Abstract 1890: Curcumin inhibits tumor angiogenesis by suppressing protooncogene RBM3 expression

RBM3 is the first RNA binding protein to be identified as a protooncogene. In previous studies, we have shown that RBM3 overexpression results in increased stability and translation of vascular endothelial growth factor (VEGF) mRNA. Furthermore, knockdown of RBM3 in HCT-116 colon cancer xenografts resulted in reduction in VEGF expression and in tumor vasculature. Similarly, curcumin the active ingredient in the spice turmeric reduced the growth of HCT-116 tumor xenografts and inhibited VEGF expression. We have now extended these observations and show that RBM3 is a critical player in tumor angiogenesis, and that curcumin inhibits tumor angiogenesis in part by suppressing RBM3 expression. Methods. RBM3 was stably transfected into NIH-3T3 mouse fibroblast cells to generate 3T3R cells. These cells were implanted on chorioallantoic membranes (CAM) in chicken embryos to determine their effect on angiogenesis. For in vitro studies, human vascular endothelial cells (HUVEC) were treated with VEGF and expression of RBM3 was determined. HUVEC-mediated capillary network formation was performed in polymerized ECMatrix. Cells were also treated with 30 µM curcumin and effects on gene expression determined by western blot analyses. Results. HUVEC cells treated with VEGF (50 ng/ml) resulted in a significant increase RBM3 expression, and high levels of capillary formation in the in vitro angiogenesis assay. SiRNA-mediated downregulation of RBM3 expression in the cells resulted in significant reduction in capillaries, which was overcome when RBM3 was ectopically expressed suggesting that RBM3 is essential for VEGF-mediated angiogenesis. HUVEC cells treated with curcumin (30 µM) treatment also demonstrated in a reduction in RBM3 expression and capillary formation, which was rescued when RBM3 was overexpressed. To determine whether RBM3 plays role in angiogenesis in vivo, 3T3 and 3T3R cells were implanted in Matrigel subcutaneously into chorioallantoic membrane of chicken embryos. 3T3R significantly increased the number of blood vessels when compared to wild type 3T3 cells. Treatment with curcumin also inhibited blood vessel formation. To further confirm these results, we determined gene expression patterns and microvessel density in HCT-116 tumor xenografts following intraperitonial administration of curcumin (200 μg/Kg/d for 21 d). Curcumin suppressed the expression of RBM3 and VEGF in the xenografts. Furthermore, there was a significant reduction in tumor microvasculature