Recombinant vp3 gene adenovirus-induced apoptosis in human breast cancer MCF-7 cells

Abstract

Objective To construct recombinant vp3 gene adenovirus pAD-vp3 and study its apoptosis inducing effect on human breast cancer MCF-7 cells. Methods vp3 gene was cloned and recom-bined into adenovirus vector pLP-AD-vp3 (pAD-vp3) at loxP site according to homologous recombination principle. pAD-vp3 was transformed into package cell line 293A and then into NIH3T3 cells for titer assay. The MCF-7 cells were transfected with pAD-vp3.Western blotting was used to detect the Apoptin protein expression. MTT assay was adopted to measure cellular proliferation and vp3 gene expression. Forty-eight h after transfection, flow cytometry (FCM) was used to examine apoptosis, and surface enhanced laser de-sorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS) was used to assay protein profile. Nude mice model of MCF-7 cells was set up to observe the tumor inhibition rate of pAD-vp3, and real-time PCR and TUNEL assay were used to detect vp3 gene and apoptosis respectively. Results Recombinant adenovirus vector pAD-vp3 was successfully constructed. Virus titer was 3 x 108 pfu/ml in the 293A culture supernatant. Forty-eight h after transfection, cellular inhibition rate was 63.3% in MTT assay, higher than that in blank control (P 0.05). Conclusion Recombinant adenovirus bearing vp3, pAD-vp3, was set up successfully. vp3 could induce apoptosis in MCF-7 cells in vivo and in vitro. Key words: Breast tumor; Gene therapy