抑制PI3K/Akt信号通路拮抗胰岛素诱导的子宫内膜癌细胞增殖

Inhibiting PI3K/Akt signaling pathway blocked the mitogenic effect induced by insulin in endometrial cancer cells.

目的:研究抑制磷脂酰肌醇3激酶(PI3K)/Akt信号通路对胰岛素诱导的子宫内膜癌细胞增殖的拮抗作用。方法:将无血清饥饿的子宫内膜癌Ishikawa3-H-12细胞分为空白对照组、10-6mol/L胰岛素单独刺激组以及不同剂量PI3K抑制剂-LY294002预处理后再用胰岛素刺激组。Western blot检测各组Akt磷酸化(p-Akt)水平,MTT试验观察细胞增殖情况。结果:胰岛素可引起内膜癌细胞Akt活化,刺激15min后p-Akt/Akt比值显著高于空白对照组(68.68%vs 26.21%,P<0.001)。LY294002以浓度依赖方式抑制胰岛素引起的Akt磷酸化。MTT试验显示,在药物处理24h,48h和72h 3个时间点,不同组别570nm吸光度值(OD570nm)均有显著差异(F=156.329,700.973,812.224,均P<0.001)。胰岛素组OD570nm值均高于同时间点的空白对照组(均P<0.001),胰岛素促内膜癌细胞增殖作用于48h时最为显著。LY294002可抑制胰岛素的增殖促进作用,此抑制作用具有浓度依赖性。不同剂量LY294002抑制作用的时间依赖性不同,48h时小剂量(0.1、1、10μmol/L)的抑制作用最为显著,72h时胰岛素重新呈现一定的促增殖作用;而50μmol/L LY294002可以持久抑制胰岛素的促增殖作用。结论:PI3K抑制剂LY294002可以通过抑制Akt磷酸化阻断胰岛素信号传导,拮抗后者促子宫内膜癌细胞增殖的作用。

Objective ：To explore the role of phosphatidylinositol 3-kinase（PI3K）/Akt signaling pathway in insulin-induced proliferation promotion in endometrial cancer ceils. Methods：Ishikawa 3-H-12 human endometrial cancer cells were serum-starved and then stimulated by insulin with or without LY294002 ,a specific inhibitor of PI3K. The phosphorylation level of Akt （p-Akt） was detected with Western blots. Cellular proliferation was determined with the MTF assay. Results： Stimulation of the Ishikawa 3-H-12 cells with 10^-6 mol/L insulin resulted in the activation of Akt. After 15 minutes,the p-Akt/Akt ratio was significantly higher than that in control（68.68% vs 26.21% ,P 〈0. 001 ）,and insulin-induced Akt activation was inhibited by LY294002 in a concentration-dependent manner. In MTT assay, OD570nm of different groups in 24hour,48hour and 72hour were measured. At each time point,there was significant difference among six groups（ F = 156. 329,700. 973,812. 224 ,all P 〈 0.001 ）. After insulin treatment ,the OD570nm in 24hour, 48hour and 72hour was all higher than that in control group （all P 〈 0. 001 ）. Insulin promoted the proliferation of Ishikawa3-H-12 cell ,and the effect was most sig- nificant when treated for 48hours. LY294002 inhibited the mitogenie effeet induced by insulin in a dose-dependent manner. When in low concentrations （0. 1 μmol/L,1μmol/L and 10μmol/ L）, the inhibition caused by LY294002 was most significant at 48hours after treatment. LY294002 in high dosage （50μmol/L） exerted potent inhibition against insulin. Conclusion： PI3K inhibitor-LY294002 blocks the signal transduetion of insulin in endometrial cancer cells through the inhibition of Akt activation. The blockage of PI3K/Akt pathway inhibits the proliferation promotion induced by insulin.