Inhibition of PI3K increases oxaliplatin sensitivity in cholangiocarcinoma cells

Abstract

Background: Resistance of cholangiocarcinoma to chemotherapy is a major problem in cancer treatment. The mechanism of resistance is believed to involve phosphoinositide-3- kinase (PI3K)/Akt activation. Although the platinum-containing compound oxaliplatin has been extensively used in the treatment of several solid tumors, recent data regarding its use to treat cholangiocarcinoma are ambiguous. Oxaliplatin resistance in this disease could potentially involve PI3K pathways. We, therefore, examined the effects of PI3K pathways in cholangiocarcinoma cells in modulating oxaliplatin resistance.

Results: After exposing the cholangiocarcinoma cell lines RMCCA1 and KKU100 to oxaliplatin, the levels of Akt and mTOR phosphorylation increased, as shown by western blot analysis. The WST-1 cell proliferation assay showed increased inhibition of cell growth under high concentrations of oxaliplatin. The combination of oxaliplatin with LY294002, an inhibitor of PI3K, resulted in a remarkable arrest of cell proliferation. Deactivation of mTOR by RAD001 was also synergistic with oxaliplatin, although to a lesser extent. The combination of oxaliplatin and a PI3K inhibitor also resulted in a significant induction of apoptosis, as demonstrated by the TUNEL assay.

Conclusion: Activation of PI3K might protect cholangiocarcinoma cells from oxaliplatininduced cytotoxicity. Although the inhibition of PI3K and the inhibition of mTOR both enhance oxaliplatin-induced cytotoxicity, PI3K inhibition has a greater effect. Targeting the PI3K pathway may be a useful approach to improve the chemotherapeutic sensitivity of cholangiocarcinoma.

Figure 1

Effect of oxaliplatin on cholangiocarcinoma cells. (A) RMCCA1 and (B) KKU100 cells were treated with oxaliplatin at various concentrations (0, 10, 50, 100 and 200 μM) for 48 hours. Effect on cell proliferation was measured by WST-1 and analyzed by spectrophotometric analysis (Absorbance = 450 nm). Results are reported as percentage inhibition of cell proliferation, where the optical density value from vehicle-treated cells was set as 100% of proliferation. Results are represented by the mean ± SE of three independent experiments. Phosphorylation of Akt and mTOR in (C) RMCCA1 and (D) KKU100 cells following 0–200 μM oxaliplatin treatment for 12 h and phosphorylation of Akt and mTOR in (E) RMCCA1 and (F) KKU100 cells following 100 μM oxaliplatin treatments for 0–48 h were determined by western blotting. Total Akt and mTOR were used as loading control. Representatives of the three independent experiments are shown.

Figure 2

Inhibition of Akt and mTOR increases the oxaliplatin-induced cytotoxicity in cholangiocarcinoma cell lines. The effects of Akt (LY294002), and mTOR (RAD001) inhibitors on the phosphorylation of Akt and P70S6K in (A) RMCCA1 and (B) KKU100 cells were determined by western blotting. Total Akt and P70S6K were used as loading controls. Cells were treated with 10 μM LY294002, 0.5 μM RAD001 or control vehicle (DMSO) for 24 hours. Representatives from 3 independent experiments are shown. The effects of LY294002 and RAD001 in (C) RMCCA1 and (D) KKU100 cells following treatment with or without oxaliplatin are shown. Cells were treated with 10 μM LY294002, 0.5 μM RAD001 or control vehicle (DMSO), followed by the addition of 0–200 μM oxaliplatin for 48 hours. Cell proliferation was measured by WST-1 and analyzed by spectrophotometric analysis (Absorbance = 450 nm). Results are reported as a percentage inhibition of cell proliferation, where the optical density value from vehicle-treated cells were set as 100% of proliferation and represent the mean ± SE of three independent experiments. (*, p < 0.05 versus the same concentration of oxaliplatin)

Figure 3

LY294002 modulated oxaliplatin-induced cell apoptosis. (A) Cells were added with 10 μM LY294002, 0.5 μM RAD001 or control vehicle and then treated with 0–200 μM oxaliplatin. TUNEL assay was done as described in the Methods section. The percentage of apoptotic cells in each group of treatments was demonstrated (values shown as mean ± SD, *; p < 0.05 versus the same concentration of oxaliplatin). (B) Apoptotic cells (green spots) were detected with an ApopTag Staining kit and counterstained with DAPI (blue). RMCCA1 cells were treated with Vehicle (a) and 100 μM oxaliplatin (b). Cells were treated with RAD001 plus 100 μM oxaliplatin (c) and with LY294002 plus 100 μM oxaliplatin (d) (Arrow head indicates the apoptotic cells). (C) Western blots analysis of cleaved caspase-3 and caspase-3 in each group of treatments. The blots were representative of three independent experiments.