摘要
目的:初步研究铁死亡通路的激活对人非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞放射敏感性的影响.方法:使用铁死亡激活剂Erastin处理NSCLC细胞系A549,透射电子显微镜观察细胞超微结构变化;采用细胞克隆形成实验观察铁死亡发生对A549细胞放射敏感性的影响;通过流式细胞术检测铁死亡通路的激活对A549细胞中活性氧(reactive oxygen species,ROS)水平、细胞凋亡和周期的影响;应用免疫荧光染色法观察铁死亡通路的激活对细胞DNA损伤的影响;采用Western blotting法检测铁死亡发生对细胞中γ-H2AX、GPX4、COX-2等蛋白表达水平的影响.结果:电子显微镜下可观察到Erastin处理细胞发生铁死亡特征性变化,表现为线粒体体积缩小、线粒体膜有局部破损,并且线粒体嵴大量断裂.Erastin处理致细胞放射敏感性增加,放射增敏比为1.611>1.Erastin对A549细胞的凋亡率无显著改变.Erastin处理致辐射诱导的A549细胞中ROS水平升高,G2/M期阻滞增加,γ-H2AX焦点形成增加,γ-H2AX、COX-2蛋白高表达,GPX4蛋白低表达.结论:激活铁死亡通路可提高A549细胞放射敏感性,其机制可能与铁死亡引起细胞内脂质活性氧(lipid reactive oxygen species,L-ROS)累积,细胞周期阻滞和DNA损伤增加等有关.
Objective:To identify the impact of activating ferroptosis pathway on the radiosensitivity of human non-small cell lung cancer(NSCLC)cells.Methods:NSCLC cell line A549 was pretreated with ferroptosis agonist Erastin,and the ultrastructure variations were observed by transmission electron microscope.The effect of activating ferroptosis pathway on the radiosensitivity of A549 cells was analyzed via clonogenic assay.Flow cytometry assay was used to evaluate the effects of activating ferroptosis pathway on reactive oxygen species(ROS)level,cell apoptosis and cell cycle,while immunofluorescence staining assay was performed to determine DNA damage caused by activation of ferroptosis pathway.In addition,Western blotting assay was applied to detect the protein expression levels ofγ-H2AX,GPX4 and COX-2 influenced by activation of ferroptosis pathway.Results:Erastin pretreatment induced typical characteristic variations in mitochondria,including reduced size,partially damaged membrane and numerous broken cristae.It showed that Erastin sensitized A549 cells to ionizing radiation(IR)with the sensitivity enhancement ratio reached to 1.611>1.It also suggested that Erastin boosted the IR-induced ROS,augmented the G2/M phase arrest,and increased theγ-H2AX foci formation.However,IR-induced cell apoptosis had no significant changes within each group.In addition,upregulated protein expression ofγ-H2AX and COX-2 together with downregulated GPX4 were detected in A549 cells treated with Erastin plus IR.Conclusion:Activation of ferroptosis pathway could radiosensitive NSCLC cells to IR.The potential mechanisms might be relevant to the accumulation of lipid reactive oxygen species(L-ROS)and the augmentation of G2/M phase arrest and DNA damage caused by activating ferroptosis pathway.
作者
石暘滨
罗意
刘莹莹
陆诗雨
徐英
张丽蓉
张琦
焦旸
SHI Yangbin;LUO Yi;LIU Yingying;LU Shiyu;XU Ying;ZHANG Lirong;ZHANG Qi;JIAO Yang(School of Radiation Medicine and Protection,Soochow University,Suzhou,Jiangsu 215121,China)
出处
《中国科技论文在线精品论文》
2021年第3期362-370,共9页
Highlights of Sciencepaper Online
基金
国家自然科学基金(81773226)
作者简介
石暘滨(1999—),本科生,主要研究方向:肿瘤抑制基因放射增敏作用与机制;通信联系人:焦旸,教授,主要研究方向:肿瘤抑制基因放射增敏作用与机制、新型电离辐射生物标志物的功能、放射性肺损伤相关分子机制.E-mail:jiaoyang@suda.edu.cn
