The Research Of Sirtuin 3 Enhancing The Chemotherapy Sensitivity Of Small Cell Lung Cancer NCI-H446

Small cell lung cancer(SCLC)is the most invasive of all lung cancer subtypes,and its rapid response to chemotherapeutic drugs is characteristic.Therefore,finding targeted treatment methods that can overcome chemotherapy resistance is the key to treating SCLC.P53 is mutated in most SCLCs,and the p53 after mutation has the characteristic of enhancing chemotherapy resistance.The p53 protein is regulated by a variety of post-translational modifications,such as methylation,phosphorylation,acetylation,etc.,where acetylation plays a key role in the function of p53.Acetylation and deacetylation of p53 may be potential targets for modulating chemosensitivity.Recent studies have shown that SIRT3,which is present in mitochondria,acts as the most important deacetylase in mitochondria and regulates acetylation of p53.However,whether SIRT3 can regulate post-translational modification and regulation of mutant p53 has not been studied.This study first observed and explored the mechanism of SIRT3-induced apoptosis in SCLC cells and increased the chemosensitivity of SCLC in vitro,and then verified it in vivo.By studying the regulation mechanism of SIRT3 on mutant p53,it may provide a new breakthrough point for the treatment of SCLC.Objective:1.To observe the effect of NCI-H446 cells overexpressing SIRT3 on cisplatin chemotherapy sensitivity;2.To explore the mechanism of SIRT3 enhancing the sensitivity of small cell lung cancer NCI-H446 cells to cisplatin.Methods:1.SIRT3 enhances the sensitivity of small cell lung cancer NCI-H446 cells to cisplatin in vitro.NCI-H446 cells were cultured and the experiment was set as a control(CON)group,a transfected empty vector(NC)group,a SIRT3 group,a cisplatin(CDDP)group,and a SIRT3+CDDP group.MTT determined the optimal concentration of SIRT3 combined with cisplatin;Annexin V-FITC flow cytometry to detect apoptosis and JC-1 detection of mitochondrial membrane potential;Western Blot detection of Bax,Bcl-2,Cleaved caspase-3,Cleaved caspase-9,caspase8 and other expression of apoptosis-related proteins;2.Mechanism of SIRT3 enhancing sensitivity of small cell lung cancer NCI-H446 cells to cisplatinSIRT3 activity assay(fluorescence)kit detects SIRT3 deacetylase activity;immunofluorescence determines colocalization of SIRT3 and mutant p53 in cells;immunoprecipitation assay detects binding of mutant p53 to SIRT3 and polyubiquitin protein K48 Ability;the ubiquitin proteasome inhibitor MG132 was added,and the ubiquitination and degradation of mutant p53 were detected by Western Blot.3.SIRT3 enhance the sensitivity of small cell lung cancer NCI-H446 cells to cisplatin in vivoA nude mouse model of small cell lung cancer was constructed.The nude mice were divided into CON group,SIRT3 group,CDDP group and SIRT3+CDDP group.The attenuated Salmonella carrying the SIRT3 plasmid was transferred into nude mice.The treatment was performed by intraperitoneal injection to observe the change of tumor growth.The apoptosis of tumor tissue was detected by TUNEL staining.The expression of SIRT3 and mutant p53 was detected by immunohistochemistry.The expression of SIRT3,mutant p53 and apoptosis-related protein were detected by Western Blot.Results:1.SIRT3 enhances the sensitivity of small cell lung cancer NCI-H446 cells to cisplatinThe effect of SIRT3 combined with cisplatin on cell proliferation was determined by MTT,and the optimal combined concentration was determined.The results showed that SIRT3 combined with 0.25?g/ml cisplatin significantly inhibited the proliferation of NCI-H446 cells compared with CON group(P<0.05).).Apoptosis was detected by Annexin V-FITC flow cytometry,and it was found that SIRT3+CDDP group significantly promoted apoptosis compared with CON group,NC group,SIRT3 group and CDDP group(P<0.05);JC-1 Compared with the CON group,the NC group,the SIRT3 group and the CDDP group,the SIRT3+CDDP group significantly reduced the membrane potential of intracellular mitochondria(P<0.05).Western Blot results showed that SIRT3 up-regulated the expression of proapoptotic proteins such as Bax,caspase8,Cleaved caspase-3 and Cleaved caspase-9,and down-regulated the expression of Bcl-2 apoptosis-inhibiting protein(P<0.05).2.SIRT3 promotes apoptosis of NCI-H446 cells by deacetylating mutant p53,which is degraded by ubiquitin proteasome pathway.SIRT3 activity assay(fluorescence)kit detects SIRT3 deacetylase activity to determine the validity of SIRT3 plasmid,and the overexpression of SIRT3 protein is active;immunofluorescence determines the colocalization of SIRT3 and mutant p53,immunoprecipitation assay for mutant p53 and The binding ability of SIRT3 and polyubiquitin protein K48,SIRT3 can bind to SIRT3 in the cytoplasm,directly deacetylate mutant p53,and increase ubiquitination.Add ubiquitin proteasome inhibitor MG132,and detect the mutant by Western Blot.The expression of p53 returned to the level of the control group,indicating that the reduction of mutant p53 was carried out by degradation of the ubiquitin proteasome pathway.3.SIRT3 enhance the sensitivity of small cell lung cancer NCI-H446 cells to cisplatin in vivoThis study found that SIRT3 combined with cisplatin inhibited tumor growth in vivo.After the attenuated Salmonella carrying the SIRT3 plasmid entered the nude mice,the growth curve of the tumor volume showed that although the SIRT3 group or the SIRT3+CDDP group had a significant inhibitory effect on tumor growth compared with the control group or the CDDP group,the tumor was significantly inhibited.The best therapeutic effect of growth is the SIRT3+CDDP group.The results of TUNEL staining showed that the apoptosis increased after combined therapy.The expression of apoptosis-related protein Bax and cleaved caspase-3 was increased by Western blot,the expression of Bcl-2 was decreased,and the expression of mutant p53 was also decreased.Immunohistochemistry results showed that the expression of mutant p53 was more markedly reduced after combination therapy.This indicates that SIRT3 can inhibit tumor growth,induce apoptosis and enhance chemosensitivity in vivo.Conclusions:1.SIRT3 can significantly enhance the chemosensitivity of small cell lung cancer NCI-H446 cells to cisplatin in vitro and in vivo.2.SIRT3 promotes apoptosis of NCI-H446 cells by deacetylating mutant p53,which is degraded by ubiquitin proteasome pathway.