ArticleBiologyBiochemistryBiomacromolecule-Ligand InteractionsChemical BiologyDrug DiscoverySmall MoleculesToxicologyMedicineDrugs and DevicesClinical PharmacologyNeurologyNeuropharmacologySmall-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice Identification of SMNPI against BoNT/A in MiceSingh Padma Singh Manglesh Kumar Chaudhary Dilip Chauhan Vinita Bharadwaj Pranay Pandey Apurva Upadhyay Nisha Dhaked Ram Kumar * Biotechnology Division, Defence Research and Development Establishment, Gwalior, Madhya Pradesh, India Afarinkia Kamyar Editor Univ of Bradford, United Kingdom * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: RKD. Performed the experiments: PS MKS PB NU AP. Analyzed the data: RKD MKS. Contributed reagents/materials/analysis tools: RKD. Wrote the paper: RKS MKS. In silico screening: DC VC. 2012 11 10 2012 7 10 e471109 5 2012 10 9 2012 © 2012 Singh et al2012Singh 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.Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC50 values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A). National Cancer Institute, National Institute of Health, USA acknowledged for providing chemicals bearing NSC prefix. Ms. Padma Singh is a JRF working in ICMR (Indian Council of Medical Research) scheme (Ref No: 3/1/3/JRF-2008/MPD-78, 31500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, C. baratii and C. butyricum, consist of seven immunologically distinct serotypes (A–G) are the causative agents of a life-threatening neuroparalytic disease known as botulism. The potency, longevity of BoNT intoxication has facilitated use of BoNTs as therapeutic agents [1], [2], [3], [4], [5] and the ease with which these toxins can be produced make them potential bioweapons and bioterrorism agents [6], [7]. Overdose with BoNT therapeutics can also result in systemic botulism [8]. BoNTs are the only toxin group in the six most dangerous biothreat agents (Category A agents) listed by Centers for Disease Control and Prevention (CDC) (http://www3.niaid.nih.gov/Biodefense/bandc_priority.htm). BoNTs are synthesized as ∼150-kDa single-chain protoxins that are post translationally processed by proteolytic cleavage to form a disulfide-linked dimer, composed of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC) [9]. The HC comprises a 50-kDa C-terminal domain (Hc) that participates in the binding of toxin to productive ectoacceptors on the cell surface of peripheral cholinergic nerve cells [10] and the 50-kDa N-terminal domain (Hn) of the HC facilitates the translocation of the LC across an endosomal membrane into the cytosol of the nerve cell [11]. The LCs are zinc metalloendoproteases [3.4.24] that exhibit extraordinary specificities for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: SNAP-25, VAMP/synaptobrevin and syntaxin [12], [13], [14], [15]. SNARE proteins are essential for exocytosis of neurotransmitter and cleavage of these protein(s) by BoNT inhibits the release of acetylcholine from synaptic terminals leading to neuromuscular paralysis or botulism [16]. The most effective immunotherapy for protection against BoNTs relies on vaccination with pentavalent toxoid species, although supplies are reserved for high-risk individuals [17]. Moreover vaccination of general public also restricts subsequent BoNT's therapeutic applications, if needed. There are no therapies available for BoNT mediated post neuronal intoxication. The current treatments for BoNT poisoning are limited to: (i) the administration of antitoxin(s) to neutralize and clear toxin from the circulation which is not effective in post neuronal intoxication [6] and, therefore, would be of limited use following an act of bioterror (as it is likely that victims would seek medical attention only after enervation); and (ii) mechanical ventilation which is necessary once BoNT-induced paralysis compromises thoracic muscle contraction. However, the latter form of treatment would also be impractical, even a limited act of bioterror employing BoNT(s), as critical care resources would likely to be overwhelmed. The estimated cost for treating a botulism patient with such intensive care could be as high as $350,000 [18]. Antibody therapy can be very effective; it has several limitations, including limited availability, lot-to-lot potency, variability and short window of application. Thus, the hypothesis rationalizing a small-molecule-based therapeutic approach for the treatment of BoNT/A-LC intoxication is as follows: Small drug like molecules can penetrate into the neuronal cytosol and inhibit the toxin's proteolytic activity during post neuronal intoxication. Alternatively, small-molecule inhibitors of BoNT are sought to antagonize the extracellular or intracellular toxin and can be potentially used to treat pre- and post-exposure. Additionally, if stockpiled in dry, sunlight free, temperature-controlled locations, chemically stable small-molecules would remain viable for many years. In contrast, vaccines possess comparatively shorter self-lives. Moreover, with respect to the development of small-molecule therapeutics, the BoNT/A-LC represents a top priority as it possesses the longest duration of activity in the neuronal cytosol in comparison to other BoNT-LCs known to cause botulism in human [5]. Research efforts to identify antagonists against BoNT intoxication have dramatically increased in recent years. However, the discovery and development of BoNT/A small-molecule inhibitors have been a challenging task for researchers since long. Part of the difficulty in this endeavor can be attributed to the unusually large peptide substrate-enzyme interface [19], [20] that requires a small-molecule with high affinity to effectively block substrate binding [21]. Also, the BoNT and its domains show considerable conformational flexibility, making design of effective inhibitors complicated. Despite these challenges, a number of papers have been published on the initial steps to discover and develop inhibitors of BoNT/A protease activity using different approaches. Using high throughput screening of the NCI Diversity Set as well as a series of 4-aminoquinolines, Burnett et al. [22] identified several small-molecule inhibitors of BoNT/A from which a common pharmacophore was predicted using molecular modeling [23]. Quinolinol derivatives (QAQ, NSC1010 and others) were reported to inhibit BoNT/A as determined by biochemical, cell and tissue based assay [24]. Mechanism of QAQ binding to BoNT/A-LC and mode of inhibition was studied in detail by Lai et al. [25]. Similarly, a high throughput screening of a library of hydroxamates [26] resulted in the selection of 4-dichlorocinnamic hydroxamate as a lead structure for further development [10]. Capkova et al. [27] structurally modified 2, 4-dichlorocinnamic acid hydroxamate to improve its potency. On the other hand, a computational screen of 2.5 million compounds resulted in the identification of an inhibitor with a Ki of 12 µM [28], but this value was later invalidated [21]. Computer-aided optimization of this inhibitor resulted in an analog that showed a two-fold improvement in inhibitory potency and displayed competitive kinetics by chelating the active site zinc atom [21]. Though the above approaches have resulted in the identification of a number of small-molecules as BoNT/A inhibitors, no compound has yet advanced to pre-clinical development [24], [29], [30], [31]. The majority of such molecules reportedly demonstrated to be effective in enzymatic assays [21], [23], [27], [28], [32], [33] and a few small-molecules have been tested in cell-based assays [34], [35], [36], [37]. But the information shows that small-molecules can significantly protect mammals against BoNT/A is scanty [31], [36]. We screened the ChemBridge and NSC libraries, consisting of millions of compounds of unknown function for similarity search to 8-hyroxy quinolinol lead, NSC 84096. Since some of these compounds were commercially available and their functions are currently undefined, we reasoned that novel inhibitors could be identified. Herein we report the effective small-molecule BoNT/A inhibitors with promising in vivo pharmacokinetics. Our results demonstrate that small-molecule can protect mice against pre and post BoNT/A challenge and support pursuit of small-molecule inhibitor as a cost effective alternative for treating botulism and for biodefence measures. Materials and Methods 1. Expression and Purification of Recombinant BoNT/A-LC Protein Previously, we have reported the conditions for the high level expression and purification of biologically active light chain protein of botulinum neurotoxin type A from a synthetic gene [38]. In brief, full length BoNT/A-LC gene was cloned in pQE30 vector and expressed in E. coli SG13009 at 21°C for 18 h. The rBoNT/A-LC was purified using Ni-NTA agarose and analyzed by 12% SDS-PAGE. The purified protein was characterized by western blotting and MALDI-TOF. The rBoNT/A-LC was dialyzed against 20 mM HEPES (pH 7.4) containing 200 mM NaCl, 10% glycerol (v∶v), pH 7.4 and stored at −20°C until used. 2. Assay of rBoNT/A-LC Activity on Synaptosomes 2.1. Preparation of Rat Brain Synaptosomes Crude synaptosomes were prepared from rat brain as described by Ferracci et al. [39]. Briefly, fresh rat brain (1 g) was homogenized with a teflon homogenizer in 10 ml of chilled homogenization buffer (0.32 M sucrose, 1 mM PMSF, 1 mM EDTA, and 10 mM HEPES, pH 7.5). Homogenized sample was centrifuged at 10,000 rpm for 15 min at 4°C, and supernatant (∼2 mg/ml) was collected and filtered with a 0.22 µ membrane and stored at −20°C. 2.2. Optimization of Assay The cleavage reaction was optimized with respect to concentrations of synaptosome substrate and rBoNT/A-LC, incubation time, and composition of cleavage buffer. Catalytic activity of rBoNT/A-LC protein was performed in 50 µl reaction mixture containing varying concentrations of rat brain synaptosomes and rBoNT/A-LC in reaction buffer (25 mM Tris, 100 mM NaCl, 19.2 mM glycine, 100 µg/ml BSA, 0.1 mM DTT, 10 µM ZnCl2, pH 7.5) and incubated at 37°C. For the time course analysis the reactions were stopped by adding 4× SDS-PAGE sample buffer at 1, 2, 5, 10, 20, 30, 60, 120, 180, 240, 300, 360, 420 and 480 min. The samples were analyzed by western blotting. 3. Molecular Docking Studies 3.1. Preparation of Ligands and Receptor The NCI and ChemBridge database libraries were chosen for virtual screening of small-molecule inhibitors on the basis of structure similarity searches. The structures of selected molecules were drawn by Chemsketch (ChemDraw) software (http://www.acdlabs.com) and saved as MDL mol files. The energy minimized pdb files were generated using ArgusLab 4.0.1 (http://www.arguslab.com). Ligand files in the pdb format were opened in AutoDock (4.1) for preparation. Once opened, ligand files were edited and saved in pbdqt format. The three-dimensional structure of BoNT/A-LC (PDB code 3BON) was obtained from the RCSB Protein Data Bank. All water molecules except those which participate in catalysis were removed. The rigid and flexible residues of the protein were selected, and two additional files were created; a file 3BONrigid.pbdqt and file 3BONflex.pbdqt. 3.2. Grid Generation and Running AutoGrid AutoDock requires pre-calculated grid maps, one for each atom type present in the ligand being docked. AutoGrid 4.1 was used to create autogrid .gpf, .glg, .fld and map files of atoms for protein. The Grid box was constructed around the active site residue Glu262 which plays a pivotal role in the catalytic activity of BoNT/A endopeptidase [40], [41]. The active site residues that surrounded by docking box were Phe163, Gln162, Glu164, Cys165, Lys166, Phe194, Glu224, His227, Arg231, Ala236, Ile237, Pro239, Val258, Ser259, Glu261, Arg363, Tyr366, and Zn(II). 3.3. Preparing the Docking Parameter File and Running AutoDock The final step in submitting the docking was to run the AutoDock function. To prepare this, the protein's rigid pbdqt file, the flexible pdbqt file and ligand's pdbqt file were specified. At the end of a docking process, the output file ‘.dlg’ showed the docked conformations. These conformations were compared one to another to determine similarities and they were clustered accordingly. The root mean square deviation (RMSD) was used to determine whether two docked conformations are similar enough to be in the same cluster. After that these clusters were ranked from the lowest energy to highest. 4. Screening of Inhibitors using Rat Brain Synaptosome Test compounds were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI (Bethesda, MD) and ChemBridge Corporation (San Diego, CA). BoNT/A peptide inhibitor (RRGC) was purchased from Bio Basic (Ontario, Canada). Compounds selected after virtual screening were tested in cleavage-based rBoNT/A-LC enzymatic assay. The inhibitors were dissolved in absolute DMSO and stock solutions were made up to 20 mM. Initial screening was performed in 40 µl reaction mixture containing 2.05 µg synaptosomes and 6 µM rBoNT/A-LC in cleavage buffer along with the 1 mM small-molecules. For determination of inhibitory molar ratios of small-molecules to rBoNT/A-LC, different concentrations of the small-molecules (100, 200 and 500 µM) and rBoNT/A-LC (25 pM, 200 nM and 6 µM) were used. In control assays, positive control was accomplished without test compound and rBoNT/A-LC and negative control test compound was replaced by DMSO. The incubation was carried out at 37°C for 15 min. The reactions were stopped by adding 4× SDS-PAGE sample buffer followed by heating at 100°C for 5 min. 5. Determination of the IC50 Fifty-percent inhibitory concentration (IC50) values for rBoNT/A-LC were calculated from nine concentrations of compound via dose-response measurements. The reactions were set up in duplicate as described earlier with the decreasing concentrations of inhibitors, keeping the concentration of rBoNT/A-LC constant at 10 nM. Each value is the average of two independent determinations. In all the cases, standard deviations were less than ±20%. 6. Western Blot Analysis Proteins from catalytic reactions were separated on a 13% SDS-PAGE before transfer to a 0.2 µ nitrocellulose membrane for 60 min at 100 V. After blocking in 5% skim milk/PBS for 2 h at room temperature (RT), the membrane was washed three times for 5 min at room temperature with PBST [2 mM Phosphate buffer (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20(v/v)]. Rabbit anti-SNAP-25 antibody against N-terminal SNAP-25 protein (Sigma, USA) was added at the dilution of 1∶5000 in PBS and the blot was incubated for 1 h at RT and washed four times with PBST. Goat anti-rabbit HRP conjugate antibody at a dilution of 1∶30000 into PBS was added and further incubated for 1 h at RT. Membrane was again washed with PBST four times, each of 10 min. Bound antibodies were detected by chemiluminescence using an ECL western blot kit (Biological Industry, Israel) as per manufacturer's instructions. Film was exposed for 15 s before development. The inhibitory action (%) of the molecules was evaluated by densitometric analysis of blots using GS800 densitometer and Quantity One software (BioRad, USA). 7. Evaluation of Small-molecule Inhibitors in Mouse Model Four inhibitor molecules were selected for in vivo experiment based on in vitro results. Female Balb/c mice (20–25 g weight) were used for in vivo experiments and they were divided into seventeen groups having five mice each (four groups for each molecule and one group for BoNT/A control). Mice from one group received 5LD50 of BoNT/A (in 200 µl PBS) intraperitoneally (ip). All four compounds were tested using four groups of mice as described below. To determine the toxicity of compounds, each of the molecule (1 mM) solubilized in PBS/DMSO (9∶1) was injected per mouse ip in second group. In third group, 100 µl (1 mM) of small-molecule inhibitor mixed with 5 LD50 of BoNT/A holotoxin was injected ip. Fourth group received 5 LD50 of BoNT/A, 30 min before the inhibitor injection. In last group of animal (fifth group) 100 µl each (1 mM) of small-molecules were injected 30 min prior to challenge with 5 LD50 of BoNT/A. All mice were examined for 4 days at hourly interval for survival, behavior, breath, and extraocular symptoms of botulism. All the animal experiments were approved by the Laboratory Ethical Committee on Animal Experimentation of Defence Research and Development Establishment, Gwalior, India via permission no. BT/01//DRDE/2009 and all efforts were made to minimize suffering. Results and Discussion In the wake of the events of September 11, 2001, research efforts aimed at the discovery of potent antagonists for agents of bioterrorism have increased exponentially. However, despite the plethora of new data that has emerged in the past 5 years, an established pharmacophore validated through in vivo models of exposure remains elusive. Indeed, in the case of BoNT, few studies have reported the assessment of any small-molecule antagonist in animal models [31], [36]. The development of small-molecule inhibitors as intraneuronal therapeutics is a crucial unmet need. Our initial impetus for producing recombinant LC as a reagent to be utilized in synaptosome based assay for the screening of potential inhibitors of the BoNT/A-LC protease activity. A 1401 bp DNA fragment encoding BoNT/A-LC along with 14 amino acids of translocation domain and 15 amino acids of N-terminal heavy chain was selected to produce rBoNT/A-LC as already reported in our previous study [38]. 1. Strategy and Virtual Screening We focused on serotype A since it is the most prevalent and well-studied among the various serotypes in human intoxication. We used in silico screening to identify BoNT/A inhibitors. The ChemBridge and NSC libraries, consisting of millions of compounds of unknown function, were chosen for virtual screening to chemoinformatically “mine” novel small-molecule non-peptidic inhibitors (SMNPIs). Since some of these compounds were commercially available and their functions currently undefined, we reasoned that novel inhibitors could be identified. Peptidomimetics and hydroxamic acid-based inhibitors have been developed that display inhibitory effects in the high nM range for the light chain of the BoNT serotype A (BoNT/A -LC) [26], [34], [35], [40], [42], [43]. Compounds that contain zinc-coordinating sulfhydryl moieties might potentially inhibit host zinc proteases thereby making them poor therapeutic leads. The characteristically poor pharmacokinetics of hydroxamates, their instability, and their reported toxicity, which is likely due to their inhibition of an array of metalloproteases, also make them problematic as therapeutic agents [44], [45]. We focused on the 8-hydroxy quinolinol lead NSC 84096 for database search. Selection of this compound was based on: (i) NSC 84096 is reported to be very potent and serotype A selective inhibitors [24]; (ii) it is reported to be non toxic and active in cell based and mouse phrenic nerve hemi-diaphragm (MPNHD) assays [24]; (iii) there are quinolinols in clinical trials for Alzheimer's disease and cancer [46]–[48]; and (iv) quinolinol-based drugs such as linolasept and vioform (generic name: Clioquinol) are available in the market. The compounds from the NCI database were docked into the active site in one of the three dimensional structure of BoNT/A-LC (PDB code: 3BON) [41] after removing the peptide occupying the active site. The top scoring 100 compounds were evaluated in detail; the list was narrowed to 25 compounds (based on binding energy and Ki) that interacted well with the active site Zn and demonstrated a ‘good fit’ in the BoNT/A-LC binding site (Table S1). Among these, twelve compounds were studied in detail and in silico parameters obtained were summarized in table 1. Other thirteen molecules were not available for the in vitro and in vivo studies. 10.1371/journal.pone.0047110.t001Table 1 In silico docking parameters obtained for selected Quinolinol derivatives used in the study by AutoDock 4.1. S No Compound ID Ki (nM) Binding energy Number of interactions Interaction between Vdw_hb_ desolve energy Electrostatic energy Total internal energy Torsional energy Ref RMS 1 CB6377128 539.2 pM −12.64 32 ASP203:N1, GLU197:N1(OH), LEU367:N1(S), Tyr366:OH, Phe163:NH −7.18 −0.06 −2.84 1.37 14.89 2 CB7969312 28.3 −10.3 1 TYR366-OH −8.66 −0.11 −2.78 1.92 11.79 3 CB7967495 4.26 −11.42 1 LYS166-N of C6H6 −6.75 −0.16 −2.3 1.65 14.92 4 CB7925339 1.04 −12.25 1 GLU262-OH −7.74 −0.12 −2.14 1.92 19.46 5 CB7887535 3.63 −11.51 1 TYR366-OH −7.47 −0.03 −2.04 1.37 12.04 6 CB6378306 6.07 −11.21 2 LYS166:O-CH3 −6.36 −0.17 −2.05 1.92 17.13 7 CB6376015 76.61 −9.71 5 THR365:N0,N1,O1(3) −7.38 −.05 −2.57 1.1 16.48 8 NSC1010 22.36 −10.44 1 GLU262-NH −7.08 −0.86 −2.72 1.37 10.85 9 NSC84090 2.11 −11.83 1 Glu164-NH −6.7 −0.06 −2.23 1.37 10.96 10 NSC84096 25.09 −10.37 1 Glu164-NH −8.18 0.14 −3.05 1.1 13.5 11 NSC84087 52.95 −9.93 1 Glu164-NH −6.72 0.03 −3.18 1.37 12.87 12 NSC84093 145.66 −9.33 2 Glu266-NHCys165-OH −6.67 −0.04 −3.0 1.37 16.01 The particular class of quinolinol identified in our study is reported to specifically inhibit BoNT serotype A and does not inhibit simply by chelating active-site zinc. The structures of the quinolinol derivatives under investigation contain additional basic moieties including 2-amino or 3-amino pyridine (Table S1). The presence of these structural motifs suggests that these molecules may interact with the hydrophobic pocket located in the active site of the BoNT/A-LC and interact with Tyr366 and Val258. The quinolinol moiety alone in the presence of zinc does not inhibit the proteolytic activity of BoNT serotypes A and B as described by Adler et al. [49]. Data obtained from in silico docking along with the in vitro inhibition at 100 µM concentration of quinolinol derivatives used in the study is summarized in table S2. As shown in figure 1, NSC 84087 is docked in the large hydrophobic pocket of the BoNT/A-LC active site, and its hydroxy quinoline moiety coordinates with zinc. The methoxy group of aniline ring can form a hydrogen bond with His227, which coordinates with zinc, and may contribute to the specificity and potency of this inhibitor. Additionally, the phenyl group is found to fit between Glu164 and Cys165 which are reported to participate in substrate binding [40]. This could explain the importance of hydroxy group in inhibiting BoNT/A-LC, and suggests that the quinolinols inhibit BoNT/A by blocking the active site zinc. It should be noted that the crystal structures of the complexes of known small-molecule and peptide inhibitors with BoNT/A-LC have shown that chelation to zinc is involved in the binding and inhibition of the light chain in both cases [40], [41]. 10.1371/journal.pone.0047110.g001Figure 1 Binding mode of NSC84087 into BoNT/A-LC substrate binding cleft showing ligand (grey) interacting with Zn atom (green) and other amino acids (ie HIS 227, GLU 164 and GLU 262). 2. Inhibition of rBoNT/A-LC using Synaptosome Model BoNT-LCs are remarkable among proteases for the extremely long substrate required for efficient proteolysis, whereas other microbial metalloproteases have been found to display activity against as short as dipeptides [50]. The catalytic LC domain of BoNT/A is a compact globule consisting of a mixture of α-helices, β-sheets, and α -strands with a zinc-containing metalloprotease active site bound deeply inside a large open cavity [19]. The remarkable substrate selectivity of BoNT/A for SNAP-25 has been explained to be a consequence of extensive interactions with two exosite domains distinct from the active site [51]. A model for substrate recognition has been proposed in which α-exosite binding occurs first via helix formation in the appropriate region of SNAP-25, followed by β-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage [19]. This model argues that, without exosite binding, BoNT/A-LC is a significantly less efficient enzyme, and thus these regions could be targeted for lead development. BoNT/A-LC requires a minimal SNAP-25 peptide sequence of ∼51 amino acids to achieve efficient cleavage, and optimal binding occurs with only the full length SNAP-25 [50]. The crystal structure of SNAP-25 (residues 141–204) bound to BoNT/A-LC (residues 2–420 with active site mutations E224Q, Y366F) provides an explanation for this finding as binding involves protein exosites that anchor the substrate and position the scissile bond for cleavage [19]. BoNT/A-LC recognizes multiple sites within SNAP-25; an extended surface on SNAP-25 distanced from the site of cleavage [19], [50] and residues adjacent to the scissile bond that are discontinuous and appear as pockets surrounding the cleavage site [51]. This implicates multistep recognition of SNAP-25 for cleavage by BoNT/A-LC. Intracellular BoNT/A-LC is shown to directly bind SNAP-25 on the plasma membrane. Solid phase binding showed that the N-terminal residues of BoNT/A-LC bound residues 80–110 of SNAP-25, which was also observed in cultured neurons. Association of the eight N-terminal amino acids of BoNT/A-LC and residues 80–110 of SNAP-25 also enhanced substrate cleavage by 2 folds. Two regions (80–110) and (180–197) of SNAP-25 contribute to the high affinity binding to BoNT/A-LC [20]. The LC of BoNT/A specifically cleaves the C-terminal 9 amino acids residues of SNAP-25, between residues Gln197 and Arg198 of the 206, thereby producing a 24 kDa cleaved protein [12]. Analytical techniques have been developed that directly assess SNAP-25 cleavage in cell lysates by immunoelectrophoresis [39]. We analyzed the amount of intact versus cleaved SNAP-25 western blotting to determine BoNT/A-LC activity/inhibition using rat brain synaptosomes. Screening was conducted by using a recombinant catalytic light chain of BoNT/A, produced in a soluble and stable form that can be easily expressed in E. coli at high levels and purified in large quantities necessary for screening. The extent of cleavage of SNAP-25 in synaptosomes by rBoNT/A-LC was determined by western blot analysis using monoclonal antibodies against SNAP-25. The selected compounds were initially screened for inhibition of rBoNT/A-LC mediated cleavage of synaptosomal SNAP-25 isolated from rat. NSC 84090 and Tetra peptide inhibitor RRGC were not showing any inhibition, however complete inhibition was recorded with 100 µM of CB796312, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC84096, like other previously reported compounds NSC 1010 and NSC 84096 as shown in the figure 2. Whereas CB7967495 & NSC84093 showed decrease in inhibition (87 and 85% respectively) of endopeptidase cleavage of SNAP 25 using the same concentrations of rBoNT/A-LC (200 nM). From this experiment seven new lead analogs (CB796312, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC84096,) at 100 µM were found to be exhibited complete protection of rBoNT/A-LC (200 nM) mediated SNAP-25 cleavage. Among these seven new molecules, five compounds (CB7925339, CB7887535, CB6378306, CB6376015 and NSC 84087) showed near-complete to- complete inhibition of SNAP-25 cleavage at 10 µM, while CB796312 afforded only 78% protection (Fig. 3). Surprisingly remaining one molecule CB6377128 did not show any significant inhibition of endopeptidase activity at 10 µM (Fig. 3) whereas it had shown protection at 100 µM (Fig. 2). The molecule NSC 84093 showed again decrease in protection from 85% at 100 µM (Fig. 3) to 58% at 10 µM (Fig. 2). 10.1371/journal.pone.0047110.g002Figure 2 Initial screening of different compounds by western blotting using specific antibodies. RRGC and twelve other compounds (CB7969312, CB7967495, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC1010, NSC84096, NSC84090, NSC84087 and NSC84093 were tested for their inhibition activity. To determine inhibition activity of these compounds, 2.05 µg synaptosomes prepared from rat brain was incubated with 200 nM of rBoNT/A-LC and 100 µM of each compounds solubilized in DMSO at 37°C for 15 min as described in materials and methods. Proteins were visualized using appropriate HRP-labeled second antibody by ECL. Nine molecules (CB796312, CB7925339, CB7887535, CB6378306, CB6376015, CB6377128, NSC1010, NSC84096, and NSC84087) showed complete inhibition while CB 7967495 & NSC84093 showed 87 and 85% of inhibition respectively. Significant inhibition was not observed with RRGC and NSC 84090 molecules. 10.1371/journal.pone.0047110.g003Figure 3 Determination of inhibition activity of eight selected compounds at 10 µM. In this experiment, reaction without inhibitor was used as a negative control and with RRGC (known inhibitors) for comparison. Near complete to complete inhibition of rBoNT/A-LC endopeptidase activity was observed by CB7925339, CB7887535, CB6378306, CB6376015 and NSC 84087 molecules. NSC 84093 and CB796312 showed 58 and 78% inhibition, respectively. No inhibition was observed with compound CB 6377128. As shown in figure 4, NSC84087 showed dose dependent inhibition of endopeptidase activity of rBoNT/A-LC (10 nM), the IC50 is estimated to be 40 nM (±3.7 nM). This indicates that 50% BoNT/A-LC inhibition is achieved when the inhibitor: rBoNT/A-LC molar ratio is 4∶1; however 100% inhibition was recorded at 6∶1 molar ratio. The extent of inhibition and the IC50 values of these leads were comparable to or even better than those of previously reported small-molecule inhibitors [(10000∶1) Park et al. [28]; (3000∶1) Burnett et al. 2007 [35]; (800∶1) Tang et al. 2007 [21]; (150∶1) Eubanks et al. 2007 [36]; (320∶1) Cai et al. 2010 [52]; (2000∶1) Pang et al. 2010 [31]; (3000∶1) Ruthel et al. 2011 [37]; (250∶1) Burnet et al. 2010 [32]; (350∶1) Thomson et al. 2011 [53], molar ratios given in parenthesis are of inhibitor: BoNT/A-LC]. The molar ratio between inhibitor and BoNT/A-LC molecules at 50% inhibition provides better parameter for comparison between assays, since it normalizes the neurotoxin used. The lower molar ratio in synaptosome based assay reflects the inhibitor's efficiency is more relevant to in vivo conditions. In fact, the IC50 values of the compounds increased (worsened) 4- to over 50-fold when the concentration of one of the model enzymes, β-lactamase, was increased 10-fold, from 1 nM to 10 nM [54]. The authors also suggested for the unifying mechanism to be followed for the discovery of the inhibitors. 10.1371/journal.pone.0047110.g004Figure 4 Western blot showing dose dependent inhibition of rBoNT/A-LC endopeptidase activity by NSC 84087. Three different concentrations (10, 1 and 0.10 µM) of NSC 84087 molecule were incubated separately with 10 nM of rBoNT/A-LC and 2.05 µM of rat brain synaptosomes at 37°C for 15 min as described in materials and methods. Complete inhibition was observed at 10 and 1.0 µM. Results from Western blots are representative of two independent preparations. DMSO was used in one of the reaction to observe the effect of solvent on the inhibition. Major advantages of a synaptosome based assay are: (i) it provides full length SNAP-25 substrate to β-exosite recognition and subsequent conformational changes in the enzyme to facilitate efficient substrate cleavage. It has been considered that, without exosite binding, BoNT/A is a significantly less efficient enzyme; and (ii) it simulates the microenvironment of the neurons. The other advantages are considerable reduction in the number of animals used (millions of reactions can be performed from synaptosomes isolated from single rat brain), substrate stability (synaptosomes isolated are stable for use as substrate for more than year), instrumentation requirement and cost incurred, especially when used for assessing large numbers of target molecules. We suspect the use of small peptide substrates (∼17mer peptides/fluorescent substrate which is minimum substrate required for BoNT/A) in high-throughput screening and identification of small-molecule inhibitors is the reason for in vitro and in vivo disconnect which is reported by Eubanks et al. [36]. In majority of such studies carried out for the development of small-molecule inhibitors against botulinum neurotoxins, higher potency of small-molecule inhibitors observed during evaluation in the in vitro assay systems using small peptide substrate, may have resulted due to the suboptimal activity of BoNT/A-LC. As these small substrate peptides used for in vitro screens are not long enough to simultaneously occupy cleavage site and either of the exosite, hence it is clear inhibition in fluorescent peptides based assays relies on the interactions in the enzyme active site. However, our findings argue that in the context of BoNT therapeutics, caution should be used in extrapolating in vivo potency from these assays. 3. Evaluation of Small-molecule Inhibitors in Mouse Model To be useful as therapeutics, the newly identified inhibitors must: (i) be able to enter neurons; (ii) inhibit the toxin within the neuronal cytosol; and (iii) be tolerated by animals (i.e. possess acceptable toxicity profiles). A true test or ultimate goal for inhibitors evaluated in both cell-free systems and even for cell based assays is whether their effectiveness holds true in vivo. The effectiveness of small-molecule inhibitors in the in vitro and ex vivo assays was only demonstrated when the compound was premixed with BoNT/A; thus far, pre-loading the inhibitor did not protect cells/tissues against BoNT intoxication [24]. The small-molecule inhibitor that was reported to be active in primary neurons [22] was demonstrated to show a dose-dependent inhibition of SNAP-25 cleavage in a non-pre-loading system (cells were pretreated with inhibitor for 45 minutes followed by incubation with BoNT/A in the continuous presence of inhibitor). Additionally, the inhibitors reported by Eubanks et al. [36] and Boldt et al. [34] were characterized in cell culture assays that involved mixing BoNT/A toxin and varying concentrations of inhibitor. To our knowledge, no group has been able to provide experimental evidence showing that their inhibitors work in a pre-loading system. After completion of our in vitro screening, three compounds were deemed to have suitable activity and were advanced into animal models (CB 7887535, CB6378306 and NSC 84087). The fourth compound, included for comparison, was a molecule previously well characterized and reported to inhibit specifically BoNT/A-LC (NSC 1010). To examine the lead compounds in vivo, a well-established mouse bioassay was used. This model is the Food and Drug Administration (FDA) standard for assessing BoNT levels and the universally accepted method for the study of BoNT antagonists (e.g., antibodies, small-molecules) [55]. For evaluation of inhibitory potential of small-molecules, they were injected into test animals as described in materials and methods. All animals were monitored continuously for a period of 4 days for signs of botulism, and the time of death was recorded. Of the compounds studied, one compound (NSC 84087) showed efficacy in preventing BoNT/A-induced death in all three modes of injections and an injection dosage of 100 µl of 1 mM per animal survived the BoNT challenge with no obvious signs of botulism upto 20 h (Fig. 5). All the mice of inhibitor followed by BoNT/A group, showed symptoms after 20 h and died within 30 h of injection. Mice of other two groups, BoNT/A followed by inhibitors and inhibitors toxin premixed, survived upto 48 h. In similar in vivo studies, conducted by Eubanks et al. [36] and Pang et al. [31] reported mere 36% increase time to death and 10% of mice survival after days of standard observation period, respectively. The second compound CB 7887535 extended the time to death from ∼9 to ∼20 h, corresponding to a more than 100% increase in time to death. Although this appears modest at first glance but this enhancement is also remarkable considering potency of the neurotoxin. In contrast, animals treated with NSC 1010 and CB6378306 molecules at similar doses died without any statistically significant increase in the time to death relative to control group (between 8–10 h). In all cases, no toxicity was observed from treatment with either inhibitor compound alone. 10.1371/journal.pone.0047110.g005Figure 5 In vivo evaluation of selected small-molecules in mouse model against 5LD50 of BoNT/A. NSC 1010 and CB6378306 were found incapable in inhibiting BoNT endopeptidase activity in mouse as a result of which animals treated with these groups died along with control (mice treated with 5LD50 of BoNT/A only), although mice treated with CB 7887535 molecules resulted in extended survival time from ∼9 h to ∼20 h. The group of animals either treated with 5LD50 of BoNT/A followed by NSC84087 molecules or treated with the mixture of NSC84087 & 5LD50 of BoNT/A showed survival upto 48 h. Results are expressed as an average of three independent experiments ± s.d. Summary Rat brain synaptosome model has been used to in vitro screen small-molecule inhibitors which can more accurately predict in vivo efficacy. Using this model, five highly potent non-toxic BoNT/A small-molecule inhibitors have been identified in present study. These compounds effectively inhibited the protease activity rBoNT/A-LC and the compound NSC 84087 is able to protect against BoNT/A challenge and encourages the pursuit of small-molecule BoNT inhibitors for the development of next generation botulism therapeutics. We recognize that these leads are viable drug candidates, and, they need to be optimized for other important drug characteristics including absorption, distribution, metabolism, excretion and toxicity (ADMET) for further clinical use. Supporting Information Table S1 List of compounds selected based on similarity search (1–20 New leads and 21–25 reported molecules) with binding energy and Ki obtained by docking. (DOC) Click here for additional data file. Table S2 Structural formula and results of initial screening at 100 µM of small-molecules on synaptosome against recombinant full-length BoNT/A light chain (200 nM). (DOC) Click here for additional data file. 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PLoS One. 2012 Oct 11; 7(10):e47110
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23071632PONE-D-12-2069510.1371/journal.pone.0046769Research ArticleBiologyBiochemistryBiotechnologyDevelopmental BiologyCell DifferentiationGeneticsGene ExpressionMolecular Cell BiologyGene ExpressionMedicineOncologyCancers and NeoplasmsBone and Soft Tissue SarcomasOsteoblastomaInvestigation of FGFR2-IIIC Signaling via FGF-2 Ligand for Advancing GCT Stromal Cell Differentiation FGF-2 Induces Stromal Cell DifferentiationSingh Shalini 1 Singh Mohini 1 Mak Isabella W. Y. 1 Turcotte Robert 2 Ghert Michelle 1 * 1 Department of Surgery, McMaster University, Hamilton, Ontario, Canada 2 Department of Orthopaedic Surgery, McGill University Health Centre, Montreal General Hospital, Quebec, Canada Heymann Dominique Editor Faculté de médecine de Nantes, France * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SS MG. Performed the experiments: SS MS. Analyzed the data: SS IWYM. Contributed reagents/materials/analysis tools: RT. Wrote the paper: SS. Edited the manuscript: MG. 2012 11 10 2012 7 10 e467696 7 2012 7 9 2012 © 2012 Singh et al2012Singh 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.Giant cell tumor of bone (GCT) is an aggressive bone tumor consisting of multinucleated osteoclast-like giant cells and proliferating osteoblast-like stromal cells. The signaling mechanism involved in GCT stromal cell osteoblastic differentiation is not fully understood. Previous work in our lab reported that GCT stromal cells express high levels of TWIST1, a master transcription factor in skeletal development, which in turn down-regulates Runx2 expression and prevents terminal osteoblastic differentiation in these cells. The purpose of this study was to determine the upstream regulation of TWIST1 in GCT cells. Using GCT stromal cells obtained from patient specimens, we demonstrated that fibroblast growth factor receptor (FGFR)-2 signaling plays an essential role in bone development and promotes differentiation of immature osteoblastic cells. Fibroblast growth factor (FGF)-2 stimulates FGFR-2 expression, resulting in decreased TWIST1 expression and increased Runx2, alkaline phosphastase (ALP) and osteopontin (OPN) expression. Inhibition of FGFR-2 through siRNA decreased the expression of ALP, Runx2 and OPN in GCT stromal cells. Our study also confirmed that FGF-2 ligand activates downstream ERK1/2 signaling and pharmacological inhibition of the ERK1/2 signaling pathway suppresses FGF-2 stimulated osteogenic differentiation in these cells. Our results indicate a significant role of FGFR-2 signaling in osteoblastic differentiation in GCT stromal cells. This study was supported by a Canadian Institutes of Health Research (CIHR) grant, the Hamilton Health Science New Investigator Fund, a Hamilton Health Science Early Career Award, a Juravinski Cancer Centre Foundation grant, and a McMaster University Surgical Associates grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Giant cell tumor of bone (GCT) is an aggressive osteolytic and potentially metastatic bone tumor. GCT typically prompts the formation of a local osteolytic lesion at the epiphyseal regions of the long bones such as the distal femur, the proximal tibia, and the distal radius [1]. High recurrence rates of 18–60% following aggressive surgical resection have been reported for GCT, which occasionally undergoes malignant transformation [2]–[5]. Cell culture experiments have shown that the preosteoblast-like GCT mesenchymal stromal cells are the only proliferating component of GCT, and are arrested in an immature differentiation state [6], [7]. The formation of skeletal elements is controlled by a complex network of signaling molecules that regulate the differentiation of mesenchymal stromal cells into osteoblasts and terminal differentiation into osteocytes under appropriate stimulation by hormones and local factors such as fibroblast growth factors (FGFs) [8]–[10]. FGF signaling plays an essential role in bone development, promoting proliferation of immature osteoblast/osteoprogenitor cells and increasing apoptosis upon exposure of cells to differentiation media [11], [12]. Four fibroblast growth factor receptor genes (FGFR1–4) have been identified in mammalian developmental processes. The specificity of FGFR1–4 is regulated in a tissue specific manner. FGFR-1 acts as a transducer of FGF signals in osteoblast proliferation [13]. In contrast FGFR-2 has been shown to enhance osteoblast differentiation in mesenchymal stem cells [14], [15] whereas FGFR3 and 4 are generally restricted to chondrocytes [16], [17]. Splice variants of the FGFR-2 gene are classified by their ability to bind specific ligands [18]. FGF receptor 2-IIIc (FGFR2-IIIc) has the ability to bind both FGF-1 and FGF-2 with a high affinity due to its possession of the IIIc exon [9], [18]–[21]. The FGFRs are tyrosine kinases which possess three extracellular immunoglobulin-like domains, a trans-membrane region and a cytoplasmic split tyrosine kinase domain which is activated upon FGF binding [22]. FGF binding to FGFR leads auto phosphorylation of intracellular tyrosine residues. FGFR phosphorylation facilitates the recruitment of numerous signaling proteins [23] which subsequently activates various signaling pathways downstream of FGFR, including the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway. ERK1/2 is one of the main downstream targets of activated FGFRs. In the bone environment, activation of ERK1/2 has been found to enhance osteoblast gene expression [24]. The transcription factor TWIST1 also plays an important role in bone and cranial suture development, and is expressed in skeletal mesenchymal cells, primary osteoblastic and preosteoblasts cells. Runx2 is a master osteogenic regulator and acts as an inducer and regulator of osteoblast differentiation in the osteoblast lineage [25]–[28]. We have previously observed a high expression of TWIST1 in GCT stromal cells [29]. TWIST1 is an upstream regulator of Runx2 that acts to downregulate Runx2 expression, prevent terminal osteoblastic differentiation, and plays an important role in specifically disrupting the balance in bone formation and resorption in GCT [29]. However, the mechanism through which TWIST1 regulates GCT stromal cell differentiation remains unclear. Based on our previous work, we hypothesized that FGF-2 ligand signaling through FGFR2-IIIc receptor suppresses TWIST1 expression and may have a positive effect on the commitment and differentiation of osteoblast precursor cells. In this study, our main focus was to investigate the FGFR2-IIIC signaling via FGF-2 ligand for GCT stromal cells differentiation. We have investigated the effect of FGF-2 signaling on GCT cells in osteogenic differentiation and determined the mechanisms involved in the regulation of osteoblast commitment and differentiation. We have also studied the role of FGFR2-IIIc in the regulation of the TWIST1 and Runx2 osteoblastic transcription factors and its activation of the ERK1/2 signaling in GCT stromal cells. Materials and Methods Ethics Statement We established primary cell cultures of GCT stromal cells from fresh GCT tissue obtained from four patients following the Hamilton Health Sciences and McMaster University Ethics Board approval and written patient consent. Primary cell culture and FGF-2 treatment The tissue was processed and maintained in DMEM containing 10% FBS, 2 mM glutamine and 100 U/mL streptomycin antibiotics. The resulting cell suspension together with macerated tissue was cultured in 37°C humidified air with 5% CO2. Following several successive passages, the mesenchymal stromal cells became the homogeneous cell type whereas the multinucleated giant cells were eliminated from culture. Primary cultures of the proliferating homogenous stromal tumor cell population obtained after the fifth or sixth passage (without any hematopoietic markers) and up to the tenth passage were used for experiments [30], [31]. GCT-stromal cells were incubated in media containing 50 nmol/L ascorbic acid, 3 mM inorganic phosphate (NaH2PO4) and a range of dilution from 10 to 40 ng/ml of FGF-2 (Minneapolis, MN). Human fetal osteoblast (hFOB) 1.19 cells (American Type Culture Collection, ATCC# CRL- 11372) were used as a control cell line to compare the endogenous FGFR-2IIIc expression in GCT stromal cells. The hFOB cell line is a clonal, conditionally immortalized human fetal cell line capable of osteoblastic differentiation and bone formation. Plasmid construction A TWIST1 open-reading frame was amplified by PCR from the plasmid purchased from Origene (Rockville, MD) using oligonucleotides cgcggatccgcgatgatgcaggacgtgtcc and ccggaattccggctagtgggacgcgacat, containing BamHI and EcoRI restriction sites, respectively. After BamHI and EcoRI digestion, the open-reading frame was ligated into a pcDNA3.1 vector and confirmed by sequencing. GCT cells were transfected using an electroporation method [29]. After 48-h post-transfection, cells were harvested for RNA isolation. RNA Isolation and RT-PCR Total RNA extraction was performed by TRIZOL Reagent (Sigma) according to the manufacturer's protocol. The resuspended RNA samples were treated with ribonuclease A (RNase A)-free DNaseI for 1 h at 37°C to remove residual genomic DNA. One microgram of total RNA was incubated with 2 ml primer cocktail at 68°C and subjected to reverse transcription (RT) using Superscript III reverse transcriptase (Invitrogen) for cDNA synthesis. PCR was carried out using Prime Taq Premix and reactions were carried out in the PCR thermal cycler (Applied Biosystems, Foster City, CA, USA). Expression of the ribosomal protein 18 (RPS18) was used as internal control. Primer sequences are listed in Table 1 . 10.1371/journal.pone.0046769.t001Table 1 Primers sequences designed for real time RT-polymerase chain (PCR) reaction. Gene symbol Oligonucletides ALP CATTGGCACCTGCCTTACTA ACGTTGGTGTTGAGCTTCTG RunX2 TCTGGCCTTCCACTCTCAGT AAGGTGGCTGGATAGTGCAT TWIST TACATCGACTTCCTCTACCAGGTC TAGTGGGACGCGGACATGGA OPN AGTTTCGCAGACCTGACATCCAGT TTCATAACTGTCCTTCCCACGGCT RPS18 GATGGGCGGCGGAAAATAG GCGTGGATTCTGCATAATGGT Protein Extraction and Western blot analysis for TWIST1, Runx2 and Osteopontin To detect protein expression, we isolated protein from FGF-2 treated and untreated GCT stromal cells. After 48 h of incubation, cells were lysed with NP-40 containing lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.5% NP-40) to disrupt the cell membrane and the cell lysate was centrifuged at 500 g for 5 min at 4°C. The supernatant (cytoplasmic fraction) was removed. Proteins were denatured by boiling in sample buffer, separated on 12% SDS–PAGE, transferred onto a PVDF membrane (Immobilon TM-PSQ, Millipore), and blocked overnight in 5% non-fat powdered milk in TBST (10 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1% (v/v) Tween-20). Mouse monoclonal anti-TWIST1 antibody, mouse anti-Runx2 antibody, and mouse anti-osteopontin antibody, rabbit anti ERK1/2 and rabbit anti phosphorylated ERK1/2 (1∶1,000 diluted in TBST) (Abcam) were used for protein detection. Peroxidase conjugated goat anti-mouse, and anti-rabbit IgG (1∶5,000 diluted in TBST) (Promega) was used as a secondary antibody. Western blots were quantified with Image J program. Alizarin Red staining for bone nodule formation Mineralization was assessed in FGF-2 treated GCT stromal cells on day 28 using an Alizarin Red-S staining assay. Cells were washed twice with PBS and fixed in 10% neutral buffered formalin (Fisher Scientific) for 30 min at room temperature. After washing with distilled water twice, cell were fixed and the matrix stained for 10 min with 0.02 g/mL Alizarin Red-S stain (pH 4.2) (Sigma-Aldrich) and visualized with an Olympus microscope to identify the presence or absence of bone nodule formation. ALP activity assay The cells were washed with PBS and lysed with NP-40 containing lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 5 mM MgCl2, 0.5% NP-40) solution. ALP activity of the lysates was determined using p-nitrophenol-phosphate as a substrate. In brief, 50 µl of the cell lysate supernatant was mixed with 50 µl of the substrate solution (p-nitro-phenyl phosphate substrate solution diluted in ALP buffer for ALP measurement or p-nitro-phenyl phosphate substrate solution diluted in acid phosphatase buffer and incubated at 37°C for 30 min. Stop solution (50 µl, 0.9 N NaOH) was added to each well and the absorbance was measured at 405 nm with a spectrophotometer. Total protein content in aliquots of the same samples was determined using Bradford reagent (Sigma-Aldrich). The amount of ALP activity was divided by the amount of total protein for normalization. siRNA Transfection Mesenchymal stromal cells of GCT were trypsinized and transfected with FGFR-2 small interfering RNAs (siRNAs) via electroporation. The cells were then washed and resuspended in Optimem reduce serum media (Gibco, Invitrogen, Canada). Subsequently, the cell suspension was mixed with 200 nM of FGFR-2 siRNA (Invitrogen), a positive Silencer™ siRNA control against GAPDH, or a non-specific negative control#1 (Ambion Inc.). Stromal cells with the siRNA mixture were electroporated using the Gene Pulser II electroporation apparatus (Bio-Rad laboratories) under a single-pulse protocol with optimized combination of voltage and capacitance. After 48 h of transfection, cells were harvested for RNA isolation. RPS18 was selected as housekeeping genes for normalization in real time PCR analysis. Immunofluorescence assay Cells were grown on cover slips and were fixed with 4% paraformaldehyde for 20 min at room temperature (RT), and permeabilized with 0.2% Triton x-100 for 5 min. Subsequently, these slides were incubated for 1 h at RT with antibodies (anti-Osteopontin,). Slides were further incubated in secondary antibody (Texas red conjugated goat-anti rabbit and alexa 488 goat anti-mouse antibodies) for 1 h at RT. Slides were washed and incubated with DAPI for 3 min at RT and mounted with 50% glycerol and visualized with fluorescence microscopy. XTT cell proliferation assay The XTT assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by metabolically active cells. We seeded 1000 cells with 100 µl of culture medium with FGF-2 and without FGF-2 in a 96 well plate, and after 48 h of culture the XTT assay was performed following the manufacturer's protocol (Roche Diagnostics). The absorbance was measured at 450 nm using a UV plate reader. Signaling pathway inhibition The culture medium of the GCT stromal cells was replaced with serum-free medium containing ERK inhibitor 1 nM (R&D Systems). The effects of signaling pathway inhibitor kinase (ERK)-PD98059 with FGF-2 (20 ng/ml) were used. After 24 h of exposure to FGF-2 and inhibitor in GCT stromal cells the lysate were collected and analyzed. The expressions of TWIST1, ALP, OPN and OC were evaluated using real-time polymerase chain reaction (PCR). Statistical analysis Statistical analyses for the real-time PCR were performed using the two sample independent student's t-test. The average value within each experiment was expressed relative to the expression of the internal control gene. P-value of <0.05 was considered statistically significant. Results Expression of FGF receptor 2-IIIc in GCT stromal cells We determined the endogenous expression of FGFR2-IIIb and FGFR2-IIIc in GCT stromal cells obtained from four patient samples. FGFR2-IIIb and FGFR2-IIIc expression was analyzed with semi-quantitative RT-PCR using FGFR2-IIIb and FGFR2-IIIc specific primers. FGFR2-IIIb and FGFR2-IIIc endogenous expression was detected in all 4 GCT cell lines. Ribosomal protein S18 (RPS18) was used as an internal control ( Fig. 1a ). We further analyzed the expression of FGFR2-IIIb and FGFR2-IIIc by quantitative real-time RT-PCR. Interestingly, qRT-PCR analysis revealed significantly higher expression of FGFR2-IIIc in all GCT stromal cells when compared to hFOB 1.19 control cells using the two-sample independent student's t-test (P≤0.01) ( Fig. 1b ). Furthermore, we did not observe any quantitatively significant FGFR2-IIIb expression (data not shown). An increase was detected FGFR2-IIIc expression in response to FGF-2 ligand stimulation in GCT stromal cells ( Fig. 1c ). Therefore further experiments focused on FGFR2-IIIc signaling in GCT. 10.1371/journal.pone.0046769.g001Figure 1 Semi-quantitative PCR. (a). Endogenous expression of FGFR2-IIIb and FGFR-2IIIc in GCT stromal cells using FGFR2 specific primers. (b). Real-time PCR of cDNA from GCT stromal cell lysates showing expression of FGFR2-IIIc relative to hFOB 1.19 cells (“hFOB”). Results are the average of three replicate experiments. (c). An increase in expression of FGFR-2IIIc is in response to FGF-2 ligand in GCT stromal cell in a dose dependent manner. Results are the average of three replicate experiments. FGF-2 effects on bone nodule formation and cell proliferation in GCT stromal cells To investigate the role of FGF-2 signaling on osteoblastic proliferation and bone nodule formation, cells were treated with FGF-2 supplemented media for 4 weeks. We observed that FGF-2 treated cells promoted matrix mineralization and bone nodule formation in GCT cells when compared to untreated cells using alizarin and von kossa staining ( Fig. 2a, 2b ). To determine cell viability and proliferation, XTT assays were performed on GCT stromal cells with and without FGF-2 treatment. We observed decreased mitochondrial activity in GCT cells treated with an increasing FGF-2 dosage when compared to untreated GCT stromal cells after 48 h, indicating decreased proliferation and cell viability with FGF-2 treatment ( Fig. 2c ). Based on our results of the dose dependent assay and XTT assay, we selected the 20 ng/ml FGF-2 concentration for further experiments. Our results confirmed that FGF-2 increases bone nodule formation and decreases cell proliferation in GCT stromal cells in culture. 10.1371/journal.pone.0046769.g002Figure 2 Bone nodule forming assay. (a). GCT stromal cells were induced into osteoblastic differentiation by exposure to FGF-2. Alizarin staining was used for bone nodules, and visualized by phase-contrast microscopy. (b). Von Kossa staining were used to determined GCT stromal cells osteoblastic differentiation by exposure to FGF-2. Bone nodules are visualized by phase-contrast microscopy. (c). XTT assay. A decreased number of mitochondrial active cells were seen in FGF-2 treated cells when compared to untreated GCT stromal cells. GCT untreated cells were used as a control (CT). FGF-2 promotes osteogenic marker expression in GCT stromal cells We next determined the effect of FGF-2 on the molecular expression of markers for osteogenic differentiation in GCT stromal cells. Cells were treated with 20 ng/ml FGF-2 supplemented media. As shown in Figure 3a , FGF-2 increased osteopontin (an early osteoblastic marker), osteocalcin (a late osteoblastic marker) and alkaline phosphatase (mature osteoblast marker) mRNA and protein expression in vitro and verified by quantification analysis ( Fig. 3b ). In immunofluorescence microscopy, we also observed an increase of cell-cell attachment, which is required for bone nodule formation ( Fig. 3c ). Additionally, we observed an increase in intensity of alkaline phosphatase (ALP activity) in FGF-2 treated GCT stromal cells ( Fig. 3d ). 10.1371/journal.pone.0046769.g003Figure 3 FGF-2 promotes osteogenic differentiation in GCT cells. (a). qRT-PCR analysis representing an increase in osteogenic differentiating markers ALP, OPN, and OC (p<0.05). These results are verified at the protein level by Western blot analysis. (b). Quatification analysis showing an increasing OPN protein level in all FGF2 treated cell types when compared to untreated cells. (c). Subcellular localization of OPN in GCT stromal cells was detected by immunofluorescence using an anti-OPN monoclonal antibody. DAPI (blue) staining indicated the nuclei. F = FGF-2 treated cells. (d). GCT cells were incubated with and without FGF-2 (20 ng/mL) for 48 h. The cells were then analyzed for ALP activity. Data represent mean ±SD. *p<0.05. Effect of FGF-2 on transcription factors responsible for osteoblastic differentiation To confirm that FGF-2 enhances osteogenic differentiation signaling in GCT stromal cells, the cells were treated with supplemental media containing 20 ng/ml FGF-2 and compared with untreated GCT stromal cells. Both PCR ( Fig. 4a ) and Western blot ( Fig. 4b ) data confirmed an increase in Runx2 mRNA and protein expression with FGF-2 treatment; whereas TWIST1 expression was decreased in all GCT samples ( Fig. 4a, b and c ). These results indicate that FGF-2 signaling may enhance osteoblast differentiation by suppressing TWIST1 and up regulating Runx2 in cultured GCT stromal cells. 10.1371/journal.pone.0046769.g004Figure 4 FGF-2 stimulation effects on osteoblastic transcription factors in GCT cells. Quantitative RT-PCR, Western blot and quantification analysis representing a significant decrease in TWIST1 and a significant increase in Runx2 mRNA and protein levels in GCT cells treated with FGF-2 (GCT1F, and GCT2F,) when compared to the GCT untreated (GCT1, and GCT2) FGF-2 cells. TWIST1 inhibits osteogenic differentiation To determine the role of TWIST1 in primary GCT mesenchymal stromal cell differentiation, we generated a TWIST-pcDNA 3.1 construct and transfected as described in primary GCT mesenchymal stromal cells. PCR data confirmed successful transfection and overexpression of TWIST in two GCT cell lines ( Fig. 5a ). TWIST mRNA levels were elevated >8-fold in GCT-1 and >7-fold in GCT-2 transfected cells when compared to GCT untransfected cells. We observed a decrease in FGFR-2IIIc expression in TWIST overexpressing GCT stromal cells when compared to GCT untransfected cells ( Fig. 5b ). Furthermore, we indentified a decrease in matrix mineralization with TWIST1 over-expressing cells treated with FGF-2 compared to GCT cells ( Fig. 5c ). We next determined that TWIST1 over-expression in FGF-2 stimulated GCT stromal cells suppresses ALP, OPN and OC (osteoblastic differentiation markers) at the mRNA level ( Fig. 5d ). These results reveal that TWIST1 over-expressing GCT stromal cells inhibit FGF-2 stimulated osteoblastic differentiation of GCT stromal cells, possibly by inhibiting the expression of the FGF-2 receptor, FGFR-2IIIc. 10.1371/journal.pone.0046769.g005Figure 5 TWIST1 suppresses osteogenic differentiation. (a). Quantitative RT-PCR analysis representing higher TWIST1 expression in transfected GCTT1, GCTT2 cells compared to untransfected (UT) GCT stromal cells. (b). Quantitative PCR analysis showing a 60% decrease of FGFR2-IIIc expression in GCTT1 and a 50% decrease of FGFR2-IIIc expression in GCTT2 stromal cells (*p<0.05). (c). GCT cells were transfected with TWIST1 and matrix mineralization compared to control (CT) and FGF-2 treated GCT cells. (d). TWIST1 over-expression showed a significant decrease in ALP, OPN and OC mRNA levels in GCT cells treated with FGF-2 (GCTT1, and GCTT2,) when compared to the GCT untreated (UT) FGF-2 cells. FGFR2 knockdown and osteoblastic differentiation in GCT cells To determine that FGF-2 ligand enhances osteoblast differentiation and osteogenic capacity in cultured GCT stromal cells through FGFR-2IIIc signaling, FGFR-2IIIc expression was knocked down by transient transfection of siRNA in GCT stromal cells. We depleted FGFR-2IIIc expression in the mesenchymal stromal cells of GCT in two representative primary cell lines and subsequently measured the expression level of TWIST1, Runx2, and ALP mRNA. We successfully obtained approximately 70% FGFR2-IIIc mRNA knock down in both primary cell lines ( Fig. 6a ). Knock down of FGFR2-IIIc resulted in approximately a 1.5 fold increase in TWIST1 expression and a significant decrease in Runx2 and ALP expression ( Fig. 6b , p <0.01). 10.1371/journal.pone.0046769.g006Figure 6 The effect of FGFR-2IIIc siRNA on mRNA expression in the GCT stromal cells. (a). FGFR2 siRNA transfection resulted in a 70% decrease in FGFR-2IIIc expression in GCT stromal cells. (b). Quantitative RT-PCR was used to represent the expression of ALP, Runx2 and TWIST following FGFR-2IIIc knockdown (KO) in GCT cells. Results are the average of three replicate experiments. FGFR-2IIIc knockdown resulted a 1.5 fold increase of TWIST1 expression and a significant decrease in Runx2 and ALP expression (p<0.05). FGFR-2IIIc mediates osteoblast differentiation in GCT cells through ERK1/2 signaling ERK1/2 is one of the main downstream targets of activated FGFRs. In order to determine whether the ERK1/2 signaling pathway plays a role in osteoblastic differentiation in GCT stromal cells, we investigated changes in ERK1/2 signaling with increased expression of FGFR2-IIIc. We found that FGF-2 stimulation was associated with an increase in phosphorylated ERK1/2 expression ( Fig. 7a and b ). After confirming that phosphorylated ERK1/2 expression is stimulated by FGF-2 in GCT stromal cells, next we evaluated the effects of ERK inhibitor on the expression of TWIST1 and osteoblastic differentiation markers at the mRNA level. Treatment of GCT mesenchymal stromal cells from two representative primary GCT cell lines with the ERK-inhibitor PD98059 under FGF-2 stimulation resulted in a significant reduction of ALP, OPN and OC mRNA levels, with optimized 1 nM concentration of inhibitor ( Fig. 7c ).We also observed a slight increase in TWIST1 expression in both cell lines. Our results suggest that FGFR2 signaling plays an essential role in osteoblastic differentiation in GCT stromal cells, possibly via ERK signaling. 10.1371/journal.pone.0046769.g007Figure 7 FGF2 signaling through ERK in osteoblast differentiation in GCT stromal cells. (a) Western blot analysis (b) and quantification showed that FGF-2 stimulation resulted in increased phosphorylation of ERK1/2 (p-ERK1/2), compared to untreated GCT cells. β-Actin was used as loading control. (c). Inhibition of osteoblastic gene expression by ERK inhibitor in GCT stromal cells based on real-time RT-PCR. GCT stromal cells were treated with 1 nM ERK-inhibitor PD98059 (all diluted in DMSO) for 24 h in serum-free medium. mRNAs were purified, cDNAs were synthesized, and the samples were analyzed by real-time PCR. The ΔΔCT method was used to calculate the real-time RT-PCR fold change using RPS18 mRNA as an endogenous control, and all changes in expression are relative to the control without treatment. Three independent real-time PCR runs were performed on each sample. Discussion GCT is characterized by the presence of osteoclast-like giant cells, a mononuclear component, and mesenchymal stromal cells. To date, the oncogenesis of GCT remains unknown as the neoplastic cells appear to be preosteoblastic cells that do not undergo terminal osteoblastic differentiation [2], [3], [6]. Mesenchymal stromal cells are able to differentiate into mature osteoblasts under appropriate stimulation with FGFs or other proteins [11]. However, the mechanisms by which these factors control osteogenesis have not been fully elucidated. In this study, we have identified a regulatory pathway involving FGF-2 and FGFR2-IIIc, which plays a role in osteogenic differentiation in GCT stromal cells. FGFR2 signaling plays an essential role in skeletal development in mouse and human genetics [11]. Several FGFR expression profile studies indicated that FGFR1 and FGFR2 are the main receptors expressed in osteoblasts whereas FGFR3 is the most important growth factor for chondrocytes [11], [31]. However, the expression of specific FGFRs and their role in GCT stromal cell differentiation remains unknown. Several microarray and proteomics studies were conducted using giant cell tumor of bone and FGFR2 and FGF ligands were observed in vivo [32]. We observed very high expression of FGFR2-IIIc, but not FGFR2-IIIb, in GCT stromal cells when compared to human fetal osteoblastic (hFOB) 1.19 cells, suggesting that FGFR2-IIIc plays a prominent role in these cells. Higher expression of FGFR2-IIIc in GCT stromal cells could be an important transducer of FGF signals in osteoblastic differentiation. We also observed an increase in the expression of FGFR2-IIIc in response to FGF-2 ligand in a dose dependent manner. To determine a more precise role for FGFR2-IIIc in GCT stromal cell differentiation, cells were treated with FGF-2 and it was observed that FGF-2 promotes cell differentiation in GCT stromal cells. FGF-2 treated cells actively promoted matrix mineralization when compared to untreated GCT cells. Although others have found FGF-2 signaling to enhance cell proliferation in bone cells [9], [33], the specific function of FGF-2 in cell proliferation in bone tumor cells is poorly understood. To investigate this important concept, an XTT assay was performed and we observed that FGF-2 decreased GCT stromal cell proliferation. Cells treated with FGF-2 have a significant decrease in their growth rate, which may be due to an increase in differentiation. In general, upon differentiation of various tissues and cell lines from different organisms, proliferation is uniformly suppressed [34], [35]. Based on these findings, we sought to determine whether FGF-2 would functionally promote osteoblastic differentiation signaling in GCT stromal cells. Quantitative RT-PCR analysis showed that FGF-2 induced an approximately 2-fold increase in mRNA expression of Runx2 at 48h in culture. Runx2 is one of the earliest and most specific markers of osteoblast differentiation, capable of inducing differentiation by regulating expression of numerous osteoblast-specific genes [20]. Runx2 interacts with multiple co-regulating transcription factors and signaling proteins, forming multimeric complexes. These complexes then either transactivate or repress target genes during osteogenic differentiation [36]–[38]. Thus, Runx2 is a transcription factor that plays the role of the initial and terminal “switch” responsible for osteoblastic cell differentiation. Osteopontin and ALP mRNA expression levels also increased in GCT cells after treating with FGF-2 when compared to the untreated GCT stromal cells, whereas TWIST1 mRNA expression was decreased. These findings are not surprising given that in a mouse model where the TWIST1 gene was overexpressed, Runx2 expression was undetectable during development [19], [26]. Additionally, TWIST1 has been shown to have negative involvement in osteoblastic differentiation by interfering with Runx2 function at early stages of osteogenesis [39]. Furthermore, to confirm that FGFR2-IIIc signaling is involved in osteoblastic differentiation, FGFR2-IIIc expression was knocked down in the GCT cells using siRNA. A yield of 70% FGFR2-IIIc knock down resulted in an approximately 1.5 fold increase in TWIST1 expression and a significant decrease in RunX2 and ALP mRNA expression in GCT stromal cells when compared to the untreated cells. Over-expression of TWIST1 expression in GCT stromal cells inhibits GCT cell differentiation and matrix mineralization. Altogether, our data supports a definite role for FGFR2-IIIc signaling in osteoblastic differentiation of GCT stromal cells in vitro. Several studies identified that the ERK1/2 signaling may be important in osteogenic differentiation [40]–[42]. ERK1/2 is one of the main downstream targets of activated FGFRs [12], [43]. Our data indicates that FGF-2 treatment of GCT cells resulted in activation of ERK1/2 in these cells. Our findings indicate that the increasing levels of FGF-2 are functionally correlated with FGFR2-IIIc which activates downstream ERK1/2 signaling pathways of osteoblast differentiation in GCT stromal cells. Inhibitors of ERK1/2 PD98059 abolished the increased osteoblast gene expression at the mRNA level. The FGF-2 induced osteoblastic differentiation gene expression was strongly suppressed by the ERK inhibitor. Our results suggest that FGFR2-IIIc and ERK1/2 pathways mediate, at least in part, the positive effect of FGF-2 stimulation on osteoblast differentiation in GCT stromal cells. These signaling pathways may therefore serve as targets in treatment strategies for this destructive tumor and other diseases of the musculoskeletal system. In summary, our data indicates that FGFR2-IIIc signaling enhances the osteogenic differentiation program in GCT stromal cells. In vitro experiments revealed that this effect results from mechanisms involving up regulation of Runx2 and down regulation of TWIST1 (see proposed signaling mechanism Fig. 8 ). The increasing levels of FGFR2-IIIc are functionally correlated with the differentiation process in GCT stromal cells and therefore the targeting of the FGFR2-IIIc pathway may serve as a useful tool for differentiation therapy in GCT patients. 10.1371/journal.pone.0046769.g008Figure 8 Proposed signaling mechanisms representating the osteogenic differentiation model induced by FGF-2 in GCT stromal cells. FGFR2-IIIc is activated by FGF-2, which in turn increases ERK1/2 signaling in GCT stromal cells. ERK1/2 activates Rux2 expression and suppresses TWIST1 expression, thus stimulating osteoblastic differentiation. ==== Refs References 1 Turcotte RE , Ferrone M , Isler MH , Wong C (2009 ) Outcomes in patients with popliteal sarcomas . 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==== Front Clin EpigeneticsClin EpigeneticsClinical Epigenetics1868-70751868-7083BioMed Central 1868-7083-4-92270355410.1186/1868-7083-4-9ResearchHypermethylation of the 5′ CpG island of the p14ARF flanking exon 1β in human colorectal cancer displaying a restricted pattern of p53 overexpression concomitant with increased MDM2 expression Nyiraneza Christine [email protected] Christine [email protected] Roger [email protected] Alex [email protected] Karin [email protected] Center for Human Genetics, Université Catholique de Louvain, Cliniques universitaires Saint-Luc, Avenue Hippocrate 10, Brussels, B-1200, Belgium2 Department of Pathology, Université Catholique de Louvain, Cliniques universitaires Saint-Luc, Avenue Hippocrate 10, Brussels, B-1200, Belgium3 Colorectal Surgery, School of Medicine, Université Catholique de Louvain, Cliniques universitaires Saint-Luc, Avenue Hippocrate 10, Brussels, B-1200, Belgium4 Institute of Pathology and Genetics, Avenue Georges Lemaître 25, Gosselies, 6041, Belgium2012 15 6 2012 4 1 9 9 19 1 2012 7 5 2012 Copyright ©2012 Nyiraneza et al.; licensee BioMed Central Ltd.2012Nyiraneza 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 It has been suggested that inactivation of p14ARF, a tumor suppressor central to regulating p53 protein stability through interaction with the MDM2 oncoprotein, abrogates p53 activity in human tumors retaining the wild-type TP53 gene. Differences in expression of tumor suppressor genes are frequently associated with cancer. We previously reported on a pattern of restricted p53 immunohistochemical overexpression significantly associated with microsatellite instability (MSI), low TP53 mutation frequency, and MDM2 overexpression in colorectal cancers (CRCs). In this study, we investigated whether p14ARF alterations could be a mechanism for disabling the p53 pathway in this subgroup of CRCs. Results Detailed maps of the alterations in the p14ARF gene were determined in a cohort of 98 CRCs to detect both nucleotide and copy-number changes. Methylation-specific PCR combined with bisulfite sequencing was used to evaluate the prevalence and distribution of p14ARF methylation. p14ARF alterations were then correlated with MSI status, TP53 mutations, and immunohistochemical expression of p53 and MDM2. The frequency of p14ARF mutations was extremely low (1/98; 1%), whereas coexistence of methylated and unmethylated alleles in both tumors and normal colon mucosa was common (91/98; 93%). Only seven of ninety-eight tumors (7%) had a distinct pattern of methylation compared with normal colon mucosa. Evaluation of the prevalence and distribution of p14ARF promoter methylation in a region containing 27 CpG sites in 35 patients showed a range of methylated CpG sites in tumors (0 to 25 (95% CI 1 to 13) versus 0 to 17 (95% CI 0 to 2)) in adjacent colon mucosa (P = 0.004). Hypermethylation of the p14ARF promoter was significantly correlated with the restricted p53 overexpression pattern (P = 0.03), and MDM2 overexpression (P = 0.02), independently of MSI phenotype. Although no significant correlation between p14ARF methylation and TP53 mutational status was seen (P = 0.23), methylation involving the proximal CpG sites within the 5′ CpG flanking exon 1β was present more frequently in tumors with restricted p53 overexpression than in those with diffuse p53 overexpression (range of methylated clones 17 to 36% (95% CI 24 to 36%) versus range 0 to 3% (95% CI 0 to 3%), P = 0. 0003). Conclusion p14ARF epigenetic silencing may represent an important deregulating mechanism of the p53-MDM2-p14ARF pathway in CRCs exhibiting a restricted p53 overexpression pattern. ==== Body Background The correct functioning of the p53-MDM2-p14ARF pathway requires a delicate balance between the opposing effects of its different components [1-3]. Genetic and epigenetic alterations have been shown to distort this balance in various human malignancies, allowing tumor cells to over-ride the tumor suppressor activity of the p53 protein, thereby facilitating neoplastic conversion [4]. In the vast majority of human neoplasia, including colorectal cancer (CRC), deregulation of the p53 pathway usually occurs by direct inactivation of the TP53 gene itself; this occurs mainly via point mutations [5], which usually increase the stability of the mutant p53 protein, leading to its overexpression [6]. However, a significant proportion of CRCs, which include mainly microsatellite instability-high (MSI-H) CRCs, and a subset of microsatellite-stable (MSS) sporadic CRCs, display a particular immunohistochemical p53 expression pattern characterized by an accumulation of p53 protein restricted to a limited number of tumor cells, a profile that we previously termed ‘restricted p53 overexpression’ [7]. This CRC subgroup has an extremely low frequency of TP53 mutation, and displays overexpression of MDM2 and normal expression of p21, suggesting that deregulation of p53 pathway in this CRC subgroup may be due to other alternative mechanisms than TP53 mutation. Inactivation of the p14ARF gene has been proposed as a mechanism that is functionally equivalent to an inactivating p53 mutation, in that it disrupts p53 activity in tumors retaining the wild-type TP53 gene [4], and more particularly in sporadic MSI-H CRC [8,9]. In this study, we examined whether p14ARF inactivation could be one of the mechanisms disturbing the p53 pathway in CRCs, particularly in tumors displaying a restricted p53 overexpression pattern. Therefore, we conducted detailed genetics and epigenetics analysis of the p14ARF gene in CRC tumors for which we had complete data on MSI status and DNA mismatch repair deficiency or sufficiency, and we investigated the relationships between p14ARF alterations and MSI phenotype, between p14ARF alterations and the p53 protein expression pattern and its mutational status, as well as with MDM2 protein expression. Results p14ARF gene alterations in colorectal cancer In our sample, we found that p14ARF mutations were extremely rare; we detected only a previously reported point mutation in one sample (1/98; 1%). This somatic missense mutation was detected in exon 2 and corresponds to a C→T transition on a CpG dinucleotide site, affecting the codon 121 (p.Ala121Val) for the p14ARF gene, and the codon 107 (p.Arg107Cys) for the p16/CDKN2A gene. Of the ninety-six patients, five (5%) patients, including two of the five patients with Lynch syndrome (hereditary non-polyposis colorectal cancer (HNPCC); OMIM #120435) were carriers of a polymorphic variant corresponding to a substitution of G→A in codon 148 in exon 2 (p.Ala148Thr) affecting only the p16/CDKN2A open-reading frame. Gene dosage detected no copy-number changes in any of the 98 CRCs examined. p14ARF promoter methylation in tumors and adjacent colon mucosa from patients with colorectal cancer Overall, MSP analysis within the 5′ CpG island of p14/ARF flanking exon 1β identified coexistence of methylated and unmethylated alleles in tumors and matched adjacent normal-appearing colon mucosa in 91 of the 98 patients (Figure 1 A). By contrast, a distinct methylation profile indicating heavy methylation was seen in seven of the ninety-eight (7.1%) CRCs examined. In these tumors, MSP results identified only methylated alleles in tumors, whereas matched adjacent normal colon mucosa contained both methylated and unmethylated PCR products (Figure 1 B). Figure 1 Methylation of the p14 ARF promoter in tumors and normal colon mucosa from patients with colorectal cancer (A) p14 ARFpromoter methylation analysis by methylation-specific PCR revealed coexistence of both unmethylated (U) and methylated (M) PCR products in tumor (T) and adjacent colon mucosa (N). (B) Extensive methylation of p14ARF promoter in tumors. The methylated PCR product was predominantly detected in tumor, whereas the adjacent colon mucosa produced both the unmethylated and methylated PCR products. MW, standard molecular weight, control +, positive control for methylated allele, (bisulfite-modified genomic blood DNA pretreated with the CpG methylase (M.SssI)); H2O, negative control with water only. Evaluation of density of p14ARF promoter methylation in tumors and normal colon mucosa from patients with colorectal cancer Next, we evaluated the degree of p14ARF promoter methylation, limiting the analysis to tumors and corresponding adjacent colon mucosa from 35 randomly selected patients (Table 1), including one of the seven CRCs that was identified as having heavy methylation by MSP (sample T2, Table 1). Table 1 Clinicopathological and molecular data for patients analyzed by bisulfite genomic sequencing Patient’s number Location Type of differentiation Stage MMR IHC MSI status p53 IHC TP53 mutation 1 Sigmoid Moderate IV Positive MSS D p.R248W 3 Sigmoid Moderate IIIB Positive MSS D No 11 Left Moderate IV Positive1 MSI-H D No 12 Rectum Moderate IIA Positive MSS D p.R273C 15 Sigmoid Well IV Positive MSS D p.R248Q 24 Left Moderate IIA Positive MSS D No 26 Rectum Well I Positive MSS D p.R248W 28 Rectum Moderate IV Positive MSS D No 30 Sigmoid Poor IIIB Positive MSS D p.C135R 33 Rectum Well IIA Positive MSS D No 34 Left Well IIB Positive MSS D No 35 Right Well IIA Positive MSS D p.[R158H (+)R267Q] 17 Sigmoid Well I Positive MSS D p.R248Q 2 Right Well IIA MLH1-/PMS2-† MSI-H R No 4 Right Mucinous IIIC MLH1-/PMS2-‡ MSI-H R No 5 Left Mucinous IIA MLH1-/PMS2-‡ MSI-H R No 6 Right Well IIA MSH2-/MSH6-† MSI-H R No 7 Right Poor IIA MLH-/PMS2-‡ MSI-H R No 8 Left Poor IV Positive MSS R No 9 Right Mucinous IIIB Positive MSS R No 10 Right Mucinous IIIB MLH1-/PMS2-† MSI-H R No 13 Left Well IIA Positive MSS R No 14 Left Well IV Positive MSS R No 16 Sigmoid Mucinous IIA Positive MSS R No 18 Rectum Poor IIIC MLH1-/PMS2-‡ MSI-H R No 19 Left Well I MLH1-/PMS2-† MSI-H R No 22 Right Mucinous IIIB MLH1-/PMS2-‡ MSI-H R No 23 Right Poor IIA MLH1-/PMS2-1 MSI-H R No 25 Left Mucinous IIA Positive MSS R No 29 Left Moderate IIA Positive MSS R No 20 Rectum Moderate IIA Positive MSS N No 21 Rectum Mucinous IIB Positive MSS N p.[K291X(+) H297Y] 27 Rectum Moderate IIA Positive MSS N c.672 + 1 G→A 31 Right Moderate IIIC Positive MSS N p.Q165X 32 Rectum Moderate IIIC Positive MSS N No Abbreviations: MMR, DNA mismatch repair system; MSI, microsatellite instability; MSI-H, microsatellite instability-high; MSS, microsatellite-stable; IHC, immunohistochemistry; D, diffuse pattern of p53 expression, R, restricted pattern of p53 expression, N, negative pattern of p53 expression. *MMR deficiency with unknown origin. †Lynch syndrome. ‡Sporadic MSI-H colorectal cancer with activating V600E BRAF somatic mutation, indicating MLH1 epigenetic silencing. Using BGS, we analyzed methylation within the 5′ CpG island of p14ARF flanking exon 1β, targeting a region containing 27 individual CpG sites, including all the CpG sites analyzed by MSP in this region (see Additional file 1: Figure S1). BGS showed different p14ARF promoter methylation levels among the 35 tumors and adjacent colon mucosa tested, with the highest methylation levels recorded in tumor samples (Figure 2, Figure 3). Of the 35 tumors examined, the range of fully methylated CpG was 0 to 25 and the median was 9 (95% CI 1 to 13), whereas in paired normal colon mucosa the range was 0 to 17 and the median 0 (95% CI 0 to 2) (P = 0.004). Of the thirty-five tumors, eighteen (51%) were extensively methylated (>9/27 CpG sites methylated, median of fully methylated CpG sites), six (17%) were partially methylated (>3/27 CpG sites partially methylated, median of partially methylated CpG sites), and 11 (32%) were unmethylated (Table 2). Figure 2 Heterogeneity of p14 ARFpromoter methylation in colorectal tumors. The samples analyzed are represented on the horizontal line, and the 27 CpG sites on the vertical line. For each case, the methylation status of each individual CpG site is shown: an empty block indicates that the concerned CpG site is unmethylated, a black block indicates that the concerned CpG sites is fully methylated, and a gray block indicates that the concerned CpG site is partially methylated. Figure 3 p14 ARF promoter methylation in adjacent colon mucosa. Table 2 Relationships between p14 ARF promoter methylation and clinicopathological data, p53 and MDM2 protein expression, TP53 mutational status, and microsatellite instability phenotype Clinicopathological and molecular parameters Overall (%) p14ARF promoter methylation profile Unmethylated Dense or partial methylation Pvalue Age, years, mean ± SD   64 ± 12 69 ± 10 0.20571 Gender    Male 16 (46%) 5 (45.5%) 11 (45.8%) 0.9833  Female 19 (54%) 6 (54.5%) 13 (54.2%) Type of tissue          Tumor 35 11 (31%) 24 (69%) 0.0019  Adjacent colon mucosa 35 24 (69%) 11 (31%) Tumor location  Right side 10 (29%) 3 (27.3%) 7 (29.2%) >0.05  Left side 25 (71%) 8 (72.7%) 17 (70.8%)   Differentiation    Well or moderate 22 (62.9%) 10 (90.9%) 12 (50%) 0.0270  Poor or mucinous 13 (37.1%) 1 (9.1%) 12 (50%)   Clinical stage    Stage I 3 (8.6%) - 3 (12.5%) 0.46742  Stage II 17 (48.6%) 5 (45.4%) 12 (50%)    Stage III 9 (25.7%) 3 (27.3%) 6 (25%)    Stage IV 6 (17.1%) 3 (27.3%) 3 (12.5%)   p53 immunohistochemistry    Negative pattern 5 (14.3%) 3 (27.3%) 2 (8.3%) 0.02752  Diffuse pattern 13 (37.1%) 6 (54.5%) 7 (29.2%)    Restricted overexpression 17 (48.6%) 2 (18.2%) 15 (62.5%)   MDM2 immunohistochemistry    Negative 12 (34.3%) 7 (63.6%) 5 (20.8%) 0.0223  Overexpression 23 (65.7%) 4 (36.4%) 19 (79.2%)   p21 immunohistochemistry    Loss to mild 9 (25.7%) 6 (54.5) 3 (12.5%) 0.01462  Moderate to high 26 (74.3%) 5 (45.5) 21 (87.5%)   TP53 mutational status    Mutation present 10 (28.6%) 5 (45.5%) 5 (20.8%) 0.2266  No mutation detected 25 (71.4%) 6 (54.5%) 19 (79.2%)   MSI status    MSI-H 11 (31.4%) 1 (9.1%) 10 (41.7%) 0.0539  MSS 24 (68.6%) 10 (90.9%) 14 (58.3%)   Abbreviations: MSI, Microsatellite instability, MSI-H, microsatellite instability-high; MSS, microsatellite-stable. 2Two-sided two-sample t-test. 2Two-tailed Fisher’s exact test. Although the majority of normal colon mucosa tested (69%) showed a significantly low frequency of methylation compared with matched tumor samples (P = 0.0019) (Table 2), extensive methylation was detected in the normal colon mucosa from six patients, including a patient with Lynch syndrome (N7; Figure 3) with a germline mutation in the MLH1 gene (Table 1) and five patients with sporadic CRC: two MSS tumors (N1, N29; Figure 3) and three MSI-H tumors (N4, N5, N22; Figure 3) with activating V600E BRAF somatic mutation associated with MLH1 epigenetic silencing (Table 1). Correlation between p14ARF promoter methylation, clinicopathological features, p53 pathway alterations, and microsatellite instability status in colorectal cancer Further, we compared p14ARF methylation data from the 35 randomly selected patients analyzed by BGS with their clinicopathological features and the molecular changes in their tumors. No significant association was seen between p14ARF methylation and either age or gender (Table 2). Although the majority of right-sided colon tumors (7/10) had increased p14ARF methylation, no significant association between p14ARF methylation and tumor location was seen (Table 2). Correlation analysis identified a significant association between p14ARF hypermethylation and poorly differentiated or mucinous tumors (P = 0.0270) (Table 2), but no significant association between p14ARF methylation and clinical stage was seen (Table 2). Compared with tumors exhibiting negative and diffuse patterns of p53 protein immunohistochemical expression, the tumors displaying a restricted p53 overexpression profile (15/17) showed a significant increase in p14ARF methylation (P = 0.0274) (Table 2). p14ARF methylation was also significantly associated with MDM2 overexpression (P = 0.0223) (Table 2). Most tumors exhibiting p14ARF hypermethylation showed an absence of TP53 mutation (19/24; 79%), but no significant association between p14ARF promoter methylation and TP53 mutational status was seen (Table 2). MSI-H CRCs were more frequently hypermethylated than MSI-low (MSI-L)/MSS CRCs (P = 0.0539) (Table 2). However, after stratification by p53 immunohistochemical expression pattern, the relationship between MSI status and p14ARF methylation was no longer significant (Figure 4). Figure 4 Relationship between p14 ARFpromoter methylation and microsatellite instability (MSI) status. The MSI-high (MSI-H) tumors had an overall higher frequency of p14ARF promoter methylation compared with MSS tumors, but after stratification by restricted p53 overexpression, the relationship between p14ARF methylation and MSI status was no longer significant. Quantification and distribution of p14ARF promoter methylation in tumors and normal colon mucosa from patients with colorectal cancer We evaluated the density and the distribution of methylation within the 5′ CpG island of the p14ARF promoter region and exon 1β. Using bisulfite genomic cloning and direct sequencing, we analyzed 200 clones obtained from 10 tumors and matched adjacent colon mucosa. For each clone, the methylation status of each individual CpG site was determined (Figure 5). For all 27 CpG sites evaluated, we found a significantly (P <0.0001) increased number of methylated clones in tumors (median 38%; 95% CI 25 to 41%; range 13 to 47%) compared with the adjacent normal colon mucosa (median 9%; 95% CI 5 to 13%; range 1 to 24%) (Table 3). Although most normal colon mucosa (7/10) showed only sparse methylation (Figure 5), densely methylated clones were seen in three of the ten normal colon mucosa tested (N1, N18 and N29; Figure 5). Bisulfite genomic cloning and direct sequencing also showed that methylation involving both CpG sites within the proximal and the distal region of the 5′ UTR CpG island of the p14ARF flanking exon 1β (nucleotide position −69 to position +4 relative to the translation codon ATG) is not a frequent event in CRC, but seems to occur more particularly in tumors displaying a restricted pattern of p53 overexpression, including MSI-H and MSS tumors (Figure 5). Overall, the 3′ region of exon 1β was more densely methylated (median 41%; 95% CI 38 to 43%; range 27 to 47%) than the promoter and 5′ region of exon 1β (median 22%; 95% CI 17 to 25%; range 13 to 25%) (P = 0.0001) (Table 3). However, the number of methylated clones on CpG sites within the proximal region of the 5′ CpG island of p14ARF was significantly higher in tumors displaying a restricted pattern of p53 overexpression (median 30%; 95% CI 24 to 36%; range 17 to 36%) than in tumors exhibiting a strong diffuse p53 expression pattern (median 0%; 95% CI 0 to 3%; range 0 to 3%) (P = 0.0003) (Table 3). Figure 5 Density and distribution of methylated CpG within the 5′ CpG island of p14 ARFflanking exon 1β. Depicted is the distribution of methylated CpG in tumors (up) and corresponding adjacent colon mucosa (down) from 10 patients. For each case, 10 independent clones (represented on horizontal line (a) to (j) were examined. The circles on the vertical line represent the 27 CpG (CpG 1 to 27) sites analyzed for each individual clone. Note that the translation start site is located between CpG sites 8 and 9. An empty circle indicates that the concerned CpG site is unmethylated, a black circle indicates that the concerned CpG site is methylated. For each tumor, the microsatellite instability (MSI) status and p53 immunohistochemistry are indicated. MSI-H, microsatellite instability-high; MSS, microsatellite-stable; p53 D, diffuse pattern of p53 expression, p53 R, restricted pattern of p53 overexpression. Table 3 Distribution and density of p14 ARF methylation in tumors and adjacent colon mucosa from patients with colorectal cancer CpG site Position relative to ATG % of methylated clones within the 5′ CpG island flanking exon 1β Tumor samples Normal colon mucosa Tumors with diffuse p53 overexpression pattern Tumors with restricted p53 overexpression pattern 1 −69 13 5 3 17 2 −43 17 1 0 24 3 −40 22 1 0 31 4 −35 25 4 0 36 5 −28 22 2 0 31 6 −22 19 2 0 27 7 −10 22 5 3 30 8 −7 21 3 0 30 9 +4 30 5 3 41 10 +25 41 8 37 43 11 +31 42 12 43 41 12 +34 45 12 50 43 13 +36 43 18 43 43 14 +38 34 12 33 34 15 +42 43 13 43 43 16 +47 41 15 53 36 17 +50 45 9 50 43 18 +52 43 12 57 37 19 +66 47 9 50 46 20 +80 44 9 50 41 21 +82 40 13 37 41 22 +90 38 13 27 43 23 +105 38 13 47 34 24 +114 41 19 50 37 25 +118 34 17 37 33 26 +121 31 24 37 29 27 +133 27 9 13 33 The percentage of methylated clones was calculated in all tumors (n = 10, ≥10 clones analyzed for each tumor) and adjacent normal colon mucosa (n = 10, ≥ 10 clones analyzed for each sample) for every CpG site. The percentage of methylated clones was higher in tumors median 38%, 95% CI 25-41%; range 13-47%), than in normal colon mucosa (median 9%, 95% CI 1 to 24; range 1 to 24%) (Wilcoxon rank sum test, P <0.0001). The percentage of methylated clones on proximal CpG sites was also higher in tumors with a restricted p53 overexpression pattern (median 30%, 95% CI 24 to 36%, range 17 to 36%) than in tumors with a diffuse p53 overexpression pattern (median 0%, 95% CI 0 to 3%, range 0 to 3%) (Wilcoxon rank sum test, P = 0.0003). Discussion The purpose of this study was to investigate whether alteration of p14ARF, a key regulator of p53-MDM2 interaction, plays a role in deregulating the p53 pathway in a subgroup of CRCs exhibiting a restricted pattern of p53 overexpression significantly associated with MSI-H phenotype, low TP53 mutation, and MDM2 overexpression, and inversely correlated with p21 expression loss [7]. Contrary to the usual situation in solid tumor types such as melanoma, pancreatic tumors and some lung tumors [10-12], the present study confirmed the extremely low frequency of intragenic mutations and allelic losses at the p14ARF locus in CRC [13]. Indeed, direct sequencing detected only one (previously reported) missense mutation affecting both the p14ARF and p16/CDKN2A genes [14]. Only 5% percent of the cases, including two patients with Lynch syndrome, were carriers of p.Ala148Thr, a variant considered a non-synonymous single nucleotide polymorphisms (nsSNP, rs3731249) [14,15]. Although the functional significance of this SNP has been controversial in studies of several cancer types [15-17], its role in CRC risk assessment warrants investigation because this variant occurred at an evolutionarily conserved amino acid with a low intolerance index, as predicted by the Sorting Intolerance from Tolerance (SIFT) program [18]. We evaluated epigenetic changes within the p14ARF promoter using two different methylation assays, MSP and BGS. We used MSP because it is widely recognized as a highly sensitive methylation assay, allowing detection of up to 0.01% of methylated alleles of a given CpG island [19]. However, this method provides only qualitative data, so for quantitative analysis, BGS complemented by cloning and direct sequencing, was used [20-22]. Although BGS is a high-resolution assay, this technique is less sensitive than MSP as detection requires at least 25% of the alleles to be methylated [22], suggesting a risk of disagreement between the two methods. One of our main findings was the detection of p14ARF promoter silencing as a potential cause of deregulation of the p53-MDM2-p14ARF signaling axis in a specific subgroup of CRCs. Using BGS, we fully characterized 35 of the 98 CRCs analyzed. A significant increase in p14ARF promoter methylation was evident in 24 CRCs (69%), and interestingly, 63% of the cases (15/24) were tumors exhibiting the restricted pattern of p53 overexpression (Figure 2, Table 2). The p14ARF promoter has been previously reported to be preferentially hypermethylated in CRCs retaining the wild-type TP53 gene [8,13,23], and has been particularly associated with sporadic MSI-H CRCs associated with MLH1 epigenetic silencing [8,9]. In addition to the relationship between the restricted p53 overexpression pattern and the MSI-H phenotype [7], we found that p14ARF promoter methylation was increased in CRCs with restricted p53 overexpression, irrespective of MSI status (Figure 4). This observation, along with our previous findings, shows that regardless of the MSI status, CRCs with the restricted p53 overexpression pattern exhibit a significant overlap in terms of their pathobiology, supporting the hypothesis of a common tumorigenic event [7]. In agreement with these observations, previous studies have shown that although CRCs have been reported to evolve either through the classic chromosomal instability pathway or through the alternative MSI pathway known to be significantly associated with the CpG island methylator phenotype, the mechanisms underlying these genomic instability pathways are not always independent [24,25], and a significant degree of overlap can therefore be expected in some tumors, regardless of the MSI status. Even though a high frequency of p14ARF promoter methylation has been previously reported to occur in tumors without TP53 mutations [8,13,23,26], an inverse correlation between TP53 mutations and epigenetic inactivation of p14ARF in CRCs does not always hold true [27]. In the current study, we found that although the majority of heavily methylated tumors did not have a TP53 mutation, p14ARF promoter methylation was increased in almost half of tumors (5/10) carrying TP53 mutations (Table 2). Interestingly, the most exceptional feature of these tumors was the distribution of p14ARF methylation. Using bisulfite genomic cloning and direct sequencing, we found that extensive methylation involving both the proximal and the distal CpG sites within the 5′ CpG island of p14ARF flanking exon 1β was rare in CRC generally, but occurred more frequently in CRCs displaying a restricted pattern of p53 overexpression (Table 3). In tumors showing a strong diffuse p53 expression pattern associated with missense TP53 mutations, the majority of the methylated clones exhibited partial methylation involving CpG sites downstream from the translation start site and extending throughout exon 1β (Figure 5, Table 3). This pattern of methylation was also seen in some normal colon mucosa (Figure 3; Figure 5 (N29)). Our results support previous observations by Zheng et al., who showed that partial methylation is the most common pattern of p14ARF methylation in primary sporadic CRCs [28]. Owing to the limited availability of an efficient antibody raised against the p14ARF protein, we were unable to examine p14ARF expression by immunohistochemistry in our tumor samples. However, previous experiments, mainly performed in CRC cell lines, have shown that extensive methylation of CpG sites within the 5′ CpG island and exon 1β of p14ARF is associated with transcription silencing and correlates with extremely low levels of p14ARF mRNA, whereas partial methylation correlates with intermediate mRNA expression [28,29]. Based on these findings, we suggest that the extensive methylation seen in CRCs with restricted p53 overexpression may represent an important functional defect in the p14ARF gene, but additional studies are needed to verify this hypothesis. Additionally, a significant relationship between MDM2 overexpression and increased p14ARF methylation was seen (79%; P = 0.0223). It is known that tumors with reduced p14ARF activity have higher MDM2 activity, which potentially leads to p53 inactivation [30]. Moreover, using immunohistochemistry, a strong inverse relationship between MDM2 and p14ARF inactivation has been previously found in different tumor types, including a subtype of human lung carcinoma displaying an abnormally stabilized p53 protein [31]. Therefore, it is conceivable that the increased MDM2 expression seen in CRCs with restricted p53 overexpression may reflect cellular functional consequences of p14ARF epigenetic inactivation. Interestingly, a previous study found an association between p14ARF epigenetic silencing and an abnormal cytoplasmic localization of MDM2 in primary CRC and tumor cell lines, mainly explained as a direct consequence of p14ARF loss of function [32]. In the current study, we did not find any MDM2 subcellular localization in our cohort of 98 CRCs. Functional interpretation of MDM2 immunostaining data are complicated by the existence of several isoforms, of which detection depends on the antibody used, and this may explain these discrepancies. It is widely believed that CpG islands in autosomal genes are usually unmethylated, except when associated with certain imprinted genes and with genes that undergo X-chromosome inactivation in females [33,34]. Supporting this paradigm, initial studies indicated methylation of the 5′ CpG island of the p14ARF promoter exclusively in tumor cells [13,27]. However, this view was challenged by detection of p14ARF methylation in normal colon mucosa from patients with CRC and from healthy people without clinical evidence of colon cancer [8,35,36]. In the current study, using the MSP assay, we found coexistence of unmethylated and methylated alleles in the majority of tumors and in all adjacent normal colon mucosa. A clear difference in methylation pattern between tumor and adjacent normal colon mucosa was seen only in the seven tumors (7.1%) that showed heavy methylation. The sensitivity of our MSP assay was significantly high. However, given that we used the conventional MSP assay, which provides qualitative data, we were limited by this high sensitivity, and were unable to distinguish the p14ARF methylation occurring in a small proportion of cells from the high-level methylation associated with epigenetic inactivation. Using the BGS approach, we found that the level of p14ARF methylation in normal tissues was generally below the threshold detection of the BGS assay, and was significantly increased in tumors compared with normal colon mucosa. However, hypermethylation was still present in normal colon mucosa from some patients, and more frequently in those with DNA mismatch repair deficiency associated with MLH1 gene inactivation. Indeed, hypermethylation of the 5′ CpG islands of the p14ARF and MLH1 genes in normal-appearing mucosa surrounding colorectal neoplastic lesions has been described as a ‘field cancerization’ phenomenon, which may occur before genetic alterations in the early stages of carcinogenesis [37]. Conclusion In summary, this study provides evidence that p14ARF promoter hypermethylation may represent an important cause of deregulation of the p53-MDM2-p14ARF signaling axis in a subgroup of CRCs displaying a restricted overexpression pattern of the p53 protein, associated with the wild-type TP53 gene, concomitant MDM2 overexpression, and normal p21 expression. Although this subgroup of CRCs includes the majority of MSI-H tumors (namely Lynch syndrome-related CRCs and sporadic MSI-H CRCs), methylation involving both proximal and distal CpG sites within the 5′ CpG island flanking exon 1β of p14ARF preferentially occurs in these tumors independently of MSI status. Further investigations are warranted to clarify the significance of this high-level methylation on the transcriptional activity of the p14ARF gene. The results from this work could have clinical implications, because therapeutic delivery of small p14ARF peptides has been reported to mimic the growth-inhibitory effects of full-length p14ARF expression and to restore p53 activity in cancers in which MDM2 is overexpressed or p14ARF is functionally inactivated [38]. Evaluation of the clinical relevance of such promising therapeutic measures would essentially provide a new set of more efficient treatment possibilities in patients with CRC who have tumors displaying the restricted pattern of p53 overexpression. Methods Ethics approval Tissues collection and analyses were approved by the institutional ethics committee of the Catholic University of Louvain (Faculty of Medicine UCL), and all participants provided written informed consent. Patients We examined 98 surgical resected tumors and corresponding adjacent normal colon mucosa from the cohort of patients (48 men, 50 women, mean ± SD age 64 ± 14 years) with primary CRC we reported previously [7]. For the 98 CRCs, clinicopathologic data and evaluation of the DNA mismatch repair (MMR) system (using MSI analysis, immunohistochemistry (IHC) for MMR proteins, MMR germline mutation) and somatic BRAF mutation, had been performed previously [7], but only data from the 35 patients extensively studied by bisulfite genomic sequencing (BGS) are shown in Table 1. Immunohistochemical analysis for p53, MDM2, and p21 proteins and mutational analysis for TP53 were also previously performed. Three distinct patterns of p53 expression were seen, including a restricted p53 overexpression pattern clearly distinguishable from both the negative pattern and the strong diffuse pattern [7]. MDM2 immunohistochemical expression was semi-quantitatively evaluated based on the percentage of positive tumor cells. MDM2 overexpression was recorded if a positive staining was evidenced in more than 10% of tumor cells nuclei [7]. p14ARF mutation screening and gene dosage Sequence-specific primers (according to GenBank accession number NM_058195) for exon 1β and exon 2 (common to both p16/CDKN2A and p14ARF), including the intronic flanking regions of the p14ARF gene, were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). PCR was carried out for each sample, and PCR products were then purified, sequenced, and run on an automated laser fluorescent DNA sequencer (3130XL; AB Applied Biosystems, Foster City, CA, USA). To detect large rearrangements (allelic imbalances) throughout the p14ARF locus, multiplex ligation-dependent probe amplification (MLPA) was performed using (Salsa PO24B 9p21 CDKN2A/2B region kit; MRC-Holland BV, Amsterdam, the Netherlands), in accordance with the manufacturer’s instructions. MLPA PCR products were separated by capillary electrophoresis using an automated laser fluorescent DNA sequencer (3130 XL; AB Applied Biosystems, Foster City, CA, USA). The relative quantities of the amplified probes in each sample were determined using Genotyper (Applied Biosystems, Foster City, CA, USA) and Excel (Microsoft Corp., Redmond, WA, USA) software (Gene Marker version 1.5; Softgenetics Inc, State College, PA, USA). The gene dosage quotient was generated using peak height rather than peak area as an indicator of DNA template amount [39,40]. For each sample, a gene dosage quotient score (peak height relative to control) was calculated and adjusted as follows: homozygous loss ≤ 0 to 0.19 ≤ hemizygous loss ≤ 0.7 to 0.75 ≤ wild-type ≤ 1 to 1.3 < duplication. Methylation-specific PCR Genomic DNA was extracted from frozen tumors and matched normal tissues using a standard phenol/chloroform method. Thereafter, bisulfite treatment of 300 ng of genomic DNA was performed (Applied Biosystems methylSEQr™ Bisulfite Conversion Kit) in accordance with the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Methylation-specific PCR (MSP) [19] was performed to examine p14ARF promoter methylation within a region located at least −60 base pairs relative to the translation codon, previously reported to be associated with p14ARF gene silencing in CRC [27]. Methylation in this region was evaluated using the primer sets previously described [27]. These primers pairs allowed assessment of the methylation status of six CpG dinucleotides specific for the 5′ CpG island of the p14ARF gene flanking exon 1β (see Additional file 2: Table S1). The MSP reactions were carried out in a total volume of 25 μl containing 2.5 μl of the manufacturer’s 10× PCR buffer (Roche Diagnostics, Basel, Switzerland), 1.5 μl of 25 mmol/l MgCl2, and 0.25 μl of 100 μmol/l dNTPs (dATPs, dTTPs, dCTPs and dGTPs), 1 μl of primer (10 pmol/μl for each), 1 to 1.25 U of DNA polymerase (FastStart; Roche Diagnostics, Basel, Switzerland), and 1 μl of bisulfite-modified genomic DNA. Normal human leukocyte DNA was methylated in vitro with a CpG methylase (M.SssI; New England BioLabs, Beverly, MA, USA) in accordance with the manufacturer’s instructions, and used as the MSP methylated-allele positive control. After amplification, 5 μl of PCR products were run in an 8% non-denaturing acrylamide gel with an appropriate size marker. Amplicons were visualized by ethidium bromide staining under UV illumination. Bisulfite genomic sequencing BGS primers designed to recognize both methylated and unmethylated alleles were generated based on the human contig sequence (GenBank accession number L41934) using MethPrimer software (http://www.urogene.org/methprimer/index1.html) [41]. The designed BGS primers were located within the 5′ CpG island of the p14ARF region flanking exon 1β, and were used to amplify a DNA sequence containing 27 CpG sites, including all the CpG sites targeted by the MSP primers within this region (see Additional file 1: Figure S1). Bisulfite-converted DNA samples from tumor tissue and corresponding adjacent normal tissues from 35 patients, randomly selected from our cohort of patients (Table 1), were subjected to PCR amplification using primer pair A and B (forward and reverse, respectively), followed by a nested PCR amplification with primer pair C and D (forward and reverse; Additional file 1: Figure S1). All the primer sequences used are summarized in (Additional file 2: Table S1). After PCR amplification, the BGS products were purified (Qiaquick PCR Purification Kit; Qiagen Inc., Valencia, CA, USA), and directly sequenced in both directions using primers C and D (forward and reverse; see Additional file 1: Figure S1) with a commercial kit (Big Dye Terminator Cycle Sequencing Ready Reaction Kit, version 1.3; Perkin Elmer/Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. Sequencing reaction products were purified on filter plates for high-throughput separation (Multiscreen™; Millipore Corp., Bedford, MA, USA) using dextran gel beads (SephadexTM G-50 Fine Beads; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in accordance with the manufacturer’s instructions. After purification, sequencing reaction products were run on an automated laser fluorescent DNA sequencer (3130XL; AB Applied Biosystems, Foster City, CA, USA). Results were analyzed using the sequencing analysis software for the sequencer (Version 1.5; AB Applied Biosystems, Foster City, CA, USA). For all 35 patients examined, the bisulfite sequencing chromatogram was analyzed for each individual CpG site, and a specific pattern was assigned: 1) unmethylated, in which the CpG site was fully converted into thymidine, indicating that the concerned CpG site is unmethylated on both alleles (see Additional file 3: Figure S2), 2) partial, showing an overlap of both thymidine and cytosine peaks on a sequencing chromatogram, indicating the presence of both methylated and unmethylated alleles (see Additional file 3: Figure S2 A), 3) methylated, in which the CpG site was fully methylated, indicating that the concerned CpG site is extensively methylated on both alleles (see Additional file 3: Figure S2 B). Cloning and sequencing For 10 patients, the amplified bisulfite PCR products from tumor and corresponding normal colon tissues were purified (Qiaquick PCR Purification Kit; Qiagen) and ligated into a pTZ57R/T plasmid vector using a TA cloning and bacterial transformation system (Ins TAclone™ PCR Cloning Kit). The plasmid was inserted into Escherichia coli cells, which were cultured overnight, then recombinant plasmid DNA was isolated and purified (Rapid Miniprep Plasmid Purification System; Marligen Bioscience, Ijamsville, Maryland, USA). Purified plasmid recombinant DNA was subjected to direct PCR amplification in a 25 μl reaction mixture containing 2.5 μl of the manufacturer’s 10× PCR buffer (Roche Diagnostics, Basel, Switzerland), 1.5 μl of 25 mmol/l MgCl2, and 0.25 μl of 100 μmol/l dNTPs, 1 μl of M13 forward and reverse primer (10 pmol/μl for each), 1 U of DNA polymerase (FastStart; Roche Diagnostics, Basel, Switzerland), and 1 μl of purified recombinant plasmid DNA template. Direct sequencing was performed in both directions using M13 primers with a commercial kit (Big Dye Terminator Cycle Sequencing Ready Reaction Kit, version 1.3; Perkin Elmer/Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. Sequencing reaction products were purified on filter plates for high-throughput separations Multiscreen™; Millipore Corp., Bedford, MA01730 USA) using dextran gel beads (SephadexTM G-50 Fine Beads; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and were run on an automated laser fluorescent DNA sequencer (3130XL; AB Applied Biosystems, Foster City, CA, USA). For each sample, at least 10 clones were analyzed. For each clone, the bisulfite genomic sequence was analyzed, and for each individual CpG site a methylation status was assigned. Statistical analysis We used the Pearson χ2 test (when the minimum expected value was ≥ 5) or the two-tailed Fisher’s exact test (when the minimum expected value was <5) to compare the frequency of p14ARF promoter methylation in 35 patients with CRC in relation to various clinicopathologic parameters and characteristics, including immunohistochemical expression of p53, MDM2, and p21, p53 mutational status and MSI status. Comparison in distribution and density of methylation between tumors and adjacent normal-appearing colon mucosa, and of tumor groups were assessed using the Mann–Whitney or Wilcoxon rank sum test. All statistical analyses were performed using the NCSS 2007 statistical & Power analysis software. All reported P-values were two sided, and the test was significant when the P ≤ 0.05. Abbreviations CRC: Colorectal cancer; IHC: Immunohistochemistry; MLPA: Multiplex ligation-dependent probe amplification; MSI: Microsatellite instability: MSI-H: Microsatellite instability-high; MSI-L: Microsatellite instability-low: MSP: Methylation-specific PCR; MSS: Microsatellite-stable; nsSNP: non-synonymous single nucleotide polymorphism; PCR: polymerase chain reaction; UV: Ultraviolet. Competing interests The authors have no competing interests to disclose. Authors’ contributions CN participated in the design of the study, carried out the pathological and molecular genetic studies, and drafted the manuscript. CS participated in the design of the study and collection of pathological data, and revised the manuscript. RD and AK participated in collection of patients. KD conceived and coordinated the study, and drafted the manuscript. All authors have read and approved the final manuscript. Supplementary Material Additional file 1 Figure S1 p14 ARF promoter methylation analysis by bisulfite genomic sequencing (BGS). The genomic sequence of 5′CpG island of p14ARF region flanking exon 1β was analyzed. The highlighted and numbered CpG indicates the 27 potential CpG sites analyzed. Bold arrows indicate position of forward and reverse MSP primers for methylated (MSPMF/MSPMR) and for unmethylated (MSPUF/MSPUR) alleles. Simple arrows indicate bisulfite genomic sequencing primers specific for both unmethylated and methylated sequences. The putative transcription start site, translation start site (+1), and the end of exon 1β (*) are indicated. Click here for file Additional file 2 Table S1. Primer sequences for methylation-specific PCR, bisulfite genomic sequencing, and PCR amplification of exon 1β and exon 2 of the p14 ARF gene. Click here for file Additional file 3 Figure S2. Bisulfite DNA sequencing chromatograms representing the three different methylation profiles for single CpG dinucleotide sites. (A) DNA sequences from tumor samples showing an overlap of both thymidine and cytosine peaks indicating a partial methylation on CpG site located at position −31 relative to the translation start site (top) compared with another sample showing an unmethylated profile at the same CpG site (bottom), (B) DNA sequences from tumor samples showing full methylation on CpG sites located at +31 and +42 relative to the translation start site (top) compared with another sample showing an unmethylated profile at the same CpG sites (bottom). Click here for file Acknowledgements We are particularly grateful to the staff members of the pathology and genetics departments for their excellent technical assistance. 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Clin Epigenetics. 2012 Jun 15; 4(1):9
==== Front BMC NeurosciBMC NeurosciBMC Neuroscience1471-2202BioMed Central 1471-2202-13-1152299808210.1186/1471-2202-13-115Research ArticleMicroRNA expression profile of the hippocampus in a rat model of temporal lobe epilepsy and miR-34a-targeted neuroprotection against hippocampal neurone cell apoptosis post-status epilepticus Hu Kai [email protected] Yuan-Yuan [email protected] Chen [email protected] Dong-Sheng [email protected] Hong-Yu [email protected] Dan-Ni [email protected] Li-Li [email protected] Li [email protected] Yi [email protected] Bo [email protected] Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, P. R. China2 Department of Neurology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410013, P. R. China3 Institute of Clinical Pharmacology, Central South University, Changsha, Hunan, 410078, P. R. China2012 22 9 2012 13 115 115 29 4 2012 10 9 2012 Copyright ©2012 Hu et al.; licensee BioMed Central Ltd.2012Hu 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 expression pattern and function of miRNAs in the rat model of temporal lobe epilepsy have not been well defined. Profiling miRNA expression in the rat model of temporal lobe epilepsy and investigating the function of specific miRNAs in epilepsy offers the prospect of a deeper understanding of the mechanisms of epilepsy. Methods The lithium-pilocarpine-induced status epilepticus model and the temporal lobe epilepsy model were established in Sprague–Dawley rats. Samples were analysed to detect deregulated miRNAs in the hippocampal temporal lobe, and several of these deregulated miRNAs were confirmed by qPCR. The expression of the pro-apoptotic miR-34a was detected at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. The antagomir of miR-34a was then utilised. The expression of miR-34a after targeting and the expression change of activated caspase-3 protein were examined. The effects of altering the expression of miR-34a and activated caspase-3 protein on neuronal survival and neuronal death or apoptosis post-status epilepticus were assessed. Results The miRNA microarray detected 9 up-regulated miRNAs (miR-146a, -211, -203, -210, -152, -31, -23a, -34a, -27a) and 15 down-regulated miRNAs (miR-138*, -301a, -136, -153, -19a, -135b, -325-5p, -380, -190, -542-3p, -33, -144, -542-5p, -543, -296*). Some of the deregulated miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) were confirmed using qPCR. Furthermore, an increase in expression of the pro-apoptotic miR-34a was demonstrated in the post-status epilepticus rat hippocampus. miR-34a was significantly up-regulated at 1 day, 7 days and 2 weeks post-status epilepticus and at 2 months after temporal lobe epilepsy. Experiments with the miR-34a antagomir revealed that targeting miR-34a led to an inhibition of activated caspase-3 protein expression, which may contribute to increased neuronal survival and reduced neuronal death or apoptosis. Conclusions Our study showed the expression profile of miRNAs in the hippocampus in a rat model of temporal lobe epilepsy and an increase in the expression of the pro-apoptotic miR-34a in post-status epilepticus rats. The results show that miR-34a is up-regulated during seizure-induced neuronal death or apoptosis, and targeting miR-34a is neuroprotective and is associated with an inhibition of an increase in activated caspase-3 protein. MiRNAEpilepsyHippocampusApoptosisStatus epilepticus ==== Body Background MicroRNAs (miRNAs) belong to a family of non-coding small RNAs and are emerging as important post-transcriptional regulators that either inhibit mRNA translation or direct target mRNA degradation [1]. They are critical for normal neuronal development and may be involved in many neurological diseases whose mechanisms remain to be explored. For example, miRNAs have important roles in gene regulatory networks involved in both brain development and adult neural plasticity [2]. miR-124a and miR-9 are able to regulate ES cell differentiation toward neuronal or glial lineages [3], and brain-specific miR-9 is critical in modulating the cellular behaviour of stem cell-derived neural progenitor cells (NPCs) [4]. Recent work by Agostini et al. found that pro-apoptotic miR-34a regulates neurite outgrowth, spinal morphology and function [5], and this work highlights the importance of miR-34a in neuronal differentiation and synaptogenesis. The studies described above are only a few examples of the involvement of miRNAs in the nervous system. A number of studies have investigated the role of miRNAs in neurological diseases such as epilepsy. Using the pilocarpine mouse model of epilepsy, Nudelman et al. reported that neuronal activity rapidly induces transcription of the CREB-regulated miR-132 in vivo[6]. This was the first study to demonstrate a change in a miRNA after seizures. Sano and Henshall reported that miR-34a is up-regulated during seizure-induced neuronal death [7] and concluded that prolonged seizures cause up-regulation of miR-34a in a subfield-specific, temporally restricted manner. However, they suggested that miR-34a is most likely not important for seizure-induced neuronal death in the mouse model. Recently, Kan et al. published a study of miRNA profiles in human temporal lobe epilepsy (TLE) [8] and extended the current concepts of human mesial TLE pathogenesis to the level of miRNA-mediated gene regulation. Before Kan’s work in human TLE, Aronica et al. had also published a paper focused on a single inflammation-associated miR-146a in TLE patients [9]. More recently, the work of Henshall et al. showed that miR-134a silencing produces neuroprotective and prolonged seizure-suppressive effects [10]. One of their previous studies suggested that targeting miR-132 reduces seizure-induced neuronal death and protects the hippocampus [11]. Both studies offered a new therapeutic target for the treatment of epilepsy. Epigenetic research of miRNAs associated with epilepsy is an interesting field and more findings are emerging. Most studies have focused on the function of brain-specific miRNAs following status epilepticus in the mouse model or on samples from TLE patients. The expression and functional changes of inflammation-, development- and neuronal death-associated miRNAs in epilepsy have been identified. However, studies addressing the roles of pro-apoptotic miRNAs in the mechanism of epilepsy using different models are limited. The study of miR-34a in the mouse model of epilepsy has delineated a restricted role for this pro-apoptotic miRNA. Whether miR-34a mediates seizure-induced neuronal death and how it is involved in that process in the widely used status epilepticus (SE) rat model remains to be explored. The lithium-pilocarpine-induced SE rat model is a reliable model to study status epilepticus and temporal lobe epilepsy [12,13]. Using this model, we have detected an aberrant miRNA expression pattern in the rat hippocampus and confirmed the presence of significantly deregulated miRNAs. Because miR-34a was up-regulated in this work and in a previous study [14], we hypothesized that miR-34a is up-regulated in the rat hippocampus after status epilepticus and contributes to seizure-induced neuronal death. Our results showed that miR-34a is up-regulated in rats after status epilepticus and targeting miR-34a in vivo could alleviate seizure-induced neuronal death or apoptosis and increase the number of surviving neurones in the hippocampus. This research indicates the neuroprotective effects of targeting miR-34a in seizure- induced neurone cell death or apoptosis in post-status epilepticus rats. Methods Ethical statement Animal care and sacrifice were conducted according to methods approved by the Animal Care and Use Committee, Xiangya Medical College, Central South University. All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Experimental animals, housing and husbandry Male Sprague–Dawley (SD) rats (6–8 weeks of age, weighing 230–270 g, from the Animal Unit, Central South University, P. R. China) were used. All animals were housed in a room kept on an alternating 12 hours light–dark cycle with a controlled temperature (18°C - 25°C) and humidity (50% - 60%). Food and water were available ad libitum. Study design, sample size and animal allocation The study design and animal allocation are outlined in Additional file 1. Because there was a loss of animals post-SE and some animals did not exhibit spontaneous seizures, the number of animals used in the study was somewhat larger. The study design, animal allocation and number of animals used for statistical analyses are listed in the notes of Additional file 1, and the selection of animals was random whenever it was possible. Regarding the sample size, there were 152 SD rats used in the entire study, and 102 total rats were included in the statistical analyses. Experimental procedures for the SE and TLE model Lithium chloride (125 mg/kg, i.p., Sigma, USA) was injected 18–20 hours prior to the administration of pilocarpine (20 mg/kg, i.p., Sigma). The severity of convulsions was evaluated by Racine’s scale, and only those animals that were classified with a score of 4–5 were included in this study. SE was defined as the onset of continuous generalised (Racine’s scale score 4–5) seizure activity lasting no less than 40 min. Intraperitoneal pilocarpine administration (10 mg/kg) was repeated every 30 min if there were no seizures or seizure activity was classified lower than a score of 4 on Racine’s scale. The maximum dose for pilocarpine injection was 60 mg/kg. All SE rats received chloral hydrate (10%, 3 ml/kg, i.p.) to terminate epileptic attacks. The control rats received an injection of an equal amount of normal saline instead of pilocarpine. All the rats were housed in the same environment and continuous observation of animal behaviour was performed from the successful establishment of the SE model to the time of the animal’s death. The temporal lobe epilepsy rats (2 months post-SE) were identified by a frequent attack of seizures (at least two spontaneous seizures that scored 4–5 on Racine’s scale), either by direct observation or by videotape recordings. Some animals exhibiting seizures were confirmed using electroencephalogram (EEG) recordings that displayed high frequency, high amplitude, poly-spike paroxysmal discharges. The animals were killed within 5 hours of occurrence of the last spontaneous seizure. Some animals were anesthetised using chloral hydrate (10%, 5 ml/kg, ip.) and decapitated, and hippocampi were removed quickly from the brain and frozen in liquid nitrogen. Other animals were anesthetised and perfused with saline followed by 4% paraformaldehyde. miRNA microarray analysis Total RNA for microarray analysis was extracted and purified using the mirVana™ miRNA Isolation Kit (Cat# AM1560, Ambion, Austin, TX, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). miRNA molecules in the total RNA were labelled by a miRNA Complete Labeling and Hyb Kit (Cat# 5190–0456, Agilent Technologies, Santa Clara, CA, US) following the manufacturer’s instructions. Each slide was hybridised with 100 ng of Cy3-labeled RNA using the miRNA Complete Labeling and Hyb Kit (Cat# 5190–0456, Agilent Technologies, Santa Clara, CA, US) in hybridisation Oven (Cat# G2545A, Agilent Technologies, Santa Clara, CA, US) at 55°C and 20 rpm for 20 hours according to the manufacturer’s instructions. After hybridisation, the slides were washed in staining dishes (Cat# 121, Thermo Shandon, Waltham, MA, US) with the Gene Expression Wash Buffer Kit (Cat# 5188–5327, Agilent Technologies, Santa Clara, CA, US). Slides were scanned using the Agilent Microarray Scanner (Cat# G2565BA, Agilent Technologies, Santa Clara, CA, US) and the Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US) with the default settings. Subsequently, the microarray image information was converted into spot intensity values using the Scanner Control Software Rev. 7.0 (Agilent Technologies, Santa Clara, CA, US). The signal after background subtraction was later exported directly into the Gene Spring GX11.0 software (Agilent Technologies, Santa Clara, CA, US) for quantile normalisation. The quantile algorithm is a method of normalisation that equalises the distribution of expression values of all the samples in an experiment. The mean normalised signal from biological replicates was then used for comparative expression analysis. The unpaired t-test with the Benjamini-Hochberg correction was used to identify differentially expressed miRNAs between the control and epilepsy samples. The significantly deregulated miRNAs were defined as miRNAs that have p-values less than 0.05 and with an expression fold-change greater than 1.5, whether they were up-regulated or down-regulated when compared with controls. Normalised data were hierarchically clustered by gene and used to plot the heat maps. Quantitative real-time PCR Total RNA was isolated using the Trizol reagent (Invitrogen, USA). Each reaction mixture of RT contained 2 μg of RNA template, 2 μL miR-RT primers, mixed with RNase-free H2O to 11 μL (volume A) and 5 μL RT buffer 5X, 0.5 μL 2.5 mM dNTPs, 1 μL RNase Inhibitor (40 U/μL), 0.5 μL Reverse Transcriptase (200 U/μL) (Fermentas) and RNase-free H2O to a total volume of 25 μL. The 11 μL volume A was mixed and centrifuged at 4°C, incubated for 10 min at 70°C and placed in an ice bath for 2 min. The 25 μL volume reactions were then incubated for 60 min at 42°C and for 10 min at 70°C. qPCR reaction was performed using the Platinum SYBR Green qPCR Super Mix UDG (Invitrogen, USA) and ABI Mx3000P QPCR System (Stratagene). qPCR was performed in triplicates. The 20 μL qPCR reaction contained the following: 9 μL SYBR Green Mix, 2 μL miRNA RT Product, 2 μL Bulge-LoopTM miRNA Forward Primer (5 μM) and 2 μL Bulge-LoopTM miRNA Reverse Primer (5 μM) (RiboBio Co., Ltd, China) and RNase-free H2O to a final volume of 20 μL. The qPCR reactions were incubated at 95°C for 20 s, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s and 70°C for 10 s and finally 95°C for 10 s, 60°C for 20 s and 95°C for 10 s. The relative expression level for each miRNA was calculated by the comparative CT method. The expression of the U6 small nucleolar RNA gene was used as an internal control. Bioinformatics analysis All significant differentially expressed miRNAs were analysed by bioinformatics algorithms. Potential targets of these miRNAs were predicted using the miRNA target prediction databases including TargetScan and miRanda. Functional classification was performed by Gene Ontology (GO) to determine the biological significance of these targets, and an accompanying p-value calculated by the Fisher’s Exact Test indicated which functions were over-represented in the targets. Moreover, using the KEGG pathway database, pathway analysis was performed to identify the enriched pathways of targets, and the p-value was calculated for each pathway using a hyper-geometric distribution for identification of the significance of pathways. miR-34a antagomir experiments To study the potential function of miR-34a in post-SE rat hippocampal neuronal apoptosis, a miRNA antagomir strategy was adopted. We antagonised the expression of miR-34a in post-SE rat hippocampus by using an antagomir that specifically targets miR-34a. A miR-34a antagomir or an antagomir-control (miR-RiboTM; RiboBio Co., Ltd., China) was dissolved in an artificial CSF (119 mmol/L NaCl, 3.1 mmol/L KCl, 1.2 mmol/L CaCl2, 1 mmol/L MgSO4, 0.5 mmol/L KH2PO4, 25 mmol/L NaHCO3, 5 mmol/L D-glucose, 2.2 mmol/L urea, pH 7.4) [15] at a concentration of 20 nmol/mL (1 nmol/50 μL for each rat) and infused at a very slow rate (25 μL of CSF was withdrawn for an hour and 50 μL of antagomir was infused for 2 hours) by micro- syringe into the lateral ventricle (Bregma: 0.8 mm posterior, -4.8 mm dorsoventral, -1.5 mm lateral; on the basis of the rat brain atlas of Paxinos and Watson [13]) of post-SE rats. The process began at 1 hour and ended at 4 hours after SE onset. To look for expression changes of miR-34a and its potential downstream molecules in post-SE rats that had received infusions of miR-34a antagomir and antagomir-control, rats were anesthetised using chloral hydrate (10%, 5 mL/kg, ip.) at 7 days post-SE before their hippocampal tissue was removed for detection of miR-34a expression and detection of its potential downstream molecules including activated caspase-3 protein. Western-blot analysis The cytoplasmic extracts were separated by 12% SDS-PAGE and transferred onto a PVDF membrane using the Bio-Rad system (Bio-Rad, USA) before blocking with TBST containing 5% nonfat-milk for 2 hours at room temperature with gentle shaking. Three washing steps of 10 min each were performed after blocking and incubation with the antibodies. The primary antibody was a rabbit anti-rat activated caspase-3 polyclonal antibody (Abcam, ab2302; 1:600); β-actin expression was used as an internal control. The membrane was incubated overnight at 4°C with the primary antibody and incubated with a goat anti-rabbit IgG/HRP antibody (SCBT, sc-2030; 1:40,000) at room temperature for 1 h. The protein/antibody complexes were detected using chemiluminescence reagents (ECL) (KPL, USA). Integrated optical density (IOD) values of activated caspase-3 protein and β-actin were measured. The relative expression amount of activated caspase-3 protein was determined by the ratio of activated caspase-3 protein/β-actin. Immunohistochemistry The paraffin-embedded sections were deparaffinised and rehydrated, followed by treatment with 1% hydrogen peroxide to eliminate endogenous peroxidase activity. After treatment with goat serum reagent at room temperature for 20 min, the sections were exposed to primary antibody for 2 h at room temperature and overnight at 4°C. The primary antibody was a rabbit anti-rat caspase-3 polyclonal antibody (Boster Co., China; 1:200). The sections were incubated with secondary antibody, a biotinylated goat anti-rabbit antibody (Boster Co., China; 1:200) at room temperature for 20 min, followed by incubation with a streptavidin-biotin peroxidase complex solution (Boster Co., China) at room temperature for 90 min. Subsequently, the sections were stained using diaminobenzidine, restained with hematoxylin, dehydrated and mounted. All slices were processed with a HPIAS-1000 Color Image Analysis system for imaging and analysis. Nissl staining and TUNEL assay Nissl staining was performed to detect the surviving neurones. The sections were stained with 0.5% Cresyl violet (w/v) for 10 minutes before they were dehydrated and mounted with permount. The surviving neurones were only counted if they possessed intact neuronal morphology. The TUNEL assay, which detects DNA fragmentation resulting from apoptotic signalling cascades, was performed to label apoptotic neurones. It may also label cells that have suffered severe DNA damage. Therefore, the TUNEL assay is helpful in identifying seizure-induced neuronal death in our experiments. The procedures were performed according to the manufacturer’s instructions (Roche Co., Germany). TUNEL-positive cells were defined as those demonstrating blue-purple nucleic staining. The CA1 and CA3 regions of each section were captured by microscope (Olympus, Japan). Under 40 × magnification, 6 visual fields were randomly selected for each section, and the surviving neurones and the total number of cells were counted. The survival rate was calculated as follows: number of surviving neurones/total number of cells × 100%. Additionally, 6 visual fields were randomly selected to count the number of dead neuronal cells and the total number of cells. The death rate was determined as follows: number of dead neuronal cells/total number of cells × 100%. For Nissl-staining or TUNEL-assay, three sections were examined for each rat before the means were obtained for statistical analysis. Statistical methods Statistical analysis was performed using the SPSS 13.0 software and all data were expressed as the mean ± standard deviation. Differences between multiple groups were statistically assessed by one-way ANOVA analysis, whereas differences between two groups were evaluated by the independent-samples t-test. For statistical analysis, p < 0.05 was considered to be statistically significant. Results Animal number analysed The number of animals in each group included in each analysis is reported in Additional file 1. Absolute numbers were used in the report. The animals and data not included in the analysis, as well as their reasons, are also clearly mentioned in the notes of Additional file 1. miRNA expression profile of the hippocampus in a rat model of TLE The Agilent Rat miRNA microarray used in this study covered over 350 rat miRNAs detected so far, and the sequence information is based on the Sanger miR-Base version 12.0. The miRNA microarray scanned images and the normalised primary microarray data are shown in Additional files 2 and 3, respectively. The cluster and heatmap of all detected rat miRNAs in this study are plotted and shown in Figure 1. The miRNA microarray differential analysis resulted in 24 significantly deregulated miRNAs as shown in Table 1, and the heatmap of these 24 aberrantly expressed miRNAs is plotted and shown in Figure 2. Among the 24 significantly deregulated miRNAs, 9 miRNAs (miR-146a, -211, -203, -210, -152, -31, -23a, -34a, -27a) were up-regulated and 15 miRNAs (miR-138*, -301a, -136, -153, -19a, -135b, -325-5p, -380, -190, -33, -542-3p, -144, -542-5p, -543, -296*) were down-regulated (differentially expressed miRNAs were defined by a fold-change >1.5, either up-regulated or down-regulated; *p <0.05). Figure 1 The heatmap of all rat hippocampal TLE miRNAs. Rows represent individual genes, and columns represent individual samples. The high (red), average (black) and low (green) expression levels are depicted by the colourgram (n = 6 for TLE rats and n = 6 for control). Table 1 The 24 significantly deregulated miRNAs in the TLE rat hippocampus MicroRNA Fold change Regulation pvalue rno-miR-146a 2.6958885 up 1.258E-02 rno-miR-211 2.610144 up 1.376E-02 rno-miR-203 2.109093 up 2.367E-02 rno-miR-210 1.8106444 up 1.999E-02 rno-miR-152 1.7704262 up 8.17E-04 rno-miR-31 1.6925983 up 3.811E-03 rno-miR-23a 1.5156718 up 7.727E-03 rno-miR-34a 1.4842122 up 2.398E-02 rno-miR-27a 1.4451101 up 1.133E-02 rno-miR-138* 1.5289445 down 3.512E-02 rno-miR-301a 1.5327321 down 7.925E-03 rno-miR-136 1.5330569 down 4.166E-03 rno-miR-153 1.5534221 down 1.826E-02 rno-miR-19a 1.6450298 down 1.832E-02 rno-miR-135b 1.657749 down 2.853E-02 rno-miR-325-5p 1.8619193 down 4.587E-02 rno-miR-380 1.8745395 down 1.176E-02 rno-miR-190 1.8834999 down 1.528E-02 rno-miR-542-3p 1.8835889 down 7.955E-03 rno-miR-33 1.9224428 down 3.279E-03 rno-miR-144 1.9507241 down 4.969E-02 rno-miR-542-5p 2.2845843 down 5.943E-03 rno-miR-543 3.0247948 down 1.067E-03 rno-miR-296* 3.0831256 down 2.85E-06 Notes: The Agilent rat miRNA microarray identified 24 significantly deregulated miRNAs (fold change >1.5, expression either upregulated or down-regulated; *p <0.05) in the hippocampus of the TLE rat when compared with the control (n = 6 for each group). Figure 2 The heatmap of differentially expressed hippocampal miRNAs in the TLE rat. The 24 significantly deregulated miRNAs have been rearranged in this heatmap, where 9 up-regulated and 15 down-regulated miRNAs are clearly displayed (n = 6 for TLE rats and n = 6 for control). miRNA confirmation using qPCR To validate the altered expression of miRNAs as detected by miRNA microarray, miR-146a, miR-210, miR-27a, miR-135b and miR-33 were selected for confirmation by quantitative real-time PCR. The results were consistent with that of the microarray analysis. As seen in Figure 3, the expression levels of miR-146a, miR-210 and miR-27a were up-regulated, while the expression levels of miR-135b and miR-33 were down-regulated (*p < 0.05). Figure 3 The quantitative real-time PCR validation for some expression-altered miRNAs. The five differentially expressed miRNAs (miR-146a, miR-210, miR-27a, miR-135b and miR-33) in the TLE rat hippocampus as detected by the Rat miRNA microarray were confirmed using qPCR (Data are presented as the mean ± SEM, *p <0.05; n = 6/TLE rats, n = 6/control). White bars: control; Black bars: experimental. Bioinformatics analysis There were 1315 and 8810 target genes predicted by microRNA target prediction databases TargetScan and miRanda. The GO terms significantly over-represented in deregulated miRNA targets are listed in Figure 4, where the percent and enrichment test p values for each GO term are indicated. Some of the top KEGG pathways important in the molecular mechanism of epilepsy are significantly over-represented among the deregulated miRNA targets and are listed in Additional file 4. The signalling pathways enriched in temporal lobe epilepsy are consistent with previous studies of the molecular portrait of basic epilepsy mechanisms using whole transcriptome analysis of the hippocampus [16]. Figure 4 GO terms significantly over-represented among the deregulated miRNA targets are shown. The percent and enrichment test p value for each GO term are indicated in this figure. miR-34a expression post-SE Expression of the pro-apoptotic miR-34a in the rat hippocampus was examined. The time points chosen for miR-34a detection were 1 day, 7 days, 2 weeks post-SE and 2 months (TLE). The expression pattern of miR-34a is shown in Figure 5. Pro- apoptotic miR-34a was significantly increased in post-SE rat hippocampus at all four time points chosen (*p <0.05). Its over-expression was notably higher at 1 day, 7 days and 2 weeks post-SE before it went down to a lower level at 2 months in temporal lobe epilepsy, although it was still markedly higher than control. Figure 5 The expression pattern of miR-34a is shown. The study time points were 1 day, 1 week, 2 weeks (post-SE) and 2 months (TLE). The pro-apoptotic miR-34a displayed increased expression at all four time points chosen (Data are presented as the mean ± SEM, *p <0.05; n = 6 for each group), when compared with control. miR-34a antagomir outcomes miR-34a antagomir treatment significantly reduced the expression of miR-34a in the rat hippocampus at 7 days post-SE (*p <0.05), when compared with the miR-34a antagomir-control-treated group (Figure 6A). Furthermore, the miR-34a antagomir specifically targeting miR-34a did not exert any antagonising effects on other miRNAs in the rat hippocampus at 7 days post-SE (data not shown). The use of miRNA antagomirs is a successful strategy for studies of miRNA function, both in vivo and in vitro, because antagomirs effectively and specifically inhibit endogenous miRNAs by causing their degradation with effects that persist at least one week or longer (Instructions for miR-RiboTM Antagomir and Antagomir-control; RiboBio Co., Ltd., China). Figure 6 The results of the miR-34a antagomir experiment. Inhibition of miR-34a expression by the miR-34a antagomir at 7 days post-SE in the rat hippocampus is shown in. A. The miR-34a antagomir significantly inhibited the expression of miR-34a in the post-SE rat hippocampus when compared with the antagomir-control (Data are presented as the mean ± SEM, *p < 0.05; n = 6 for each group). The expression of activated caspase-3 protein in the post-SE rat hippocampus both before and after miR-34a antagomir/antagomir-control treatments as detected by western blot analysis is shown in B. Western blot detected an up-regulation in the expression of activated caspase-3 protein at 7 days post-SE in the rat hippocampus, when compared with control (*p <0.05, n = 6 for each group). The expression of activated caspase-3 protein at 7 days post-SE in the rat hippocampus, however, decreased in the miR-34a antagomir-treated group, when compared with the antagomir-control-treated group (**p < 0.05, n = 6 for each group). Alteration of activated caspase-3 expression The Western blot results demonstrated an up-regulation of activated caspase-3 protein expression at 7 days post-SE in the rat hippocampus, when compared with the control (*p <0.05) (Figure 6B). Moreover, the expression level of activated caspase-3 protein at 7 days post-SE in the rat hippocampus decreased after miR-34a antagomir treatment, when compared with the miR-34a antagomir-control-treated group (*p <0.05) (Figure 6B). Immunohistochemistry confirmed the western blot results shown in Figure 7, where the activated caspase-3 protein was significantly over-expressed in the post-SE rat hippocampal CA1 region and CA3 regions (Figure 7F and Figure 7J) when compared with the control (Figure 7E and Figure 7I). Moreover, activated caspase-3 protein expression was significantly down-regulated in the post-SE rat hippocampal CA1 and CA3 regions after miR-34a antagomir treatment (Figure 7G and Figure 7K) when compared with the antagomir-control-treated group (Figure.7H and Figure 7L). Figure 7 Activated caspase-3 protein in the post-SE rat hippocampus. Overview: A-D; CA1 region: E-H; CA3 region: I-L. Scale bars: 300 μm (A-D); 30 μm (E-L). Alterations in neuronal survival and neuronal death Nissl-staining was performed to detect surviving neurones, and the TUNEL-assay was used to label apoptotic neurones by detecting the DNA fragmentation that results from apoptotic signalling cascades. It may also label cells that have suffered severe DNA damage. TUNEL is therefore helpful for identifying seizure-induced neuronal death in our experiments. In the post-SE rat hippocampus, neuronal survival decreased, whereas neuronal death increased (at 7 days post-SE) (Table 2, Figure 8 and Figure 9). As seen, neuronal survival was reduced in the post-SE rat hippocampal CA1 and CA3 regions (Figure 9F and Figure 9J; Figure 9E and Figure 9I as control), and neuronal death increased in the CA1 and CA3 regions (Figure 8F and Figure 8J; Figure 8E and Figure 8I as controls). Table 2 Neuronal death (apoptosis) and neuronal survival at 7 days post-SE in the rat hippocampus Neurones Control Epilepsy Antagomir Antagomir-control   CA1 CA3 CA1 CA3 CA1 CA3 CA1 CA3 Percent Death 17.91 ± 4.78% 20.57 ± 5.02% 48.86 ± 12.39% a 40.39 ± 11.42% a 30.49 ± 8.91% b 24.80 ± 5.80% b 49.35 ± 12.74% 40.69 ± 10.97% Percent Survival 88.15 ± 10.02% 78.89 ± 8.92% 47.32 ± 11.93% a 51.25 ± 14.80% a 72.81 ± 10.60% b 68.28 ± 11.54% b 46.26 ± 9.50% 52.05 ± 14.70% Notes: In rat hippocampal CA1 and CA3 regions, the number of dead neurones or apoptotic cells was significantly increased and the number of surviving neurones was markedly decreased at 7 days post-SE, as detected by the TUNEL-assay and Nissl-staining (aP < 0.01, epilepsy vs control, n = 6 /group). In the same regions, however, neuronal death or apoptosis was decreased and neuronal survival was increased significantly after miR-34a antagomir or antagomir-control treatment (bP < 0.01, miR-34a antagomir vs antagomir-control, n = 6 /group). The survival rate is determined by the number of surviving neurones/ number of total cells × 100%, whereas the neuronal death or apoptotic rate is determined by the number of dead neurones or apoptotic cells/ number of total cells × 100%. Figure 8 Neurone cell death or apoptosis visualised by the TUNEL-assay in the post-SE rat hippocampus. Overview: A-D; CA1 region: E-H; CA3 region: I-L. Scale bars: 300 μm (A-D); 30 μm (E-L). Figure 9 Neurone survival visualised by Nissl-staining in the post-SE rat hippocampus. Overview: A-D; CA1 region: E-H; CA3 region: I-L. Scale bars: 300 μm (A-D); 30 μm (E-L). miR-34a antagomir treatment had an inhibitory effect on activated caspase-3 protein expression and led to an increase in neuronal survival as well as a decrease in seizure-induced neuronal death at 7 days post-SE in the rat hippocampus (Table 2, Figure 8 and Figure 9). The number of surviving neurones was markedly increased in the CA1 and CA3 regions (Figure 9G and Figure 9K; Figure 9H and Figure 9L as controls) when compared with the antagomir-control-treated group. Additionally, there was a significant decrease in neuronal cell death after miR-34a antagomir treatment in the post-SE rat hippocampal CA1 and CA3 regions (Figure 8G and Figure 8K; Figure 8H and Figure 8L as controls) when compared with the control. Discussion Because a number of previous studies have investigated the role of miRNAs in epilepsy [6-11,14,17], it is important to provide a discussion of our own data in light of previously published profiles and results. Different models may yield different study outcomes, and comparisons of results between different or even the same models are necessary. Similar to our study, Song et al. used the classical status epilepticus rat model and reported differential expression of miRNAs in the hippocampus in a rat model of TLE [17]. Yet, the number of samples used for miRNA microarray analysis (TLE rats = 3, control rat = 1) in their study did not strictly conform to the limits needed for statistical analysis. Furthermore, the standards to screen differentially expressed miRNAs in their study (fold-change: three ratios >1 plus at least two ratios >1.5 for up-regulated miRNAs; three ratios <1 plus at least two <0.67 for down-regulated miRNAs) did not meet the stringent conditions of the method for analysing miRNA microarray raw data; the overall inclusion of all data, where miRNA signal intensities and all microarray values are integrated for statistics, appears to be preferable. Therefore, we investigated the miRNA expression profile of TLE in the rat hippocampus using a more rigorous design of animal numbers (TLE rats = 6, control = 6) and method of analysing microarray raw data. The results from our study were different to some extent from Song’s report. There were some common differentially expressed miRNAs in the two studies; e.g., both detected an up-regulation of the inflammation-associated miR-146a and the apoptotic miR-23a. However, many differentially expressed miRNAs detected varied between the two studies. For example, the pro-apoptotic miR-34a was detected in our study as a significantly deregulated miRNA, and further investigation of the expression and function of miR-34a in the mechanism of epilepsy was performed. To extend the animal studies to human research, Kan et al. recently detected miRNA profiles in human TLE and reported marked aberrant expression of miRNAs in mTLE patients [8]. In their study, autopsy control patients and two mTLE patient groups were compared, and segregated miRNA signatures for three different patient groups were revealed. Analysis of the changes of individual miRNAs identified 165 miRNAs with up- or down-regulated expression, and 51 of these miRNAs exhibited a fold change >2.0. Interestingly, the significantly deregulated inflammation-associated miR-146a detected in a TLE patient by Aronica et al. [9] and in the rat model of TLE by Song et al. [17] and our research was not identified in Kan’ work. This could be due to different standards for the selection of TLE patients or varied criteria for the surgery selection of TLE patients. In addition, different standards to screen for significantly deregulated miRNAs may also contribute to a difference in results. Because the human species exhibits a much larger pool of miRNAs than rats, it is natural that miRNA profiling in human TLE patients may result in a larger number of differentially expressed miRNAs than in rats with TLE. Some deregulated miRNAs were identified both in the TLE patients of Kan’s work and in our rat TLE model, such as miR-27a, miR-190, miR-203 and miR-301a. Moreover, Kan’s findings suggest that miRNA changes in mTLE affect the expression of immunomodulatory proteins and further facilitate immune responses. Accordingly, the KEGG pathway analysis of differentially expressed miRNAs in our research also identified that pathways in signal transduction and immune system were significantly enriched. They are consistent with opinion that immune cells and their inflammatory mediators play an important role in the pathophysiology of seizures and epilepsy [18]. Studies by the Henshall group [10,11] identified the functions of several miRNAs in epilepsy by using mouse models. In a recent paper, Henshall et al. reported that miR-134, a miRNA implicated in the control of dendritic spine morphology, was up-regulated in a mouse model of status epilepticus, and silencing of miR-134 expression reduced neurone dendrite spine density and rendered mice refractory to seizures [10]. The Henshall group also reported that the expression level of another miRNA, miR-132, was increased following status epilepticus. Targeting miR-132 in vivo depleted miR-132 levels in the mouse hippocampus and reduced the seizure-induced neuronal death [11]. Furthermore, Sano et al. reported miR-34a up-regulation during seizure-induced neuronal death in a mouse model [7] and concluded that miR-34a up-regulation in subfield-specific, temporally restricted manner is most likely not important for seizure-induced neuronal death. The strategy of these studies commonly includes the selection of a target miRNA followed by expression analysis and the use of an antagomir. The target miRNAs chosen were mainly brain-specific and possess functions in either dendritic spine morphogenesis or neuronal cell death, as these aspects are important factors for the development of epilepsy. The immune response and astrocytes have also been identified to play a role in epileptogenesis, yet miRNA functional studies concerning this aspect of the mechanism of epilepsy are few and more studies are needed. The Sano study did not detect a protective effect of the miR-34a antagomir in the kainic acid-induced status epilepticus mouse model, although they showed an up-regulation of miR-34a in hippocampal subfields after status epilepticus. This is somewhat different from our results, as we have not only found an up-regulation of miR-34a in the post-status epilepticus rat hippocampus but also detected a neuro-protective effect of targeting miR-34a during seizure-induced neuronal death. The reasons for the difference in these results are multi-faceted, but the most important reason is likely due to the different epilepsy models used. There are various animal models with chronic brain dysfunction that are thought to reflect processes underlying human epilepsy. These chronic models of epilepsy include the kindling model of TLE and post-status epilepticus models of TLE. Pilocarpine and kainate models replicate features of human TLE and can be used as animal preparations for understanding the basic mechanisms of epileptogenesis [13]. However, differences between the models, species and strains do exist in that the processes underlying epileptogenesis and the extent of neuronal damage differs [19]. Furthermore, the degree to which miR-34a can induce apoptosis may vary between different models. For example, there is evidence for miR-34a having varying effects in the promotion of apoptosis [20,21]. As up-regulation of miR-34a caused by prolonged seizures in vivo has already been shown, we propose that miR-34a may have a more important role in the lithium- pilocarpine-induced status epilepticus in rats. In this model, neuronal injury leads to miR-34a up-regulation, and the protective effect of the miR-34a antagomir in our study may be related to the ability of miR-34a to induce apoptosis in post-status epilepticus rats. Because seizure-induced neuronal death may be more prominent when miR-34a has much stronger pro-apoptotic function, targeting miR-34a may result in subsequent neuroprotective effects. The potential mechanism of miR-34a regulation of downstream targets needs further mention. miR-34a up-regulation in a transgenic mice model of Alzheimer’s disease resulted in a higher expression level of activated caspase-3 protein by inhibiting bcl-2 translation, suggesting that bcl-2 is an important target for miR-34a [22]. This is consistent with results from previous studies that identified bcl-2 as a miR-34a target [23-26]. Because abnormal expression of miR-34a may contribute to the pathogenesis of Alzheimer’s disease, it is speculated that seizure-induced up-regulation of miR-34a may also play a role in the pathophysiology of epilepsy, at least in part by affecting bcl-2 expression. Prolonged experimental seizures can cause apoptosis and activate caspases and bcl-2 family proteins [27]. These apoptosis-associated genes may contribute to mechanisms underlying the development and maintenance of epilepsy, and modulating these genes can alter hippocampal damage after seizures. miR-34a as an epigenetic factor may play a role in modulating apoptosis-related genes such as bcl-2 and the caspases after status epilepticus. We focused on the effects of antagonising miR-34a on the expression of the end-phase apoptosis executor, the caspase-3 gene, to show that pro-apoptotic miR-34a and caspase-3 expression levels are related. Seizure-induced neuronal death was less prominent after treatment with the miR-34a antagomir in the post-status epilepticus rat hippocampus. What accounts for the neuroprotective effect is most likely due to a milder inhibitory effect of reduced miR-34a expression on bcl-2 translation, resulting in a lowered expression level of activated caspase-3 protein and subsequent reduction in seizure-induced neuronal death. Conclusions In conclusion, our results demonstrate the expression profile of miRNAs in the hippocampus in a rat model of TLE and the pattern of expression increase of the pro-apoptotic miR-34a in post-SE rats. This study also demonstrated up-regulation of miR-34a in seizure-induced neuronal death or apoptosis and showed the neuro-protective effect of targeting miR-34a. The latter may be related to a lowered expression level of the activated caspase-3 protein. The present research provides insight into the involvement of miRNAs in the mechanism of epilepsy. Abbreviations MiRNAs: MicroRNAs; NPCs: Neural progenitor cells; AEDs: Anti-epileptic drugs; SE: Status epilepticus; TLE: Temporal lobe epilepsy; EEG: Electroencephalogram; GO: Gene ontology; XIAP: X-linked inhibitor of apoptosis protein. Competing interests The authors declare that they have no competing interests. Authors’ contributions KH contributed to the study design and established the SE model, extracted the miRNAs and contributed in paper writing. YYX contributed to western blot analysis, immnohistochemistry, TUNEL-assay and Nissl-staining. CZ and HYL carried out the quantitative real-time PCR. DSO contributed to the statistical analyses and paper writing. DNS and LLL performed the bioinformatic analysis. LF and YL carried out the miRNA antagomir experiment. BX contributed in study design, supervised the study and contributed in paper writing. