Background/Aim: Cholangiocarcinoma (CCA) is an aggressive cancer for which standard treatments are still ineffective. This study demonstrated the antiproliferative and anti-metastatic activity of metformin, an anti-diabetic drug, in CCA cells. Materials and Methods: Cell proliferation, migration/invasion and anoikis resistance were determined. The underlying mechanisms were identified using western blotting and immunocytofluorescence. Results: Metformin significantly suppressed proliferation of CCA cells in a dose- and time-dependent manner, regardless of glucose present in the medium. A low dose of metformin significantly increased anoikis and inhibited migration/ invasion of CCA cells that was in concert with the decrease of vimentin, matrix metalloproteinase (MMP)-2 and -7. Activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) by phosphorylation together with suppression of nuclear translocation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-ĸB) were the underlying mechanisms for these effects. Conclusion: Metformin is a potent antiproliferative and anti-metastatic agent against human CCA cells. These findings encourage the repurposing of metformin in clinical trials to improve CCA treatment.

Background/Aim: Pyruvate kinase M2 (PKM2) is an enzyme that is predominantly overexpressed in various types of cancer. The role of PKM2 in liver fluke-associated cholangiocarcinoma (CCA) remains unclear. This study aimed to investigate the antitumor activity of shikonin, a PKM2 inhibitor, in CCA cells. Materials and Methods: Immunohistochemistry and immunoblotting were used to determine PKM2 expression in CCA tissues and cells. Antiproliferative effects of shikonin were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation and trypan blue exclusion assays. The anti-metastatic activity of shikonin was determined using the Boyden chamber assay. Mechanisms by which shikonin inhibited CCA progression were determined. Results: PKM2 was overexpressed in CCA compared to normal bile duct epithelial cells. Shikonin significantly inhibited growth, and migration of CCA cells while inducing their death. A mechanistic study revealed that antitumor effects of shikonin in CCA cells depended on increased production of reactive oxygen species. Conclusion: Shikonin may be a novel therapeutic agent for patients with CCA.

Cholangiocarcinoma (CCA) is a dismal cancer. At present, there is no effective chemotherapeutic regimen for CCA. This may be due to the marked resistance of CCA to chemotherapy drugs, for which a mechanism remains unknown. Nuclear factor-κB (NF-κB) is constitutively activated in a variety of cancer cells, including CCA. It has been shown to play roles in growth, metastasis, and chemoresistance of cancer. In the present study, we examined whether NF-κB is involved in the chemoresistance of CCA and whether dehydroxymethylepoxyquinomicin (DHMEQ), an effective NF-κB inhibitor, can overcome the drug resistance of CCA. Two CCA cell lines, KKU-M213 and KKU-M214, were treated with DHMEQ and/or chemotherapeutic drugs. Cell viability, apoptosis, and the expressions of the ATP-binding cassette (ABC) transporters were compared. The combination of chemotherapy drugs, 5-fluorouracil, cisplatin, and doxorubicin, with DHMEQ significantly enhanced the cytotoxicity of all chemotherapeutic drugs compared to DHMEQ or drug alone. Furthermore, the mRNA level of ABCB1, a multidrug-resistant protein, was significantly decreased in the 5-fluorouracil combined with DHMEQ-treated cells. These findings suggest that the inhibition of NF-κB by DHMEQ enhanced the chemoresponsiveness of CCA cells, possibly by reducing the expression of ABC transporter. Inhibition of NF-κB may be a potential chemodrug-sensitizing strategy for chemoresistant cancer such as CCA.

BACKGROUND: It is well known that 1α,25-Dihydroxyvitamin D₃(1,25[OH]₂D₃) restrains cell proliferation and induces differentiation and apoptosis in normal and tumor cells. The authors of this report recently demonstrated that 1,25(OH)₂D₃ effectively inhibits the proliferation of cholangiocarcinoma (CCA) cell lines. The antitumor activity and the underlying mechanism of 22-oxa-D₃, an analog of vitamin D, in mice and in tissue cultures from patients with CCA were further explored in the current study. METHODS: Cell growth and cell cycle distribution were examined in CCA cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Mice were injected subcutaneously with 4 × 10⁶ CCA cells at both flank sides and intraperitoneal injections with phosphate-buffered saline or 22-oxa-D₃(15 μg/kg/day) for 17 days thereafter. Tumors were removed the next day. The expression levels of cyclin D1 and the cyclin-dependent kinase inhibitor p21 were determined by Western blot analysis and immunohistochemistry. Growth inhibition of 22-oxa-D₃ in fresh tissue samples from patients with CCA was analyzed by using a histodrug response assay. RESULTS: 22-Oxa-D₃ effectively suppressed the growth of CCA cell lines in a time-dependent and dose-dependent manner. 22-Oxa-D₃ arrested CCA cells at G1 phase to S phase by the suppression of cyclin D1 expression and the up-regulation of p21. Supplementation of 22-oxa-D₃ to CCA-inoculated mice significantly inhibited tumor growth without hypercalcemia or serious side effects. The treatment also induced cellular apoptosis in tissue samples from patients with CCA. CONCLUSIONS: 22-Oxa-D₃ effectively suppressed tumor growth in CCA-inoculated mice and induced cellular apoptosis in tissue samples from patients with CCA. The current data encourage further investigation of 1,25(OH)₂D₃ or its analogues as therapeutic agents in the treatment of patients with CCA. Cancer 2010. © 2010 American Cancer Society.