Adenovirus-mediated tissue-targeted expression of the CDglyTk gene for the treatment of breast cancer

Abstract

The aim of this study was to evaluate the selective killing efficacy of adenovirus (Ad)-mediated double suicide genes driven by the kinase domain-containing receptor (KDR) promoter in human breast cancer cells and vascular endothelial cells. Two Ad-mediated double suicide gene systems [with the two suicide genes, thymidine kinase (TK) and cytosine deaminase (CD)] with the KDR promoter (Ad-KDRP-CDglyTK) and the cytomegalovirus (CMV) promoter (Ad-CMV-CDglyTK) were established and transfected into the KDR-expressing MCF7 human breast cancer, EC304 human vascular endothelial and LS174T human colon carcinoma, which does not express KDR, cell lines. The selective killing efficiency and specificity of the double suicide gene system were measured in vitro by the analysis of cellular proliferation and assayed in vivo by subcutaneous injection of MCF7 cells into nude mice. The microvessel density (MVD) in the transplanted tumor was determined by immunohistochemical staining of CD34 cells. Our results showed that the transgenic CDglyTK genes were expressed in three cell lines (MCF7, ECV304 and LS174T) infected with Ad-CMV-CDglyTK. However, of the cells infected with Ad-KDRP-CDglyTK, the transgenic CDglyTK gene was only expressed in the KDR-expressing MCF7 and ECV304 cells, but not in the KDR-deficient LS174T cells. Cell proliferation was significantly reduced in a dose-dependent manner by pre-treatment with ganciclovir (GCV) and 5-fluorocytosine (5-FC) in MCF7 and ECV304 cells with transfected KDRP-CDglyTK genes and the three cell lines transfected with the CMV-CDglyTK genes. Similar results were not observed in the LS174T cells with transfected KDRP-CDglyTK genes. The results of this study show that the tumor-targeted expression of CDglyTK driven by the KDR promoter has a high specificity and performance. The killing effect of the CD/TK fusion gene in the target cells was significantly increased compared with the single suicide gene. The cell cycle of MCF7 and ECV304 cells transfected with KDRP-CDglyTK genes was arrested at the S phase following treatment with the prodrugs. The tumors formed by the MCF7 cells with the double suicide gene system were much smaller and the MVD of the tumor tissue was significantly decreased compared with the control. This study demonstrates that tumor‑targeted expression of the CDglyTK gene driven by the KDR promotor may be a novel strategy for the gene therapy of human breast cancer.