Microcystin-leucine arginine induces the proliferation of cholangiocytes and cholangiocarcinoma cells through the activation of the Wnt/ß-catenin signaling pathway.

Background: Microcystin-leucine arginine (MC-LR) is a cyanobacterial hepatotoxic toxin found in water sources worldwide, including in northeastern Thailand, where opisthorchiasis-associated cholangiocarcinoma (CCA) is most prevalent. MC-LR is a potential carcinogen; however, its involvement in liver fluke-associated CCA remains ambiguous. Here, we aimed to evaluate the effect of MC-LR on the progression of CCA via the Wnt/ß-catenin pathway in vitro. Methods: Cell division, migration, cell cycle transition, and MC-LR transporter expression were evaluated in vitro through MTT assay, wound healing assay, flow cytometry, and immunofluorescence staining, respectively. Following a 24-h treatment of cultured cells with 1, 10, 100, and 1,000 nM of MC-LR, the proliferative effect of MC-LR on the Wnt/ß-catenin signaling pathway was investigated using immunoblotting and qRT-PCR analysis. Immunohistochemistry was used to determine ß-catenin expression in CCA tissue compared to adjacent tissue. Results: Human immortalized cholangiocyte cells (MMNK-1) and a human cell line established from opisthorchiasis-associated CCA (KKU-213B) expressed the MC-LR transporter and internalized MC-LR. Exposure to 10 nM and 100 nM of MC-LR notably enhanced cells division and migration in both cell lines (P < 0.05) and markedly elevated the percentage of S phase cells (P < 0.05). MC-LR elevated PP2A expression by activating the Wnt/ß-catenin signaling pathway and suppressing phosphatase activity. Inhibition of the ß-catenin destruction complex genes (Axin1 and APC) led to the upregulation of ß-catenin and its downstream target genes (Cyclin D1 and c-Jun). Inhibition of Wnt/ß-catenin signaling by MSAB confirmed these results. Additionally, ß-catenin was significantly expressed in cancerous tissue compared to adjacent areas (P < 0.001). Conclusions: Our findings suggest that MC-LR promotes cell proliferation and progression of CCA through Wnt/ß-catenin pathway. Further evaluation using invivo experiments is needed to confirm this observation. This finding could promote health awareness regarding MC-LR intake and risk of CCA.

Anticancer properties and metabolomic profiling of Shorea roxburghii extracts toward gastrointestinal cancer cell lines.

BACKGROUND: Gastrointestinal cancer (GIC) ranks as the highest cause of cancer-related deaths globally. GIC patients are often diagnosed at advanced stages, limiting effective treatment options. Chemotherapy, the common GIC recommendation, has significant disadvantages such as toxicity and adverse effects. Natural products contain substances with diverse pharmacological characteristics that promise for use in cancer therapeutics. In this study, the flower of renowned Asian medicinal plant, Shorea roxburghii was collected and extracted to investigate its phytochemical contents, antioxidant, and anticancer properties on GIC cells. METHODS: The phytochemical contents of Shorea roxburghii extract were assessed using suitable methods. Phenolic content was determined through the Folin-Ciocalteu method, while flavonoids were quantified using the aluminum chloride (AlCl3) method. Antioxidant activity was evaluated using the FRAP and DPPH assays. Cytotoxicity was assessed in GIC cell lines via the MTT assay. Additionally, intracellular ROS levels and apoptosis were examined through flow cytometry techniques. The correlation between GIC cell viability and phytochemicals, 1H-NMR analysis was conducted. RESULTS: Among the four different solvent extracts, ethyl acetate extract had the highest phenolic and flavonoid contents. Water extract exhibited the strongest reducing power and DPPH scavenging activity following by ethyl acetate. Interestingly, ethyl acetate extract demonstrated the highest inhibitory activity against three GIC cell lines (KKU-213B, HepG2, AGS) with IC50 values of 91.60 µg/ml, 39.38 µg/ml, and 35.59 µg/ml, while showing less toxicity to normal fibroblast cells. Ethyl acetate extract induced reactive oxygen species and apoptosis in GIC cell lines by downregulating anti-apoptotic protein Bcl-2. Metabolic profiling-based screening revealed a positive association between reduced GIC cell viability and phytochemicals like cinnamic acid and its derivatives, ferulic acid and coumaric acid. CONCLUSIONS: This study highlights the potential of natural compounds in Shorea roxburghii in the development of more effective and safer anticancer agents as options for GIC as well as shedding light on new avenues for cancer treatment.

Overexpression of microRNA-205-5p promotes cholangiocarcinoma growth by reducing expression of homeodomain-interacting protein kinase 3.

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.

High prevalence of chronic kidney disease and its related risk factors in rural areas of Northeast Thailand.

