靶向抑制胰岛素受体底物1表达增强人乳腺癌细胞放射敏感性的机制研究

Overexpression of insulin receptor substrate 1 (IRS-1) has been found in human breast cancer cells. We have found that targeting knockdown of IRS-1 expression in human breast cancer cell sensitized its radiosensitivity to γ-ray irradiation in our former experiments. But its mechanism is still not clear. This project aims to study the radiosensitization effect and its machenism in human breast cancer cells on three levels: in vitro, in vivo and in clinical specimens by targeting knock-down of IRS-1 expression. To study the radiosensitization effect in human breast cancer cells by targeting knockdown of IRS-1 expression using colony formation assay in vitro and using nude mice xenograft tumor model in vivo. To detect the cell cycle distribution in human breast cancer cells and in the tissue of nude mice xenograft tumor with flow cytometry by staining the cells with Propidium Iodide (PI). To measure the apoptosis in human breast cancer cells with flow cytometry by staining the cells with Propidium Iodide (PI) and Annexin V-FITC in vitro, while using TUNEL Assay to examine the apoptosis in the tissue of nude mice xenograft tumor in vivo. To investigate the DNA double strand break (DSB) repair induced by γ-ray irradiation in human breast cancer cells with different expression levels of IRS-1 using Comet Assay. To detect the signal molecullar protein expression associated to the pathways of apoptosis and DNA DSB repair using Western-Blot. To check the IRS-1 expression in the clinical specimens using Western-Blot and immunohistochemistry and to analyse the relationship between IRS-1 protein level and the curative effect of radiotherapy, and then to explore the feasibility of taking IRS-1 as a new radiosensitization biomarker by targeting knockdown of IRS-1 expression in human breast cancer cells.

胰岛素受体底物1（IRS-1）在人乳腺癌细胞内高表达，前期研究发现靶向抑制乳腺癌细胞IRS-1表达，增加其对γ射线的放射敏感性。本项目从体外细胞、体内荷瘤裸鼠模型和临床标本三个水平研究靶向抑制IRS-1表达对乳腺癌细胞的放射增敏作用及机制：应用细胞集落形成法和裸鼠肿瘤生长曲线研究靶向抑制IRS-1表达对乳腺癌细胞的放射增敏作用；流式细胞仪检测体外细胞和体内裸鼠肿瘤组织细胞周期（PI染色）分布及体外实验细胞凋亡（PI和Annexin V-FITC染色）情况，TUNEL法检测裸鼠肿瘤组织凋亡情况；彗星实验检测不同IRS-1表达水平乳腺癌细胞γ射线诱导DNA DSB修复情况；Western-Blot检测细胞及肿瘤组织凋亡相关信号分子及DNA损伤修复相关信号分子表达；检测临床标本IRS-1表达，分析不同IRS-1表达水平与放疗疗效间关系；探讨靶向抑制IRS-1表达作为乳腺癌放射增敏新靶标的可行性。

乳腺癌是女性发病率最高的恶性肿瘤，部分乳腺癌尤其是三阴性乳腺癌（TNBC）因缺乏靶向治疗措施以及辐射抵抗致使其对放/化疗疗效不佳。本研究首先探索了靶向抑制胰岛素受体底物1（IRS-1）表达对雌/孕激素受体阳性的人乳腺癌MCF-7和T-47D细胞放射增敏作用的机制，采用集落形成试验、Western blot法和免疫荧光法检测发现，IRS-1敲低表达通过抑制HR修复通路中修复蛋白BRAC1 foci形成而使DNA DSBs分子标志物γ-H2AX表达及其foci形成增加，降低受照细胞对DSBs的修复能力；IRS-1敲低表达在低激素水平下对MCF-7细胞的放射增敏作用与抑制β-catenin核转位有关。在此基础上，本研究选择了3株人TNBC MDA-MB-231、MDA-MB-468和Hs578T细胞，探索Wnt/β-catenin通路在调控TNBC细胞放射敏感性中的作用以及WHO推荐的抗绦虫首选药物氯硝柳胺对TNBC细胞的放射增敏作用及其机制，体外细胞试验采用Western blot法、免疫荧光法和实时荧光定量PCR法检测证实，IR诱导了TNBC细胞Wnt/β-catenin通路的激活；采用集落形成试验证实，β-catenin敲低表达能显著提高TNBC细胞的放射敏感性，而β-catenin过表达及给予Wnt3a则显著提高了细胞的辐射抗性，表明Wnt/β-catenin通路的激活在人TNBC细胞的辐射抵抗中发挥重要作用。体外细胞试验和荷瘤鼠动物实验表明，氯硝柳胺通过抑制IR诱导的Wnt/β-catenin通路的激活，显著提高TNBC细胞的放射敏感性，增强射线局部照射对裸鼠体内TNBC细胞生长的抑制作用。体外细胞试验表明，氯硝柳胺还能通过抑制TNBC细胞受照后早期（0.5h和2h）NHEJ和HR修复通路中的重要修复蛋白ATM、DNA-PKcs、53BP1、BRCA1、RPA70和KU80的磷酸化或核内募集抑制IR诱导的DNA损伤反应，从而抑制DSBs修复、提高IR诱导的染色体断裂；进一步采用绿色荧光蛋白报告基因法检测证实，氯硝柳胺能明显抑制NHEJ和HR介导的DSBs修复。. 本研究结果将为乳腺癌靶向放射增敏新药的研发提供新的靶点和启示，并为TNBC的放射治疗提供新的高效、低毒的放射增敏剂，开发氯硝柳胺这一“老药”的新用途，具有重要的研究意义和应用前景。