蛋白激酶B抑制剂CCT128930对人骨肉瘤U2-OS细胞凋亡与自噬的影响  

Effects of Protein Kinase B Inhibitor CCT128930 on the Apoptosis and Autophagy of Human Osteosarcoma U2-OS Cells

作　　者：周运生[1] 王凤泽[2] 

机构地区：[1]泰安市中心医院放疗科,山东泰安271000 [2]泰山医学院生命科学学院,山东泰安271016

出　　处：《中国药房》2016年第13期1767-1770,共4页China Pharmacy

基　　金：国家自然科学基金资助项目(No.81272683)

摘　　要：目的:研究蛋白激酶B抑制剂CCT128930对人骨肉瘤U2-OS细胞凋亡与自噬的影响。方法:体外培养U2-OS细胞,经0(空白对照,下同)、10、20μmol/L CCT128930作用24 h后采用DNA片段末端标记法观察U2-OS细胞的凋亡情况;经0、5、10、20μmol/L CCT128930作用24 h后采用Western blot法检测细胞中半胱氨酸氨基转移酶3(Caspase-3)、Caspase-8、Caspase-9、Caspase切割底物PARP蛋白表达及自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比例;将质粒绿色荧光蛋白-微管相关蛋白轻链3(GFP-LC3)转染U2-OS细胞,经0、5、10μmol/L CCT128930作用24 h后采用荧光观察GFP-LC3融合蛋白在细胞内的表达;采用MTT法和Western blot法检测20μmol/L氯喹、CCT128930及二者联合作用于细胞24 h后的细胞活力及Caspase-3、PARP蛋白的表达。结果:与空白对照比较,CCT128930作用后U2-OS细胞凋亡率升高(P<0.01),Caspase-3、Caspase-8、Caspase-9、PARP蛋白表达增强,LC3-Ⅱ/LC3-Ⅰ比例升高(P<0.05或P<0.01),GFP-LC3融合蛋白表达增强;与氯喹联用后,使U2-OS细胞活力和Caspase-3、PARP蛋白表达均下降。结论:CCT128930可诱导U2-OS细胞发生凋亡和自噬,抑制自噬可能促进CCT128930对U2-OS细胞的毒性作用。OBJECTIVE：To study the effects of protein kinase B inhibitor CCT128930 on the apoptosis and autophagy of human osteosarcoma U2-OS cells. METHODS：U2-OS cells were cultured in vitro,and the apoptosis of U2-OS cell was observed after treated with 0（blank control,similarly hereinafter）,10 and 20 μmol/L CCT128930 for 24 h. After treated with 0,5,10 and20 μmol/L CCT128930 for 24 h,the protein expression of Caspase-3,Caspase-8,Caspase-9 and Caspase cutting substrate PARP,the proportion of autophagy-related protein LC3- Ⅱ/LC3- Ⅰ were detected by Western blot method. Plasmid GFP-LC3 transfected U2-OS cells;after treated with 0,5 and 10 μmol/L CCT128930 for 24 h,immunofluorescence was used to detect the expression of GFP-LC3 protein. MTT assay and Western blot assay were used to detect the viability,the expression of Caspase-3 and PARP protein of U2-OS cells after treated with 20 μmol/L chloroquine and CCT128930 alone or combination for 24 h. RESULTS：Compared with blank control,the apoptotic rate of U2-OS cells increased after CCT128930 treatment（P〈0.01）,and the expression of Caspase-3,Caspase-8,Caspase-9 and PARP protein increased;the proportion of LC3-Ⅱ/LC3-Ⅰ increased（P〈0.05 or P〈0.01）;the expression of GFP-LC3 protein increased. CCT128930 combined with chloroquine reduced the viability of U2-OS cells,the protein expression of Caspase-3 and PARP. CONCLUSIONS：CCT128930 can induce the apoptosis and autophagy of U2-OS cells. Blocking autophagy can enhance the toxicity of CCT128930 to U2-OS cells.

关 键 词：蛋白激酶B CCT128930 细胞凋亡 细胞自噬 人骨肉瘤U2-OS细胞 

分 类 号：R965[医药卫生—药理学]