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The microRNA miR-205-5p has diverse effects in different malignancies, including cholangiocarcinoma (CCA), but its effects on CCA progression is unclear. Here we investigated the role and function of miR-205-5p in CCA. Three CCA cell lines and human serum samples were found to have much higher expression levels of miR-205-5p than seen in typical cholangiocyte cell lines and healthy controls. Inhibition of miR-205-5p suppressed CCA cell motility, invasion and proliferation of KKU-213B whereby overexpression of miR-205-5p promoted cell proliferation and motility of KKU-100 cells. Bioinformatics tools (miRDB, TargetScan, miRWalk, and GEPIA) all predicted various miR-205-5p targets. Experiments using miR-205-5p inhibitor and mimic indicated that homeodomain-interacting protein kinase 3 (HIPK3) was a potential direct target of miR-205-5p. Overexpression of HIPK3 using HIPK3 plasmid cloning DNA suppressed migration and proliferation of KKU-100 cells. Notably, HIPK3 expression was lower in human CCA tissues than in normal adjacent tissues. High HIPK3 expression was significantly associated with longer survival time of CCA patients. Multivariate regression analysis indicated tissue HIPK3 levels as an independent prognostic factor for CCA patients. These findings indicate that overexpression of miR-205-5p promotes CCA cells proliferation and migration partly via HIPK3-dependent way. Therefore, targeting miR-205-5p may be a potential treatment approach for CCA., (© 2023. The Author(s).)

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

Cholangiocarcinoma (CCA) is one of the oxidative stress-driven carcinogenesis through chronic inflammation. Insulin receptor substrate 1 (IRS1), an adaptor protein of insulin signaling pathways, is associated with the progression of many inflammation-related cancers. This study hypothesized that oxidative stress regulates IRS1 expression and that up-regulation of IRS1 induces CCA progression. The localizations of IRS1 and an oxidative stress marker (8-oxodG) were detected in CCA tissues using immunohistochemistry (IHC). The presence of IRS1 in CCA tissues was confirmed using immortal cholangiocyte cells (MMNK1), a long-term oxidative-stress-induced cell line (ox-MMNK1-L), and five CCA cell lines as cell culture models. IRS1 was overexpressed in tumor cells and this was associated with a shorter patient survival time and an increase in 8-oxodG. IRS1 expression was higher in ox-MMNK1-L cells than in MMNK1 cells. Knockdown of IRS1 by siRNA in two CCA cell lines led to inhibition of proliferation, cell cycle progression, migration, invasion, stemness, and oxidative stress resistance properties. Moreover, a transcriptomics study demonstrated that suppressing IRS1 in the KKU-213B CCA cell line reduced the expression levels of several genes and pathways involved in the cellular functions. The findings indicate that IRS1 is a key molecule in the connection between oxidative stress and CCA progression. Therefore, IRS1 and its related genes can be used as prognostic markers and therapeutic targets for CCA therapy.

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment., (© 2022. The Author(s).)

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation., (© 2021. The Society for In Vitro Biology.)

Opisthorchis viverrini (Ov) infection causes hepatobiliary diseases and is a major risk factor for cholangiocarcinoma. While several omics approaches have been employed to understand the pathogenesis of opisthorchiasis, effects of Ov infection on the host systemic metabolism and fecal microbiota have not been fully explored. Here, we used a 1 H NMR spectroscopy-based metabolic phenotyping approach to investigate Ov infection-induced metabolic disturbances at both the acute (1 month postinfection, 1 mpi) and chronic (4 mpi) stages in hamsters. A total of 22, 3, and 4 metabolites were found to be significantly different in the liver, serum, and urine, respectively, between Ov+ and Ov- groups. Elevated levels of hepatic amino acids and tricarboxylic acid (TCA)-cycle intermediates (fumarate and malate) were co-observed with liver injury in acute infection, whereas fibrosis-associated metabolites (e.g., glycine and glutamate) increased at the chronic infection stage. Lower levels of lipid signals ((C H 2 ) n and C H 2 CH 2 CO) and higher levels of lysine and scyllo -inositol were observed in serum from Ov+ hamsters at 1 mpi compared to Ov- controls. Urinary levels of phenylacetylglycine (a host-bacterial cometabolite) and tauro-β-muricholic acid were higher in the Ov+ group, which coexisted with hepatic and mild kidney fibrosis. Furthermore, Ov+ animals showed higher relative abundances of fecal Methanobrevibacter (Archaea), Akkermansia , and Burkholderia-Paraburkholderia compared to the noninfected controls. In conclusion, along with liver and kidney pathologies, O. viverrini infection resulted in hepatic and mild renal pathologies, disturbed hepatic amino acid metabolism and the TCA cycle, and induced changes in the fecal microbial composition and urinary host-microbial cometabolism. This study provides the initial step toward an understanding of local and systemic metabolic responses of the host to O. viverrini infection.

