Rapamycin-induced autophagy sensitizes A549 cells to radiation associated with DNA damage repair inhibition.
Rapamycin-induced autophagy sensitizes A549 cells to radiation associated with DNA damage repair inhibition.
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DOI：
10.1111/1759-7714.12332
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发表时间：
2016-07
期刊：
影响因子：
2.9
通讯作者：
Qian K
中科院分区：
文献类型：
--
作者：
Li Y;Liu F;Wang Y;Li D;Guo F;Xu L;Zeng Z;Zhong X;Qian K
据报道，放疗后癌细胞的自噬增加。然而，目前尚不清楚放射引起的自噬增加是否会影响DNA损伤修复并使癌细胞敏感。本研究研究了雷帕霉素（一种诱导自噬的雷帕霉素抑制剂的哺乳动物靶点）对人肺腺癌A549细胞的放射增敏作用。用不同浓度的雷帕霉素处理A549细胞。采用甲基噻唑-四氮唑法测定细胞活力。采用集落形成法检测放疗后A549细胞的存活分数值。透射电镜观察自噬体。此外，Western blot检测自噬蛋白LC3和p62、DNA损伤蛋白γ-H2AX和DNA损伤修复蛋白Rad51、Ku70和Ku80的变化。实时聚合酶链反应检测Rad51、Ku70和Ku80信使核糖核酸（mRNA）的表达水平。雷帕霉素抑制A549细胞增殖呈剂量和时间依赖性。抑制浓度（IC） 10剂量的雷帕霉素可诱导A549细胞自噬。与单独使用雷帕霉素或辐射相比，雷帕霉素联合辐射显著降低了细胞的集落形成能力。与单一方案相比，雷帕霉素和放疗联合增加了γ-H2AX表达水平，降低了Rad51和Ku80表达水平。然而，无论放射程度如何，雷帕霉素治疗均未引起Rad51、Ku70和Ku80 mRNA水平的任何变化。这些发现表明，增加的自噬使肺癌细胞对辐射敏感。
Autophagy has been reported to increase in cancer cells after radiation. However, it remains unknown whether increased autophagy as a result of radiation affects DNA damage repair and sensitizes cancer cells. In this study, the radiosensitization effect of rapamycin, a mammalian target of rapamycin inhibitor that induces autophagy, on human lung adenocarcinoma A549 cells was investigated. A549 cells were treated with different concentrations of rapamycin. Cell viability was evaluated by methyl‐thiazolyl‐tetrazolium assay. Survival fraction values of A549 cells after radiotherapy were detected by colony formation assay. Autophagosome was observed by a transmission electron microscope. Furthermore, Western blot was employed to examine alterations in autophagy protein LC3 and p62, DNA damage protein γ–H2AX, and DNA damage repair proteins Rad51, Ku70, and Ku80. Rad51, Ku70, and Ku80 messenger ribonucleic acid (mRNA) expression levels were examined by real‐time polymerase chain reaction. Rapamycin suppressed A549 cell proliferation in dose and time‐dependent manners. An inhibitory concentration (IC) 10 dose of rapamycin could induce autophagy in A549 cells. Rapamycin combined with radiation significantly decreased the colony forming ability of cells, compared with rapamycin or radiation alone. Rapamycin and radiation combined increased γ–H2AX expression levels and decreased Rad51 and Ku80 expression levels, compared with single regimens. However, rapamycin treatment did not induce any change in Rad51, Ku70, and Ku80 mRNA levels, regardless of radiation. These findings indicate that increasing autophagy sensitizes lung cancer cells to radiation.