摘要
目的:探索4EGI-1对黑素瘤A375细胞中AKT通路的激活作用及后者对4EGI-1抑制A375细胞增殖的影响。方法:用20、40、60μmol/L的4EGI-1处理黑素瘤A375细胞12、24 h,Western boltting检测4EGI-1对A375细胞中AKT磷酸化的影响;A375细胞分别受4EGI-1(40μmol/L)、BEZ235(5μmol/L)、4EGI-1(40μmol/L)+BEZ235(5μmol/L)联合处理,CCK-8实验和流式细胞术分别检测4EGI-1、BEZ235单独或联合处理对A375细胞增殖和细胞周期的影响,Western boltting检测对细胞周期相关蛋白Cyclin D1表达的影响。结果:4EGI-1促进A375细胞中AKT的磷酸化,且具有剂量依赖性;而BEZ235能够拮抗4EGI-1对AKT的促磷酸化作用。与4EGI-1组和BEZ235组相比,联合组的A375细胞增殖抑制率显著升高[24 h时,4EGI-1组和BEZ235组抑制率分别为(7.6±1.2)%和(2.4±3.1)%,而联合组抑制率为(19.8±4.3)%;P<0.05],S期细胞比例显著降低(4EGI-1组和BEZ235组S期细胞比例分别为25.65%和25.82%,联合处理组细胞S期为13.08%;P<0.05),Cyclin D1表达显著降低(4EGI-1组和BEZ235组Cyclin D1相对表达值为0.81±0.04和0.76±0.04,联合处理组细胞Cyclin D1相对表达值为0.25±0.03;P<0.05)。结论:AKT反馈激活拮抗4EGI-1对黑素瘤A375细胞的抑制作用,联合使用AKT抑制药物能够增强4EGI-1对A375细胞的生长抑制作用。
Objective: To investigate the effect of 4EGI-1 on AKT activity in melanoma cell A375 and its role in 4EGI-1-induced growth inhibition of the cells. Methods: After treated with indicated concentrations of 4EGI-1( 20,40,60μmol /L) for 12 h,A375 cells were lysed to analyze AKT phosphorylation by using immunoblotting. A375 cells were also treated with 4EGI-1 and BEZ235 alone or in combination to assess their effects on cell proliferation through the CCK-8 assay. Their influences on cell cycle were determined using flow cytometry and the expression of Cyclin D1 was examined by immunoblotting. Results: 4EGI-1 induced AKT phosphorylation in A375 cells dose-dependently. While the inhibition rates of 4EGI-1 and BEZ235 on A375 cells were( 7. 6 ± 1. 2) % and( 2. 4 ± 3. 1) % respectively,the rate increased significantly to( 19. 8 ± 4. 3) % when both compounds were used together. The percentage of cells in S phase cells were25. 65% and 25. 82% after treated with 4EGI-1 or BEZ235. It decreased to 13. 08% in A375 cells exposed to both4EGI-1 and BEZ235,indicating that BEZ235 enhanced the inhibitory effect of 4EGI-1 on A375 growth. BEZ235 also enhanced the inhibitory effect of 4EGI-1 on cyclin D1 expression. The relative expression levels of cyclin D1 in 4EGI-1group and BEZ235 group were 0. 81 ± 0. 04 and 0. 04 ± 0. 76 respectively,it was reduced to 0. 25 ± 0. 03 when the cells were cultured in the presence of both compounds. Conclusion: Akt phosphorylation induced by 4EGI-1 counteracts its growth inhibition on A375 cell. Combination of 4EGI-1 and Akt inhibitor is a more effective strategy to inhibit the growth of A375 cells.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2015年第4期438-442,共5页
Chinese Journal of Cancer Biotherapy
基金
贵州省中药现代化专项项目资助(No.20125018)~~
