Breast Cancer Res Treat (2012) 134:943–955 953 Fig. 6 Ellagic acid interacted with the ATP-binding sites of VEGFR- 2 kinase domain. The LigandFit algorithm in Discovery Studio 2.1 was applied to predict the binding mode of ellagic acid within ATP- binding domain of VEGFR-2 kinase. As anticipated, ellagic acid could stably bind to the ATP-binding pocket near the hinge region, which connected N- and C-lobes within VEGFR-2 catalytic domain. Speciﬁcally, ellagic acid formed hydrogen bonds with residues Lys866 and Glu883. Besides, ellagic acid could form strong p–p interactions with Phe1045 Discussion Extensive laboratory evidences supported that angiogenesis can be detected throughout the onset, growth, and metas- tasis in breast cancer [33]. Developing nontoxic anti- angiogenesis agents has thus become a promising strategy in breast cancer prevention and treatment. Ellagic acid is a dietary-derived polyphenol which has been widely reported to possess anti-cancer properties. In this study, we ﬁrst focused on the inhibitory effects of ellagic acid on several steps of angiogenesis at the cellular level. It was found that ellagic acid could markedly inhibit angiogenesis-associated activities including proliferation, migration/invasion, and capillary formation on VEGF-stimulated endothelial cells. Besides, previous studies indicated that angiogenesis in vivo involved not only endothelial cells but also their surrounding cells [25]. Thus, chick aortic arch model was then used to mimic the in vivo process of angiogenesis. As anticipated, the sprouts formation stimulated by VEGF from ellagic acid-treated aorta was weak and scattered compared with the vehicle-treated groups. Nevertheless, the above in-vitro models lack the biological complexity of vascular system in vertebrate animals. Supporting evi- dences concerning in vivo anti-angiogenesis effects of ellagic acid then came from chick embryonic CAM model and MDA-MB-231 tumor xenograft model. Ellagic acid obviously inhibited blood vessels formation on CAM model and signiﬁcantly suppressed tumor growth accom- panied by a reduced MVD on tumor tissues in MDA- MB-231 xenograft model. Notably, we found that ellagic acid did not pose signiﬁcant cytotoxicity to HUVECs at any tested concentrations based on LDH assay, indicating that the inhibitory effects of ellagic acid was not likely due to toxicity at the cellular level. In addition, vascular recurrence after ellagic acid removal on CAM models and no obvious toxic pathologic changes in normal tissues of mice xenograft model further demonstrated that ellagic acid could exert anti-angiogenesis effects with limited toxicity. These evi- dences might provide an experimental basis for further study of ellagic acid in safe clinical application. We then veriﬁed the intrinsic mechanisms for the above anti-angiogenesis activities of ellagic acid. Inhibition of VEGFR-2 has been served as a prosperous strategy for angiogenesis therapeutic intervention [34]. By functionally coupling with VEGF, VEGFR-2 would undergo auto- phosphorylation mainly at Tyr1175 sites within its intra- cellular kinase domain and then initiate a series of downstream signal transductions to endothelial cells [35]. Our ﬁndings revealed that ellagic acid could suppress Tyr1175 phosphorylation of VEGFR-2 stimulated by VEGF as well as VEGFR-2 tyrosine kinase activity. Mul- tiple VEGFR-2 downstream signaling mediators such as ERK, AKT, JNK, and eNOS were also involved in regu- lation of endothelial cells survival and proliferation. According to previous supporting evidences [36, 37], P42/ 44 ERK activation attributes to an increased proliferation on endothelial cells; AKT is responsible for the survival of endothelial cell; JNK activation induces proliferation and migration on endothelial cells by promoting nuclear acti- vation of c-Jun; and eNOS plays a key role in vascular permeability and migration activities on endothelial cells. Our results demonstrated that multiple MAPK signaling mediators including p-ERK, p-AKT, p-JNK, and eNOS were inhibited by ellagic acid. In addition, it was also found that ellagic acid dose dependently inhibited MMPs activity, conﬁrming the previous ﬁndings that decreased MMPs activity might be also responsible for interfering with the binding of VEGF to VEGFR-2, and therefore inhibiting the neo-angiogenesis process [38]. Furthermore, ROS was reported as a downstream signaling of VEGFR-2 and served as a survival mediator in supporting endothelial cells proliferation [28]. Our results showed that the ROS level was signiﬁcantly decreased after ellagic acid admin- istration, which might be a consequence event of decreased VEGFR-2 activity. All these results showed that ellagic acid treatment suppressed the VEGFR-2 pathways. Mean- while, the ﬁndings in vitro were also consistent with our in vivo results, indicating that the reduced MVD value may owe to the suppression activities of ellagic acid on the expression of phosphorylated VEGFR-2 and its down- stream signaling molecules. To thoroughly understand how ellagic acid interacted with VEGFR-2 to exert anti-angiogenesis effects, we fur- ther examined the structure-based interaction between 123