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Study design, sample size and animal allocation. The experiments, grouping design, sample size and animal number for statistical analyses are detailed in this file. Click here for file Additional file 2 Agilent Rat miRNA microarray scanned images. The 12 Agilent Rat miRNA microarray scanned images are shown in this file. Click here for file Additional file 3 Agilent Rat miRNA microarray raw data. The 12 Agilent Rat miRNA microarray raw data are shown in this file. Click here for file Additional file 4 The KEGG pathway analysis results. The significantly enriched pathways of deregulated miRNA targets, as predicted by the KEGG pathway database, are shown in this file. Click here for file Acknowledgements This work was supported by the National Natural Science Foundation of China (81100967, 81071048, 81000553), Specialized Research Fund for the Doctoral Program of Higher Education (20110162120002), and Fundamental Research Funds for the Central Universities of China (2011QNZT152). ==== Refs Bartel DP MicroRNAs: target recognition and regulatory functions Cell 2009 136 2 215 233 10.1016/j.cell.2009.01.002 19167326 Liu C Zhao X MicroRNAs in adult and embryonic neurogenesis Neuro- molecular Med 2009 11 3 141 152 152 Krichevsky AM Sonntag KC Isacson O Kosik KS Specific microRNAs modulate embryonic stem cell-derived neurogenesis Stem Cells 2006 24 4 857 864 10.1634/stemcells.2005-0441 16357340 Delaloy C Liu L Lee JA Su H Shen F Yang GY Young WL Ivey KN Gao FB MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors Cell Stem Cell 2010 6 4 323 335 10.1016/j.stem.2010.02.015 20362537 Agostini M Tucci P Steinert JR Shalom-Feuerstein R Rouleau M Aberdam D Forsythe ID Young KW Ventura A Concepcion CP Han YC Candi E Knight RA Mak TW Melino G microRNA-34a regulates neurite outgrowth, spinal morphology, and function Proc Natl Acad Sci USA 2011 08 52 21099 21104 22160706 Nudelman AS DiRocco DP Lambert TJ Garelick MG Le J Nathanson NM Storm DR Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo Hippocampus 2010 20 4 492 498 19557767 Sano T Reynolds JP Jimenez-Mateos EM Matsushima S Taki W Henshall DC MicroRNA-34a upregulation during seizure-induced neuronal death Cell Death Dis 2012 3 e287 10.1038/cddis.2012.23 22436728 Kan AA van Erp S Derijck AA de Wit M Hessel EV O’Duibhir E de Jager W Van Rijen PC Gosselaar PH de Graan PN Pasterkamp RJ Genome-wide microRNA profiling of human temporal lobe epilepsy identifies modulators of the immune response Cell Mol Life Sci 2012 69 18 3127 3145 10.1007/s00018-012-0992-7 22535415 Aronica E Fluiter K Iyer A Zurolo E Vreijling J van Vliet EA Baayen JC Gorter JA Expression pattern of miR-146a, an inflammation-associated microRNA, in experimental and human temporal lobe epilepsy Eur J Neurosci 2010 31 6 1100 1107 10.1111/j.1460-9568.2010.07122.x 20214679 Jimenez-Mateos EM Engel T Merino-Serrais P McKiernan RC Tanaka K Mouri G Sano T O’Tuathaigh C Waddington JL Prenter S Delanty N Farrell MA O’Brien DF Conroy RM Stallings RL Defelipe J Henshall DC Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects Nat Med 2012 [Epub ahead of print] 10.1038/nm.2834 Jimenez-Mateos EM Bray I Sanz-Rodriguez A Engel T McKiernan RC Mouri G Tanaka K Sano T Saugstad JA Simon RP Stallings RL Henshall DC miRNA Expression profile after status epilepticus and hippocampal neuroprotection by targeting miR-132 Am J Pathol 2011 179 5 2519 2532 10.1016/j.ajpath.2011.07.036 21945804 Curia G Longo D Biagini G Jones RS Avoli M The pilocarpine model of temporal lobe epilepsy J Neurosci Methods 2008 172 2 143 157 10.1016/j.jneumeth.2008.04.019 18550176 Löscher W Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. 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Int J Physiol Pathophysiol Pharmacol 2009 1 2 97 115 21383882
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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23082150PONE-D-12-1725310.1371/journal.pone.0047218Research ArticleBiologyModel OrganismsAnimal ModelsMouseNeuroscienceCognitive NeurosciencePainBehavioral NeuroscienceMedicineNon-Clinical MedicineHealth Care PolicySexual and Gender IssuesMedical SociologySocial and Behavioral SciencesSociologySexual and Gender IssuesThe Effect of Social Stress on Chronic Pain Perception in Female and Male Mice Social Stress, Chronic Pain Perception and GenderAghajani Marjan 1 2 3 Vaez Mahdavi Mohammad Reza 1 2 * Khalili Najafabadi Mohsen 1 Ghazanfari Tooba 2 3 1 Department of Physiology, Faculty of Medical Sciences, Shahed University, Tehran, Iran 2 Equity and Health Research Department, Shahed University, Tehran, Iran 3 Department of Immunology, Faculty of Medical Sciences, Shahed University, Tehran, Iran Ardehali Hossein Editor Northwestern University, United States of America * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: MA MRVM MKN TG. Performed the experiments: MA MRVM. Analyzed the data: MA MRVM MKN TG. Contributed reagents/materials/analysis tools: MA MRVM MKN TG. Wrote the paper: MA MRVM. 2012 17 10 2012 7 10 e4721819 6 2012 10 9 2012 © 2012 Aghajani et al2012Aghajani 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 current investigations on social stress primarily point to the negative health consequences of being in a stressful social hierarchy. The repetitive nature of such stressors seems to affect behavioral response to pain both in rodents and humans. Moreover, a large discrepancy in the possibility of social stresses affecting pain perception in the two genders exists. The present study examined the effect of chronic social stress on nociceptive responses of both sexes by implementing of food deprivation, food intake inequality and unstable social status (cage-mate change every 3 days) for a period of 14 days in 96 Balb/c mice. In this regard we injected 20 µl formalin 2% into the plantar surface of hind paw at the end of stress period and scored pain behaviors of all subjects, then serum concentrations of proinflammatory cytokines were measured. Our results showed that there was significant difference in chronic phase of formalin test following implementation of food deprivation and inequality (P<0.05) as compared to control group, so that pain perception was decreased considerably and this decline in inequality exposed subjects was well above isolated ones (P<0.05); whereas unstable social situation did not affect pain perception. Moreover, IL-1 and IL-6 concentrations in serum of stressed mice of both genders were well above control group (p<0.05). Finally, despite chronic pain perception in control and unstable male subjects was larger than females; the decrease of chronic pain perception in male stressed animals (poverty and inequality experienced subjects) was much more than stressed females. These results revealed that although food deprivation and social inequality can induce hypoalgesia, some socioeconomic situations like social instability don't affect pain sensation, whereas there were similar increases of proinflammatory cytokines level in all socially stressed subjects. In addition, males display larger hypoalgesic responses to inequality as compared with females. This research was provided by financial support and supervision of Shahed Medical University (grant number: 87/14/A/P) but this funding source had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. ==== Body Introduction Health is not just the outcome of genetic or biological processes but is also influenced by the social and economic conditions in which we live. These influences have become known as the ‘social determinants of health’. Inequalities in social conditions give rise to unequal and unjust health outcomes for different social groups [1]. Poverty and social inequality can be intrinsically alienating and distressing, and of particular concern are the direct and indirect effects of poverty on the development and maintenance of emotional, behavioral and psychological problems [2], [3]. Pain is an important sensorial modality with an elevated degree of complexity and subjectivity that involves not only the transduction of noxious stimuli, but also cognitive and emotional features [4], [5]. Several studies have shown the association of the social stresses with the experience of pain [6]–[8]. As in some experiments on animal species, increase in nociceptive thresholds (hypoalgesia) has been found after exposure to social stressors [9]. Nevertheless, other studies have reported that repeated exposure to such stressors can potentiate acute pain perception in humans and animals [10]–[14]. It has been shown both short-term and intermittent food deprivation diminishes acute nociception in laboratory rodents. However, neither prolonged dietary restriction effects nor prolonged pain has received as much attention [15]. Numerous animal models exist for the exploration of mechanism(s) and mediators of persistent pain in particular [16] and such studies in rodents addressing the link between the pain and social stresses are likely to be relevant in humans [17]. Until a few years ago, a large majority of studies used the terms “sex” and “gender” interchangeably. However, an important distinction has been made between the 2 terms: “sex” refers to biological differences between women and men according to their reproductive organs, whereas “gender” refers to a broader and more complex psychological, environmental, socio-cultural, and political framework that encompasses the characteristics ascribed to each sex that are generally accepted and influenced by society (gender role) [18]. Over the years, laboratory and human research has focused on the biological factors that could potentially mediate gender-related differences in pain responses. The role of psychological and social determinants has also been amply investigated [18]–[21]. Sociopsychological explanations for this gender pattern have focused on the greater exposure of women to psychosocial adversity, on women's reporting a variety of recurrent pains than men, as well as differences in responses to stress, which in turn reflect gender differences in role expectations [22]. Thus, the first aim of the present study was to investigate the impact of social stresses on chronic pain perception. Our previous reports provide an impetus that animals as well as humans sense differences in social situations via a bio-psycho-neuro-social phenomenon [23], [24]; in the present study we investigated whether behavioral response to pain could be modulated after implementing different kinds of social stressors. Since recent researches indicate that nervous systems of males and females differently process and react to pain [25], another objective of our study was to evaluate the role of various social factors that may contribute to gender differences in pain sensitivity in laboratory animals. Experimental Procedures 2.1. Animal model and experimental protocol 96 naive, adult male and female inbred mice of the Balb/c strain (aged 8–10 weeks) were the experimental subjects which were purchased from the Pastour Institute (Karaj, Iran). Ambient temperature was maintained between 22 and 24°C, and the vivarium was maintained under a 12∶12 h light/dark cycle. On arrival, the subjects of each gender were weighted and then randomly assigned to six experimental groups comprising each of eight mice (totally 12 groups). Before the study, the mice were assimilated into the new home for two weeks (there were placed 2 mice of same genders per cage) with no limitation for food (rodent laboratory chow (Daam and Toyur Food Co.) containing by weight: 23.4% protein, 4.5% fat,5% fiber, 7.3% ash, 50% utilizable carbohydrate, and vitamins) and water. Food intake was measured daily and the obtained average amount of ad libitum food consumption was 4.5 gram/day for each mouse. Twelve groups of both genders were set in two rooms: eight groups in one and the remainder in another (isolated) room. After two weeks of assimilating period, three conditions consisting of different social situations [food deprivation, affliction of inequality in food deprived animals (encountering with groups that had free access to diet) and cage-mate change (unstable social status)] were applied to below groups during further 2 weeks: 1. Control group This group had free access to diet without any deprivation, inequality and change of cage-mate. 2. Food Deprivation (See) group (FD+See) According to definition of Van Haasteren [26] each mouse in this group received one third (1.5 gram/day) of their normal daily food intake between 09:00 and 11:00 h, and experienced food inequality by sensing that other animals were feeding and smelling food odor, but without any cage-mate change. 3. Food Deprivation (Isolated) group (FD+Isolate) As same as second group each mouse received 1.5 gr/day food but without experience of social instability (cage-mate change) and any inequality as they were placed in isolated room. 4. Food Deprivation plus Cage-mate Change (See) group (FD+See+CC) As well as food deprivation and inequality, these animals experienced social instability (cage-mate change every 3 days). 5. Food Deprivation plus Cage-mate Change (Isolated) group (FD+Isolate+CC) They had similar condition like the fourth group but no experience of food inequality (This group as same as third group were placed in isolated room). 6. Cage-mate Change group (CC) These animals just faced with social instability (cage-mate change every 3 days) and there was no experience of food deprivation and inequality. In this unstable paradigm [27], one of two mice from each cage was swiped and introduced to another cage in which another resident mouse had experienced this change 3 days ago (such social instability paradigm was also performed in groups 4 and 5). 24 hours after implementing these social stressors (i.e. at day 15 of the study), the animals were weighted again and then they underwent pain assessment which is described in the following section. It should be noted that food deprivation induces states distinct from physiological situation of caloric restriction [15]. At the end of experiments, the animals were anesthetized and their cardiac blood was collected [28] in order to further investigation about changes of proinflammatory cytokines that will clarify to which extent the observed changes in pain perceptions are attributable to social stresses (it should be noted that we used Balb/c Strain because of their similarity in genetic, just for assessing the effects of social stress on immunological factors [29]), and then they were perished by the high dose of anesthetic agent. The experimental protocols and procedures described in this research were approved by Institutional Animal Care and Use Committee (IACUC) at the Medical University of Shahed (Iran) and complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Moreover, all experiments followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data [30]. Finally, we decided not to determine the phase of the estrous cycle in females because two studies showed that there is no difference in nociceptive responses to the formalin test during these stages [31], [32]. 2.2. Nociceptive assay Experiments took place between 12:00 and 16:00. 20 µl of 2% formalin was injected into the plantar surface of the right hind-paw. Mice were standing on a glass floor within Plexiglas observation cylinders (25 cm diameter; 22.5 cm high) and habituated to these cylinders to be tested concurrently for 30 minutes before the formalin injection. Then they were briefly removed, injected and replaced in the cylinder and their pain response was scored every 15 second for 60 minute according to below sampled scales: 0- no pain : normal weight born on injected paw 1- favoring : injected paw in contact with the floor, but full weight not on the paw 2- lifting : injected paw elevated 3- licking : licking or biting the injected paw The observational period was divided into 20 blocks of 3 minutes each (at which we firstly calculated the mean pain score of each minute and then the mean pain score of each block), so we had a biphasic diagram at which a first peak of behavioral response to pain (typically licking behavior of the formalin-injected paw) during the first 6 minute interval reflected the behavioral response to acute pain; whereas the second part of the curve represented a persistent pain (the average scale from minutes 15 to 35 was considered as chronic phase [33]). In between the two curves, there was an inter-phase in which paw-licking behavior was almost reduced to zero [34]. 2.3. Immunological assay After formalin test, mice were anesthetized slightly with ether and then while we sensed heart beats from apex, blood samples were obtained from ventricle. For measurement of cytokine concentrations clotted blood samples were centrifuged immediately at 3000 rpm for 10 minutes and serums stored at −70°C. Circulating immunoreactive IL-6 and IL-1 levels were measured using commercially available quantitative enzyme-linked immunosorbent assays (R&D Systems Europe, Abingdon, UK) [35]. The assays did not measure biological activity of the cytokines. Standard sensivity assays were used and the manufacturers reported the sensivity thresholds in serum as 0.7 pg/ml and 1.5 pg/ml for IL-6 and IL-1, respectively. All measurements were made by a single trained individual to avoid interobserver variation. 2.4. Statistical analysis Statistical analyses were performed using the Sigma Stat software (Systat Sofware, Inc., Point Richmond, CA, USA). Analyses included Mann-Whitney U test for non-parametric data (gender differences), two-way ANOVA test (the effect of each social stressor on the mean nociceptive score in each phase (I, interphase, II) of the formalin test) and one-way ANOVA test (statistical significance for cytokines concentrations) for multiple comparisons, followed by the post hoc Tukey–Kramer test for parametric distribution. Additionally, the results of pain behaviors after formalin injection were analyzed with a one-way ANOVA for repeated measures (10 blocks) to test difference between all blocks in each group. A significance level of P<0.05 was used in all cases. Data are presented in the text and in all figures as means ± SEM. Results 3.1. Body weight Control and all experimental animals were similar in body weight before procedure. As shown in Figure 1 , body weight of groups which experienced food poverty merely or coincided with food inequality (with or without cage-mate change) for 14 days, was less than their initial body weight (p<0.05), and there was no significant difference between male and females in this regard. Unstable social status resulted in weight loss but this decline was not considerable both in males and females (male: p = 0.081; female p = 0.054); this served to indicate that just food poverty and inequality paradigm has caused obvious weight loss. 10.1371/journal.pone.0047218.g001Figure 1 Body weight changes in male mice exposed to chronic psychosocial stress. Implementing food poverty merely or coincided with food inequality (with or without cage-mate change) have caused significant decrease in body weight of both males and females (there is no difference between these two genders). FD+See: Food Deprived and inequality experienced group, FD+Isolate: Food Deprived group without inequitable situation, FD+CC+See: Food Deprived group which also experienced inequality and cage-mate change simultaneously, FD+CC+Isolate: Food Deprived and cage-mate change experienced group without inequitable situation, CC = the group which just experienced cage-mate change. *p<0.05: beginning of study vs. end of chronic social stress (Values are means ± SEM; n = 6–8 animals in each group). 3.2. Comparison of chronic pain perception between control and socially stressed males and females A repeated measure one-way ANOVA analysis detected the characteristic biphasic curve of the formalin-induced behavioral response in control groups. Classically, a first peak of Licking behavior during the first 6 minute block reflects the behavioral response to acute pain, whereas the second part of the curve represents persistent (chronic) pain. Interestingly, this biphasic response was not observed in animals which underwent food deprivation merely or coincided with food inequality ( Figures 2 – 5 ); according to these findings pain behavior had no increase at the second phase of formalin test in food deprived and inequality experienced animals which showed a significant effect of these social stressors on creation of hypoalgesia (p<0.05). For confirmation this point, we used repeated measurement one-way ANOVA in order to compare 3-min blocks in a group; so it revealed there was no significant difference between 3-min blocks of formalin test's second phase in food deprived and inequality experienced mice (groups 2 to 5). In animals which just experienced cage-mate change (unstable social status) a biphasic curve after injection of formalin was created as same as control groups; representing that chronic pain perception was not affected by this type of social stressor ( Figure 6 ). 10.1371/journal.pone.0047218.g002Figure 2 Effect of food deprivation and inequality without cage-mate change on pain behavior during the formalin test. The observation period is divided into 20 blocks of 3 minutes each. Unlike control male and female subjects, biphasic curve was not observed in animals which underwent food deprivation and inequality simultaneously (Data are means ± SEM, females; Control and FD+See: n = 8, males; Control: n = 7 and FD+See: n = 6). 10.1371/journal.pone.0047218.g003Figure 3 Effect of food deprivation merely, without inequality and cage-mate change on pain behavior during the formalin test. The observation period is divided into 20 blocks of 3 minutes each. In all stressed animals as compared to controls, pain behaviors did not appear in chronic phase of formalin test (Data are means ± SEM, females; Control and FD+Isolate: n = 8, males; Control and FD+Isolate: n = 7). 10.1371/journal.pone.0047218.g004Figure 4 Effect of food deprivation, inequality and cage-mate change on pain behavior during the formalin test. The observation period is divided into 20 blocks of 3 minutes each. In all stressed animals as compared to controls, pain behaviors did not appear in chronic phase of formalin test (Data are means ± SEM, females; Control and FD+See+CC: n = 8, males; Control and FD+See+CC: n = 7). 10.1371/journal.pone.0047218.g005Figure 5 Effect of food deprivation and cage-mate change without food inequality on pain behavior during the formalin test. The observation period is divided into 20 blocks of 3 minutes each. Pain behavior of stressed animals was not revealed during the chronic phase of formalin test as compared to controls (Data are means ± SEM, females; Control and FD+Isolate+CC: n = 8, males; Control and FD+Isolate+CC: n = 7). 10.1371/journal.pone.0047218.g006Figure 6 Effect of cage-mate change merely without food deprivation and inequality on pain behavior during the formalin test. The observation period is divided into 20 blocks of 3 minutes each. Implementing cage-mate change merely did not affect pain behaviors during the chronic phase of formalin test as same as controls (Data are means ± SEM, females; Control and FD+Isolate+CC: n = 8, males; Control and FD+Isolate+CC: n = 7). As shown in Figure 7 , chronic pain perception both in males and females not only was affected by food deprivation but also by food inequality; expressing that implementing of such stressors has caused significant decrease of chronic pain sensation (hypoalgesia) compared with control group (p<0.001).This decline in food deprived mice along with inequitable situation was greater than isolated food deprived animals, ((FD (See) vs FD (Iso): p<0.001 and FD+CC (See) groups vs FD+CC (Iso) groups: p<0.001). However, as mentioned before, the second phase response in 6th group (merely experienced unstable situation) did not show significant difference with control group (male: p = p = 0.932; female: p = 0.98). 10.1371/journal.pone.0047218.g007Figure 7 Effect of different social stressors on second-phase responses to formalin. The score of pain behaviors between 15 and 35 min after formalin injection is plotted, along with the standard error of the mean (n = 6–8 in each group). As it is obvious, unlike food poverty and inequality, cage-mate change did not influence chronic pain perception as like controls. Moreover, food deprived and inequality experienced males perceive pain as less as females. *p<0.05: significant difference between stressed and controls of females. #p<0.05: significant difference between stressed and controls of males. & p<0.05: significant difference between males and females. 3.3. Comparison of chronic pain perception between males and females As expected ( Figure 7 ), a significant main effect of gender emerged (p = 0.032) reflecting the generally greater pain perception exhibited by control males relative to control females. Moreover, pain perception of male subjects which just experienced unstable situation, was well above in comparison with similar group of females (p = 0.013). Comparison of male and females of food deprived animals which experienced inequitable situation with or without cagemate change, showed males were hypoalgesic than females (FD+See and FD+CC+See animals: p<0.001); however, there was no significant difference between isolated subjects which underwent food poverty with or without cagemate change (FD+Isolated animals: p = 0.16, FD+CC+Isolted animals: p = 0.308). Figure 8 reveals that the level of pain decrease toward control subjects, in food deprived and inequality experienced males is well above its decrease in similar stressed females (p<0.05). 10.1371/journal.pone.0047218.g008Figure 8 The level of pain decrease in socially stressed animals toward control groups. It is clear that male subjects which experienced food inequality, revealed further hypoalgesic response as compared to females. Data are means ± SEM. *p<0.05: significant difference between stressed and controls of females. #p<0.05: significant difference between stressed and controls of males. 3.4. Comparison of serum cytokines levels between control and experimental groups As shown in Figure 9 , evaluation of IL-6 concentration in serum of experimental subjects both in females and males showed IL-6 levels in all animals which underwent food deprivation, food inequality and cage mate change have increased significantly as regards control mice (p<0.05) and this increase in males and females which experienced all three stresses is well above other stressed animals; although this difference toward some of these groups is not significant. In addition, beside high levels of IL-1 concentration ( Figure 10 ) in stressed mice as compared to controls (p<0.05); IL-1 concentration in serum of food deprived animals which experienced unstable social status and unequality simultaneously, was also more than other stressed animals of both genders (p<0.05). 10.1371/journal.pone.0047218.g009Figure 9 Effect of of different social stressors on serum concentration of proinflammatory cytokines (Interleukin-6) in mice. Data are means ± SEM. *p<0.05: significant difference between stressed and controls of females. #p<0.05: significant difference between stressed and controls of males. 10.1371/journal.pone.0047218.g010Figure 10 Effect of of different social stressors on serum concentration of proinflammatory cytokines (Interleukin-1) in mice. Data are means ± SEM. *p<0.05: significant difference between stressed and controls of females. #p<0.05: significant difference between stressed and controls of males. Discussion The purpose of this study was to investigate how the male and female animals differ in their experience of experimental chronic pain and whether long-term social stresses differentially affect this response. To achieve these goals, adult male and female mice were subjected to a model of chronic social stress which offers the opportunity to investigate about differences of pain perception in association with social stresses and gender. We found that, all male and female subjects which underwent food poverty and inequality with or without social instability were hypoalgesic in chronic phase of formalin test as compared to control animals. However, social instability merely didn't affect chronic pain perception after subcutaneous injection of 2% formalin in both genders. In addition, this hypoalgesic response in food deprived and inequality experienced males was well above females. 4.1. Effects of chronic social stresses on chronic pain perception in both genders Pain research has shown that (1) not all noxious stimuli are processed centrally or peripherally in the same way and (2) not all aversive stimuli are capable of eliciting an analgesic response and can in fact elicit a hyperalgesic response [36]. Studies have shown that being in chronic pain rather than acute pain express elevated pain behaviors in the presence of an aversive stimulus [37]. However, little has been previously reported about the effects of chronic social stress on persistent pain, despite different studies have shown the prevalence of pain in people who experience a long term period of stress, is much less [34], [38], [39]. Pain can be characterized by its duration (from momentary to chronic), location (e.g., muscle, viscera), or cause (e.g., nerve injury, inflammation). Characterization of pain by duration may be arbitrary (i.e., when does pain become chronic?), but is useful because most significant human pain conditions are long-lasting, whether referred to as persistent or chronic [16]. The majority of experimental pain results obtained in animals are consistent with those obtained in humans. These findings have been observed using various experimental tests and nociceptive modalities, but have often been investigated in the formalin test [32], [40], [41]. Rodent hind paw inflammation is a commonly used model of persistent inflammatory pain in which hind paw injection of formalin or capsaicin is used to assess intense, short-lasting (minutes to tens of minutes) persistent pain [16]. The results of this study are consistent with the notion that even food poverty and inquality can be stressful, in turn provoking a physiological reaction akin to that which occurs upon perception of a physical threat [42] one component of which can be hypoalgesia. Anatomical, pharmacological and behavioral evidence from stressed-induced analgesia studies revealed that amygdale, periaqueductal grey (PAG) and rostral ventromedial medulla (RVM) as critical structures, contribute to descending inhibitory pain pathways and lesions of these structures attenuate the conditioned stressed-induced analgesia response which can be mediated by opioid receptors [38], [42]. Opioids hypoalgesic effects are particularly prominent in inflammatory conditions [43]. Implementing of food deprivation and inequality for a period of two weeks resulted in significant decrease in body weight, which was more pronounced in female mice; so it seems that such weight loss can be in accordance with limitation of calorie during deprivation that can lead to a potentiated hypoalgesic response [44]. Studies of malnourished children have shown that Protein-Energy Malnutrition may lead to oxidative stress, which can lead to increased activity of proinflammatory cytokines [45], this finding is in accordance with our following researches about increase of serum proinflammatory cytokines concentration (IL-6, IL-1) beside oxidative stress markers (data has not been published) such as MDA (Malon-Di-Aldehyde), especially in food deprived and inequality undergoing mice. It seems more probably that attenuated sensivity of immune cells to glucocorticoids [46] after implementing mentioned social stressors have caused extra levels of proinflammatory cytokines. So, inflammation of peripheral tissue as well as increasing the number of nociceptor endings and disrupting the peri-neural barrier which facilitates the access of opioid agonists to their receptors [47], leads to increased synthesis and axonal transport of opioid receptors in DRG neurons, resulting in their up-regulation and enhanced G-protein coupling at peripheral nerve terminals [48]. The body produces its own powerful pain-modulating neurotransmitters. It is stated chronic stressors combined with a diet low in protein, can create deficiencies in the three most critical of these pain-modulators: serotonin, gamma-amino-butyric acid (GABA), and endorphin. In Practical Pain Management, the use of diet and amino acid supplementation in promoting optimal levels of serotonin, GABA and the endogenous opiates have been discussed, as it was emphasized that if adequate protein is consumed, endorphin levels may remain high enough to effectively modulate pain [49], [50]. However in present study we took in consideration that hypoalgesic response after food deprivation (which may lead to katabolism and decrease of body's total protein) is likely related to activation of endogenous opiates, so further investigation are needed to explain this contradiction. Stress can affect pain perception differentially, as accession of hypealgesia or hopoalgesia depends on the type of stressor as well as its intensity and duration [51]. It was stated that highly unstable situation does not predict elevated basal cortisol concentrations in an individual [52]. As we saw in our study, pain behavior in unstable group (cage-mate changed) did not differ from control animals; so we can presume that our social instability paradigm did not release opioids to create stress-induced hypoalgesia, despite serum concentrations of IL-1 and IL-6 have increased at this group and it seems more probably that hypoalgesic response after food deprivation and inequality is regulated via pathways different from proinflammatory cytokines; so further mechanistic invesigations are needed to reveal what is the cause of this contradiction. 4.2. Gender differnces in chronic pain perception after implementing social stresses Clinically it is well documented that women are more likely than men to report a variety of recurrent pains which are often described as being more severe and frequent compared to men [53]. Therefore, numerous laboratory studies have been conducted to try to understand the mechanisms underlying these difference [25], [53], [54]. However, very few studies have investigated gender differences in chronic pain models such as nerve injury and persistent inflammation [19]. Concerning gender differences, male rodents exhibit larger hypoalgesic responses to environmental stress - a natural trigger that serves to activate the same descending analgesia pathways that are acted upon by centrally acting opiate drugs. Such findings suggest that both ascending and descending pathways involved in the experience of pain are influenced by hormonal or other gender-related factors [22]. We observed herein that male mice display significantly more hypoalgesia than females following food deprivation and inequality and it seems males are more prone to adverse effect of social inequality. The reasons for these discrepancies remain unknown, but the possibility of hormonal factors to affect the magnitude of analgesic responses should not be overlooked. The potential importance of gonadal hormones in accounting for gender differences in opioid-induced antinociception has been examined in numerous investigations [55]. Although there are discrepancies in the literature, a number of reports suggest that gonadal hormones play a clear role in opioid antinociception [32], [56]. Indeed it is stated that gonadal steroid hormone binding sites are ubiquitously distributed throughout central nervous system regions involved in pain perception and pain inhibition, such as the periaqueductal gray, rostroventral medulla, and spinal cord dorsal horn [22]. While the reasons for a lack of an effect of menstrual cycle phase on gender differences in pain sensitivity are not known, one possibility relates to a putative threshold effect of gonadal hormones on pain sensitivity. For example, even in the early follicular phase of the menstrual cycle when estrogen and progesterone are at their lowest, women still exhibit significantly greater hormone levels than men [57]. Despite some studies have shown estrogens seem to play a role in inducing hyperalgesia and pain [58], [59], analgesic effects of estrogen and progesterone in animals have been documented [60], but consistency in the pattern and direction of the relationship between hormones and nociception is lacking and underlying mechanisms have yet to be elucidated [57], [61]. Accordingly, it seems high levels of these gonadal hormones in females as compared to males can be a reason for high persistent pain perception in control females in our study; however, since it is shown that the largest effects of esterogen and/or progestrone occur during the interphase of formalin test [40], we can suggest that the discrepancy between food deprived and inequality experienced males and females in persistent pain response can be influenced by several biological (genes and hormones) and other confounding socio-environmental factors, not just by gonadal hormones diversity. 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Am J Physiol Regul Integr Comp Physiol 291 : R245 –R256 .16484434
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PLoS One. 2012 Oct 17; 7(10):e47218
==== Front ScoliosisScoliosisScoliosis1748-7161BioMed Central 1748-7161-7-112264057410.1186/1748-7161-7-11ReviewSoft braces in the treatment of Adolescent Idiopathic Scoliosis (AIS) – Review of the literature and description of a new approach Weiss Hans-Rudolf [email protected] Mario [email protected] Orthopedic Rehabilitation Services, Alzeyerstr. 23, Gensingen, D-55457, Germany2 Orthomed Scolicare, Orthopedic Technical Services, Alzeyerstr. 23, Gensingen, D-55457, Germany2012 28 5 2012 7 11 11 18 2 2012 23 4 2012 Copyright ©2012 Weiss and Werkmann; licensee BioMed Central Ltd.2012Weiss and Werkmann; 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 use of soft braces to treat scoliosis has been described by Fischer as early as 1876. With the help of elastic straps, as the authors suggested, a corrective movement for individual curve patterns should be maintained in order to inhibit curve progression. Today this concept has been revived besides soft 3 point pressure systems. Some shortcomings have been revealed in literature in comparison with hard braces, however the concept of improving quality of life of a patient while under brace treatment should furtherly be considered as valuable. Purpose of this review is to gather the body of evidence existent for the use of soft braces and to present recent developments. Method A review of literature as available on Pub Med was performed using the key words ‘scoliosis’ and ‘soft brace’ at first. The search was expanded using ‘scoliosis’ and the known trademarks (1) ‘scoliosis’ and ‘SpineCor’, (2) ‘scoliosis’ and ‘TriaC’, (3) ‘scoliosis’ and ‘St. Etienne brace’, (4) ‘scoliosis’ and ‘Olympe’. The papers considered for inclusion were new technical descriptions, preliminary results, cohort studies and controlled studies. Results When searching for the terms ‘scoliosis’ and ‘SpineCor’: 20 papers have been found, most of them investigating a soft brace, for ‘scoliosis’ and ‘TriaC’: 7 papers displayed, for ‘scoliosis’ and ‘St. Etienne brace’: one paper displayed but not meeting the topic and for ‘scoliosis’ and ‘Olympe’: No paper displayed. Four papers found on the SpineCor™ were of prospective controlled or prospective randomized design. These papers partly presented contradictory results. Two papers were on soft Boston braces used in patients with neuromuscular scoliosis. Discussion There is a small but consistent body of evidence for the use of soft braces in the treatment of scoliosis. Contradictory results have been published for samples treated during the pubertal growth spurt. In a biomechanical analysis the reason for the lack of effectiveness during this period has been elaborated. Improved materials and the implementation of corrective movements respecting also the sagittal correction of the scoliotic spine will hopefully contribute to an improvement of the results achievable. Conclusions The treatment of scoliosis using soft braces is supported by some papers providing a small body of evidence. During the growth spurt the use of soft braces is discussed contradictory. There is insufficient evidence to draw definite conclusions about effectiveness and safety of the intervention. ==== Body Background The use of soft braces to treat scoliosis has first been described by Fischer (1876, cited by Schanz [1], see also Additional file 1). With the help of elastic straps, as the authors suggested, a corrective movement for individual curve patterns should be maintained in order to inhibit curve progression (Figure 1). Figure 1 Left the soft brace as described by Fischer 1876 and a soft brace of current standard as distributed today (middle and right). Unfortunately no picture from the rear exists for the Fischer brace, however the adjustment of the corrective ribbon from the front seems rather identical. (With kind permission by Pflaum, Munich). However soft braces have been forgotten for long: Hard braces have been proposed by Wullstein (1902) not long after the first publications on soft braces [2]. Later the Milwaukee brace has been proposed [3], the Chêneau brace [4,5] and the Boston brace [6], all of them hard braces with reasonable impact on the quality of life of the patients treated. While the Milwaukee brace was less effective [4], early outcome studies have described the Chêneau brace [4,5] and the Boston brace as effective in the prevention of curve progression during growth [6]. Prospective controlled multicenter [7] and long-term studies [8] have shown the Boston brace to be effective, but there were also outcome studies on the Chêneau brace clearly demonstrating that in-brace correction and compliance are crucial to the outcome of brace treatment [9]. During the late 80’es and early 90’es in France soft braces with the 3-point pressure approach have been described like the St. Etienne brace and the Olympe [10,11], but these have not been investigated furtherly. During the 90’es soft brace treatment was re-developed [12]. The soft brace as presented by Coillard [12-14], comparable to the brace described by Schanz [1], was indroduced another time and the first results were published in 2003 [13]. A cohort of patients from 4 – 14 years of age have been treated successfully with the help of this brace. Later on more positive results have been published [14]. However, as Coillard and Rivard have pointed out the group of patients at risk for progression (first signs of maturation, premenarchial) showed to have the least positive effects [14]. In two independend prospective controlled studies the soft brace as presented by Coillard [12-14] has been shown to be less effective than hard braces [15,16]. This fact has been analyzed and the unsatisfactorily correction of the sagittal profile in the soft brace as presented by Coillard [12-14] has been accused to be the reason for this [17]. As a matter of fact a relordosation of the lumbar spine is not induced when using the soft brace as presented by Coillard [12-14]. Therefore the compressive forces generated in this application may destabilize the spine, while a restoration of the sagittal profile can stabilize and even correct scoliosis as has been described by van Loon in 2008 [18]. Other reviews, experimental studies or case series seem to support these findings [19-26]. As it seems, to improve the results of soft braces during the high risk phase of the pubertal growth spurt, the sagittal plane seems to be important to focus on in order to improve the results of treatment also in the population at risk for progression (Figure 2). Figure 2 Universal Spine Orthosis as developed 2007. This brace uses shifting of shoulder and pelvic girdle against each other as well as a restoration of the sagittal profile (right). (With kind permission by Pflaum, Munich). Purpose of this development was to improve the corrective movements in frontal plane as have been described [12-14] and add a considerable correction of the sagittal plane in order to stabilize the spine while it is compressed by the elastic straps, wrapped around the entire trunk. However, some shortcomings [15-17] have been revealed in literature in comparison with hard braces, however the concept of improving quality of life of a patient while under brace treatment should furtherly be considered as valuable. Purpose of this review is to gather the body of evidence existent for the use of soft braces and to present recent developments. Method A review of literature as available on Pub Med was performed (January 31st. 2012) using the key words ‘scoliosis’ and ‘soft brace’ at first. The search was expanded using ‘scoliosis’ and the known trademarks (1) ‘scoliosis’ and ‘SpineCor’, (2) ‘scoliosis’ and ‘TriaC’, (3) ‘scoliosis’ and ‘St. Etienne brace’, (4) ‘scoliosis’ and ‘Olympe’. A case report is presented to demonstrate the in-brace correction achievable in a recent development. Results Using the key words ‘scoliosis’ and ‘soft brace’ 16 articles displayed, only, four of them meeting the topic. Two were referring to the treatment of patients with IS, two were on the treatment of neuromuscular scoliosis. The expanded search revealed the following: (1) ‘scoliosis’ and ‘SpineCor’: 20 papers have been found, most of them investigating a soft brace. (2) ‘scoliosis’ and ‘TriaC’: 7 papers displayed. (3) ‘scoliosis’ and ‘St. Etienne brace’: one paper displayed but not meeting the topic (4) ‘scoliosis’ and ‘Olympe’: No paper displayed. The papers considered for inclusion were new technical descriptions, preliminary results, cohort studies and controlled studies [12-16,27-44]. Four papers found on the SpineCor™ were of prospective controlled or prospective randomized design [15,16,39,42]. Two papers were on soft Boston braces used in patients with neuromuscular scoliosis [45,46]. Recent developments The idea of using soft braces and reducing the impact braces have on the patients is appreciated. However as there seem to be certain shortcomings, these should be ruled out to increase patients’ safety and enlarge the range of indications. The corrective movement in frontal plane as described by Fischer [1] and later by Coillard [12-14] should be preserved while lumbar lordosis should be augmented with the help of a newly designed soft brace. The first step into this direction was the development of a soft brace called “Universal Spine Orthosis (USO)™” (patent pending) in 2007, which has not been applied widely. The harness for adjustment of the corrective ribbons provided using the soft brace as presented by Coillard [12-14] was replaced by a small plastic fixation element where the elastic ribbons can be adjusted to. This fixation element is adjusted to augment lumbar lordosis (Figure 2). The USO™ allows to adjust the corrective ribbons in multiple ways for different purposes and therefore can be used for other indications than scoliosis treatment as well. However the USO, like other softbraces in use today [12-14] is not easy to adjust and also with this softbrace the patients may experience some problems in everday use (eg. clothes change, toilet use). Therefore we found further developments necessary to (1) make the brace easier to adjust, (2) easier to use and (3) to implement the corrective movements necessary in 3D. Within the Scoliologic™ ‘Best Practice’ program of physiotherapy we have a simple 3D system of postural corrections. This consists of (1) pelvic girdle correction, (2) shifting of shoulder girdle against pelvic girdle, (3) spiral shoulder girdle correction and (4) correction of the sagittal profile [21,22]. These are also the main principles of correction we find in the Scoliologic™ ‘Best Practice’ hard braces (Chêneau light™, Gensingen brace™) leading to high in-brace corrections (Figure 3 and 4) as has been documented recently [22-24]. The application of the Scoliologic™ ‘Best Practice’ braces has been shown to have the least rate of surgery [25], when compared to other studies respecting the SRS inclusion criteria. Figure 3 Corrective movement during a ‘Power Schroth’ exercise. (With kind permission by Pflaum, Munich). Figure 4 Corrective movement in the Gensingen brace™. In immature patients and single curve patterns an overcorrection is not rare. (With kind permission by Pflaum, Munich) Therefore it was the aim of our recent development to implement all these principles of correction and by the same time make the new soft brace development smaller and more easy to adjust. The result of this development is demonstrated in the following. A new soft brace for 3D correction of scoliosis – the Spinealite™ Correcting ribbon materials Contrary to the correcting ribbon material as used in the soft brace as presented by Coillard [12-14] we apply materials with much less elasticity (Figure 5). In principle the materials we use are of endelastic entity and do not lose the tension force after a few weeks time of wearing. The correction as adjusted can be maintained, however as there is no unlimited freedom of movement like in the soft brace as presented by Coillard [12-14], this brace is not as comfortable to wear. However less comfort is outwighted by a constant force of correction leading to the best possible results as achievable with soft braces considering that correction in brace treatment is crucial to the outcome [9,26]. Figure 5 Aspects of the Spinealite™ soft brace attached for a 3CL pattern according to the ALS classification. Adjustment The adjustment of the Spinealite™ is pattern dependent (Figure 6). We derive the adjustment of the system from the ALS Classification (Augmented Lehnert-Schroth Classification). The shift is adjusted for all curvature patterns containing a thoracic curvature (3CH, 3CN, 3CTL, 3CL, 4C). In these cases the shoulder girdle is retracted on the thoracic convex side and shifted over to the thoracic concave side. In patterns without a significant lumbar curve (3CH, 3CN, 3CTL) the pelvic attachment is made more central, in patterns containing a significant lumbar curve (3CL,4C, 4CL, 4CTL) the attachment to the pelvic part of the system has to be made more lateral in order to allow a lifting of the hemipelvis on the side of the lumbar convexity (Additional file 2). Figure 6 The ALS classification. (With kind permission by Pflaum, Munich). The adjustment is described in more detail in the accompanying product description. Case report of the in-brace correction possible in the Spinealite™ A 12.6 year old girl from Russia presented in the practice of the senior author for brace treatment. The girl was 2 months postmenarchial with a Cobb angle of 28° thoracic and 22° lumbar in summer 2011 when she was treated with a Chêneau brace for the first time (Angle of Trunk Rotation [ATR] thoracic 8°, lumbar 3°). The in-brace correction in the 1st. brace for a double curve pattern was fairly good to 10° thoracic and 11° lumbar (Figure 7). After 6 months she had outgrown the first brace and returned for a second brace. The curve has been stable with 27° thoracic and 22° lumbar. As at the time of the last presentation the clinical pattern tended more to a single thoracic pattern (Angle of Trunk Rotation [ATR] thoracic 7°, lumbar 0°) it was decided to treat the girl with a 3CN type of brace from the Gensingen library. Figure 7 The patient presented in the case report in her 1st. Gensingen brace™. A 3CL Gensingen brace™ was adjusted leading to an acceptable correction. The initial angle of curvature was 28° and in brace correction was 10°. As the patient reported some problems with compliance in hard braces it was decided to offer the Spinealite™ soft brace additionally so she could change braces wearing the Spinealite™ for 12 hrs. over daytime and the Gensingen brace™ for 12 hrs. in the evenings and at night. Both braces were able to overcorrect the main thoracic curvature to −16° (Figure 8 and Figure 9). However the patient felt comfortable in the hard brace without any pains and uncomfortable in the soft brace when full correction was applied. So for the start we reduced the correction to a comfortable position and asked the parents to increase the correction every week as the ribbons can be easily be readjusted at home. Figure 8 The patient presented in the case report in her 2nd. Gensingen brace™. A 3CN Gensingen brace™ was adjusted leading to an overcorrection. The initial angle of curvature was 27° and in brace correction was −16°. The little amount the apical vertebra is rotated shows a high flexibility of the curve. Nevertheless, an overcorrection has only been achieved when the correction was changed to single curve correction without addressing the lumbar curve. Figure 9 The patient presented in the case report in her Spinealite™ soft brace. Spinealite™ soft brace was adjusted leading to an overcorrection. The initial angle of curvature was 27° and in brace correction was −16°. The lumbar counter curve in frontal plane does not seem to be corrected much, however the axial load of this system when applied to the maximum correction obviously leads to an increased rotation. Therefore we would not allow the use of this system alone without hard brace and we will always propose regular clinical controls. A video showing the adjustment of correction in this new soft brace is available [47]. Discussion There is a small body of evidence for the use of soft braces in the treatment of scoliosis [12-17,27-44]. Contradictory results have been published for samples treated during the pubertal growth spurt [15-17,30,32,34]. In a biomechanical analysis the reason for the lack of effectiveness during this period has been elaborated [17]. Improved materials and the implementation of corrective movements respecting also the sagittal correction of the scoliotic curve will contribute to an improvement of the results achievable (Figure 5 and 9). At this moment we apply the new soft brace system (Spinealite™) together with high corrective hard braces, 12 hrs. each, however with increasing numbers of patients and increased experience we do hope to offer this new soft brace as the sole form of treatment for adolescents during growth in the near future. Although there is a small body of papers on soft brace treatment as found in literature [12-17,27-44] we would not expose our patients to the risk of sole softbrace treatment as some of the papers reveal contradictory outcomes [15-17,30,32,34] and restrictions to use certain types of braces with respect to curve patterns [30]. At this stage there is no evidence that the Spinealite™ can improve the outcome of soft bracing, however the principle of applying corrective movements as described by Fischer [1] and later by Coillard [12-14] has been found to be beneficial to some extent. The trunk shift in combination with the other 3D corrective movements is a powerful corrective force as can be seen on Figure 4. The addition of a sagittal corrective movement theoretically should enable to improve the outcome of soft bracing additionally [18-20], but this finally has to be proven in future studies. This new soft brace has been shown to be able to correct a scoliosis to an extent comparable to high corrective hard braces. Therefore we expect beneficial outcomes when the brace can be worn as prescribed [9,26]. In principle the Fischer brace, SpineCor and the Spinealite are using a certain corrective movement mainly derived from physical therapy approaches, while the only other soft brace on the market today, the TriaC is using the standard three-point system and can only be applied for certain single curves. The Spinealite, contrary to the Fischer brace and the SpineCor, only uses one dorsal compressive force (Figure 10), allowing a lordosation of the trunk in the middle between the two attachment areas (Shoulder and thigh), which is at the thoracolumbar junction / high lumbar region. This corrective movement in sagittal plane has been shown to be beneficial for curve correction [18]. Additionally, the materials used are less elastic than in the SpineCor preventing free mobility (also into the deformity), but this exactly may be viewed as the difference between a corrective brace and a t-shirt. Figure 10 Simplified model of the trunk compression as applied in soft braces using the corrective movement principle. While in the Fischer brace and in the SpineCor ventral and dorsal compression forces are applied, the Spinealite only uses one dorsal compressive force (Figure 10), allowing a lordosation of the trunk in the middle between the two attachment areas (Shoulder and thigh), which is at the thoracolumbar junction / high lumbar region. The full correction as possible should not be applied from the very start. A slight corrective movement should be visible in frontal and sagittal plane which can be increased every week. The application of this new system, however requires an experienced clinician able to distinguish between the different curve patterns of the ALS classification used for a proper adjustment of both, hard- and soft brace. The Spinealite™ soft brace / biofeedback device is using certain corrective movements which have been described earlier on [21,22,48-51], however, according to a recent review it should be emphasized that the power of exercises should not be overestimated [52], see Figure six from that article. Nevertheless, we propose to perform the Scoliologic™ ‘Best Practice’ program as described by Borysov and Borysov extensively [51]. Conclusions 1. The treatment of scoliosis using soft braces is supported by some papers providing a small body of evidence. 2. During the growth spurt the use of soft braces is discussed contradictory. 3. There are shortcomings with respect to limitations of indication. 4. There is insufficient evidence to draw definite conclusions about effectiveness and safety of the intervention (soft brace treatment). Consent Written informed consent was attained by the patients and parents to permit the publication of the clinical pictures. Competing interests HRW is advisor of Koob-Scolitech, Abtweiler, Germany and has applied for patents. MW declares to have no competing interest. Authors’ contribution HRW: Review of the data base, hand review, manuscript writing, patient acquisition. MW: Patient acquisition, technical support, prototype production according to the adjustment plan, brace / biofeedback device adjustment to the patient. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Some pages from Schanz [[1]] showing, that at the beginning of the last century there already was a wide knowledge about the possibility of soft brace adjustment. Various kinds of soft braces were known as well as combinations of hard braces with soft (elastic) parts. Click here for file Additional file 2 Short description of the Spinealite™ application. Click here for file Acknowledgements I wish to thank Pflaum Company for permitting the publication of pictures taken or modified from the book with the title Weiss HR: Best practice in conservative scoliosis care. 4th edition 2012. ==== Refs Schanz A Die statischen Belastungsdeformitäten der Wirbelsäule mit besonderer Berücksichtigung der kindlichen Wirbelsäule 1904 Stuttgart: Enke 158 Weiß HR Wirbelsäulendeformitäten – Konservatives Management 2003 München: Pflaum Lonstein JE Winter RB The Milwaukee brace for the treatment of adolescent idiopathic scoliosis. A review of one thousand and twenty patients J Bone Joint Surg Am 1994 76 8 1207 1221 8056801 von Deimling U Wagner UA Schmitt O [Long-term effect of brace treatment on spinal decompensation in idiopathic scoliosis. A comparison of Milwaukee brace--Chêneau corset] Z Orthop Ihre Grenzgeb 1995 133 3 270 273 10.1055/s-2008-1039447 7610709 Hopf C Heine J Long-term results of the conservative treatment of scoliosis using the Cheneau brace Z Orthop Ihre Grenzgeb 1985 123 3 312 322 10.1055/s-2008-1045157 4050044 Emans JB Kaelin A Bancel P Hall JE Miller ME The Boston bracing system for idiopathic scoliosis. Follow-up results in 295 patients Spine 1986 11 8 792 801 10.1097/00007632-198610000-00009 3810295 Nachemson AL Peterson LE Effectiveness of treatment with a brace in girls who have adolescent idiopathic scoliosis. A prospective, controlled study based on data from the Brace Study of the Scoliosis Research Society J Bone Joint Surg Am 1995 77 6 815 822 7782353 Danielsson AJ Hasserius R Ohlin A Nachemson AL A prospective study of brace treatment versus observation alone in adolescent idiopathic scoliosis: a follow-up mean of 16 years after maturity Spine 2007 32 20 2198 2207 10.1097/BRS.0b013e31814b851f 17873811 Landauer F Wimmer C Behensky H Estimating the final outcome of brace treatment for idiopathic thoracic scoliosis at 6-month follow-up Pediatr Rehabil 2003 6 3–4 201 207 14713586 Ollier M Olympe (Orthèse Lyonnaise MassuesPression Elastique) “Strech Brace“ Paper at the 19th annual meeting 1991 Modena: GEKTS, October 18th – 19th Daler S Mouilleseaux B Diana G Orthèse élastique trois points pour le traitement des scolioses lombaires idiopathiques évolutives de l’adolescent Paper at the 21st annual meeting 1991 Geneva: GEKTS, October 15th – 16th Coillard C Leroux MA Zabjek KF Rivard CH Reductibility of idiopathic scoliosis during orthopedic treatment Ann Chir 1999 53 8 781 791 10584390 Coillard C Leroux MA Zabjek KF Rivard CH SpineCor–a non-rigid brace for the treatment of idiopathic scoliosis: post-treatment results Eur Spine J 2003 12 2 141 148 12709852 Coillard C Circo AB Rivard CH SpineCor treatment for early scoliosis: 15 to 25° 5th International Conference on the Conservative Management of Spinal Deformities 2008 Athens, April 2–5 Weiss HR Weiss GM Brace treatment during pubertal growth spurt in girls with idiopathic scoliosis (IS): a prospective trial comparing two different concepts Pediatr Rehabil 2005 8 3 199 206 16087554 Wong MS Cheng JC Lam TP Ng BK Sin SW Lee-Shum SL Chow DH Tam SY The effect of rigid versus flexible spinal orthosis on the clinical efficacy and acceptance of the patients with adolescent idiopathic scoliosis Spine 2008 33 12 1360 1365 10.1097/BRS.0b013e31817329d9 18496349 Weiss HR SpineCor vs. natural history - explanation of the results obtained using a simple biomechanical model Stud Health Technol Inform 2008 140 133 136 18810014 van Loon PJ Kühbauch BA Thunnissen FB Forced lordosis on the thoracolumbar junction can correct coronal plane deformity in adolescents with double major curve pattern idiopathic scoliosis Spine 2008 33 7 797 801 10.1097/BRS.0b013e3181694ff5 18379408 Weiss HR Dallmayer R Gallo D Sagittal counter forces (SCF) in the treatment of idiopathic scoliosis: a preliminary report Pediatr Rehabil 2006 9 1 24 30 16352502 Weiss HR Rigo M The chêneau concept of bracing - actual standards Stud Health Technol Inform 2008 135 291 302 18401099 Weiss HR Befundgerechte Physiotherapie bei Skoliose Edited by: Ingeborg Liebenstund 2011 Pflaum: Munich Pflaum Physiotherapie 3 Weiss HR Best Practice in conservative scoliosis care 2012 4 Munich: Pflaum Weiss HR “Brace Technology” Thematic Series - the Gensingen brace(TM) in the treatment of scoliosis. Scoliosis 2010 5 22 10.1186/1748-7161-5-22 20942970 Weiss HR Korsettversorgung bei Skoliose Orthopädie Technik 2011 62 7. 488–498 Werkmann M Weiss HR Rate of surgery in patients under treatment with a Chêneau light brace using the SRS inclusion criteria Scoliosis 2012 7 Suppl 1 O45 Clin J Aubin CÉ Sangole A Labelle H Parent S Correlation between immediate in-brace correction and biomechanical effectiveness of brace treatment in adolescent idiopathic scoliosis Spine (Phila Pa 1976) 2010 35 18 1706 1713 10.1097/BRS.0b013e3181cb46f6 21330954 Veldhuizen AG Cheung J Bulthuis GJ Nijenbanning G A new orthotic device in the non-operative treatment of idiopathic scoliosis Med Eng Phys 2002 24 3 209 218 10.1016/S1350-4533(02)00008-5 12062179 Bulthuis GJ Veldhuizen AG Nijenbanning G Clinical effect of continuous corrective force delivery in the non-operative treatment of idiopathic scoliosis: a prospective cohort study of the TriaC-brace Eur Spine J 2008 17 2 231 239 Epub 2007 Oct 10 10.1007/s00586-007-0513-9 17926071 Wynne JH The Boston Brace and TriaC systems Disabil Rehabil Assist Technol 2008 3 3 130 135 10.1080/17483100801903988 18465395 Zeh A Planert M Klima S Hein W Wohlrab D The flexible Triac-Brace for conservative treatment of idiopathic scoliosis. An alternative treatment option? Acta Orthop Belg 2008 74 4 512 521 18811036 Grivas TB Kaspiris A European braces widely used for conservative scoliosis treatment Stud Health Technol Inform 2010 158 157 166 20543417 Hasler CC Wietlisbach S Büchler P Objective compliance of adolescent girls with idiopathic scoliosis in a dynamic SpineCor brace J Child Orthop 2010 4 3 211 218 10.1007/s11832-010-0249-7 21629374 Coillard C Circo AB Rivard CH SpineCor treatment for Juvenile Idiopathic Scoliosis: SOSORT award 2010 winner Scoliosis 2010 5 25 10.1186/1748-7161-5-25 21067608 Gammon SR Mehlman CT Chan W Heifetz J Durrett G Wall EJ A comparison of thoracolumbosacral orthoses and SpineCor treatment of adolescent idiopathic scoliosis patients using the Scoliosis Research Society standardized criteria J Pediatr Orthop 2010 30 6 531 538 10.1097/BPO.0b013e3181e4f761 20733415 Negrini S Minozzi S Bettany-Saltikov J Zaina F Chockalingam N Grivas TB Kotwicki T Maruyama T Romano M Vasiliadis ES Braces for idiopathic scoliosis in adolescents Spine (Phila Pa 1976) 2010 35 13 1285 1293 20461027 Szwed A Kołban M Jałoszewski M Results of SpineCor dynamic bracing for idiopathic scoliosis Ortop Traumatol Rehabil 2009 11 5 427 432 19920284 Coillard C Circo A Rivard CH A new concept for the non-invasive treatment of Adolescent Idiopathic Scoliosis: the Corrective Movement principle integrated in the SpineCor System Disabil Rehabil Assist Technol 2008 3 3 112 119 10.1080/17483100801903913 18465393 Coillard C Alin C Rivard CH Treatment of early adolescent idiopathic scoliosis using the SpineCor System Stud Health Technol Inform 2008 135 341 355 18401103 Coillard C Vachon V Circo AB Beauséjour M Rivard CH Effectiveness of the SpineCor brace based on the new standardized criteria proposed by the scoliosis research society for adolescent idiopathic scoliosis J Pediatr Orthop 2007 27 4 375 379 10.1097/01.bpb.0000271330.64234.db 17513955 Coillard C Leroux MA Badeaux J Rivard CH SPINECOR: a new therapeutic approach for idiopathic scoliosis Stud Health Technol Inform 2002 88 215 217 Review 15456035 Griffet J Leroux MA Badeaux J Coillard C Zabjek KF Rivard CH Relationship between gibbosity and Cobb angle during treatment of idiopathic scoliosis with the SpineCor brace Eur Spine J 2000 9 6 516 522 10.1007/s005860000175 11189920 Coillard C Circo A Rivard C A prospective randomized study of the natural history of idiopathic scoliosis versus treatment with the SpineCor brace Scoliosis 2012 7 Suppl 1 O24 10.1186/1748-7161-7-S1-P24 Coillard C Circo A Rivard C Effectiveness of the SpineCor treatment for large scoliotic curves compared to moderate and small curves Scoliosis 2012 7 Suppl 1 O25 10.1186/1748-7161-7-S1-O25 Herrero C Herrero E SpineCor treatment – the Spanish experience. First results Scoliosis 2012 7 Suppl 1 O39 10.1186/1748-7161-7-S1-O39 Letts M Rathbone D Yamashita T Nichol B Keeler A Soft Boston orthosis in management of neuromuscular scoliosis: a preliminary report J Pediatr Orthop 1992 12 4 470 474 10.1097/01241398-199207000-00010 1613089 Leopando MT Moussavi Z Holbrow J Chernick V Pasterkamp H Rempel G Effect of a Soft Boston Orthosis on pulmonary mechanics in severe cerebral palsy Pediatr Pulmonol 1999 28 1 53 58 10.1002/(SICI)1099-0496(199907)28:1<53::AID-PPUL9>3.0.CO;2-2 10406051 Weiss HR The Spinealite™ Soft Brace. You Tube Video http://www.youtube.com/watch?v=bkNP7juQ7VY Weiss HR Hollaender M Klein R ADL based scoliosis rehabilitation--the key to an improvement of time-efficiency? Stud Health Technol Inform 2006 123 594 598 PubMed Abstract | Publisher Full Text 17108494 Weiss HR Klein R Improving excellence in scoliosis rehabilitation: a controlled study of matched pairs Pediatr Rehabil 2006 9 3 190 200 PubMed Abstract 17050397 Weiss HR Spinal deformities rehabilitation-state of the art review Scoliosis 2010 5 28 PubMed Abstract | BioMed Central Full Text DOI:dx.doi.org | PubMed Central Full Text 10.1186/1748-7161-5-28 21184673 Borysov M Borysov A Scoliosis short-term rehabilitation (SSTR) according to ‘Best Practice’ standards - are the results repeatable? Scoliosis 2012 7 1 10.1186/1748-7161-7-1 22251672 Weiss HR Physical therapy intervention studies on idiopathic scoliosis-review with the focus on inclusion criteria Scoliosis 2012 7 4 10.1186/1748-7161-7-4 22277541
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==== Front J Occup Med ToxicolJ Occup Med ToxicolJournal of Occupational Medicine and Toxicology (London, England)1745-6673BioMed Central 1745-6673-7-122272072410.1186/1745-6673-7-12ResearchAssessment of the influence of whole body vibration on Cochlear function Moussavi-Najarkola Seyyed-Ali [email protected] Ali [email protected] Ramazan [email protected] Mojdeh [email protected] Mehdi [email protected] Department of Occupational Health, School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran2 Department of Occupational Health, Health promotion research center, Zahedan University of Medical Sciences (ZUMS), Zahedan, Iran3 Department of Anatomical Sciences, School of Medical Sciences, Tarbiat Modares University (TMU), Tehran, Iran4 Department of Audiology, School of Rehabilitation, Iran University of Medical Sciences (IUMS), Tehran, Iran2012 21 6 2012 7 12 12 16 8 2011 4 5 2012 Copyright ©2012 Moussavi-Najarkola et al.; licensee BioMed Central Ltd.2012Moussavi-Najarkola 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 Whole body vibration (WBV) is a potentially harmful consequence resulting from the dissipation of energy by industrial machineries. The result of WBV exposure on the auditory system remains unknown. The objective of the present research was to evaluate the influence of WBV on cochlear function, in particular outer hair cell function. It is hypothesized that WBV impairs cochlear function resulting in decreased Distortion Product Otoacoustic Emission (DPOAE) levels (Ldp) in rabbits subjected to WBV. Methods Twelve rabbits were equally divided into vibration and control groups. Animals in vibration group were exposed to 1.0 ms-2 r.m.s vertical WBV at 4–8 Hz for 8 h/day during 5 consecutive days. Outer hair cell function was assessed by comparing repeated-measurements of DPOAE levels (Ldp) across a range of f2 frequencies in rabbits both exposed and unexposed to WBV. DPOAE level shifts (LSdp) were compared across ears, frequencies, groups, and times. Results No differences were seen over time in DPOAE levels in the non-exposed rabbits (p = 0.082). Post-exposure Ldp in rabbits exposed to WBV were significantly increased at all test frequencies in both ears compared to baseline measures (p = 0.021). The greatest increase in Ldp following exposure was seen at 5888.5 Hz (mean shift = 13.25 dB). Post-exposure Ldp in rabbits exposed to WBV were not significantly different between the right and left ears (p = 0.083). Conclusion WBV impairs cochlear function resulting in increased DPOAE responses in rabbits exposed to WBV. DPOAE level shifts occurred over a wide range of frequencies following prolonged WBV in rabbits. Cochlear functionWhole body vibrationDistortion product otoacoustic emissionsRabbit's hearingDPOAEs ==== Body Background Many workers are unavoidably subjected to whole body vibration (WBV) due to the nature of assigned duties. WBV is frequently found in various industries and environments. Whole-body vibration (WBV) is caused by vibration transmitted through the seat or the feet by workplace machines and vehicles with frequencies of concern ranging from 0.5 to 80 Hz [1]. General health and well-being effects of WBV exposure on the human body have been studied over a number of years [2]. Both animal and human studies have shown that exposure to high levels of vibration can have serious effects on the human body, causing damage to a variety of vital organs [3]. WBV exposure may result in musculoskeletal impairments, central or peripheral nervous disorders [4]. Moreover, sympathic and gastrointestinal disorders are reported due to WBV [5]. Occupational deafness may be caused or aggravated by the additive effects of several environmental factors, especially vibration [6]. A paucity of studies has been conducted on the assessment of the influence of WBV exposure on cochlear function at non-realistic levels typically found in industrial settings. Okada et al. (1972) cited that temporary threshold shift (TTS) occurred after both 20 and 60 min of exposure to vibration with an acceleration of 500 cm/s and a frequency of 5 Hz, which is regarded as a resonance frequency of human body [7]. Yokoyama et al. (1974) showed that there was no significant change in threshold sensitivity after exposure to vibration alone [8]. While the literature on whole-body vibration is inconclusive, Hamernik et al. (1980) suggested that vibration may induce or increase hearing loss or cochlear damages. Based on their opinion, low frequencies (<100 Hz), although relatively ineffective in initiating an auditory response, can vibrate the membranous labyrinth if levels are high enough [9]. Hamernik et al. (1981) reported that vibration alone had essentially no effect on threshold [10]. While, Hamernik et al. (1989) showed that only stronger vibration exposure conditions (30-Hz, 3 g r.m.s) can alter the dependent measures of hearing and can alter the shape of the permanent threshold shift (PTS) audiogram [11]. Soliman et al. (2003) reported that the exposure to vibration only led to enhancement of both DPOAE amplitude and signal to noise ratio [12]. Bochnia et al. (2005) showed that vibration-induced damages to the inner ear structures may cause a worsening of hearing, especially at low and medium frequencies [13]. Therefore, a significant gap is evident in understanding the result of WBV exposure on cochlear function at realistic levels typically found in industrial settings. Distortion Product Otoacoustic Emissions (DPOAEs) are sounds measured in the ear canal that reflect mechanical activity of outer hair cells [14]. In animals, DPOAEs can be used to screen hearing by providing an objective means of confirming healthy cochlear function [15]. DPOAEs are measured in response to two simultaneously presented primary tones, f1 and f2, where f2 is slightly higher in frequency and at a level equal to or less than f1[16]. DPOAEs are likely generated from at least two locations on the basilar membrane, the overlapping region between f1 and f2, nearer to the f2 place, and the cubic distortion place (2f1-f2) [17]. DPOAEs are most commonly measured as Distortion Product diagrams (DP-grams) that depict DPOAE levels (Ldp) as a function of f2 for a selected combination of primary-tone levels L1 and L2[18,19]. The objective of the present study was to evaluate the influence of WBV on cochlear function, in particular outer hair cell function. It is hypothesized that WBV impairs cochlear function resulting in decreased DPOAE levels (Ldp) in rabbits subjected to WBV. Materials and methods Laboratory animal model and animal house condition Three months old, healthy male New Zealand White rabbits (weighing from 1800 to 2200 g; mean 2000 g) selected from Pasture Institute of Iran were divided into two groups as control (C) and vibration (WBV) groups. The sample size for the minimal effect size was calculated to be 5, while 10% should be added for probable death [20]. Thereby, total sample size was calculated at 5.5 and rounded to 6 (for each group). In the present study, 12 healthy rabbits were selected among 15 rabbits based on their hearing ability measured by DPOAE responses and were divided into two groups. Rabbits were maintained in a conditioned animal house at 20-22°C temperature, 30-70% relative humidity, and 10 times/h air exchange. Animals were kept on a 12-h light/12-h dark cycle. Required space for each rabbit by 2000 g body weight was considered about 0.14 m2 according to toxicology reference conditions [20]. Rabbits were allowed free access to food (Pars laboratory animal chow) and tap water. "General Principles of Helsinki Law related to Laboratory Animal" were followed. Exposure protocol Experimental animals were subjected to 1.0 ms-2 r.m.s (root mean square) WBV in z-axis at 4–8 Hz for 8 h per day during 5 consecutive days by putting them into an exposure chamber on a vibrating platform, while control animals were treated identically except exposure to WBV. Experimental protocol was set as: pre- DP-gram (baseline; day 0), rest periods (3 days on days 1 to 3), exposure periods (only for vibration rabbits; WBV exposure on days 4 to 8), first post- DP-gram (immediately following WBV exposure); rest period (3 days; days 9 through 11), and second post- DP-gram (72 h following WBV exposure). WBV exposure chamber Six experimental rabbits were exposed to vertical WBV with definite characteristics by putting them into a 50 × 50 × 50 cm transparent poly carbonate Plexiglas chamber on a self constructed vibrating platform (Figure 1). Vertical vibration (in z-axis) was chosen to achieve a larger pathway, longer stability and more impact for passing WBV along the body and implemented according to ISO-2631 (1997). Like other general vibrating systems, this system also consisted of three components including mass (total mass of chamber, 8 spring shock absorbers, metal plate dimensioned 50 × 50 cm, mounts, rabbits' weights, and 4 compressed plastic shock absorbers was equal to around 45 kg), stiffness (spring and shock absorbers), and damping. Vibrating platform was formed from a three-phase body vibrator (Model M3/65, ITAL VIBREH Company; Italy) for generating vibration and an inverter (Model 0.37 KW IG5A-4; LG Company; Korea) to obtain to desired characteristics of WBV. Air displacement was about 10 times/h by allocating 20 openings with a 3 cm diameter at the lateral faces and floor as well as 2 windows dimensioned 10 × 15 cm at the ceiling. Laboratory background noise was monitored systematically during experiments with a Casella CEL-490 sound level meter located near the exposure chamber. Background noise in the animal house and lab was found to be below 20±2 dBA SPL. Figure 1 Plexiglas exposure chamber inserted on a vibrating platform. The vibrating system consisted of mass, stiffness (spring and shock absorbers), and damping, with a total mass of 45 kilograms. DPOAE examinations At the end of the exposure period, rabbits were anaesthetized by intramuscular injection of 60% Ketamine (40 mg/kg, i.m.) and 40% Xylazine (10 mg/kg, i.m.) mixture. Before DPOAE measurement, animals were examined otologically to exclude any infection and/or remove ear channel blocking wax. At the time of DPOAE recordings, middle ear function was examined by tympanometry test (226-Hz tympanometry, with an 85 dB sound pressure level: SPL, and 400 daPa/s; Madsen-Zodiac 901; GN Otometrics, Münster Company, Germany). The criteria for normal middle-ear function were set as Type-A tympanogram, middle ear pressure values between −100 and +50 daPa, and middle ear compliance values between 0.3 and 2.0 ml. DPOAE analyzer (DPOAE 4000 I/O Model; made of HOMOTH Company; Germany) were used for recording the outer hair cells function in both ears of the animals. DPOAEs were measured in an acoustic room with background noise level less than 3 dBA SPL. Two pure primary tones (L1 = 75 and L2 = 65 dB SPL; L1-L2 = 10 dB SPL) with f2/f1 = 1.25 were used to measure DPOAEs at f2 frequencies ranging from 500 Hz to 10 kHz. Ten f2 frequencies were measured as the best auditory sensitivity responses in NZW rabbits due to classical conditioning of the nictitating membrane (NM) response [21]. The criterion for normal DPOAE was defined so that the difference between the emission level and the noise-floor levels (SNR) was above 6 dB SPL. Before DPOAEs, signal levels were calibrated in the ear canal by an emission probe microphone. The contents of stimuli were summed, and the summed energy in the 2f1–f2 frequency buffer was served to estimate DPOAE amplitudes. DPOAE levels (Ldp) on three occasions were examined, and their respective level shifts (LSdp) were compared between control and WBV groups. Constant body temperature was controlled during the DPOAE examinations for avoiding intervention in measurements. Statistical analysis Two-sample Kolmogorov-Smirnov analysis was used to assess the normality of collected data. Power analysis was used to calculate the minimum sample size required to get a significant result (H0:x¯=6). Repeated measures analysis of variance (ANOVA) was used to compare Ldp across test sessions within each group. One-way ANOVA was used to compare Ldp between groups at each test session, and post-hoc comparisons were adjusted when necessary using Tukey’s Honestly Significant Difference. Paired-sample T-test was used to compare Ldp between the right and left ears. Independent-Sample T-test was used to compare Ldp across pre- and post-exposure times. Differences were considered significant with p < 0.05. Results Collected data was confirmed to be normal in both the control and WBV groups (C.I. = 0.95; Z = 328; p < 0.001). The sample size of the study design was adequate to achieve significance at an effect size of 83.6% of the normal signal. Pre- and post-exposure DPOAE analyses revealed that no differences were seen over time in DPOAE levels (Ldp) in the non-exposed rabbits (F = 4.72; p = 0.082) (Figure 2a,b). Ldp were not significantly different between the right and left ears (t = 3.13; p = 0.076), nor were they different across frequencies (F = 6.21; p = 0.063). Figure 2 DPOAE levels and noise floor levels in control rabbits. Ldp and Lnf were measured in control and WBV exposed rabbits, with L1 = 75 dBA, L2 = 65 dBA and a f2/f1 ratio of 1.25. a: right ear; b: left ear. Each point represents mean±1 SD from 6 rabbits. DPOAE level (Ldp) analyses showed that the pre-exposed Ldp of rabbits in WBV group were found to be equal to those measured in control rabbits (p = 0.089) (Figure 3a,b), while post-exposure Ldp in rabbits exposed to WBV were significantly increased at all test frequencies in both ears as compared to the respective controls (t = 3.48; p = 0.035) or in rabbits prior to exposure (t = 5.25; p = 0.021). The greatest post-exposure Ldp was seen at 5888.5 Hz (mean Ldp, day 8 = 49.72 dB; mean Ldp, day 11 = 46.19 dB). Post-exposure Ldp in rabbits exposed to WBV were not shown to be significantly different between the right and left ears (t = 5.78; p = 0.083). Figure 3 DPOAEs levels and noise floor levels in WBV exposed rabbits. Experimental conditions are identical to those described in Figure 2. First and second DPOAE level shifts (LSdp) in WBV rabbits were found to be significantly different from those measured in the respective controls (p = 0.019 and p = 0.023 respectively) (Figure 4a,b). LSdp following exposure to WBV were significantly different across times (F = 4.77; p = 0.031). The greatest first and second LSdp (the greatest increases in Ldp, day 8 and Ldp, day 11) in rabbits following exposed to WBV were shown at 5888.5 Hz (mean first level shift = 13.25 dB, mean second level shift = 10.8 dB). LSdp in rabbits subjected to WBV were not significantly different between the right and left ears (p = 0.075). Figure 4 First and second DPOAEslevel shifts (LSdp) in control and WBV exposed rabbits.a: right ear; b: left ear. Discussion DPOAE levels (Ldp) in vibration rabbits were increased at a vast range of frequencies, mostly at mid-to-high frequencies (i.e., Ldp increased slowly from 588 Hz to 5888.50 Hz, then decreased steeply to 9855 Hz). There are, therefore, two important findings in this study: 1) WBV resulted in DPOAE level (Ldp) increases, and; 2) the greatest change in DPOAE level (Ldp) occurred at 5888.5 Hz. Consistently, Soliman et al. (2003) showed that 4-weeks-exposure to vibration only in guinea pigs led to enhancement of DPOAE amplitudes [12]. Martin et al. (1977) reported that NZW rabbits’ auditory sensitivity is maximal in the mid-to-high frequency range and rapidly decreased in the lower and higher frequencies due to the conditional nictitating membrane (NM) response [21]. Deviating from our finding, Soliman et al. (2003) found the maximum DPOAE response amplitudes in WBV guinea pigs at 1006 Hz [12]. Brown (1987) believed that DPOAE levels (Ldp) tend to be largest at the frequency of the highest hearing sensitivity in the animal species [22]. Soliman et al. (2003) concluded that more damage to the inner hair cells than the outer hair cells is the reason for increased DPOAE amplitudes in WBV exposed animals [12]. These increments were believed to be related to the affected IHCs by loss of afferent input which reduced the activity in the efferent olivocochlear bundle as well as the presence of normal OHCs that amplified the generation of DPOAEs [12]. Similar studies proposed different effects of WBV exposure on the cochlear function through a variety of causes. Okada et al. (1972) reported temporary threshold shift (TTS) after vibration exposure, which were suggested to occur at the resonance frequency of human body [7]. Temkin (1973) showed that vibration is responsible for increasing cochlear damage from noise exposure in mice [10]. Hamernik et al. (1989) found that histological changes in the extent of the outer hair cell loss were responsible for the cochlear function shifts that occurred following vibration exposure conditions [11]. Bochnia et al. (2005) asserted that vibration-induced changes were seen in all the examined inner ear areas, whereas hair-cell damage was more often seen in the apex, spreading gradually to the base and from the circumference (outer hair cells of the third row) to the modiolus [13]. Hamernik et al. (1980) found that a damaged cochlea and vibrated membranous labyrinth were the main causes for vibration-induced cochlear function changes after low-frequency vibration [9]. Consistent with the results of this study, several factors were found to be associated with the enhanced DPOAE response amplitudes such as hypoxia [23], low frequency electromagnetic fields [24,25], and induced labyrinthitis [26,27], and some ototoxic drugs [26]. By contrast, some other studies reported that the DPOAE response amplitudes were significantly depressed following a number of factors including the administration of ototoxic drugs [28,29], acoustic trauma or noise overexposure [29,30], Meniere’s disease [31], sudden idiopathic sensorineural hearing loss [32], acoustic neuroma [33], presbycusis [34], and hereditary hearing disorders [35]. DPOAE levels (Ldp) were found to change with the time after exposure. Ldp was elevated on day 8, then decreased to a level slightly higher than baseline on day 11. Similar reversible and temporary differences were reported after interrupting the exposure to different noxious agents such as noise overexposure or acoustic trauma [30], ototoxic drugs [28], sudden idiopathic sensorineural hearing loss [32], and thermoprobe lesioning [36]. These increases in DPOAE levels (Ldp) might be attributed to the temporary and reversible effect of the vibration exposure as a basal cochlear lesion progressed through the frequency region being monitored. Consistently, other data confirm that the temporary increase in DPOAE amplitudes occurring before reductions can be interpreted as an improvement of the general condition of the exposed rabbits over time [26,37]. This could be interpreted that with continued DPOAE monitoring, the emissions would eventually return to baseline values as indicted by the decrease in the LSdp between days 8 and 11. This also may be related to the presence of a lesion more basal than the frequency region being monitored [26] and the reversible recovery from temporary OHCs fatigue [12]. This released OHCs from the suppression leads to DPOAE amplification, so that DPOAE somewhat returns to the normal values after recovery from vibration, coinciding with disappearance of vacuolation from IHCs. This will results in the return of olivocochlear bundle activity, with normalization of OHC activity [12]. DPOAE levels (Ldp) in vibration-exposed rabbits were not found to be significantly different across the ears. The same vibration exposure, as well as the presence of a little distance between the rabbit's seat and the vibration generator seemed to be the main reason for the identical findings on two ears. Consistent with this result, some studies confirmed that DPOAE amplitudes were the same on right and left ears [2,3,12]. Contrary to this finding, pitch discrepancy (binaural diplacusis) were reported across the ears while presenting the same frequency stimulus [38], and tone-evoked DPOAE amplitudes were somewhat larger in the left ear [39]. Efferent activity seemed to be involved in the systematic binaural discrepancies of DPOAE response magnitudes on right and left ears in humans [40]. First and second DPOAE level shifts (LSdp) in rabbits subjected to vibration were found to be distinctly larger than those measured in rabbits not exposed to vibration. Similar finding appeared in guinea pigs at LSdp following a 4-week vibration exposure that could be attributed to the normal OHCs, severely vacuolated IHCs, and edematous and vacuolated supporting cells [12]. Conclusion WBV impairs cochlear function resulting in increased DPOAE responses in rabbits. DPOAE level shifts occurred over a wide range of frequencies following prolonged WBV. WBV caused first DPOAE level shifts on day 8 which transformed to second DPOAE level shifts on day 11 because of partial reversible recovery following interruption of exposure. Increased understanding of the physiology of enhanced DPOAE levels (Ldp) in rabbits will require a parallel histological study. Competing interests The authors declare that they have no competing interests. Authors' contributions SAMN, AK, RM and MS contributed to the conception, design and drafting of this manuscript. SAMN also carried out the audiometry tests, participated in the sequence alignment of the drafted manuscript, performed experiments and analyzed audiometry data. MA performed the calibration and setting of the DPOAEs device prior to audiometry test. All authors read and approved the final manuscript. Acknowledgement We would like to specially thank Professor Roger P. Hamernik for sincerely and friendly critical comments and technical support. We gratefully thank Professor Richard D. Kopke for helpful comments in early steps of starting this project. 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23119004PONE-D-12-1436510.1371/journal.pone.0048402Research ArticleBiologyBiotechnologyDrug DiscoverySmall MoleculesMolecular Cell BiologySignal TransductionSignaling CascadesAkt Signaling CascadeInsulin Signaling CascadePhospholipid Signaling CascadeProtein Kinase Signaling CascadeSignaling in Selected DisciplinesOncogenic SignalingMechanisms of Signal TransductionMembrane Receptor SignalingMedicineDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryObstetrics and GynecologyBreast CancerOncologyCancer TreatmentChemotherapy and Drug TreatmentCancers and NeoplasmsBreast TumorsOncology AgentsBiotechnologyOncologyPharmacologyMedicine and Health SciencesOncologyCancers and NeoplasmsBreast TumorsBreast CancerBiology and Life SciencesCell BiologyCell ProcessesCell ProliferationBiology and Life SciencesCell BiologyCell ProcessesCell Cycle and Cell DivisionBiology and Life SciencesBiochemistryProteinsPost-Translational ModificationPhosphorylationBiology and Life SciencesCell BiologyCell ProcessesCell Cycle and Cell DivisionCell Cycle InhibitorsMedicine and Health SciencesEndocrinologyDiabetic EndocrinologyInsulinBiology and Life SciencesBiochemistryHormonesInsulinMedicine and Health SciencesOncologyCancer TreatmentMedicine and Health SciencesOncologyCancers and NeoplasmsBreast TumorsTargeting 3-Phosphoinoside-Dependent Kinase-1 to Inhibit Insulin-Like Growth Factor-I Induced AKT and p70 S6 Kinase Activation in Breast Cancer Cells Targeting PDK1 in Breast CancerBaxi Sangita M. 1 Tan Wei 1 Murphy Sean T. 2 Smeal Tod 1 Yin Min-Jean 1 * 1 Oncology Research, Pfizer Worldwide Research and Development, San Diego, California, United States of America 2 Medicinal Chemistry, Pfizer Worldwide Research and Development, San Diego, California, United States of America Tan Ming Editor University of South Alabama, United States of America * E-mail: [email protected] Interests: Authors are employees and stock holders of Pfizer, Inc. There are no other relevant declarations relating to employment, consultancy, patents, products in development or marketed products. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: MJY SMB TS. Performed the experiments: MJY SMB WT. Analyzed the data: SMB WT MJY. Contributed reagents/materials/analysis tools: MJY STM. Wrote the paper: SMB MJY. 2012 31 10 2012 10 4 2017 7 10 e4840218 5 2012 25 9 2012 © 2012 Baxi et al2012Baxi 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.Binding of IGF to IGF-IR activates PI3K to generate PIP3 which in turn recruits and activates proteins that contain a pleckstrin homology (PH) domain, including AKT and PDK1. PDK1 is highly expressed in breast tumor samples and breast cancer cell lines. Here we demonstrate that targeting PDK1 with the potent and selective PDK1 inhibitor PF-5177624 in the IGF-PI3K pathway blocks breast cancer cell proliferation and transformation. Breast cancer cell lines MCF7 and T47D, representing the luminal ER positive subtype and harboring PIK3CA mutations, were most responsive to IGF-I induction resulting in upregulated AKT and p70S6K phosphorylation via PDK1 activation. PF-5177624 downregulated AKT and p70S6K phosphorylation, blocked cell cycle progression, and decreased cell proliferation and transformation to block IGFR-I induced activation in breast cancer cells. These results may provide insight into clinical strategies for developing an IGFR-I inhibitor and/or a PDK1 inhibitor in luminal breast cancer patients. These authors have no support or funding to report. ==== Body Introduction The insulin-like growth factor (IGF) system is a complex set of interactions comprised of the ligands IGF-I and IGF-II, their corresponding receptors (IGFR-I and IGFR-II), IGF binding proteins 1–6 (IGFBPs), insulin receptor substrate (IRS), and related downstream pathways. The IGF signaling pathway plays a critical role in cellular proliferation and inhibition of apoptosis. Multiple studies using cultured breast cancer cells and xenograft or transgenic mouse models have demonstrated a critical role for IGF-IGFR signaling in breast cancer progression and metastasis [1], [2], [3], [4]. Many components of the IGF axis are altered in circulation and serve as important markers for prognosis and diagnosis in breast cancer patients [5], [6], [7]. In addition, activation of the IGF axis is implicated in the development of resistance to targeted therapies in breast cancer patients [8], [9], [10], [11]. Therefore, inhibition of IGF signaling pathways should be considered as potential targeted therapies for breast cancer treatment. Several small compound inhibitors and monoclonal antibodies targeting the IGF pathway have been investigated preclinically and/or are currently in early clinical development; these studies have provided evidence of anti-tumor activities in breast cancers [12], [13]. Binding of IGF to IGF-I receptor (IGF-IR) stimulates conformational change of the receptor and receptor tyrosine kinase activation, recruits and phosphorylates intracellular adaptor proteins such as IRS family members and SHC, and results in the activation of the PI3K pathway [12]. PI3Ks phosphorylate the D3 position of membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an important secondary messenger in the recruitment and activation of proteins that contain a pleckstrin homology (PH) domain, including AKT and 3′-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a 63-kDa Ser/Thr kinase with a catalytic domain near its N terminus and a pleckstrin homology domain at its C terminus. The pleckstrin homology domain is necessary for targeting PDK1 to the plasma membrane in order to phosphorylate the T-loop sites of numerous substrates, such as AKT at residue threonine-308 (T308). This T-loop activation at T308, along with phosphorylation of the serine-473 (S473) residue by mTORC2, fully activates AKT to induce downstream signaling pathways important for tumor progression [14], [15]. PDK1 has also been shown to phosphorylate p70S6K, isoforms of PKCs, and many other kinase substrates resulting in activation of downstream signaling and cell proliferation [14], [16]. The oncogenic activity of aberrant PI3K pathway signaling through PDK1 has been extensively studied. Hypomorphic mutation of PDK1 in PTEN+/− mice markedly protects these animals from developing a wide range of tumors [17]. Overexpression of PDK1 is sufficient to transform mammary epithelial cells [18] as well as potentiate ErbB2-induced transformation and migration [19], while down-regulation of PDK1 levels inhibits cell proliferation, survival, migration and metastasis of human breast cancer cells [20], [21]. In addition, knockdown of endogenous PDK1 in PIK3CA mutant breast cancer cells suppresses anchorage-independent growth, indicating a functional dependence on PDK1 in these cells [22]. Furthermore, PDK1 is highly expressed in a majority of human breast cancers and cell lines. Over 70% of invasive breast carcinomas express activated PDK1 at a moderate to high level [23], while 20% of breast tumors have five or more copies of the gene encoding PDK1 [19]. Additionally, elevated phosphorylation of PDK1 was associated with PIK3CA mutations in human breast tumor samples [22]. Consistent with the finding in tumor samples, PDK1 levels were also elevated in most breast cancer cell lines tested [19], [22]. Therefore, targeting PDK1 in the IGF-PI3K pathway may provide an additional opportunity for breast cancer treatment. In this study, we demonstrate that the selective and potent PDK1 inhibitor, PF-5177624, inhibits IGF-I stimulated AKT phosphorylation at residue T308 and the subsequent phosphorylation of downstream signaling molecules such as p70S6K. Inhibition of PDK1 activity is sufficient to induce anti-tumor activity in breast cancer cells such that PF-5177624 inhibits cell proliferation and cell transformation in these cells. Our data suggest that a selective and potent PDK1 inhibitor is likely to inhibit IGF-I driven tumorigenesis in breast cancer cells and moreover, that a PDK1 inhibitor should be evaluated as a therapeutic for breast cancer patients with elevated IGF-I activation. 10.1371/journal.pone.0048402.g001Figure 1 IGF-I stimulated PDK1 activity in MCF7 and T47D cells. Breast cancer cell lines MCF7 and T47D were cultured in normal growth media (supplemented with 10% FBS) or serum starved for 24 hours. Growth factors EGF (A), TNFα (B), IGF-I (C), or insulin (D) were added to the culture media for 15 minutes after starvation. Cells were subsequently harvested and lysed, and lysates were subjected to SDS-PAGE. Western blot analysis was performed to examine the phosphorylation levels of AKT and p70S6K. GAPDH was included in each western blot analysis as a protein loading control. Materials and Methods PDK1 Inhibitors PF-5177624 was synthesized as previously described [25]. PF-5177624 was dissolved in DMSO for all cellular assays. 10.1371/journal.pone.0048402.g002Figure 2 PDK1 inhibitor PF-5177624 decreased IGF-I induced AKT and p70S6K phosphorylation. All IGF-1 stimulation experiments were subject to serum starvation for 24 hours prior to compound treatment or IGF-1 addition. MCF7 and T47D cells were stimulated with IGF-I at various time points, and phosphorylation levels of IGFR-I, AKT and p70S6K were determined (A). The structure of PF-5177624 is shown in (B). MCF7 or T47D cells were pre-treated with the PDK1 inhibitor, PF-5177624 at 0.2 uM, 1 uM, or 5 uM for two hours prior to stimulation with IGF-I as noted. Cell lysates were prepared and subjected to SDS-PAGE. Western blot analysis were performed to determine the phosphorylation levels of IGFR-I, AKT, and p70S6K (C), as well as PARP cleavage (E). (D) Cells were cultured in a 96-well microtiter plate and treated with PF-5177624. After two hours of compound treatment, cells were stimulated with IGF-I (15 minutes) and cell lysates were analyzed by pAKT (T308) ELISA assay to determine the IC50 value of PF-5177624. The data shown for the cell treatments and western blots analysis are representative of at least two experiments. Cell Culture BT20, HCC1954, MCF7, T47D, and MCF-10-2A cell lines were purchased from the American Type Culture Collection and cultured according to ATCC instructions. The gene mutation status of all cell lines was obtained from the Sanger COSMIC database: http://www.sanger.ac.uk/. Prior to stimulation, cells were cultured without serum for 24 hours. Cells were stimulated with EGF (100 ng/ml, Calbiochem/EMD Chemical), TNF-α, (50 ng/ml, R&D Systems), IGF-I (200 ng/ml, R&D Systems), or insulin (100 nM, Sigma-Aldrich) as indicated. 10.1371/journal.pone.0048402.g003Figure 3 PF-5177624 blocked IGF-I induced cell cycle progression in MCF7 and T47D cells. Cells were serum-starved for 24 hours in order to synchronize cells at the G0/G1 stage. Cells were pre-treated with DMSO or PF-5177624 for 2 hours prior to addition of IGF-I for 6, 18, 24, 48, or 72 hours. Cells were subsequently harvested, fixed, and stained with PI and cell cycle profiles were obtained by flow cytometry. Bar graphs indicating the percentages of cells in the various cell cycle stages at the 72 hour time point are shown in (A) and (B). Phosphorylation of Histone H3 was determined on cells treated with 5 µM PF5177624 for 24, 48, and 72 hours and normalized to DMSO treatment at the same time points as shown in (C). MCF7 and T47D cells were also incubated with BrdU and subject to FACS to measure incorporation of new DNA. The fold decrease of BrdU incoporation relative to DMSO treatment at the same time point are shown in (D). The t-test was performed to determine if there were differences in samples treated with compound versus DMSO at the same time point; *  =  p<0.05, **  =  p<0.001, ***  =  p<0.005. 10.1371/journal.pone.0048402.g004Figure 4 PF-5177624 inhibited cell proliferation and cell transformation in MCF7 and T47D cells. Cells were cultured in complete medium and treated with PF-5177624 for 72 hours prior to addition of rezasurin to determine the IC50 value of cell proliferation inhibition by PF-5177624 (A). Soft agar assays were performed in MCF7 (B and C) and T47D cells (D and E). The images from representative dishes and microscopy are shown to demonstrate colony numbers and morphology (B and D). Colony numbers were also counted (C and E). Immunoblotting Cells were lysed in lysis buffer (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) prior to use. Protein concentration was determined using the BCA Protein Assay (Pierce/Thermo Fisher Scientific) per the 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 from Cell Signaling Technology, Inc with the exception of GAPDH (Santa Cruz Biotechnology) and phospho-p70S6K T252 (custom order). All western experiments were repeated at least two times. Cell Cycle Profiling Cells were plated in six-well plates (100,000 cells/well) and allowed to adhere overnight prior to serum-starvation. After 24 hours of serum-starvation, compound was added for 2 hours and cells were then subjected to IGF-I stimulation for 6, 18, 24, 48, or 72 hours. At each time point, cells were collected, fixed, and permeablized using the Cell Cycle Phase Determination Kit (Cayman Chemical) following the manufacturer’s protocol. Samples were stored at −20°C until staining DNA with propidium iodide. Samples were also stained by anti-phosphorylated Histone H3 and anti-Bromodeoxyuridine (BrdU) to determine the number of cells in the mitotic and S phase stages. Anti-pH3 Fluor488-conjugated, anti-BrdU FITC-conjugated antibodies and BrdU were obtained from BD Bioscience and Sigma-Aldrich, respectively. Cells were labeled with 33 µM BrdU for 30 minutes before subjected to BrdU staining. A minimum of 10000 events were collected per sample on a BD FACSCalibur (BD Biosciences). Data analysis was done with FCS Express (De Novo Software). All assays were run in duplicate, and have been repeated at least twice. pAKT T308 ELISA Cells were plated at 50,000 cells/well in a 96-well microtiter plate and allowed to adhere overnight. Cells were serum starved for 24 hours prior to compound treatment; PF-5177624 was added to each well (3-fold serial dilution starting at 10 µM) for two hours, followed by stimulation with IGF-I (15 minutes, 200 ng/ml) as noted. After compound treatment and stimulation, cells were lysed and lysate transferred to the ELISA plate. The pAKT T308 chemilumiscent ELISA was performed per the manufacturer’s directions (Cell Signaling Technology, Inc). All assays were run in duplicate, and have been repeated at least twice. Cell Proliferation Cells were plated at 3,000 cells/well in a 96-well microtiter plate and allowed to adhere overnight. PF-5177624 was added to each well (3-fold serial dilution starting at 10 µM) and incubated for three days. At 72 hours post-compound addition, Resazurin (250 µg/ml final concentration, Sigma-Aldrich) was added to each well. Plates were incubated at 37°C for six hours and then analyzed for florescence (emission 590 nm, excitation 560 nm). All assays were run in duplicate, and have been repeated at least twice. Soft Agar Assay MCF7 (50,000/well) and T47D (20,000/well) cells were plated for anchorage independent growth with compound in 0.35% BD Difco Noble agar (BD Diagnostic Systems) over a bottom layer of 0.5% BD Difco Noble agar containing MEM or RPMI, respectively. Cultures were maintained for a minimum of four weeks by weekly addition of compound in fresh agar (0.35%) containing medium. Microscope images of colony morphology were taken prior to colony visualization by addition of iodonitrotetrazolium chloride (1 mg/ml, Sigma-Aldrich) for 18 hours. Colonies were counted using the colony count function on the AlphaImager Gel Imaging System (Alpha Innotech/Protein Simple). All assays were run in duplicate, and have been repeated at least twice. Results IGF-I stimulates PDK1 Activity in Breast Cancer Cells The mTORC2 complex directly phosphorylates AKT at S473 while PDK1 phosphorylates AKT at T308 [14], [24]. Therefore, pAKT-S473 is an indicator for PI3K/mTOR activity and pAKT-T308 is an indicator for PDK1 activity induced by RTKs. Similarly, specific phosphorylation sites in p70S6K correlate to the specific stimulatory activity of upstream kinases. Phosphorylation of p70S6K at T389 indicates activity stimulated by the mTORC1 complex, and phosphorylation at T252 indicates activation by PDK1 directly [14]. To investigate which RTK would stimulate the highest PDK1 activity, breast cancer cell lines HCC1954, BT20, MCF7, and T47D were treated with IGF-I, insulin, EGF or TNF-α, and phosphorylation levels of AKT and p70S6K were determined by western blot analysis (Figure 1 and Figure S1). All four tested breast cancer lines harbor PIK3CA mutations yet basal AKT phosphorylaton levels are not similar and induced AKT and p70S6K phosphorylation by RTKs are also different. The basal phosphorylation levels of both AKT and p70S6K are not significantly high in MCF7 and T47D cells, and are stimulated by EGF, IGF-I, and insulin but not by TNF-α. Among these four growth factors, IGF-I induced the highest activation of pAKT in MCF7 and T47D cells (Figure 1C). Furthermore, IGF-I was not able to induce phosphorylation of AKT and p70S6K in an untransformed mammary epithelial cell line, MCF-10-2A (Figure S1E). These results suggest that PIK3CA mutations in MCF7 and T47D cells do not lead to constitutive activation of downstream signaling but may enhance RTK stimulation signaling by activating high levels of phosphorylation of AKT and p70S6K in these cell lines. The highest phosphorylation levels of p70S6K at both T389 and T252 was observed primarily in MCF7 samples stimulated by EGF, IGF-I, and insulin. In T47D cells, however, phosphorylation of p70S6K upon IGF-1 stimulation is observed at T389 but not at T252. Taken together, these results suggest that PDK1 may play a critical role in IGF-I activated AKT and S6K phosphorylation in MCF7 and T47D cells. IGF-I induced Phosphorylation of AKT and p70S6K is Inhibited by a PDK1 Inhibitor As previously reported, we have identified a potent and selective PDK1 inhibitor, PF-5177624 [25]. PF-5177624 has a Ki value around 1 nM against PDK1 kinase activity in a biochemical assay and >100-fold selectivity against other PI3K/AKT pathway kinases. To further confirm our hypothesis that PDK1 plays a critical role in IGF-induced signaling in breast cancer cells, we used PF-5177624 (Figure 2B) to challenge the IGF-I induced signaling pathway. When MCF7 cells were treated with IGF-I, activation signals started at 15 minutes and were sustained up to 18 hours, but started to decline at 24 hours. Phosphorylation of IGFR at Y1135 increased ∼4–6 fold, AKT phosphorylation at both the S473 and T308 sites increased 8–12 fold, phosphorylation of p70S6K at T389 increased 2–3 fold, and phosphorylation of p70S6k at T252 increased around 2 fold (Figure 2A, Figure S2). In T47D cells treated with IGF-I, phosphorylation of IGFR at Y1135 increased 4–7 fold and AKT phosphorylation (S473 and T308) increased 3–6 fold, both for up to 24 hours. However, phosphorylation of p70S6K at T389 increased only 1.5–2 fold between 15 minutes and 2 hours and returned to basal levels after hour 6, while phosphorylation at T252 was not stimulated by IGF-I addition in T47D cells (Figure 2A, Figure S2). Upon PF-5177624 pre-treatment for two hours, IGF-I induced pAKT (both S473 and T308) and pP70S6K (both T389 and T252) were markedly reduced in MCF7 (Figure 2C, Figure S2, and Table S1), and resulted in the increase of an apoptosis response as indicated by greater PARP cleavage in a dose-dependent manner at the three later time points (Figure 2E). The PDK1 inhibitor PF-5177624 did not inhibit the phosphorylation of IGFR in neither MCF7 nor T47D cells, indicating that the downregulation in phosphorylation of AKT and p70S6K is due to the specific inhibition of PDK1 (Figure 2C and Figure S2). PF-5177624 did inhibit IGF-I induced AKT phosphorylation at both the S473 and T308 sites in T47D cells, though inhibition was not observed at the 18 hour time point. Since phosphorylation of T252 in p70S6K was only minimally induced by IGF-I addition, PF-5177624 only inhibited pS6K at T389 in T47D cells (as shown in Figure 2C, Figure S2, and Table S2). Interestingly, although PF-5177624 inhibited pAKT (both S473 and T308) and pS6K (T389) in T47D cells, this inhibition did not induce PARP cleavage in T47D cells (Figure 2E). A quantitative ELISA to determine the phosphorylation of AKT at T308 was performed in IGF-I stimulated MCF7 and T47D cells and indicated that PF-5177624 dose-dependently inhibited pAKT at T308 with IC50 values of ∼400 nM and ∼700 nM in MCF7 and T47D, respectively (Figure 2D). PF-5177624 was also able to inhibit the phosphorylation of AKT and S6K in MCF7 and T47D cells grown under normal conditions (Figure S3). Therefore, a PDK1 inhibitor inhibited IGF-I induced activation of downstream signaling, including the phosphorylation of AKT and p70S6K, and resulted in the induction of PARP cleavage in MCF7 cells. A PDK1 Inhibitor Blocks IGF-I induced Cell Cycle Progression Next, we looked at cell cycle progression to investigate whether the PDK1 inhibitor PF-5177624 was able to block IGF-I induced cell growth. MCF7 and T47D cells were serum starved for 24 hours to synchronize cells in the G0/G1 phase and then treated with PF-5177624 or DMSO two hours prior to IGF-I stimulation (200 ng/ml). Cells were harvested at the indicated time points post-stimulation and subjected to FACS analysis to determine cell cycle progression under IGF-I stimulation conditions. In MCF7 cells, IGF-I induced DNA synthesis with an increasing S phase starting at 18 hours and one cell cycle was completed at approximately 48 hours (Figure S4A). Addition of PF-5177624 in MCF7 cells decreased the cell population in the S phase and increased those in the G2/M block (Figure 3A and Table S3). In T47D cells, cell cycle progression induced by IGF-I started at 18 hours and lagged until 72 hours (Figure S4B); addition of PF-5177624 decreased the cell population in S phase and increased G0/G1 arrest (Figure 3B and Table S3). BrdU incorporation and phosphorylated Histone H3 staining assays were also performed to further confirm the decrease in S phase in MCF7 and T47D cells and G2/M block in MCF7 cells (Figure 3C and 3D). These results indicate that IGF-I stimulated different cell cycle progression profiles for MCF7 and T47D cells, and inhibition of PDK1 activity by PF-5177624 resulted in different patterns of cell cycle progression blockade whereby PF-5177624 leads to G2/M block in MCF7 cells and G0/G1 arrest in T47D cells. Furthermore, PF-5177624 also induced a subG1 cell population in both cell lines, suggesting that inhibition of PDK1 may induce cell apoptosis in both MCF7 and T47D cells (Figure 3, Figure 4 and Table S3). We have shown that PF-5177624 can induce PARP cleavage in MCF7 but not in T47D cells (Figure 2E), which suggests that it is likely that the apoptosis mechanism in T47D may be through an alternate mechanism other than PARP. The different cell cycle blockade patterns could be due to differences in downstream signaling inhibition by PF-5177624 in MCF7 and T47D cells, as we demonstrated that p70S6K phosphorylation at both T389 and T252 was stimulated by IGF-I and inhibited by PF-5177624 in MCF7 cells, but that there was not significant phosphorylation of p70S6K at T252 by IGF-I stimulation in T47D cells (Figure 2C). The PDK1 Inhibitor PF-5177624 Inhibits Cell Proliferation and Cell Transformation of Breast Cancer Cells A cell proliferation assay was performed to determine whether PF-5177624 would demonstrate inhibition of cell growth under normal conditions (Figure 4A). IC50 values determined from the pAKT (T308) ELISA and cell proliferation assays treated with PF-5177624 are relatively similar, indicating a good correlation between kinase activity and cell proliferation. Furthermore, an anchorage independent growth assay was performed to evaluate whether inhibition of PDK1 activity would block cell transformation of MCF7 and T47D cells. Both MCF7 and T47D cells form colonies when cultured in soft agar, and addition of PF-5177624 decreased both colony size and colony numbers in the soft agar assay (Figure 4B–4E). The concentration needed to decrease colony formation was lower than the cell proliferation assay, and this observation is similar to previous reports in the literature where inhibition of PDK1 has a more profound effect on inhibition of cell transformation than cell proliferation [26]. We have demonstrated that PDK1 plays a critical role in the activation of AKT and p70S6K in MCF7 cells which may in part explain the better potency by PF-5177624 in inhibition of colony formation in MCF7 as compared to T47D cells. Discussion PDK1 is downstream of PI3K and elevated phosphorylation of PDK1 has been associated with PIK3CA mutations in human breast tumor samples [22]. Therefore, we examined breast cancer cell lines with PIK3CA mutations in this study to investigate whether inhibition of PDK1 activity had antitumor effects in breast cancer cells subjected to growth factor stimulation. The breast cancer cell lines HCC1954 and BT20 are representative of triple negative breast cancer cells (TNBCs), while MCF7 and T47D cells represent luminal breast cancer cells. Although all four of these cell lines harbor a PIK3CA mutation, phosphorylation of AKT and p70S6K in MCF7 and T47D cells is not constitutively high and can be induced by IGF-I and other growth factors. These results highlight the difference in PI3K-AKT signaling in different breast cancer subtypes, and imply that growth factor stimulation may play an important role in luminal breast cancer cells harboring a PIK3CA mutation. Phosphorylation of AKT at T308 and p70S6K at T252 are indicators of PDK1 activity. Our data clearly show that IGF-I induced high levels of phosphorylation of AKT and p70S6K and that this activity could be modulated by inhibition of PDK1 activity in MCF7 cells. These data are also in line with recent publications demonstrating a role for IGF-IR/PDK1 signaling in the mediation of breast cancer cell growth [27], [28]. Cross-talk between the ErbB/HER family and the IGFR-I receptor signaling pathway has been described and suggested as a possible mechanism of resistance in breast cancer patients treated with the anti-HER2/neu antibody trastuzumab [10], [29]. IGFR-I signaling has also been implicated in the regulation of metastasis and invasion of breast cancer cells independent of tumor cell growth [2], [30]. However, IGFR-I upregulation of cell growth and cell cycle progression in ER positive breast cancer cells has not been well described with the exception of more recent publications [27], [31]. Results from our study further confirm that IGFR-I mediates cell proliferation through PDK1 activity in luminal breast cancer cells, and moreover, extend the evidence that the PI3K/AKT pathway is regulated by IGF-I stimulation in luminal but not triple negative breast cancer cells. This information is important and will assist development of clinical trial strategies in the design of combination therapies in different breast cancer subtypes. Collectively, there are multiple pieces of evidence indicating that PDK1 plays an important role in mediating cell proliferation, transformation, migration and other oncogenic mechanisms as previously described. In this study, we further demonstrated that IGF-I induced cell proliferation is mediated by PDK1 activity in luminal breast cancer cells. Therefore, the driving mechanism of PDK1 mediated tumorigenesis may be cell type dependent or may be variable based on the genetic background of specific tumor cells. For example, a recent study used knockdown of PDK1 by an RNAi approach but did not observe a block in prostate or leukemia tumor development in PTEN-deficient transgenic mouse models [32]. As multiple kinases have been implicated as substrates of PDK1 [14], it is also likely that a PDK1 inhibitor, such as PF-5177624, would block IGF-I induced cell transformation by inhibiting multiple PDK1 substrates collaboratively in breast cancer cells. Moreover, another recent study suggests that direct interaction of PDK1 and IGFR-I in MCF7 cells may also provide insight into how a PDK1 inhibitor might block IGF-I induced signaling [27]. Several PDK1 inhibitors have been described [26], [33], [34]; however, these compounds lack cellular potency and reasonable kinase selectivity profiles. Here we demonstrate that a relatively potent and selective PDK1 inhibitor, PF-5177624, is able to inhibit IGF-I induced phosphorylation of downstream signaling molecules, block cell cycle progression, and decrease cell proliferation and transformation in luminal breast cancer cells. Although PF-5177624 does not have the desirable pharmacokinetic properties to perform animal studies, it serves as a good tool to investigate PDK1-mediated pathways. Further optimization of this series of PDK1 compounds is needed to conduct in vivo studies. In conclusion, we have demonstrated that IGF-I signaling through the activation of PDK1 plays a critical role in the induction of cell cycle progression in luminal breast cancer cells. Furthermore, using the PDK1 inhibitor PF-5177624, we confirmed that inhibition of AKT and p70S6K can block IGF-I induced cell proliferation and transformation in MCF7 and T47D cells. These results provide evidence that inhibition of PDK1 may have greater anti-cancer therapeutic benefits in breast cancer patients with higher IGF-I/IGFR-I levels, and that a combination therapy featuring a PDK1 inhibitor and an IGFR-I inhibitor may have synergistic effects in this patient population. Supporting Information Figure S1 Breast cancer cell lines HCC1954 and BT20 were cultured in normal growth media (with 10% FBS) or serum starved for 24 hours. Growth factors EGF (A), TNFα (B), IGF-I (C), or insulin (D) were added to the culture media for 15 minutes after starvation. Cells were subsequently harvested and lysed, and lysates were subjected to SDS-PAGE. Western blot analysis was performed to examine the phosphorylation levels of AKT and p70S6K. An untransformed breast cell line, MCF-10-2A, was cultured in growth media with or without FBS and treated with IGF-I for 15 minutes after starvation. Cell lysates were evaluated by western blot to examine the phosphorylation levels of AKT and p70S6K (E). GAPDH was included in each western as protein loading control. (TIF) Click here for additional data file. Figure S2 The digital density of protein band in each western blot analysis was determined by FluorChem Q software for the blots shown in Figure 2A (A) and Figure 2C (B). Relative activity of pIGFR, pAKT, and pS6K was determined by normalization of the density of the phosphorylated protein bands to GAPDH and total IGFR, AKT, and S6K. Data are from a representative experiment in MCF7 (A) and T47D (B). Similarly, relative activity of pIGFR, pAKT, and pS6K after PF-5177624 treatments was calculated, and plotted by normalization to DMSO treated samples, and the graphs of 2 hour treatment were shown for MCF7 (C) and T47D (D). The t-test was performed to determine if there were any difference compared to the DMSO treatment group; *  =  p<0.05, **  =  p<0.001, ***  =  p<0.005. (TIF) Click here for additional data file. Figure S3 MCF7 and T47D cells cultured in normal growth media were treated with 0.2, 1 or 5 µM of PF-5177624 at various time points, and phosphorylation levels of IGFR-I, AKT and p70S6K were determined by western blot. (TIF) Click here for additional data file. Figure S4 Cells were serum-starved for 24 hours in order to synchronize cells at stage G0/G1. Cells were pre-treated with DMSO or PF-5177624 for 2 hours prior to addition of IGF-I for 6, 18, 24, 48, or 72 hours. Cells were subsequently harvested, fixed, and stained with PI and cell cycle profiles were obtained by flow cytometry. DNA content is shown in MCF7 (A) and T47D (B). (TIF) Click here for additional data file. Table S1 The detailed relative activity changes of pIGFR-I, pAKT and pS6K by PF-5177624 treatment in MCF7 cells at all time points are summarized. (TIF) Click here for additional data file. Table S2 The detailed relative activity changes of pIGFR-I, pAKT and pS6K by PF-5177624 treatment in T47D cells at all time points are summarized. (TIF) Click here for additional data file. Table S3 The detailed cell population at the various cell cycle stages at each time point are summarized in MCF7 and T47D cells. (TIF) Click here for additional data file. The authors would like to thank Jacques Ermolieff for leading the PDK1 project, and Marlena Walls for her help with experiments. ==== Refs References 1 Jackson JG , Zhang X , Yoneda T , Yee D (2001 ) Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines . Oncogene 20 : 7318 –7325 .11704861 2 Sachdev D , Zhang X , Matise I , Gaillard-Kelly M , Yee D (2010 ) The type I insulin-like growth factor receptor regulates cancer metastasis independently of primary tumor growth by promoting invasion and survival . 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PLoS One. 2012 Oct 31; 7(10):e48402
