| 3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol (I3C), the putative anticancer agent present in vegetables of the Brassica genus. In this study, the effect of DIM on in vitro markers of angiogenesis was investigated. We found that DIM produced a concentration-dependent decrease in proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells (HUVECs), which are significant at only 5 muM DIM. The antiproliferative effect was accompanied by a G1 cell cycle arrest in actively proliferating HUVECs in 24 h, and enhanced apoptosis of HUVECs in 48 h. Further study revealed that DIM down-regulated expression of cyclin-dependent kinase 2 and 6 (CDK2, CDK6), and up-regulated expression of CDK inhibitor, p27KIP1, in HUVECs. The up-regulation of p27KIP1 mRNA was not due to a transcriptional activation, as DIM failed to up-regulate p27KIP1 transcripts, and p27KIP1 promoter reporter failed to respond to DIM. However, CDK2 and CDK6 mRNA levels were both down-regulated by DIM, consistent with the reduction of their protein levels.; In addition, we found that DIM can inhibit ERK1/2 phosphorylation induced by a variety of growth factors, including vascular endothelial cells growth factor (VEGF), in a concentration- and time-dependent manner, with a 80% inhibition by 25 muM DIM treated for 2 h, in HUVECs. Furthermore, Ras-GTP content, which dramatically increased after HUVECs were challenged by either growth factors or serum, was found to be reduced by nearly 80% with 25 muM DIM administration, which in turn resulted in the reduced activities of Raf and MEK, culminating in the drop of ERK1/2 activation. Over-expression of GTPase mutant Ras G12V (constitutively active) in HUVECs reversed the inhibitory effect of DIM on ERK1/2 activation. These data provide evidence that DIM inhibits VEGF-mediated signal transduction, which might interfere with its downstream biological effects necessary for angiogenesis.; To examine whether DIM also exhibits anti-angiogenic activities in vivo, we conducted Matrigel plug assay in a rodent model and chicken chorioallantoic membrane (CAM) assay in fertilized eggs. (Abstract shortened by UMI.)... |