Background: Helicobacter pylori (HP) has been detected in the hepatobiliary tract of cholangiocarcinoma (CCA) patients in regions both endemic and non-endemic for Opisthorchis viverrini (OV) infection. However, whether H. pylori infection promotes CCA development remains unknown. We investigated CCA development in hamsters induced by a combination of infection with H. pylori and administration of N-nitrosodimethylamine (NDMA) and compared findings with those in an OV plus NDMA group., Materials and Methods: Eighty-five hamsters were divided into four groups: (1) normal, (2) administered NDMA, (3) infected with cagA + H. pylori and administered NDMA (HN group), and (4) infected with OV and administered NDMA (ON group). Animals were euthanized at 3 and 6 months post-infection. Histopathological changes of liver and the expression of markers associated with carcinogenesis were studied., Results: At 3 months post-infection (p.i.), cholangitis and lymphoid follicles without tumor appearance were noted in the HN group, whereas extensive fibrosis was seen in members of the ON group, 10% of which had developed tumors. At 6 months p.i., 10% of hamsters administered NDMA alone had developed CCA, whereas in the HN and ON groups, 20% and 60% of hamsters, respectively, had developed CCA. Cytokeratin-19 (CK19) expression was observed in the CCA tissues of both the HN and the ON groups, confirming the bile duct origin of the CCA cells. CCA development in the HN group might be inflammation-mediated, as suggested by overexpression of HMGB1, PCNA, IL-8, and 8-OxodG in CCA tissues., Conclusion: cagA + H. pylori infection and carcinogen intake can induce CCA development with slow progression., (© 2021 John Wiley & Sons Ltd.)

Objective: Choledocholithiasis (CDL), a potential risk for cholangiocarcinoma (CCA) development, is often a consequence of bacterial infection. Thus, the microbial population that contributes to CDL might also be involved in CCA development. We compared the microbiome in bile fluid of CDL patients and CCA patients., Methods: Bile samples were collected from CDL (n = 30) and CCA (n =30) patients. Microbial profiling was performed individually by the sequencing of V3-V4 regions of the 16S rRNA gene., Results: Enterobacter, Pseudomonas, and Stenotrophomonas species were much more abundant in bile samples from CCA compared to CDL (p.

Background/aim: Forkhead box O4 (FOXO4) has been demonstrated to be a tumor suppressor and proposed as target for treatment of a variety of cancer types. However, the role of FOXO4 in cholangiocarcinoma (CCA), a dangerous cancer of bile-duct epithelium, has rarely been explored., Materials and Methods: The proliferative rate of CCA cell lines KKU-213B, KKU-055 and KKK-D068 was investigated using the sulforhodamine B (SRB) assay. Levels of FOXO4, cyclin E1 (CCNE1), CCNE2, cyclin-dependent kinase 2 (CDK2) and cell division cycle 25A (CDC25A) expression were measured using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). The cell-cycle profile was explored using flow cytometry., Results: The SRB assay demonstrated that KKU-213B expressed low levels of FOXO4 but its proliferative rate was highest of all cell lines tested. Interestingly, ectopic expression of FOXO4 significantly suppressed proliferation of KKU-213B cells. Cell-cycle analysis revealed that the cell population in the G 0 /G 1 phase was significantly higher in FOXO4-transfected KKU-213B cells than in controls. RT-qPCR analysis demonstrated that the levels of expression of genes that play a role in the G 1 /S transition, namely CCNE1, CCNE2, CDK2 and CDC25A, were significantly lower in FOXO4-transfected KKU-213B cells compared to controls., Conclusion: FOXO4 suppressed CCA cell proliferation partly via down-regulating the expression of genes involved in the G 1 /S transition, leading to G 0 /G 1 arrest. Our findings suggest that induction of FOXO4 expression might be an alternative approach for the treatment of CCA., (Copyright © 2020 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cholangiocarcinoma (CCA), a malignancy of biliary epithelium, is related to liver stem cell deregulation. FoxAs are a group of transcription factors that play critical roles in liver stem cell differentiation. In this study, the expression levels of FoxAs (i.e., FoxA1, FoxA2 and FoxA3) were detected in intrahepatic CCA tissues and the functions of FoxAs were studied in CCA cell lines. FoxA1 and FoxA2 were mainly localized in the nuclei of normal bile duct (NBD) cells and some of the cancer cells. Low expression of FoxA1 in CCA tissues (72%) was significantly correlated with poor prognosis. FoxA3 expression of CCA cells was localized in the nucleus and cytoplasm, whereas it was slightly detected in NBDs. High expression of FoxA3 in cancer tissues (61%) was significantly related to high metastasis status. These findings suggest the opposing roles of FoxA1 and FoxA3 in CCA. Moreover, the FoxA1-over-expressing CCA cell line exhibited a significant reduction in proliferative and invasive activities compared to control cells. Knockdown of FoxA3 in CCA cells resulted in a significant decrease in proliferative and invasive activities compared with control cells. Taken together, in CCA, FoxA1 is down-regulated and has tumor suppressive roles, whereas FoxA3 is up-regulated and has oncogenic roles., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Zinc finger protein 423 (ZNF423) is a transcriptional factor involved in the development and progression of cancers but has not yet been examined in cholangiocarcinoma (CCA), an oxidative stress-driven cancer of biliary epithelium. In this study, we hypothesized that oxidative stress mediated ZNF423 expression regulates its downstream genes resulting in CCA genesis. ZNF423 protein expression patterns and 8-oxodG (an oxidative stress marker) formation in CCA tissues were investigated using immunohistochemical analysis. The results showed that ZNF423 was overexpressed in CCA cells compared to normal bile duct cells adjacent of the tumor. Notably, ZNF423 expression was positively correlated with 8-oxodG formation. Moreover, ZNF423 expression in an immortalized cholangiocyte cell line (MMNK1) was increased by hydrogen peroxide-treatment, suggesting that oxidative stress induces ZNF423 expression. To investigate the roles of ZNF423 in CCA progression, ZNF423 mRNA was silenced using specific siRNA in CCA cell lines, KKU-100 and KKU-213. Silencing of ZNF423 significantly inhibits cell proliferation and invasion of both CCA cell lines. Taking all these results together, the present study denoted that ZNF423 is an oxidative stress-responsive gene with an oncogenic property contributing to the regulation of CCA genesis.

