950 Breast Cancer Res Treat (2012) 134:943–955 Fig. 3 Ellagic acid blocked neo-angiogenesis in chick-associated models. a Ellagic acid dose dependently suppressed sprout formation on the organotypic model of chick aortic ring. Chick aortic rings were embedded in Matrigel and treated with different concentrations of ellagic acid stimulated by VEGF. The changes of sprout formations around various aorta samples were observed on the 3rd day (left panel; a 0 lM, b 2.5 lM, c 5 lM, d 10 lM). The index was deﬁned as a percentage of the untreated control (right panel, values are represented as means ± SD, n = 6, *P \ 0.05, **P \ 0.01 versus untreated control). b The inhibitory effects of ellagic acid on sprout formation could be reversed after removal of ellagic acid from chick Ellagic acid inhibited breast cancer growth and neoangiogenesis in vivo To investigate in vivo anti-angiogenesis effects of ellagic acid, breast cancer xenograft was built to evaluate whether ellagic acid could suppress tumor-induced angiogenesis in vivo. Previous studies indicated that MDA-MB-231 cell line was the ﬁrst choice as pre-clinical breast cancer model owing to its high aggressive nature either in vitro or in vivo [29]. Thus, immunodeﬁcient mice bearing MDA-MB-231 xenografts were treated daily with or without ellagic acid by intraperitoneal administration for 25 days. After mice were sacriﬁced, tumors and tissues were taken out for further analysis. Representative mice with MDA-MB-231 xenografts and tumor masses are shown in Fig. 5a. It was 123 aortic ring. Aortic ring was initially fed with both VEGF and ellagic acid (10 lM) for 48 h (left panel), and then continually treated with VEGF after removal of ellagic acid for an additional 48 h (right panel). Images were representative of three independent experiments. c Ellagic acid could inhibit the microvessels formation on in vivo chick embryonic CAM model. Images were representative of three independent experiments (left panel). The index was deﬁned as the mean number of visible microvessel branch with the deﬁned area of drug-containing pellets on each CAM model (right panel, values are represented as means ± SD, n = 6, *P \ 0.05, **P \ 0.01 versus untreated control) found that treatment with ellagic acid signiﬁcantly led to suppression of MBA-MD-231 tumor volumes when com- pared with the control group treated with vehicle, sug- gesting that ellagic acid could inhibit tumor growth in vivo. To further examine whether ellagic acid could suppress breast cancer growth by inhibiting angiogenesis, tumor tissues were stained with speciﬁc antibodies against CD31, P-VEGFR2 (Tyr 1175), P-AKT, and P-JNK in Fig. 5b. CD31 is a widely used endothelial marker for quantifying angiogenesis by calculating microvessel density (MVD) [30]. We found that vessels in vehicle-treated group were more numerous and stained more intensely compared with 100 mg/kg ellagic acid-treated group. In addition, ellagic acid treatment could obviously down-regulate expressions of P-VEGFR2, P-Akt, and P-JNK, further demonstrating