(-)-表儿茶素通过调控A549R细胞自噬改善放疗敏感性的作用机制

Mechanism of EC Regulation of Autophagy in A549R Cells to Improve Radiotherapy Sensitivity

目的 探讨(-)-表儿茶素[(-)-Epicatechin, EC]通过联合放疗(Radiotherapy, RT)改善非小细胞肺癌细胞放疗敏感性的作用机制。方法 体外培养A549人非小细胞肺癌细胞,构建放疗抵抗株A549R。通过细胞计数试剂盒8(Cell Counting Kit-8,CCK-8)检测细胞增殖活力,脱氧核苷酸末端转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL)染色观察细胞凋亡情况,Transwell实验检测细胞迁移能力。蛋白质免疫印迹(Western blot)检测凋亡相关蛋白(Bcl-2、Bax、Cleaved Caspase-3)和自噬相关蛋白(Beclin、LC3II/I)的表达。免疫荧光染色检测Cleaved Caspase-3和LC3的表达。单丹磺酰尸胺(Monodansylcadaverine, MDC)染色检测自噬体的形成。结果 EC联合RT抑制细胞增殖和迁移能力、诱导细胞凋亡和自噬、下调Bcl-2的表达、上调Bax、Cleaved Caspase-3、Beclin和LC3Ⅱ/I的表达,而加入自噬抑制剂(Chloroquine, CQ)或mTOR信号通路激活剂MHY1485处理后部分减弱了EC联合RT处理对细胞的作用。结论 EC可作为放疗增敏剂,通过抑制mTOR信号通路诱导A549R细胞自噬和凋亡改善放疗敏感性。

Objective To investigate the mechanism of(-)-epicatechin(EC)in improving the radiosensitivity of non-small cell lung cancer cells by combining radiotherapy(RT).Methods A549 human non-small cell lung cancer cells were cultured in vitro to construct a radiotherapy-resistant cell line A549R.Cell proliferation activity was detected by Cell Counting Kit-8(CCK-8).The cell apoptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining.Transwell assay was used to detect cell migration ability.The expressions of apoptosis-related proteins(Bcl-2,Bax,Cleaved Caspase-3)and autophagy-related proteins(Beclin,LC3II/I)were detected by Western blot.The expressions of Cleaved Caspase-3 and LC3 were detected by immunofluorescence staining.Monodansylcadaverine(MDC)staining was used to detect the formation of autophagosomes.Results EC combined with RT inhibited cell proliferation and migration,induced cell apoptosis and autophagy,down-regulated the expression of Bcl-2,up-regulated the expressions of Bax,Cleaved Caspase-3,Beclin and LC3II/I,while the effect of EC combined with RT was partially attenuated by the treatment with autophagy inhibitor(chloroquine,CQ)or mTOR signaling pathway activator MHY1485.Conclusion EC can be used as a radiosensitizer to improve the radiosensitivity of A549R cells by inhibiting mTOR signaling pathway to induce autophagy and apoptosis.