Fluorouracil (5-FU) is the first-line chemotherapeutic drug for cholangiocarcinoma (CCA), but its efficacy has been compromised by the development of resistance. Development of 5-FU resistance is associated with elevated expression of its cellular target, thymidylate synthase (TYMS). E2F1 transcription factor has previously been shown to modulate the expression of FOXM1 and TYMS. Immunohistochemical (IHC) analysis revealed a strong correlated upregulation of FOXM1 (78%) and TYMS (48%) expression at the protein levels in CCA tissues. In agreement, RT-qPCR and western blot analyses of four human CCA cell lines at the baseline level and in response to high doses of 5-FU revealed good correlations between FOXM1 and TYMS expression in the CCA cell lines tested, except for the highly 5-FU-resistant HuCCA cells. Consistently, siRNA-mediated knockdown of FOXM1 reduced the clonogenicity and TYMS expression in the relatively sensitive KKU-D131 but not in the highly resistant HuCCA cells. Interestingly, silencing of TYMS sensitized both KKU-D131 and HuCCA to 5-FU treatment, suggesting that resistance to very high levels of 5-FU is due to the inability of the genotoxic sensor FOXM1 to modulate TYMS expression. Consistently, ChIP analysis revealed that FOXM1 binds efficiently to the TYMS promoter and modulates TYMS expression at the promoter level upon 5-FU treatment in KKU-D131 but not in HuCCA cells. In addition, E2F1 expression did not correlate with either FOXM1 or TYMS expression and E2F1 depletion has no effects on the clonogenicity and TYMS expression in the CCA cells. In conclusion, our data show that FOXM1 regulates TYMS expression to modulate 5-FU resistance in CCA and that severe 5-FU resistance can be caused by the uncoupling of the regulation of TYMS by FOXM1. Our findings suggest that the FOXM1-TYMS axis can be a novel diagnostic, predictive and prognostic marker as well as a therapeutic target for CCA.

Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer., Competing Interests: The authors have declared that no competing interests exist.

Early B cell factor 1 (EBF1) is a transcription factor involved in the differentiation of several stem cell lineages and it is a negative regulator of estrogen receptors. EBF1 is down-regulated in many tumors, and is believed to play suppressive roles in cancer promotion and progression. However, the functional roles of EBF1 in carcinogenesis are unclear. Liver fluke-infection-associated cholangiocarcinoma (CCA) is an oxidative stress-driven cancer of bile duct epithelium. In this study, we investigated EBF1 expression in tissues from CCA patients, CCA cell lines (KKU-213, KKU-214 and KKU-156), cholangiocyte (MMNK1) and its oxidative stress-resistant (ox-MMNK1-L) cell lines. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was used as an oxidative stress marker. Our results revealed that EBF1 expression was suppressed in cancer cells compared with the individual normal bile duct cells at tumor adjacent areas of CCA tissues. CCA patients with low EBF1 expression and high formation of 8-oxodG were shown to correlate with poor survival. Moreover, EBF1 was suppressed in the oxidative stress-resistant cell line and all of CCA cell lines compared to the cholangiocyte cell line. This suggests that prolonged oxidative stress suppressed EBF1 expression and the reduced EBF1 level may facilitate CCA genesis. To elucidate the significance of EBF1 suppression in CCA genesis, EBF1 expression of the MMNK1 cell line was down-regulated by siRNA technique, and its effects on stem cell properties (CD133 and Oct3/4 expressions), tumorigenic properties (cell proliferation, wound healing and cell migration), estrogen responsive gene (TFF1), estrogen-stimulated wound healing, and cell migration were examined. The results showed that CD133, Oct3/4 and TFF1 expression levels, wound healing, and cell migration of EBF1 knockdown-MMNK1 cells were significantly increased. Also, cell migration of EBF1-knockdown cells was significantly enhanced after 17β-estradiol treatment. Our findings suggest that EBF1 down-regulation via oxidative stress induces stem cell properties, tumorigenic properties and estrogen responses of cholangiocytes leading to CCA genesis with aggressive clinical outcomes., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor interstitial fluid contains tumor-specific proteins that may be useful biomarkers for cancers. In this study, we identified proteins present in cholangiocarcinoma interstitial fluid. Proteins derived from three samples of tumor interstitial fluid and paired samples of adjacent normal interstitial fluid from cholangiocarcinoma patients were subjected to two-dimensional liquid chromatography with tandem mass spectrometry. Candidate proteins were selected based on a greater than twofold change in expression levels between tumor interstitial fluid and normal interstitial fluid. Upregulation of six proteins in tumor interstitial fluid, including S100 calcium binding protein A6 (S100A6), S100 calcium binding protein A9, aldo-keto reductase family 1 member C4, neuropilin-1, 14-3-3 zeta/delta, and triosephosphate isomerase was assessed by western blot and immunohistochemistry. Their potential as markers was evaluated in human cholangiocarcinoma tissue arrays, and in serum using enzyme-linked immunosorbent assay. Expression of S100A6 was higher in tumor interstitial fluid than in normal interstitial fluid and showed the highest positive rate (98.96%) in cholangiocarcinoma tissues. Serum levels of S100A6 did not differ between cholangitis and cholangiocarcinoma patients, but were significantly higher than in healthy individuals ( p < 0.0001). In cholangiocarcinoma cases, S100A6 level was associated with vascular invasion ( p = 0.007) and could distinguish cholangiocarcinoma patients from healthy individuals as effectively as the carbohydrate antigen 19-9. In addition, potential for drug treatment targeting S100A6 and other candidate proteins was also demonstrated using STITCH analysis. In conclusion, proteomics analysis of tumor interstitial fluid could be a new approach for biomarker discovery, and S100A6 is a potential risk marker for screening of cholangiocarcinoma.

