热疗中热休克蛋白90对26S蛋白酶体的调控机制

Mechanism of heat shock protein 90 for regulating 26S proteasome in hyperthermia

目的探讨热疗中热休克蛋白90(HSP90)对26S蛋白酶体的调控机制。方法建立42℃热疗的Hep G2肝癌细胞系模型,评价不同热疗持续时间(0、3、6、12、24 h)对细胞内活性氧簇(ROS)和细胞增殖的影响;并对热疗导致Hsp90α和26S蛋白酶体的影响进行分析。结果热疗后细胞内ROS的含量增加(F=28.958,P<0.001),且热疗对细胞的增殖抑制程度随着时间的延长显著增强(F=621.704,P<0.001);热疗导致胞内Hsp90α表达量增加(F=27.403,P=0.035),26S蛋白酶体表达量明显降低(F=164.174,P<0.001),活性也下降(F=133.043,P<0.001);干扰HSP90α后,26S蛋白酶体也减少(F=180.231,P<0.001)。结论随着热疗时间延长,细胞内ROS含量增加,热应激和ROS共同导致蛋白持续变性而消耗HSP90,使没有足够的HSP90对26S蛋白酶体组装和稳定导而导致变性蛋白蓄积发生未折叠蛋白反应使细胞死亡。

Objective To investigate the mechanism by which heat shock protein 90(HSP90) regulates 26 S proteasome in hyperthermia. Methods Hyperthermic Hep G2 cell models established by exposure of the cells to 42 ℃ for 3, 6, 12, and 24 h were examined for production of reactive oxygen species(ROS) and cell proliferation, and the changes in Hsp90α and 26 S proteasome were analyzed. Results ROS production in the cells increased significantly after hyperthermia(F=28.958, P<0.001),and the cell proliferation was suppressed progressively as the heat exposure time extended(F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level(F=164.174, P<0.001) and activity(F=133.043, P<0.001) of 26 S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26 S proteasome(F=180.231, P<0.001). Conclusion The intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26 S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.