Non-viral Suicide Gene Therapy: Cytosine Deaminase Gene Directed by VEGF Promoter and 5-fluorocytosine as a Gene Directed Enzyme/prodrug System in Breast Cancer Model

The present study investigated the potential of vascular endothelial growth factor (VEGF) promoter to derive cytosine deaminase (CD) transfected by polyamidoamine (G4-PAMAM) dendrimers to 4T1 murine breast cancer cell line as gene-directed enzyme/prodrug therapy. The VEGF promoter and cytosine deaminase gene were cloned into the pEGFP-N1vector from the genomic DNA of 4T1 and E. coli, respectively. The frequency of transfection for VEGF-CD-pEGFP-N1 and pEGFP-N1- CD treated groups was 35±3 and 36±4, respectively. MTT assay was perform to evaluate the cytotoxic effects of converted 5-flurocytosine on 4T1 cells. Also, the optimal concentration of 5-FC in 4T1 cells transfected by VEGF-CD-pEGFP-N1 plasmid was evaluated. The GFP expression of transfected 4T1 cells by VEGF-CD-pEGFP-N1were observed by fluorescent microscopy and flowcytometry. Results demonstrated that the suicide CD gene was successfully expressed in 4T1 cells determined by RT-PCR and GFP expression. A concentration of 200 μg/ml 5-FC was identified as optimal dose of prodrug. Furthermore, the CD/5-FC enzyme/prodrug system not only demonstrated toxicity on transformed 4T1 cells but also exerted a ‘bystander effect’ determined by MTT assay. The results showed that by 35% transfection with VEGF-CD–pEGFP-N1and CD-pEGFP-N1 plasmids, 80% and 90% inhibition of the cells growth occurred, respectively.