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-N1 vector from the genomic DNA of 4T1 and E. coli, respectively. The frequency of transfection for VEGF-CD-pEGFP-N1 and pEGFPN1-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-N1 were observed by fluorescent microscopy and flow cytometry. Results demonstrated that the suicide CD gene was successfully expressed in 4T1 cells determined by RT-PCR and GFP expression. A concentration of 200 mu g/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-N1 and CD-pEGFP-N1 plasmids, 80 % and 90 % inhibition of the cells growth occurred, respectively.