The Mechanism Of A Novel C-Src Inhibitor Oxfendazole On Non-small Cell Lung Cancer

BackgroundRecent studies have indicated that lung cancer is one of the most common malignant tumors in the world,with the highest morbidity and mortality.It was responsible for 1.8 million new cases in 2012(12.9%of all cancers)and caused 1.6 million deaths(19.4%of all cancers).Lung cancer was proved to be the most common cancer worldwide several decades ago:with 660,000 new cases estimated in 1980,but it had risen to 1.8 million new cases in 2012,in which 58%occurred in less developed regions.In lung cancer,the incidence of NSCLC(non-small cell lung cancer)patients is about 85%,and lung adenocarcinoma is the most frequent histological subtype,accounting for nearly 40%of all lung cancer cases.Prior to the use of targeted therapy and immunotherapy,patients with advanced lung cancer had a poor prognosis.Platinum doublet chemotherapy,which was the standard of care for all patients with incurable locally advanced or metastatic NSCLC,achieved a response rate of 19%,and a median overall survival of 7.9 months.Modern trials with targeted treatments have resulted in significantly better outcomes.Recent reports have demonstrated that c-Src is altered and highly activated in NSCLC,especially in adenocarcinomas.The level of c-Src kinase activity has been reported to correlate with tumor size in NSCLC.Furthermore,the mitogenic effect of both nicotine and asbestos in NSCLC cells is likely to involve in the activation of c-Src.These observations indicate that c-Src activity is frequently associated with the malignant progression of a variety of NSCLC cells.Because c-Src is also known to be an important mediator of upregulated receptor tyrosine kinases,including HER2 and EGFR,therapeutic strategies to inhibit c-Src activity are currently being developed.Recently,several inhibitors of activated c-Src in cancer cells have been examined in clinical trials for the treatment of not only lung but also breast,colon,and prostate cancer.In preclinical studies,NSCLC cells that were treated with dasatinib(BMS-354825)demonstrated decreased cell growth and changes in downstream signalings that resulted in a reduced capability for invasion.These reports suggest that c-Src could be an attractive target for treatment in a particular subset of patients with NSCLC.There are nine non-receptor-type protein tyrosine kinases that show a high level of similarity in their primary structures and in the structures of their functional domains.Together,they are called the src family.They seem to have common sites specific for oncogene activation.Recent findings suggest that the kinases are closely associated with cell surface molecules and they mediate extracellular signals through the activation of their tyrosine kinase activity.Cisplatin is a common chemotherapeutic drug in the treatment of lung cancer,but patients frequently develop resistance to it partly because of the overactivated or overexpressed c-Src.Objective1.To find 'old drug' that can effectively inhibit NSCLC through drug screening.2.To evaluate the anti-NSCLC mechanism of oxfendazole(OFD).3.Tyrosine kinase c-Src family proteins are recognized as proto-oncogenes in many tumors and can be used as potential targets for antitumor drugs.The purpose of this study is to evaluate whether OFD can be used as a novel c-Src inhibitor and to elucidate its specific mechanism.4.To evaluate the killing effect of OFD combined with chemotherapeutic drugs on NSCLC cells.According to the above results,we proposed new anti-NSCLC therapies with a single drug or combination with other anti-tumor drugs.Methods1.CCK-8 assay was used to screen drugs that could significantly inhibit the cell survival of NSCLC from a FDA-approved drug library.2.CCK-8,flow cytometry,immunoblotting and tumor xenograft models were used to detect the anti-NSCLC activity of OFD.3.The effect of OFD on c-Src activation in NSCLC cells were detected by immunoblotting,molecular docking and cytokine stimulation.Furthermore,the overexpression plasmid of c-Src was constructed to further detect whether c-Src affected the anti-NSCLC effect of OFD.4.To determine the application prospects of OFD in the treatment of NSCLC by combining with chemotherapeutic drugs.Results1.OFD was screened out to effectively inhibit the survival of NSCLC cells through drug screening.2.Further experiments showed that OFD displayed good anti-NSCLC activity in vitro and in vivo.In addition,flow cytometry analysis showed that OFD could induce the cell cycle arrest at G0/G1 phase in NSCLC cells.Meanwhile,immunoblotting showed that OFD could significantly down-regulate the expression of cyclin-related proteins CDK4,CDK6 and p-Rb,and up-regulate the expression of p21 and p27.Additionally,luciferase assay showed that OFD could inhibit the transcriptional activity of transcription factor E2F1.3.OFD inhibited the activation of c-Src in a variety of NSCLC cells,and down-regulated the phosphorylation of c-Src in a concentration-dependent manner.OFD also inhibited the activation of c-Src in NSCLC cells induced by EGF and TGF?.4.CCK-8 assay showed that OFD significantly inhibited the growth of different NSCLC cell lines,while overexpression of c-Src partially inhibited the anti-NSCLC effect of OFD.At the same time,molecular docking also shows that OFD can be well docked into the active pocket of c-Src.5.CCK-8 and immunoblotting assays showed that OFD could synergistically enhance the cytotoxicity of chemotherapy drugs to NSCLC by reducing the activation of c-Src.ConclusionIn this study,we found that OFD potently suppressed the activation of c-Src in NSCLC cells,and inhibited the cell survival of NSCLC cells.In addition,overexpression of c-Src decreased the effects of OFD on NSCLC cells.Further studies showed that OFD induced cell cycle arrest at G0/G1 phase,downregulated CDK signaling in NSCLC cells,including CDK4,CDK6,p-Rb and E2F1,and upregulated the expression of p21 and p27.In addition,OFD enhanced the cytotoxicity of cisplatin and taxol against NSCLC cells by reducing c-Src activation,which suggested that OFD may be repurposed for NSCLC therapy in clinic in combination with other drugs,like cisplatin and taxol.