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

Figure 1 β-escin enhanced the sensitivity of cholangiocarcinoma cells to chemotherapeutic drugs. A: MDR cell line QBC939/5-FU cells were treated with different concentrations of 5-FU, CDDP, VCR, and MMC for 24 h. Cell viability measured by MTT assays; B-C: Effects of β-escin (20 μmol/L) in combination with chemotherapeutics (40 μmol/L) on QBC939 cells and QBC939/5-FU cells. Cell viability measured by MTT assays after treatment for 24 h. ^aP < 0.05 vs control. MDR: Multidrug resistance; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; 5-FU: 5-fluorouracil; VCR: Vincristine sulfate; MMC: Mitomycin C; CDDP: Cisplatin.

Figure 2 Expression of P-gp down-regulated by β-escin. A: Expression of MRP mRNA analyzed using real-time PCR after treatment with β-escin for 24 h; B: Expression of MDR mRNA analyzed using real-time PCR (upper panel) and agarose gel electrophoresis analysis (lower panel) after treatment with β-escin for 24 h. Data were normalized to GAPDH; C: Expression of P-gp protein detected by ICC. Zoom: × 400; D: Expression of P-gp protein detected by western blot. β-Actin used as loading control. E: β-escin. ^aP < 0.05, ^bP < 0.01 vs control. PCR: Polymerase chain reaction; MDR: Multidrug resistance; MRP: MDR-related protein; P-gp: P-glycoprotein; ICC: Immunocytochemistry.

Figure 3 Down-regulation of P-glycoprotein induced by β-escin via inhibiting the activation of the GSK3β/β-catenin pathway. A: The activation of the Wnt/β-catenin pathway assessed by TCF/LEF responsive luciferase reporter activity in QBC939 and QBC939/5-FU cells after treatment with β-escin. Data were shown as the mean ± SD of three independent experiments; B: Western blot analysis of GSK3β and β-catenin expression in QBC939 and QBC939/5-FU cells treated with a series of concentrations of β-escin; C: Western blot analysis of GSK3β and P-gp in QBC939 and QBC939/5-FU cells after different treatments for 24 h. β-actin used as loading control. E: β-escin (20 μmol/L); W: Wnt3a (50 μg/L); C: Control (PBS). ^aP < 0.05, ^bP < 0.01 vs control. TCF: T-cell factor; LEF: Lymphoid enhancer factor.