摘要
目的观察DNA损伤应答抑制剂及其联合常规化疗药物(顺铂等)在卵巢癌耐药细胞株OVCAR-8中的作用,研究其化疗致敏效应。方法利用针对DNA损伤应答关键信号蛋白质的抑制剂,与顺铂等连用处理卵巢癌细胞,分析这些药物处理方式对卵巢癌细胞的杀伤能力。MTT法检测不同药物作用后OVCAR-8的增殖抑制情况;免疫荧光法检测OVCAR-8中磷酸化组蛋白2A变体(γH2AX)和p53结合蛋白1(53BP1)的表达,观察二者在DNA损伤位点的募集和形成灶点的能力。结果毛细血管扩张共济失调突变蛋白(ATM)/ATM和Rad 3相关蛋白(ATR)抑制剂与顺铂联用能抑制损伤修复机制的活化,明显减弱OVCAR-8细胞的增殖活力(P<0.01),促进其凋亡;在羟基脲和Wortmannin联合处理时,OVCAR-8细胞的ATR转导信号(如γH2AX)减弱,细胞生存率明显降低(P<0.05);多聚ADP-核糖聚合酶(PARP)抑制剂与顺铂联合处理OVCAR-8细胞未发现明显的增敏作用(P>0.05)。结论适当的DNA损伤应答抑制剂有潜力提高常规化疗药物的抗肿瘤效果,以达到快速清除肿瘤细胞和防止产生耐药性的效果。
Objective To assess the sensitisation effects of DDR inhibitors combined with conventional chemotherapeutics agents(cisplatin et al)in a drug-resistant ovarian cancer cell line(OVCAR-8).MethodsInhibitors of DDR regulators with cisplatin were applied to challenge OVCAR-8,and evaluated the DNA damage response(DDR)and cytotoxic effects of different combination of chemicals.Inhibition of proliferation to OVCAR-8of different drugs was evaluated by MTT assay.The activation of phosphorylation of histone family 2A variant(γH2AX)and p53 binding protein 1(53BP1)in OVCAR-8were evaluated by immunofluorescence to observe their ability of recruitment and forming foci at DNA damage site.Results We observed that combined treatment of ataxia-telangiectasia mutated(ATM)/ATM and Rad 3-related(ATR)inhibitor and cisplatin can suppress the activation of damage repair mechanisms and weakened the proliferative activity of OVCAR-8cells(P〈0.01);ATR pathway was suppressed and the signal ofγH2AX weakened and cell survival rate significantly reduced when combination therapy of HU and Wortmannin(P〈0.05);poly ADP-ribose polymerase(PARP)inhibitor could not enhance chemosensitivity in OVCAR-8 cells when combined with cisplatin(P〈0.05). Conclusion We substantiated that appropriate inhibitors of DNA damage response may have a potential to improve the anti-tumor effect of conventional chemotherapy drugs and prevent drug resistances.
出处
《四川大学学报(医学版)》
CAS
CSCD
北大核心
2016年第3期316-320,336,共6页
Journal of Sichuan University(Medical Sciences)
基金
四川省科技厅应用基础计划项目(No.2012JY0011)
四川省杰出青年学术技术带头人资助计划(No.2012JQ0005)资助
关键词
卵巢肿瘤
顺铂耐药
DNA损伤应答
抑制剂
Ovarian cancer
Cisplatin resistance
DNA damage response
Inhibitors