Intermediate analogues 2-bromomethyl-1,3-dimethoxyanthraquinone , 2-hydroxymethyl-1,3-dimethoxyanthraquinone , 2-formyl-1,3-dimethoxyantharquinone , 1,3-dimethoxy-2-methylanthraquinone , and 1,3-dihydroxy-2-methylanthraquinone  also exhibited moderate cytotoxicity against MCF-7 and K-562 cancer cell lines, respectively. Other structurally related compounds like 1,3-dihydroxyanthraquinone , 1,3-dimethoxyanthraquinone  were also showed good cytotoxicity. The target compound damnacanthal (1) was found to be the most cytotoxic against the MCF-7 and K-562 cancer cell lines, with IC50 values of 3.80 ± 0.57 and 5.50 ± 1.26, respectively. The structures of all compounds were elucidated with the help of detailed spectroscopic techniques.Naturally occurring anthraquinones, damnacanthal ( Damnacanthus subspinosus and Morinda parvifolia have long been used in traditional medicine for the treatment of cancer . The use of an excess amount (1.5 equiv.) of PCC also gave the undesired 1-hydoxy-3-methoxyanthraquinone-2-carboxylic acid. Upon treating compound 7 with AlCl3/CH2Cl2, nordamnacanthal (2) was obtained in 28% yield. The detailed reaction conditions are shown in The synthesis of nordamnacanthal (none (5) ,31,32 annone (5) . Compounve yield ,31. Comp1) was accomplished by first acetylated the precursor compound 3 with (CH3)2SO4 and potassium carbonate to afforded monoacetylated derivative 8. Upon methylation of compound 8 with K2CO3/CH3I compound 9 was obtained, which then brominated with NBS to yield 1-methoxy-3-aectoxy-2-bromomethyl-1-methoxyathraquinone (10) .A single crystal of compound The compounds were dissolved in dimethylsulfoxide  to get a stock solution of 1 mg/mL and stored at 4 °C.2 and 90% humidity throughout the study. Cell viability was assessed by trypan blue exclusion method where both MCF-7 and K562 cell lines were collected, washed with PBS and mixed with 1:1 ratio of trypan blue dye and the cell number and viability were evaluated using haemacytometer under inverted light microscope. Only cell with viability higher than 95% will be subjected to MTT cell viability assay.Adherent human estrogen dependent breast carcinoma MCF-7 and suspension human chronic myelogenic leukemia K-562 cell lines were obtained from ATCC, USA. Both MCF-7 and K562 were cultured in RPMI-1640 supplemented with 10% FBS and incubated at 37 °C, 5% CO5 cells/mL and incubated overnight at 37 °C, 5% CO2 at humidified atmosphere of 95% air. When cells were reached 70% confluence then subjected to the cell viability assay. Medium containing the seeded MCF-7 cell was discarded. Treatment of both cultures were carried out by first adding 100 µL of RPMI-1640 medium supplemented with 10% of FBS into all wells except row A of the 96 well plates . Then, 100 µL of each diluted compound (60 µg/mL) was added into row A and row B. A series of two-fold dilutions of compounds were carried out from row B to row G to give concentrations of 30, 15, 7.5, 3.75, 1.375 and 0.687 µg/mL, while row H was left as untreated control cell. For K-562 culture plate, 100 µL of cells (2 × 105 cells/mL) was added into all wells. For MCF-7 culture plate, 100 µL of RPMI-1640 medium was added into all wells. The plates were incubated in 37 °C, 5% CO2 and 90% humidity incubator for 72 h. After incubation period, 20 µL of MTT  was added into all wells and the plates were further incubated for 4 h. Then, 170 µL of supernatant was aspirated from every well plate. The plates containing K-562 culture were centrifuged at 200 × g for 5 min prior to medium aspiration. The resulting formazan crystals in each well were solubilized by 100 µL of DMSO  followed by incubation for 20 min. Finally, the plates were read at 570 nm and 630 nm as reference wavelength by using µQuant ELISA Reader . The results of the compound-treated cells were compared with the standard doxorubicin. Each compound and control was assayed in triplicates in three independent experiments. The percentage of inhibitions were calculated by using Graph pad and expressed in µg/mL or µM and listed in The effect of all compounds on cell viability of MCF-7 and K-562 cell lines were determined using the colorimetric 3--2,5-diphenyltetrazolium bromide (MTT) assay . MCF-7 c1) and nordamnacanthal (2) was achieved. The key difference from the previously synthesis was the conversion of hydroxymethylanthraquinones 7 or 12 into the corresponding aldehyde, which was achieved by the use of optimized mild conditions with PCC as oxidant. The synthesis in good yield of 2-ethoxymethyl-3-hydroxy-1-methoxyanthraquinone (11) was achieved through a short sequence. The structure activity relationship suggested that methoxy, formyl, bromomethyl and hydroxyl groups are important for the cytotoxicity and selectivity in these substituted anthraquinones. These side chain modifications give an idea for further efforts to increase the therapeutic potential of this class of compounds. The synthesis of damnacanthal (


TM, cancer cell line encyclopedia (CCLE), and cancer therapeutics response portal (CTRP) were analyzed. The correlation of expression levels of candidate and IC50 of candidate genes-targeted drugs were verified on NCI-N87 and MKN-45 GC cell lines. We found that RARA, THRA, CACNB1, and TOP2A are drug sensitive biomarkers of ERBB2-targeted treatment with FDA-approved drugs. All these genes act through Myc signaling pathway. Myc is the downstream hub gene of both ERBB2 and RARA. The expression of RARA, THRA, and CACNB1 were negatively correlated with Myc activation, while ERBB2 and TOP2A positively correlated with Myc activation. SH3BGRL3, SH3BGRL, and NRG2 were identified as potential ligands of ERBB2. The ERBB2+ GC with RARA amplification demonstrated better prognosis than those without RARA amplification, while overexpression of NRG2 and SH3BGRL correlated with poor prognosis in ERBB2+ GC. About 90% of ERBB2+ GC was compatible with chromosome instability (CIN) subtype of TCGA, which overlaps with intestinal-type GC in Lauren classification. In validating experiments, combination of Lapatinib and all-trans retinoic acid (ATRA) synergistically suppresses cell growth, and accompanied by decreased expression of MYC. In conclusions, we identified several predicting biomarkers for ERBB2-targeted therapy and corresponding histological features of ERBB2+ GC. Combination of ERBB2 antagonist or RARA agonist may be effective synergistic regimens for ERBB2+ GC.Exploring ERBB2-related pathways will help us finding sensitive molecules and potential combined therapeutic targets of ERBB2-targeted therapy for ERBB2+ gastric cancer (GC). In this study, we performed a cross-databases study focused on ERBB2+ GC. The data of ERBB2+ GC deposited in the cancer genome atlas (TCGA), gene expression omnibus (GEO), InBio Map In Chin by FISH . In gene by FISH . A phase by FISH . Because by FISH . Current by FISH . FindingERBB2 is located in chromosome 17q21.2, where some common oncogenes or tumor suppressor genes, such as TOP2A, TAU, p53, and HIC-1 are located . Our pre1) and cBioPortal database2 (3 (TM database4 (5) (6)  . The inf (5) (6) . Kyoto E2.Human GC cell lines NCI-N87 and MKN45 were purchased from the type Culture Collection of Chinese Academy of Sciences . All cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) at 37°C in a humidified incubator with 95% air and 5% COFirstly, 5000/well NCI-N87 or MKN45 cells were placed in 96 well plates (100 μl/well). Cells were treated by different concentrations of Lapatinib  and All-Trans Retinoic Acid , respectively. CCK-8(10 μl/well) was used and OD value was measured at 450 nm by spectrophotometry  at different time points.The cytoplasmic protein and nuclear protein were extracted, respectively by extraction kit  according to the protocols. Western blot was performed as previously described . The antP-value less 0.05 was considered statistically significant.EdgeR package in R language was used to screen differentially expressed genes . We plotP-value . Four out of these 47 genes are the molecular targets of FDA-approved drugs. They are CACNB1  for amlodipine, TOP2A  for etoposide, RARA for ATRA and THRA  for tiratricol. These genes were selected as potential targets of combination therapy with ERBB2-targeted drugs.Among 413 cases of GC from TCGA dataset, ERBB2+ GC accounted for 14.29% (59/413 cases), and the others were ERBB2- GC (354/413 cases). We compared the differential expressed genes and copy number variations between ERBB2+ and ERBB2- GC groups. Forty-seven differential expressed genes were selected based on the likelihood ratio (LR) and Figure 1B). These genes were located in adjacent region of chromosomal 17q . In ERBB2+ GC, the rate of gene amplification was 57.63% (34 out of 59) for RARA, 62.71% (37 out of 59 (62.71%) for THRA, 50.85% (30 out of 59) for TOP2A, and 54.24% (32 out of 59) for CACNB1. The elevated mRNAs were also identified for cases with gene amplification, compared to cases without gene amplification .Gene amplification of RARA, THRA, CACNB1, and TOP2A was often accompanied by ERBB2 amplification , THRA is −1.85 , and CACNB1 is −1.89 . TOP2A and ERBB2 were positively associated with Myc activation . These results implied that agonists of RARA, THRA, and CACNB1 or inhibitors of TOP2A or ERBB2 might be combined together for the treatment of ERBB2+ GC.Gene set enrichment analysis (GSEA) is a visualized method of the gene ontology analysis, which could help researchers finding a group of functional genes in a signaling pathway. Through GSEA, we found that all amplified genes of 17q are Myc-related genes. Of those, RARA, THRA, and CACNB1 were negatively correlated with Myc activation, which revealed minus value of enrichment score (ES). For instance, the ES value of RARA is −1.93  are three candidate ligands of ERBB2. Both RARA and ERBB2 are upstream genes in Myc pathway . To clarify the clinical significance of amplified genes in ERBB2+ GC, these genes in two datasets from TCGA and GEO were further analyzed. The tendency of better prognosis was noted in RARA amplification group, compared to non-RARA amplification group of ERBB2+ GC in both TCGA cohort , and GEO cohort . In ERBB2+ GC of TCGA cohort, high levels of NRG2 and SH3BGRL were significantly related to poor prognosis , but not for SH3BGRL3 . These results implied the ligand function of NRG2 and SH3BGRL in cancer progression of ERBB2+ GC.The association and pathway of amplified genes were analyzed by InBio MapP = 0.031, Figure 4A, left). The ERBB2-amplified cell lines also showed lower IC50 (μM) to drug Afatinib than other cell lines . The CNV of ERBB2 or RARA