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23139753PONE-D-12-1053810.1371/journal.pone.0047672Research ArticleBiologyBiochemistryEnzymesEnzyme KineticsEnzyme RegulationProteinsProtein InteractionsRegulatory ProteinsBiomacromolecule-Ligand InteractionsBiophysicsProtein ChemistryHuman Epididymis Protein-4 (HE-4): A Novel Cross-Class Protease Inhibitor HE4 Inhibits a Wide Range of ProteasesChhikara Nirmal 1 Saraswat Mayank 1 ¤ Tomar Anil Kumar 1 Dey Sharmistha 1 Singh Sarman 2 Yadav Savita 1 * 1 Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India 2 Department of Lab Medicine, All India Institute of Medical Sciences, New Delhi, India Abrams William R. 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: NC MS SY. Performed the experiments: NC AKT. Analyzed the data: NC MS SD SY. Contributed reagents/materials/analysis tools: SD SS. Wrote the paper: NC AKT MS SY. ¤ Current address: Centre for Bioanalytical Sciences, Dublin City University, Glasnevin, Dublin-9, Ireland 2012 5 11 2012 7 11 e4767213 4 2012 18 9 2012 © 2012 Chhikara et al2012Chhikara 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.Epididymal proteins represent the factors necessary for maturation of sperm and play a crucial role in sperm maturation. HE-4, an epididymal protein, is a member of whey acidic protein four-disulfide core (WFDC) family with no known function. A WFDC protein has a conserved WFDC domain of 50 amino acids with eight conserved cystine residue. HE-4 is a 124 amino acid long polypeptide with two WFDC domains. Here, we show that HE-4 is secreted in the human seminal fluid as a disulfide-bonded homo-trimer and is a cross-class protease inhibitor inhibits some of the serine, aspartyl and cysteine proteases tested using hemoglobin as a substrate. Using SPR we have also observed that HE-4 shows a significant binding with all these proteases. Disulfide linkages are essential for this activity. Moreover, HE-4 is N-glycosylated and highly stable on a wide range of pH and temperature. Taken together this suggests that HE-4 is a cross-class protease inhibitor which might confer protection against microbial virulence factors of proteolytic nature. This work was supported by financial grants from the Department of Science and Technology (DST), Government of India. The authors thank the Council of Scientific and Industrial Research (CSIR), New Delhi for the fellowship granted to NC. The authors also thank Department of Science and Technology (DST), Government of India, for providing fellowship to AK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Human and chimpanzee genomes are similar to the extent of nearly 90% but, there are differences which impart uniqueness to the human species as well as chimpanzees and reveal the intrinsic genetic differences in the expressions of genes. A sudden or adaptive evolution in some of the genes or genomic regions might be playing some essential part in these differences. Recent comparative analysis of human and chimpanzee genome sequences identified some 16 regions with high density of rapidly evolving genes [1]. One such region contains genes encoding whey acidic protein (WAP) domain proteins. This region on human chromosome 20q13 is called WAP four-disulfide core domain (WFDC) locus containing 14 genes encoding WFDC type proteinase inhibitors [2]. Apart from these 14 genes from the same locus there are at least four other proteins having WFDC domain but are present at different chromosomes (Ch. 16, 17 and X chromosome) [3]–[6] Amplification of the 20q12–13 region has been documented in breast and ovarian carcinoma [7]–[8]. Consistent with these studies, SLPI and elafin are known to be expressed in various carcinomas and implicated in initiation or progression of tumorigenesis [9]–[13]. Most of the members of the family with the exception of SLPI, elafin, KAL1, EPPIN and ps20 have not been examined at the protein level. SLPI, elafin and ps20, have been reported to be expressed in different cell types including airway epithelium and mucosal secretions from tissues including male reproductive tract, respiratory tract as well as in inflammatory cells like T-cells and macrophages [14]–[16]. Two types of functions attributed to this family of proteins are regulation of proinflammatory mediators and anti-bacterial or anti-fungal activity [17]–[18]. Anti-infective activity of SLPI, elafin, and pro-infection attributes of ps20 regarding HIV have also been uncovered [19]. Another member of the family, WFDC-2 or HE-4 (human epididymis protein-4) was found to be the most frequently upregulated in ovarian carcinomas [20]. This protein is also known as Epididymal secretory protein E4, Major epididymis-specific protein E4 and putative protease inhibitor WAP5. WFDC-2 gene product was originally thought to be a protein specifically expressed in the epididymis and was dubbed as a tissue marker for the same [21]. Later, it was found to be expressed in the oral cavity, respiratory tract, female genital tract and distal renal tubules. Evidence of its expression in the colonic mucosa has also been found [22]. Evidence of HE-4 expression in various tumor types of the lung including lung adenocarcinoma has also been reported [16]. HE-4 levels in the serum have been suggested to be a sensitive marker for ovarian cancer and protein used as a histological marker for ovarian cancer [23]–[25]. On the basis of structural and sequential similarity of HE-4 with other WAP proteins like SLPI and elafin it was suggested that the protein might have antiprotease activity within the male reproductive, oral and respiratory tract. Other than this suggestion of HE-4 working as an antiprotease, no work has been done to elucidate the role and/or structure of the protein. Even this claim projecting it as a protease inhibitor has never been tested. It seems plausible that HE-4 will have as yet undetermined pivotal roles in human physiology. An investigation of this demand detailed studies at the protein level. A prerequisite for the above said objective is a rapid and efficient method to generate sufficient amounts of protein. This paper reports an accessible and efficient purification of HE-4 protein from human seminal fluid. We have further characterized HE-4, and found it to be a cross-class protease inhibitor and show a significant binding with all the proteases tested. Results HE-4 purified from seminal fluid exists as a trimer HE-4 was purified from normozoospermic pooled human seminal plasma. Heparin-sepharose unbound fraction was further fractionated on DEAE-sephacel. Seminal plasma fractionated into four main peaks on DEAE-sephacel (Fig. 1 A, B). All the peaks were further fractionated by sephadex G-75 size exclusion chromatography (data shown only for peak I of DEAE) and HE-4 immnuoreactivity was found in peak III of G-75 fractionation (Fig. 1E, lane II). Western blot of purified protein and whole seminal plasma showed a band of approximately 14 kDa (Fig. 1E) which was confirmed by SDS-PAGE of the same in reducing conditions (Fig. 1D lane III). Under non-reducing conditions, HE-4 was found to migrate as a single band of approximately 42 kDa (Fig. 1D lane II). This suggests that HE-4 exists as a disulphide bonded trimer in human seminal plasma and disulfide bond reduction provide a monomer of 14 kDa. Identity of HE-4 was also confirmed by LC-MS/MS (Table 1). Mascot score of identification for HE-4 was 123 and four peptides were matched. One previous study has reported HE-4 to be N-glycosylated in saliva [26]. We analyzed the glycosylation of HE-4 with PAS staining method (Fig. 2A). We treated HE-4 with PNGase F under denaturing conditions and compared it with the native protein. An approximate shift of 2.5 kDa in the migration of deglycosylated HE-4 in SDS-PAGE was observed compared to native protein (Fig. 2B). This confirmed that HE-4 in seminal fluid is N-glycosylated. 10.1371/journal.pone.0047672.g001Figure 1 Purification of HE-4 protein from human seminal plasma using affinity, ion exchange and gel filtration chromatography. (A) Elution profile of anion exchanger DEAE sephacel. The peaks formed as a function of X-axis as elution volume in ml and Y-axis is mA at 280 nm. The second (….) line represents the gradient of NaCl. The first peak with black base given is the one where we found HE-4. (B) 12.5% SDS-PAGE of peaks obtained from anion exchanger DEAE sephacel. Lane I: Protein marker. Lane II: Elution profile of First peak in which we obtained HE-4 with other impurities. Lane III, IV and V: Elution profile of second, third and fourh peak respectively. (C) Elution profile of Sephadex G-75. The peaks were obtained as a function of X axis as elution volume in mL and Yaxis is mA at 280 nm. Buffer used for elution is 50 mM Tris-HCl pH 8.0 and 0.2 M NaCl. (D) 12% SDS-PAGE of the third peak of elution profile of sephadex G-75. Lane I: Protein Marker. Lane II: Elution of the third peak in non-reducing condition and Lane III: Elution profile of a third peak in reducing conditions. (E) Immunodetection of HE-4 in crude seminal plasma and of the third peak of elution profile of sephadex G-75. Lane I: Crude seminal plasma. Lane II: Purified protein (IIIrd peak of sephadex G-75). (F) The standard curve for Sephadex G-75 and lines drawn for the position of HE-4 in the standard curve. 10.1371/journal.pone.0047672.g002Figure 2 Glycosylation analysis of HE-4. (A) PAS staining of HE-4 protein. (B) 12.5% SDS-PAGE of glycolsylated and unglycosylated form of HE-4 Lane I: Protein marker. Lane II: Glycosylated HE-4. Lane III: Unglycosylated HE-4. 10.1371/journal.pone.0047672.t001Table 1 Identified Peptides of HE-4 by LC-MS/MS. Matched Peptide Score Mr(expt) Mr(calc) K.VSCVTPNF 37 922.8927 922.4219 R.DQCQVDSQCPGQMK.C 51 1680.9627 1679.6702 R.DQCQVDSQCPGQMK.C+Oxidation (M) 51 1697.4527 1695.6651 K.CCSAGCATFCSLPNDK.E 67 1848.5127 1846.7107 WFDC-2 protein Homo sapiens (Human): Mass: 13953 Score: 123 Queries matched: 4 emPAI: 1.39. Effect of treatment with various chemicals on solution behavior and possible aggregation pattern of native HE-4 was analyzed with dynamic light scattering (DLS). Purified HE-4 was incubated with solutions of varying pH (from acidic to alkaline) and different chemicals including various salts and reducing agents at different concentrations. The hydrodynamic radius (Rh) of purified HE-4 (Tris-HCl buffer pH 8.0 in which protein was purified) was found to be 3.18±0.07 nm. This corresponds to approximately 46 kDa, and in complete agreement with migration of HE-4 in SDS-PAGE. Different pH had varied effect on HE-4 Rh values (Fig. S1A). At low pH, the Rh was higher which returned to native values as pH increased. Metal ions can contribute to aggregation of proteins [27] therefore we also recorded Rh values in the presence of various divalent metal ions and we have arranged them in decreasing order in the graph (Fig. S1A). Notably highest values were recorded in the presence of magnesium and iron salts while copper and calcium salts values were closer to the Rh native protein. Twenty mM ZnCl2 reduced the Rh value below native protein Rh value, and introduction of EDTA to the same sample in twice the molar ratio increased the Rh value, and brought it closer to the native protein. Altering pH and presence of metal ions change the solution behavior of HE-4, and may even cause the protein to adopt different conformations. As the concentration of detergents and various reducing agents increased, HE-4 Rh values decreased to less than that of the native protein (Fig. S1B, C and D). These DLS results, when considered with other data reported in the study support the conclusions drawn in this section. Sequence analysis of HE-4 Multiple sequence alignments (MSA) of HE-4 across multiple species were performed using ClustalW2 on EBI web server (Fig. 3). WFDC-2 protein sequences from different species show high similarity (Fig. 3C). Each domain consists of eight Cys residues which are fully conserved. Besides, there are other-many conserved residues a close examination of alignment reveals that these conserved residues also follow a common pattern of spacing. Comparison of the spacing between common conserved residues in each domain finds the following pattern: K(1)G(1)CP(7/11)C(3)C(2)D(2)C(4)KCC(2)GC(3/4)C(2)P, where the number in bracket represents the length of spacing. Both WAP domains of HE-4 were separately further aligned with WAP domains of other WFDC family members having anti-protease activities assigned to them (pig elafin, human elafin and human SLPI-WAP2) to gain information about function of HE-4 (Fig. 3A, B). It, reveals that in addition to C residues, K-121/72/85, P-122/73/86, G-123/74/87, P-126/77/90, M-134/85/98, N-136/87/100, P-137/88/101, P-138/89/102, N-139/90/103, D-142/95/108, K-150/103/116, G-154/107/120, G-157/110/123 and P-163/116/129 (Representations, Amino acid-position in pig elafin sequence/position in human elafin/position in human SLPI) are fully conserved, and all these may be crucial for anti-protease activity of these domains. Alignment of these domains with HE-4-WAP1 shows that out of 22 conserved residues, 3 residues including second cysteine are not conserved (C-132/83/96, M-134/85/98 and P-137/88/101), and five others are replaced by similar amino acid residues (as indicated by ‘:’ & ‘.’ in alignment). Similarly, some amino acids are not conserved, and some are substituted by similar amino acids in HE-4-WAP2. When we adjusted the optimum alignments, of HE-4-WAP1 and WAP2 domains with known antiprotease activity WAP domains, in such a way that all eight cysteine residues are perfectly conserved, and the similar pattern was observed in both alignments. Overall, six amino acid residues were found fully conserved (For HE-4-WAP1: K32, G34, P37, D52, K60, P73 and For HE-4 WAP 2: K76, G78, P81, D100, K108, P122). 10.1371/journal.pone.0047672.g003Figure 3 Multiple sequence alignment of HE-4 with other WAP protein and HE-4 of different species. At the end of each line of amino acid sequence, amino acid residue numbers are given and after that % homology is written. (A) Sequence alignment of HE-4 WAP 1 domain with other antiproteases like elafin and WAP-2 domain of SLPI. (B) Sequence alignment of HE-4 WAP2 domain with other antiproteases like elafin and SLPI. (C) Multiple sequence alignment of HE-4 protein of human with HE-4 protein of different species. HE-4 is a cross-class protease inhibitor Protease inhibition activity of HE-4 was tested according to the method of Lee and Lin [28]. Hemoglobin was used as a substrate and percentage inhibition was calculated as described in material and methods. Trypsin, chymotrysin, prostate specific antigen (PSA), proteinase K, pepsin and papain were employed for the experiment, and HE-4 showed dose-dependent inhibition of all of these proteases (Fig. 4A). The dose dependence was more for papain and pepsin and less so for other proteases. Blue native electrophoresis was performed to determine the physical complex formation of HE-4 with all the proteases (Fig. 5). All the proteases tested showed a higher molecular weight complex with HE-4 compared to HE-4 or proteases alone. 10.1371/journal.pone.0047672.g004Figure 4 Antiprotease activity of HE-4 at different concentration and at different condition against various serine, cystine and aspartic proteases. (A) Percentage inhibition of various proteases like: trypsin, chymottrypsin, PSA, proteinase K, pepsin and papain by HE-4. X-axis is the HE-4 concentration in µg/ml and Y-axis is the % inhibition of respective proteases. (B) Effect of temperature (25°C–100°C) on the protease inhibition activity of HE-4 against trypsin. (C) Effect of pH (2–10) on the protease inhibition activity of HE-4 against trypsin. (D) Effect of different chemicals like: ZnCl2 SDS and β-mercaptoethanol on the protease inhibition activity of HE-4 using trypsin as model protease. EDTA was added to ZnCl2 pre-treated HE-4 to check whether it can rescue the decrease in activity induced by ZnCl2. SDS control is the % inhibition of trypsin by 5% SDS and 5% SDS point is the activity of SDS-pretreated HE-4 against trypsin. +ve control is HE-4 inhibition of trypsin without any treatment and −ve control is without HE-4, therefore 0% inhibition. (E) Effect of varying concentrations of EDTA on rescue of trypsin % inhibition decrease by ZnCl2 treatment of HE-4. (F) Effect of varying concentration of DTT on trypsin inhibitory activity of HE-4. 10.1371/journal.pone.0047672.g005Figure 5 Blue native gel of HE-4 after 2 hr. incubation with different proteases. Proteases and HE-4 alone were also run to compare with the complexes formed with them. 1: Marker 45 kDa Ovalbumin, A: HE-4, B: Trypsin, C: Trypsin+HE-4, D: Chymotrypsin, E: Chymotrypsin+HE-4, F: Proteinase K, G: Proteinase K+HE-4, H: PSA, I: PSA+HE-4, J: Pepsin, K: Pepsin+HE-4, L: Papain, M: Papain+HE-4. Effect of pH, temperature and other chemicals on HE-4 activity was tested using trypsin as a model protease. HE-4 had slightly better trypsin inhibitory activity after incubation at pH 2 and 10 while, at pH 4–6 activity was more or less similar (Fig. 4C). Effect of temperature on HE-4 inhibition of trypsin was undertaken, and activity was measured after incubation of HE-4 at different temperatures. HE-4 inhibited trypsin the most at 25°C and inhibitory activity decreased to 81% at 60°C but even after boiling at 100°C for 10 min, HE-4 retained 29% inhibition (Fig. 4B) suggesting a highly stable tertiary structure resistant to heat. This is further supported by the observation that HE-4 retained 64% inhibition of trypsin in the presence of SDS. Only 5% SDS itself showed minor inhibition of trypsin activity but much less compared to SDS-treated HE-4 (figure 4D). This suggests that SDS does not denature HE-4 significantly upto 5% concentration. Presence of β-mercaptoethanol (10% v/v) abolished the activity of HE-4 (Figure 4D). A study of effect of disulfide bond reduction on HE-4 activity against trypsin was undertaken using different concentrations of DTT. There was a significant decline in the inhibitory activity of HE-4 against trypsin (29.12%) already at 0.25 mM DTT (Figure 4F). In presence of 1 mM DTT, HE-4 completely lost its inhibitory activity (Figure 4F). This suggests that disulfide bonds based structure stabilization is essential for the protease inhibition. EDTA had no effect on inhibition while 2 mM ZnCl2 reduced the activity to 83% and curiously, introduction of twice the molar ratio of EDTA to zinc supplemented sample restored the activity a little (Fig. 4D). Activity of HE-4 against trypsin, which decreased with ZnCl2 could be rescued by adding increasing concentrations of EDTA (Figure 4E). Interaction of HE-4 with various proteases as seen by surface plasmon resonance HE-4 interactions with serine proteases were confirmed with surface plasmon resonance (SPR) and kinetic constants were calculated. HE-4 was immobilized on research grade CM5 chip using EDC/NHS chemistry as described in methods. Different proteases were flowed over the chip in varying concentrations, ranging from 75–300 nM. Concentration of proteases above this range was shown to reduce the signal, possibly due to “Hook effect”. Among all the proteases tested, the highest affinity (KD) and association constant (KA) was found to be for proteinase K followed by chymotrysin, PSA and then trypsin (Table 2). Trypsin had the lowest association and dissociation constants among all the proteases tested. 10.1371/journal.pone.0047672.t002Table 2 Binding affinity of HE-4 with different proteases. Protease HE-4 was immobilized on CM5 chip Proteases were immobilized on CM5 chip HE-4 was immobilized on CM5 chip Proteases were immobilized on CM5 chip KD (M) KA (M−1) KD (M) KA (M−1) Kd(S−1) [Koff] Ka (M−1 S−1) [Kon] Kd(S−1) [Koff] Ka (M−1 S−1) [Kon] Proteinase K 4.65×10−8 2.15×107 5.05×10−8 1.85×107 9.48×10−6 204 10.36×10−6 230 Chymotrypsin 7.56×10−8 1.32×107 7.62×10−8 1.17×107 4.31×10−6 57.1 5.26×10−6 64.8 Trypsin 4.46×10−4 2.24×104 5.34×10−4 1.96×104 1.02×10−5 0.0229 1.62×10−5 0.0384 PSA 1.06×10−5 9.40×104 1.19×10−5 9.23×104 1.0×10−5 0.941 1.27×10−5 1.658 In separate experiments HE-4 or proteases were immobilized on CM5 chip and various proteases or HE-4 was flowed over it. Converse study was also performed with SPR, where all serine proteases were on the chip and HE-4 was flowed. No significant changes were observed in binding affinity of the proteases and HE-4 (Table 2). Unfortunately, we could not determine the kinetic constants for papain and pepsin as we faced an unexpected problem of negative sensograms (Fig. 6A, B). Although, negative sensograms are not uncommon they are usually ascribed to the differences in buffer composition pre and post injection [29]. In the present study, there were no differences in the buffer compositions therefore we sought to further investigate the reason of signal dropping below baseline. For this, papain was incubated with increased concentration of HE-4 (HE-4: papain; 1∶1 to 5∶1) at pH 8.5 for 1 hr, and as a control HE-4 and papain was incubated separately for 1 hr at room temperature. Later mixtures were resolved on 14% SDS-PAGE under reducing conditions. HE-4 and papain, when incubated alone, showed band at their own molecular weight, but when they are incubated together two new bands appeared in the mixture below HE-4 band as seen in fig. 7A. In SDS-PAGE, we observed two bands that are probably the cleavage product of HE-4 by papain because with increasing concentrations of HE-4, the band at approximately 10 kDa increase in intensity while the original band of HE-4 does not increase in intensity which would be explainable by HE-4 cleavage by papain. This was confirmed with western blot which showed a low molecular weight HE-4 band lower than full length HE-4 (Figure 7C Lane 2). This explains the negative sensogram of papain (Fig. 6B) suggesting that after initial interaction with HE-4 (see the upward spike), papain cleaves and releases HE-4 from the chip bringing sensogram below baseline. In case of pepsin, we were surprised by the results as seen in fig. 7B when pepsin is incubated with HE-4(HE-4: papain; 1∶1 to 5∶1) at pH 5.0 for 1 hr it undergoes self-cleavage, as evident by the band present at approximately 20 kDa and with increasing concentration of HE-4, the decrease in intensity of pepsin (full size) band. This band at 20 kDa was definitely of pepsin as western blot using HE-4 antibodies (Figure 7C Lane 3) revealed that there was no band of HE-4 at that position. This only partially explains the sensogram of pepsin in fig. 6A and does not explain why the signal went down below the baseline unless there was also the minor cleavage of HE-4 which was not detectable in SDS-PAGE or WB because SPR would be more sensitive to even small amount of cleavage. The sensograms of other proteases were characteristic of a normal protein-protein interaction (Figure 8). 10.1371/journal.pone.0047672.g006Figure 6 SPR sensograms of HE-4 interaction with papain and pepsin. Concentrations of HE-4 were fixed at 10 µM. Concentrations of papain and pepsin were 75 nM. Injection time of papain and pepsin is indicated by an arrow. (A) Pepsin-HE-4 interaction. (B) Papain-HE-4 interaction. 10.1371/journal.pone.0047672.g007Figure 7 14% SDS-PAGE to resolve HE-4 and pepsin, papain incubated alone or together for the same duration. (A) 14% SDS-PAGE (silver stained) Lane1: Molecular weight marker. Lane2: Fresh HE-4. Lane3: HE-4 after 1 hr incubation. Lane 4: Fresh papain. Lane5: papain after 1 hr incubation. Lane 6- Lane 10 papain and HE-4 in 1∶1–1∶5 (10 µg∶10 µg-10 µg∶50 µg) ratio after 1 hr incubation (in 50 mM tris-HCl buffer, pH 8.5). (B) 14% SDS-PAGE (silver stained) Lane1: Molecular weight marker. Lane2: Fresh HE-4 (10 µg). Lane3: HE-4 after 1 hr incubation (10 µg). Lane 4: Fresh pepsin (10 µg). Lane5: pepsin (10 µg) after 1 hr incubation. Lane 6- Lane 10 pepsin and HE-4 in 1∶1–1∶5 ratio (10 µg∶10 µg–10 µg∶50 µg) after 1 hr incubation (in 50 mM sodium acetate buffer, pH 5.0). (C) Immunodetection of HE-4 after incubating HE-4 alone and with pepsin and papain for the same duration. Lane1: HE-4 (10 µg) after 1 hr incubation. Lane 2: HE-4 (10 µg) after 1 hr incubation with papain (10 µg) and Lane3: HE-4 (10 µg) 1 hr incubation with pepsin (10 µg). 10.1371/journal.pone.0047672.g008Figure 8 SPR sensograms of HE-4 interaction with serine proteases. HE-4 immobilised on CM5 chip as described in methods. Concentration of HE-4 was fixed at 10 µM. Concentrations of serine proteases (from top to bottom) were 300, 150, and 75 nM respectively. (A) Trypsin-HE-4 interaction. (B) Chymotrypsin-HE-4 interaction. (C) PSA- HE-4 interaction. (D) Proteinase K- HE-4 interaction. Discussion HE-4 is upregulated in ovarian carcinoma and also expressed in a variety of lung tumors. There are several reports in the literature which suggest important functions of HE-4: over-expression in ovarian carcinoma, over-expression in prostate cancer model mice with PTEN inactivation, interaction with pleiotrophin (PTN) which regulates angiogenesis and is involved in tumor formation as well as the upregulation of HE-4 during the expected window of receptivity in the endometrium under the control of progesterone in primates [20], [30]–[32]. All of these evidences point towards an important role of HE-4 in human physiology but to date no study has tried to investigate the role or structure of HE-4 in normal or pathological conditions. We have developed a rapid and efficient method for purification of native HE-4 from human seminal fluid, which will help study the function and structure of HE-4. HE-4 is glycosylated and highly stable protein HE-4 exists as a disulfide bonded trimer in human seminal fluid as inferred from SDS-PAGE in reducing and non-reducing conditions. This is not unheard of in proteins and many human and some viral proteins employ intermolecular disulfide linkages to form tertiary structure [33]–[34]. HE-4 has eight predicted disulfide bonds per monomer of protein as it is a small protein, these bonds and intermolecular disulfide linkages would be expected to give it a compact structure resistant to denaturing agents. Accordingly, it found that HE-4 is resistant to pH, heat and even SDS in protease inhibition assay taking trypsin as a model protease. Seminal fluid has a high concentration of zinc and it regulates the function of several seminal fluid proteins like PSA and sememnogelin [35].Therefore, we measured Rh value and activity of HE-4 in the presence of zinc and we observed lower Rh than that of purified native protein in Tris buffer. Introduction of EDTA in twice the molar ratio of zinc increased the Rh value a little bringing it closer to native HE-4. Trypsin inhibition activity of HE-4 reduced slightly in the presence of zinc (mean: 83%) while the addition of EDTA (twice the molar ratio) to the Zn supplemented HE-4 recovered the activity a little (mean: 85.6%) as shown in figure 4D. The activity lost by 2 mM ZnCl2 could be completely rescued by addition of increasing concentrations of EDTA (Figure 4E). At this stage evidence for mechanism of effect of zinc on activity and structure of HE-4 is inconclusive. However, this idea is being pursued further in our laboratory. Disulfide bonds play an important role in maintaining the native conformation of a protein, which in turn provide stability/resistance towards pH and temperature treatments. HE-4 contains 8 disulfide bonds. Therefore, it was of interest to evaluate the effect of DTT reduction on the trypsin inhibitory activity of HE-4. Even at .05 mM there was a significant reduction in trypsin inhibitory activity of HE-4 and at 1 mM HE-4 lost all its activity against trypsin (Figure 4F). Disulfide bond reduced HE-4 was oxidized and refolded (as described in methods) but HE-4 was unable to restore the activity and at 16 hr. time-point only 3% activity was restored. PNGase F treatment of HE-4 produced a shift of approximately 2.5 kDa which is considerable given the molecular weight of the monomer (Figure 2B). This also partly explains the observed heat and pH resistance of the protein. Asn44 has been reported to be glcyosylated in salivary HE-4 previously [36], and no other glycosylation site has been either predicted or reported. This implies that Asn44 which lies in the N-terminal WAP domain is heavily glycosylated. One previous study has reported HE-4 to be a secreted glycosylated protein of approximately 25 kDa in two ovarian carcinoma cell lines [32]. This difference with seminal fluid form of protein could be cell line specific, or it could be another isoform of the protein resulting from alternative splicing [36]. HE-4 can undergo alternative splicing to yield four different isofroms other than full length protein. Some of these isoforms has only N-terminal WAP domain and some C-terminal WAP domain while three of these isoforms own unique sequence not found in others. So this 25 kDa isoform found in ovarian cancer lines could be different from the presumably full length (based on molecular weight from SDS-PAGE) protein we have in seminal fluid, and it might even lack trimerization property, or have a different glycosylation pattern. Importance of glycosylation in functions of HE-4 (seminal fluid or other sites) requires further studies which are underway in the laboratory. HE-4 is a disulfide-bonded trimer and bioinformatic analysis alone does not correctly predict its function A consensus sequence was obtained by aligning HE-4 sequences form different species (Fig. 3C). Though the high degree of similar conservation of residues and spacing between them points towards a similarity in functional aspects of both domains, but we cannot deny the fact that length difference between first and second cysteine residues may contribute to the different functions of these domains or the ability to covalently oligomerise, which requires inter-molecular disulfide bridges. WFDC domain of elafin and one of the two WFDC domains of SLPI is known to impart anti-proteinase activity to both of these proteins. Therefore, it is a widely held notion that all of the members of the family will have anti-proteinase activity. Sequence analysis by Bingle et al showed that only these two members have the same spacing between cysteines essential for protease inhibition [19]. Authors of the paper argue that no other member of the WFDC family contains the same spacing, so it is not necessary that they will have anti-proteinase activity. A separate study by Hu et al show that both wild type and WAP mutated KAL-1 enhanced amidolytic activity of uPA (urokinase-type plasminogen activator) [37] which are consistent with the prediction made by Bingle et al. This study adds a new dimension in this discussion about function of WFDC family members as we have observed antiprotease activity in HE-4. The possible reason for this discrepancy might be this spacing playing some role in inhibitory activity of SLPI and elafin. Both of these proteins are monomers while, we found HE-4 to be a disulfide bonded trimer so this rearrangement of the structure might give HE-4 unusual properties not predicted by sequence analysis. Moreover, we found that disulfide bond reduction abolishes the protease inhibition by HE-4, so we suggest that, in case of HE-4, this protease inhibition does not depend upon any of the two domains (N- and C-terminal WAP domain) alone as whole trimer seems to be necessary for the inhibition. Although one can speculate that HE-4 might be an example of inhibitors where single domains are repeated and linked together (as in ovomucoid) and this new single chain inhibitor can inhibit many different proteases [38]. The possibility of three monomers linked together by disulfide bridges point towards a compact, more rigid structure, and might resemble mechanism of ecotin in which, a homodimer is active, and both monomers provide the protease binding surface [39]. Further studies are needed to understand the mechanism of inhibition of a wide range of proteases by HE-4 which will highlight the residues crucial for inhibition; however, proximity of a variety of residues induced by trimerization might be necessary. Although we do not exclude the possibility that monomer folding pattern of HE-4 as compared to SLPI and elafin is not markedly different, so some common residues might be beneficial for protease inhibition by these proteins. Multiple sequence alignment was performed with both the WAP domains of HE-4 with WAP domains of WFDC family members having known protease inhibition activity (Fig. 3A, B). Some key amino acids which are suggested to be necessary for antiprotease activity of these domains are not conserved in HE-4, however, there is more than 70% conservation overall. More importantly, it is not always feasible to justify function of HE-4 simply on the basis of conservation of critical residues in primary sequence. Factors such as the overall structure of the domain, exposure of some specific amino acids and types of residues lining the active site may be more vital, and contribute to the activity as may be true in this study. HE-4 is cross-class protease inhibitor which is cleaved by papain and induces autolysis of pepsin in vitro HE-4 inhibited a range of serine proteases like trypsin, chymotrypsin, PSA and proteinase K as well as cysteine proteases like papain and aspartyl proteases like pepsin. The physical complex formation of HE-4 with all these proteases was confirmed with blue native electrophoresis where complexes were migrating less compared to HE-4 or proteases alone (Figure 5). Blue native electrophoresis was chosen because some of these protease like trypsin are basic proteins therefore they do not migrate towards anode and are lost. Fig. 4A shows the protease inhibition assay results starting at 5 µg/ml concentration of HE-4 up to 50 µg/ml, and at 50 µg/ml concentration, it inhibits almost completely all the tested proteases. SLPI inhibits trypsin and chyomtrypsin with Ki which is not markedly different for both of these proteases [40] while HE-4 has low affinity towards trypsin and considerably higher affinity towards chymotrypsin (Table 2) as determined by SPR. This highlights different inhibition profile for HE-4 and SLPI. SLPI is thought to neutralize the excess neutrophil protease activity in upper airways. HE-4 is also expressed in sub-mucosal glands of respiratory tissues, but although SLPI and HE-4 both are found in the same tissue, the precise cells expressing both these proteins are mutually exclusive [16]. This suggests them to be under different regulatory control which point towards slightly different functions of both these proteins. HE-4 has a strong affinity towards PSA at pH 5.0 using 75–300 nm PSA (KD = 1.06×10−5 M & KA = 9.40×104 M−1) and suggests that this protein might be involved in regulation of kallikrein activity cascades. Protein C inhibitor (PCI), α-2 macroglobulin (A2M) and α1-anti chymotrypsin (ACT) are three main inhibitors of PSA reported in human seminal fluid. Concentrations of these inhibitors in seminal fluid are low compared to PSA [41]–[43] suggesting there must be other inhibitors, which serve as main inhibitors. HE-4 seems to be one such inhibitor. PSA has been implicated in liquefaction of human seminal fluid upon ejaculation and HE-4 probably regulates the activity of PSA in semen and might protect sperm against excessive PSA activity. HE-4 inhibited proteinase K (75–300 nm) with highest affinity among the proteases tested as apparent by highest KA = 2.15×107 M−1 & KD = 4.65×10−8 M at pH5.0 (Table 2). Proteinase K is a member of subtilisin like protease family, and it has been shown that structures of most members is conserved as a core with insertions and deletion confined to surface loops [44]. This suggests that HE-4 might be a broad spectrum inhibitor of microbial subtilisin like proteases although they need. Many pathogenic fungi utilize subtilisin-like serine proteases as their virulence factors [45]–[48] and HE-4 as a part of the host response might be conferring protection in the sites of its expression one of which is the respiratory tract, others being oral cavity and urogenital tract. We could not determine the kinetics constants for HE-4 with pepsin and papain due to the negative sensogram obtained. On further investigation, we found that papain when incubated with HE-4, presents two more bands in SDS-PAGE below the molecular weight of HE-4. With increasing concentration of HE-4, the HE-4 band remains more or less same in intensity while one of the bands below it (approx 8 kDa) seems to increase in intensity and intensity of papain band also remains the same. This band of cleaved HE-4 was confirmed by western blot (Figure 7C) using HE-4 antibodies. This leads us to propose that papain cleaves HE-4 to generate low molecular weight fragments. The sensogram of papain injection over immobilized HE-4 on CM5 chip shows initial interaction and subsequent dropping of signal below baseline which is in accordance with papain cleavage of HE-4. In protease inhibition assay, HE-4 shows considerable inhibition of papain which leads us to suggest that even after cleavage, the fragment(s) of HE-4 remain(s) bound to papain inhibiting its proteolytic activity. This was confirmed with blue native electrophoresis as papain incubated with HE-4 presented only one band of high-molecular weight (Figure 5). Contrastingly, when pepsin was incubated with HE-4, it seemingly underwent self-cleavage, as evidenced by the appearance of approximately 20 kDa band which increased in intensity with increasing concentration of HE-4 with simultaneous reduction in intensity of full length pepsin band. Pepsinogen undergoes autolysis, and yield active pepsin (approximately 35 kDa) at acidic pH, while we observed a 20 kDa fragment upon incubation with HE-4. Moreover, in both the incubation experiments, we had taken control of incubating the papain and pepsin alone at appropriate pH 8.5 and 5 respectively, and these fragments of HE-4 cleavage by papain and self-cleavage of pepsin did not appear when these proteases were incubated alone. This led us to conclude that HE-4 induces the self cleavage of pepsin and papain cleaves HE-4 and the fragment of HE-4 remains active. In blue native electrophoresis we observed only one band of HE-4 (full length) when pepsin and HE-4 were incubated together (Figure 5). This suggests that pepsin cleavage also does not dissociate HE-4 from pepsin. Cysteine proteases are implicated in a variety of processes in mammalian physiology. This inhibition of papain by HE-4 fragment has greater implications for cell biology. Papain like cysteine proteases has a conserved core structure [49], and highly conserved catalytic site formed by three residues Cys25, His159, and Asn175 (papain numbering) [50]. HE-4 might inhibit other members as well, or they like papain, might be involved in cleavage of HE-4, and producing fragment, which seems to be active, although it remains speculative until further studies. IA3, PI-3 and equistatin, all inhibitors of cysteine proteases like papain with different specificities and targets have been isolated from different animal and microbial sources [51]. Cruzain is a papain like protease of trypanosoma cruzi, causative agent of chagas disease and its inhibitors are being looked into as possible drug development leads [52]. Structure elucidation of HE-4 and its inhibition mechanism of papain despite cleavage might help design potent inhibitors. While, on the other hand, discovery of self cleavage of pepsin when combined with protease inhibition of pepsin by HE-4 in protease inhibition assay point that HE-4 renders pepsin inactive by inducing the self-cleavage. Induced self- cleavage of a protease by a protease inhibitor has not been reported in literature previously to the best of our knowledge. This is an exceedingly curious instance, and further characterization of this phenomenon is underway in the laboratory. To confirm whether HE-4 preparation is contaminated with any endogenous protease co-purifying with HE-4, we assayed HE-4 alone with hemoglobin and BAPNA and compared to other proteases used in this study. No protease activity was found in the purified HE-4 confirming that this self-cleavage of pepsin is due to HE-4. Aspartic proteases are extremely valuable drug targets including the retroviral family and fungal aspartic proteases. Although there are structural differences between retroviral and eukaryotic pepsin-like proteases, there are similarities as well; the cleavage site loops are homologous, the Asp dyad is located in the interface region and N-terminal lobe of pepsin like enzymes are structurally similar to viral subunits [53]–[54]. HE-4 might help design better aspartic protease inhibitors in the future. Finally, we can say this is the first report to establish that HE-4 is a highly stable protein which shows cross-class protease inhibition. A broad spectrum protease inhibition points towards a role in innate immunity conferring protection against microbial virulence factor of proteolytic nature. Seminal fluid HE-4 might be different from HE-4 found in other tissues like ovarian cancer cells because of alternative splicing or different glycosylation. Its ability to trimerize might not be present in all the isoforms. Materials and Methods Sample collection Human semen samples, only normospermic (sperm count >20 million/ml, sperm motility >50%), were collected from Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi, after written informed consent and the approval of the study protocol from ethics sub-committee/ethics committee of AIIMS (permit number T-03/01-04-2009). Semen samples were first subjected to liquefaction at room temperature (RT) for 30 min. Semen sample was centrifuged at 1300 g for 15 min at 4°C to separate sperm from seminal plasma. Later, for further clarification of seminal plasma supernatant was centrifuged at 7000 g for 15 min. at 4°C. Isolation and purification of HE-4 The supernatant, diluted with 50 mM Tris–HCl, pH7.5, containing 150 mM NaCl, was loaded on heparin–sepharose CL-6B (GE-Healthcare, Uppsala, Sweden) column. The unbound fraction was pooled separately and applied on DEAE-Sephacel column. 50 mM Tris–HCl (pH 8.0) was used as equilibration/binding buffer. After extensive washing, DEAE-sephacel bound proteins were eluted with NaCl linear gradient (0–0.5 M) in equilibration buffer. The first peak, obtained at 0.1 M NaCl, was pooled and concentrated up to 20 mg/ml by ultrafiltration (Millipore USA). Final purification of protein was achieved by size exclusion chromatography on sephadex G-75 (Sigma- Aldrich, USA) column, pre-equilibrated with 50 mM Tris–HCl (pH8.0, containing 150 mM NaCl). Fractions eluted at the flow rate of 6 ml/hr were measured at 280 nm, and pooled separately for each peak and concentrated by ultrafiltration using 3 kDa membrane cut-off. 12.5% SDS-PAGE was performed as previously described [55] to analyze approximate molecular weight and purity of the protein. Finally, gel was stained with colloidal coomassie brilliant blue (CBB). Kinetic analysis Antiprotease activity of HE-4 against different proteases and effect of various treatments Inhibitory activity of different serine proteases like trypsin, chymotrypsin, PSA, proteinase K and cysteine proteases like papain and aspertyl proteases like pepsin were measured with the modified method described by Lee and Lin [28]. The sample assay was as the following: 250 µl of HE-4 trimer purified from human seminal plasma was pre-incubated, at 37°C, with the same volume of proteases dissolved in 0.1 M glycine–NaOH buffer (pH 9.5) for 20 min. All the enzymes were purchased from Sigma, except PSA which was purified manually as previously described [56]. Papain and pepsin were dissolved in 50 mM ammonium acetate buffer (pH 6.5) and 50 mM sodium acetate buffer (pH 5.0) respectively. 500 µl of 1% solution of hemoglobin dissolved in the same buffer (Sigma- Aldrich, USA) was added to it, and the mixture was incubated for 40 min. The reaction was stopped by adding 2 ml of 5% trichloroacetic acid. Samples were centrifuged at 15,000 g for 10 min., and the absorbance of the supernatant was measured at 280 nm. The enzyme standard assay was as follows: 250 µl of a sample was replaced by distilled water. The chosen enzyme concentrations gave an increase of absorbance at λ°280 of approximately 0.005OD U/min. With purified protein (HE-4), a control assay to detect the activity of endogenous proteases was also performed: 250 µl of an enzyme solution was replaced by distilled water. The percentage of inhibition was calculated as follow: Additionally, effect of temperature, pH and different chemicals on trypsin inhibition activity of HE-4 was also analyzed. For that we first incubated the 50 µg/ml of protein (HE-4) at different temperature in the range of 25°C–100°C, pH(2–10) and with different chemicals like SDS, β-mercaptoethanol, EDTA, ZnCl2 and ZnCl2+EDTA for 1 hr and the following experiments were performed as described above. In case of SDS HE-4 was incubated with 5% SDS for 2 hours before being added to trypsin and checking the activity as described above. For SDS control, inhibition of trypsin by only 5% SDS without the HE-4 was also checked as shown in fig. 4D as SDS-control. To determine the effect of zinc on HE-4 activity, HE-4 was pre-incubated with 2 mM ZnCl2 for 2 hr. and then activity was checked. Then in the aliquots of the same sample, different concentrations of EDTA were added and activity was checked. The effect of DTT reduction on inhibitory activity of HE-4 was examined after incubation of HE-4 with different concentrations of DTT (0.05–1.0 mM) in 25 mM NH4HCO3 for 15 min at 56°C. The reaction was terminated by adding iodoacetamide at twice the amount of each DTT concentration and the residual inhibitory activity against trypsin was determined as described above. Refolding and oxidation assay was performed as described previously [57] to check whether activity can be restored after reduction. Briefly, HE-4 was incubated at 37°C for 4 hr with 0.5 M phosphate buffer and 10 mM DTT then reaction was stopped by adding iodoacetamide (IAA) so that final concentration of IAA is 20 mM. It was dialyzed against 0.1 M KCl-HCl buffer (pH 2.0) for 3 hr. Then it was followed by dialysis against 0.01 M of the same buffer for 16 hr. at 4°C. The dialysed protein was rapidly diluted 100times with bufferA (100 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA (pH 8.5), 1 mM GSH and 0.5 mM GSSG). The mixture was kept at 25°C for refolding to occur. Aliquots were withdrawn at different time intervals (0, 1, 2, 4, 8, 16 hours) and trypsin inhibitory activity was determined as described above. Blue Native gel electrophoresis Blue Native electrophoresis was performed as described previously [58]. Briefly, all proteases were incubated with equal amount of HE-4 for 2 hours at RT. Then loading Buffer (15% glycerol, 50 mM Bistris/HCl, pH 7.0) was added to complex mixtures. Different proteases and HE-4 alone were also run in separate lane to make the comparison with complex with HE-4 and protease alone. 5–18% acrylamide gradient gel was used for separation of complexes and 50 mM tricine, 15 mM bis-tris/HCl pH 7.0 was used as cathode buffer with coomassie blue G-250 (0.002 or 0.02%). Gel was started with 0.02% coomassie G-250 in cathode buffer and after running for 1 hr it was replaced with same buffer but having 0.002% coomassie G-250 instead. 50 mM bis-tris/HCl pH 7.0 was used as anode buffer and it remained the same for the whole run. Blue native gel was performed at 4–7°C. Electrophoresis was started at 100 Volts until samples were within stacking gel. When sample reached resolving gel 15–17 mA current was applied. Gel was run for total 3–4 hr. Evaluation of binding potential and binding constants using SPR binding studies The protein sensor chip was prepared by immobilization of HE-4 on a research grade CM5 chip (Biosensor AB, Uppsala, Sweden) according to the manufacturer's recommendations. SPR binding studies were performed using a BIAcore 2000TM (Biacore International, AB, and Uppsala, Sweden). 60 µl of HE-4 (30 µg/ml) in 10 mM sodium acetate buffer (pH5.0) was injected to the flow cell for 4 min at the rate of 5 µl/min and washed for 60 min at the rate of 20 µl/min. The analytes at different concentrations were injected for 4 min at a rate of 10 µl/min. The protein samples were diluted in sodium acetate buffer pH5.0. Typically, 90 µl of different dilutions of protein were injected at a flow rate of 30 µl/min. The same buffer was passed over the sensor surface at the end of sample injection, to allow dissociation. After a 3 min dissociation phase, the sensor surface was regenerated by 30 µl of 50 mM NaOH and 1 M NaCl. The response was monitored as a function of time (sensogram) at 25°C. Binding studies were done by plotting the changes in response unit (RU) values with time (Sensograms). Kinetic constants of different proteases were calculated from the association and dissociation phases with BIA evaluation software version 3.0. Similarly converse study was also performed with SPR where all serine proteases were on the chip and HE-4 was flowed. Supporting Information Figure S1 Effect of different reagents and conditions on the oligomerization of HE-4 was measured by DLS. Plot was drawn as a function of concentration of reagents and conditions as a function of X axis with hydrodynamic radii of protein on Y axis. (A) Effect of pH 2–10 empty circle) and different salt: MgCl2, FeCl2, CuCl2, CaCl2, ZnCl2 and ZnCl2+EDTA (filled circle). And (B) Effect of DTT (filled circle) and CHAPS (empty circle) in varying concentration (1%–5%). (C) Effect of SDS (filled circle) and Tween-20(empty circle) in varying concentration (2.5%–10% and 0.005%–0.05%) respectively. (D) Effect of Triton X-100(empty circle) and β-mercaptoethanol (filled circle) in varying concentration (0.1%–1.0% and the 5%–10%) respectively. (TIF) Click here for additional data file. Supplemental Methods S1 (DOC) Click here for additional data file. ==== Refs References 1 Chimpanzee Sequencing and Analysis Consortium (2005 ) Initial sequence of the chimpanzee genome and comparison wth the human genome . Nature 437 : 69 –87 .16136131 2 Hurle B , Swanson W (2007 ) NISC Comparative Sequencing Program (2007 ) Green ED (2007 ) Green Comparative sequence analyses reveal rapid and divergent evolutionary changes of the WFDC locus in the primate lineage . 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PLoS One. 2012 Nov 5; 7(11):e47672
==== Front Front MicrobiolFront MicrobiolFront. Microbio.Frontiers in Microbiology1664-302XFrontiers Media S.A. 2313343910.3389/fmicb.2012.00387MicrobiologyPerspective ArticleIn silico 3D structure analysis accelerates the solution of a real viral structure and antibodies docking mechanism Miki Motohiro 12Katayama Kazuhiko 1*1Department of Virology II, National Institute of Infectious DiseasesTokyo, Japan2Denka-Seiken Co., LtdNiigata, JapanEdited by: Hironori Sato, National Institute of Infectious Diseases, Japan Reviewed by: Masaru Yokoyama, National Institute of Infectious Diseases, Japan; Sam-Yong Park, Yokohama City University, Japan *Correspondence: Kazuhiko Katayama, Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan. e-mail: [email protected] article was submitted to Frontiers in Virology, a specialty of Frontiers in Microbiology. 06 11 2012 2012 3 38714 8 2012 18 10 2012 Copyright © Miki and Katayama.2012 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.Norwalk virus (NoV) is responsible for most outbreaks of non-bacterial gastroenteritis. NoV is genetically diverse and show antigenically variable. Recently, we produced a monoclonal antibody called 5B-18 that reacts broadly with NoV genogroup II (GII). We suspected the 5B-18 binds to a conformational epitope on 3D structure of virion. X-ray crystallography showed us that 5B-18 binds to NoV at the P domain, which protrudes from the capsid surface of the virion. However, there seems to be no space that would allow the IgG to approach the virion. To solve this problem, we used cryo-electron microscopy to examine NoV GII virus-like particles (VLPs). The P domain rises up higher in NoV GII than in NoV GI, and it seems to form an outer layer around the virion. Finally, using in silico modeling we found the 5B-18 Fab arms and NoV P region are quite flexible, so that 5B-18 can bind the NoV virion from bottom of P domain. This study demonstrates the shortcomings of studying biological phenomenon by only one technique. Each method has limitations. Multiple methods and modeling in silico are the keys to solving structural problems. Norwalk virusmonoclonal antibodyx-ray crystallographyin silico modelingcryo-electron microscopy ==== Body THE BASICS OF NORWALK VIRUS Norwalk virus (NoV) is responsible for most of the outbreaks of non-bacterial gastroenteritis in developed countries and, it is thought, in developing countries as well. Yet, although NoV was identified more than 30 years ago, we know little about their pathogenicity and basic virology (Guix et al., 2007). Studies of NoV have been hampered by the lack of a cell-culture system or a small animal model in which the virus will grow, except murine norovirus that is classified as NoV genogroup V (Wobus et al., 2006). NoV belongs to the family Caliciviridae. The genus Norovirus has only one species, Norwalk viruses, with five genogroups (GI–GV). Genogroup GI and II cause most human infections, and they are further subdivided into numerous genotypes (GI.1–8 and GII.1–17; Zheng et al., 2006). The NoV genome is a 7.3 to 7.7-kb positive-sense, polyadenylated, single-stranded RNA molecule. It contains three open reading frames (ORFs): ORF1 encodes a non-structural polyprotein, and ORF2 and ORF3 encode the major and minor capsid proteins, VP1 and VP2, respectively (Jiang et al., 1992; Lambden et al., 1993). Without an in vitro system for propagating the virus, the antigenicity of NoV has been inferred from studies of virus-like particles (VLPs). Nucleic acid-free VLPs self-assemble when the capsid protein is expressed in a baculovirus expression system (Figure 1A). The VLPs are assumed to have a similar morphology and, thus, antigenicity as that of the native virions (Jiang et al., 1992). Cryo-electron microscopy (cryo-EM) and x-ray crystal structures of the prototype norovirus VLP (GI.1, Norwalk virus) showed that the VLPs form a T = 3 icosahedral structure (Prasad et al., 1994, 1999). FIGURE 1 The cryo-EM image of GII.10 VLP and the P dimer structure fit to the cryo-EM image by in silico modeling. The cryo-EM structure of NoV GII.10 VLP was constructed with 180 molecules of VP1 protein (A). The cross-section of GII.10 VLP (B). P domains (blue and green) rise up from shell domain (yellow), and it seemed to construct an outer layer of the capsid. Diagram based on the x-ray crystal structure of the GII.10 P domain-Fab complex shows Fab bound to the lower side of the P1 sub domain. Top side indicates the outside of the VLP, and the shell side contains the VLP core that is called shell. A white arrow indicates the GII.10 P dimer; the monomer is colored blue and purple. Black arrow shows MoAb 5B-18 Fab region. Green represents the heavy chain, and yellow represents the kappa chain. The part of oval shadow represents VLP shell (C). The x-ray crystallography was fit into the cryo-EM structure by in silico modeling (D). The GII.10 P domain (apo P domain structure) into the A/B dimer subunit (light blue and pink, respectively) and C/C dimer subunit (cyan). However, structures of large protein complexes are difficult to determine by x-ray analysis. We sought to understand the structure of the virion and how it interacts with antibodies by combining data from x-ray diffraction, cryo-EM, and in silico modeling. A MONOCLONAL ANTIBODY REACTS BROADLY WITH NoV GII NoV is generally detected by RT-PCR with degenerate primers or an ELISA with NoV-specific antibodies. Many polyclonal and monoclonal antibodies used in the ELISA kits were developed in mice or rabbit immunized with norovirus VLPs (Hansman et al., 2011). Recently, we produced a monoclonal antibody called 5B-18 that reacts broadly with NoV GII (Hansman et al., 2012). In fact, 5B-18 is used as a NoV GII broad-range capture antibody in a commercial ELISA kit [NV-AD(III) SEIKEN NoV antigen ELISA] and in an immunochromatography (IC) kit (Quick naviNoro IC kit, both from Denka-Seiken, Japan). The 5B-18 monoclonal antibody was produced by immunizing a mouse with norovirus VLPs. Several monoclonal antibodies bind to the shell (S) domain (Yoda et al., 2003; Li et al., 2010), and others bind to the protruding (P) domain (Lindesmith et al., 2012). We suspect that 5B-18 also binds to S or P domain on the surface of the NoV virion. However, no high-resolution structural details of the antibody binding to the VLPs, S domain or P domain are available. X-RAY CRYSTALLOGRAPHY OF THE BINDING SITE The 5B-18 binds major NoV genotypes, such as GII.4 and GII.3, and the minor NoV genotypes GII.10 and GII.12 strongly. We suspect 5B-18 binds to a conserved epitope on the NoV capsid surface. We wanted to define the recognition site of 5B-18 and the NoV minor genotype GII.10 P domain, and we began with x-ray crystallography, one of the gold standard for protein structural studies. We expressed the NoV GII.10 P domain in the Escherichia coli strain BL21 (DE3). The P domain was purified and stored in gel filtration buffer. Next we prepared of 5B-18 Fab fragment by immunizing a mouse with NoV GII.4-strain 445 VLPs (GenBank accession number DQ093064; Denka-Seiken, Japan). To prepare crystals of the bound complex, purified GII.10 P domain and Fab were mixed in a 1.4:1 ratio. Crystals were grown by the hanging-drop vapor-diffusion method, mixing the protein and reservoir solution (40% [vol/vol] polyethylene glycol [PEG] 400, 5% [wt/vol] PEG 3350, and 0.1 M acetic acid, pH 5.5) in a 1:1 ratio. Crystals grew over 1 week at 20°C. One GII.10 P domain-Fab complex crystal diffracted x-rays to a resolution 3.3Å, and we solved the structure by molecular replacement with a GII.10 P domain monomer (PDB ID 3ONU) and a mouse Fab (PDB ID 1WEJ) as search models. Molecular replacement indicated an asymmetrical unit contained two P domain monomers and two 5B-18 Fabs, each with a kappa and a heavy chain (Figure 1C; Hansman et al., 2012). The binding of the P domain and the Fab involved nine hydrogen bonds. Of these, eight linked the P1 subdomain to the kappa chain, and one linked the P1 subdomain and the heavy chain. More specifically, the amino acids in the P1 subdomain amino acids that interacted with the 5B-18 Fab were as follows (in each pair, the amino acids are for the P1 domain and Fab, respectively): Tyr533 and Tyr92 (one bond), Thr534 and Gly93 (three bonds), Thr534 and Trp97 (one bond), Leu535 and Tyr32 (one bond), Glu496 and Tyr92 (one bond), and Asn530 and Ser94 (one bond). Finally, Val433 and Asn52 in the heavy chain formed one hydrogen bond (Hansman et al., 2012). CONFIRMATION OF 5B-18 BINDING With the x-ray crystallographic analysis, we found the 5B-18 antibody bound to a hidden site on the P domain that is located inside of the shell of NoV particle. However, in a previous study, the NoV GI structure indicated that bottom of the P domain was completely covered by the shell of NoV particle (Figure 1C). If the structure of GII is the same as GI, then 5B-18 could not bind GII. These results presented an apparent paradox for the 5B-18 binding mechanism. To resolve the paradox, we set out to identify the binding residue in the capsid. From the crystallographic analysis, we knew that the 5B-18 Fab formed hydrogen bonds with residues at three sites in the P1 subdomain, called A, B, and C (Figure 2A). By aligning the amino acid sequences of representatives from NoV GII genotypes, we discovered that Val433 (site A) was the most variable. Other genotypes had threonine, serine, asparagine, leucine, or methionine at this position. Thr534 (site C) was mostly conserved: the only other amino acid at this position was a serine. Glu496 (site B), Asn530 (site C), Tyr533 (site C), and Leu535 (site C) were all highly conserved among the representative GII genotypes. FIGURE 2 Schematic diagram of NoV VP1 protein and the amino acid alignment of NoV GII VP1 sequences. Capsid (VP1) sequences from 11 GII genotypes were aligned, and the GII.10 capsid sequence was used as the consensus. The GII.10 P domain residues that interacted with the 5B-18 Fab involved three sites on the P domain termed A, B, and C. The six GII.10 P domain residues that interacted with the 5B-18 Fab are indicated by light blue rectangles (A). Left panel of (B): a schematic of the VP1 deletion mutants. The construct 1 includes the N-terminal region and shell domain of VP1, and amino acids 4–223 from the N-terminal methionine of VP1. The P1-1 and P2 domain constructs without binding site A are construct 2 (amino acids 224–426), with binding site A is construct 4 (amino acids 224–439). Construct 3 has all binding sites A, B and C (amino acids 427–548). Construct 5 deletes binding site A from construct 3 (amino acids 440–548). Right panel of (B): western blotting results with 5B-18. Samples indicated as VLP, NC (negative control), mutant construct 1 (#1), #2, #3, #4 and #5. The black arrow represents VLP band at 58 kDa, and the white arrows represent #3 and #5 products at 13 kDa. To confirm that 5B-18 binds the A, B, and C regions, we divided the GII.10 capsid domain into three major subdomains: N, S, P1-1 P2, and P1-2. We prepared five constructs (1–5), expressed them in an E. coli expression system, and identified a liner epitope of 5B-18 by western blotting (Figure 2B). Construct 3, a P1-2 region (i.e., A, B, and C), showed the strongest band signal, and construct 5 (i.e., B and C) showed a positive band. The intensity of the band from construct 5 was only about half the strength of construct 3 because it did not contain epitope A. However, construct 4 included only A, and constructs 1 and 2 also were not detected. Thus, the three 5B-18 epitopes A, B, and C were confirmed to be part of the binding epitope. Next, we determined if 5B-18 binds to other NoV GII VLPs (Figure 2A). We prepared and purified six kinds of GII VLPs that were 809 (GII.3), 104 (GII.4), 445 (GII.6), 026 (GII.10), Hiro (GII.12), and GII.13 VLPs as aligned in Figure 2A. The GII VLPs that had all 5B-18 epitopes A, B, and C were captured by the anti-GII VLPs rabbit serum that was pre-coated on ELISA plate and detected with 5B-18 and horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary antibodies. When the cut-off value was under 0.2, 5B-18 detected all kinds of GII VLPs in a dose-dependent manner (data not shown). These results suggested that 5B-18 binds to a variety of GII VLPs. In fact, the commercial ELISA and IC kits use 5B-18 (Denka-Seiken, Japan), and we have practical results showing that 5B-18 detects various infectious NoV GII in stool samples. COMBINING CRYO-EM AND IN SILICO MODELING TO SOLVE A PARADOX We had a simple question. Are the structures of NoV GI VLP and GII VLP the same or not? For G1 VLP, there is no space where the 5B-18 can access and bind the bottom of P domain. If the GII VLP had same conformation as the GI VLP, the lower part of the P domain would be buried under the virion shell (Figure 1C). However, 5B-18 binds and detects GII VLPs and GII infectious viruses. These conflicting facts suggested that the structures of the GII VLPs and infectious GII virions were different than the GI VLP structure. However, structure determinations by x-ray crystallography have many challenges and limitations, and we suspected this might be one of those cases. To answer the question, we turned to cryo-EM and in silico modeling. We reconstructed the overall structure of GII.10 VLPs and 5B-18 Fabs from the x-ray structural data. To determine if the GII VLP had enough space to allow binding, we also used in silico modeling to fit the P and 5B-18 Fabs structures that had been derived by x-ray crystallography. The GII.10 VLPs formed homogeneous, monodisperse particles in ice. By reference-free class averages and at 10Å resolution (0.5 FSC criterion), these icosahedral particles had several notable features, including spike-like structures extending from the vertices (Figure 1B), and at the three- and fivefold axes, significant amounts of the surface of the S domain were exposed (Figure 1A). The GII.10 VLP P domain formed a second outer shell that seems to be separated from the S domain by about 15Å (Figure 1B). Thus, unlike the GI VLPs and virions, GII VLPs and virions seem to have a space between the shell and bottom of P domain, indicating that the two genotypes have different structures. Furthermore, the electron density was much weaker at the tip of the P domain (the P2 subdomain) than at the base. This observation is consistent with published reconstructions of calicivirus particles (Bhella et al., 2008; Bhella and Goodfellow, 2011) and indicates that the P domains have considerable heterogeneity. Next we attempted to fit the GII.10 P domain and P domain-Fab complex structures into the GII.10 VLP cryo-EM structure. At 10Å resolution, the GII.10 P domain monomers in the VLP were easily distinguished. We manually fitted the crystal structures of the GII.10 P domain and P domain-Fab complex into the GII.10 VLP cryo-EM map, using published reports of GV.1 P domain dimers and the GV.1 cryo-EM map (Taube et al., 2010) as guides. We refined the approximate alignment with the Fit-in-Map function in UCSF Chimera (Pettersen et al., 2004) to a cross-correlation coefficient of 0.94 (Figure 1D) with excellent results. The x-ray structure of the GII.10 P domain dimer (PDB ID 3ONU) unambiguously fitted the corresponding density in the cryo-EM map (Figure 1D). Only some loops of the P2 subdomain did not fit. They had only weak electron density, and their tips were less ordered than the S domain and P1 domains in the cryo-EM reconstruction. These subdomains are probably more flexible. P1, but not the P2, subdomains in the VLP appeared to be connected to the P domain dimers. Next we fitted the x-ray structure of the P domain from the P domain-Fab complex into the reconstructed A/B dimer subunit and found that the 5B-18 binding site was close to an adjacent dimer of P domain (Figure 1C). At the twofold axes, the 5B-18 Fab was hindered by the S domain, which also provided an obstacle to assembly of the neighbor P domain dimer. However, when the P domain was fitted into the C/C dimer subunit, the 5B-18 Fab was in contact with the P domain dimer and slightly interfered with part of the S domain at the fivefold axes. Thus, the antibody binding site overlapped with part of the P1 subdomain. Thus, this model predicted an unstable structure in which the VLP could not bind with the 5B-18 antibodies. How could this be? There are several possibilities. First, 5B-18 might bind at sites on the P domain that are only transiently exposed. Second, 5B-18 might bind to defects in the P domain. Finally, the Fab arms of 5B-18 might be very flexible. IgG flexibility is not unknown. For example, a neutralizing antibody 9C12 binds to hexon, the major coat protein of adenovirus, at a ratio of 240 antibody molecules to one virus particle or one antibody per hexon trimer (Varghese et al., 2004). By dynamic light scattering and negative-stain EM, electron-dense material coats the virus, but it was not aggregated at neutralizing ratios. In images reconstructed from cryo-EM, the viral surface was covered by electron density from the 9C12 antibody. Two Fab arms bridge two peripentonal hexons. One has a normal Fab shape and fitted the models well (Harris et al., 1998). The other arm has a somewhat distorted structure. A low-density tail extends to a third hexon that forms a minor alternate binding site. The normal arm binds to a unique site in the asymmetric unit of the virus. It has no alternate binding sites because a penton, rather than a hexon, is positioned at the icosahedral fivefold axis. In addition, the angle between the long axes of the Fabs was <115° that was found in the uncomplexed IgG1 (Harris et al., 1998). Thus, flexibility is important for the bivalent binding of 9C12. ESTIMATING THE FLEXIBILITY IN THE STRUCTURE The findings from the 9C12 study were informative for our 5B-18 paradox. 5B-18 could reach the bottom of the P domain if the Fab domain could bend and escape the P1 subdomain or star-like structure on the shell. 5B-18 IgG bound equally well with intact and partially broken GII.10 VLPs. To determine if 5B-18 binds to intact or broken particles, we took advantage of a characteristic of norovirus VLPs: they are less stable and appear to be broken at high pHs (Ausar et al., 2006). Therefore, we looked at 5B-18 binding at different pHs. At low and neutral pHs (5.3, 6.3, and 7.3), the GII.10 VLPs were mostly homogenous in size and unbroken, but at higher pHs (8.3 and 9.3), they were less homogenous and partially broken. 5B-18 IgG bound to GII.10 VLPs at different pH values with nearly identical efficacies, regardless of the fraction of damaged particles. At pH 5.3, 6.3, and 8.3, the titer was 512,000. At pH 9.3, it was 1,024,000, and at pH 7.3, it was 2,048,000 (optical density cutoff of 0.2; Hansman et al., 2006). We also determined size distribution of the VLPs by dynamic light scattering in each pH conditions. VLPs were shown single peak on diameter 38 to 50 nm (data not shown). These results suggest that 5B-18 appears detects nominally intact GII.10 VLPs. We studied the 5B-18 binding mechanism by x-ray crystallography, molecular virology, and cryo-EM. We combined the results in in silico modeling that simulates molecular dynamics and is a reliable method for revealing fluctuations in protein structure. Each technique complemented the other by filling in for data that was lacking from the others. Interestingly, the 5B-18 study suggests that VLP and viral virion have structure flexibility and that IgG molecule have flexible arms. They co-work each other and bind. In silico modeling is clearly a powerful tool for enhancing our understanding of basic viral processes. 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. We thank P. Kwong and his lab members for their guidance in this work and for critical discussions about structural analysis and K. Nagayama and K. Murata for assistance with the cryo-electron microscopy and for discussions. We also thank H. Sato for giving us this great opportunity to publish our studies. ==== Refs REFERENCES Ausar S. F. Foubert T. R. Hudson M. H. Vedvick T. S. Middaugh C. R. (2006 ). Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature. J. Biol. Chem. 281 19478 –19488 16675449 Bhella D. Gatherer D. Chaudhry Y. Pink R. Goodfellow I. G. (2008 ). Structural insights into calicivirus attachment and uncoating. J. Virol. 82 8051 –8058 18550656 Bhella D. Goodfellow I. G. (2011 ). 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==== Front TrialsTrialsTrials1745-6215BioMed Central 1745-6215-13-1572294362710.1186/1745-6215-13-157Study ProtocolWELL.ME - Wellbeing therapy based on real-time personalized mobile architecture, vs. cognitive therapy, to reduce psychological distress and promote healthy lifestyle in cardiovascular disease patients: study protocol for a randomized controlled trial Compare Angelo [email protected] Vassilis [email protected] Vontas [email protected]ña Wendy Moreno [email protected] Enrico [email protected] Enzo [email protected] Efstathopoulos [email protected] Michele [email protected] Human Factors and Technologies for Health - HTC Centre; Dept. of Human and Social Sciences, University of Bergamo, Piazzale S. Agostino 2, 24129, Bergamo, BG, Italy2 Medical School, National University of Athens, Athens, Greece3 Technopolis ICT Business Park, Thessaloniki, Greece4 SOROS GABINETE, Socia ProgramasEuropeos, Europe, Spain5 Istituto Auxologico Italiano; Catholic University of Milan, Milan, Italy6 Medical Department, Bracco SpA; IULM - University, Fondazione Bracco, Milan, Italy7 NoemaLife Spa, Bologna, Italy2012 3 9 2012 13 157 157 4 3 2012 17 7 2012 Copyright ©2012 Compare et al.; licensee BioMed Central Ltd.2012Compare 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 There is compelling evidence that psychological factors may have the same or even greater impact on the possibility of adverse events on cardiac diseases (CD) than other traditional clinical risk factors. Anxiety and depression are predictors of short- and long-term adverse outcomes, increased risk for higher rates of in-hospital complications, re-infarction, malignant arrhythmias, and mortality in CD patients. Despite researchers finding that cognitive behavior therapy (CBT) reduced depressive and anxiety symptoms, the fact that such results are maintained only in the short term and the lack of maintenance of the long-term affects the absence of changes in lifestyles, preventing the possibility of a wide generalization of results. Recently wellbeing therapy (WBT) has been proposed as a useful approach to improve healthy lifestyle behaviors and reduce psychological distress. Methods/design The present randomized controlled study will test WBT, in comparison with CBT, as far as the reduction of symptoms of depression, anxiety and psychological distress, and the improvement of lifestyle behaviors and quality of life in cardiac patients are concerned. Moreover, innovations in communication technologies allow patients to be constantly followed in real life. Therefore WBT based on personalized mobile technology will allow the testing of its effectiveness in comparison with usual WBT. Discussion The present study is a large outpatient study on the treatment of co-morbid depression, anxiety, and psychological distress in cardiac patients. The most important issues of this study are its randomized design, the focus on promotion of health-related behaviors, and the use of innovative technologies supporting patients’ wellbeing in real life and in a continuous way. First results are expected in 2012. Trial registration ClinicalTrials.gov Identifier: NCT01543815. AnxietyDepressionPsychological distressCognitive behavior therapyWellbeing therapyMobile technologyCardiac disease ==== Body Background Cardiac disease (CD) is the major cause of death worldwide, accounting for approximately 16.7 million deaths each year, mainly from heart attack and stroke. Furthermore, such a figure is most likely to increase approximately to 25 million deaths by 2020, given current trends. Yet fatalities represent only the tip of the iceberg; the greater burden of cardiovascular diseases, affecting an estimated 128 million people, is attributable to non-fatal cardiovascular events and their long-term consequences. Although there are well-accepted national guidelines for both primary and secondary prevention of cardiac illness, little attention has been devoted to the impact of psychological risk factors on cardiovascular disease. Nonetheless, there is wide evidence that psychological risk factors may cause equal or even more adverse events than other traditional clinical risk factors [1-4]. In patients with ischemic heart disease, anxiety and depression are predictors of adverse short- and long-term outcomes [5,6]. Patients suffering from anxiety or depression during hospital admission are at increased risk for higher rates of in-hospital complications such as recurrent ischemia, re-infarction, and malignant arrhythmias [7,8]. They also suffer higher mortality and re-infarction rates months to years after their initial cardiac event [8-11]. Affective disorders including clinical depression and anxiety are common in patients with congestive heart failure. Prevalence findings show that the prevalence rates of (all subtypes) anxiety and depression are 18.4% and 28.6%, respectively [12], and depression is a significant predictor of worse evolution for heart failure (HF) patients [13]. Furthermore, the occurrence of such disorders significantly impacts the quality of life, medical outcomes, and the use of the healthcare service. A number of potential mechanisms have been proposed to justify such an impact, including autonomic nervous system dysfunction, inflammation, cardiac arrhythmias, and altered platelet function. The relationship between emotional distress and the experience of fatigue in patients with HF may have a devastating effect on a patient’s ability to cope with and manage daily activities, including self-care and adherence to recommended treatment. Reports show that anxiety is associated with mental fatigue, whereas depression is associated with the reduction of activity, low motivation, and the assumption of lifestyles which are dysfunctional towards a healthy status. Moreover physical fatigue is affected by symptomatic distress [14]. In everyday practice it is important to consider that a high NYHA classification together with emotional problems may contribute to anxiety or depression, while social support and active relationships may positively influence the psychological health of patients with heart failure [15]. In particular, baseline distress assessed in primary care (odds ratios (OR) 5.51; 95% confidence intervals (CI) = 2.56 to 11.62) appears to be an independent predictor of distress at 9-monthfollow-up. Recent findings show that anxiety and low social support were independently associated with HF-related re-admission, which indicates the need for their inclusion in the assessment and management of HF [16]. Moreover, data show that depression ((HR = 1.81) and social isolation (HR = 2.25) may be mortality predictors independently of demographics, clinical predictors, and treatment [17]. Failure to understand and address psychological risk factors for coronary heart disease(CHD) events may be one reason for which CHD morbidity and mortality remain so high. Anxiety disorders and depression are among the most prevalent psychiatric disorders [18]. Given the prevalence of anxiety and depression in the general population and in patients with CHD, the potential public health impact for preventing the development and progression of CHD by properly appreciating the nature of the link between anxiety or depression and CHD is enormous [19,20]. Thus, in CVD patients it is clinically relevant to assess the patient’s psychological profile and treat his/her emotional conditions causing an increased risk for major adverse cardiovascular events. Psychological interventions for anxiety and depression Major behavioral and drug trials conducted in the last 15 years have focused on the best treatment of depression in cardiac patients [21]. Cognitive behavioral therapy (CBT) is often successfully used in the treatment of depressive disorders. Although the adoption of CBT obtained good results in reducing depression, no beneficial effects for cardiac events in 2 years was reached inpatients receiving the intervention (24.2%) when compared with a control group undergoing a usual treatment (24.1%). Because dysfunctional cognition plays a relatively minor role in atypical depression, treatments other than cognitive-behavioral interventions may be more beneficial to CHD patients with atypical depression or exhaustion [22]. Moreover, CBT is a well-documented, evidence-based treatment appropriate for the treatment of anxiety, which should be started at the beginning of cardiac treatment to ensure that patients fully understand their condition [23]. Despite researchers finding that CBT reduces depressive and anxiety symptoms, the limited size of studies prevents a wide generalization of the results [24]. Recently wellbeing therapy (WBT) has been proposed as a useful approach to improve healthy lifestyle behaviors and reduce psychological distress. WBT aims at enhancing psychological wellbeing on the basis of Ryff’s [25] six dimensions: autonomy, personal growth, environmental mastery, purpose in life, positive relations, and self-acceptance. Previous studies documented the efficacy of this psychotherapy in treating patients with mood and anxiety disorders, and in preventing relapse in recurrent depression [26-28]. Moreover, recent results showed that a sequential combination of CBT and WBT yield significant and persistent benefits in cyclothymic disorder [29]. Methods/Design Hypotheses Starting from current literature, we hypothesize that WBT and WBT version based on a personalized mobile technology approach will allow for a reduction of symptoms of depression, anxiety, and psychological distress in cardiac patients. Moreover and therefore, we assume an improvement in lifestyle behaviors (for example, quitting smoke, increased physical activity, better medication adherence) and quality of life when compared to CBT and usual care (UC). Setting The study will be conducted in collaboration with the National Kapodistrian University of Athens, Greece, and Consorci Sanitari de Terrassa (CST), Spain, during the 3-year EU collaboration project FOR ALL (‘Universal Service for Managing and Monitoring Cardiac and Psychological Health of European Cardiac Patients’, Project No. C-029399). Subjects Patients with the following eligibility criteria have been included in the present study: cardiac disease; a current diagnosis of at least one of the following: major or minor depression, dysthymia, anxiety according to DSM-IV criteria and HADS criteria, Mini-Mental State Examination score higher than 24;written informed consent was requested from the patient. Exclusion criteria included: uncertain prognosis for 12 months due to other conditions; acute coronary disease in the last 2 months; existence of another life-threatening illness of the patient (such as active cancer, chronic kidney failure); severe neurological problem (brain syndrome/orientation problem/difficult peripheral neuropathy); severe mental illness (active psychosis/suicide risk/severe dementia); linguistic limitations (such as stuttering/untreated audio impairment); a significant functional problem (such as unconsciousness/connection to respiration device/confinement to a wheelchair or bed/severe walking disability/need of help with basic daily activities); objective limit endangering liability for participation in the seven meetings (such as remote living location/convict/drug addiction). All eligible patients will be approached by trained clinical psychologists during their hospitalization period and will be screened for depression, anxiety, and psychological distress (Screening phase). Patients with elevated scores on such scales will be invited for a baseline interview to further identify possible exclusion criteria. After this interview, patients satisfying inclusion criteria are further informed about the design of the study and asked to give their informed consent (Evaluation phase). Informed consent will be sought for:(a) using anonymous data from the questionnaires for reports and scientific publications; and (b) for informing the general practitioner (GP) about the study results. Patients will be eventually included in the study and randomized (Randomization phase) to WBT, or WBT-personalized mobile technology platform (MobWBT), either CBT or the care as UC. The phases of the recruitment process are described in Figure 1. Figure 1 Schematic outline of flow chart of WELL.ME study. During the Screening phase, patients with high anxiety, depression, and psychological distress will be detected. At Evaluation, inclusion and exclusion criteria will be assessed. After this, the first randomization in three arms (CBT, WBT, and CU)will be done. After the 7 weeks of treatment, the second randomization will be done in WBT arms, obtaining two sub-arms: WBT and MobWBT. Randomization Randomization will be performed by statistical experts of the team study using a computerized random number generator at http:// http://www.randomization.com. Randomization will occur after the baseline measurements and, only in WBT group, after the first randomization (Figure 1). To obtain equal numbers in both conditions, a block randomization design is chosen. After the completion of the inclusion procedure, the clinical psychologist will obtain the file containing the condition to which the patient is allocated. Design The comparison among WBT, CBT, and CU will be assessed in a three-arm randomized controlled clinical trial. After this first randomization, patients in WBT group will be randomized across WBT and MobWBT. Intervention Wellbeing therapy (WBT) and WBT based on mobile technology tool (MobWBT) WBT is based on Ryff’s cognitive model of psychological wellbeing [25]. This model was selected on the basis of its easy applicability to clinical populations [28]. WBT is divided into three phases (Table 1). Table 1 Wellbeing therapy protocol for conventional format (outpatients based tools) and for real-time personalized mobile technologies (Mobile Technology Tool) Phases Focus Objectives Outpatient-based tool Mobile technology tool Initial phase Identifying and setting episodes of wellbeing into situational context Report the circumstances surrounding the episodes of wellbeing rated on a scale of 0 to 100, with zero being absence of wellbeing and 100 being the most intense wellbeing Diary Mobile diary, personalized by the baseline assessment results and by an adaptive learning algorithm depending on patient’s answers during mobile monitoring         Visual analogical scale for wellbeing         Assessment     Monitor the quality of experience associated with daily situations (work, leisure, and so on)         Identification of instances of wellbeing and of optimal experiences     Intermediatephase Remove the obstacles to sustained psychological wellbeing on self-monitoring of moments and feelings of wellbeing and graded task assignments Identify thoughts and beliefs Graded task assignments of undertaking particular pleasurable activities for a certain time each day Electronic personalized task assignment by the baseline assessment results and by an adaptive learning algorithm depending on patient’s answers during mobile monitoring     Leading to premature interruption of wellbeing             Visual analogical scale for wellbeing         Assessment     Identify the areas of psychological wellbeing which are unaffected by irrational or automatic thoughts and which are saturated with them         Reinforce and encourage activities that are likely to elicit wellbeing and optimal experiences     Final phase Be able to readily identify moments of wellbeing Dimensions of psychological wellbeing are progressively introduced Graded task assignments of undertaking particular pleasurable activities for a certain time each day Mobile diary, personalized by the baseline assessment results and by an adaptive learning algorithm depending on patient’s answers during mobile monitoring   Be aware of interruptions to wellbeing feelings (cognitions)           Guide the patient from an impaired level to an optimal level according to the above six dimensions   Electronic personalized task assignment by the baseline assessment results and by an adaptive learning algorithm depending on patient’s answers during mobile monitoring   Follow optimal experiences         Meeting the challenge that may entail optimal experiences is emphasized               Visual analogical scale for wellbeing         Assessment After the first 7 weeks, patients in the WBT arm will be randomized into two arms: WBT and MobWBT. Patients in the WBT group will not receive any treatments, while patients in MobWBT will be followed by a WBT-Mobile technology tool for 8 weeks (Table 1). Figure 2 shows the MobWBT technology platform. The MobWBT technology platform is based on an elaboration of datasets collected from psychological monitoring in real time and used to train a machine learning algorithm to recognize psychological wellbeing conditions. The algorithm is downloaded onto the smartphone and the training and testing process will be repeated to improve its predictive ability. The final aim of MobWBT technology platform is to identify in realtime the psychological wellbeing condition and deliver personalized WBT techniques precisely at the moment of greatest need. Figure 2 Wellbeing therapy based on real-time personalized mobile technologies. Cognitive behavioral therapy (CBT) CBT techniques for anxiety and depression will be performed [30] using a manual protocol. CBT-Depression is based on cognitive restructuring and behavioral techniques. Such techniques towards curing depression focus on the identification of specific problems linked to behavior and thinking moods. The therapist uses structured learning techniquesto teach patients how to monitor and write down their negative thoughts and mental images. The goal is to recognize how those ideas affect the patients’ mood, behavior, and physical condition. Therapists also teach important coping skills, such as problem solving and planning pleasurable experiences. CBT-Anxiety addresses negative patterns and distortions in the way we look at the world and at ourselves. The goal of cognitive behavioral therapy for anxiety is to identify and correct these negative thoughts and beliefs. The process involves three steps: Identifying negative thoughts, challenging negative thoughts, replacing negative thoughts with realistic thoughts. Usual care (UC) In UC no extra intervention is provided. Patients receive care as defined in the usual management program (six annual visits with primary care nurse and cardiologist to monitor disease progression). Patients will be screened througha set of questionnaires to assess depression and anxiety every 2 months to record their psychological symptoms. If scores on these questionnaires indicate a moderate to severe level, a notification is sent to theirGP. However, when necessary patients may always consult their GP and receive treatment for depression/anxiety by the GP, or (after a referral) by a mental health specialist. Treatment procedures Both WBT and CBT will be conducted for eight 45 minutes sessions. The first two sessions will be conducted in the same week. The remaining six sessions will be conducted over a period of 6 weeks (one session per week). The total therapy duration for each patient will be 7 weeks. Psychotherapy will be provided by clinical psychologists and psychiatrists trained on specific well-assessed treatment protocol. The Medical Ethics Committee of National Kapodistrian University of Athens and the Consorci Sanitari de Terrassa (CST) approved the study protocol and informed consent. Psychotherapy will be provided individually twice a week. Measurements Besides the psychological screening baseline at month 1 (T0), all patients will be assessed at months 6 (T1), 12 (T2) and 24 (T3) for follow-up. Table 2 shows an overview of variables measured at each time point. Moreover, at the baseline assessment, questions on demographic variables (for example, age, marital status, work, educational level, socioeconomic status), psychiatric history (for example, previous diagnosis of depression and/or anxiety, family history regarding psychiatric diagnosis), and/or health behaviors (for example, alcohol use, smoking habits, physical activity) will be included. Clinical variables and diagnosis of other chronic diseases will be obtained from medical records. Table 2 Variables measured at each time point   PHQ-9 GAD-7 DCPR PSI SF-12 PGWBI LHFQ T0 X X X X X X X T1 X X X X       T2 X X X X X X X T3 X X X X X X X Primary outcome measures Healthy lifestyle Diet, physical activity, weight, blood lipids, glucose, and insulin were measured at baseline and at months 6, 12, and 24. Depression and anxiety The Patient Health Questionnaire-9 (PHQ-9) is a short self-report questionnaire based on the nine symptoms of major depression, as defined in the Diagnostic and Statistical Manual (DSM-IV). The scale has a good overall accuracy, sensitivity, and specificity in a general primary care population [31]. A cutoff of 7can be used to indicate possible depression [32]. The Generalized Anxiety Disorder-7 scale (GAD-7)has shown good reliability and validity to detect generalized anxiety disorder as well as other anxiety disorders in primary care patients [33]. A score >7can be used to indicate a possible anxiety disorder. The Diagnostic Criteria for Psychosomatic Research (DCPR) [34] represents a diagnostic and conceptual framework that aims at translating psychosocial variables derived from psychosomatic research into operational tools. A set of 12syndromes was developed: disease phobia, thanatophobia, health anxiety, illness denial, persistent somatization, functional somatic symptoms after a psychiatric disorder, conversion symptoms, anniversary reaction, irritable mood, type A behavior, demoralization, and alexithymia. The Psychosocial Index (PSI) [35] is a simple self-rated instrument including 55 items for assessing stress, psychological distress, abnormal illness behavior, and wellbeing. Psychological Wellbeing Scale (PWB) [25] is used in the original 20-item per scale version devised to evaluate six dimensions of well-being: (1) autonomy; (2) environmental mastery; (3) personal growth; (4) positive relationships with others; (5) purpose in life; and (6) self-acceptance. Quality of life/Health status Health Survey (SF-12) is a health status questionnaire composed by two components (mental and physical) measuring functional status, wellbeing, and general health; higher scores indicate a better health status [36]. The scale was developed to be used in a variety of chronic diseases [37]. The Psychological General Wellbeing Index (PGWBI)is an index to measure the level of subjective psychological wellbeing that has been validated by decades of clinical practice. PGWBI has been developed as a tool to measure self-representations of intra-personal affective or emotional states reflecting a sense of subjective wellbeing or distress, thus capturing what we could call a subjective perception of wellbeing [38,39]. The original PGWBI consists of 22 self-administered items, rated on a six-point scale, which assess psychological and general wellbeing of respondents in six health-related quality of life (HRQoL) domains: anxiety; depressed mood; positive wellbeing; self-control; general health; and vitality. Each item may score 0 to 5, referring to the last 4weeks of the subject’s lifetime. Each domain is defined by a minimum of three to a maximum of five items. The scores for all domains can be summarized into a global summary score, which reaches a theoretical maximum of 110 points, representing the best achievable level of wellbeing [38], a sort of ‘state of bliss’. A number of studies are cross-sectional and longitudinal psychometric validation and correlation with a large number of other indexes of medical and mental health, through different contexts (communities, institutions, hospitals) [40]. The average PGWB total score of results from studies of the population is between 80 and 81 points. Minnesota Living With Heart Failure Questionnaire (LHFQ) [41] measures patients’ perception of the effects of HF in their lives. It is a questionnaire initially prepared to be self-administered, formed by 21 items that contemplate the physical, socioeconomic, and psychological limitations frequently reported by patients connected with their HF. Patients’ self- assessment is quantified by the sum of answers of the 21 items. The scale of answers for each question ranges from 0(‘no’) to 5(‘too many’), where0represents ‘no limitations’ and 5 represents ‘maximum limitation’. Higher scores indicate worse HRQOL. Ethical principles The study of this protocol does not involve the administration of drugs. However, the investigator is responsible for ensuring that the study is conducted in accordance with the principles defined by the 18th World Medical Assembly (Helsinki, 1964) and subsequent amendments established by the 29th (Tokyo, 1975), 35th (Venice, 1983), 41st (Hong Kong, 1989), and the 48th World Medical Assembly (Somerset West, South Africa, 1996), and 52nd (Edinburgh, Scotland, 2000) General Assembly. The study will also be conducted in accordance to the Ministerial Decree of 15 July 1997 transposing the text of the rules of Good Clinical Practice for human trials of medical products within the EEC. Planned statistical analyses The demographic and clinical characteristics of the two study groups will be compared at baseline to verify their homogeneity. To do this analysis of variance (ANOVA) for continuous variables and chi-square test of Mantel-Haenszel test for discrete variables will be used. Completer and intent-to-treat analysis will be accomplished to compare treatments both in primary and secondary outcomes. First prevalence of depression, anxiety, and psychological distress will be displayed in frequency tables and both conditions (control and intervention) will be compared using the chi-square (Fisher’s exact test when appropriate) and Student’s t-test, for discrete and continues variables respectively. The effect of the intervention will be determined using multilevel analyses (mixed effect regression models) to compare baseline and follow-up measures of all continuous data. In all these analyses a P value of <0.05 will be considered statistically significant. Sample size calculation In this study the change in scores inPHQ-9 and GAD-7 are chosen as primary outcome measures to determine the effect of a disease management approach. A difference of 0.5 standard deviations is considered necessary to find a clinically significant effect of the intervention. In order to detect this difference and assuming a 80% power, a minimum of 80 patients is needed in each condition [42]. When assuming that 20% of participants dropout, quite a normal figure in this type of research, a minimum of 80 patients per condition is needed to maintain sufficient power. We expect that in order to reach such a number, anticipating a response rate of 70% of which 20% is eligible, a total of 600 patients will have to be screened. Discussion This article describes the background, objectives, and design of a large randomized controlled trial that will test the effectiveness of WBT to treat depression, anxiety, psychological distress, and to improve healthy lifestyle in cardiac disease patients in comparison with CBT and CU. Moreover, the research protocol will test the effectiveness of MobWBT in comparison with WBT. Previous research on cardiac patients has already shown that depression and anxiety are common co-morbidities in these patient populations, and are negatively associated with healthy lifestyle and health status, and positively with morbidity, mortality, and healthcare costs [8-11,17,18]. However, randomized studies on the effects of treating depression, anxiety, and psychological distress are very limited, thus preventing the wide generalization of results [24]. Recently WBT has been proposed as a useful approach to improve healthy lifestyle behaviors and reduce psychological distress. Therefore, this study was developed to test the effectiveness of a WBT approach for co-morbid depression and anxiety in these patient populations. Moreover, innovations in communication technologies allow patients to be followed continuously in real life. Therefore, through WBT based on personalized mobile technology it will be possible to test the effectiveness of these technologies in comparison with usual WBT. Conclusions In conclusion, both depression and anxiety are common diseases in cardiac patients that may provoke a considerable worsening of the cardiac pathology condition. However, research of possible treatment strategies to alleviate this additional burden is limited. Therefore, the WELL.ME study tests the use of WBT to treat co-morbid depression and anxiety in patients with cardiac disease. Trial status The WELL.ME study trial was conceived and designed in 2007. At the time this manuscript was submitted full approval by the Medical Ethics Committee had been obtained. Abbreviations CBT: Cognitive behavioral therapy; CD: Cardiac disease; CHD: Coronary heart disease; CI: Confidence interval; CSM-IV: Diagnostic and Statistical Manual, 4th edition; DCPR: Diagnostic Criteria for Psychosomatic Research; GAD-7: Generalized Anxiety Disorder 7; GP: General practitioner; HF: Heart failure; HRQoL: Health-related quality of life; M.I.N.I.: Mini International Neuropsychiatric Interview; MobWBT: WBT version based on personalized mobile technology; NYHA: New York Heart association; PGWBI: Psychological General Wellbeing Index; LHFQ: Minnesota Living with Heart Failure Questionnaire; OR: Odds ratio; PHQ-9: Patient Health Questionnaire 9; PSI: Psychosocial Index; PWB: Psychological Wellbeing Scale; SPSS: Statistical Package for Social Sciences; UC: Usual care; WBT: Well-being therapy. Competing interests The authors declare that they have no competing interests. Authors’ contributions AC and VK in collaboration with VA and MC designed the study. All authors have been involved in writing this manuscript and have approved the final manuscript and its submission. Funding EU - eTen Program Grant 2007. ==== Refs Kubzansky LD Kawachi I Going to the heart of the matter: do negative emotions cause coronary heart disease? 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==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23145157PONE-D-12-1770710.1371/journal.pone.0049347Research ArticleBiologyBiochemistryMetabolismLipid MetabolismMedicineClinical Research DesignCross-Sectional StudiesEpidemiologyCardiovascular Disease EpidemiologyEpidemiological MethodsNon-Clinical MedicineHealth Care PolicyHealth InformaticsRelationship between Resting Pulse Rate and Lipid Metabolic Dysfunctions in Chinese Adults Living in Rural Areas Resting Pulse Rate and Lipid Metabolic DysfunctionWang Chong-jian 1 2 * Li Yu-qian 3 Li Lin-lin 1 Wang Ling 1 Zhao Jing-zhi 4 You Ai-guo 5 Guo Yi-rui 1 Li Wen-jie 1 1 Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, Henan, PR China 2 Research Centre, CHU Sainte-Justine, Montreal, Quebec, Canada 3 Department of Clinical Pharmacology, School of Pharmaceutical Science, Zhengzhou University, Zhengzhou, Henan, PR China 4 Department of Endocrinology, Military Hospital of Henan Province, Zhengzhou, Henan, PR China 5 Department of Disease Control and Prevention, Henan Provincial Center for Disease Control and Prevention, Zhengzhou, Henan, PR China Makishima Makoto Editor Nihon University School of Medicine, Japan * E-mail: [email protected] Interests: The authors have declared that no conflict of interest exists. Conceived and designed the experiments: CJW. Performed the experiments: YQL LLL LW WJL. Analyzed the data: JZZ AGY YRG. Contributed reagents/materials/analysis tools: YQL LLL JZZ AGY YRG. Wrote the paper: CJW LW. 2012 7 11 2012 7 11 e4934719 6 2012 10 10 2012 © 2012 Wang et al2012Wang 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 Resting pulse rate has been observed to be associated with cardiovascular diseases. However, its association with lipid metabolic dysfunctions remains unclear, especially resting pulse rate as an indicator for identifying the risk of lipid metabolic dysfunctions. The purpose of this study was to examine the association between resting pulse rate and lipid metabolic dysfunctions, and then evaluate the feasibility of resting pulse rate as an indicator for screening the risk of lipid metabolic dysfunctions. Methods A cross-sectional survey was performed, and 16,926 subjects were included in this study from rural community residents aged 35–78 years. Resting pulse rate and relevant covariates were collected from a standard questionnaire. The fasting blood samples were collected and measured for lipid profile. Predictive performance was analyzed by receiver operating characteristic (ROC) curve. Results A significant correlation was observed between resting pulse rate and TC (r = 0.102, P = 0.001), TG (r = 0.182, P = 0.001), and dyslipidemia (r = 0.037, P = 0.008). In the multivariate models, the adjusted odds ratios for hypercholesterolemia (from 1.07 to 1.15), hypertriglyceridemia (1.11 to 1.16), low HDL hypercholesterolemia (1.03 to 1.06), high LDL hypercholesterolemia (0.92 to 1.14), and dyslipidemia (1.04 to 1.07) were positively increased across quartiles of resting pulse rate (P for trend <0.05). The ROC curve indicated that resting pulse rate had low sensitivity (78.95%, 74.18%, 51.54%, 44.39%, and 54.22%), specificity (55.88%, 59.46%, 57.27%, 65.02%, and 60.56%), and the area under ROC curve (0.70, 0.69, 0.54, 0.56, and 0.58) for identifying the risk of hypercholesterolemia, hypertriglyceridemia, low HDL hypercholesterolemia, high LDL hypercholesterolemia, and dyslipidemia, respectively. Conclusion Fast resting pulse rate was associated with a moderate increased risk of lipid metabolic dysfunctions in rural adults. However, resting pulse rate as an indicator has limited potential for screening the risk of lipid metabolic dysfunctions. This research was supported by the National Key Basic Research Program of China (Grant NO: 2012CB526709), National Natural Science Foundation of China (Grant NO: U1204823), China Postdoctoral Science Foundation (Grant NO: 20100471003 & 201104401), and Medical Scientific Research Foundation of Health Department of Henan Province (Grant NO: 201004042 & 201204051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Previous studies have shown that resting pulse rate is associated with the development of cardiovascular diseases, and elevated resting pulse rate is an important indicator of increased morbidity and mortality among people with hypertension, coronary artery disease, and diabetes mellitus [1]–[5]. A recent epidemiological study has shown that resting pulse rate is associated with an increased risk of metabolic syndrome [6]. However, relatively few studies have evaluated the correlation between resting pulse rate and the risk of lipid metabolic dysfunctions, particularly in rural adults. In addition, no study has explored whether resting pulse rate could be used as an indicator to identify the risk of lipid metabolic dysfunctions in rural adults. Lipid metabolic dysfunction is a widely recognized risk factor for cardiovascular diseases, which is the leading cause of death in both developed and developing countries [7], [8]. The World Health Organization (WHO) estimates that lipid metabolic dysfunctions are associated with more than half of global cases of ischemic heart disease and more than four million deaths per year [9]. Emerging evidence demonstrates that lipid metabolic dysfunctions can be prevented and controlled, which will be helpful to reduce morbidity and mortality of cardiovascular diseases and other relevant diseases [10], [11]. Therefore, identifying relevant factors that may predict the risk of lipid metabolic dysfunction is an important approach toward improved understanding and prevention of this disease, especially within high-risk population [12]. We examined the correlation between resting pulse rate and the risk of lipid metabolic dysfunctions in a large-scale epidemiological survey of rural Chinese adults. We also evaluated the feasibility of resting pulse rate as an indicator for screening the risk of lipid metabolic dysfunctions. 10.1371/journal.pone.0049347.t001Table 1 Characteristics of study population stratified by resting pulse rate (n = 16,926). Variables Resting pulse rate, beats/min P value ≤67 (n = 4,341) 68–74 (n = 4,517) 75–81 (n = 3,946) >81 (n = 4,122) Age (years), mean (±sd) 52.87 (10.80) 52.78 (10.83) 53.28 (10.86) 54.70 (11.23) 0.001 Female, n (%) 2,033 (46.83) 2,771 (61.35) 2,619 (66.37) 2,895 (70.23) 0.001 Education, n (%) 0.001 No education 680 (15.66) 823 (18.22) 688 (17.44) 815 (19.77) Primary school 1,471 (33.89) 1,556 (34.45) 1,342 (34.01) 1,483 (35.98) Middle school 1,748 (40.27) 1,696 (39.36) 1,553 (36.36) 1,498 (36.34) High school or more 442 (10.18) 442 (9.79) 363 (9.20) 326 (7.91) Marital status, n (%) 0.020 Married/cohabitation 3,940 (90.83) 4,113 (91.12) 3,621 (91.79) 3,699 (89.78) Divorced/widowed/unmarried 401 (9.17) 404 (8.88) 325 (8.21) 423 (10.22) Physical activity, n (%) 0.001 Low 1,185 (27.30) 1,376 (30.46) 1,253 (31.75) 1,447 (35.10) Moderate 874 (20.13) 1,002 (22.18) 858 (21.74) 894 (21.69) High 2,282 (52.57) 21.39 (47.35) 1,835 (46.50) 1,781 (43.21) Mean individual income (annual), n (%) 0.057 <1000 CNY 1,763 (40.61) 1,730 (38.30) 1,496 (37.91) 1,637 (39.71) 1000 ∼ CNY 1,347 (31.03) 1,429 (31.64) 1,252 (31.73) 1,331 (32.29) ≥2000 CNY 1,231 (28.36) 1,353 (30.06) 1,198 (30.36) 1,154 (28.00) Current smoking, n (%) 1,204 (27.74) 955 (21.14) 733 (18.58) 674 (16.35) 0.001 Current drinking, n (%) 584 (13.45) 482 (10.67) 368 (9.33) 345 (8.37) 0.001 More high-fat diet, n (%) 214 (4.93) 252 (5.58) 225 (5.70) 238 (5.77) 0.298 More vegetable and fruit intake, n (%) 2,235 (51.81) 2,264 (50.12) 1,964 (49.77) 2,030 (49.30) 0.101 Family history of hypercholesterolemia, n (%) 422 (9.72) 468 (10.36) 416 (10.54) 461 (11.18) 0.104 Waist circumference (cm), mean (±sd) 82.45 (9.70) 83.26 (10.06) 83.57 (10.42) 84.98 (10.82) 0.001 Pulse pressure (mmHg), mean (±sd) 46.30 (13.84) 46.88 (12.95) 48.28 (12.77) 51.30 (13.30) 0.001 Glucose (mmol/L), mean (±sd) 5.46 (1.08) 5.57 (1.22) 5.78 (1.65) 6.06 (2.02) 0.001 TC (mmol/L), mean (±sd) 4.46 (0.90) 4.58 (0.93) 4.57 (0.92) 4.62 (0.99) 0.001 TG (mmol/L), mean (±sd) 1.61 (1.09) 1.68 (1.13) 1.76 (1.19) 1.82 (1.25) 0.001 HDL-C (mmol/L), mean (±sd) 1.18 (0.26) 1.17 (0.26) 1.16 (0.26) 1.15 (0.27) 0.001 LDL-C (mmol/L), mean (±sd) 2.57 (0.75) 2.64 (0.77) 2.60 (0.76) 2.60 (0.81) 0.001 Hypercholesterolemia, n (%) 157 (3.62) 216 (4.78) 185 (4.69) 265 (6.43) 0.001 Hypertriglyceridemia, n (%) 707 (16.29) 817 (18.09) 823 (20.86) 941 (22.83) 0.001 Low HDL hypercholesterolemia, n (%) 1,356 (31.43) 1,472 (32.59) 1,316 (33.35) 1,416 (34.35) 0.033 High LDL hypercholesterolemia, n (%) 68 (1.57) 80 (1.77) 55 (1.39) 90 (2.18) 0.040 Dyslipidemia, n (%) 1,798 (41.42) 1,972 (43.66) 1,805 (45.74) 1,905 (46.22) 0.001 Abbreviations: sd, standard deviation; CNY: China Yuan; TC  =  total cholesterol; TG  =  triglyceride s; HDL  =  high density lipoprotein; LDL  =  low density lipoprotein. Methods Subjects A population-based cross-sectional survey was performed, and subjects were selected randomly from eligible candidates listed in the residential registration record from rural district in Henan Province. The eligibility of the candidate was defined as those who were stable residents for at least 10 years in the areas aged 35–78 years, and were free from the following conditions: 1) severe psychological disorders, physical disabilities, cancer, chronic kidney disease, Alzheimer’s disease, or dementia, within 6 months; or 2) currently diagnosed with tuberculosis, acquired immune deficiency syndrome (AIDS), and other infectious diseases. After cancer (n = 48), chronic kidney disease (n = 152), physical disabilities (n = 8), tuberculosis (n = 12), and other infectious diseases (n = 9) were excluded, 17,042 subjects who met the criteria were enrolled in the study. Of the eligible participants, 116 (0.68%) subjects were excluded because of missing information on resting pulse rate (n = 69), lipid profile (n = 47). Ultimately, 16,926 subjects were selected for the present analysis. The procedure of the study was approved by the Zhengzhou University Medical Ethics Committee, and written informed consent was obtained from all participants. 10.1371/journal.pone.0049347.t002Table 2 Odds ratios and 95% confidence intervals of lipid metabolic dysfunctions according to quartiles of resting pulse rate (n = 16,926). Variables Resting pulse rate, beats/min P for trend ≤67 (n = 4,341) 68–74 (n = 4,517) 75–81 (n = 3,946) >81 (n = 4,122) Hypercholesterolemia Crude OR(95%CI) 1.00 1.06(0.93–1.20) 1.10(1.03–1.18) 1.14(1.05–1.22) 0.001 Adjusted OR(95%CI) * 1.00 1.07(0.93–1.22) 1.12(1.04–1.20) 1.15(1.07–1.23) 0.001 Hypertriglyceridemia Crude OR(95%CI) 1.00 1.09(1.02–1.17) 1.14(1.10–1.18) 1.15(1.11–1.20) 0.001 Adjusted OR(95%CI) * 1.00 1.11(1.04–1.18) 1.14(1.09–1.17) 1.16(1.12–1.20) 0.001 Low HDL hypercholesterolemia Crude OR(95%CI) 1.00 1.02(0.99–1.05) 1.03(1.01–1.05) 1.05(1.02–1.08) 0.006 Adjusted OR(95%CI) * 1.00 1.03(1.01–1.07) 1.05(1.02–1.07) 1.06(1.03–1.10) 0.005 High LDL hypercholesterolemia Crude OR(95%CI) 1.00 0.92(0.77–1.11) 1.10(1.01–1.18) 1.11(1.01–1.19) 0.018 Adjusted OR(95%CI) * 1.00 0.92(0.78–1.08) 1.12(1.04–1.20) 1.14(1.08–1.18) 0.012 Dyslipidemia Crude OR(95%CI) 1.00 1.03(1.00–1.06) 1.04(1.02–1.06) 1.05(1.03–1.08) 0.009 Adjusted OR(95%CI) * 1.00 1.04(1.01–1.07) 1.05(1.03–1.07) 1.07(1.04–1.10) 0.006 * Adjusted for age, sex, education, marital status, individual income, smoking, drinking, fat intake, vegetable and fruit intake, family history of hypercholesterolemia, central obesity, physical activity, pulse pressure and medication use. Measurement of Pulse Rate and Covariates Pulse rate and blood pressure were measured by electronic sphygmomanometer (Omron HEM-770A, Japan) in the sitting position three times. During the process of measurement, a standardized protocol was adapted from procedure recommended by the American Heart Association [13]. Participants were advised to avoid alcohol, cigarette smoking, coffee, tea, and excessive exercise for at least 30 minutes prior to having their pulse rate and blood pressure read. Relevant covariates that might be expected to affect pulse rate and lipid metabolic dysfunctions were selected and collected using a standard questionnaire administered by trained staff, including of demographic characteristics (age and sex), socioeconomic status (educational level, marital status, and individual annual income), family and individual disease history (hypertension, diabetes, heart disease, cancer, chronic kidney disease, stroke, tuberculosis, and AIDS), and dietary and lifestyle (smoking, drinking, fat intake, vegetable and fruit intake, and physical activity). Body weight and height were measured twice in light indoor clothing without shoes to the nearest 0.1 kg and 0.1 cm, respectively. Waist circumference (WC) was measured twice at the mid-point between the lowest rib and the iliac crest to the nearest 0.1 cm, after inhalation and exhalation. Central obesity based on WC (Male: WC≥90 cm; Female: WC≥80 cm) was defined according to WHO criteria for the Asia-Pacific population [14]. The interview included questions related to the diagnosis and treatment of hypercholesterolemia. All data were collected by specially trained physicians and public health workers using standardized methods with stringent levels of quality control. An overnight fasting blood specimen was collected in a vacuum tube containing EDTA for measurement of lipid profile. Blood specimens were centrifuged at 4°C and 3000 rpm for 10 minutes, and the plasma was transferred and stored at −20°C for biochemical analyses. Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were analyzed enzymatically by automatic biochemical analyzer (Hitachi 7080, Tokyo, Japan) with use of commercial reagents. Low-density lipoprotein cholesterol (LDL-C) level was calculated by use of the Friedewald equation for participants with TG level <4.52 mmol/L (400 mg/dl): LDL-C = TC-(HDL-C)-TG/5 [15]. Ascertainment of Outcomes Lipid metabolic dysfunctions were defined as a self-reported history of hypercholesterolemia and undiagnosed dyslipidemia. Lipid metabolic dysfunctions in participants without a prior diagnosis of hypercholesterolemia were categorized according to the China Adult Dyslipidemia Prevention Guide (2007 Edition) criteria as follows [16]: hypercholesterolemia was defined as the level of TC ≥6.22 mmol/L (240 mg/dl); hypertriglyceridemia was defined as the level of TG ≥2.26 mmol/L (200 mg/dl); low HDL hypercholesterolemia was defined as the level of HDL-C <1.04 mmol/L (40 mg/dl); and high LDL hypercholesterolemia was defined as the level of LDL-C ≥4.14 mmol/L (160 mg/dl). The subjects were considered as dyslipidemia if one of TC, TG or HDL-C were dysfunctions based on the above diagnostic criteria. All study participants were asked to bring their prescription medications during the clinic visit. A self-reported history of hypercholesterolemia was confirmed by the use of hypolipidemic agents (AHFS code: 24: 06.04, 24: 06.05, 24: 06.06, 24: 06.08, 24: 06.92). In addition, the hospital charts of all hospitalized cases of hypercholesterolemia were reviewed. Statistical Analysis For statistical analysis, in order to facilitate comparison with previously reports, values of resting pulse rate were stratified into quartile: Quartile 1 (Q1≤67 beats/min), Quartile 2 (Q2 68–74 beats/min), Quartile 3 (Q3 75–81 beats/min), and Quartile 4 (Q4>81 beats/min). For continuous variables, differences groups were determined by one-way ANOVA. Categorical variables were evaluated using Mantel-Haenszel statistics of chi-square (χ2) test, and the trend chi-square test was used to measure the dose-response relationship. The Pearson correlation coefficient was used to analyze the linear association between resting pulse rate and lipid metabolic dysfunctions. Univariate and multivariate logistic regression models were built to quantify the risk of lipid metabolic dysfunctions adjusting for possible confounders and socioeconomic variables. Covariates included age (continuous), sex (two categories), education level (four categories), marital status (two categories), individual income (three categories), smoking (yes vs. no), drinking (yes vs. no), fat intake (two categories), vegetable and fruit intake (two categories), family history of hypercholesterolemia (yes vs. no), physical activity (three categories), waist circumference (two categories), pulse pressure (two categories), and medications use (yes vs. no). Receiver Operating Characteristic (ROC) curve was used to assess predictive performance using the same data with multivariate logistic regression analysis. Area under ROC curve (AUC) was also utilized to compare the combined sensitivity and specificity among different categories of the subjects. All analyses were conducted using SAS 9.1 (SAS Institute, USA). All reported P-values were two-sided, and P-values less than 0.05 were considered to be statistically significant. 10.1371/journal.pone.0049347.t003Table 3 Predictive performance of resting pulse rate to screen lipid metabolic dysfunctions in rural adult subjects (n = 16,926). Sensitivity (%, 95% CI) Specificity (%, 95% CI) PPV (%, 95% CI) NPV (%, 95% CI) Hypercholesterolemia 78.95 (66.12–88.59) 55.88(53.17–58.45) 6.77 (5.73–8.84) 98.54 (96.85–99.01) Hypertriglyceridemia 74.18 (67.78–79.89) 59.46 (56.67–62.21) 24.01 (21.41–27.38) 93.02 (89.01–96.13) Low HDL hypercholesterolemia 51.54 (46.81–56.22) 57.27 (54.12–60.44) 35.56 (31.28–40.98) 72.04 (67.52–77.82) High LDL hypercholesterolemia 44.39 (39.18–47.33) 65.02 (62.45–68.89) 47.45 (43.18–50.79) 62.86 (59.25–65.78) Dyslipidemia 54.22 (50.11–58.26) 60.56 (57.22–63.93) 48.89 (48.10–56.12) 65.61 (59.14–65.93) Results Table 1 shows the baseline characteristics of study population stratified by resting pulse rate. In general, age, waist circumference, pulse pressure, and glucose level were higher, and lipid metabolic dysfunctions risk factors such as lower-educational level, divorced or widowed, positive family history of hypercholesterolemia, less physical activity and vegetable and fruit intake, and more high-fat diet were more prevalent among subjects with higher resting pulse rate. Current smoking and drinking were inversely related to pulse rate (Z = −13.10 and −7.87, P<0.01). The prevalence of hypercholesterolemia, hypertriglyceridemia, low HDL hypercholesterolemia, high LDL hypercholesterolemia, and dyslipidemia is significantly increased with higher resting pulse rate (Z = 1.64–45.36, P<0.01). A statistical relationship was observed between resting pulse rate and TC (r = 0.102, P = 0.001), TG (r = 0.182, P = 0.001), and dyslipidemia (r = 0.037, P = 0.008), but the correlation coefficient is weak. Although a negative correlation was detected between resting pulse rate and HDL-C (r = −0.026, P = 0.322), it was not statistically significant. In addition, no significant relationship existed between resting pulse rate and LDL-C (r = 0.026, P = 0.321). Table 2 summarizes the unadjusted and adjusted odds ratios and 95% confidence interval of lipid metabolic dysfunctions according to quartiles of resting pulse rate. The results showed that resting pulse rate was positively associated with the risk of lipid metabolic dysfunctions through a dose-response effect (P for trend <0.05). When adjusted for age, education, marital status, individual income, smoking, drinking, fat intake, vegetable and fruit intake, family history of hypercholesterolemia, central obesity, physical activity, pulse pressure, and medication use, the adjusted odds ratios for hypercholesterolemia (from 1.07 to 1.15), hypertriglyceridemia (1.11 to 1.16), low HDL hypercholesterolemia (1.03 to 1.06), high LDL hypercholesterolemia (0.92 to 1.14), and dyslipidemia (1.04 to 1.07) were significantly increased across quartiles of resting pulse rate for rural adults. Table 3 presents the optimum sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for resting pulse rate to screen lipid metabolic dysfunctions in rural adults. The results indicated that resting pulse rate as an indicator has relatively low sensitivity and specificity for identifying the risk of hypercholesterolemia (78.95% sensitivity and 55.88% specificity), hypertriglyceridemia (74.18% and 59.46%), low HDL hypercholesterolemia (51.54% and 57.27%), high LDL hypercholesterolemia (44.39% and 65.02%), and dyslipidemia (54.22% and 60.56%) at the optimum cut-off point values, respectively. Further analysis showed that resting pulse rate has limited potential for screening an increased risk of hypercholesterolemia (AUC = 0.70±0.04, 95% CI: 0.68–0.72), hypertriglyceridemia (AUC = 0.69±0.02, 95% CI: 0.66–0.71), low HDL hypercholesterolemia (AUC = 0.54±0.02, 95% CI: 0.51–0.56), high LDL hypercholesterolemia (AUC = 0.56±0.02, 95% CI: 0.55–0.57), as well as dyslipidemia (AUC = 0.58±0.02, 95% CI: 0.56–0.61). Discussion There were two fundamental questions that were answered in this study: 1) whether a relationship exists between resting pulse rate and lipid metabolic dysfunctions, and 2) whether resting pulse rate could be used as an indicator for screening the risk of lipid metabolic dysfunctions in rural adults. Firstly, the study analyzed the correlation coefficient between resting pulse rate and lipid metabolic dysfunctions. The results showed that a statistical relationship was detected between resting pulse rate and TC (r = 0.102), TG (r = 0.182), and dyslipidemia (r = 0.037), but no significant correlation was observed with HDL-C (r = −0.026) and LDL-C (r = 0.026). Previous results from American and Israeli Industries Study supported our findings [17]–[19]. In addition, similar relationships were also observed between resting pulse rate and lipid metabolic dysfunctions by Freitas and his colleagues [20], but the correlation was weaker than results from their study (TC: r = 0.189, TG: r = 0.215, HDL-C: r = 0.035, and LDL-C: r = 0.118). Alternatively, univariate regression analyses showed that resting pulse rate was positively associated with risk of lipid metabolic dysfunctions through dose-response effect (P for trend <0.05). After adjusting for possible confounders, similar results were also observed by multivariate logistic regression analysis. This finding was generally in agreement with a previous study, and fast resting pulse rate was positively associated with an increased risk of hypertriglyceridemia [21]. However, the positive association between resting pulse rate and lipid metabolic dysfunctions was not found in a Japanese study [22], but a dose-response effect was observed between resting pulse rate and lipid metabolic dysfunctions in their study, which was similar to our findings. The reason for this could be explained by the different ethical, racial, or geographic background. Previous studies showed that resting pulse rate provides an overall index of autonomic tone, and elevated resting pulse rate may reflect an imbalance in the autonomic nervous system favouring sympathetic activation [23], [24]. Enhanced sympathetic activity has been linked to lipid metabolic dysfunctions, high blood pressure, insulin sensitivity, and the metabolic syndrome [18], [25]–[27]. Our findings support the biological plausibility of a positive association between resting pulse rate and lipid metabolic dysfunctions. Secondly, this study considered accuracy performance by examining discrimination. Sensitivity and specificity are important when testing whether a predictor can accurately discriminate positive and negative outcomes [28]. The ideal indicator should have both high sensitivity and high specificity [29], [30]. The results showed that resting pulse rate as an indicator has low sensitivity and specificity for identifying true positive or negative patients in rural adults. Since AUC provides a superior performance index in addition to superior accuracy, it is often used to evaluate the predictive accuracy of classifiers [31], [32]. This study also used AUC values for performance comparisons of different levels of resting pulse rate. The results indicated that resting pulse rate has limited potential for screening an increased risk of lipid metabolic dysfunctions. The results were similar to those of a recent published study from Brazilian population [20]. Overall, our findings suggested that resting pulse rate as an indicator for identifying the risk of lipid metabolic dysfunctions had fairly poor accuracy and reliability. Although this study was the first to evaluate the correlation between resting pulse rate and lipid metabolic dysfunctions in rural adults, some limitations should be noted. The cross-sectional design does not offer support to causality statements and, therefore, prospective studies from different populations are necessary to describe more accurately the longitudinal relationship between resting pulse rate and lipid metabolic dysfunctions. Secondly, the results were based on a sample design and, hence, incorporating multi-center data should be considered in future research. Thirdly, only resting pulse rate was used to identify those at high risk of lipid metabolic dysfunctions in this study, and combination with other cardiovascular risk factor could screen more adequately subjects at increased risk to develop lipid metabolic abnormalities [5]. In addition, the absence of insulin measures to screen more clearly the relationship between resting pulse rate and glucose metabolism should be considered in future research [20]. Another possible limitation is that the cut-off of resting pulse rate was defined using quartile [33]. This approach was chosen because resting pulse rate stratified into quartiles has been applied in most previously epidemiological studies [3], [5], [20]; this allowed us to compare our results with previously published reported. Despite these limitations, the results are based on a large population-based epidemiologic study after adjusting for potential confounders, and the exposure assessment of resting pulse rate has been carried out systematically in this study, which precludes differential reporting in relation to the outcome. Conclusion In summary, our findings demonstrated that fast resting pulse rate was associated with a moderate increased risk of lipid metabolic dysfunctions in rural adults, and that fast pulse rate at rest might raise the risk for the development of lipid metabolic dysfunctions. In addition and more importantly, this data revealed that resting pulse rate as an indicator has limited potential for screening an increased risk of lipid metabolic dysfunctions, which suggested that resting pulse rate might not be utilized as a perfect risk indicator of lipid metabolic dysfunctions in rural adults. The authors would like to thank the participants, the coordinators, and administrators for their supports during the study. To Mr. Mark Dickson (Doctoral Candidate) and Mr. Muanda FT (Doctoral Candidate), the authors would like to express their gratitude for their critical reading of the manuscript. ==== Refs References 1 Palatini P , Thijs L , Staessen JA , Fagard RH , Bulpitt CJ , et al (2002 ) Predictive value of clinic and ambulatory heart rate for mortality in elderly subjects with systolic hypertension . Arch Intern Med 162 : 2313 –21 .12418945 2 Hsia J , Larson JC , Ockene JK , Sarto GE , Allison MA , et al (2009 ) Resting heart rate as a low tech predictor of coronary events in women: prospective cohort study . BMJ 338 : b219.4 .19193613 3 Anselmino M , Ohrvik J , Rydén L , Euro Heart Survey Investigators (2010 ) Resting heart rate in patients with stable coronary artery disease and diabetes: a report from the euro heart survey on diabetes and the heart . 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==== Front Int J Mol SciInt J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 10.3390/ijms131013438ijms-13-13438ReviewMultiple Sclerosis: The Role of Cytokines in Pathogenesis and in Therapies Amedei Amedeo 123*Prisco Domenico 24D’Elios Mario Milco 1231 Department of Internal Medicine, University of Florence, Largo Brambilla 3, Florence 50134, Italy; E-Mail: [email protected] Department of Biomedicine, Patologia Medica Unit, Azienda Ospedaliero-Universitaria Careggi, Largo Brambilla 3, Firenze 20134, Italy; E-Mail: [email protected] Center of Oncologic Minimally Invasive Surgery, University of Florence, Largo Brambilla 3, Florence 50134, Italy4 Department of Medical and Surgical Critical Care, University of Florence, Largo Brambilla 3, Florence 50134, Italy* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel./Fax: +39-055-4271495.2012 19 10 2012 13 10 13438 13460 02 8 2012 01 10 2012 11 10 2012 © 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.2012This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0).Multiple sclerosis, the clinical features and pathological correlate for which were first described by Charcot, is a chronic neuroinflammatory disease with unknown etiology and variable clinical evolution. Although neuroinflammation is a descriptive denominator in multiple sclerosis based on histopathological observations, namely the penetration of leukocytes into the central nervous system, the clinical symptoms of relapses, remissions and progressive paralysis are the result of losses of myelin and neurons. In the absence of etiological factors as targets for prevention and therapy, the definition of molecular mechanisms that form the basis of inflammation, demyelination and toxicity for neurons have led to a number of treatments that slow down disease progression in specific patient cohorts, but that do not cure the disease. Current therapies are directed to block the immune processes, both innate and adaptive, that are associated with multiple sclerosis. In this review, we analyze the role of cytokines in the multiple sclerosis pathogenesis and current/future use of them in treatments of multiple sclerosis. multiple sclerosiscytokinesT helper cells (Th)Interleukin-17 (IL-17)Interferons (IFNs) ==== Body 1. Introduction Multiple sclerosis (MS), the clinical features and pathological correlate for which were first described by Charcot, is a chronic neuroinflammatory disease with unknown etiology and variable clinical evolution. Because the neuroinflammatory process usually starts in patients in their early twenties, some authors hypothesized a slow infection during adolescence as a possible (co)factor of MS. Many viruses have been suspected and studied in this regard, but so far no specific virus has been found to be the MS cause [1,2]. Finally, one of the current MS treatments is with IFN-which was originally discovered as a broad spectrum antiviral agent [3,4]. Multiple sclerosis strikes twice as many women than men and worldwide, about 2–3 million MS patients are mainly found in Europe and in countries with Caucasian immigration, such as USA, Australia and Northern Asia [5,6]. The possible influence of the geographic environment has been studied using monozygotic twins that obtain the MS prevalence from their prepubertal geographic destination [7]. Although neuroinflammation is a descriptive denominator in MS based on histopathological observations, namely the penetration of leukocytes into the central nervous system (CNS), the clinical symptoms of relapses, remissions and progressive paralysis are the result of losses of myelin and neurons [8,9]. In analogy with other multifactorial diseases, MS is influenced by genetic and environmental factors. Therefore, in the absence of etiological factors as targets for prevention and therapy, the definition of molecular mechanisms that form the basis of inflammation, demyelination and toxicity for neurons have led to a number of treatments that slow down disease progression in specific patient cohorts, but that do not cure the disease. The perfect MS drug should reverse the processes of neuroinflammation, demyelination and neuronal loss, but such substances do not exist. Therapies have been refined beyond anti-inflammatory glucocorticosteroids. The introduction of IFN-, copolymer and, more recently, natalizumab, monoclonal antibody (Moab) against a1b4 integrin, has considerably improved the life of many MS patients, But intrinsic therapeutic limitations and severe side effects associated with these drugs, stimulate basic and clinical researchers to define innovative and more patient-compliant treatment strategies. With the latest tools of molecular biology, such as whole genome sequencing, genome-wide association studies [10,11], gene expression profiling coupled with protein analysis [12,13], we may be able, in the future, to define common identifiers for specific cohorts of MS patients, to perform better and earlier diagnoses and, hopefully, discover etiological factors. However, this is presently not yet possible and therapies are directed to block the immune processes, both innate and adaptive, that are associated with MS. In this review we analyze the role of cytokines in the MS pathogenesis and current/future use of them in treatments of multiple sclerosis. 