Mechanism Of Enhancing The Sensitivity Of Non-small Cell Lung Cancer To Apatinib Via Inhibition Of Autophagy

BackgroungLung cancer has the highest incidence in the world.Among them,non-small cell lung cancer(NSCLC)accounts for about 85%of all lung cancers,and most patients have metastasized at the time of initial diagnosis.Apatinib is a small molecule inhibitor of vascular endothelial growth factor recEPtor 2(VEGFR2)and has been shown to have an exact anticancer effect on advanced non-small cell lung cancer through antitumor angiogenesis.Related studies have shown that tumor autophagy is closely related to tumor angiogenesis.Therefore,it is of great value to study the mechanism of autophagy affecting the sensitivity of non-small cell lung cancer to Apatinib.Objectives(1)To study the effects of Apatinib on the proliferation,apoptosis and autophagy of non-small cell lung cancer cells.(2)To explore the direct effect of autophagy on tumor cells in Apatinib-treated non-small cell lung cancer.(3)To explore the effect of autophagy on vascular endothelial cells in Apatinibtreated non-small cell lung cancer.(4)To study the effects of Apatinib combined with CQ on non-small cell lung cancer in vivo by using immunodeficiency mouse non-small cell lung cancer xenograft model.Methods(1)Apatinib was used to treat A549 and PC9 at different concentrations or different durations of exposure.Cell proliferation and apoptosis levels were measured by MTT,colony formation,flow cytometry,and Western blot.(2)Apatinib was used to treat A549 and PC9 at different concentrations or different durations.The autophagosomes formed by the cells were observed by transmission electron microscopy,and LC3 and Beclin-1 were detected by Western blot.(3)A549 or PC9 were divided into 4 groups,they are Control group,Apatinib group,CQ group,and Apatinib combined with CQ group.Western blot and flow cytometry were used to detect apoptosis level.(4)The cell supernatants of the above 4 groups were collected to act on HU VEC cells.Transwell test and scratch test were used to detect cell migration and invasion ability,tube formation experiment was used to detect angiogenesis ability,qPCR was used to detect tumor cell VEGFA mRNA levels,and ELISA was used to detect cell supernatant VEGFA levels.(5)Transfection of small interfering RNA Beclin-1 down-regulates the expression of autophagy-related gene Beclin-1 and test its effect by Western blot and qPCR;CQ or si Beclin-1 was used to inhibit autophagy,and the effect of inhibiting autophagy on JAK2/STAT3/VEGFA signaling pathway was detected by Western blot;(6)Subcutaneous tumor formation in nude mice was divided into Control group,Apatinib group,CQ group,and Apatinib combined with CQ group,and tumor volume of nude mice was measured.The changes of JAK2/STAT3/VEGFA signaling pathway was detected by Western blot.Results(1)Apatinib inhibits the growth of NSCLC cells and induces its apoptosis in a concentration-dependent and time-dependent manner.(2)Apatinib promotes autophagy in NSCLC cells in a concentration-dependent and time-dependent manner.(3)Inhibition of autophagy enhance the inhibitory effect of Apatinib on NSCLC cells.(4)Inhibition of autophagy can enhance the anti-angiogenesis effect of Apatinib.(5)Inhibition of autophagy can enhance the anti-angiogenesis effect of Apatinib through the JAK2/STAT3/VEGFA signaling pathway.ConclusionsApatinib acts on non-small cell lung cancer cells,which can inhibit proliferation,induce apoptosis,and promote autophagy.Being Combined with autophagy inhibitors can enhance the antiangiogenic effect of Apatinib through the JAK2/STAT3/VEGFA signaling pathway.