摘要
目的:探讨氯喹(CQ)联合放疗对非小细胞肺癌(NSCLC)细胞增殖、侵袭、迁移和凋亡的影响及其主要机制.方法:体外培养A549和PC-9细胞,分为C对照组、CQ(8µmol/L)单给药处理组、IR(4 Gy/8 Gy)单放疗处理组、CQ(8µmol/L)+IR(4 Gy/8 Gy)联合处理组.CQ预处理1 h,进行放疗处理.采用集落克隆实验和EdU结合实验检测各实验组细胞的增殖情况;Transwell实验检测各实验组细胞的侵袭和迁移能力;流式细胞术检测各实验组细胞的细胞凋亡率;电镜观察各实验组细胞的自噬水平;Western blotting实验检测各实验组细胞的自噬和凋亡相关蛋白的表达.结果:集落克隆实验和EdU结合实验结果显示,相比于单CQ处理或单放疗处理组,CQ联合放疗可显著抑制A549和PC-9细胞增殖(P<0.01);Transwell实验结果显示CQ联合放疗可显著抑制A549和PC-9细胞侵袭和迁移(P<0.01);流式细胞仪检测结果显示CQ联合放疗增加A549和PC-9细胞凋亡(P<0.05);电镜结果显示CQ能够抑制放疗诱导的自噬(P<0.01);Western blotting结果显示CQ联合放疗组的Caspase3水平上调而Bcl-2水平下降,LC3Ⅱ和p62的蛋白表达水平增加的较单CQ或单放疗处理组更为明显(P<0.01).结论:CQ抑制放疗诱导的自噬,CQ联合放疗抑制NSCLC细胞的增殖、侵袭和迁移,促进NSCLC细胞的凋亡.
Abstract
Objective:To investigate the inhibitory effects of chloroquine(CQ) combined with radiation therapy on the proliferation and metastasis of non-small cell lung cancer(NSCLC) cells,and its main mechanism.Methods:A549 and PC-9 cells were cultured in vitro,and divided into the control group,CQ(8 µmol/L) single administration treatment group,IR(4 Gy/8 Gy) single radiotherapy treatment group and CQ (8 µmol/L)+IR (4 Gy/8 Gy) combined treatment group.The CQ was pretreated for 1 h,and radiotherapy treatment was performed.The colony cloning assay and EdU incorporation assay were used to detect the proliferation of cells,the Transwell assay was used to detect the invasive and migratory ability of cells,the flow cytometry was used to detect the apoptosis rate of cells,the electron microscopy was used to observe the autophagy level of cells,and the Western blotting was used to detect the expression of autophagy and apoptosis-related proteins of cells in each experimental group.Results:The results of colony cloning assay and EdU incorporation assay showed that the CQ combined with radiotherapy significantly inhibited the proliferation of A549 and PC-9 cells compared with the CQ or radiotherapy alone treatment group(P<0.01).The results of Transwell assay showed that the CQ combined with radiotherapy significantly inhibited the invasion and migration of A549 and PC-9 cells(P<0.01).The results of flow cytometry showed that the CQ combined with radiotherapy increased the apoptosis of A549 and PC-9 cells(P<0.05).The results of electron microscopy showed that the CQ could inhibit radiotherapy-induced autophagy(P<0.01).The results of Western blotting showed that the Caspase 3 levels were up-regulated,while Bcl-2 levels were decreased in the CQ combined with radiotherapy group,and the increasing of protein expression levels of LC3Ⅱand p62 were significantly more than those in the single drug or single radiotherapy treatment group(P<0.01).Conclusions:CQ inhibits radiotherapy-induced autophagy,and CQ combined with radiotherapy inhibits the proliferation,invasion and migration,and promotes the apoptosis of NSCLC cells.
基金项目
大健康研究院区域性疾病联合研究中心专项资金资助项目(2023bydjk002)
安徽省高校自然科学研究重点项目(KJ2021A0715)
维普
万方数据