The Research On The Role Of TOPK In Apoptin-induced Apoptosis In Tumor Cells

Objective: The aim of the present study was to assess the efficacy of TOPK in Apoptin-induced apoptosis in tumor.Explore whether TOPK could phosphorylate Apoptin directly and confirm the phosphorylation sites.Methods: 1.The plasmids for lentivirus packaging,pc DNA3.1,and pc DNA3.1-HATOPK were prepared by plasmid extraction.The PTD4-apoptin(P4A)protein was prepared by induced expression in prokaryotes and purified by Ni-sepharose.2.A variety of human tumor cells were cultured,and the expression level of TOPK was detected by Western Blot.Colon cancer cells HCT116,gastric cancer cells SGC7901 and hepatocellular carcinoma cells Hep G2 were selected for the following experients.The P4 A protein was incubated with these cells.MTT was used to detect the effect of P4 A protein on cell viability;Western Blot was used to detect the expression of apoptosis related protein c-caspase3.3.TOPK was knocked down in HCT116&SGC7901 by lentivirus-medicated shRNA system and had selected by puromycin for five days to set up the stable silenced cell lines.After incubated with P4 A protein,we investigated whether knockdown of TOPK had effect on apoptin-induced apoptosis in SGC7901 cells and HCT116 cells.4.pcDNA3.1-HA-TOPK was transiently transfected into hepatocellular carcinoma cells HepG2.After incubated with P4 A protein,we investigated whether overexpression of TOPK had an effect on apoptin-induced apoptosis in HepG2 cells.5.In vitro kinase assay was carried out to verify the phosphorylation of Apoptin protein by active TOPK and then the amino acid sequence was analyzied by NetPhos2.0,those serines and threonines with high scores were selected and related peptides were synthesized.In vitro kinase assays were carried out to detect the phosphorylation of peptides by active TOPK.Results: 1.The plasmids lentivirus packaging were co-transfected into 293 T cells and the lentivirus was successfully obtained.The eukaryotic expression plasmid pcDNA3.1-HA-TOPK was prepared.The purified P4 A protein was prepared successfully.2.TOPK expression varies in different tumor cells,highly expressed in HCT116 cells as well as SGC7901 cells,moderately expressed in Hela cells,and lowly expressed in MKN45 and HepG2 cells.HCT116 cells,SGC7901 cells,HepG2 cells were selected for the following experiments.P4 A incubation induced the increased expression of ccaspase3 in the three tumor cells,which indicated that these cells were sensitive to P4 A treatment.3.TOPK was knocked down successfully in HCT116 cells and SGC7901 cells.After incubated with P4 A,compared with the sh Mock cells,the cell viability was increased in the shTOPK cells.The expression of c-caspase3 was decreased in the shTOPK cells.These results indicated that knocking down TOPK had impaired the sensitivity of P4 A in HCT116 cells and SGC7901 cells.4.pcDNA3.1-HA-TOPK was successfully transfected in HepG2 cells.After incubated with P4 A,compared with the untreated group,the cell viability was decreased and the expression of c-caspase3 was increased.Compared with P4 A alone or pcDNA3.1&P4A,the proliferation inhibition rate and the expression of c-caspase3 were higher in the pcDNA3.1-HA-TOPK&P4A.These results indicated that overexpressed TOPK had heightened the sensitivity of P4 A in HepG2 cells.5.In vitro kinase assays indicated that active TOPK could phosphorylate P4 A directly,and Ser89,Ser93,Thr8,Thr20,Thr27,Thr56,Thr108,Thr114 were potential phosphorylation sites.Conclusion: Knockdown of TOPK effectively impired apoptin-induced apoptosis in SGC7901 cells and HCT116 cells.Overexpression of TOPK enhanced apoptininduced apoptosis in HepG2 cells.Active TOPK could phosphorylate P4 A directly,and Ser89,Ser93,Thr8,Thr20,Thr27,Thr56,Thr108,Thr114 were potential phosphorylation sites.