Background: The combination of Opisthorchis viverrini (OV) infection and chemical carcinogen induces cholangiocarcinoma (CCA) in hamsters via inflammation-mediated mechanisms. Thus, suppression of inflammatory cells at the initial stages of CCA development would be of benefit. We aimed to investigate whether IL-17-producing CD4+ T cells (Th17) and CD4+ Foxp3+ T cells (Treg) are involved in the early stages of CCA genesis and can be targeted for suppression by melatonin., Methods: Inflammation, an initial stage of CCA development, was induced in hamsters by a combination of O. viverrini infection and N-nitrosodimethylamine (NDMA) administration. Melatonin (50mg/kg) was additionally administered to one group for the 30days of the experiment. Liver tissue-resident T cells were investigated using immunostaining, western blotting, and real-time PCR., Results: OV+NDMA-induced CCA tissues showed significantly higher numbers of inflammatory cells, especially eosinophils, bile duct proliferation and IL-17+ cell infiltration compared to normal livers. Expression of Foxp3 was localized in the bile duct epithelial cells, and especially in the bile duct hyperplasia. Accumulation of CD4+ and IL-17+ cells and intense staining of the Foxp3+ marker were consistent with their protein levels. Infiltration of IL-17+ inflammatory cells and Foxp3+ cells, as well as increases in their transcription expression levels, were significantly lower in the melatonin-treated group. In contrast, increased CD4+ cell infiltration and TNF-α expression were also observed through melatonin treatment., Conclusion: Melatonin exerts an immunomodulatory effect, suppressing eosinophils and Th17 cells and expression of Foxp3, but enhancing CD4+ cells and TNF-α. This suggests that melatonin may be used for CCA chemoprevention., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Background: Cholangiocarcinoma (CCA), a malignant tumor of the biliary epithelium, is a tumor with an ineffective diagnosis and poor prognosis. We have previously identified an overexpression of orosomucoid 2 (ORM2) along with CCA development in hamsters using a proteomics technique., Objective: To evaluate plasma ORM2 as a candidate risk biomarker for CCA in humans., Methods: Overexpression of ORM2 in CCA patients was assessed by western blotting and immunohistochemistry. The diagnostic efficacy of ORM2 was investigated in the plasma of patients with hepatobiliary diseases - including 46 cholangitis patients and 70 CCA patients - compared with 20 healthy individuals, using enzyme-linked immunosorbent assay (ELISA)., Results: Overexpression of ORM2 was observed in the cytoplasm of bile duct tumor cells and in the adjacent normal hepatocytes at a much higher intensity than in normal bile duct cells. Western blot analysis revealed that its expression was significantly higher in the plasma of CCA patients compared with that of healthy individuals (P < 0.01). ELISA showed that plasma ORM2 levels were significantly elevated in CCA and cholangitis groups compared with healthy individuals (P < 0.0001). The sensitivity and specificity of ORM2 in distinguishing CCA patients from healthy patients were 92.86% and 73.68%, respectively., Conclusions: Plasma ORM2 could serve as a new risk marker for CCA.