2. The Role of T Cells-Associated Cytokines in MS Pathogenesis About 15 years ago, Opdenakker and colleagues assembled the available information from histopathological data and inflammation-associated components to create the REGA (Remnant Epitopes Generate Autoimmunity) model to understand MS and to place MS therapies into a new context. In this model, the proteinases, notably matrix metalloproteinases (MMP), cleave substrate proteins into autoimmune peptides that (re)activate specific T cells [14] (Figure 1). The model used MMP-2 and MMP-9 as examples of proteinases, because previous publications linked MMP-9 in the cerebrospinal fluid with multiple sclerosis and other neuroinflammatory diseases and because myelin basic protein (MBP) was cleaved into immune-dominant peptides by MMP-9 [15,16]. Although most recognized for these two aspects, the REGA model contained much more information related to autoimmune diseases (e.g., about regulatory cytokines) that was instrumental and will remain important for the future drug development [14]: (a) it addressed the critical point that (unspecific) inflammatory reactions with myeloid cells may be primordial and a possible target in MS. In 1994, this was rather controversial and led to some opposition in an era when most MS research was centered on T cells. (b) It translated the central stage of inflammation in the autoimmune process into molecules and addressed the importance of balances between pro-inflammatory versus anti-inflammatory primary cytokines in the disease process. Interleukin-1β (IL-1β), TNFα (Tumor Necrosis Factor-α) and IFN-γ (Interferon-γ) were included as pro-inflammatory cytokines and IFN-α/β and TGF-β (Transforming growth factor β) as anti-inflammatory cytokines. (c) It addressed the role of myeloid antigen presenting cells in the activation of T cells. Moreover, MBP-specific T cells isolated from MS patients and encephalitogenic T cells recovered from immunized animals have confirmed that T cells play a central role in the MS pathology [17–19]. EAE (Experimental Autoimmune Encephalomyelitis), the most studied MS animal model, is an acute or chronic-relapsing, acquired, inflammatory and demyelinating autoimmune disease. The animals are injected with the whole or parts of various proteins that make up myelin. These proteins induce an autoimmune response in the animals—that is, the animal’s immune system mounts an attack on its own myelin as a result of exposure to the injection. The animals develop a disease process that closely resembles MS in humans. EAE has been induced in a number of different animal species including mice, rats, guinea pigs, rabbits, macaques, rhesus monkeys and marmosets. For various reasons including the number of immunological tools, the availability, lifespan and fecundity of the animals and the resemblance of the induced disease to MS, mice and rats are the most commonly used species. The animals are inbred to reliably produce susceptibility to EAE in the animals. As with humans and MS, not all mice or rats will have a natural propensity to acquire EAE. Moreover, different breeds will develop different forms of EAE, some of which act as good models for the different human forms of MS. Different EAE forms are also used as models for the different stages of MS. Even though EAE is the best MS model, it is not multiple sclerosis and a number of significant assumptions are made when proposing EAE as an animal model for MS. Several proteins or parts of proteins (antigens) are used to induce EAE including: Myelin Basic Protein (MBP), Proteolipid Protein (PLP), and Myelin Oligodendrocyte Glycoprotein (MOG), but, importantly, EAE can also be induced by adoptively transferring an expanded population of myelin-specific encephalitogenic CD4+ (T helper (Th)) cells [20], precisely, selfreactive Th1 clones [21]. In the 1990s, Mosmann and Coffman postulated that Th cells can be classified into two distinct subsets, Th1 and Th2. Th1 cells produce large quantities of IFN-γ, driven by IL-12, which promotes cellular immunity directed against intracellular pathogens. Alternatively, Th2 cells, which secrete IL-4, IL-5, IL-13, and IL-25, are essential in the destruction of extracellular parasites and the mediation of humoral immunity [22,23]. Increased levels of Th1 cytokines are particularly evident during EAE/MS relapse, whereas increased Th2 cytokines are found during remission in MS patients [24]. Clinical and hematological symptoms are exacerbated in relapsing/remitting MS patients following the IFN-γ administration, and this is also observed in other Th1-type diseases, whereas it is less apparent in Th2 diseases [25,26]. In other words, Th1 cells were earlier thought to be pathogenic T cells, whereas Th2 cells were thought to confer an anti-inflammatory potential, constituting protective T cells in both MS and EAE [27–30]. However, this clear-cut immune-dysregulation of the Th1/Th2 balance in EAE and MS may be part of a hidden complex of interactions underlying EAE and MS [31]. The Th1-driven nature of the MS disease was challenged by the discovery that IFN-γ and IFN-γ-receptor-deficient mice, as well as mice that lack other molecules involved in Th1 differentiation, such as IL-12p35, IL-12 receptor β2 (IL-12Rβ2, and IL-18, were not protected from EAE, but instead were more susceptible to the disease [32–36]. Unexpectedly, mice deficient in IL-12α (IL12p35, a component of the Th1 paradigm, are vulnerable to EAE. Similarly, IL-12Rβ2-deficient mice develop more severe clinical manifestations of EAE, whereas IL-12p40-deficient mice are resistant to EAE [34,35,37]. These contradictory data indicate that an imbalance in the Th1/Th2 milieu cannot explain the overall immunopathogenic mechanisms underlying multiple sclerosis. 2.1. Immunopathogenic Function of IL-17 When the p19 (a novel cytokine heavy-chain homolog of the IL-6 subfamily) chain is linked to the p40 chain, a subunit of IL-12 (another IL-12 subunit is the p35 chain), it forms a novel cytokine designated IL-23. Cua and colleagues verified that IL-23, but not IL-12, is essential for the induction of EAE by generating IL-23p19 knockout (KO) mice and comparing them with IL-12p35 KO mice [38]. Furthermore, an IL-17-producing T cell subset, driven and expanded by IL-23, can pathogenically induce EAE when adoptively transferred into naive mice [39,40]. These IL17-producing T cells were dramatically reduced in the CNS of IL-23p19-deficient mice. These results suggested that IL-17-producing CD4+ T cells are a distinct and novel Th subset that exacerbates autoimmunity, and designated them Th17 cells [41,42]. The Th17 discovery further clarifies the cytokine MS profile [43] and, recently, the levels of IL-17 produced by MBP-stimulated peripheral blood cells were shown to correlate with the active lesions in MS patients [44]. Like other Th subsets, the Th17 lineage is activated by a specific cytokine milieu. However, IL-23 cannot produce Th17 cells de novo from naïve T cells, and the IL-23 receptor (IL-23R) is not expressed on naïve T cells [45]. TGF-β up-regulates IL-23R expression, thereby conferring responsiveness to IL-23. These data confirm that TGF-β is a key cytokine in the commitment to Th17 expansion [46]. In mice, TGF-β together with IL-6 can activate antigen-responsive naïve CD4+ T cells to develop into Th17 cells [47]. In humans, naïve CD4+ cells exposed to IL-6, TGF-β, and IL-21 can develop into Th17 cells; and the IL-23 production plays a role in maintaining these Th17 cells [48,49]. Altogether, Th17 cells require IL-23, TGF-β, IL-6, and IL-1 for their generation. Th17 cells produce IL-17 (A and F), which are upregulated in chronic lesions [50,51], and IL-22, which is also involved in the MS pathogenesis. Also, microarray studies of lesions in MS patients demonstrated an increased IL-17 expression, confirming that Th17 cells play an important role in the development of inflammation and damage of the CNS. Patients with MS have greater numbers of IL-17-mRNA-expressing mononuclear cells in the cerebrospinal fluid (CSF) than in the blood. Previously, no increase in the numbers and expression of IL-17 mRNA by mononuclear cells isolated from the CSF was observed in MS patients, but higher levels of IL-17 mRNA were observed in the CSF than in the blood, with the highest levels in the blood detected during clinical exacerbations [52]. These data confirm the pivotal role of IL-17 in MS both peripherally and centrally. 2.2. Reciprocal Interactions of Cytokines Produced by T Cells IL-1R KO mice have impaired Th17 cells and are protected from EAE [53]; also, IL-1β increases the progression of relapse onset in MS [54], implying a role for IL-1β in the development of EAE and MS. EAE was abolished by a virus-expressing IL-4 but not IL-10 in chronic relapsing EAE. Therefore, the cytokine environment was converted from a disease-promoting IL-23-producing condition to a disease-limiting IL-4-producing condition by the local expression of IL-4 from a Herpes simplex virus vector delivered to the brain [55]. Moreover, the increased expression of IL-4 in glial cells was associated with the reduced EAE severity [56], suggesting that the upregulation of Th2 cytokines inhibits the propagation of the EAE/MS inflammation promoted by Th17. TGF-β is a key cytokine in the generation of the regulatory T cells (Tregs), that inhibit the autoimmune response and protect against inflammatory injury. Tregs are involved in the regulation of Th1/Th2 and Th17 cells. Therefore, the generation of pathogenic Th17 cells induce autoimmunity, the generation of Tregs inhibit autoimmune tissue injury [57]. Although EAE was once considered a classical Th1 disease, it has been proposed that it is predominantly Th17 driven. The data about IL-17 and IFN-γ in MS genesis, indicate that their roles may depend on the nature of the immune response and that the IL-17 may overcome the inhibitory effect of IFN-γ, which generally prevents inflammation at the brain [58]. When pure Th17 cells, polarized with TGF-β to deplete any IFN-γ production, are adoptively transferred to mice, they do not induce EAE, suggesting that the reciprocal interactions among Th17-related cytokines enroll and activate the involvement of associated immune cells. Interestingly, when Th17 cells are combined with Th1 cells, they can fully induce EAE disease [59]. Liu and colleagues also demonstrated that the loss of STAT3 by T cells results in an intrinsic developmental defect that renders STAT3−/− mice resistant to CNS inflammatory diseases. STAT3 is required for the production of IL-17 by Th17 cells, the generation of double positive T cells expressing IL-17 and IFN-γ, and T cell trafficking into CNS tissues. This suggests that STAT3 may be a therapeutic target for modulating CNS autoimmune diseases [60]. A recent study showed significant differences in the regulation of inflammation in the brain and spinal cord, depending on different Th17/Th1 ratios, by demonstrating that specific T-cell populations targeting different myelin epitopes are characterized by different Th17/Th1 ratios in EAE [61]. Also, the Th1 have the potential to reciprocally regulate Th17 cells during EAE. IL-21 is a type I four-α-helix bundle cytokine that belongs to the IL-2 family and functions as a “growth hormone”—like cytokine. In detail, IL-21 plays a pivotal role in the expansion and differentiation of the Th17 lineage [62]. During clonal expansion, IL-21 also promotes IL-23R expression in differentiated Th17 cells, which plays an important role in the stabilization of the Th17 lineage in the presence of IL-23 [63]. Although no effects were observed when IL-21 was administered after EAE progression, the IL-21 administration boosted natural killer (NK) cell functions before the induction of EAE, including the secretion of IFN-γ [64]. Alternatively, IL-27 (an IL12/IL23 family member) is a negative regulator of Th17 cell differentiation and can prevent inflammatory demyelination in the EAE model [65]. IL-27 drives the expansion and differentiation of IL-10-producing Tregs by inducing the expression of three key molecules: the basic leucine-zipper transcription factor Maf (generally known as c-Maf), the IL-21, and ICOS (an inducible T-cell costimulator structurally and functionally related to CD28. Moreover, IL-27-driven c-MAF expression transactivates the production of IL-21, which favor the expansion of IL-27-induced Tr1 cells. ICOS also promotes IL-27-driven Tregs. Each of these elements is essential, because the loss of c-MAF, IL-21 signaling, or ICOS reduces the frequency of IL-27-induced Treg differentiation (Figure 2) [66]. Exacerbation of EAE was demonstrated in IL-27-deficient mice, and interestingly, IL-27 treated mice had markedly reduced CNS inflammatory infiltration, indicating the downregulation of Th17 phenomena [67]. Recently, a novel effector T-cell subset, Th9 cells, has been identified, and the ability of this T-cell subset to induce EAE is currently being investigated. Jager and colleagues generated specific Th17, Th1, Th2, and Th9 cells in vitro to directly characterize their encephalitogenic potency after adoptive transfer. They found that Th1, Th17, and Th9 cells, but not Th2 cells, induce EAE. Interestingly, each T-cell subset induced disease in a distinct pathological manner, suggesting that the different effector Th subsets that induce EAE do so differently and implying that the pathological heterogeneity in MS lesions might be partly attributable to various characteristics of myelinreactive effector T cells [68,69]. The authors also suggested that MS might be a disease caused by multiple distinct myelinreactive effector cells. The disease induced by Th17 cells in some animals exhibited symptoms atypical of EAE, including ataxia, severe imbalance, and weight loss associated with high mortality. Some animals had a mixture of atypical and typical EAE symptoms. When cells were recovered from the CNS, it appeared that the transferred Th9 cells produced IFN-γ. The identities of the other cell populations did not seem to drift after their in vivo transfer [69]. It has recently been demonstrated that cultured in the presence of TGF-β, Th17 cells produce IL-9. Th17 cells generated in vitro with IL-6 and TGF-β and ex vivo-purified Th17 cells both produced IL-9. Also, the IL-9 neutralization and IL-9R deficiency attenuated the disease, and this correlated with reductions in Th17 cells and IL-6-producing macrophages in the CNS. These data confirmed the IL-9 role in the EAE development/progression and suggested IL-9 as a Th17-derived cytokine that contributes to inflammatory disease [70]. 3. Cytokines and Innate Immune Cells in MS Myelin is presented in the circulation, and other CNS antigens are thought to be expressed in the cervical lymph nodes, which can trigger the conversion of myelin-specific T cells to pathogenic T cells. Adhesion molecules, the integrins, allow these myelin-specific T cells to penetrate the blood–brain barrier (BBB) under inflammatory conditions, and in this way, activated and memory T cells can enter the CNS [71]. Myelin-specific T cells migrate into the CNS and the movement of antigen-presenting cells (APCs) into the CNS is essential for lymphocyte reactivation and the initiation of the inflammatory cascade in the EAE development [72]. Subsequently, inflammatory and immune cells, such as granulocytes and macrophages, are attracted into the CNS parenchyma, where they mediate tissue inflammation and tissue damage [73]. The brain was formerly considered an immune-privileged organ, but today, we understand that any damage to the CNS can activate immune cells in situ, particularly microglial cells, that have a key role in maintaining the autoimmune responses in the CNS [74]. Also, microglial cells upregulate the expression of MHC (major histocompatibility complex) and costimulatory molecules to initiate the generation of the inflammatory milieu. Dendritic cells (DCs) seem to play a critical role in antigen presentation to invading T cells and in the of cytokines/chemokines release, thereby guiding into the lesion the entry of monocytes and lymphocytes [75]. Whereas the CD4+ cells recruit macrophages, which release proinflammatory cytokines (IL-1, IL-6, TNF-α), destructive molecules (nitric oxide) and MMPs, the CD8+ T cells directly attack MHC class I-expressing cells (oligodendrocytes and neurons) [76,77]. TNF receptor 1 (TNFR1 but not TNFR2 signaling is critical for demyelination and the limitation of T-cell responses during immune-mediated CNS disease [78]. This complicated process triggers the recruitment of innate immune cells, especially macrophages and microglia, which in turn mediate demyelination, axonal damage, and lesions. In autopsy samples from MS patients, the expression of IL-17 is evident in perivascular lymphocytes and in astrocytes and oligodendrocytes located in the active areas of CNS lesions. IL-17R is also identifiable in acute and chronic plaques of MS patients, suggesting the enrichment of Th17 and CD8+ T cells in active MS lesions [79]. Also, microarray analysis of MS lesions has demonstrated increased transcripts of genes encoding inflammatory cytokines, particularly IL-6, IL-17, and IFN-γ. A significant increase in IL-23 mRNA and protein expression is found in lesion tissues and activated macrophages, microglia and especially mature DCs have been shown to be important sources of IL-23p19 [80]. There is also evidence that MS endothelial cells express high levels of IL-17R and are more permeable to IL-17. This microenvironment favors the differentiation of naïve CD4+ T cells into Th17 cells, that transmigrate efficiently across BBB endothelial cells (BBB-ECs), leading to the destruction of human neurons and initiating CNS inflammation [81]. Similarly, the expression of IL-17R and IL-22R on BBBECs has been examined in MS lesions, and IL17 and IL-22 have been shown to disrupt BBB tight junctions in vitro and in vivo. IL-6 transsignaling may also play a role in the autoimmune inflammation of the CNS, mainly by regulating the early expression of adhesion molecules, possibly via cellular networks at the BBB [82]. Ifergan and colleagues demonstrated that a subset of monocytes migrate across the inflamed human BBB and differentiate into DCs under the influence of BBB-secreted TGF-β and GM-CSF (granulocyte macrophage colony-stimulating factor). These DCs can produce IL-12p70, TGF-β, and IL-6 and promote the expansion of Th1 and Th17 cells. The abundance of such DCs in situ is strongly associated with microvascular BBB-ECs within acute MS lesions and with a significant number of Th17 cells in the perivascular infiltrate [83]. Astrocytes play significant physiological roles in CNS homeostasis and act as a bridge between the CNS and the immune system. Astrocytes also contribute to the complex interactions during CNS inflammation. IL-17 functions in a synergistic manner with IL-6 to induce IL-6 expression in astrocytes. Astrocytes upregulate the expression of IL-17 and IFN-γ in T cells, which is consistent with the capacity of astrocytes to express IL-23 subunit p19 and the common IL12/IL23 subunit p40, but not IL-12 subunit p35 [84]. Recently, increased IL-17RA expression in the CNS of mice with EAE and in both astrocytes and microglia in vitro [85] has been demonstrated. Also, the suppressor of cytokine signaling 3 (Socs3 participates in IL-17 functions in the CNS as a negative feedback regulator, in fact mouse models of Socs3 small interfering RNA (siRNA) knockdown and Socs3 deletion, showed enhanced IL-17 and IL-6 signaling in astrocytes, indicating that astrocytes can act as a target of Th17 cells and IL-17 in the CNS [86]. Similarly, in mice deficient of Act1, critical for IL17 signaling, the Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils and macrophages. Therefore, astrocytes are critical in IL-17–Act1-mediated leukocyte recruitment during EAE [87]. Interestingly, the data obtained by a monkey MS model established that macrophages respond to the Th1 milieu and neutrophils respond to Th17 cytokines. Also, the study showed dense accumulations of T/B cells and macrophages/microglia at the sites of perivascular and parenchymal lesions in the neocortex and subcortical white matter, indicating that the inflammatory response, especially activation of macrophages and microglia, may be regulated differently in the gray matter areas of the primate brain [88]. In summary, DC-like cells in the peripheral tissues and microglia in the CNS are responsible for cytokine polarization and the Th17 expansion. The complex interactions of Th17 cells with different cells, such as y microglia, astrocytes, neutrophils and macrophages, all contribute to the MS immunopathogenesis. 4. Current and Future Clinical Applications of Cytokine-Mediated Treatments Our understanding of the MS patho-physiology has led to the development of novel therapeutic strategies. Since the early 1990s, disease-modifying drugs have been introduced for the selective MS management, including IFN-β and glatiramer acetate (GA), which have become the standard treatment for relapsing/remitting MS [89] but in this paragraph we analyze, in detail, three domains of novel immune-mediated therapeutics used for MS; the first domain includes immunosuppressive/immunomodulator agents, such as mitoxantrone, laquinimod (ABR-215062, cladribine (Mylinax), and teriflunomide. The second domain includes immune-modulatory agents: (a) cytokine inhibitors such as IFN-β; (b) agents that deplete specific immune cell subsets, such as alemtuzumab (a human mAb targeting CD52 expressed especially by T cells) [90,91] and rituximab (which targets CD20 to deplete B cells) [90,92]; (c) agents that selectively block coreceptors and costimulators, such as daclizumab (an anti-CD25 mAb that inhibits activated T cells and induces regulatory immune cells) [93]. The third domain includes neuroprotective agents associated with immunomodulation, including broad-spectrum immunomodulators such as statins, PPAR agonists (e.g., pioglitazone, gemfibrozil), the sex hormone estriol (E3, fumarate and minocycline), all of which have been effective in the MS treatment. IFN-β has been clinically introduced to treat MS patients based on its ability to shift a Th1-mediated response to a Th2 phenotype. However, microarray studies have indicated that a number of genes in MS patients are upregulated by the cytokines associated with the differentiation of T cells into Th1 rather than into Th2, suggesting that this shift may not be the only therapeutic mechanism of IFN-β in MS [94]. IFN-β therapy also reduces IL-23 mRNA levels [95] and inhibits human Th17 cell differentiation, so the Th17 axis could be another target of IFN-β therapy [96]. IFN-β-mediated IL-27 production by innate immune cells has been shown to play a critical role inhibiting Th17 cells in [97]. In addition, the therapeutic effect of IFN-β is probably attributable to the induction of the regulatory cytokine IL-10 [95]. A high IL-17F level in the serum of people with relapsing/remitting MS is associated with the failure of IFN-β therapy. This characteristic of IFN-β might contribute to an exploration of some logical biomarkers for predictive assessment of the response to a popular therapy for MS [98]. IFN-β inhibits the Th17 expansion in MS, and this might contribute to an improvement in the clinical symptoms. The effectiveness of this inhibition, however, requires intact IFN-γ signaling in T cells. In a recent study, Conti and colleagues reported that both mRNA and cell surface expression of the signaling chain of the IFN-γ receptor (IFN-γR2 and its cognate tyrosine kinase JAK2 are enhanced in peripheral blood Th17 cells and clones from patients with active MS compared with those with inactive MS. IFN-γ decreased the frequency of Th17 peripheral cells and proliferation of Th17 clones from patients with active MS; also the stimulation of healthy donors PBMCs in Th17-polarizing conditions resulted in the enhancement of JAK2 expression and accumulation of cell surface IFN-γR2. The role of JAK2 in the modulation of IFN-γR2 was demonstrated as its transduction prevented rapid internalization and degradation of IFN-γR2 in JAK2-deficient γ2A cells. In others words, these data identify JAK2 as a critical factor that stabilizes IFN-γR2 surface expression in Th17 cells from patients with active MS, making them sensitive to IFN-γ [99]. Although B cells may have a dual role in the pathogenesis of MS, they contribute to the induction of the autoimmune response but also mediate the resolution of the CNS inflammatory infiltrate [100,101]. A recent study demonstrated that supernatants transferred from IFN-β-treated B cells inhibited Th17 cell differentiation and suppressed the secretion of IL-17A. Likewise, IFN-β also induces in B cells the IL-10 secretion which may mediate their regulatory potency [102]. Thus, IFN-β exerts its therapeutic effects at least in part by targeting B cells’ functions that contribute to the autoimmune pathogenesis of MS, which may uncover extra mechanisms of the B-cell contribution to the autoimmune effects and provide novel targets for future selective MS treatment [103]. Glatiramer acetate (GA); Copaxone; copolymer 1 exerts a clinical response in MS patients via its modulation of IFN-γ and IL-4 by reducing the expression of IFN-γ and ensuring the stable expression of IL-4 in anti-CD3/CD28-stimulated PBMCs [102]. Moreover, GA enhances the suppressive effects of Tregs in MS [104]. Studies of human DCs have shown that GA modulates the production of inflammatory mediators without affecting DC maturation or immune-stimulatory potential. DCs exposed to GA secrete low levels of the Th1-polarizing factor IL12p70 [105] and induce IL4-secreting effector Th2 cells such as IL-10 [106]. These results show that APCs, including DCs, are essential for the GA-mediated shift in Th-cell phenotypes and indicate that DCs are an important target of the immunomodulatory effects of GA. Mitoxantrone, a cytotoxic drug with immunomodulatory properties, is used to treat progressive MS forms [107]. Mitoxantrone increases the ex vivo production of the Th2 cytokines IL-4 and IL-5, but with no significant changes in IFN-γ, TNF-α, IL-10, or IL-17 expression by CD4+ T cells, indicating that the immune-modulation afforded by mitoxantrone treatment in MS acts through the enhancement of Th2-type cytokines [108]. Currently, a head-to-head race for approval had initially developed between two under-the-spotlight oral immunomodulatory agents—fingolimod and cladribine [109]. Fingolimod (FTY720/Gilenya, Novartis), an S1PR modulator [110], is under the spotlight because it has completed phase III trials [111] and has been approved by the FDA (Food and Drug Administration) as the first oral, first-line treatment for relapsing MS [112,113]. S1PR is mainly expressed by immune cells, neuronal cells, endothelial cells, and smooth muscle cells [114–117] and have key roles in angiogenesis, neurogenesis, and the regulation of immune cell trafficking [118–120]. The immunomodulatory effect of fingolimod acts in two pathways: inhibits the function of S1PR, which facilitates the CC-chemokine receptor 7-(CCR7-) mediated retention of T cells in the lymph nodes, including naïve and memory T cells. In this way, it reduces the infiltration of inflammatory cells into the CNS [121,122] and reduces the numbers of autoreactive Th17 cells that are recirculating via the lymph and blood to the CNS [123–125]. prohibits neuroinflammation via the modulation of the S1PR1 expressed on oligodendrocytes, neurons, astrocytes, and microglia [126,127]. Another oral immunomodulatory drug, cladribine (2-chlorodeoxyadenosine) (which has been withdrawn for US market for MS), is a synthetic chlorinated deoxyadenosine analog [128] that is activated by intracellular phosphorylation in specific cell types, resulting in preferential and sustained reduction of peripheral T and B cells, mimicking the immunedeficient status of hereditary adenosine deaminase deficiency [129]. Orally administered cladribine shows significantly efficacy in MS patients [130]. Oral cladribine reduces relapses by 55%–58% and has an impact on disability progression and all MRI outcome markers in patients with relaxing/remitting MS [130–132]. Nevertheless, to exactly weigh the benefits of both novel immunomodultory agents against the potential risks is necessary and must be monitored continually. These advances in identifying unique therapeutic targets for MS have instigated numerous phase II and phase III clinical trials, for example, trials of various mAbs, including those directed against CD52 (alemtuzumab), CD25 (daclizumab), and CD20 (rituximab), and trials of disease-modifying therapies, such as teriflunomide, laquinimod, and fumarate [133]. For example, alemtuzumab targets the surface molecule CD52 on all T-cell populations and other cellular components of the immune system, such as thymocytes, B cells, and monocytes [134]. Minocycline, an oral semisynthetic tetracycline antibiotic, can penetrate the CNS and has interesting pleiotropic biological functions and neuroprotective effects, including in demyelinating diseases such as MS [135]. A study about the impact of oral minocycline on clinical and MRI outcomes and serum immune molecules during the 24 months of open-label minocycline treatment evidenced that no relapses occurred between months 6 and 24, and the levels of the p40 subunit of IL12 were elevated during the 18 months of treatment, which might have counteracted the pro-inflammatory effects of IL-12R [136]. 5. Conclusions Multiple sclerosis is the most common disabling CNS disease in young adults. It is characterized by recurrent relapses and/or progression, which are attributable to multifocal brain and/or spinal cord inflammation [137]. The effector Th cells and correlated cytokines, especially IL-17, play a well-recognized role in the initiation of autoimmune tissue inflammation, and have an established association with the pathogenesis of this disorder [28]. Komiyama and colleagues demonstrated that EAE was significantly suppressed in IL17−/− mice, and was manifested as delayed onset, reduced maximum severity, with ameliorated histological changes and early recovery [138]. However, the outcomes have varied when the differentiation and/or functions of Th17 cells have been blocked in clinical trials of human autoimmune diseases, with notable success only in psoriasis and Crohn’s disease, but negative results in relapsing/remitting MS. The strategy of inhibiting the Th17 response has had even less support in preclinical studies in animal models [139]. These data raise the questions of whether MS is mediated solely by Th1 cells or solely by Th17 cells, whether it is mediated by both pathways, or whether perhaps it is mediated by neither pathway [137]. There is growing evidence that autoreactive T cells (particularly Th1 and Th17 cells) participate in the MS patho-physiology. Although the exact roles of Th1 and Th17 cells in the development of MS lesions are not well understood, it appears that both these effector T-cell populations can cause CNS inflammation and demyelinating lesions in MS and EAE [140,141]. Our increasing understanding of the immunopathogenic roles of Th1, Th2, Th17 cells and Tregs in MS should facilitate the development of novel immune-modulatory therapeutic approaches to MS [142,143]. Currently approved disease-modifying treatments achieve their effects primarily by blocking the pro-inflammatory response in a nonspecific manner. Their limited clinical efficacy calls for a more differentiated and specific therapeutic approach. We can confidently say that IFN-β, GA, and mitoxantrone are fairly clinically effective for MS patients. The addition of minocycline has also shown benefits in the MS treatment. More immune-modulatory therapeutic agents are currently in clinical trials, including fingolimod (FTY720, alemtuzumab, and rituximab add-on therapies) [144]. The extensive clinical application of these possible new immune-modulatory therapeutic agents will be under close scrutiny in the near future. In conclusion, from basic research efforts and preclinical findings in EAE models it is clear that possibilities exist for better MS treatments. The designs of simple and good clinical studies by caring neurologists, a courageous attitude by the pharmaceutical industry and political support for these new developments will be needed if we want to improve the life-quality of MS patients and reduce the social costs needed for this chronic disease. Although no prevention or cure for MS exists, patients may count on limited drug discovery successes from the past and on continued efforts to find new, more efficient and better tolerated ways of fighting this disease. However, efficiency in drug development can be enhanced on the basis of combination therapies, whereas side effects may be decreased by better formulations and lower doses in combination therapies. Acknowledgements We thank Italian Ministry of University and Research and Ente Cassa di Risparmio di Firenze for supporting our studies. Figure 1 The Remnant Epitopes Generate Autoimmunity (REGA) model. Multiple sclerosis is a multifactorial autoimmune disease of unknown etiology. Different factors (host genetics, environmental determinants, and especially the immune system) can influence the disease progression. Figure 2 T helper cell differentiation. When naïve CD4+ T cells, classified by absence of CD25 and high levels of CD62L, encounter specific antigens, they can differentiate into different effector subsets. It is likely that several “master” transcription factors, individually required for T-cell differentiation towards one of the end effector stages, are initially expressed upon engagement of the TCR with costimulatory receptors. Each transcription factor drives a specific set of genes required for lineage commitment and the expression of signature cytokines and negatively affects alternative pathways. However, the microenvironment is the driving force that determines the outcome of the differentiation course. Th1 cells are established in the presence of IFN-γ and IL-12 and signaling via STAT1 and STAT4, resulting in the expression of the master transcription factor T bet. Th2 cells depend on IL-4 and STAT6 for the increased expression of GATA3, whereas the simultaneous presence of TGF-β results in the development of Th9 cells, utilizing an undefined master transcription factor. The presence of TGF-β, with IL-2 signaling via STAT5, is known to generate, at least in vitro, inducible Treg, which utilize Foxp3. Also, it is TGF-β in combination with IL-6 signaling via STAT3 that drives the expression of RORγt, resulting in the differentiation of Th17 cells. ==== Refs References 1 Noseworthy J.H. Progress in determining the causes and treatment of multiple sclerosis Nature 1999 399 A40 A47 10392579 2 Giraudon P. Bernard A. Chronic viral infections of the central nervous system: Aspects specific to multiple sclerosis Rev. Neurol. (Paris) 2009 165 789 795 19656540 3 Billiau A. Edy V.G. 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Int J Mol Sci. 2012 Oct 19; 13(10):13438-13460
==== Front BMC SurgBMC SurgBMC Surgery1471-2482BioMed Central 1471-2482-12-S1-S2210.1186/1471-2482-12-S1-S22Research ArticleObservational study: daily treatment with a new compound “tradamixina” plus serenoa repens for two months improved the lower urinary tract symptoms Iacono Fabrizio [email protected] Domenico [email protected] Ester [email protected] Antonio [email protected] Giuseppe [email protected] Bruno [email protected] Department of Urology – University Federico II of Naples, Via S. Pansini, 5 – 80131 Naples – Italy2 Department of General, Geriatric, Oncologic Surgery and Advanced Technologies,-University “Federico II” of Naples. Via Pansini, 5 - 80131 – Naples, Italy2012 15 11 2012 12 Suppl 1 Selected articles from the XXV National Congress of the Italian Society of Geriatric SurgerySilvestro Canonico, Bruno Amato and Beatrice SensiThis supplement has not been supported by sponsorship or other external funding.S22 S22 Copyright ©2012 Iacono et al; licensee BioMed Central Ltd.2012Iacono 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 Lower urinary tract symptoms (LUTS) are associated with great emotional costs to individuals and substantial economic costs to society. This study seeks to evaluate the effect of a new natural compound “Tradamixina plus Serenoa Repens” in order to improve lower urinary tract symptoms. Methods 100 patients (≥45years) who had had LUTS/BPH for >6 mo at screening and with IPSS -The international Prostate symptom scores- ≥13 and maximum urinary flow rate (Qmax) ≥4 to ≤15 ml/s. were recruited. The compound “Tradamixina plus Serenoa Repens” (80 mg of Alga Ecklonia Bicyclis, 100 mg of Tribulus Terrestris and 100 mg of D-Glucosamine and N-Acetyl-D-Glucosamine plus 320 mg of Serenoa Repens) was administered daily for 2 months. At visit and after 60 days of treatment patients were evaluated by means of detailed medical urological history, clinical examination, laboratory investigations (total PSA), and instrumental examination like urolfowmetry. Efficacy measures included IPSS-International Prostate Sympto, BPH Impact Index (BII), Quality-of-Life (QoL) Index. Measures were assessed at baseline and end point (12 wk or end of therapy) and also at screening, 1 and 4 wk for IPSS, and 4 wk for BII. Statistical significance was interpreted only if the results of the preceding analysis were significant at the 0.05 level. Results After 2 months of treatment the change from baseline to week 12 relative to “Tradamixina plus Seronea Repens” in total IPSS and Qol was statistically significant. Differences from baseline in BII were statistically significant for “Tradamixina plus Seronea Repens” above all differences in BII were also significant at 4 wk (LSmean ± SE: -0.8 ± 0.2). In the distribution of subjects over the PGI-I and CGI-I response categories were significant for”Tradamixina plus Seronea Repens” (PGI-I: p = 0.001; CGI-I). We also observed a decrease of total PSA. Conclusion The daily treatment with a new compound “Tradamixina plus Serenoa Repens” for 2 months improved the male sexual function , it improved the bother symptoms which affect the patient’s quality of life , improved uroflowmetric parameters, and we also observed a decrease of serum PSA level. 10-11 May 2012 XXV National Congress of the Italian Society of Geriatric Surgery Padova, Italy ==== Body Background Lower urinary tract symptoms (LUTS) are associated with great emotional costs [1] to individuals and substantial economic costs to society [2]. The prevalence and severity of LUTS increases with age [3], and the progressive growth of the aged population group has broadened the societal impact of LUTS. LUTS comprise storage symptoms (daytime urinary frequency, nocturia, urgency, urinary incontinence) voiding symptoms (slow stream, splitting or spraying, intermittency, hesitancy, straining, terminal dribble), and post micturition symptoms (sensation of incomplete emptying, post micturition dribble) [4] In EPIC, 62.5% of 7210 men in the five countries surveyed reported one or more LUTS; the prevalence of individual LUTS in men increased with age. A greater percentage of men reported storage symptoms (51.3%) vs. voiding (25.7%) or post micturition (16.9%) symptoms. Nocturia, defined by the ICS as waking one or more times to urinate during the night, was the most commonly reported symptom (48.6% of men); terminal dribble (14.2% of men) and sensation of incomplete emptying (13.5% of men) were the most commonly reported voiding and post micturition symptoms, respectively [5]. A large-scale multinational study revealed that 90%of men aged 50 to 80 suffer from potentially troublesome LUTS [3]. Questionnaire data from 1,271 men with LUTS indicated that many men have storage and voiding symptoms [6]. The same study demonstrated that voiding symptoms were the most common male LUTS, but that storage symptoms made up four of the five most bothersome LUTS. Although LUTS are also highly prevalent in women, their frequent comorbidity with prostatic disease in men adds complexity to the management of male LUTS [6]. Benign prostatic hyperplasia (BPH) is a pathologic process that contributes to, but is not the sole cause of, lower urinary tract symptoms (LUTS) in aging men. Despite intense research efforts in the past five decades to elucidate the underlying etiology of prostatic growth in older men, cause-and effect relationships have not been established. For example, androgens are a necessary but not a clearly causative aspect of BPH. Previously held notions that the clinical symptoms of BPH are due simply to a mass-related increase in urethral resistance are too simplistic. It is now clear that a significant portion of LUTS is due to age –related detrusor dysfunction. Bladder outlet obstruction itself may induce a variety of neural alteration in the bladder, which contributes to symptomatology. Moreover bothersome LUTS may be seen on men with polyuria, sleep disorders, and a variety of systemic medical conditions unrelated to the prostate- bladder unit. BPH is but one cause of the LUTS in aging men commonly, and probably incorrectly, referred to as prostatism. BPH is a classical age-related disease and present in 20% of men at the age of 40 years, with progression to 70% at the age of 60 years [7]. The clinical relevance of this disorder is underscored by the fact that up to 50% of elderly men develop lower urinary tract symptoms (LUTS) due to BPH/BPE, and that transurethral resection of the prostate remains one of the most frequent interventions in elderly men, with a lifetime risk for surgery of around 25–30% [7]. Histopathologically, BPH is characterized by an increased number of epithelial and stromal cells around the urethra with an excessive nodular growth localized to the points where ejaculatory ducts enter into the transitional or periurethral zones of the prostate. At the cellular level, alterations including basal cell hyperplasia (enlarged, hypertrophic basal cells), increased stromal mass (particularly the amount of smooth muscle cells), enhanced extracellular matrix deposition, reduced elastic tissue, more infiltrating lymphocytes around ducts, acinar hypertrophy and more luminal corpora amylacea and calcifications in the form of prostatic calculi [8]. Periurethral nodules in BPH compress the urethra and may cause urodynamic obstruction. Such an obstruction can lead to LUTS as well as secondary changes that may ultimately require surgical intervention, such as bladder hypertrophy, urinary tract infection development of post-void residual volume, upper urinary tract changes and urinary retention. The observed increase in cell number may be due to epithelial and stromal proliferation or to impaired programmed cell death leading to cellular accumulation. Androgens, estrogens, stroaml, epithelial interactions, growth factors, and neurotransmitters may play a role, either singly or in combination in the etiology of the hyperplastic process. The prostate receives innervations from the sympathetic and the parasympathetic nerve system. The sympathetic system (noradrenergic nerves) is responsible for expelling prostatic fluid into the urethra during ejaculation [9], and the parasympathetic system (cholinergic nerves) increases the rate of secretion [10]. Moreover, the neuronal system has been shown to regulate prostatic function and growth [11]. Neuronal systems with effects on the prostate include the alpha adrenergic [12], the beta adrenergic [13] the cholinergic [14], the enkephalinergic [15], the peptidergic [16] and the nitrinergic system [13]..Sympathetic signaling pathways are important in the pathophysiology of LUTS, as reviewed subsequently. In addition, there is increasing evidence that sympathetic pathways may be important in the pathogenesis of the hyperplastic growth process. Alpha blockade, in some model systems can induce apoptosis [17,18] .α –adrenergic pathways can also modulate the smooth muscle cell phenotype in the prostate[19]. All the components of the rennin-angiotensin system are present in prostatic tissue and may be active in BPH [20]. The alpha-1-adrenoreceptor is the prime determinant for urethral resistance causing outflow obstruction and LUTS [21]. Based on this observation, an important cornerstone of medical management of LUTS due to BPH/BPE is based on alpha-1-adrenergic-receptor (adrenoreceptor) blockade to reduce urethral resistance. This study seeks to evaluate the effect of a new natural compound “tradamixina “ made of Alga Ecklonia Bicyclis, Tribulus Terrestris and D-Glucosamine plus Serenoa Repens in order to improve lower urinary tract symptoms. Seven phlorotannins were isolated and characterized from an edible marine brown alga Ecklonia Bicyclis, along with three common sterol derivatives (fucosterol, ergosterol, and cholesterol) according to the comprehensive spectral analysis of MS and NMR data. Compounds 7-phloro eckoland ,6,6′-bieckoll in phlorotannin derivatives were obtained for the first time with their respective high yields. Any bioactive report of compound Fucodiphloroethol G was found nowhere up to date [22]. Ecklonia bicyclis has radical scavenger activity 10-100 times more powerful than any other polifenol terrestris plants, which have only 3-4 fenolic and rings that are commonly considered among the most effective antioxidant molecules. Common polyphenols are soluble in water also and have a relatively short half-life introduced into the body. All phlorotannins had antioxidant properties in vitro, especially, compounds 6,6′-bieckol which showed significant activities compared to the other phlorotannins [23]. In the Ecklonia bicyclis there are molecules that are able to reduce the inflammatory response, partially neutralizing the inflammatory damage caused by ROS and in part by slowing the gaming lipoxygenase and inhibiting the formation of prostaglandin E2, a powerful inflammatory mediator. Jung Wk [24] revealed that Ecklonia bicyclis inhibits LPS-induced NO and PGE2 production in a concentration-dependent manner and inhibits inducible iNOS and COX-2 in BV2 microglia without significant cytotoxicity. Ecklonia bicyclis treatment significantly reduced NF-kB translocation and DNA-binding in LPS-stimulated BV2 microglia. This effect was mediated through the inhibition of the degradation of the inhibitor kappaB and by inhibition of the mitogen-activated protein kinase (MAPK) phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. This data also indicate that Ecklonia bicyclis extracts exert anti-inflammatory effects by suppressing proinflammatory cytokines. Collectively, these results suggest that Ecklonia bicyclis suppresses the induction of cytokines by LPS, as well as iNOS and COX-2 expression, by blocking NF-kappaB and MAPK activation. These findings provide mechanistic insights into the anti-inflammatory and neuroprotective actions of Ecklonia bicyclis in BV2 microglia [24]. Won -Kyo Jung[25] revealed that also only dieckol suppresses LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced the generation of proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Moreover, dieckol significantly reduced LPS-induced nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinases (MAPKs) activation, as well as reactive oxygen species (ROS) production. Taken together, the inhibition of LPS-induced NO and PGE2 production might be due to the suppression of NF-κB and p38 MAPK signal pathway and, at least in part, by inhibiting the generation of ROS. (25) S.-K. Kim revealed extracts reduced the concentrations of IL-4 and IL-5 by 66% and 84%, respectively, and resulted in a 73% reduction in the secretion of TNF-α . Ecklonia bicyclis extracts enhanced IFN-γ production, but not to a significant degree [26]. The protodioscin is a steroidal saponin, which is about 45% of the extract obtained from aerial parts of Tribulus Terrestris. Its particular steroidal structure has an androgen-mimetic action, binding and activating the receptor of testosterone. So this substance is able to increase the endogenous production of testosterone, dihydrotestosterone, a hormone luteinizing hormone (LH), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S). Because of these effects in experimental animals there is an increase in spermatogenesis and the frequency of matches. In the rabbit in particular it has been shown that the compound stimulates the release of nitric oxide (NO) by vascular endothelium of the corpora cavernosa thereby having a pro-erectile effect. The mechanism behind this effect appears to involve the pathway of steroid hormones. Although in humans protodioscin is used for the treatment of erectile dysfunction [27,28]. In a placebo-controlled study on a group of young volunteers serum levels of testosterone, androstenedione and luteinizing hormone were detected after administration of Tribulus Terrestris at doses of 10 and 20 mg / kg. After 4 weeks of treatment, these values were similar to those of untreated [29]. Biovis contains polymers of d-glucosamine and n-acetyl-d-glucosamine that act on both the non-adrenergic and non-colinergic system (NANC) and on the endothelial cell system as a strong nitric oxide synthetase (NOS) stimulator [30]. Serenoa Repens has been approved in France and Germany for treatment of BPH [31]. The mechanism of action of Serenoa Repens has been investigated in several in vitro or indirect in vivo studies and has not been definitively defined. The mechanism may include alteration of cholesterol metabolism, anti-oestrogenic, anti-androgenic (including 5a-reductase inhibitor activity), anti-inflammatory effects, and a decrease in available sex hormone binding globuline [32,34]. Methods An observational study was conducted from May 2011 to May 2012, at our Department of Urology. We enrolled 100 patients. Eligible men were ≥45 yrs. of age who had had LUTS/BPH for >6 mo at screening and with IPSS (The international Prostate symptom scores) ≥13 and maximum urinary flow rate (Qmax) ≥4 to ≤15 ml/s. Exclusion criteria were: drug assumption :α-blocker , Serenoa Repens , dutasteride, finasteride; hyper- and hypothyroidism, neurogenic syndrome (multiple sclerosis, multiple atrophy, Parkinson’s disease, tumors, stroke, disk disease, spinal cord disorders, polyneuropathy, uraemia), lower pelvic surgery (oncological pelvic surgery, lower urinary and genital tract surgery), prostate cancer. The compound “Tradamixina plus Serenoa Repens” (80 mg of Alga Ecklonia Bicyclis, 100 mg of Tribulus Terrestris and 100 mg of D-Glucosamine and N-Acetyl-D-Glucosamine plus 320 mg of Serenoa Repens) was administered daily for two months. At visit patients were evaluated by means of detailed medical urological history, clinical examination, laboratory investigations (total PSA), and instrumental examination like uroflowmetry. Efficacy measures included IPSS-International Prostate Symptom Scores (primary measure), BPH Impact Index (BII), and week 1 IPSS (mIPSS) questions beginning with ‘‘Since your last visit.’’ IPSS storage and voiding subscores, nocturia question (question 7), and IPSS Quality-of-Life (QoL) Index were also assessed. Measures were assessed at baseline and end point (12 wk or end of therapy) and also at screening, 1 and 4 wk for IPSS, and 4 wk for BII. The Patient and Clinician Global Impression of Improvement (PGI-I and CGI-I, respectively) instruments and the subject-rated Treatment Satisfaction Scale–BPH (TSS-BPH) [22], evaluated from 0% (greater) to 100% (lower) satisfaction, were administered at end point. Uroflowmetry was performed using standard calibrated devices at the screening, baseline, and end point visits. Valid Qmax measurements required prevoid total bladder volume (assessed by ultrasound) of ≥150 to ≤550 ml and voided volume (Vvoid) of ≥125 ml. Bladder capacity was calculated post hoc as the sum of Vvoid and postvoid residual (PVR) volume. Safety was evaluated based on subject-reported adverse events (AE), PVR, clinical laboratory parameters (total PSA). The primary efficacy objective was evaluating the change in total IPSS from baseline to end point. Efficacy was analysed in all subjects. At last continuous efficacy measures, uroflowmetry, and PVR were evaluated as a change from baseline to week 12/last-observation carried-forward end point. All other efficacy analyses (including all tradamixina results) were assessed at the 0.05 significance level without adjustment for multiplicity. A fixed-sequence testing procedure was implemented to control type I error in analyses of primary and key secondary outcomes for “Tradamixina plus Serenoa Repens” using the following pre-specified order: total IPSS at end point, total IPSS after 4 wk, BII at end point, mIPSS after 1 wk, and BII after 4 wk. Statistical significance was interpreted only if results of the preceding analysis were significant at the 0.05 level. Results were presented independent of the fixed sequence, as all end points tested under this procedure achieved statistical significance. All other efficacy analyses were assessed at the 0.05 significance level without adjustment for multiplicity. PGI-I- Patient Global Impression of Improvement- was analysed using the Cochran –Mantel-Haenszel test adjusted for baseline LUTS severity .Changes from baseline to end of therapy in Qmax, PVR, and clinical laboratory parameters were analysed using a ranked analysis of variance (ANOVA) with a term for treatment group. Treatment group differences for average urinary flow rate (Qave), Vvoid, and bladder capacity were performed as post hoc analyses. Results and discussion Mean age was 64 yrs. of age (10.2% ±75 yr of age). At randomisation, IPSS was ≥20 in 30% of subjects, and Qmax was <10 ml/s in 54%. The change from baseline to week 12 relative to “Tradamixina plus seronea Repens” in total IPSS was statistically significant p = 0.001 (Table 1). Least squares mean (LSmean) plus or minus standard error (SE) differences in IPSS were significant for “Tradamixina plus Serenoa Repens” at 1 wk (mIPSS: -1.5 ± 0.5; p = 0.003 ) and 4 wk (-2.2 ± 0.6; p < 0.001 ). Changes in IPSS subscores and nocturia are shown in table 1. Differences from baseline in BII were statistically significant for “Tradamixina plus Seronea Repens” ( p = 0.003), above all differences in BII were also significant at 4 wk (LSmean ± SE: -0.8 ± 0.2;p < 0.001 ). For the IPSS QoL Index, significant improvements at 12 wk were reported ( p = 0.022).(Table 2). The TSSBPH overall satisfaction score at end point was significantly low (indicating higher satisfaction) ( p = 0.005).In the distribution of subjects over the PGI-I and CGI-I response categories were significant for “Tradamixina plus Seronea Repens” (PGI-I: p = 0.001; CGI-I: p = 0.004). More subjects and their clinicians perceived improvements in LUTS at end point . Improvements in Qmax were significantly great (p = 0.009) (Table 3). For PVR, mean reductions from were observed with “Tradamixina plus Serenoa Repens”, but were not statistically significant. We also observed a decrease of total PSA (Table 4). “Tradamixina plus Serenoa Repens” improved total IPSS score and PGI-I and CGI-I scores because the Ecklonia bicyclis with its anti-inflammatory action and antioxidant effects, suppress LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced the generation of proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Moreover, dieckol significantly reduced LPS-induced nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinases (MAPKs) activation, as well as reactive oxygen species (ROS) production [25]. It improved the bother symptoms. BPH is associated with changes of innervations, and biological mediator production and release in the central zone of the prostate [35]. In particular, there is a decrease of nerves of the enkephalinergic [36] and nitrinergic systems [37,38] both mediating smooth muscle cell relaxation [37]. Compared to normal tissue, nNOS expression has been observed to decrease in the transitional zone of BPH tissue. On the other hand, inducible NOS (iNOS), produced after stimulation by immune and inflammatory cytokines and free radicals, has been reported to increase selectively in the stroma of patients suffering from BPH [36]. This observation is probably due to increased proinflammatory conditions in BPH. The importance of the NOS system for the prostate stroma is further supported by experiments with constitutive active NOS dependent guanylylcyclases. cGMP analogues have been shown to activate the Proteinkinase G II and to inhibit proliferation of human smooth muscle cells in vitro [37]. Two enzymes, the neuronal (NOS-I) and the endothelial (NOS-III) isoforms, are Ca2_-dependent and constitutively expressed.The third enzyme is an inducible Ca2_-independent isoform (NOS-II), expressed after stimulation with Escherichia coli lipopolysaccharide (LPS) and/or different cytokines, such as interferon-γ (IFNγ), interleukin-1, or tumor necrosis factor- NOS-II induction occurs at the transcriptional level and is mediated by the early activation of some nuclear transcriptional factors, including NFkB [38]. A large body of experimental evidence suggests that physiological levels of NO, similar to those produced by the basal activity of NOS-I or NOS-III, prevent induction of NOS-II mRNA expression through the suppression of NFkB activation [39,40]. As a consequence, NOS-II gene expression takes place after LPS/cytokine stimulation, provided that the NOS-I- or NOS-III-generated NO is reduced below a threshold value in a short time [41]. A recent report [42,43] shows that NOS-II inducers (e.g. LPS and IFNγ) consistently elicit a rapid inactivation of NOS-I by tyrosine phosphorylation, an event leading to a decrease of basal NO levels. A recent study [44] reports that inhibition of NOS-I can be achieved via activation of cytosolic phospholipase A2 (cPLA2), a large molecular mass member of the family of PLA2 enzymes. The activities of NOS-I and cPLA2 are both regulated by increases in the intracellular concentration of free Ca2 ([Ca2]i). Not surprisingly, enhancing the [Ca2]i caused a parallel increase in both activities and accumulation of respective products, NO and arachidonic acid (AA). Inducible nitric oxide synthase (iNOS) is expressed in a variety of cell types, in particular in inflammatory cells, in response to diverse pro-inflammatory stimuli. This process requires critical levels of arachidonic acid (AA), generated by constitutive phospholipase A2 (PLA2), promoting tyrosine kinase-dependent phosphorylation, and inhibition, of constitutive NOS. Lowering basal NO levels is indeed critical for the activation of NF-κB, and thus for the expression of genes (e.g. iNOS) regulated by this transcription factor. It is interesting to note that NO and AA, two small lipid soluble molecules, rapidly cross the plasma membrane thereby allowing the triggering of the above responses in distal cells. That is, constitutive NO might inhibit NF-κB activity in the same cells (e.g. astrocytes) in which it is generated, as well as in other cells that do not express constitutive NOS (e.g. microglia). NO from cells unable to respond to pro-inflammatory stimuli (e.g. neurons) will also contribute to these effects. Along the same line, AA released by pro-inflammatory molecules in specific cell types (e.g. astrocytes) might suppress constitutive NOS activity in the same cells as well as in other cells (e.g. neurons). Thus, AA produced at the very early stages of the inflammatory response is a likely critical signal switching the regulation of the “NO tone” from physiological (i.e. mediated by constitutive NOS) to pathological (i.e. mediated by iNOS). This second phase of the inflammatory response is often accompanied by the onset of deleterious effects in the tissue in which a critical role is played by iNOS-derived NO (directly or indirectly, i.e. via formation of peroxynitrite) as well as by products of the AA cascade. In summary, the relative amounts of NO and AA, released by their constitutive enzymes, produce autocrine and paracrine effects regulating the onset of an inflammatory response in which, in addition to other factors, NO and AA are extensively released by their inducible enzymes [45]. Biovis contains polymers of d-glucosamine and n-acetyl-d-glucosamine that act both on the non-adrenergic and non-colinergic system (NANC) and on the endothelial cell system as a strong nitric oxide synthetase (NOS) stimulator [46]. This explains why there is an improvement of Qave and Qmax. In fact while α-blocker drugs facilitated the opening of the bladder neck thanks to the presence of alpha receptors, Tradamixina improved the NOS action on its receptor, so for the reasons above also improved LUTS. Serenoa Repens also has anti-inflammatory effects. As described above, α1-adrenoceptor antagonists are commonly used in the treatment of men with voiding symptoms (urinary obstruction, pollakiuria and urinary incontinence) secondary to BPH. Goepel et. Al. have shown that Serenoa Repens might have α1-adrenoceptor inhibitory properties. Serenoa Repens significantly affects pharmacological receptors, such as the α1-adrenoceptor and the muscarinic receptor in the lower urinary tract, to relieve the irritative and obstructive symptoms of dysuria due to BPH and LUTS [34]. In addition to traditionally used medications, like α1-adrenoceptor antagonists, antimuscarinics, 5α-reductase inhibitors, and phytotherapy, several new therapeutic agents, such as selective β3-adrenoceptor agonists, are potentially useful for treating LUTS suggestive of BPH, particularly for storage symptoms secondary to outflow obstruction [47]. Thus, the effects of SPE on these receptors in the lower urinary tract might be pharmacologically relevant. To date, more than 11 placebo-controlled trials and 4 active-controlled trials with SPE in men with BPH have been conducted. Patient numbers were usually limited and the evaluation periods were relatively short, so it would be difficult to evaluate the effect of SPE and ascertain the efficacy of SPE in BPH patients. However, some placebo-controlled studies and comparisons to α1-blockers have recently been conducted with relatively long-term treatments and sufficient numbers of patients [48-50]. BPH causes dysuria and residual urine via a mechanical stoppage due to hypertrophy of prostatic tissue and via a functional stoppage caused by α1-adrenoceptor hypertonia of prostatic smooth muscle. Previous studies have demonstrated that Serenoa Repens had a number of pharmacological effects: 1) an antiandrogenic effect — inhibition of 5α-reductase I and II and inhibition of binding of dihydrotestosterone (DHT) to the cytosolic androgen receptors, 2) an anti-inflammatory effect, 3) an anti-proliferative effect, and 4) significant binding of pharmacological receptors existing in the lower urinary tract. In BPH there is a decreased ratio between androgen and estrogen, and tribulus terrestris by increasing total testosterone serum level restores the ratio. Table 1 International prostate symptom score (IPSS) total,storage and voiding subscores and nocturia question (IPSS question 7). IPSS total: Change from baseline ,LS mean ±SE -6,3±0,5 p= 0.001 Storage subscore 1 Change from baseline ,LS mean ±SE -2,2±0,2 p= 0.055 Voiding subscore2 Change from baseline ,LS mean ±SE -4,1± 0.3 p< 0,001 Nocturia question3 Change from baseline ,LS mean ±SE -0,5± 0.13 p= 0,080 1) IPSS question: 2+4+7 2) IPSS question: 1+3+5+6 3) IPSS question:7 Table 2 International Prostate Symptom Score Quality of Life Index, Treatment Satisfaction scale, and Patient or Clinician Global Impression of Improvement Scores at 12 wks. compared to baseline IPSS total Qol index Change from baseline ,LS mean ±SE -1,3±0,1 TSS-BPH overall mean±SD 26,9±17,7 PGI-I no. Very much better 58 Much better 35 A little better 5 No change 2 A little worse 0 Much worse 0 Very much worse 0 CGI-I no. Very much better 41 Much better 30 A little better 15 No change 14 A little worse 0 Much worse 0 Very much worse 0 Table 3 Uroflowmetry and postvoid residual volume Qmax ml/s: Baseline 9.9±3.6 Mean change 2,4±5.5 Q ave ml/s Baseline 5,9±2,1 Mean change 1,6±3,2 V void ml Baseline 257.7±94.3 Mean change 8.2±101.5 Bladder capacity ( ml ) Baseline 310±109.4 Mean change 3,5±113.8 PVR (ml) Baseline 53,3±50,4 Mean change -4,6±47,0 Qmax = maximum flow rate; Qave = average flow rate; Vvoid = voided volume; PVR = postvoid residual volume. Table 4 Total PSA Baseline 3.6±2,4 Mean change 1,1±0,2 Conclusion The daily treatment with a new compound “Tradamixina” (100 mg of Alga Ecklonia Bicyclis, 80 mg of Tribulus Terrestris and 80 mg of D-Glucosamine and N-Acetyl-D-Glucosamine) plus 320 mg Serenoa Repens for two months improved the male sexual function. It improved the bother symptoms which affect the patient’s quality of life, improved uroflowmetric parameters, and we observed a decrease of serum PSA level. These effects are due to its antioxidant, anti-aging action, and the bother symptoms due to its anti-inflammatory action. Infact it neutralizes the action of ROS, LPS, COX2, NFkβ, probably also reducing the concentrations of TNF-α, MMP-1. The decrease of PSA is due to the anti-inflammatory action. This result can be the basis for future research. List of abbreviations AA: Arachidonic acid; AE: Adverse events; BII: BPH Impact Index; BPH: Benign prostatic hyperplasia; CGI-I: The Clinician Global Impression of Improvement; COX-2: Cyclooxygenase-2; cPLA2: Cytosolic phospholipase A2; DHEA: Dehydroepiandrosterone; DHEA-S: Dehydroepiandrosterone sulfate; IL-1β: Interleukin; iNOS: Inducible nitric oxide synthase; IPSS: International Prostate Symptom; IPSS: International Prostate Symptom; LH: Hormone luteinizing hormone; LUTS: Lower urinary tract symptoms; MAPKs: Mitogen-activated protein kinases; MMP-1: Matrix metalloproteinase-1; NANC: Non-adrenergic and non-colinergic system; NF-κB: Nuclear factor κB; NO: Nitric oxide; NOS: Nitric oxide synthetase; NOS-I: Neuronal Nitric oxide synthetase isoforms I; NOS-III: Endothelial Nitric oxide synthetase isoforms III; PGE2: Prostaglandin E2; PGI-I: Patient Global Impression of Improvement; PGI-I: The Patient and Clinician Global Impression of Improvement; PVR: Postvoid residual; Qave: Urinary flow rate; QoL: Quality-of-Life; ROS: Reactive oxygen species; TNF-α: Tumor necrosis factor; TSS-BPH: Treatment Satisfaction Scale–BPH; Vvoid: Voided volume Competing interests The authors declare that they have no competing interests. Authors’ contributions FI, DP: conception and design, interpretation of data, given final approval of the version to be published; EI, AR, GR: acquisition of data, drafting the manuscript, given final approval of the version to be published; BA: critical revision, interpretation of data, given final approval of the version to be published. Acknowledgements This article has been published as part of BMC Surgery Volume 12 Supplement 1, 2012: Selected articles from the XXV National Congress of the Italian Society of Geriatric Surgery. 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BMC Surg. 2012 Nov 15; 12(Suppl 1):S22
==== Front BMC SurgBMC SurgBMC Surgery1471-2482BioMed Central 1471-2482-12-S1-S110.1186/1471-2482-12-S1-S1Research ArticlePeripheral blood mono-nuclear cells implantation in patients with peripheral arterial disease: a pilot study for clinical and biochemical outcome of neoangiogenesis Amato Bruno [email protected] Rita [email protected] Corte Gianni Antonio [email protected] Giovanni [email protected] Tommaso [email protected] Guido [email protected] Roberto [email protected] Antonio [email protected] Giovanni [email protected] Pio [email protected] Alessandro [email protected] Claudio [email protected] Department of General, Geriatric, Oncologic Surgery and Advanced Technologies University of Naples Federico II, Via S. Pansini,5 – 801311 Napoli, Italy2 Department of Medicine and Surgery, University of Salerno, Italy3 Dept of Medical and Surgical Sciences, University Magna Graecia, Catanzaro, Italy4 Department of Molecular Medicine, Padova University Hospital, Italy, Via Giustiniani n.2, 35126 Padova, Italy2012 15 11 2012 12 Suppl 1 Selected articles from the XXV National Congress of the Italian Society of Geriatric SurgerySilvestro Canonico, Bruno Amato and Beatrice SensiThis supplement has not been supported by sponsorship or other external funding.S1 S1 Copyright ©2012 Amato et al; licensee BioMed Central Ltd.2012Amato 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 Substantial progresses in the management of peripheral arterial disease (PAD) have been made in the past two decades. Progress in the understanding of the endothelial-platelet interaction during health and disease state has resulted in better antiplatelet drugs that can prevent platelet aggregation, activation and thrombosis during angioplasty and stenting. A role in physiological and pathological angiogenesis in adults has been recently shown in bone marrow–derived circulating endothelial progenitors (BM-DCEPs) identified in the peripheral blood. These findings have paved the way for the development of therapeutic neovascularization techniques using endothelial progenitors. Methods This pilot study includes five patients, aged 60 to 75, with a history of claudication and recruited from September 2010 to February 2011 at the A.O.U. Federico II of Naples. PBMNCs have been implanted three times in the limb with the worst ABI value in all the patients included in the study. The clinical follow up was performed during the subsequent 12 months from the beginning of the treatment. Results In four patients there was a regression of ulcerative lesions. One patient’s condition improved after the first implantation but later did not respond to the further treatments. All patients achieved a pain relief as judged by the numeric pain scale. Pain relief remained satisfactory in three patients for one year. Pain gradually returned to the pre-treatment level in two patients. All patients referred an ameliorating in their quality of life expressed even by an improvement in claudication free walking distance. These improvements are reflected also by intra-arterial digital subtraction angiography (IADSA) that shows an improvement of arterial vascularization. Conclusions The data from this study suggest an efficacy of BM-DCEPs implantation in terms of improvement of the vascularization and quality of life in patients affected by Peripheral Arterial Disease. Nevertheless a double-blind placebo-controlled study is needed to confirm our findings. 10-11 May 2012 XXV National Congress of the Italian Society of Geriatric Surgery Padova, Italy ==== Body Background Peripheral arterial disease (PAD) is a pathologic condition associated with arteriosclerosis. The symptoms of PAD mainly affect the lower limbs resulting in intermittent claudication and rest pain. Strategies to treat the limb ischemia and its resulting symptoms include both pharmacologic therapy and invasive procedures. Despite the available therapies, 25% of patients still progress each year to limb amputations. Recently, bone marrow derived circulating endothelial progenitors (BM-DCEPs) have been identified in peripheral blood showing a role in physiological and pathological angiogenesis in the elderly. Preclinical studies showed BM-DCEPs to be useful when implanted in the ischemic limb for treatment of PAD. Based on the above mentioned observation, peripheral blood mononuclear cells implantation (PB-MNCs) has been used as therapeutic strategy for critical limb ischemia (CLI). Although encouraging results have been obtained by using those therapies, the underlying mechanism is still not completely known. This is based on stimulation of angiogenesis by extracellular and cellular components. This pilot study has been conducted to evaluate the efficacy of implanted PBMNCs on clinical outcomes in patients at a symptomatic stage of PAD. We also focused on molecular markers of neo-angiogenesis to elucidate the real mechanism underlying the creation and stabilization of neo-vessels and in which measure the circulating endothelial progenitors (CEPs) and muscle cells are involved. Methods Five patients (three males) aged 60 to 75 (mean 65) with a history of claudication were recruited from September 2010 to February 2011 at the A.O.U. Federico II of Naples to participate in this pilot study, according to the Ethical Committee of Federico II University of Naples. Written informed consent was obtained before study participation. Patients who met the following inclusion/exclusion criteria were eligible for inclusion: those with symptomatic bilateral PAD (Fontaine scale ≥IIB - CFW distance ≤ 100 mt), aged 60 -75, where PAD has been diagnosed according to the clinical criteria and ABI < 0.6 (calculated as the worst value recorded at tibial anterior or posterior artery), with one or more stenosis of at least 50% in an artery of the lower limbs shown by duplex, angio-RM or intra-arterial digital subtraction angiography (IADSA) not eligible for endovascular revascularization treatments and with at least 2 comorbidities (e.g., hypertension, hyperlipaemia, obesity and/or carotid, coronary obstructions). Exclusion criteria were: a) estimated survival less than 6 months; b) acute stage of severe limb ischemia with severe inflammatory process affecting the patient’s life that required limb amputation to avert grave outcome; c) lymphopenia and/or thrombocytopenia and/or hemophilia; d) diabetes; e) chronic inflammatory diseases; f) connective tissues diseases; g) acute infectious processes; h) fever, physical trauma or surgery in the previous 45 days; i) acute illness, such as acute coronary or limb ischemia within 16 weeks; j) cancer. A detailed medical history was compiled for all patients with special attention given to cardio-vascular risk factors. The presence of arterial hypertension was defined with a blood pressure ≥ 140/90 mmHg in at least two measurements or current treatment with anti-hypertensive drugs. The presence of dyslipidemia was defined as total cholesterol >200 mg/dl, LDL-cholesterol >100 mg/dl, triglycerides >150 mg/dl, or current treatment with lipid-lowering drugs. The presence of diabetes was defined by concentrations of fasting plasma glucose ≥ 126 mg/dl or current treatment with oral antidiabetics and/or insulin. All of the patients included in the study were treated three times with PBMNCs (one injection every two months for a 6 month treatment) implantation in the limb with the worst ABI value. Tissue samples from the gastrocnemius of the interested leg were collected before the first cells implantation (T1) and before the third cycle of cells implantation to detect the markers of neoangiogenesis reported in Fig. 1. Blood samples were collected at the following times: T0 – enrollment; T1 before the implantation; 1 and 2 weeks and then 1 month after the first implantation; T2 before the second implantation; 1 and 2 weeks and then 1 month after the second implantation; T3 before the third implantation; 1 and 2 weeks and then 1 month after the third implantation. Fig. 1 Follow up scheme to evaluate the patient’s response to the treatment. The clinical follow up was performed during the subsequent 12 months from the beginning of the treatment at the followings time points: T0 – enrolment; 1 and 2 weeks and then 1 month after the first implantation; 1 and 2 weeks and then 1 month after the second implantation; 1 and 2 weeks and then 1 month after the third implantation; after 12 months from the time of enrolment. The following parameters were considered: 1) clinical history reporting a) onset of complication during the follow up, b) require of specific surgical treatment for PAD, c) return or progression of symptoms of claudication, d) evaluation of trophic lesions [1] documented also by photography, e) evaluation of the pain (using the pain numeric scale 0-10); f) a multi-parametric questionnaire on the quality of life analysing the previous 4 weeks named SF-36 [2]; 2) improvement in walking (Fontaine scale) measured as claudication-free walking distance (CFWD) and maximal walking distance (MWD), using the Skinner-Gardner protocol and the 6-minute walk distance test; 3) measurement of the ABI (ankle-brachial pressure index or Windsor index); 4) pheripheral arterial diameter and blood flow characteristics (peak, volume, pulse systolic rates of blood flow, indices of vessels stiffness) by Duplex Ultrasonography; 5) evolution of the stenosis and neoangiogenesis documented by intra-arterial digital subtraction angiography (IADSA) (Fig.1). Results During the one year follow-up no complications or need of surgical treatments were registered. In four patients there was a regression of ulcerative lesions after the second treatment with a complete resolution of the lesions one month after the third treatment. A 73 year old male patient condition improved after the first implantation but then did not respond to further treatments. In this patient necrosis was seen in two sites; on the tips of the left big toe and middle fingers and in three sites on the right leg. The necrotic tissue on the top of the left big toe fell off spontaneously and the ulcer undermined deeper in the necrotic area epithelialized and healed eventually after one year. Unfortunately, he died 14 months after the enrolment due to a cardiovascular complication. Relief of pain as judged by the numeric pain scale was achieved in all patients in a week and continued up to one month in four patients. Pain relief remained satisfactory in three patients for one year. However pain gradually returned to the pre-treatment level in two patients. All patients referred an ameliorating in their quality of life expressed even by an improvement in claudication free walking distance more evident in three of five patients. The ABI surprisingly did not reach the expected improvement, remaining in a mean of 0.6 (mean baseline values 0.4), and not showing a direct relation between the clinical features and the instrumental detections. Therefore, We should be careful when using ABI as follow up and as a diagnostic parameter to monitor neo-angiogenesis. However the number of patients is too small to assume that our results are definitive. IADSA in three patients showed an apparent increase of visible arteries after treatment (Figure 2). Such a long-term effect could be obtained to bona fide angiogenesis induced by repeated implantation of PB-MNCs. Fig. 2 Left, at enrolment, Right, after treatment. Discussion and conclusions Peripheral vascular disease (PVD) is known to affect 10% to 15% of the adult population in developed countries and is often associated with coronary artery disease. Arteriosclerosis obliterans (ASO) is the most common cause of PVD affecting the lower limbs. The two cardinal symptoms of limb ischemia are intermittent claudication and rest pain. Intermittent claudication (IC), defined as a symptomatic deficiency in blood supply to the exercising muscle that is relieved with rest, is generally a reliable indicator of occlusive arterial disease. This disorder results from an imbalance between supply and demand of blood flow that fails to satisfy ongoing metabolic requirements. Rest pain occurs in patients with critical limb ischemia and often coincides with ischemic ulceration and/or gangrene. Treatment of PVD includes pharmacotherapy, percutaneous transluminal angioplasty, and vascular surgery. The treatment chose depends on the severity of symptoms and the arteries involved. However, 30% to 50% of patients with critical limb ischemia require limb amputation within one year because of a poor response to treatment [3]. Recent progress in understanding the mechanisms underlying vascular formation in adults as well as during embryogenesis has opened up a therapeutic avenue for patients without any current options [4]. Initial therapeutic approaches were aimed at delivering angiogenic factors, such as vascular endothelial growth factors (VEGF) and fibroblast growth factor-2, to ischemic tissues by using recombinant proteins or vectors encoding these factors [5,6]. A number of preclinical studies reported improvement of perfusion by such methods in animal models with limb ischemia [7,8]. Although the initial nonrandomized clinical trials showed beneficial effects, the results of controlled clinical trials were not consistent. More recently, bone marrow–derived circulating endothelial progenitors (BM-DCEPs) were identified in the peripheral blood [9,10] and have been shown to contribute to both physiological and pathological angiogenesis in adults [11,12]. These findings have led to the development of therapeutic neovascularization techniques using endothelial progenitors. Preclinical studies indicated that implantation of bone marrow mononuclear cells (BM-MNC), which contain endothelial progenitors, into ischemic limbs was very effective [13-15]. The results of the first clinical trial showed that implantation of BM-MNC significantly improved the tissue oxygen concentration and blood flow in ischemic limbs, resulting in a decrease of rest pain and the involution of ischemic ulcers [16]. Although promising results have been obtained, the mechanism by which cell therapy improves limb ischemia is largely unknown. Because direct incorporation of implanted cells into newly formed vessels is reported to be relatively rare, it has been assumed that angiogenic factors secreted by implanted cells are responsible for the efficacy of cell therapy [17,18]. Implantation of mononuclear cells increased the production of the angiogenic cytokines in muscle cells. A deficiency of the angiogenic cytokines in muscle cells blunted the ability of implanted cells to increase vascularization, suggesting that muscle cells and not mononuclear cells were important as a source of the angiogenic cytokines. Subsequently, Tateno et al. discovered that angiogenic cytokines, especially IL-1beta, were associated with the response to treatment. Many previous studies by other groups suggested that angiogenic factors secreted by implanted cells play a critical role in therapeutic neovascularization [17,18]. In contrast, Tateno's group in vitro and in vivo studies demonstrated that the implanted mononuclear cells did not secrete sufficient cytokines for neovascularization but, instead, stimulated muscle cells to produce IL-1beta. This is consistent with the observation that most of the implanted cells disappeared from the ischemic tissues as early as 3 days after implantation, which is before the reconstruction of the vascular system started. Thus, it is likely that muscle cells but not implanted cells are a major source of angiogenic cytokines in ischemic limbs [19]. Recently, a Japanese study analyzed the long-term outcome of therapeutic neovascularization with PB-MNCs. Overall, improvement of ischemic symptoms was observed in 60% to 70% of the treated patients. The annual major amputation rate decreased to 10%, and the mortality rate was reduced to 20% at 2 years and 30% at 3 years in their patients. Their results, together with previous reports suggest that the performance of therapeutic neovascularization with PB-MNCs might be safe and effective for patients with critical limb ischemia. Although this study was not placebo-controlled and therefore cannot assess the efficacy and safety of cell therapy only with their results, they conclude that therapeutic angiogenesis with PB-MNCs is a safe and potentially effective treatment for critical limb ischemia [20]. Although the results of this pilot study suggest an efficacy of BM-DCEPs implantation in terms of improvement of the vascularization and quality of life in patients affected by peripheral arterial disease, double-blind placebo-controlled studies are needed to confirm our findings. Such a study is currently in progress. List of abbreviations CEAP: (Clinical-Etiology-Anatomy-Pathophysiology); CHIVA: Cure Conservatrice et Haemodinamique de l’Insuffisance Veineuse en Ambulatorie procedure; LMWH: Low Molecular Weight Heparin Competing interest The authors declare that they have no competing interests. Authors’ contributions BA: conception and design, interpretation of data, given final approval of the version to be published; RC: acquisition of data, drafting the manuscript, given final approval of the version to be published; ADC: acquisition of data, drafting the manuscript, given final approval of the version to be published; GM: acquisition of data, drafting the manuscript, given final approval of the version to be published; TB: acquisition of data, drafting the manuscript, given final approval of the version to be published; GC: acquisition of data, drafting the manuscript, given final approval of the version to be published; RR: acquisition of data, drafting the manuscript, given final approval of the version to be published; AB, CT: acquisition of data, drafting the manuscript, given final approval of the version to be published; GA, AP: conception and design, given final approval of the version to be published. Acknowledgements This article has been published as part of BMC Surgery Volume 12 Supplement 1, 2012: Selected articles from the XXV National Congress of the Italian Society of Geriatric Surgery. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcsurg/supplements/12/S1. ==== Refs Procházka V Gumulec J Jalůvka F Salounová D Jonszta T Czerný D Krajča J Urbanec R Klement P Martinek J Klement GL Cell therapy, a new standard in management of chronic critical limb ischemia and foot ulcer Cell Transplantation 2010 19 1413 24 10.3727/096368910X514170 20529449 Norstanstig J Gelin J Hensater M Taft C Walking performance and health-related quality of life after surgical or endovascular invasive versus non-invasive treatment for intermittent claudication--a prospective randomised trial Eur J Vasc Endovasc Surg 2011 42 2 220 7 10.1016/j.ejvs.2011.02.019 21397530 Dormandy JA Rutherford RB Management of peripheral arterial disease (PAD). TASC Working Group. TransAtlantic Inter-Society Concensus (TASC) J Vasc Surg 2000 31 S1 S296 10666287 Carmeliet P Mechanisms of angiogenesis and arteriogenesis Nat Med 2000 6 389 395 10.1038/74651 10742145 Takeshita S Zheng LP Brogi E Kearney M Pu LQ Bunting S Ferrara N Symes JF Isner JM Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model J Clin Invest 1994 93 662 670 10.1172/JCI117018 7509344 Harada K Grossman W Friedman M Edelman ER Prasad PV Keighley CS Manning WJ Sellke FW Simons M Basic fibroblast growth factor improves myocardial function in chronically ischemic porcine hearts J Clin Invest 1994 94 623 630 10.1172/JCI117378 7518840 Ferrara N Alitalo K Clinical applications of angiogenic growth factors and their inhibitors Nat Med 1999 5 1359 1364 10.1038/70928 10581076 Yla-Herttuala S Alitalo K Gene transfer as a tool to induce therapeutic vascular growth Nat Med 2003 9 694 701 10.1038/nm0603-694 12778168 Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Witzenbichler B Schatteman G Isner JM Isolation of putative progenitor endothelial cells for angiogenesis Science 1997 275 964 967 10.1126/science.275.5302.964 9020076 Shi Q Rafii S Wu MH Wijelath ES Yu C Ishida A Fujita Y Kothari S Mohle R Sauvage LR Moore MA Storb RF Hammond WP Evidence for circulating bone marrow-derived endothelial cells Blood 1998 92 362 367 9657732 Asahara T Masuda H Takahashi T Kalka C Pastore C Silver M Kearne M Magner M Isner JM Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization Circ Res 1999 85 221 228 10.1161/01.RES.85.3.221 10436164 Rafii S Lyden D Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration Nat Med 2003 9 702 712 10.1038/nm0603-702 12778169 Ikenaga S Hamano K Nishida M Kobayashi T Li TS Kobayashi S Matsuzaki M Zempo N Esato K Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model J Surg Res 2001 96 277 283 10.1006/jsre.2000.6080 11266284 Shintani S Murohara T Ikeda H Ueno T Sasaki K Duan J Imaizumi T Augmentation of postnatal neovascularization with autologous bone marrow transplantation Circulation 2001 103 897 903 10.1161/01.CIR.103.6.897 11171801 Li TS Hamano K Suzuki K Ito H Zempo N Matsuzaki M Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats Am J Physiol Heart Circ Physiol 2002 283 H468 H473 12124190 Tateishi-Yuyama E Matsubara H Murohara T Ikeda U Shintani S Masaki H Amano K Kishimoto Y Yoshimoto K Akashi H Shimada K Iwasaka T Imaizumi T Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial Lancet 2002 360 427 435 10.1016/S0140-6736(02)09670-8 12241713 Urbich C Dimmeler S Endothelial progenitor cells: characterization and role in vascular biology Circ Res 2004 95 343 353 10.1161/01.RES.0000137877.89448.78 15321944 Kinnaird T Stabile E Burnett MS Epstein SE Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences Circ Res 2004 95 354 363 10.1161/01.RES.0000137878.26174.66 15321945 Tateno K Minamino T Toko H Akazawa H Shimizu N Critical roles of muscle-secreted angiogenic factors in therapeutic neovascularization Circ Res 2006 98 1194 1202 10.1161/01.RES.0000219901.13974.15 16574905 Moriya Junji Minamino Tohru Tateno Kaoru Shimizu Naomi Kuwabara Yoichi Sato Yasunori Saito Yasushi Komuro Issei Long-term outcome of therapeutic neovascularization using peripheral blood mononuclear cells for limb ischemia Circ Cardiovasc Interv 2009 2 245 254 10.1161/CIRCINTERVENTIONS.108.799361 20031722
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BMC Surg. 2012 Nov 15; 12(Suppl 1):S1
==== Front Case Rep TransplantCase Rep TransplantCRIM.TRANSPLANTATIONCase Reports in Transplantation2090-69432090-6951Hindawi Publishing Corporation 2319825610.1155/2011/368623Case ReportSuccessful Treatment of Refractory Wart with a Topical Activated Vitamin D in a Renal Transplant Recipient Moscarelli Luciano *Annunziata Filomena Mjeshtri Anduela Paudice Nunzia Tsalouchos Aris Zanazzi Maria Bertoni Elisabetta Renal Unit, Careggi University Hospital, Viale Pieraccini 18, 50139 Florence, Italy*Luciano Moscarelli: [email protected] Editor: K. Kawamura 2011 3 1 2012 2011 36862317 11 2011 26 12 2011 Copyright © 2011 Luciano Moscarelli et al.2011This 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.Warts are benign proliferations of the skin and mucosa caused by infection with human papillomavirus. They are commonly treated with destructive modalities such as cryotherapy with liquid nitrogen, local injection of bleomycin, electrocoagulation, topical application of glutaraldehyde, and local and systemic interferon-β therapy. These treatment modalities often cause pain and sometimes scarring or pigmentation after treatment. We herein report a case with a right index finger wart, which was successfully treated with a topical activated vitamin D. ==== Body 1. Introduction Warts are a benign proliferation of the skin and mucosa caused by infection with human papillomavirus (HPV). HPV is ubiquitous, and renal transplant recipients (RTRs) may never totally clear HPV infections, which are the most frequently recurring infections. This infection is important because of its link to the development of certain skin cancers, in particular, squamous cell carcinoma. Regular surveillance, sun avoidance, and patient education are important aspects of the management strategy. Warts are usually treated by traditional destructive modalities such as cryotherapy with liquid nitrogen, local injection of bleomycin, electrocoagulation, topical application of glutaraldehyde, and local and systemic interferon-β therapy [1–3]. However, the tolerance of patients to these treatment modalities is poor, because they often cause pain, especially in children, and sometimes scarring or pigmentation after treatment. No treatment has been uniformly effective, and warts are often refractory, especially in immunocompromised patients where their quality of life is threatened. Here, we report an RTR with a right index finger wart, which was successfully treated with a topical activated vitamin D. 2. Case Report A 41-year-old woman with unknown native kidney disease received a renal transplant from deceased donor in January 2009. She was on treatment with immunosuppressive therapy based on tacrolimus, steroid, and mycophenolate mofetil. She presented 19 months after transplantation a wart on right index finger (Figure 1(a)) which obtained partial clearance after 6-month treatment with cryotherapy and elettrocoagulation but it regrew rapidly. We attempted treatment with simple local application of activated vitamin D (gauze wet with calcitriol 0.5 μg solution) at least two times a day (during the morning and the next night). The patient was advised to reapply a gauze wet with calcitriol 0.5 solution after each handwashing. Three months later, the wart disappeared without pain or other side effects (Figure 1(b)), and it has not recurred within the 9 months since the disappeared. The medication was well tolerated. No adverse effects or abnormal serum test results, including elevated serum calcium level, were observed. 3. Discussion Incidence of warts in RTRs varies from 8% to 55% depending on the patient's characteristics, the time since transplantation, and immunosuppressive protocols [4]. Skin biopsy and identifying the type of HPV are required to diagnose precisely. Unfortunately, we could not take biopsies from the affected lesion. Therefore, our diagnosis was only based on the clinical appearance. The vitamin D system has multiple physiological and pharmacological effects mediated by action of the vitamin D receptors (VDRs). Recently, VDR activators (VDRAs) have been shown to inhibit cell replication and have immunomodulatory properties. An important observation was reported which suggested that toll-like receptor (TLR) activation of human macrophages upregulated expression of vitamin D receptor and vitamin D-1-hydroxylase genes, leading to induction of the antimicrobial peptide [5]. This suggests an association of TLRs and vitamin-D-mediated innate immunity [5]. Previously the topical application of vitamin D derivatives has become a first-line therapy in the routine treatment of chronic plaque psoriasis as well as for palmoplantar keratosis [6]. A combination of isotretinoin and calcitriol has been reported as the most effective therapy for HPV-associated precancerous and cancerous skin lesions [7]. The effect of vitamin D derivatives was speculated to be derived from its potential to regulate epidermal cell proliferation and differentiation and to modulate cytokine production [8]. Our case report demonstrates for the first time to our knowledge that local application of activated vitamin D is an effective and well-tolerated supplementary treatment of recalcitrant wart. A new focus of interest is the levels of activated vitamin D to be reached, particularly in relation to local cellular growth regulation [9]. These levels may provide an explanation for the striking effect of the activated vitamin D and the minimal effect of its the simple application seen in this study. In spite of the proposed mechanisms, any treatment of warts may be confounded by a potent placebo effect. Hence, we realize the need for further placebo-controlled studies before any final conclusions can be reached. However, the lack of regression of the wart before being treated by other modality in the same patient seems to suggest a local rather than a systemic or placebo effect. Figure 1 Refractory wart on right index finger (a). It was treated using local application of calcitriol 0.5 μg solution and disappeared completely three months later (b). ==== Refs 1 Sterling JC Handfield-Jones S Hudson PM Guidelines for the management of cutaneous warts British Journal of Dermatology 2001 144 1 4 11 11167676 2 Allam JP Hagemann T Bieber T Novak N Successful treatment of therapy-resistant plantar verrucae vulgares with systemic interferon-β Journal of Dermatology 2004 31 7 582 583 15492428 3 Gibbs S Harvey I Sterling J Stark R Local treatments for cutaneous warts: systematic review British Medical Journal 2002 325 7362 461 464 4 Blohme I Larko O Skin lesions in renal transplant patients after 10-23 years of immunosuppressive therapy Acta Dermato-Venereologica 1990 70 6 491 494 1981421 5 Liu PT Stenger S Li H Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response Science 2006 311 5768 1770 1773 16497887 6 Barker JNWN Ashton RE Marks R Harris RI Berth-Jones J Topical maxacalcitol for the treatment of psoriasis vulgaris: a placebo- controlled, double-blind, dose-finding study with active comparator British Journal of Dermatology 1999 141 2 274 278 10468799 7 Majewski S Skopinska M Bollag W Jablonska S Combination of isotretinoin and calcitriol for precancerous and cancerous skin lesions The Lancet 1994 344 8935 1510 1511 8 Egawa K Ono T Topical vitamin D3 derivatives for recalcitrant warts in three immunocompromised patients British Journal of Dermatology 2004 150 2 374 376 14996120 9 Osborne JE Hutchinson PE Vitamin D and systemic cancer: is this relevant to malignant melanoma? British Journal of Dermatology 2002 147 2 197 213 12174089
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Case Rep Transplant. 2011 Jan 3; 2011:368623
==== Front J Anim Sci BiotechnolJ Anim Sci BiotechnolJournal of Animal Science and Biotechnology1674-97822049-1891BioMed Central 2049-1891-3-262295830810.1186/2049-1891-3-26ReviewProgress of genome wide association study in domestic animals Zhang Hui [email protected] Zhipeng [email protected] Shouzhi [email protected] Hui [email protected] Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Harbin, 150030, People's Republic of China2 College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, People's Republic of China2012 22 8 2012 3 1 26 26 31 12 2011 14 8 2012 Copyright ©2012 Zhang et al.; licensee BioMed Central Ltd.2012Zhang 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.Domestic animals are invaluable resources for study of the molecular architecture of complex traits. Although the mapping of quantitative trait loci (QTL) responsible for economically important traits in domestic animals has achieved remarkable results in recent decades, not all of the genetic variation in the complex traits has been captured because of the low density of markers used in QTL mapping studies. The genome wide association study (GWAS), which utilizes high-density single-nucleotide polymorphism (SNP), provides a new way to tackle this issue. Encouraging achievements in dissection of the genetic mechanisms of complex diseases in humans have resulted from the use of GWAS. At present, GWAS has been applied to the field of domestic animal breeding and genetics, and some advances have been made. Many genes or markers that affect economic traits of interest in domestic animals have been identified. In this review, advances in the use of GWAS in domestic animals are described. Domestic animalsGenome wide association study (GWAS)Quantitative trait loci (QTL)Single-nucleotide polymorphism (SNP) ==== Body Introduction The concept and means to identify genes related to complex traits at the genome-wide level can be traced back to the 1990s. Mapping of quantitative trait loci (QTL) was the preferred approach to detect genetic variation for economically important traits at the genome-wide level. To date, thousands of QTLs for numerous traits have been reported (http://www.animalgenome.org/QTLdb/). However, most of these reported QTLs were detected using microsatellite markers with low map resolution and the confidence interval (CI) covers more than 20 cM [1], even a whole chromosome [2]. Therefore, it is difficult to detect the important genes for traits of interest based on the information. The identification of causal mutations that underlying QTLs has been challenging in domestic animals. The genome wide association study (GWAS) is a new technique for the identification of causal genes for important traits in livestock. The GWAS uses sequence variations (mainly single nucleotide polymorphisms, SNPs) in the whole genome, together with the phenotype and pedigree information, to perform association analysis and to identify genes or regulatory elements that are important for the traits of interest. GWAS has become feasible in humans as well as in domestic animals as a result of the development of large collections of SNPs and the development of cost-effective methods for large-scale SNP analysis. Compared with traditional QTL mapping strategies, GWAS confers major advantages both in the power to detect causal variants with modest effects and in defining narrower genomic regions that harbor causal variants [3]. GWAS is an ideal technique to discover the major genes for complex traits and is a novel way to study the genetic mechanism of complex traits. In this paper, we reviewed the progress of GWAS in domestic animals. Progress of GWAS in domestic animals GWAS was first used in the analysis of human disease and great progress was made. GWAS was extended to the field of domestic animal genetics and breeding when genomic sequences were available for several domestic species and large numbers of SNPs were discovered as a by-product of sequencing or in subsequent re-sequencing. There are many kinds of commercial SNP chip available for cattle (50,000 SNPs; Illumina BovineSNP50 BeadChip), dogs (22,362 SNPs; Illumina CanineSNP20 BeadChip), sheep (56,000 SNPs), pigs (60,000 SNPs; Illumina PorcineSNP60 BeadChip), horses (54,602 SNPs; Illimina EquineSNP50 BeadChip) and chickens (60,000 SNPs; Illumina ChickenSNP60 BeadChip). Although the application of GWAS to domestic animals has only occurred relatively recently, there have been a series of results reported, especially from the analysis of the genetic mechanisms of quantitative traits. An assumption made in the analysis of GWAS is that significant associations can be detected because the SNPs are in linkage disequilibrium (LD) with the causative mutations for the traits of interest. The high density of SNP markers in the chip used in GWAS was sufficient to identify the LD between SNP markers and causative mutations. During the past few years, several examples of successful GWAS in domestic animals, including cattle, pigs, horses, dogs, sheep and chickens, have been reported (Table 1). Table 1 Summary of reported GWAS on domestic animals No. Trait Chip Animal Method Significant level Result Ref 1 Milk yield 50K 62,343 Holstein Friesian cows sired by 798 sires Mixed linear models P < 0.001 Identified 362 significant SNPs [4] 2 Milk yield 50K 767 Holstein bulls Single and Multiple trait regression analysis P < 0.001 Identified 169 significant SNPs [5] 3 Milk yield 50K 2,093 Chinese Holstein from the Holstein cattle farms in Beijing, China Single locus regression analysis Bonferroni P < 0.05 level Identified 105 significant SNPs including two SNPs located close to the DGAT1 gene (160bp apart) and within the GHR gene, respectively [6] 4 Milk yield 50K 1,039 bulls with pedigree information from Danish Jersey cattle Regression analysis Bonferroni correction P < 1.5e−6 Identified 98 significant SNPs [7] 5 Milk quality traits 50K 780 Holstein sons of 142 sires Bayesian analysis (BayesA) P ≤ 0.001 Identified 73-461 significant SNPs (depending on trait) [8] 6 Milk quality traits 50K 3,356 Japanese Black cattle from Yamagata Prefecture GRAMMAR-CG method Bonferroni correction P < 1.28e-6 (5%); P < 2.57e-7 (1%) Identified 32 significant SNPs mainly in region of 49-55 Mb on BTA19 containing FASN gene [9] 7 Milk quality traits 50K 1,905 Dutch Holstein Friesian cows from 398 commercial farms throughout the Netherlands Two step single SNP association analysis using general linear model and animal model FDR < 0.05 Identified 54 significant regions mainly on BTA14, 19, and 26 containing ABCG2, PPARGC1A, ACSS2, DGAT1, ACLY, SREBF1, STAT5A, GH, FASN, SCD1and AGPAT6 [10] 8 Milk quality traits 50K 1,912 Holstein-Friesian cows from 398 commercial herds throughout the Netherlands General linear model FDR <0.01 Identified several important regions mainly on BTA5, 6, 11 and 14 [11] 9 Fertility trait 10K 10 low-fertility and 10 high-fertility bulls of Pure Holstein Regression analysis P < 0.01 Identified 97 significant SNPs including one SNP in Integrin beta 5 gene [12] 10 Fertility trait 50K 267 Holstein cows Generalized linear mixed model P < 0.05 Identified 27 significant SNPs [13] 11 Fertility trait 50K 2,531 Danish and Swedish Holstein bulls Mixed model analysis Bonferroni correction P < 2.16e-5 (BTA1); P < 7.41e-5 (BTA28) Identified 74 significant SNPs mainly on BTA 3, 5, 10, 13, 19, 20, and 24 [14] 12 Fertility trait 50K 2,062 Danish and Swedish Holstein bulls Mixed model analysis Bonferroni correction P < 2.16e-5 (BTA1); P < 7.41e-5 (BTA28) Identified several important regions on BTA4, 6, 12, 18, 20, and 25 [15] 13 Growth trait 50K 150 sires representing 7 breeds including Angus, Charolais, Gelbvieh, Hereford, Limousin, Red Angus and Simmental ANOVA test FDR < 0.01 Identified 231 significant SNPs [16] 14 Growth trait 10K; 50K 852 steers from 7 different pure breeds including Angus, Murray Grey, Shorthorn, Hereford, Brahman, Santa Gertrudis and Belmont Red Regression analysis P <0.001 Identified 75 significant SNPs mainly on BTA3, 5, 7, and 8 [17] 15 Meat quality and carcass traits 50K 940 Beef cattle Regression analysis P <0.001 Identified 87 significant SNPs for meat quality traits and 127 significant SNPs for carcass traits [18] 16 Classical bovine spongiform encephalopathy (BSE) 50K Holstein cows including 143 BSE affected (case) and 173 unaffected (control) collected in Southern England Chi-square test P < 5e-5 Identified one SNP on chromosome 1 at 29.15 Mb and another locus on chromosome 14 [19] 17 Bovine Tuberculosis (TB) 50K 14,013 Irish Holstein- Friesian cows Regression analysis Bonferroni correction P < 1.21e-6 Identified 3 significant SNPs in a 65kb genomic region on BTA 22 containing SLC6A6 gene [20] 18 Bovine Paratuberculosis 50K Italian and American Holstein cows including Group A with 590 cases vs 600 controls and Group B with 590 cases vs 117 controls GRAMMAR-CG method P < 5e-5 Identified 6 significant SNPs on BTA 1, 12 and 15 and several other significant SNPs on BTA 1, 6, 7, 13, 16, 21, 23 and 25 [21] 19 Dominant White Phenotype and Bilateral Deafness 770K Seven white and 79 spotted German Fleckvieh General Linear Model (GLM) -log10Praw = 254.4; -log10PBonferroni-adjusted = 248.6 Identified a most significant region on BTA 22 containing MITF gene [22] 20 Androstenone 60K 987 pigs divergent for androstenone concentration from a commercial Duroc- based sire line QFAM test FDR of q-value ≤ 0.05 Identified 37 significant SNPs mainly on SSC1 and SSC6 [23] 21 skatole levels 60K 891 pigs from a composite Duroc sire line QFAM test FDR of q-value ≤ 0.05 Identified 16 significant SNPs on SSC6: 0-6Mb [24] 22 Boar taint and related sex steroids 60K 1,251 purebred Norwegian Landrace and 918 purebred Duroc male pigs ANOVA test P < 0.05 Identified g 28 regions related to boar taint [25] 23 Boar taint and fertility traits 60K 1,533 purebred Landrace and 1,027 purebred Duroc male pigs ANOVA test P < 0.05 Identified 34 significant regions mainly on SSC1, 2, 3, 4, 7, 13, 14 and 15 [26] 24 Knobbed acrosome defect 60K 14 Finnish Yorkshire boars affected with KAD and 21 controls Chi-square test Permutation correction P < 0.0002 Identified a significant 0.7 Mb region on SSC15 containing STK17b and HECW2 genes [27] 25 Body Composition and Structural Soundness Traits 60K 412 Large White line pigs and 408 pigs from a Large White ×  Landrace cross Bayes C Bootstrap correction 0.001 < P < 0.002 Identified several important genes including MC4R, IGF2, CHCHD3, BMP2 and HOXA [28] 26 Fat area 60K 150 crossbred pigs [Pietrain × (German Large White ×  German Landrace)] ANOVA test P < 1.0e-5 Identified 663 important genes [29] 27 Brown coat colour 60K Brown pigs (n = 121) vs non-brown-coated pigs (n = 745) Chi-square test Permutation test Identified TYRP1 gene [30] 28 Racing distance 50K 118 elite Thoroughbred racehorses divergent for race distance aptitude Chi-square test Bonferroni correction Punadj. = 1.61e-9; PBonf. = 6.58e-5 Identified a significant 690 kb region on ECA18 containing MSTN gene [31] 29 Dwarfism 50K Ten Friesian dwarf horses vs ten controls Chi-square test Bonferroni correction P < 1.72e-6 Identified a significant region on ECA14: 3.8-5.4 Mb containing PROP1 gene [33] 30 Lavender Foal Syndrome (LFS) 50K Egyptian Arabian including 7 affected foals, their 31 relatives, as well as 114 controls Fisher’s exact test P < 0.05 Identified a significant region containing RAB27A and MYO5A genes [34] 31 Recurrent laryngeal neuropathy (RLN) 50K 234 cases (196 Warmbloods, 20 Trotters, 14 Thoroughbreds, and 4 Draft horses), 228 breed-matched controls Chi-square test Bonferroni correction P < 1.09e-6 (significant); P < 2.11e-5 (suggestive) Identified two significant SNPs on ECA21 and ECA31 [35] 32 Horn morphology 50K 107 Soay sheep from the neighbouring island of Soay Chi-square test Keff correction of P < 1.859e-6 Identified a significant region on Chr10 including RXFP2 gene [36] 33 Inherited Rickets 50K Corriedale sheep including 17 affected and 3 carriers IBD analysis P < 0.05 Identified a 6Mb region on Chr6 including DMP1 gene [40] 34 Degenerative myelopathy (DM) 50K Pembroke Welsh corgi including 38 DM-affected cases and 17 controls Chi-square test P < 0.01 Identified a region of 28.91-29.67 Mb on CFA31 including SOD1 gene [43] 35 Canine atopic dermatitis (cAD) 20K 48 Golden Retrievers including 25 with atopic dermatitis and 23 healthy controls Chi-square test P < 0.001 Identified 35 significant SNPs [46] 36 Arrhythmogenic right ventricular cardiomyopathy (ARVC) 50K 65 ARVC-affected boxer dogs vs 100 controls Chi-square test P < 0.05 Identified a region of CFA17: 32,256,760-32, 388,077bp containing STRN gene [47] 37 Intervertebral Disc Calcification 20K Dachshund dogs including 48 cases and 46 controls Chi-square test Permutation test using 100,000 permutations Identified a region of CFA12: 36.8-38.6Mb with 36 significant SNPs [48] 38 Fatness 3K 720 birds from two populations including F2: Broiler ×  Fayoumi and F2: Broiler × Leghorn One-way ANOVA test P < 0.01 Identified 39 significant SNPs mainly on GGA1, 2, 3, 4, 7, 8, 10, 12, 15 and 27 [49] 39 Body weight 60K 278 individuals from F2 population crossed by Silky Fowl × White Plymouth Rock Linear regression analyses Bonferroni correction P < 1.92e-6 (significant); P < 3.85e-5 (suggestive) Identified 9 significant SNPs on GGA4: 71.6-80.2 Mb including LDB2 gene [50] 40 Growth traits 60K 489 birds from F2 population crossed by WRR × XH Generalized least square analysis Bonferroni correction P < 4.08e-8 (highly significant); P < 2.04e-6 (significant); P < 4.08e-5 (suggestive) Identified 68 significant SNPs and 23 genes for 18 growth traits [51] 41 Egg production and quality 60K 385 White leghorn and 361 brown-egg dwarf layers Fisher’s combined probability method Bonferroni correction P < 1.5 e-6, Identified 8 significant SNPs and two genes including GRB14 and GALNT1 [52] Cattles More than ten papers described the use of GWAS for several economically important traits in cattle, including milk yield, milk quality, fertility, growth, meat quality and carcass traits, were reported. For milk yield in dairy cattle, there were four GWAS reports, and a total of 734 SNPs with significant effects on milk yield were detected [4-7]. These SNPs were mainly on chromosomes 8, 9, 10, 11, 13, 25 and 29 and a significant SNP was located close to the DGAT1 gene (160bp apart). For the milk quality trait (eg. fatty acid composition, protein percentage, fat percentage), there were also four GWAS reports, and 547 SNPs on chromosomes 5, 6, 11, 14, 19 and 26 were significantly associated with milk quality [8-11]. The genes, identified from the GWAS results, that might be important for milk quality traits included ABCG2PPARGC1AACSS2DGAT1ACLYSREBF1STAT5AGHFASNSCD1 and AGPAT6. Another four GWAS reported 198 significant SNPs related to the fertility trait such as fertilization rate, clastocyst rate and calving [12-15]. These SNPs were mainly on chromosomes 3, 4, 5, 6, 10, 12, 13, 18, 19, 20, 24 and 25, and the important genes detected from the GWAS results were collagen type I alpha 2 and integrin beta 5. The results indicated that the incubation of bull spermatozoa with antibodies against integrin beta 5 significantly decreased their ability to fertilize oocytes suggesting that the bovine sperm integrin beta 5 protein play an important role during fertilization and could serve as a positional or functional marker of fertility in the bull. Snelling et al. [16] and Bolormaa et al. [17], respectively, reported GWAS on the cattle growth trait (eg. body weight and height), and a total of 306 significant SNPs were detected. These significant SNPs were mainly on chromosomes 3, 5, 7 and 8. There has been only one GWAS study on cattle meat quality, reported by Bolormaa et al. [18]. In total, 940 beef cattle were used in this study and 87 SNPs with significant effects on meat quality (intramuscular fat percentage) were detected. This GWAS also detected 127 SNPs with significant effects on carcass traits (longissimus muscle and rump fat). Classical bovine spongiform encephalopathy (BSE) was a disease that invariably cause fatal in cattle and has been implicated as a significant human health risk. A GWAS on BSE was carried out using the SNP50 beadchip in Holstein cows [19]. The results of this study revealed that the a SNP on chromosome 1 at 29.15 Mb was associated with BSE disease and another locus on chromosome 14, within a cluster of SNPs showed a trend toward significance. The genes within these regions might be important for BSE and need to be further investigated. Bovine tuberculosis (TB) was a significant veterinary and financial problem in many parts of the world. Finlay et al. carried out a GWAS on bovine tuberculosis using Irish dairy herd and the results indicated that 3 SNPs in a 65kb genomic region on BTA 22 were significantly associated with tuberculosis susceptibility [20]. The SLC6A6 gene within this region might be important for tuberculosis. Another GWAS report was also focused on tuberculosis using two populations of Holstein cows and 6 SNPs on chromosomes 1, 12 and 15 in one population and several SNPs on chromosomes 1, 6, 7, 13, 16, 21, 23 and 25 in another population were detected for their significant association with Paratuberculosis [21]. The genes related to these significant SNPs might be important for Paratuberculosis in cattle. The 770K SNP chip for Bovine was a high density (HD) bead array from Illumina, containing 777,000 SNP markers. This high density SNP chip allows a variety of applications including genome wide selection and identification of quantitative trait loci. Philipp et al. carried out a GWAS using this HD bead array in German Fleckvieh Cattle to detect the mutations associated with Dominant White Phenotype and Bilateral Deafness [22]. The results of this study revealed a most significantly associated region on bovine chromosome (BTA) 22. There were 13 genes in this significant region, including MITF, which was essential for the development and post-natal survival of melanocytes. The further sequence analysis of this gene revealed that there was a missense mutation in exon 7 that was associated with Dominant White Phenotype and Bilateral Deafness. Pigs An example of a GWAS on androstenone levels in male pigs was reported by Duijvesteijn et al. [23]. They used the Illumina Porcine 60K SNP Beadchip and genotyped 987 pigs divergent for androstenone concentration from a commercial Duroc-based sire line. The association analysis, which involved 47,897 SNPs, revealed that androstenone levels in fat tissue were significantly affected by 37 SNPs mainly on porcine chromosomes 1 and 6. On chromosome 6, a large region of 10 Mb was shown to be associated with androstenone, and this region covered several candidate genes that are potentially involved in the synthesis and metabolism of androgens. The chromosome 6 might be an important chromosome in the determination of androstenone levels. Skatole is another component of boar taint, in addition to androstenone. Ramos et al. [24] carried out a GWAS for skatole using the same animals as Duijvesteijn et al. [23]. The results indicated that 16 SNPs located on the proximal region of chromosome 6 were significantly associated with skatole levels but no obvious candidate genes could be pinpointed in the region. Using GWAS and LDLA (linkage disequilibrium and linkage analysis) analysis, Grindflek et al. found 28 chromosome regions related to boar taint in commercial Landrace and Duroc breeds [25]. These chromosome regions were mainly on chromosomes 1, 2, 3, 5, 6, 7, 10, 11, 13, 14 and 15. Further study was carried out using 1,533 purebred Landrace and 1,027 purebred Duroc and a total of 34 regions were found significantly associated with boar taint and fertility traits. These 34 regions were mainly on chromosomes 1, 2, 3, 4, 7, 13, 14 and 15 [26]. Sironen et al. reported a GWAS on infertility (knobbed acrosome defect, KAD) trait in the Finnish Yorkshire pig population using the PorcineSNP60 Genotyping BeadChip, and the KAD-associated region was identified within 0.7 Mbp on porcine chromosome 15 [27]. There were two genes, STK17b and HECW2, located within this region. The sequencing in the protein coding region of these two genes revealed two SNPs within HECW2 gene, but no polymorphisms were detected within STK17b gene. One nonsynonymous SNP identified within the HECW2 gene was further genotyped for all 14 KAD-affected and 10 control boars. All KAD-affected boars were homozygous for this SNP, but also four control boars had the same homozygous allele, indicating that this SNP was unlikely to be the causal mutation. Fan et al. used Illumina’s PorcineSNP60 BeadChip to perform a GWAS on 820 commercial female pigs that were phenotyped for backfat, loin muscle area and body conformation in addition to traits of foot and leg (FL) structural soundness [28]. A total of 51,385 SNPs were used in the GWAS and a number of candidate chromosomal regions were discovered; some of them corresponded to QTL regions reported previously. In these regions, some well-known candidate genes for the traits of interest were identified, such as MC4R (for backfat) and IGF2 (for loin muscle area), and a number of novel promising genes were reported, including CHCHD3 (for backfat), BMP2 (for loin muscle area, body size and several FL structure traits), and some HOXA family genes (for overall leg action). Functional clustering analyses classified the genes into categories related to bone and cartilage development, muscle growth and development or the insulin pathway, which suggested that the traits were regulated by common pathways or gene networks that exert roles at different spatial and temporal stages. Fatness is one of the important economic factors in pork production, and also associated with serious diseases in humans. Ponsuksili et al. applied a GWAS to traits of hepatic gene expression, focusing on transcripts with expression levels that correlated with fatness traits in a porcine model [29]. A total of 150 pigs were studied for transcript levels in the liver. The 24K Affymetrix expression microarrays and 60K Illumina single nucleotide polymorphism (SNP) chips were used in the study. A total of 663 genes, whose expression levels being significantly correlated with the trait “fat area”, were detected. The association between the genome-wide SNPs and expression of these 663 genes was analyzed and the result revealed 4,727 expression quantitative trait loci (eQTL). Brown coat color is another important economic trait in pigs, and a GWAS was performed by Ren et al. using the Illumina PorcineSNP60 BeadChips on Tibetan and Kele pigs [30]. By means of a haplotype-sharing analysis, the critical region was refined to a 1.5-Mb interval on chromosome 1 that encompasses only one pigmentation gene: tyrosinase-related protein 1 (TYRP1). Mutation screens of sequence variants in the coding region of TYRP1 revealed a strong candidate causative mutation (c.1484_1489del). The protein-altering deletion showed complete association with the brown coloration across Chinese-Tibetan, Kele, and Dahe breeds. It occurred exclusively in brown pigs and was absent from all non-brown-coated pigs from 27 different breeds. The findings provide compelling evidence that brown coloration in the three Chinese indigenous pig breeds is caused by the same ancestral mutation in TYRP1. Horses It is widely recognized that inherited variation in physical and physiological characteristics of the horse is responsible for the variation in individual aptitude for racing distance, and that muscle phenotypes in particular are important. A genome-wide SNP-association study for optimum racing distance was performed using the EquineSNP50 Bead Chip genotyping array in a cohort of 118 elite Thoroughbred racehorses divergent for race distance aptitude [31]. The GWAS result indicated that the most significant SNP was located on chromosome 18 about 690 kb from the gene encoding myostatin (MSTN). Together with previous results [32], this indicated that the MSTN gene may be a major factor affecting racing distance in horses. Dwarfism is also an important trait in horses. Orr et al. performed a GWAS on dwarfism in Friesian horses using 34,429 SNPs, and the most significant SNP was located close to a gene implicated in human dwarfism [33]. Lavender foal syndrome (LFS) is a lethal inherited disease of horses that has a suspected autosomal recessive mode of inheritance. Brooks et al. reported a GWAS for LFS using a small sample of 36 horses segregating for LFS [34]. These horses were genotyped using a newly available SNP chip containing 56,402 SNPs. The GWAS results indicated that the region containing two functional candidate genes encoding ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A) was significantly associated with LFS. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30. A PCR–RFLP result indicated that all affected horses were homozygous for this mutation. This locus might be the causal mutation for LFS in horses. Another disease known as recurrent laryngeal neuropathy (RLN), is also important in horses. It causes abnormal respiratory noise during exercise and can impair performance. Dupuis et al. carried out a GWAS using the Illumina Equine SNP50 BeadChip in 234 cases (196 Warmbloods, 20 Trotters, 14 Thoroughbreds, and 4 Draft horses), 228 breed-matched controls, and 69 parents [35]. The result indicated that two loci reached suggestively significant level in Warmbloods, respectively on chromosomes 21 and 31. The two signals were driven by the enrichment of a “protective” haplotype in controls compared with cases. This result indicated that these two signals are important for RLN in horses. Sheep The first report of the use of GWAS in sheep was made on horn types by Johnston et al. [36]. A genome-wide association study was conducted using 36,000 SNPs and determined the main genetic candidate for horns to be RXFP2, an autosomal gene with known involvement in determining primary sexual characteristics in humans and mice [37-39]. Evidence from additional SNPs in and around RXFP2 supports a new model of horn-type inheritance in Soay sheep, and for the first time sheep with the same horn phenotype but different underlying genotypes can be identified. In addition, RXFP2 was shown to be an additive quantitative trait locus (QTL) for horn size in normal-horned males, accounting for up to 76% of the additive genetic variation in this trait. This finding contrasts markedly with GWAS of quantitative traits in humans and some model species, where it is often observed that mapped loci only explain a modest proportion of the overall genetic variation. The other study of GWAS in sheep was reported by Zhao et al. [40], who in the same year used the same Illumina OvineSNP50 BeadChip as Johnston et al. [36]. This study was focused on the inheritance of rickets in Corriedale sheep. A GWAS was carried out on 20 related sheep, comprising 17 affected individuals and 3 carriers. A homozygous region that included 125 consecutive SNP loci was identified in all 17 affected sheep, covering a region of 6 Mb on ovine chromosome 6. There were 35 genes in this region; the gene for dentin matrix protein 1 (DMP1) was sequenced and a nonsense mutation, 250C/T, was identified on exon 6. This mutation introduced a stop codon (R145X) and could truncate C-terminal amino acids. Genotyping by PCR–RFLP for this mutation showed that all 17 affected sheep had the “T T” genotype; the 3 carriers were “C T”; 24 phenotypically normal related sheep were either “C T” or “C C”; 46 unrelated normal control sheep from other breeds were all “C C”. The other SNPs in DMP1 were not concordant with inherited rickets and can all be ruled out as candidates. Previous research has shown that mutations in the DMP1 gene are responsible for autosomal recessive hypophosphatemic rickets in humans [41]. Dmp1 knockout mice exhibit rickets phenotypes [42]. Therefore the R145X mutation in DMP1 is thought to be responsible for inherited rickets in Corriedale sheep. Dogs Degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Awano et al. carried out a GWAS using 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls [43]. This produced the strongest associations with markers on chromosome 31 in a region containing the canine SOD1 gene. SOD1 was considered to be a regional candidate gene from the results of previous studies in human and mice [44,45]. Re-sequencing of SOD1 in normal and affected dogs revealed a G to A transition and homozygosity for the A allele was associated with DM in five dog breeds. The result indicated that the SOD1 gene is important for DM in dogs. Canine atopic dermatitis (cAD) is a common disease in dogs, and the first GWAS was reported by Wood et al. using the Illumina Canine SNP20 array [46]. The study used affected and unaffected Golden Retrievers to carry out the GWAS, and one SNP was over the log5 threshold and 35 SNPs were over the log3 threshold. Further validation studies of the top 40 SNPs from the GWAS results were performed using Sequenom genotyping of larger numbers of cases and controls across eight breeds. Two SNPs were associated with cAD in all breeds tested, and these two SNPs were located in intergenic regions. The effects of these two SNPs were independent of each other, indicating that further fine mapping and re-sequencing was required for these areas. Another 12 SNPs were shown by Sequenom genotyping to be associated with cAD, but these were not important in all breeds. The results of this study suggested that GWAS would be a useful approach to identify genetic risk factors for cAD. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is inherited most frequently as an autosomal dominant trait with incomplete age-related penetrance and variable clinical expression. A GWAS for ARVC was carried out by Meurs et al. using the canine 50k SNP array in adult Boxer dogs, which identified several regions significantly associated with ARVC, of which the strongest SNP resided on chromosome 17 [47]. Fine-mapping and direct DNA sequencing identified an eight base pair deletion in the 3' untranslated region (UTR) of the striatin (STRN) gene on chromosome 17 that was associated with ARVC in the Boxer dog. Further analysis indicated that the deletion affected a stem loop structure of the mRNA. Dogs that were homozygous for the deletion had a more severe form of disease, on the basis of a significantly higher number of ventricular premature complexes. The results of this study suggested that STRN may serve as a novel candidate gene for ARVC. Intervertebral disc calcification and herniation commonly affect Dachshunds. The number of calcified discs at 2 years of age, determined by radiographic evaluation, is a good indicator of the severity of disc degeneration and thus serves as a measure of the risk of developing intervertebral disc herniation. A GWAS analysis was carried out to identify genetic variants associated with intervertebral disc calcification in Dachshunds [48]. In total, 48 cases with > =6 disc calcifications or that had been treated surgically for disc herniation and 46 controls with 0–1 disc calcifications were genotyped using the Illumina CanineHD BeadChip. A region on chromosome 12 from 36.8 to 38.6 Mb containing 36 significant SNPs was identified in the GWAS analysis. The results of this study suggested that the genetic variations in the region on chromosome 12 may be important for the development of intervertebral disc calcification in Dachshunds. Chickens The first GWAS in chickens was reported by Abasht and Lamont using 3,000 SNPs on the whole genome in two F2 populations; the results indicated that there were 15 and 24 markers significantly associated (P < 0.01) with abdominal fatness (AF) in the two F2 populations, respectively [49]. These SNPs were on 10 chromosomes (1, 2, 3, 4, 7, 8, 10, 12, 15 and 27). Further analysis revealed that these SNPs were considered to be associated with QTL with cryptic alleles. This study revealed cryptic alleles to be an important factor in heterosis for fatness observed in two F2 populations of chickens, and suggested that epistasis was the common underlying mechanism for heterosis and cryptic allele expression. There was a GWAS about chicken body weight [50]. A total of 26 SNP effects related to 9 different SNPs were significantly associated with body weight at 7–12 weeks of age. These significant SNPs were mainly in a region of the chicken chromosome 4 approximately 8.6 Mb in length (71.6–80.2 Mb). The LIM domain-binding factor 2 (LDB2) gene in this region had the strongest association with body weight for weeks 7–12, and with average daily gain for weeks 6–12. This gene may be important in the regulation of body weight in the chicken. Another GWAS about chicken growth was reported by Xie et al [51]. A total of 257 SNP effects involving 68 SNPs and 23 genes were detected for 18 traits with genome-wide significance [51]. Among these identified SNPs or regions, the 1.5 Mb region (173.5–175 Mb) of chicken chromosome (GGA) 1 was the most important for chicken growth traits and genes in this region may be important for chicken growth. The egg production and quality traits were important in layer chickens. Liu et al. carried out a GWAS on chicken egg production and quality traits using two populations including White Leghorn and Brown-Egg Dwarf Layers. The result indicated that there were 8 SNPs significantly associated with egg production and quality traits [52]. Among these significant SNPs, several were located in known genes including GRB14 and GALNT1 that can impact the development and function of ovary. Conclusions In summary, there was a great progress of GWAS in domestic animals and some genes for economically important traits have been identified. However, the main problem lies in the inconsistencies among the results of these GWAS reports for the same trait, which may be mainly attributed to many aspects such as population size, density of the markers (SNPs), population genetic structure, choice of statistical models, as well as type I and II errors. To achieve the accurate estimation of SNP effects on traits of interest in a GWAS, larger population size and higher density of the markers (SNPs) were required. Currently, SNP chips were widely applied in GWAS and enhanced the identification of QTL for traits of interest in domestic animals. Compared with SNP chips, sequencing could provide nearly all information about the variations, including SNP, copy number variation (CNV) and the deletion/insertion, et al., on the whole genome in detected population. Along with the reduction in sequencing cost, it is possible that all individuals in the tested populations might be sequenced and genotyped and GWAS might be carried out in this platform then. In the future, GWAS in domestic animals will focus on the identification of causative mutations for economically important traits. The findings will inevitably facilitate the understanding of the genetic architecture of complex traits in domestic animals and practical improving the breeding programmes. Abbreviations QTL, Quantitative trait loci; GWAS, Genome wide association study; SNP, Single-nucleotide polymorphism; CI, Confidence interval; LD, Linkage disequilibrium; LDLA, Linkage disequilibrium and linkage analysis; KAD, Knobbed acrosome defect; FL, Foot and leg; eQTL, Expression quantitative trait loci; LFS, Lavender foal syndrome; RLN, Recurrent laryngeal neuropathy; DM, Degenerative myelopathy; cAD, Canine atopic dermatitis; ARVC, Arrhythmogenic right ventricular cardiomyopathy; AF, Abdominal fatness; CNV, Copy number variation. Competing interests The authors declare that they have no competing interests Authors’ contributions HZ collected the information used in the manuscript and drafted the manuscript. ZW collected the information and helped to draft the manuscript. SW collected the information and helped to draft the manuscript. HL co-led the conception and design of the study, participated in the collection of the information and contributed to writing the manuscript. All authors read and approved the final manuscript. Acknowledgements This research was supported by China Agriculture Research System (No. CARS-42), National 863 project of China (No. 2010AA10A102), National 973 Project of China (No.2009CB941604) and Program for Innovation Research Team in University of Heilongjiang Province (No.2010td02). ==== Refs Soller M Weigend S Romanov MN Dekkers JC Lamont SJ Strategies to assess structural variation in the chicken genome and its associations with biodiversity and biological performance Poult Sci 2006 85 2061 2078 17135660 Schreiweis MA Hester PY Moody DE Identification of quantitative trait loci associated with bone traits and body weight in an F2 resource population of chickens Genet Sel Evol 2005 37 677 698 10.1186/1297-9686-37-7-677 16277974 Hirschhorn JN Daly MJ Genome-wide association studies for common diseases and complex traits Nat Rev Genet 2005 6 95 108 15716906 Hayes BJ Bowman PJ Chamberlain AJ Savin K van Tassell CP Sonstegard TS Goddard ME A validated genome wide association study 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yes
J Anim Sci Biotechnol. 2012 Aug 22; 3(1):26
