The in vitro study on the infection of oncolytic adenovirus CNHK600-IL-24 to breast cancer cells

Objective To construct an oncolytic adenovirus CNHK600-IL-24,and to evaluate its tumor-specific proliferation and killing capacity in vitro. Methods The IL-24 gene was cloned into adenovirus shuttle vector SG502-ΔCR2,and CNHK600-IL-24 was obtained by cotransfection of SG502-INS-IL-24 and pPE3 plasmids and subsequent recombination in 293 cells.Fluorescence microscopy and viral replication experiments were performed to evaluate the proliferation ability in breast cancer cell line MDA-MB-231 and normal fibroblastic cell line MRC-5.MTT assay was performed to determine the inhibitory effect on the two cell lines.ELISA was performed to detect IL-24 in the supernatant after the virus infection.And we detected the intracellular IL-24 protein by Western blot analysis.Finally,the flow cytometry was used to detect cell apoptosis. Results The oncolytic adenovirus CNHK600-IL-24 was correctly constructed and confirmed by restriction DNA sequence analysis and PCR.The titer of CNHK600-IL-24 reached 1.9×1010 pfu/mL.CNHK600-IL-24 proliferated and replicated more efficiently in MDA-MB-231 cells than in MRC-5 cells.CNHK600-IL-24 could kill MDA-MB-231 cells at very low MOI,while the killing capacity to MRC-5 cells was rather weak.The amount of IL-24 increased obviously 48 hours postinfection in MDA-MB-231 cells,but for MRC-5 cells,the level of IL-24 maintained low.The flow cytometry analysis showed that CNHK600-IL-24 induced apoptosis in the MDA-MB-231 cells,but the apoptosis of MRC-5 cells was low. Conclusions The high-titer oncolytic adenovirus CNHK600-IL-24 was successfully constructed and purified.CNHK600-IL-24 could proliferate specifically in breast cancer cells and lysed them.