热休克蛋白90抑制剂对LoVo细胞生长及细胞周期的影响

The effects of heat shock protein 90 inhibitor 17-AAG on the proliferation and cell cycles of LoVo cells

背景与目的:热休克蛋白90(heat shock protein90,Hsp90)是体内重要的伴侣分子,体内许多重要的蛋白质是其底物蛋白质,但其本身不能降解底物蛋白,只能与底物相互作用,起到分子伴侣的作用。Hsp90抑制剂能抑制肿瘤细胞增殖,诱导肿瘤细胞凋亡,导致周期阻滞,破坏Hsp90与底物的结合及促进Hsp90底物蛋白质降解。为探讨Hsp90与生存素(Survivin)同时作为治疗靶点的效应及可能机制,本实验观察Hsp90抑制剂17-烯丙胺-17-脱甲氧格尔得霉素(17-AAG)对人结肠癌LoVo细胞增殖、细胞周期以及对周期相关蛋白Survivin水平的影响。方法:用四氮唑盐还原法(MTT法)检测17-AAG对LoVo细胞增殖影响;Propidium Iodide(PI)染色检测细胞周期变化;Western blot检测Hsp90底物蛋白Survivin的变化。结果:17-AAG呈时间-剂量-依赖性抑制LoVo细胞增殖。100、500、和800ng/ml17-AAG作用于LoVo细胞24h后,细胞增殖抑制率分别为21.00%、40.81%、60.34%,作用48h后,细胞增殖抑制率分别为27.29%、48.17%、80.97%,作用72h,细胞增殖抑制率分别为34.45%、67.81%和88.42%。17-AAG导致LoVo细胞周期改变,100和500ng/ml17-AAG作用72h,G0/G1期分别占(61±3)%及(74±3)%,LoVo细胞阻滞于G0/G1期,未加药处理的LoVo细胞G0/G1期比例仅为(48.2±0.8)%(P<0.001)。500ng/ml17-AAG作用于LoVo细胞72h可部分清除Hsp90底物蛋白Survivin(P<0.001)。结论:17-AAG呈时间-剂量依赖性抑制LoVo细胞增殖,阻滞LoVo细胞于G0/G1期,并能部分清除Survivin。

Background and purpose： Hsp90 is cell chaperone protein that interacts with many proteins, but itself can not degrade its client proteins. Hsp90 inhibitor can inhibit tumor cell proliferation, induce cell apoptosis, cell growth arrest and increase the degradation of Hsp90 client proteins like Survivin. In order to explore the co-effects of Hsp90 inhibitor 17-AAG and Survivin on LoVo cells and the possible mechanisms, we observed the effects of 17- AAG on proliferation and cycles of LoVo cells and the protein level of Survivin. Methods： LoVo cells were treated with 17-AAG. The cell proliferation inhibition rate was evaluated by MTT assay. The cell cycle was detected by flow cytometry. The expression of Hsp90 client protein Survivin was detected by Western blot. Results： 17-AAG time- dose-dependently inhibit the proliferation of LoVo cells, after 100 ng/ml,500 ng/ml and 800 ng/ml 17-AAG exposure for 24 hrs, the cell proliferation inhibition rate was 21.00%, 40.81%, 60.34% respectively, after exposure for 48 hrs, the cell proliferation inhibition rate was increased to 27.29%, 48.17%, 80.97% respectively, after exposure for 72 hrs, the cell proliferation inhibition rate was to 34.45%, 67.81%, 88.42%; 17-AAG arrested cell cycle, when LoVo cells were exposed to 100 ng/ml 17-AAG for 72 hrs, the cell ratio of G0/G1 phase was（61±3）%, when to 500 ng/ml for 72 hrs, cell ratio of G0/G1 phase was increased to（74±3）%, compared to（48.2±0.8）% LoVo cells without 17-AAG（P〈0.001）. Hsp90 client protein Survivin was derived by 17-AAG from LoVo cells. Conclusion： 17-AAG can time-dose-dependently inhibit proliferation of LoVo cells, induces cell growth arrest and increase the degradation of Hsp90 client protein Survivin.