Ruethairat Sriraksa

CpG-island methylation study of liver fluke-related cholangiocarcinoma

Background:Genetic changes have been widely reported in association with cholangiocarcinoma (CCA), while epigenetic changes are poorly characterised. We aimed to further evaluate CpG-island hypermethylation in CCA at candidate loci, which may have potential as diagnostic or prognostic biomarkers.Methods:We analysed methylation of 26 CpG-islands in 102 liver fluke related-CCA and 29 adjacent normal samples using methylation-specific PCR (MSP). Methylation of interest loci was confirmed using pyrosequencing and/or combined bisulfite restriction analysis, and protein expression by immunohistochemistry.Results:A number of CpG-islands (OPCML, SFRP1, HIC1, PTEN and DcR1) showed frequency of hypermethylation in >28% of CCA, but not adjacent normal tissues. The results showed that 91% of CCA were methylated in at least one CpG-island. The OPCML was the most frequently methylated locus (72.5%) and was more frequently methylated in less differentiated CCA. Patients with methylated DcR1 had significantly longer overall survival (Median; 41.7 vs 21.7 weeks, P=0.027). Low-protein expression was found in >70% of CCA with methylation of OPCML or DcR1.Conclusion:Aberrant hypermethylation of certain loci is a common event in liver fluke-related CCA and may potentially contribute to cholangiocarcinogenesis. The OPCML and DcR1 might serve as methylation biomarkers in CCA that can be readily examined by MSP.

Differential expression of IL-8 and IL-8 receptors in benign, borderline and malignant ovarian epithelial tumours

INTRODUCTION Ovarian Cancer is the leading cause of death from gynecological malignancy. The poor prognosis is mainly due to presentation at a late stage and poor response to therapy. Much research is needed to identify diagnostic and prognostic biomarkers as well as therapeutic targets for ovarian cancer. Interleukin-8 is expressed by many tumour types and is known to have mitogenic, motogenic and angiogenic effects on tumour cells. AIMS The aim of this study was to investigate the expression of IL-8 and IL-8 receptors (IL-8RA and IL-8RB) in different histological subtypes of ovarian tumours, as potential prognostic biomarkers in ovarian tumours. MATERIALS AND METHODS Immunohitochemistry was used to study the expression of IL-8 and IL-8 receptors in 115 ovarian tumours including 21 benign tumours, 25 borderline tumours and 69 carcinomas of serous, clear cell, endometrioid and mucinous types. The correlation of expression profile, tumour type, stage, and progression free survival and overall survival was statistically analysed. RESULTS IL-8 and IL-8 receptors were expressed in all types of tumours with variable intensity and subcellular distribution. There was a statistically significant correlation between levels of expression and tumour stage and tumour type, being mostly significant in serous tumours. No correlation with patient progression free survival or overall survival was found. CONCLUSION This is the first study investigating the expression of IL-8 and IL-8 receptors using immunohistochemistry in different types of ovarian tumours, including benign and borderline tumours. IL-8 and IL-8RA are potential prognostic biomarkers and therapeutic targets in ovarian cancer, particularly in ovarian serous carcinoma.

The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma

Interleukin-8 (IL-8) is a pro-inflammatory cytokine which exerts its effects via binding to 2 receptors, CXCR1 and CXCR2 and is known to promote angiogenesis, mitogenesis and motogenesis in cancer. IL-8 is over expressed in endometrial carcinoma, but the expression of CXCR1 and CXCR2 in endometrial carcinoma has not been previously investigated. The aim of this study was to determine the expression of IL-8 receptors in endometrial carcinoma. The expression of CXCR1 and CXCR2 was studied in endometrial carcinomas and normal endometrium by immunohistochemistry in 101 tumours. IL-8 and IL-8 receptor expression was also studied by Real-time quantitative PCR (RT-qPCR) in 17 tumours in comparison to normal endometrium. The expression profile was correlated to the clinico-pathological features of the tumours. Immunohistochemistry showed CXCR1 and CXCR2 were expressed in all cases of endometrial carcinoma, with CXCR1 showing stronger expression. There was a statistically significant correlation between CXCR2 staining intensity and tumour grade (P=0.012) and disease free survival (P=0.015) independently. On RT-qPCR, 14/17, 15/17 and 16/17 tumours showed significant increase in IL-8, CXCR1 and CXCR2 expression levels in comparison to normal endometrium, with median fold increase of 42-fold, 51-fold and 27-fold, respectively. This is the first report of the expression of IL-8 receptors in endometrial carcinoma and the results show an association between IL-8 and IL-8 receptors and the pathogenesis of endometrial carcinoma, and represent potential prognostic biomarkers and therapeutic targets.

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

Histone Deacetylases and their Inhibitors as Potential Therapeutic Drugs for cholangiocarcinoma - Cell Line findings

Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of the epigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found with cholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACs class I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) and an immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, as well as a classical chemotherapeutic drug 5-fluorouracil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. IC50 and IC20 were then analyzed for each agent and cell line. Moreover, synergistic potential of VPA or SAHA in combination with 5-FU at subtoxic dose (IC20) of each agent was also evaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition was analyzed by Students t-test. The result demonstrated that HDACs were expressed in all studied cell types. Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less sensitive to classical chemotherapeutic 5-FU was highly sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU significantly inhibited cell proliferation in CCA cell lines compared to single agent treatment (P ≤ 0.01), while sequentially combined treatments were less effective. The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.

