Perifosine通过抑制PI3K/Akt途径调节人胶质瘤U251细胞增殖、凋亡与自噬

Perifosine regulates human brain glioma U251 cell proliferation,apoptosis and autophagy through suppression of PI3K / Akt pathway

目的:探讨Akt激酶抑制剂perifosine对人胶质瘤U251细胞凋亡、细胞周期和自噬的影响,确定perifosine诱导的细胞自噬与其促进胶质瘤细胞凋亡的相关性。方法:Perifosine处理U251细胞后,采用MTT法检测细胞活力;流式细胞术分析perifosine对U251细胞周期的影响;Annexin V-FITC/PI双标法检测perifosine对胶质瘤细胞凋亡的作用;免疫印迹法检测细胞内P21、P27和cyclin B1等细胞周期调控相关蛋白以及caspase-9、PARP等细胞凋亡相关蛋白的表达水平;通过观察细胞内自噬标志分子LC3-II的分布与表达来确定perifosine对自噬的诱导作用。结果:Perifosine剂量依赖性地抑制U251细胞活力,能够通过抑制cyclin B1的表达而阻滞胶质瘤细胞周期于G_2期。Perifosine促进了U251细胞内caspase-9和PARP的剪切,抑制survivin表达,从而诱导胶质瘤细胞发生凋亡。同时,perifosine与自噬抑制剂氯喹联用后,U251细胞凋亡数量明显增加。结论:Perifosine能够抑制U251细胞的增殖,同时诱导细胞发生凋亡与自噬,抑制自噬促进了perifosine诱导的胶质瘤细胞凋亡。

AIM： To investigate the effect of perifosine,an inhibitor of protein kinase B（ PKB/Akt）,on the cell cycle,apoptosis and autophagy in human brain glioma U251 cells,and to determine the relationship between perifosine-induced autophagy and apoptosis of glioma. METHODS： The cell growth inhibition was determined by MTT assay.The cell cycle distribution of U251 cells was examined by flow cytometry. The cell apoptosis was analyzed by Annexin VFITC apoptosis detection kit. The protein expression of P21,P27,cyclin B1,caspase-9 and PARP was examined by Western blot analysis. The distribution and expression of LC3-II,an autophagy marker,was observed to determine the effect of perifosine-induced autophagy. RESULTS： Perifosine inhibited the cell viability in a dose-dependent manner. In perifosine-treated U251 cells,the cell cycle was arrested in G_2 phase and the expression of cyclin B1 was inhibited. Perifosine induced apoptosis of U251 cells through activation of caspase-9 cleavage,PARP cleavage and survivin inhibition. In addition,suppression of autophagy by chloroquin,an inhibitor of autophagy,increased the number of apoptotic cells. CONCLUSION： Perifosine inhibits cell proliferation and triggers apoptosis and autophagy in human U251 cells. Blocking autophagy magnifies perifosine-induced glioma cell apoptosis.