Drug Res (Stuttg) 2021; 71(07): 395-406
DOI: 10.1055/a-1488-6054
Original Article

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

Manouchehr Emamian
1  Department of Genetics & Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
2  Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
,
Akbar Abbaspour
2  Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
,
Tina Shahani
1  Department of Genetics & Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
2  Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
4  Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
,
Alireza Biglari
1  Department of Genetics & Molecular Medicine, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
4  Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
,
Ali Sharafi
2  Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
3  Department of Pharmaceutical Biotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
4  Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
› Institutsangaben
Funding A-12–848–15; provided by Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan, Iran.

Abstract

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.



Publikationsverlauf

Eingereicht: 31. März 2021

Angenommen: 20. April 2021

Publikationsdatum:
28. Juni 2021 (online)

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