and their responses to targeted drugs Lapatinib or Afatinib of GC cell lines were listed in Table 1. Cell line NCI-N87 were more sensitive to ERBB2-targeted drugs, which is related to dual amplification of ERBB2 and RARA, while MKN7 was not sensitive to ERBB2-targeted drugs, which is related to non-amplification of RARA.Since the gene copy number, mRNA level and IC50 value to ERBB2-targeted drugs of multiple cancer cell lines were deposited in CCLE database, the drug responsive status of 17 GC cell lines was analyzed. Among those, four cell lines  were ERBB2-amplified cell lines that showed lower IC50 (μM) to drug Lapatinib than other GC cell lines  and Afatinib . Similarly, overexpression of RARA mRNA was significantly associated with lower IC50 to Lapatinib  and Afatinib . However, there was no significant correlation for NRG2 and SH3BGRL mRNA expression and drug sensitivity . Nevertheless, GC cases with overexpression of ERBB2 and NRG2 (ERBB2-H/NRG2-H) were more sensitive to Lapatinib, compared to low gene expression of ERBB2-L/NRG2-L group . However, there was no significant difference between ERBB2-H/SH3BGRL-H group and ERBB2-L/NRG2-L group for drug Afatinib .ERBB2 mRNA overexpression was often significantly related to lower IC50 to Lapatinib . Group 3 is better than that in Group 2 for sensitivity to Lapatinib .The relationship of simultaneous overexpression of NRG2, ERBB2, and RARA with Lapatinib sensitivity was listed in 9, which provided opportunity to analyze correlation between genomic information and clinicopathological characteristics. For 59 cases of ERBB2+ GC, the pathology reports and slide images of 46 cases were uploaded in database . Fifty-three cases out of 59 ERBB2+ GC (89.83%) belong to chromosome instability (CIN) molecular subtype, and the others include three Epstein-Bar Virus-related (EBV) subtype, two genome stable (GS) subtype, and one microsatellite instability (MSI) subtype . Based on their pathological reports of ERBB2+ GC, all of those are intestinal-type GC in traditional Lauren classification, such as tubular adenocarcinoma, papillary adenocarcinoma, and a few mucinous adenocarcinomas.The clinical details and histology of TCGA cohort were recorded in cBioPortal databaseTable 2), cell line NCI-N87 (ERBB2-H/RARA-H) and MKN-45 (ERBB2-L/RARA-L) were selected for further validating experiments. The IC50s of Lapatinib (targeted to ERBB2) and ATRA (targeted to RARA) were assayed by CCK8 method on NCI-N87 and MKN-45 cell lines. As showed in Figure 6A, cell line NCI-N87 was more sensitive than cell line MKN-45 to drug Lapatinib , while there was no significant difference of IC50s of ATRA in both GC cell lines . By further CI analysis, synergistic effect of ATRA (25 μM) and Lapatinib (between 0.005 and 0.5 μM) was observed on NCI-N87 cell line, but not in MKN-45 cell line (Table 3).To verify the effect on Myc signaling pathway, the cytoplasmic protein and nuclear protein were extracted separately, and examined expressing levels after incubating Lapatinib, ATRA or both. As results, after 2 h incubation of Lapatinib (0.05 μM), ATRA (25 μM), or both on NCI-N87 and MKN-45 cells, obvious down-regulation of MYC protein levels of cytoplasm and nucleus was found in Lapatinib treated NCI-N87 cells, but bot ATRA did. Importantly, incubation of Lapatinib and ATRA simultaneously further reduced nuclear protein level of MYC in NCI-N87 cells . The similar effects were also observed on NCI-N87 cells after 48 h incubation by Lapatinib and ATRA, or both . However, these effects were not observed in MKN-45 cells .Based on data analysis of multiple GC cell lines  of ERBB2 have been proposed. The latter has been reported as a ligand for ERBB3/ERBB4 . SlatterIt has been reported that there are some histopathological features of ERBB2+ GC . ERBB2 pIn our validating study, we confirmed that GC cell line with amplified ERBB2 and RARA (NCI-N87) was sensitive to Lapatinib relative to GC cell without amplified ERBB2 and RARA (MKN-45). The sensitivity to ATRA on both cell lines did not show significant difference, which may attribute to some alternative pathways for ATRA-mediated apoptosis . We founFigure 7). Moreover, combination of RARA agonist ATRA with ERBB2-targeted drug disclosed synergistic anticancer effect for ERBB2+ GC in vitro.In summary, by cross-database analysis, GC with increased expression of ERBB2, RARA, and NRG2 simultaneously should be taken as sensitive category to Lapatinib therapy. The current study proposed that NRG2-ERBB2-MYC is cross-interfaced with RARA-MYC pathway. Intervention of both routes will regulate hub gene Myc, and then inhibit cell growth for ERBB2+ GC (YY, XH, and ZZ were involved in concept and design. ZX, JW, and JZ acquired the data and performed data mining. ZX, JJ, and RY performed the experiments. WC and YY supported the analysis. All authors wrote, reviewed, and revised the manuscript.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.


Structures of these compounds were elucidated based on their spectral properties. While some of these sesquiterpene coumarins showed modest cytotoxic activity against COLO205, KM12, A498, UO31, and TC32 cancer cell lines, selective cytotoxicity of anatolicin (8) and 14′-acetoxybadrakemin (7) were observed at nanomolar level against the UO31 kidney cancer cell line.Seven known sesquiterpene coumarins and a new sesquiterpene coumarin, anatolicin ( Heptaptera anatolica (Boiss.) Tutin.Cancer is the second leading cause of death worldwide according to WHO statistics . Cancer Ferula (Apiaceae) species have been used for the treatment of various tumors since ancient times; Dioscorides refers to the use of such drugs in the De Materia Medica + .Air-dried and coarsely powdered roots (100 g) of 4 cells per well for both the UO31 and A498 cell line into a 384-well “assay plate” and then incubated for 24 h. DMSO solutions of the test materials (8 μL) were diluted 1:25 with medium (192 μL) and then subjected to five 2:1 serial dilutions (100 μL each) on a 96-well plate. Duplicate 40 μL aliquots of each sample concentration were then transferred to a 384-well “dilution plate”, which could accommodate the duplicate samples from two 96-well plates. A 5 μL aliquot of each solution on the dilution plate was transferred to the cell cultures on the 384-well assay plate to give a final volume of 50 μL and a DMSO concentration of 0.4%. Cells were incubated for 48 h at 37 °C in the presence of the test samples and then treated with the tetrazolium salt XTT . Viable cells reduced the XTT to a colored formazan product, and after an additional 4 h incubation period, the amount of formazan produced was quantified by absorption at 450 nm using the absorption at 650 nm as a reference. Sanguinarine was used on each plate as a positive control. Similar methodology and seeding densities were used for COLO205, KM12 (colon), A673, and TC32 (Ewing sarcoma) cell lines.The primary assay used for this study was a two-day, two-cell-line XTT bioassay , an in vHeptaptera anatolica yielded seven known and one new sesquiterpene coumarin. Although cytotoxic activity of sequiterpene coumarins were described in the literature earlier [7) and anatolicin (8)) toward UO31 kidney cancer cell line had not been reported previously.Investigation of the dichloromethane extract of the roots of  earlier , high po


Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the major miRNAs and proteins extracted from the exosomes were similar among the four cell lines. However, the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs  and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours. Exosomes are small extracellular vesicles released from almost all cell types, including immune cells and tumour cells , as the Exosomes have also been suggested to play important roles in the microenvironmental crosstalk of human haematopoietic tumours, including leukaemia and lymphoma ,8. It haLymphoma is a haematopoietic malignancy originating from lymphoid cells, and it is categorised into more than 80 distinct subtypes . Among tIn recent years, naturally occurring canine tumours were regarded as potential models of human tumours. In particular, canine lymphoma shares many characteristics of human NHL, including clinical presentation, immunophenotypic composition, chemotherapeutic protocols, and response to treatment ,21. TherBased on these backgrounds, we considered that comprehensive analyses of both miRNA and protein profiles within exosomes derived from canine lymphoid tumour cell lines could provide useful insights for understanding the molecular profiles and biological functions of exosomes derived from human lymphoid tumours. The aim of this study was to comprehensively analyse the miRNA and protein profiles within the exosomes released from canine lymphoid tumour cells.The size distributions of exosomes isolated from four canine lymphoid tumour cell lines, CLBL-1, GL-1, UL-1, and Ema, are shown in At first, the miRNA profiles of exosomes and parent cells were investigated via small RNA sequencing analysis. A minimum of 20 million raw reads were generated for each sample see . The numThen, hierarchical clustering using the amounts of miRNA in CLBL-1, GL-1, and UL-1 was conducted. This analysis yielded three clusters composed of exosomes and parent cells of each cell line . In addiThe top ten miRNAs contained in exosomes and parent cells are listed in In the comparison of the amounts of miRNAs between cells and exosomes, the amounts of 39, 20, and 24 miRNAs were significantly different in CLBL-1, UL-1, and Ema, respectively (q < 0.01) . Among tThe difference in the amount of miR-350 between exosomes and parent cells was confirmed by RT-qPCR . HoweverThe results of separating exosomal proteins from each cell line by SDS-PAGE are shown in The top twenty proteins that were detected in each cell line by LC-MS/MS are listed in The exosomal miRNA profiles were also compared between the VCR-S cell lines (CLBL-1 and GL-1) and the VCR-R cell line (UL-1) see . In dataNTRK2, MAPK8, BCOR, and PIK3R1 genes).Among these miRNAs, the significant differences in the amounts of miR-151, miR-8908a-3p, and miR-486 were confirmed by RT-qPCR using the four cell lines including Ema, which is resistant to VCR . The amoFollowing the LC-MS/MS analysis, proteins that were detected only in VCR-S or VCR-R cell lines were also extracted . Among tIn the present study, the miRNA and protein profiles within exosomes derived from four canine lymphoid tumour cell lines were comprehensively analysed by small RNA sequencing and LC-MS/MS. The novel findings of this