吴茱萸碱激活结肠癌细胞自噬抑制其增殖的研究

Evodiamine activates autophagy and inhibits the proliferation in colon cancer cell

目的探讨吴茱萸碱(evodiamine,Evo)对结肠癌HCT-116细胞自噬、增殖的影响及其可能的分子机制。方法 CCK-8法检测Evo对HCT-116细胞增殖的影响;Evo(3、6μmol/L)处理48 h后,MDC法检测细胞内自噬小泡的数量,DHE法检测细胞内活性氧(ROS)的量,Western blotting法检测细胞自噬相关蛋白和腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(m TOR)通路相关蛋白的表达;Evo(6μmol/L)分别与自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)和凋亡抑制剂Z-DEVD-FMK共同作用48 h后,Western blotting法检测细胞内自噬及凋亡相关蛋白的表达。结果与对照组比较,Evo对HCT-116细胞呈现浓度依赖性的增殖抑制作用;Evo(3、6μmol/L)使HCT-116细胞内的ROS量升高、自噬小泡数量增加、自噬相关蛋白LC3表达增加、p62表达降低、p-AMPK及m TOR表达增加;Evo与自噬抑制剂联合给药后,细胞内LC3蛋白表达减少,而有活性的Caspase-3表达增加,Evo与凋亡抑制剂联合给药后,细胞内LC3表达增加,而有活性的Caspase-3表达被抑制。结论 Evo能够通过AMPK/m TOR通路激活自噬、抑制HCT-116细胞增殖,且细胞自噬与凋亡呈现互补作用。

Objective To investigate the effect of evodiamine（Evo） on the autophagy and proliferation of colon cancer HCT-116 cells and the underlying mechanism.Methods The effect of Evo on proliferation of HCT-116 cells was detected by CCK-8 method.After being processed with Evo（3 and 6 μmol/L） for 48 h,the number of autophagic vesicles were detected by MDC method.The amount of ROS in HCT-116 cells was measured by DHE assay,and the protein related with autophagy and AMPK/m TOR pathway was detected by Western blotting.After the treatment of Evo（6 μmol/L） combined with autophagy inhibitor 3-MA（3-methyladenine） or apoptosis inhibitor Z-DEVD-FMK respectively for 48 h,Western blotting was used to detect the expression of autophagy and apoptosis-related protein in HCT-116 cells.Results Compared with the control group,Evo inhibited the proliferation of HCT-116 cells in a dose-dependent manner; After treated with Evo（3 and 6 μmol/L） for 48 h,the amount of intracellular ROS and autophagic vesicles were increased,the protein expression levels of LC3,p-AMPK,and m TOR were increased while the expression of p62 was increased.After being treated with Evo and autophagy inhibitor,the protein expression of LC3 was decreased while activated Caspase-3 was increased; Combination of Evo and apoptosis inhibitor increased the expression of LC3 and inhibited the expression of activated Caspase-3.Conclusion Evo can activate autophagy of HCT-116 cells through AMPK/m TOR pathway and inhibit the proliferation,and the effect of autophagy and apoptosis on cells are complementary.