Endogenous production of nitric oxide contributes to proliferation effect of vascular endothelial growth factor-induced malignant melanoma cell

The objectives of this study were to observe the effect of overexpression of vascular endothelial growth factor (VEGF) on the proliferation of the malignant melanoma (MM) cell line A375, and to study the role of nitric oxide (NO) in this process and the mechanism of VEGF induced-A375 cell proliferation. The VEGF(165) cDNA was transfected into A375 cells by electroporation. VEGF mRNA and protein in A375 cells were detected by RT-PCR and ELISA. The proliferation of A375 cells was assessed by cell counting and MTT assay. Protein expression of iNOS, eNOS and nNOS was detected by Western blotting. NO production in A375 cell supernatant was measured by the nitrate reductase method. VEGF mRNA in A375 cells was significantly increased 72 h and 96 h after transfection of VEGF(165) cDNA, as were VEGF protein, NO and iNOS levels. However, protein expression of eNOS and nNOS was not detected in either transfected or untransfected cells. Proliferation of A375 cells transfected with VEGF(165) cDNA was enhanced. The nitric oxide synthase inhibitor L-NAME could dose-dependently inhibit the proliferation of A375 cells evoked by VEGF. These results indicate that VEGF enhances the expression of iNOS in A375 cells and results in an increase in NO formation, which may be important in the process of VEGF-induced proliferation of A375 cells.