study are summarised in In small RNA sequencing, the number of reads mapped to miRNA and the mapping rate to miRNA were comparatively lower in both exosomes and parent cells of Ema than the other three cell lines. These results suggested that the total amount and proportions of miRNAs in total RNA within exosomes or parent cells were lower in Ema than the other 3 cell lines, since the same amount of RNA of Ema was used as the other 3 cell lines. In the hierarchical clustering analysis and PCA plots for three cell lines, three distinct clusters composed of the exosomes and parent cells of each cell line were observed, and the results of these analyses were similar when the data from Ema were included. Therefore, it was indicated that the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells.Small RNA sequencing also revealed that five major miRNAs  extracted from exosomes or parent cells were similar among the four cell lines. Previous studies have reported that exosomes derived from tumour cells contain miRNAs of the let-7 family ,23. It hPIK3R3 gene [In the comparison of the amounts of miRNAs between exosomes and parent cells, significant differences were observed for 39, 20, and 24 miRNAs in CLBL-1, UL-1, and Ema, respectively, whereas there was no significant difference in GL-1. Among these miRNAs, the significant differences in the amounts of miR-350 between exosomes and parent cells were confirmed in all four cell lines by RT-qPCR, which is one of the novel findings of the present study. These results suggested that miR-350 might be a molecule selectively incorporated into exosomes in lymphoid tumour cells. The predicted target genes of miR-350 did not include those previously reported to be associated with the pathophysiology of tumour cells. However, miR-350 was reported to promote apoptosis through down-regulation of 3R3 gene –9, exosoLC-MS/MS analysis revealed that exosomes derived from each cell line contain various types of protein. Most of the proteins abundantly contained in exosomes were common among the four cell lines, including those related to cytoskeleton, such as β-actin, tubulins, and heat shock proteins. In addition, CD63 or CD81 were also detected in exosomes derived from each cell line. Exosomal markers have been reported to include members of the tetraspanin family  and heat shock proteins  ,26. It wNTRK2, a member of neurotrophic tyrosine receptor kinase family. The expression of NTRK2 was reported to be down-regulated in patients with breast cancer with a poor prognosis [MAPK8  and BCOR. The MAPK8 gene is a member of the MAP kinase and JNK family, and involved in various cellular processes including cell proliferation, differentiation, and apoptosis [BCOR gene encodes a co-repressor of BCL6, a transcriptional repressor that is required for formation of germinal centres [PIK3R1, one of the oncogenes that promotes cell proliferation and tumour cell invasion [In the comparison of the amounts of miRNA within the exosomes, the amounts of miR-151, miR-8908a-3p, and miR-486 were confirmed to be different between VCR-S cell lines and VCR-R cell lines by RT-qPCR. The amounts of miR-151 and miR-8908a-3p were significantly lower in VCR-S cell lines, while miR-486 was significantly more abundant in these cell lines. In a previous report of the comparison of exosomal miRNA profiles between drug sensitive and resistant lymphoma cells, the expression levels of miR-99a-5p and miR-125b-5p were significantly higher in chemotherapy resistant patients than in chemotherapy sensitive patients . Howeverrognosis . The exprognosis . The tarpoptosis ,32. The  centres ,34 and s centres . The tarinvasion . Based oinvasion . TherefoAmong the proteins detected by LC-MS/MS in the present study, CD82 was detected in the exosomes of VCR-S cell lines but not in those of VCR-R cell lines, and the difference in its amount was confirmed by western-blotting. In addition, CD82 was not detected in proteins extracted from parent cells of CLBL-1 and GL-1, suggesting that CD82 was selectively delivered into exosomes in these cell lines. Since expression of CD82 within exosomes has not been reported in lymphoid tumours, this is a newly identified exosomal protein that might be associated with drug resistance in these tumours. CD82 has been reported to suppress tumour metastasis  and be aIn conclusion, most of the miRNAs and proteins abundantly contained in exosomes are common among the four cell lines, but the miRNA profiles in exosomes reflect those of parent cells and differ among cell lines. In addition, miR-151, miR-8908a-3p, miR-486, and CD82 proteins were differentially abundant within the exosomes between VCR-S and VCR-R cell lines. Further investigations are needed to elucidate the biological functions of these molecules in the crosstalk between tumour cells and tumour microenvironment.2.Four canine lymphoid tumour cell lines  were used in this study: CLBL-1, a canine B-cell lymphoma cell line ; GL-1, a7 cells  and 2×107 cells (Ema) cultured for 24h in growth medium without foetal bovine serum. We used reduced number of Ema cells because they proliferate faster than other cell lines and become confluent during cultivation if 3×107 cells were used at the start of the cultivation for exosome isolation. Exosomes were isolated from cell culture media using the Total Exosome Isolation (from cell culture media) , and exosome protein and RNA were prepared using the Total Exosome RNA and Protein Isolation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The number and sizes of isolated exosomes were measured using NanoSight NS300 system . The concentrations of exosome protein samples were measured using Micro BCA Protein Assay (ThermoFisher Scientific), and the concentrations and size distributions of exosomal RNA samples were measured using Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer . Total RNA of each parent cell line was extracted using miRNeasy Mini Kit , and concentration and integrity were measured as described above. Each total RNA sample was prepared in duplicate.Exosomes were isolated from 3×10Small RNA sequencing libraries were prepared with 156 ng of total RNA using NEB Next Multiplex Small RNA Library Prep Kit . RNA sequencing was performed in duplicate using NextSeq500  with High Output Kit (Illumina) as stranded, single 36-base reads following the manufacturer’s instruction.http://www.targetscan.org/) with Bowtie2 (version 2.2.9) [https://ddbj.nig.ac.jp/DRASearch/submission?acc=DRA006696).Raw BCL data for each sample were de-multiplexed with bcl2fastq (version 2.18.0.12) and were stored in independent FASTQ files. The sequence data were trimmed with Trimommatic (version 0.36)  to cleann 2.2.9) . The depn 2.2.9) ,50. ClusT values of three independent experiments performed in triplicate. CT values were determined using the second derivative maximum method, in which a CT value is expressed as the cycle number at which the second derivative was at its maximum. After validation, target genes of the miRNAs were predicted using miRbase (http://www.mirbase.org/) [The amounts of miRNAs extracted from the small RNA sequencing data were validated by RT-qPCR using TaqMan MicroRNA Assay . The candidate miRNAs selected for validation are listed in the se.org/) ,52.Protein profiles of exosomes were analysed by LC-MS/MS. An EASY-Spray column  was employed for separation of each exosomal protein sample at the flow rate of 300 nl/min. The amount of exosomal protein used was 2 μg. A quadrupole tandem mass spectrometer  was used in positive ion mode for analytic detection. The raw MS spectra data were queried against the NCBI Canine protein sequence database using the MASCOT database search engine, and peptides were quantified according to the spectral counts.Expressions of the candidate proteins extracted from the LC-MS/MS data were verified by western blotting. One μg of protein extracted from exosome or parent cells was separated by SDS-PAGE and blotted onto a PVDF membrane. The membranes were blocked in 5% skimmed milk and incubated with primary antibodies against CD82, HSP90B, or β-actin. HSP90B was selected as the internal control of the exosomal protein. Then, the membranes were incubated with secondary antibodies. The antibodies, dilutions, and incubation temperatures are shown in the In the differential expression analysis using EdgeR, a false discovery rate  of less than 0.01 was considered statistically significant. One-way ANOVA followed by Tukey’s post-hoc test was performed for multiple comparisons of miRNA quantities in the RT-qPCR using the STATMATE  software, and P-values of less than 0.05 were considered statistically significant.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 FigSize distributions of exosomes of CLBL-1 (a), GL-1 (b), UL-1 (c), and Ema (d).(TIF)Click here for additional data file.S2 Fig“18S” indicates the peak corresponds to 18S ribosomal RNA, and “28S” indicates that corresponds to 28S ribosomal RNA.(TIF)Click here for additional data file.S3 FigExosomes and parent cells clustered similarly for each cell line and the profiles are different among cell lines. Orange dots (exosomes) and red dots (parent cells) correspond to CLBL-1, violet dots (exosomes) and blue dots (parent cells) to GL-1, grey dots (exosomes) and black dots (parent cells) to UL-1, and yellow dots (exosomes) and green dots (parent cells) to Ema.(TIF)Click here for additional data file.S4 FigThe names of miRNAs whose amounts were significantly larger in exosomes than parent cells are shown in red, and those whose amounts were significantly smaller in exosomes than parent cells are shown in blue.(TIF)Click here for additional data file.S5 FigLane M is the protein ladder. Lanes 1–4 correspond to the exosomal proteins extracted from CLBL-1, GL-1, UL-1, and Ema, respectively, and lanes 1’-4’ correspond to exosomal proteins precipitated with trichloroacetic acid extracted from CLBL-1, GL-1, UL-1, and Ema, respectively.(TIF)Click here for additional data file.S6 FigIn exosomes (a), the amounts of 11 miRNAs were significantly lower in VCR-S cell lines than VCR-R cell line, and those of 5 miRNAs were significantly higher in VCR-S cell lines than VCR-R cell line. In parent cells (b), the amounts of 8 miRNAs were significantly lower in VCR-S cell lines than VCR-R cell line, and those of 7 miRNAs were higher in VCR-S cell lines than VCR-R cell line.(TIF)Click here for additional data file.S7 FigThe figures of the same membrane were shown in (a) and (b), but exposure times were different between these figures. In (TIF)Click here for additional data file.