In Thailand, chronic kidney disease (CKD) screening was reported in 2009 with an overall prevalence of 17.5% and the highest at 22.2% in the northeastern region. This study aimed to find out CKD prevalence of the Kidney Disease Improving Global Outcomes criteria and their related risk factors in the rural community. A population-based study was conducted in the rural sub-districts of northeastern Thailand. Data of socio-demographic status, lifestyle, underlying diseases, blood pressure, and body mass index were recorded. Blood and urine analysis was conducted along with ultrasonography of kidneys. Specimen collection and analyses were repeated after 3 months, and the factors associated with CKD were studied by logistic regression analysis. A total of 2205 participants with a mean age of 57.8 ± 11.7 years and female predominance (66.7%) completed the study. The prevalence of CKD was 26.8%, i.e., stages 1 (7.3%); stage 2 (9.0%); stage 3a (6.0%); stage 3b (2.8%); stage 4 (1.4%); and stage 5 (0.3%). Hypertension, diabetes mellitus, and renal stones were the major underlying diseases. Only 3.5% of the participants were aware of having CKD. An increase in age, male, unemployment, current smoking, diabetes, hypertension, underweight, anemia, hyperuricemia, and leukocytosis were significantly associated factors with the disease. The study revealed that CKD has developed as a significant public health problem in rural northeastern Thailand and one out of every four people has CKD. Therefore, early interventions are essential for the proper management and prevention of CKD.

Repeated Ivermectin Treatment Induces Ivermectin Resistance in Strongyloides ratti by Upregulating the Expression of ATP-Binding Cassette Transporter Genes.

Ivermectin (IVM) is a widely used anthelmintic. However, with widespread use comes the risk of the emergence of IVM resistance, particularly in strongyloidiasis. Adenosine triphosphate (ATP)-binding cassette (ABC) transporter genes play an important role in the IVM-resistance mechanism. Here, we aimed to establish an animal experimental model of IVM resistance by frequent treatment of Strongyloides ratti with subtherapeutic doses of IVM, resistance being evaluated by the expression levels of ABC transporter genes. Rats infected with S. ratti were placed in experimental groups as follows: 1) untreated control (control); 2) treated with the mutagen ethyl methanesulfonate (EMS); 3) injected with 100 µg/kg body weight of IVM (IVM); 4) treated with a combination of EMS and IVM (IVM+EMS). Parasites were evaluated after four generations. Extent of IVM resistance was assessed using IVM sensitivity, larval development, and expression of ABC genes. By the F4 generation, S. ratti in the IVM group exhibited significantly higher levels of IVM resistance than did other groups according to in vitro drug-sensitivity tests and inhibition of larval development (IC50 = 36.60 ng/mL; 95% CI: 31.6, 42.01). Expression levels of ABC isoform genes (ABCA, ABCF, and ABCG) were statistically significantly higher in the IVM-resistant line compared with the susceptible line. In conclusion, IVM subtherapeutic doses induced IVM resistance in S. ratti by the F4 generation with corresponding upregulation of some ABC isoform genes. The study provides a model for inducing and assessing drug resistance in Strongyloides.

Opisthorchis viverrini Infection Induces Metabolic and Fecal Microbial Disturbances in Association with Liver and Kidney Pathologies in Hamsters.

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 1H 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 ((CH2)n and CH2CH2CO) 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-ß-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.

CagA+ Helicobacter pylori infection and N-nitrosodimethylamine administration induce cholangiocarcinoma development in hamsters.

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.

Profiling of Bile Microbiome Identifies District Microbial Population between Choledocholithiasis and Cholangiocarcinoma Patients.

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.

N-glycosylation profiling of serum immunoglobulin in opisthorchiasis patients.

Alteration of immunoglobulin glycosylation correlates with inflammatory diseases and infectious diseases including parasitic infections. Immunoglobulin glycosylation patterns may be implicated in disease development and have also been proposed as diagnostic tools for several diseases. Previous studies have reported the immunoglobulin profiles in experimental animals and in patients infected with the carcinogenic human liver fluke, Opisthorchis viverrini. However, the N-glycosylation profiles of immunoglobulins and their subclass-specific glycoforms in opisthorchiasis patients have never been elucidated. Here, N-glycosylation patterns of immunoglobulins and their subclass-specific glycoforms in sera of O. viverrini-infected patients were investigated using triple quadrupole mass spectrometry coupled with multiple reaction monitoring. Peptide fragmentation was utilized to quantify the immunoglobulin glycoforms normalized to the unique peptide of each subclass. Overall, serum levels of IgG and IgA in O. viverrini patients were significantly increased compared to uninfected controls. Twenty-seven glycoforms were detected based on analysis of detached glycans in all immunoglobulin subclasses. The abundance of immunoglobulin glycopeptides in serum of opisthorchiasis patients deviated significantly from controls. Immunoglobulin glycosylation patterns were associated with both pro- and anti-inflammatory properties. In conclusion, O. viverrini infection alters the serum immunoglobulin glycosylation profile and these changes could distinguish between O. viverrini-infected individuals and healthy controls. SIGNIFICANCE: We demonstrated that both quantities and glycoforms of serum immunoglobulin subclasses were altered in Opisthorchis viverrini-infected individuals as investigated by the QqQ-MS-MRM method. Patterns of immunoglobulin with a specific glycoform might contribute to immune responses to O. viverrini infection.

