Rap1 Stimulates VEGF and IL-8 Secretion in Oral Squamous Cell Carcinoma
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Rap1 is a small GTP-binding protein that has been reported to upregulate vascular endothelial growth factor (VEGF) and lnterleukin 8 (IL-8) transcription in prostate carcinoma cells. VEGF and IL-8 induce angiogenesis, which has a significant role in tumor growth and metastasis. Objective: The aim of this study was to determine whether Rap1 induced VEGF and IL-8 secretion in human oral squamous cell carcinoma cell lines. The hypothesis is that active Rap1 leads to the secretion of VEGF and IL-8 in the cancer cells, which provides a mechanism for regulation of tumor growth and metastasis. Methods: Cell lysates and conditioned media were harvested from ten human oral squamous cell carcinoma cell lines at - 60-70% confluence. The cell lines were evaluated for active Rap1 levels via immunoprecipitation of Rap1-GTP, and for total Rap1 by immunoblot analysis. The conditioned media was used to measure VEGF and IL-8 secretion by using an enzyme-linked immunosorbent assay (ELISA). Two cell lines were transfected with Rap-GAP, which inactivates endogenous Rap1. Conditioned media and cell lysates were harvested from the transiently transfected cell lines. Results: The level of active Rap1 expression in the cancer cells correlated with VEGF and IL-8 secretion. Cell line UMSCC74-A had the highest amount of active Rap1 and VEGF and IL-8 secretion. Transfection with RapGAP in UMSCC-11A and UMSCC-74A, led to lower levels of VEGF and IL-8 secretion. Over expression of Rap1 in UMSCC-11A resulted in increased secretion of these two angiogenic factors. Although there was a correlation between active Rap1 and VEGF and IL-8 secretion, no correlation was found between total Rap1 (active and inactive) and the two cytokines. Conclusions: In oral squamous cell carcinomas, Rap1 activation leads to increased VEGF and IL- 8 secretion.