==== Front Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 10.1155/2012/139045Research ArticleHerbal Supplement Ameliorates Cardiac Hypertrophy in Rats with CCl4-Induced Liver Cirrhosis Li Ping-Chun 1 2 Chiu Yung-Wei 3 4 Lin Yueh-Min 5 Day Cecilia Hsuan 6 Hwang Guang-Yuh 2 Pai Peiying 7 Tsai Fuu-Jen 8 Tsai Chang-Hai 9 Kuo Yu-Chun 10 Chang Hsiao-Chuan 11 Liu Jer-Yuh 12 13 *Huang Chih-Yang 8 10 14 * 1Division of Cardiovascular Surgery, China Medical University Hospital, Taichung 40402, Taiwan 2Department of Life Science, Tunghai University, Taichung 40704, Taiwan 3Emergency Department and Center of Hyperbaric Oxygen Therapy, Tungs' Taichung MetroHarbor Hospital, Taichung 43503, Taiwan 4Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan 5Department of Pathology, Changhua Christian Hospital, Changhua 50006, Taiwan 6Department of Nursing, MeiHo University, Pingtung 91202, Taiwan 7Division of Cardiology, China Medical University Hospital, Taichung 40402, Taiwan 8Graduate Institute of Chinese Medical Science, China Medical University, Taichung 40402, Taiwan 9Department of Healthcare Administration, Asia University, Taichung 41354, Taiwan 10Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan 11Department of Biotechnology, Asia University, Taichung 41354, Taiwan 12Center for Molecular Medicine, China Medical University Hospital, Taichung 40402, Taiwan 13Graduate Institute of Cancer Biology, China Medical University, Taichung 40402, Taiwan 14Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan*Jer-Yuh Liu: [email protected] and *Chih-Yang Huang: [email protected] Editor: Y. Ohta 2012 6 11 2012 2012 13904531 5 2012 31 7 2012 7 8 2012 Copyright © 2012 Ping-Chun Li et al.2012This 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.We used the carbon tetrachloride (CCl4) induced liver cirrhosis model to test the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy and the effectiveness of Ocimum gratissimum extract (OGE) and silymarin against cardiac hypertrophy. We treated male wistar rats with CCl4 and either OGE (0.02 g/kg B.W. or 0.04 g/kg B.W.) or silymarin (0.2 g/kg B.W.). Cardiac eccentric hypertrophy was induced by CCl4 along with cirrhosis and increased expression of cardiac hypertrophy related genes NFAT, TAGA4, and NBP, and the interleukin-6 (IL-6) signaling pathway related genes MEK5, ERK5, JAK, and STAT3. OGE or silymarin co-treatment attenuated CCl4-induced cardiac abnormalities, and lowered expression of genes which were elevated by this hepatotoxin. Our results suggest that the IL-6 signaling pathway may be related to CCl4-induced cardiac hypertrophy. OGE and silymarin were able to lower liver fibrosis, which reduces the chance of cardiac hypertrophy perhaps by lowering the expressions of IL-6 signaling pathway related genes. We conclude that treatment of cirrhosis using herbal supplements is a viable option for protecting cardiac tissues against cirrhosis-related cardiac hypertrophy. ==== Body 1. Introduction Patients with advanced cirrhosis have consistently been diagnosed with cardiac dysfunction under the condition of hyperdynamic circulation [1]. Increased cardiac output and reduced systemic vascular resistance are both signs of this condition [2–4]. Although cardiac dysfunction in patients with cirrhosis and potential clinical implications have long been known [5], little is understood regarding the molecular mechanism of action involved in cirrhosis-associated alteration in cardiac structure and function, especially cardiac hypertrophy. Cirrhosis is known as a possible cause of portal vein constriction which may induce the activation of vasopressin, angiotensin II (Ang II), and the sympathetic nervous system [6]. Cardiac hypertrophy is induced by such direct mechanical wall stress as well as paracrine/autocrine factors such as Ang II, which in turn activates specific signaling pathways, for instance, mitogen-activated protein kinases (MAPKs) and calcineurin. These can cause cardiac hypertrophy and increase of related gene expressions, such as proto-oncogenes c-Fos and c-JUN, genes which encode atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), and structural genes β-myosin heavy chain (β-MHC) and skeletal α-actin [7]. Ang II is associated with increased plasma levels of proinflammatory cytokines such as interleukin-6 (IL-6) [8], which is an effective stimulator of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in cardiac hypertrophy [7]. However, the role of these protein markers and transcriptional factors in cardiac hypertrophy and remodeling in vivo has not been examined in cirrhosis-associated hypertrophy. Carbon tetrachloride (CCl4) is frequently used to induce experimental cirrhosis in rats [9]. This model has recently been used to investigate the role of lipophilic bile acids and examine cardiac gene expression profiles in cirrhotic cardiomyopathy [10, 11]. Silymarin, a standardized extract of the milk thistle (Silybum marianum L. Gaertner), contains three biochemicals: silybin, silydianin, and silychristin and has a long tradition as a herbal remedy [12]. Ocimum gratissimum extract (OGE), a commonly used herb in folk medicine, is rich in antioxidants and possesses many therapeutic functions [13–21]. Both herbal extracts have been shown using the CCl4 model to inhibit liver cirrhosis [22]. Therefore the motive for this experiment is to use the CCl4-induced liver cirrhosis model to understand the molecular mechanism of action involved in cirrhosis-associated cardiac hypertrophy, as well as to test effectiveness of silymarin and OGE against cardiac damage and hypertrophy. 2. Materials and Methods 2.1. Preparation of OGE Leaves of Ocimum gratissimum were harvested and washed with distilled water followed by homogenization with distilled water using polytron. The homogenate was incubated at 95°C for 1 hour (h) and then filtered through two layers of gauze. The filtrate was centrifuged at 20,000 g, 4°C for 15 minutes (min) to remove insoluble pellets and the supernatant (OGE) was thereafter collected, lyophilized, and stored at −20°C until use. The final extract (OGE) was composed of 11.1% polyphenol (including 0.03% catechins, 0.27% caffeic acid, 0.37% epicatechin, and 3.27% rutin). 2.2. Animals and Treatment Forty male wistar rats weighing 200–240 g were purchased from the National Animal Center and housed in conventional cages with free access to water and rodent chow at 20–22°C with a 12-hour light-dark cycle. All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Chung Shan Medical University (IACUC, CSMU) for the care and use of laboratory animals. The rats were divided evenly into five groups of 8 rats and treated intraperitoneally with CCl4 (8% CCl4/corn oil, 1 mL/kg body weight (BW) twice a week, Monday and Thursday) for 8 weeks, as described by Hernández-Muoz et al. [23], with some modifications. At the same time, the rats were treated with various dosages of OGE (0–0.04 g/kg BW), or silymarin orally (0.2 g/kg BW, four times a week, Tuesday, Wednesday, Friday, and Saturday) [24, 25]. The control rats were treated with corn oil (1 mL/kg BW) and fed a normal diet. At the end of the experiment, blood and heart were immediately obtained after the animals were sacrificed. 2.3. Histological Examinations The heart was fixed in 10% formalin, processed using routine histology procedures, embedded in paraffin, cut in 5 μm sections, and mounted on a slide. The samples were stained with hematoxylin and eosin for histopathological examination. 2.4. Preparation of Tissue Extract All procedures were performed at 4°C. The heart samples were lysed by 30 strokes using a Kontes homogenizer at a ratio of 100 mg tissue/1 mL lysis buffer. The lysis buffer consisted of 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1 mM PMSF, 10 μg/mL leupeptin, 1 mM sodium orthovanadate, 1% (v/v) 2-mercaptoethanol, 1% (v/v) Nonidet P40, and 0.3% sodium deoxycholate. These homogenates were centrifuged at 100,000 g for 1 h at 4°C. The supernatant was stored at −70°C for Western blot assay. 2.5. Electrophoresis and Western Blot Tissue extract samples were prepared as described above. Sodium do deco sulfate-polyacrylamide gel electrophoresis is carried out as described by Laemmli [26] using 10% polyacrylamide gels. After samples are electrophoresed at 140 V for 3.5 h, the gels are equilibrated for 15 min in 25 mM Tris-HCl, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol. Electrophoresed proteins are transferred to nitrocellulose paper (Amersham, Hybond-C Extra Supported, 0.45 Micro) using Hoefer Scientific Instruments Transpher Units at 100 mA for 14 h. The nitrocellulose paper was incubated at room temperature for 2 h in blocking buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20, and 3% (v/v) fetal bovine serum. Antibodies BNP, phospho-GATA binding protein 4 (p-GATA4), nuclear factor of activated T cells (NFAT), mitogen-activated protein kinase kinase 5 (MEK5), extracellular signal-regulated protein kinase 5 (ERK5), phospho-extracellular signal-regulated protein kinase 5 (p-ERK5), phospho-Janus kinase (p-JAK), signal transducer and activator of transcription 3 (STAT3), α-tubulin purchased from Santa Cruz Biotechnology, Inc. (CA, USA), and IL-6 purchased from Abcam Inc. (MA, USA) are diluted to 1 : 2000 in antibody binding buffer containing 100 mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20, and 1% (v/v) fetal bovine serum. Incubations were performed at room temperature for 3.5 h. The immunoblots were washed three times in 50 mL blotting buffer for 10 min and then immersed for 1 h in the second antibody solution containing horseradish peroxidase goat anti-rabbit or anti-mouse IgG (Promega, WI, USA), which were diluted in binding buffer to 1000-fold, for various antibodies. After washing with blocking buffer, the membrane was visualized using chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.6. Statistical Analysis The experimental results are expressed as the mean ± SE. Data were assessed using analysis of variance (ANOVA) followed by a Student-Newman-Keuls correction to adjust the significance level to avoid a type I error. Student's t-test was used in the comparison between groups. A P value less than 0.05 was considered statistically different. 3. Results 3.1. Changes in Heart Weight of CCl4-Induced Cirrhosis-Associated Cardiac Hypertrophy Throughout the experimental period of 8 weeks, there was no difference in body weight of rats within the 5 groups. At the end of the experiments when rat livers were measured, liver fibrosis was observed in the CCl4-treated group, as compared to the control group which was given olive oil. And for the groups treated with OGE or silymarin, a protective effect was observed: liver fibrosis was significantly ameliorated compared to the CCl4-treated group (data pending publication). In comparison, Table 1 shows that the whole heart weight (WHW), left ventricle weight (LVW), and their ratio to the body weight of the CCl4-treated group were significantly higher than the control group. For groups treated with 0.02 g/kg BW OGE and treated with 0.2 g/kg BW silymarin, weights of the heart remained equal to the control group. However, for the group treated with 0.04 g/kg BW OGE, the weight values had a less significant decrease compared to the CCl4-treated only group. 3.2. Changes in Diameter and Thickness and Histological Structure of Left Heart Ventricle of CCl4-Induced Cirrhosis-Associated Cardiac Hypertrophy The left ventricle diameter of the CCl4-treated group was significantly larger and the walls were moderately thicker than the control group (Figure 1 upper panel and Table 2), but a change of that scale in ventricle diameter was not present in the OGE and silymarin cotreated groups. The left most picture in Figure 1 (lower panel) shows the appearance of a normal heart: one with a unified tissue pattern. However, hearts treated with CCl4 had clearly lost its tissue integrity, but such a change was clearly not observed in groups cotreated with 0.02 g/kg BW OGE and silymarin. 3.3. The Expression of Cardiac Hypertrophy Related Genes in the Heart of CCl4-Treated Rats The expression of cardiac hypertrophy related genes, such as BNP, p-GATA4, and NFAT4, were also tested [7]. Their figures were increased in the CCl4-treated group as compared to the control group (Figures 2 and 3). In the groups cotreated with 0.02 g/kg BW OGE or silymarin, the expression of BNP, p-GATA4, and NFAT returned to control level. The results of the 0.04 g/kg BW OGE-treated group were consistent with the above figures, in that their expressions were decreased, but not back to control levels. 3.4. The Expression of IL-6 Signaling Pathway Related Genes in the Heart of CCl4-Treated Rats We wanted to test for IL-6 signaling pathways because studies have shown that cardiac hypertrophy can be attributed to IL-6 related cytokines [7]. Western blotting analysis shows that the expressions of IL-6, MEK5, ERK5, and p-ERK5 were increased in the CCl4-treated group as compared to the control group (Figure 4). In the groups cotreated with 0.02 g/kg BW OGE or silymarin, the expression of IL-6, MEK5, ERK5, and p-ERK5 returned to control level. The expressions were also lowered in the 0.04 g/kg BW OGE-treated group, but not back to the levels of the control group. The expressions of other IL-6 signaling pathway genes, p-JAK and STAT3, were tested, the data shows that both their expressions were increased in the CCl4-treated group as compared to the control group (Figure 5). In the groups cotreated with 0.02 g/kg BW OGE or silymarin, the expressions of p-JAK and STAT3 returned to control levels, except for the 0.04 g/kg BW OGE group, which were lowered but not back to the control levels. This result is consistent with the data above. 4. Discussion Numerous reports center on the involvement of IL-6 and the related cytokines in cardiac hypertrophy [7] as an inducer of downstream pathways. IL-6 is a typical cytokine which was found to have a potent hypertrophic effect on cardiomyocytes [27], as the overexpression of this cytokine has been linked to hypertrophic myocardium injury [28]. In the present study, our data showed that the expressions of IL-6 increased in CCl4-induced cirrhosis rats detected with occurrence of cardiac hypertrophy, which suggests that the cirrhosis-associated cardiac hypertrophy may be related with the IL-6 signaling pathway in the CCl4-treated rats. IL-6 is involved in multiple intracellular signaling pathways, particularly the MEK5-ERK5 pathway [29–32], which plays a critical role in the induction of eccentric cardiac hypertrophy that can progress to dilated cardiomyopathy and sudden death [33, 34], and the JAK-STAT3 pathway, which promotes the increase of cell dimensions [35–37]. Since the experiments suggest a relationship between CCl4-induced cirrhosis-associated cardiac hypertrophy and IL-6, we decided to analyze the mechanism concerning OGE and silymarin and how it may inhibit cardiac hypertrophy through the inhibition of IL-6 extracellular signals. Western blotting analysis shows that the expressions of IL-6, MEK5, ERK5, and p-ERK5 were increased in the CCl4-treated groups as compared to the control (Figure 4) and were partially restored to control levels when cotreated with OGE or silymarin. Moreover, the expressions of p-JAK and STAT3 were increased in the CCl4-treated group (Figure 5) and restored by OGE or silymarin cotreatment, as in the above gene expressions. Taken together, these findings indicate that both the JAK-STAT3 and the MEK5-ERK5 pathways related genes were overexpressed by IL-6 expression in response to CCl4-induced cirrhosis-associated cardiac hypertrophy (Figure 6), which confirms the importance of the two pathways and also demonstrates that their overexpression may be reversed by OGE or silymarin treatment thus lowering liver cirrhosis and reducing the chance of cardiac hypertrophy. An interesting note is that silymarin, which has rarely been demonstrated to treat cardiac hypertrophy [38], suggests that some common elements between herbal preparations, such as their antioxidant properties, may be responsible for treatment against liver cirrhosis-induced cardiac damage. Cardiac hypertrophy can be classified as physiological and pathological hypertrophy [7], with the physiological being a natural bodily response to maturation, pregnancy, and exercise, and the pathological being a response to pathological stress signals, such as inflammation, cardiac injury, or exposure to toxicity. In our study, we found that many genes was responded to cardiac hypertrophy by CCl4 induction, including MEK5, ERK5, JAK2, STAT3, NFAT3, GATA4, and fetal gene BNP, which are used as a pathological marker [39–42] (Figure 6). Since pathological hypertrophy is also associated with observable loss of tissue integrity, which we also found in CCl4-treated rats, this suggests that CCl4 induced cirrhosis-associated cardiac hypertrophy may belong to pathological hypertrophy and can also be explored further as a pathological model. There is a peculiar phenomenon that a 0.02 g/kg BW dose of OGE had a significant inhibition effect on CCl4-induced cardiac hypertrophy and on the related gene expressions than a 0.04 g/kg BW dose. A possible explanation suggests that the saturation of the higher dose could have lowered the effectiveness of the treatment. 5. Conclusions In summary, CCl4-induced cirrhotic cardiac damage can occur through the IL-6 signaling pathway which leads to eventual cardiac hypertrophy. OGE and silymarin can protect cardiac cells from CCl4-induced damage possibly by inhibiting the expression of the IL-6 signaling pathway related genes. Moreover, we also found in further research that CCl4 induced cardiac damage can induce the FASL signaling pathway and the TGF-β signaling pathway, which may lead to cell apoptosis and eventual cardiac fibrosis (pending publication). It seems that multiple mechanisms are involved in the CCl4 induced cardiac damage. However, in the present study, we suggest that OGE and silymarin in the form of herbal supplements are a viable option for the protection of cardiac tissues against cirrhosis-related cardiac hypertrophy. Acknowledgments The authors thank Dr. Edwin L. Cooper for reading the paper and making suggestions. This work was supported by grants from the National Science Council, China (NSC 98-2320-B-039-042-MY3), by the Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004) and in part by the (CMU97-203 and CMU98-asia-12), Taiwan. Figure 1 Cardiac pathologic analysis in the heart of CCl4-treated rats. Herbs and CCl4 were given as described in Materials and Methods. The top panels show the heart of the macroscopic cross-section. The bottom panels show high magnification (×400) of tissue structure. LV: left heart ventricle; RV: right heart ventricle. Figure 2 The expressions of BNP by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE, n = 8. *P < 0.05 as compared with control group. # P < 0.05 as compared with the CCl4-treated group. Figure 3 The expressions of NFAT3 and phosphorylated-GATA4 by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE, n = 8, *P < 0.05 as compared with control group, and # P < 0.05 as compared with CCl4-treated group. Figure 4 The expressions of IL6 and its downstream signaling proteins MEK5, ERK5, and phosphorylated-ERK5 by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE, n = 8, *P < 0.05 as compared with control group, and # P < 0.05 as compared with CCl4-treated group. Figure 5 The expressions of JAK-Stat3 pathway by Western blotting analysis (a) and quantitative analysis (b) in the heart of CCl4-treated rats. The individual severity rates in rats were expressed as mean ± SE, n = 8. *P < 0.05 as compared with control group. # P < 0.05 as compared with CCl4-treated group. Figure 6 The summary of the mechanism of CCl4-induced cirrhosis-associated cardiac hypertrophy. Our data demonstrated that Ocimum gratissimum and silymarin extracts attenuate cardiac cells from CCl4 induced damage possibly by lowering liver cirrhosis which reduces the chance of cardiac hypertrophy maybe via inhibiting IL-6 signaling pathway activation. Table 1 Changes in body weight and organ weight of CCl4-induced cirrhosis-related cardiac hypertrophy.   Aa B C D E   (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) BW (g) 425 ± 16.475 402 ± 8.920 385 ± 6.547 388 ± 10.823 420 ± 19.272 WHW (g) 1.041 ± 0.015 1.173 ± 0.031* 0.975 ± 0.023# 1.089 ± 0.026 1.023 ± 0.015# LVW (g) 0.813 ± 0.010 0.898 ± 0.018* 0.745 ± 0.028# 0.777 ± 0.021 0.767 ± 0.023# WHW/BW (%) 2.467 ± 0.095 2.918 ± 0.093* 2.535 ± 0.065# 2.813 ± 0.067 2.461 ± 0.101# LVW/BW (%) 1.922 ± 0.050 2.233 ± 0.045* 1.933 ± 0.054# 2.190 ± 0.062 1.844 ± 0.085# LVW/WHW (%) 0.781 ± 0.011 0.767 ± 0.012 0.764 ± 0.020 0.779 ± 0.015 0.751 ± 0.030 a Group A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and 0.02 g/kg of OGE, Group D is given CCl4 and 0.04 g/kg of OGE, and Group E is given CCl4 and 0.2 g/kg of silymarin. The individual severity rates in rats were expressed as mean ± SE. BW: body weight; WHW: whole heart weight; LVW: left ventricle weight. *Significant differences from Group A, P < 0.05. #Significant differences from Group B, P < 0.05. Table 2 Changes in diameter and thickness of left heart ventricle of CCl4-induced cirrhosis-related cardiac hypertrophy.   Aa B C D E   (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) Diameter of LV (mm) 8.17 ± 0.00 10.67 ± 0.22* 8.50 ± 0.19# 9.33 ± 0.22# 8.83 ± 0.11# Thickness of LV (mm) 3.83 ± 0.11 4.43 ± 0.15* 3.87 ± 0.12# 4.17 ± 0.11 3.83 ± 0.11# Thickness/diameter (mm) 0.42 ± 0.01 0.42 ± 0.01 0.46 ± 0.02 0.45 ± 0.02 0.43 ± 0.01 a Group A is given olive oil and water, Group B is given CCl4 and water, Group C is given CCl4 and 0.02 g/kg of OGE, Group D is given CCl4 and 0.04 g/kg of OGE, and Group E is given CCl4 and 0.2/kg g of silymarin. The individual severity rates in rats were expressed as mean ± SE. 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Evid Based Complement Alternat Med. 2012 Nov 6; 2012:139045
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23236477PONE-D-12-2315310.1371/journal.pone.0051340Research ArticleMedicineCritical Care and Emergency MedicineMultiple Organ FailureRespiratory FailureSepsisVentilatory SupportInfectious DiseasesViral DiseasesCytomegalovirus InfectionHerpes SimplexPulmonologyRespiratory InfectionsLower Respiratory Tract InfectionsVentilatory SupportCytomegalovirus and Herpes Simplex Virus Effect on the Prognosis of Mechanically Ventilated Patients Suspected to Have Ventilator-Associated Pneumonia Impact of CMV and HSV on Ventilated PatientsCoisel Yannael 1 * Bousbia Sabri 2 Forel Jean-Marie 3 Hraiech Sami 3 Lascola Bernard 2 Roch Antoine 3 Zandotti Christine 2 Million Matthieu 2 Jaber Samir 1 Raoult Didier 2 Papazian Laurent 3 1 Service d’Anesthésie-Réanimation Saint Eloi, Centre Hospitalier Universitaire, and INSERM Unité 1046, Université Montpellier 1, Montpellier, France 2 UMR CNRS 7278, Université Aix-Marseille, Marseille, France 3 Réanimation des Détresses Respiratoires et Infections Sévères, Hôpital Nord, Assistance Publique des Hôpitaux de Marseille, and UMR CNRS 7278, Université Aix-Marseille, Marseille, France Costa Cristina Editor University Hospital San Giovanni Battista di Torino, Italy * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: YC LP SB BL. Performed the experiments: YC SB JMF SH BL AR CZ MM LP. Analyzed the data: YC LP SB. Wrote the paper: YC LP SJ DR. 2012 7 12 2012 7 12 e513401 8 2012 7 11 2012 © 2012 Coisel et al2012Coisel 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.Objective Cytomegalovirus (CMV) and herpes simplex virus (HSV) are common viruses that can affect critically ill patients who are not immunocompromised. The aim of this study was to determine whether the identification of CMV and/or HSV in mechanically ventilated critically ill patients suspected of having pneumonia was associated with an increased mortality. Design Prospective epidemiological study. Setting Medical intensive care unit of a tertiary medical center. Patients Ninety-three patients with suspected pneumonia. Interventions Patients with suspected pneumonia had bronchoalveolar lavage and blood samples taken to confirm the diagnosis. Antigenemia was used to detect CMV in the blood. Bronchoalveolar lavage samples were submitted to testing using quantitative real-time Polymerase Chain Reaction. Measurements and Main Results We identified 22 patients with a CMV infection, 26 patients with an HSV infection and 45 patients without CMV or HSV infection (control group). Mortality at day 60 was higher in patients with a CMV infection than in patients from the control group (55% vs. 20%, P<0.01). Mortality at day 60 was not significantly increased in the group with HSV infection. Duration of ICU stay and ICU mortality were significantly higher in patients with CMV infections when compared to patients from the control group, whereas ventilator free days were significantly lower in patients with CMV infections when compared to patients from the control group. Conclusions In critically ill patients, a CMV infection is associated with an increased mortality. Further interventional studies are needed to evaluate whether treatment could improve the prognosis. This work was supported by a grant from the Programme Hospitalier de Recherche Clinique 2006. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction It has been shown for some time that cytomegalovirus (CMV) and herpes simplex virus (HSV) can cause severe disease in immunocompromised patients, either via reactivation of a latent viral infection (the most frequent cause) or via the acquisition of a primary viral infection [1]. More recently, CMV and HSV have been recognized as being pathogenic in critically ill patients who are not receiving immunosuppressive agents [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. However, the impact of a CMV or an HSV infection on outcome is still debated [2], [10], [13], [14], [17], [18], [19], [20], [21], [22], [23], [24]. Only a few studies have concomitantly evaluated the impact of CMV and HSV on prognosis [2], [7], [12]. In a recent retrospective cohort study of intensive care unit (ICU) patients, Miggins et al. [7] reported that an increased risk of death was associated with several viral species, including CMV and HSV. In a previously published study [2], no such impact on outcome was found for HSV infections. Moreover, the recent use of real-time PCR may have modified the epidemiology of such infections [8], [25]. We hypothesized that the identification of CMV in blood and/or bronchoalveolar lavage (BAL) samples could be associated with a higher mortality rate when compared to controls that do not develop a viral infection. The aim of the present study was therefore to prospectively evaluate the impact that CMV or HSV has on outcome. A control population of patients not developing these viral infections was used as a comparison. This study was performed in a cohort of mechanically ventilated ICU patients that were suspected of having pneumonia. 10.1371/journal.pone.0051340.t001Table 1 Characteristics of patients upon admission to the ICU. Overall Population HSV infection group CMV infection group Control group p (n = 93) (n = 26) (n = 22) (n = 45) Age (yr), median [IQR] 63[52–73] 64[54–73] 69[61–75]* 59[43–69] 0.024 Male gender, n (%) 55 (59) 14 (54) 14 (64) 27 (60) 0.78 SAPS II, median [IQR] 45 [31–55] 50[36–58] 40[31–53] 44[31–55] 0.43 Direct admission from the community, n (%) 65 (69) 19 (73) 10 (45) 36 (80) 0.43 Reason for ICU admission  Acute respiratory failure 40 (43) 9 (34) 13 (59) 18 (40) 0.36  Acute exacerbation of chronic respiratory insufficiency 11 (12) 3 (12) 4 (18) 4 (9)  Neurologic failure 11 (12) 3 (12) 0 (0) 8 (18)  Septic shock 9 (10) 4 (15) 1 (5) 4 (9)  Postoperative respiratory failure 8 (8) 1 (4) 2 (9) 5 (11)  Cardiogenic shock 4 (4) 1 (4) 1 (5) 2 (4)  Hemorrhagic shock 2 (2) 2 (8) 0 (0) 0 (0)  Miscellaneous 8 (9) 3 (12) 1 (5) 4 (9) Immunosuppression on ICU admission, n (%)  No immunosupression 84 (90) 23 (88) 21 (95) 40 (89) 0.46  Chemotherapy 2 (2) 0 (0) 1 (5) 1 (2)  Long-term corticosteroids 6 (7) 2 (8) 0 (0) 4 (9)  HIV 1 (1) 1 (4) 0 (0) 0 (0) Prior ARDS, n (%) 27 (29) 8 (31) 10 (46) 9 (20) 0.10 SAPS II, simplified acute physiologic score II; ARDS, acute respiratory distress syndrome; * p<0.05 vs. No HSV – No CMV. 10.1371/journal.pone.0051340.t002Table 2 Characteristics of patients at the time of diagnosis. All HSV infection group CMV infection group Control group p (n = 93) (n = 26) (n = 22) (n = 45) Duration of mechanical ventilation prior to suspicion of pneumonia (days), median [IQR] 2 [2]–[10] 8 [2]–[12] * 7 [2]–[14] * 2 [2]–[5] 0.004 CPIS score, median [IQR] 4 [3]–[5] 4 [3]–[5] 3 [2]–[5] 4 [3]–[5] 0.57 SOFA score (total), median [IQR] 7 [5]–[9] 6 [3]–[9] 7 [5]–[9] 8 [6]–[10] 0.43 Prior antibiotics, n (%) 46 (50) 13 (50) 8 (36) 25 (56) 0.34 Enteral nutrition, n (%) 47 (51) 15 (58) 13 (59) 19 (42) 0.30 Closed-suction system, n (%) 29 (31) 7 (27) 8 (36) 14 (31) 0.78 Nasogastric tube, n (%) 66 (71) 20 (77) 16 (73) 30 (67) 0.64 Sedation, n (%) 76 (82) 23 (89) 20 (91) 33 (73) 0.13 NMBA, n (%) 26 (28) 8 (31) 8 (36) 10 (22) 0.45 Anti H2, n (%) 0 (0) 0 (0) 0 (0) 0 (0) 1 Sucralfate, n (%) 1 (1) 0 (0) 0 (0) 1 (2) 0.58 Antacids, n (%) 1 (1) 1 (4) 0 (0) 0 (0) 0.27 Proton-pump inhibitors, n (%) 84 (90) 24 (92) 19 (86) 41 (91) 0.76 Statin, n (%) 9 (10) 2 (8) 4 (18) 3 (7) 0.30 Strict glycemic control, n (%) 10 (10) 4 (15) 2 (9) 4 (9) 0.67 Massive blood transfusion, n (%) 14 (15) 4 (15) 6 (27) 4 (9) 0.14 Intrahospital transfer, n (%) 57 (61) 17 (65) 13 (59) 27 (60) 0.88 Corticosteroids for septic shock, n (%) 37 (40) 12 (46) 12 (55) 13 (29) 0.10 Corticosteroids for ARDS, n (%) 8 (9) 1 (4) 3 (14) 4 (9) 0.48 Activated protein C, n (%) 2 (2) 1 (4) 0 (0) 1 (2) 0.66 Reintubation, n (%) 14 (15) 5 (19) 5 (23) 4 (9) 0.26 CPIS, clinical pulmonary infection score; SOFA, sequential organ failure assessment score; NMBA, Neuromuscular blocking agents; massive blood transfusion, replacement of a patient’s total blood volume in less than 24 hours; * p<0.05 vs. No HSV – No CMV. Materials and Methods A. Ethics Statement The study was approved by the Local Ethics Committee of the Université de la Méditerranée (Marseille, France; approval n° 07-026) which waived the need for written consent, because the protocol did not impact on patient management and complied with standard care in our unit. However, all patients and/or next of kin were informed. B. Study Setting and Population This prospective study was performed in the medical ICU of Sainte-Marguerite University Hospital in Marseille, France. Over a one-year period, all consecutive patients (18 years or older) were prospectively included if they were mechanically ventilated and suspected of having pneumonia. None of the patients were included in a recently published study from the same group [23]. Suspicion of pneumonia was based on the appearance of a new pulmonary infiltrate on chest radiographs, associated with at least 2 of the following criteria: fever >38°C or hypothermia <36°C; white blood cell count >10×109/L or <4×109/L; purulent tracheal secretions; or a decrease in the PaO2/FiO2 ratio [26], [27]. Fiberoptic bronchoscopy examination was performed in each patient within the first 12 h of suspecting pneumonia. Bronchoalveolar lavage was performed as previously described [27]. BAL samples were tested by RT-PCR, and standard cultures were performed to identify the bacteria and fungi present in the blood and BAL samples [28]. 10.1371/journal.pone.0051340.g001Figure 1 Viral load on bronchoalveolar lavage for herpes simplex virus ( Figure 1A ) and cytomegalovirus ( Figure 1B ) according to mortality at day 60. 10.1371/journal.pone.0051340.t003Table 3 Virological results. All HSV infection group CMV infection group Control group (n = 93) (n = 26) (n = 22) (n = 45) HSV status, n(%)  IgM HSV 3 (3) 2 (8) 1 (5) 0 (0)  IgG HSV 81 (87) 24 (92) 19 (86) 38 (84)  BAL RT-PCR 31 (33) 25 (96) 6 (27) 0 (0) CMV status, n(%)  IgM CMV 8 (9) 0 (0) 8 (36) 0 (0)  IgG CMV 72 (77) 18 (69) 20 (91) 34 (76)  BAL RT-PCR 16 (17) 0 (0) 16 (73) 0 (0)  Antigenemia 10 (11) 0 (0) 10 (46) 0 (0) BAL, bronchoalveolar lavage; RT-PCR, real time polymerase chain reaction. 10.1371/journal.pone.0051340.t004Table 4 Outcomes. All HSV infection group CMV infection group Control group p (n = 93) (n = 26) (n = 22) (n = 45) Mortality at day 60, n (%) 32 (34) 11 (42) 12 (55)* 9 (20) 0.012 ICU Mortality, n (%) 32 (34) 11 (42) 12 (55)* 9 (20) 0.012 Duration of mechanical ventilation (days), median [IQR] 14 [7]–[29] 14.5 [10]–[26] 19.5 [13]–[44] † 10 [3]–[25] 0.009 VFD at day 28 (days), median [IQR] 5 [0–22] 5.5 [0–23] 0 [0–0]† 18 [0–26] 0.001 VFD at day 60 (days), median [IQR] 37 [0–54] 36.5 [0–55] 0 [0–25]† 50 [11.5–58] 0.001 Duration of ICU stay (days), median [IQR] 16 [9]–[30] 18 [11]–[30] 25.5 [15]–[43] † 13 [7–28.5] 0.037 Shock, n (%) 40 (43) 10 (39) 17 (77)* ‡ 13 (30) 0.001 Acute renal failure, n (%) 24 (26) 6 (23) 11 (50)* 7 (16) 0.01 Bacteremia, n (%) 19 (20) 3 (12) 10 (46)* 6 (13) 0.004 ARDS, n (%) 18 (19) 5 (19) 6 (27) 7 (16) 0.52 Bacterial VAP, n (%) 12 (13) 2 (8) 4 (18) 6 (13) 0.55 ICU, intensive care unit; ARDS, acute respiratory distress syndrome; VAP, ventilator-associated pneumonia; BAL, bronchoalveolar lavage; VFD, ventilator-free days. * p<0.01 vs. the No HSV – No CMV group; † p<0.05 vs. the No HSV – No CMV group; ‡ p<0.016 vs. the HSV group. C. Diagnosis of Pneumonia One of the investigators made daily rounds in the ICU to identify eligible patients, to determine the onset of pneumonia based on the diagnostic criteria described below, and to record relevant data from the medical records (bedside flow sheets) and the hospital’s mainframe computer, which housed microbiological test results. All chest radiographs were analyzed prospectively by at least two of the investigators. Confirming a diagnosis of bacterial pneumonia required a suspicion of pneumonia and a BAL quantitative culture that grew at least one microorganism at a concentration ≥104 colony-forming units (cfu)/mL. D. Baseline Assessment and Data Collection Each patient’s hospital chart was prospectively implemented, and the following data were recorded during admission to the ICU: age, sex, Simplified Acute Physiologic Score II (SAPS II) [29], presence of co-morbidities, and presence of previous immunosuppression or previous Acute Respiratory Distress Syndrome (ARDS). In addition, on the day of sampling, we recorded the Sepsis-related Organ Failure Assessment (SOFA) score [30], the time spent on a mechanical ventilator from admission to the suspicion of pneumonia, and the CPIS score (Clinical Pulmonary Infection Score) modified by Luna [31]. Other relevant clinical characteristics and outcomes complicating the ICU stay following inclusion (ARDS, bacteremia, bacterial ventilator-associated pneumonia (VAP), acute renal failure, shock, ventilator free days and mortality) were also recorded throughout the ICU stay. 10.1371/journal.pone.0051340.g002Figure 2 Meta-analyse of the mortality associated with Cytomegalovirus (CMV) Diagnosis methods are detailed in table 5 . 10.1371/journal.pone.0051340.g003Figure 3 Meta-analyse of the mortality associated with Herpes Simplex Virus (HSV) Diagnosis methods are detailed in table 6 . 10.1371/journal.pone.0051340.t005Table 5 Diagnosis Methods used to diagnose CMV infection. CMV Sample Diagnosis methods Domart 1990 [39] Blood, urine Viral culture Cook 1998 [35] Lower respiratory tract, tracheal aspiration, blood, skin Viral culture Kutza 1998 [37] Blood PP65 antigenemia, PCR Heininger 2001 [4] Blood, lower respiratory tract Viral culture, PCR Cook 2003 [2] Blood, tracheal aspiration Serology, viral culture Jaber 2005 [14] Blood PP65 antigenemia Limaye 2008 [13] Blood PCR Von Muller 2006 [47] Blood, tracheal aspiration, urine Serology, PP65 antigenemia, viral culture in blood, tracheal aspiration and urine Ziemann 2008 [24] Blood PCR Chiche 2009 [23] Blood, lower respiratory tract Serology, PP65 antigenemia, viral culture in BAL Chilet 2010 [34] Blood, tracheal aspiration PCR Heininger 2011 [17] Blood, tracheal aspiration PCR Coisel 2012 Blood, lower respiratory tract Serology, BAL-PCR, PP65 antigenemia BAL, bronchoalveolar lavage; PCR, polymerase chain reaction. 10.1371/journal.pone.0051340.t006Table 6 Diagnosis Methods used to diagnose HSV infection. HSV Sample Diagnosis methods Cook 1998 [35] Lower respiratory tract, tracheal aspiration, blood, skin viral culture Bruynseels 2003 [9] Lower respiratory tract, throat viral culture Cook 2003 [2] Tracheal aspiration viral culture Ong 2004 [38] Lower respiratory tract, throat PCR Engelmann 2007 [36] Lower respiratory tract, tracheal aspiration, throat PCR, viral culture, direct immunofluorescence Luyt 2007 [10] Lower respiratory tract, tracheal aspiration, bronchial biopsies BAL-PCR, BAL-viral culture, cytology Linssen 2008 [18] Lower respiratory tract PCR De Vos 2009 [8] Lower respiratory tract PCR Scheithauer 2010 [19] Lower respiratory tract, tracheal aspiration PCR Smith 2010 [12] Tracheal aspiration PCR Bouza 2011 [48] Lower respiratory tract viral culture Coisel 2012 Blood, lower respiratory Tract Serology, BAL-PCR BAL, bronchoalveolar lavage; PCR, polymerase chain reaction. E. Serologies and Antigenemia Viral serology (IgM and IgG) for cytomegalovirus (CMV) and herpes simplex (HSV) were performed using conventional serological methods with an enzyme linked immunosorbent assay (ELISA). Antigenemia for cytomegalovirus was evaluated using a CINA complete kit (Argene SA, Verniolle, France). Briefly, erythrocytes were lysed by mixing 2 mL of EDTA blood with 8 mL of erythrocyte lysing solution and then centrifuged at 300×g for 10 min. Erythrocyte lysis and centrifugation was repeated twice. Then, the supernatant was discarded and the leukocyte pellet was resuspended in 1 mL of phosphate buffered saline (PBS), counted, and then diluted to 2×106 cells/mL. One hundred µL (200,000 cells) were cytocentrifuged at 900 rpm for 3 min on glass slides and air-dried. The slides were fixed with a paraformaldehyde solution for 10 min and washed 3 times with PBS. The cells were then incubated for 30 min at 37°C with a mixture of two monoclonal antibodies (1C3 and AYM). After washing with PBS, the slides were incubated for 30 min at 37°C with a secondary antibody conjugated with fluorescein. The slides were then subjected to 3 final washes with PBS, and examined under a fluorescent microscope. The results are given as the number of positive cells per 200,000 cells. Blood PCR was not routinely done when the study was designed. F. Identification of CMV and HSV by PCR on BAL The presence of CMV and HSV was tested with a quantitative real-time PCR. Viral nucleic acids were extracted from 200 µL of BAL fluids with a MDX workstation using a QIAamp Virus BioRobotMDx Kit (Qiagen, Courtaboeuf, France), as recommended by the manufacturer’s instructions. Quantitative real time PCR for CMV and HSV was performed using a LightCycler® instrument (Roche Diagnostics, Meylan, France) with the QuantiTect Probe PCR Kit (Qiagen). HSV was tested with the PolF (5′-GGGCCAGGCGCTTGTTGGTGTA-3′) and the PolR (5′-CATCACCGACCCGGAGAGGGAC-3′) primer set (Eurogentec, Seraing, Belgium), and the specific TaqMan probe (6FAM-CCGCCGAACTGAGCAGACACCCGCGC-TAMRA) (Applied Biosystems, Courtaboeuf, France). The presence of CMV was tested with the pp65F (5′- GCAGCCACGGGATCGTACT -3′) and the pp65R (5′-GGCTTTTACCTCACACGAGCATT-3′) primer set, and the specific TaqMan probe (6FAM-CGCGAGACCGTGGAACTGCG-TAMRA). The reaction was carried out in 20 µL, in a final volume containing 10 µL of QuantiTect master mix, 0.2 µM of probe, 0.2 mM of each primer, and 4 µL of DNA. The qPCR was initiated by an enzyme-activation incubation at 95°C for 15 min to activate DNA Polymerase, followed by 40 cycles of denaturation at 95°C for 10 s and an annealing-extension step at 60°C for 1 min. Serial dilutions, ranging from 102 to 105 copies/mL, of synthesized sequences that correspond to the targeted viral genes, were used as positive controls. These dilutions were also used to determine the viral load in positive BAL fluids. A CMV and HSV negative specimen was used as a negative control. G. Definitions The CMV infection group was defined as patients suspected of having pneumonia with positive CMV DNA detection in BAL fluid and/or positive antigenemia and/or the presence of IgM for CMV. We did not differentiate between endogenous reactivation or exogenous infection as the cause of the active infection. The HSV infection group was defined as patients suspected of having pneumonia associated with positive HSV DNA detection in BAL fluid, or the presence of IgM for HSV in patients without CMV identification by antigenemia, real-time PCR or specific IgM. The “control group” was defined by the absence of a CMV or HSV infection in patients suspected of having pneumonia. A bacterial coinfection required the presence of at least one bacteria at a concentration exceeding 104 cfu/mL in the BAL fluid. H. Study Outcomes 1. Primary outcome The primary outcome was mortality for both viruses, evaluated at day 60. 2. Secondary outcomes Secondary outcomes were the ICU mortality, the day-28 mortality, the number of days with mechanical ventilation, the number of ventilator-free days (days alive and with a successful weaning from mechanical ventilation for at least 48 hrs) between day 1 and day 28, and between day 1 and day 60 [32]. I. Statistical Analysis Data are expressed as the median with an interquartile range (IQR) or as number of events (percentage). Continuous variables were compared using a Kruskall-Wallis one-way analysis of variance on ranks, with a pairwise multiple comparisons procedure using Dunn’s method. The chi-squared test was used to compare categorical variables. All reported P values are two-sided. For all statistical tests used, a p value of <0.05 was considered significant. A Bonferroni method was applied for multiple comparisons when necessary (leading to a significant p value of <0.016 when applied). Multiple logistic regressions were used to adjust the day 60 mortality regarding 2 pre-defined variables (SAPS II score on admission and SOFA score the day of BAL). Results A. Patients Characteristics 1. All patients During the study period, ninety-three consecutive patients met the inclusion criteria and were prospectively included in the study. Less than one third of all patients were ventilated for ARDS. Ten percent of all patients were previously immunosuppressed (Table 1). 2. Virological status Twenty-six patients (28%) were included in the HSV group, 22 (24%) in the CMV group, and 45 patients (48%) were negative for both HSV and CMV (control group). Patients from the CMV group were older (Table 1). As shown in Table 2, there was no significant difference between the three groups at the time of diagnosis, excepting a longer duration of mechanical ventilation in patients from the CMV and the HSV groups before the realization of the BAL as compared with the control group. 3. Virological diagnosis Eighty-one patients (87%) had IgG for HSV and 72 patients (77%) had IgG for CMV. Thirty-one patients had a positive BAL for HSV using RT-PCR (from 6.2×103 to 9.4×109 copies/mL, figure 1A) and 16 patients had a positive BAL for CMV using RT-PCR (from 9.9×103 to 3.1×107 copies/mL, figure 1B). Table 3 shows that antigenemia and RT-PCR were positive for 46% and 73% of the patients exhibiting an active CMV infection, respectively. Eight of the patients with a positive antigenemia for CMV also had a positive RT-PCR for CMV. Finally, all but four patients from the CMV infection group had a positive RT-PCR for CMV performed on BAL samples and/or a positive antigenemia. Only one patient from the HSV group presented IgM with a negative RT-PCR for HSV on BAL. Six patients from the CMV group also had a positive RT-PCR for HSV. By definition, no patient from the HSV group had a positive antigenemia or RT-PCR for CMV. A concomitant confirmed bacterial lung infection was present in 11 patients from the HSV group (42%), 11 (50%) from the CMV group and 16 (36%) from the control group (p = 0.52). Gram-negative bacilli represented respectively 79%, 62% and 72% of the bacteria cultured from BAL. B. Outcomes (Table 4) Mortality at day 60 was higher in patients with a CMV and/or a HSV infection [48% (CI 95 from 35% to 62%)] compared to patients from the control group [20% (CI from 11% to 34%)] (p<0.005). More specifically, mortality at day 60 was higher in patients with a CMV infection [55% (CI 95 from 35% to 73%)] compared with control patients [20% (CI from 11% to 34%)] (p<0.01). This difference remained significant after adjusting for age, SAPS II score on admission and SOFA score on the day of diagnosis. Mortality at day 60 was not significantly higher in the HSV group [42% (CI from 26% to 61%)] compared to the control group. As shown in Figure 1, there was no relationship between mortality at day 60 and the viral load for both the CMV group and the HSV group. Ventilator-free days at D28 and D60 were significantly lower in patients developing an active CMV infection than in patients from the control group. The duration of ICU stay and the ICU mortality rate were significantly higher in patients developing an active CMV infection than in patients from the control group (Table 4). However, there was no difference between the CMV group and the HSV group regarding these parameters. There was no correlation between the viral load and the number of ventilator-free days at D28 and D60 (data not shown). Complications such as bacteremia, acute renal failure or shock were significantly more frequent in the CMV group (Table 4). In contrast, there were no increases in the rate of bacterial VAP or ARDS following virus identification in the CMV group when compared to the other two groups. Discussion The present study suggests that an active CMV infection in critically ill patients increases both crude and adjusted mortalities at day 60. CMV infection was also associated with less ventilator free days at day 28 and day 60, and an increased duration of ICU stay compared with patients without CMV and HSV identification. Infection with a Herpesviridae family virus, namely CMV and HSV, is very common in the general population, whether they are immunosuppressed or not [33], [34], [35], [36], [37], [38]. In critically ill patients, the incidence of both active CMV and HSV infection is matter of controversy [14], [23]. Moreover, many studies were performed in trauma or surgical patients. Serological positivity for CMV reported in critically ill patients ranged from 13% [39] to 100% [40]. Respiratory samples positive for CMV ranged from 0% [41] to 13% [4], antigenemia ranged from 0% [42] to 17% [14], and even 85% in one study [23]. However, the use of open lung biopies found that up to 50% of patients with ARDS were infected with CMV [16]. These differences could be explained by different diagnostic methods for the detection of CMV, including viral culture, antigenemia and PCR assays [22]. Previous studies used culture-based assays (low sensitivity and time-consuming), whereas more recent studies have used antigenemia (more sensitive and quantitative results) or PCR assays [13]. Nevertheless, none of these methods have been validated in ICU patients. Moreover, our results should take in account the relative lack of sensitivity and specificity of some of these diagnostic methods (serology for example). It was likely that some patients with positive virus may actually have infection, whereas other with positive samples may just be false positive. The newest diagnostic methods have not been validated in ICU patients. However, in immunocompromised patients, techniques such as PCR and antigenemia present an adequate diagnostic accuracy [43], [44]. CMV reactivation in intensive care patients is not trivial. Indeed, in a study using a murine model, Cook et al. showed that CMV reactivation caused abnormal tumor necrosis factor-α expression and induced abnormal pulmonary fibrosis, both of which were prevented with ganciclovir [45]. Reactivation of CMV could lead to an increased duration of ventilation or ICU stay in non-immunosuppressed patients in an intensive care setting [2], [14], [23], [24], [46], [47]. A human study found an independent correlation between CMV reactivation and morbidity in non-immunosuppressed patients [17], however, there was no correlation with mortality. Another human study found a significant increased mortality rate in patients expressing CMV, but could not demonstrate a cause-effect relationship [20]. In our study, we could identify factors associated with positive CMV samples, but causative links between both had not been addressed. To our knowledge, this is the first study indicating that an active CMV infection in critical care patients increased crude and adjusted mortality at day 60. Our results are concordant with those of Heininger et al. [4], who found that the mortality rate tended to be higher in patients with active CMV infections, with a significant increase in ICU length of stay in survivors. Limaye [13] also found and association between CMV reactivation and a composite end point (prolonged hospitalization or death). In our unit, all patients with an active CMV infection were treated with gancyclovir, which make it difficult to conclude regarding the efficacy of this treatment. Only an interventional trial could conclude if CMV is definitely responsible for a longer duration of mechanical ventilation/LOS. Indeed, a longer duration of exposure to mechanical ventilation could be associated with an increased risk to identify CMV without any impact on prognosis. This is unlikely because in the present study, patients from the control group were ventilated invasively for a longer period than the time to identify CMV in the CMV group. Figures 2 and 3 represent two meta-analyses of the mortalities associated with CMV and HSV. Even if the diagnostic criteria and the studied populations are very different from one study to another (Tables 5 and 6), these analyses suggest that both CMV and HSV are associated with increased mortality rates. This study strongly suggests that CMV reactivation in critically ill patients is associated with increased mortality. 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PLoS One. 2012 Dec 7; 7(12):e51340
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23236406PONE-D-12-2213910.1371/journal.pone.0050919Research ArticleBiologyImmunologyImmunologic SubspecialtiesTransplantationMedicineClinical ImmunologyImmunologic SubspecialtiesTransplantationGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BInfectious diseasesViral diseasesHepatitisHepatitis BOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaSurgeryTransplant SurgeryThe Survival Benefit of Liver Transplantation for Hepatocellular Carcinoma Patients with Hepatitis B Virus Infection and Cirrhosis Survival after LT for HCC with HBVZhang Qing 1 Chen Xinguo 1 Zang Yunjin 1 Zhang Li 3 Chen Hong 1 Wang Letian 1 Niu Yujian 1 Ren Xiuyun 1 Shen Zhongyang 1 * Shang Lei 2 * 1 Institute of Liver Transplantation, General Hospital of Chinese People's Armed Police Force, Beijing, China 2 Department of Health Statistics, Faculty of Preventive Medicine, Fourth Military Medical University, Xi'an, China 3 First Department of Surgery, Shaanxi Provincial Corps Hospital of Chinese People's Armed Police Force, Xi'an, China Bouchard Michael Editor Drexel University College of Medicine, United States of America * E-mail: [email protected] (ZS); [email protected] (LS)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ZS QZ. Performed the experiments: QZ XC YZ LZ. Contributed reagents/materials/analysis tools: HC LW XR. Technical support: ZS LS XC YZ HC. Critical revision of the manuscript for important intellectual content: ZS. Study supervision: ZS LZ LW. Drafting of the manuscript and revision of the manuscript for total content: QZ. Study design and statistical analysis: LS. Patient enrollment: XC YZ LZ. Follow up patients for data collection: YN. Data collection: XR. 2012 7 12 2012 7 12 e5091925 7 2012 25 10 2012 © 2012 Zhang et al2012Zhang 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 precise predictive survival model of liver transplantation (LT) with antiviral prophylaxis for hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and cirrhosis has not been established. The aim of our study was to identify predictors of outcome after LT in these patients based on tumor staging systems, antitumor therapy pre-LT, and antiviral prophylaxis in patients considered to be unfit by Milan or UCSF criteria. Methods From 2002 to 2008, 917 LTs with antiviral prophylaxis were performed on patients with HBV-cirrhosis, and 313 had concurrent HCC. Results Stratified univariate and multivariate analyses demonstrated that independent predictors for poor survival were tumor size >7.5 cm (P = 0.001), tumor number >1 (P = 0.005), vascular invasion (P = 0.001), pre-LT serum alpha-fetoprotein (AFP) level ≥1000 ng/ml (P = 0.009), and pre-LT aspartate aminotransferase (AST) level ≥120 IU/L (P = 0.044). Pre-LT therapy for HCC was an independent predictor of better survival (P = 0.028). Based on CLIP and TNM tumor staging systems, HCC patients with HBV-cirrhosis who met the following criteria: solitary tumor ≤7.5 cm, or ≤4 multifocal nodules, the largest lesion ≤5 cm and total tumor diameter ≤10 cm, or more nodules with the largest lesion ≤3 cm, and pre-LT serum AFP level <1000 µg/L and AST level <120 IU/L without vascular invasion and lymph node metastasis who were unfit for UCSF, had survival rates of 89% at 5 years. There was a 47% 5-year survival rate for patients with HCC exceeding the revised criteria. Conclusions The current criteria for LT based on tumor size, number and levels of AFP and AST may be modestly expanded while still preserving excellent survival after LT. The expanded criteria combined with antiviral prophylaxis and pre-LT adjuvant therapy for HCC may be a rational strategy to prolong survival after LT for HCC patients with HBV-associated cirrhosis. The authors have no support or funding to report. ==== Body Introduction Hepatitis B is endemic to China [1]. Of the 350 million individuals worldwide infected with the hepatitis B virus (HBV), one-third resides in China, with 130 million carriers and 30 million chronically infected people [2], [3]. The chronically infected individuals may be either asymptomatic or have chronic inflammation of the liver that leads to cirrhosis over a period of several years. HBV infection dramatically increases the incidence of hepatocellular carcinoma (HCC), the most common primary malignant cancer of the liver [4]. Furthermore, HBV-induced cirrhosis (HBV-cirrhosis) is the most common cause of HCC. In China, most HCC patients also have HBV-related cirrhosis [5]. The relationship between HCC with HBV-associated cirrhosis has long been recognized, and the primary therapeutic modality for HCC is surgical extirpation. Unfortunately, only a small number of patients are suitable for liver resection because of the advanced stage of tumors at the time of diagnosis, as well as the frequent development of tumors in a background of HBV-associated cirrhosis with poor liver function. It has been established that liver transplantation (LT) with antiviral prophylaxis is the only therapeutic option for simultaneously treating HCC and HBV-associated cirrhosis [6]–[9], and it is accepted that LT is superior to hepatic resection in early HCC with cirrhosis [10]–[13]. Mazzaferro et al. reported good LT outcomes for small HCC ([Milan] criteria: (solitary tumor ≤5 cm, or three or fewer lesions none >3 cm) with cirrhosis, with 4-year overall and recurrence-free survival rates of 85% and 92%, respectively [8]. Recently, a set of expanded criteria for tumor staging was proposed that was associated with excellent survival after OLT. HCC patients who met UCSF criteria (solitary tumor ≤6.5 cm or 3 nodules with the largest lesion ≤4.5 cm, and a total tumor diameter ≤8 cm) after LT had 1- and 5-year survival rates of 90% and 75.2%, respectively [14], which were similar to the survival rates in patients without HCC. Nevertheless, for patients with HBV-associated HCC, there are usually more aggressive tumors and elevated hepatitis activity that could lead to hepatocyte necrosis, as well as HBV-associated cirrhosis with poor liver function. Despite several criteria showing excellent outcomes for LT for HCC [6], [8], [15]–[17], those criteria only focused on the size and number of tumors or pathologic tumor staging. They did not consider HCC induced by various other etiologies, other tumor factors, or liver markers, such as pre-LT serum alpha-fetoprotein (AFP) levels, Child-Pugh scores or liver function indicators, as determinants of HCC patient outcome. Nevertheless, there is no clear consensus for HBV-associated HCC, especially for advanced HCC patients with HBV- cirrhosis who may still have a favorable outcome after LT. Factors affecting outcome in patients with aggressive HCC have been extensively studied [14], [18], [19]. It has been shown that tumor size, tumor number, pathologic tumor differentiation, the presence or absence of vascular invasion, lymph node metastases, pre-LT serum alpha-fetoprotein (AFP), liver function lever, and preoperative tumor treatment are prognostic variables that have a clear impact on outcome [7], [8], [14], [20]–[22]. Tumor TNM staging for predicting survival of HCC patients has also been considered in the past. Recently, some studies [23], [24] have claimed that the Cancer of the Liver Italian Program (CLIP) staging system is one of the best staging systems in predicting survival in patients with advanced HCC compared to the Japanese, and AJCC TNM, and TNM sixth edition. The others lacked any prognostic parameters of liver dysfunction or AFP. A staging system that combines tumor factors, tumor marker(s) and hepatic function is the best predictor of prognosis of HCC patients, especially for HCC with HBV-associated cirrhosis. Some studies have reported that tumor diameter, poor tumor differentiation, vascular invasion, AFP level, HBV reinfection and prophylaxis were independent predictors of outcome [18], [25], [26]. Although most studies have shown that HBV infections carry a high risk of recurrence after resection or LT [27], prophylactic use of hepatitis B immunoglobulin (HBIG) combined with the nucleoside analogue lamivudine can markedly decrease the reinfection rate of HBV by suppressing HBV replication [28], [29]. Some reports have demonstrated that dual prophylaxis for HBV after LT reduces the risk of HBV reinfection and improves patient survival [30]. Therefore, it is possible that a subset of HBV-associated HCC patients who exceed Milan or UCSF criteria may still have a favorable outcome after LT. In patients with HBV-related HCC, the tumors are usually large and aggressive and accompanied by elevated inflammatory activity that can lead to aggressive hepatocyte necrosis. In spite of the proposed expanded criteria such as UCSF [31], Pittsburgh [31] or UNOS to select HCC patients for LT [32], no universally accepted criteria have been established to select suitable HCC patients with HBV-cirrhosis for LT. The aim of this study was to establish criteria to select suitable HCC patients with HBV-cirrhosis for LT with antiviral prophylaxis and anti-HBV treatment. Materials and Methods Patient population Between July 2002 and December 2008, 917 LTs with antiviral prophylaxis were performed for HBV cirrhosis at the Institute of Liver Transplantation, General Hospital of Chinese People's Armed Police Force, China (according to the China Liver Transplant Registry: https://www.cltr.org/). Of these 917 patients, 313 patients who were diagnosed with HCC and HBV-induced cirrhosis, underwent LT and had complete follow up information were enrolled in this study. HCC patients with extra-hepatic tumor metastasis, including lymph node metastasis or inferior vena cava tumor thrombus by imaging diagnosis before LT, were excluded from this study. Because the majority of cases who exceeded the Milan or UCSF criteria without tumor downgrading therapy pre-LT had been transplanted before 2004, the cases with a total tumor diameter of tumor nodules >12 cm were excluded from this study. In addition, patients with HBV co-infection with hepatitis C were also excluded from this study. The baseline characteristics of the 313 patients are summarized in Table 1. 10.1371/journal.pone.0050919.t001Table 1 The main clinical and pathological characteristics of the study patients. Variables N (%) Gender Male 288 (92.0) Female 25(8.0) Age (year) ≤50 151(48.2) >50 162(51.8) HBeAg Negative 204(65.2) Positive 109(34.8) HBV-DNA (×103 IU/ml) <1 118(37.7) <2500 145(46.3) ≥2500 39 (96.5) Child-Pugh score A 118(37.7) B 131(41.9) C 64(20.4) ALT (IU/L) 1N, 134(42.8) ≥1N, <2N 104(33.2) ≥2N, <3N 37(11.8) ≥3N 38(12.1) AST(IU/L) 1N, 104(33.2) ≥1N, <2N 116(37.1) ≥2N, <3N 47(15.0) ≥3N 46(14.7) ALP(IU/L) 1N, 204(65.2) ≥1N, <2N 85(27.2) ≥2N 24(7.7) Tumor size (cm) ≤3 115(36.7) >3, ≤5 121(38.7) >5, ≤7.5 40(12.8) >7.5 37(11.8) Number of tumor nodules Single 214(68.4) 2 56(17.9) 3 15(4.8) 4, 5(1.6) >4 23(7.3) Tumor differentiation I (well) 13(4.2) II (moderate) 278(88.8) III (poor) 22(7.0) Serum AFP level (ng/ml) ≤500 247(84.0) 500–1000 16(5.1) 1000–2000 23(7.3) 2000–5000 15(4.8) ≥5000 12(3.8) Venous invasion Absent 272(86.9) Present 41(13.1) Lymph node invasion Absent 295(94.2) Present 18(5.8) Pre-LT antitumor therapy Absent 48(15.3) Present 96(30.7) Fit Milan criteria? Yes 179(57.2) No 134(42.8) Fit UCSF criteria, but unfit Milan? Yes 42(13.4) No 271(86.6) Post-LT HBsAg reinfection Negative 293(93.6) Positive 20(6.4) Rejection Absent 285(91.1) Present 28(8.9) Post-LT treatment for recurrence Absent 259(82.7) Present 54(17.3) In the current study, there were 288 men and 25 women, aged 25 to 70 years, with a median age of 49.65 years. The mean tumor size was 4.37 cm (±2.7, range 0.3–12.0). AFP levels and liver function indicators were obtained within 1 month before LT. The median AFP level was 1,016 µg/L (range 1.62 to 60,500 µg/L). The median ALT, AST and ALP level were 93.8 IU/L (range 8 to 3196 IU/L), 109.7 IU/L (range 17 to 4409 IU/L) and 114.8 IU/L (range 23 to 643 IU/L), respectively. Of the 313 patients, 122 (38.9%) had normal AFP levels (<20 IU/L). HBV DNA levels obtained within 3 months before OLT were available in 302 of 313 patients. The median HBV DNA level was 2,500 IU/ml (range, 11.4–91200). All patients were tested positive for HBsAg. In addition, 96 of 313 patients were given pre-LT treatments: 82 cases of transarterial chemoembolization, of which 4 were combined with percutaneous ablations and 7 were followed with hepatic resection, 8 cases of percutaneous ablations with radio frequency, 2 percutaneous ablation cases with ethanol injection, and 10 resection only cases before LT (when there was intrahepatic recurrence of hepatocellular carcinoma). No tumor adjuvant treatment was given after LT unless tumor recurrence was detected. In an additional analysis, the entire cohort of 313 patients with HCC were divided into 3 groups: 197 (57.2%) fit the Milan criteria group, 42 (13.4%) did not fit the Milan, but did fit the UCSF criteria (Milan-UCSF) group, and 92 (29.4%) in the group with patients who exceeded UCSF criteria(>UCSF), of which 38 (41.3%) were given pre-LT treatments for downgrading therapy or decreasing the risk of tumor dissemination during the long waiting period for LT. This retrospective study was performed incompliance with principles of the Helsinki Declaration, and institutional guidelines. Diagnosis and evaluation As a part of the pre-transplant workup for HBV in recipients, infection with HBV pre-LT was routinely checked by the following viral markers: hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), hepatitis B core antibody, hepatitis Be antigen (HBeAg), and hepatitis B virus deoxyribonucleic acid (HBV DNA) levels. Pre-transplant HBV infection was defined as serum HBsAg and/or HBV DNA positivity. HBV status was assessed before and after transplantation with HBV markers detection and HBV DNA PCR assay. Tests to determine viral mutation were also conducted to identify resistance to lamivudine or adefovir. In order to differentiate HBV recurrence from graft rejection, a percutaneous or transjugular liver biopsy was performed. HCC was diagnosed pre-LT by measuring serum AFP levels, and by a combination of 2 abdominal imaging techniques (ultrasound, computed tomography [CT], positron emission tomography [PET], or magnetic resonance imaging [MRI]). Final diagnosis of HCC and cirrhosis in the explanted livers was determined by pathological examination. Routine post-LT examinations included abdominal ultrasonography, X-ray imaging, serial serum AFP levels, and whole-body CT scans, as deemed necessary. During LT follow-up, early tumor recurrence or metastasis was assessed by AFP level and abdominal ultrasonography once a month by whole-body CT or MRI examinations, and by bone scintigraphy every 3–6 months. To histologically confirm recurrence, a biopsy was conducted if necessary. Liver function was routinely checked pre-LT and post-LT. The explanted livers were fixed in formalin and examined by two experienced pathologists. The number of tumors, tumor size (maximum diameter of tumor nodules), the presence of vascular invasion, perihepatic lymph node invasion, and the degree of differentiation (well, moderately, and poorly differentiated) were recorded. The total tumor diameter for patients with multiple tumor nodules was calculated as the sum of the maximal diameter of each lesion. The total tumor diameter could not be calculated in patients with numerous tumor nodules too small to measure. Three hundred and thirteen patients were identified and were staged using tumor staging systems: the Cancer of the Liver Italian Program (CLIP) and TNM classification (UICC/AJCC,2010) [23]. The pathologic tumor stage (T) was determined according to the TNM staging system. For patients with known solitary or multicentric tumors detected by two imaging methods, tumor size was calculated using tumor nodules identified in the liver explants. Antiviral protocols All patients were routinely given hepatitis B immunoglobulins and nucleoside analogues (lamivudine, adefovir, or entecavir) based on the antiviral protocol shown in Table 2. 10.1371/journal.pone.0050919.t002Table 2 Antiviral prophylaxis for HBV reinfection after LT. Patients with high risk of HBV reinfection Patients with low risk of HBV reinfection (HBV-DNA ≥105 copies/ml, or HBeAg [+]) (HBV-DNA <105copies/ml, or HBeAg[−]) Pre-LT: nucleoside analogues, qd 2–4w Pre-LT: nucleoside analogues, qd 0–2 w Intraoperative: HBIG 4000 IU, iv Intraoperative: HBIG 2000 IU, iv Post-LT: HBIG 1000 IU, iv, qd, 1–7d Post-LT: HBIG 1000 IU, iv, qd, 1–7d After 7 days, HBIG 1000 IU, iv, once a week; or HBIG 400 IU, im, qd or qod or twice a week After 7 days, HBIG 1000 IU, iv, once a week; or HBIG 400IU, im, qd or qod or twice a week Adjust frequency of HBIG administration to reach target therapeutic concentration Adjust frequency of HBIG administration to reach target therapeutic concentration. Target therapeutic concentration Post-LT Target therapeutic concentration Post-LT ≤6 months post-LT: anti-HBs titre ≥500 IU/L ≤6 months post-LT: anti-HBs titre ≥300 IU/L 6–12 months post-LT: anti-HBs titre ≥200 IU/L/ 6–12 months post-LT: anti-HBs titre ≥200 IU/L ≥12 months post-LT: anti-HBs titre ≥100 IU/L ≥12 months post-LT: anti-HBs titre ≥100 IU/L Immunosuppressive therapy During LT, all patients were administered a drug regimen based on the calcineurin-inhibitor combined with mycophenolate mofetil (MMT) and prednisone. Prednisone was gradually withdrawn within 3 months after LT, and sirolimus was initiated 3 months after LT. During follow-up, patients were given long-term treatments with tacrolimus or cyclosporin A alone or combined with either MMT or sirolimus. Statistical analysis Overall and disease/recurrence-free survival analyses were performed with the Kaplan-Meier method, and the survival time was calculated from the day of operation to either the day of death or the most recent follow-up visit. Group survival curves were compared using the log-rank test (Mantel-Cox). Clinical variables and univariate correlations between overall survival and recurrence-free survival were determined using the Chi-square test and the Spearman rank test, respectively. In addition, all variables were analyzed for independent significance using multivariate step-wise Cox regression analysis. Statistical calculations were performed by SPSS 11.0 statistical software. The significance level was defined as two-sided (P<0.05). Results In the study, 313 adult LT patients with HBV-associated HCC and cirrhosis had complete follow-up. The median follow-up period was 65.0 months (61.9±28.3; range 2–120). The main clinical and tumor pathology characteristics of the 313 patients are shown in Table 1. After LT, 20 of the 313 patients (6.40%) were found to have been re-infected with HBV hepatitis (20 cases were serum HBsAg positive, 6 were HBeAg positive, and 14 were HBV DNA probe positive). Thirteen of 20 patients had HBV reinfection, of which 4 were withdrawn from antiviral prophylaxis due to HCC occurrence, 2 were withdrawn by themselves without doctor's orders, and 7 patients were withdrawn from hepatitis B immune globulin treatment (HBIG) 2 years after LT. Histologic Data and Tumor Staging Systems The pathologic tumor characteristics of 313 HCC patients based on the TNM classification (sixth edition; T1-4 stage) and CLIP tumor staging systems are summarized in Table 3. One-hundred and forty of the 313 (44.8%) patients had CLIP 0 or 1, of which 125 had solitary lesions ranging from 1 to 7 cm in diameter, and 15 were multifocal tumor lesion (no more than 3 lesions). In 178/313 (56.9%) patients at stage T1 had solitary lesions of 4.16 cm (±2.329) in diameter. In 82/313 (26.2%) patients with CLIP 2, 43 tumors were solitary, 36 were multifocal tumor lesions (≤4 tumor lesions), and 3 had >4 tumor nodules, while in 85/313 (27.2%) patients at stage T2 with tumor sizes ≤5 cm, 6 tumors were solitary, 61 were multifocal tumor lesions (≤4 tumor lesions), and 18 had >4 tumor nodules. In 51/313 (16.3%) patients with CLIP3, 23 were solitary, 20 were multifocal lesions (≤4 tumor lesions), and 8 had >4 tumor nodules. In 32/313 patients (10.2%) at stage T3 with a median tumor size of 7.58 cm (±3.234) in diameter, 10 patients (T3a) had multifocal tumors (<3 lesions), 22 patients were T3b of whom 13 patients had solitary, 5 had multifocal tumors (≤4 tumor lesions), and 4 had >4 tumor nodules). In 28/313 (8.9%) patients with CLIP4, 14 were solitary, 5 were multifocal tumors (≤4 tumor lesions), and 9 had >4 tumor nodules. In 12/313 (3.8%) patients with CLIP5, 9 were solitary, 1 was a multifocal tumor lesion (≤4 tumor lesions), and 2 had >4 tumor nodules. In 18/313 patients (5.8%) at stage T4 with a median tumor size of 6.64 cm (±3.846) in diameter, 14 were solitary and 4 were multifocal tumors (≤3 lesions). 10.1371/journal.pone.0050919.t003Table 3 The pathologic characteristics of HCC patients based on CLIP and TNM staging systems. Variables N (%) Tumot Size (cm) Portal vein or hepatic vein invasion Lymph node invasion Mean Std. Deviation T1 178(56.9) 4.16 2.329 0 (0.0) 0 (0.0) T2 85(27.2) 3.11 1.442 0 (0.0) 0 (0.0) T3 (3a+3b) 32(10.2) 7.58 3.234 22 (68.7) 0 (0.0) T4 18(5.8) 6.64 3.846 6(33.3) 18(100.0) CLIP criteria CLIP 0 54(17.3) 3.63 1.354 0 (0.0) 1(5.6) CLIP 1 86(27.5) 3.57 1.637 0 (0.0) 4(22.2) CLIP 2 82(26.2) 4.00 2.410 0 (0.0) 4(22.2) CLIP 3 51(16.3) 4.74 3.052 8(15.7) 3(16.7) CLIP 4 28(8.9) 7.12 3.710 10(35.7) 5(27.7) CLIP 5 12(3.8) 7.90 4.025 10(83.3) 1 (5.6) The majority of patients (89.1%) had moderately differentiated HCC (histologic grade II), while 13 (4.2%) had well-differentiated (histologic grade I), and 21 (6.7%) had poorly differentiated HCC (histologic grade III). Vascular invasion was present in 41 patients (13.1%). Among these patients, 28 had invasion of the main portal vein, portal vein branch or hepatic vein (22 patients with T3b and 6 patients with T4, of which 8 patients were CLIP3, 10 patients were CLIP4, and 10 patients were CLIP5 (Table 3). Thirteen patients had only microvascular invasions. These data, including a case of perihepatic lymph node invasion detected by a pathologist after LT, are shown in Table 3. The overall and tumor recurrence-free survival rates of HCC patients based on the TNM classification (T1-4) and the CLIP tumor staging systems are shown in Table 4. The Kaplan-Meier curves showed clearly different survival rates for patients scored according to the CLIP 1–5, and T1–4 staging systems with high statistical significance (P<0.05) in all cases. Moreover, there were highly statistically differences in the overall survival or tumor recurrence-free survival between T2 and T3 patients (P<0.001 in both cases). The overall 1-, 3-, and 5-year survival rates for the patients with T2 were 96%, 90% and 81%, respectively, while patients with T3 were 51%, 34% and 26% for the 1-, 3-, and 5-year survival rates, respectively. For patients with CLIP 3, the 1-, 3-, and 5-year survival rates were 86%, 76%,and 71%, respectively. 10.1371/journal.pone.0050919.t004Table 4 Univariate analysis of patient overall survival and recurrence-free survival based on the TNM and CLIP staging systems. Variables Overall survival rate (%) P-value Tumor recurrence-free survival rate (%) P-value 1 year 3 years 5 years 1 year 3 years 5 years T1 95 92 90 93 91 91 T2 96 90 81 0.023 83 79 79 0.018 T3 (3a+3b) 51 34 26 0.000 33 24 24 0.000 T4 72 50 44 0.000 48 35 35 0.000 CLIP criteria CLIP 0 98 92 90 92 92 90 CLIP 1 100 98 94 0.304 96 96 96 0.350 CLIP 2 93 85 77 0.012 81 72 72 0.012 CLIP 3 84 76 71 0.000 76 70 70 0.008 CLIP 4 67 60 53 0.000 54 54 54 0.000 CLIP 5 40 20 20 0.000 22 22 22 0.000 Patient survival and recurrence During follow up, 86/313 patients (27.5%) died. Of the 86 patients, 70 (81.3%) died from tumor recurrence, and 16 (21.6%) died from other causes (1 case of sepsis, 3 of pulmonary infection, 2 of liver failure from rejection, 4 of liver failure from biliary passage complication, 1 of recurrent hepatitis, 1 of graft versus host disease [GVHD], 1 of acute myocardial infarction, 1 of cerebrovascular accident, 1 of hemorrhagic shock, and 1 case had a traffic accident). Recurrence of HCC was the most common cause of death after LT. The median tumor recurrence-free survival time of the 313 patients was 59 months. Of the 313 patients, univariate analysis showed that the overall 1-, 3-, - and 5 -year survival rates were 90%, 84%, and 78.3%, respectively, and the 1-, 3-, and 5- -year tumor recurrence-free survival rates were 82%, 78% and 78%, respectively. Patients in the Milan group or the Milan-UCSF group had good survival after LT, with 3- and 5-year overall survival rates of 95% and, 91% or 91% and 79%, respectively. There was HCC recurrence in 78/313 patients (24.9%) with a median time to recurrence of 11 months (range, 1–49). With regards to the sites of the first tumor recurrence, 28/78 cases (35.9%) were intrahepatic, 32/78 cases (41.0%) were in the lung, 5/78 (6.4%) cases were in the bone, 2/78 (2.6%) cases in the head, 3/78 (3.8%) were in the adrenal gland, 1/78 (1.3%) cases were in the peritoneum (0.1%), and 5/78 of the patients (6.4%) had both intrahepatic and extrahepatic recurrence, as well as 2 patients with new-onset malignant tumor in the stomach or esophagus over 3 years after LT. Postoperative HCC therapy was given to 54/313 patients (17.3%) who were diagnosed with tumor recurrence during LT follow-up. Therapy consisted of radiotherapy (18 cases, 33.3%), transarterial chemoembolization (3 cases, 5.5%), percutaneous ablations (4 cases, 7.4%), and reoperation (14 cases: 10 resection [18.5%] and 4 LT [7.4%]), as well as combinations of 2 or 3 HCC therapies (15 cases, 27.8%). Univariate analysis of prognostic factors for overall survival and tumor recurrence-free survival Prognostic factors for overall survival identified through univariate analysis are reported in Table 5. Among the pre-LT factors, tumor size (>5 cm), tumor number (≥2), poor tumor differentiation, vascular invasion and lymph node invasion, high serum AFP level, and poor liver function (ALT and AST levels) were all significant risk factors affecting overall survival post-LT. Patients with serum AFP levels ≤1000 µg/L did better post-LT, with 1-, 3-, and 5-year overall survival rates of 94%, 80% and 80%, respectively (P = 0.011) compared to patients with serum AFP levels >1000. Patients with ALT levels (<1N) did better post-LT, with 1-, 3-, and 5- year overall survival rates of 97%, 91%,and 86%, respectively (P = 0.004) compared to patients with ALT levels (>1N). In contrast, patients with AST levels (≥3N) were associated with poor overall survival post-LT, with 1-, 3-, and 5-year overall survival rates of 71%, 62%, and 60% (P = 0.000), respectively. All patients received antivirus treatment, and the HBV reinfection rate (6.4%) had a significant association with overall survival post-LT (P = 0.049). 10.1371/journal.pone.0050919.t005Table 5 Univariate analysis of patient characteristics and overall survival risk factors. Variables Overall survival rate (%) P-value 1 year 3 years 5years 7years Gender Male 90 83 78 72 Female 92 88 88 88 0.288 Age (year) ≤50 89 82 81 76 >50 91 95 76 70 0.605 HBeAg Negative 90 82 78 75 Positive 90 86 79 68 0.807 HBV-DNA(×103 IU/ml) <1 92 87 81 75 >1 90 83 78 74 0.885 Child-Pugh score A 92 84 77 75 B 87 84 82 73 0.901 C 92 84 76 73 0,929 ALT(IU/L) 1N, 97 91 86 82 ≥1N,<2N 86 78 71 65 0.004 ≥2N, <3N 84 81 73 73 0.112 ≥3N 89 76 76 68 0.053 AST(IU/L) 1N, 97 93 84 84 ≥1N,<2N 89 82 79 69 0.065 ≥2N, <3N 93 86 80 75 0.376 ≥3N 71 62 60 60 0.000 ALP(IU/L) 1N, 95 88 81 75 ≥1N, <2N 82 77 74 71 0.233 ≥2N 73 67 67 67 0.064 Tumor size (cm) ≤3 97 90 86 80 >3, ≤5 98 93 86 78 0.959 >5, ≤7.5 83 72 66 66 0.006 >7.5 50 44 44 44 0.000 Number of tumor nodules Single 88 84 82 80 2 96 82 64 47 0.010 3 100 100 100 100 0.096 4, 80 80 80 48 0.313 >4 91 77 71 71 0.390 Tumor differentiation I (well) 100 100 100 100 II (moderate) 90 84 79 73 0.051 III (poor) 77 68 60, 60, 0.013 Serum AFP level (ng/ml) ≤500 94 88 83 77 500–1000 94 80 80 80 0.778 1000–2000 74 70 56 56 0.011 2000–5000 73 60 60 60 0.023 ≥5000 58 50 50 25 0.000 Venous invasion Absent 95 89 84 78 Present 48 39 33 33 0.000 Lymph node invasion Absent 91 86 81 76 Present 72 50 44 33 0.000 Pre-LT antitumor therapy Absent 50 32 29 29 Present 95 88 80 75 0.000 Post-LT HBsAg reinfection Negative 90 83 80 77 Positive 95 90 56 42 0.049 Rejection Absent 90 83 78 72 Present 93 89 85 85 0.232 The prognostic factors for the 1-, 3-, 5- and 7-year tumor recurrence-free survival rates identified through univariate analysis are reported in Table 6. Based on the Log Rank and Kaplan-Meier analyses, tumor size (>5 cm) (Fig. 1A), tumor number (≥2) (Fig. 1B), the presence of vascular invasion (Fig. 1C), poor tumor differentiation (Fig. 1D), lymph node invasion (Fig. 1E), pre-LT high serum AFP level (>1000 ng/ml) (Fig. 1F), ALT (≥3N) and AST level (≥3N) (Fig. 1G) were all significantly associated with poor recurrence-free survival after LT. In addition, we observed that the post-LT HBV re-infection rate (P = 0.027) was significantly associated with tumor recurrence. 10.1371/journal.pone.0050919.g001Figure 1 Univariate analysis of factors affecting of tumor recurrence-free survival rates of HCC patients in the study: (A) tumor size, (B) tumor number, (C) vascular invasion, (D) tumor differentiation, (E) lymph node invasion, (F) pre-LT serum AFP level, (G) pre-LT Serum ALT levels, and (H) pre-LT Serum AST levels. 10.1371/journal.pone.0050919.t006Table 6 Univariate analysis of patient characteristics and tumor recurrence-free survival risk factors. Variables Tumor recurrence-free survival rate (%) P-value 1 year 3 years 5years 7years Gender Male 88 88 88, 88 Female 81 78 77, 75 0.254 Age (year) ≤50 82 80 80, 77 >50 82 77 76, 75 0.663 HBeAg Negative 82 79 78 76 Positive 82 78 78, 75 0.916 HBV-DNA(×103 IU/ml) <1 84 79 79, 74 >1 82 79 79, 77 0.939 Child-Pugh score A 81 79 78, 74 B 83 79 79, 77 0.890 C 83 76 76 76 0.885 ALT(IU/L) 1N, 87 86 86, 86 ≥1N, <2N 81 72 71, 63 0.002 ≥2N, <3N 78 75 75, 75 0.091 ≥3N 70 70 70, 70 0.017 AST(IU/L) 1N, 90 89 88, 81 ≥1N, <2N 82 76 76, 74 0.034 ≥2N, <3N 84 78 78, 78 0.207 ≥3N 62 59 59, 59 0.000 ALP(IU/L) 1N, 86 81 81, 77 ≥1N, <2N 75 74 72, 72 0.129 ≥2N 69 69 69, 69 0.104 Tumor size (cm) ≤3 86 86 86, 86 >3, ≤5 91 85 85, 83 0.078 >5, ≤7.5 75 69 66, 57 0.006 >7.5 44 44 44, 44 0.000 Number of tumor nodules Single 84 82 82, 80 2 71 63 63, 52 0.004 3 100 100 100, 100 0.088 4, 60 60 60, 60 0.268 >4 77 72 72, 72 0.357 Tumor differentiation I (well) 100 100 100, 100 II (moderate) 82 79 78, 76 0.051 III (poor) 73 62 62 0 0.013 Serum AFP level (ng/ml) ≤500 87 83 83, 80 500–1000 88 80 80, 80 0.941 1000–2000 61 61 61, 61 0.005 2000–5000 56 56 56, 56 0.006 ≥5000 42 42 42, 42 0.000 Venous invasion Absent 88 84 84, 81 Present 36 32 32, 32 0.000 Lymph node invasion Absent 84 81 81, 78 Present 48 35 35, 35 0.000 Pre-LT antitumor therapy Absent 33 27 27, 27 Present 84 80 79, 79 0.000 Post-LT HBsAg reinfection Negative 82 80 80, 77 Positive 80 53 53, 53 0.027 Rejection Absent 81 78 77, 75 Present 89 86 86, 86 0.281 However, there was no significant difference in either the overall survival (P = 0.901, P = 0.929) or tumor recurrence-free survival (P = 0.890, P = 0.885) post-LT between Child-Pugh score A, B or C (Table 5 and 6). Remarkably, no significant difference in overall survival (P = 0.647) or tumor recurrence-free survival (P = 0.596) were shown between patients with >4 tumors and patients with ≤4 tumors. Independent prognostic factors for overall survival and tumor recurrence-free survival Independent prognostic factors for overall survival identified through multivariate analysis are reported in Table 7. Multivariate analysis showed that tumor size >7.5 cm (P = 0.001, hazard ratio [HR] = 3.528; 95% confidence interval [CI]: 1.717–7.246), tumor number >1 (P = 0.004, HR = 2.196; CI 1.285–3.752), the presence of vascular invasion (P = 0.002, HR = 2.740; CI 1.450–5.177), the pre-LT serum AFP level ≥1000 ng/ml (P = 0.010, HR = 2.083; CI: 1.192–3.641) and an AST level ≥120 IU/L (P = 0.044, HR = 2.061; CI:1.021–4.160) were all independent predictors of poor survival post-LT. However, the ALT level was not an independent predictor for overall survival post-LT by multivariate analysis. 10.1371/journal.pone.0050919.t007Table 7 Multivariate analysis of patient characteristics and overall survival risk factors. Variables B SE Wald Sig. Exp(B) 95.0% CI for Exp(B) Lower Upper Age(≤50 v>50 y) 0.178 0.248 0.517 0.472 1.195 0.735 1.943 Venous invasion 1.008 0.325 9.637 0.002 2.740 1.450 5.177 Lymph node invasion 0.623 0.391 2.536 0.111 1.864 0.866 4.010 Pre-LT tumor therapy −0.643 0.293 4.831 0.028 0.526 0.296 0.933 Tumor size (≤7.5 v>7.5 cm) 1.261 0.367 11.783 0.001 3.528 1.717 7.246 Tumor number (1 v>1) 0.786 0.273 8.279 0.004 2.196 1.285 3.752 AFP(<1000 v ≥1000 ng/ml) 0.734 0.285 6.642 0.010 2.083 1.192 3.641 ALT (≤40 v>40 IU/L) −0.055 0.410 0.018 0.893 0.946 0.424 2.114 AST (<120 v ≥120 IU/L) 0.723 0.358 4.071 0.044 2.061 1.021 4.160 Independent prognostic factors for recurrence-free survival identified through multivariate analysis are reported in Table 8. Multivariate analysis showed that independent predictors of poor recurrence-free survival were tumor size >7.5 cm (P = 0.001, HR = 3.309; CI 1.607–6.814), a tumor number >1 (P = 0.005, HR = 2.154; CI 1.260–3.682), the presence of vascular invasion (P = 0.001, HR = 2.788; CI 1.496–5.196), and a pre-LT serum AFP level ≥1000 ng/ml (P = 0.009, HR = 2.094; CI 1.200–3.653). The Cox proportional hazard model also showed that the higher the tumor size or the pre-LT serum AFP level, the higher the risk ratio, and there was no the relation with tumor number. Patients with pre-LT antitumor therapy had a significantly lower likelihood of recurrence-free survival (P = 0.011, HR = 0.484; CI 0.277–0.845). 10.1371/journal.pone.0050919.t008Table 8 Multivariate analysis of patient characteristics and tumor recurrence-free survival risk factors. Variables B SE Wald Sig. Exp(B) 95.0% CI for Exp(B) Lower Upper Age (≤50 v>50 y) 0.230 0.252 0.834 0.361 1.258 0.769 2.060 Venous invasion 1.025 0.318 10.417 0.001 2.788 1.496 5.196 Lymph node invasion 0.605 0.388 2.431 0.119 1.831 0.856 3.918 Pre-LT tumor therapy −0.611 0.293 4.360 0.037 0.543 0.306 0.963 Tumor size (≤7.5 v>7.5 cm) 1.197 0.369 10.537 0.001 3.309 1.607 6.814 Tumor number (1 v>1) 0.767 0.274 7.865 0.005 2.154 1.260 3.682 AFP(<1000 v ≥1000 ng/ml) 0.739 0.284 6.773 0.009 2.094 1.200 3.653 ALT (≤40 v>40 IU/L) 0.312 0.304 1.052 0.305 1.366 0.753 2.477 AST (<120 v ≥120 IU/L) 0.587 0.308 3.627 0.057 1.799 0.983 3.291 Pre-LT antitumor therapy Among 313 patients, univariate analysis showed that the overall survival (P = 0.000) and recurrence-free survival rates (P = 0.000) of patients with pre-LT antitumor therapy were better than those of patients with no pre-therapy for HCC (Tables 5 and 6; Fig. 1H). Multivariate analysis (Tables 7 and 8) showed that pre-LT antitumor therapy was an independent predictor of good survival (P = 0.028, HR = 0.526; CI: 0.296–0.933) and recurrence-free survival (P = 0.037, HR = 0.543; CI: 0.306–0.963), Among those in the exceeding UCSF criteria group, statistical analysis showed that 38 patients with pre-LT antitumor therapy had a better overall survival (P = 0.000) and recurrence-free survival rates (P = 0.000, Fig. 2) than those of patients without pre-LT antitumor therapy for HCC. The 1-, 3-, and 5-year overall survival rates of patients who exceeded the UCSF criteria with pre-LT antitumor therapy were 92%, 87% and 83%, respectively, while patients without pre-LT antitumor therapy had rates of 56%, 38% and 35%,, respectively. 10.1371/journal.pone.0050919.g002Figure 2 Kaplan-Meier analysis of the recurrence-free survival rates of patients with and without antitumor therapy pre-LT in the exceeding UCSF group. Proposed Modified Tumor Staging Classification Based on our observations, we defined an expanded set of criteria for HCC patients with HBV- cirrhosis that was associated with excellent survival after LT. These criteria included: solitary tumor <7.5 cm, ≤4 nodules with the largest lesion ≤6.5 cm or multiple nodules (>4) with the largest lesion ≤3 cm, and a pre-LT serum AFP level ≤1000 ng/ml and a AST level <120 IU/L (3N) without vascular invasion of the major portal vein branches or lymph node metastasis. Among 313 patients with HCC, statistical analysis showed that the 3- and 5-year overall survival rates of patients who fit the revised criteria were 95% and 90%, respectively, essentially identical to the survival rates in patients who fit the Milan or UCSF criteria in this study, with 5-year overall survival of 91% or 88%, respectively. Among the exceeding UCSF criteria group, the Log Rank analysis showed that 19 patients fit our revised criteria, and all of them had a good overall survival (P = 0.002) and recurrence-free survival (P = 0.001), with 5-year overall survival and recurrence-free survival rates of 89% and 82%, respectively (Table 9). Patients who exceeded our revised criteria had 5-year overall survival and recurrence-free survival rates of 47%, and 45%, respectively (Fig. 3). Eight of the 19 patients had pre-LT antitumor therapy. There were no significant differences in overall survival post-LT between patients who fit our revised criteria and patients in the Milan group (P = 0.444) or the Milan-UCSF group (P = 0.866) (Table 9). 10.1371/journal.pone.0050919.g003Figure 3 Kaplan-Meier analysis of the overall survival rates of patients who were fit and unfit for the revised criteria in the exceeding UCSF group. 10.1371/journal.pone.0050919.t009Table 9 The overall survival and recurrence-free survival rates of patients who fit the Milan, Milan-UCSF, and revised criteria in the exceeding UCSF group. Variables Overall survival rate (%) P-value Tumor recurrence-free survival rate (%) P-value 1 year 3 years 5 years 1 year 3 years 5 years Fit revised criteria 100 89 89 94 82 82 Fit Milan 98 95 91 0.444 93 90 89 0.484 unfit Milan, but fit UCSF 95 90 79 0.866 88 83 80 0.753 Discussion The prognostic assessment of HCC patients with HBV-cirrhosis is complicated by factors such as liver function, HBV infection, and tumor characteristics [5]. It is currently known that survival and recurrence post-LT are affected by tumor characteristics such as tumor size, tumor number, differentiation, vascular invasion, lymph node metastasis, and pre-LT serum AFP levels [14], [18], [19]. However, these risk factors have not been adequately assessed in studies with large enough sample sizes, and there is no universally accepted suitable selection policy for LT in HCC with HBV- cirrhosis. The present study addressed this deficiency by performing a stratified analysis of 313 HCC patients with HBV -cirrhosis who underwent LT with antiviral therapy. Univariate analysis and multivariate analysis in the current study showed that independent predictors of tumor recurrence and poor LT outcome for HCC patients with HBV-associated cirrhosis were tumor size >7.5 cm, tumor number >1, the presence of vascular and lymph node invasion, and pre-LT serum AFP levels ≥1000 ng/ml, and AST levels ≥120 IU/L. In addition, pre-LT antitumor therapy remained a significant independent factor for survival, with a 5-year survival of 80% with pre-LT therapy. This finding is consistent with previous reports [33], [34] and supported the use of downstaging therapy pre-LT for HCC patients with HBV-associated cirrhosis. We suggest that the important predictors in determining outcome post-LT for HCC patients with HBV-associated cirrhosis are not just the pathologic tumor factors, but also the AFP levels, liver function levels and pre-LT antitumor therapy. The prevailing criteria such as Milan and UCSF are limited for HCC patients when only the factors of tumor size and tumor number are included. Therefore, we recommend expanding the selection criteria for LT of HCC patients with HBV-associated cirrhosis to include pre-LT therapy for HCC. In the current study, the overall survival rates for the 313 patients after LT were 90%, 84% and 78.3% at 1, 3, and 5 years, respectively. It is generally accepted that adjuvant antiviral treatment for LT patients with HBV-related HCC can prevent HBV reinfection [25], [35]–[37]. In the present study, comprehensive antiviral prophylaxis with hepatitis B immune globulins (HBIg) and lamivudine, adefovir or entecavir were given to all the patients, of whom only 6.40% had HBV reinfection after LT. This result is similar to other reports [22], [38]–[39], and indicates that antiviral prophylaxis can prevent HBV reinfection and that antiviral therapy is effective in improving survival of LT patients with HCC. It was reported that the outcome for patients with advanced HCC is related not only to tumor stage, but also to the extent of liver dysfunction [20]–[23]. Fidel-David et al [23] showed that the TNM (sixth edition) classification system alone was not useful for predicting overall survival, but CLIP staging systems were good informative staging systems in predicting survival in patients with advanced HCC. Five prognostic strata were defined according to a score derived from the analysis of variables related to cirrhosis (Child-Pugh score), tumor morphology, AFP level, and portal vein thrombosis. In this study, the 5-year survival rates of patients in the current study with CLIP 0–2 or stage T1–2 were similar to the survival rates of Milan criteria reported by Mazzaferro et al. [8] (83% at 4 years) for similar tumor stages. However, for patients who exceeded the Milan or UCSF criteria, there were 25% patients with CLIP 1–2, and 32.6% patients with CLIP 3, while there were 54.3% patients with stage T1–2. The overall survival rates of 1-, 3- and 5-years for the patients with CLIP3 were 86%, 76% and 71%, respectively, while for the patients with stage T3, the overall survival rates were 51%, 34% and 26%, respectively. The result showed that HCC patients with CLIP >2 may still have a favorable outcome after LT. Remarkably, our results also showed that there were no significant differences in the overall survival or recurrence between patients with >4 tumors lesions and patients with ≤4 tumors lesions. Furthermore, our results showed that HCC patients with stage T2 had a good overall survival, with 81% 5-year overall survival rates versus 26% in patients with stage T3a. According to the TNM staging systems (UICC/AJCC, 2010), patients who had multiple tumors, any of which were ≤5 cm in diameter, were considered to be at stageT2, while patients with multiple tumors, any of which are >5 cm, were considered to be at stage T3a. Therefore, we recommend expanding the selection criteria for LT of HCC patients with HBV-associated cirrhosis to include multifocal tumors (>3) with a limit in tumor size based on T2. Our study also suggests that excellent survival can be achieved in HCC patients with CLIP3 and T2 who meet our proposed criteria: solitary tumor ≤7.5 cm, ≤4 multifocal nodules with the largest lesion ≤5 cm and a total tumor diameter ≤10 cm or multiple nodules with the largest lesion ≤3 cm, and a pre-LT serum AFP level <1000 ng/ml and a AST level <120 IU/L without vascular invasion of the major portal vein branches or lymph node metastasis. In the study, HCC patients who fit our revised criteria, but exceeding UCSF criteria, had survival rates of 89% at 5 years, and did better than those patients who exceeded our revised criteria, with survival rates of 47% at 5-years Moreover, the overall survival rates in patients who fit our revised criteria were similar to that in patients who fit the Milan or UCSF criteria (91% or 79%). There have been several previous studies that have provided some evidence for predicting survival outcomes after LT [18]–[20], [33], [34], [40], [41]. Except for several reports on small HCC with good survival using criteria such as Milan [8] or UCSF [14], et al. Marsh et al [42] reported that a subgroup of PT4 patients with 4 or more nodules, none greater than 3 cm, had a 5-year tumor-free survival rate of 80%. However, Tan et al. [43] reported that patients with HCC less than 8 cm (multifocal in 10 patients) who underwent LT had disease-free survival rates of 80% and 63% at 1 and 3 years, respectively. McPeake et al. [44] showed less favorable results for patients with larger or multifocal tumors. The limitation of their results might be due to a lack of detailed information on the size and number of lesions in the multifocal HCC, as well as on the pre-LT AFP levels, liver function levels and antitumor therapy pre-LT. ALT and AST levels in patients with chronic liver disease are considered markers of inflammation that reflect the etiopathogenetic mechanism of hepatocyte necrosis [45], while the Child-Pugh score is considered an index of liver cirrhosis that reflects the severity of the clinical condition [46], [47]. When liver cells are damaged or dying, ALT and AST leak into the bloodstream. The resulting presence of these enzymes in serum is a clinical expression of a biologic activity of multicentric carcinogenesis [48]. Some reports have shown that high AST is an accurate predictor of tumor recurrence or poor outcome [45], [49]. High AST levels are predictors of recurrence because inflammation in the liver can stimulate production of some adhesion molecules on cancer cells in the remnant liver, and cause postoperative recurrence [49]. The current study demonstrated that pre-LT AST ≥3N levels were independent predictors of poor outcome in univariate analyses and were shown to be sensitive predictors of prognosis for LT in HCC patients with HBV-associated cirrhosis. However, Child-Pugh scores were not shown to be significant predictors of survival by univariate and multivariate analyses. The findings can be explained by the fact that repeated and severe inflammation and cellular necrosis enhance proliferation of HBV, as well as accelerate the development of HCC and the formation of micro-metastases by increasing the rate of random mutations. In addition, changes of enzymes and proteins in biochemical reactions of tumor cells often predict tumor development and progression [50]. Thus, based on the univariate and multivariate analyses, AST (<3N) as a significant predictor with an 80% 5-year overall survival rate was included in the expanded criteria in this study. Fidel-David et al [23], reported that AST is an independent prognostic factors for the overall survival of advanced HCC. We believe that the administration of adjuvant anti-inflammatory therapy with the appropriate anti-HBV treatment may improve AST levels. In addition, among those patients who exceeded UCSF criteria in this study, the majority of HCC patients received chemoembolization or combined with percutaneous ablations therapy for downstaging of tumors before LT. Those patients with pre-LT antitumor therapy had a better overall survival rate at 5-year (83%) than that of patients without pre-LT antitumor therapy (35%). This finding suggest that it is necessary to widely use antitumor therapy pre-LT for downstaging of tumors and to decrease the risk of tumor dissemination in HCC patients who exceed UCSF criteria during the increased waiting time for LT. From the results of the current study, this procedure did not influence the effect of operation in HCC patients with cirrhosis pre-LT whose hepatic function might have been damaged by chemoembolization or combined with percutaneous ablations therapy. Thus, our results support the use of pre-LT antitumor therapy in expanding the selection criteria to offer the potential benefit of LT to some advanced HCC patients with HBV-associated cirrhosis who would otherwise be excluded from LT. Our results appear to differ from several previous studies reporting worse survival after LT for patients with solitary tumors over 6.5 cm or patients with 3 multifocal tumors of sizes >4.5 cm. Furthermore, the TNM stage needs to be precisely evaluated by pathologists. However, it is sometimes impossible to obtain clear-cut tumor characteristics preoperatively without a biopsy of the lesion which can introduce risk of tumor seeding along the biopsy tract by liver biopsy. The prevailing criteria such as Milan and UCSF are limited for predicting post-LT overcomes because they only include factors such as tumor size and tumor number. In conclusion, we focused on cut-offs for tumor burden and re-calculated the statistics based on the results of using expanded criteria according to CLIP and TNM. We propose the adaption of expanded selection criteria for HCC patients with HBV-associated cirrhosis pre-LT: solitary tumor ≤7.5 cm, ≤4 multifocal nodules with the largest lesion ≤5 cm and a total tumor diameter ≤10 cm or more nodules with the largest lesion ≤3 cm, and a pre-LT serum AFP level ≤1000 ng/ml and a AST level <120 IU/L without vascular invasion of the major portal vein branches or lymph node metastasis. Such expanded selection criteria combined with antiviral prophylaxis and pre-LT therapy for HCC and inflammation may be a rational strategy to prolong survival after LT for HCC patients with HBV-associated cirrhosis. 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==== Front Case Rep RadiolCase Rep RadiolCRIM.RADIOLOGYCase Reports in Radiology2090-68622090-6870Hindawi Publishing Corporation 10.1155/2012/642062Case ReportIntramedullary Chondrosarcoma of Proximal Humerus Yadav Pratiksha *Thakkar Dolly Thind S. S. Department of Radiodiagnosis, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Maharashtra, Pune 411006, India*Pratiksha Yadav: [email protected] Editors: P. García González and A. Vade 2012 2 12 2012 2012 64206213 10 2012 30 10 2012 Copyright © 2012 Pratiksha Yadav et al.2012This 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.Primary chondrosarcoma is the third most frequent primary malignancy of bone after myeloma and osteosarcoma. It is ranging from slow growing nonmetastasising lesions to highly aggressive lesions. We report a case of primary intramedullary chondrosarcoma of proximal humerus. A 60-year-old female presented with pain and hard swelling involving the left arm for 5 months. Radiograph showed a lucent expansile intramedullary lesion with matrix calcification and associated soft tissue mass. CT confirmed the finding. MRI showed a lobulated lesion which is hyperintense on T2WI with low signal fibrous septae. Increased tracer uptake was seen on bone scan. Histopathology confirmed the radiology diagnosis. The patient underwent wide resection and endoprosthetic reconstruction of proximal humerus. ==== Body 1. Case Report Primary chondrosarcoma is the third most common primary malignant tumor of bone after myeloma and osteosarcoma. It is most commonly seen between 30 and 70 years of age. We report a case of a sixty-year-old female who presented with gradually progressive pain and swelling over the proximal part of left arm since 5 months associated with restricted flexion, extension, and abduction of left shoulder. On inspection, loss of normal contour of left shoulder due to a diffuse swelling and asymmetric pectoral girdle (Figure 1). Skin over the surface was normal, with mild prominence of the veins. On palpation, it is tender and measuring approximately 6 cm in length and 4 cm in breadth. It was bony hard in consistency and fixed in nature with immobile skin over the swelling. Clinical diagnosis was of a neoplastic musculoskeletal pathology. Radiographs of left shoulder showed an ill defined, expansile, and osteolytic lesion involving the cortical and medullary region of neck and proximal shaft of left humerus with a wide zone of transition (Figures 2 and 3). Few specks of calcifications were seen within it. It shows endosteal scalloping with cortical break and adjacent soft tissue component. In addition, a calcified nodular opacity was seen in the peripheral left lung mid zone. As computed tomography (CT) is useful in defining the bony anatomy, integrity of the cortex surrounding a lesion, and calcifications within, a helical CT scan of 5 mm thickness was done from the superior margin of left shoulder to mid arm level. It revealed that the osteolytic expansile lesion was seen with endosteal scalloping and cortical thinning. It contains matrix calcification, break in cortices at multiple sites with adjacent anterolateral soft tissue component, and specks of calcification within it (Figure 4). The deltoid muscle in its anterior portion was thinned out and displaced. • MRI is known to best depict the tissue character, delineate the extent of bone marrow involvement, and pinpoint the effect of soft tissue masses on surrounding neurovascular structures. MRI of left arm was done on 1.5 Tesla machine using T1WI, T2WI, and GRE sequences in coronal, axial, and sagittal planes. There is a well-defined lobulated lesion which is predominantly hypointense on T1WI and hyperintense on T2WI with low signal septae (Figures 5, 6, 7, and 8). The glenohumeral joint space was normal and neurovascular bundle was not affected. Subsequently bone scan was run to look for any metastatic lesion elsewhere as 10–20% of chondrosarcomas are known to metastasise. The lesion in humerus revealed increased uptake of tracer in 1st phase and high soft tissue pooling in 2nd phase. A focal tracer uptake seen in left mandible was probably related to dental pathology. Diffuse inhomogeneous increased tracer uptake was seen in the 3rd phase (Figure 9). The nodular opacity in left mid zone that was seen on the X-ray was presumed to be a calcified granuloma as it showed no activity on bone scan. FNAC sample showed pleomorphic nuclei with vacuolated cytoplasm in chondromyxoid background (Figure 10). Justification of diagnosis was made by comparing the possible features of chondrosarcoma with our case. As ENNEKINGS SYSTEM FOR STAGING: • STAGE- II B, • GRADE-HIGH, • SITE-EXTRACOMPARTMENTAL, • METASTASES-NONE. After a preanaesthetic assessment, the patient was taken up for surgery under general anaesthesia for wide resection and endoprosthetic reconstruction of proximal humerus. 2. Discussion Chondrosarcomas account for the third most common primary tumour of the bone, after myeloma and osteosarcoma [1]. This primary sarcoma of bone in adults has a male predominance and is seen between the 3rd and 7th decade of life, more common in male, as male : female ratio is 1.5 : 1. Usual clinical presentation of chondrosarcoma is pain, tenderness, with or without a mass, and a slow growth over an average duration of 1-2 year. The characteristic feature of chondrosarcomas is to produce coalescent cartilage lobules of varying sizes with often a necrotic or cystic centre [2]. Chondrsarcoma is graded from 1 (low) to 3 (high). Low grade chondrsarcoma is very close in appearance to enchondroma and osteochondroma and has occasional binucleated cells. High grade chondrsarcoma have increased cellularity, atypia, and mitoses [3]. 2.1. Skeletal Distribution The commonest sites are the pelvic bones, femur, humerus, and ribs followed by other sites such as the trunk, skull, and facial bones. Hands and feet are rarely involved. Peculiar forms are known to develop on laryngeal cartilage, base of the skull, or in soft tissue. Chondrosarcomas can occur on preexisting lesions. Central chondrosarcoma predominates in long bones and peripheral tumours in the pelvis and vertebrae. 2.2. Imaging Plain films allow depicting the location of the lesion to identify the cartilaginous nature as well as its aggressiveness. The most frequent type of lesion is central chondrosarcoma. The tumour begins in the metaphysis and extends to the diaphysis. It is a well-defined lytic lesion, associated with endosteal scalloping, cortical thinning, or thickening. High-grade tumours show irregular margins. Calcifications of the tumoral matrix may be punctate, flocculent, or have a ring-like pattern they can be small, or disseminated, dense, or subtle. Their absence is frequent in aggressive types. In the soft tissue, the mass is frequently huge and palpable when tumour has an extension. CT scan has a diagnostic role as it shows the bony destruction, the small calcifications, and the intra- and extraosseous extent. In typical forms, MRI shows a lobulated lesion with a low or intermediate signal on T1-weighted images and a high-signal intensity on T2 [4]. MRI shows the medullary involvement and the soft-tissue mass precisely. In diffusion, low-grade lesions show a lobulated pattern with enhanced septations after intravenous injection of contrast media. High-grade tumours do not have septations and show a more diffuse, heterogeneous enhancement. Benign and low-grade tumors cannot be differentiated by the MRI appearance of the matrix alone. 2.3. Differential Diagnosis The main differential diagnosis in chondromas is specially in the differentiation between a benign chondroma and a low-grade central chondrosarcoma [5]. Features suggestive of a malignant lesion are pain, proximal location or a location on the axial skeleton, size being greater than 5 cm, a lobulated aspect, an ill-defined margin, endosteal erosion, and bone destruction with an extraosseous component [6, 7]. Biopsy is necessary to make the diagnosis. A metaphyseal lesion could suggest a chondromyxoid fibroma, while an epiphyseal lesion could suggest a chondroblastoma or a giant cell tumour (Figures 11 and 12). Fibrous dysplasia or a bone infarction can be misdiagnosed as chondrosarcomas; the lack of cortical erosion or of soft-tissue mass would suggest something other than a chondrosarcoma. New immunohistochemistry techniques contribute to the differentiation of malignant lesions [8, 9]. More rarely, a lytic lesion can be considered with a lytic form of osteosarcoma or fibrosarcoma, a plasmocytoma or a metastasis. 2.4. Chondrosarcomas: Variants 2.4.1. Periosteal Chondrosarcomas [10] This is a rare form representing 1-2% of all cases of chondrosarcomas. The growth of the tumour begins at the surface of the bone (usually metaphysis of the distal femur or proximal humerus) and develops in the soft tissues as a lobulated mass. The lesion is usually well differentiated and grows slower than central forms. The cortex is never normal, either eroded or often thickened by the tumour, but never destroyed. Ring-like calcifications can be disseminated or localised within the mass. Medullary involvement evaluated on CT or MRI is rare and limited. Uncalcified tumoral nodules are hypodense on CT and show a high signal on T2-weighted MRI. Satellite nodules can be depicted and separated from the principal lesion. The outcome is generally favourable after an appropriate surgical resection. The differentiation from an osteochondroma is generally easy. The diagnosis of periosteal chondroma can be made by histology alone. Patients are younger and lesions are smaller, not painful, and are located more distally on the skeleton. The periosteal osteosarcoma is more often located on the diaphysis and has reactionary cortical spiculations. 2.4.2. Mesenchymal Chondrosarcoma This entity represents 2-3% of all chondrosarcomas and combines an undifferentiated cell component with well-differentiated cartilaginous areas [11]. The diagnosis is only made on this biphasic aspect. The average age of the patients is 26 years. Common skeletal sites are the femur, pelvic bones, ribs, and vertebrae. Extraosseous site involvement such as brain, meninges, or soft tissues is seen in about one-third of cases. The prognosis is poor, with early pulmonary, bony, and lymph nodes metastases. The tumours are large, destructive lesions with a purely lytic pattern. Calcified masses can be found. The lesion appears of low signal intensity on T1-weighted MRI and heterogeneous high signal on T2 images [12]. Multidrug chemotherapy used in osteosarcomas can be combined with surgery and radiotherapy, but the 10-year survival is only 28%. 2.4.3. Clear Cell Chondrosarcoma In rare forms (2%) of chondrosarcoma, these lesions are distinguished by their cytology, epiphyseal location in long bones, and slow evolution [13]. There is a male predominance and patients are in the third to fifth decade. Clinical symptoms are pain and swelling may last up to almost 23 years. Some tumours may be an incidental finding; pathological fractures have been reported in one-quarter of cases. The commonest sites are the femur, humerus and tibia. This low-grade tumour shows a geographic lytic epiphyseal lesion with extension to the metaphysis. The margins can be well defined, but indistinct or sclerotic margins have also been described. Calcifications of the tumoral matrix are not always present. There is no extension to the soft tissues. Periosteal reactions are unusual. CT may be useful to depict lobulated margins and calcified matrix. MRI shows a well-delineated low signal on T1-weighted images and heterogeneous high signal on T2 images. The main differential diagnosis is the chondroblastoma, which is a smaller lesion in younger patients. The other differential diagnoses include giant cell tumours and other epiphyseal tumours. The treatment is radical surgery. The prognosis is good with a 5-year survival of 92% [2], even though metastases are found in 15% of cases (lung, brain, and bones). 2.4.4. Dedifferentiated Chondrosarcoma This form represents 10–12% of all chondrosarcomas [2]. It is characterised by a special histology and very poor prognosis. Pain and swelling are the usual clinical symptoms as well as pathological fractures [14]. The commonest locations are the femur, the acetabulum, and the proximal humerus. These metaphyseal or diaphyseal lesions are rapidly destructive. Osteolytic lesion is associated with calcifications resulting in the biomorphic pattern [2]. A huge soft-tissue mass without calcifications, seen on CT or MRI, is also indicative for this diagnosis. Imaging helps to direct biopsy of the lytic area in order to improve the histological diagnosis. The treatment involves surgery and adjuvant chemotherapy or radiotherapy (Figures 13, 14, and 15). The prognosis is very poor, with an overall 5-year survival rate of only 8–13%. The metastases appear in the lungs but also in unusual sites such as the adrenal gland, brain, and liver. 2.4.5. Secondary Chondrosarcoma Twelve percent of all chondrosarcomas are developed in a preexisting lesion. It may be secondary to a solitary osteochondroma, osteochondromatosis, enchondromatosis (Ollier's disease), fibrous dysplasia, Paget's disease, irradiated bone, or synovial chondromatosis [2]. In osteochondromatosis, the risk of sarcomatous transformation is 5–25%; it is 25–50% in enchondromatosis and nearly 100% in Maffucci's syndrome [14]. The increased size of an enchondroma, lytic area with cortical destruction, associated with pain or fracture are features suggestive of malignant transformation. An enlarging exostosis associated with pain, the appearance of a less mineralized zone in the cartilage cap, calcifications in the soft tissues, and the thickening of the cap (>1 cm) on CT and MRI suggest sarcomatous transformation [15]. Authors' Contribution P. Yadav has analysed the case by radiograph, CT, and MRI with the assistance and useful support of D. Thakkar and has also advised bone scan and biopsy which has confirmed the diagnosis. S. S. Thind, the Head of the Department of Radiodiagnosis, has given his clear concepts and critical ways of looking at different aspects of the case, encouraging the rest of the authors to proceed with the case workup. Acknowledgments The authors are grateful to the Department of Orthopedics and Pathology at Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune, Maharashtra, India, for their extended support, without whom this entire case work up would have been impossible. Figure 1 The picture is showing a 60 years old female who came with the c/o pain and swelling in upper arm. Figure 2 A 60- year-old female came with the c/o pain and swelling in upper arm. Radiograph of left shoulder shows lobulated, and expansile, osteolytic lesion involving the head, neck and proximal shaft of humerus including medullary and cortical region. Few specks of calcification were seen. With involvement of soft tissue. Figure 3 Axial radiograph of left shoulder confirms the findings of the AP radiograph and shows soft tissue swelling with few specks of calcification (arrow). Figure 4 Axial CT images show osteolytic, expansile lesion causing endosteal scalloping with thinning of cortex. Break in the bony cortex at few places (large arrow) with calcific specks seen in soft tissue (small arrow). Figure 5 60-year-old female with swelling in upper arm. MRI was done on 1.5 T Siemen's machine. Coronal MRI, STIR T2WI showing a well-defined lobulated hyperintense lesion with fibrous septae in the medulla and cortex. Involvement of the soft tissue on medial and lateral aspect (arrow). Figure 6 60-year-old female with swelling in upper arm. MRI was done on 1.5 T Siemen's machine. Coronal T2WI showing hyperintense lobulated mass with fibrous septae (arrow). Figure 7 60-year-old female with swelling in upper arm. MRI was done on 1.5 T Siemen's machine. Coronal T1WI showing large lobulated lesion which is hypointense in signal intensity. Figure 8 Sagittal T2W images showing hyperintense lobulated mass. Figure 9 Bone scan: anterior and posterior images. Increased tracer uptake in the lesion. Figure 10 Histopathology shows pleomorphic nuclei with vacuolated cytoplasm in chondromyxoid background. Figure 11 Excision of lesion was done with affected soft tissue and bone. Figure 12 Excised mass lesion. Figure 13 Prosthesis surgery. Figure 14 Postoperative radiograph. Figure 15 Postsurgery picture. ==== Refs 1 Gelderblom H Hogendoorn PCW Dijkstra SD The clinical approach towards chondrosarcoma The Oncologist 2008 13 3 320 329 2-s2.0-43549101708 18378543 2 Krishnan K Dahlin’s Bone Tumors: General Aspects and Data on 11087 Cases 1996 5th edition Philadelphia, Pa, USA Lippincot-Raven Publishers 3 http://www.bonetumor.org/ 4 Varma DG Ayala AG Carrasco CH Guo SQ Kumar R Edeiken J Chondrosarcoma: MR imaging with pathologic correlation Radiographics 1992 12 4 687 704 2-s2.0-0026885435 1636034 5 Welkerling H Kratz S Delling G Ewerbeck V Differentiation of enchondroma and low-grade chondrosarcoma-clinicopatological and radiological findings in 34 cases Sarcoma 2002 6, article S9 6 Geimaerdt MJA Hermans J Bloem JL Usefulness of radiography in differentiating enchondroma from gentral grade I chondrosarcoma American Journal of Roentgenology 1997 169 1097 1104 9308471 7 Murphey MD Flemming DJ Boyea SR Bojescul JA Sweet DE Temple HT Enchondroma versus Chondrosarcoma in the appendicular skeleton: differentiating features Radiographics 1998 18 5 1213 1237 2-s2.0-0032160137 9747616 8 Eefting D Geimaerdt MJA Le Cessie S Taminiau AHM Hogendoom PCW Diagnostic impact of histologic parameters in differentiating enchondroma from grade I central chondrosarcoma Sarcoma 2002 6, article S1 9 Bovee JV Kok P Van den Broek LJ Hogendoom PCW Immunohistochemistry as a tool to distinguish osteochondroma and grade I peripheral chondrosarcoma Sarcoma 2002 6, article S1 10 Vanel D De Paolis M Monti C Mercuri M Picci P Radiological features of 24 periosteal chondrosarcomas Skeletal Radiology 2001 30 4 208 212 2-s2.0-0035032954 11392294 11 Forest M Tomeno B Vanel D Orthopedic Surgical Pathology: Diagnosis of Tumors and Pseudotumoral Lesions of Bones and Joints 1998 Edinburgh, UK Churchill Livingstone 12 Ly JQ Mesenchymal chondrosarcoma of the maxilla American Journal of Roentgenology 2002 179 4 1077 1078 2-s2.0-0036783708 12239076 13 Fechner RE Mills EM Fechner RE Mills EM Cartilaginous lesions Tumors of the Bones and Joints 1993 8 3rd edition Washington, DC, USA Armed Forces Institute of Pathology 14 Anract P Tomeno B Forest M Chondrosarcomes dédifférenciés. Etude de treize cas cliniques et revue de la littérature Revue de Chirurgie Orthopédique et Traumatologique 1994 80 669 680 15 Hudson TM Springfield DS Spanier SS Benign exostoses and exostotic chondrosarcomas: evaluation of cartilage thickness by CT Radiology 1984 152 3 595 599 2-s2.0-0021227535 6611561
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Case Rep Radiol. 2012 Dec 2; 2012:642062
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23251568PONE-D-12-2010710.1371/journal.pone.0051537Research ArticleBiologyBiochemistryHistologyImmunologyImmunopathologyModel OrganismsAnimal ModelsRatMolecular Cell BiologyGene ExpressionChemistryChemical BiologyInorganic ChemistryMedicineGastroenterology and HepatologyAcute Toxicity and Gastroprotection Studies of a New Schiff Base Derived Copper (II) Complex against Ethanol-Induced Acute Gastric Lesions in Rats Gastroprotection Studies of Copper (II) ComplexHajrezaie Maryam 1 3 Golbabapour Shahram 1 3 Hassandarvish Pouya 1 Gwaram Nura Suleiman 2 A. Hadi A. Hamid 2 Mohd Ali Hapipah 2 Majid Nazia 3 Abdulla Mahmood Ameen 1 * 1 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia 2 Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia 3 Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia Roberts David D. Editor Center for Cancer Research, 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: MH SG MAA. Performed the experiments: MH SG PH NSG. Analyzed the data: SG NM. Contributed reagents/materials/analysis tools: MH AHAH HMA NSG. Wrote the paper: SG MAA. 2012 10 12 2012 7 12 e5153711 7 2012 8 11 2012 © 2012 Hajrezaie et al2012Hajrezaie 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 Copper is an essential element in various metabolisms. The investigation was carried out to evaluate acute gastroprotective effects of the Copper (II) complex against ethanol-induced superficial hemorrhagic mucosal lesions in rats. Methodology/Principal Findings Rats were divided into 7 groups. Groups 1 and 2 were orally administered with Tween 20 (10% v/v). Group 3 was orally administered with 20 mg/kg omeprazole (10% Tween 20). Groups 4–7 received 10, 20, 40, and 80 mg/kg of the complex (10% Tween 20), respectively. Tween 20 (10% v/v) was given orally to group 1 and absolute ethanol was given orally to groups 2–7, respectively. Rats were sacrificed after 1 h. Group 2 exhibited severe superficial hemorrhagic mucosal lesions. Gastric wall mucus was significantly preserved by the pre-treatment complex. The results showed a significant increase in glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), and Prostaglandin E2 (PGE2) activities and a decrease in malondialdehyde (MDA) level. Histology showed marked reduction of hemorrhagic mucosal lesions in groups 4–7. Immunohistochemical staining showed up-regulation of Hsp70 and down-regulation of Bax proteins. PAS staining of groups 4–7 showed intense stain uptake of gastric mucosa. The acute toxicity revealed the non-toxic nature of the compound. Conclusions/Significance The gastroprotective effect of the Copper (II) complex may possibly be due to preservation of gastric wall mucus; increase in PGE2 synthesis; GSH, SOD, and NO up-regulation of Hsp70 protein; decrease in MDA level; and down-regulation of Bax protein. The authors would like to thank the University of Malaya for funding this research, RG373/11HTM and HIR-MOHE (F000009-21001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Ulcer is an open sore or lesion, usually found on the skin or mucous membrane of the body. A peptic ulcer is a lesion occurs at the lining of the stomach or duodenum, where hydrochloric acid and pepsin are present. In the past, it was thought that lifestyle factors, such as stress and diet caused ulcers. Later, researchers found that stomach acids (i.e., hydrochloric acid) and pepsin took part in ulcer formation. Many researches have shown that most ulcers (80% of gastric ulcers and 90% of duodenal ulcers) contribute significantly to bacterium Helicobacter pylori. Among the three factors (lifestyle, hydrochloric acid and pepsin, and H. Pylori) important in ulcer development, H. pylori is the primary cause of the gastric ulcer [1], [2]. Stress is an important etiological factor in the incidence of gastric ulcer. Free radicals, in stress-involved gastrointestinal injuries, may inactivate synthetize of mucosal prostaglandin (by accumulating H2O2, an inhibitor in synthesis of the prostaglandin, which propitiates the generation of reactive oxygen species [3]. Lipid-derived free radicals such as conjugated dienes and lipid hydroperoxides cause oxidative reactions. The process of lipid peroxidation is mediated by the interaction between hydroxyl radicals and the cell membrane, during which the lipid-derived free radicals produce [4]. Recently, a numerous researches have been inclined to find effective synthetic chemical compounds with gastroprotective properties [5], [6], [7]. 10.1371/journal.pone.0051537.g001Figure 1 Chemical structure of Cu(BrHAP)2. Copper is an essential trace element in human metabolism, but does not exist in ionic form in biological systems. The amount of copper in the body is measured in complexes with organic compounds. The process of chelating metals smuggles them to bypass the intestinal wall and to enter into the mainstream of nutrient flow and usage in the body. Copper complexes such as copper aspirinate and copper tryptophanate, remarkably increase healing rate of ulcers and wounds [8]. The Copper complexes shorten the healing period of gastric ulcers for five days and promote the wound healing process at the same time retaining anti-inflammatory activity [8] while non-steroidal anti-inflammatory drugs (e.g. ibuprofen and enefenamic acid) suppress wound healing process.In recent years, copper coordination compounds with schiff-base ligands have been extensively studied.It seems that their reactivity with dioxygen or its reduced derivativesistheirunique biological property [9]. Some of schiff base derived copper compounds causesevere cytotoxicity, through the generation of reactive oxygen species which can damage different biomolecules [10]. However, they may be considered as efficient catalyst for the oxidation of different substrates [11]. Many of these complexes have been investigated for their effective scavengers of superoxide radicals [12], acting as antioxidants.This study introduced the Copper (II) complex derived from N,N’dimethyl ethylene diamine and 2-hydroxyacetophenone schiff base ligand, its synthesis and its chemical characterization. Also, the study evaluated acute toxicity and gastroprotective activity of the complex against absolute ethanol-induced acute hemorrhagic mucosal lesions in rats. 10.1371/journal.pone.0051537.t001Table 1 Elemental analysis and spectral characterization for the ligand and its metal complex. Ligand Elemental Analysis Analytical Calculated: C, 69.87; H, 8.80; N, 13.58. Found: C, 68.48; H, 8.98; N, 14.98. IR (KBr disc cm−1) υ(Ar–OH), 3436sb; υ(C–H), 2836s; υ(C = N), 1628s; υ(C–C), 1438s; υ(C–N), 1166s; UV-Vis (DMSO), λmax (ε, Mol−1cm−1): 279 nm (3091.79, π-π*); 303 nm (3152.21, CT) 1H-NMR (DMSO-d6) 2.23 (s, 3H, CH3), 2.28 (6H, 2CH3), 2.49 (t, 2H, 2CH2), 6.78–7.02 (4H, ArH), 10.7 (s, 1H, phenolic) 13C-NMR (DMSO-d6) 16.72 (CH3), 45.54 (2CH3), 45.86, 57.53 (2CH2), 167.91 (C = N) ArC: [125.15 (CH), 128.48(CH), 141.33 (CH), 1148.29 (CH), 149.82 (C)] Complex Elemental Analysis Analytical Calculated: C, 40.29; H, 5.07; N, 7.83 Found: C, 40.88; H, 5.20; N, 7.23. IR (ATR cm−1) 2843.53 m,2800.66 m ν(C-H), 1102.37 m ν(C-N), 1474.35 m, 1436.35 m ν(C-C), 1605.47 m, 1593.89 ν(C = N), 518.09 m ν(M-N), 473.95ν(M-O). UV-Vis (DMSO) 296 (π→π*); 362 (n→π*); 376 (LMCT); 610 (d→d*). Materials and Methods Experimental Section All of the chemicals used in this study were obtained from Fluka and Aldrich and used as received without further purification. Schiff bases were synthesized by condensation reaction. The two ethanolic solutions were stirred for 2 h or 5 h after dissolving it. The ethanol was then evaporated using Rota evaporator. Infrared spectra were obtained using KBr discs (4000–400 cm−1) on Perkin –Elmer FT-IR spectrometer. Elemental analysis (C, H, N) were performed using a Flash EA 1112 Series elemental analyzer in University of Technology Malaysia. Synthesis of the Copper Complex 2-hydroxyacetophenone (0.21 g, 1.65 mM) was weighed accurately and was dissolved in 25 mL of absolute ethanol dripping to an ethanolic solution and N,N′dimethylethyldiamine (0.14 g, 1.65 mM) at room temperature and was refluxed for 3 h. A clear yellow oily product was formed after the evaporation process. The product was isolated in liquid form by adding a few drops of diethyl ether/n-hexane and the purity was confirmed by TLC. Stoichiometric amount of the synthesized schiff base ligand (1.65 mM) was dissolved in 25 mL absolute ethanol, an equimolar quantity of Copper (I) bromide (0.21 g, 1.65 mM) in a minimum amount of ethanol was added. A blue precipitate was formed. The precipitate was filtered, washed with ethanol and dried in vacuum desiccators. Recrystallization was performed in ethanol. The crystal structure of the compound X-ray [13] is as shown in the proposed structure in Figure 1. Elemental analysis and spectral characterization for the ligand and its metal complex are presented in the Table 1. 10.1371/journal.pone.0051537.g002Figure 2 Histological sections in acute toxicity test (H & E staining, 20x). Histological sections of liver (first row) and kidney (second row) in acute toxicity test. Rats treated with 5 mL/kg vehicle (10% Tween 20) (A and D). Rats treated with 500 mg/kg (5 mL/kg) the Copper (II) complex (B and E). Rats treated with 2000 g/kg (5 mL/kg) the Copper (II) complex (C and F). There is no significant differences in structures of liver and kidney between treated and control groups. 10.1371/journal.pone.0051537.g003Figure 3 Macroscopical appearance of the gastric mucosa in rats. The negative control group (A) has no injury of gastric mucosa. The ulcer control group (B) shows sever injuries in the gastric mucosa. Absolute ethanol produced extensive visible hemorrhagic necrosis of gastric mucosa. The reference control group (omeprazole, 20 mg/kg) (C) shows mild injuries in the gastric mucosa comparing to the injuries seen in group 2. Rats received 10 mg/kg of the complex (D) has moderate injuries in the gastric mucosa. The extract reduces the formation of gastric lesions induced by absolute ethanol. Rats received 20 mg/kg of the complex (E) shows mild injuries in the gastric mucosa. Rats received 40 mg/kg of the complex (F) shows mild injuries in the gastric mucosa. Rats received 80 mg/kg of the complex (G) does not show any injuries of the gastric mucosa instead flattening of gastric mucosa is visible (white arrow). Black arrows point to the superficial hemorrhagic mucosal lesions. 10.1371/journal.pone.0051537.g004Figure 4 Effects of the complex on gastric ulcer area, inhibition percentage and alcian blue binding capacity. Alcian blue binding capacity is defined as Gastric wall mucus (GWM). Groups 1 to 3 represent the negative control group, the ulcer control group and the reference control group (omeprazole, 20 mg/kg), respectively. The experimental groups received 10, 20, 40 and 80 mg/kg of the complex are presented as groups 4–7, respectively. All values are expressed as mean ± standard error mean. Mean difference is significant, in GWM, 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. Inhibition of gastric lesions (%) is indicated in brackets above the columns. 10.1371/journal.pone.0051537.g005Figure 5 Effect of the complex on glutathione (GSH), superoxide dismutase (SOD) and nitric oxide (NO) (A,B and C). Groups 1 to 3 represent the negative control group, the ulcer control group and the reference control group (omeprazole, 20 mg/kg), respectively. The experimental groups received 10, 20, 40 and 80 mg/kg of the complex are presented as groups 4–7, respectively. All values are expressed as mean ± standard error 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.0051537.g006Figure 6 Effects of the complex on malondialdehyde (MDA), prostaglandin E2 (PGE2) and protein concentration (A, B and C). Groups 1 to 3 represent the negative control group, the ulcer control group and the reference control group (omeprazole, 20 mg/kg), respectively. The experimental groups received 10, 20, 40 and 80 mg/kg of the complex are presented as groups 4–7, respectively. All values are expressed as mean ± standard error 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.0051537.g007Figure 7 Histological study of gastric mucosal damage in rats (20x). In the negative control group 1 (A), no disruption to the surface epithelium is observed. The ulcer control group (B) has severe disruption to the surface epithelium (black arrows) and necrotic lesions penetrate deeply into mucosa and extensive edema of submucosal layer (yellow arrows) and leucocyte infiltration (blue arrows) are present. The reference control group (omeprazole, 20 mg/kg) (C), mild disruption of the surface epithelium mucosa is present but deep mucosal damage is absent. Reduction of submucosal edema and leucocytes infiltration is seen. Rats received 10 mg/kg of the complex (D) has moderate disruption of surface epithelium with submucosal edema and leucocytes infiltration of submucosal layer. Rats received 20 mg/kg of the complex (E) shows mild to moderate disruption of surface epithelium. In rats received 40 mg/kg of the complex (F), there is mild disruption to the surface epithelium. Reduction of submucosal edema and leucocytes infiltration of the submucosal layer are shown. Rats received 80 mg/kg of the complex (G) has no disruption to the surface epithelium. Chemicals In this study, omeprazole (obtained from UMMC Pharmacy) was used as the reference anti-ulcer medicine. The medicine was dissolved in 10% Tween 20 (Merck Schuchardt OHG, 85662 Hohenbrunn, Germany). A dilution of (10% v/v) Tween 20 was used as the vehicle for dosing all of the rats (5 mL/kg) and were administered orally to the rats in a dosage of 20 mg/kg body weight (5 mL/kg) according to the recommendation of Mahmood et al. [14]. Experimental Animals Acute toxicity study Healthy male and female Sprague-Dawley rats (6–8 weeks old) were obtained from the Animal House, Faculty of Medicine, University of Malaya, Kuala Lumpur (Ethics No. PM/27/07/2010/MAA (R). The rats were weighed between 150–180 g. The animals were given standard rat pellets and tap water ad libitum. The acute toxicity study was used to determine a safe dose for the Copper (II) complex. 36 rats (18 males and 18 females) were randomly assigned into 3 groups labeled as vehicle (10% Tween 20), and 100 mg/kg and 2000 mg/kg of the Copper (II) complex (10% Tween 20) [15]. The animals were fasted overnight prior to the dosing. Food was withheld for another 3 to 4 h after dosing. The animals were under observation for 0.5, 2, 4, 8, 24 and 48 h after administration to monitor any onset of clinical or toxicological symptoms. Mortality, if any, was recorded over a period of 2 weeks. The animals were sacrificed on day 15. Histology, hematological and serum biochemical parameters were determined according to the OECD [15]. This study was approved by the Ethics Committee for animal experimentation, Faculty of Medicine, University of Malaya, Malaysia Ethic No. PM/07/05/2011/MAA (R). Throughout experiments, all of the animals received humane care according to the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences. 10.1371/journal.pone.0051537.g008Figure 8 Effect of the complex on gastric tissue glycoprotein-PAS staining (20x). The negative control group (A), the ulcer control group (B), the reference group (omeprazole, 20 mg/kg) (C), rats received 10 mg/kg of the complex (D), rats received 20 mg/kg of the complex (E), rats received 40 mg/kg of the complex (F), and rats received 80 mg/kg of the complex (G). Magenta color in the apical epithelial cells in the treated groups with the compound (groups 4–6) shows gradual increase in mucosal secretion of gastric glands. The intense secretion of mucus in gastric glands is demonstrated in group 7. The arrow points to the glycoprotein accumulation. 10.1371/journal.pone.0051537.g009Figure 9 Immunohistochemical analysis of expression of Hsp70 proteins (20x). The negative control group (A), the ulcer control group (B), the reference group (omeprazole, 20 mg/kg) (C), rats received 10 mg/kg of the complex (D), rats received 20 mg/kg of the complex (E), rats received 40 mg/kg of the complex (F), and rats received 80 mg/kg of the complex (G). Immunohistochemistry staining of Hsp70 shows over-expression of Hsp70 protein in the experimental groups (D-G). The arrow points to the Hsp70 protein accumulation. 10.1371/journal.pone.0051537.g010Figure 10 Immunohistochemical analysis of expression of Bax proteins (20x). The negative control group (A), the ulcer control group (B), the reference group (omeprazole, 20 mg/kg) (C), rats received 10 mg/kg of the complex (D), rats received 20 mg/kg of the complex (E), rats received 40 mg/kg of the complex (F), and rats received 80 mg/kg of the complex (G). Immunohistochemistry staining of Bax proteins shows down-expression of Bax protein in D-G. The arrow points to the Bax protein accumulation. Gastric ulcer study Sprague Dawley healthy adult male rats were obtained from Experimental Animal House, Faculty of Medicine, University of Malaya; Ethic No. PM/28/09/2011/MAA (R). The rats (weighed between 225–250 g) were divided randomly into 7 groups consisting of 6 rats each group. Each rat was caged individually with wide-mesh wired bottoms to prevent coprophagia during the experiment. The animals were maintained on a standard pellet diet and tap water ad libitum. Gastric ulcer-induced by ethanol Prior to the experiment, the rats were fasted for 24 h, according to a recommended method [14]. They had free access to drinking water till 2 h before the experiment. Gastric ulcer was induced by orogastric intubation of absolute ethanol (5 mL/kg) according to the method recommended by Abdulla et al. [14]. Group 1 and group 2 were orally administered with 10% Tween 20 (5 mL/kg). Group 3 received an oral dose of 20 mg/kg omeprazole in 10% Tween 20 (5 mL/kg), as the reference control group. The experimental groups were orally administered with the Copper (II) complex dissolved in 10% Tween 20 (5 mL/kg) at different dosages of 10, 20, 40 and 80 mg/kg (Groups 4, 5, 6 and 7, respectively). Then, 1 h after this pre-treatment, group 1 was orally administered with 5 mL/kg 10% of Tween 20.Groups 2–7 were received absolute ethanol orally (5 mL/kg). The rats were euthanized 60 min later (based on the previously used method [16]) with an overdose of xylazine and ketamine anesthesia and their stomachs were immediately excised. Macroscopic appearance of acute gastric mucosal lesion Lesions of the gastric mucosa appear as elongated bands of superficial hemorrhagic mucosal lesions parallel to the long axis of the stomach. Gastric mucosa of each rat was thus examined for the damages. The length and width of the hemorrhagic mucosal lesions (mm) were measured with a planimeter (10 × 10 mm2 = ulcer area) under a dissecting microscope (1.8x). The hemorrhagic mucosal lesions area was measured by counting the number of small squares (2 mm × 2 mm) covering the length and the width of each lesion band. The sum of the areas of the lesions for each stomach was applied in a calculation of the UA where the sum of small squares × 4 × 1.8 = UA (mm2) were calculated according to the recommendation of Zahra et al. [17]. The inhibition percentage (I%) was calculated by the following formula according to the recommendation of Abdulla et al. [18]. Gastric wall mucus determination The glandular segments of the stomach were removed, weighed and assessed to determine gastric wall mucus in rats [19]. Each segment was transferred immediately to a 1% alcian blue solution (in sucrose solution, buffered with sodium acetate at pH 5).Rinsing with sucrose solution, the excess dye was removed. The dye complexed with the gastric wall mucus was extracted by magnesium chloride solution. A 4 mL aliquot of blue extract was then shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged then the absorbance of the aqueous layer was measured at 580 nm. The quantity of alcian blue extracted per gram of glandular tissue was then calculated. Preparation of stomach homogenate 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 [20]. 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. Antioxidant activities of stomach homogenate The GSH levels of the gastric tissues were measured using gastric tissue supernatant with the GSH kit (Cayman Chemical Co., Mich, USA), according to the manufacturer’s instructions. The SOD activities in the gastric tissues were determined, according to the manufacturer’s protocol, using the SOD assay kit (Cayman Chemical Co., Mich, USA). Using a commercial kit (Cayman Chemical Co., Mich, USA), the NO levels of gastric tissues were measured according to the manufacturer’s instructions. Enzymatic Activities of Stomach Homogenate Measurement of membrane lipids peroxidation (MDA) The rate of lipoperoxidation in the gastric mucous membrane is valued through the measurement of MDA using the TBARS test. The stomachs were washed with normal saline to minimize any interference of hemoglobin with free radicals and to remove blood adhered to the mucous membrane. The stomachs were homogenized with potassium phosphate buffer (10% (w/v). A total of 250 µL was then stored at 37°C for 1 h. Then 400 µL of 35% perchloric acid was added. The mixture was centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was mixed with 400 µL of 0.6% thiobarbituric acid and incubated at 95–100°C for 1 h. After cooling, the absorbance was measured at 532 nm. A standard curve was used for the calculation by 1,1,3,3-tetrametoxypropane. The results were expressed nM of MDA/mg of protein. The concentration of the proteins was measured using the method described by Bradford assay [21]. The measurement of total protein in the stomach sample is based on the interaction between the Coomassie Blue G250 dye and proteins. The interaction of high molecular weight proteins with the dye causes a shift in the ionic charge of the dye to the anionic form, with strong absorbance at 595 nm. To run the assay, a proper volume of albumin standard, distilled water, buffer solution and each sample were added to the wells. For a sample preparation, 2 µL of a sample and 38 µL of the buffer solution were added to a well. Then, 200 µL Bradford's solutions (diluted 5×) were added to the well. After 5 min, the absorbance was recorded at the wavelength of 595 nm [21]. Measurement of PGE2 formation The gastric mucosa was weighed, minced with scissors, and homogenized at 4°C in PBS buffer, accordingly. Homogenates were centrifuged at 13 400 g for 10 min. The supernatants were subjected to a 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. [22]. Histological Evaluation of the Gastric Lesions Hematoxylin and eosin staining technique Specimens of the gastric tissue were fixed in 10% buffered formalin and were processed in the paraffin tissue-processing machine (Leica, Germany). Sections of the stomach were sectioned at 5 µm and stained with hematoxylin and eosin for histological evaluation [23]. Study of mucosal glycoproteins Sections of the glandular portion of the rat stomach of each group were stained with PAS stain to observe mucus production and to evaluate changes in both acidic and basic glycoproteins [24]. Immunohistochemical evaluation Slides of the tissue sections heated at 60°C for 25 min in a hot-air oven (Venticell, MMM, Einrichtungen, Germany). The tissue sections were de-paraffinized in xylene and were rehydrated using graded alcohol. Antigen retrieval process was performed in 10 mM sodium citrate buffer boiled in microwave. Immunohistochemical staining was conducted according to manufacturer's protocol (Dakocytomation, USA). Briefly, endogenous peroxidase was blocked by peroxidase block (0.03% hydrogen peroxide containing sodium azide). The tissue sections were washed gently with the washing buffer. Then the sections were incubated with Hsp70 (1∶500) or Bax (1∶200) biotinylated primary antibodies for 15 min. The sections were rinsed gently with wash buffer and place in the buffer bath. The slides were then placed in a humidified chamber. Sufficient amount of streptavidin–HRP (streptavidin conjugated to horseradish peroxidase in PBS containing an anti-microbial agent) was incubated with the sections for 15 min. Then, the tissue sections were rinsed gently in the washing buffer and were place in the buffer bath. DAB-substrate-chromagen was incubated with the sections for 5 min, following washing and counterstaining with hematoxylin for 5 sec. The sections were then dipped in weak ammonia (0.037 M/L) 10 times and were rinsed with distilled water prior to the mounting of cover slips. Positive findings of the immunohistochemical staining are shown in brown color under a light microscope. Statistical Analysis All of the values were reported as mean ± SEM. The statistical significant differences between groups were assessed using one-way ANOVA. A value of p<0.05 was considered significant. Results Acute Toxicity Study In the acute toxicity study, the animals were treated with the Copper (II) complex at a dosage of 100 mg/kg and 2000 mg/kg and were kept under observation for 14days. All of the animals were alive and did not manifest any sign of toxicity at these dosages. There was no detectable sign of hepatic or renal toxicity in the treated groups as compared with the control group (Figure 2 and Table S1). Macroscopic Evaluation of Gastric Lesions Gross comparison of the stomachs among the groups is illustrated in Figure 3.The results showed that rats in the groups 4–7 had significantly reduced areas of gastric mucosal lesions formation compared to rats in group 2 (p<0.05). The Copper (II) complex significantly suppressed the formation of the hemorrhagic mucosal lesions and interestingly flattened the gastric mucosal folds in group 7 (Figure 3G). Furthermore, absolute ethanol-induced mucosal lesions were significantly reduced in term of size and severity among the rats received the pre-treatment of the Copper (II) complex. The gastroprotective activity of the Copper (II) complex in the ethanol-induced acute hemorrhagic mucosal lesions model is shown in Figure 4. The inhibition of hemorrhagic mucosal lesions in groups 4–7 was remarkable in comparison with the group 3 (Figure 3 and Figure 4). Gastric Wall Mucosal Evaluation Gastric wall mucus was significantly reduced in the group 2. Rats pre-treated with the Copper (II) complex showed meaningful inhibition of the depletion of gastric wall mucus. In the group 2, the gastric wall mucosal was significantly decreased following the ethanol administration. But, the pre-treatment with the Copper (II) compound significantly inhibited this reduction (Figure 4). Antioxidant Activities of Stomach Homogenate Rats in the group 2 showed significant reductions in the levels of GSH, SOD and NO as compared to those in the group 1, whilst, rats in the groups 3–7 showed compensatory increases (Figures 5). Biochemical Assays of MDA and PGE2 in Gastric Tissue Homogenate Administration of absolute alcohol significantly surged the MDA level of the gastric homogenate in comparison to group 1. The MDA level of the gastric tissue in the groups 3–7 was decreased as compared with group 2 (Figure 6A). In the gastric mucosa homogenate, the level of PGE2 was decreased in group 2(compared to the group 1). Groups 3–7 showed increases in the PGE2levels as compared to group 2 (Figure 6B). These results indicated that pre-treated with the complex increased the level of PGE2 in the tissue homogenate as shown in the groups 4–7, demonstrating the possible protective property of the compound against acute hemorrhagic mucosal lesions (Figure 6B). Protein concentration in gastric homogenate was significantly decreased in the group 2 compared with the group 1. Administration of the complex significantly increase the protein content of gastric homogenate compared with the group 1 (Figure 6C). Histological Evaluation of Gastric Lesions Histological observation of the ethanol-induced gastric mucosal lesions in group 2, showed comparatively extensive damage to the gastric mucosa, and edema and leukocytes infiltration of the submucosal layer (Figure 7). Rats received pre-treatment with the Copper (II) complex showed comparatively better protection of the gastric mucosa by reduction in ulcer area, edema and leukocyte infiltration of the submucosal layer (Figure 7). Periodic Acid Schiff (PAS) of Mucosal Glycoproteins Increased in the level of PAS staining of gastric mucosa in groups 4–7 in comparison to group 2 indicated the increase in the glycoprotein content of the gastric mucosa. Group 4–7 reversed the decrease in PAS staining induced by ethanol as shown in Figure 8. Immunohistochemistry Immunohistochemical results showed that rats in the groups 4–7 had over-expression of Hsp70 protein (Figure 9). The expression of Hsp70 protein in group 2 was down-regulated as compared to the group 4–7 (Figure 9). Immunohistochemical staining of Bax protein demonstrated that rats in the groups 4–7 had down-expression of Bax protein (Figure 10). Ethanol, as shown in the group 2, up-regulated the expression of Bax, whilst pre-treatment with the complex decreased the expression of this protein in the groups 4–7 by (Figure 10). Discussion The IR spectra of the complex possesses very strong characteristic absorption bands in the region of 1585–1656 cm−1 attributed to the C = N stretching vibration of the schiff base imino functional group [25], [26]. The spectra for the complex show M–N bands at a lower wavelength in the range of 454–556 cm−1 [7], [27]. The electronic spectra for the complex is obtained in DMSO solvent and showed absorption bands in three-distinct regions. The first region, ranging from 221 to approximately 229 nm, is characteristic for the electronic inter ligand π→π* transitions [28], while the second characteristic wavelength, in the region of 281 nm to approximately 322 nm, is the second inter ligand n→π transition [29]. The third distinct region, ranging from 407 nm to approximately 498 nm, is the characteristic for the LMCT from the nitrogen atom to the transition metal center [30]. Acute toxicity test did not show any signs of toxicity and mortality over a period of 14daysand emphasized on that the Copper (II) complex was safe to be used. No hepatic toxicity and no renal toxicity were seen through the biochemistry and histology results. Ethanol-induced gastric mucosal lesions made it possible to evaluate gastroprotective activity [31]. Ethanol is metabolized in the body and releases superoxide anions and hydroperoxy free radicals. Oxygen-derived free radicals are implicated in the mechanism of acute and chronic ulceration in the stomach [32]. The genesis of ethanol-induced gastric lesions showed a multifactorial origin with a decrease in gastric mucus, and was associated with the significant production of free radicals, leading to increased lipid peroxidation which in turn caused damage to cells and cell membranes [33]. The results of the present study showed that the Copper (II) complex possessed an effective anti-ulcer activity against ethanol-induced hemorrhagic mucosal lesions in rats. The compound increased the gastric wall mucus, inconsistent with the results previously reported by Salga et al. [6]. The gastroprotective effect of the compound appeared to be mediated partly through the preservation of gastric mucus secretion. Omeprazole, a proton pump inhibitor, offered a fairly protected gastric mucosa has been widely used as an acid inhibitor agent in treatment of disorders related to gastric acid secretion [34]. Omeprazole, besides its anti-secretory effect, and effectiveness in acid-dependent ulcer models, is effective in acid independent models, like ethanol-ulcer model, and exert mucosal protection in non-anti-secretory doses [35]. Changes in gastric motility is important in development and prevention of experimental gastric lesions [18]. In the present study, the flattening of the mucosal folds suggested that the gastroprotective effect of the compound was due to decrease in gastric motility. The Flattening of the folds increased the mucosal area exposed to the necrotizing agents, and reduced the volume of the gastric irritants on rugal crest [16], [18]. Increase in the level of GSH appeared consistent with the adaptation phenomenon, the higher the GSH level, the less the damage occurred. GSH and other antioxidants prevented tissue damage by keeping reactive oxygen species at their physiological levels [36]. SOD is able to convert superoxide to hydrogen peroxide, and subsequently, the catalase converts hydrogen peroxide to water. Reduction in the activity of SOD was observed in the gastric mucosa homogenate in the ulcerated rats. This is likely that SOD is utilized in the decomposition of superoxide anion generated by lipid peroxidation. The decrease in the activity of SOD in the hemorrhagic lesions might be resulted from a number of deleterious effects. Pre-treatment with the Copper (II) complex increased the activity of SOD which suggested this effect as a gastroprotective method of the complex in reduction of lipid peroxidation. On the contrary, previous study showed that the activity of SOD increased in the ethanol treated group compared to the normal rats [37]. The Copper (II) complex maintained the level of SOD similar to the normal control group. It seemed that the Copper (II) complex could decrease the oxidative stress condition caused by ethanol. Ethanol effectively reduced the level of nitric oxide NO in the gastric mucosa and slowed the flow of gastric blood, thereby caused the development of hemorrhagic lesions and consequently led to solubilisation of gastric mucus constituents. These actions resulted in an increased flow of Na+ and K+, an increase in pepsin secretion, loss of H+ ions and histamine into the lumen [38], [39]. NO had the ability of inhibiting the neutrophil infiltration to provide a protective barrier for the gastric mucosa against ethanol [40]. The Copper (II) complex enhanced the production activity of NO. Similarly, synthesis of NO protected the gastric mucosa against damage induced by various ulcerogenic models [41]. This finding introduced the ability of the Copper (II) complex to increase the level of NO content as a gastroprotective agent. As mentioned before, oxidative damage is an important reason for cell membrane damage. MDA is the final product of lipid peroxidation and is used to determine lipid peroxidation levels [42]. Our results showed that gastric tissue homogenate in the pre-treated rats with the Copper (II) complex exhibited significant antioxidant activity by decreasing the levels of MDA and by elevating the levels of PGE2 in response to oxidative stress. Lipid peroxidation is known as important pathophysiological event in a variety of diseases [43]. So, the level of MDA is a biomarker for oxidative stress [44]. PGE2 is crucial in the regulation of gastric mucus secretion. PGE2showed protective effects against various gastric injury models [45]. Ethanol is able to reduce the mucosal PGE2 content [46]. PGE2asthe most abundant gastrointestinal prostaglandin, regulates functions of the gut, including motility and secretion [47]. PGE2exerts a protective action on the stomach through the activation of E prostanoid receptors [48]. Cytoprotection by prostaglandin is unrelated to the inhibition of gastric acid secretion since prostaglandin increases the resistance of gastric mucosal cells to the necrotizing effect of strong irritants. Prostaglandins maintain the cellular integrity of the gastric mucosa, and maybe beneficial in the treatment of a variety of diseases in which gastric mucosal injury is present [46]. The results of the current study reveal that protection against gastric mucosa lesions and inhibition of leucocytes infiltration into the gastric wall happened in the rats pre-treated with the Copper (II) complex. Group 2 showed that activation and infiltration of neutrophils, initiate the formation of the lesion. Similarly, Abdulla et al. reported that the reduction of neutrophil infiltration into ulcerated gastric tissue enhanced the prevention of gastric ulcers in rats [18]. Absolute alcohol extensively damages the gastric mucosa, leading to increased neutrophil infiltration into the gastric mucosa. Neutrophils mediate lipid peroxidation through the production of superoxide anions [49]. Neutrophils are the major source for inflammatory mediators and can release potent reactive oxygen species such as superoxide, hydrogen peroxide and myeloperoxidase derived oxidants. This study showed that mucosal level of PGE2 was significantly enhanced by the Copper (II) complex. The enhancement indicated a gastroprotective effect of the Copper (II) complex. Prostaglandins influence virtually every component stimulating mucus and bicarbonate secretion, maintaining mucosal blood flow, enhancing the resistance of epithelial cells to injury induced by cytotoxins and inhibiting leukocyte recruitment [50]. There is a variety of factors influence gastric ulcer prevention. For instance, mucus and bicarbonate secretion seems important in the ulcer preventing process because the mucus/bicarbonate layer protects newly formed cells from acid and peptic injury [51]. PAS staining exhibits characteristic carmine staining of stomach regions that secrete mucopolysaccharides. Group 7 showed intense secretion of mucus in gastric glands. Mucus production is one of the main mechanisms of local gastric mucosal defense [52]. Hsp70 proteins defend cells from oxidative stress or heat shock. Ethanol generates reactive oxygen species (ROS) which normally inhibits the expression of Hsp70 and enhances the expression of Bax. Hsp70 prevents these partially-denatured proteins from aggregating, and allows them to refold. Hsp70 is a 70 kDa protein from the Hsp family present on mammalian cells. It is the most conserved and abundantly produced protein in response to different forms of stress [53], such as heat, toxic agents, infection and proliferation [54]. These proteins are responsible to protect cellular homeostatic processes from environmental and physiologic injuries by preserving the structure of normal proteins and repairing or removing damaged proteins. Over-expression of Hsp70 proteins and down-regulation of Bax proteins were shown in this study that suggest these regulations a gastroprotective method of the Copper (II) complex against ethanol-induced superficial hemorrhagic mucosal lesions in rats. Conclusions The Copper (II) complex derived from N,N’dimethyl ethylene diamine and 2-hydroxyacetophenone schiff base ligand did not cause any sing of acute toxicity in rats (100 mg or 2000 mg/kg). The compound could significantly protect the gastric mucosa against ethanol-induced injury. Antioxidant activities of GSH, SOD and NO were significant increase in the gastric mucosal homogenate, while there was a remarkable decrease in MDA. Assay of PGE2in the gastric tissue homogenates revealed significant increase in the PGE2 level in the pre-treated group with the complex as compared with the group 2. Such protection was ascertained grossly by significant increase in the gastric wall mucus in comparison with the ulcer control group. Also the reduction of hemorrhagic mucosal areas in the gastric wall as well as the reduction or inhibition of edema and leukocytes infiltration of the submucosal layers were shown histologically. Immunohistochemistry staining of Hsp70 and Bax proteins showed up-regulation of Hsp70 protein and down-regulation of Bax protein in rats pre-treated with the Copper (II) complex. Increased the PAS staining of gastric mucosa of treated animals in comparison to group 2 indicated the increase in glycoprotein content and the Copper (II) complex reversed the decrease in PAS staining induced by ethanol. 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PLoS One. 2012 Dec 10; 7(12):e51537