Purpose: To discover protein markers in chronic/advanced opisthorchiasis for the early detection of Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA)., Experimental Design: Liver tissues derived from normal hamsters and those with chronic/advanced opisthorchiasis (n = 5 per group) were subjected to 2DE and LC-MS/MS. Candidate protein expression was confirmed in hamster models and human CCA tissue microarray (TMA) using immunohistochemistry and Western blot., Result: Proteomics analysis detected 14-3-3 eta only in infected hamsters, not in uninfected controls. Immunohistochemistry and Western blot analysis confirmed low expression of 14-3-3 eta in normal hamster livers and demonstrated increased expression through time in infected livers. This protein was also observed in parasite organs, especially during the chronic phase of opisthorchiasis. Moreover, increased expression of 14-3-3 eta, relative to normal hamster livers, was observed during the early stage of CCA induced by OV infection and administration of N-nitrosodimethylamine. Immunohistochemical analysis of human TMA revealed that 14-3-3 eta was highly expressed in CCA (84.23%, 187/222 cases) but was not found in hepatocellular carcinoma or healthy liver tissues., Conclusions and Clinical Relevance: 14-3-3 eta protein has potential as a screening and early diagnostic marker for CCA., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3 T MRI and (1)H MRS were useful for quantitative investigation of the serial development of hepatobiliary changes in Opisthorchis viverrini infection in hamsters, and the differential diagnosis of cholangiocacinoma (CCA) development from bile duct changes and normal condition is unclear. In this study, we investigated the serial development of hepatobiliary changes and CCAgenesis in O. viverrini-infected and N-nitrosodimethylamine (NDMA) treated hamsters (ON group) using 3 T MRI and (1)H MRS and the results were compared with those either in the O. viverrini-infected group (OV group) and uninfected normal controls. In the ON group, CCAs were first found at 9 weeks post-infection, with sizes of ~2 mm. The typical MR signal characteristics of CCA were hypo- and occasionally isointensity signal on T1-weighted images, and mild-moderate to hyper-intensity signal on T2-weighted images compared to the liver parenchyma. T2-weighted images with fat suppression revealed dilatation of the intra- and extrahepatic bile ducts, and often defined the anatomical level of biliary obstruction, cystic lesions, liver abscesses, and CCA which was starting seen of these noticeable abnormalities at 5 weeks onwards. The results of fibrosis grading using MR images showed a positive correlation (r=0.90, P<0.038 by Spearman's rank correlation test) with those of the histopathological grading. In addition, 3.0 T (1)H MRS showed elevated choline and decreased lipids levels in the liver tissues of the ON group. In conclusion, MRI and (1)H MRS are useful for the quantitative investigation of the serial development of hepatobilary changes and CCA in hamsters, and are potentially useful as early diagnostic tools for CCA., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The diagnosis of cholangiocarcinoma (CCA) is often challenging, leading to poor prognosis. CCA arises via chronic inflammation which may be associated with autoantibodies production. This study aims to identify IgG antibodies directed at self-proteins and tumor-associated antigens. Proteins derived from immortalized cholangiocyte cell line (MMNK1) and CCA cell lines (M055, M214 and M139) were separated using 2-dimensional electrophoresis and incubated with pooled plasma of patients with CCA and non-neoplastic controls by immunoblotting. Twenty five immunoreactive spots against all cell lines-derived proteins were observed on stained gels and studied by LC-MS/MS. Among these, heat shock protein 70 (HSP70), enolase 1 (ENO1) and ribonuclease/angiogenin inhibitor 1 (RNH1) obtained the highest matching scores and were thus selected for further validation. Western blot revealed immunoreactivity against HSP70 and RNH1 in the majority of CCA cases and weakly in healthy individuals. Further, ELISA showed that plasma HSP70 autoantibody level in CCA was significantly capable to discriminate CCA from healthy individuals with an area under the receiver operating characteristic curve of 0.9158 (cut-off 0.2630, 93.55% sensitivity and 73.91% specificity). Plasma levels of IgG autoantibodies against HSP70 were correlated with progression from healthy individuals to cholangitis to CCA (r = 0.679, P<0.001). In addition, circulating ENO1 and RNH1 autoantibodies levels were also significantly higher in cholangitis and CCA compared to healthy controls (P<0.05). Moreover, the combinations of HSP70, ENO1 or RNH1 autoantibodies positivity rates improved specificity to over 78%. In conclusion, plasma IgG autoantibodies against HSP70, ENO1 and RNH1 may represent new diagnostic markers for CCA.

Human liver fluke, Opisthorchis viverrini (Ov), is the major risk factor of cholangiocarcinoma (CCA) in northeastern Thailand. Our approach focuses on genetic progression and molecular changes in the carcinogenic pathway of liver fluke-associated CCA aimed at assessing patients at risk of CCA and using chemoprevention as the secondary cancer prevention to reduce the incidence of CCA. This review summarizes altered gene expressions, biomolecules and their modification, i.e. DNA adducts, oxidized proteins, oxysterols and fibrotic markers in hamster- and human-CCA. Potential risk biomarker(s) and chemopreventive agent(s) criteria and selection were based on results from experimental and epidemiological studies identifying hepatobiliary disease, including CCA. Laboratory results reveal that oxidative stress induced by Ov infection leads to bimolecular damage, tissue remodeling especially periductal fibrosis and alteration of gene expressions, which could be involved in all steps of CCA carcinogenesis. Some of these molecules are reported to change their levels in opisthorchiasis, periductal fibrosis diagnosed by ultrasonography and CCA. Chemoprevention in experimental CCA tumorigenesis is discussed. These multiple risk biomarkers could now be explored for screening including chemopreventive intervention of subjects living in endemic areas where the prevalence of opisthorchiasis remains high., (© 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. Chronic inflammation such as parasite infection and primary sclerosing cholangitis can be an etiological factor of cholangiocarcinoma. Using a proteomic approach and double-fluorescent staining, we identified high expression and colocalization of albumin and cytokeratin-19 in liver fluke-associated cholangiocarcinoma tissues, compared with normal livers from cholangiocarcinoma patients and cadaveric donors, respectively. Albumin was detected not only in cells of hyperplastic bile ducts and cholangiocarcinoma, but also in liver stem/progenitor cell origin, such as canal of Hering, ductules, and ductular reactions, suggesting the involvement of stem/progenitor cells in cholangiocarcinoma development. To clarify the involvement of liver stem/progenitor cells in cholangiocarcinoma, we examined several stem/progenitor cell markers (CD133, CD44, OV6, and Oct3/4) in cholangiocarcinoma tissues analyzed by immunohistochemical staining, and measured 8-oxodG levels by using HPLC-ECD as an inflammation-related DNA lesion. In addition, a stem/progenitor cell factor Bmi1, 8-nitroguanine (formed during nitrative DNA damage), DNA damage response (DDR) proteins (phosphorylated ATM and γ-H2AX), and manganese-SOD (Mn-SOD) were analyzed by immunohistochemistry. Stem/progenitor cell markers (CD133, OV6, CD44, and Oct3/4) were positively stained in 56, 38, 47, and 56% of 34 cholangiocarcinoma cases, respectively. Quantitative analysis of 8-oxodG revealed significantly increased levels in CD133- and/or Oct3/4-positive tumor tissues compared to negative tumor tissues, as well as 8-nitroguanine formation detected by immunohistochemistry. In the cases of CD44- and/or OV6-positive tissue, no significant difference was observed. Cholangiocarcinoma patients with CD133- and/or Oct3/4-positive tumor tissues showed significantly lower expression of Mn-SOD and higher DDR protein, γ-H2AX. Moreover, CD133- and/or Oct3/4-positive cholangiocarcinoma patients had significant associations with tumor histology types, tumor stage, and poor prognoses. Our results suggest that CD133 and Oct3/4 in cholangiocarcinoma are associated with increased formation of DNA lesions and the DDR protein, which may be involved in genetic instability and lead to cholangiocarcinoma development with aggressive clinical features., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC)., Methods: Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N-nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA., Results: In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-κB gene expression., Conclusion: ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis.

