岩白菜素通过下调PI3K-AKT-mTOR通路抑制恶性胶质瘤的恶性生物学行为

Bergenin inhibits malignant biologic behaviour of glioblastoma through regulating PI3K-AKT-mTOR pathway

目的探究岩白菜素对恶性胶质瘤细胞的抑制作用及可能机制。方法常规培养U251和U87细胞,以一定浓度梯度(0、0.5、1、2、4、8μM)的岩白菜素与U251和U87细胞共孵育,MTT法检测细胞增殖能力,平板克隆形成实验检测集落形成能力,Transwell小室迁移和侵袭实验检测纵向迁移能力,划痕愈合实验检测横向迁移能力,Hoechst细胞凋亡检测试剂盒检测凋亡,免疫印迹检测PI3K-AKT-mTOR通路的变化,MTT检测与PI3K抑制剂共孵育后岩白菜素对U251细胞抑制作用的变化。同时利用MTT评估岩白菜素对正常人脑胶质HEB细胞的细胞毒性。结果与一定浓度梯度的岩白菜素共孵育后,MTT法检测发现U251和U87细胞的增殖能力以时间依赖性和浓度依赖性下降,平板克隆形成实验检测发现U251和U87细胞的集落形成能力浓度依赖性下降,Transwell小室迁移和侵袭实验检测发现U251细胞的纵向迁移和侵袭能力浓度依赖性下降,划痕愈合实验发现U251细胞的横向迁移能力浓度依赖性下降,Hoechst细胞凋亡检测试剂盒检测发现与一定浓度的岩白菜素共孵育后凋亡细胞增多,免疫印迹检测发现,总PI3K、总AKT及总mTOR没有改变,但是磷酸化的PI3K、磷酸化的AKT及磷酸化的mTOR呈浓度依赖性下降,PI3K抑制剂并不能增强岩白菜素对U251细胞的抑制作用。岩白菜素对HEB无明显细胞毒性。结论岩白菜素可以特异性抑制恶性胶质瘤细胞的恶性生物学行为,作用机制与减少PI3K-AKT-mTOR通路的磷酸化有关。

Objective to explore the action and possible mechanism of bergenin suppression to malignant glioma cells.Method U251 and U87 cells were cultivated routinely and then treated with bergenin(0、0.5、1、2、4、8μM).Next,the cells were detected by MTT assay to test the viability,plate clone formation assay to test colony formation ability,transwell small chamber migration and invasion assay to test longitudinal migration and invasion ability,scratch healing assay to test lateral migration ability,Hoechst apoptosis detection kit to test apoptosis,and western blot to test the change of PI3K-AKT-mTOR pathway,MTT assay was utilized to test whether PI3K inhibitor can increase the inhibition effect of bergenin on U251.Cytotoxicity of bergenin on normal human brain glial cell line HEB was assessed by MTT.Results After the cells treated with certain concentrate gradient bergenin,the U251 and U87 cell viability declined in a time and concentration dependent way by MTT assay,the colony formation ability of U251 and U87 cells was suppressed in a concentration dependent manner by plate clone formation assay,the longitudinal invasion and migration ability were inhibited in a concentration dependent manner through transwell chamber migration and invasion assay,and simultaneously the lateral migration ability was also restrained in same way.Meanwhile,the apoptosis of U251cells increased when the cells was dealed with bergenin via Hoechst apoptosis detection kit.Subsequent western blot assay found the total PI3K,AKT and mTOR expression quantity do not change,but the phosphorylated PI3K,AKT and mTOR expression quantity declined in a concentration dependent way,PI3K inhibitor can not increase the inhibitory effect of bergenin on U251.Bergenin had no cytotoxicity on HEB cells.Conclusion Bergenin can specially inhibit the malignant biological behavior of malignant glioma cells and the mechanism have relation with regulating PI3K-AKTmTOR pathway.