University of South Florida Morsani College of Medicine, Tampa, FL ; Radiation Oncology Branch, National Cancer Institute, Bethesda, MD.
Transl Oncol. 2013 Jun 1;6(3):355-62. doi: 10.1593/tlo.13163. Print 2013 Jun.
The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according γH2AX foci. PP242 exposure did not influence the initial level of γH2AX foci after irradiation but did significantly delay the dispersal of radiation-induced γH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair.
雷帕霉素（mTOR）的机械靶标是调节基因翻译的关键激酶，已被认为是放射增敏的潜在靶点。本研究的目的是比较别构 mTOR 抑制剂雷帕霉素和竞争性 mTOR 抑制剂 PP242 的放射增敏活性。基于免疫印迹分析，雷帕霉素仅部分抑制 mTOR 复合物 1（mTORC1）活性，对 mTOR 复合物 2（mTORC2）没有影响，而 PP242 抑制了含有 mTOR 的两种复合物的活性。单独照射对 mTORC1 或 mTORC2 活性没有影响。集落形成实验用于定义 mTOR 抑制剂对体外放射敏感性的影响。在所评估的两种肿瘤细胞系中，PP242 在照射前 1 小时处理可增加放射敏感性，而雷帕霉素则没有作用。照射后添加 PP242 也增强了两种肿瘤系的放射敏感性。为了研究放射增敏的机制，根据 γH2AX 焦点评估 DNA 双链断裂的诱导和修复。PP242 暴露不影响照射后 γH2AX 焦点的初始水平，但明显延迟了辐射诱导的 γH2AX 焦点的分散。与肿瘤细胞系相反，正常人类成纤维细胞系的放射敏感性不受 PP242 影响。最后，在携带 U251 异种移植瘤的小鼠中给予 PP242 可增强放射诱导的肿瘤生长延迟。这些结果表明，在临床前肿瘤模型中，PP242 可增强肿瘤细胞的体外和体内放射敏感性，并表明这种作用涉及 DNA 修复的抑制。
