Rap1GAP Inhibits VEGF Autocrine And Paracrine In Melanoma

Purpose:To establish the Rap1GAP over expression melanoma cell system, and research its effects on melanoma’s tumorgenesis and mechanism. Research the relationship between Rap1GAP overexpression and VEGF’s synthesis and secretion in malignant melanoma cells. Investigate the Rap1GAP’s inhibition VEGF effects mechanism. And explore VEGF’s affection on malignant melanoma cells. Research the function of VEGF paracrine in melanoma cells with Rap1GAP over expression and the effects on the vascular endothelial cells in melanoma.Methods:Transfection the malignant melanoma cells by plasmid pcDNA-Rap1GAP-Flagged and plasmid pcDNA 3.1. Subcutaneous injection the cells into Balb/c null mice, to establish tumorgenesis models. Compare the tumor size in different Rap1GAP expression. Test the number of new born vessels in different tumors by immunohistochemistry. And test the expression levels of HIF-αand VEGF by western blot. Active or inhibit transfected melanoma cells’Rap1 and its downstream signal molecules by Epac, PD98059 and LY294002. In different levels of VEGF and Rap1GAP expression, test the proliferation of cells by MTT, migration by transwell test and zymography, apoptosis by TUNEL with or without tool drugs interface. Test the concentration of VEGF in melanoma cells cultured medium. Cross training the endothelial cells by transfected melanoma cells cultured medium with or without Rap1GAP over expression. And investigate the Rap1GTP levels in endothelial cells in different culture medium.Result:The malignant melanoma cells with Rap1GAP over expression formed lower size tumor compare to normal cells. In over expression Rap1GAP tumors, HIF-αand VEGF expression were lower than the malignant melanomas with normal Rap1GAP expression. In vitro experiments also show Rap1GAP inhibits HIF-αand VEGF expression as well as in vivo. Epac increases VEGF and p-ERK levels in malignant melanoma cells, however, both Rap1GAP and PD98059 are able to cancel its affections. VEGF is able to increase Rap1 activity by ERK and Akt pathway activation, which is also inhibited by Rap1GAP. The further research show that in melanoma cells, VEGF inhibits apoptosis related proteins including Caspase 3 and Caspase 9, increases Cyclin D1 to improve proliferation, and actives MMP-9 and MMP-2. Those results are accord with cell biological research. However, all affections of VEGF mentioned is able to be inhibited by Rap1GAP. VEGF concentration significant decreased in the melanoma cells with Rap1GAP over expression. And after cross training, endothelial cells cultured in the medium from Rap1GAP over expression melanoma cells has lower Rap1GTP levels.Conclusion:Rap1GAP inhibits malignant melanoma tumorgenis. As VEGF induce angiogenesis and vascular mimic in melanoma, decreasing expression of VEGF and HIF-αmay play an important role in its affection. Malignant melanoma cell has VEGF autocrine effects which work through Akt and ERK pathway. VEGF stimulates melanoma cells’ VEGF secretion that forms a positive feedback called "VEGF loop". And it leads to cell proliferation out of control, and finally tumorgenesis. Phosphorylated ERK inhibits VEGF mRNA hydrolyzation, which may increase VEGF in translation process. Akt pathway plays an important role in malignant melanoma tumorgenesis, which also increases VEGF mRNA level by improving HIF-αexpression. Rap1GAP inhibits VEGF expression in both of transcription and translation. What’s more, Rap1GAP also inhibits VEGF’s effects by inhibition of downstream proteins of VEGF. The melanoma cells are able to paracrine VEGF to induction and chemotaxis the endothelial cells to melanoma. As a result angiogenesis in melanoma, it forms enough vessels to supply nutrition. The mechanism of angiogenesis in melanoma may be related to activation in melanoma.