IDR-E804 is a cell-permeable indirubin derivative that blocks the STAT-3 signaling pathway
]. Previous studies have demonstrated that IDR-E804 is a promising anti-cancer agent because it is able to inhibit the proliferation and induce the apoptosis of various human cancer cells
]. IDR-E804 has also been shown to be a potent, reversible, and ATP-competitive inhibitor of the kinase activities of Src, Cdk1/cyclin E, Cdk2/cyclin A, and Cdk1/cyclin
]. This compound has also been shown to reduce the phosphorylation levels of Src, JAK1, and STAT-3 in MDA-MB-468 human breast cancer cells
]. In addition to the inhibitory activities of Src, Cdk1/cyclin E, Cdk2/cyclin A, and Cdk1/cyclin, the apoptotic effect of IDR-E804 has been shown to occur in response to the down-regulation of anti-apoptotic proteins Mcl-1 and survivin. These anti-cancer effects of IDR-E804 in various human cancer cells led us to investigate the role of IDR-E804 in angiogenesis, which is essential for cancer development
The present study provides evidence that IDR-E804 is a VEGFR-2 inhibitor that inhibits angiogenesis and tumor progression. Our work focused on the inhibitory effects of IDR-E804 on the proliferation, migration and tube formation in HUVECs, which are critical steps involved in endothelial angiogenesis. Due to the inhibition of VEGFR-2 phosphorylation and activation, IDR-E804 reduced the ERK and AKT signaling pathway in HUVECs (Figure
). We also found that IDR-E804 directly inhibited the kinase activity of purified VEGFR-2, a novel activity of IDR-E804 that has not yet been characterized. To the best of our knowledge, this is the first study to demonstrate the inhibitory effect of IDR-E804 on angiogenesis via inhibition of VEGF/VEGFR-2 signaling, at least in part. We also demonstrated that IDR-E804 effectively suppresses the growth of CT-26 colorectal cancer grafts via inhibition of angiogenesis as well as acceleration of apoptosis (Figures
). Since previous results indicated that IDR-E804 inhibits Src kinase activity and STAT-3 phosphorylation in various cancers which are critical for angiogensis
], it is possible that IDR-E804 inhibits angiogenic process via Src and/or STAT-3 signaling pathways, but the detail action of IDR-E804 on Src and STAT-3 activation in HUVECs requires further investigation.
Our in vitro
studies with HUVECs demonstrated that IDR-E804 inhibited the proliferation, migration and capillary-like structure formation in VEGF-stimulated HUVECs (Figure
). A similar phenomenon was observed in the rat aortic ring assay (Figure
), suggesting that IDR-E804 inhibits microvessels formation. Endothelial cell signaling in response to VEGF is well established
]. VEGF is one of the most potent tumor angiogenic factors that promotes the proliferation and migration of endothelial cells and increases vascular permeability
]. It is well known that VEGF and its receptors are important mediators during different steps of angiogenesis in cancer
]. For its multifunction, VEGF activates a diverse and integrated network of signaling pathways. These different signaling cascades play different roles in the biological functions and also cross-talk to each other. The survival effect of VEGF activation is primarily mediated by the AKT pathway. AKT not only inhibits the pro-apoptotic proteins and apoptotic caspases, but also up-regulates the anti-apoptotic proteins
]. ERK signaling has been implicated in the proliferation, survival, and protection against receptor-mediated apoptosis of endothelial cells
]. Moreover, ERK signaling is thought to stimulate angiogenesis by promoting endothelial cell motility
]. In the present study, the phosphorylation of the AKT and ERK of HUVECs was increased upon VEGF stimulation and IDR-E804 significantly reduced AKT and ERK phosphorylation. These results indicate that E804 inhibits proliferation and migration of endothelial cells via
inhibition of AKT and ERK.
The present study indicates that IDR-E804 (20 μM) effectively suppressed tumor volume and tumor weight (Figure
D) without adverse effects on mouse body weight (Figure
inhibition of angiogenesis as well as acceleration of apoptosis in allograft colon tumor mice (Figure
). Previous studies demonstrated that IDR-E804 induced apoptosis in transformed MDA-MB-468 human breast cancer cells, but did not induce apoptosis in normal MCF-10A cells. In addition, Mcl-1 and survivin expression is dramatically reduced in response to IDR-E804 and apoptosis is induced in breast cancer cells
]. Our immunohistochemistry results revealed that the generation of new blood vessels and Ki-67 positive cells in the treated group were reduced when compared with the control groups (Figure
). Moreover, the numbers of TUNEL positive cell were significantly higher in IDR-E804 treated tumor sections (Figure
). These findings indicate that IDR-E804 induced the apoptosis in cancer cells as well as tumor tissues in mice.
Previous studies demonstrated that indirubin (C16
) and indirubin-3’-monoxime (C16
), an indirubin derivative, are potential drug candidates for angiogenesis related diseases
]. Indirubin inhibited prostate tumor growth mainly through antitumor angiogenesis via blocking VEGFR-2 mediated STAT-3 signaling pathway in endothelial cell
]. In addition, indirubin-3’-monoxime also blocked the proliferation, migration, and capillary-like structure formation of HUVECs
]. Comparing previous study, IDR-E804 exerted anti-angiogencic activity in HUVECs at lower concentration than indirubin and indirubin-3’-monoxime. The inhibited concentration of indirubin and indirubin-3’-monoxime against the proliferation, migration as well as tube formation of HUVECs was 25-100 μM and 2.5-20 μM, respectively
]. Although indirubin blocked the phosphorylation of VEGFR-2, the concentration which blocks the phosphorylation of VEGFR-2 was 25-100 μM whereas IDR-E804 was 1-10 μM. Although additional work is needed to elucidate the relationship between the structure and anti-angiogenic activity of indirubin or other indirubin derivatives, IDR-E804 might have powerful potential to inhibit angiogenesis than indirubin and indirubin-3’-monoxime.
It should be noted that this in vivo study has a few limitations. Since we tested only a single dose of IDR-E804 for the in vivo mouse experiments, detailed dose-response effect of IDR-E804 on angiogenesis and tumor growth in vivo needs to be investigated. In addition, preclinical studies to test the safety of IDR-E804 in vivo should be conducted to determine if it is suitable for further use as an anti-cancer and anti-angiogenic agent.