Apoptosis-inducing effect of adenovirus-mediated Apoptin on human large cell lung cancer NCI-H460 cells

Abstract:

Objective：To construct recombinant adenovirus vector（pAdxsi-GFP-VP3） containing VP3 gene and to investigate the apoptosis-inducing effect of VP3 gene on human large cell lung cancer NCI-H460 cells. Methods：VP3 gene was gained by enzyme digestion with pcDNA3.0-VP3 recombined plasmid as a template and was diverted to shuttle vector. The pAdxsi-GFP-VP3 was ob－tained by connecting with pAdxsi vector. After enzymatic digestion and verification by sequencing，virus amplification and purification were conducted，and the titer of the recombinant adenovirus was determined by biological and physical assays finally. The pAdxsi-GFP-VP3 was used to transfect human large cell lung cancer NCI-H460 cells with optimum multiplicity of infection. Expression of Apoptin in large cell lung cancer cells was respectively detected by reverse transcription polymerase chain reaction（RT-PCR） and Western blot. Cell ultrastructure at different periods of time after the transfection was observed by transmission electron microscopy（TEM）. Ef－fect of Apoptin on cell proliferative vitality was determined by MTT assay. Apoptotic rate and cell cycle in NCI-H460 cells after transfection were measured by flow cytometry（FCM）. Results：The pAdxsi-GFP-VP3 was successfully constructed by enzyme diges－tion and sequencing identification and the virus titer was 1.6×109 PFU/ml. RT-PCR results showed the mRNA expression of Apoptin in each experimental group at 48 h after the transfection. Western blot confirmed that Apoptin protein was highly expressed in each experimental group at 72 h after the transfection. The typical morphological changes in early apoptosis such as cell shrinkage，chromatin condensation and apoptotic bodies were observed under TEM. MTT assay indicated that the proliferative activity of trans－fected NCI-H460 cells was remarkably decreased and present－ed in a time-dependent manner（F=93.384，P=0.000） and there were statistical significances among different groups（F=18.427，P=0.003）. FCM detection showed that the expression of Apoptin in NCI-H460 cells after the transfection could induce cell apoptosis with a gradual increase in the apoptotic rate over time（F=47.307，P=0.000；F=303.237，P=0.000）. Moreover，the proliferation of NCI-H460 cells was slowed down；the proportion of cells was decreased at S phase（F=80.240，P=0.000） and was blocked at G2/M phase after the transfection compared with that of control group（F=110.080，P=0.000）． Conclusion：Our data demonstrate that Apoptin can effectively induce apoptosis of NCI-H460 cells，which may lay an ex－perimental basis for applying Apoptin in human NSCLC gene therapy.