Microcystin-leucine arginine induces the proliferation of cholangiocytes and cholangiocarcinoma cells through the activation of the Wnt/β-catenin signaling pathway
Suppakrit Kongsintaweesuk,
Sirinapha Klungsaeng,
Kitti Intuyod,
Anchalee Techasen,
Chawalit Pairojkul,
Vor Luvira,
Somchai Pinlaor,
Porntip Pinlaor
Affiliations
Suppakrit Kongsintaweesuk
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; Medical Sciences Program, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Sirinapha Klungsaeng
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Kitti Intuyod
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Anchalee Techasen
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; School of Medical Technology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Chawalit Pairojkul
Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Vor Luvira
Department of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Somchai Pinlaor
Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Porntip Pinlaor
Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; School of Medical Technology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand; Corresponding author. School of Medical Technology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
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/β-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/β-catenin signaling pathway was investigated using immunoblotting and qRT-PCR analysis. Immunohistochemistry was used to determine β-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/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 < 0.001). Conclusions: Our findings suggest that MC-LR promotes cell proliferation and progression of CCA through Wnt/β-catenin pathway. Further evaluation using in vivo experiments is needed to confirm this observation. This finding could promote health awareness regarding MC-LR intake and risk of CCA.