Aberrant DNA Methylation at Genes Associated with a Stem Cell-like Phenotype in Cholangiocarcinoma Tumors

Genetic abnormalities of cholangiocarcinoma have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterized. We have profiled the DNA methylomes of 28 primary cholangiocarcinoma and six matched adjacent normal tissues using Infiniums HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly and epigenetically regulated in this tumor type. Using a linear model for microarray data, we identified 1610 differentially methylated autosomal CpG sites, with 809 hypermethylated (representing 603 genes) and 801 hypomethylated (representing 712 genes) in cholangiocarcinoma versus adjacent normal tissues (false-discovery rate ≤ 0.05). Gene ontology and gene set enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in cholangiocarcinoma including homeobox genes and target genes of PRC2, EED, SUZ12, and histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of cholangiocarcinoma (n = 102). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in cholangiocarcinoma. These data have implications for cholangiocarcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. Cancer Prev Res; 6(12); 1348–55. ©2013 AACR.

Verification of complete bisulfite modification using Calponin-specific primer sets.

OBJECTIVES To verify complete bisulfite modification. DESIGN AND METHODS Bisulfite modified DNA was screened by PCR with Calponin-specific primer sets prior to detecting methylation status of 14-3-3sigma by methylation-specific PCR. RESULTS False positive methylation was obtained from incomplete bisulfite modification. The lower detection limit of wild type Calponin-specific primer is approximately 0.375 ng of unmodified DNA. CONCLUSIONS Verification of complete bisulfite modification by Calponin-specific primer sets can reduce misinterpretation of methylation.

Validation of methylation-sensitive high resolution melting for the detection of DNA methylation in cholangiocarcinoma

OBJECTIVES To validate methylation-sensitive high resolution melting (MS-HRM) for detection of DNA methylation. DESIGN AND METHODS Methylation of two independent loci, OPCML and DcR1, was analyzed in cholangiocarcinoma and adjacent normal samples by using MS-HRM, methylation-specific PCR and pyrosequencing. RESULTS There was significant agreement between methods at both loci. CONCLUSIONS MS-HRM represents the excellent potential and reliability for quantifying DNA methylation levels in clinical samples.

TRAIL in Combination with Subtoxic 5-FU Effectively Inhibit Cell Proliferation and Induce Apoptosis in Cholangiocarcinoma Cells.

In the past decade, the incidence and mortality rates of cholangiocarcinoma (CCA) have been increasing worldwide. The relatively low responsiveness of CCA to conventional chemotherapy leads to poor overall survival. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has emerged as the most promising anti-cancer therapeutic agent since it is able to selectively induce apoptosis of tumor cells but not normal cells. In this study, we aimed to investigate the therapeutic effect of TRAIL in CCA cell lines (M213, M214 and KKU100) compared with the immortal biliary cell line, MMNK1, either alone or in combination with a subtoxic dose of 5-fluorouracil (5-FU). We found that recombinant human TRAIL (rhTRAIL) was a potential agent which significantly inhibited cell proliferation and mediated caspase activities (caspases 8, 9 and 3/7) and apoptosis of CCA cells. The combined treatment of rhTRAIL and 5-FU effectively enhanced inhibition of CCA cell growth with a smaller effect on MMNK1. Our finding suggests TRAIL to be a novel anti-cancer therapeutic agent and advantage of its combination with a conventional chemotherapeutic drug for effective treatment of CCA.

The frequency and significance of WT-1 expression in serous endometrial carcinoma

Serous endometrial carcinoma is an aggressive type of endometrial carcinoma. Wilms tumor gene 1 (WT-1) is commonly expressed in ovarian serous carcinomas and considered a diagnostic marker of these tumors. However, it is generally believed that WT-1 is rarely expressed by endometrial serous carcinoma. The aim of this study was to evaluate the frequency and significance of WT-1 expression in endometrial serous carcinoma. We studied the expression of WT-1 in formalin-fixed, paraffin-embedded tumor sections from 77 cases of endometrial serous carcinoma. Thirty-four tumors showed positive expression for WT-1 (44%). There was a statistically significant association between the presence of WT-1 expression and disease-free survival (DFS), where patients with tumors expressing WT-1 had a shorter DFS compared with those with no WT-1 expression (P = .031; median DFS, 15 and 38 months, respectively). By multivariate Cox regression analysis, DFS was independent from other clinicopathological data (tumor stage, presence of lymphovascular space invasion, cervical involvement, and extrauterine spread), indicating that WT-1 expression is independently associated with DFS. Our study shows that WT-1 is expressed in a considerable percentage of endometrial serous carcinomas, suggesting a role for WT-1 in the pathology of these tumors. This has therapeutic significance, as WT-1 is an emerging target for immunotherapy. Moreover, our results show that WT-1 has prognostic value, being predictive of DFS. As a potential prognostic marker and therapeutic target, we recommend that WT-1 expression should be included in histopathologic reports of endometrial serous carcinoma.