Development of gene-mediated radiotherapy for tumors

基因介导的肿瘤放疗的发展

基本信息

批准号：
13470187
负责人：
TSUZUKI Teruhisa
金额：
$ 9.28万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470187/
关键词：
DNA damage DNA repair Cell death Synthetic oligomer DNA recombination Antigene Radio sensitivity Gene intervention DNA組み換え

项目摘要

Rad51 is a key component of the homologous recombination repair pathway. Previous experiments with the antisense oligonucleotides, designed to down-regulate Rad51 gene expression, resulted in leading to increased radiosensitivity of mouse glioma cell lines after high doses of radiation and an increased survival rate for glioma-bearing mice treated with the Rad51 antisense molecule prior to irradiation. As part of our efforts to devise strategies for enhancing the effectiveness of radiation therapy, we explored the use of RNAi(RNA interference) as a means of attenuating Rad51 expression in mouse teratocarcinoraa F9 cells in a gene therapy context. After transfection of the Rad51 siRNA(short interfering RNA), cells were cultured for two days, then irradiated by X-ray at a dose of 2 Gy. Radiosensitivity of the cells was analyzed,by MTT assay. The amount of Rad51 protein in Rad51 siRNA transfected cells was shown to approximately 30-40% of the level s expressed in control cells. The increased sensitivity of the cells to X-ray irradiation was evident when the cells were transfected with Rad51 siRNA, compared to mock-transfected cells. Furthermore, the effect of sensitization with Rad51 siRNA transfection was also observed when the cells were treated with an anti-tumor drug, cisplatin, which bind and break DNA. The concentration of cisplatin, which reduced cytotoxysicity to 50%, became 0.2μM from 0.4μM. Simultaneous exposure of the cells to X-ray irradiation and cisplatin exerted additive effect to the growth inhibition of F9 cells.These results show the critical role of Rad51 in cellular responses to radiation as well as an anti-cancer drug, cisplatin, and indicate the potential use of Rad51-targeted RNAi in tumor radiosensitization.

Rad51 是同源重组修复途径的关键组成部分，之前的反义寡核苷酸实验旨在下调 Rad51 基因表达，结果导致小鼠神经胶质瘤细胞系在高剂量辐射后的放射敏感性增加，并提高了存活率。在放射治疗前用 Rad51 反义分子治疗患有神经胶质瘤的小鼠，作为我们努力制定增强放射治疗有效性策略的一部分，我们探索了使用 Rad51 反义分子。 RNAi（RNA 干扰）作为基因治疗背景下减弱小鼠畸胎癌 F9 细胞中 Rad51 表达的一种方法转染 Rad51 siRNA（短干扰 RNA）后，将细胞培养两天，然后在 100 ℃ 下用 X 射线照射。 2 Gy剂量下，MTT法检测Rad51 siRNA转染细胞中Rad51蛋白含量。与对照细胞中表达水平的大约30-40%相比，与模拟转染的细胞相比，当用Rad51 siRNA转染细胞时，细胞对X射线照射的敏感性明显增加。当用抗肿瘤药物顺铂处理细胞时，还观察到用Rad51 siRNA转染，其结合并破坏顺铂的浓度，从而降低了顺铂的浓度。细胞毒性从 0.4μM 提高到 50%，变为 0.2μM。同时将细胞暴露于 X 射线照射和顺铂对 F9 细胞的生长抑制产生附加作用。这些结果也表明 Rad51 在细胞对辐射的反应中发挥着关键作用。作为一种抗癌药物顺铂，并表明了 Rad51 靶向 RNAi 在肿瘤放射增敏中的潜在用途。