PU-H71对增加宫颈癌细胞辐射敏感性的研究

Study of the effect of PU-H71 on increasing radiosensitivity of cervical cancer cells

目的探讨热休克蛋白90(Hsp90)抑制剂PU-H71联合X射线照射对辐射抗性人宫颈癌细胞的作用。方法利用生物信息学分析Hsp90基因在宫颈癌组织和癌旁组织中的表达。通过分次照射法(2 Gy/次, 共30次)获得辐射抗性的宫颈癌细胞系HeLa RR和SiHa RR, 将细胞分为对照组(二甲基亚砜处理)、单纯照射组、PU-H71组(0.5 μmol/L PU-H71处理)、PU-H71+照射组(0.5 μmol/L PU-H71处理24 h后给予照射)。利用克隆形成法检测细胞存活。细胞处理后1、6、24 h, 用免疫荧光法检测γH2AX聚焦点。细胞处理后1、2、6、12、24 h, 利用蛋白质印迹法检测Rad51的表达;细胞处理后2 h, 检测磷酸化的DNA依赖蛋白激酶催化亚基(p-DNA-PKcs)的蛋白质表达。细胞处理后48 h, 流式细胞术检测细胞凋亡。结果 PU-H71增强了辐射抗性宫颈癌细胞对X射线的敏感性。与单纯照射组相比, PU-H71+照射组HeLa RR和SiHa RR细胞, 在10%存活下的辐射增敏比(SER)分别为1.36和1.27, 而凋亡率分别提高了约72.1%和63.1%。PU-H71使辐射诱导的γH2AX聚焦点的持续时间延长, 抑制了DNA依赖蛋白激酶催化亚基(DNA-PKcs)的磷酸化, 从而限制非同源末端连接(NHEJ)修复, 同时延迟了同源重组修复。结论 PU-H71通过抑制DNA双链断裂修复途径, 增加了辐射抗性宫颈癌细胞的辐射敏感性, 有望作为一种增强宫颈癌放射治疗疗效的放射增敏剂。

Objective To investigate the effect of heat shock protein 90(Hsp90)inhibitor PU-H71 combined with X-ray on radioresistant human cervical cancer cells.Methods The expression levels of Hsp90 gene between cervical cancer tissues and adjacent tissues were analyzed by bioinformatics.Radioresistant cervical cancer cell lines HeLa RR and SiHa RR were obtained by fractional irradiations(2 Gy per fraction,30 fractions).The cell lines were divided into the control group(treated with dimethyl sulfoxide),irradiation alone group,PU-H71 group(treated with 0.5μmol/L PU-H71),and PU-H71+irradiation group(irradiation at 24 h after treatment with 0.5μmol/L PU-H71).Cell survival was detected by clonal formation assay.Immunofluorescence assay was used to detectγH2AX foci at 1,6,and 24 h after cell treatment.The expression level of Rad51 protein at 1,2,6,12,and 24 h after cell treatment was detected using Western blot.The expression level of phosphorylated DNA-dependent protein kinase catalytic subunit(p-DNA-PKcs)was measured at 2 h after cell treatment.Cell apoptosis at 48 h after cell treatment was assessed by flow cytometry.Results PU-H71 enhanced the sensitivity of radioresistant cervical cancer cells to X-ray.Compared with the irradiation alone group,the radiation sensitization ratios(SER)of HeLa RR and SiHa RR cells at 10%survival were 1.36 and 1.27,and the apoptosis rates were increased by approximately 72.1%and 63.1%in the PU-H71+irradiation group,respectively.PU-H71 delayed the duration ofγH2AX foci induced by X-ray,inhibited the phosphorylation of DNA-dependent protein kinase catalytic subunit(DNA-PKcs),thus preventing non-homologous end joining(NHEJ)repair and delaying homologous recombination repair.Conclusion PU-H71 increases the radiosensitivity of radioresistant cervical cancer cells by inhibiting the repair pathway of DNA double-strand break,which is expected to be a radiosensitizer to enhance the efficacy of radiotherapy for cervical cancer.