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23272116PONE-D-12-1820210.1371/journal.pone.0051549Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologySignal TransductionSignaling CascadesAkt Signaling CascadeCell DeathMedicineOncologyBasic Cancer ResearchMetastasisCancer PreventionOtorhinolaryngologyHead and Neck CancersEpithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance and Metastatic Potential in Adenoid Cystic Carcinoma EMT Is Required for Anoikis Resistance in ACCJia Jun 1 2 Zhang Wei 1 2 Liu Jian-Ying 1 Chen Gang 1 2 Liu Hui 1 Zhong Hao-Yan 1 2 Liu Bing 2 Cai Yu 1 2 Zhang Jia-Li 1 3 Zhao Yi-Fang 1 2 * 1 The State Key Laboratory Breeding Base of Basic Science of Stomatology and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China 2 Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, China 3 Department of Pathology, School and Hospital of Stomatology, Wuhan University, Wuhan, China Viglietto Giuseppe Editor University Magna Graecia, Italy * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JJ WZ GC Y-FZ. Performed the experiments: WZ J-YL HL H-YZ. Analyzed the data: JJ WZ GC. Contributed reagents/materials/analysis tools: BL YC J-LZ. Wrote the paper: JJ WZ GC Y-FZ. 2012 14 12 2012 7 12 e5154925 6 2012 2 11 2012 © 2012 Jia et al2012Jia 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.Human adenoid cystic carcinoma (ACC) is characterized by diffused invasion of the tumor into adjacent organs and early distant metastasis. Anoikis resistance and epithelial mesenchymal transition (EMT) are considered prerequisites for cancer cells to metastasize. Exploring the relationship between these processes and their underlying mechanism of action is a promising way to better understand ACC tumors. We initially established anoikis-resistant sublines of ACC cells; the variant cells revealed a mesenchymal phenotype through Slug-mediated EMT-like transformation and displayed enhanced metastatic potential both in vitro and in vivo. Suppression of EMT by knockdown of Slug significantly impaired anoikis resistance, migration, and invasion of the variant cells. With overexpression of Slug and Twist, we determined that induction of EMT in normal ACC cells could prevent anoikis, albeit partially. These findings strongly suggest that EMT is indispensable in anoikis resistance, at least in ACC cells. Furthermore, we found that the EGFR/PI3K/Akt pathway acts as the common regulator for EMT-like transformation and anoikis resistance, as confirmed by their specific inhibitors. Gefitinib and LY294003 restored the sensibilities of anoikis-resistant cells to anoikis and simultaneously impaired their metastatic potential. In addition, the results from our in vivo model of metastasis suggest that pretreatment with gefitinib promotes mouse survival by alleviating pulmonary metastasis. Most importantly, immunohistochemistry of human ACC specimens showed a correlation between the overexpression of Slug and EGFR staining. This study has demonstrated that Slug-mediated EMT-like transformation is required by human ACC cells to achieve anoikis resistance and their metastatic potential. Targeting the EGFR/PI3K/Akt pathway holds potential as a preventive strategy against distant metastasis of ACC. This work was supported by the grants from The National Natural Science Foundation of China (81102054, 30973330, 81170977). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Adenoid cystic carcinoma (ACC) is one of the most common malignancies of the major and minor salivary glands [1] that accounts for approximately 15%–25% of all malignant salivary gland carcinomas [1], [2]. Typically, ACC grows slowly, but it spreads relentlessly into adjacent tissues and develops distant metastasis frequently to the lungs, bone, and soft tissues. Most patients with ACC (80%–90%) die within 10–15 years after being diagnosed due to high rates of recurrence and distant metastasis [3]. Although numerous studies have identified the factors related to the prognosis and outcome of ACC, little is known about the underlying molecular mechanisms that control its ability to spread to distant organs. A better understanding of the biological process involved in ACC metastasis is therefore urgently needed. Metastasis is a multistep process in which cancer cells spread from the primary site to distant organs through the lymphatic or circulatory system [4], [5]. In normal epithelial cells, loss of cell–cell or cell–matrix interactions triggers a form of apoptosis known as anoikis, which inhibits the survival of cancer cells in the circulation [6]. Thus, their survivability in anchorage-independent environments, such as dissemination in the circulatory system, is considered a prerequisite for cancer cells to metastasize. Epithelial mesenchymal transition (EMT) describes a series of rapid changes in the cellular phenotype in which epithelial cells down-modulate their adhesion structures, alter their polarity, and adopt a mesenchymal morphology [7]. EMT is essential for the formation of mesodermal tissue from early embryonic epithelial cells during development, and it is associated with wound healing, tissue inflammation, and organ fibrosis in adults [7], [8]. Accumulating evidence suggests that an EMT-like transformation contributes to tumor progression in most cases, including human ACC [9], [10]. Recent studies have shown that transformation not only endows cancer cells with motility to detach from neighboring cells but also promotes anoikis resistance in cancer cells in anchorage-independent circumstances [11], [12]. The crosstalk between integrin–extracellular matrix (ECM) and growth factors involved in EMT also exists between the pathways related to anoikis resistance [13], [14]. More recent studies have demonstrated that cancer cells undergo an EMT-like transformation in the peripheral circulation of patients with carcinomas [15]–[17], strongly suggesting that EMT contributes to anoikis resistance. Exploring the relationship between anoikis resistance and EMT and their common mechanism of action is thus a promising way of better understanding metastasis. In this study, we established anoikis-resistant variants of ACC cells to investigate the involvement of EMT-like transformation in the acquisition of anoikis resistance. We found that Slug-mediated EMT promotes cell motility and contributes to the acquisition of anoikis resistance. We also found that activation of the epidermal growth factor receptor (EGFR, ErbB-1, HER1)/phosphoinositide-3 kinase (PI3K)/Protein Kinase B (PKB, Akt) signaling pathway is the common mechanism of EMT and anoikis resistance in activated ACC cells. Using a pharmacological inhibitor of EGFR, we succeeded in delaying pulmonary metastasis in nude mice injected with these variant cells and promoting their survival, which suggests that targeting the EGFR/PI3K/Akt pathway holds potential in preventing metastasis of human ACC. Materials and Methods Chemicals and Antibodies Poly(2-hydroxyethyl methacrylate) [poly(HEMA)], dimethyl sulfoxide (DMSO), Hoechst 33258, LY294002, and G418 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gefitinib was purchased from Selleck Chemicals (Houston, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GIBCO (Carlsbad, CA, USA). Primary antibodies against human phospho-EGFR (Tyr1068), phospho-PI3K p85 (Tyr458), phospho-Akt (Ser473), Slug, cleaved caspase-3, cleaved caspase-9, and cleaved poly(ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Danvers, MA). Primary antibodies against PI3K p85, Akt, Bax, Bcl-2, EGF, matrix metalloproteinase 9 (MMP9), and actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies against E-cadherin, N-cadherin, α-smooth muscle actin, vimentin, and Twist were purchased from Epitomics, Inc. (Burlingame, CA, USA). Recombinant EGF was purchased from Cell Signaling Technology as well. Cell Culture and Generation of Anoikis-Resistant Variants The high (ACC-M) and low (ACC-2) metastatic cell lines of human salivary ACC were obtained from the China Center for Type Culture Collection [18] and maintained in DMEM supplemented with 10% FBS (v/v), 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Poly(HEMA) was prepared by dissolving it in 95% ethanol (v/v) to a concentration of 20 mg/ml and subsequently added to cell culture wells at a density of 5 mg/cm2. The dishes were then allowed to dry overnight. Anoikis-resistant ACC cells (ACCAr) were generated by sequential cycles of culture using poly(HEMA)-treated (suspended) and untreated (adhered) dishes. Briefly, ACC monolayers were trypsinized, transferred to poly(HEMA)-treated dishes for 3 days of culture and then to untreated dishes, and allowed to replicate for 1 day. The variants were harvested after six rounds of culture and then labeled as ACCAr cells. Clinical Samples and Immunohistochemistry Fifty pathologically confirmed human ACC specimens with six corresponding pericancerous normal salivary gland tissues were collected at the Hospital of Stomatology, Wuhan University. All specimens were fixed in 4% buffered paraformaldehyde and embedded in paraffin. The procedures were performed in accordance with the National Institutes of Health guidelines for the use of human tissues. The study was approved by the review board of the Ethics Committee of the Wuhan University Hospital of Stomatology. Written informed consent was obtained from all study participants. Immunohistochemical analysis was performed according to our previous procedures [18], as described in Protocol S1. Transfection and Infection with Lentivirus pEGFP-N1-SNAI2-GFP (pEGFP-Slug) and pEGFP-N1-TWIST1-GFP (pEGFP-Twist) were purchased from Genechem (Shanghai, China). The constructs were confirmed to be correct by restriction enzyme digestion and sequencing. The stably transfected ACC-2 cell lines of pEGFP-Twist and pEGFP-Slug were selected by G418 (500 µg/ml) for 2 weeks and confirmed by reverse transcription (RT)-PCR and Western blot analysis. For siRNA-mediated inhibition, three siRNA sequences against human Slug (5′-AACTGGACACACATACAGTG-3′, 5′-CAGACCCATTCTGATGTAAAG-3′, and 5′-GAGGAAAGACTACAGTCCAAG-3′) were cloned into pBRsi-hU6 lentiviral vector systems (Slug siRNA 1, Slug siRNA 2, and Slug siRNA 3). The negative control (NC) siRNA and lentiviral vector package were provided by Genechem. The RNA interference (RNAi) efficiency of the three constructs was confirmed by RT-PCR and Western blot analysis. According to the results, Slug siRNA 2 was selected for the formal experiments (Figure S3). Cell Growth Analysis and Viability Measurement MTT assay was performed to measure the growth curves of ACC and ACCAr cells. Briefly, 2000 cells/well were plated onto 96-well plates and incubated for 24, 48, and 72 h with 100 µl of DMEM. Then, 10 µl of MTT (5 mg/ml) was added to each well, which was incubated for another 4 h. Afterwards, the supernatant was discarded with 100 µl of dimethyl sulfoxide added to each well. Absorbance was assessed at 490 nm using a 96-well microplate reader (Bio-Tek). Cell viability was measured with a Vi-CELL Cell Viability Analyzer (Beckman Coulter, Fullerton, CA) based on trypan blue exclusion (TBE) assays. Determination of Apoptosis Apoptosis induction by the detachment in ACC or ACCAr cells was determined according to our previous procedures [18] as follows: (a) morphological evaluation by Hoechst staining; (b) quantitation of cytoplasmic histone-associated DNA fragments with Cell Death Detection ELISAPLUS; and (c) Western blot analysis for Bax/Bcl-2 ratio, caspase-3, caspase-9, and PARP cleavage. Wound Healing Assay ACC and ACCAr cells were seeded in six-well culture plates (Corning Life Sciences, NY) and allowed to grow to 90% confluence. The center of the cell monolayers was scraped with a sterile micropipette tip to create a gap of constant width. After 12 h, the cells that migrated into the gap were counted after fixation and observed under a phase microscope (Leica). Migration and Invasion Assays The ability of ACC and ACCAr cells to pass through filters was measured using a transwell Boyden chamber system (Corning Life Sciences) containing a polycarbonate filter (6.5 mm diameter; pore size of 8 µm). Matrigel was diluted to 200 µg/ml and applied to the top side of the filter in the cell invasion assay. In contrast, the filter was not coated in the cell migration assay. ACC and ACCAr cells were seeded in the upper chamber at a density of 104 cells/well in 100 µl of serum-deprived medium, whereas 10% FBS medium was applied to the lower chamber as chemoattractant. After incubation for 24 h at 37°C, the cells in the upper surface of the membrane were carefully removed with a cotton swab, and migrated cells were fixed with methanol, stained with eosin, and then photographed and quantified. Soft Agar Assay For soft agar assay, 2000 trypsinized cells were seeded in 0.4% low-melting-point agarose (Takara, Kyoto, Japan) on top of a 1% agarose layer, and scans were taken 11 days later. The number of macroscopic colonies was determined using ImageJ software (http://rsb.info.nih.gov/ij/index.html). Immunofluorescence Analysis for Cells The localization of E-cadherin and Vimentin was detected by indirect immunofluorescence analysis. In brief, ACC cells were grown on glass coverslips with indicated treatment. Then, cells were washed with PBS, fixed in 100% methanol at −20°C, and blocked in 10% non-immune goat serum for 1 h at room temperature. Then cells were incubated with indicated primary antibody with dilution of 1∶200 overnight at 4°C followed by incubation with Dylight488-conjugated goat anti-mouse IgG (1∶400, Jackson, West Grove, PA) for 1 h at room temperature. The nuclei were stained with DAPI and the coverslips were mounted on a microscope slide with embedding medium. The cells were observed and photographed with a fluorescence microscope (Leica). Quantitative Real-Time RT-PCR Quantitative real-time RT-PCR was performed to evaluate the mRNA expression levels of EMT-related genes, growth factors, and MMPs of the ACCAr cells. Total RNA was isolated with TRIzol Reagent (Invitrogen). Aliquots (1 µg) of RNA were reverse transcribed to cDNA (20 µl) with oligo(dT) and M-MuLV reverse transcriptase (Fermentas, Glen Burnie, MD). One-fifth of the cDNA was used as a template for PCR using SYBR Premix Ex Taq™ (Perfect Real Time) kits (Takara, Kyoto, Japan) in an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). 18s rRNA was selected as an internal control for each experiment. See Protocol S1 for the primer sequences designed for PCR. Western Blot Analysis The proteins in corresponding cells were collected, and the concentration of protein in supernatants was estimated using BCA assay (Pierce Chemical, Rockford, IL). Subsequently, 40 mg of protein was separated on 10% SDS-polyacrylamide gels and electroblotted on polyvinylidene fluoride membranes (Roche Applied Science, Germany). The blots were blocked overnight with 5% nonfat dry milk and probed with primary antibodies at dilutions recommended by the suppliers. Immunoblots were detected by horseradish peroxidase-conjugated secondary antibody (Pierce) using a chemiluminescence kit (Pierce) and photographed. Mouse Model of Pulmonary Metastasis Female BALB/c nude mice (18–20 g; 6–8 weeks old) were purchased from the Center for Animal Experiment of Wuhan University in pressurized ventilated cages according to institutional regulations. All studies were approved and overseen by the Institutional Animal Care and Use Committee, Center for Animal Experiment, Wuhan University. ACC-2, ACC-2 pretreated with EGF (10 ng/ml) for 1 h, ACC-2Ar, and ACC-2Ar cells pretreated with gefitinib (1 µM) for 1 h (2×106 in 0.1 ml of PBS) were collected from subconfluent culture and inoculated intravenously into the caudal vein of the mice. Mice were inspected daily and euthanized by CO2 when clinical symptoms became apparent. Lung tissues were exteriorized carefully, fixed in 4% buffered paraformaldehyde, and embedded in paraffin. Metastatic lesions in the lungs were counted by visual inspection of hematoxylin and eosin-stained histological tissue sections. The sizes of metastases and the area of lung tissue per section were determined with ImageJ software. Statistical Analysis All values were expressed as the mean ± SE of three independent experiments. Data analyses were conducted using OriginPro 8.6.0 (OriginLab Corporation, Northampton, MA). Image analyses for soft agar assays were conducted using ImageJ software (http://rsb.info.nih.gov/ij/index.html). One-way ANOVA, the Student–Newman–Keuls test, and Spearman’s rank correlation test were used for statistical analysis. p<0.05 was considered statistically significant. Results Establishment of Anoikis-Resistant Sublines of ACC Cells Dishes treated with poly(HEMA) were used to induce anoikis-resistant sublines in both ACC cell lines. ACC-2Ar and ACC-MAr, referred to as the anoikis-resistant sublines of ACC-2 and ACC-M cells, respectively, were established after six alternating cycles of culture under suspension and adhesion (Figure 1A). Cells were inoculated in the dishes coated with poly(HEMA) to identify whether the variants could escape the apoptosis induced by detachment. TBE assay showed that the cell viabilities of anoikis-resistant variants were much higher than those of parental cells (Figure 1B). After culture in suspension for 24 h, several apoptotic morphological features, such as apoptotic bodies, cell shrinkage, and chromatin condensation, were observed in normal ACC cells, but they were not detected in ACCAr cells (Figure 1C). Quantitative assessment using Cell Death Detection ELISAPLUS showed that the anoikis fractions of ACC-2Ar and ACC-MAr cells were 0.887 and 0.501, respectively, compared with 0.256 and 0.170 in the parental cells (p<0.05) (Figure 1D). The results of anchorage-independent growth assay in soft agar indicated that ACCAr cells acquired the ability to survive in an anchorage-independent environment (Figure 1E). The cleavage of caspase-3, caspase-8, caspase-9, and poly (ADP-ribose) polymerase (PARP) as well as the expression of Bcl-2 and Bax were subjected to Western blot analysis to further investigate the mechanism underlying cell death. As shown in Figure 1F, the cleavage of capase-3 and that of caspase-9 were significantly augmented in normal ACC cells; however, cleavage of caspase-8 was not detected (data not shown), indicating that detachment induced anoikis via an intracellular apoptotic pathway. In addition, the Bcl-2/Bax ratio was diminished in normal ACC cells. In summary, the ACC cells cultured in suspension conditions for several rounds succeeded in acquisition the characteristic of anoikis resistance. 10.1371/journal.pone.0051549.g001Figure 1 Establishment of anoikis-resistant sublines of ACC cells. A, Schematic of establishing anoikis-resistant sublines of ACC by cycles of culture under suspension and adhesion. B, The cell viabilities of ACC and ACCAr in suspension were measured using TBE assay (Mean ± SE, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). C, Hoechst 33258 staining was performed to detect the apoptosis of ACC and ACCAr cells that cultured in suspension. D, DNA fragments of ACC and ACCAr in suspension were detected using Cell Death Detection ELISAPLUS (Mean ± SE, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). E, Anchorage-independent growth of ACC and ACCAr cells was analyzed using soft agar assay. F, Expression levels of cleaved PARP, cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 were determined using Western blot analysis (**p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). Anoikis-Resistant Sublines of ACC are more Aggressive in Metastasis In Vitro As the acquisition of anoikis resistance is generally correlated with increased ability of migration and invasion in the progression of tumors, we measured the migration and invasion of ACC cells using wound healing and transwell Boyden chamber assays. ACC-2Ar and ACC-MAr both showed enhanced migration and invasion compared with normal ACC cells (Figure 2A–D). The effects of cell proliferation on migration were excluded because the proliferation curve of ACCAr cells showed an even lower proliferation rate in both the ACC-2 and ACC-M variants (Figure S1). Our results revealed that the mRNA expression levels of vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9), and stromal cell-derived factor-1 alpha (SDF-1α) were up-regulated in ACC cells as they gained anoikis resistance (Figure 2E), which can account for the increased migration and invasion abilities of the variant cell lines. Taken together, these results revealed that the ACCAr cells selected by suspended culture displayed much more aggressive potential than their parental cells both in vitro. 10.1371/journal.pone.0051549.g002Figure 2 Enhanced migration and invasion of ACCAr cells. A and B, The migration of ACC and ACCAr was analyzed using wound healing and transwell Boyden system assays. C, The invasion of ACC and ACCAr was measured using a transwell Boyden system coated with Matrigel. D, Quantification of migrated and invaded cells in wound healing assays and transwell migration/invasion assays. The results were represented as relative ratio to ACC-2 (Mean ± SE, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). E, The relative mRNA expression levels of VEGF, MMP9, and SDF-1α were analyzed by real-time RT-PCR. The results were represented as relative ratio to ACC-2 (Mean ± SE, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). ACCAr Cells Undergo EMT-Like Transformation in Attachment Culture Simultaneous with the detection of the migration and invasion abilities of ACCAr cells, their morphological transformation from cobblestones to an elongated, spindle-shaped appearance was observed (Figure 3A). As the acquisition of anoikis resistance is widely considered to generally be accompanied by morphological EMT-like changes, we decided to further verify the nature of this transformation. As shown in Figure 3A, E-cadherin was correctly localized at the adherent junctions in normal ACC cells, but they were lost in the ACCAr cells. Simultaneously, vimentin as a mesenchymal marker notably increased in ACC-2Ar cells, compared with its negative expression in ACC-2 cells (Figure 3A). Moreover, the results from western blots and qPCR showed the expression levels of several epithelial markers were down-regulated with the up-regulation of mesenchymal proteins at the mRNA and protein levels (Figure 3B and C, Figure S2). The expression level of vimentin in ACC-M cells was higher than that in ACC-2 cells, which might be responsible for the more aggressive nature of ACC-M cells compared with ACC-2 cells [19], [20]. However, the ACCAr cells would lose their mesenchymal features when cultured in adhesion for 6 days, indicating that EMT-like transformation might be a transient process. 10.1371/journal.pone.0051549.g003Figure 3 EMT-like transformation in ACCAr cells. A, A mesenchymal morphology as well as epithelial (E-cadherin) and mesenchymal (vimentin) markers were detected in ACC-2Ar and ACC-MAr. B, Protein expression levels of EMT-related genes in ACCAr cells were measured using Western blot analysis. C, The mRNA expression levels of E-cadherin, N-cadherin, and vimentin in ACCAr cells were determined using qRT-PCR. D, The mRNA expression levels of Snail, Slug, and Twist in ACCAr cells were determined using qRT-PCR. E, The mRNA expression levels of bone morphogenetic protein 4 (BMP4) and connective tissue growth factor (CTGF) in ACCAr cells were determined using qRT-PCR. The results of qRT-PCR were represented as relative ratio to ACC-2 cells (Mean ± SE, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). Several transcription factors that repress epithelial genes, such as those encoding E-cadherin and cytokeratins, via E-boxes in the corresponding promoters include Twist and members of the Snail protein family. We then assessed the mRNA and protein expression levels of these transcription factors, which played critical roles in the EMT process (Figure 3B and D). Our results showed that Slug was significantly up-regulated, Twist slightly increased, and the expression level of Snail did not significantly change in ACCAr cells. In addition, we measured previously reported EMT-related cytokines, and the qPCR results revealed that the mRNA expression of connective tissue growth factor (CTGF) was up-regulated, however no change in bone morphogenetic protein 4 (BMP4) was determined (Figure 3E). In summary, our results suggest that EMT-like transformation is strongly induced by the acquisition of anoikis resistance in ACC cells. Most importantly, we found that ACC-2Ar showed more prominent anoikis resistance, migration, invasion, and EMT-like transformation compared with its parental cells than the ACC-M cell line. Thus, ACC-2Ar and its parental cells were chosen for further investigation. Activation of the EGFR/PI3K/Akt Signaling Pathway Moderates the Acquisition of Anoikis Resistance and EMT-Like Transformation Previous research has shown that growth factors, such as transforming growth factor-beta 1 (TGF-β1), epithelial growth factor (EGF), and hepatocyte growth factor (HGF), among others, induce EMT-like transformation and anoikis suppression in epithelial cells [21], [22]. Thus, we assessed those EMT inducers at the mRNA expression level. As shown in Figure 4A, EGF was strongly induced in the process of establishment of ACCAr cells, which was further confirmed by Western blot analysis. In addition, the phosphorylation level of its receptor, EGFR, was augmented simultaneously (Figure 4B). Furthermore, stimulation with EGF (10 ng/ml) for 48 h was sufficient to induce EMT-like changes in normal ACC cells, strongly suggesting the essential role of the EGFR signaling pathway in EMT induction of ACCAr cells (Figure 4C and D). Gefitinib (1 µM), a specific inhibitor for EGFR, effectively suppressed the activation of EGFR and reverted the spindle-shaped appearance of ACC-2Ar cells back to their epithelial shape in adhesion (Figure 4C and D). Moreover, treatment with gefitinib negatively affected the apoptosis evasion of variants in detachment and their migration capabilities (Figure 4E and F). 10.1371/journal.pone.0051549.g004Figure 4 Involvement of EGFR/PI3K/Akt pathway in acquisition of anoikis resistance and EMT-like transformation in ACCAr cells. A, The mRNA expression levels of EGF in ACCAr cells were measured using qRT-PCR, and results were expressed as relative ratio to ACC-2 cells (Mean ± SEM, **p<0.01 versus ACC-2, ## p<0.01 versus ACC-M). B, The protein expression levels of EGFR, PI3K, Akt, and their phosphorylated forms in ACC and ACCAr cells were determined using Western blot analysis. C, The transformed ACC-2 cells (both ACCAr and EGF-treated cells) were treated with gefitinib (1 µM) and LY294002 (10 µM) for 12 h before detection. The parental ACC-2 cells were used as Control. The expression levels of the phosphorylated form of EGFR, Akt, and E-cadherin, vimentin, and Slug were subjected to Western blot analysis. D, A mesenchymal morphology as well as epithelial (E-cadherin) and mesenchymal (vimentin) markers were detected in ACC cells with indicated treatment as describe above. E, The apoptosis rate in suspension was analyzed using Cell Death Detection ELISAPLUS (Mean ± SE, *p<0.05, **p<0.01 versus ACC-2; ## p<0.01 versus ACC-2Ar). F, The migration of ACC cells with indicated treatment was analyzed using transwell Boyden system. Previous studies have demonstrated that the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways are up-regulated by EGFR activation, leading to EMT and cellular migration [10], [23], [24]. The key kinases of these signaling cascades were subjected to Western blot analysis. The results showed that the PI3K/Akt pathway was activated in the variants and that the phosphorylation status of extracellular regulated protein kinases (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK) did not change. Furthermore, several pharmacological inhibitors were used to further confirm the involvement of the corresponding signaling pathway, showing that specific inhibitors of MAPK/ERK kinase (MEK) (U0126), p38 (SB203580), and JNK (SP600125) did not affect transformation and aggression in ACC-2Ar cells (data not shown). However, suppression of the PI3K/Akt signaling pathway with LY294002 (10 µM) abrogated the augmentation of Slug expression induced by EGFR activation and restored E-cadherin levels in ACC-2Ar cells, accompanied by a reversal from their spindle-shaped cell morphology toward an epithelial appearance (Figure 4C and D). Moreover, inhibition of PI3K/Akt signaling inhibited enhanced migration and invasion in ACC-2Ar cells (Figure 4F). Collectively, these results demonstrated that activation of EGFR induced the acquisition of anoikis resistance in ACCAr cells, led to EMT-like transformation, and enhanced metastatic potential mainly via the EGFR/PI3K/Akt signaling pathway. Gefitinib Suppresses the Enhanced Metastasis of Anoikis-Resistant Sublines of ACC In Vivo To determine the enhanced metastatic capability of ACCAr and the preventive effects of gefitinib against the metastatic abilities of these cells in vivo, we injected cells with distinct pretreament (ACC-2Ar was selected for the marked improvement in its migration and invasion abilities compared with ACC-MAr) into nude mice intravenously to establish an in vivo metastasis model. Our results indicated that the ACC-2Ar cells significantly shortened the life span of mice compared with normal ACC-2 cells. After the mice were sacrificed, each organ was imaged and retained. Growth of the anoikis-resistant subline cells was detected in the lung, reflecting the metastatic tendency of ACC. The incidence of metastasis was measured by the number of pulmonary metastatic clones (Figure 5A and B). Consistent with our in vitro studies, ACC-2Ar showed much enhanced metastatic ability in the model and displayed a much shorter life span (Figure 5C and D). Most importantly, pretreatment with gefitinib suppressed the formation of metastatic lesions in mice (Figure 5A and B) and significantly lengthened the life span of mice intravenously injected with ACC-2Ar cells by almost 5 days (Figure 5C and D). All above data revealed the enhanced metastatic abilities of the transformed cells that could be blocked by the EGFR inhibitor gefitinib, suggesting the potential preventive effects against ACC metastasis by targeting EGFR pathway. 10.1371/journal.pone.0051549.g005Figure 5 Prevention of ACCAr and EGF-induced metastasis in vivo by gefitinib. For in vivo metastasis analyses, the pulmonary metastatic models of mice were established by injected with ACC cells with indicated treatment by tail vein. The ACC-2 cells were pretreated with EGF (10 ng/ml) for 48 h, and gefitinib (1 µM) for 1 h, as well as ACC-2Ar were used in the assays. ACC-2 parental cells were also injected as Control. A, Representative hematoxylin and eosin-stained histological sections of lungs from the mice injected with ACC cells. B, Quantitative analysis of pulmonary metastasis from mice as described above. Lesions of 0.1 mm or higher were counted from 20 slides per treatment (Mean ± SE, n = 5 for each group, *p<0.05 versus ACC-2). C, Survival curve of the mice with indicated treatments. The survival analysis were performed by OriginPro (n = 5 for each group, *p<0.05 versus ACC-2). D, Survival times of the mice as indicated treatments were expressed as Mean and SD. Slug-Mediated EMT-Like Transformation is Required for Anchorage-Independent Growth, Migration, and Invasion in ACCAr Cells As EMT-like transformation was accompanied by the formation of anoikis resistance in both ACCAr and EGF-stimulated ACC cells, it was reasonable for us to investigate whether EMT was required for the acquisition of anoikis resistance in these cells. RNAi-mediated knockdown of Slug reversed EMT in these variant cells, manifesting as the morphological reversal of ACC-2Ar to its cobblestone shape, compared with the NC group (Figure 6A and B). As the major epithelial marker, E-cadherin was observed to relocalize at the adherens junctions of the cells and restored protein expression levels (Figure 6A and B). Meanwhile, the expression of vimentin was significantly down-regulated after ACCAr and EGF-treated cells were infected with Slug siRNA (Figure 6A). 10.1371/journal.pone.0051549.g006Figure 6 Requirement of Slug-mediated EMT in acquisition of anoikis resistance and migration/invasion. ACC-2Ar and ACC-2 were transfected with Slug siRNA and NC siRNA. The ACC-2-transfected cells were then stimulated with EGF (10 ng/ml). A, The protein expression levels of Slug, E-cadherin, and vimentin were determined using Western blot analysis. B, The morphology and expression of E-cadherin were analyzed by immunofluorescence. C, Apoptosis in suspension was measured using Cell Death Detection ELISAPLUS (Mean ± SE, **p<0.01 versus ACC-2Ar with NC siRNA treatment). D, Anchorage-independent growth was analyzed using soft agar assay. Quantitative analyses were performed by counting the visible clones using ImageJ. The results were expressed as relative ratio to ACC-2 with pEGFP transfection (Mean ± SE, **p<0.05 versus ACC-2 with NC siRNA transfection, ##p<0.05 versus ACC-2Ar with NC siRNA transfection). E, ACC-2 cells were transfected with NC siRNA and Slug siRNA, respectively, and then cultured in suspension. The EGFP protein was carried by the lentivirus as marker. The number of EGFP-positive cells was counted by flow cytometry and represented as relative ratio to ACC-2 cells cultured in adhesion (Mean ± SE, *p<0.05 versus ACC-2 in adhesion). F, Migration (top panel) and invasion (low panel) were determined using transwell Boyden systems coated without and with Matrigel, respectively. The apoptosis induced by detachment in variants and EGF-treated cells was analyzed using Cell Death Detection ELISAPLUS. As shown in Figure 6C, apoptosis evasion in ACCAr and EGF-treated cells was significantly blocked by Slug siRNA. Furthermore, the survivability of these cells in anchorage-independent conditions was measured using soft agar assay. The results indicated that depletion of Slug markedly suppressed the anchorage-independent growth of both ACCAr and EGF-treated cells (Figure 6D). In addition, to analyze the role of EMT in the formation process of anoikis resistance, we cultured ACC-2 cells infected with Slug or negative control (NC) siRNA (labeled with GFP as biomarker), as previously described with rounds of suspension and adhesion. The results revealed that GFP-positive cells under fluorescence microscope diminished with the culture rounds, indicating that these cells, which were unable to undergo EMT, were undergoing apoptosis in suspension (Figure 6E). Finally, the aggressive characteristics of ACCAr or EGF-treated cells were analyzed using migration and invasion assays performed in a transwell system. The results showed that knockdown of Slug significantly suppressed migration or invasion of these cells, confirming that EMT plays an essential role in the metastasis of ACC (Figure 6F). Taken together, all above results suggested that Slug-mediated EMT-like transformation was essential for the anoikis resistance of the variants, as well as enhanced migration and invasion. Overexpression of Twist or Slug is Enough to Induce EMT and Acquisition of Anoikis Resistance Having confirmed that Slug-mediated EMT is essential to anoikis resistance, migration, and invasion in ACC cells, we therefore investigated whether EMT induced by overexpression of Slug or Twist could generate anoikis-resistant abilities in ACC cells. Thus, we constructed and transfected overexpression plasmids of Twist and Slug into ACC-2 cells. After selecting the stable clones, we observed dramatic morphological changes compared with the controls (pEGFP) that were similar to the transformation in ACCAr cells (Figure 7A). Moreover, we detected up-regulation of vimentin and down-regulation of E-cadherin, indicating that EMT can be induced by the overexpression of Twist or Slug (Figure 7B). We then assessed the anoikis-resistant abilities in suspension and migration/invasion capabilities in vitro of these transformed cells. The results demonstrated that these clones were more fragile to detachment-induced apoptosis compared with the ACCAr cells but much blunter compared with parental ACC cells under such conditions (Figure 7C). Similar results were also found for anchorage-independent growth in soft agar assay (Figure 7D). Migration and invasion assays performed with a transwell Boyden system showed increased cell numbers across the semipermeable membrane compared with the parental ACC cells (Figure 7E). These results indicated that forced EMT in ACC cells by overexpression of EMT-related transcription factors contributed to the acquisition of anoikis resistance and migration/invasion although that its effects on anchorage-independent growth declined compared with ACCAr cells. 10.1371/journal.pone.0051549.g007Figure 7 Prevention of apoptosis in ACC cells in suspension by EMT induction. The ACC-2 cells were transfected with pEGFP, pEGFP-Twist, and pEGFP-Slug. A, The morphology and location of E-cadherin were analyzed by immunofluorescence. B, The protein expression levels of Slug, Twist, E-cadherin, and vimentin were determined using Western blot analysis. C, Apoptosis in suspension was measured using Cell Death Detection ELISAPLUS (Mean ± SE, *p<0.05 versus ACC-2 with pEGFP transfection, #p<0.05 versus pEGFP-Twist group). D, Anchorage-independent growth was analyzed using soft agar assay Quantitative analyses were performed by counting the visible clones using ImageJ. The results were expressed as relative ratio to ACC-2 with pEGFP transfection (Mean ± SE, **p<0.05 versus ACC-2 with pEGFP transfection, ##p<0.05 versus pEGFP-Twist group). E, Migration and invasion were determined using transwell Boyden systems coated without and with Matrigel, respectively. F, The mRNA expression levels of VEGF, MMP9, and SDF-1α were analyzed using qRT-PCR, and the results were expressed as relative ratio to ACC-2 transfected with pEGFP (*p<0.05 versus ACC-2 pEGFP group, **p<0.01 versus ACC-2 pEGFP group). Expression of EMT-Related Genes in ACC and its Correlation with EGFR Activation To make our present work more clinically significant, we investigated the natural status of EGFR, as well as the expression levels of EMT-related genes in ACC tissues. Representative immunohistochemical results for the selected cases are shown in Figure 8A. EGFR was moderately stained in the cytoplasm of most ACC cells, especially in the solid type, which is considered to be the most aggressive of the three pathological types of ACC. We next identified the expression levels of EMT-related genes in ACC tissues via serial sections. Membranous or cytoplasmic staining of E-cadherin in ACC was much lower than that in normal salivary glands, and even lost in some cases. In addition, Slug was strongly stained in the nucleus of tumor cells in ACC, suggesting that EMT frequently occurs in ACC. Serial section immunohistochemistry revealed that loss of E-cadherin was generally linked with the expression of Slug, and Spearman’s rank test showed that E-cadherin staining and Slug were negative correlated (p = 0.0263, Figure 8B). The significant correlation between EGFR and Slug was confirmed using Spearman’s rank test (p = 0.0481, Figure 8B). MMP9, as a marker of metastasis or invasion, was also analyzed. The results from serial sections showed that intensive staining of MMP9 in the cytoplasm was consistent with the positive staining of EGFR. All these results indicate that EMT, manifesting as loss of E-cadherin and nuclear expression of Slug, is significantly correlated with the positive staining of EGFR, which potentially helps promote metastasis in ACC. 10.1371/journal.pone.0051549.g008Figure 8 Expression levels of EGFR, Slug, E-cadherin, and MMP9 in ACC tissues. A, Representative immunohistochemical staining of EGFR, Slug, E-cadherin, and MMP9 in human adenoid cystic carcinoma and negative gland tissue (NSG). B, Spearman’s correlation were used to determine the relationship between EGFR and Slug (left panel) and that between Slug and E-cadherin (right panel) (p<0.05). Histoscore based on quantification as described in the method and statistics with OriginPro. Discussion In this study, we investigated the role of EMT-like transformation in the acquisition of anoikis resistance in ACC cells and determined the therapeutic effects of targeting the EGFR/PI3K/Akt signaling pathway in the metastasis of human ACC. Metastasis of human ACC is a major cause of mortality [2]. The prognosis for patients with ACC depends mainly on the control of distant metastasis rather than on the success of locoregional cure [25]. Metastasis is a complex multistep process that includes local invasion, intravasation, survival in the circulation, arrest in capillaries, extravasation, and, finally, outgrowth to produce macrometastasis to distant organs [5], [26]. Resistance against anoikis is considered a prerequisite for cancer cells to survive in the circulation [8]. The present study detected an EMT-like transformation in ACCAr cells, in accordance with previous studies showing that most tumor cells in the circulation, usually termed circulating tumor cells (CTCs), lost their epithelial markers and underwent EMT-like transformation [27]. Thus, the method for detection and isolation of CTCs in patients based on the capture of epithelial cell adhesion molecules [28] should be modified according to our study. Generally, EMT is described as a process that endows cancer cells with motility to shed from primary sites and intravasate into the circulation [7]. The present study confirmed that EMT-like transformation also contributed to the survival of tumor cells in the circulation, highlighting the complex and multiple roles of EMT in cancer metastasis. Although several studies have shown that there are more CTCs in the circulation of patients with cancer [29], [30], the incidence of metastasis is quite small. The results from our study suggest that only those transformed cells can survive in the circulation and finally form micrometastases to distant organs. The transcription factors that repress the expression of E-cadherin, such as Twist, Snail, and Zeb1, among others, have been described as central mediators in EMT-like transformation [31], [32]. In our experiments, Slug proved to be a crucial transcription factor in EMT-like transformation. Distinct from other EMT-related transcription factors, Slug is considered to contribute to the function of stem cells via the c-kit signaling pathway or by suppressing p53 transcriptional activities [33]–[35]. A recent study reported that Slug and Sox9 were sufficient to convert differentiated luminal cells into stem cells and contributed to the tumorigenic and metastasis-seeding abilities of human breast cancer cells, suggesting that Slug has a more important role in cancer progression [36]. Moreover, our unpublished data also showed that the expression of CD44, a marker of cancer stem cells, was increased in these anoikis-resistant variants and that overexpression of Slug in ACC cells was sufficient to up-regulate the expression of this molecule. To confirm the essential role of Slug in EMT-like transformation and the acquisition of anoikis resistance in ACC cells, we suppressed Slug expression using RNAi. Down-regulation of Slug significantly increased the apoptosis rate of variant cells in suspension, reduced growth in anchorage-independent conditions, and reversed the EMT-like transformation, indicating that Slug-mediated transformation is required for the acquisition of anoikis resistance in ACC cells. Moreover, by blockade of Slug elevation in parental ACC cells, we succeeded in alleviating the formation of anoikis resistance in parental ACC cells. Moreover, we investigated whether induction of EMT by forced overexpression of Slug or Twist could rescue the cells from death in suspension. Overexpression of Slug or Twist was sufficient to induce EMT-like transformation, enhanced the migration and invasion abilities of ACC cells, and contributed to anoikis resistance in ACC cells, albeit only partially, suggesting that other survival pathways are involved in anoikis resistance in these variant cells. Previous studies have demonstrated in many cases that growth factor signaling pathways mediating the EMT process might also contribute to anoikis resistance [37], [38]. In the present study, we proved that the EGFR/PI3K/Akt signaling pathway acts as the common regulator for EMT-like transformation and the acquisition of anoikis resistance in ACC cells. qPCR and Western blot analysis confirmed the elevation of EGF, and the phosphorylation of EGFR notably increased in ACCAr cells compared with their parental cells, indicating the probable role of EGF/EGFR in variant ACC cells. Thus, we stimulated ACC cells with EGF and found that EGF was sufficient to induce EMT-like transformation and prevent tumor cells from detachment-induced apoptosis. To uncover the underlying mechanism through which EGF induces such processes, we investigated the probable pathways previously reported as the downstreams of EGFR. As numerous studies have shown that the activation of EGFR promotes cancer invasion and metastasis in a PI3K/Akt- and/or MEK/ERK-dependent manner [10], [21], [39], [40], we measured the activation of Akt and ERK in our experiments. We found that phosphorylation of PI3K, p85, and Akt was significantly increased in detachment-selected and EGF-treated cells and that the phosphorylation level of ERK did not significantly change in both conditions. Furthermore, pretreatment with the PI3K-specific inhibitor LY294002 completely reversed the EMT-like transformation induced by EGF or anoikis-resistant variant cells via the down-regulation of Slug and alleviated the survivability of these cells in suspension. According to these findings, we hypothesized that the therapeutic mechanism against EGFR activation is a promising means of preventing metastasis of human ACC. Therefore, we selected gefitinib, a highly selective, reversible inhibitor of the tyrosine kinase domain associated with EGFR that has demonstrated efficacy in patients harboring EGFR overexpression or mutations [41]–[43], for our experiments. Having confirmed the solid effects of this pharmacological inhibitor on the reversal of EMT-like formation and suppression in anoikis via in vitro studies, we therefore established a nude mouse model of pulmonary metastasis to examine its therapeutic effects on the metastasis of ACC cells in vivo. The results showed that pretreatment with gefitinib significantly decreased the pulmonary metastatic formation abilities of the anoikis-resistant variant cells and promoted survival of the mice. All these findings strongly suggest that targeting EGFR/PI3K/Akt holds promising therapeutic efficacy in human ACC. To make the above-mentioned findings more clinically significant and therapeutically meaningful, we then investigated the natural expression status of EGFR signaling and the EMT inducer Slug in ACC specimens. Previous studies have reported that EMT-related genes, such as Snail, Twist, and Sip1, are frequently observed in cases of human ACC [44], [45] and proved to be associated with poor outcomes and high metastasis rates in patients [44], [46], [47]. Our results showed that EGFR overexpression is correlated with the expression of Slug, indicating that aberrant activation of EGFR signaling induces EMT and further promotes tumor metastasis. In addition, we investigated E-cadherin and vimentin to validate whether EMT occurred in the patients with ACC. Our immunohistochemical staining results revealed that loss of E-cadherin and acquisition of vimentin frequently occurred in ACC specimens, especially in cells adjacent to the stroma, suggesting that the interaction between cancer cells and mesenchymal cells may contribute to these processes. In conclusion, this study has confirmed that induction of EMT-like transformation is required for the acquisition of anoikis resistance in ACC cells. Our research has also revealed that activation of the EGFR/PI3K/Akt signaling pathway is responsible for both EMT-like transformation and anoikis resistance in ACC, pointing to the potential therapeutic value of targeting such pathways in preventing distant metastasis of human ACC. Supporting Information Figure S1 Anoikis resistant variants of ACC cells showed alleviated proliferation compared to parental cells. MTT assays were performed to measure the proliferation rate of ACC cells. (TIF) Click here for additional data file. Figure S2 The mRNA expression of epithelial and mesenchymal markers in anoikis resistant variants of ACC cells. (TIF) Click here for additional data file. Figure S3 Confirmation of the efficacy of RNA interference against Slug. (TIF) Click here for additional data file. Protocol S1 (DOC) Click here for additional data file. ==== Refs References 1 Ellington CL, Goodman M, Kono SA, Grist W, Wadsworth T, et al.. 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PLoS One. 2012 Dec 14; 7(12):e51549
==== Front Genet Vaccines TherGenet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central 1479-0556-10-42271612510.1186/1479-0556-10-4ResearchStructure based sequence analysis & epitope prediction of gp41 HIV1 envelope glycoprotein isolated in Pakistan Jafri Syyada Samra [email protected] Saliha [email protected] Syed Babar [email protected] Masaud [email protected] University of the Punjab, Lahore, Pakistan2 Government College University, Faisalabad, Pakistan3 International Islamic University Islamabad, Islamabad, Pakistan2012 20 6 2012 10 4 4 15 5 2012 12 6 2012 Copyright ©2012 Jafri et al.; licensee BioMed Central Ltd.2012Jafri 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 Gp41 is an envelope glycoprotein of human immune deficiency virus (HIV). HIV viral glycoprotein gp41, present in complex with gp120, assists the viral entry into host cell. Over eighty thousands individuals are HIV infected in Pakistan which makes about 0.2% of 38.6 million infected patients worldwide. Hence, HIV gp41 protein sequences isolated in Pakistan were analyzed for the CD4 and CD8 T cells binding epitopes. Results Immunoinformatics tools were applied for the study of variant region of HIV gp41envelope protein. The protein nature was analyzed using freely accessible computational software. About 90 gp41 sequences of Pakistani origin were aligned and variable and conserved regions were found. Four segments were found to be conserved in gp41 viral protein. A method was developed, involving the secondary structure, surface accessibility, hydrophobicity, antigenicity and molecular docking for the prediction and location of epitopes in the viral glycoprotein. Some highly conserved CD4 and CD8 binding epitopes were also found using multiple parameters. The predicted continuous epitopes mostly fall in the conserved region of 1–12; 14–22 and 25–46 and can be used as effective vaccine candidates. Conclusions The study revealed potential HIV subtype a derived cytotoxic T cell (CTL) epitopes from viral proteome of Pakistani origin. The conserved epitopes are very useful for the diagnosis of the HIV 1 subtype a. This study will also help scientists to promote research for vaccine development against HIV 1 subtype a, isolated in Pakistan. Human immunodeficiency virusPakistangp41EpitopesBioinformatics ==== Body Introduction An envelope virus HIV-1 expresses a surface glycoprotein mediating the attachment and fusion of virus with cellular membranes. HIV carries nearly 70 spikes [1] and is transmitted through mucosal secretions during sexual intercourse. CD4+T cells present in lymphoid organs and blood is the main site of infection. During mid-1990s, first X-Ray crystal structure of GP-41 was solved. GP-41 mediates fusion of target cells to HIV-1. Understanding of its structure provides the understanding of virus entry into the host and describes the mode of action of compounds that block this process. As the infection cycle is initiated by the fusion of viral proteins with cell membranes, followed by the release of viral genome and proteins into the host. HIV-1 follows a multi-step process to enter into the host. This multi-step entry process provides active targets for the development of new therapeutic agents to block this entry. Designing of specific agents which can create hindrance in the entry of viral protein at each step are of considerable importance and substantial progress has been made in understanding the entry of HIV in host cell. GP-41 interacts with GP-120 non-covalently forming an oligomeric structure. Crystallographic and physical data suggests trimeric GP-41 – Gp-120)3 form of this oligomeric structure. It is postulated that GP-41 facilitates the fusion of viral cell membrane with the target’s membrane and undergoes major conformational rearrangements in a “spring-loaded mechanism” elaborated for influenza hemagglutinin [2]. HIV-1 is thought to be the major cause of infection in Pakistan. A core is present in the “sprung” conformation of GP-41[3,4] which is formed by an extended triple-stranded α-helical coiled coil. Outside of the coil is packed in reverse direction by carboxy-terminal α-helix bringing carboxy and amino terminals close to each other at long rod end. It is found that GP-41 is in stable state in the form of sprung conformation. Vaccine designing is a complicated process in envelop proteins due to the presence of several forms with distinct conformations. Mature oligomer may not have most of the epitopes on unprocessed oligomeric or monomeric envelop molecule. Materials and methods Sequences searching NCBI protein database was used for the retrieval of gp 41, HIV1 subtype a proteins sequences of. 194 aa sequences were selected out of 200 retrieved from database in FASTA format. Alignment and conservancy Multiple alignment of sequences and conservancy was found using offline ClastralW tool [5] useful for large no of sequences. T-cell epitopes of HIV gp41 protein prediction Selected sequences were used for T-Cell epitope mapping using Epijen online software. A*0201, A*0301, A*1101 and B*07 were four HLA alleles used for predicting epitopes which have been reported to be recognize in more than 90% of the world population, regardless of ethnicity. Secondary structure prediction SOPMA library [6], which is freely available server, was used for secondary structure prediction (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). Tertiary structure prediction through homology modeling Modeller v9.10 [7] was used for predicting tertiary structure and Chimera software was used for displaying different patterns like secondary structure, and physiological properties of protein sequences. PDB Structure 1 F23 was used as a template for homology modeling. Evaluation of homology model To check the stereochemical quality of the HIV gp41 model, The Procheck suite of programs was used to construct Ramachandaran plot [8] for model validation. Phylogenetic analysis The evolutionary history was inferred using the Neighbor-Joining method [9]. Bootstrap method [10] was used to check the reliability of results. The evolutionary distances were computed using the Poisson correction method [11] and are in the units of the number of amino acid substitutions per site. The analysis involved 9 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA5 [12]. Results Sequence alignment and conservancy Phylogenetic analysis shows that HIV gp41of Pakistani origin is sharing common ancestry with Russia, China and Uganda while has distant relationship with India and its other neighboring countries (Figure 1a). Three amino acids in the gp41 sequence i.e. Threonine, Alanine and Aspartate at positions 13, 22 and 32 respectively are showing most frequent mutations (Figure 1b). Figure 1 HIV gp41 amino acid sequence: a, Phylogenetic Analysis of HIV gp41 Sequences, b, Position 13, 22 and 32 shown in Blue are most frequently mutated gp41amino acids. T-cell epitopes of HIV gp41 protein prediction T-cell epitopes were predicted using Epigen online software on the basis of IC50 value. HLA 0201 showed minimum IC50 value, ensuring maximum binding affinity among all residues (Table 1). Epitopic residues with lowest IC50 predicted values are shown in Figure 2a. Table 1 Predicted T cell epitopes T Cell Epitope HLA 0201 Starting Position Peptide –logC50 (M) IC50 Value (nM) 14 LLHSLIEEA 9.075 0.84 7 EINNYTTLL 8.611 2.45 HLA 0301 30 EQDLLELDK 7.296 50.58 18 LIEEAQNQQ 6.854 139.96 A* 1101 30 EQDLLELDK 7.497 31.84 32 DLLELDKWA 7.018 95.94 B* 07 34 LELDKWASL 6.934 116.41 28 KNEQDLLEL 6.254 557.19 Figure 2 a, 3D Model showing Epitopic region of gp41with maximum affinity, b, Tertiary structure of gp41 contains 2 helices. Molecular characterization of gp41 Various servers were used to find Glycosylation sites in envelope protein. No such sites were found in gp41 sequence [13-15]. N-glycosylation sites are searched as Asn-X-Ser or Asn-X-Thr sequences, where X is any amino acid residue. Secondary and tertiary structure prediction Secondary structure contains 93.48% helices and 6.52% turns but contains no extended sheets as predicted by SOPMA. Tertiary structure of gp41 was constructed using Moeller v 9.10. Chimera was used for model visualization. It was observed that its structure contains 2 helices covering most of the region, and coils but has no Beta pleated sheet (Figure 2b). Using Procheck server Ramachandaran plot was constructed to verify the validity of 3D structure. 93.2% residues were lying in the most favorable region while 6.8% residues were present in additionally allowed region. No residue was observed in generously allowed or disallowed region. Phylogenetic analysis The evolutionary history was inferred using the Neighbor-Joining method [9]. The optimal tree with the sum of branch length = 7.60693736 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (200 replicates) are shown next to the branches [10]. The evolutionary distances were computed using the Poisson correction method [11] and are in the units of the number of amino acid substitutions per site. The analysis involved 9 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 45 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 [12]. Discussion In this study 194 sequences were randomly selected from 200 total no of sequences available at the NCBI database. Mutations were observed in all the aligned sequences and it was found that these mutations are more frequent at 3 positions. These amino acid positions are 13, 22 and 32. At position 13, instead of Threonine (T), Serine (S) and Asparagine (N) were observed in most of the cases. Serine (S) was observed instead of Alanine (A) at position 22 in many sequences while instead of Aspartic Acid (D), Glutamic acid (E) was observed at position 32 in many sequences. Mutations were also found at some other positions but these mutations were not frequent and occurred seldom when all the sequences were compared. 1–12 and 33–46 regions were found conserved in all the sequences except two sequences. While two regions, 16–21 and 23–31, were absolutely conserved in all the sequences. Amino acid composition of the sequence was checked and it was observed that Tryptophan is having maximum percentage i.e. 204.23% while serine was present in in least amount i. e. 105.09%. Tertiary structure of HIV gp41 was predicted on the basis of homology modeling using MODELLER software. PDB structure 1 F23 was used as a template for homology modeling. HIV gp41 was molecularly characterized using various online servers and it was observed that it has no Glycosylation site or Myrisylation site while it has 0.75% Protein Kinase A sites and 0.58% Casein Kinase 2 Sites. T cell epitopes, A*0201, A*0301, A*1101 and B*07, were predicted using Epijen online software. These epitopes were HLA alleles and have been reported in more than 90% of the world population, regardless of ethnicity. IC50 values were calculated and IC50 value was found least for HLA 0201showing their higher affinity as compared to other alleles. While in rest of the epitopes IC50 value was quite high showing very low affinity. Evolutionary relationship was checked among HIV gp41 sequence of various countries. Pakistan and India share common ancestor but this result is not reliable. Very reliable results were obtained that Uganda shares a common ancestor with China, Russia, Africa, Saudi Arab and Afghanistan and also that Africa shares a common ancestry with Saudi Arab and Afghanistan. No reliable results were obtained about the ancestry of HIV gp41 sequence of India, Pakistan and Nigeria. Conclusion The study revealed potential HIV subtype a derived cytotoxic T cell (CTL) epitopes from viral proteome of Pakistani origin. The conserved epitopes are highly useful for the diagnosis of the HIV 1 subtype a. This study will also help scientists to promote research for vaccine development against HIV 1 subtype a to save Pakistani population from potential threats of HIV. Competing interest The authors declare that they have no competing interest. Authors’ contribution SSJ and MS designed the study. MS and SBJ performed the immunoinformatics analysis and rafted the manuscript. SK critically reviewed the manuscript. All authors have read and approved the final manuscript. Acknowledgment We are thankful to ISCB-RSG-Pakistan for giving us opportunity to work with expert bioinfomraticians in Virtual Internship Program 2011. ==== Refs Dennis R Burton: A vaccine for HIV type 1: The antibody perspective Proc Natl Acad Sci USA 1997 94 10018 10023 10.1073/pnas.94.19.10018 9294155 Bullough PA Hughson FM Skehel JJ Wiley DC Structure of influenza haemagglutinin at the pH of membrane fusion Nature (London) 1994 371 37 44 10.1038/371037a0 8072525 Chan DC Fass D Berger JM Kim PS Core structure of gp41 from the HIV envelope glycoprotein Cell 1997 89 263 273 10.1016/S0092-8674(00)80205-6 9108481 Weissenhorn W Dessen A Harrison SC Skehel JJ Wiley DC Atomic structure of the ectodomain from HIV-1 gp41 Nature (London) 1997 387 426 430 10.1038/387426a0 9163431 Chenna R Sugawara H Koike T Lopez R Gibson TJ Higgins DG Thompson JD Multiple sequence alignment with the Clustal series of programs Nucleic Acids Res 2003 31 13 3497 3500 http://www.rfcgr.mrc.ac.uk/Registered/Webapp/emboss-w2h/ 10.1093/nar/gkg500 12824352 Geourjon C Deléage G SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments Comput Appl Biosci 1995 11 6 681 684 8808585 Fiser A Sali A Modeller: generation and refinement of homology-based protein structure models Meth Enzymol 2003 374 461 491 14696385 Ramachandran GN Ramakrishnan C Sasisekharan V Stereochemistry of polypeptide chain configurations J Mol Biol 1963 7 95 99 10.1016/S0022-2836(63)80023-6 13990617 Saitou N Nei M The neighbor-joining method: A new method for reconstructing phylogenetic trees Mol Biol Evol 1987 4 406 425 3447015 Felsenstein J Confidence limits on phylogenies: An approach using the bootstrap Evolution 1985 39 783 791 10.2307/2408678 Zuckerkandl E Pauling L Bryson V, Vogel HJ Evolutionary divergence and convergence in proteins Evolving Genes and Proteins 1965 Academic, New York 97 166 Tamura K Dudley J Nei M Kumar S MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 Mol Biol Evol 2003 24 1596 1599 17488738 Bairoch A Bucher P Hofmann K The PROSITE database, its status in 1997 Nuc Aci Res 1997 25 217 223 10.1093/nar/25.1.217 Hubbard SC Ivatt RJ Synthesis and processing of asparagine-linked oligosaccharides Annu Rev Biochem 1981 50 555 583 10.1146/annurev.bi.50.070181.003011 7023366 Bause E Structural requirements of N-glycosylation of proteins. 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Genet Vaccines Ther. 2012 Jun 20; 10:4
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23285066PONE-D-12-1920610.1371/journal.pone.0052491Research ArticleBiologyBioethicsAnimal StudiesDevelopmental BiologyMorphogenesisRegenerationOrganism DevelopmentRegenerationStem CellsMesenchymal Stem CellsNeural Stem CellsModel OrganismsAnimal ModelsRatMolecular Cell BiologyCellular TypesStem CellsMesenchymal Stem CellsNeural Stem CellsNeuroscienceDevelopmental NeuroscienceNeural Stem CellsNeurobiology of Disease and RegenerationMedicineNeurologyParkinson DiseaseEctopic Pregnancy-Derived Human Trophoblastic Stem Cells Regenerate Dopaminergic Nigrostriatal Pathway to Treat Parkinsonian Rats Human Trophoblast Stem Cells Treat Parkinson RatsLee Tony Tung-Yin 1 Tsai Cheng-Fang 2 Hsieh Tsung-Hsun 3 Chen Jia-Jin Jason 3 Wang Yu-Chih 1 Kao Mi-Chun 1 Wu Ruey-Meei 4 Singh Sher 5 Tsai Eing-Mei 1 2 * Lee Jau-Nan 1 * 1 Department of Obstetrics and Gynecology and Center of Excellence for Environmental Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 2 Graduate Institute of Medicine, Kaohsiung Medical University College of Medicine, Kaohsiung, Taiwan 3 Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan 4 Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan 5 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Hsieh Patrick Ching-Ho Editor Institute of Clinical Medicine, National Cheng Kung University, Taiwan * E-mail: [email protected] (JNL) (ET); [email protected] (EMT) (JL)Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JNL EMT. Performed the experiments: TTYL THH JJJC RMW CFT YCW MCK. Analyzed the data: JNL EMT THH SS. Contributed reagents/materials/analysis tools: JNL EMT THH SS. Wrote the paper: JNL EMT TTYL. 2012 21 12 2012 7 12 e524914 7 2012 14 11 2012 © 2012 Lee et al2012Lee 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 Stem cell therapy is a potential strategy to treat patients with Parkinson’s disease (PD); however, several practical limitations remain. As such, finding the appropriate stem cell remains the primary issue in regenerative medicine today. We isolated a pre-placental pluripotent stem cell from the chorionic villi of women with early tubal ectopic pregnancies. Our objectives in this study were (i) to identify the characteristics of hTS cells as a potential cell source for therapy; and (ii) to test if hTS cells can be used as a potential therapeutic strategy for PD. Methods and Findings hTS cells expressed gene markers of both the trophectoderm (TE) and the inner cell mass (ICM). hTS cells exhibited genetic and biological characteristics similar to that of hES cells, yet genetically distinct from placenta-derived mesenchymal stem cells. All-trans retinoic acid (RA) efficiently induced hTS cells into trophoblast neural stem cells (tNSCs) in 1-day. Overexpression of transcription factor Nanog was possibly achieved through a RA-induced non-genomic c-Src/Stat3/Nanog signaling pathway mediated by the subcellular c-Src mRNA localization for the maintenance of pluripotency in tNSCs. tNSC transplantation into the lesioned striatum of acute and chronic PD rats not only improved behavioral deficits but also regenerated dopaminergic neurons in the nigrostriatal pathway, evidenced by immunofluorescent and immunohistological analyses at 18-weeks. Furthermore, tNSCs showed immunological advantages for the application in regenerative medicine. Conclusions We successfully isolated and characterized the unique ectopic pregnancy-derived hTS cells. hTS cells are pluripotent stem cells that can be efficiently induced to tNSCs with positive results in PD rat models. Our data suggest that the hTS cell is a dynamic stem cell platform that is potentially suitable for use in disease models, drug discovery, and cell therapy such as PD. This work was partly supported by grants of KMUH99-9I04 and NSC 99-2628-B-037-009-MY3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Parkinson’s disease (PD) is caused by the dysfunction or the loss of dopaminergic neurons in the nigrostriatal pathway of the midbrain, making it the second most common neurodegenerative disorder in humans. Current pharmacological drugs only provide symptomatic relief but do not retard the disease progression. Consequently, cell replacement by using human fetal mesencephalic tissue [1], [2] or embryonic stem (ES) cell-derived dopaminergic neurons [3], [4], [5] remains an important therapeutic strategy; however, several practical limitations exist, such as shortage of cell sources, variations in outcomes, adverse effects, and socio-ethical issues [6]. Placenta-derived mesenchymal stem (PDMS) cells have also shown promise; but outcomes remain uncertain [7]. In the search for a suitable cell source, we isolated and identified the ectopic pregnancy-derived human trophoblast stem (hTS) cells from the early chorionic villi of a tubal ectopic pregnancy. The characteristics of hTS cells suggest it as a potential alternative source of pluripotent stem cells for the treatment of PD and of other neurodegenerative diseases. We investigated the microenvironmental factors that affect pluripotency and proliferation of hTS cells. In women, fertilization occurs in the fallopian tubes, where the distinction between the inner cell mass (ICM) and the trophectoderm (TE) [8] and the switch from totipotency to pluripotency takes place during embryogenesis [9]. When an ectopic pregnancy occurs in the fallopian tube, the cellular processes may continue until clinical intervention. Before intervention, however, the microenvironmental factors might affect cell differentiation. For example, the pleiotropic cytokine LIF expresses significantly higher levels in the fallopian tubes than that in the endometrium [10], descending from the ampulla to the isthmus [11]. LIF levels may elevate up to 2- to 4-fold in ectopic pregnancies [11]. Functionally, LIF activates transcription factors, Oct4 and Nanog, for the maintenance of pluripotency in ES cells [12], [13]. On withdrawal of LIF, cell proliferation continues but the caudal-related homeobox transcription factor Cdx2 is activated, driving ES cells differentiation into trophectoderm fate [14]. Nevertheless, the significance of LIF on trophoblastic development is largely unknown. In this study, we clarified the inter-relationships of LIF, Oct4, Nanog and Cdx2 on pluripotency and proliferation of hTS cells. Next, we investigated if retinoic acid (RA) can effectively induce hTS cell differentiation to a neural cell. RA is a well-recognized signaling molecule, involved in the development, regeneration, and maintenance of the nervous system [15]. RA promotes the generation of DA neurons and the enhancement of axon outgrowths of neurites in hES cells [16]. Conventionally, RA may function as a paracrine signal or an autocrine signal, entering the nucleus via retinaldehyde dehydrogenases (RALDHs) to bind RA receptors (RARs) or retinoid X receptors (RXRs) to activate the retinoic acid-response element (RARE) for gene transcription. Notably, the striatum expresses the highest endogenous levels of RA in the brain [17]. In our study, we found that RA efficiently induced differentiation of hTS cells into dopaminergic trophoblastic neural stem cells (tNSCs). We then investigated whether tNSCs were effective in a PD rat model. We found that intracranial transplantation of tNSCs substantially regenerated the dopaminergic nigrostriatal pathway and functionally alleviated parkinsonian motor deficits in both acute and chronic PD rats. Together, these results suggest that hTS cells are a viable pluripotent cell source that circumvents socio-ethical concerns for the treatment of PD. Materials and Methods hTS Cell Culture and Differentiation Tiny villous tissues (Fig. S1A) were well-minced in serum-free α-MEM (Sigma-Aldrich, St. Louis, MO) followed by trypsinization with 0.025% trypsin/EDTA (Sigma-Aldrich) (15 min) that was halted by adding α-MEM containing 10% FBS. Adherent cells were cultured in α-MEM, 20% FBS, and 1% penicillin-streptomycin at 37°C in 5% CO2. Particularly, the doubling time of cell growth was 7.67 hr. Cell antibodies, primers, and differentiations used in this study are shown in Supporting Information (Table S1, Table S2). Plasmids Plasmid construct (F1B-GFP) used in this study is described in the Supporting Information (Text S1). hTS cells were co-transfected with a DNA mixture of F1B-GFP to yielded over 95% of transfection rate. RT-PCR, Western Blots, Southern Blots, Immunoprecipitation, IP Assay, and Flow Cytometry Methods performed in this work are described previously [18] and details are available in the Supporting Information (Text S1). Chromatin Immunoprecipitation (ChIP) Cells were serum-deprived for overnight and treated with RA (10 µM) for 30 min. The mixtures of protein G beads (Roche) and lysate were incubated with rocking at 4°C for 2 hr. After removing the beads by centrifugation, the lysate was added with IgG (Santa Cruz) and primary anti-Stat3 antibody (1∶1,000, Santa Cruz) in accompany with new protein G beads. The mixtures were incubated with rocking overnight at 4°C. The immunoprecipitation complex was washed by RIPA lysis buffer and subjected for polymerase chain reaction (PCR) using Nanog promoter primer: forward, GACAGCCCCCACTTAACAAA and reverse, GCTTTTTCCCTCTGGCTCTT). Microarray Data Analysis Cells, with or without treatment of RA (10 µM), were cultured overnight and total RNAs were extracted using TRIzol reagent (Invitrogen) and subjected for Affymetrix microarray by using Affymetrix Human Genome U133 plus 2.0 GeneChip according to the manufacturer’s guidance (Santa Clara, CA, http://www.affymetrix.com) performed at Genomic Medicine Center of National Taiwan University College of Medicine, Taipei, Taiwan). Data analysis was performed by using softwares of MetaCore, GeneSpring GX (version 7.3), and IPA Ingenuity System (version 8.8). Immunofluorescence For immunocytochemistry, after fixation with 4% paraformaldehyde in PBS (room temperature; 5 min) and washes, cells were permeabilized with 2% FBS/0.4% Triton X-100 in PBS (15 min), followed by 5% FBS blocking solution (2 hr) and rinsed three times. After incubation with specific primary antibody in PBS at 4°C overnight, appropriate FITC or PE or Texas Red or DyLight 488 or DyLight 594 conjugated secondary antibody was added for 1 hr at room temperature. By DAPI staining, nucleus (5 min) cells were subjected for microscopy. For immunohistochemistry, brain sections were fixed in 4% paraformaldehyde (2 hr) and dehydrated in 30% sucrose in 0.02 M PBS (2 days) followed by processes in a freezing microtome (Leica). Coronal sections (30 µm) were treated with 0.3% H2O2 and Ready-To–Use blocking solution (IHC-101b, Bethyl Laboratories, Montgomery, TX) for 15 min followed by incubation with primary anti-TH antibody (1∶1,000, Temecula, CA) at 4°C. Anti-rabbit second antibody (IHC-101d, Bethyl Laboratories) was used with DAB kit (Bethyl) for microscopic analysis. Immunofluorescence Tissue Analysis Coronal brain sections were immunostained with primary antibodies: TH (1∶250; H-196, sc-14007; Santa Cruz), NeuN (1∶100; MAB377; Millipore, Temecula, CA), and GFAP (1∶200; BSB 5566, Declere; Bio SB, Santa Barbara, CA) in accompany with FITC-anti-TH, Cy-3-anti-NeuN, or Cy-3-anti-GFAP. Data were analyzed by TissueGnostics (TissueGnostics GmbH, Vienna, Austria). Evaluation was made by comparison between the lesioned (or the cell-implanted) side and the normal side. Cells with bizarre size or intensity of GFAP outside the normal (e.g., artifact or unusually heavy stain area or intensity in cell overlapping with Wilson’s pencils) were excluded. Animal Studies and Behavioral Assessments Animal preparations were licensed and well-described previously [19], (Text S1). Before experiment, the apomorphine-induced rotation test after 6-OHDA injection was pre-evaluated weekly in PD rats to achieve a stable hemiparkinsonian status to avoid bias. In acute PD rats (body weight, 225–250 g), cells (3×106 cells/5 µl/5 min) were transplanted into the lesioned striatum at site (AP +1.0 mm, Lat +2.7 mm, Dep 6.4 mm) under anesthesia. The cell viability remained stable between 96% and 98% during the implantation procedures. In chronic PD rats, the body weight of rats was controlled between 560±65 g at pre-test and 548±46 g at post-test. Cells (1.5×106) were grafted at the same site. To obtain the brain sections, rats were anesthetized by sodium pentobarbitone (60 mg/kg i.p., Apoteksbolaget, Sweden) and trans-cardially perfused with saline (50 ml, 0.9% w/v) followed by ice-cold paraformaldehyde (200 ml, 10% w/v in 0.02 M PBS) were performed at 18- and 12-weeks in the acute and chronic PD rats, respectively. Brain sections were subjected for immunocytochemistry, and immunofluorescence tissue analysis as indicated. All behavioral assessments were performed as described previously [19], (Text S1). Ethics Statement Patient consent and sourcing of human tissue were approved by the Institutional Review Board on Human Subjects Research and Ethics Committees of Kaohsiung Medical University Hospital (KMUH-IRB-950140). The animal experiments were conducted according to the guideline and Approval of Animal Use Protocol of National Cheng Kung University (IACUC Approval No. 98010). The NCKU IACUC guidelines aim to reduce animal suffering by use of anesthesia, use of analgesics, and provide nutritional and fluid support. Statistical Analysis Data obtained from RT-PCR and flow cytometry were calculated by Student’s t-test. For behavioral assessments, data were expressed as mean ± SEM. In acute PD rats, data of apomorphine-induced rotation tests were analyzed by using repeated measure analysis of variance (ANOVA) tests (SPSS Release 12.0 software) and applied least significant difference test (LSD) post hoc comparisons after repeated measure ANOVA tests between two groups. In chronic PD rats, paired Student’s t-test was used to compare the two groups. p-value <0.05 was considered statistically significant. Results Ectopic Pregnancy Gives Rise to hTS Cells A unique population of adherent cells was isolated from the early chorionic villi of embryos (gestational age: 5–7 weeks) in tubal ectopic pregnancies (Text S1). Neither feeder cells nor multiple inducers were used in the cell culture and the differentiation of hTS cells, as distinct from hES cells [3], [4], [5], circumventing potential side-effects and contaminations. After growth factor depletion, the main differentiated lineage of trophoblasts should be mature syncytial multinuclear trophoblasts. However, we only isolated mononuclear cytotrophoblast cells that adhered to the dish, as mature cells were removed in the isolation process. The population doubling time of hTS cells was 8 hr, which is distinct from the 48 hr population doubling time of the first trimester placenta-derived cytotrophoblast [20]. These cells expressed markers for hES cell-related specific stage embryonic antigen (SSEA)-1, −3 and −4 with immunolocalization identical to cytotrophoblasts in the villus (Fig. 1A). By immunocytochemistry, the cells expressed pluripotency transcription factors Oct4, Sox2, Nanog and Cdx2 in the nuclei at passage 9 and 17 (Fig. 1B, Fig. S1B). 10.1371/journal.pone.0052491.g001Figure 1 Characteristics of hTS Cells. (A) Histology of an ectopic pregnancy at fallopian tube by HE staining (left large panel). Bla: blastocyst, V: chorionic villi, FT: fallopian tube. Immunocytochemistry of SSEAs (red, arrow) showed SSEA-1 in the cytoplasm (left upper); SSEA-3 in the nucleus (middle upper); and SSEA-4 in both the cytoplasm and the membrane (right upper), corresponding to cytotrophoblasts in the ectopic chorionic villus (lower panel). (B) Immunofluorescent Oct4 and Cdx2 at passage 9 (P-9), Oct4 and Nanog expressed in the nuclei at passage 9 (P-9) and 17 (P-17) (left column). Scale bar: 20 µm. Expression of Cdx2, Oct4, Sox2, and Nanog in the nuclei in the amplified cells (middle and right columns). Scale bar: 10 µm. (C) Expression of specific genes of both trophectoderm (TE) and ICM by RT-PCR. (D) hTS cells expressed genes of three germ layers before induction (left column) and after appropriate induction (right column). (E) For cellular homogeneity, Oct4-positive cells in P-9 and P-17 with 100% and 97.3%, respectively, by TissueGnostics analysis. n = total cell number counted. (F) Flow cytometric analysis at passage 15, cells expressed positive Oct4, Nanog, Sox2, and Cdx2 were 97.9%, 98.7%, 95%, and 94%, respectively. (G) Southern blots showed no obvious change in telomere length at passages 3 (P-3) and 7 (P-7). (H) Distinction between hTS cells and PDMS cells in global gene expressions (total 54,675 genes; p<0.05 and fold change >2) with a homogenous histogram. (I) Different relative intensity values of genes (4,864 genes) between PDMS and hTS cells. Blue box indicating 50% of total genes and error bars indicating 25% and 75%. (J) Venn diagram illustrated the numbers of immue-related genes (upper panel) and mesenchymal genes (lower panel) in PDMS and hTS cells by microarray analysis. Overlap: common genes; Bilateral regions: unique genes. The hTS cells expressed specific genes of the TE (i.e., Cdx2, Fgfr-2, Eomes, and BMP4), the ICM (i.e., Oct4, Nanog, FGF4, and Sox2) (Fig. 1C), as well as the three primary germ layers (Fig. 1D). We then evaluated for cellular homogeneity. With TissueGnostics analysis, we found that these cells expressed 100% and 97.3% Oct4-positive cells at passages 9 and 17, respectively (Fig. 1E). Flow cytometry with Oct4, Sox2, Nanog and Cdx2 at passage 15 demonstrated 97.9%, 95.0%, 98.7%, and 94.0% positive cells, respectively (Fig. 1F). Flow cytometry with surface markers CD44, CD73, CD105 and HLA-ABC at passages 3 and 9 also demonstrated over 97% positive (Fig. S1C). These results show that hTS cells are a highly homogenous population. Flow cytometric analysis demonstrated that hTS cells expressed mesenchymal, but not hematopoietic, stem cell characteristics (Fig. S1D). Upon appropriate induction (Table S2), they gave rise to a variety of specific cell phenotypes such as osteoblasts, chondrocytes, myocytes, and adipocytes (Fig. S1E). Neither change in karyotype (46, XY) by chromosome analyses (Fig. S1F) nor in telomere length by Southern blots (Fig. 1G) was observed in a series of cell cultures. We defined these isolated cells as hTS cells. An important question is whether hTS cells are distinct from PDMS cells. To investigate, we utilized the Affymetrix platform to compare global gene expressions between them. The results showed that the gene distribution differed not only with their histogram patterns (Fig. 1H), but also with their intensity values (Fig. 1I). Of the total 54,675 genes, 4,864 genes (8.9%) were distinct between them, including molecular and cellular functions and physiological system development (Table S3). Furthermore, both exhibited unique genes with respect to immune and mesenchymal characteristics (Fig. 1J and Table S4). Therefore, we concluded that hTS cells represented a unique TE-derived stem cell population distinct from PDMS cells obtained from the uterus. LIF Maintains the Proliferative Capability of hTS Cells Previous studies show that gradient levels of LIF exist in the fallopian tube [11] and that in the absence of LIF, ES cells differentiate into the TE fate [14]. To mimic the environment, we investigated the effect of LIF on hTS cells. We treated hTS cells with different concentrations of LIF (i.e., 500, 250, and 125 IU/ml) for 3-days each. Of the pluripotency-associated transcription factors, LIF (500 IU/ml) induced overexpressions of Oct4 and Sox2, but not Nanog and Cdx2 by RT-PCR (Fig. 2A). By flow cytometry, withdrawal of LIF (500 IU/ml) overexpressed Nanog and Cdx2; however, it suppressed Oct4 and Sox2 in a dose-dependent manner (Fig. 2B, Fig. 2C, and Fig. S2A). As the hTS cells travel from a high LIF environment to a low LIF environment along the fallopian tube, the relative Nanog/Cdx2 ratio increases (>2-fold), but the Oct4/Cdx2 ratio decreases (Fig. 2D). By using siRNAs and shRNAs, we also show that knockout of Nanog promoted expressions of Cdx2, and knockout of Cdx2 promoted Nanog (Fig. 2E, Fig. 2F, and Fig. S2B). Similar to the reciprocal relationship between Oct4 and Cdx2 in ES cells [21], we showed the existence of a reciprocal relationship between Nanog and Cdx2 in hTS cells. Expression of Cdx2 is required for maintaining the trophoblastic phenotype. These results suggested that Oct4 is involved in the maintenance of pluripotency at the early TE cells and Nanog enhanced the pluripotent capability in hTS cells at a later stage during transportation in the tube. 10.1371/journal.pone.0052491.g002Figure 2 Regulation of Pluripotency Transcription Factors by LIF. (A) Expressions of Nanog, Cdx2, Sox2 and Oct4 mRNAs after treating different concentrations of LIF (B) By flow cytomery, withdrawal of LIF overexpressed Nanog and (C) enhanced expression of Nanog and Cdx2 but suppressed Oct4 and Sox2. (D) Physiological gradient of LIF levels from ampulla toward isthmus with an increased Nanog/Cdx2 ratio, but a decreased Oct4/Cdx2 ratio in a dose-dependent manner. (E) A reciprocal relationship between Nanog and Cdx2 evidenced by pretreatment with siRNAs (10−8 M, Sigma) and (F) with shRNAs (10−8 M, Sigma), respectively. RA Generates Trophoblast Neural Stem Cells in vitro We then attempted to generate neural cells by treating hTS cells with RA over time (i.e., 1-, 3-, 5- and 7-days). A population of neural stem cells (NSCs) was generated with phenotypes similar to those neural restricted precursor subtypes described in previous studies [22], including glial restricted precursors (GRP), neuronal restricted precursors (NRP), multipotent neural stem cells (MNS), astrocytes (AST), and undefined trophoblast giant cells (TGC) (Fig. 3A, left panel). Notably, the composition of these phenotypes remained relatively stable within 5 days of RA treatment. However, at 7 days, most of the NSCs became undefined TGC (Fig. 3A, right panel), compatible with previous reports [23]. The precursor cells expressed neurofilament protein, nestin, and astrocyte-specific glial fibrillary acidic protein (GFAP) measured at day-3 and day-5 after treatment (Fig. 3C). Immunocytochemically, we found that they expressed NSC-specific markers, for example: GFAP and fibroblast growth factor receptor-1 (FGFR1) for GRP; βIII-tubulin and microtubule-associated protein 2 (MAP2) for NRP; and GFAP and vimentin for MNS (Fig. 3C). We found consistency in cellular characteristics of the RA-induced NSCs, hereby defined as trophoblast neural stem cells (tNSCs) in the following studies. 10.1371/journal.pone.0052491.g003Figure 3 Biological Characteristics of tNSCs. (A) Frequency of NSC sub-phenotypes after RA (10 µM) induction over time: 1-, 3-, 5- and 7-days at first, second, third, and fourth row, respectively. n: total cells counted. GRP: glial restricted precursors; NRP: neuronal restricted precursors; MNS: multipotent neural stem cells; and TGC: undetermined trophoblast giant cells. (B) Flow cytometric comparison of RA-induced nestin, GFAP, and neurofilament expressions between 3-day and 5-day. Data represent mean ± SD (n = 3). (C) Immunocytochemical identification of GRP, NRP and MNS with specific markers. (D) Expression of RA-associated genes before and after RA induction for 1-day by RT-PCR. (E) RA-induced overexpression of Nanog by flow cytomertic and (F) immunocytochemical analysis (n = 60 for each item) by Student t-test. (G) Withdrawal of LIF significantly promoted expression of Nanog induced by RA (10 µM) by flow cytometry. *: p<0.01 compared to the control. All data indicate mean ± SD (n = 3). In tNSCs, RA induction promoted expressions of NSC-related markers (e.g., nestin, neurofilament, Ngn3, MAP-2, NeuroD, CD133, and Oct4), of RA receptors (e.g., RARβ, RARγ, RXRα, and RXRβ), and most importantly, of RA-synthesizing enzymes RALDH-2 and -3 (Fig. 3D). The expression of RALDH-2 and -3 in hTS cells and tNSCs was unique because hES cells or even the retinol-treated ES cells do not express RALDH-2 [24]. By flow cytometry and immunocytochemistry, results showed that RA induced significant overexpression of Nanog and Oct4 but less so Cdx2 in hTS cells (Fig. 3E and Fig. 3F). To this end, we showed that RA efficiently induced hTS cells into tNSCs, maintaining a steady state within 5 days. Withdrawal of LIF Promotes Nanog Expression for Pluripotency of tNSCs Given that LIF can interplay with RA on neural differentiation of ES cells [25], we examined the effect of LIF on the RA-induced Nanog expression in tNSCs. To mimic the microenvironment of the fallopian tube, we first incubated hTS cells with LIF in variable concentrations (i.e., 500, 250, 125 IU/ml). On the 3rd day, we added RA (10 uM) for 1-day before analysis. Flow cytometry showed that a higher level of LIF significantly repressed the RA-induced Nanog (Fig. 3F and Fig. S3). These results suggested that as hTS cells move towards the isthmus, the better the RA’s ability to maintain cellular pluripotency by Nanog expression. RA Induces Nanog through Non-genomic Signaling Pathway How does RA induce Nanog expression? We explored the molecular mechanisms of RA-induced Nanog in hTS cells. Previous studies report that eukaryotic Initiation factor 4B (eIF4B) is involved in the initiation phase of eukaryotic translation [26] through binding to internal ribosome entry site (IRES) [27]. c-Src mRNA has also been reported to contain an IRES element [28]. Activation of eIF4B might also be associated with c-Src mRNA activity for translation initiation [26], [29]. In the developing striatum and hippocampus, an increased Src kinase activity coincides not only with the peak period of neuronal differentiation and growth [30], but also the characteristics of self-renewal [31]. We found that RA promoted production of eIF4B in between 1–4 hr of incubation but faded away at 24 hr (Fig. 4A). This action was inhibited with eIF4B siRNA (Fig. 4B). We found that RA induced a rapid and transient expression of c-Src mRNA, peaking at 15 min (Fig. 4C), followed by production of c-Src protein at 1 hr (Fig. 4D). Notably, RA failed to induce c-Src mRNA elevation by qPCR at 1-day treatment (Fig. 4E). 10.1371/journal.pone.0052491.g004Figure 4 A Non-genomic RA Signaling Pathway. (A) Time course of RA induced production of eIF4B. (B) Activation of c-Src was inhibited by using eIF4B siRNA. (C) RA (10 µM) induced a rapidly transient elevation of c-Src mRNA peaking at 15 min in hTS cells. Data represent mean ± SD (n = 3); *:p<0.01 in Student’s t test. (D) RA induced fast production of c-Src and phosphorylation of Stat3 in 1 hr by Western blots. β-actin: control. (E) qPCR assay showed that RA did not induce expression of c-Src mRNA at 1 day incubation and expression c-Src mRNA was not affected by using 2 different eIF4B siRNAs. (F) Stat3 directly interacted with c-Src by IP assay. (G) c-Src siRNA inhibited expression of Stat3. (H) c-Src inhibitor PP1 analog (4 µM) inhibited the RA-induced phosphorylation of Stat3 and RA induced overexpression of Nanog in hTS cells by Western blots. (I) RA induced binding interaction of Stat3 and Nanog promoter by ChIP assay. (J) Nanog expression was inhibited by Stat3 siRNA. (K) Schematic of the RA-induced c-Src/Stat3/Nanog pathway via subcellular c-Src mRNA localization in hTS cells. Dotted line indicates undetermined mechanism(s). Subsequently, we found that active c-Src bound directly to signal transducer and activator of transcription 3 (Stat3) (Fig. 4F) by phosphorylation at site Tyr705 to produce protein, which was inhibited by c-Src siRNA (Fig. 4G). This action was also inhibited by a selective c-Src inhibitor PP-1 analog (Fig. 4H). We identified a direct action of Stat3 on the Nanog gene promoter by chromatin immunoprecipitation (ChIP) assay (Fig. 4I). Nanog was produced in 4 hr, which was inhibited by PP1 analog (Fig. 4H) and by Stat3 siRNA (Fig. 4J). For hTS cells, we propose the possibility of an RA-activated non-genomic eIF4B/c-Src/Stat3/Nanog signaling pathway through subcellular c-Src mRNA localization (Fig. 4K). The mechanism of RA-induced activation of eIF4B and the direct relationship between eIF4B and c-Src will require further study. How the RA-activated Nanog maintains hTS cell pluripotency will also require further study. Cell Therapy Regenerates Nigrostriatal Pathway and Improves Behavioral Deficits in Acute and Chronic PD Rats To determine the efficacy of cell therapy and the cell fate potential of tNSCs in animal studies, we grafted the cells into the brain of PD rats. We produced 6-hydroxydopamine (6-OHDA)-caused PD rats (Text S1). In acute PD rats, we performed the apomorphine-induced rotation test every 3-weeks up to 12-weeks post-cell therapy. Then we examined GFP-tagged hTS cells for cell fate at 18-weeks post-cell therapy. In chronic PD rats, we bred PD rats for over one year (average 12.3 months) in order to mimic the pathologically progressive nature of PD. After cell therapy, behavioral tests and validation of regeneration of dopaminergic nigrostriatal pathway were performed. In Acute PD Rats We transfected hTS cells with F1B(-540)-GFP plasmid construct (kindly provided by Dr. I. M. Chiu) to yield a success rate of over 95%. For cell therapy, acute PD rats were divided into three groups: group (a) received the GFP-tagged 1-day RA-induced tNSCs (n = 4); group (b) received GFP-tagged 5-day RA-induced tNSCs (n = 4); and group (c) received PBS solution as control (n = 4). We transplanted the tNSCs into the lesioned striatum (STR) under anesthesia. We performed the apomorphine-induced rotation test every 3 weeks post-cell therapy (Fig. 5A). The results of group (a) showed a significant improvement in rotational behavior from 3-weeks to 12-weeks post-cell therapy. The results of group (b) showed significant improvement during the initial 6-weeks, but the improvement was gradually lost after the 6th week. No improvement was observed in control group (c). 10.1371/journal.pone.0052491.g005Figure 5 Transplantation of tNSCs in Acute PD Rats. (A) Apomorphine-induced rotation tests. Group (a) (blue line, n = 4) received 1-day RA-induced tNSCs. Group (b) (green line, n = 4) received 5-day RA-induced NSCs. Group (c) indicated control (red line, n = 4). Statistic analysis by repeated measure ANOVA: p-value = 0.001. LSD post hoc comparisons after repeated measure ANOVA in between two groups: *: p<0.05; **: p<0.01. (B) Regeneration of TH-positive cells at the STR (upper) and the SNC (lower) of lesioned side 18-week after cell therapy. Scale bar: 100 µm. (C). Amplified dopaminergic neuronal circuitry in the lesioned SNC (upper). A comparative normal feature shown in the lower panel. (D) No TH-positive immunoreactivity was found in the lesioned STR (upper panel) and subthalamic nucleus (STN, lower panel) of group (b) and (E) group (c). Arrow: needle track. (F) Presence of GFP-tagged tNSCs (arrow) at the STR (upper panel) and the SNC (lower panel) 18-weeks post-cell therapy. Next we verified the recovery of the nigrostriatal pathway by immunocytochemical studies. In group (a), abundant newly generated TH-positive neurons appeared in the lesioned substantia nigra pars compacta (SNC) with multiple outgrowths to form neural circuitries with the surrounding host tissues (Fig. 5B and Fig. 5C). We did not observe such phenomena in group (b) (Fig. 5D) and in control group (c) (Fig. 5E). No immuno-suppressive agent was used and no teratoma formation was found. To investigate the viability of those implanted GFP-tagged tNSCs in the brain, PD rats were sacrificed at 18-weeks post-implantation and their brain sections were examined by immunofluorescent analysis. The results revealed that the GFP-tagged tNSCs remained sporadically in clusters in the lesioned STR and SNC (Fig. 5F). In Chronic PD Rats During the breeding period, we performed the apomorphine-induced rotation test monthly to ascertain the rats’ PD state until cell therapy. Behavioral assessments were performed every 3 weeks post-cell therapy, including the apomorphine-induced rotation test, the bar test for akinesia, the stepping test for rigidity, and the footprint analyses for postural imbalance and gait disorder as described previously [19]. Group I (n = 6) was the control, while Group II (n = 6) received 1-day RA-induced tNSCs. In group II, we observed a significant improvement of the apomorphine-induced rotations from 3 weeks to 12 weeks post-cell therapy (Fig. 6A), similar to the previous acute PD rats study. The bar test showed that the grasping time of the affected forelimb was significantly shortened at 3-weeks and continued to improve at 12-weeks (Fig. 6B). Assessments by step length (Fig. 6C), stride length (Fig. 6D), walking speed (Fig. 6E), and base of support (Fig. 6F) revealed significant improvement from 3-weeks to 12-weeks post-cell therapy. These studies were performed on a transparent walkway recorded by video camera (Fig. 6G). These results indicated that cell therapy with tNSCs was able to regenerate the dopaminergic nigrostriatal pathway and functionally improved the behavioral impairments in both acute and chronic PD rats. 10.1371/journal.pone.0052491.g006Figure 6 Behavioral Assessments in Chronic PD Rats. (A) Apomorphine-induced rotations analysis revealed a significant improvement in chronic PD rats that received tNSCs cell therapy (Group II, n = 6, black round line) compared to the control group (Group I, n = 6, blank round line). (B) Bar tests (sec) improved at 3-week post-cell therapy. (C) Shortened step length and (D) stride length were significantly improved after 3-week post-cell therapy in PD rats. (E) Significant improvement in walking speed (cm/sec) in PD rats at 3-week post-cell therapy. (F) Significant shortening in base of support (mm) was seen at 3-week post-cell therapy. (G) A well-designed cage with a video camera for footprint analyses. (a): in normal rats, (b): in PD rats pre-cell therapy, and (c): post-cell therapy. Asterisk: compared to the control. Student’s t test: *:p<0.05; **: p<0.01; and ***: p<0.001. tNSCs Regenerate the Dopaminergic Nigrostriatal Pathway in vivo Next, we compared the regeneration of the dopaminergic nigrostriatal pathway after cell therapy with tNSCs by immunofluorescence tissue analysis between acute and chronic PD rats. We investigated brain sections of post-cell therapy acute PD rats (n = 2 at 1-week post-injury, n = 2 at 6-weeks post-injury, n = 2 as sham control for post-injury, n = 6 at 12-weeks post-cell therapy, and n = 2 as sham control for cell therapy) and post-cell therapy chronic PD rats (n = 2 at 12-weeks post-cell therapy and n = 2 as sham control for cell therapy). In the post-injury PD rats at 6-weeks, the 6-OHDA caused progressive neural degeneration in the SNC, resulting in various cavities immunohistochemically (Fig. 7A). Quantitative analysis showed that the number of DA neurons in the SNC reduced to 48% at 1-week post-injury and to 13% at 6-weeks post-injury compared to the intact side (Fig. 7B and Fig. S4). In the STR, DA neurons were reduced to 78% at 1-week post-injury and to 4% at 6-weeks post-injury (Fig. 7B). In acute PD rats at 12-weeks post-cell therapy, tNSCs generated numerous DA neurons at the cavity wall with TH-positive nervous terminals projecting into the cavity (Fig. 7A, insert). We found that DA neurons had regenerated to 67% in the SNC and to 73% in the STR. 10.1371/journal.pone.0052491.g007Figure 7 in vivo Regeneration of TH(+) and GFAP(+) Cells with Less Immuno-responses. (A) Immunohistofluoresence analysis. TH(+) and NeuN(+) motor neurons (arrow) in the SNC of control (left upper). Decreased TH(+) (arrow) at 1-week after 6-OHDA injury (right upper). Apparent reduction in TH(+) neurons with disarrangement of TH-positive neural terminals (green granules), and various degenerative cavity formation (red hollow circle) at 6-week post-injury (left lower). After transplantation at 12-weeks, TH(+) neurons (arrow) at wall of the degenerative cavity (red hollow circle; insert) with TH(+) neural terminals (green color) projecting into the cavity (right lower). (B) Number of TH(+) cells at 1- and 6-weeks reduced to 48% and 13% in the lesioned SNC (red) and 78% and 4% in the lesioned STR (light blue), respectively, post-injury. After transplantation, TH(+) cells re-grew up to 67% and 73% in the lesioned SNC and STR, respectively (right panel). Data analyzed by the software Tissuequest 2.0 (TissueGnostics Gmbh, Vienna, Austria). (C) Regeneration of dopaminergic neurons in the lesioned SNC (lower panel) with amplification (left upper, insert a) compared with the intact side (right upper, insert b). (D) Transplantation of tNSCs at 12-weeks yielded 78% of recovery rate in TH-positive neurons in the lesioned SNC compared to the intact side. Data represent mean ± SD (n = 3). (E) Degeneration of TH-FITC(+) and GFAP-Cy-3(+) Wilson’s pencils (blank arrow) at 6-week post-injury in the lesioned STR (left column). At 12-weeks post-cell therapy (right column), several GFAP(+) cells (arrow) appeared inside the fine fibers of re-established Wilson’s pencils (blank arrow). (F) For immunohistofluorescence imaging analysis, cells were counted in the gate (left scatter plots) determined by the location of cell size (8–10 µm in diameter) and its corresponding intensity of GFAP-Cy-3. Gate (red scatter plot): glial cells counted; black scatter plots: exclusive cells with bizarre size; blue scatter plots: cells with abnormal GFAP intensity. In the STR, the GFAP(+) cells were 65.5% in the lesioned side before treatment and became 93.9% after cell therapy compared to the intact side (right panel). Data represent mean ± SD (n = 3). Student t-test. (G) hTS cells implanted into the SCID mice (n = 6) raised only minor immunoreactions and without tumorigenesis observed. Myxoid-like bizarre cells (black arrow), muscle fibers (blank arrow), and needle track (NT). (H) Flow cytometric analysis of hTS cells, tNSCs, and hES cells. High expression of HLA-ABC in hTS cells and tNSCs, but less in hES cells. No HLA-DR expressed among them. Immune-related gene expressions, including CD14, CD44, CD33, CD34, CD105, and CD133, were compared among them. In chronic PD rats at 12-weeks post-cell therapy, immunohistochemical study revealed that DA neuronal circuitries were substantially regenerated in the therapeutic side of the SNC, mirroring the intact side (Fig. 7C), consistent with our previous studies. The recovery of DA neurons reached 78% in the SNC (Fig. 7D). Glial cells are known to play as mediators in guiding the migration of neurons to their destinations or as sources of neural regeneration [32]. We found that in the STR, 6-OHDA in chronic PD rats caused not only degeneration of both GFAP(+) cells and DA neurons, but also disarrangement of the striatopallidal fibers (pencils of Wilson) and nigral axons. These phenomena were improved after tNSC therapy, showing numerous GFAP(+) cells embedded in the fine myelinated fibers (Fig. 7E). The GFAP(+) cells regenerated from 66% at 6-weeks post-injury to 94% at 12-weeks post-cell therapy in the lesioned STR (Fig. 7F). These results indicated that transplantation of tNSCs regenerated the dopaminergic nigrostriatal pathway in chronic PD rats. No teratoma formation was found in both the acute and the chronic PD rats. Longer term studies beyond 18-weeks post-transplantation may be useful. tNSCs Exhibit Immune Advantages for Transplantation Translation of stem cells into cell therapies requires the understanding of their antigenicity and immunogenicity for any clinical application to be useful. We first implanted the hTS cells into male severe combined immunodeficient (SCID) mice intramuscularly for 6–8 weeks. Histologically, no teratoma was found; but we observed myxoid-like bizarre cells between the muscle fibers (Fig. 7G). hTS cells appear to indicate immune advantages compared to hES cells with respect to teratoma formation [33], [34]. We then compared the expressions of immune-related genes among hTS cells, tNSCs, and hES cells. In vitro, HLA-ABC (MHC-I) was expressed highly in hTS cells (99.4%) and in tNSCs (99.7%), but lower in hES cells (12.9%); however, no surface HLA-DR (MHC-II) was expressed among them (Fig. 7F). No major difference was found among the three cells for innate immune system marker CD14, for effector-memory T-cells marker CD44, and for human endothelial cell proliferation marker CD105. Surprisingly, hES cells and tNSCs expressed the immature hematopoietic and endothelial cell marker CD34, linked to neurogenesis and angiogenesis [35], while hTS cells expressed significantly less. Both hTS and hES cells expressed a myeloid leukemia-associated marker CD33 and cancer stem cell marker CD133, while tNSCs expressed significantly less. Discussion The search for an alternative cell source for treatment of PD has been challenging [1], [2], [3], [4], [5], [6]. Here, we isolated hTS cells from preimplantation embryos in women with ectopic pregnancy. hTS cells are not only distinct from PDMS cells, but also exhibit hES cell-like qualities in pluripotency and viability in proliferation. Given that hTS cells originate from the TE and not from the ICM of the blastocyst, hTS cells circumvent the socio-ethical issues that surround the use of hES cells. Furthermore, ectopic pregnancies account for 1 to 2% of all pregnancies in industrialized countries and even higher in developing countries; thus, providing a locally abundant source of stem cells with relatively closer genetic make-up for patients in need of cell therapy. It is interesting to note that hTS cells express both the markers of the ICM and TE. In murine embryos, Oct4 protein is detected in the ICM but not the TE. However, in porcine and bovine blastocysts, Oct4 protein is detected in both the ICM and the TE [36]. In human blastocysts, there is 31 times higher concentration of Oct4 mRNA in the ICM than that in the TE [37]. In hTS cells, we show co-expressions of pluripotent markers such as Oct4 and SSEA-4 along with TE-specific marker Cdx2. This may be specific to ectopic pregnancy-derived hTS cells and possibly due to the microenvironment of a tubal ectopic pregnancy. Further study of this microenvironment is required. At the preimplantation stage, extrinsic factors like LIF [9] may influence the epigenetic gene regulation in embryos. Not surprisingly, LIF affected the fate choice of hTS cells similar to its action on ES cells [9], [25]. Mimicking the microenvironment in the fallopian tube, we showed that withdrawal of LIF in hTS cells downregulated Oct4, but upregulated Nanog and Cdx2. This fact suggested that Oct4 was initially responsible for the pluripotent state. However, hTS cell pluripotency became dependent of Nanog at a later stage, consistent with the notion that Nanog plays a crucial role in cell fate specification following the formation of the blastocyst [38]. The elevation of both Nanog and Nanog/Cdx2 ratio maybe responsible for the maintenance of pluripotency in hTS cells. Further study of this direct relationship is required. The overexpression of Cdx2 indicates hTS cell’s trophoblastic phenotype as it enhances the formation of TS cells in ES cells [14]. RA was capable of determining the neuronal fate choice of hTS cells. hTS cells expressed RALDH-2 and -3, which enables the metabolism of retinol into RA [15], while ES cells did not [25]. We have found some evidence to suggest that RA-enabled hTS cell differentiation into stable tNSCs through a non-genomic eIF4B/c-Src/Stat3/Nanog signaling pathway via subcellular c-Src mRNA localization. The fast transient production of c-Src was possibly related to the RA-trigged machinery in local protein synthesis rather than conventional genetic processes. We suggest that the RA-activated eIF4B is possibly and indirectly associated with subcellular c-Src mRNA localization in hTS cells. How RA induced eIF4B activation remains to be further evaluated. Critical details of the regulatory mechanisms between eIF4B and c-Src will require further study. A more comprehensive knowledge of this pathway in tNSC proliferation may provide helpful insight to why Src-kinase activity increases at the peak period of neurogenesis in the developing striatum and hippocampus [30] and why the disruption of Stat3 signaling contributes significantly to neuronal death in PD [39]. A potential therapeutic advantage of tNSCs is its proliferation of a variety of restricted neural precursor subtypes. Of which, GRP and astrocytes may be attributed to the regeneration of both DA neurons and glial cells in the lesioned nigrostriatal pathway, substantially promoting dopaminergic neurogenesis [6]. Another advantage is that tNSCs facilitated more site-specific integration rather than regional incorporation, as we observed neural regeneration only in the lesioned sites. Application of tNSCs in other central nervous system diseases must be researched. Previous studies report that hES cell-derived DA neurons do not survive in the grafted striatum and fail to improve the behavioral deficits in PD rats [40]. Cell therapy with tNSCs regenerated the dopaminergic nigrostriatal pathway and functionally improved the behavioral impairments in both acute and chronic PD rats. It is interesting to note from our animal studies that 1-day RA-induced tNSCs maintain long term effectiveness while the 5-day RA-induced tNSCs showed behavioral improvement in the beginning, but failed to be effective after 6-weeks. We believe that the transplanted 5-day RA-induced tNSCs were near the end of their effective window; that is, when most of the tNSCs transition into undefined giant cells. Contrastingly, the 1-day RA-induced tNSCs were extremely viable and once transplanted, maintained its regenerative effect. It may be helpful that tNSCs were implanted into the striatum of the brain, where cells would favorably meet an RA-enriched microenvironment [17], facilitating continuous proliferation in vivo. Moreover, the implanted tNSCs increased glial cells in the striatum, which is compatible with previous reports that RA induces the expression of GFAP [41] and that GFAP(+) progenitor cells may give rise to neurons and oligodendrocytes throughout the CNS [42]. The use of hTS cells in cell therapy may be optimal within an efficacy window: 1-day after RA-induction to tNSCs. In chronic PD rats, tNSCs regenerated about two-thirds of the DA neurons in the nigrostriatal pathway at 12-weeks post-cell therapy. This positively suggests that tNSCs may also be effective for treatment of patients that have suffered PD symptoms over a long period of time. However, the appropriate dosage of tNSCs for treatment of PD remains to be evaluated. Our investigation of hTS cell immune characteristics began with the intramuscular transplantation of hTS cells in SCID mice. Although we observed bizarre cells, we did not observe any teratoma formation. In subsequent animal studies, we neither used immunosuppressed mice nor immunosuppressants and no teratoma formation appeared. It has been reported that neural progenitor stem cells have immune privilege and have survived rejection in allografts [43]. It is interesting to note that by differentiation of hTS cells to tNSCs, RA-induced changes in expression of immune-related markers. For example, while the expression of CD34(+) increased, expressions of CD133(+) decreased. The biological significance is unclear. Nevertheless, it has been shown that autologous transplantation with CD34(+) immune-selected grafts is feasible in children with high-risk neuroblastoma [44], linking positive expression to graft feasibility. Also consistent with a recent report [45], lower CD133 expression in tNSCs may decreased the probability of tumorigenesis, as CD133(+) cells possess the capacity for unlimited self-renewal and can trigger brain tumor initiation [34]. Although it is promising that hTS cells were successfully employed for graft acceptance without teratoma formation in SCID mice and tNSCs in PD rats, further studies are warranted to investigate the possible immune responses of hTS cells or their derivatives after transplantation. In conclusion, hTS cells are a promising cell source for regenerative medicine. The source from tubal ectopic pregnancies is abundant. The culture requires no feeder layer. The induction to tNSCs is not only simple (RA) but also fast (1-day). tNSCs also show early indications of immune-privilege. hTS cells and hTS cell-derived tNSCs may be remarkable candidates for shortening research timeframes, creating new disease models, and also for the treatment of patients with PD and other neurodegenerative diseases. Supporting Information Figure S1 (A) Microscopic feature of ectopic pregnancy-derived chorionic villi dissected. (B) At passage 9 and 17, hTS cells co-expressed Oct4 (red) and Nanog (green) in a homogeneous distribution. Scale bar: 20 µm. Nucleus: blue DAPI color. (C) Flow cytometry with Oct4, Sox2, Nanog and Cdx2 at passage 15 demonstrated 97.9%, 95.0%, 98.7%, and 94.0% positive cells, respectively. (D) Flow cytometric analyses revealed that hTS cells express markers of mesenchymal stem cells but not markers of hematopoietic stem cells. (E) Differentiation of hTS cells into specific phenotypes, including osteoblasts, chondrocytes, myocytes, and adipocytes by appropriate inductions. (F) Chromosome analyses revealed that normal karyotypes (44,XY) did not change at passages 3, 10, and 15 of cultivation in hTS cells. (TIF) Click here for additional data file. Figure S2 (A) Withdrawal of LIF suppressed Oct4 and Sox2, but overexpressed Cdx2 by flow cytometry in hTS cells. (B) A reciprocal relationship between Nanog and Cdx2 evidenced by pretreatment with siRNAs (10−8 M, Sigma) by flow cytometry. Data represent mean ± SD, n = 3. (TIF) Click here for additional data file. Figure S3 Flow cytometry showed that RA (10 µM) enhanced Nanog expression by withdrawal of LIF levels from 500, 250, 125, toward 0 IU/ml after 3-day incubation in hTS cells. (TIF) Click here for additional data file. Figure S4 To qualify the TH(+)NeuN(+) neurons in the SNC pre- and post-cell therapy, coefficient of determination between TH-FITC and NeuN-Cy-3 was measured by immunohistofluorescent scatter plots in the chronic PD rats. Normal SNC: R2 = 0.72 (left upper); 6-OHDA damaged SNC (1-week), R2 = 0.77 (right upper); 6-OHDA damaged SNC (6-weeks), R2 = 0.25 (left lower); SNC after tNSCs transplantation (12-weeks), R2 = 0.66 (right lower). Result shown represents the average of 2 rats. (TIF) Click here for additional data file. Table S1 All antibodies, primers, and reagents used in this study. (DOC) Click here for additional data file. Table S2 Recipes used for cell differentiations. (DOC) Click here for additional data file. Table S3 Significant distinction in biological functions in 4,864 distinguishable genes (p<0.005, fold change >2) between hTS cells and PDMS cells. (DOC) Click here for additional data file. Table S4 Comparison of immune-related gene expression in relative intensity value between hTS cells and PDMS cells from Metasearch. (DOC) Click here for additional data file. Text S1 Detailed experimental procedures and supporting references. (DOC) Click here for additional data file. We thank Dr. Robert Silman and Mr. Yuta Lee for comments on the preparation of manuscript, Dr. I. M. Chiu for providing F1B(-540)-GFP plasmid construct, C. P. Chen for PDMS cells, Y. S. Liu for hES cells and Ms. H. T. Lee for technical assistance. ==== Refs References 1 Freed CR , Greene PE , Breeze RE , Tsai WY , DuMouchel W , et al (2001 ) Transplantation of embryonic dopamine neurons for severe Parkinson’s disease . N Engl J Med 344 : 710 –719 .11236774 2 Olanow CW , Goetz CG , Kordower JH , Stoessl AJ , Sossi V , et al (2003 ) A double-blind controlled trail of bilateral fetal nigral transplantation in Parkinson’s disease . 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23285066
PMC3528662
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2021-01-05 16:47:09
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PLoS One. 2012 Dec 21; 7(12):e52491