Four passages are described: the absence of cells in 2005 when the research started in France; the presence of supernumerary embryos available for research in Israeli IVF biobanks; the production of the I6 stem cell bank in Israel; the importation and laboratory biobanking of the cells in France. Human embryonic stem cell lines can never be completely disentangled from reproduction.The first French clinical trial using human embryonic stem cells for regenerative purposes was launched in 2014, using the I6 stem cell line that was imported from Israel. From Israel to France, national reproductive policies and practices inform how basic scientists produce, manage and circulate cells across countries. Building on an interdisciplinary co-production involving two social scientists and a life scientist, this article suggests that biobanks  Stem Cells published an article which opened the path towards the first phase one clinical trial using huES in France to treat infarcted hearts . Unlike the Israeli stem cell sector, the French scientific stem cell context is still understudied from an anthropological perspective, which explains the difference in references with the Israeli case studied by Sigrid Vertommen.Sigrid Vertommen is a historian and political scientist who conducts fieldwork-based research on the political economy of assisted reproduction in Israel/Palestine. Her contribution to the article is informed through 99 semi-structured interviews with the various stakeholders who have shaped Israel's stem cell policies and practices  and through a close reading of institutional documents by the National Bioethics Committee, the Israeli Academy of Sciences and Humanities and the Ministry of Health. This data collection largely took place in the context of her doctoral research at Ghent University in Belgium, however, additional interviews were conducted with the Israeli stem cell researcher who was involved in the production of the I6 stem cell line.Michel Pucéat is a life scientist, a developmental biologist, who has been involved in the stem cell field since the authorization to use HUES cells in France in 2005. The laboratory he runs has used the stem cell line that is the topic of study in this article. His contribution to this article is based on his involvement in stem cell research nationally and internationally. He has given access to primary data, continuously discussed them with Noémie Merleau-Ponty and Sigrid Vertommen and shared information as well as has participated to the writing of this article.et al. . She continues her critique thatgenerally, STS work has been less successful than political science in finding places for human beliefs and imagination, and in accounting for significant economic, technological and social disparities in the practices of world-making; nor has science studies paid much attention to what happens when particular epistemic and material constructions of the world circulate through societies configured by very different historical and material constraints. Jasanoff , 28These suggestions inspire our study of how the I6 line is made and circulated through global scientific practices within repronational contexts.The different steps involved in the production of the I6 line from an IVF embryo and its storage in various biobanks and countries entail several types of “exchange regimes” . For the I6 cell line in Israel, this process started right after the derivation of the line from the embryo. Then, after their transfer to France, the French scientists kept growing the cells and continued passaging them. Cells can thus prolong their life and be expanded as a stock, and they can be available for multiple usages. Cells are grown in a medium and some of them are frozen and stored for future uses, while others are experimented on by adding reagents that interact with them. Frozen vials can be shipped to different laboratories and countries through “Material Transfer Agreements” (MTA) that have different contents depending on the institutions and the various regulations implied. The notion of “passage” also exists in Science and Technology Studies.The meanings of the word “passages” in this article are multiple. In biology, a passage of cells is the process of splitting them from one dish to several dishes in order to amplify their number. This is a mandatory process for huES cells to grow and this is the meaning implied by Gottweis, Salter and Waldby . Similarly, human embryos are defined in a lose way, which allows different actors to have different interpretations. In legal and bioethical texts, Raschini (approximation catégorielle) that leave spatial and temporal frontiers of meaning unlimited or open for transformation, such as the notion of “potential human person.” Indeed, in France, huES science is regulated through a legal, bioethical and public reproductive framework that views embryos as neither complete objects of science nor complete subjects of humanity (Merleau-Ponty Human embryonic stem cells are undifferentiated cells extracted from blastocysts (day 5 or 6 embryos). The derivation and cell culture of stem cell lines is the foundation for the production of materials for bioscientific laboratories specialized in stem cell research. In 1998, the first human embryonic stem cell line was derived in the USA using an embryo from and in collaboration with Israel  However, this debate, which is old and more complex than the ‘who or what’ question, is far from being resolved and might never come to a satisfying solution for all.The Second World War and the Nazi machinery of eugenics and extermination has profoundly oriented French bioethics towards protecting nascent human lives , the cells come from reproductive clinics where patients keep frozen supernumerary embryos they can give to research. These embryos are protected by a set of regulations stemming from a tradition of bioethical discussions as old as the first French birth resulting from IVF in 1982. Indeed, the French National Consultative Bioethics Committee (CCNE) was created in 1983 by President François Mitterrand. As described through the committee's website, it aims at “elucidating scientific progress, raising new issues challenging society and observing change from the perspective of ethics.” Its first recommendation CCNE  addressees Memmi . This pres Memmi . Howeveres Memmi . Since 1Taking all these ethical and reproductive considerations on the potential personhood of human embryos into account, France first banned research on human embryos or their cells. In the late 1990s and early 2000s, French scientists could not derive cell lines from IVF embryos or import any of them from abroad, as the National Biomedical Agency would not authorize such procedures. In 2005, the ban on huES research was smoothed to support research with therapeutic goals, however, due to the previous legislation, no stem cell lines were available in the country. Line derivation is not an easy task and requires significant human and financial resources. Line derivation is a project which the French team who published the cardiac differentiation protocol in 2007 had no intention to pursue. The research was authorized because of its therapeutic goal, not because it was meant to derive cell lines. Relying on international connections, Israel was one of the few countries where line derivations had been developed during previous years. Why specifically this country? This is where the best expertise had been set. Trying to understand what enabled this expertise opens a wide range of topics that cannot be reduced to technical skills only. By focusing on two passages in Israeli biobanks, i.e. the abundant presence of supernumerary IVF embryos and their availability for research purposes as well as the production and storage of the I6 line, we unpack the particularity of Israel's reproductive-embryonic interface.home-grown Israeli stem cell lines. Benjamin Reubinoff may have been the first Israeli researcher to establish stem cell lines in 2000, but he did it abroad with Alan Trounson and his colleagues at Monash University in Melbourne, and then returned them back to Israel.I spent a few months in Wisconsin to learn about the derivation techniques, and then I came back to Israel. We used five frozen embryos, six days old blastocysts that were left over from fertility treatment, only three resulted in a successful derivation, the I3, I4 and I6 lines. These were the first Being home-grown or native-born is an important reference in Israeli society, which in colloquial Hebrew is proudly referred to as being a “Sabra.” A Sabra is a Jew who is born on Israeli territory rather than in diasporic exile. The term, referring to the cactus fruit that is known to be tough and prickly on the outside and sweet on the inside, first appeared in the 1930s and was used by the Zionist movement to celebrate the “New Jew” that emerged in Mandatory Palestine. Unlike the diasporic “Old Jew” who was often depicted as weak and powerless in Zionist discourse, the “New Jew” was portrayed as a fighter, a kibbutz member or farmer who showed strong, healthy and productivist physical qualities :es Almog .The I6 “Sabra” stem cell line constitutes an interesting entry point to understand the particularity of Israel's reproductive-embryonic sector, which for over two decades has been internationally acclaimed for its scientific excellence and commercial success. Since the field of stem cell research was established in 1998, Israeli scientists such as Benyamin Reubinoff, Joseph Itskovitz-Eldor, Michal Amit, Karl Skorecki, Dalit Ben-Yosef and Nissim Benvenisty have been at its “frontier” Vogel . In 2002maya b’alma or mere water :In the Jewish ethos life precedes every law. There is no law that is stronger than life itself. This is an important ethos. […] Here in Israel life supersedes everything else. People would go a long way in order to preserve life, to give life, to help cure. So life has a different meaning here.Rather than being antagonistic forces, Israel's religious and scientific communities have thus forged a remarkable alliance in its policy views and recommendations on embryonic research The coordinator of the Women and Medical Technologies Program of the Israeli feminist organisation Isha L’Isha, added :There is kind of a joke among researchers. They say that most of the eggs and embryos that are circulating in global stem cell research around the world are coming from IVF clinics in Haifa.Four of the five original cell lines that were developed in 1998 by Thomson and his research team were created out of spare IVF embryos from the fertility clinic in Rambam Medical Center in Haifa, headed by Joseph Itskovitz-Eldor colonial perspective.This “pioneering” ethos is also present in Israeli discourses and practices of human embryonic stem cell research, where the belief in medical and scientific progress tends to overhaul fears of bioethical and societal dystopias. For instance, when asking one Israeli stem cell researcher in what kind of legal climate the early stem cell discoveries took place in Israel, she replied :In those days it was the Technion that approved or disapproved the movement of the I6 line. The material transfer agreements at the Technion are not very restrictive, compared to other research institutions. This is because we want the knowledge and materials to be used by other institutions, so that they can further develop.Unlike in many other countries where the export of stem cell lines is decided on a national level, in Israel this decision is made by the individual research institute without public discussion or the involvement of national committees on medical ethics. As Itskovitz and Amit were hopeful and determined to quickly develop clinical applications in the field of cell repair, they were also keen to export Israeli stem cells for biomedical