944 invasion into breast tumor masses, there are at least six different angiogenesis-associated growth factors secreted, among which vascular endothelial growth factor (VEGF) is the most potent angiogenesis stimulator [5, 6]. The speciﬁc action of the VEGF on the endothelial cells is mainly mediated by two types of receptor tyrosine kinases (RTKs), namely VEGFR-1 (Flt-1) and VEGFR-2 (KDR in human/ Flk-1 in mice) with high afﬁnities. Of the two receptors, VEGFR-2 plays a more important role in mediating the mitogenesis and permeability of endothelial cells. Activa- tion of VEGFR-2 contributes to phosphorylation of mul- tiple downstream signals including ERK, JNK, and AKT that subsequently promote proliferation, migration, and tube formation of endothelial cells. Considering anti- angiogenesis therapy is to target endothelial cells that support tumor growth rather than cancer cells themselves, VEGFR-2 has become an important therapeutic target for cancer anti-angiogenesis therapy [7–10]. VEGFR-2 belongs to the most aggressive RTKs. Agents now targeting RTKs in oncology can be roughly divided into three categories—antibodies targeting RTK ligands (i.e., targeting VEGF), antibodies targeting receptors themselves (i.e., targeting VEGFR-2), and small molecular inhibitors targeting VEGFR-2 kinase domain [11]. The recombinant of the three-dimensional crystal structure of VEGFR-2 kinase domain provided a basis for structure- based design of small molecular inhibitors. Structurally, VEGFR-2 consists of 1,356 amino acids in humans, and can be separated into three domains: the extracellular VEGF-binding domain consisting of seven immunoglobu- lin-like segments, the transmembrane domain, and the intracellular catalytic domain possessing tyrosine kinase activity. Upon speciﬁcally binding to VEGF, VEGFR-2 would undergo dimerization within the extracellular domain and autophosphorylation within the intracellular catalytic domain by consuming ATP, accompanied by following activation of downstream signaling cascades that stimulate angiogenesis. The ATP-binding site and less conserved surrounding sites are thus especially important for anti-VEGFR2 agent design [12, 13]. Various orally active small molecular inhibitors of VEGFR-2 are now in clinical trials including sunitinib, vandetanib, and sorafenib [14]. Disappointedly, long- duration treatment with these agents might be accompanied by distinct adverse effects such as hemorrhage, hyperten- sive crisis, and gastrointestinal perforation [15]. Therefore, there has been renewed interest in natural inhibitors that could block VEGFR activation. Many natural phenolic compounds or their speciﬁc derivatives are found pos- sessing potent anti-cancer properties [16–18]. Among them, ellagic acid (4,40,5,506, 60-hexahydroxydiphenic acid 2,6,2060-dilactone) is a representative small molecular pol- yphenol widely found in fruits and vegetables. Extensive 123 Breast Cancer Res Treat (2012) 134:943–955 studies have reported that ellagic acid exerts potent anti- oxidant effects either by directly acting as an antioxidant or by activating cellular antioxidant enzyme systems [19, 20]. In addition, ellagic acid was also found to be effective in anti-carcinogenesis through inhibiting tumor cell prolifer- ation, inducing apoptosis, breaking DNA binding to car- cinogens, and most importantly, disturbing angiogenesis processes required for tumor growth [21]. However, the mechanisms involved were not thoroughly elucidated. In this study, the effects of ellagic acid on inhibiting breast cancer angiogenesis were validated in vitro and in vivo. Mechanistic study further indicated that ellagic acid could signiﬁcantly inhibit VEGFR-2 kinase activity and block its signaling pathway both in vitro and in vivo. Meanwhile, the structure-based interaction between ellagic acid and VEGFR-2 was found to be stable conformation based on in-silico analysis which revealed that hydrogen bond and aromatic interactions were formed. Materials and methods Cell culture and drug preparation The human breast cancer cell line MDA-MB-231 was purchased from the American Type Culture Collection (ATCC), and maintained in L-15 medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 lg/ml). Human umbilical vein endothelial cells (HUVEC) were cultivated in gelatinized culture plates in M199 medium supplemented with 15% FBS, 1% PS, 50 lg/ml endothelial cell growth supplement (ECGS, BD Bioscience) and 100 lg/ml heparin at 37 C in a humidiﬁed atmosphere containing 5% CO2. Ellagic acid (C98%, Sigma-Aldrich, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO, ﬁnal concentration is 0.1%) was diluted in 19 PBS to prepare required concentrations. In each treatment, the cells were treated with vehicle or with various concentrations of ellagic acid in the presence of 20 ng/ml human VEGF (Pepro Tech, Rocky Hill, NJ). Cell counting assay HUVECs were seeded in a 6-well plate with a density of 2 9 104 cells per well at 37 C and 5% CO2 for overnight attachment. Then the cultivated medium was discarded and replaced with fresh serum-free medium with or without drug involvement in the presence of VEGF. At the time intervals of 12, 24, and 48 h, cells were harvested for counting in the presence of trypan blue. All samples were assayed in triplicate to generate proliferation curves as described.