==== Front Case Rep PsychiatryCase Rep PsychiatryCRIM.PSYCHIATRYCase Reports in Psychiatry2090-682X2090-6838Hindawi Publishing Corporation 10.1155/2012/368039Case ReportCombined Case of Blood-Injury-Injection Phobia and Social Phobia: Behavior Therapy Management and Effectiveness through Tilt Test Ferenidou Fotini 1 *Chalimourdas Theodoros 1 Antonakis Velissarios 2 Vaidakis Nikolaos 1 Papadimitriou Georgios 1 11st Department of Psychiatry, Eginition Hospital, University of Athens Medical School, Vas. Sofias 72, 11528 Athens, Greece21st Cardiology Clinic, Hippokration Hospital, University of Athens Medical School, Vas. Sofias 114, 11527 Athens, Greece*Fotini Ferenidou: [email protected] Editors: J. S. Brar, L. Dell'Osso, F. Jollant, E. Jönsson, and Y. Kaneda 2012 13 12 2012 2012 36803912 11 2012 1 12 2012 Copyright © 2012 Fotini Ferenidou et al.2012This 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 efficacy of behavior therapy based mainly on real-life exposure situations as well as applied tension was examined for a combined case of blood-injury-injection (BII) phobia and social anxiety disorder. Treatment involved 28 behavior therapy sessions, while applied tension technique was also described and practiced. The specific contribution of social skills techniques, fantasy, and real-life situations exposure was examined in a single case design. The subject was a 39-year-old male with anxiety symptoms when confronting an audience, as well as symptoms of the autonomic nervous system (bradycardia and syncope), which were better explained by BII. All self-report measures regarding fear, social phobia, and anxiety were reduced after behavior therapy and remained maintained at followup, while BII decreased further after applied tension techniques. The contribution of behavior therapy to the overall outcome of the case is considered significant for many reasons that are discussed in the pape. ==== Body 1. Introduction Blood-injury-injection phobia (BII) is quite a common phobia. Estimates of lifetime prevalence of BII phobia range 3.5% for men and women [1] to as high as 4.9% for women [2], and unlike individuals with other specific phobias, 75% of those with BII phobia report a history of fainting in response to phobic stimuli. The most outstanding characteristic of blood phobics is their unique physiological pattern when confronted with the phobic stimuli. The fainting response is characterized as a vasovagal syncope and has been described in the literature as a two-phase, or biphasic (also called diphasic) response to BII stimuli [3, 4]. The initial phase involves an increase in heart rate and blood pressure as is typical of the fight-flight component of an anxiety response [5], whereas the second phase is characterized by bradycardia and hypotension leading to reduced cerebral blood flow and ultimately fainting [4], although this pattern is not always observed [6]. Fainting in response to BII stimuli is a significant source of concern for many patients suffering from blood phobia and can aggravate avoidance. According to the DSM-IV-TR [7], the fear that is cued by the presence or anticipation of the feared stimulus (e.g., receiving an injection or seeing blood) is deemed unreasonable and excessive. If the situation is not avoided altogether, the afflicted person endures the phobic object (e.g., blood) or situation (e.g., going to the doctor) with intense anxiety or distress. Consequently, significant interference with the person's normal routine, including occupational and social functioning, is observed and is a necessary diagnostic criterion [8]. The treatment technique generally recognized as efficacious for BII phobia is Applied Tension (AT), which combines a muscle tension technique with in vivo exposure [6, 9, 10]. The tension technique utilizes repeated tense and release sequences of the skeletal muscles to counteract cardiovascular and autonomic changes implicated in BII-related syncope. Versions of this technique have been used for some time for combating orthostatic hypotension in patients as well as fainting in various settings such as blood donation [11–14]. Kozak and Montgomery [15] are generally regarded as the first to have devised AT as a behavioral treatment strategy for BII phobia. Their aim was to target the syncopal episode rather than the initial anxious arousal, which is typically observed as the first stage of the biphasic response. The tensing of skeletal muscles involved in this technique led to increases in blood pressure and heart rate, and was thereby thought to counteract the two major autonomic phenomena involved in the vasovagal syncope: hypotension through peripheral vasodilation and bradycardia by massive vagal excitation. In addition, skeletal muscle tension (particularly in the extremities) promoted increased blood flow to the heart and brain, thereby preventing the fainting response [16]. Social Anxiety Disorder (SAD) or Social Phobia, as defined in DSM-IV-TR [7], is a marked and persistent fear of one or more social or performance situations in which the person is exposed to unfamiliar people or to possible scrutiny by others. The individual fears that he or she will act in a way (or show anxiety symptoms) that will be humiliating or embarrassing. Activities such as meetings or interactions with strangers, attending social gatherings, formal presentations, and those requiring assertive behavior are commonly feared by individuals with social phobia [17]. The recent National Comorbidity Survey [18] reported a lifetime prevalence of 13.3% for social phobia and its prevalence appears to be increasing [19]. Social phobia is highly comorbid with other anxiety disorders, depression, and substance abuse, and it significantly increases the risk for these disorders [20]. It runs a chronic course and is associated with significant impairments in functioning and overall quality of life, as well as an increased risk of suicidal ideation and attempts [21–24]. Research on SAD has demonstrated that cognitive, behavioral, and physiological components are all present and interact in the presentation of this disorder [25]. For instance, individuals with SAD tend to engage in more self-monitoring of their own behavior, focus on potential threats, believe that individuals will notice their anxiety symptoms and interpret them negatively, and are more likely to perceive their behaviors negatively than individuals without social anxiety [17, 26–28]. Due to the cognitive, behavioral and physiological components of SAD, numerous studies have investigated the efficacy of cognitive-behavioral therapy (CBT) on SAD. In fact, CBT is the most widely used and researched treatment for SAD [29]. Research has consistently shown CBT to be an efficacious treatment for SAD when administered either individually or in a group format [30–32]. In order to make a good match between the phobic patient and treatment method one can scrutinize the available techniques and decide which of the three anxiety components each method is focused on. So, for example, social skills training, reinforced practice, flooding, modeling, and contact-desensitization can be viewed as focusing on the behavioral component. Techniques such as stress-inoculation training and systematic rational restructuring concentrate on the cognitive component, while the physiological component is the primary target for relaxation methods (e.g., applied relaxation), anxiety management training, systematic desensitization and biofeedback. Naturally, some of the techniques contain aspects of more than one component but the above categorization is based on each technique's primary characteristic [33]. 2. Case Presentation 2.1. Subject The subject was—at the beginning of treatment—a 39-year-old male, married with one child. He was working as a Lecturer at the University and was the third child of a 5-member family. His parents were divorced since 1985, when the patient was 13 years old. He reported a prior history of anxiety symptoms during his lectures in front of an audience, which were often accompanied by syncope and serious injuries. Since syncope cannot be explained by social phobia, it was considered necessary to perform an autonomous nervous system exam. Tilt test was performed and found abnormal results (quite important reduce in heart rate). Additionally, several medical history facts (drop in blood pressure when being present in procedures that included blood) substantiated BII phobia. No prior psychiatric or medical history was reported. Regarding his family history, his father was hospitalized in a psychiatric institution twice, involuntarily, when the patient was 7 and 15 years old. Furthermore, his uncle—his father's brother—was referred as “the village's crazy guy”, showing quite often “abnormal” behavior. Equivalent mental problems were referred regarding his paternal grandmother (behavioral problems) and his other uncle (from his mother's side-alcoholic and violent). His mother reported BII, which she managed to get over after her first baby delivery. The subject was going under psychoanalytic psychotherapy for the last two years, with mild improvement. He had never received any pharmacological compounds regarding his problem, before or during behavior therapy. 2.2. Measures The State-Trait Anxiety Inventory [34] is comprised of 40 items divided evenly between state anxiety and trait anxiety. The authors reported reliability for trait anxiety was 0.81; as expected, figures were lower for state anxiety (0.40). Internal consistency ranges between 0.83 and 0.92. The Fear Questionnaire [35] is a one-page self-rating form described to monitor change in phobic patients. The form yields four scores: main phobia, global phobia, total phobia, and anxiety-depression. The total phobia score is composed of agoraphobia, social, and blood-injury subgroups. The form is short, reliable, and valid. The Liebowitz Social Anxiety Scale (LSAS) is the first clinician-rating scale developed for the assessment of social phobia [36] and was designed to assess the range of social interaction and performance situations that individuals with social phobia may fear and/or avoid [37]. Its 24 items are divided into two subscales that address social interactional (11 items) and performance (13 items) situations. The clinician asks the patient to rate fear and avoidance during the past week on 0–3 Likert-type scales; however, the clinician is given latitude to question the patient's responses and adjust the ratings accordingly. Thus, the LSAS provides six subscale scores: total fear, fear of social interaction, fear of performance, total avoidance, avoidance of social interaction, and avoidance of performance. An overall total score is often calculated by summing the total fear and total avoidance scores, and this index is the one most commonly employed in studies of the pharmacotherapy of social phobia. The Beck Anxiety Inventory (BAI), created by Beck and Steer, is a 21-item multiple-choice self-report inventory that measures the severity of anxiety in adults and adolescents. Because the items in the BAI describe the emotional, physiological, and cognitive symptoms of anxiety but not depression, it can discriminate anxiety from depression. Each of the items on the BAI is a simple description of a symptom of anxiety in one of its four expressed aspects: (1) subjective (e.g., “unable to relax”), (2) neurophysiologic (e.g., “numbness or tingling”), (3) autonomic (e.g., “feeling hot”), or (4) panic-related (e.g., “fear of losing control”) [38]. A tilt table test, occasionally called upright tilt testing, is a medical procedure often used to diagnose dysautonomia or syncope. Patients with symptoms of dizziness or lightheadedness, with or without a loss of consciousness (fainting), suspected to be associated with a drop in blood pressure or positional tachycardia are good candidates for this test. The procedure tests for causes of syncope by attempting to cause syncope by having the patient lie flat on a special table or bed while connected to ECG and blood pressure monitors. The table then creates a change in posture from lying to standing [39]. 2.3. Procedure The subject was referred to the second author for treatment of social phobia. He underwent 28 weekly behavioral therapy sessions based on exposure to real-life experiences (the total therapy time frame was 7 weeks). Twenty sessions had to do with social phobia management, 8 had to do with BII management and their duration was approximately 45 minutes each. During visits 1 and 2, history taking took place, while during visits 3, 4, and 5 the therapist went through behavioral analysis, psychoeducation and target specification. Behavioral analysis included the stimulants as well as the physical, cognitive, and behavioral reactions that they trigger. Stimulants were his position as an observational subject in front of an audience (during his lectures or when he expresses his opinion), as well as when he faces colleagues that belong to a higher academic level than his. Additionally, pictures and thoughts that had to do with blood seemed to provoke the same reaction. Physical reactions were sweating, muscle tension, tachypnea, dizziness, scotodinia, syncope, while cognitive reactions were thoughts such as: “I am inadequate, I will not make it, I will faint and be ridiculed.” Finally, behavioral reactions (avoidance and coping strategies) had to do with sitting versus standing, avoiding eye contact, not expressing his opinion, whispering instead of speaking out, or even exiting the room. The above-mentioned reactions had a great impact on his job (professional restrictions, thinking of quitting his job) as well as restrictions in his social life. During visits 6–9, role playing, relaxation techniques, and imaginable exposure to anxiety stimulants took place, while visits 10–15 included gradual exposure to real-life stimulants. First, exposure took place at his work place, for example, giving difficult lectures in front of a small audience, in front of Ph.D. students or other university teachers that he well knew and gradually in front of professors of higher academic level. The subject received assertiveness training and sessions 15 to 18 included exposure to other situations. He was asked to be loud while talking on the phone, at the super market, to make frequently questions to people he did not know (e.g., on the street) and to even risk feeling shame and losing acceptance from others. Taking into consideration the fact that social phobia alone cannot explain syncope, as well as information from the patient's history, blood-injury-injection (BII) phobia seemed to be also present obstructing behavior therapy. BII—as mentioned above—is usually followed by a two-phase vasovagal response. First, due to the action of the sympathetic nervous system, heart rate, and blood pressure increase, but after a while the parasympathetic nervous system takes action followed by sudden reduce in heartrate and blood pressure. The degree to which the cardiovascular system responses, differentiates patients from healthy population. Bradycardia—as a response to the fearful stimulant—may lead to syncope, while fear is sustained since possible syncope leads to anxiety. Applied tension seems to be an effective treatment for blood phobia [40]. The rationale for applied tension is very simple, and after describing the diphasic pattern the patient is given the following explanation. As the second phase of the diphasic response consists of a rapid drop in BP the cerebral blood flow is also reduced and the patient feels dizzy, and eventually faints. In order to reverse this development the patient needs to learn a coping skill that can be applied quickly and easily in almost any situation. One coping skill that produces an increase in BP and cerebral blood flow is applied tension. This consists of two parts; first learning to tense the gross body muscles, and second learning to identify the earliest signs of the drop in BP, and using these as a cue to apply the tension technique. By being exposed to different blood-injury stimuli under the therapist's supervision, the patient will gradually grow more and more efficient in identifying these early signs and applying the tension [41]. The tension technique was as follows: the patient was instructed to tense the muscles of the arms, the chest, and the legs, and keep tensing until a feeling of warmth is rising in the face (10–20 sec). Then she/he lets go of the tension and returns to the starting level, but does not relax further. After a pause of 20–30 sec the patient does the tension again, and then releases it. This procedure is repeated 5 times, and as homework assignment the patient is instructed to perform 5 tension-release cycles 5 times a day. The technique is supposed to take place in combination with real-life situations and has as a goal reverse of the two-phase vasovagal response. Therefore, syncope is prevented and patient's confidence is reinforced. Sessions 19 and 20 included the above-mentioned information and education, accompanied by special brochures. Finally, sessions 21 to 28 included real-life exposure to blood and blood related procedures. Specifically, the patient—together with his therapist—observed blood collection and blood donation procedures in a hospital and came in touch with syringes and needles. He even had his own blood collected, became a blood donor and watched a surgical procedure with success (without feeling any of the previously mentioned symptoms). 2.4. Outcome The subject was administered the battery of questionnaires described above just before the beginning of treatment, after the end of treatment (after session 28), and at 6 and 18 months posttreatment. Total results of the questionnaires are presented in Figure 1. The results of the questionnaires separately can be found in Figures 2, 3, 4, and 5. As can be seen, his self-reported fear, social phobia and anxiety were reduced after behavior therapy and were maintained at followup. BII was quite reduced after exposure techniques and applied tension methods. Additionally, tilt test was performed three times during behavior therapy sessions, but was stopped due to important reduce in heart rate. Specifically, it took place at the beginning of therapy (1st session), in the middle (16th session) and in the end of treatment (28th session). The first tilt test was stopped at the first minute due to bradycardia, low blood pressure and syncope. The second tilt test was also stopped during the first minute due to similar symptoms (although syncope did not occur), and the third tilt test was stopped during the 3rd minute due to symptoms of the autonomic nervous system. During the fourth tilt test (18 months posttreatment), patient remained symptom free and hemodynamically stable throughout the whole procedure (20 minutes duration). Finally, BII was quite reduced after exposure techniques and applied tension methods. 3. Discussion Behavior therapy was successful in reducing this subject's social phobia as well as BII phobia. Although the sole contribution of behavior therapy to his improvement is not possible to determine given the inclusion of psychoanalytic sessions 2 years before, the contribution of behavior therapy to the overall outcome is considered significant for many reasons. First, his self-reported social phobia, blood injury phobia and social anxiety on all measures as well as general symptoms of anxiety (as measured by the Beck Anxiety Inventory) decreased further following behavior therapy. Second, he was able to take over as a president of a control committee on organic foods, a responsibility he used to avoid before, because of the stress it provoked him. Third, he was a candidate of the workforce committee of his workplace (he had to introduce himself to many people, talk about himself and his achievements), where he was finally elected as president. Fourth, he represented many times abroad the Ministry he was working for with quite success. His own comments regarding behavior therapy and exposure in particular were as follows: “The method was liberating, since I realized that I was trapped in a game where I had to get confirmation from everybody;” “After some (therapy technique) repetitions I felt really free and I realized that true freedom comes when someone is free from his fears.” His comments regarding relaxation included: “The first relaxation experience was amazing. I truly relaxed and realized that it was years since I felt calm and self-concentrated;” “It helped me deal with the anxiety of expecting something, for example, before the beginning of a class I try to practice relaxation techniques in order to deal with the permanent anxiety—which wouldn't let me think clearly—and not overestimate things.” Finally his comments regarding training on assertiveness skills were: “ During class I try—and most of the times I succeed—to be myself without trying to imitate my “serious” professors; the way my body moves, my smooth voice, my low profile and my friendly mood towards students, have a relaxing impact on me since I no more try to be someone else rather than myself;” “ I do not hesitate to admit that I do not know something, which is quite soothing for me since I do not need to know everything.” According to the social skills deficit model, people with social anxiety lack important skills to interact effectively with others. Consequently, they experience fewer interpersonal rewards and more punishments, leading them to avoid social interactions when possible, thereby further limiting their ability to acquire effective social skills. Accordingly, comprehensive treatment for social anxiety would provide explicit training to help patients acquire and use appropriate social skills and thereby reverse the negative cycle [42]. Research on SAD has demonstrated that cognitive, behavioral, and physiological components are all present and interact in the presentation of this disorder [25]. To summarize the experimental studies on social phobia in psychiatric patients, one could say that the effects of treatment are rather modest. All the studies reviewed found significant changes on different self-report measures. No consistent results pointing to the superiority of any method studied has emerged and results seem to depend on the anxiety component that is prominent in each patient [33]. Blood-injury-injection (BII) phobia presents with a unique anxiety response that often involves blood pressure drops and pronounced bradycardia, which can culminate in fainting. Although the current recommended treatment for BII phobia is Applied Tension (AT) surprisingly little empirical evidence is available on the additive efficacy of tension beyond exposure alone. Literature search yielded five controlled treatment studies for BII phobia, all from one research group. Beyond AT, these studies also tested Exposure only (E), Tension only, Applied Relaxation (AR), or a combination of AR and AT. Based on self-reported levels of anxiety, in-session avoidance and fainting, AT was superior over other conditions; however, when considering pre- to posttreatment effect sizes on BII-related questionnaires, E outperformed all other treatments. In addition, AT did not yield better results on physiological measures, and individuals with BII fears improved similarly within studies across treatment groups, regardless of fainting status. Heterogeneity in patient populations (e.g., extent of fainting proneness), differential targeting of BII phobia manifestations, and small sample sizes may explain some of the variability in findings [16]. Although the present case study shows significant results for BII therapy with a combination of blood related procedures exposure and applied tension, further research is needed to determine the efficacy of treatment techniques for BII phobia patients with and without fainting history. Figure 1 Questionnaires' scores pre- and posttreatment and followup. Figure 2 Marks & Mathews fear Questionnaire pre- and posttreatment and follow up. Figure 3 Liebowitz Social Anxiety Scale (LSAS) pre- and posttreatment and followup. Figure 4 Spielberger State-Trait Anxiety Inventory pre- and posttreatment and follow up. 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23304602
PMC3532914
CC BY
2021-01-05 10:42:14
yes
Case Rep Psychiatry. 2012 Dec 13; 2012:368039
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23300886PONE-D-12-2547210.1371/journal.pone.0053176Research ArticleBiologyAnatomy and PhysiologyMusculoskeletal SystemMolecular Cell BiologySignal TransductionSignaling PathwaysLeptin Signal TransductionCell GrowthMedicineAnatomy and PhysiologyEndocrine SystemEndocrine PhysiologyHormonesNutritionObesityRheumatologyOsteoarthritisSurgeryOrthopedic SurgeryLeptin Induces Cyclin D1 Expression and Proliferation of Human Nucleus Pulposus Cells via JAK/STAT, PI3K/Akt and MEK/ERK Pathways Leptin Induces Nucleus Pulposus Cell ProliferationLi Zheng 1 Shen Jianxiong 1 * Wu William Ka Kei 2 Yu Xin 1 Liang Jinqian 1 Qiu Guixing 1 Liu Jiaming 1 1 Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College, Beijing, China 2 Department of Medicine and Therapeutics, Institute of Digestive Diseases, LKS Institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Englert Christoph Editor Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Germany * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: ZL JS. Performed the experiments: ZL XY. Analyzed the data: ZL J. Liang J. Liu. Contributed reagents/materials/analysis tools: ZL JS GQ. Wrote the paper: ZL WKKW. 2012 31 12 2012 7 12 e5317620 8 2012 26 11 2012 © 2012 Li et al2012Li 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.Increasing evidence suggests that obesity and aberrant proliferation of nucleus pulposus (NP) cells are associated with intervertebral disc degeneration. Leptin, a hormone with increased circulating level in obesity, has been shown to stimulate cell proliferation in a tissue-dependent manner. Nevertheless, the effect of leptin on the proliferation of human NP cells has not yet been demonstrated. Here, we show that leptin induced the proliferation of primary cultured human NP cells, which expressed the leptin receptors OBRa and OBRb. Induction of NP cell proliferation was confirmed by CCK8 assay and immunocytochemistry and Real-time PCR for PCNA and Ki-67. Mechanistically, leptin induced the phosphorylation of STAT3, Akt and ERK1/2 accompanied by the upregulation of cyclin D1. Pharmacological inhibition of JAK/STAT3, PI3K/Akt or MEK/ERK signaling by AG490, Wortmannin or U0126, respectively, reduced leptin-induced cyclin D1 expression and NP cell proliferation. These experiments also revealed an intricate crosstalk among these signaling pathways in mediating the action of leptin. Taken together, we show that leptin induces human NP cell cyclin D1 expression and proliferation via activation of JAK/STAT3, PI3K/Akt or MEK/ERK signaling. Our findings may provide a novel molecular mechanism that explains the association between obesity and intervertebral disc degeneration. The authors have no support or funding to report. ==== Body Introduction The high morbidity of low back pain causes severe incapacity that increases medical expense and impacts the workforce, posing high socioeconomic costs [1]. Effective treatment of low back pain is therefore a matter of great public concern. Athough the etiology of low back pain is multifactorial, intervertebral disc degeneration (IVD) is thought to be a major cause [2]. IVD is a process that is influenced by genetic predisposition, lifestyles (e.g. occupation, smoking, alcohol consumption), co-morbidities (e.g. obesity and diabetes), and aging [3]. Several biomechanical parameters, such as height, fluid pressurization, dissipation, stiffness, and flexibility, are implicated in the initiation and progression of IVD [4]. Other factors, such as formation of cell cluster and the proliferation of fibrocartilaginous tissue, may also take part in IVD [5]. Thus far, the cause of increased cell proliferation in IVD remains unclear. First described in 1994, leptin (the 16 kDa product of the OB gene) is a peptide hormone secreted mainly by adipose tissues [6]. It is also produced by a variety of cells including placental cells and gastric epithelial cells [7]. Fibrocartilaginous tissues, including articular cartilage and intervertebral disc, hace been recently recognized as other sources of leptin [8]. Serum leptin levels are positively associated with body weight, implicating the involvement of this hormone in the regulation of food intake [9]. In addition, leptin is implicated in the modulation of other physiological processes, such as angiogenesis, wound healing, central and peripheral endocrine actions, and renal and pulmonary functions [10]. Emerging evidence suggest that leptin may function as a growth factor to stimulate cell proliferation in a tissue-dependent manner [11]. For instance, exogenous leptin induces sustained proliferative responses in prostate and lung eptithelial cells, pancreatic beta cells as well as breast and gastric cancer cells [12]. A recent study has shown that human herniated disc tissues and rat NP cells express leptin and its functional receptor [13]. Leptin also stimulates the proliferation of rat NP cells in vitro [14]. Nevertheless, it remains unclear whether leptin can induce human NP cell proliferation. Moreover, the mechanism of leptin-induced NP cell proliferation has not yet been elucidated in human or animals. Leptin exerts its action through its cell membrane receptor that belongs to class I cytokine receptor family. Thus far, six receptor isoforms, designed as OBRa to OBRf, have been identified [15]. However, only OBRb (the long isoform of the OBR) contains intracellular motifs required for the initiation of intracellular signaling [16]. OBRb mediates the action of leptin via multiple signaling pathways, including Janus kinase/signal transducers and activators of transcription (JAK/STAT), mitogen-activated protein kinases (MAPK), protein kinase C (PKC), nitric oxide (NO), and cyclic AMP pathways [17]. In other cell types, leptin also activates extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K) in a SHP-2- and IRS-dependent manner, respectively [18]. The complexity of leptin signaling is increased by the crosstalk among these signaling pathways. The involvement of these pathways in leptin signaling in NP cells remains unknown. Obesity is an established risk factor for IVD in which leptin may play a significant role in its pathogenesis. In the present study, we determined the effect of leptin on the proliferation of primary cultured human NP cells and delineated the underlying molecular mechanism. Results Morphology, Immunofluorescence Characterization and Real-time Quantitative PCR Validation of Primary Cultured Human NP cells After approximately 1 week, the primary cultured human NP cells reached almost complete confluence. The cells were polygonal, with multiple cytoplasmic processes and granular cytoplasm (Fig. 1A). Immunofluorescence for collagenase type II and cytokeratin 19 was observed in these cells. Figure 1C and 1D shows a typical field observed in these cultures. CA12, which was recently decribed by Minogue et al as a marker gene for human NP cells, was found to have significantly higher levels of expression in NP cells and NP tissue than in AF tissue and chondrocytes (Fig. 1D). IBSP and FBN1, which were also recently decribed by Minogue et al as negative markers gene for human NP cells, was found to have significantly low levels of expression in NP cells and NP tissue than in AF tissue and chondrocytes (Fig. 1E and 1F). 10.1371/journal.pone.0053176.g001Figure 1 Morphology, immunofluorescence characterization and real-time quantitative PCR validation of primary cultured human NP cells. (A) Phase-contrast photomicrograph of primary NP cells cultured in vitro for about 1 week, just before reaching complete confluence. Original magnification, ×40. (B) Real-time RT-PCR analysis of novel NP cell marker gene CA12 in NP cells, chondrocytes, NP and AF. Real-time RT-PCR analysis was performed in triplicate and the expression levels of CA12 mRNAs were normalized to GAPDH mRNAs. Error bars represent standard deviration. (C) Fluorescence microscopy images showing collagenase type II were observed in NP cells. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40× objective. (D) Fluorescence microscopy images showing cytokeratin 19 were observed in NP cells. Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40× objective. Real-time RT-PCR analysis of novel negative NP cell marker gene IBSP (E) and FBN1 (F) in NP cells, chondrocytes, NP and AF. Real-time RT-PCR analysis was performed in triplicate and the expression levels of IBSP and FBN1 mRNAs were normalized to GAPDH mRNAs. Human NP cells Expressed Leptin Receptors The expression of two isoforms of leptin receptors, namely OBRa and OBRb, was determined in primary cultured human NP cells by RT-PCR and Real-time RT-PCR. Results indicated that the mRNA expression of both isoforms was detected in human NP cells (Fig. 2A and 2B), hinting at the possibility that NP cells are capable of receiving leptin signals, at least in part, via these two OBRs. 10.1371/journal.pone.0053176.g002Figure 2 Leptin stimulates NP cells growth. (A) The mRNA expression of OBRa and OBRb were detected in human NP cell with RT-PCR. The amplified cDNA fragments, 116 bp GAPDH, 609 bp OBRb, and 200 bp OBRa, together with DNA marker were electrophoresed. (B) Real-time RT-PCR analysis of OBRa and OBRb mRNA in NP cells. Real-time RT-PCR analysis was performed in triplicate and the expression levels of OBRa and OBRb mRNAs were normalized to GAPDH mRNAs. Error bars represent standard deviration. (C) For the dose-development studies NP cells were treated with either complete culture medium only or varying concentrations of leptin (1–1500 ng/ml) for 48 or 72 h. (D) For the time-development studies, NP cells were treated either complete culture medium only or leptin (10 ng/ml) for varying time intervals (24–96 h). (E) To further compare the cell viabilities before and after leptin treatment, NP cells were serum starved before treatment (day -1), and 1 day later treated with 10 ng/ml leptin, serum in serum-free media or vehicle (control group) over 2 days. Values are presented as mean ±SD (n = 4). The statistically differences compared with the control are noted as *p<0.05, and ***p<0.001. Data represent three independent experiments. Leptin Induced Human NP cell Proliferation In serum-replete conditions, increasing concentrations of leptin (1–1500 ng/ml) significantly increased NP cell proliferation in a dose-dependent manner, with the maximal response at 1000 ng/ml at 48 h (Fig. 2C). Time-dependent induction of NP cell proliferation by leptin (10 ng/ml), a concentration within the range of plasma concentrations found in obese individuals, was observed with the maximal response at 96 h (Fig. 2D). The pro-survival effect of leptin on the loss of NP cell viability induced by serum deprivation was also examined. In this set of experiments, NP cells were pre-incubated in serum-free medium for 1 day before leptin stimulation in which serum deprivation was continuously maintained. As shown in Figure 2E, serum deprivation reduced the number of viable NP cells in all groups. Nevertheless, the number of viable cells in the leptin-treated group was signifiantly higher than that of the serum-free group but was not significantly different from the serum-replete group. This finding suggests that, in addition to its proliferative effect, leptin may maintain NP cell survival in face of nutrient starvation. The proliferative effect of leptin was confirmed immunohistochemical staining of PCNA and Ki-67 in NP cells treated with or without leptin (Fig. 3A, 3B). As shown in Figure 3C, 3F, there was a significant increase in the percentage of PCNA-positive and Ki-67-positive NP cells in the group treated with 10 ng/ml leptin as compared with the control group. Treatment with leptin (10 ng/ml) significantly increased PCNA and Ki-67 mRNA level in a time-dependent manner, with a maximal response at 48 h or 24 h respectively (Fig. 3D, 3G). 10.1371/journal.pone.0053176.g003Figure 3 Immunohistochemical staining of NP cells against PCNA and Ki-67. After 1-day serum deprivation, NP cell were treated vehicle (control), 10 ng/ml leptin in serum-free medium for 48 h. Immunohistochemical staining of NP cells against PCNA (A) and Ki-67(B). Nuclei were stained with DAPI, shown in blue. Images were acquired using laser scanning confocal microscopy under a 40× objective. PCNA-positive (C) and Ki-67(D) percentages in cultured NP cells 48 h after different concentrations of leptin and vehicle (control). Values are presented as mean ±SD. As compared with control, **p<0.01. (C–D) Real-time RT-PCR analysis of PCNA and Ki-67 mRNA expression in NP cells following leptin treatment for 0, 12, 24, 48 h, or 72 h. Real-time RT-PCR analysis was performed in triplicate and the expression levels of PCNA and Ki-67 mRNAs were normalized to GAPDH mRNAs. Error bars represent standard deviration. (E–F) NP cells were serum starved for 24 h, and then treated with vehicle (−), 10 ng/ml leptin (+), 10 µM U0126 (U0), 40 µM AG490 (AG) or 250 nM wortmannin (Wort) for 48 h. PCNA and Ki-67 mRNA expression were detected with Real-time RT-PCR analysis using GAPDH as an internal control. Error bars represent standard deviration. Leptin Induced STAT3, Akt and ERK Phosphorylation in Human NP cells Leptin has been shown to instigate intracellular signaling via activation of JAK2/STAT3, PI3K/Akt, and MEK/ERK pathways in other cell types. In the present study, the effect of leptin on the activities of these pathways in human NP cells was determined by Western blot for total and phosphorylated STAT3, Akt, and ERK1/2. As shown in Figure 4, leptin stimulation time-dependently increased the phosphorylation of STAT3, Akt and ERK1/2 without altering the total protein levels. The induction of STAT3, Akt and ERK1/2 phosphorylation could be observed as early as 5 min after leptin stimulation and the maximal stimulation occurred at 30 min, 15 min and 5 min post-stimulation, respectively. These findings indicate that leptin could readily activate JAK2/STAT3, PI3K/Akt, and MEK/ERK pathways in human NP cells. 10.1371/journal.pone.0053176.g004Figure 4 Leptin activates phosphoryolations of STAT3, Akt, and ERK1/2 in NP cells. After 1-day serum deprivation, leptin (10 ng/ml) was added into the serum-free medium of NP cells for 5 min, 15 min and 30 min, and then the protein amouts of phosphorylated forms of Akt (p- Akt) (A), ERK1/2 (p- ERK1/2) (B) or STAT3 (p- STAT3) (C) were detected with western blotting analysis. GAPDH was also detected for a loading control. Pharmacological Inhibition of JAK2/STAT3, PI3K/Akt, and MEK/ERK Pathways Prevented Leptin-induced NP cells Proliferation To examine the possible involvements of JAK2/STAT3, PI3K/Akt, and MEK/ERK pathways in leptin-induced NP cell proliferation, NP cells were treated with or without AG490 (JAK inhibitor), wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor), alone or in combination, in the absence or presence of leptin. Results from CCK8 cell proliferation assays showed that AG490 or U0126 remarkably reduced leptin-induced NP cell proliferation whereas wortmannin exerted only modest inhibition. However, co-inhibition of PI3K and MEK with wortmannin and U0126 could almost completely block the proliferative effect of leptin in NP cells as in other combined treatment groups (AG490+wortmannin, AG490+U0126, AG490+wortmannin+U0126). In contrast, these inhibitors per se did not significantly alter NP cell proliferation, indicating that inhibition of JAK2/STAT3, PI3K/Akt, and MEK/ERK pathways specifically blocked the proliferative effect of leptin (Fig. 5). 10.1371/journal.pone.0053176.g005Figure 5 Pharmacological inhibitors of JAK, PI3K/Akt, and MEK/ERK1/2 prevent NP cells growth from leptin induction. After 1-day serum deprivation, NP cell were treated vehicle (control), 10 ng/ml leptin (Lep), 40 µM AG490 (AG), 250 nM wortmannin (Wort), 10 µM U0126 (U0126), or 10 ng/ml leptin together with 40 µM AG490 (Lep+AG), 250 nM wortmannin (Lep+Wort), 10 µM U0126 (Lep+U0126), 40 µM AG490 and 10 µM U0126 (Lep +U0126+ AG), 40 µM AG490 and 250 nM wortmannin (Lep+AG+Wort), 10 µM U0126 and 250 nM wortmannin (Lep+U0126+Wort), 10 µM U0126, 40 µM AG490 and 250 nM wortmannin (Lep+U0126+ Wort+AG) in serum-free medium for 48 h. Values are presented as mean ±SD (n = 4). The statistically differences compared with the control are noted as *p<0.05, and ***p<0.001. Data represent three independent experiments. Crosstalk Among JAK/STAT3, PI3K/Akt, and MEK/ERK Pathways in Leptin-stimulated NP cells The data presented so far indicates that JAK/STAT3, PI3K/Akt, and MEK/ERK pathways mediated the mitogenic effect of leptin in NP cells. Whether there is crosstalk among these three signaling pathways remained unclear. Western blot analysis indicates that U0126, AG490 and wortmannin significantly reduced leptin-induced ERK1/2, STAT3 and Akt phosphorylation, respectively. Interestingly, in addition to its effect on STAT3 phosphorylation, JAK2 inhibitor AG490 also partially reduced phosphorylation of ERK1/2 but not Akt induced by leptin. In contrast, MEK inhibitor U0126 reduced phosphorylation of ERK1/2, STAT3 and Akt while PI3K inhibitor wortmannin specifically reduced Akt phosphorylation induced by leptin (Fig. 6). 10.1371/journal.pone.0053176.g006Figure 6 Crosstalk among JAK/STAT3, PI3K/Akt, and MEK/ERK pathways in leptin-stimulated NP cells. After 1-day serum deprivation and then 30-min pre-treatment of vehicle (−), 10 µM U0126 (U0126), 40 µM AG490 (G) or 250 nM wortmannin (Wort), NP cells were treated with vehicle (−) or 10 ng/ml leptin (+) in serum-free media for 30 min respectively. Following, the protein amounts of phosphorylated of Akt (p- Akt) (A), ERK1/2 (p- ERK1/2) (B) or STAT3 (p- STAT3) (C) were detected with western blotting analyses. The same blots were stripped and reprobed with antibodies specific for total proteins of Akt, ERK1/2, or STAT3. GAPDH was also detected for a loading control. Data represent three independent expriments. Leptin Induced Cyclin D1 Expression in a JAK-, PI3K-, and MEK-dependent Manner Increased cyclin D1 expression is known to promote cell cycle progression during G1-S transition. Here we examined the possible involvement of cyclin D1 in leptin-induced NP cell proliferation and its relationship with the JAK/STAT3, PI3K/Akt, and MEK/ERK pathways. Western blot and Real-time RT-PCR analysis show that leptin time-dependently increase cyclin D1 protein and mRNA expression in human NP cells, with the both maximal response at 72 h. Furthermore, inhibitors of JAK (AG490), PI3K (wortmannin) or MEK (U0126) blocked leptin-induced cyclin D1 protein and mRNA expression (Fig. 7). 10.1371/journal.pone.0053176.g007Figure 7 Leptin induces NP cells’cyclin D1 expression and pharmacological inhibitors of JAK, PI3K/Akt, and MEK/ERK1/2 prevent NP cells’cyclin D1 expression from leptin induction. (A) After 1-day serum deprivation, NP cells were incubated in serum-free media containing 10 ng/ml leptin for varying time intervals (12–96 h), and then the amouts of cyclin D1 protein were detected with western blotting analysis using GAPDH as an internal control. (B) NP cells were serum starved for 24 h, and then treated with vehicle (−), 10 ng/ml leptin (+), 10 µM U0126 (U0), 40 µM AG490 (AG) or 250 nM wortmannin (Wort) for 48 h. The amouts of cyclin D1 protein were detected with western blotting analysis using GAPDH as an internal control. (C) Real-time RT-PCR analysis of cyclin D1 mRNA expression in NP cells following leptin treatment for 0, 12, 24, 48 h, 72 h, or 96 h. Real-time RT-PCR analysis was performed in triplicate and the expression levels of cyclin mRNAs were normalized to GAPDH mRNAs. Error bars represent standard deviration. (D) NP cells were serum starved for 24 h, and then treated with vehicle (−), 10 ng/ml leptin (+), 10 µM U0126 (U0), 40 µM AG490 (AG) or 250 nM wortmannin (Wort) for 48 h. Cyclin D1 mRNA expression were detected with Real-time RT-PCR analysis using GAPDH as an internal control. Error bars represent standard deviration. Discussion Increasing epidemiological evidence has supported that obesity is closely associated with IVD [19]. The cellular and molecular mechanism of obesity-related IVD, however, remains unclear. In this respect, leptin, a hormone with increased circulating levels in obese patients, has been implicated in the pathogenesis obesity-related IVD. We first characterized NP cells by assessing the morphology, the expression of collagenase type II, cytokeratin 19, CA 21, IBSP and FBN1. The results showed that NP cells in this study possessed the above these characteristics of NP cells, which was consistent with previous research [20], [21]. In this study, we demonstrated that leptin directly stimulated proliferation of human NP cells which expressed leptin receptors OBRa and OBRb. The mitogenic effect of leptin was supported by CCK-8 assay, immunostaining for two proliferative markers PCNA and Ki-67 as well as induction of cyclin D1. In this regard, disc cell proliferation is known to be associated with IVD and is likely the cause of cluster formation. In fact, proliferating cells have been found to be defective in the synthesis of normal matrix components and thereby promoting disc degeneration. These findings suggest that increased NP cell proliferation induced by leptin may be one of the possible mechanisms underlying the detrimental influence of obesity on the development of IVD; on the other hand, the effect induced by leptin on NP cell may be fine for normal intervertebral disc. Although leptin functions as a growth factor in various types of cells, the exact mechanism of leptin-induced cell proliferation is not fully understood. In the present study, we showed that leptin-induced NP cell proliferation was accompanied with increased phosphorylation of STAT3, Akt and ERK1/2 and upregulation of cyclin D1. Inhibition of JAK, PI3K or MEK also reduced leptin-induced cyclin D1 expression and NP cell proliferation. These findings suggest that leptin induces cyclin D1 Expression and proliferation of human NP cells via JAK/STAT, PI3K/Akt and MEK/ERK pathways. Cyclin D1 is an important mediator that controls cell cycle transition from G1-to-S-phase [22]. In this regard, leptin has been shown to induce cyclin D1 in human breast and endometrial cancer cells as well as hepatocarcinoma cells[23]–[25]. To this end, the regulation of cyclin D1 and cell proliferation by JAK/STAT, PI3K/Akt and MEK/ERK signaling has been widely reported, especially in cancer biology studies [26]. Here, we demonstrate that the concomitant activation of JAK/STAT, PI3K/Akt and MEK/ERK pathways is required for induction of cyclin D1 and cell proliferation by leptin in human NP cells. In fact, other cytokines, such as PDGF, bFGF and IGF-I have been found to stimulate the proliferation of disc cells via the ERK and Akt signaling pathways and are implicated in the development of IVD [27]. Leptin acts via transmembrane receptors, which are structurally similar to the class I cytokine receptor family. Leptin receptor OBR is produced in several alternatively spliced forms designated OBRa to OBRf [15]. These OBRs are expressed in a variety of tissue including lung, kidney, liver, stomach and articular cartilage [28], [29]. Among these OBRs, OBRb is the best studied and believed to be the major signal mediator of leptin. Activation of leptin receptors has been previously shown to stimulate JAK2/STAT3 pathway, PI3K/Akt and MEK/ERK pathways in other cell types [25]. Nevertheless, only sporadic studies have attempted to delineate their crosstalk. A previous study has shown that MEK inhibition blocked the activation of PI3K/Akt induced by leptin in human papillary thyroid cancer cells [30]. In line with this finding, our data suggest that leptin-induced Akt phosphorylation was dependent on MEK activity. Another important finding emerging from this work is that there is a reciprocal regulation between JAK2/STAT3 and MEK/ERK pathways, in which U0126 (MEK inhibitor) abolished STAT3 phosphorylation while AG490 (JAK inhibitor) partially reduced ERK phosphorylation induced by leptin in human NP cells. This finding is in discrepancy with those reported by Trinko et al. showing that U0126 blocked leptin-induced phosphorylation of ERK1/2 but not STAT3 in the central nervous system of rats [31]. Yin et al. also showed that JAK inhibition but not MEK inhibition prevented the growth stimulation of breast cancer cells by leptin [32]. The mechanism underlying these discrepancies, however, warrants further investigation. One of the drawbacks of this study is the lack of age-matched non-degenerate discs as control. NP cells derived from LDD patients may not necessarily reflect the in vivo scenario. This study only evaluated the effect of leptin on LDD NP cells. The comparion of the reaction of LDD and normal NP cells to leptin stimulation should provide further information for the potential involvement of leptin in LDD development. In conclusion, the evidence presented in this work indicates that leptin stimulates proliferation of human NP cells. The mitogenic effect of leptin is mediated through upregulation of cyclin D1 via concomitant activation of JAK/STAT3, PI3K/Akt, or MEK/ERK pathways that may become potential targets for pharmacological intervention. Our data also reveal an unreported signaling crosstalk among JAK/STAT3, PI3K/Akt, or MEK/ERK pathways in mediating the action of leptin. These molecular alterations constitute a possible link between obesity and an increased risk for IVD. Materials and Methods Ethics Statement All of the experimental protocols were approved by the Clinical Research Ethics Committee of the Peking Union Medicial College Hospital. Human lumbar IVD samples obtained from patients undergoing discectomy following approval from the Clinical Research Ethics Committee of the Peking Union Medicial College Hospital and fully informed written consent of patients. Reagents Inhibitors were purchased from the following sources: JAK inhibitor AG490 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide), MEK inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) and PI3K inhibitor Wortmannin were purchased from Sigma. All other chemicals and reagents were purchased from Sigma unless otherwise specified. Isolation and Primary Culture of Human NP cells The human NP cells were dissected from patient disc surgical specimens (8 donors, 31–40 years, and average Thompson degeneration grade 2–3). All of these NP tissues was isolated from patients underwent surgeries for disc degeneration (L4/5) and not disc herniation, and therefore contact between these tissues and cells outside of the disc, these are macrophages, endothelial cells and other immune cells, were minimal or nonexistent. No granulation tissue was present. Two cell types populate the human NP: notochordal cells and chondrocyte-like cells [33]. As the notochordal cells slowly decline in abundance and appear to be absent after 10 years of age [34], NP tissues of human adolescents and adults consist of only chondrocyte-like cells. NP cells were isolated and cultured as previously described [35], [36]. Tissues specimens were first washed thrice with PBS, NP was separated from the AF using a stereotaxic microscope, then cut into pieces (2–3 mm3), and NP cells were released from the NP tissues by incubation with 0.25 mg/ml type II collagenase for 12 h at 37°C in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Grand Island, NY). After isolation, NP cells were resuspended in DMEM containing 10% FBS (GIBCO, NY, USA), 100 µg/ml streptomycin, 100U/ml penicillin and 1% L-glutamine, and then incubated at 37°C in a humidified atmosphere with 95% air and 5% CO2. The confluent cells were detached by trypsinization, seeded into 35-mm tissue culture dishes in complete culture medium (DMEM supplemented with 10% FBS, 100 µg/ml streptomycin and 100U/ml penicillin) in a 37°C, 5% CO2 environment. The medium was charged every 2 days. NP cells cultured in vitro within 10 days, the second passage was used for subsequent experiments. Reverse Transcription (RT)-polymerase Chain Reaction (PCR) for Detection of OBRa/b Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufactuer’s instructions. The RNA was quantified by absorbance at 260 nm. cDNA was sythesized from 2 µg of total RNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Oligo (dT)18 was used as the RT primer for the reverse transcription of mRNAs. Briefly, 2 µg total RNA with 1 µl dNTP and 1 µM Oligo (dT)18 in a total volume of 8 µl were denatured at 65°C for 5 min, immediately cooled on ice, and incubated with the reverse transcriptase reaction mixture supplemented with 1U RNase inhibitor in a total volume of 20 µl at 37°C for 60 min to generate the first-stand cDNA. The reaction was terminated by incubation at 55°C for 5 min and rapidly cooled on ice. The reverse-transcribed cDNA was further PCR-amplified by specific primers in 20 µl PCR mixture (cDNA, 0.25 mM NTP, 1 µM forward and reverse primers, 0.5 U Tap) for 35 cycles. The primer sets for detection of OBR and GAPDH are listed in Table 1. 10.1371/journal.pone.0053176.t001Table 1 Nucleotide sequences of primers used in Real-time RT-PCR. Gene Forword Primer Reverse Primer Tm (°C) Productsize (bp) CA12 CGTGCTCCTGCTGGTGATCT AGTCCACTTGGAACCGTTCACT 60 70 OBRa TTGTGCCAGTAATTATTTCCTCTT AGTTGGCACATTGGGTTCAT 56 200 OBRb CCAGAAACGTTTGAGCATCT CAAAAGCACACCACTCTCTC 56 609 Cyclin D1 AAC TACCTGGACCGCTTCCT CCACTT GAGCTTGTTCACCA 56 204 PCNA AGTGGAGAACTTGGAAATGGAA GAGACATGGAGTGGCTTTTGT 56 154 Ki-67 TCCTTTGGTGGGCACCTAAGACCTG TGATGGTTGAGGTCGTTCCTTGATG 56 156 IBSP CCAGAGGAAGCAATCACCAAA GCACAGGCCATTCCCAAA 60 68 FBLN1 CCTTCGAGTGCCCTGAGAACTA ACCGATGGCCTCATGCA 60 74 GAPDH TCAACGACCACTTTGTCAAGCTCAGCT GGTGGTCCAGGGGTCTTAC 56 116 Real-time PCR Total mRNA was extracted from cells by using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RNA was isolated with chloroform and isopropanol, washed with ethanol, and dissolved in water. Quantification of RNA was based on spectrophotometric analysis at 260/280 nm with values between 1.8 and 2.0 confirmed the purity of the RNA samples. A 2-µg sample of total RNA was reverse-transcribed with 200 U of MMLV reverse transcriptase (Invitrogen) using Oligo(dT) primers in a 20 µL reaction mixture following the manufactures’ instructions. Relative transcript levels of OBRa, OBRb, CA21, Cyclin D1, PCNA and Ki-67 mRNA were determined by real-time PCR using the iQ5 Real-Time PCR Detection System (Bio-Rad, California, USA). The real-time PCR reaction was composed of 1x SYBR Green fluorescent dye (Takara, Dalian, China), 1 µl forward primers (10 µM), 1 µl reverse primers (10 µM), 1x qPCR mix, 1 µl cDNA. The sequences of the specific primers are shown in Table. 1. To produce the melting curve, the reactions were subject to one step at 95°C for 30 s followed by 45 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 30 s. The relative gene expression was assessed by the ΔΔCt method. GAPDH was used as an internal control. Determination of NP cell Proliferation by Cell Counting Kit-8 (CCK8) Assay NP cells were seeded in 96-well plates at the density of 1000 cells per well with 100 µl of complete culture medium. After adhesion for 24 hours, the medium was change to DMEM/F-12 supplemented with or without 5% (v/v) FBS and recombinant human leptin (Sigma-Aldrich, Oakville, ON, Canada) was added to the medium to final concentrations ranging from 1 ng/ml to 1000 ng/ml. The cells were then cultured for another 24, 48 or 96 h. Cells that did not exposed to leptin were used as controls and the wells to which only culture medium was added served as blanks. At the end of leptin stimulation, the supernatant was removed, and 100 µl of DMEM/F12 medium containing 10 µl of CCK8 was added to each well for another 3 h at 37°C. The culture plates were then shaken for 10 min and the optical density (OD) values were read at 450 nm. Immunohistochemistry for Proliferative Markers PCNA and Ki-67 Coverslips were placed into 24-well plate and then NP cells were plated and treated with or without 10 ng/ml leptin for 48 h. Afterwards, medium was removed and the cells were washed twice with PBS and fixed with 3.5% formaldehyde for 30 min at 37°C. The cells were rinsed with PBS for 3 times, permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min and blocked with 3% (w/v) BSA and 0.05% (v/v) Tween 20 in PBS for 30 min at room temperature. After blocking, the cells were incubated overnight at 4°C with primary antibody. The antibodies used were as follows: rabbit monoclonal anti-PCNA antibody (dilution ratio 1∶500, Bioworlde, USA) and rabbit monoclonal anti-Ki-67 antibody (dilution ratio 1∶500, Bioworlde, USA). The cells were then treated with fluorescent anti-rabbit secondary antibody (1∶500, Bioworlde, USA) for 2 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were acquired with a Leica TCS SP2 confocal microscopy (Leica, Mannheim, Germany) using the Leica Confocal Software. Western Blot Analysis for Cyclin D1 and Total and Phosphorylated STAT3, ERK1/2 and Akt Western blot was performed as described with modifications [37]. Briefly, total cellular proteins were extracted from NP cells with the lysis buffer and separated on 10% SDS-PAGE gel. After electrophoresis, proteins were electrotranferred onto the nitrocellulose membrane (Millipore, MA, USA). The membrane was then blocked with 5% (w/v) skim milk in TBST (20 nM Tris-HCL(pH 7.6), 137 nM NaCl, and 0.1% Tween-20), probed with appropriate primary antibodies at 4°C overnight and bound with HRP-conjugated secondary antibodies at room temperature for 1 h. Chemiluminescent signal was detected using ECL kit (Millipore, MA, USA) and autoradiography. Statistical Analysis Results were expressed as means ± SD of multiple experiments. Statistical analysis was performed with Student’s t-test for comparison between two groups or an analysis of variance (ANOVA) followed by the Turkey’s t-test for comparison of multiple groups. P values less than 0.05 were considered statistically significant. We thank Prof. Hong Zhao and Prof. Shugong Li for assitance in disc samples collection. Finally, we would like to thank all the volunteers who took part in this study. ==== Refs References 1 Katz JN (2006 ) Lumbar disc disorders and low-back pain: socioeconomic factors and consequences . J Bone Joint Surg Am 88 Suppl 221 –24 .16595438 2 Millecamps M , Tajerian M , Naso L , Sage EH , Stone LS (2012 ) Lumbar intervertebral disc degeneration associated with axial and radiating low back pain in ageing SPARC-null mice . Pain 153 : 1167 –1179 .22414871 3 Tzaan WC , Chen HC (2011 ) Investigating the possibility of intervertebral disc regeneration induced by granulocyte colony stimulating factor-stimulated stem cells in rats . 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Spine J 7 : 437 –443 .17433782 14 Zhao CQ , Liu D , Li H , Jiang LS , Dai LY (2008 ) Expression of leptin and its functional receptor on disc cells: contribution to cell proliferation . Spine (Phila Pa 1976) 33 : E858 –864 .18978578 15 Fruhbeck G (2006 ) Intracellular signalling pathways activated by leptin . Biochem J 393 : 7 –20 .16336196 16 Cottrell EC, Mercer JG (2012) Leptin receptors. Handb Exp Pharmacol: 3–21. 17 Lam QL , Lu L (2007 ) Role of leptin in immunity . Cell Mol Immunol 4 : 1 –13 .17349207 18 Pai R , Lin C , Tran T , Tarnawski A (2005 ) Leptin activates STAT and ERK2 pathways and induces gastric cancer cell proliferation . Biochem Biophys Res Commun 331 : 984 –992 .15882975 19 Samartzis D , Karppinen J , Mok F , Fong DY , Luk KD , et al (2011 ) A population-based study of juvenile disc degeneration and its association with overweight and obesity, low back pain, and diminished functional status . J Bone Joint Surg Am 93 : 662 –670 .21471420 20 Dahia CL , Mahoney E , Wylie C (2012 ) Shh signaling from the nucleus pulposus is required for the postnatal growth and differentiation of the mouse intervertebral disc . PLoS One 7 : e35944 .22558278 21 Minogue BM , Richardson SM , Zeef LA , Freemont AJ , Hoyland JA (2010 ) Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation . Arthritis Rheum 62 : 3695 –3705 .20722018 22 Shimura T (2011 ) Acquired radioresistance of cancer and the AKT/GSK3beta/cyclin D1 overexpression cycle . J Radiat Res 52 : 539 –544 .21881296 23 Saxena NK , Vertino PM , Anania FA , Sharma D (2007 ) leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3 . J Biol Chem 282 : 13316 –13325 .17344214 24 Catalano S , Giordano C , Rizza P , Gu G , Barone I , et al (2009 ) Evidence that leptin through STAT and CREB signaling enhances cyclin D1 expression and promotes human endometrial cancer proliferation . J Cell Physiol 218 : 490 –500 .18988190 25 Chen C , Chang YC , Liu CL , Liu TP , Chang KJ , et al (2007 ) Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathway . Endocr Relat Cancer 14 : 513 –529 .17639064 26 Creamer BA , Sakamoto K , Schmidt JW , Triplett AA , Moriggl R , et al (2010 ) Stat5 promotes survival of mammary epithelial cells through transcriptional activation of a distinct promoter in Akt1 . Mol Cell Biol 30 : 2957 –2970 .20385773 27 Pratsinis H , Kletsas D (2007 ) PDGF, bFGF and IGF-I stimulate the proliferation of intervertebral disc cells in vitro via the activation of the ERK and Akt signaling pathways . 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PLoS One 6 : e27180 .22076135 32 Yin N , Wang D , Zhang H , Yi X , Sun X , et al (2004 ) Molecular mechanisms involved in the growth stimulation of breast cancer cells by leptin . Cancer Res 64 : 5870 –5875 .15313931 33 Trout JJ , Buckwalter JA , Moore KC (1982 ) Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus . Anat Rec 204 : 307 –314 .7181135 34 Roughley PJ (2004 ) Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix . Spine (Phila Pa 1976) 29 : 2691 –2699 .15564918 35 Studer RK , Vo N , Sowa G , Ondeck C , Kang J (2011 ) Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-alpha . Spine (Phila Pa 1976) 36 : 593 –599 .21178846 36 Navone SE , Marfia G , Canzi L , Ciusani E , Canazza A , et al (2012 ) Expression of neural and neurotrophic markers in nucleus pulposus cells isolated from degenerated intervertebral disc . 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PLoS One. 2012 Dec 31; 7(12):e53176
==== Front Comput Math Methods MedComput Math Methods MedCMMMComputational and Mathematical Methods in Medicine1748-670X1748-6718Hindawi Publishing Corporation 10.1155/2012/549102Research ArticleBayes Clustering and Structural Support Vector Machines for Segmentation of Carotid Artery Plaques in Multicontrast MRI Guan Qiu 1 Du Bin 1 Teng Zhongzhao 2 Gillard Jonathan 2 Chen Shengyong 1 *1College of Computer Science, Zhejiang University of Technology, Hangzhou 310023, China2Department of Radiology, University of Cambridge, Hills Road, Cambridge CB2 0SP, UK*Shengyong Chen: [email protected] Editor: Carlo Cattani 2012 19 12 2012 2012 5491026 10 2012 19 11 2012 Copyright © 2012 Qiu Guan et al.2012This 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.Accurate segmentation of carotid artery plaque in MR images is not only a key part but also an essential step for in vivo plaque analysis. Due to the indistinct MR images, it is very difficult to implement the automatic segmentation. Two kinds of classification models, that is, Bayes clustering and SSVM, are introduced in this paper to segment the internal lumen wall of carotid artery. The comparative experimental results show the segmentation performance of SSVM is better than Bayes. ==== Body 1. Introduction Cardiovascular diseases (CVDs) are the leading cause of death globally according to the recent statistics of the World Health Organization. Atherosclerosis, a kind of systematic inflammatory disease, is estimated to be responsible for CVDs to a great extent. Therefore, there are considerable interests in characterizing atherosclerotic plaques for proper treatment planning. Research in the past 20 years indicates that plaque vulnerability is very relative to its structure, such as the lumen condition, atherosclerotic components within the plaque [1–5]. As the fundamental step, artery wall should be segmented accurately. Meanwhile, explicit detection of wall is very important to locate each component inside the plaque correctly, which is also very significant for the subsequent procedures such as component analysis. Automated analysis of plaque composition in the carotid arteries has been presented by many researchers. Different imaging techniques always bring out distinct characteristic of image, which will restrict different applicable approach to approach of segmentation. Among current standard imaging techniques in clinical, in vivo multicontrast MRI technique has been generally validated to be used to quantify the composition of plaque effectively [6]. Most segmentation methods based on this kind of imaging technique are generally based on manual extraction of numerous contours. Automatic segmentation not only makes the combination of different multicontrast-weighted MR Image possible, but also can further make full use of the advantages of different image to improve the accurate rate of classification of component within lumen. Other impressive experiments are also carried out by taking use of model-based clustering and fuzzy clustering [7], maximum-likelihood classifier and nearest-mean classifier [8], morphology-enhanced probability maps [9], and k-means clustering [10]. Most of these methods are based on voxel-wise statistical classification, and the manual analysis cannot be completely replaced by them. An automatic method which was used to segment the carotid artery plaques in CT angiography (CTA) [11] has potential to replace the manual analysis. Firstly, the vessel lumen was segmented. Subsequently, classifier was trained to classify each pixel. However, this algorithm is needed to be improved to deal with the multicontrast-weighted MR Image. Furthermore, in order to provide a more accurate and objective ground truth, a simultaneous segmentation and registration model [12] is necessary in registration. This method is an active contour model based on simultaneous segmentation and registration which is belong to mutual-information-based registration [13]. Therefore, researches concerning segmentation of plaques are essential. The paper is organized as follows. Significance of studying carotid artery plaque and current research contributions are briefly presented in Section 1. Section 2 is mainly focus on describing major and special preprocessing such as ill-illumination uniforming and image registration. Two kinds of model used to segment the wall boundary are descried in detailed in Section 3. Section 4 focuses on two algorithms to segment the lumen, and a conclusion and further work are presented in Section 5. 2. Testing Image Set The complete process of plaque analysis system is organized as below, which is composed of four modules. Firstly, carotid artery region should be separated from the original MRI image and then move on to the preprocessing parts including noise removal and illumination uniform. After that, the lumen and the outer wall in the images are obtained in turn. The latter operations are related with extracting and modeling essential plaque components, and mechanical analysis based on FSI (fluid-structure interaction) theory will be also introduced to estimate the risk extent of a plaque. The steps in Figure 1 will be discussed in detail in this paper. 2.1. Acquisition of Testing Image Set Images used in our research are acquired by a MRI scanner named GE SIGNA. Taking Figure 2(a) for instance, it can be found that carotid arteries marked by two rectangles are closely surrounded by other tissues as muscles, fat, bones, and other vessels in the 512 mm × 512 mm MRI image. In order to handle carotid artery alone as shown in Figure 2(b), small ROI of each artery region should be firstly segmented from the original scanning image by picking out the artery centroid which size is 81 mm × 81 mm. The reduction of interested region effectively avoids disturbing from other tissues and also improves the computing speed. The detail of MRI acquisition has already been published in [14]. Briefly speaking, patients undergo high resolution MRI of their carotid arteries in a 1.5 Tesla MRI system (named as Signa HDx GE Healthcare, Waukesha, WI, USA) with a 4-channel phased-array neck coil (named as PACC, Machnet BV, Elde, The Netherlands). Artifact resulted from movement is minimized by using a dedicated vacuum-based head restraint system (VAC-LOK Cushion, Oncology Systems Limited, UK). It is used to fix the head and neck of patient in a comfortable position to avoid occurrence of artefact. After an initial coronal localizer sequence is sampled and tested, 2-dimensional (2D) axial time-of-fight (TOF) MR angiography is performed to identify the location of the carotid bifurcation and the region of maximum stenosis. Axial images are acquired through the common carotid artery 12 mm (4 slices) below the carotid bifurcation to a point 12 mm (4 slices) distal to the extent of the stenosis identified on the TOF sequence. This kind of method ensures that the whole region of carotid plaque is completely imaged. To describe the characteristic of different MRI sequence, the following parameters are used: T1 weighted (repetition time/echo time: 1  ×  RR/7.8 ms) with fat saturation, T2 weighted (repetition time/echo time: 2  ×  RR/100 ms) with fat saturation, proton density weighted (repetition time/echo time: 2  ×  RR/7.8 ms) with fat saturation, and short-time inversion recovery (repetition time/echo time/inversion time: 2  ×  RR/46/150 ms). The window of view of each MR image is 10 cm × 10 cm, and size of data matrix is 512 × 512. The spatial resolution achieved of each pixel is 0.39 mm × 0.39 mm. In Figure 2(a), two small ROIs marked by red rectangles are carotid arteries each size of RIO is 81 mm × 81 mm. Figure 2(b) is the amplified images of these two areas. 2.2. Preprocessing Due to the inhomogeneity of coil, the intensity of each image should be adjusted to be relative uniform to obtain relative consistent gray scale for the subsequent segmentation based on clustering. The region (14 mm × 14 mm), which lies in the center of the vessel, is selected as the interesting region. The contrast of the image is increased by a linear transformation, (1) u1=u0−mM−m×255, where u 0 is the initial intensity, u 1 is adjusted intensity, and  M  and  m  are the maximum intensity and minimum intensity of the original image. The adjusted results of intensity uniform are shown in Figure 3. 2.3. Image Registration According to the characteristics of MR image, the contour of lumen is clearly presented in the sequence of T1 which is blood suppressed for short. In Figure 4, mark two feature points in images (a) and (b) as red points. Normally, the luminal bifurcation and narrowest location are selected as marking points for registration. Generally speaking, the image is indistinct as shown in Figure 4. Therefore it is very difficult to mark feature points in some images. In order to deal with this problem, the registration method proposed in this paper is based on prior-constrained segmentation of carotid artery under DOG scale space. As seen from the name, the segmentation algorithm implies two parts. First, inspired by SIFT algorithm, the advantage of difference of Gaussian (DOG) scale space is introduced to catch the edges that seem ambiguous in the original image scale, which is the scale derivative of Gaussian scale space along the scale coordinate. Second, given a simple prior knowledge that the artery wall is near round, a given thickness of carotid artery wall is set to restrict the searching area. Prior shape is critical information for external wall segmentation. The steps to get the wall boundary are shown in Figure 5. Then through minimizing the energy function using a gradient flow, we can achieve the goal of simultaneous segmentation and registration [12]. On the one hard, this new method can reduce the influence of noise on the original images and lead to improved registration, on the other hand it also can improve the precision segmentation, especially for segmentation the blurred images. Given two images I 1 and I 2; C 1 is the object contour of I 1, and C 2 is the object contour of I 2. Establish mapping C 2 = g(C 1). The steps of simultaneous segmentation and registration method are listed as follows. Step 1 Initialize C 1,  g, and C 2. Step 2 Optimize the registration parameters to obtain the optimal mapping function  g. Step 3 Evolute C 1 to obtain the optimum partition line of the current image I 1, and obtain the optimal split line of the current image  I  by C 2 = g(C 1). Step 4 Reach the maximum number of iterative steps, or before and after the two results of the iteration are less than the threshold value then the algorithm stops, ended; otherwise turn to Step 2. 3. Modelling To compare the results of different algorithm of modeling, two kinds of model which are based on Bayes classification algorithm and SSVM (structural support vector machines) are carried out in this paper. 3.1. Building of Training Set From MRI slices with matching histological slices, slices 12 and 25 are selected to generate the training set for segmentation. Images of those two slices are manually segmented based on registered histological results and relative intensity. A total of 549 pixels (each pixel contains 4 densities representation with total 4 different contrast weight) are selected randomly in the investigation. From these segmentation results, each pixel is determined to belong to one of the 4 issue types including lipid (denoted as Z 1), normal issue (denoted as Z 2), calcification (denoted as Z 3), and others (including lumen or outer issue, denoted as Z 4). The training set is used to generate the probability function which is used to determine the probability of tissue type of each pixel in the model based on Bayes classification. 3.2. Model Based on Bayes Classification The most important part of the segmentation algorithms is to determine the probabilities of each pixel. These probabilities represent the likelihood that the tissue of the pixel at the current location is lipid, calcification, normal issue, or others. Maximum classifier is used to determine which issue type the pixel belongs to. Figure 6 gives the flow-chart of our maximum decision probability functional classifier. Where I→ is one pixel of multicontrast weighted MR images transformed by preprocessing, gi(I→) is the decision function, and P(Zi∣I→) is class-conditional probability density function (pdf). By comparing values of four functions, if gi(I→) is the maximum probability value of one pixel, then pixel I→ belongs to Z i and is labeled i. 3.3. Model Based on SSVM Recently, structured prediction has already attracted much attention, and many approaches have also been developed based on it. Structured learning is one of the main approaches of structured prediction, which not only studies the problems with well-structured inputs and outputs but also reveals strong internal correlations. It is formulated as the learning of complex functional dependencies between multivariate input and output representations. Structured learning has significant impact in addressing important computer vision tasks. Figure 7 gives the flowchart of SSVM to obtain gray information. The flowchart of the iterative training of SSVM is given in Figure 8. 4. Comparison The results of segmentation of slices 28 and 34 MR images based on Bayes and SSVM are illustrated in Figure 9. As seen in Figure 9, the segmentation result in term of classification algorithm reveals that the performance of SSVM is much better than that of Bayes due to the former including structural information, and smoothing effect of segmentation of SSVM is also obvious. The results presented by image are inadequate to make evaluations. Here a parameter named misclassification rate is defined to judge the accuracy of each algorithm. In the experiment of this paper, a selected slice MR image is corrupted by global intensity varying from 20% to 40% and adding 1%–9% noise. Misclassification rate, an evaluating criterion, is defined as the ratio of misclassified pixels to total number of pixels of this class. It is formulated as (2) as follows: (2) e(i)=fp+fnn, where e(i) is the misclassification rate of tissue i;  fp  is the false positive responses (pixel belongs to tissue  i but is classified as other tissues);  fn  is the false negative responses (pixel does not belong to tissue i but is classified as tissue type i);  n  is the total number of pixels of tissue type i. The misclassification rate of lumen obtained by Bayes and SSVM algorithm is listed in Table 1. From the statistics shown in Table 1, it can be seen that the misclassification rate caused by SSVM is much lower than that of Bayes. That stands for the performance of SSVM outperforms that of Bayes, especially while the level of noise is higher. 5. Conclusion To summarize, the work in this paper is focus on the first several steps of carotid artery plaque analysis, including preprocessing of MR image, model-based segmentation of lumen, plaque, and external wall. Two kinds of model, Bayes and SSVM, are separately constructed and applied to the detection of internal wall. Receivable boundaries can be both obtained by two algorithms, the results of experiment shows the segmentation performance of SSVM is better than that of Bayes, especially, while the level of noise in image is higher. But there are still some improvements need to be done in the future to break the limitations of the current work. Firstly, improve Bayes to better performance by increasing structural information. Secondly, introduce sequence image tracking technique in research to improve the performance of human interaction to specify the center of lumen. Further effort should focus on estimation of artery location in each MRI slice and take advantage of information gained from previous slice to pick out the artery centroid of current image. Moreover, several other algorithms need to be testified and compared with them when dealing with plaques. Acknowledgments The work was supported in part by the National Science Foundation of China (NSFC no. 61173096, 61103140, and 51075367), Doctoral Fund of Ministry of Education of China (20113317110001), and Zhejiang Provincial S and T Department (2010R10006, 2010C33095). Figure 1 Flow of operations. Figure 2 ROI extraction: (a) original MRI image, (b) extracted images. Figure 3 Preprocessing of selected slices of MR images: (a) a set of original images, (b) resultant images after contrast normalization. Figure 4 Handle marking points for registration: (a) MR images, (b) manual outline, (c) result of registration. Figure 5 Flowchart of multiscale PCA. Figure 6 Flowchart of maximum decision probability functional classifier. Figure 7 Flowchart of SSVM to obtain gray information. Figure 8 Flowchart of the iterative training of SSVM. Figure 9 Two segmentation results of selected slice using multicontrast MR images: (a) testing MR images; (b) automatic segmentation results of Bayes classifier; (c) automatic segmentation results of SSVM process. Table 1 Misclassification rate of lumen for Bayes and SSVM. Noise Misclassification rate Bayes SSVM 1% 3.5 2.6 3% 5.3 4.8 5% 6.5 6.3 7% 10.6 8.5 9% 16.9 9.6 ==== Refs 1 Teng Z He J Degnan AJ Critical mechanical conditions around neovessels in carotid atherosclerotic plaque may promote intraplaque hemorrhage Atherosclerosis 2012 223 2 321 326 22762729 2 Teng Z Degnan AJ Chen S Gillard JH Characterization of healing following atherosclerotic carotid plaque rupture in acutely symptomatic patients: an exploratory study using in vivo cardiovascular magnetic resonance Journal of Cardiovascular Magnetic Resonance 2011 13 1, article 64 3 Chen SY Guan Q Parametric shape representation by a deformable NURBS model for cardiac functional measurements IEEE Transactions on Biomedical Engineering 2011 58 3 480 487 2-s2.0-79952136167 20952325 4 Chen SY Zhang J Zhang H Myocardial motion analysis for determination of tei-index of human heart Sensors 2010 10 12 11428 11439 2-s2.0-78650285617 22163536 5 Chen SY Zhang J Guan Q Liu S Detection and amendment of shape distortions based on moment invariants for active shape models IET Image Processing 2011 5 3 273 285 2-s2.0-79959726461 6 Trivedi RA U-King-Im J Graves MJ Multi-sequence in vivo MRI can quantify fibrous cap and lipid core components in human carotid atherosclerotic plaques European Journal of Vascular and Endovascular Surgery 2004 28 2 207 213 2-s2.0-3242774635 15234703 7 Adame IM van der Geest RJ Wasserman BA Mohamed MA Reiber JHC Lelieveldt BPF Automatic segmentation and plaque characterization in atherosclerotic carotid artery MR images Magnetic Resonance Materials in Physics, Biology and Medicine 2004 16 5 227 234 2-s2.0-4444294114 8 Clarke SE Beletsky V Hammond RR Hegele RA Rutt BK Validation of automatically classifiedmagnetic resonance images for carotid plaque compositional analysis Stroke 2006 37 1 93 97 16339462 9 Liu F Xu D Ferguson MS Automated in vivo segmentation of carotid plaque MRI with morphology-enhanced probability maps Magnetic Resonance in Medicine 2006 55 3 659 668 2-s2.0-33644790879 16470594 10 Karmonik C Basto P Vickers K Quantitative segmentation of principal carotid atherosclerotic lesion components by feature space analysis based on multicontrast MRI at 1.5 T IEEE Transactions on Biomedical Engineering 2009 56 2 352 360 2-s2.0-63849173887 19272944 11 Vukadinovic D Rozie S van Gils M Automated versus manual segmentation of atherosclerotic carotid plaque volume and components in CTA: associations with cardiovascular risk factors International Journal of Cardiovascular Imaging 2012 28 4 877 887 2-s2.0-79956273552 21614484 12 Chen Y Thiruvenkadam S Huang F Gopinath KS Brigg RW Simultaneous segmentation and registration for functional MR images 1 Proceedings of the 16th International Conference on Pattern Recognition 2006 Québec, Canada 747 750 13 Pluim JPW Maintz JBA Viergever MA Mutual-information-based registration of medical images: a survey IEEE Transactions on Medical Imaging 2003 22 8 986 1004 2-s2.0-0043028206 12906253 14 Sadat U Weerakkody RA Bowden DJ Utility of high resolution MR imaging to assess carotid plaque morphology: a comparison of acute symptomatic, recently symptomatic and asymptomatic patients with carotid artery disease Atherosclerosis 2009 207 2 434 439 2-s2.0-70450237302 19520370
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Comput Math Methods Med. 2012 Dec 19; 2012:549102
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23301071PONE-D-12-2760710.1371/journal.pone.0053431Research ArticleBiologyModel OrganismsAnimal ModelsRatMolecular Cell BiologySignal TransductionSignaling CascadesApoptotic Signaling CascadeStress Signaling CascadeCellular Stress ResponsesMedicineCritical Care and Emergency MedicineNeurointensive CareResuscitationNeurologyCerebrovascular DiseasesIschemic StrokeHypothermia Protects the Brain from Transient Global Ischemia/Reperfusion by Attenuating Endoplasmic Reticulum Response-Induced Apoptosis through CHOP Hypothermia Attenuating Apoptosis through CHOPLiu Xiaojie Wang Mingshan * Chen Huailong Guo Yunliang Ma Fuguo Shi Fei Bi Yanlin Li Ying Department of Anesthesiology, Qingdao Municipal Hospital, Shandong, China Lewin Alfred Editor University of Florida, United States of America * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: MW. Performed the experiments: XL HC YL. Wrote the paper: XL. Contributed laboratory: YG. Contributed rats: FM. Provided materials: FS. Provided analysis tools: YB. 2013 3 1 2013 8 1 e5343131 8 2012 28 11 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.Endoplasmic reticulum (ER) stress has been implicated in the pathology of cerebral ischemia. Apoptotic cell death occurs during prolonged period of stress or when the adaptive response fails. Hypothermia blocked the TNF or Fas-mediated extrinsic apoptosis pathway and the mitochondria pathway of apoptosis, however, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study aimed to elucidate whether hypothermia attenuates brain cerebral ischemia/reperfusion (I/R) damage by suppressing ER stress-induced apoptosis. A 15 min global cerebral ischemia rat model was used in this study. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in hippocampus CA1 were assessed after reperfusion of the brain. The expressions of C/EBP-homolo gous protein (CHOP) and glucose-regulated protein 78 (GRP78) in ischemic hippocampus CA1 were measured at 6, 12, 24 and 48 h after reperfusion. The results showed that hypothermia significantly attenuated brain I/R injury, as shown by reduction in cell apoptosis, CHOP expression, and increase in GRP78 expression. These results suggest that hypothermia could protect brain from I/R injury by suppressing ER stress-induced apoptosis. This work was supported by Qingdao Municipal Science and Technology Bureau 11-2-3-2-5-nsh Shandong Provincial Health Department 2001CAICKAF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Hypothermia has been recognized as an effective method in reducing brain injury caused by a variety of neurological insults and may play an important role in emergency brain resuscitation of patients with ischemic stroke, head trauma and cardiac arrest [1], [2], [3], [4], [5]. The neuroprotective effects of mild hypothermia have been well documented in experimental models [6], [7], [8]. The precise mechanisms by which mild hypothermia protects the brain remain to be further elucidated. It is reported that hypothermia prevents cell death by multiple pathways. Among them, hypothermia attenuate cytochrome C (CytC) release and apoptosis [9] including the tumor necrotic factor (TNF) pathway or Fas-mediated extrinsic apoptosis pathway [10]. Earlier studies have found that hypothermia decreases P53 protein levels in the brain which activates genes of apoptosis and pro-apoptotic proteins including Bak,Bax, and PUMA. The Bcl-2 family is much influenced in the way that the pro-apoptotic proteins are inhibited which should have lead to the formation of pores in the mitochondria’s membrane, allowing the release of the cytochrome C to the cytosol, activating caspases, culminating in neuronal death [11], [12], [13]. Zhao [14] reported that biphasic CytC release occurred after transient global ischemia and mild hypothermia protects against ischemic damage by blocking the second phase (12 h, 24 h) of CytC release, possibly by blocking caspases activity. From the above studies, hypothermia can reduce the mitochondria pathway of apoptosis which also known as intrinsic apoptosis pathway. Both the extrinsic and intrinsic apoptosis pathways ultimately activate caspases and then culminate in neuronal death. However, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. Recent studies have illustrated the relationship between theendoplasmic reticulum stress(ER) stress and the mitochondria -mediated apoptosis pathway, especially the relationship between bcl-2 family and ER stress [15]. One of the upstream signals that activate these pathways is referred to ER stress. ER is the site for protein synthesis and folding, and also involved in calcium homeostasis and lipid biosynthesis. Many stimuli such as ischemia and hypoxia might perturb ER function resulting in accumulation of unfolded proteins in the ER lumen. This was also known as unfolded proteins response (UPR). Three ER transmembrane receptors named doublestranded RNA-dependent protein kinase-like ER kinase (PERK); inositol requiring enzyme-1 (IRE1) and activating transcription factor (ATF6) are activated to restore ER functions [16]. Firstly, ER chaperones [78-kDa glucose regulated protein (GRP78)] and ER-associated degradation of unfolded proteins as the initially a protective response of UPR. If the stress is prolonged UPR might activate enhancer binding protein homologous protein (chop), caspase-12 and c-Jun N-terminal kinase protein (JNK) [17]. Studies have showed chop is the downstream of all three ER stress pathways and plays an important role in endoplasmic reticulum stress [18]. Prolonged ER stress is implicated in the pathogenesis of ischemia and CHOP plays an important role in the cerebral ischemic damage induced by neuronal death. Up to now, there are no prior studies examining the effects of hypothermia on the ER stress induced apoptotic pathway after transient global ischemia, so we explored these apoptotic events after transient global ischemia, and investigated the effects of hypothermia hypothermia against transient global ischemia through ER stress pathway in the transient global cerebral ischemia model. Materials and Methods 2.1 Subjects The study was approved by Qingdao Municipal Hospital Animal Ethics Committee. All the experimental procedures were conducted in conformity with the guidance suggestions for the care and use laboratory animals formulated by the Ministry of Science and Technology of the People’s Republic of China [19]. A total of 273 male Wistar rats (220 to 260 g) supplied by Qingdao Institute of Drug Control were used. Of these, 129 rats were withdrawn from the study for various reasons: 4 died from anesthesia; 27 died during electrocautery of vertebral arteries; 68 died during global ischemia; 19 died during the post surgical period. And 11 rats were paralyzed in vetebral arteries electrocauterizaed. The other 144 rats were randomly divided into global cerebral ischemia/reperfusion group (n = 48), hypothermia group (n = 48) and sham group (n = 48). Core body temperatures were monitored with a rectal probe throughout the surgical procedure. At 6, 12, 24 and 48hours after reperfusion, 6 rats from each group were sacrificed for histological staining, tunnel staining and immunohistochemistry. The other 6 rats were sacrificed for western blot analysis. GRP78 and CHOP expression were measured by immunohistochemistry and western blot analysis. 2.2 Surgeries Rats were starved for 12 h prior to surgery, and then anesthetized by injecting of 10% chloral hydrate (0.35 ml/100 g) intraperitoneally. A model of transient, bilateral hemispheric ischemia was established according to previously described methods [20]. Briefly, vertebral arteries were electrocauterized to induce permanent occlusion. The common carotid arteries were exposed, isolated, and marked using a cotton thread loop. After 24 hours, the exposed bilateral common carotid arteries of the anesthetized rats rats were blocked by artery clamps .After 15 min of ischemia, the clamps were removed to allow reperfusion. A needle electrode was inserted subcutaneously from the parietal region, and connected with the ED-Swan Mark electroencephalo-gram system (Nihon Kohden, Tokyo, Japan). The criteria of successful model were that rats presented unconscious, bilateral dilation of pupils, loss of spontaneous voluntary movements and the righting reflex throughout the ischemia and initial reperfusion periods. As the rats were anesthetized, we mainly rely on EEG. For rats in the hypothermic group, core temperature was decreased to 32–34°C by spraying 100% alcohol onto the rat’s body, and was brought back to normal with a light and a heating pad. Hypothermia maintained for 3 hours after reperfusion. For rats in the sham group, 4 vessels were exposed but without occlusion. 2.3 (HE) Staining, Immunohistochemistry and TUNEL Method The rats (n = 6 at each time point) were deeply anesthetized and intracardially perfused with 0.9% NaCl, followed by 4% paraformaldehyde (PFA). Brains were removed and brain tissues at the coronal plane from 1 to 4 mm posterior to the optic chiasma were harvested. After being fixed for 2 hours, washed for 4 hours, dehydrated with gradient ethanol, cleared with xylene, the brain tissues were embedded with paraffin and cut into 4 µm thickness for hematoxylin and eosin (HE) staining, immunohistochemical staining and TUNEL assay. Anti-rat GRP78 and anti-rat chop polyclonal primary antibody (IgG) were purchased from Santa Cruz Biotechnology. The paraffinized sections were blocked to endogenous peroxidase activity by incubation in distilled water containing 3% hydrogen peroxide for 10 min. Antigen retrieval was performed, using a 0.01 mol/L citrate buffer (pH 6.0) in high pressure cooker for 10 min. Anti GRP78 and anti chop antibody used at a dilution of 1∶150 and 1∶100, respectively, in 2% BSA/PBS were added on the slides and incubated at 37°C for 1 hour. GRP78 and chop were detected with HRP-Polymer anti-Rabbit IgG for 1 h at room temperature. The peroxidase binding sites were detected by staining with diaminobenzidine (DAB). Finally, staining was performed using hematoxylin for 3 secs and observed by microscopy. TUNEL method was performed as described previously [21]. Briefly; Paraffin sections were de-waxed and hydrated. Followed by 3%H202 at room temperature for 10 minutes. The sections were then digested with fresh diluted proteins K(1∶200) at 37°C for 10 minutes. Then, 1 µl TdT and DIG. d-UTP, together with 18 µl marker buffer, were added to each section at 37°C for 2 hours. The sections were then blocked at room temperature for 30minutes,followed by incubation with biotinylated anti-digoxin antibody(1∶200) at 37°C for 30 minutesSABC(1∶100) at 37°C for 30 minutes, DAB coloration, and observation by microscopy.. 2.4 Western-blots Animals were perfused transcardially with normal saline, the brains were quickly removed and the hippocampal CA1region was rapidly dissected. The bilateral hippocampus tissues were separated and homogenized with RIPA Lysis Buffer (Beyotime Biotechnology, China). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China). Equal amounts of protein samples (total protein extracts, after centrifugation at 12,000 g at 4°C for 5 min. Then, the protein was mixed with buffer and heated at 99°C for 5 min. For western blot analysis an equal amount of protein (50–100 µg) was loaded in each well and subjected to 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred from the gel to polyvinylidinene fluoride (Millipore, Bedford, MA, USA) membranes and blocked in 5% non-fat dry milk prepared in 1× TBST. The membranes were incubated with the primary antibodies overnight at 4°C. The following primary antibodies were used: GRP78(1∶200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CHOP (1∶100, Santa Cruz Biotechnology, CA, USA) or GAPDH antibody (1∶1000, Zhongshan Goldenbridge Biotechnology, China). After washing primary antibodies with 1×TBST, the membranes were incubated with appropriate secondary antibodies for 2 h at room temperature. The blots were developed using ECL (Beyotime Institute of Biotechnology, China). 2.5 Statistics All data were expressed as mean ± standard deviation (SD) and analyzed by SPSS 13.0. Repeated measures ANOVA was used as appropriate for comparison between different groups followed by post hoc test for multiple comparisons. P<0.05 was considered as statistically significant. Results 3.1 Hippocampus Neuronal Morphology Compared with sham group, the number of normal neurons in hippocampus CA1 area decreased in ischemia and hypothermia group. And the number of neurons with nuclear pyknosis increased significantly in hippocampus CA1 area from ischemia and hypothermia group. What’s more, compared with hypothermia group, the neuronal degeneration in ischemia group is more severe at each time points from ischemia group. At reperfusion 48 hours, the number of survival neurons in hypothermia group is more than in ischemia group (Fig. 1). 10.1371/journal.pone.0053431.g001Figure 1 Neurons in hippocampus CA1 area. (a) The picture showed the neurons in hippocampus CA1 area. The neurons in sham group (A) displayed regular appearance with large and round nuclei but pyknosis was observed in ischemia (C) and hypothermia group (B). (b) Compared with sham group(89.3±6.1) (A), the number of normal neuronal is fewer and the neurons of morphologic abnormality is more in ischemia group(47.3±4.5) (C). The number of survival neurons in hypothermia group(64.5±7.5) (B) is more than that in ischemia group. /400×visual field. The neurons in sham group (A) displayed regular appearance with large and round nuclei but pyknosis was observed in ischemia(C) and hypothermia group (B). Compared with sham group (89.3±6.1) (A), the number of normal neuronal is fewer and the neurons of morphologic abnormality is more in ischemia group (47.3±4.5) (C). The number of survival neurons in hypothermia group (64.5±7.5) (B) is more than that in ischemia group. /400×visual field. 3.2 The Expression of GRP78 and Chop The expression of chop and GRP78 protein neurons was found in hippocampus CA1 after cerebral ischemia reperfusion (Fig. 2, 3). In normothermic and mild hypothermic ischemia groups, the optical densities of chop and GRP78 in hippocampus CA1 neurons at 6, 12, 24 and 48hour reperfusion following 15 minute ischemia (15 min/6 h, 15 min/12 h, 15 min/24 h, 15 min/48 h respectively) were showed in Fig. 2, 3. Mild hypothermia decreased the expression of GRP78 protein and increased the expression of chop protein. Immunohistochemistry showed the GRP78 was barely detected in sham group (A). The expression of GRP78 in hypothermia group (B) is much stronger than that in ischemia group (C) at reperfusion 24 hours. /400×visual field. 10.1371/journal.pone.0053431.g002Figure 2 Expression of GRP78 in hippocampus CA1. (a) Immunohistochemistry showed the GRP78 was barely detected in sham group (A). The expression of GRP78 in hypothermia group (B) is much stronger than that in ischemia group (C) at reperfusion 24 hours. /400×visual field (b) Western blot analysis showed that the GRP78 was barely detected in sham group. In brains of ischemia group, it was increased 6 hour after 15 min of ischemia and gradually decreased thereafter; however, the degree of increase was much bigger in the hypothermia brains. (c) Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly increased GRP78 after 15 minutes of ischemia (P<0.05 compared with ischemia brains at the same time points, 6 rats from each group at every time points were used for analysis). 10.1371/journal.pone.0053431.g003Figure 3 Expression of chop in hippocampus CA1. (a) Immunohistochemistry showed the chop was barely detected in sham group (A).The expression of chop in hypothermia group (B) is much weaker than that in ischemia group (C) at reperfusion 24 hours. /400×visual field (b) Western blot analysis showed that the chop was barely detected in sham group. In brains of ischemia group, it was increased 6 hour after 15 minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. (c) Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P<0.05 compared with ischemia brains at the same time points. 6 rats from each group at every time points were used for analysis). Western blot analysis showed that the GRP78 was barely detected in sham group. In brains of ischemia group, it was increased 6 hour after 15 min of ischemia and gradually decreased thereafter; however, the degree of increase was much bigger in the hypothermia brains. Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly increased GRP78 after 15 minutes of ischemia (P<0.05 compared with ischemia brains at the same time points, 6 rats from each group at every time points were used for analysis). Immunohistochemistry showed the chop was barely detected in sham group (A). The expression of chop in hypothermia group (B) is much weaker than that in ischemia group (C) at reperfusion 24 hours. /400×visual field. Western blot analysis showed that the chop was barely detected in sham group. In brains of ischemia group, it was increased 6 hour after 15 minutes of ischemia and gradually decreased thereafter; however, the degree of increase was much smaller in the hypothermia brains. Quantitative analysis of Western blotting showed that hypothermia after ischemia significantly decreased chop after 15 minutes of ischemia (P<0.05 compared with ischemia brains at the same time points. 6 rats from each group at every time points were used for analysis). 3.3 TUNEL TUNEL positive staining was absent in the sham groups (Fig. 4), and there were few TUNEL positive cells among the hypothermia group (Fig. 4). However, many positive cells could be observed in the ischemia group (Fig. 4). Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using TUNEL staing. The sham group showed a large number of neurons and almost no TUNEL-positive cells (5.1±1.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons was decreased and substantial TUNEL-positive cells were detected. The number of TUNEL-positive cells in hypothermia group (34.4±4.2) (B) is more than in ischemia group (40.5±5.7) (C), /400×visual field. 10.1371/journal.pone.0053431.g004Figure 4 Neuronal apoptosis in CA1 region of hippocampus induced by global cerebral ischemia. Detection of apoptosis in hippocampus CA1 pyramidal neurons was carried out using Tunel staing. The sham group showed a large number of neurons and almost no TUNEL-positive cells (5.1±1.2) (A). In ischemia (C) and hypothermia (B) groups, the number of neurons were decreased and substantial TUNEL-positive cells were detected. The number of TUNEL-positive cells in hypothermia group (34.4±4.2) (B) is more than in ischemia group (40.5±5.7) (C), /400×visual field. Discussion It has been reported that hypothermia blocked the TNF pathway of apoptosis or extrinsic Fas-mediated apoptosis pathway and the mitochondria pathway of apoptosis. However, whether hypothermia can block endoplasmic reticulum mediated apoptosis is never known. This study confirms that mild hypothermia for 3 h after global ischemia protects hippocampus neurons from a global ischemic insult, and demonstrates that this protection following global ischemia is associated with reduced apoptosis as assessed by TUNEL staining. Furthermore, we showed for the first time that mild hypothermia was associated with increased GRP78 expression and decreased chop expression suggesting a mechanism for the observed neuroprotection. ER is the site for protein synthesis and folding, and also involved in calcium homeostasis and lipid biosynthesis. Cerebral ischemia causes severe ER stress that results in ER function disruption and unfolded proteins accumulation in the ER lumen called unfolded protein reaction UPR [22], ultimately leads to cell death [23]. Apoptosis initiated by ER stress is largely dependent on the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Brain I/R injury up-regulates the expression of ER stress markers such as CHOP and GRP78 [24] that results in ER stress-associated apoptosis [25]. Similar to their studies, our results also demonstrated that I/R injury increases CHOP expression, GRP78 induction and apoptosis in the hippocampus after cerebral ischemia. The ER chaperone GRP78 (glucose-regulated protein of 78 kDa) with antiapoptotic properties is a central regulator of ER homeostasis, and its up-regulation is widely used as a sentinel marker for ER stress under pathologic conditions. As a major ER chaperone, GRP78 facilitates protein folding, preventing intermediates from aggregating and promoting misfolded protein for proteasome degradation [20]. Our result has revealed the activation of GRP78 started at 6 h after ischemia and reached the peak at 24 h after ischemia attack. Compared with the ischemia group, the amount of GRP78 expression in hypothermia group is much more than that in ischemia group which is in. consistent with the finding of Masayuki Aoki [26], [27]. There was a significant reduction in the number of TUNEL-positive cells in the hippocampus CA1 in the hypothermia group rats at 48 h of reperfusion in our study. Therefore, we believe that the increased GRP78 expression contributes to the hypothermia-induced neuroprotection in hippocampus area against cerebral ischemia. It has been reported that CHOP−/−mice exhibit reduced apoptosis in response to ER stress and have smaller infarcts than wild-type animals subjected to bilateral carotid artery occlusion [28], [29]. Thus, CHOP is the downstream of ER stress induced apoptosis [30], [31]. Further, our results showed that mild hypothermia significantly decreased the activation of CHOP at 24 h of reperfusion. In agreement with the study of Wang, our study showed the increased expression of GRP78 attenuated the induction of CHOP during ER stress and reduced ER stress-induced apoptosis when hypothermia was applied after global ischemia [32]. Previous studies have suggested that CHOP sensitizes cells to ER stress induced-apoptosis through down-regulation of the anti-apoptotic factor B cell lymphoma-2 (Bcl-2) and up-regulation of reactive oxygen species (ROS) [14], [23]. However, numerous studies have certificated the neuroprotection of hypothermia by reducing the oxidative stress, lipid peroxidation, glutamate excitotoxicity together with increased the anti-apoptotic protein Bcl-2 [33]. We hypothesize that some molecules of CHOP pathway, such as Bcl-2 and ROS, may contribute to the decreased TUNEL-positive cells by hypothermia. We already have known that the permeabilization of the mitochondrial outer membrane (MOM) and the consequent release of cytochrome c are regulated by a group of proteins known as the Bcl-2protein families. Collectively, the Bcl-2 protein family monitors incoming stress signals and orchestrates the initiation of the mitochondrial or intrinsic pathway to apoptosis. Bcl-2 has previously shown that it’s suppress cell death by inhibiting free radical production and prevent the release of cytochrome C from mitochondria and block caspase activation [34], [35], [36]. But the intrinsic relationship among them induced by hypothermia following global ischemia needs to be further studied. ==== Refs References 1 Alberts MJ , Lyden PD , Zivin JA , Brott TJ , Brass LM , et al (1995 ) Emergency brain resuscitation . Ann Intern Med 122 : 622 –627 .7887560 2 Jiang JY , Yu MK , Zhu C (2006 ) Effect of long-term mild hypothermia or short-term mild hypothermia on outcome of patients with severe traumatic brain injury . J Cereb Blood Flow Metab (26) 771 –776 . 3 Kochanek PM , Safar PJ (2003 ) Therapeutic hypothermia for severe traumatic brain injury. JAMA . 289 : 3007 –3009 . 4 McIntyre LA , Fergusson DA , Hébert PC , Moher D , Hutchison JS (2003 ) Prolonged therapeutic hypothermia after traumatic brain injury in adults. JAMA . 289 : 2992 –2999 . 5 The Hypothermia after Cardiac Arrest Study Group (2002 ) Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. 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==== Front Diagn PatholDiagn PatholDiagnostic Pathology1746-1596BioMed Central 1746-1596-7-1702321698110.1186/1746-1596-7-170Short ReportLuminal B tumors are the most frequent molecular subtype in breast cancer of North African women: an immunohistochemical profile study from Morocco El Fatemi Hinde [email protected] Sanae [email protected] Sofia [email protected] Kaoutar [email protected] My Abdelilah [email protected] Abdelaziz [email protected] Omar [email protected] Afaf [email protected] Department of Pathology, Hassan II teaching hospital, Fez, Morocco2 Department of Gynecology, Hassan II teaching hospital, Fez, Morocco3 Department of Oncology, Hassan II teaching hospital, Fez, Morocco2012 7 12 2012 7 170 170 15 10 2012 4 12 2012 Copyright ©2012 EL FATEMI; licensee BioMed Central Ltd.2012EL FATEMI; 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 Breast cancer may be classified into luminal A, luminal B, HER2+/ER-, basal-like and normal-like subtypes based on gene expression profiling or immunohistochemical (IHC) characteristics. The aim of our study is to show the molecular profile characteristic of breast cancer in the North African population of Morocco. This work showed preliminary results and correlations with clinicopathological and histological parameters. Three hundred and ninety primary breast carcinomas tumor tissues were immunostained for ER, PR, HER2, CK5/6, CK8/18 and Ki67 using paraffin tissue. Methods We reviewed 390 cases of breast cancer diagnosed on January 2008 to December 2011 at the Department of pathology, Hassan II teaching hospital, Fez, Morocco. Age, size tumor, metastatic profile, node involvement profile, histological type and immunohistochemical profile were studied. Results The average age was 46 years; our patients were diagnosed late with a high average tumor size. Luminal B subtype was more prevalent (41.8%), followed by luminal A (30.5%), basal-like (13, 6%), Her2-overexpressing (9, 2%), and unclassified subtype (4.9%). Conclusion This study showed that molecular classification and biological profile may be different according to geographical distribution, to encourage further studies to know the genomic profile of tumors and the environment. Virtual slide http://www.diagnosticpathology.diagnomx.eu/vs/1675272504826544 Breast cancerMolecular classificationLuminal B subtypeStagingAntibodiesImmunohistochemistry ==== Body Background Breast cancer is a heterogeneous disease such that they may have different prognoses and respond to therapy differently despite similarities in histological types, grade and stage. Based on the presence or absence of expression of the estrogen receptor (ER), breast cancer is divided in two groups: ER+ and ER-. Genetic expression profile has identified two subtypes of the ER+ tumors: luminal A and luminal B. ER- tumors also include two subtypes, the HER2+ and the basal type. These subtypes differ in their biology and both demonstrate short disease-free periods after treatment and poorer outcome. In Morocco, it’s the first cancer in women and is currently a major public health problem. The molecular classification in breast carcinomas is now based upon gene expression analysis using DNA microarrays and allows to identify at least five groups: luminal A, luminal B, HER2-overexpressing, basal-like and normal breast-like [1-3]. However, large-scale subtyping using gene expression profiling from formalin-fixed, paraffin-embedded samples is not currently feasible and remains very expensive. Therefore, immunohistochemical markers have been used as surrogates tools for DNA microarray in subtyping breast cancer [4,5]. Several studies used routinely panels of immunohistochemical markers to classify breast cancers into subtypes similar to those previously defined using gene expression analyses [6]. The recent study was realized by Prat et al. [4]. They defined several immunohistochemical subtypes: (luminal A, Luminal B, HER2- enriched, basal-like) and a normal breast-like group that show significant differences in incidence, survival and response to therapy. Luminal A (ER positive (ER+) and/or PR positive (PR+), Her2 negative (Her2-)) with ki67<14%, luminal B (ER + and/or PR+ with ki67>14%, Her2 positive or negative (Her2+/-), Her2+/ER − subtype (Her2+, ER−, PR−) and basal-like (ER−, PR−, Her2−, Cytokeratin 5/6 positive (CK5/6+) and/or Her1+ (EGFR)). Tumors which were negative at immunohistochemical staining for all markers (ER, PR, Her2, Her1, and CK5/6) were considered unclassified subtype [4]. According to this classification, we performed immunohistochemical staining for ER, PR, Her2, Her1, CK8/18, basal CK5/6 and KI67 in paraffin sections from blocks of breast cancer. The aim of the present study was to estimate the prevalence of breast cancer subtypes in patients of the north east region of Morocco, and to correlate between clinical and pathological characteristics. Methods Patients this study was approved by Ethical Committee of Hassan II teaching hospital Center. A total of three hundred and ninety patients diagnosed with invasive breast carcinoma were examineted. Clinical information was retrieved from the medical records. Breast cancers were classified according to the World Health Organization (WHO, 2003) [7] while histological grading and staging were performed according to Modified Bloom-Richardson classification [8] and American Joint Committee on Cancer (AJCC) [9], respectively. Immunohistochemical study Tumors sections were deparaffinized and rehydrated. Peroxide blocking was done with 0.4% H2O2. Antigen retrieval was achieved by heat retrieval using a pressure cooker. After washing, the slides were treated with protein blocking agent (UltraTech HRP, Immunotech) then incubated with the following primary antibodies: anti-human ER (ER1D5, Immunotech), PR (PR10A9, Immunotech), CK5/6 (D5/16B4, Cell Marque), CK8/18 (RTU-5D3, Novocastra), Ki67 (SP6 Cell Marque). After rinsing with PBS, the slides were incubated with a secondary biotinylated antibody (Immunotech). The slides were then rinsed with PBS. Sections were then incubated with streptavidin-peroxidase reagent. Staining for the slides was developed with Amino-Ethyl-Carbazole (Ultra Tech AEC, Immunotech) and then the slides were counterstained with hematoxylin, hydrated, and mounted. For Her2, immunohistochemical was carried out using with HercepTest (A0485, Dako) according to the commercial instructions for use. Immunohistochemical surrogate biomarkers of molecular classification Immunohistochemical subtypes were defined as follows: Luminal A (ER + and/or PR+, Her2-, KI67<14%), luminal B (ER + and/or PR+, Her2+/-, Ki67>14%), basal-like (ER-, PR-, Her2-, and CK5/6+, CK14+), Her2+/ER-, and unclassified subtype (negative for all markers) (Table 1). CK8/18 expression was used for confirmation the luminal subtypes. Table 1 Immunohistochemical characterization of molecular subtypes of breast cancer Molecular subtype Immunohistochemical characterization Luminal A ER (+) et Her-2 (-) et CK8/18 (+), Ki67<14% Luminal B ER (+) et Her-2 (+) ou ER (+), Her-2 (-) et Ki67>14% Her-2 ER (-) et Her-2 (+) Basal ER (-) et Her-2 (-) et CK5/6 (+) Positive controls were included in each staining run and consisted of breast cancers known to express each of the antigens of interest. Cases were considered positive for ER and PR according to standardized guidelines using a cut-off of ≥1% stained tumour nuclei. Fluorescence in situ hybridization (FISH) study All Her2 score 2+ cases were analyzed by FISH. They were performed using the PathVysion HER2 DNA Probe (Abbott Vysis Inc., Downers Grove, IL) according to the manufacturer’s protocol. The probe cocktail included the LSI HER-2/neu probe and the CEP17 probe. Fluorescence signals were analyzed and digitalized using the CytoVisionTM image analysis system (Applied Imaging International Ltd., Newcastle-Upon-Tyne, UK). Between 60 and 100 nuclei were scored for each case. Signal ratios (HER2: CEP17) ≥ 2, 2 were classified as amplified. In the absence of positive FISH data, tumors scored 2+ by IHC were considered as negative for HER-2. Her2 was scored based on a 0 to 3 scale according to the criteria set by ASCO (American Society of Clinical Oncology/College of American Pathologists) [10]. Scores 0 and 1+ were considered as negative; score 2+ was considered borderline; and score 3+ was considered as strongly positive. FISH was performed on the borderline cases (score 2+). KI67 In breast cancer, immunohistochemical assessment of the proportion of cells staining for the nuclear antigen Ki67 has become the most widely used method for comparing proliferation between tumor samples. Potential uses include prognosis, prediction of relative responsiveness or resistance to chemotherapy or endocrine therapy, estimation of residual risk in patients on standard therapy and as a dynamic biomarker of treatment efficacy in samples taken before, during, and after neoadjuvant therapy, particularly neoadjuvant endocrine therapy. Cases were considered positive for ki67 according to standardized guidelines using a cut-off of ≥14% stained tumour nuclei with moderate intensity (Figure 1). Figure 1 Distribution by the proliferation index assessed by Ki 67. Statistical analysis Statistical analysis was performed in the Department of Epidemiology, of the Faculty of Medicine and Pharmacy of Fez and was carried out using Epi-Info (3.3.2). Results and discussion The study was achieved on 390 patients diagnosed with infiltrating breast cancer and managed at the Department of pathology in Hassan II University Hospital in Fez. The patient’s average age at diagnosis was 46 years (ranging from 22 to 92 years). The tumor clinical stage on first diagnosis, according to American Joint Committee on Cancer Staging Systems, showed that 47 women (14.5%) are at stage I, 108 (33%) are at stage II, 114 (35%) are at stage III and 57 (17.5%) at stage IV. After histological analysis, the tumor average size was 3.7 ± 2.6 cm (ranging from 0.2 to 16 cm). Most of these tumors (87, 2%) were diagnosed as invasive carcinoma (IC NOS) while 5, 4% were invasive lobular carcinomas (ILC), 2% were metaplastic carcinoma (MC), and few patients had cancers of rare histology (5, 4%), which were summarized as “other types” in our study. The histological grade distribution for luminal B were grade II (59.9%), grade III (30.6%) but few patients only were grade I (9,6%) with p<0,0001. Vascular emboli were detected in 71.2% of patient’s luminal B. The status of lymph nodes was determined for 326 patients among which 68, 8% had positive lymph nodes and 12.5% had distant metastasis. The immunohistochemical study showed that 67, 1% patients were ER positives, 63, 7% were PR positive, and 24, 7% were Her2 positive. Therefore, Luminal B subtype was more prevalent (41.8%), followed by luminal A (30.5%), basal-like (13, 6%), Her2-overexpressing (9, 2%), and unclassified subtype (4.9%). The luminal B subtypes present a higher median tumor size (T2 and T3) as like basal-like and Her2-overexpressing. IHC subtypes were significantly different by histological grade (p< 0.00001). The luminal B also represented a higher percentage of cases with histological grade II and III (90, 5%) like, basal-like and Her2-overexpressing subtypes (90, 2% and 96% respectively) and a very low percentage of tumors with histological grade I (9,6%, 9,8% and 4%, respectively). The Luminal B subtype had 71, 2% vascular emboli, approach Her2-overexpressing subtypes (75,6%). In this study, the luminal B tumors had average percentage of lymph node metastasis (68, 8%) and distant metastasis (12, 5%). Table 2 Twenty-two patients (12.2%) died because of cancer-related events during the follow-up. Among these patients, 25% belong to basal-like, 22% belong to unclassified subtype group. 20% to Her2-overexpressing group, 17% to luminal B and only 5% in luminal A. Table 2 Prevalence of intrinsic subtypes and clinico-pathological characteristics subtype luminal A luminal B Her-2 Basallike Unclassified Histological type %            IDC Nos 30,5 41,1 10,9 13,9 3,6  ILC 47,6 42,9 4,8 0,0 4,8 Other types 25 8,3 16,7 50 0,0 P<0,00001           Histological grade%             I  28,3 9,6 9,8 4 15,8   II  59,2 59,9 56,1 32 26,3   III   12,5 30,6 34,1 64 57,9 P<0,0001           Vascular emboli %            Positive 60,8 71,2 75,6 64,4 63,2 negative  39,2 28,8 24,4 35,3 36,8 p=0,29           Intraductal carcinoma %             Yes 55 39,5 41,5 25,5 78,9   No 45 60,5 58,5 74,5 21,1   P=0,0013           Tumour size (cm)             T1 42,9 31,8 9,1 12,5 25   T2 34,5 44,76 45,5 46,9 25   T3 14,3 18,8 27,3 15,6 12,5   T4 8,4 4,7 18,2 25 37,5 P=0,0003           Necrosis %           Present 8,5 17,8 22,2 22,6 10,5 Absent 91,5 82,8 77,8 77,4 89,5 P=0,0698           Positive Lymph nodes % 52,4 68,8 86,7 81,3 - P=0,0066           Distant metastasis %           Yes 4,5 12,5 17,9 26,7 28,6 No 95,5 87,5 82,1 73,3 71,4 P=0,0030           Molecular studies of breast cancer revealed biological heterogeneity of the disease and opened new perspectives for personalized therapy. While multiple gene expression-based systems have been developed, current clinical practice is largely based upon conventional clinical and pathologic criteria. Digital image analysis (DA) with multivariate statistics of the data opens new opportunities in this field [11]. Recently, gene expression studies, using microarray technology, confirmed that the heterogeneity of clinical response could be correlated with different molecular profiles of breast cancers [1]. Arvydas and al reported a series of tissue microarrays of 109 patients with breast ductal carcinoma, were stained for a set of 10 IHC markers (ER, PR, HER2, Ki67, AR, BCL2, HIF-1α, SATB1, p53, and p16) [12]. This study demonstrates that factor analysis of multiple IHC biomarkers measured by automated DA is an efficient exploratory tool clarifying complex interdependencies in the breast ductal carcinoma IHC profiles. Major factor of the aggressive disease behaviour (i-Grade) is characterized by opposite loadings of ER/PR/AR/BCL2 and Ki67/HIF-1α. The i-Grade factor scores represent integral quantitative characteristics that reveal bimodal distribution and are strongly associated with the histological grade and relevant intrinsic subtypes. In HR-positive tumours, the aggressiveness of the tumour is best reflected by the combination of Ki67 and ER, rather than Ki67 and BCL2. Inverse relation between HER2 and PR expression in the HR-positive tumours which, along with the inverse relation between Ki67 and ER, may shed the light into the differential information conveyed by the ER and PR expression. Remarkably, SATB1 along with HIF-1α reflected the second major factor of variation in patients with breast cancer; furthermore, in the HR-positive group they were inversely associated with the HR and BCL2 expression and represented the major factor contributing to the variation in the IHC data set. However, this factor was not associated with the clinicopathologic categories studied. Biological meaning of this variation remains unclear: HIF-1α and SATB1 may convey important biological messages other than the aggressiveness of the disease reflected by Ki67 expression and histological grade. Meanwhile, the authors, support the notion that the suggested prognostic significance of SATB1 may be related to its inverse relation to the ER expression. Finally, this analysis confirms high expression levels of p16 in Triple-negative tumours [12]. In our study, based on recent updated IHC subtype definition by Prat et al. [4], we estimated the prevalence of breast cancer subtypes in patients from the north east Moroccan region and established the correlations between clinico-pathological characteristics. The patients recruited in our university hospital were younger than in western series; the average age at diagnosis was 46.8 years. In terms of clinical staging, only 14.5% patients were diagnosed at stage I, while 33% were at stage II, 35% at stage III and 17.5% at stage IV. On the other hand, after histological analysis the average tumor size was 3.7 cm and 75% of cases measured more than 2 cm. A majority of tumors were stage II or III. Our data showed that more than 50% of patients of all subtypes presented positive lymph nodes and more than 12% of cases had distant metastasis on first diagnosis. This could be due to late consultation during the progression of the disease in our region as well as to lack of the Medicare coverage, lack of screening mammography program and women’s awareness trainings particularly in rural area. The predominant histology type in this study was invasive cancer (87.4%), similar to most breast cancer studies worldwide. Overexpression of the protein and/or amplification of the HER2 gene have been reported in approximately 20 to 30% of breast cancers, similar to what was found in our patients (24,7%). Her2+ tumors are associated with either poor prognosis or with response to trastuzumab [13]. Our results showed a distribution of breast cancer subtypes non similar to what was reported by other immunohistochemical studies [4]. In this study, the prevalence of luminal B (41, 8%) and is the more frequent subtype. Considering the fact that all patients came from a university hospital the possible effects of a selection bias should be regarded. Cancer registry available includes only data from the university hospital and not all North African population of Morocco. Triple-negative subtype represented approximately 13, 6% of our series and 78% of them were basal-like tumors. These results were similar to what was found in other studies [4,14]. The basal-like group was defined by immunohistochemistry, as being negative for ER, PR, and Her2 and positive for Ck5/6, CK14 and/or Her1 (EGFR). It is important to study whether EGFR is overexpressed in patients with breast cancer since these patients can be given specific EGFR molecule tyrosine kinase inhibitors such as gefitinib and lapatinib [15,16]. There are only a few reports regarding the overexpression of EGFR, with these studies indicating 8-36% of breast cancers over express this protein. However, systematic studies appraising EGFR gene amplification and mutations in the same set of cases among Chinese female patients with breast cancer are absent [17-19]. EGFR gene mutations are infrequent in breast cancers. This suggested that EGFR mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy for breast cancers [20]. In literature, the luminal B tumors are associated with poor recurrence-free and disease-specific survivals in all adjuvant systemic treatment categories including hormone therapy; the identification of specific signaling pathways driving luminal B biology is paramount to improve treatment. Sircoulomb et al. and Holland et al. have independently identified the ZNF703 gene, located in chromosomal region 8p12, as preferentially amplified in luminal B tumours [21]. The natural history of breast cancer involves progression through defined pathological and clinical stages, starting with ductal hyperproliferation, with subsequent evolution into in situ and invasive carcinomas, and finally into metastatic disease. The majority of invasive breast cancer develop over long periods of time from certain pre-existing benign lesions. The best characterized premalignant lesions recognized today are referred to as atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS). Douglas and al, [22] reported that Lobular neoplasia (LN) and invasive lobular carcinoma (ILC) are detected together frequently in the same specimen and location of the tumor--in up to 90% of cases of ILC [23]. Invasive carcinomas were present with LN in 45.6% of cases, with similar rates of association with invasive carcinoma and ILC (47.2%). Bratthauer and Tavassoli stratified the LNs as "lobular intraepithelial neoplasias" (LIN) and evaluated the frequency of association between LIN subtypes (1, 2, and 3) and invasive carcinoma. The percentage of LIN 1 (equivalent to ALH) that was associated with invasive carcinoma was 14%, and 89% of these tumors were IDCs. In the patients with LIN 3 (equivalent to LCIS), the frequency of association with IDC and ILC was 23% and 86%, respectively. The authors concluded that the advance from LIN 1 to LIN 3 was linked to a 64% increase in the frequency of invasive carcinoma and a greater than 70% rise in the likelihood of ILC [24]. Apoptosis is a frequent phenomenon in breast cancer and it can be detected by light microscopy in conventional histopathological sections or by special staining techniques. Balance between expression status of anti-apoptotic and pro-apoptotic proteins determines cells to be alive or not. The key event of apoptosis occurrence is cascade activation of caspases, and inhibitor of apoptosis proteins (IAPs) play a important role in caspase inhibition. XIAP is the most potent caspase inhibitor and Smac is one of the antagonists of XIAP. Down regulation of XIAP expression or applying exogenous Smac mimics can sensitize tumor cells, especially for breast cancer cells, to chemotherapeutics and promote apoptosis [25,26]. In immunohistochemistry, XIAP and Smac were positive in cytoplasm of tumor cells with strong or moderate intensity, respectively. The positive ratio of XIAP (84.3%, 86/102) was more higher than that of Smac (33.3%, 34/102), and immunoscore of XIAP was higher than Smac in IDC too (P < 0.0001). It was noteworthy that 44 IDC samples were nuclear positive for XIAP, but none was for Smac. And cytoplasm positive status of XIAP nuclear positive group was stronger than the negative group (P = 0.030, 0.047). Otherwise, Smac immunoscore was prevalent in HER2 positive group than negative group (P < 0.0001). Remaining data revealed that the expression status of XIAP/Smac was not correlated with patient age, tumor size, lymph node status, histologic grading, expression of ER and PR. XIAP was a potent protein for apoptosis inhibition and Smac was an important negative regulator of the former. Disturbed balance of expression between XIAP and Smac probably contributed to carcinogenesis and XIAP positive nuclear labeling was a sign of unfavourable prognosis in breast invasive ductal carcinoma [27]. Others studies have demonstrated that claudin-6 functions as a cancer suppressor in human MCF-7 breast cancer cells. The growth inhibitory effect could be attributed to inhibition of cell proliferation and induction of apoptosis. Claudins (CLDNs) constitute a family of integral membrane proteins and have been identified as prominent structural components of tight junction ( TJ ) strands [28,29]. TJs are junctional complexes which mediate cell-to-cell adhesion in epithelial and endothelial cellular sheets [30], and which affect cell polarity and tight junction formation [28]. Guo and al reported that the apoptosis signal-regulating kinase 1 (ASK1) expression is low in breast cancer, and the levels of ASK1 mRNA and protein expression are correlated with that of claudin-6. They have identified a novel mechanism responsible for the pro-apoptosis function of claudin-6, and ASK1 may become a target for breast cancer treatments. The integrins, a family of transmembrane glycoproteins, play a major role in invasive and metastatic processes. Integrins are involved in cell adhesion in both cell-extracellular matrix and cell-cell interactions, and particularly, beta 1 integrin is involved in proliferation and differentiation of cells in the development of epithelial tissues. The putative role of beta 1 integrin expression on survival and metastasis in patients with breast invasive ductal carcinoma (IDC) was studied by Santos and al [31]. His study showed that beta1 integrin can be a marker of poor prognosis in breast cancer. Beta 1 integrin was overexpressed in 32.8% of IDC and was related with HER-2 and VEGF expression, and it had a significant relationship with metastasis and death , survival analysis showed that the overexpression of this protein is very significant in specific survival (number of months between diagnosis and death caused by the disease). Immunohistochemistrey is important to determinate the tumors of unknown origin (5-15%) and specially breast cancer metastasis (1.5%). Mammaglobin (MAG) antibody in the immunohistochemical panel for the detection of tumors of unknown origin contributed to the detection of metastasis of breast cancer. The diagnostic strategy with the highest positive predictive value (88%) included hormone receptors and mammaglobin in serial manner [32]. Conclusions We have shown that simple IHC-based classification of breast tumors can be helpful. Since the predictive power of IHC criteria appears to be similar to that of gene expression analysis, this information can be used to improve therapeutic decisions, mainly for luminal B, Her2- overexpressing and basal-like subtypes. The luminal B subtype was associated with a poor prognosis and unfavorable clinico-pathological characteristics. In addition, findings concerning tumors stages are alarming and highlight the importance of early screening and the urgent need to improve women’s awareness of breast cancer in our region. Our results should be confirmed by large studies to be conducted in other institutions and hospitals including patients coming from different regions of Morocco. Abbreviations HER2: Epithelial human receptor 2; ER: Estrogen receptor; PR: Progesterone receptor; IHC: Immunohistochemistry; CK: Cytokeratin; WHO: World Health Organization; AJCC: American Joint Committee on Cancer; FISH: Fluorescence in situ hybridization; IC NOS: Invasive carcinoma not otherwise specified; ILC: Invasive lobular carcinoma; MC: Metaplastic carcinoma; DA: Digital image analysis; EGFR: Epidermal growth factor receptor; ADH: Atypical ductal hyperplasia; ALH: Atypical lobular hyperplasia; DCIS: Ductal carcinoma in situ; LCIS: Lobular carcinoma in situ; LN: Lobular neoplasia; LIN: Lobular intraepithelial neoplasia; ASK1: Apoptosis signal-regulating kinase 1. Competing interests The authors declare that they have no competing interests. Authors’ contributions All authors analyzed, interpreted and approved the final manuscript. Funding This study received no specific grant from any funding agency in the public, commercial or not-for-profit sectors ==== Refs Perou CM Sorlie T Eisen MB Molecular portraits of human breast tumours Nature 2000 406 747 752 10.1038/35021093 10963602 Sorlie T Perou CM Tibshirani R Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications Proc Natl Acad Sci USA 2001 98 10869 10874 10.1073/pnas.191367098 11553815 Sorlie T Molecular portraits of breast cancer: tumour subtypes as distinct disease entities Eur J Cancer 2004 40 2667 26675 10.1016/j.ejca.2004.08.021 15571950 Aleix P Parker JS Olga K Cheng F Chad L Herschkowitz JI Xiaping H Perou CM Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer Breast Cancer Res 2010 12 R68 10.1186/bcr2635 20813035 Bhargava R Striebel J Beriwal S Prevalence, morphologic features and proliferation indices of breast carcinoma molecular classes using immunohistochemical surrogate markers Int J Clin Exp Pathol 2009 2 444 455 19294003 Tang P Wang J Bourne P Molecular classifications of breast carcinoma with similar terminology and different definitions: are they the same? 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==== Front Case Rep MedCase Rep MedCRIMCase Reports in Medicine1687-96271687-9635Hindawi Publishing Corporation 10.1155/2012/735026Case ReportBrain Metastasis as an Initial Manifestation of Ovarian Carcinoma: A Case Report Tuncer Zafer Selçuk 1 Boyraz Gokhan 2 *Yazıcıoğlu Aslıhan 2 Selcuk İlker 2 Yücel Senem Özge 2 1Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey2Department of Obstetrics and Gynecology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey*Gokhan Boyraz: [email protected] Editor: Michail Varras 2012 22 12 2012 2012 7350262 11 2012 11 12 2012 Copyright © 2012 Zafer Selçuk Tuncer et al.2012This 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.Brain metastasis is a rare and late manifestation of ovarian carcinoma. A 30-year-old woman initially presenting with neurologic symptoms was later found to have mucinous ovarian carcinoma. The patient had a 6 cm adnexal mass with multiple millimetric brain metastatic lesions. Following a surgical staging laparotomy procedure, the patient received brain irradiation and systemic chemotherapy. ==== Body 1. Introduction Brain metastasis from ovarian carcinoma is very uncommon; only less than 600 cases have been documented to date in the literature and it might be seen as a late manifestation of an ovarian tumor [1]. It usually occurs in patients who have a prolonged survival following chemotherapy regimen [2]. However, a limited number of ovarian carcinoma cases were reported with the initial diagnose of brain metastasis by the neurologic signs and symptoms in the literature. Thus, another case of brain metastasis from an ovarian tumor as an initial manifestation is presented here with the hope of improving diagnosis and management of such patients. 2. Case Presentation A 30-year-old gravida 2, para 2 woman admitted to our hospital with complaints of headache, nausea, vomiting, and right-sided blurred vision. She did not report any previous medical history or malignancy. Her neurologic examination revealed a right optic disc edema suggesting a posterior orbital mass. Her cranial computerized tomography (CT) scan showed multiple lesions that are a 6 mm mass on the right parietal lobe, a 16 mm mass on the left occipital, and another 7 mm mass on the left temporal lobe (Figures 1 and 2). All the lesions were hyperintense and surrounded by edema which suggests a metastatic cancer. Her cranial magnetic resonance imaging (MRI) also confirmed similar findings suggestive of a metastatic cancer to the brain. For identification of the primary malignancy, she initially underwent thoracic and abdominopelvic CT. Upper abdominal CT scan revealed indistinctly bordered and heterogeneously contrast accumulating multiple lesions less than 15 mm in the liver. There was a pelvic tumor of 62 × 53 mm sized and solid cystic in nature located in the right adnexa. The pelvic mass was round shaped and thin capsulated that contains irregular septations and a solid component of 38 × 24 mm on MRI scan (Figure 3). The right pelvic lymph nodes were slightly enlarged. No ascites were detected in the pelvic cavity. The endoscopic evaluation of the upper and the lower gastrointestinal tract and the ultrasonographic evaluation of the breast was found to be normal. The Pap test was reported to be negative for malignancy. Her serum tumor marker levels (CA 125, CA 15-3, CA 19-9, CEA, and AFP) were within normal limits. The patient underwent a diagnostic laparoscopy for further evaluation of the adnexal mass. Biopsy of the adnexal mass and the liver was obtained and the pathologic examination revealed an ovarian mucinous cystadenocarcinoma with metastatic lesions of liver. The patient then was subjected to a laparotomy for a surgical staging procedure including total abdominal hysterectomy, bilateral salpingooophorectomy, bilateral pelvic and para-aortic lymphadenectomy, and infracolic omentectomy and appendectomy. Her postoperative course was uneventful and she was discharged at the 6th postoperative day. The patient was determined to have a stage IV ovarian carcinoma with brain metastasis. An adjuvant therapy including whole brain irradiation (a total dose of 30 Gy in 10 fractions and 3 Gy per fraction) with simultaneous dexamethasone and systemic chemotherapy (two lines of six cure 400 mg/m2 carboplatin plus 175 mg/m2 paclitaxel with three weeks interval) was administered postoperatively. While documenting the patient, she was still alive 1.5 years after the initial diagnosis. 3. Discussion Ovarian cancer still remains as one of the leading causes of cancer related deaths in women. The majority of women with ovarian carcinoma have nonspecific pelvic and abdominal symptoms. Ovarian carcinoma is often diagnosed at an advanced stage but the disease rarely extends outside the peritoneal cavity. Central nervous system involvement in ovarian carcinoma is a very rare and late complication with an incidence of approximately 1% [2]. Common sources of brain metastases are lung, breast, renal and colorectal carcinoma, and malignant melanoma [1]. Mayer et al. reported only 5 cases of brain metastasis in 567 tissue biopsies performed on patients with ovarian carcinoma, accounting for an incidence of 0.9% [3]. Mucinous type of ovarian carcinoma incidence was even lower with a rate of 0.35% in the same study. The presented patient represents the only case with brain metastasis among 372 cases of ovarian carcinoma operated in the last 10 years at the institution. Metastasis is an important issue for tumor treatment and has great effects on survival rates. 5–30% of ovarian cancers are metastatic malignancies and colorectal adenocarcinoma is one of the outstanding causes of that situation. Colonic adenocarcinoma has similar features with primary ovarian carcinoma and can mimic it so colonic adenocarcinoma is the foremost misdiagnosed entity [4, 5]. Immunohistochemistry is very helpful for the differential diagnosis; CK20, CK7, and CDX2, especially, are important intermediate filament proteins used for attaining the accurate diagnosis. CK20 is normally expressed in the gastrointestinal epithelium [6]; however, CK7 is detected in normal tissue and tumors of the lung, breast, ovary, biliary tract, and endometrium [7]. A patient with a pathology report of CK20+/CK7− is predicting a colorectal cancer in approximately 75–95% of the cases [6, 7]. Since these two markers have limitations, CDX2 is highly expressed in colorectal cancers (61–100%) and rarely expressed (0–100%) in primary ovarian mucinous tumors. CK7, CK20, and CDX2 are useful markers for the discrimination of metastatic mucinous ovarian tumors and primary mucinous ovarian tumors [8]. Our patient was CK7 positive and CK20 and CDX2 negative stained. The differential diagnosis of brain metastases includes primary intracranial tumor, brain abscess, cerebral infarction, and cerebral hemorrhage [9]. However, recent studies showed that the incidence of brain metastasis has increased to 2–4% over time [2]. One of the possible reasons for the observed increase during the past decades includes changes in the natural course of ovarian carcinoma due to the improved control of intra-abdominal disease with surgery and chemotherapy. That results in a longer survival and allows distant metastasis to implant and grow, in addition to that, the availability of better imaging techniques also facilitates the diagnosis of distant metastasis [2]. The germline mutations BRCA1/2 cause women to have a tendency for cancer. With another point of view, up to 10% of ovarian cancer patients carry BRCA1/2 mutations [10]. BRCA1 mutation carriers have a big risk of early onset developing breast cancer with a lifetime risk of 56% to 84% and the estimated risk of developing ovarian cancer in BRCA1 mutation carriers ranges from 36% to 63%, whereas BRCA2 mutation carriers have an increased risk for both breast and ovarian cancer with an estimated risk of ovarian cancer ranging from 10% to 27%. There is a risk of developing a metachronous ovarian cancer for women with early onset breast cancer (younger than 40 years old) or who have a family history of breast or ovarian cancer or women who are BRCA1 mutation carriers [11]. Although ovarian cancer is frequently confined to the peritoneal cavity, distant and visceral metastasis can occur like our patient. That situation may have a tendency for some patient groups; Gourley et al. analysed Scottish women with BRCA1/2 mutations who are suffering from ovarian cancer without a history of previous breast cancer and showed that they had a frequency for visceral metastasis than matched control nonhereditary ovarian cancer patients who are negative for family or personal history of BRCA1/2. It is especially related to BRCA1 mutation carriers and 92% of these patients developed visceral metastasis; on the other hand only 16% of control group was complicated with that situation. This is also indicative for brain metastasis. As far this aspect of the literature is considered there is an intense relationship between visceral metastasis and BRCA1/2 mutation carriers for that reason BRCA1/2 mutation analysis should be performed for ovarian cancer patients who develop visceral metastasis [12]. Our patient was 30 years old but she is with no suggestive family history of breast cancer; having a visceral metastasis and being under 40 years old should be indicative for a genetic counsceling and BRCA mutation screening for her. The most common presenting symptoms of brain metastasis is headache, nausea, and vomiting which are related to increased intracranial pressure [9]. However, the presenting symptom of the patient, blurred vision, was possibly due to papilla edema. Our patient's having relatively small pelvic mass without ascites has probably precluded early diagnosis of ovarian carcinoma before the development of brain metastasis. The lesions of the brain metastasis were multiple in most of the reported cases in the literature [2]. A similar finding was also detected for our patient. Chen et al. found that the majority of the patients were under 65 years old and the age of patient at the time of diagnosis was not related to the survival period while the median survival rate for patients >65 and <65 is 41.9 and 8.9 months, respectively. The most common symptom and sign were headache and motor disfunction, respectively. Single lesion and surgical management of the lesion with combination therapy were favourable for these groups with better outcomes of prognosis [13]. Despite improvements in surgery and chemotherapy for ovarian carcinoma, survival is still limited. In most of the cases with cerebral metastasis, the prognosis is even poorer. Once the brain metastasis develops, the prognosis is very poor; the main therapeutic aim is to palliate and help the patient for a moderate status, and the median survival time is 3–6 months; 15% of patients are alive at the end of a year [14]. The prognosis for patients who have isolated brain metastasis is somehow a bit better than the patients with additional other organ metastasis. Brain involvement generally occurs and becomes clinically apparent after the diagnosis of ovarian cancer but a few cases have been described in the literature for simultaneous diagnosis of brain metastasis and ovarian cancer. Surgery, radiotherapy and chemotherapy could only lengthen the survival of the patient if combined [15]. Surgery, whole brain radiation therapy (WBRT), stereotactic radiosurgery (SRC), gamma knife surgery (GKS), chemotherapy, and alternative combination of these methods could be applied for metastatic brain lesions which were described in the literature before. Anupol et al. used gamma knife surgery (GKS) for a patient and had a good outcome; GKS treats multiple lesions at the same time, is easy to perform for recurrent and new lesions, and has a short hospital stay with less risk of deficit [16]. The surgical resection of the brain metastasis if possible and whole brain radiation therapy (WBRT) results in a prolonged survival time than surgery or WBRT did alone [17]. Systemic chemotherapy is also important for initial treatment of ovarian cancer especially taxol and carboplatin regimes may provide good initial approach after surgical staging procedure of primary tumor. Our patient was 30 years old with simultaneously diagnosed multiple metastatic brain lesions of ovarian cancer, so a surgical approach to brain lesions would not be approved. She was treated with surgical staging with the removal of the primary tumor in the abdomen, systemic chemotherapy (taxol and carboplatin) with brain radiation therapy. However, the cause of death in the majority of the patients was related to both brain metastasis and abdominal spread of disease [2]. And although brain metastasis is uncommon in ovarian carcinoma, it may develop even in the early phase of the disease. Disclaimer Authors did not receive any financial support for the preparation of this paper. Authors did not have communications with any company during the preparation of this paper. Figure 1 Cranial CT scan: lesion on the right parietal lobe. Figure 2 Cranial CT: left occipital cortical lesion. Figure 3 Pelvic MRI: the right adnexal mass. ==== Refs 1 Piura E Piura B Brain metastases from ovarian carcinoma ISRN Oncology 2011 2011 527453 2 Pectasides D Aravantinos G Fountzilas G Brain metastases from epithelial ovarian cancer. The Hellenic Cooperative Oncology Group (HeCOG) experience and review of the literature Anticancer Research 2005 25 5 3553 3558 2-s2.0-23344439254 16101179 3 Mayer RJ Berkowitz RS Griffiths CT Central nervous system involvement by ovarian carcinoma: a complication of prolonged survival with metastatic disease Cancer 1978 41 2 776 783 2-s2.0-0017850859 630551 4 Tavassoli FA Devilee P World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Breast and Female Genital Organs 2003 Lyon, France IARC Press 5 Bae JH Lee AW Tong SY Park YG Namkoong SE Park JS Preoperative and postoperative characteristics of metastatic ovarian cancers: experiences with 112 Korean women Journal of Korean Medical Science 2009 24 114 119 19270823 6 Moll R Lowe A Laufer J Franke WW Cytokeratin 20 in human carcinomas: a new histodiagnostic marker detected by monoclonal antibodies American Journal of Pathology 1992 140 2 427 447 2-s2.0-0026561237 1371204 7 Rubin BP Skarin AT Pisick E Rizk M Salgia R Use of cytokeratins 7 and 20 in determining the origin of metastatic carcinoma of unknown primary, with special emphasis on lung cancer European Journal of Cancer Prevention 2001 10 1 77 82 2-s2.0-0035090777 11263595 8 Vang R Gown AM Wu LSF Immunohistochemical expression of CDX2 in primary ovarian mucinous tumors and metastatic mucinous carcinomas involving the ovary: comparison with CK20 and correlation with coordinate expression of CK7 Modern Pathology 2006 19 11 1421 1428 2-s2.0-33750304107 16980943 9 Jin J Zhou X Liang X A study of patients with brain metastases as the initial manifestation of their systemic cancer in a Chinese population Journal of Neuro-Oncology 2011 103 3 649 655 2-s2.0-79959782055 20978821 10 Malander S Ridderheim M Måsbäck A One in 10 ovarian cancer patients carry germ line BRCA1 or BRCA2 mutations: results of a prospective study in Southern Sweden European Journal of Cancer 2004 40 3 422 428 2-s2.0-1642554820 14746861 11 Domchek SM Jhaveri K Patil S Risk of metachronous breast cancer after BRCA mutation-associated ovarian cancer Cancer . In press 12 Gourley C Michie CO Roxburgh P Increased incidence of visceral metastases in Scottish patients with BRCA1/2-defective ovarian cancer: an extension of the ovarian BRCAness phenotype Journal of Clinical Oncology 2010 28 15 2505 2511 2-s2.0-77956400006 20406939 13 Chen PG Lee SY Barnett GH Use of the radiation therapy oncology group recursive partitioning analysis classification system and predictors of survival in 19 women with brain metastases from ovarian carcinoma Cancer 2005 104 10 2174 2180 2-s2.0-27644448816 16208705 14 Deutsch M Beck D Manor D Brandes J Metastatic brain tumor following negative second-look operation for ovarian carcinoma Gynecologic Oncology 1987 27 1 116 120 2-s2.0-0023274165 3570045 15 Kumar L Barge S Mahapatra AK Central nervous system metastases from primary epithelial ovarian cancer Cancer Control 2003 10 3 244 253 2-s2.0-0038390307 12794622 16 Anupol N Ghamande S Odunsi K Driscoll D Lele S Evaluation of prognostic factors and treatment modalities in ovarian cancer patients with brain metastases Gynecologic Oncology 2002 85 3 487 492 2-s2.0-0036089312 12051879 17 Cohen ZR Suki D Weinberg JS Brain metastases in patients with ovarian carcinoma: prognostic factors and outcome Journal of Neuro-Oncology 2004 66 3 313 325 2-s2.0-1342291008 15015663
