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Background/aim: Cholangiocarcinoma is a lethal cancer, and current chemotherapeutic drugs are not very effective. Recent studies reported that cholangiocarcinoma cells were sensitive to adenosine. One adenosine analog, 8-chloroadenosine (8-CA), was shown to be more potent than adenosine and induced apoptosis in leukemia cells. This study examined effects of 8-CA in cholangiocarcinoma cells and immortalized cholangiocytes., Materials and Methods: Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell invasion was examined by transwell assay. Cell cycle and cell death were evaluated by flow cytometry. Colorimetric absorbance assay was used to assessed RNA and protein synthesis as well as mitochondrial membrane potential. Protein levels were examined by western blot analysis. Animal experiment was performed in Balb/cAJcl-Nu mice., Results: 8-CA reduced cholangiocarcinoma cell growth, prevented colony formation and caused endoplasmic reticulum stress and cell-cycle arrest. Eventually, apoptosis was induced. However, treatment with 8-CA did not interfere with RNA synthesis or protein synthesis and did not alter mitochondrial membrane potential. Combination of 8-CA with several chemotherapeutic drugs in vitro was less effective than 8-CA alone and the drugs alone, except for the combination of 8-CA with hydroxychloroquine, which had an additive effect on RMCCA-1 cells. However, further in vivo study showed that treatment with 8-CA alone inhibited tumor growth more than treatment with a combination of 8-CA with hydroxychloroquine., Conclusion: 8-Chloroadenosine inhibited CCA cells by inducing endoplasmic reticulum stress and apoptosis. In vivo study showed that 8-CA inhibited cholangiocarcinoma tumor growth better when administered alone as compared to a combination with hydroxychloroquine., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: Cholangiocarcinoma (CCA) is a cancer of the bile duct with a poor prognosis. The present study examined the ability of curcumin to sensitize apoptosis in the TNF-related apoptosis-inducing ligand (TRAIL)-resistant CCA cell lines of HuCCA-1 and KKU-213A., Methods: Apoptosis was measured using a TUNEL assay. Protein expression was determined by immunoblotting. Membrane death receptor 5 (DR5) was detected by flow cytometry. Protein complex was examined by co-immunoprecipitation., Result: Curcumin potentiated TRAIL-induced apoptosis in both cell lines, indicating the sensitization to TRAIL-induced apoptosis by curcumin. Additionally, curcumin increased DR5 expression and membrane localization; however, the curcumin/TRAIL combination did not result in further increases in DR5 expression and membrane localization in either cell line. Moreover, the curcumin/TRAIL combination reduced DR5/decoy receptor 2 (DcR2) complexes in both cell lines, suggesting that curcumin may enhance TRAIL-induced apoptosis by disrupting DR5/DcR2 interaction. In addition, levels of the anti-apoptotic complex DR5/ DDX3/GSK3β were reduced by the curcumin/TRAIL combination in HuCCA-1 but not in KKU-213A cells. This study also demonstrated that the DR5/DcR2 and DR5/DDX3/GSK3β complexes could be observed under basal conditions, suggesting that these anti-apoptotic complexes may contribute to TRAIL-resistant phenotypes in both cell lines. Pretreatment with the antioxidant N-acetylcysteine attenuated curcumin-enhanced apoptosis by TRAIL, indicating that curcumin sensitized TRAIL-induced apoptosis through an oxidative stress-dependent mechanism., Conclusion: The present study demonstrates the potential of using curcumin in combination with TRAIL to yield better TRAIL therapy outcomes in TRAIL-resistant CCA.

