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
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篇名: | 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 |
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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、20 μmol/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、20 μmol/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、10 μmol/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20 μmol/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。 |
ABSTRACT: | 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 and 20 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. |
期刊: | 2016年第27卷第13期 |
作者: | 周运生,王凤泽 |
AUTHORS: | ZHOU Yunsheng,WANG Fengze |
关键字: | 蛋白激酶B;CCT128930;细胞凋亡;细胞自噬;人骨肉瘤U2-OS细胞 |
KEYWORDS: | Protein kinase B; CCT128930; Apoptosis; Autophagy; Human osteosarcoma U2-OS cells |
阅读数: | 244 次 |
本月下载数: | 2 次 |
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