Our previous report showed that platelet-derived growth factor (PDGF) and related genes were upregulated in a Syrian hamster model and could be detected in all human cholangiocarcinoma (CCA) tissues. We therefore hoped that PDGF could be used as a diagnostic and prognostic marker. We analyzed 78 samples of human CCA and adjacent tissues for PDGF and related gene expression, and localized PDGF protein expression. The mechanism of anti-cancer drugs on PDGF and related genes or proteins in CCA cell lines (OCA17, M156, and KKU100) was studied through MTT cell viability assay, quantitative real-time PCR, and immunoblotting. Mutagenesis of the PDGFRA coding region was analyzed. Moreover, the PDGFRA in sera of CCA patients and healthy controls was investigated. PDGFA was found to be upregulated in CCA tissue (84.6 %). Positive PDGFA immunohistochemical staining was significantly correlated with status (P = 0.000), stage of CCA (P = 0.013), metastasis (P = 0.017), and short survival rate (P = 0.005), and the multivariate analysis confirmed that PDGFA positive immunostaining had a higher likelihood of the risk of death (HR = 2.907, P = 0.016). For DNA point mutation of the PDGFRA sequence, silent mutations were found at tyrosine kinase 2 V824V (exon 18) and A603A (exon 13), and a missense mutation in S478P (exon 10); there was only a missense mutation in S478P (29 %) that has significant correlation with the histopathological grading (P = 0.037) and positive immunoreactive PDGFA (P = 0.021). In vitro cell line study by immunowestern blotting found that sunitinib malate had an inhibitory effect on the PDGFA pathway by decreasing p-PDGFRA, AKT, and p-AKT expression. The serum level of PDGFA in CCA patients was significantly higher than those of healthy control by 1.4-fold (P = 0.014). The present results suggest that PDGFA and PDGFRA may be used for CCA prognosis and/or as diagnostic candidate markers.

Chronic opisthorchiasis caused by Opisthorchis viverrini infection is characterized by advanced periductal fibrosis leading to hepatobiliary diseases (HBD), including cholangiocarcinoma (CCA). We aimed to determine fibrotic markers to differentiate HBD status including opisthorchiasis, benign biliary disease (BBD) and CCA. Candidate fibrotic markers in plasma of healthy individuals (n = 14) and patients with opisthorchiasis (n = 32, pre- and post-treatment with praziquantel), BBD (n = 31), CCA (n = 37) and other types of tumors (n = 14) were measured by ELISA and zymography. Plasma levels of hydroxyproline (HYP), collagen I, MMP-7 and TIMP2 in opisthorchiasis patients were significantly higher than those in healthy individuals, and MMP9/TIMP2 balance may be associated with tissue resorption after praziquantel treatment. HYP and TIMP-2 levels were significantly correlated with periductal fibrosis status evaluated by ultrasonography. Plasma HYP level of CCA patients was the highest among HBD patients (p < 0.05). ROC curves revealed HYP, MMP-7 and collagen I levels significantly distinguished opisthorchiasis, BBD and CCA (p < 0.001). Odd ratio (OR) analysis demonstrated these markers in opisthorchiasis were predictable for BBD risk (p < 0.05; OR = 28.50, 10.12 and 4.63 for collagen I, MMP-7 and HYP, respectively), and the risk was reduced by praziquantel treatment. Interestingly, only plasma HYP level in BBD was predictable for CCA risk (OR = 3.69; p = 0.020). In conclusion, plasma HYP, collagen I and MMP-7 may be useful as novel predictive markers of opisthorchiasis-related BBD, and HYP may be used as a diagnostic marker for CCA., (Copyright © 2011 UICC.)

