Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation

Saturday, 06/10/2012  |   Herb or Compound  |  no comments

Kwak H-J et al International Journal of Cancer. Volume 118, Issue 11, pages 2711–2720, 1 June 2006 DOI: 10.1002/ijc.21641
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component in the root and rhizome of Rheum palmatum, is a tyrosine kinase inhibitor with a number of biological activities, including antitumor effects. Here, we examine the effects of emodin on vascular endothelial growth factor (VEGF)-A-induced angiogenesis, both in vitro and in vivo. In vitro, emodin dose-dependently inhibits proliferation, migration into the denuded area, invasion through a layer of Matrigel and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated with VEGF-A. Emodin also inhibits basic fibroblast growth factor-induced proliferation and migration of HUVECs and VEGF-A-induced tube formation of human dermal microvascular endothelial cells. Specifically, emodin induces the cell cycle arrest of HUVECs in the G0/G1 phase by suppressing cyclin D1 and E expression and retinoblastoma protein phosphorylation, and suppresses Matrigel invasion by inhibiting the basal secretion of matrix metalloproteinase-2 and VEGF-A-stimulated urokinase plasminogen activator receptor expression. Additionally, emodin effectively inhibits phosphorylation of VEGF-A receptor-2 (KDR/Flk-1) and downstream effector molecules, including focal adhesion kinase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, Akt and endothelial nitric oxide synthase. In vivo, emodin strongly suppresses neovessel formation in the chorioallantoic membrane of chick and VEGF-A-induced angiogenesis of the Matrigel plug in mice. Our data collectively demonstrate that emodin effectively inhibits VEGF-A-induced angiogenesis in vitro and in vivo. Moreover, inhibition of phosphorylation of KDR/Flk-1 and downstream effector molecules is a possible underlying mechanism of the anti-angiogenic activity of emodin. Based on these data, we propose that an interaction of emodin with KDR/Flk-1 may be involved in the inhibitory function of emodin toward VEGF-A-induced angiogenesis in vitro and responsible for its potent anti-angiogenic in vivo. © 2006 Wiley-Liss, Inc.
Angiogenesis, the formation of new vessels from preexisting vasculature, is an essential process in a variety of physiological and pathological conditions, including wound healing, embryonic development, chronic inflammation, cancer and metastasis.1, 2, 3, 4, 5 Complex sequential steps are involved in angiogenesis, such as basement membrane degradation by proteases, endothelial cell proliferation and migration, formation of capillary tubes and survival of newly formed blood vessels.6 Angiogenesis is tightly regulated by an intricate balance between stimulators and inhibitors.6 Among these, vascular endothelial growth factor (VEGF)-A, a soluble angiogenic factor produced by many tumors as well as normal cell lines, including vascular smooth muscle cells, lung epithelium and pituitary folliculo-stellate cells, plays a key role in regulating normal and pathologic angiogenesis.7
VEGF-A is a potent mitogen for endothelial cells, and an important mediator of angiogenesis, inducing endothelial cell proliferation, protease expression and migration, as well as subsequent organization of cells to form a capillary tube.8, 9, 10 In particular, a number of studies have shown that VEGF-A is the most important angiogenic factor closely associated with neovascularization in human tumors. Recent reports have disclosed increased VEGF-A mRNA in tumor cell lines. The VEGF-A level is an important prognostic marker of tumor angiogenesis.10, 11 VEGF-A induces angiogenesis via binding to its two receptor tyrosine kinases, KDR/Flk-1 and Flt-1, expressed on endothelial cells. KDR/Flk-1 is required mainly for mitogenic and chemotactic responses, whereas Flt-1 contributes to endothelial cell morphogenesis.12, 13 Recently, the intracellular signaling pathways mediating these effects downstream of KDR/Flk-1 activation have been identified. KDR/Flk-1 induces proliferation through activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway leading to gene transcription14 and endothelial cell survival through phosphatidylinositol 3-kinase activation, resulting in increased lipid phosphatidylinositol (3,4,5)P3 and activation of several important intracellular molecules, such as Akt and the small GTP-binding protein, Rac.15 The Akt pathway additionally triggers endothelial nitric oxide synthase (eNOS) activity16, 17 to generate NO, leading to vascular permeability and cellular migration. Other signal transduction molecules implicated in KDR/Flk-1-dependent cytoskeletal regulation and cell migration include p38 mitogen-activated protein kinase (MAPK) and p125 focal adhesion kinase (FAK).18, 19
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a tyrosine kinase inhibitor isolated from Rheum palmatum, is an active constituent of Chinese herbs.20 The compound sensitizes HER-2/neu-overexpressing lung cancer cells, has antitumor effects on neuroectodermal and represses transformation and metastasis-associated properties of HER-2/neu-overexprssing breast cancer cells.20, 21, 22 Emodin additionally exerts anti-inflammatory effects on endothelial cells by inhibiting tumor necrosis factor -induced activation of nuclear factor-kappa B in human umbilical vein endothelial cells (HUVECs).23 Given that numerous endogenous factors and pharmacological agents that regulate cancer progression and inflammation additionally affect angiogenesis, it seems quite likely that emodin would also affect angiogenesis. In the present study, we investigated whether emodin has anti-angiogenic activity especially on the VEGF-A-induced angiogenesis, and if so, the critical mechanism of action.
Our data show that emodin significantly inhibits VEGF-A-induced angiogenesis in vitro and in vivo. Emodin effectively blocks VEGF-A-induced proliferation, migration, invasion and tube formation of HUVECs in vitro, and markedly inhibits neo-angiogenesis of chick chorioallantoic membrane (CAM) and mouse Matrigel in vivo. Blockade of VEGF-A-induced tyrosine phosphorylation of KDR/Flk-1 and downstream signaling molecules by emodin may be responsible for its potent anti-angiogenic activity. The results strongly highlight the efficacy of this compound in controlling neovessel formation in diseases induced by angiogenesis.

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