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 β-escin in c o m b i n a t i o n w i t h c h e m o t h e ra p y o n C C A c e l l s. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of β-escin and common chemotherapeutics on the proliferation of human CCA cells(QBC939, Sk-Ch A-1, and MZ-Ch A-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/β-catenin pathway. The protein levels of P-gp, p S9-GSK3β, p T216-GSK3β, GSK3β, β-catenin, and p-β-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 β-escin compared with either agent alone(P < 0.05). In addition, the combination of β-escin(20 μmol/L) with 5-FU and VCR was synergic with a combination index < 1. Further investigation found that the m RNA and protein expression of P-gp was downregulated by β-escin. Moreover, β-escin induced GSK3β phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of β-catenin. Interestingly, activation of the GSK3β/β-catenin pathway induced by Wnt3 a resulted in upregulation of P-gp, which was effectively abolished by β-escin, indicating that β-escin down-regulated P-gp expression in a GSK3β-dependent manner.CONCLUSION: β-escin was a potent reverser of P-gpdependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3β/β-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.