Fbxo6 Conferred Drug-sensitization To Cisplatin Via Inhibiting The Activation Of Chk1 In Non-small Cell Lung Cancer

Objective: Fbxo6 is a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF(Skp1/Cdc53-Cullin1/F-box).Previous studies demonstrated that Fbxo6 facilitated the growth and proliferation but inhibited the apoptosis and invasion of gastric cancer cells.More recent work has established important roles of Chk1 not only in DNA damage response but also in unperturbed cell,which may regulate DNA damage checkpoints,cell cycle arrest,DNA repair and cell death.However,the role of Fbxo6 in non-small cell lung cancer(NSCLC)is still not clear.Since Fbxo6 expression was identified in two NSCLC cell lines in previous study,we hypothesized that Fbxo6 also played a role in the development of NSCLC.Methods: Firstly,immunohistochemistry was used to detect the expression of 183 nonsmall cell lung cancer tissue samples and to analyze the relationship between the expression and clinicopathological factors,especially prognosis.In addition,we used stable transfection,instantaneous gene knockout and Chk1 specific inhibitor AZD7762 to change the expression level of Fbxo6 and inhibit the expression of Chk1.The biological effects of Fbxo6 in nonsmall cell lung cancer cell lines were studied by Western Blot,MTT and colony formation experiments.The A549 and A549/DDP cells were treated with cisplatin,changes of apoptosis and autophagy induced by A549 and A549/DDP cells were detected by protein imprinting assay.In the process of further studying the specific mechanism,we used immunoprecipitation and ubiquitination experiments to detect the molecules interacting with Fbxo6.Finally,we detected the changes of γH2AX under different treatment conditions of drugs and Fbxo6 by immunofluorescence assay.Results: It was found that the expression of Fbxo6 was correlated with early TNM stage(P=0.004),and the prognosis of patients with non-small cell lung cancer with highexpression of Fbxo6 was better.Western Blot showed that Fbxo6 down-regulated the expression of P-Chk1,Cdk4 and Cyclin D1.MTT and Colony formation assays showed that Fbxo6 inhibited the proliferation of non-small cell lung cancer cells.In addition,after treated with cisplatin,γH2AX indicated that Fbxo6 inhibited the repair reaction after DNA damage,and the overexpression of Fbxo6 after drug treatment accelerated the repair process of gamma H2 AX.After cisplatin-induced apoptosis,A549 and A549/DDP cells were interfered and transfected,respectively.It was found that Fbxo6 promoted the apoptotic level of non-small cell lung cancer cells.At the same time,cisplatin can activate the cell to produce stronger autophagy.After bi-directional regulation of Fbxo6 expression,Fbxo6 inhibits the autophagy of non-small cell lung cancer cells by Western blot analysis,and enhanced the chemosensitivity induced by cisplatin.Immunocoprecipitation results showed that Fbxo6 interacted with Chk1,reduced Chk1 by ubiquitination and inhibited Chk1 and Chk1 phosphorylation.The use of Chk1 specific inhibitors counteracted the increased chemosensitivity of cisplatin induced by Fbxo6.Conclusion: Fbxo6 can inhibit tumor proliferation,repair damage,autophagy and promote apoptosis by binding with Chk1,thus enhancing the chemosensitivity of cisplatin.Fbxo6 may be an effective therapeutic target to overcome the chemosensitivity of cisplatin.