research by cooperating with stem cell laboratories in other countries :es Gross , 582. Ines Gross , 244. InLast time, few months ago when I had to arrange an MTA for the cells, the price was 1300 dollars plus shipping costs, which is really not that much if you come to think of all the tender love and care and money that we have put in deriving these cells.The stem cell sector constitutes a key sector in Israel's bio-economy that since the late seventies has been positioning itself as a major player in the global market of biomedical research and development, with a special focus on healthcare  remarked:To understand the production of the I6 line one needs to understand the particularity of Israel's repronational IVF-stem cell interface. Similarly, the reception of these cells in France is not only about importing a global material, but also nationalizing it.What happens when French biologists ask for an authorization to import huES from abroad? They have to fill in a lot of documentation for the National Biomedical Agency to verify the legitimacy of the demand. The National Biomedical Agency asks whether the lines are registered at the UK Stem Cell Bank or the National Institutes of Health in the USA (NIH). These two institutions play a powerful and active role in standardizing practices, through which a bioscientific community works for the promotion of a new field of research . Furthermore, article 16 and 16-8 of the Civil Code must be respected, stating the “primacy of the person,” forbidding any “violation of its dignity” and “guaranteeing respect for the human being from the beginning of its life.” Regarding The clauses on the “primacy of the person,” its “dignity” or the “beginning of life,” however, are more difficult to verify. These notions relate to a specific French framework that we addressed at the beginning of this article. The logic behind the notion of “potential human person” sustained an open but protective view on human embryos. However, this notion depends on the national context, for example, if we think of the United Kingdom where research is authorized until 14 days of development on what is called the “pre-embryo” Franklin  as well Interestingly, the authorization to import a huES line, given by the National Biomedical Agency, based on the above law articles, is neither the recognition of other bioethical frames nor a complete recognition of a standardized global material for research. The authorization is a process of nationalization that reinvents the cells as French by framing their importation in specific words. The Civil Code's ontological vocabulary has no reference to a sociological reality outside of France. These words can be seen as drivers for new meanings. When cells enter the French territory, they are inscribed in a set of institutional practices that give them new meaning relating to French history, bioethics and legislation. This ontological production is not only discursive but has very practical consequences.The Israel number 6 cell line was nationalized by regulating laboratory biobank management. Even if the process of derivation has completely transformed cells which are no longer embryos, even if the cells come from a different country with a different history, economy and religion, these cells gain a status of specialness as soon as they enter French territory. An inspector of the National Biomedical Agency commented that their traceability must be “total.” Penal rules are very clear, and anyone who breaks these rules risks a fine of up to 30 000 euros and two years of imprisonment . “It is a very delicate context relating to the Human and human reproduction,” commented the inspector during an interview with Merleau-Ponty (28th of July 2015). This sentence tightly binds stem cells to their IVF past. It also stresses the delicate balance between research goals and French representations on human embryos.Cells’ traceability can be established in several ways. For example, experiments using huES must be written in special laboratory notebooks, while all other experiments with different materials, human and non-human, can be written in the same notebook. Storage procedures must be clearly identified, and laboratories are required to provide maps of their facilities and clear identification of where the cells are stored. Whilst special incubators for cell culture are not mandatory, the basic science laboratory which cultured the I6 did so in a specific one, as the laboratory space dedicated to cell culture was big enough. Similarly, the huES bank, the physical tank, was in a special room away from all the other cells, both human and animal. Moreover, when the inspector narrated how he inspects a laboratory, he stressed that he does not look at the other cells’ management  but concentrates all his attention on the huES lines. Once within French territory these cells are connected to a new ontological apparatus which inscribes them in a context that has nothing to do with personal biographies or Israeli layers, but which relates to French bioethics.When the press release referred to the French clinical trial as having used “human embryonic stem cells,” it certainly enacted a “disentangled” version of stem cell science from reproductive dynamics Glasner . HoweverFurthermore, looking at which cells were used also opens up a history of travel across two national frameworks, the French and the Israeli one. Following transnational connections of biobanks from France to Israel and back, we have delineated how the cells are co-produced within different repronational contexts while circulating across the world. If the derivation of a human embryonic stem cell line implies a biological transformation of the human embryo, as well as the use of these cells in protocols which have little to do with reproduction, this article has shown that the reproductive framings of these cells inform their regenerative future. Moreover, the global circulation of these cells is not a disappearance of local contexts which are, on the contrary very much involved in the transnational logics.The French basic science laboratory that started its research in 2005 relied on international collaborations as French bioethics and regulations had prevented the production of stem cell lines based on a specific reproductive understanding of embryos as “potential human persons.” The cells used in the scientific protocols came from Israel. The embryo that was given to produce the stem cell line was transformed into a biological material named “Israel number 6” in a context where numerous supernumerary embryos were available to donation and stem cell science associated with bioethical leniency and academic entrepreneurialism. The international character of the I6 stem cell line is an entanglement of national contexts (the French and the Israeli one) in which reproduction plays a central role, albeit in different ways. The production and circulation of the line from one country to the other materializes through the usage of several types of conservation sites, which accompany the passaging of the cells.Id., 18). When biologists tick boxes on the French importation permits, they connect cells to French reproductive regulations that have little to do with the Israeli IVF-stem cell interface that co-produced the cells. When the inspector of the National Biomedical Agency explains how he visits stem cells labs and verifies notebooks and biobank management, he justifies his practice by referring to “the Human and human reproduction,” reproducing a French nationalized version of reproduction in the stem cell lab. Each cell passage within biology practices is the reproduction of sameness and difference, as the cells divide into two versions of themselves and microDNA changes might occur. Passaging cells across sites, institutions and countries is about circulating a common material for research (the I6 line) while modifying its understanding in two different repronational contexts and a global market.The meaning of passage refers not only to the ways in which scientists build networks and solve problems Callon , but alset al. et al. This focus on long-term genealogy contributes to the ongoing critique of the bench to bedside view of bioscientific translation (Wainwright


Escherichia coli (E. coli) from the B2 phylogenetic group is implicated in colorectal cancer (CRC) as it possesses a genomic island, termed polyketide synthetase (pks), which codes for the synthesis of colibactin, a genotoxin that induces DNA damage, cell cycle arrest, mutations and chromosomal instability in eukaryotic cells. The aim of this study was to detect and compare the prevalence of E. coli expressing pks (pks+E. coli) in CRC patients and healthy controls followed by investigating the virulence triggered by pks+E. coli using an in-vitro model. Mucosal colon tissues were collected and processed to determine the presence of pks+E. coli. Thereafter, primary colon epithelial (PCE) and colorectal carcinoma (HCT116) cell lines were used to detect cytopathic response to the isolated pks+E. coli strains. Our results showed 16.7% and 4.3% of CRC and healthy controls, respectively were pks+E. coli. Further, PCE displayed syncytia and cell swelling and HCT116 cells, megalocytosis, in response to treatment with the isolated pks+E. coli strains. In conclusion, pks+E. coli was more often isolated from tissue of CRC patients compared to healthy individuals, and our in-vitro assays suggest these isolated strains may be involved in the initiation and development of CRC. Globally, colorectal cancer (CRC) is the second most common type of cancer in females and third most common type of cancer in males with 694,000 fatal cases recorded in 2012. Similarly in Malaysia, the National Cancer Patient Registry reported CRC as the second most common cancer in both males and females with a total of 4,501 cases diagnosed and reported from 2008–2013 , 10Bacteic group . This gein (clb) , 12], , , . I. Ipks+Entestine . Moreovehe colon .pks+E. coli CRC patients had their tumors on the distal part of the colon. Similar outcome was reported in which pks+E. coli was found to more often colonize the distal colon as compared to the proximal colon . T. Tin-vitytoplasm . These cytoplasm . This prpks+E. coli might represent the capacity of the specific genotoxin to initiate the process of tumorigenesis. Studies have demonstrated that signs of tumorigenesis such as genomic instability, chromosome aberrations, increased mutation frequency and hallmarks of cellular senescence were observed by the surviving cells upon exposure to pks+E. coli , [, [pks+EA damage , 25. Thepks+E. coli might also be responsible for the exacerbation of tumorigenesis, rather than just the initiation process. Previous study observed that direct infection of fibroblast cells with pks+E. coli promoted cancer cell proliferation and expansion [pks+E. coli and CRC, a study using an in vivo model was performed by Arthur et al. whereby they mono-associated mice with pks+E. coli with AOM (azoxymethane—a known carcinogen) treatment [pks+E. coli might be involved in exacerbating tumorigenesis. Although this results does not completely confirm the association of pks+E. coli and the initiation and/or exacerbation of tumorigenesis, we could only state that the CPE produced is merely a preliminary result that requires further study and the different type of CPE obtained may be due to activation of different metabolite and/or pathway that ultimately lead to either initiation of tumorigenesis or exacerbation of tumorigenesis.Meanwhile, for HCT 116 cell line, the CPE observed may demonstrate that the genotoxin from xpansion . In ordereatment . Combinepks+E. coli, there have been no study thus far that confirmed the presence of specific target site aimed by pks+E. coli. However, it has been documented by previous study that the