芬苯达唑通过激活自噬抑制肺腺癌细胞增殖及侵袭、迁移能力

Fenbendazole inhibits the proliferation,migration and invasion of lung adenocarcinoma cell by activating autophagy

目的 探究芬苯达唑对肺腺癌细胞增殖、迁移、侵袭的影响以及自噬与迁移、侵袭的关系。方法 将人肺腺癌A549、H358细胞分为对照组及芬苯达唑(1、2.5、5、10、20μmol/L)组。用CCK-8法及细胞计数法检测细胞增殖水平,用划痕实验及Transwell实验检测细胞迁移、侵袭能力,通过RT-qPCR及蛋白免疫印迹法检测上皮–间充质转化(EMT)相关蛋白的m RNA及蛋白表达水平,通过转录组测序并分析自噬相关基因及其表达量。用自噬抑制剂氯喹(5、10μmol/L)与芬苯达唑(5μmol/L)处理细胞后再通过划痕及Transwell实验检测细胞迁移、侵袭能力。结果 与对照组相比,芬苯达唑各浓度处理组能显著抑制A549和H358细胞活性及迁移、侵袭能力(P<0.05、0.01、0.001);随着芬苯达唑药物浓度增加,N-钙黏蛋白(N-cadherin)、细胞角蛋白(Cytokeratin)、纤维连接蛋白(FN1)m RNA相对表达逐渐下降(P<0.05、0.01、0.001);紧密连接蛋白(Occludin)、波形蛋白(Vimentin)m RNA表达水平在低浓度时较高,随着芬苯达唑药物浓度增加Occludin、Vimentin m RNA表达水平降低(P<0.05、0.01、0.001)。与对照组相比,随着芬苯达唑药物浓度增加,N-cadherin、Cytokeratin、FN1、Occludin蛋白相对表达量显著降低,而Vimentin蛋白相对表达水平随药物浓度降低而增高(P<0.05、0.01、0.001)。基因差异热图显示,芬苯达唑处理后自噬相关基因(MAP1LC3B、ATG5、ULK3、Bax、ATG16L1)表达量增高,芬苯达唑的浓度增加使自噬相关蛋白重组人自噬效应蛋白(Beclin-1)、微管相关蛋白轻链3(LC3)-II/LC3-I相对蛋白表达量升高,选择性自噬接头蛋白P62表达量明显降低(P<0.05、0.01、0.001)。在使用了氯喹阻断自噬后,可以逆转芬苯达唑引起的细胞增殖、迁移及侵袭(P<0.05、0.01、0.001)抑制作用。结论 芬苯达唑可以通过促进自噬从而抑制了肺腺癌细胞的增殖、迁移和侵袭能力。

Objective To explore the effects of fenbendazole on the proliferation,migration and invasion of lung adenocarcinoma cells and the relationship between autophagy and migration invasion.Methods A549 and H358 cells were divided into control group and fenbendazole group(1,2.5,5,10,and 20μmol/L).Cell proliferation level was detected by CCK-8 method and cell counting method,cell migration and invasion ability was detected by scratch assay and Transwell assay,mRNA and protein expression levels of epithelial-mesenchymal transformation(EMT)related proteins were detected by RT-qPCR and western blot.Autophagy related genes and their expression levels were analyzed by transcriptome sequencing.The cells were treated with autophagy inhibitors chloroquine(5,10μmol/L)and fenbendazole(5μmol/L),and then the migration and invasion ability of the cells were detected by scratch and Transwell assay.Results Compared with control group,different concentration fenbendazole treatment groups significantly inhibited the activity,migration and invasion ability of A549 and H358 cells(P<0.05,0.01,0.001).With the increase of fenbendazole concentration,the relative mRNA expressions of N-cadherin,Cytokeratin,and FN1 decreased gradually(P<0.05,0.01,0.001).Occludin and Vimentin mRNA expression levels were higher at low concentration,and decreased with the increase of fenbendazole concentration(P<0.05,0.01,0.001).Compared with control group,the relative expression of N-cadherin,Cytokeratin,FN1,and Occludin decreased significantly with the increase of fenbendazole concentration.The relative expression level of Vimentin protein increased with the decrease of drug concentration(P<0.05,0.01,0.001).The thermal map of gene difference showed that the expression of autophagy related genes(MAP1LC3B,ATG5,ULK3,Bax,ATG16L1)increased after fenbendazole treatment,and the expression of autophagy related proteins Beclin-1 and LC3-II/LC3-I increased with the increase of fenbendazole concentration.The expression of P62 showed a decreased trend(P<0.05,0.01,0.001).After chloroquine was used to block autophagy,the inhibitory effects of fenbendazole on cell proliferation,migration and invasion could be reversed(P<0.05,0.01,0.001).Conclusion Fenbendazole can inhibit the proliferation,migration,and invasion abilities of lung adenocarcinoma cells by activating autophagy.