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、60 μmol/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（40 μmol/L）、BEZ235（5 μmol/L）、4EGI-1（40 μmol/L）+BEZ235（5 μmol/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CyclinD1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），CyclinD1表达显著降低（4EGI-1组和BEZ235组CyclinD1相对表达值为0.81±0.04和0.76±0.04，联合处理组细胞CyclinD1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, 60 μmol/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 were 2565% and 25.82% after treated with 4EGI-1 or BEZ235. It decreased to 13.08% in A375 cells exposed to both 4EGI-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-1 group 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.

贵州省中药现代化专项项目资助（No. 20125018）