process of pathogenesis due to pks+E. coli may occur as follows. Initially, there must be direct contact between the live bacteria with the relevant cells to induce the toxicity of pks+E. coli [pks toxin, pks+E. coli, has the ability to adhere and invade the colonic epithelial cell through a receptor known as carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) [pks+E. coli will later release its toxin to the surrounding environment leading to the changes in the morphology of cells that can be observed as CPE that will cause premature cellular senescence and tumorigenesis [pks+E. coli. This abnormal overproduction of growth factors will bind to the receptor of the signalling pathways that will subsequently initiate the cascade effect of the pathway and lead to tumorigenesis [Although there are many research conducted with regards to E. coli . This pkCEACAM6) . pks+E.igenesis . Cells tigenesis , 26.pks+E. coli in distal colon as compared to proximal colon, this may be due to the difference in the embryologic origins as distal colon derives from hindgut while proximal colon derives from midgut [As for the presence of more m midgut . This dipks+E. coli is more commonly isolated from the tumor and normal colon mucosa in Malaysian CRC patients being treated at UMMC as compared to the healthy controls. Additionally, all the pks+E. coli isolates from Malaysian CRC patients showed CPE in two distinct cell lines in-vitro, suggesting that these bacteria might play a role in the initiation as well as promotion of carcinogenesis. These findings may perhaps represent only one additional factor potentially contributing to colon carcinogenesis in Malaysia. There were limitations in this study such as the insufficient number of samples that led to inadequate comparison between CRC patients and normal healthy patients. Thus, in addition to assessing specific bacteria and the microbiome, future Malaysian studies should focus on the role of dietary intake and biological factors in a more balanced ratio of CRC patients and normal healthy patients together with an in-vivo animal model to further assess the factors contributing to CRC and the colon carcinogenicity of Malaysian strains of pks+E. coli.In conclusion, we demonstrated that S1 AppendixQuestionnaire; (A) In Bahasa Malaysia (B) In English.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


SOCS1 and SOCS3 have been associated with tumour progression and response to treatments in different kinds of cancers, including GBM. In this study, cell lines of IDH-wildtype GBM from primary cultures were obtained, and the role of SOCS1 and SOCS3 in the radiotherapy response was analysed. Fifty-two brain aspirates from GBM patients were processed, and six new cell lines of IDH-wildtype GBM were established. These new cell lines were characterized according to the WHO classification of CNS tumours. SOCS1 and SOCS3 expression levels were determined, at mRNA level by Q-PCR, at protein level by immunocytochemistry, and Western blot analysis. The results showed that SOCS1 and SOCS3 are overexpressed in GBM, as compared to a non-tumoral brain RNA pool. SOCS1 and SOCS3 expression were reduced by siRNA, and it was found that SOCS3 inhibition increases radioresistance in GBM cell lines, suggesting a key role of SOCS3 in radioresistant acquisition. In addition, radioresistant clonal populations obtained by selective pressure on these cell cultures also showed a significant decrease in SOCS3 expression, while SOCS1 remained unchanged. Furthermore, the induction of SOCS3 expression, under a heterologous promoter, in a radiotherapy resistant GBM cell line increased its radiosensitivity, supporting an important implication of SOCS3 in radiotherapy resistance acquisition. Finally, the treatment with TSA in the most radioresistant established cell line produced an increase in the effect of radiotherapy, that correlated with an increase in the expression of SOCS3. These effects of TSA disappeared if the increase in the expression of SOCS3 prevented with an siRNA against SOCS3. Thus, SOCS3 signal transduction pathway (JAK/STAT) could be useful to unmask new putative targets to improve radiotherapy response in GBM.Glioblastoma multiforme (GBM) is a poor prognosis type of tumour due to its resistance to chemo and radiotherapy.  Glioblastoma multiforme (GBM) is the most common malignant tumour of the central nervous system (CNS) in the adult population, its incidence being around 2–3 people per 100.000 in USA and Europe. It is considered a very aggressive and lethal tumour, because there is not an effective therapy to date, thereby, being an incurable type of cancer [de novo”, and low-grade gliomas that become GBM, denominated secondary GBM. Both are histologically identical, but primary GBM has worse prognostic than secondary GBM [GBM is usually divided in two groups, depending on their origin: primary GBM’s which are developed “dary GBM ,4.IDH-wildtype GBM and IDH-mutant GBM; IDH-wildtype GBM has a worse prognosis, with a survival rate around 10 to 15 months, it often has mutations in the TERT promoter (76%), and sometimes harbours alterations in TP53 (27%) and PTEN (24%). IDH-mutant GBM has a better prognosis and the survival rate is increased to 31 months. In this type of tumours, the mutations of TERT promoter are rare (26%) and TP53 and PTEN alterations are also present in a low percentage [Recently, the World Human Organization (WHO) has updated the classification of CNS tumours, and has included molecular marks, which enable to identify the gliomas, and also distinguish between primary and secondary GBM. This classification has established The current therapy for GBM includes resection surgery, followed by radio and chemotherapy, frequently given together to obtain a synergistic effect. The radiotherapy treatment consists of five consecutive sessions of 2Gy at a 6Gy/min dose rate per week for six weeks, the total treatment being 60Gy . AdditioMGMT (0–6 methylguanine-DNA Methyltransferase) gene expression. MGTM is a protein involved in the DNA repair system, which is able to avoid DNA damage caused by TMZ or BCNU [Despite of these aggressive treatments, the survival rate is only increased in a few months, because GBM has different ways to acquire resistance to chemo and radiotherapy, either activating DNA repair system or producing alterations in the cell cycle and apoptosis regulation ,10. Resi or BCNU ,12. HoweSOCS3 has been proposed as a malignant prognostic biomarker [SOCS1 and SOCS3 in GBM has been studied as putative factors involved in radiotherapy resistance [SOCS1 and SOCS3 proteins are members of the Suppressors of Cytokine Signalling (SOCS) family. Both are implicated in the signal regulation of JAK/STAT pathway, which is involved in cell proliferation and apoptosis . Commonliomarker , and thesistance . Likewissistance ,20.SOCS1 and SOCS3 expression [On the other hand, histone deacetylases inhibitors (iHDACs) have been proposed as new anti-cancer agents , due to pression .IDH-wildtype GBM, established from primary cultures of GBM patients, have been obtained and characterized according to the current WHO classification. In addition, they have been tested for their chemo and radiotherapy sensitivity. The results showed that SOCS1 and SOCS3 are overexpressed in primary GBM, and also demonstrate a relationship between SOCS3 expression and radiotherapy resistance acquisition, suggesting the utility of SOCS3 and its signal transduction pathway as a new source of therapeutic targets.In this study, six human cell lines of GBM primary cell cultures were obtained from brain aspirates of patients who have been diagnosed with GBM. These brain aspirates came from resection surgery of patients older than 18 years old, which had signed the informed consent. The procedures for obtaining the tissue samples were developed in accordance with the national ethical and legal standards, and following the guidelines established in the Declaration of Helsinki (2000). The research project was conducted under the written approval of the Ethic Committee of Clinical Research (CEIC) of the “Hospital General Universitario de Elche (Spain)”, and in collaboration with the Hospital Biobank, which are included in the Valencian Network of Biobanks.2). Maintenance of cell cultures consisted in regular passes, and media replacement until they reach a growth rate similar to cell lines.The brain aspirates were centrifuged at 1.000 rpm, 10 min. The pellets obtained were resuspended in 3 volumes of Erythrocyte Lysis buffer (Qiagen), and incubated for 30 min, at 4°C. Then, they were again centrifuged in the same conditions, and the final pellets were resuspended in 10 ml of Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (Gibco) containing 10% Fetal Bovine Serum, qualified, heat inactivated (Gibco), and 1% of streptomycin and penicillium mixture (Biowest). This media was denominated completed DMEM F12. These cellular suspensions were sieved using the Sieve tissue grinder kit (Sigma Aldrich). The sieved solution was centrifuged at 1.000 rpm, 5 min. Then, it was performed an enzymatic digestion with hyaluronidase (4.5 U/ml), and collagenase II (0.09 mg/ml) for 30 min, at 37°C. Finally, cellular suspensions were centrifuged at 1.000 rpm, 5 min and resuspended with complete DMEM F12 media. The cell suspension was placed in a T25 flask and kept into an incubator with a controlled atmosphere  (Gibco) during 10 min, and they were permeabilized using 0.2% TRITON X-100 in DPBS for 10 min. In order to avoid unspecific unions blocking was carried out with a solution of 1% Bovine Serum Albumin in DPBS for 1 hour. At that point, cells were incubated with antibodies: anti-GFAP , anti-SOCS1 , or anti-SOCS3  into a humidify chamber overnight, at 4°C. Primary antibodies were used to a 1/250 dilution. Next day, cells were incubated with a secondary antibody (Molecular Probes) used to a 1/1.000 dilution, and with DAPI for 2 hours, at room temperature. In parallel, the same protocol was performed for negative controls, but in this case, these cells were incubated only with the secondary antibody, without primary antibody. The immunostained cells were visualized by a Leica TCS SP2 confocal microscope (Leica Microsystems).Cell lines were fixed using neutral formalin and included into paraffin to build TMA (Tissue Microarrays) sections. Then, sections were deparaffined, rehydrated and incubated with primary antibodies anti-P53  or anti-PTEN . Subsequently, the immunocytochemistry assay was performed with streptavidin-biotin technique, using the Immunostainer Techmate 500 (Dako), and the results were visualized by Envision System (DakoCytomation).DNA isolation was performed from frozen dry pellets of each cell line, using the DNAeasy Blood & Tissue kit (Qiagen). To carry out the ARN isolation, cells were stored in RNAeasy Lysis Buffer (Qiagen) plus 1% β-Mercaptoethanol (Sigma Aldrich), then the kit RNAeasy Plus (Qiagen) was used to isolate RNA, according to the manufacturer’s instructions.Protein extracts were obtained and Western blot analysis was performed following protocols previously describe . For thiIDH1, IDH2 and TERT genes were detected by PCR, following the protocol previously described [The single nucleotide polymorphism of escribed ,28. In aMGMT promotor in the cell lines was performed using SALSA MS-MLPA ME011.B1 Mismatch Repair Genes kit (MRC-Holland) according to the protocol provided by the manufacturer. The quantity of DNA used was 200 ng, the PCR products were resolved in ABI-PRISM 3500 Genetic analyser (Applied Biosystem). The data from the electrophoretic separation were analysed using GeneScan Software (Applied Biosystems), and Microsoft Excel spreadsheet templates provided by the manufacture. To determine the methylation status, the criteria proposed by Jeuken et al [The methylation status of en et al  have