Jujuboside A Inhibited NSCLC Tumorigenes Is Via Activating Fat4-HIPPO-YAP Pathway

Lung cancer is one of the most common leading causes of cancer-related deaths worldwide because of its high incidence and mortality rates among all cancer.Non-small-cell lung cancer(NSCLC),accounts for approximately 80%of all lung cancer cases.Chemotherapy is the main treatment for cancer because most patients have advanced disease at diagnosis.However,drug resistance and early metastasis limit the clinical efficacy of chemotherapy.Therefore,one promising approach to reduce lung cancer mortality is the identification of preneoplastic lesions and administration of chemopreventive agents to reverse,suppress,or arrest lung tumorigenesis.In vitro studies,we have shown that Jujuboside A can specificity cause lung cancer cell Senescence,apoptosis as well as proliferation suppressed,and does not affect the normal cells.Further research shows that the anti-tumor function of the Jujuboside A is mainly derived from the HIPPO pathway activated,Jujuboside A combine with Fat4 protein on the cell membrane,activate the HIPPO pathway.However,there is no literature reported Jujuboside A about the therapeutic effect and mechanism of lung cancer.Based on the research status,we also adopts the urethane induced lung cancer mice model to evaluate Jujuboside A chemical prevention effect,and illustrate its related mechanisms and pathways.Methods:1.The determination of Jujuboside A on cell viability and the expression level of YAP in NSCLC cellsThe NSCLC cell line A549?H1299?H1975 and human embryonic lung fibroblast cell line MRC-5 were detected by MTT assay after the administration of Jujuboside A in 2 to 5 days,which would help to confirm the drug concentration and time.Meanwhile,the mRNA and protein expression of YAP were measured by Real time PCR and Western Blot.2.The regulation of Jujuboside A on the protein expression level of YAPBased on the results of MTT assay,the effect of Jujuboside A on HIPPO pathway in A549 cells was assayed by Western Blot.The activation of Fat4,a membrane protein on A549 cells,was detected by protein in heat transfer experiment.3.The regulation of YAP protein and function studiesTo ensure the regulation of Jujuboside A in HIPPO pathway,the downstream proteins of YAP were measured.Besides,the function of stemness,senescence,proliferation and apoptosis were detected by flow cytometry,and confocal.4.To investigate the function change of si-Fat4 H1975 cell lineAfter silenced Fat4 protein,the function of stemness,senescence,proliferation and apoptosis were detected by flow cytometry,and confocal.5.Research on animal models of lung cancer to ascertain the chemical prevention effect of Jujuboside AThe lung cancer mice model induced by urethane was established to ascertain the chemical prevention effect of Jujuboside A under the administration of 5 mg/kg,10 mg/kg,20 mg/kg in 22 weeks.The change of the animal body weight and the number of pulmonary tumor nodules were recorded,pulmonary pathological changes were inspected by HE staining,the distribution YAP in lung was detected by immunohistochemistry,the peripheral blood mononuclear cell(PBMC)in macrophages were assayed by flow cytometry.Results:1.Jujuboside A inhibited cell viability of NSCLC cells and reduced the expression level of YAPAfter drug administration in 2-5 days,Jujuboside A showed significant inhibition on NSCLC cells.According to the MTT assay results,to ensure more than 80%of the cells viability,the drug concentration was determined as 0,25,50,100 ?M in 5 days.2.Jujuboside A bonded Fat4 to activate the HIPPO pathwayAccording to the results,Jujuboside A bonded Fat4 to activate the HIPPO pathway,therefore reduced the expression of YAP.3.Jujuboside A suppressed the downstream pathway of YAP by activating HIPPO pathway.The results showed that Jujuboside A regulated all the downstream proteins of YAP as a result of the HIPPO pathway activation.The lung cancer cells dyed by ?-galactosidase exhibited the phenomenon of aging,and the aging-related protein p16 was increased.The surface marker CD133/CD44 detected by flow cytometry showed that stemness degree in lung cancer cells were decreased as well as stemness marker protein Nanog.Through detection methods of EdU,plate colony and flow cytometry,the proliferation ability and proliferation cycle related proteins Cyclin D1 were declined in lung cancer cell.The flow cytometry and Western blot results of the apoptosis related proteins revealed that cell apoptosis was increased.4.Si-Fat4 in H1975 cell line prevent Jujuboside A from reducing the expression of YAPAfter reduced the expression of Fat4 protein,Jujuboside A couldn' t reduced the expression of YAP,and the functional change of YAP had been reversed.5.The chemical prevention effect of Jujuboside A on animal models of lung cancer in the function and mechanismThe urethane induced lung cancer animal experiment,Jujuboside A significantly reduced the rate of lung nodules.The body weight of mice in Jujuboside A group is apparently heavier than the control group.In addition,the spleen and thymus weight index showed that Jujuboside A can enhance the immunity of animals.The Immunohistochemical results showed that Jujuboside A can significantly reduce the YAP expression in lungs.At the same time,the number of PBMC which were transformed M1 macrophages were larger.Conclusion:Results showed that Jujuboside A might bond with Fat4 to activate the HIPPO pathway,finally reduce the protein expression of YAP.With the decline of YAP,the YAP downstream proteins,proliferation,apoptosis and dye related proteins were all reduced,which would have a role on blocking the tumor cell cycle,inducing tumor cell apoptosis,reducing tumor cell dry condition.Moreover,Jujuboside A possibly activated the immune ability to reduce the tumor occurrence.Therefore,this study can provide a proof that Jujuboside A would have certain effect for lung cancer chemoprevention.