Ribozyme mediated cleavage of cell-associated isoform of vascular endothelial growth factor (VEGF) inhibits liver metastasis of a pancreatic cancer cellline

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Stromal angiogenesis is an important factor for progression of malignant neoplasms. we used hammerhead ribozymes against vascular endothelial growth factor (VEGF) gene transcripts to downregulate cell-associated VEGF189 isoform function in a pancreatic cancer cell line MIA-PaCa2. The b-actin promoter-driven ribozyme sequences were introduced into the cells by lipofection. We evaluated their effects on growth and neovascularisation of the cell lines. Metastatic potential was evaluated by the injection of transformants into the splenic pulp of nude mice. Anti-VEGF189 ribozyme specifically and efficiently cleaved the VEGF189 mRNA isoform but not other types of isoforms in vitro. MIA-Paca2 introduced with anti-VEGF189 ribozyme did not show any alteration of growth rate under tissue culture conditions compared to MIA-Paca2 introduced with disabled ribozyme. Production of tissue type plasminogen activator decreased in the human umblical vascular endothelial cells cocultured with the MIA-Paca2 cell line introduced with anti-VEGF189. When the transformants were subcutaneously transplanted, tumour volume of the ribozyme-introduced MIA-Paca2 xenografts was significantly smaller than that of disabled ribozyme-introduced cell line xenografts (P<0.01, Dunnett's test). No metastasis was apparent of the MIA-Paca2 introduced with anti-VEGF189, while disabled ribozyme transfected MIA-Paca2 show significant liver metastasis (P<0.05, Fisher's exact test). These results suggested that the VEGF189 mRNA plays an important role in growth and metastatic potential through alteration of angiogenic balance in cancer tissue.