beeRetrotranscription of RNA to cDNA was made with 1 μg of RNA, using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific), according to the protocol provided by manufacturer.MGMT (Hs01037698_M1), SOCS1 (Hs00705164_s1) or SOCS3 (Hs02330328_s1), 10 μl TaqMan Gene Expression Master Mix  and 4 μl of cDNA obtained by retrotranscription. Endogenous control used was GAPDH-VIC (ThermoFisher Scientific). As a reference sample, an RNA pool from normal brain  tissue from 5 human adults was used. (BioChain). For all studies, 3 technical replicates were performed, and negative controls were carried out with water. Q-PCR assays were performed in the 7.300 Real Time PCR System (Applied Biosystems).Q-PCR was carried out in 96 wells plates, with a master mix containing 1μl of specific gene assay: The drug used for chemotherapy was BCNU. Cells were plated, treated with 50μM BCNU for 24 hours and harvested for cellular viabilities studies.Radiotherapy was performed in the Radiotherapy Oncology Unit of the “Hospital General Universitario de Elche”, using a VARIANT 2100C linear accelerator. The dose was 7Gy, with a dose rate of 6Gy/min.For both assays, cells were plated in 6 well plates and kept into an incubator with controlled atmosphere, for 24 hours. The radio or chemotherapy treatment was supplied, and cells were incubated again in the same conditions for an additional period of 24 hours.To assess the drug effect, viability studies were performed using the Muse Count & Viability Assay Kit; radiation effect was determined by Muse Cell Cycle Assay Kit (MERK). The results were visualized by a MUSE Cell Analyzer (MERK).The GB39 and G242 cell lines were plated in 100 mm cell culture plates. Weekly, they were irradiated with a 5Gy dose until reach an accumulated dose of 45Gy. Only few isolated cells survived to this process. Each isolated cell was recovered using the Cloning cylinders glass (Sigma Aldrich) and plated in a 96 wells plate. When these cells started to proliferate, they were transferred to a 24 wells plate, and then to a 6 wells plates. Finally, clonal radioresistant populations were obtained from the isolated cells.SOCS1 and SOCS3 expression inhibition was carried out by transfection with specific small interfering RNA (siRNA). The transfection was made with specific siRNA of SOCS1  and SOCS3  to a 10 μM concentration, mixed with 5 μl of Lipofectamine RNAiMAX (Invitrogen) diluted in OPTI-MEN (Gibco). As a negative control, the same solution was used with non-specific siRNA . Transfected cells lines were maintained in this solution plus complete DMEM F12 media without antibiotic for 48 hours at 37°C in a humified 5% CO2/air atmosphere.SOCS3 expression, we used a synthetic plasmid which had the SOCS3 Open Reading Frame (ORF) and a resistance cassette of Hygromycin under a heterologous promoter provided by Sinobiological (HG11315-UT). Transfection with a control plasmid was also performed to assess whether the transfection has an intrinsic effect. Cells were transfected with 15 ng or 25 ng of the plasmid using Lipofectamine 2.000 (Invitrogen) diluted in OPTI-MEN (Gibco) and grown in culture media plus Hygromycin to select only the transfected cells. Then, the culture was divided into 3 aliquots, one for the SOCS3 expression studies, and the other two for cell cycle distribution studies one of them was irradiated, while the other was not.In order to upregulated SOCS3, the cells were pre-treated for 16 h with different doses of TSA (25–200 nM) before proceeding to irradiation.To induce the increase in the expression of the endogenous levels of SOCS1 and SOCS3 expression and cell cycle distribution data. Normal distribution of the data was tested by Shapiro-Wilk test and homoscedasticity was tested by Levene Test. Data from cell cycle distribution and percentages of SubG1/G2M showed a non-normal distribution. Data from expression studies showed a normal distribution, and homogeneity of variances. Parametric and non-parametric comparation test were used as required.Mean and SD values were calculated for To paired comparisons between irradiated and non-irradiated phases of cell cycle, and between the percentages of SubG1/G2M of irradiated and non-irradiated cells Mann Whitney test was performed.In the expression analysis, the followed criteria to consider alterations of expression were the proposed by Schmittgen & colls . They esP-Values <0.05 were considered to be statistically significant. The software used to carry out the statistical analysis was SPSS 24.0 (IBM Software).Fifty-two brain aspirates were collected during this study. All of them were diagnosed as GBM by the Anatomy Pathology Unit. The brain aspirates were processed, and primary cultures were stablished from them. RNA, DNA and cryopreserved cells were obtained systematically from different passages. Finally, six of these primary cultures which had a quickly and stable growth rate and kept their molecular features, were selected for this study. The behaviour of these six primary cultures was similar to those of a cell line. These new cell lines were named HGUE -GB (Glioblastoma)-number of culture, for example: HGUE-GB-16. However, for this article they have been named GB+number of culture; for example, GB16, to facilitate easy understanding of Figures and Figures legends.Fig 1), and it was confirmed that all established cell lines had preserved their glial origin.To confirm whether these six new cell lines had preserved their glial origin, immunocytochemistry assays were performed to detect the expression of glial fibrillar acidic protein (GFAP), since this protein is considered a glial marker, and particularly of astrocytes . ImmunolIDH1, IDH2 and TERT were determined by Sanger sequencing, and the electropherograms obtained were studied to detect the described mutations: in IDH1 R132, in IDH2 R172, and the C225T or C250T TERT promoter region [IDH1 or IDH2 were found in the six GBM cell lines . However, all of them harbour a C250T mutation in the TERT promoter . The GB16 cell line showed this mutation in heterozygosis.These new GBM cell lines were classified according to the guidelines proposed by WHO in their 2016 CNS tumours classification , the “Hor region ,32. No mFig 2D, 2I, 2N, 2S, 2Y and 2AD). Nevertheless, the cell lines showed positive immunoreactivity to PTEN antigen , indicating that PTEN expression was conserved.P53 modifications were also analysed by immunocytochemistry, and all established GBM cell lines had alterations in this protein, showed by P53 staining in their nuclei . Furthermore, the expression of MGMT was studied by Q-PCR to associate the methylation status with its expression level. As expected, only the GB16 cell line showed expression of MGMT mRNA by Q-PCR, taking into consideration the MGMT promoter methylation status. GB16 also exhibited overexpression of MGMT mRNA, compared to RNA pool from non-tumoral brain tissue (Fold Change (FC) = 1.7±0.15).The methylation status of the r in GBM , so bothS2 Fig).Finally, we characterized the response of these GBM cell lines to BCNU, a drug used in chemotherapy. The most chemosensitive cell line was GB42, as BCNU induced a 49.9% ± 3.7 cell death, followed by the cell lines GB39, GB40 and GB48, which were slightly affected, their viability being reduced by 25.2% ± 1.8, 19.5% ± 1.1 and 27.9% ± 2.1, respectively. The higher rate of survival was achieved by GB16, as only the 12.6% ± 2.3 of its cells were affected by BCNU, so it was considered a GBM resistant cell line to chemotherapy , suggesting that the first effect of radiotherapy is to arrest cell cycle in the G2/M phase of the cell cycle, in GBM cell lines. To determine the fate of cells blocked in phase G2/M, viability studies were performed 72h after irradiation. The results showed an increase in the percentage of dead cells similar to the percentage of cells blocked in phase G2/M, 24h after irradiation, suggesting that the initially blocked cells end up dying later on .To evaluate the radiotherapy effect, the cell lines were irradiated to a 7Gy dose (6Gy/min rate). Then, 24 hours after treatment, the cell cycle distribution of different cells (control and irradiated) were compared to determine the response to radiotherapy. Significative changes in the G2/M phase, due to irradiation, plus the increase of cells in SubG1, since this phase is associated to cell death (Δ (SubG1 + G2/M) IR-CTR). Any cell line reached a radiotherapy response higher than 50%, the most affected being the GB42 cell line (49.8% ± 1.2 affected cells), and the most radioresistant being the GB39 cell line (21.2% ± 1.6 affected cells). The rest of cell lines showed middle percentages of response.From the data showed above, the effect of radiotherapy was calculated as the increase of arrested cells in the G1 + G2/M) IR-CTR) for each cell line was similar to the percentages indicated above for the same cell line growing with 10% fetal bovine serum .The radiosensitivity of cell lines was also tested after grew up the cells in serum-free media, and the value of  was isolated. Then, a retrotranscription and Q-PCR assay were carried out. FC values were calculated to evaluate the expression differences between GBM and non-tumoral brain tissue.To study the expression profile of SOCS1 and SOCS3, when compared to an RNA pool from non-tumoral brain tissue. The SOCS1 expression was between 2 and 5 times higher, and the SOCS3 expression increased in GBM cell lines between 3 and 4.5 times .The GBM cell lines overexpressed S5B Fig).Furthermore, a Western blott analysis was carried out to confirm the overexpression of SOCS3 in the GBM cell lines. In this case, total protein extracts from the cell lines were obtained, and then a Western blotting was performed using antibodies to SOCS3 and GAPDH (as endogenous control). All cell lines overexpressed SOCS3 when compared to a protein pool from non-tumoral brain tissue .To confirm the expression data of SOCS1 and SOCS3 mRNA expression, GBM cell lines were exposed to 7Gy, and 24 hours after irradiation cells were harvested, and total RNA was isolated. In parallel, control non-irradiated cells were collected to be used as an expression control. No significant changes were observed in SOCS1 and SOCS3 mRNA levels after irradiation .In order to study the effect of radiotherapy on SOCS1 and SOCS3 mRNA expression was significative affected after exposure to radiotherapy, time-course and dose-response experiments were performed. In this case, the study was carried out in the most radioresistant cell line (GB39), and in the most radiosensitive one (GB42). Non-treated cell lines were used as expression control, and RQ values were calculated from them. Changes in SOCS1 and SOCS3 mRNA expression were not detected in GB39 nor GB42 at different times . Moreover, changes on SOCS1 and SOCS3 expression were undetected in dose-response experiments , with the only exception of the GB39 cell line, where its expression was reduced by half, in comparison with the control (RQ = 0.5 ±0.03) at the highest radiotherapy dose.To determine whether SOCS1 and/or SOCS3 play a role in GBM radioresistance, SOCS1 and SOCS3 expression was inhibited using siRNA. First, we studied that transfection with non-specific siRNA (siRNA.NS) did not altered SOCS1 and SOC3 expression, nor cell cycle distribution. The results did not show significative differences between control and siRNA.NS transfected cells in any cases .To determine whether SOCS1 and SOCS3 was significative reduced. Cell cycle distribution was also studied in each sample  in order to evaluate the effect of radiotherapy .Subsequently, parallel assays of expression pre-irradiation and cell cycle post-irradiation were carried out. In this case, we compared the mRNA expression between siRNA.NS transfected cells and siRNA.SOCS1 or siRNA.SOCS3 transfected cells, which demonstrated that expression of SOCS1 was reduced by 70% in