Background: The incidence of cholangiocarcinoma (CCA) is increasing worldwide and current single chemotherapeutic drug treatments are ineffective. CX-4945 and cisplatin are currently in clinical trial for CCA treatment., Materials and Methods: We assessed the effects of the sequence of administration of CX-4945 and cisplatin applied in combination treatments on their efficacy in CCA cells in vitro. CCA cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Apoptosis was examined using flow cytometry. The percentage of cells positive for phosphorylated H2A histone family member X (γ-H2AX) were measured using both flow cytometry and immunofluorescence., Results: CCA cell viability was reduced to 50% after 24 h of treatments with CX-4945 and cisplatin as single agents. Interestingly, treatment with cisplatin 6 h prior to CX-4945 treatment induced significantly more DNA damage and apoptosis than CX-4945 treatment followed by cisplatin. Unexpectedly, CX-4945 treatment followed by cisplatin was less effective than single treatment in RMCCA-1 CCA cells. In addition, a 1:1 ratio of each drug was the most effective combination in these cells., Conclusion: These data demonstrate that the combination of CX-4945 and cis platin acts additively when cisplatin is applied first, at least in part due to increased DNA damage and apoptosis. Furthermore, treatment with CX-4945 prior to cisplatin treatment reduces the efficacy of this drug combination in CCA cells., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Cholangiocarcinoma is a lethal disease with increasing incidence worldwide. New therapeutic compounds are urgently needed for this disease. Here, the inhibitory effect of adenosine on cholangiocarcinoma cells was studied., Materials and Methods: Western blot analysis was used to study autophagy and flow cytometry to analyze cell death and the cell cycle., Results: Cholangiocarcinoma and immortalized cholangiocytes responded to adenosine differently, and adenosine inhibited cholangiocarcinoma cell growth by inducing autophagy. Adenosine failed to activate adenylyl cyclase in cholangiocarcinoma cell lines, but activated this enzyme in immortalized cholangiocytes. Adenosine treatment activated AMPK and led to phosphorylation of its downstream proteins including ULK and Raptor. In addition, autophagy induced by adenosine appeared to be a survival mechanism. The combination of adenosine with autophagy inhibitors greatly increased cell death, as compared to the use of either agent alone. Interestingly, immortalized cholangiocytes were more resistant to adenosine., Conclusion: Adenosine may have potential for application in cholangiocarcinoma treatment., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cholangiocarcinoma (CCA) is a malignant biliary tract epithelium tumor. The programmed death-1 (PD-1)/programmed receptor-ligand 1 (PD-L1) signaling pathway has been implicated as an immune escape mechanism in several cancers. The present study aimed to assess the expression of PD-L1 on human CCA cell lines and its potential role in suppressing CD8 + T- cell function. A panel of intrahepatic CCA cell lines was evaluated for immune regulatory checkpoint ligands and inflammation markers. Effects of pro-inflammatory cytokine, interferon gamma (IFN-γ), on the expression of immune regulatory checkpoint ligands and inflammation markers were determined. The PD-L1 function was measured by co-culturing CCA cells with lymphocytes. Most of the selected Thai CCA cell lines, including HuCCA-1, RMCCA-1, KKU-100, and KKU-213, expressed higher PD-L1 than normal cholangiocyte MMNK-1 and ANK-1 cells. Both PD-L1 and cyclooxygenase-2 (COX-2) expressions were highest in HuCCA-1 cells. A 48 h treatment with IFN-γ increased the expression of PD-L1 and COX-2 in CCA cells. The expression of CTLA-4 ligands, including H7-1 and H7-2, did not change after IFN-γ treatment. Rofecoxib, a specific COX-2 inhibitor, mitigated IFN-γ-induced PD-L1 expression. After 48 h co-incubation, CD8 + T-cell apoptosis was increased as compared to the control group. Pretreatment of CCA cells with IFN-γ further increased CD8 + T-cell apoptosis. Pembrolizumab, an anti-PD-1 antibody, mitigated CCA cell escape phenomenon. The inhibition of T-cell-mediated immune response via the PD-L1/PD-1 axis are evidenced in intrahepatic CCA. Immunotherapy with checkpoint inhibitor offers a potentially therapeutic strategy for CCA patients; however, further in vivo and clinical studies are required., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aberrant Notch and Wnt signaling are known drivers of cholangiocarcinoma (CCA), but the underlying factors that initiate and maintain these pathways are not known. Here, we show that the proline-rich homeodomain protein/hematopoietically expressed homeobox (PRH/HHEX) transcription factor forms a positive transcriptional feedback loop with Notch3 that is critical in CCA. PRH/HHEX expression is elevated in CCA, and depletion of PRH reduces CCA tumor growth in a xenograft model. Overexpression of PRH in primary human biliary epithelial cells is sufficient to increase cell proliferation and produce an invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 reveals that unlike Notch3, PRH directly activates canonical Wnt signaling. These data indicate that hyperactivation of Notch and Wnt signaling is independent of the underlying mutational landscape and has a common origin in dysregulation of PRH. Moreover, they suggest new therapeutic options based on the dependence of specific Wnt, Notch, and CDK4/6 inhibitors on PRH activity. SIGNIFICANCE: The PRH/HHEX transcription factor is an oncogenic driver in cholangiocarcinoma that confers sensitivity to CDK4/6 inhibitors., (©2019 American Association for Cancer Research.)

