An identical trend was observed in rat aortic ring assay, suggesting that I3M has antiangiogenic effects on endothelial cells. The Matrigel plug assay mimics normal, Icotinib physiological conditions perfectly for the quantitative evaluation of neo angiogenesis, yet also demonstrates many of the top features of tumor angiogenesis. Angiogenic growth factors are locally released from a growing tumefaction to induce endothelial cell proliferation and migration and extracellular matrix degradation, that is required to allow invasion and vessel formation. Our research showed that I3M nearly abolished angiogenesis in this assay. These strongly declare that I3M inhibits angiogenesis not only in vitro but also in vivo. VEGF is a key mediator of tumor angiogenesis that functions mainly through VEGFR 2. VEGFR 2 is the primary receptor within the VEGF signaling pathway that regulates endothelial cell proliferation, migration, differentiation, tube formation, and angiogenesis. We examined whether Gene expression I3M inhibits the activation of VEGFR 2, to know the molecular mechanism of the I3M mediated anti-angiogenic impact. As shown in Figure 5A, VEGFR 2 was phosphorylated subsequent addition of exogenous VEGF to HUVECs. Pretreatment of the cells with I3M notably blocked the VEGF induced phosphorylation of VEGFR 2 without affecting the overall VEGFR expression levels, suggesting that I3M is definitely an inhibitor of VEGFR 2. The process by which angiogenesis is inhibited by I3M was initially investigated by measuring the VEGFR 2 activation. We found that I3M specifically inhibited the kinase activity of purified VEGFR 2, a novel activity of I3M that has not been characterized. As far Everolimus clinical trial even as we are aware, this is actually the first study to demonstrate the inhibitory effect of I3M on angiogenesis via inhibition of VEGF/VEGFR 2 signaling. How I3M inhibits VEGFR 2 kinase activity remains as yet not known. It has previously been shown that its analogues and indirubin selectively hinder CDKs by competing with ATP for binding to the catalytic site of the kinase. Indirubins can also be strong ATP competitive inhibitors of GSK 3. Based on these previous reports and the that I3M inhibits the kinase activity of purified VEGFR 2, I3M could be an efficient ATP competitive inhibitor of VEGFR 2 kinase. We examined whether I3M involves these signal pathways in HUVECs, since previous indicated that I3M affect the signal pathways of bFGF and NF kB which are participating angiogenesis. I3M reduced the phosphorylation of FGFR 1 although not NF kB activation. Depending on these findings, we consider that I3M could downregulate angiogenesis via FGFR 1 signal paths and the blocking VEGFR 2, at the least a component. In summary, our reports show that I3M functions as an inhibitor of the VEGFR 2 signaling pathway, leading to inhibition of angiogenesis. Our data suggest a fresh mechanism of action for I3M and its possible use as an anticancer and antiangiogenic agent.