Galectin-1 is a beta-galactoside-binding lectin to function in cell adhesion, proliferation, differentiation, and might be involved in tumor progression and metastasis. In the present study, the expression kinetics of galectin-1 during the tumorigenesis of a parasite Opisthorchis viverrini infection-induced cholangiocarcinoma (CCA) was investigated in model animal hamsters, and the expression was confirmed in human CCA cases. It was found that galectin-1 was overexpressed at mRNA and protein levels with the tumor progression. The mRNA expression was elevated in very early stage during tumorigenesis and the increase was time dependent. Galectin-1 protein expression profiles indicated that the increased expression was mainly located in the epithelium of extensively proliferated and hyperplasia small bile ducts at early stage of CCA development in model animal and mainly in the extensive tumor stroma tissues in both model animals and human CCA cases at later stage. The analysis of correlation of the overexpression with clinicopathology in human cases suggested that high expression of galectin-1 was associated with advanced stage and metastasis and with shorter cumulative overall survival of the patients. Multivariate Cox regression analysis revealed that galectin-1 expression was of independent prognostic significance for CCA. Our results suggest that galectin-1 is likely involved in the tumorigenesis and expected to serve as a tumor stroma marker in diagnosis and prediction of metastasis and poor prognosis of the opisthorchiasis-associated CCA.

Carbonylation is an irreversible and irreparable protein modification induced by oxidative stress. Cholangiocarcinoma (CCA) is associated with chronic inflammation caused by liver fluke infection. To investigate the relationship between protein carbonylation and CCA progression, carbonylated proteins were detected by 2D OxyBlot and identified by MALDI-TOF/TOF analyses in pooled CCA tissues in comparison to adjacent nontumor tissues and normal liver tissues. We identified 14 highly carbonylated proteins in CCA tissues. Immunoprecipitation and Western blot analyses of individual samples confirmed significantly greater carbonylation of serotransferrin, heat shock protein 70-kDa protein 1 (HSP70.1), and α1-antitrypsin (A1AT) in tumor tissues compared to normal tissues. The oxidative modification of these proteins was significantly associated with poor prognoses as determined by the Kaplan-Meier method. LC-MALDI-TOF/TOF mass spectrometry identified R50, K327, and P357 as carbonylated sites in serotransferrin, HSP70.1, and A1AT, respectively. Moreover, iron accumulation was significantly higher in CCA tissues with, compared to those without, carbonylated serotransferrin. We conclude that carbonylated serotransferrin-associated iron accumulation may induce oxidative stress via the Fenton reaction, and the carbonylation of HSP70.1 with antioxidative property and A1AT with protease inhibitory capacity may cause them to become dysfunctional, leading to CCA progression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cholangiocarcinoma is a crucial health problem in northeast Thailand. Although rare, it is a highly fatal disease and the prognosis of CCA patients is very poor. To determine if expression of specific genes is useful for diagnosis and prognosis for CCA. We examined the expression of HSP70, HSP90, RB1, cyclin D1, and HDAC6 in 50 resections of human CCA tissues by quantitative real-time PCR. The expression of HSP70, RB1, and HDAC6 was "dominant down-regulation," while the expression of cyclin D1 and HSP90 was "dominant up-regulation." There were no correlations between RB1, cyclin D1, HSP90, and clinicopathological parameters such as status, histology type, histological grading, stage of CCA, and metastasis. A significant association was found between HDAC6 and CCA staging (p = 0.000), CCA gross type and HSP70 (p = 0.046) as well as RB1 expression (p = 0.046). Patients with down-regulation of HSP70 had significantly poorer prognosis than those in the up-regulation group (p = 0.002). Expression of HSP70 may be useful as a new prognostic marker for CCA.

Chronic inflammation induced by liver fluke (Opisthorchis viverrini) infection is the major risk factor for cholangiocarcinoma (CCA) in Northeastern Thailand. Increased levels of proinflammatory cytokines and nuclear factor kappa B that control cyclooxygenase-2 and inducible nitric oxide activities, disturb the homeostasis of oxidants/anti-oxidants and DNA repair enzymes, all of which appear to be involved in O. viverrini-associated inflammatory processes and CCA. Consequently oxidative and nitrative stress-related cellular damage occurs due to the over production of reactive oxygen and nitrogen species in inflamed target cells. This is supported by the detection of high levels of oxidized DNA and DNA bases modified by lipid peroxidation products in both animal and human tissues affected by O. viverrini-infection. Treatment of opisthorchiasis patients with praziquantel, an anti- trematode drug was shown to reduce inflammation-mediated tissue damage and carcinogenesis. The principal mechanisms that govern the effects of inflammation and immunity in liver fluke-associated cholangiocarcinogenesis are reviewed. The validity of inflammation-related biomolecules and DNA damage products to serve as predictive biomarkers for disease risk evaluation and intervention is discussed., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Proteomic analysis was performed to search for the diagnostic biomarkers of the early stage of cholangiocarcinoma (CCA). For this purpose, CCA was experimentally induced in hamsters by the combination of N-nitrosodimethylamine (NDMA) treatment and Opisthorchis viverrini (OV) infection. Pooled plasma of normal control, NDMA-treated, OV-infected and OV+NDMA (ON) treated group was separated by 1-D PAGE, and the trypsin-digested bands were analyzed with LC-MS/MS. Among 82 overexpressed proteins, the study focused on 26 proteins overexpressed in ON group because CCA development was almost exclusively found in this group. A further selection was made based on the protein overexpression on day 21, the precancerous stage. Orosomucoid 2 (Orm2) was overexpressed in OV and ON groups and kinesin 18A (KIF18A) was overexpressed in the ON group. The overexpression levels were verified by real-time RT-PCR and western blotting in the liver and plasma. The transcription and translation levels of these two candidate molecules increased significantly at 21 days post-treatment before tumor development. Immunohistochemistry revealed KIF18A was expressed in the epithelial cells of newly formed small bile ducts, some inflammatory cells and hepatocytes. These results suggest that Orm2 and KIF18A could be the potential biomarkers for early diagnosis of CCA.

