抑制热休克蛋白70表达可增强宫颈癌细胞对顺铂化疗的敏感性

Inhibiting HSP70 expression enhances cisplatin sensitivity of cervical cancer cells

目的探讨抑制宫颈癌Hela229细胞内热休克蛋白(HSP)70表达对顺铂(DDP)化疗敏感性的影响。方法 3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)及集落克隆法测定各因素对Hela229细胞的抑制作用;溴化丙啶(PI)单染法及DAPI检测细胞的凋亡率;JC-1染色法测定线粒体膜电位(△Ψm)的变化;Western blotting测定相关蛋白的表达;建立裸鼠动物模型测定各因素体内的抑瘤作用。结果宫颈癌细胞较正常宫颈细胞高表达HSP70;当HSP70抑制剂与顺铂合用,细胞的存活率较单用顺铂显著下降(P<0.01);PI与DAPI染色实验表明HSP70抑制剂能增加顺铂诱导的细胞凋亡率;JC-1染色结果显示,HSP70抑制剂处理后顺铂组细胞线粒体膜电位发生了明显的降低。Western blotting实验结果显示HSP70蛋白表达抑制剂与顺铂合用较单用顺铂显著增加促凋亡蛋Bax/caspase-3表达及caspase-3活化;明显降低抑制凋亡相关蛋白Bcl-2表达。裸鼠移植肿瘤模型结果表明HSP70抑制剂与顺铂合用能增强顺铂对模型小鼠肿瘤的抑制作用(P<0.01)。结论抑制HSP70表达可增强宫颈癌细胞对顺铂的敏感性,机制可能是通过抑制HSP70,进而调节凋亡相关蛋白的表达,促进宫颈癌细胞凋亡。HSP70有望成为宫颈癌基因治疗的一个新靶点。

Objective To investigate the relationship between sensitivity to cisplatin(DDP) and the expression of HSP70 in cervical cancer cells in vitro. Methods Cervical cancer Hela229 cells treated with different concentrations of DDP and the HSP70 inhibitor(PFT-μ) were examined for cell viability using MTT assay and colony forming ability. The cell apoptosis was analyzed by flow cytometry with propidium iodide staining and DAPI staining, and JC-1 staining was used to determine mitochondrial membrane potential. The expressions of HSP70, Bcl-2, Bax and caspase-3 were measured with Western blotting.A nude mouse model bearing Hela229 cell xenograft was used to evaluate the effect of DDP and PFT-μ on tumor growth.Results Hela229 cells expressed a higher level of HSP70 than normal cervical cells. The combined use of PFT-μ significantly enhanced the inhibitory effect of DDP(P<0.01) and increased the cell apoptosis in Hela229 cells. JC-1 staining demonstrated that DDP combined with PFT-μ more obviously reduced mitochondrial membrane potential. DDP combined with PFT-μ more strongly lowered Bcl-2 expression and increased the expressions of casepase-3 and Bax than DDP alone. In the nude mouse model, PFT-μ significantly enhanced DDP sensitivity of Hela229 cell xenografts(P<0.01). Conclusions Inhibition of HSP70 expression can enhance the sensitivity of cervical cancer cell to DDP both in vivo and in vitro possibly by promoting cell apoptosis, suggesting the potential of HSP70 as a new target for gene therapy of cervical cancer.