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Case Rep Med. 2012 Dec 22; 2012:735026
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23320098PONE-D-12-1444910.1371/journal.pone.0053645Research ArticleBiologyBiochemistryDrug DiscoveryBiotechnologyDrug DiscoveryMolecular Cell BiologyMedicineDiagnostic MedicinePathologyGeneral PathologyBiomarkersDrugs and DevicesDrug Research and DevelopmentDrug DiscoveryOncologyOncology AgentsEpithelial-Mesenchymal Transition Markers and HER3 Expression Are Predictors of Elisidepsin Treatment Response in Breast and Pancreatic Cancer Cell Lines EMT and HER3 Predicts Elisidepsin SensitivityTeixidó Cristina 1 Marés Rosó 1 Aracil Miguel 2 Ramón y Cajal Santiago 3 Hernández-Losa Javier 3 * 1 Molecular Pathology Group, Vall d’Hebron Research Institute, Universidad Autonoma of Barcelona, Barcelona, Spain 2 Pharmamar, Madrid, Spain 3 Pathology Department, Vall d’Hebron University Hospital, Universidad Autonoma of Barcelona, Barcelona, Spain Han Zhaozhong Editor Vanderbilt University, United States of America * E-mail: [email protected] Interests: Miguel Aracil is an employee for Pharmamar Company, which developed Elisidepsin. There are no other relevant declarations relating to employment, consultancy, patents, products in development or marketed products. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: CT SRC JHL. Performed the experiments: CT RM. Analyzed the data: CT JHL. Contributed reagents/materials/analysis tools: CT RM MA SRC JHL. Wrote the paper: CT JHL. 2013 8 1 2013 8 1 e5364516 5 2012 3 12 2012 © 2013 Teixidó et al2013Teixidó 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.Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment. This work has been partially funded by Pharmamar Company and by CENIT grant (CEN-2009-1016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734), a synthetic cyclic peptide originally isolated from the marine mollusk Elysia rufescens [1], is a cytotoxic anticancer agent [2], [3], [4]. Elisidepsin does not exhibit a linear cytotoxic dose-response and acts independently of the multidrug resistant status of various tumor cell lines [5]. The primary mechanisms of action of elisidepsin have not been identified, although multiple cellular targets have been described, many of which, due to the hydrophobic nature of the compound, are associated with the cell membrane [6], [7], [8], [9]. One of the several targets that are proposed to be involved in the cellular response to elisidepsin treatment is the human epidermal growth factor receptor family (HER) with several in vitro studies identifying HER3 and the downstream signaling pathway PI3K-AKT as major determinants of the cytotoxic activity of elisidepsin [10], [11]. Moreover, it has recently been postulated that elisidepsin induces the redistribution of HER3 from the plasma membrane to intracellular vesicles without comparable effects on HER1 and HER2, suggesting that it is HER3 that plays a key role in determining sensitivity to the drug [9]. On the other hand, specifically in relation to epithelial cells, one of the best-described processes that affects the composition of the cell membrane is that of the epithelial-mesenchymal transition (EMT), which is where cells downregulate their cell-cell junctions and acquire spindle cell morphology [12], [13]. The EMT plays an important role in development [14], [15], particularly in gastrulation and neural crest migration [14]. A critical component is the loss of type I cadherins that maintain stable cell-cell contacts through adherens junctions and desmosomes [16], [17]. To preserve cellular shape and polarity, the intracellular domains of cadherins connect to the actin cytoskeleton through α-catenin and β-catenin [18], [19], [20]. In most cases, this is associated with transcriptional repression of E-cadherin [21], [22], which in turn increases cell invasiveness [13], [22], [23], [24]. Several specific repressor factors have been identified, such as the zinc-finger domain-containing Snail and Slug factors [25], and the basic helix-loop-helix factor Twist, all of which can bind to the so-called E-boxes within the cadherin-1 (CDH1) gene promoter [25], [26]. Their function is regulated by oncogenic pathways, particularly by AKT, glycogen synthase kinase 3β (GSK-3β), NF-κB, RAS and SRC, some of which have been described as potential elisidepsin targets [15], [27], [28]. In this scenario, until the proposed above targets are validated, robust models that permit the identification novel predictive biomarkers are essential. To this end, and due to the increasing evidence supporting a role for the EMT in the progression of many cancer types, with critical roles in invasion and metastatic dissemination, we decided to study both HER3 and EMT as new predictive markers of elisidepsin treatment sensitivity in a panel of breast and pancreatic cell lines. In this report, we show that continued exposure to elisidepsin is correlated with a downregulation of epithelial markers in four different cancer cell types (pancreatic, breast, lung and colon). This behavior is further accompanied by several morphological and signaling changes, resulting in the upregulation of mesenchymal markers. Furthermore, we investigated the effect of the drug on the expression of HER proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing and knocking-down HER3 receptor. Finally, we identified HER3 expression as the most important sensitivity marker of elisidepsin studied. Results Cancer Cell Line Sensitivity to Elisidepsin We performed cell viability assays in a panel of 12 cell lines (6 breast cancer cell lines and 6 pancreatic carcinoma cell lines) to determine if there was a correlation between epithelial or mesenchymal expression markers and cell sensitivity to elisidepsin. Cells were treated with increasing concentrations of the compound for 72 h. The half maximal (50%) inhibitory concentration (IC50) values for elisidepsin, as measured by a crystal violet assay using a spectrophotometer, ranged from 0.075 to 14 µM within the cell line panel (Fig. 1A). 10.1371/journal.pone.0053645.g001Figure 1 Elisidepsin sensitivity. A) Elisidepsin IC50s were determined in a panel of breast (left) and pancreatic (right) cancer cell lines using a crystal violet assay. Cells were exposed to elisidepsin for 72 h. Results are shown as the mean ± SD of at least three independent experiments. B) Cell proliferation in parental and subtoxic elisidepsin-treated cells. Cumulative numbers of cell divisions [shown as population doubling level (PDL)] are shown for MCF-7 and MiaPaCa-2 cells until passage 5. Proliferation of MCF-7 (IC50∶0.4 µM) and MiaPaCa-2 (IC50∶14 µM) cells was suppressed when elisidepsin was added to the culture at subtoxic doses (0.2 and 1 µM, respectively). The number of MiaPaCa-2 and MCF-7 seeded cells were 1.25×105 and 1.4×105, respectively. Each growth curve was performed at least twice with similar results, SDs are shown, and each time point was performed in duplicate. P, passage. According to the results of a previous paper from our lab and others [27], [28], only those cells with an IC50 value under or equal to 1 µM are considered sensitive to the elisidepsin. MDA-MB-231, PANC-1 and MiaPaCa-2 cell lines were the only cell lines that had an IC50 value higher than 1 µM (6.5, 7.5 and 14 µM, respectively). The other cell lines were classed as being sensitive to the drug (with IC50 values ranging from 0.075 to 0.6 µM). The effect of elisidepsin is not considered to be time-dependent as no significant difference in the ratio of IC50 values was seen by Sewell et al. [7] following 1 h exposure and continuous exposure. However, when we treated the cells with continuous exposure to a subtoxic dose (i.e. lower than the IC50) the cells grew more slowly than the parental ones (Fig. 1B). Recent studies have shown that the potent cytotoxic activity of elisidepsin is exerted very rapidly through insertion of the drug molecule into the plasma membrane, which causes a drastic loss in membrane integrity [8]. However, we found that, despite elisidepsin-induced loss of membrane integrity, those cells that remained alive after treatment could recover and proliferate again (Fig. S1). This was shown by treating MCF-7 cancer cell lines with 1 µM elisidepsin for 4 h, removing the drug and measuring proliferation at different time points. More than 50% of cells died after 4 h drug treatment but when the media was replaced the cells recovered and their viability increased. Correlation between EMT Markers and Elisidepsin Cell Sensitivity In order to evaluate EMT protein expression levels and correlate them with the sensitivity of the cell lines to elisidepsin, we performed different analyses using western blot, immunofluorescence and immunohistochemistry (IHC) in a panel of 12 cell lines. The protein expression of E-cadherin, β-catenin, vimentin, Slug, Snail and Twist-1 were assessed by immunocytochemical and western blot analysis, while the protein expression of E-cadherin, β-catenin, and vimentin were evaluated by immunohistochemical analysis. We aimed to determine whether the various elisidepsin-sensitive cancer cell lines shared similar basal levels of EMT genes. In the breast cancer cell lines we found E-cadherin expression in the sensitive cell lines. All cell lines had detectable expression of β-catenin, whereas Slug expression was variable and not related to their sensitivity to elisidepsin. Furthermore, Snail expression was only found in MDA-MB-435, and all the cell lines that exhibited levels of Twist-1 and vimentin were less sensitive to the drug (Figs. 2A–C). In contrast, elisidepsin-sensitive pancreatic carcinoma cell lines expressed E-cadherin and β-catenin, whereas the less sensitive cells expressed Slug. Lastly, Snail, Twist-1 and vimentin expression was found in sensitive and insensitive cell lines alike (Figs. 3A–C). To summarize, E-cadherin protein was significantly expressed in the sensitive cell lines independently of their tumoral origin (Mann-Whitney test: p  = 0.0364; Fig. S2), and vimentin was significantly expressed in the less sensitive ones (Mann-Whitney test: p  = 0.0364). On the other hand, Twist-1 and Snail proteins were found in all less sensitive cell lines (Mann Whitney test: p  = 0.0636 and p  = 0.1000, respectively), with the exception of two sensitive cell lines that were positive for vimentin expression (CFPAC and AsPC-1), one sensitive cell line that was positive for Twist-1 expression (CFPAC) and another one that was positive for Snail expression (SKBR3). 10.1371/journal.pone.0053645.g002Figure 2 Expression of EMT markers associated with elisidepsin sensitivity in breast cancer cell lines. Protein expression levels of different EMT markers were evaluated by immunocytochemistry (A), western blot (B) and IHC (C). A) Immunocytochemistry of two epithelial (E-cadherin and β-catenin) and four mesenchymal markers (vimentin, Slug, Snail and Twist). Magnification 100x. B) E-cadherin, β-catenin, Slug, Snail, Twist, vimentin and β-actin (loading control) were detected by western blot analysis using 50 µg of total protein. C) Basal levels of E-cadherin, β-catenin and vimentin were analyzed by IHC. Magnification 20x. Each experiment was performed at least in duplicate. 10.1371/journal.pone.0053645.g003Figure 3 Expression of EMT markers associated with elisidepsin sensitivity in pancreatic cancer cell lines. E-cadherin, β-catenin, vimentin, Slug, Snail and Twist basal expression levels were evaluated by immunocytochemistry (A) and western blot (50 µg of protein/lane) (B). Magnification 100x. Membranes were stripped and reprobed with anti-β-actin as an internal control. C) E-cadherin, β-catenin and vimentin protein expression levels were evaluated by IHC. Magnification 20x. These analyses were performed in duplicate. HER3 Expression Levels Correlate with Elisidepsin Cell Sensitivity The primary mechanisms of action of elisidepsin remain to be elucidated but we and other groups have found that after 4 h treatment with 1 µM elisidepsin, HER3 receptor levels are downregulated in a panel of different cell lines, including lung, breast, melanoma and colon carcinomas [10], [11]. To determine if HER3 protein expression levels correlate with the sensitivity of the cell lines to elisidepsin, we performed IHC (Fig. 4A) and western blot analysis (Fig. 4B) in all cell lines. Cell lines that were less sensitive to elisidepsin had little to no HER3 while sensitive cell lines expressed significantly increased levels of this protein (Mann-Whitney test: p  = 0.0091; Fig. S3). In addition, others members of the HER family were checked by western blot (Fig. 4B) but no correlations with elisidepsin sensitivity were found with HER1, HER2 and HER4 (Mann-Whitney test: p  = 0.7273, p  = 0.5182 and p  = 0.8909, respectively). 10.1371/journal.pone.0053645.g004Figure 4 HER3 expression levels correlate with cell sensitivity to elisidepsin. A) Cell pellets were fixed in formalin, embedded in paraffin and a HER3 IHC was performed. Cell lines more sensitive to elisidepsin had significant HER3 levels. Magnification 40x. B) Basal expression levels of HER family members were analyzed by western blot; an association between HER3 expression and elisidepsin sensitivity was observed (Mann-Whitney test: p  = 0.0091; Fig. S3). Cell lines less sensitive to elisidepsin (MDA-MB-231, PANC-1 and MiaPaCa-2) did not show significant HER3 protein levels, while PANC-1 and MiaPaCa-2 cell lines show levels of other HER family members. No correlation was observed with HER1, HER2 and HER4 expression levels (Fig. S3). These protein expression levels were analyzed in duplicate and 50 µg of protein of cell lysate were loaded in each lane. Acquired Resistance to Elisidepsin Induces an EMT Phenotype Three elisidepsin-resistant cancer cell lines [one breast (MCF-7) and two pancreatic (HPAC, AsPC-1)] were generated by continuous exposure to increasing concentrations of the drug (see Material and Methods). Cancer cell lines were exposed to elisidepsin at a starting concentration of its IC50. Elisidepsin concentration was increased every week until cells became resistant to the drug, after approximately 12 months in the case of MCF-7, and after approximately 4 months in the case of HPAC and AsPC-1. The morphology of the resistant cancer cell lines was modified after continuous exposure to the drug when compared to that of the parental cell lines (data not shown). Our hypothesis was that the loss of epithelial markers observed in our panel of cancer cell lines could be responsible for the resistance to elisidepsin treatment, which would in turn result in the acquisition of mesenchymal markers in these cells. We then performed western blot analysis of the cancer cell lines with acquired resistance and compared them to the corresponding parental control cells. We identified that the three different cancer cell types with acquired resistance to elisidepsin had altered basal levels of EMT markers (Fig. 5A). All resistant cell lines showed decreased E-cadherin, γ-catenin and increased vimentin and Twist-1 expression. β-catenin expression was downregulated in the resistant HPAC and AsPC-1 cancer cell lines but upregulated in the MCF-7. In contrast, levels of Slug and Snail were upregulated in the resistant cancer cell lines HPAC and AsPC-1 but no differences were found in the breast carcinoma MCF-7 cell line. 10.1371/journal.pone.0053645.g005Figure 5 Acquired resistance to elisidepsin induces an EMT phenotype. A) Cells were lysed, proteins were extracted and western blots were performed with equal amounts of cell lysate (50 µg protein). Expression of epithelial (E-cadherin, β-catenin, γ-catenin)- and mesenchymal (vimentin, Slug, Snail, Twist)-associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. β-actin was used as an internal control. These western blots were performed in triplicate. B) Expression levels of HER1, HER2, HER3, HER4, pAkt, and pMAPK were analyzed by western blot using 50 µg of protein cell lysate. The membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. C, control; R, resistance. We also performed the same approach in different resistant cell lines from colon and lung (HCT116 and A549, respectively) with similar results (Fig. S4). Analysis by western blot confirmed that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, we wanted to see if these cells also showed different expression levels of HER family members and proteins of their signaling pathways. We observed that the levels of all HER family members and their downstream signaling partners were downregulated in all resistant cancer cell lines (Figs. 5B and S4). A suppression of downstream signaling was similarly seen in the breast and pancreatic resistant cell lines, and the same expression pattern was also observed in other colon and lung resistant cell lines, highlighting the relevance of this phenomena. Modulation of HER3 Affects Cancer Cell Line Sensitivity to Elisidepsin Based on previous studies from our group and others demonstrating that elisidepsin downregulates the HER3 receptor tyrosine kinase and that high expression of HER3 is prevalent in a broad number of different tumor cells, we investigated if modulation of protein expression levels of the HER3 receptor affects sensitivity to elisidepsin in a panel of tumor cell lines with variable expression of this receptor. To examine this experimentally, we utilized a shRNA construct to stably reduce HER3 expression in a panel of cell lines (Fig. 6). Stable clones of HER3 shRNA and LUC shRNA (control) vector-transfected cells were selected and examined for expression of HER3. As expected, levels of HER3 were significantly reduced in HER3-transfected cells, but not in those containing the pLKO LUC shRNA vector alone, indicating that the decrease in HER3 was not due to non-specific effects of introducing shRNA into the cells. Next, cell viability assays were performed to analyze elisidepsin sensitivity in the generated cells. Figure 6 shows that cells that have reduced levels of HER3 due to shRNA-mediated knockdown of its expression showed loss of sensitivity to elisidepsin treatment in comparison to control cell lines. 10.1371/journal.pone.0053645.g006Figure 6 Loss of HER3 expression decreases the sensitivity to elisidepsin treatment. Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in SKBR3 (A), MCF-7 (B), MDA-MB-231 (C), MDA-MB-435 (D), BT474 (E), BxPC-3 (F), HPAC (G) and AsPC-1 (H) cells. HER3 expression was downregulated with shRNA (grey squares); LUC shRNA transfected cells were used as the control (black diamonds). Mean, SD, and IC50 values are shown from three independent experiments. Cell viability was measured using a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 µg of protein to confirm their levels of HER3 expression. To investigate whether ectopic HER3 expression affects the elisidepsin sensitivity of low HER3-expressing cells, the levels of HER3 were increased by transfecting cells with a cDNA encoding HER3, which resulted in increased sensitivity of the cells to elisidepsin. In comparison to control cells transfected with a LUC vector, decreased cell viability was noted in HER3-transfected cells (Fig. 7). Altogether, these results suggest that ectopic HER3 expression sensitizes these cells to elisidepsin treatment. 10.1371/journal.pone.0053645.g007Figure 7 Upregulation of HER3 increases elisidepsin sensitivity. Cell viability after treatment with various concentrations of elisidepsin for 72 h was determined in PANC-1 (A), MiaPaCa-2 (B), MDA-MB-435 (C) and MDA-MB-231 (D) cells. Stable cell lines with an upregulation of HER3 expression (with the pIRES HER3) are shown with white circles while black diamonds are used for LUC-transfected control cells (with the pIRES-LUC). Mean, SD, and IC50 values are shown from three independent experiments. Cell viability was measured by a crystal violet assay. Before performing the viability experiments, all cell lines were checked by western blot using 50 µg of protein to confirm their levels of HER3 expression. Discussion Elisidepsin is a novel marine compound with a potent cytotoxic activity in various tumor cell lines. The mechanisms of actions of this compound remain poorly understood, although several targets have been proposed to be involved in the cellular response to elisidepsin treatment, such as fatty acid-containing ceramides, fatty acid 2-hydroxylase (FA2H), lysosomes, lipid rafts and epithelial growth factor receptors, including the HER receptors [10], [29], [30], [31], [32], [33]. In the present study we explored whether basal levels of EMT markers and HER receptor proteins could be predictive markers for elisidepsin treatment. The role of the cell membrane as an important target of elisidepsin was studied in breast and pancreas cancer cell lines. Basal levels of EMT protein expression markers showed a significant correlation with the cell viability response to elisidepsin treatment in a panel of 12 different cancer cell lines. The epithelial marker E-cadherin protein was significantly expressed in the sensitive cell lines (p  = 0.0364) while expression of the mesenchymal markers vimentin, Twist-1 and Snail, was found in all cell lines with reduced sensitivity to the drug. Furthermore, this study showed that continuous exposure to elisidepsin correlates with a downregulation of epithelial markers in 4 different cancer cell types (breast, pancreas, lung and colon). Loss of epithelial markers was further evidenced by the detection of morphological changes in the cells. These changes, which were observed after continuous long-term exposure of different cell types to elisidepsin, suggest that the drug is able to modify the composition of the plasma membrane. This behavior was further accompanied by signaling changes, resulting in the upregulation of mesenchymal markers. This analysis confirmed that acquired resistance to elisidepsin is associated with a switch to the EMT state. On the other hand, regarding HER family receptors, we observed an association between HER3 protein expression and sensitivity to elisidepsin treatment in a variety of cell lines (p  = 0.0091). The other members of the HER family were also checked by western blotting and we did not find any significant correlation. Interestingly, HER4 expression was observed in 4 out of 5 elisidepsin-sensitive breast cancer cell lines, and further studies that include more breast cancer cell lines are necessary to establish the potential predictive marker of the HERs for elisidepsin sensitivity in breast cancer models. Cell lines that were less sensitive to elisidepsin had lower or undetectable levels of HER3 in comparison with sensitive cell lines, supporting a hypothetical role for HER3 in the cellular response to this drug, although other authors propose that HER3 is part of a secondary process involving cell membrane alterations due to elisidepsin treatment [9] or to a reduction in the proliferation rate in those cells with less HER3 expression. Importantly, we could not discard the possibility that elisidepsin may affect other signaling pathways, such as those of the transforming growth factor-β (TGF-β) family, which potentially plays a role in both EMT and HER3 expression. Malignant cells commonly possess overactive signal transduction cascades that provide potential selective targets for antitumor drugs [34]. Based on previous studies in which we observed elisidepsin treatment-induced HER3 downregulation [11], in addition to the fact that high expression of the HER3 receptor tyrosine kinase is prevalent in tumor cells, including cancers of the breast, ovary and prostate [35], [36], [37], [38], [39], [40], [41], [42], we correlated basal protein expression levels of HER3 with sensitivity to elisidepsin in a panel of tumor cell lines with variable expression of this receptor and found that downregulation of HER3 exerted a protective effect against elisidepsin cytotoxicity. In fact, HER3 significantly increases cell sensitivity in all cell lines studied, supporting previous indications that HER3 could be a good predictive marker of cell sensitivity to elisidepsin. In summary, we present solid evidence that sensitivity to elisidepsin correlates with HER3 receptor expression. However, it remains to be elucidated why elisidepsin affects HER3 and why its effects depend on HER3 expression. Materials and Methods Chemicals Elisidepsin (Figure S5) was obtained from PharmaMar (Madrid, Spain) as a lyophilized powder. It was reconstituted in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Taufkirchen, Germany)/ethanol (1∶1) as a 1 mM stock solution, which was stored in aliquots at −20°C. Drug dilutions were freshly prepared before each experiment in order to avoid degradation. Cells and Cell Culture Cell lines were obtained from the American Type Culture Collection (VA, USA). The following cell lines were maintained in RPMI 1640 with 4 mM L-glutamine: MDA-MB-435, MDA-MB-231, MCF-7 and SKBR3 (breast carcinoma), and AsPC-1 and BxPC-3 (pancreas carcinoma). The following cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine and 4.5 g/L glucose: HCT116 (colon carcinoma), MDA-MB-468 (breast carcinoma), and HPAC, PANC-1 and MiaPaCa-2 (pancreas carcinoma). A549 (lung carcinoma) was maintained in Ham's F-12 medium supplemented with 1 mM L-glutamine. Finally, DMEM:Ham's F12 (1∶1 mixture) supplemented with 1 mM L-glutamine was used to maintain BT474 (breast carcinoma). HPAC resistant to elisidepsin 8 µM and AsPC-1 resistant to elisidepsin 4.5 µM were generated by continuous exposure to the drug for ∼4 months, while HCT 116 resistant to elisidepsin 100 µM, A549 resistant to elisidepsin 25 µM, and MCF-7 resistant to elisidepsin 4 µM were generated by continuous exposure to the drug for ∼1 year. The media for all cell lines were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 10 mM HEPES and were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2. Western Blots Immediately prior to use in western blotting, cultured cells were lysed and collected in lysis buffer. Lysates were centrifuged and supernatants were collected for protein concentration determination using the Bradford (Bio-Rad Protein Assay, Munich, Germany) method. Equal amounts of protein were separated by 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels, electrophoresed at 100 V and electroblotted onto polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA) at 0.4 A at room temperature. Blots were blocked in 5% nonfat dry milk in phosphate-buffered saline (PBS) for 1 h at room temperature. After blocking, membranes were incubated overnight with primary antibodies against HER1 (F4, Sigma-Aldrich); HER2 (CB11, BioGenex, Fremont, CA, USA); HER3 (2F12, NeoMarkers, Fremont, CA); HER4 (111B2), Akt (#9272), γ-catenin (#2309) and Snail (L70G2; all from Cell Signaling, Beverly, MA, USA); MAPK (C-14), Slug (H-140) and Twist-1 (H-81; all from Santa Cruz, Heidelberg, Germany); β-actin (A2228, Sigma-Aldrich); vimentin (V9, Dako, Sant Just Desvern, Spain); and E-cadherin and β-catenin (Novocastra, Badalona, Spain). After incubation with horseradish peroxidase-conjugated secondary antibodies, antigen-antibody complexes were visualized using enhanced chemiluminescence (Amersham Biosciences, Dreieich, Germany). Western blots were repeated in independent conditions at least twice and representative blots are shown. Densitometrical quantification of autoradiograms was performed using ImageJ software (version 1.41o, National Institutes of Health, Bethesda, MD) by normalizing to the intensity of β-actin in each sample and are expressed in arbitrary densitometric units. Immunocytochemistry Cells were seeded on coverslips at 60% confluence, fixed in 4% formaldehyde/PBS, permeabilized in 100% methanol for 20 min, and blocked in 2% BSA for 1 h. Fixed cells were incubated with anti-E-cadherin (clone 36B5, 1∶50) and anti-β-catenin (Novocastra, 1∶50), anti-Snail (Cell Signaling, 1∶50), anti-Vimentin (Dako, 1∶50), and anti-Slug and anti-Twist-1 (Santa Cruz, 1∶50) overnight at 4°C. After washing with PBS, cells were incubated for 1 h in blocking buffer at room temperature with either 1∶800 Alexa Fluor 546 rabbit anti-mouse IgG (Invitrogen, Barcelona, Spain) or 1∶200 Alexa Fluor 647 mouse anti-human (Invitrogen). Cells were washed twice with PBS and once with distilled water. Finally, cells were mounted in Citifluor (Leicester, UK) before observation and analysis with fluorescence microscopy. Immunohistochemical Staining IHC using the avidin-biotin-peroxidase technique was performed for each antibody. Five-micron-thick sections were cut from formalin-fixed, paraffin-embedded cell pellets and placed on poly-L-lysine-coated glass slides. Sections were deparaffinized in xylene and rehydrated in graded alcohol. Endogenous peroxidase was blocked by immersing the sections in 0.1% hydrogen peroxidase in absolute methanol for 20 min. For antigen retrieval, the tissue sections were heated in a pressure cooker in 10 mM citric acid monohydrate, pH 6.0, for 5 min, and then incubated with primary antibodies for 60 min at room temperature. IHC was performed with the Benchmark XT slide stainer (Ventana Medical Systems, Inc, Tucson, AZ, USA). The primary antibodies and dilutions used were: anti-HER3 [generated by Dr. Pandiella (IBMCC, Salamanca, Spain), 1∶75], anti-E-cadherin (Dako, pre-diluted), anti-β-catenin (Novocastra, 1∶50) and anti-vimentin (Dako, pre-diluted). All slides were hematoxylin counterstained, dehydrated, and mounted. Negative controls were performed by omitting the primary antibody and showed minimal non-specific signal. Cell Growth Assay Cells were plated overnight at a density of 50,000 cells/well. Cell lines were treated with various concentrations of elisidepsin for 72 h. At least 3 wells were used for each condition and cell viability was measured by a crystal violet assay. Briefly, cells were fixed after each treatment in 1% glutaraldehyde for 20 min, washed twice in PBS, stained with 0.1% crystal violet for 30 min and then washed with abundant deionized water. Colorant was recovered using 5% acetic acid and optical density was measured at 590 nM with an ELISA plate reader. Plasmids and Cell Transfection The pIRES-HER3 and the pIRES-Luciferase (LUC) were kindly donated by Dr. Scaltriti (Vall d’Hebron University Hospital Research Institute, Barcelona, Spain). The pIRES-LUC was used as a control for transfection. The pIRES vectors confer hygromycin resistance. Cells were transfected for 12 h with Jet Pei (Polyplus-Transfection, Illkirch, France). To eliminate untransfected cells and generate stably expressing HER3 cell lines, medium supplemented with hygromycin (Sigma-Aldrich) was added 24 h after transfection, and cells underwent selection for 10 days. Lentivirus shRNA Production and Transduction Short hairpin RNAs (shRNAs) were used for inhibiting HER3 expression in different cancer cell lines. The following sequences were used: shHER3_3.1F: GATCCAAGAGCGACTAGACATCAAGCTTCAAGAGAGCTTGATGTCTAGTCCCTCTTTTTTTACGCGTG, shHER3_3.1R: AATTCACGCGTAAAAAAAAGAGCGACTAGACATCAAGCTCTCTTGAAGCTTGATGTCTAGTCGCTCTTG; shHER3_3.3F: ATCCGCCAATACCAGACTGTACTTCAAGAGAAGTACAGTGTCTGGTATTGGTTTTTTACGCGTG, and shHER3_3.3R: AATTCACGCGTAAAAAACCAATAC CAGACACTGTACTCTCTTGAAGTACAGTGTCTGGTATTGGCG. The different sequences were cloned into the lentiviral vector pLKO.1 (Sigma-Aldrich). The pLKO.1-shRNA LUC was used as a control for transfection. All vectors encode puromycin resistance. Plasmids pVSVG and pCMVΔR8.91 for the expression of packaging and envelope proteins were kindly provided by Dr. Peeper (VU University Medical Center, Amsterdam, The Netherlands). Two plates seeded with 1.5×106 HEK 293T cells were co-transfected in DMEM 10% FBS with 2 µg of pLKO.1, 2 µg of pCMVΔR8.91 and 2 µg of pVSVG and incubated overnight. Cells were washed and incubated in 10% CO2 with medium containing 5% FBS. After 48 h, the virus-containing supernatant was recovered and filtered with 0.45 nM filters (Sarstedt, Nümbrecht, Germany). Titration was performed by infecting cells with the recovered viral particles in the presence of 4 µg/mL polybrene (Sigma-Aldrich). Western blot was used to assess the silencing efficacy of the two shHER3 sequences, and the most effective one (shHER3.3) was chosen. To obtain cell lines with stable depletion of HER3, infected cells were selected with puromycin for three days. Statistical Analysis Statistical studies were performed with the Statistical Package for the Social Sciences (SPSS 15.0; SPSS, Chicago, IL). The Mann-Whitney test was used to find associations of the parameters analyzed between two previously selected groups, Sensitive (includes cell lines with an elisidepsin IC50≤1 µM) and Less Sensitive (includes cell lines with an elisidepsin IC50>1 µM). Cell growth data are expressed as the mean ± standard deviation (SD). Statistical significance was set at a two-tailed p value of 0.05. Supporting Information Figure S1 MCF-7 cells can recover after elisidepsin treatment. A) Cells were treated with 1 µM of elisidepsin for 4 h, the culture medium was changed and cells were maintained in the fresh medium for 4, 24, 48 and 72 h. HER1-4 protein expression levels were analyzed by western blot using 50 µg of protein from total MCF-7 cell lysates loaded in SDS-PAGE gels. Membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. B) Cells were treated with 1 µM of elisidepsin for 4 h and proliferation was measured by a crystal violet assay at different time points (white squares) and compared to untreated cells (black diamonds). Results are expressed as the mean ± SD of two independent experiments. C, control. (TIF) Click here for additional data file. Figure S2 Statistical analysis of EMT basal expression levels of breast and pancreas cancer cell lines. Levels of ErbB3 protein were quantified using western blot analysis (see Material and Methods) by densitometry. The graph represents the relative ErbB3 expression in elisidepsin-sensitive (IC50≤1 µM) and -resistant (IC50>1 µM) cell lines. The Mann-Whitney test showed a statistically significant p value of 0.015. (TIF) Click here for additional data file. Figure S3 Elisidepsin cell sensitivity is associated with HER3 expression levels. Levels of HER1, HER2, HER3 and HER4 protein were quantified with western blot analysis (Fig. 4) and subsequent densitometry. Cells that have an elisidepsin IC50 value of ≤1 µM were considered sensitive to the drug. The graph represents the HER family members expression relative to elisidepsin sensitivity. A statistically significance relationship between HER3 expression levels and elisidepsin sensitivity was found (Mann-Whitney test: p  = 0.0091) but not with the other members. (TIF) Click here for additional data file. Figure S4 Generation and characterization of elisidepsin-resistant cell lines from colon and lung. A) Cells were lysed, proteins were extracted and western blots performed with an equal amount of cell lysate (50 µg protein). Expression of epithelial (E-cadherin, β-catenin, γ-catenin)- and mesenchymal (vimentin, Slug, Snail, Twist)-associated proteins differentiates between elisidepsin-sensitive and elisidepsin-resistant cell lines. β-actin was used as an internal control. These western blots were performed in triplicate. B) Expression levels HER1, HER2, HER3, HER4, pAkt, and pMAPK were analyzed by western blot using 50 µg of protein cell lysate. The membranes were stripped and reprobed with anti-β-actin to verify equal protein loading. HCT 116 (C) and A549 (D) elisidepsin-sensitive cancer cell lines were rendered resistant by persistent exposure to increasing concentrations of elisidepsin. Cells were treated with elisidepsin at the indicated concentrations for 72 h and cell viability was measured using a crystal violet assay. Error bars show the SD of three replicate experiments. C, control; R, resistance. (TIF) Click here for additional data file. Figure S5 Chemical structure of elisidepsin. (TIF) Click here for additional data file. We would like to thank Dr. Atanasio Pandiella for providing the HER3 antibody and for helpful discussions during the preparation of the manuscript. ==== Refs References 1 Hamann MT , Otto CS , Scheuer PJ , Dunbar DC (1996 ) Kahalalides: Bioactive Peptides from a Marine Mollusk Elysia rufescens and Its Algal Diet Bryopsis sp.(1) . J Org Chem 61 : 6594 –6600 .11667527 2 Wosikowski K , Schuurhuis D , Johnson K , Paull KD , Myers TG , et al (1997 ) Identification of epidermal growth factor receptor and c-erbB2 pathway inhibitors by correlation with gene expression patterns . 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PLoS One. 2013 Jan 8; 8(1):e53645
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23308143PONE-D-12-1679510.1371/journal.pone.0053087Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCell DeathMedicineDrugs and DevicesDrug Research and DevelopmentOncologyCancers and NeoplasmsLung and Intrathoracic TumorsNon-Small Cell Lung CancerBasic Cancer ResearchOncology AgentsFrondoside A Suppressive Effects on Lung Cancer Survival, Tumor Growth, Angiogenesis, Invasion, and Metastasis Frondoside A a Novel Lung Anticancer AgentAttoub Samir 1 * Arafat Kholoud 1 Gélaude An 2 Al Sultan Mahmood Ahmed 1 Bracke Marc 2 Collin Peter 3 Takahashi Takashi 4 Adrian Thomas E. 5 De Wever Olivier 2 1 Department of Pharmacology & Therapeutics, Faculty of Medicine & Health Sciences, U. A. E. University, Al-Ain, United Arab Emirates 2 Laboratory of Experimental Cancer Research, University Hospital, Gent, Belgium 3 Coastside Bio Resources, Stonington, Maine, United States of America 4 Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan 5 Department of Physiology, Faculty of Medicine & Health Sciences, U. A. E. University, Al-Ain, United Arab Emirates Chellappan Srikumar P. Editor H. Lee Moffitt Cancer Center & Research Institute, United States of America * E-mail: [email protected] Interests: Peter Collin is director, laboratory manager, employee and stock-holder of Coastside Bio Resources, a Maine, USA Corporation. Thomas Adrian and Peter Collin are co-inventors of a United States patent describing Frondoside A and other sea cucumber glycosides as putative anti-cancer agents, and may benefit financially if Frondoside A becomes a drug for human cancers. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: SA ODW. Performed the experiments: SA KA AG MAAS TEA. Analyzed the data: SA MB TEA ODW. Contributed reagents/materials/analysis tools: PC TT. Wrote the paper: SA ODW TEA MB TT. 2013 8 1 2013 8 1 e5308713 6 2012 27 11 2012 © 2013 Attoub et al2013Attoub 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.A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer. This work was financially supported by the FMHS grant number NP/08/27 (SA), the UAE University grant under a contract no. 01-04-8-11/09 (SA), the Terry Fox Fund for Cancer Research (SA and TA), the UAEU-NRF 09/10 grant number 21MO72 (SA), and the Maine Technology Institute, Gardiner, Maine, USA, and the National Cancer Institute, RAPID Program (PC). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Lung cancer is the most common form of cancer with one of the highest mortality rates in the world. Targeted therapies for selected subgroups of patients constitute a remarkable progress in the treatment of lung cancer. However, despite these advances, controversies remain, patients die, and a cure remains elusive [1]. Natural compounds are emerging as a new generation of anticancer agents with limited toxicity in cancer patients [2], [3]. They can have high value in tumors resistant to classical chemotherapies or resistant to tyrosine kinase inhibitors such as gefitinib Sea cucumbers have been valued for hundreds of years in the Chinese diet as a food delicacy, as well as a medicine for a wide variety of diseases. In the United States and Canada, sea cucumber tissues are dried, pulverized and encapsulated as nutraceuticals for over-the-counter dietary health supplements, primarily directed at inflammatory conditions in humans and companion animals [4]. Frondoside A is a triterpenoid glycoside isolated from the Atlantic cucumber, Cucumaria frondosa. (See [5] for chemical structure). Recent studies demonstrate that low concentrations of Frondoside A inhibit the growth and induced apoptosis of human pancreatic, leukemia and breast cancer cells via caspase activation [6]–[8]. The chemotherapeutic agents currently in use for lung cancer are still unsatisfactory due to associated co-lateral toxicity and drug-induced resistance [9]–[11] which motivate our investigation of the impact of Frondoside A on human non-small cell lung cancer survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Materials and Methods Cell culture and reagents Human lung cancer cells LNM35 (NSCLC) [12], A549 and NCI-H460-Luc2 (Caliper LifeSciences, US) were maintained in RPMI 1640 (Invitrogen, Paisley, UK), human melanoma MDA-MB-435, human mammary adenocarcinoma cells MCF-7, and human hepatoma cells HepG2 were maintained in DMEM (Invitrogen, Paisley, UK). All media were supplemented with antibiotics (penicillin 50 U/ml; streptomycin 50 µg/ml) (Invitrogen, Cergy Pontoise, France) and with 10% fetal bovine serum (FBS, Biowest, Nouaille, France). EndoGRO™ Human Umbilical Vein Endothelial Cells (HUVECs) (Millipore, Temecula, CA) were maintained in EndoGRO™-MV-VEGF Complete Media Kit (Millipore, Temecula, CA). Cisplatin was purchased from Sigma-Aldrich (Sigma-Aldrich, Saint-Quentin Fallavier, France). Frondoside A was purified from Cucumaria frondosa, harvested near Stonington, Maine and the purity (99.9%) confirmed by NMR as previously described [13], [14]. Cellular viability Cells were seeded at a density of 5,000 cells/well into 96-well plates. After 24 h, cells were treated for another 24 h with different concentrations of Frondoside A (0.01–5 µM), in triplicate. Control cultures were treated with 0.1% DMSO. The effect of Frondoside A on cell viability was determined using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison, USA), based on quantification of ATP, which signals the presence of metabolically active cells. The luminescent signal was measured using the GLOMAX Luminometer system. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%. Caspase 3/7 activity LNM35 cells were seeded at the density of 5,000 cells/well into 96-well plate and treated with Frondoside A (1–2.5 µM) for 2 and 24 h, in triplicate. Caspase-3/7 activity was measured using a luminescent Caspase-Glo 3/7 assay kit following the manufacturer's instructions (Promega Corporation, Madison, USA). Caspase reagent was added and the plate was mixed using an orbital shaker and incubated for 2.5 h at room temperature. Luminescence was measured using a GLOMAX Luminometer system. Wound healing motility assay LNM35 cells were grown in six-well tissue culture dishes until confluence. Cultures were incubated for 10 min with Moscona buffer. A scrape was made through the confluent monolayer with a plastic pipette tip of 1 mm diameter. Afterwards, the dishes were washed twice and incubated at 37°C in fresh RPMI containing 10% fetal bovine serum in the presence or absence of the non-toxic concentrations of Frondoside A (0.1–0.5 µM). At the bottom side of each dish, two arbitrary places were marked where the width of the wound was measured with an inverted microscope (objective ×4) (Olympus 1X71, Japan). Motility was expressed as the average ± S.E.M of the difference between the measurements at time zero and the 6, 24 and 30 h time period considered. Matrigel invasion assay The invasiveness of the lung cancer cells LNM35 treated with Frondoside A (0.1–1 µM) was tested using BD Matrigel Invasion Chamber (8-µm pore size; BD Biosciences, Le Pont de Claix, France) according to manufacturer's protocol. The PI3 kinase inhibitor LY294002 (20 µM) was used as a positive inhibitor of cellular invasion. Briefly, Cells (1×105 cells in 0.5 mL of media and the indicated concentration of Frondoside A) were seeded into the upper chambers of the system, the bottom wells in the system were filled with RPMI supplemented with 10% fetal bovine serum as a chemo-attractant and then incubated at 37°C for 24 h. Non-penetrating cells were removed from the upper surface of the filter with a cotton swab. Cells that have migrated through the Matrigel were fixed with 4% formaldehyde, stained with DAPI and counted in 25 random fields under a microscope. The assay was carried out in duplicate and repeated three times for quantitative analysis. Chorioallantoic membrane (CAM) angiogenesis assay This assay was performed as described previously [15], with some modifications. Briefly, fertilized eggs were incubated for 3 days at 37, 8°C with a humidity of 48%. On day 4, albumen was removed to detach the shell from the developing CAM and a window was made in the eggshell, exposing the CAM, and covered with a breathing film (suprasorb F®). The eggs were returned to the incubator until day 10, prior to application of the test compounds. Test compound and control compound (DMEM without 10% FBS) dissolved in DMEM without 10% FBS were poured onto separate sterile discs (12 mm diameter), which were allowed to dry under sterile conditions. A solution of cortisone acetate (125 µg/disc) was poured onto all discs to prevent an inflammatory response. Test discs probed with recombinant human bFGF (Peprotech) served as a control for angiogenesis stimulation. On each CAM, the disc containing control compound and the disc containing test compound were placed at a distance of 1 cm. The windows were covered and the eggs were incubated until day 14, before assessment of angiogenesis. Therefore, the eggs were flooded with 10% buffered formalin and the eggs were kept at room temperature for at least 20 minutes. The CAM, the area of the discs included, was placed in a petri dish with 10% buffered formalin. The plastic discs were removed and phase-contrast pictures of the area of the plastic discs were taken. The vascular index was measured as described previously [16]. Vascular intersections on a grid containing three concentric circles (6, 8 and 10 mm diameter with as center the center of the disc) were counted. The angiogenic index = (t-c)/c, with t the number of intersections in the area covered by the test disc and c the number of intersections in the area covered by the control disc in the same egg. The Mann-Whitney U-test was used for statistical analysis (p<0.05). Vascular tube formation assay Assessment of in vitro capillary formation used Matrigel (Becton Dickinson, Le Pont de Claix, France). Matrigel is a squamous cell carcinoma basement membrane matrix composed primarily of collagen IV, laminin, entactin, and heparan sulfate proteoglycans. The Matrigel matrix was thawed, gently mixed to homogeneity using cooled pipettes, and diluted v/v with the EndoGRO™-MV-VEGF Complete Media Kit medium (Millipore, Temecula, CA, USA). Matrigel, 50 µl/well, supplemented with angiogenic peptides and other effectors was used to coat the wells of 96-well plates. The plate was then incubated for one hour at 37°C to allow the matrix solution to solidify prior to treatment. HUVECs (at a density of about 4×104 cells/well and the indicated concentration of Frondoside A) were plated to each well and incubated for 8 h at 37°C in 0.1 mL of EndoGRO™-MV-VEGF Complete Media Kit medium (Millipore, Temecula, CA, USA). Then cells were photographed using an inverted phase contrast photomicroscope. The tubular network growth area was compared in control and inhibitor-treated Matrigel matrix. Tube formation was quantified by counting the number of tube-like structures formed in each well. The effect of Frondoside A on viability of the HUVEC was determined using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison, USA), as previously described for the cancer cells. Tumor growth and metastasis assay The animal experiments were performed in accordance with the protocol approved by the animal ethics committee and the Institutional Animal Care at the Faculty of Medicine & Health Sciences/UAE University. Six-week-old athymic NMRI female nude mice (nu/nu, Charles River, Germany) were housed in filtered-air laminar flow cabinets and handled under aseptic conditions. Procedures involving animals and their care were conducted in conformity with Institutional guidelines that are in compliance with Faculty of Medicine & Health Sciences, national and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; and NIH Guide for Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985). LNM35 cells (1×106 cells in 200 µl PBS) were injected subcutaneously into the lateral flank of the nude mice. One week after inoculation, when tumors had reached the volume of approximately 150 mm3, animals (six in each group) were treated in the first protocol for 25 days with Frondoside A (0.01 and 1 mg/kg/day, ip) or carrier solution (control) in order to determine the effect of Frondoside A alone on tumor growth and metastasis. In the second protocol, animals were treated for only 10 days with the lowest dose of Frondoside A (0.01 mg/kg/day, ip), cisplatin (1 mg/kg/day, ip), or with combined Frondoside A and cisplatin treatment. Control animals were treated with carrier solution. Tumor dimensions and animal weights were measured every 3 days. Tumor volume (V) was calculated using the formula: V = 0.4×a×b2, with “a” being the length and “b” the width of the tumor. After sacrifice, the tumors and axillary lymph nodes were excised and weighed. Immuno-histochemical determination of CD31/platelet-endothelial cell adhesion molecule 1 (PECAM-1) for Microvessel Density The effect of Frondoside A on angiogenesis was evaluated using CD31 immuno-staining. The tumor tissues were quickly frozen in isopentane at −130°C and stored at −70°C until further processing. Eight-µm frozen sections were fixed in acetone, and incubated overnight with a CD31 antibody (clone MEC13.3, 1∶100) (BD Pharmingen, San Jose, CA, USA). Slides were then washed three times in PBS and incubated with secondary antibody labeled with rhodamine (goat anti-rat 1∶100) for one hour at room temperature. The area occupied by CD31-positive microvessels and total tissue area per section were compared between treated and control mice. All analyses were performed in a blind fashion. Results were expressed as means ± S.E.M. of the number of experiments. The difference between experimental and control values were assessed by ANOVA followed by Dunnett's post-hoc multiple comparison test. Tumor growth and metastasis studies were analyzed using the unpaired Student's t-test. P<0.05 indicate a significant difference. Results Effect of Frondoside A on cellular viability As shown in fig. 1 , Frondoside A concentrations (0.01–5 µM) caused a concentration-dependent decrease in cell viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 cells over 24 hours. The IC50 concentrations (producing half-maximal inhibition) at 24 h were in the range of 1.7 and 2.5 µM Frondoside A for all cell lines. 10.1371/journal.pone.0053087.g001Figure 1 Inhibition of cellular viability by Frondoside A. Exponentially growing LNM35 (A), A549 (B), NCI-H460-Luc2 (C), MDA-MB-435 (D), MCF-7 (E), and HepG2 (F) cells were treated with vehicle (0.1% DMSO) and the indicated concentrations of Frondoside A. Viable cells were assayed as described in Materials and Methods. All experiments were repeated at least three times. Columns, mean; bars, S.E.M. **Significantly different at P<0.01, ***Significantly different at P<0.001. Frondoside A induces caspase-3/7 activation Caspase-3/7 activity is essential in apoptotic cell death pathways. The relative activity of caspases 3/7 was analyzed in LNM35 cells treated for 2 and 24 h with Frondoside A (1–2.5 µM), and normalized to the number of cells per well. As shown in Fig. 2 , caspase 3/7 activity increased by 2.5- and 10.8-fold in LNM35 cells treated for 24 h with Frondoside A 1 and 2.5 µM respectively. Similar effect was observed after treatment with Frondoside A (1–2.5 µM) for 2 h (data not shown). 10.1371/journal.pone.0053087.g002Figure 2 Induction of caspase-3/7 activity was analyzed in LNM35 cells treated for 24 h with Frondoside A (1–2.5 µM), normalized to the number of viable cells per well and expressed as fold induction compared with the control group. *Significantly different at P<0.05. Impact of Frondoside A on LNM35 xenografts To confirm the pharmacological relevance of our in vitro data, the anticancer activity of Frondoside A was investigated in vivo in athymic mice inoculated with LNM35 lung cancer cells. The growth of the LNM35 human tumor xenografts was monitored every third or fourth day for 25 consecutive days after daily i.p. injection of 0.01 mg and 1 mg/kg of Frondoside A. Treatment with the lowest dose of Frondoside A (0.01 mg/kg/day) reduced the volume of the LNM35 xenografts by 41% ( Fig. 3A ). A similar difference was also found in tumor weight at the end of the experiment (2.1+/−0.3 g versus 4.5+/−0.8 g; P<0.05; Fig. 3C ). Treatment with the highest dose (100 times more) of Frondoside A (1 mg/kg/day) reduces about 43.9% tumor volume of the LNM35 xenografts ( Fig. 3B ). Almost similar difference was found in tumor weight at the end of the experiment (3+/−0.5 g versus 4.5+/−0.8 g; Fig. 3D ). This experiment clearly demonstrated that the lowest dose of Frondoside A (0.01 mg/kg/day) is optimal for the inhibition of tumor growth. There were no manifest undesirable effects of Frondoside A treatment on animal behaviour or body weight in either experiment ( Fig. 3E and 3F ). In addition, there were no visible abnormalities at necropsy, or any other obvious signs of toxicity as previously described by our team [7]. 10.1371/journal.pone.0053087.g003Figure 3 Frondoside A induces regression of established LNM35 xenografts. A) & B) Tumor volume of LNM35 xenografts inoculated subcutaneously in nude mice and treated with Frondoside A (0.01 and 1 mg/kg, intra-peritoneal injections, respectively) or control carrier solution alone, for a total of 25 days. Data points represent the mean ± S.E.M. of 6 mice per group. C) & D) Tumor weight obtained from the same control and treated nude mice. Data points represent the mean ± S.E.M. of 6 mice per group. Columns, mean; bars, S.E.M. E) & F) Body weight of these mice. Data points represent the mean ± S.E.M. of 6 mice per group. *Significantly different at P<0.05. Inhibition of angiogenesis by Frondoside A in the xenografted tumors and the CAM assay in vivo and in the capillary-like structures in vitro Angiogenesis is an attractive target in cancer therapy not only because it supplies oxygen and nutrients for the survival of tumor cells but also provides the route for metastatic spread of these cancer cells. First, we demonstrate that in the proliferating areas at the periphery of the tumor, microvessel density (measured by CD31 staining) was significantly reduced by Frondoside A (0.01 mg/kg/day) ( Fig. 4, right panel ) in comparison with the control-treated tumors ( Fig. 4, left panel ). Next, we used the CAM assay involving the coordination and integration of multicellular responses during development of the chick embryo to confirm this potential anti-angiogenic effect of Frondoside A. As shown in Fig. 5 and 6 , bFGF (2 µg/ml), stimulated 15% new vessel formation with angiogenic indices statistically different compared with the control DMEM medium. Frondoside A (100 and 500 nM) inhibited basal angiogenesis in a concentration-dependent manner with respectively 7% and 12% inhibition of angiogenic index compared to control ( Fig. 5 and 6 ). The formation of new blood vessels induced by bFGF was also completely suppressed by Frondoside A (500 nM) ( Fig. 5 and 6 ). Finally, to assess whether the anti-angiogenic effect of Frondoside A involves a direct interaction of the compound with endothelial cells, we conducted comparative studies on the formation of capillary-like structures in vitro, using HUVECs plated on Matrigel-coated plates. As shown in Fig. 7A , human endothelial cells have the ability to form capillary structures when seeded and cultured on top of Matrigel substrate. Control cells move from their initial uniform pattern of dispersed cell layers and associate to form a network of cell clusters connected by long, multicellular processes leading to the formation of tube-like structures. Addition of non-toxic concentrations of Frondoside A (0.01–1 µM) resulted in a marked inhibition of this spontaneous angiogenic phenotype ( Fig. 7A, and 7B ). Frondoside A induced inhibition of this spontaneous angiogenic phenotype occurred without significant reduction of cell viability ( Fig. 7C ). Taken together, these data confirm a strong anti-angiogenic potential of Frondoside A. 10.1371/journal.pone.0053087.g004Figure 4 The anti-angiogenic activity of Frondoside A in the xenograft tumor: Immuno-histochemical staining of lung xenograft tumors for CD31 (microvessel density). 10.1371/journal.pone.0053087.g005Figure 5 The anti-angiogenic activity of Frondoside A in the Chorioallantoic membrane (CAM) assay in vivo: CAM was treated with control (serum-free medium), 100 nM Frondoside A, 500 nM Frondoside A, 2 µg/mL bFGF, 2 µg/mL bFGF+500 nM Frondoside A and the vascularization of test discs was photographed. 10.1371/journal.pone.0053087.g006Figure 6 Quantification of CAM angiogenesis assay: Bars indicate angiogenic indices (%) of CAM's probed with 2 µg/mL bFGF, 100 nM Frondoside A, 500 nM Frondoside A, 2 µg/mL bFGF+500 nM Frondoside A. Data represents mean ± SD (Mann-Whitney U-test, *: p<0.05, ***: p<0.01). In each experiment, six eggs were tested per condition. 10.1371/journal.pone.0053087.g007Figure 7 Impact of Frondoside A on the formation of capillary-like structures by HUVECs in vitro. A) Patterns of angiogenesis induced by human umbilical vein endothelial cells (HUVEC) cultured on Matrigel matrix in 96-well plates in the absence or presence of the Frondoside A. B) Quantification of tubular morphogenesis induced in HUVEC cells cultured in the absence or presence of Frondoside A. Tube formation was determined by the length of tube-like structures containing connected cells. Data are mean ± S.E.M. from three separate experiments. Asterisks indicate that significantly different values were obtained in the presence of the indicated inhibitors vs. the corresponding control stimulation. ***Significantly different at P<0.001. C) HUVEC cells were treated with vehicle (0.1% DMSO) and the indicated concentrations of Frondoside A. Viable cells were assayed as described in Materials and Methods. All experiments were repeated at least three times. Columns, mean; bars, S.E.M. Frondoside A also impairs lung cancer cell migration and invasion in vitro and metastasis in vivo Cancer progression is associated with the abrogation of normal controls that limit cell migration and invasion, eventually leading to metastasis. Lung cancer patients are at high risk of recurrence in the form of metastatic disease. Metastasis starts with cell migration in the primary tumor, leading to local tissue invasion and entry into lymph or blood vessels. The ability of Frondoside A to reduce cellular migration was investigated using a classic in vitro wound healing model. Frondoside A reduced cellular migration of LNM35 cells in a concentration- and time-dependent manner ( Fig. 8A ). Similarly, Frondoside A impaired the invasion of LNM35 cells in matrigel invasion assay ( Fig. 8B ). Frondoside A induced inhibition of cellular migration and matrigel invasion occurred without significant reduction of cell viability ( Fig. 1A ). 10.1371/journal.pone.0053087.g008Figure 8 Frondoside A also impairs lung cancer cell migration and invasion in vitro and metastasis in vivo. A) Wounds were introduced in LNM35 confluent mono-layers cultured in the presence or absence (control) of Frondoside A (0.1–0.5 µM). The mean distance that cells travelled from the edge of the scraped area for 6, 24, and 30 h at 37°C was measured in a blinded fashion, using an inverted microscope (4× magnifications). Data are means ± S.E.M. of two independent experiments. B) LNM35 cells were incubated for 24 h in the presence or absence of Frondoside A (0.01–1 µM) and LY294002 (20 µM). Cells that invaded into Matrigel were scored as described in Materials and Methods. Columns, mean; bars, S.E.M. Lymph nodes metastasis weight of established human lung cancer xenografts treated with Frondoside A 0.01 mg/kg (C) and 1 mg/kg (D) every day for 25 days. Columns, mean; bars, S.E.M. *Significantly different at P<0.05, **Significantly different at P<0.01, ***Significantly different at P<0.001. Next, we assessed the metastatic behavior of the human pulmonary cell line LNM35 by examining axillary lymph nodes. Histological examination of the lymph nodes in LNM35-bearing mice revealed the presence of LNM35 cells in all lymph nodes from both the control and Frondoside A treated mice (data not shown). In the control-treated group, the mean lymph nodes weight was 548.5+/−112.8 mg compared with 256+/−43.3 and 268.8+/−55.7 mg in the groups treated with Frondoside A 0.01 mg and 1 mg/kg/day, respectively ( Fig. 8C and 8D ). Frondoside A enhances the anticancer activity of cisplatin As shown in Fig. 9A , daily administration of Frondoside A to nude mice reduced the growth of LNM35 human tumor xenografts by 40.3% at day 10. Inhibition of LNM35 tumor xenograft growth by Frondoside A was comparable with that produced by the DNA-damaging anticancer agent cisplatin (46.9%). Combined treatment of Frondoside A with cisplatin resulted in a remarkable potentiation (67.6%; P<0.05) of the cisplatin therapeutic effect ( Fig. 9A ). There were no manifest side effects of the combined treatment on animal behaviour or body weight ( Fig. 9B ). 10.1371/journal.pone.0053087.g009Figure 9 Antitumor activity of Frondoside A alone or in combination with the anticancer drug, cisplatin, against human lung tumor xenografts in athymic nude mice. A) Frondoside A enhances cisplatin efficacy against NSCLC LNM35 cells growing as xenografts. B) Impact of Frondoside A and cisplatin alone or in combination on body weight. *Significantly different at P<0.05. Discussion Despite advances in molecular biology of lung cancer, improved diagnosis, and even optimal target therapies, the current protocols for the treatment of lung cancer are still insufficient to produce striking clinical benefits and the cure of lung cancer patients remains unsuccessful. The current targeted therapy drugs develop resistance and they are very expensive and not available to the majority of lung cancer patients in the world. Many drugs have relatively low activity; few patients are cured, with a brief, if any, increase in survival [17]. The history of anti-cancer agents, such as paclitaxel, shows that the mechanism of action was identified several years after its clinical efficacy was demonstrated. Despite not knowing the drug's mechanism of action, we believe that the in vitro as well as the strong in vivo anti-cancer effects of Frondoside A should translate in the clinic to a new potent anti- lung cancer agent. In the present study, we investigated the impact of Frondoside A on lung cancer cells progression. We showed that Frondoside A caused concentration-responsive (0.01–5 µM) decreases in viability of LNM35, A549, and NCI-H460-Luc2 cells over 24 hours through the caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM Frondoside A. These results are consistent with previously published results that low concentration of Frondoside A inhibits the growth of human pancreatic cancer cells and induced apoptosis through caspase 9/3/7, increased bax, decreased bcl-2 and mcl-1, and arrested cell cycle and up-regulated p21 [8], induced apoptosis in human leukemia cells via caspase activation [6], and significantly decreases the viability of the estrogen receptor (ER)-negative MDA-MB-231 breast cancer cells by inducing apoptosis via the Caspase9-Caspase3/7 intrinsic pathway [7]. It has also been demonstrated that Frondanol A5, an impure extract of Cucumaria frondosa skin from which Frondoside A is originally derived, induced growth inhibition at S and G2-M phase with a decrease in Cdc25c and an increase in p21WAF1/CIP1 with significant apoptosis associated with H2AX phosphorylation and caspase-2 cleavage in the colon cancer-derived HCT116 cells [4]. A second paper also demonstrated that Frondanol-A5P, a polar precipitate sub-fraction of Frondanol-A5, inhibited proliferation and induced G2/M phase cell cycle arrest in two pancreatic cancer cells with decreased expression of cyclin A, cyclin B, and cdc25c [18]. This anticancer effect of Frondoside A is not tissue specific, and in this context we demonstrated that Frondoside A induced a concentration-dependent decrease in cell viability of the melanoma MDA-MB-435, the breast MCF-7, and the hepatoma HepG2 cells. To assess the effects of Frondoside A on lung tumor development in vivo, LNM35 xenografts were made in immuno-suppressed mice. We demonstrated that intraperitoneal administration of Frondoside A caused a strong regression of established tumors. Maximum inhibition was obtained with the low dose of 0.01 mg/kg/day for 25 days. A 100 times higher dose gives similar effects indicating that increasing the dose will not improve the anti-cancer efficacy of Frondoside A. The cancer inhibitory effect of Frondoside A has been observed previously on AsPC-1 pancreatic and MDA-MB-231 xenografts in athymic mice [7], [8]. This anti-tumor effect of Frondoside A may be partly due to its immuno-stimulatory effect [13] and/or anti-angiogenic effect. In this study, we demonstrated for the first time, that Frondoside A is a strong anti-angiogenic agent. It reduces the microvessel density (measured by CD31 staining) in the xenografted tumor treated with Frondoside A and also significantly reverse basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Similarly, Frondoside A completely suppressed capillary structure formation on Matrigel substratum by human endothelial cells. Angiogenesis is an attractive target in cancer therapy not only because it supplies oxygen and nutrients for the survival of tumor cells but also provides the route for metastatic spread of these cancer cells. Cancer progression is associated with abrogation of the normal controls that limit cell migration and invasion, leading eventually to metastasis. As metastasis is the major cause of death in cancer patients, the development of new treatment regimens that reduce invasion and metastasis is highly important in cancer therapy. In this context, we demonstrated that Frondoside A induced a highly significant time- and concentration-dependent inhibition of cell migration, invasion in vitro and metastasis in vivo. These results are in agreement with our previous study showing that Frondoside A at concentrations that are not cytotoxic to the cells exerts a strong inhibitory effect on both the migratory and invasive properties of MDA-MB-231 breast cancer cells [7] and with another recent study demonstrating that Frondoside A inhibits breast cancer metastasis to the lungs [19]. Building on aforementioned results, and knowing from clinical trials that single agent treatments rarely result in clinical benefits to cancer patients, and that combination therapy are necessary for effective treatment of tumors, we investigated the therapeutic advantage of combination of cisplatin, (a first line treatment of lung cancer), with Frondoside A in nude mice bearing LNM35 xenografts. We found that Frondoside A enhances the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer and perhaps other cancers too. We thank Dr. Mien-Chie Hung from University of Texas MD Anderson Cancer Center for providing the MDA-MB-435 cells and Dr. Katarina Hostanska from Department of Internal Medicine, Institute for Complementary Medicine, University Hospital Zurich, Switzerland for providing the A549 cells. ==== Refs References 1 Mann J (2002 ) Natural products in cancer chemotherapy: past, present and future . Nat Rev Cancer 2 : 143 –148 .12635177 2 Molinski TF , Dalisay DS , Lievens SL , Saludes JP (2009 ) Drug development from marine natural products . Nat Rev Drug Discov 8 : 69 –85 .19096380 3 Kuno T , Tsukamoto T , Hara A , Tanaka T (2012 ) Cancer chemoprevention through the induction of apoptosis by natural compounds . Journal of Biophysical Chemistry 3 : 156 –173 . 4 Janakiram NB , Mohammed A , Zhang Y , Choi CI , Woodward C , et al (2010 ) Chemopreventive effects of Frondanol A5, a Cucumaria frondosa extract, against rat colon carcinogenesis and inhibition of human colon cancer cell growth . 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yes
PLoS One. 2013 Jan 8; 8(1):e53087
==== Front Case Rep Emerg MedCase Rep Emerg MedCRIM.EMCase Reports in Emergency Medicine2090-648X2090-6498Hindawi Publishing Corporation 10.1155/2012/342760Case ReportMarathon Runner with Acute Hyponatremia: A Neurological Disorder Kormann R. 1 Philippart F. 2 3 Hubert S. 1 *Bruel C. 2 1Internal Medicine, Groupe Hospitalier Paris Saint Joseph, 75014 Paris, France2Intensive Care Unit, Groupe Hospitalier Paris Saint Joseph, 75014 Paris, France3Paris Descartes University, 75006 Paris, France*S. Hubert: [email protected] Editors: C.-C. Lai, M. Sand, and H. P. Wu 2012 31 5 2012 2012 3427609 2 2012 7 3 2012 Copyright © 2012 R. Kormann et al.2012This 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.We report the case of an athletic 49-year-old female who has run the 2011 Marathon of Paris and was addressed to the hospital for a confusion. The investigations revealed a cerebral edema complicating a severe hyponatremia secondary to an exercise-associated hyponatremia (EAH). Using 3% hypertonic saline solution, the evolution the patient rapidly improve allowing discharge after 7 days. We then discuss the importance of EAH in long-term efforts. ==== Body 1. Introduction Marathons, and more widely long-term efforts attract more and more nonprofessional runners (popular festive events and charity runs). Due to this modification of population behavior, pathologies associated with high-level sport practices tend to take a huger place in emergency clinical practice. Among them, exercise-associated hyponatremia (EAH) is a very important one. 2. Case Presentation We report the case of an athletic 49-year-old female with no medical records who has run the 2011 Marathon of Paris (42.195 Km). In view of the marathon, the patient started to train a few months ahead as per two to three runs weekly. She completed the marathon within 5 h 30, with an average speed of 7.5 Km/h, on a warm day (27 degrees celsius). Along the run, she drunk 4 L of mineral water (5 mg/L NaCl), and had some energy bars at the various feeding stations. Four hours after completion of the marathon, the patient felt dizzy and nauseous, with a strong asthenia and encompassed three vomiting periods associated with disorientation and confusion. She was then admitted to the emergency room 7 hours after the afore-mentioned symptoms occurred. Upon admission, the patient was conscious but disoriented, clouded, with a Glasgow coma score of 13. She was apyretic and presented no circulatory failure. The neurological examination revealed acute spreadover of osteotendinous reflexes the four limbs, no systematic deficiencies and cutaneous plantar reflexes in flexion, isochoric and reactive pupils. Her body weight was 53.4 Kg (normal average body weight of 50 Kg). The patient was then transferred to the intensive care unit (ICU) for diagnosis and treatment. Four hours later, the patient had a generalized tonic-clonic seizure that resolved following 1 mg clonazepam IV injection. The cerebral CT-scan showed a diffused supratentorial cerebral oedema (Figure 1). The initial biological parameters were natremia 121 mmol/L, chloremia 88 mmol/L, protidemia 70 g/L, glycemia 8.5 mmol/L, kalemia 3.3 mmol/L, bicarbonates 18 mmol/L, blood urea nitrogen 3.7 mmol/L, creatinemia 68 μmol/L, blood urea nitrogen 2.9 mmol/L, uric acid 214 mmol/L, Glycemia 6.6 mmol/L, calcemia 1.8 mmol/L, phosphorus 0.87 mmol/L, magnesemia 1.04 mmol/L, CPK 14486 UI/L, Hb 10.1 g/dL, platelet count 172 G/L, and leukocytes 11.06 G/L. The calculated osmolality was 260 mosm/kg and the measured osmolality was 246 mosm/kg. Arterial blood gases showed a respiratory alkalosis to compensate an increased plasmatic anion gap metabolic acidosis, with initial blood lactate 10.5 mmol/L. The initial collection of a urine sample showed an urinary osmolality of 489 mosm/L. We report a natriuresis and kaliuresis of 86 and 75 mmol/L, respectively. The measure of the free cortisol was 1250 nmol/L at 8 am, and 1350 nmol/L following the ACTH test, by which therefore eliminate an adrenal origin of hyponatremia. The patient was given a calculated bolus of isotonic saline as per the Adrogue formula. As no further clinical improvements followed, with a persistent hyponatremiaof 121 mmol/L, the patient was given a treatment based on 3% hypertonic saline solution. Natremia was recorded and indicated 128 mmol/L and 136 mmol/L at 4 hours and 7 hours after beginning of the treatment, respectively. Despite a rapid correction of natremia abnormalities, biological improvement was associated with the resolution of the neurological perturbations without apparition of new neurological symptoms. The patient was discharged after 7 days following complete resolution of clinical and biological disorders. 3. Discussion The clinical and biological context is a physical exercise-associated hyponatremia(EAH) as defined by a plasmatic sodium concentration below 135 mmol/L 24 h following a prolonged physical activity [1, 2]. Marathons attract plenty of nonprofessional runners like our patient who ran her first marathon at the age of 49. Risk factors in favor of EAH occurrence can be merged into two groups: (i) runner condition-associated factor (female, low body weight, lack of training, slow runner, excessive drinking consumption during the run, and NSAIDS consumption); (ii) environment-related factors (prolonged physical activity of over 4 h, warm air temperature, and drinks availability along the run [3]). This syndrome, first described in 1981 [1], is closely related to an evolution in high-level sport practices (marathon; triathlon) and the concept of abundant hydration during the activity in order to reduce dehydratation and hypernatremiarisks [4]: an overdrinking consumption during and after the run, and the related occurrence of a syndrome of inappropriate secretion of antidiuretic hormone (SIADH). The excessive drinking consumption during activity is the primary and critical risk factor associated to EAH occurrence [4, 5]. Then, it has been shown that the risk to develop an EAH is correlated to an increase of body weight during the physical activity as observed in the present patient (+3,4 Kg). There are no indications of a thirst deregulation among runner, as thirst is a late adaptation mechanism in this particular regulation system [6]. Despite this, the hyperhydratation during activity on its own cannot resume hyponatremia. The kidneys can excrete 750 to 1500 mL/h of water in normal physiological conditions [7]. ADH is a hormone secreted by the hypothalamus and is subject to stimulation upon low charges (1-2%) in the plasmatic osmolality [8]. In addition to the osmotic stimulation, other mechanisms inducing the central production of ADH have been described: nausea, vomiting organic stress, pain and endorphins, volemic status, central temperature increase, or hypoxia [2, 4, 6–9]. The excess of ADH production is responsible for potentially serious hyponatremiaas described by Siegel et al. [10] and recognized as a key factor for the EAH occurrence [2, 4, 7, 9, 10]. Moreover, It has been suggested that cytokines release (IL6) could also be involved in the nonosmotic stimulation of ADH [7, 9]. A rapid decrease of natremia (<128 mmol/L) is correlated with the emergence of serious clinical alterations, notably neurological disorders following brain swelling [3], as described in the present patient. Patients associated with symptomatic EAH and subsequent serious neurological (confusion, vigilance disorders, coma, and tonic-clonic seizures) or pulmonary (acute respiratory failure) disorders should imperatively be transferred to ICU with a repeated surveillance as the natremia. Acute hyponatremia treatment can benefit of a novel concept associated with a remarkable therapeutic efficiency [2, 4, 7, 9, 10]. Treatment of serious EAH conditions involve the administration of a 3% hypertonic saline solution at 1 mL/kg/h, that is subsequently adjusted following natremia status. Increases of natremia to 1 mmol/L/h during the first 6 h, 9 mmol/L during the first 24 h, and 18 mmol/L during the first 48 h are accepted. Ideally, natremia should not evolve over 20–25 mmol/L during the first 48 h [5, 10]. The isotonic saline solution appears inefficient for the treatment of EAH as described in a previous study [4] and as observed in the present case, in which no noticeable positive biological response was detected following initial administration of saline. The EAH prevention relies on information regarding hyperhydratation risks during the run and the necessity to control water supplies in a range of 400–700 mL/h, depending on body weight and weather conditions [5]. Beltrami has described the historical link between overdrinking during a prolonged and intensive sport activity and the occurrence of hyponatremia. This overdrinking behavior seems to be supported by sport drinks selling companies. Moreover, these sport drinks (enriched solutions with mineral salts and ions, slightly less hypotonic than mineral water) do not prevent EAH, neither is the sodium supplier by systematic per os intake [3]. 4. Conclusion In summary, EAH is associated to a SIADH which the severity is as high as the hydric supply during and after the physical activity, occurring during or in the few hours following the activity. It could sometimes evolve as critical emergency situation with signs of neurological disorders, and the only efficient treatment consists of the administration of hypertonic saline solution. The prevention strategy consists in informing runners on good hydration practices during the run and the hyperhydratation-associated risks. Figure 1 Noninjected CT-scan realized on the day of admission. ==== Refs 1 Noakes TD Goodwin N Rayner BL Branken T Taylor RKN Water intoxication: a possible complication during endurance exercise, 1985 Wilderness & Environmental Medicine 2005 16 4 221 227 2-s2.0-33644876429 16366205 2 Siegel AJ Verbalis JG Clement S Hyponatremia in marathon runners due to inappropriate arginine vasopressin secretion The American Journal of Medicine 2007 120 5 461.e11 461.e17 2-s2.0-34247268567 3 Hew-Butler T Ayus JC Kipps C Statement of the second international exercise-associated hyponatremia consensus development conference, New Zealand, 2007 Clinical Journal of Sport Medicine 2008 18 2 111 121 2-s2.0-40549143064 18332684 4 Noakes TD Sharwood K Speedy D Three independent biological mechanisms cause exercise-associated hyponatremia: evidence from 2,135 weighed competitive athletic performances Proceedings of the National Academy of Sciences of the United States of America 2005 102 51 18550 18555 2-s2.0-29444435800 16344476 5 Noaken TD Wilson G Gray DA Lambert MI Dennis SC Peak rates of diuresis in healthy humans during oral fluid overload South African Medical Journal 2001 91 10 852 857 2-s2.0-0035168531 11732457 6 Verbalis JG Disorders of body water homeostasis Best Practice & Research 2003 17 4 471 503 7 Rosner MH Kirven J Exercise-associated hyponatremia Clinical Journal of the American Society of Nephrology 2007 2 1 151 161 2-s2.0-34247188519 17699400 8 Schrier RW Body water homeostasis: clinical disorders of urinary dilution and concentration Journal of the American Society of Nephrology 2006 17 7 1820 1832 2-s2.0-33745863535 16738014 9 Siegel AJ Exercise-associated hyponatremia: role of cytokines The American Journal of Medicine 2006 119 7, supplement 1 S74 S78 2-s2.0-33745689811 16843089 10 Siegel AJ Hypertonic (3%) sodium chloride for emergent treatment of exercise-associated hypotonic encephalopathy Sports Medicine 2007 37 4-5 459 462 2-s2.0-34247895666 17465635
23326709
PMC3542923
CC BY
2021-01-05 10:32:16
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Case Rep Emerg Med. 2012 May 31; 2012:342760
==== Front J Transl MedJ Transl MedJournal of Translational Medicine1479-5876BioMed Central 1479-5876-10-2402321707810.1186/1479-5876-10-240ReviewOpportunities and challenges of disease biomarkers: a new section in the journal of translational medicine Wang Xiangdong [email protected] Peter A [email protected] Department of Respiratory Medicine, Biomedical Research Center, Zhongshan Hospital Qing-Pu Branch, Fudan University, Shanghai, China2 Clinical Bioinformatics, Lund University Hospital, Lund, Sweden3 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA2012 5 12 2012 10 240 240 29 8 2012 1 11 2012 Copyright ©2012 Wang; licensee BioMed Central Ltd.2012Wang; 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.Disease biomarkers are defined to diagnose various phases of diseases, monitor severities of diseases and responses to therapies, or predict prognosis of patients. Disease-specific biomarkers should benefit drug discovery and development, integrate multidisciplinary sciences, be validated by molecular imaging. The opportunities and challenges in biomarker development are emphasized and considered. The Journal of Translational Medicine opens a new Section of Disease Biomarkers to bridge identification and validation of gene or protein-based biomarkers, network biomarkers, dynamic network biomarkers in human diseases, patient phenotypes, and clinical applications. Disease biomarkers are also important for determining drug effects, target specificities and binding, dynamic metabolism and pharmacological kinetics, or toxicity profiles. ==== Body The word “biomarker” describes a traceable and characterized substance that is an indicator of biological morphology, processes and function. Disease biomarkers are used to diagnose various phases of diseases, monitor severities of diseases and responses to therapies, and are predictors of prognosis of patients and likely responses to therapy. One of criteria to evaluate the value of disease biomarkers is the disease-associated specificity, sensitivity, traceability, stability, repeatability and reliability. Increasing numbers of biomarkers are being discovered and identified from preclinical research, but only a few have been found to be useful clinically. The Journal of Translational Medicine opens a new Section of Disease Biomarkers to bridge identification and validation of gene or protein-based biomarkers, network biomarkers, dynamic network biomarkers in human diseases, patient phenotypes, and clinical applications. The Section intends to accelerate the discovery and development of human disease-specific biomarkers for the early diagnosis, evaluation of diseases and predictions of responses to therapy. The Section will be an important and critical platform to underscore the significance and value of Disease Biomarkers and promote innovation and development of disease-specific biomarkers by integrating multidisciplinary aspects of science. The following are aspects of the planned biomarker studies: a) Benefitting drug development: Disease biomarkers can play critical roles not only in clinical applications but also in drug discovery and development as related to drug efficacy or toxicity, allowing selection of “the right drugs and the right patients” [1]. For example, more than 90% of new anti-cancer drugs in clinical trials have failed marketing approval, often due to poor efficacies and efficiencies, high toxicities, drug resistance or unexpected safety issues. The lack of disease-specific biomarkers is one of major challenges, often making it difficult to predict drug efficacy. The Section of Disease Biomarkers will accelerate the procedure of biomarker identification and validation for determining drug effects, target specificities and binding, dynamic metabolism and pharmacological kinetics, toxicity profiles, or side-effects. In addition, Disease Biomarkers will emphasize the importance of molecular and cellular responses to drugs under pathophysiological conditions, creating a new concept of “the right biomarker, the right drug and the right patient”. This will benefit our understanding of molecular mechanisms of interactions between biomarkers, drugs and diseases. Optimal biomarkers should allow monitoring drug efficacy and safety and individual responses to specific treatments. Patients evaluated and treated at the early stage of disease may enhance business decisions related to drug development, facilitating regulatory approval for new therapies [2]. Quantification and safety of biomarkers are necessary for final decisions related to new drug development and applications. b) Integration of multidisciplinary sciences: Identification, validation, development and marketing of disease-specific biomarkers need to integrate processes in molecular biology, new biotechnologies, clinical sciences, regulatory policies, and clinical applications. “Omic” science and technology play important roles in the identification and discovery of biomarkers. The Omic scope includes genomics, proteomics, metabolomics, pharmacogenomics, transcriptomics, and other high-throughput methodologies. Selected biomarker candidates can be validated using computational biology, high-throughput image analysis, molecular genetics, specimens from human tissue banks, mathematical medicine and biology, protein expression and profiling, and systems biology. Clinical bioinformatics have been suggested as a new way to combine clinical measurements and signs with human tissue-generated bioinformatics, helping to understand the role of selected biomarker candidates in clinical settings, disease development and progression, and therapeutic strategies, and mapping relationships of drug and biomarker candidates with clinical examinations, pathology data, biochemical analysis, imaging, and therapies [3]. For example, locations of targeted biomarkers are validated in human is tissues, using imaging data of tumor regions in various lung compartments. Selectivity and precision of proteomic analyses were performed in patients with chronic lung diseases and cancer [4]. Panels of disease-specific protein biomarkers that define the disease stage were selected in chronic obstructive pulmonary disease. By targeted proteomic analysis, a digital evaluation score system was developed for assessing the severity of the disease, clinical information, and lung function [5,6]. A number of new integrated scientific areas have been created during the identification and development of disease-specific biomarkers. Genomic medicine was proposed to bring biomarkers into the mainstream of clinical practice and improve therapies and quality of patient life, although such strategies are in the very early stages. Systems clinical medicine has been defined as one of new strategic areas for development of disease biomarkers, involving integration of systems biology, clinical phenotypes, high-throughout technologies, bioinformatics and computational science in order to improve diagnosis, prognosis and therapies of diseases [7]. Next generation sequencing for genomic analysis of individual genomes has been used for single nucleotide polymorphism discovery and estimates of allele frequency, gene ontology analysis of the target genes, analysis of the microRNAs expression, sequencing platforms for mRNA biomarker analysis, genome-wide analysis, and the exploration of the proteome, all of which should lead to the identification of useful protein biomarkers. c) Significance of molecular imaging in validation of biomarkers: It has been suggested that selected biomarker candidates need to be validated in human tissues, (e.g. measuring the expression of targeted mRNA and proteins in pathological tissues of patients) as well as correlating the over-expression of targeted candidates with dysfunction of organs or tissues. Tissue microarray analysis is a powerful tool for validation of biomarker candidates, especially with an algorithm-tissue array co-occurrence matrix analysis for quantifying cellular phenotypes based on textural regularity by local inter-pixel configurations [8]. In addition, matrix-assisted laser desorption/ionization mass spectrometry imaging is used for spatial distribution and relative abundance of molecules directly in tissues in order to improve the quality of molecular images and differentiate tissue regions that cannot be morphologically defined. It has been suggested that such imaging may be beneficial for disease diagnosis and prognosis, biomarker discovery and drug therapy, even though there are still many barriers. As an example, clinical phenotypes and severities of chronic obstructive pulmonary disease were measured by the parametric response map, a voxel-wise image analysis technique of whole-lung using computed tomography that results in more accurate diagnosis of individual patients, together with integration of clinical information [9]. Numerous imaging technologies have been applied for biomarker validation and development, including near infrared imaging, neuronuclear imaging, whole-body diffusion magnetic resonance imaging, specific biomarker detection on the cell surface in real time, positron emission tomography, molecular contrast-enhanced ultrasound imaging, and acceptor fluorescence anisotropy that measures variations in hetero- fluorescence resonance energy transfer deriving from protein-protein interactions. d) Network biomarkers and dynamic network biomarkers: Multiple biomarkers were found to improve the prediction of death from cardiovascular causes during a more than 10 year follow-up of patients, suggesting that simultaneous presence of biomarkers in cardiovascular disease as well as presence of established risk factors substantially improves the risk stratification for death from cardiovascular disease in elderly men [10]. Network biomarkers and dynamic network biomarkers represent new types of biomarkers with interactions between proteins or genes that can be monitored and evaluated at different stages and time points during development of disease [11]. The amount of high-throughput genomic and proteomic data from patients has been rapidly increasing and is expected to correlate with clinical phenotypes, disease severities, therapeutic responses, and prognoses. Gene regulatory networks or protein interaction networks may describe functions for the panel of relevant network biomarkers. Dynamic network biomarkers can demonstrate changes at various stages in diseases and seem to have disease specificity. Disease biomarkers need to be validated by integration with clinical informatics, which translates clinical descriptive information on signs, symptoms, biochemical analyses, imaging and therapies into digital data [5,6]. Clinical bioinformatics may be helpful in discovering disease-specific, stage-specific, severity-specific and/or therapy-predictive biomarkers. e) Challenges in development: As one of major technologies for biomarker identification and discovery, genomics and proteomics as well as sequencing still face a large number of challenges, (e.g., analysis of the individual’s genome in the context of extensive population-based data, phenotypic significance and specificity, duration and severity, of disease, genomic or proteomic variation, combinations of genomic, expression-based, metabolomic, and proteomic data, and the value of disease biomarkers for making clinical decision). Some tissue microarrays have been questioned due to study sizes, reduced throughput, variability, and expenses related to the observations. A recent study combined genome-wide association with forced expiratory volume in 1 second and the ratio of forced expiratory volume in 1 second to forced vital capacity, identified 16 new genomic regions showing association with pulmonary function [12]. Those biomarkers were selected from 48,201 individuals of European ancestry, with follow up of top associations in an additional 46,411 individuals. However, there is further need to better understand the biomarkers associated with molecular mechanisms involving pulmonary function and validate selected targets in order to reduce lung dysfunction. Conclusions The Section of Disease Biomarkers is expected to accelerate the process from identification to validation, research to development, resulting in improved design of clinical trials. There should also be positive impacts on governmental approval, and clinical applications to policy and regulation, and relevance to “personalized” medicine and to public health. The Section will publish articles related to development of advanced biotechnologies for biomarker discovery, the biomarkers associated with the early detection of diseases, and monitoring of disease severity and duration as well as patient responses to therapies, predictions of patient outcomes and life qualities, the clinical trial and evaluation of biomarkers, and regulation and ethics of disease biomarkers. Competing interests The authors declare no competing interests. Authors’ contributions XDW and PW have the equal contributions to the preparation of manuscript. All authors read and approved the final manuscript. ==== Refs Kelloff GJ Sigman CC Cancer biomarkers: selecting the right drug for the right patient Nat Rev Drug Discov 2012 11 201 204 10.1038/nrd3651 22322254 Hampel H Frank R Broich K Teipel SJ Katz RG Hardy J Herholz K Bokde AL Jessen F Hoessler YC Sanhai WR Zetterberg H Woodcock J Blennow K Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives Nat Rev Drug Discov 2010 9 7 560 574 10.1038/nrd3115 20592748 Wang XD Liotta L Clinical bioinformatics: a new emerging science J Clin Bioinforma 2011 1 1 10.1186/2043-9113-1-1 21884620 Marko-Varga G Végvári Á Rezeli M Prikk K Ross P Dahlbäck M Edula G Sepper R Fehniger TF Understanding drug uptake and binding within targeted disease micro-environments in patients: a new tool for translational medicine Clin Transl Med 2012 1 8 10.1186/2001-1326-1-8 Chen H Wang Y Bai C Wang XD Alterations of plasma inflammatory biomarkers in the healthy and chronic obstructive pulmonary disease patients with or without acute exacerbation J Proteomics 2012 75 10 2835 2843 10.1016/j.jprot.2012.01.027 22343073 Chen H Song Z Qian M Bai C Wang XD Selection of disease-specific biomarkers by integrating inflammatory mediators with clinical informatics in AECOPD patients: a preliminary study J Cell Mol Med 2012 16 6 1286 1297 10.1111/j.1582-4934.2011.01416.x 21883889 Wu D Rice CM Wang XD Cancer bioinformatics: a new approach to systems clinical medicine BMC Bioinformatics 2012 13 71 10.1186/1471-2105-13-71 22549015 Yan D Wang P Knudsen BS Linden M Randolph TW Statistical methods for tissue array images - algorithmic scoring and co-training Ann Appl Stat 2012 6 3 1280 1305 10.1214/12-AOAS543 22984376 Galbán CJ Han MK Boes JL Chughtai KA Meyer CR Johnson TD Galbán S Rehemtulla A Kazerooni EA Martinez FJ Ross BD Computed tomography-based biomarker provides unique signature for diagnosis of COPD phenotypes and disease progression Nat Med 2012 10.1038/nm.2971 (PMID: 23042237) Zethelius B Berglund L Sundström J Ingelsson E Basu S Larsson A Venge P Arnlöv J Use of multiple biomarkers to improve the prediction of death from cardiovascular causes N Engl J Med 2008 358 20 2107 2116 10.1056/NEJMoa0707064 18480203 Wang XD Role of clinical bioinformatics in the development of network-based Biomarkers J Clin Bioinforma 2011 1 28 10.1186/2043-9113-1-28 22024468 Artigas MS Loth DL Wain LV Gharib SA Obeidat M Genome-wide association and large-scale follow up identifies 16 new loci influencing lung function Nat Genet 2011 43 1082 1090 10.1038/ng.941 21946350
23217078
PMC3543303
CC BY
2021-01-04 22:06:59
yes
J Transl Med. 2012 Dec 5; 10:240
==== Front PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 23326583PONE-D-12-2546210.1371/journal.pone.0054125Research ArticleBiologyImmunologyImmunityInflammationMolecular cell biologySignal transductionMembrane Receptor SignalingNucleotide Receptor SignalingSignaling in cellular processesProtein kinase C signalingMedicineClinical ImmunologyATP Mediates NADPH Oxidase/ROS Generation and COX-2/PGE2 Expression in A549 Cells: Role of P2 Receptor-Dependent STAT3 Activation ATP Induces ROS-Dependent COX-2 ExpressionCheng Shin-Ei 1 Lee I-Ta 1 2 Lin Chih-Chung 2 Wu Wan-Ling 1 Hsiao Li-Der 1 Yang Chuen-Mao 1 * 1 Department of Physiology and Pharmacology and Health Aging Research Center, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan 2 Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan Viola Joao PB. Editor National Cancer Institute (INCA), Brazil * E-mail: [email protected] Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SEC ITL CCL CMY. Performed the experiments: SEC WLW LDH. Analyzed the data: CMY. Contributed reagents/materials/analysis tools: SEC WLW CMY. Wrote the paper: ITL CCL CMY. 2013 11 1 2013 8 1 e5412520 8 2012 6 12 2012 © 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.Background Up-regulation of cyclooxygenase (COX)-2 and its metabolite prostaglandin E2 (PGE2) are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE2 release remain unclear. Principal Findings Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin), PKC (Gö6983, Gö6976, Ro318220, and Rottlerin), ROS (Edaravone), NADPH oxidase [diphenyleneiodonium chloride (DPI) and apocynin], Jak2 (AG490), and STAT3 [cucurbitacin E (CBE)] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47phox, Jak2, STAT3, and cPLA2 markedly reduced ATPγS-induced COX-2 expression and PGE2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. Significance Taken together, these results showed that ATPγS induced COX-2 expression and PGE2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies. This work was supported by NSC98-2321-B-182-004, NSC99-2321-B-182-003, NSC98-2314-B-182-021-MY3, and NSC98-2320-B-255-001-MY3 from National Science Council, Taiwan; EMRPD1A0831, EMRPD1A0841, EMRPD1B0311, and EMRPD1B0321 from Ministry of Education, Taiwan; and CMRPG391032, CMRPG381523, CMRPD170493, CMRPD180372, CMRPD1B0381, and CMRPG3B1091 from Chang Gung Medical Research Foundation, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body Introduction Lung inflammation is a pivotal event in the pathogenesis of chronic obstructive pulmonary disease and asthma [1]. Cyclooxygenases (COXs) are responsible for the formation of prostaglandins (PGs), which are involved in inflammatory responses [2]. COX-2 is primarily an inducible isoform whose expression can be up-regulated by cytokines, mitogens, and endotoxins in many cell types [2]. It is highly expressed in inflamed tissues and believed to produce PGs involved in inflammatory processes [3]. Moreover, the physiological relevance of the purinergic signaling network for airway defenses is emerging through cumulating reports of abnormal ATP and adenosine levels in the airway secretions of patients with asthma and chronic pulmonary obstructive diseases. The consequences for airway defenses range from abnormal clearance responses to the destruction of lung tissue by inflammation [4]. Thus, to clarify the mechanisms of COX-2 induction by ATP in lung epithelium was recognized as a new therapeutic approach in the management of respiratory diseases. ATP transports chemical energy within cells, is produced by cellular respiration and is used by enzymes and structural proteins in many cellular processes [5]. Extracellular ATP is an important mediator of intercellular communication via the activation of purinergic P2X and P2Y receptors mediated through ion channels and GTP binding protein coupled receptors, respectively [6]. Growing evidence indicates the involvement of ATP and purinoceptors in the pathogenesis of lung diseases [5], [6]. ATP has been shown to induce COX-2 expression [7], [8], and then causes the inflammatory responses. However, the mechanisms by which ATP induced COX-2 expression in A549 cells are not completely understood. Oxidative stress is an important factor in the pathogenesis of respiratory diseases. Excessive ROS can directly damage cellular macromolecules, resulting in cell cycle arrest and/or cell death [9]. NADPH oxidase is an enzymatic source for the production of ROS under various pathologic conditions [10]. Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47phox, p67phox, and p40phox. The p47phox regulatory subunit plays a critical role in acute activation of NADPH oxidase; phosphorylation of p47phox is thought to relieve inhibitory intracellular interactions and permit the binding of p47phox to p22phox, thereby increasing NADPH oxidase activation [10]. ROS have been shown to regulate COX-2 expression and induce inflammation [11]. In addition, protein kinase C (PKC) has been involved in the transduction of signals for cell proliferation and differentiation [12]. Some studies have indicated that the expression of COX-2 is mediated by the activation of PKC [13], [14]. PKC has also been shown to stimulate NADPH oxidase activity and ROS generation [15]. Here, we investigated the role of PKC in ATP-induced ROS generation and COX-2 expression. Signal transducer and activator of transcription (STAT)3 belongs to the STAT family. STAT3 was first identified and cloned from mouse liver cDNA library in the study of IL-6 signaling [16]. Like its relatives, STAT3 is inactive in nonstimulated cells, but is rapidly activated by various cytokines and growth factors [16]. The phosphorylation of STAT3 at Tyr705 is most commonly mediated by Janus kinases (Jaks), especially Jak2 [17]. COX-2 expression has also been shown to be mediated via Jak2/STAT3 activation in various cell types [18], [19]. These findings imply that these signaling components Jak2/STAT3 might be also implicated in the expression of COX-2 induced by ATP in A549 cells. Therefore, ATP may play a potential role in regulation of expression of inflammatory genes, such as COX-2 and thereby promote inflammatory responses. We report here for the first time that ATPγS-induced COX-2 expression was mediated through a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3-dependent pathway in A549 cells. Methods Materials Anti-cPLA2, anti-PKCα, anti-PKCι, anti-PKCμ, anti-STAT3, anti-Jak2, anti-β-actin, and anti-p47phox antibodies were from Santa Cruz (Santa Cruz, CA). Anti-COX-2 antibody was from BD Transduction Laboratories (San Diego, CA). Adenosine 5′-O-(3-thiotriphosphate) (ATPγS), Gö6983, Gö6976, GF109203X, Ro318220, Rottlerin, PPADS, suramin, AG490, CBE, and arachidonic acid were from Biomol (Plymouth Meeting, PA). All other chemicals and enzymes were obtained from Sigma (St. Louis, MO). Edaravone (MCI-186) was from Tocris Bioscience (Ellisville, MO). CellROX™ Deep Red Reagent and CM-H2DCFDA were from Invitrogen (Carlsbad, CA). Cell Culture A549 cells (human alveolar epithelial cell carcinoma) were purchased from the American Type Culture Collection (Manassas, VA) and grown as previously described [20]. Western Blot Analysis Growth-arrested A549 cells were incubated with ATPγS at 37°C for the indicated time intervals. The cells were washed, scraped, collected, and centrifuged at 45000×g at 4°C for 1 h to yield the whole cell extract, as previously described [20]. Samples were denatured, subjected to SDS-PAGE using a 12% running gel, transferred to nitrocellulose membrane, incubated with an anti-COX-2 or anti-cPLA2 antibody for 24 h, and then incubated with an anti-mouse horseradish peroxidase Ab for 1 h. The immunoreactive bands were detected by ECL reagents and analyzed by using a UN-SCAN-IT Gel 6.1 program (Silk Scientific, Inc., Orem, UT). Real-time PCR Total RNA was extracted using TRIzol reagent. mRNA was reverse-transcribed into cDNA and analyzed by real-time RT-PCR. Real-time PCR was performed using SYBR Green PCR reagents (Applied Biosystems, Branchburg, NJ) and primers specific for COX-2 and GAPDH mRNAs. The levels of COX-2 expression were determined by normalizing to GAPDH expression. Isolation of Cell Fractions Cells were harvested, sonicated for 5 s at output 1.5 with a sonicator (Misonix Inc., Farmingdale, NY), and centrifuged at 8000 rpm for 15 min at 4°C. The pellet was collected as the nuclear fraction. The supernatant was centrifuged at 14000 rpm for 60 min at 4°C to yield the pellet (membrane fraction) and the supernatant (cytosolic fraction). Determination of NADPH Oxidase Activity by Chemiluminescence Assay Cells grew onto 6-well culture plates, after exposure to ATPγS for the indicated time intervals, were gently scraped and centrifuged at 400×g for 10 min at 4°C. The cell pellet was re-suspended in 35 µl/per vial of ice-cold RPMI-1640 medium (Gibco BRL, Grand Island, NY), and the cell suspension was kept on ice. To a final 200 µl volume of pre-warmed (37°C) RPMI-1640 medium containing either NADPH (1 µM) or lucigenin (20 µM), 5 µl of cell suspension (0.2×105 cells) was added to initiate the reaction followed by immediate measurement of chemiluminescence in an Appliskan luminometer (Thermo®) in out-of-coincidence mode. Appropriate blanks and controls were established, and chemiluminescence was recorded. Neither NADPH nor NADH enhanced the background chemiluminescence of lucigenin alone (30−40 counts per min). Chemiluminescence was measured continuously for 12 min, and the activity of NADPH oxidase was expressed as counts per million cells. Measurement of Intracellular ROS Accumulation The intracellular H2O2 levels were determined by measuring fluorescence of DCF-DA. A549 cells were washed with warm HBSS and incubated in HBSS containing 10 µM DCFH-DA at 37°C for 45 min. Subsequently, HBSS containing DCFH-DA was removed and replaced with fresh cell medium. Cells were then incubated with various concentrations of ATPγS. Cells were washed twice with PBS and detached with trypsin/EDTA, and the fluorescence intensity of the cells was analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA) at 495-nm excitation and 529-nm emission for DCF. In addition, CellROX™ Deep Red Reagent is a fluorogenic probe designed to reliably measure ROS in living cells. The cell-permeable CellROX™ Deep Red dye is nonfluorescent while in a reduced state and upon oxidation exhibits excitation/emission maxima at 640/665 nm. A549 cells were treated with ATPγS for the indicated time intervals, CellROX™ Deep Red Reagent was added at a final concentration of 5 µM to the cells, and then incubated for 30 min at 37°C. Subsequently, medium was removed and the cells were washed three times with PBS. The resulting fluorescence was measured using a fluorescence microscope (Zeiss, Axiovert 200M). Transient Transfection with siRNAs The small interfering RNA (siRNA) duplexes corresponding to human cPLA2, PKCα, PKCι, PKCμ, p47phox, Jak2, and STAT3 and scrambled siRNA were from Invitrogen (Carlsbad, CA). Transient transfection of siRNAs was carried out using Metafectene transfection reagent. siRNA (100 nM) was formulated with Metafectene transfection reagent according to the manufacturer’s instruction (Biontex Lab. GmbH, Planegg/Martinsried, Germany). Measurement of PGE2 Generation A549 cells were cultured in 6-well culture plates. After reaching confluence, growth-arrested cells were treated with ATPγS for the indicated time intervals at 37°C. The medium were collected and stored at −80°C until being assayed. PGE2 was assayed using a PGE2 enzyme immunoassay kit (Cayman) according to the manufacturer’s instructions. Analysis of Data All the data were estimated using the GraphPad Prism Program (GraphPad, San Diego, CA). Data were expressed as the mean±SEM and analyzed with a one-way ANOVA followed with Tukey’s post-hoc test at p<0.05 level of significance. All the experiments were performed at least three times. Results ATPγS Induces COX-2 Expression via a PKCs Signaling PKCs have been shown to be involved in proliferation and differentiation [12]. Some studies have indicated that the expression of COX-2 is mediated by the activation of PKCs [13], [14]. Here, we investigated the role of PKCs in ATPγS-induced COX-2 expression. As shown in Fig. 1A, pretreatment with the inhibitor of non-selective PKC (Ro318220), Ca2+-dependent PKC (Gö6983 and Gö6976), or selective PKCδ (Rottlerin) markedly attenuated ATPγS-induced COX-2 expression in A549 cells. COX-2 is the enzyme which converts arachidonic acid to PGH2, which can be further metabolized to prostanoids, including PGE2, prostacyclin (PGI2), and thromboxane A2 (TXA2) [21]. Pretreatment with these inhibitors also attenuated ATPγS-induced COX-2 mRNA expression and PGE2 generation (Fig. 1B). Translocation of PKC from the cytosol to the membrane is necessary to activation of PKC [12]. Next, we investigated whether ATPγS could stimulate PKCs translocation in A549 cells. As shown in Fig. 1C, ATPγS and PMA (a PKCs activator) stimulated the translocation PKCα, PKCι, and PKCμ from the cytosol to the membrane in a time-dependent manner. Moreover, to further ascertain the role of PKCs in ATPγS-induced COX-2 protein expression, as shown in Figs. 1D and E, transfection with siRNAs of PKCα, PKCι, and PKCμ down-regulated PKCα, PKCι, and PKCμ protein expression, respectively, and then reduced ATPγS-induced COX-2 expression and PGE2 production. These data demonstrated that PKCs play an important role in ATPγS-induced COX-2 expression in A549 cells. 10.1371/journal.pone.0054125.g001Figure 1 ATPγS regulates COX-2 expression via PKCs in A549 cells. Cells were pretreated with Gö6983, Gö6976, Ro318220, or Rottlerin for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE2 release. (C) Cells were treated with ATPγS (100 µM) for the indicated time intervals or PMA (1 µM) for 15 min. The cytosolic and membrane fractions were prepared and analyzed by western blot using an anti-PKCα, anti-PKCι, or anti-PKCμ antibody. β-actin and Gαs were used as a marker protein for cytosolic and membrane fractions, respectively. (D, E) Cells were transfected with siRNA of scrambled, PKCα, PKCι, or PKCμ, and then incubated with ATPγS for 6 h. The expression of PKCα, PKCι, PKCμ, and COX-2 were analyzed by western blot. The media were collected and analyzed for PGE2 release. Data are expressed as mean±S.E.M. of three independent experiments. *p<0.5; # p<0.01, as compared with the cells exposed to ATPγS alone (A, B) or the cells transfected with scrambled siRNA and exposed to ATPγS alone (D, E). NADPH Oxidase/ROS are Involved in ATPγS-induced COX-2 Expression NADPH oxidase is an enzymatic source for the production of ROS under various pathological conditions [11]. ROS has been shown to induce COX-2 expression associated with inflammation [11]. Thus, the role of NADPH oxidase/ROS generation in ATPγS-induced COX-2 expression was investigated. As shown in Fig. 2A, pretreatment of A549 cells with NADPH oxidase inhibitors [DPI and apocynin (APO)] or a ROS inhibitor (Edaravone) significantly abrogated ATPγS-induced COX-2 protein expression. In addition, pretreatment with these inhibitors also attenuated ATPγS-induced COX-2 mRNA expression and PGE2 generation (Fig. 2B). To further ascertain that generation of ROS was involved in ATPγS-induced COX-2 expression, a fluorescent probe, DCFH-DA, was used to determine the generation of ROS in A549 cells. As illustrated in Figs. 2C and D, ATPγS induced a significant increase in NADPH oxidase activity and ROS generation within 15 min, reached a peak within 60 min, and slightly declined within 120 min. Pretreatment with Eadaravone, DPI, and APO attenuated ATPγS-induced NADPH oxidase/ROS generation. On the other hand, we used CellROX™ Deep Red Reagent to confirm the generation of ROS in ATPγS-stimulated A549 cells. As shown in Figs. 2E and F, ATPγS induced ROS generation in a time-dependent manner, which was also attenuated by pretreatment with Edaravone, DPI, or APO in these cells. Activated NADPH oxidase is a multimeric protein complex consisting of at least three cytosolic subunits of p47phox, p67phox, and p40phox. It has been demonstrated that p47phox organizes the translocation of other cytosolic factors, hence its designation as “organizer subunit” [22]. Here, we found that ATPγS induced a significant translocation of p47phox from the cytosol to the membrane (Fig. 2G). The role of p47phox in ATPγS-mediated responses was also confirmed by transfection with p47phox siRNA which down-regulated p47phox protein expression, and then attenuated COX-2 expression and PGE2 production induced by ATPγS in A549 cells (Figs. 2H and I). These results indicated that NADPH oxidase activation and ROS generation play critical roles in ATPγS-induced COX-2 expression in A549 cells. 10.1371/journal.pone.0054125.g002Figure 2 ATPγS induces NADPH oxidase-dependent COX-2 expression in A549 cells. Cells were pretreated with Edaravone, DPI, or APO for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE2 release. Cells were labeled with DCF-DA (10 µM), and then incubated with ATPγS (100 µM) for (C) the indicated time intervals or (D) pretreated with Edaravone (10 µM), DPI (10 µM), or APO (100 µM) for 1 h, and then stimulated with ATPγS (100 µM) for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured and NADPH oxidase activity was determined. Cells were treated with (E) ATPγS for the indicated time intervals or (F) pretreated with Edaravone (10 µM), DPI (10 µM), or APO (100 µM) for 1 h, and then stimulated with ATPγS (100 µM) for 1 h. After incubation, CellROX™ Deep Red Reagent was added at a final concentration of 5 µM to the cells, and then incubated for 30 min at 37°C. Subsequently, medium was removed and the cells were washed thrice with PBS. The resulting fluorescence was measured using a fluorescence microscope. (G) Cells were incubated with ATPγS (100 µM) for the indicated time intervals. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47phox antibody. (H, I) Cells were transfected with siRNA of scrambled or p47phox, and then incubated with ATPγS for 6 h. The levels of p47phox and COX-2 expression were analyzed by western blot. The media was collected and analyzed for PGE2 release. Data are expressed as mean±S.E.M. of three independent experiments. *p<0.5; # p<0.01, as compared with the cells exposed to ATPγS alone (A, B, D), the cells exposed to vehicle alone (C), or the cells transfected with scrambled siRNA and exposed to ATPγS alone (H, I). ATPγS Induces NADPH Oxidase Activation and ROS Production via PKCs PKCs have also been shown to stimulate NADPH oxidase activity and ROS generation [15]. Thus, we investigated whether ATPγS stimulated NADPH oxidase activation and ROS production via PKCs activation in A549 cells. As shown in Figs. 3A and B, pretreatment with Ro318220, GF109203X, Gö6983, Gö6976, or Rottlerin markedly inhibited ATPγS-stimulated NADPH oxidase activity and H2O2 and/or ROS generation. In addition, pretreatment with these inhibitors also reduced p47phox translocation from the cytosol to the membrane (Fig. 3C). These data suggested that PKC plays a key role in ATPγS-stimulated NADPH oxidase activation and ROS production in A549 cells. 10.1371/journal.pone.0054125.g003Figure 3 ATPγS induces PKC-dependent ROS generation in A549 cells. (A) Cells were labeled with DCF-DA (10 µM), pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured (gray bar). In addition, NADPH oxidase activity was determined (white bar). (B) Cells were pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. After incubation, ROS generation was determined by using CellROX™ Deep Red Reagent as described in Fig. 2E. (C) Cells were pretreated with Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47phox antibody. Data are expressed as mean±S.E.M. of three independent experiments. # p<0.01, as compared with the cells exposed to ATPγS alone. ATPγS Induces COX-2 Expression via P2 Receptors Extracellular nucleotides regulate ion transport and inflammatory responses of the lung epithelium by activation of P2 receptors [12]. To investigate whether ATPγS could induce COX-2 expression, PKCs translocation, and ROS generation via P2 receptors, the P2Y and P2X receptor antagonists, suramin and PPADS were used. As shown in Figs. 4A and B, pretreatment with PPADS or suramin markedly inhibited ATPγS-induced COX-2 protein and mRNA expression and PGE2 production. ATPγS-stimulated ROS production, NADPH oxidase activity, and p47phox translocation was also inhibited by pretreatment with PPADS or suramin in A549 cells (Figs. 4C–E). In addition, pretreatment with PPADS or suramin also reduced PKCα, PKCι, and PKCμ translocation from the cytosol to the membrane in response to ATPγS (Fig. 4F). These data demonstrated that ATPγS induces COX-2 expression via P2 receptors in A549 cells. 10.1371/journal.pone.0054125.g004Figure 4 ATPγS induces P2 receptor-dependent COX-2 expression, PKC translocation, and ROS generation in A549 cells. Cells were pretreated with PPADS or suramin for 1 h, and then treated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE2 release. (C) Cells were labeled with DCF-DA (10 µM), pretreated with PPADS or suramin for 1 h, and then incubated with ATPγS for 1 h. The fluorescence intensity (relative DCF fluorescence) was measured (gray bar). In addition, NADPH oxidase activity was determined (white bar). (D) Cells were pretreated with PPADS or suramin for 1 h, and then incubated with ATPγS for 1 h. After incubation, ROS generation was determined by using CellROX™ Deep Red Reagent as described in Fig. 2E. (E) Cells were pretreated with PPADS (10 µM) or suramin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The membrane and cytosolic fractions were prepared and analyzed by western blot using an anti-p47phox antibody. (F) Cells were pretreated with PPADS (10 µM) or suramin (10 µM) for 1 h, and then treated with ATPγS for 15 min. The cytosolic and membrane fractions were prepared and analyzed by western blot using an anti-PKCα, anti-PKCι, or anti-PKCμ antibody. Data are expressed as mean±S.E.M. of three independent experiments. # p<0.01, as compared with the cells exposed to ATPγS alone. Jak2/STAT3 are Involved in ATPγS-induced COX-2 Expression STAT3 is a transcription factor that is activated by many cytokines and growth factors and plays a key role in cell survival, proliferation, and differentiation [23]. The phosphorylation of STAT3 at Tyr705 is most commonly mediated by Jaks, especially Jak2 [17]. Thus, we also evaluated whether Jak2 and STAT3 were involved in ATPγS-induced COX-2 expression in A549 cells. As shown in Figs. 5A and B, pretreatment with the inhibitors of Jak2 (AG490) and STAT3 (CBE) reduced ATPγS-induced COX-2 protein and mRNA expression and PGE2 production. Moreover, ATPγS stimulated Jak2 and STAT3 phosphorylation in a time-dependent manner (Fig. 5C). In response to cytokines and growth factors, STAT family members are phosphorylated by receptor-associated kinases and then form homo- or heterodimers that translocate into the nucleus, where they act as transcription activators [23]. Next, we showed that ATPγS markedly induced STAT3 translocation in a time-dependent manner in A549 cells (Fig. 5D), which was inhibited by pretreatment with AG490, CBE, PPADS, and suramin in A549 cells (Fig. 5E). We further confirm the roles of Jak2 and STAT3 in ATPγS-induced responses by using siRNAs of Jak2 and STAT3. Here, we showed that ATPγS-induced COX-2 expression and PGE2 generation was reduced by transfection with siRNA of Jak2 or STAT3 (Figs. 5F and G). These results showed that ATPγS induces COX-2 expression via a P2 receptor/Jak2/STAT3 signaling in A549 cells. 10.1371/journal.pone.0054125.g005Figure 5 ATPγS induces COX-2 expression via Jak2/STAT3 in A549 cells. Cells were pretreated with AG490 or CBE for 1 h, and then incubated with ATPγS for (A) 6 h or (B) 2 h. The levels of COX-2 (A) protein and (B) mRNA were analyzed by western blot and real-time PCR, respectively. (A) The media were collected and analyzed for PGE2 release. (C) Cells were treated with ATPγS (100 µM) for the indicated time intervals. The cell lysates were analyzed by western blot using an anti-phospho-Jak2, anti-phospho-STAT3, anti-STAT3, or anti-β-actin antibody. Cells were pretreated (D) without or (E) with AG490 (10 µM), CBE (10 µM), PPADS (10 µM), or suramin (10 µM) for 1 h, and then incubated with ATPγS (100 µM) for (D) the indicated time intervals or (E) 60 min. The cytosolic and nuclear fractions were prepared and analyzed by western blot using an anti-phospho-STAT3 or anti-STAT3 antibody. GAPDH and Lamin A were used as a marker protein for cytosolic and nuclear fractions, respectively. (F) Cells were transfected with siRNA of scrambled, Jak2, or STAT3, and then incubated with ATPγS for 6 h. The levels of Jak2, STAT3, and COX-2 expression were analyzed by western blot. The media were collected and analyzed for PGE2 release. Data are expressed as mean±S.E.M. of three independent experiments. # p<0.01, as compared with the cells exposed to ATPγS alone (A, B), the cells exposed to vehicle alone (C), or the cells transfected with scrambled siRNA and exposed to ATPγS alone (F, G). ATPγS Stimulates Jak2/STAT3 Activation via a PKCs/ROS Signaling We further investigated whether PKCs and NADPH oxidase/ROS were involved in ATPγS-stimulated Jak2 and STAT3 activation in A549 cells. We found that ATPγS-stimulated Jak2 and STAT3 translocation and phosphorylation was reduced by pretreatment with Edaravone, APO, DPI, Ro318220, GF109203X, Gö6983, Gö6976, or Rottlerin and transfection with siRNAs of p47phox, PKCα, PKCι, and PKCμ (Figs. 6A and B). These results demonstrated that ATPγS stimulates Jak2 and STAT3 activation via PKC/NADPH oxidase/ROS in A549 cells. 10.1371/journal.pone.0054125.g006Figure 6 ATPγS regulates PKC/ROS-dependent Jak2 and STAT3 activation in A549 cells. (A) Cells were pretreated with APO (100 µM), DPI (10 µM), Edaravone (10 µM), Gö6983 (10 µM), Gö6976 (10 µM), GF109203X (3 µM), Ro318220 (10 µM), or Rottlerin (10 µM) for 1 h, and then incubated with ATPγS for 1 h. The cytosolic and nuclear fractions were prepared and subjected to Western blot analysis using an anti-phospho-STAT3 or anti-STAT3 antibody. (B) Cells were transfected with siRNAs of scrambled, p47phox, PKCα, PKCι, and PKCμ, and then incubated with ATPγS for 1 h. The cell lysates were subjected to western blot analysis using an anti-phospho-Jak2, anti-phospho-STAT3, or anti-β-actin antibody. Data are expressed as mean±S.E.M. of three independent experiments. # p<0.01, as compared with the cells exposed to scrambled siRNA+ATPγS. ATPγS Induces COX-2/PGE2 Expression via a cPLA2/AA Pathway Indeed, cytosolic phospholipase A2 (cPLA2) is also involved in PGE2 production. As shown in Fig. 7A, ATPγS markedly induced cPLA2 and COX-2 expression in a time-dependent manner in A549 cells. However, ATPγS had no effect on COX-1 expression in A549 cells. We also found that ATPγS-induced COX-2 expression was inhibited by transfection with siRNA of cPLA2 or COX-2 (Fig. 7B). Moreover, COX-2 siRNA had no effects on ATPγS-induced cPLA2 protein expression (Fig. 7B). These data suggested that ATPγS induced COX-2 expression via a cPLA2-dependent pathway. We further found that arachidonic acid (AA) markedly enhanced COX-2, but not COX-1 expression in A549 cells (Fig. 7C). Finally, we observed that AA and ATPγS induced PGE2 production (Fig. 7D). Moreover, ATPγs-induced PGE2 production was reduced by transfection with siRNA of cPLA2 or COX-2 (Fig. 7E). These results suggested that ATPγS induced PGE2 production via a cPLA2/AA/COX-2 pathway in A549 cells. On the other hand, we found that ATPγS-induced cPLA2 expression was reduced by the inhibitors of PKCs, ROS, Jak2, and STAT3 in A549 cells (data not shown). Thus, we suggested that ATPγ induced PGE2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA2/AA/COX-2 pathway in A549 cells. 10.1371/journal.pone.0054125.g007Figure 7 ATPγS induces COX-2 expression via a cPLA2/AA signaling. (A) Cells were treated with ATPγS for the indicated times. The expression of cPLA2, COX-2, or COX-1 was determined by Western blot. (B) Cells were transfected with cPLA2 or COX-2 siRNA, and then treated with ATPγS for 24 h or 6 h. The expression of cPLA2 or COX-2 was determined by Western blot. (C) Cells were treated with AA for the indicated times. The expression of COX-2 or COX-1 was determined by Western blot. (D) Cells were treated with ATPγS or AA for the indicated times. The production of PGE2 was measured. (E) Cells were transfected with cPLA2 or COX-2 siRNA, and then treated with ATPγS for 6 h. The production of PGE2 was measured. Data are expressed as mean±S.E.M. of three independent experiments. # p<0.01, as compared with the cells exposed to vehicle alone (D) or scrambled siRNA+ATPγS (E). Discussion Asthma and COPD are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. ATP is a major signaling molecule in the patients with asthma and COPD [4], [5]. ATP elicits its actions by engaging cell surface purinoceptors, and substantial preclinical evidence suggests that targeting these receptors will provide novel approaches for the treatment of asthma and COPD [4], [5]. Patients with COPD show evidence of increased release of ROS leading to oxidative stress [24]. On the other hand, several lines of evidence suggest that high levels of PGs, synthesized by COX-2, are involved in inflammatory responses [25]. The molecular mechanisms by which ATP induces COX-2-dependent PGE2 generation are not fully understood in A549 cells. The present study clearly demonstrated that COX-2 expression induced by ATPγS was mediated through a P2 receptor/PKC/NADPH oxidase/Jak2/STAT3/cPLA2 pathway. Genetic silencing through transfection with siRNA of cPLA2, PKCα, PKCι, PKCμ, p47phox, Jak2, or STAT3 or pretreatment with the inhibitors of P2 receptors, PKCs, NADPH oxidase, Jak2, and STAT3 abrogated ATPγS-induced COX-2 expression and PGE2 release. Therefore, P2 receptor activation by ATPγS causes inflammatory responses through ROS and PGE2 production. Moreover, PKC, NADPH oxidase, Jak2, and STAT3 were also involved in ATPγS-induced COX-2 expression in A549 cells (Fig. 7). Extracellular adenosine 5'-triphosphate (eATP) is ubiquitously used for cell-to-cell communication [5]. The low level of eATP that exists in a “halo” surrounding resting cells signals the presence of neighboring living cells. Larger increases in eATP that are associated with cell death serve as a key “danger” signal in inflammatory processes [26]. Various aspects of purinergic signaling have been demonstrated in different cell types [12], [27], [28]. PKC represents a family of more than 11 phospholipid-dependent Ser/Thr kinases that are involved in a variety of pathways that regulate cell growth, death, and stress responsiveness [29]. PKC isoforms are divided into three categories according to the cofactors that are required for optimal phospholipid-dependent catalytic activity [29]. ATP has been shown to regulate PKC activation [12], [30]. In addition, PKC plays a key role in regulating COX-2 induction [14], [31]. Indeed, we showed that COX-2 expression and PGE2 production in response to ATPγS were significantly reduced by transfection with siRNAs of PKCα, PKCι, and PKCμ or pretreatment with the inhibitors of PKCs in A549 cells. Translocation to the membrane is necessary to activate PKC [12]. This notion is confirmed by our observation that ATPγS stimulated PKCs translocation from the cytosol to the membrane. These results suggested that ATPγS plays an important role in PKCs activation leading to COX-2/PGE2 expression in A549 cells. Cells and tissues are routinely subjected to sublethal doses of various oxidants, either exogenously through environmental exposure or endogenously through inflammatory processes [24], [29]. The biological function of NADPH oxidase enzymes might be attributable to the production of ROS [24]. Activation of the NADPH oxidase, i.e. activation of gp91phox, requires stimulus-induced membrane translocation of cytosolic proteins, including the small GTPase Rac and the two specialized cytosolic proteins p67phox and p47phox, each containing two SH3 domains [32], [33]. In this process, p47phox translocates to the membrane by itself, whereas p67phox is recruited via p47phox [34], [35]: they constitutively associate via the interaction of the C-terminal SH3 domain of p67phox with the p47phox C-terminus [36], [37]. Thus, p47phox plays a central role in the membrane translocation. Indeed, our results confirmed that ATPγS-induced COX-2 expression and PGE2 synthesis was reduced by pretreatment with a ROS inhibitor (Edaravone) and the inhibitors of NADPH oxidase (DPI or APO) or transfection with p47phox siRNA. Pretreatment with DPI or APO inhibited ATPγS-induced ROS generation. These results suggested that NADPH oxidase-dependent ROS generation was involved in ATPγS-induced COX-2/PGE2 expression. Although the signaling pathways underlying ATPγS-regulated NADPH oxidase have not been completely defined, involvement of PKC in NADPH oxidase activation has been reported in various cell types [15], [38], [39]. This note is confirmed by our observation that ATPγS-induced NADPH oxidase activity, ROS generation, and p47phox translocation was inhibited by pretreatment with the inhibitors of PKCs. Among the purinoreceptors, P1Rs (now known as A1, A2, and A3 receptors) respond to adenosine but not to ATP, whereas all P2Rs (P2XR or P2YR) respond to ATP, some also respond to ADP, uridine 5'-triphosphate, or uridine 5'-diphosphate [5], [26]. In the present study, we found that ATPγS regulated COX-2/PGE2 expression, PKC activation, and ROS generation via P2 receptor in A549 cells by pretreatment with the inhibitors of P2 receptors. These data suggested that ATPγS may cause lung and airway inflammation via the P2 receptor-dependent COX-2/PGE2 induction. STATs are a class of transcription factors bearing SH2 domains that become activated upon tyrosine phosphorylation [23], [40]. STAT3 is a transcription factor that is activated by many cytokines and growth factors and plays a key role in cell survival, proliferation, and differentiation [23], [40]. The phosphorylation of STAT3 at Tyr705 is most commonly mediated by Jaks, especially Jak2 [17]. COX-2 expession has also been shown to be mediated via STAT3/Jak2 activation [18], [19]. Moreover, this is confirmed by our data that pretreatment with the inhibitor of Jak2 or STAT3 markedly inhibited ATPγS-induced COX-2 expression and PGE2 generation in A549 cells. Oxidative stress has been shown to increase the activity of transcription factors, such as STAT3 [40]. Here, we found that NADPH oxidase-dependent ROS production was involved in ATPγS-stimulated Jak2 and STAT3 phosphorylation. Thus, ROS may be critical for the inflammatory responses triggered by ATPγS, through the up-regulation of redox-sensitive transcription factors and hence the expression of proinflammatory genes. Further understanding of the effects and roles of ROS in cellular functions as amplification of proinflammatory and immunological responses, signaling pathways, activation of transcription factors, and gene expression will provide important information regarding pathological processes contributing to chronic lung diseases. In summary, as depicted in Fig. 8, our results showed that ATPγS induced ROS production through a P2 receptor/PKCs/NADPH oxidase signaling, in turn initiated the activation of Jak2 and STAT3. Activated STAT3 was recruited to the promoter region of COX-2 leading to an increase of COX-2 expression associated with PGE2 release. Therefore, the inhibitors of P2 receptors may be proven useful in diminishing ATPγS-induced lung inflammation and chronic pathology. 10.1371/journal.pone.0054125.g008Figure 8 Schematic diagram illustrating the proposed signaling pathway involved in ATPγS-induced COX-2 expression and PGE2 generation in A549 cells. ATPγS activates the P2 receptor/PKC/NADPH oxidase pathway to enhance ROS generation, which in turn initiates the activation of Jak2 and STAT3, and ultimately induces COX-2-dependent PGE2 generation in A549 cells. We thank Ms. Chi-Yin Lee for her technical assistance. ==== Refs References 1 Lin CC , Lee CW , Chu TH , Cheng CY , Luo SF , et al (2007 ) Transactivation of Src, PDGF receptor, and Akt is involved in IL-1β-induced ICAM-1 expression in A549 cells . J Cell Physiol 211 : 771 –780 .17299794 2 Li H , Bradbury JA , Dackor RT , Edin ML , Graves JP , et al (2011 ) Cyclooxygenase-2 regulates Th17 cell differentiation during allergic lung inflammation . Am J Respir Crit Care Med 184 : 37 –49 .21474648 3 Carey MA , Germolec DR , Langenbach R , Zeldin DC (2003 ) Cyclooxygenase enzymes in allergic inflammation and asthma . 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PLoS One. 2013 Jan 11; 8(1):e54125