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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 activation of AKT pathway in melanoma A375 cells and the effect of 4EGI-1 on A375 cell proliferation. Methods: The melanoma A375 cells were treated with 20, 40 and 60μmol / L of 4EGI-1 for 12,24 h, and the effect of 4EGI-1 on AKT phosphorylation in A375 cells was detected by Western blotting. A375 cells were treated with 4EGI-1 / L), BEZ235 (5μmol / L), 4EGI-1 (40μmol / L) and BEZ235 (5μmol / L). CCK-8 and flow cytometry were used to detect the effect of 4EGI-1 and BEZ235 alone or in combination A375 cells proliferation and cell cycle, Western blotting detection of cell cycle related protein Cyclin D1 expression. Results: 4EGI-1 promoted the phosphorylation of AKT in A375 cells in a dose-dependent manner, while BEZ235 antagonized the phosphorylation of AKT induced by 4EGI-1. Compared with 4EGI-1 group and BEZ235 group, the inhibition rates of A375 cells in the combination group were significantly increased ([7.6 ± 1.2]% vs (2.4 ± 3.1)%] in the 4EGI-1 and BEZ235 groups at 24 h (19.8 ± 4.3)%, P <0.05]. The proportion of cells in S phase was significantly decreased (25.65% and 25.82%, respectively) in SEG-1 group and BEZ235 group S phase was 13.08%; P <0.05), the expression of Cyclin D1 was significantly decreased (relative expression value of Cyclin D1 was 0.81 ± 0.04 and 0.76 ± 0.04 inEGI-1 group and BEZ235 group, and the relative expression value of Cyclin D1 in combined treatment group was 0.25 ± 0.03; P <0.05). CONCLUSION: AKT feedback activation inhibits the inhibitory effect of 4EGI-1 on melanoma A375 cells. Combination of AKT inhibitory drugs can enhance the growth inhibitory effect of 4EGI-1 on A375 cells.