β-escin reverses multidrug resistance through inhibition of the GSK3β/β-catenin pathway in cholangiocarcinoma

AIM: To develop a safe and effective agent for cholangiocarcinoma (CCA) chemotherapy. METHODS: A drug combination experiment was conducted to determine the effects of beta-escin in combination with chemotherapy on CCA cells. 3-(4,5--dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of beta-escin and common chemotherapeutics on the proliferation of human CCA cells (QBC939, Sk-ChA-1, and MZ-ChA-1). Immunocytochemistry was used to detect the expression of P-glycoprotein (P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/ beta-catenin pathway. The protein levels of P-gp, pS9-GSK3 beta, pT216-GSK3 beta, GSK3 beta, beta-catenin, and p-beta-catenin were further confirmed by western blotting. RESULTS: The drug sensitivity of QBC939 and QBC939/ 5-fluorouracil (5-FU) cells to 5-FU, vincristine sulfate (VCR), or mitomycin C was significantly enhanced by beta-escin compared with either agent alone (P < 0.05). In addition, the combination of beta-escin (20 mol/ L) with 5-FU and VCR was synergic with a combination index < 1. Further investigation found that the mRNA and protein expression of P-gp was downregulated by beta-escin. Moreover, beta-escin induced GSK3 beta phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of beta-catenin. Interestingly, activation of the GSK3 beta/ beta-catenin pathway induced by Wnt3a resulted in upregulation of P-gp, which was effectively abolished by beta-escin, indicating that beta-escin down-regulated P-gp expression in a GSK3 beta-dependent manner. CONCLUSION: beta- escin was a potent reverser of P- gpdependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3 beta/ beta- catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA. via