Yang Lina, Liu Yuanyuan, Sun Chao, Yang Xinrui, Yang Zhen, Ran Juntao, Zhang Qiuning, Zhang Hong, Wang Xinyu, Wang Xiaohu
Institute of Cell Biology, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000, P.R. China ; Department of Heavy Ion Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu 730000, P.R. China ; Department of Radiotherapy, Gansu Province Tumor Hospital, Lanzhou, Gansu 730005, P.R. China.
Department of Heavy Ion Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu 730000, P.R. China.
Oncol Lett. 2015 Nov;10(5):2856-2864. doi: 10.3892/ol.2015.3730. Epub 2015 Sep 18.
Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5-50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of ATM and ATR did not rescue the A549 cells subjected to ionizing irradiation. Therefore, future studies on DNA-PKcs, ATM and ATR may lead to novel specific therapies that supplement general radiotherapy for the treatment of lung cancer.
非小细胞肺癌（NSCLC）由于其DNA损伤修复系统，对传统射线表现出放射抗性。通过减少那些持续增强受损DNA修复的因素，NSCLC可能对放射治疗敏感。在本研究中，用NU7026和CGK733分别处理正常肺成纤维细胞MRC-5和肺癌A549细胞，它们分别是DNA依赖性蛋白激酶催化亚基（PKcs）、共济失调毛细血管扩张症突变基因（ATM）和共济失调毛细血管扩张症及Rad3相关基因（ATR）的抑制剂，随后进行X射线和碳离子照射。分别用MTT法、集落形成试验、免疫荧光和流式细胞术分析处理后细胞的细胞毒性活性、细胞存活率、DNA损伤修复能力、细胞周期阻滞和凋亡率。分别通过逆转录定量聚合酶链反应和蛋白质印迹法检测ATM、ATR和DNA-PKcs基因的转录和翻译水平。结果表明，在用抑制剂预处理后进行电离辐射，A549细胞的放射敏感性和DNA修复能力降低，凋亡细胞百分比和细胞周期G/M期阻滞细胞百分比显著增加。与对照和X射线处理的细胞相比，用碳离子照射处理的A549细胞中，ATM、ATR、DNA-PKcs和DNA双链断裂生物标志物磷酸化组蛋白H2AX的表达水平在转录或翻译水平均上调。此外，用5-50μM的NU7026或CGK733处理对MRC-5细胞未产生任何明显的细胞毒性，并且DNA-PKcs抑制剂增强A549细胞放射敏感性的作用比ATM和ATR抑制剂更强。这些发现表明ATM和ATR在辐射诱导的细胞死亡中作用较小，因为ATM和ATR的上调并未挽救遭受电离辐射的A549细胞。因此，未来对DNA-PKcs、ATM和ATR的研究可能会导致新的特异性疗法，作为肺癌综合放射治疗的补充。
