Study On Flubendazole Promotes Autophagy And Induces Apoptosis In Non-small Cell Lung Cancer Cells

BACKGROUNDLung cancer is one of the most common malignant tumors in China and even in the world,of which non-small cell lung cancer(NSCLC)constitutes 85%of all lung cancer.Due to the increase in the number of smokers and the aggravation of environmental pollution,the incidence and mortality of lung cancer are rapidly growing worldwide.So it is of great significance to explore effective treatments for the patients with lung cancer.Flubendazole is an FDA-approved anthelmintic drug as a member of benzimidazole family.It works by binding with the tubulin of parasite and has a good insect repellent effect.Recent researches showed that flubendazole is a potential anti-tumor drug that can inhibit cancer cell proliferation,including myeloma,leukemia,glioma,breast cancer,colorectal cancer,melanoma,esophageal squamous cell carcinoma,etc.However,no research has been conducted on NSCLC.In this study,the effect of flubendazole on NSCLC was studied by using A549 and H460 cells,and its mechanism was discussed.METHODS1.A549 and H460 cells of NSCLC were treated with different concentrations of flubendazole(0,0.0625,0.125,0.25,0.5,1,2,4?mol/L)for 48 hours,and the cell viability was detected by CCK-8 assay.2.A549 and H460 cells were treated with 2?mol/L flubendazole for 12,24 and 48 hours.The levels of autophagy-related proteins p62 and LC3 were detected by Western Blot.3.Adenovirus with double-label fluorescence(HBAD-mRFP-GFP-LC3)was transfected into A549 and H460 cells,which were divided into control group,flubendazole-treated group,flubendazole combined with hydroxychloroquine treated group,hydroxychloroquine-treated group and rapamycin-treated group.The autophagic flux was observed and photographed under fluorescence microscope.4.A549 and H460 cells were treated with different concentrations of flubendazole(0,0.125,0.5,2?mol/L)for 48 hours,the apoptosis rate of cells were measured by Annexin V-FITC/PI staining combined with flow cytometry respectively.RESULTS1.Flubendazole inhibited A549 and H460 cells in a dose-dependent manner.The higher the concentration,the more obvious the inhibition.2.After treated with 2?mol/L flubendazole for 12,24 and 48 hours,the level of p62 decreased and LC3 II/I ratio increased,suggesting that the intracellular autophagy level was increased.3.The mRFP-GFP-LC3 transfected cells showed that the level of autophagy in the control group was the lowest.In flubendazole-treated group and rapamycin-treated group,the fluorescence was mainly red after fluorescence merge,suggesting that autophagy was activated.When flubendazole and hydroxychloroquine were used together,the expression of green fluorescence was higher than that of the flubendazole-treated group.After the fluorescence merge,the fluorescence was red-yellow and the yellow fluorescence was dominant.Hydroxychloroquine-treated group was mainly yellow fluorescence,which means that autophagic flux was blocked.4.After treated with different concentrations of flubendazole for 48 hours,the apoptosis rates of A549 and H460 cells were higher than that of the control group.As the concentration of flubendazole increased,the apoptosis rates were also increased,showing a dose-dependent relationship.CONCLUSIONWe demonstrated in vitro experiments that flubendazole can inhibit the proliferation of NSCLC and reduce the viability of A549 and H460 cells.Further research found that flubendazole can up-regulate the expression of LC3 II/I,down-regulate p62 levels,activate the autophagy flow in A549 and H460 cells,and promote autophagy of cells.Flubendazole can also induce apoptosis in A549 and H460 cells,and it is positively correlated with drug concentration.Therefore,flubendazole may be a potential treatment for lung cancer.