放射线诱导BRCA1出核效应对PARP抑制剂的增敏作用

Sensitization of PARP inhibitor through ionizing radiation induced BRCA1 nuclear export

目的探讨放射线诱导的乳腺癌1号基因(BRCA1)出核效应对同源重组介导的DNA双链断裂损伤(DSB)修复功能的影响及对聚腺苷二磷酸核糖聚合酶(PARP)抑制剂的增敏作用。方法采用亚细胞纯化及蛋白印迹法检测乳腺癌细胞株MCF7经放射线处理后BRCA1的亚细胞定位,同时采用免疫荧光法检测细胞核磷酸化的H2AX组蛋白(γ-H2AX)、Rad51核焦点形成。流式细胞技术检测细胞凋亡、克隆形成实验检测体外细胞存活情况,并制作裸鼠皮下移植瘤模型,分为4组,分别为:对照组、放射+DMSO组、假照+ABT-888(ABT-888为PARP抑制剂)组、放射+ABT-888组,每5 d为1个治疗周期,周期第1天给予2 Gy照射或假照射。第2~5天给予溶剂(对照组、放射+DMSO组)或20 mg/(kg·d)的ABT-888(假照+ABT-888组),总共给予4个周期的治疗。检测各组的体内抑瘤作用。结果经4 Gy放射处理后,MCF7胞核BRCA1表达量减少,仅为对照组的41%,但胞浆内表达量明显增多,是对照组的2.14倍(P<0.01);同时MCF7细胞经4 Gy放射处理后ABT-888诱导的Rad51核焦点形成阳性细胞也较对照组明显减少(7%vs.30%,P=0.01)。放射线和ABT-888联合应用导致MCF7细胞凋亡率增高(19%),与对照组(5%)、放射+DMSO组(9%)或假照+ABT-888组(6.2%)相比较差异有统计学意义(P<0.01)。裸鼠移植瘤模型中放射+ABT-888组对肿瘤生长抑制作用明显优于假照+DMSO组或假照+ABT-888组(P<0.01)。结论放射线可诱导BRCA1出核并增强PARP抑制剂的抗肿瘤作用。

Objective To investigate the sensitization of PARP inhibitor through ionizing radiation （IR） induced BRCA1 nuclear export, and the consequent defieney in DNA double strand break - repair function. Methods The loca- tion of BRCA1 in nuclear or/and cytoplasmic compartments was assessed by subcellular fractionation in MCF7 cells after IR. The formation of γ- H2AX and RadS1 foei were detected by immunofluorescence staining. After labeled with Annexin V - FITC, apoptotic cells were analyzed by flow cytometry. In vivo anti - tumor activity was evaluated in MCF7 xenograf- ted SCID mice. Results Nuclear BRCA1 of IR in treated cells were 41% that of mock irradiated cells. Cytoplasmic BRCA1 of IR in treated cells were 2.14 folds compare to that of mock irradiated cells. ABT - 888 induced 7% and 30% Rad51 positive cell in IR treated or mock irradiated cells, respectively. Combined treatment of IR and ABT - 888 induced 19% apoptosis cells, significantly higher than IR and ABT - 888 alone. The combination of IR with ABT - 888 in MCF7 xenograft model resulted in a superior therapeutic response compared to the single - agent alone. Conclusion Ionizing radiation can induced BRCA1 nuclear export and sensitize PARP inhibitor.