siRNA.SOCS1 transfected cells when compared to siRNA.NS . However, the effect of irradiation was the same on siRNA.NS and siRNA.SOCS1 cells . Therefore, the radiotherapy response was not affected by SOCS1 expression reduction.The mRNA expression of SOCS3 was reduced by 73% in siRNA.SOCS3 transfected cells . Furthermore, the irradiation caused a significative differential effect in siRNA.NS and siRNA.SOCS3 transfected cells . The siRNA.SOCS3 transfected cells were more radioresistant than the cells transfected with siRNA.NS, since their percentage of cells in the SubG1 and G2/M phases post-irradiation was smaller than the number of cells in these phases in the siRNA.NS transfected cells . These data reveal that SOCS3 expression inhibition provides resistance to radiotherapy in GBM cell lines.The expression of Clonal radioresistant populations were obtained from the most radioresistant cell line (GB39), and from the most radiosensitive cell line (GB42). After an accumulated 45Gy (5Gy/week) dose treatment, 10 clonal survival populations were isolated from GB39, and 6 from GB42. Finally, 6 clonal survival populations from GB39, and 1 from GB42 were obtained.Fig 7A and 7D). The expression of SOCS1 and SOCS3 at mRNA level was determined in all of them. While the expression of SOCS1 was unmodified , the expression of SOCS3 was significantly lower in all clonal radioresistant populations in contrast to their parental cell lines . These results reinforce the data obtained by siRNA.As expected, cell cycle analysis showed that the isolated clonal populations were more radioresistant than their parental cell line . In order to achieve the upregulating of SOCS3 mRNA expression was evaluated by Q-PCR, and the data obtained confirmed that cells transfected with 15ng and 25 ng of SOCS3 plasmid increased the expression of SOCS3, when compared to cells transfected with control plasmid. The cells transfected with 25 ng of plasmid showed four times increase in SOCS3 mRNA level, and the cells transfected with 15 ng showed three times increase , which suggest that the amount of plasmid correlates with the increase of gene expression.The increase of SOCS3 mRNA expression showed a greater response to irradiation, compared to control cells. The percentage of cells in SubG1 and G2/M phases was twice as much in cells transfected with SOCS3 plasmid than in cells transfected with control plasmid , and the radiosensitive of GB39 cell line also increased in the same way .Finally, we wondered whether increasing endogenous SOCS3 expression, a siRNA experiment blocking SOCS3 expression was performed, with or without TSA pre-treatment, keeping on this way the SOCS3 mRNA levels downregulated. In these conditions, the radiosensitization effects of TSA disappeared .To demonstrate that the TSA-induced increase in radiosensitivity showed in the radioresistant cell line was due to the increase in SOCS3 and the percentages of SubG1+G2/M remained unchanged for all TSA concentrations tested .The effect of TSA was also tested in the most radiosensitivity cell line (GB42) following the same conditions used for GB39. In this case, the endogenous levels of The progression of GBM is fast and difficult to control, due to its chemo and radioresistance. Hence, it is very important to obtain accurate models to study this type of cancer. For that purpose, there are many cell lines which mimic GBM, such as U87MG or LN229 (ATCC collection), but these cell lines are far away from the patient state and they usually have been modified to achieve an immortalized status. The six new human GBM cell lines established in this study resemble more the patient state, because not only they were originated from them but also, they preserve their glial nature and molecular features of the initial tumour and they lack any artificially induced modification. Other advantage of using these cell lines from primary cultures is the possibility to recreate their progression and go back and forth in their development to study gene expression or physiological properties, as DNA, RNA and cryopreserved cells have been stored at different passages.IDH-wildtype GBM, which is the most aggressive GBM group, according to 2016 WHO SNC classification. Moreover, they harbour TERT mutations in their promoters, and alterations in P53, two characteristics that support the fact that they are indeed primary GBM [IDH-wildtype GBM well characterized according to WHO are these cell lines, so they can be used as a proper model of primary GBM to study the malignant features of these tumours.The molecular studies of these cell lines have confirmed that they were included in the MGMT promoter methylated, and unlike the rest of cell lines, it expresses this gene at the mRNA level. Furthermore, we demonstrated the relationship between MGMT promoter methylation status and chemoresistance, which has been postulated in many previous studies [In relation to chemotherapy, all these cell lines showed a middle response to BCNU treatment, reinforcing their classification as primary GBM. The most resistant cell line affected only by 12.6% ± 2.3, was GB16. Epigenetic results indicated that this cell line has the  studies . However studies ,35.1 and S phases, and increasing the percentage of cells in G2/M phase. Thus, the statistically significant increase of arrested cells in the G2/M phase was stablished as a hallmark to determine the radiotherapy effect on these cell lines. According to this value, the GBM cell lines showed a low radiotherapy response, and never reached more than 50%, indicating again that they belong to the worst prognostic group.Moreover, radiotherapy affected the distribution of the cells in the different phases of the cell cycle, reducing the cellular percentage in the GSOCS genes have been associated with carcinogenesis and tumour progression [SOCS1 and SOCS3 have been postulated as candidates to regulate the radio and chemoresistance acquisition in other types of cancer, such as prostate cancer [SOCS1 and SOCS3 in radiotherapy resistance in GBM. These genes inhibit the cytokine response in many cell types, including CNS, and are involved in the immune response [gression . Particue cancer ,38, or ce cancer , but theresponse . SOCS prresponse .SOCS1 and SOCS3 were overexpressed in the six GBM cell lines analysed. This increase in mRNA expression has been previously reported in breast cancer where SOCS family plays a role in tumour proliferation [SOCS1 expression by a siRNA did no change the radiotherapy response in any of the six cell lines, and the isolated clonal resistant populations did not show any modification in the level of SOCS1 mRNA expression, indicating that SOCS1 is not directly related to radioresistance acquisition in GBM, as it was postulated by Zhou et al [SOCS1 was ectopically expressed, because it was silenced in U87 cells by promoter methylation [SOCS1 promoter is not methylated. In contrast, the inhibition of SOCS3 expression by siRNA significantly increased radioresistance of the six cell lines studied, and also there was a downregulation of SOCS3 mRNA levels in all the isolated clonal radioresistant populations, suggesting again that SOCS3 plays a key role in GBM radioresistance. Zhou et al indicated that the depletion of SOCS3 induced radiosensitivy, though in their study SOCS3 wasn’t directly silenced, and radiotherapy response was tested only in U87 cell line which expressed a phosphorylation-deficient DN STAT3 allele [SOCS3 are in agreement with previous studies that have hypothesised the relationship between the methylation of the SOCS3 promoter and poor prognosis in GBM [SOCS3 could lead to an increase in the JAK/STAT pathway activation, which would contribute to acquisition of features of tumour invasion and metastasis [feration . The expferation . Therefoou et al . The reahylation . Meanwhi3 allele , and this in GBM , and itss in GBM ,20. Furttastasis .SOCS3 in radioresistance acquisition, SOCS3 expression was induced under a heterologous promoter in the most radioresistant established cell line (GB39). Upregulation of SOCS3 produced radiosensitization in this cell line demonstrating again, that SOCS3 participates in GBM radioresistance. Finally, the endogenous expression of SOCS3 was also upregulated by TSA treatment, and again the most radioresistant cell line showed an increase in its radiosensitivity, in a TSA dose-depended way. This effect of TSA disappeared when the increase in SOCS3 expression induced by TSA was blocked by SOCS3.siRNA. Previous studies in myeloproliferative neoplasms and colorectal cancer, indicated that TSA treatment increases the SOCS3 expression, and this increase repressed the cell growth in both type of cancers [SOCS3 expression was associated to a lower activation of JAK/STAT pathway, reinforcing the key role of SOCS3 and JAK/STAT pathway in radioresistance acquisition proposed in this work.To further support the implication of  cancers ,42. ThesSOCS3 expression in radiotherapy resistance acquisition in GBM, so the downregulation of SOCS3 expression by siRNA increased the radiotherapy resistance in all the cell lines tested. In the same sense, a statistically significant downregulation of SOCS3 mRNA was observed in all the isolated radioresistant clonal population, when compared with the parental cell line. On the other hand, the induction of SOCS3 expression under a heterologous promoter in the most resistant established cell line induced its radiosensitization. Finally, the pre-treatment with TSA in the stablished cell lines produced an increase in SOCS3 expression levels and a parallel radiosensitization of these cells. Interestingly, the effects of TSA are completely abrogated when the increase in SOCS3 mRNA produced by this drug is blocked by SOCS3 siRNA.The data obtained in this study demonstrate the implication of SOCS3 plays a key role in radioresistance acquisition in GBM, and reveal the possible implication of the JAK/STAT pathway, which is inhibited by SOCS, in response to radiotherapy. These findings show that SOCS3 and its signal transduction pathway could be considered as a source of putative new targets to overcome GBM radioresistance.Taken together, the results probe that S1 FigMGMT promoter of GBM cell lines: GB16 (a), GB37 (b), GB39 (c), GB 40 (d), GB42 (e), GB 48 (f).Methylation status of  Black bars show control methylated CpG islands. Yellow lines show the threshold to consider a methylated CpG island, following the Jeuken et al [MGMT. In the abscissa axis, all promoter of genes studied by SALSA MS-MLPA ME011.B1 Mismatch Repair Genes kit are shown.en et al  criteria(TIF)Click here for additional data file.S2 FigBars show the percentage ± SD of cell death in each cell line.(TIF)Click here for additional data file.S3 FigBars show the percentage ± SD of cell death in each cell line.(TIF)Click here for additional data file.S4 Fig1 + G2/M) IR-CTR) ± SD.Bars show the effect of radiotherapy as the (Δ (SubG(TIF)Click here for additional data file.S5 Fig(a) Expression profile of SOCS1 and SOCS3 in primary GBM cell lines. Fold change (FC) values ± SD of SOCS1 mRNA expression (black bars), and SOCS3 mRNA expression (white bars) are shown for each cell line represented in the X-axis. (b): Western blot analysis of SOCS3 expression on total protein extracts from established cell lines. The molecular sizes of the bands are shown to the right.(TIF)Click here for additional data file.S6 Fig1 and G2/M phases (a) and mRNA expression (b) for control (CTR) and transfected with non-specific siRNA (siRNA.NS) cells.Representation of the 95% confidence interval of % of cells in the SubG(TIF)Click here for additional data file.S7 FigSOCS3 mRNA expression in the different experiments (a). Striped bars represent mean percentages ± SD of cells in SubG1 and G2/M phases (b). (n≥3).White bars show mean ± SD of (TIF)Click here for additional data file.