Breast Cancer Res Treat (2012) 134:943–955 were used for the drawing of chemical structures. The LigandFit algorithm employed the strategy including initial generation of ligand orientations within VEGFR-2 catalytic domain followed by molecular dynamics (MD)-based simulated annealing, and ﬁnal reﬁnement by energy mini- mization. The crystal structure of VEGFR-2 was obtained from Protein Data Bank (http://www.rcsb.org/pdb/) with the PDB ID of 1Y6B. The water molecules in VEGFR-2 were removed. For the docking purpose, the ATP site within VEGFR-2 was deﬁned as the ligand-binding site following the template of 1Y6B, and ellagic acid was docked into VEGFR-2 with proper parameter setting. Speciﬁcally, starting from the initial conﬁguration, 100 different orientations of ellagic acid were randomly gen- erated and docked into the ATP pocket of VEGFR-2. All the 100 docking poses were presented simultaneously for the analysis of the interactions between ellagic acid and VEGFR-2. A docking without any output pose was con- sidered as a failure. Results Ellagic acid inhibited proliferation of HUVECs induced by VEGF To examine the anti-angiogenic effects of ellagic acid in vitro, proliferation of VEGF-induced HUVECs was detected ﬁrst [22]. Previous studies reported that cell number decreased while MTT formazan formation increased in the presence of polyphenol-associated com- pound (kaempferol) [23]. Therefore, cell counting assay was conducted rather than MTT assay in this study. As shown in Fig. 1a, the proliferation of endothelial cells stimulated by VEGF was markedly decreased after ellagic acid treatment ranging from 2.5 to 20 lM. Besides, HUVECs not containing VEGF showed obscure changes in cell proliferation, indicating extracellular VEGF acted as a strong attractant for endothelial cells proliferation. Then we treated endothelial cells with ellagic acid at different time intervals of 12, 24, and 48 h. The results presented in Fig. 1b showed that ellagic acid signiﬁcantly suppressed endothelial cell proliferation in a time course. As detected by BrdU incorporation assay (Fig. 1c), DNA synthesis of HUVECs was also signiﬁcantly inhibited by ellagic acid in a dose-dependant manner. To validate whether or not ellagic acid would result in toxicity effects on HUVECs, LDH cytotoxicity assay was carried out. As shown in Fig. 1d, ellagic acid brought little toxic effects on HUVECs. Meanwhile, there were little abnormal mor- phological changes of endothelial cells after ellagic acid administration for 48 h (Fig. 1e). We came to the conclu- sion that ellagic acid at non-cytotoxic doses could 947 signiﬁcantly inhibit proliferation of VEGF-stimulated endothelial cells. Ellagic acid suppressed VEGF-induced migration and tube formation of HUVECs Then, we studied the potential of ellagic acid in blocking the migration and tube formation abilities of HUVECs [24]. First, transwell assay demonstrated that the invasive cells to the lower chamber were signiﬁcantly decreased with the increasing concentration of ellagic acid (Fig. 2A), indicating that the migration ability of HUVECs was dose dependently suppressed by ellagic acid. Meanwhile, the inﬂuence of ellagic acid on migration ability of endothelial cells was also assessed by wound-healing assay. As shown in Fig. 2B, the gap width of control group narrowed more obviously than that of ellagic acid-treated group from 0 to 24 h, reﬂecting that ellagic acid could time dependently weaken the migration capability of endothelial cells. Then the effects of ellagic acid on tube-formation ability of HUVECs were tested on Matrigel. In Fig. 2C, HUVECs showed well-formed tubular structure in the absence of ellagic acid. Capillaries were gradually abrogated due to increasing ellagic acid concentrations. Almost 80% destruction of tube network was observed when HUVECs were incubated with ellagic acid at 10 lM. Taken together, ellagic acid suppressed the migratory process and tube formation activities of endothelial cells in the presence of VEGF. Ellagic acid blocked angiogenesis in chick-associated models To mimic the in vivo angiogenesis situation, the organo- typic assay in chick aortic arch model was then conducted by measurement of endothelial cell outgrowth [25]. It was found that sprouts around ellagic acid-treated rings were shorter and fewer cells migrated into the matrix, suggesting that ellagic acid could inhibit the sprout length and density after stimulation of VEGF (Fig. 3a). We further examined whether the inhibitory effects of ellagic acid were revers- ible. As shown in Fig. 3b, removal of ellagic acid con- tributed to sprout recurrence around the aortic ring, indicating that ellagic acid brought little toxic effects on normal vessel tissues. Besides, a different chick model system by in vivo chick embryonic CAM assay showed that the number of blood vessels gradually decreased with increasing concentrations of ellagic acid accompanied by VEGF stimulation (Fig. 3c). Overall, it was demonstrated that ellagic acid could inhibit sprouts formation from chicken aorta model and microvessels formation in CAM model. 123