Breast Cancer Res Treat (2012) 134:943–955 951 Fig. 4 Ellagic acid attenuated VEGFR-2 tyrosine kinase activity and VEGFR-2 signaling pathway. a Ellagic acid reduced the phosphor- ylation of VEGFR-2 in VEGF-stimulated HUVECs. Endothelial cells were pre-cultured with ellagic acid at different concentrations for 24 h with the stimulation of VEGF (20 ng/ml). Expressions of VEGFR-2 and P-VEGFR2 were then examined by western blotting assay. It was found that P-VEGFR2 expression was markedly reduced, while the total level of VEGFR-2 was unaffected after the treatment of ellagic acid. b Ellagic acid suppressed VEGFR-2 kinase activity. VEGFR-2 and various concentrations of ellagic acid were incubated in kinase reaction buffer in 96-well plate coated with a poly-Glu-Tyr substrate. Phosphorylation of the substrate was moni- tored with a puriﬁed phosphotyrosine speciﬁc monocolonal antibody that ellagic acid played an important role in suppressing angiogenesis at least partly through VEGFR-2 signaling pathways. Meanwhile, no signiﬁcant differences of body weights were detected among all the groups, indicating that ellagic acid administration brought little toxic effects on mice. Furthermore, normal tissue sections were embedded in parafﬁn and stained by hematoxylin and eosin (H&E) for toxicological analysis. No apparent pathological abnor- malities were found in the normal tissues including heart, liver, spleen, lung, and kidney. Therefore, we could draw the conclusion that there were no signiﬁcant adverse effects of ellagic acid in vivo, and the inhibitory effects of ellagic conjugated to horseradish peroxidase followed by chromogenic reaction with horseradish peroxidase substrate. Data are presented as a percentage of the control (values represent means ± SD, n = 3). c Ellagic acid inhibited VEGFR-2 downstream signaling molecules, including P-ERK/ERK, P-AKT/AKT, P-JNK/JNK, and e-NOS in a dose-dependant manner. Besides, the expression of MMP-2 and MMP-9 were also inhibited after ellagic acid administration. d The HUVECs intracellular ROS level was detected by DCFH-DA staining assay. The results showed that the intracellular ROS level was signiﬁcantly decreased after ellagic acid treatment (values represent means ± SD, n = 6, *P \ 0.05, **P \ 0.01 versus untreated control) acid on cancer growth might not be due to systemic toxicity in mice. The result in Fig. 5c could provide an experi- mental basis for further study of ellagic acid in safe clinical application. Ellagic acid located at the ATP-binding sites of VEGFR-2 kinase domain We next analyzed the binding pattern between ellagic acid and VEGFR-2 kinase domain to further understand how ellagic acid exerted anti-angiogenesis effects via VEGFR-2 and its signaling pathways. The ATP site within VEGFR-2 kinase domain was thus deﬁned as the ligand-binding site 123