Microcystin-leucine Arginine Induces the Proliferation of Cholangiocytes and Cholangiocarcinoma Cells Through the Activation of the Wnt/β-catenin Signaling Pathway.

BackgroundMicrocystin-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/β-catenin pathway in vitro.MethodsCell division, migration, cell cycle transition, and MC-LR receptor expression were evaluated in vitro through MTT, wound healing assays, 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/β-catenin signaling pathway was investigated using immunoblotting and qRT-PCR analysis. Immunohistochemistry confirmed increased β-catenin expression in CCA tissue compared to adjacent tissue.ResultsHuman immortalized cholangiocyte cells (MMNK-1) and a human cell line established from opisthorchiasis-associated CCA (KKU-213B) expressed the MC-LR receptor and internalized MC-LR. Exposure to 10 nM and 100 nM of MC-LR notably enhanced 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/β-catenin signaling pathway and suppressing phosphatase activity. Inhibition of the β-catenin destruction complex genes (Axin1 and APC) led to the upregulation of β-catenin and its downstream target genes (Cyclin D1 and c-Jun). Inhibition of Wnt/β-catenin signaling by MSAB confirmed these results. Additionally, β-catenin was significantly expressed in cancerous tissue compared to adjacent areas (P < 0.05).ConclusionsOur findings suggest that MC-LR promotes cell proliferation and progression of CCA through Wnt/β-catenin pathway. Further evaluation using in-vivo experiments are warranted to confirm this observation. This finding could promote health awareness regarding MC-LR intake and risk of CCA.