952 Breast Cancer Res Treat (2012) 134:943–955 Fig. 5 Ellagic acid inhibited growth and neoangiogenesis on MDA- MB-231 breast cancer xenografts. a Nude mice bearing MDA-MB- 231 tumor were treated daily with the vehicle or ellagic acid at 50 or 100 mg/kg/day by intraperitoneal administration for 25 days. Repre- sentative mice with MDA-MB-231 xenografts and tumor masses were shown. Besides, it was found that treatment with ellagic acid obviously suppressed tumor volumes compared to the vehicle control group, indicating that ellagic acid could signiﬁcantly inhibit the MBA-MD-231 tumor growth in vivo (values represent means ± SD, n = 6, *P \ 0.05 versus vehicle group). b Tumor tissues were prepared for immunohistochemistry detection with speciﬁc antibodies against CD31, P-VEGFR2, P-AKT, and P-JNK. It was found that ellagic acid could obviously decrease tumoral microvessel density (MVD) indexed by CD31 in comparison with vehicle treatment. Meanwhile, ellagic acid treatment could obviously attenuate expres- sions of P-VEGFR2, P-Akt, and P-JNK, further demonstrating that ellagic acid played an important role in suppressing angiogenesis at least in part via VEGFR-2 signaling pathways in vivo. c Ellagic acid resulted in little toxicity effects in vivo. No signiﬁcant differences of body weights were detected among all the groups (upper panel). Besides, it was found that ellagic acid did not cause apparent pathological abnormalities in the normal tissues including heart, lung, liver, spleen, and kidney, suggesting that there were no signiﬁcant adverse effects of ellagic acid in vivo (down panel) based on the homology of 2-anilino-5-aryloxazole [31, 32]. As anticipated, ellagic acid could stably locate at the ATP- binding pocket near the hinge region, which connected N- and C-lobes within VEGFR-2 catalytic domain (Fig. 6). Three active residues, i.e., Lys866, Glu883, and Phe1045 at the ATP pocket were shown essential for the binding of ellagic acid with VEGFR-2. Particularly, ellagic acid could form hydrogen bonds with active residues Lys866 and Glu883, which were located at the hinge region and played an important role in identifying VEGFR-2 ligands. Besides, ellagic acid could form strong p–p interactions with Phe1045. The binding pattern of ellagic acid within VEGFR-2 provided structural insight for development of small natural inhibitors. 123