蛋白酶体抑制剂Marizomib联合顺铂对人子宫颈癌细胞的细胞毒性及凋亡的影响

Effects of proteasome inhibitor marizomib combined with cisplatin oncytotoxicity and apoptosis induction in human cervical cancer cells

目的探讨蛋白酶体抑制剂Marizomib(Mzb)联合顺铂对人子宫颈癌细胞的抗肿瘤效应及其机制。方法应用人子宫颈癌细胞系SiHa、C33A和子宫颈上皮永生化细胞H8,通过CCK-8、克隆形成实验、Western blot法和流式细胞术,分析Mzb联合顺铂对子宫颈癌细胞的细胞毒性和凋亡的影响。结果CCK-8结果显示,Mzb浓度在0、0.001~0.5μmol/L时,H8细胞的增殖活性分别为100%、101.92%~28.73%,SiHa细胞的增殖活性分别为100%、100.48%~3.54%,C33A细胞增殖活性分别为100%、83.7%~17.08%。顺铂(0、0.3~40μmol/L)与Mzb(0.01μmol/L)联用后,SiHa细胞增殖活性分别为98.41%、88.06%~55.04%,C33A细胞增殖活性分别为65.58%、61.04%~44.49%。克隆形成实验结果显示,Mzb浓度分别为0、0.01、0.025μmol/L时,SiHa细胞克隆数分别为535、391、318,C33A细胞克隆数分别为472、293、172。与子宫颈上皮永生化细胞H8相比,Mzb显著抑制子宫颈癌细胞增殖并呈剂量依赖性(P<0.05),与顺铂联用后效果更显著(P<0.05)。Western blot结果显示,Mzb促进SiHa和C33A细胞内多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)和Caspase 3裂解(P<0.05),与顺铂联用后效果更显著。流式细胞术结果显示,顺铂组SiHa和C33A细胞凋亡率分别为5.8%、21.5%;Mzb组细胞凋亡率分别为6.8%、28%,两药联合组细胞凋亡率分别为8.8%、33.7%。说明Mzb促进子宫颈癌细胞凋亡,与顺铂联用后凋亡更显著(P<0.05)。结论Mzb对体外子宫颈癌细胞有较强的抗肿瘤效应,能够敏感化子宫颈癌细胞对顺铂的治疗。在治疗子宫颈癌的临床试验中,顺铂联合Mzb可能是可行且有效的治疗选择。

Purpose To investigate the antitumor effect and its mechanism of proteasome inhibitor marizomib(Mzb)combined with cisplatin(CDDP)on human cervical cancer cells.Methods Effects of Mzb combined with CDDP on cytotoxicity and apoptosis on the human cervical cancer cell lines SiHa,C33A and immortalized cervical epithelial cell line H8 were detected through CCK-8 and colony formation assay,as well as Western blot and flow cytometry.Results When the concentration of Mzb ranged from 0,0.001-0.5μmol/L,CCK-8 results showed that the proliferation activities of H8 cells were 100%,101.92%-28.73%,100%,100.48%-3.54%in SiHa cells,and 100%,83.7%-17.08%in C33A cells,respectively.The proliferation activities of SiHa cells were 98.41%,88.06%-55.04%,and 65.58%,61.04%-44.49%in C33A cellswhen CDDP(0,0.3-40μmol/L)was combined with Mzb(0.01μmol/L).The results of clone formation experiment showed that the clone numbers were 535,391,318 in SiHa cells,and 472,293,172 in C33A cells,respectively when the concentration of Mzb were 0,0.01μmol/L and 0.025μmol/L.Therefore,compared with immortalized cervical epithelial cell line H8,Mzb significantly inhibited the proliferation of cervical cancer cells in a dose-dependent manner(P<0.05),and the effect was more significant in thecombination with CDDP(P<0.05).Western blot results showed that Mzb alone promoted the cleavage of poly ADP-ribose polymerase(PARP)and Caspase 3 in SiHa and C33A cells(P<0.05),and the effect was more significant in thecombination with CDDP.The results of flow cytometry showed that the apoptosis rates of SiHa and C33A cells were 5.8%and 21.5%in CDDP group,6.8%and 28%in Mzb group,8.8%and 33.7%in combination group,respectively.These results indicated that Mzb promoted the apoptosis of cervical cancer cells,and the apoptosis was more significant after thecombination with CDDP(P<0.05).Conclusion Mzb has a strong antitumor effect on cervical cancer cellsin vitro,and can sensitize cervical cancer cells to CDDP treatment.CDDP in combination with Mzb may be one of feasible and effective therapeutic options in the clinical trials for treating cervical cancer.