Expression of FOXO4 Inhibits Cholangiocarcinoma Cell Proliferation In Vitro via Induction of G0/G1 Arrest.

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 G0/G1 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 G1/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 G1/S transition, leading to G0/G1 arrest. Our findings suggest that induction of FOXO4 expression might be an alternative approach for the treatment of CCA.

Anti-parasitic Drug Ivermectin Exhibits Potent Anticancer Activity Against Gemcitabine-resistant Cholangiocarcinoma In Vitro.

BACKGROUND/AIM: The antiparasitic drug, ivermectin (IVM), exerts anticancer activities in diverse cancer types. However, its anticancer activity against cholangiocarcinoma (CCA), especially the drug-resistant phenotype, has not yet been explored. MATERIALS AND METHODS: IVM was tested for its anticancer activity against gemcitabine-sensitive (KKU214) and gemcitabine-resistant (KKU214GemR) CCA cell lines in vitro using the sulforhodamine B and clonogenic assays as well as cell-cycle analysis. RESULTS: IVM treatment inhibited cell proliferation and colony formation of both KKU214 and KKU214GemR in a dose- and time-dependent manner. KKU214GemR cells were more sensitive than KKU214 to IVM treatment. IVM treatment caused S-phase cell-cycle arrest and also cell death as indicated by an increase of sub-G0/G1 population in KKU214GemR cells treated with IVM for 48 h. CONCLUSION: IVM exerts anti-CCA activities and gemcitabine-resistant KKU214GemR cells are more sensitive to IVM treatment. Thus, IVM might be useful as an alternative treatment for CCA, especially in patients who do not respond to gemcitabine.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.

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.

Co-occurrence of opisthorchiasis and diabetes exacerbates morbidity of the hepatobiliary tract disease.

Complications arising from infection with the carcinogenic liver fluke Opisthorchis viverrini cause substantial morbidity and mortality in Thailand and adjacent lower Mekong countries. In parallel, the incidence rate of diabetes mellitus (DM) is increasing in this same region, and indeed worldwide. Many residents in opisthorchiasis-endemic regions also exhibit DM, but the hepatobiliary disease arising during the co-occurrence of these two conditions remains to be characterized. Here, the histopathological profile during co-occurrence of opisthorchiasis and DM was investigated in a rodent model of human opisthorchiasis in which diabetes was induced with streptozotocin. The effects of excretory/secretory products from the liver fluke, O. viverrini (OVES) on hepatocyte and cholangiocyte responses during hyperglycemic conditions also were monitored. Both the liver fluke-infected hamsters (OV group) and hamsters with DM lost weight compared to control hamsters. Weight loss was even more marked in the hamsters with both opisthorchiasis and DM (OD group). Hypertrophy of hepatocytes, altered biliary canaliculi, and biliary hyperplasia were more prominent in the OD group, compared with OV and DM groups. Profound oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine, proliferating cell nuclear antigen, and periductal fibrosis characterized the OD compared to OV and DM hamsters. Upregulation of expression of cytokines in response to infection and impairment of the pathway for insulin receptor substrate (IRS)/phosphatidylinositol-3-kinases (PI3K)/protein kinase B (AKT) signaling attended these changes. In vitro, OVES and glucose provoked time- and dose-dependent effects on the proliferation of both hepatocytes and cholangiocytes. In overview, the co-occurrence of opisthorchiasis and diabetes exacerbated pathophysiological damage to the hepatobiliary tract. We speculate that opisthorchiasis and diabetes together aggravate hepatobiliary pathogenesis through an IRS/PI3K/AKT-independent pathway.

Proteomics detection of S100A6 in tumor tissue interstitial fluid and evaluation of its potential as a biomarker of cholangiocarcinoma.

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.

Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line.

