共同抑制mTORC2和热休克蛋白90对多发性骨髓瘤细胞凋亡的影响

Research on multiple myeloma cell apoptosis by inhibition of mTORC2 and chaperon pathways

目的探讨共同抑制mTORC2信号通路和热休克蛋白90对多发性骨髓瘤（MM）细胞AKT蛋白表达及细胞凋亡的影响。方法采用雷帕霉素（20nmol／L）、17-烯丙胺-17-脱甲氧格尔德霉素（17-AAG）（600nmol／L）分别及两药联合处理MM细胞株U266、KM3细胞0、8、24、48h，MTT法检测其对细胞增殖的影响；流式细胞术检测其对细胞凋亡及细胞周期的影响；Westernblot法检测其对p-AKT（ser473）、P—AKT（thr450）、P．S6（$235／236）及AKT蛋白表达的影响。结果与空白对照组比较，雷帕霉素、17一AAG分别及两药联合后均可抑制U266、KM3细胞增殖，尤以联用后抑制作用最为明显（P值均〈0．05）；均可使细胞周期阻滞在G。期，尤其在作用48h时周期阻滞最明显（P值均〈0．01）；处理48h后空白对照组、雷帕霉素组、17-AAG组、两药联用组KM3细胞的凋亡率分别为（12．21±0．89）％、（18．88±1．83）％、（21．04±0．60）％、（60．07±2．13）％，U266细胞的凋亡率分别为（8．72±0．15）％、（16．45±0．65）％、（17．14±0．59）％、（54．25：t=1．76）％，与空白对照组比较差异均有统计学意义，两药联用组促凋亡作用更为明显（P值均〈O．01）。雷帕霉素作用48h后可抑制mTORC2信号通路；单用雷帕霉素或17-AAG时可降低AKT蛋白的表达，两药联用作用更为明显（P值均〈0．01）。结论共同抑制mTORC2和HSP90活性可降低AKT蛋白的表达，在体外可明显促进MM细胞株U266、KM3细胞的凋亡。

Objective To explore apoptosis of multiple myeloma （MM） cells and its mechanism by the combined inhibition of mTORC2 signaling pathway and heat shock protein 90. Methods The effects of Rapamycin, 17-AAG and the combination on proliferation of MM cell lines U266 and KM3 were assessed using MTT at different time points （0, 8, 24, 48 hour）. Cell apoptosis and cell cycle distribution were measured by flow cytometry. The specific proteins p-AKT （ser473）, p-AKT （thr450）, p-S6 （$235/236） and AKT were detected by Western blotting. Results Rapamycin, 17-AAG and the combination suppressed the proliferation of MM cell lines U266 and KM3, especially the combination of Rapamycin and 17-AAG synergistically inhibited the proliferation （P〈0.05）; Rapamycin induced G, arrest both at 24 and 48 hours, 17-AAG also induced G~ arrest, especially at 48 hours （P〈0.01）; Rapamycin, 17-AAG alone decreased the expression of AKT and induced MM cell apoptosis to some extent （P〈0.01）; Chronic rapamycin treatment inhibited mTORC2; Inhibition of both mTORC2 and chaperon pathways degraded AKT and induced MM cell apoptosis, which was significantly higher than that of any single agent （P〈 0.01 ）. Conclusion Inhibition of both mTORC2 and chaperon pathways decreased the expression of AKT to induce apoptosis of MM cells in vitro.