Apoptosis-inducing Effect Of Adenovirus-mediated Apoptin On Human Large Cell Lung Cancer NCI-H460Cells

Objective To construct recombinant adenovirus vector containingVP3gene and investigate the apoptosis-inducing effect of VP3gene onhuman large cell lung cancer NCI-H460cells.Methods VP3gene was gained by enzyme digestion withpcDNA3.0-VP3recombined plasmid as a template and diverted to shuttlevector. The pAdxsi-GFP-VP3was obtained by connecting with pAdxsivector. After enzymatic digestion and verification by sequencing, virusamplification and purification were conducted, and the titer of therecombinant adenovirus was determined by biological and physical assaysfinally. The pAdxsi-GFP-VP3was used to transfect human large cell lungcancer NCI-H460cells with optimum multiplicity of infection. Expressionof Apoptin in large cell lung cancer cells was respectively determined byreverse transcription polymerase chain reaction (RT-PCR) and Western Blotassay. Cell ultrastructure at different periods of time after transfection wasobserved by transmission electron microscopy (TEM). Effect of Apoptin on cell proliferative vitality was detected by MTT assay. Apoptotic rate and cellcycle in NCI-H460cells after transfection were measured by flow cytometry(FCM).Results The pAdxsi-GFP-VP3was successfully constructed byenzyme digestion and sequencing identification, and the virus titer was1.6×109PFU/ml. RT-PCR results showed the mRNA expression of Apoptinin each experimental group at48hours after transfection. Western blot assayconfirmed that Apoptin protein was highly expressed in eachexperimental group at72hours after transfection. The typical ultrastructuralchanges in infected cells were found with TEM，for example cell shrinkage,chromatin condensation and apoptotic bodies. MTT assay indicated that theproliferative activity of transfected NCI-H460cells was remarkablydecreased and presented time-dependent (P<0.05). FCM detection showedthat the expression of Apoptin in NCI-H460cells after transfection couldinduce cell apoptosis with a gradual increase in the apoptotic rate over time(P<0.05). Moreover, the proliferation of NCI-H460cells was slowed down,and the proportion of cells at S phase was decreased (P<0.05) and that atG2／M phase was blocked after transfection compared with control group(P<0.05).Conclusion Our data demonstrate that Apoptin can effectivelyinduce apoptosis of NCI-H460cells, which may lay an experimental basisfor further study on the application of Apoptin in human NSCLC gene therapy.