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol's mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that FOS up-regulation was related to CCA pathogenesis. This study is the first providing more information and molecular mechanisms about global transcriptome alterations and oncogenic enhancement of chronic alcohol exposure in normal cholangiocytes.

Previous studies showed that glyphosate stimulates breast cancer cell growth via estrogen receptors. The present study investigated the effect of glyphosate on the estrogen signaling pathway involved in the induction of cholangiocarcinoma (CCA) cell growth. HuCCA-1, RMCCA-1 and MMNK-1 were chosen for comparison. The effects of glyphosate on cell growth, cell cycle and molecular signaling pathways were measured. The results showed that HuCCA-1 cells expressed estrogen receptor alpha (ERα), while ERα was not detected in RMCCA-1 and MMNK-1 cells. ERα was mostly expressed in cytoplasmic compartment of HuCCA-1 cells. Estradiol (E2) (10 -11 -10 -5  M) induced cell proliferation in HuCCA-1 but not in RMCCA-1 and MMNK-1 cells. Glyphosate at the same concentration range also induced HuCCA-1 cell proliferation. The S phase of the cell cycle, and protein levels of the cyclin family were significantly increased after treatment of glyphosate or E2. Both compounds also induced the expression of proliferative signaling-related proteins including ERα, VEGFR2, pERK, PI3K(p85), and PCNA. These effects of glyphosate and E2 were abolished by the ER antagonist, 4-hydroxytamoxifen and U0126, a MEK inhibitor. The data from this study indicate that glyphosate can induce cell growth in ERα positive CCA cells through non-genomic estrogen receptor/ERK1/2 signaling pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium associated with Opisthorchis viverrini, primary sclerosing cholangitis and hepatitis viral infection. In the global population, men have higher incidence rates for CCA than women; thus, a gender disparity in the progression of chronic inflammation of the biliary duct leading to malignancy may involve the effects of estrogen (E2). Genistein (GE), a prominent phytoestrogen found in soy products, is an estrogen receptor β (ERβ) agonist and a tyrosine kinase inhibitor. The present study investigated the effects of GE on the growth of CCA cells by cell viability assay. The effects on signaling proteins were detected by western blot analysis and ELISA. Gene expression was examined by RT-qPCR. Two human intrahepatic CCA cell lines, HuCCA‑1 and RMCCA‑1, were utilized. GE (50‑200 µM) reduced the viability of the two cell lines, and also inhibited the activation of epidermal growth factor receptor (EGFR) and AKT, as evidenced by decreasing protein levels of phosphorylated (p)-EGFR (Tyr1173) and p‑AKT (Ser473), respectively. GE altered the mitogen‑activated protein kinase signaling cascade by mediating decreased protein levels of p‑p38 and increased protein levels of p‑ERK1/2. GE significantly decreased the levels of interleukin 6 (IL6) and induced the expression of inducible nitric oxide synthase (iNOS). GE also downregulated the expression of p‑ERα (Ser118) protein and ERα mRNA levels. Finally, GE induced the downregulation of the protein levels of ERβ. Of note, E2 deprivation potentiated the GE-induced reduction of p‑EGFR (Tyr1173) and total AKT proteins and production of IL6, and mediated the downregulation of GE-induced iNOS protein. In conclusion, GE inhibited the growth of human CCA cell lines by reducing the activation of EGFR and AKT, and by attenuating the production of IL6. E2 and ER were also involved in the growth-inhibitory effect of GE in CCA cells.

Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1-40 µM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth.

Andrographis paniculata is an important herbal medicine widely used in several Asian countries for the treatment of various diseases due to its broad range of pharmacological activities. The present study reports that A. paniculata extracts potently inhibit the growth of liver (HepG2 and SK-Hep1) and bile duct (HuCCA-1 and RMCCA-1) cancer cells. A. paniculata extracts with different contents of major diterpenoids, including andrographolide, 14-deoxy-11,12-didehydroandrographolide, neoandrographolide, and 14-deoxyandrographolide, exhibited a different potency of growth inhibition. The ethanolic extract of A. paniculata at the first true leaf stage, which contained a high amount of 14-deoxyandrographolide but a low amount of andrographolide, showed a cytotoxic effect to cancer cells about 4 times higher than the water extract of A. paniculata at the mature leaf stage, which contained a high amount of andrographolide but a low amount of 14-deoxyandrographolide. Andrographolide, not 14-deoxy-11,12-didehydroandrographolide, neoandrographolide, or 14-deoxyandrographolide, possessed potent cytotoxic activity against the growth of liver and bile duct cancer cells. The cytotoxic effect of the water extract of A. paniculata at the mature leaf stage could be explained by the present amount of andrographolide, while the cytotoxic effect of the ethanolic extract of A. paniculata at the first true leaf stage could not. HuCCA-1 cells showed more sensitivity to A. paniculata extracts and andrographolide than RMCCA-1 cells. Furthermore, the ethanolic extract of A. paniculata at the first true leaf stage increased cell cycle arrest at the G0/G1 and G2/M phases, and induced apoptosis in both HuCCA-1 and RMCCA-1 cells. The expressions of cyclin-D1, Bcl-2, and the inactive proenzyme form of caspase-3 were reduced by the ethanolic extract of A. paniculata in the first true leaf stage treatment, while a proapoptotic protein Bax was increased. The cleavage of poly (ADP-ribose) polymerase was also found in the ethanolic extract of A. paniculata in the first true leaf stage treatment. This study suggests that A. paniculata could be a promising herbal plant for the alternative treatment of intrahepatic cholangiocarcinoma., (Georg Thieme Verlag KG Stuttgart · New York.)

Cholangiocarcinoma (CCA) remains to be a major health problem in several Asian countries including Thailand. The molecular mechanism of CCA is poorly understood. Early diagnosis is difficult, and at present, no effective therapeutic drug is available. The present study aimed to identify the molecular mechanism of CCA by gene expression profile analysis and to search for current approved drugs which may interact with the upregulated genes in CCA. Gene Expression Omnibus (GEO) was used to analyze the gene expression profiles of CCA patients and normal subjects. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology enrichment analysis was also performed, with the KEGG pathway analysis indicating that pancreatic secretion, protein digestion and absorption, fat digestion and absorption, and glycerolipid metabolism may serve important roles in CCA oncogenesis. The drug signature database (DsigDB) was used to search for US Food and Drug Administration (FDA)-approved drugs potentially capable of reversing the effects of the upregulated gene expression in CCA. A total of 61 antineoplastic and 86 non-antineoplastic drugs were identified. Checkpoint kinase 1 was the most interacting with drug signatures. Many of the targeted protein inhibitors that were identified have been approved by the US-FDA as therapeutic agents for non-antineoplastic diseases, including cimetidine, valproic acid and lovastatin. The current study demonstrated an application for bioinformatics analysis in assessing the potential efficacy of currently approved drugs for novel use. The present results suggest novel indications regarding existing drugs useful for CCA treatment. However, further in vitro and in vivo studies are required to support the current predictions. [ABSTRACT FROM AUTHOR]

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α′ catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings. [ABSTRACT FROM AUTHOR]