1. Tarbiat Modares University 2. Tarbiat Modares University
Abstract
Introduction
Targeted expression of killer genes in tumor cells ensures specific elimination of cancer cells with minimal effect on normal cells. utilizing a cancer specific promoter fused with the killer gene offers particular opportunities for selective targeting of cancer cells. the natural killing ability of pseudomonas aeruginosa exotoxin a (eta) makes it an attractive candidate for eradication of tumor cells. interleukin8 receptor called cxcr1 is overexpressed in breast cancer cells, thus cxcr1 promoter can be used as breast cancer specific promoter.the objective of this study was to compare the expression rate of pseudomonas aeruginosa exotoxin a gene under the control of cxcr1 promoter in breast adenocarcinoma cell line mda-mb-231 and in normal breast epithelial cell line mcf-10a and to determine the killing activity of this construct in these cell lines as the representative of cancerous breast cells and normal breast cells respectively.
Methods
We used the pgl4.14 vector to construct a novel plasmid (pgl4.14 cxcr1-eta) for eukaryotic expression of pseudomonas aeruginosa exotoxin a (eta). mda-mb-231 and mcf10a cell lines were transfected with this construct, using lipofectamine 2000. the eta gene expression was measured by real-time pcr. the cytotoxic effect of this protein on the cells were determined by mtt assay.
Results
Rt-pcr was conducted to determine the expression levels of eta gene in the cells. rt-pcr results showed that, under the cxcr1 promoter, expression of eta was 81% in mda-mb-231 cells and 38% in mcf-10a cells. the cytotoxic effect of eta on cancerous and normal cell lines was determined by mtt assays. according to the results of mtt assay, in the mda-mb-231 cell line more than 90% cell death was observed when these cells transfected by pgl4. cxcr1-eta construct. nevertheless mcf-10a cells exhibited about 34% dead cell concentrations.
Conclusion
Our results confirmed that introducing the pseudomonas aeruginosa exotoxin a gene under the control of cxcr1 promoter into breast cells can selectively kill cancerous cells with minimal effect on normal cells, thus these two elements might be promising tools for breast cancer gene therapy.
Keywords
Targeted killer gene therapy, breast cancer cells, pseudomonas aeruginosa exotoxin a, cxcr1 promoter
