The Mechanism Study Of Apoptosis Induced By Noval Naphthalimide Compound NA17 In Non-small-cell Lung Cancer NCI-H460 Cells

Lung cancer is one of the leading causes of cancer-related mortality worldwide,nearly 80%to 90%of patients with lung cancer are non-small cell lung cancer(NSCLC).Over the past 40 years,the overall survival of patients with advanced lung cancer was less than a year due to the invalid treatment.The advent of molecular targeted therapies has inspired new confidence in the treatment of lung cancer.However,molecular targeted therapy based on the target EGFR did not improve the status of high mortality in patients with advanced lung cancer.Therefore,the key to develop the clinically effective drugs is to find a new therapeutic target based on the molecular genetic background of lung cancer,especially in non-small cell lung cancer.Inactive of tumor suppressor gene p53 has become a common cause of non-small cell lung cancer morbidity and treatment ineffective.Restoring p53 function in non-small cell lung cancer has attracted the attention of domestic and foreign scholars as effective targeted therapy.A series of novel naphthalimide derivatives have been synthesized in our laboratory.The results of the pp53-TA-Luc reporter gene showed that the noval naphthalimide derivative NA17 could significantly increase the activity of p53 and effectively inhibit the growth of tumor cell lines.However,the target and molecular mechanism of action of NA17 is not clear.In this study,a variety of experimental methods in the field of molecular pharmacology were carried out to deeply research the anti-tumor mechanism of NA17,and the target of NA17 was identified,providing theoretical basis and data reference for the following-up study of the compound.1.The anti-tumor activity of NA17 was screened by MTT assay in vitro.The cell viability of NA17 against tumor cell lines(NCI-H460,BEL-7404,HCT116,MCF-7,Hep G2,MGC-803)and normal cell lines(HL-7702,WI-38)showed NA17 exhibited broad antitumor activity,with significant inhibition proliferation on multiple tumor cells but low cytotoxicity to normal cells.there is a better selectivity.Different types of non small cell lung cancer cell lines(NCI-1975,NCI-358,NCI-827)showed different sensitivity to NA17,which is mainly related to the different degree of activation of endogenous p53.In addition,reducing the expression of endogenous p53 by RNA interference can significantly reduce the sensitivity of tumor cells to NA17,indicating that NA17 played an anti-tumor effect by activating p53.2.Western blot(WB)results showed that NA17 induced phosphorylation of p53 serine 392 site(p-p53 s392)in a concentration-dependent manner.The results of subcellular distribution,DNA damage detection,Coip-MS and protein immunoblotting experiment showed that NA17 activated wild-type p53 in NCI-H460 cells by directly interacting with p53 in the cytoplasm,rather than by DNA damage or increasing endogenous expression of p53.3.The effect of NA17 on cell cycle was examined by flow cytometry.The results showed that NA17 arrested NCI-H460 cells at G1 phase.The expression level of G1 phase-related regulatory proteins were detected by WB,and the results showed that NA17 could affect the activity of cyclin-CDK4 complex by up-regulating the expression of p21 which is G1 check-point regulation protein,inducing cell G1 phase arrest effectively.4.The effect of NA17 on apoptosis was detected by Hoechst33258 staining,AIF immune-fluorescence assay and caspase-3 activation.The results showed that NA17 could induce the early apoptosis of cells.AIF subcellular distribution experiment results,downregulation of Bcl-xl and upregulation of Bim,Bax and Bak which are key apoptotic proteins in mitochondria suggested that NA17 induced apoptosis by endogenous apoptotic pathways.Further studies revealed that targeted accumulation of phosphorylation of p53(p-p53)in mitochondria by NA17 resulted in conformational activation of Bak,leading to cell apoptosis.In this study,the pharmacological activity and mechanism of NA17 were systematically studied.The results showed that NA17 directly interacted with p53 and triggered phosphorylation of p53 at serine 392 site,phosphorylation of p53 targeted accumulation in mitochondria to play a role in cycle arrest and apoptosis,thereby exerted antitumor effect.