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.

Opisthorchiasis is the major public health problem in the endemic areas of Thailand and Laos because Opisthorchis viverrini infection causes serious hepatobiliary diseases including CCA. The molecular mechanism of the CCA carcinogenesis induced by the infection remains obscure. To reveal the potential genes and signaling pathways to involve in the carcinogenesis, the present study investigated the expression of c-Ski, an oncogene, and two TGF-β signaling pathway relative genes, TGF-β and Smad4, during the development of CCA induced by O. viverrini infection in hamster model, and in human opisthorchiasis associated CCA. The results showed that the expression of c-Ski gene was greatly up-regulated during the carcinogenesis of CCA in hamster model. The overexpression of c-Ski was confirmed by immunohistological staining result which showed the increased expression of c-Ski protein in cytoplasm of the epithelial lining of hepatic bile ducts. Moreover, the immunohistological staining of the specimens of human opisthorchiasis associated CCA revealed the up-regulated expression of c-Ski and Smad4 proteins in the cytoplasm of the epithelial lining of hepatic bile ducts and stomal fibrosis respectively. The expression of TGF-β and Smad4 were up-regulated, which expression kinetics was time-dependent of CCA development. These results suggest that c-Ski is likely involved in the carcinogenesis of CCA induced by O. viverrini infection through regulating TGF-β signaling pathway.

Infection by Opisthorchis viverrini, a risk factor for cholangiocarcinoma (CCA) may act through chronic inflammation, oxidative stress and lipid peroxidation (LPO)-related damage and growth stimuli. 1,N6-etheno-2'-deoxyadenosine (epsilondA), and 3,N4-etheno-2'-deoxycytidine (epsilondC), markers for LPO-derived DNA damage were highly increased in white blood cell and urine of O. viverrini-infected Thai patients. In order to investigate tissue specificity etheno adducts were measured in a cholangiocarcinogenesis model, in O. viverrini-infected hamsters that had received N-nitrosodimethylamine (NDMA, 12.5 ppm in dw) for 2 months. epsilondA- and epsilondC-levels were analyzed in paraffin-embedded liver sections by a novel immunohistochemical method, from 21 up to 180 days post-O. viverrini-infection. In inflamed areas of the liver, etheno adducts were localized in the nuclei of inflammatory cells and in the epithelial lining of the bile duct. Semi-quantitative image analysis showed higher adduct levels in the liver of O. viverrini-infected hamsters, treated with or w/o NDMA when compared with untreated controls. Levels were found highest in the liver of O. viverrini-infected plus NDMA-treated hamsters. Adducts increased in an age-dependent manner from O. viverrini-infection until CCA development. Increased adduct formation paralleled histopathological changes in plasma alkaline phosphatase (ALP) activity, bile duct hyperplasia, dysplasia, precancerous lesions, and CCA appearance. Also elevated expression of alkyladenine DNA glycosylase (AAG), which excises 1,N6-ethenoadenine (epsilonA) was linked to higher adduct formation, suggesting imbalanced repair. Our results implicate accumulation of inflammation-related, promutagenic DNA damage in target tissue and possibly imbalanced repair in the onset of cholangiocarcinogenesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Parasite infection of Opisthorchis viverrini is a major risk factor for cholangiocarcinoma. Our previous immunohistochemical studies showed that O. viverrini infection induced oxidative DNA lesions in the bile duct epithelium during cholangiocarcinoma development. The current study assessed the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an oxidative DNA lesion, in the urine and leukocytes of O. viverrini-infected subjects and cholangiocarcinoma patients. Forty-nine O. viverrini-infected patients, 55 cholangiocarcinoma patients, and 17 healthy controls were enrolled in the study. We measured 8-oxodG levels in the urine and leukocytes of these subjects using an electrochemical detector coupled to high-performance liquid chromatography. O. viverrini-infected patients were assessed before treatment and 2 months and 1 year after praziquantel treatment. Urinary 8-oxodG levels were significantly higher in cholangiocarcinoma patients (6.83 +/- 1.00 microg/g creatinine) than in O. viverrini-infected patients (4.45 +/- 0.25 mug/g creatinine; P < 0.05) and healthy subjects (3.03 +/- 0.24 microg/g creatinine; P < 0.01) and higher in O. viverrini-infected subjects than in healthy subjects (P < 0.01). The urinary 8-oxodG levels in O. viverrini-infected patients significantly decreased 2 months after praziquantel treatment and were comparable with levels in healthy subjects 1 year after treatment. Urinary 8-oxodG levels were significantly correlated with leukocyte 8-oxodG levels, plasma nitrate/nitrite levels, and aspartate aminotransferase activity. In conclusion, this study, in addition to our previous studies, indicates that 8-oxodG formation by parasite infection may play an important role in cholangiocarcinoma development. Urinary 8-oxodG may be a useful biomarker to monitor not only infection but also carcinogenesis.