Cholangiocarcinoma (CCA) is a deleterious bile duct tumor with poor prognosis and is relatively resistant to chemotherapy. Therefore, alternative or supplementary agents with anticancer and chemosensitizing activities may be useful for the treatment of CCA. A novel anthocyanin complex (AC) nanoparticle, developed from extracts of cobs of purple waxy corn and petals of blue butterfly pea, has exhibited chemopreventive potential in vivo. In the present study, the anti-CCA activities of AC and their underlying molecular mechanisms were investigated further in vitro using a CCA cell line (KKU213). The potential use of AC as a chemosensitizer was also evaluated in a gemcitabine-resistant CCA cell line (KKU214GemR). It was demonstrated that AC treatment suppressed proliferation of KKU213 CCA cells in dose- and time-dependent manners. AC treatment also induced apoptosis and mitochondrial superoxide production, decreased clonogenicity of CCA cells, and downregulated forkhead box protein M1 (FOXM1), nuclear factor-κB (NF-κB) and pro-survival protein B-cell lymphoma-2 (Bcl-2). The expression of endoplasmic reticulum (ER) stress-response proteins, including protein kinase RNA-like ER kinase, phosphorylated eIF2α, eukaryotic initiation factor 2α and activating transcription factor 4, also decreased following AC treatment. It was also identified that AC treatment inhibited KKU214GemR cell proliferation in dose- and time-dependent manners. Co-treatment of KKU214GemR cells with low doses of AC together with gemcitabine significantly enhanced efficacy of the latter against this cell line. Therefore, it is suggested that AC treatment is cytotoxic to KKU213 cells, possibly via downregulation of FOXM1, NF-κB, Bcl-2 and the ER stress response, and by induction of mitochondrial superoxide production. AC also sensitizes KKU214GemR to gemcitabine treatment, which may have potential for overcoming drug resistance of CCA.

Melatonin suppresses eosinophils and Th17 cells in hamsters treated with a combination of human liver fluke infection and a chemical carcinogen.

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.

Differential Protein Expression Marks the Transition From Infection With Opisthorchis viverrini to Cholangiocarcinoma.

Parts of Southeast Asia have the highest incidence of intrahepatic cholangiocarcinoma (CCA) in the world because of infection by the liver fluke Opisthorchis viverrini (Ov). Ov-associated CCA is the culmination of chronic Ov-infection, with the persistent production of the growth factors and cytokines associated with persistent inflammation, which can endure for years in Ov-infected individuals prior to transitioning to CCA. Isobaric labeling and tandem mass spectrometry of liver tissue from a hamster model of CCA was used to compare protein expression profiles from inflammed tissue (Ovinfected but not cancerous) versus cancerous tissue (Ov-induced CCA). Immunohistochemistry and immunoblotting were used to verify dysregulated proteins in the animal model and in human tissue. We identified 154 dysregulated proteins that marked the transition from Ov-infection to Ov-induced CCA, i.e. proteins dysregulated during carcinogenesis but not Ov-infection. The verification of dysregulated proteins in resected liver tissue from humans with Ov-associated CCA showed the numerous parallels in protein dysregulation between human and animal models of Ov-induced CCA. To identify potential circulating markers for CCA, dysregulated proteins were compared with proteins isolated from exosomes secreted by a human CCA cell line (KKU055) and 27 proteins were identified as dysregulated in CCA and present in exosomes. These data form the basis of potential diagnostic biomarkers for human Ov-associated CCA. The profile of protein dysregulation observed during chronic Ovinfection and then in Ov-induced CCA provides insight into the etiology of an infection-induced inflammation-related cancer.

Coinfection with Helicobacter pylori and Opisthorchis viverrini Enhances the Severity of Hepatobiliary Abnormalities in Hamsters.

Persistent infection with Opisthorchis viverrini causes hepatobiliary abnormalities, predisposing infected individuals to cholangiocarcinoma (CCA). In addition, Helicobacter pylori is highly prevalent in most countries and is a possible risk factor for CCA; however, its role in enhancing hepatobiliary abnormality is unclear. Here, we investigated the effects of coinfection with H. pylori and O. viverrini on hepatobiliary abnormality. Hamsters were divided into four groups: (i) normal, (ii) H. pylori infected (HP), (iii) O. viverrini infected (OV), and (iv) O. viverrini and H. pylori infected (OV+HP). At 6 months postinfection, PCR and immunohistochemistry were used to test for the presence of H. pylori in the stomach, gallbladder, and liver. In the liver, H. pylori was detected in the following order: OV+HP, 5 of 8 (62.5%); HP, 2 of 5 (40%); OV, 2 of 8 (25%). H. pylori was not detected in normal (control) liver tissues. Coinfection induced the most severe hepatobiliary abnormalities, including periductal fibrosis, cholangitis, and bile duct hyperplasia, leading to a significantly decreased survival rate of experimental animals. The greatest thickness of periductal fibrosis was associated with a significant increase in fibrogenesis markers (expression of alpha smooth muscle actin and transforming growth factor beta). Quantitative reverse transcription-PCR revealed that the highest expression levels of genes for proinflammatory cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) were also observed in the OV+HP group. These results suggest that coinfection with H. pylori and O. viverrini increased the severity of hepatobiliary abnormalities to a greater extent than either single infection did.

Plasma orosomucoid 2 as a potential risk marker of cholangiocarcinoma.

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.