946 Gelatin zymography Supernatants from a HUVEC culture system with or without ellagic acid treatment were collected for MMPs activity analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. One milligram per milliliter of gelatin was prepolymerized on a 10% polyacrylamide gel as a substrate. Electrophoresis was carried out at 4 C. The gel was washed with washing buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, and 2.5% Triton X-100), followed by incubation with a buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl2, 0.02% NaN3, and 1 lM ZnCl2) at 37 C for 16 h and visualized with Coomassie Blue R-250. Measurement of reactive oxygen species 2070-Dichloroﬂuorescein diacetate (DCFH-DA, Sigma, St. Louis, MO) was used to measure ROS formation. After exposed to different concentrations of ellagic acid for 24 h, endothelial cells were then incubated in DCFH-DA con- taining medium (ﬁnal concentration: 10 lM) at 37 C for 20 min. Cells were washed with PBS three times to remove DCFH-DA that not entered in cells. The ﬂuorescence was visualized immediately at wave lengths of 485 nm for excitation and 530 nm for emission by inverted ﬂuores- cence microscope. Total green ﬂuorescence intensities of each well were quantiﬁed using image analysis software. Chick aortic ring models In brief, the aortic arches were dissected from 12-day-old chick embryos, cut into rings and embedded into Matrigel in a 6-well plant. After overnight incubation at 37 C, rings were added with serum-free medium containing ellagic acid in the presence of VEGF. Sprouts were formed within 48–72 h. Images were photographed at 59 magniﬁcation of a Zeiss inverted microscope. The extent of sprouts for- mation from chick aortic ring was quantiﬁed using image- pro software. Chick embryo chorioallantoic membranes (CAM) models 5-Day-old fertilized chicken eggs were incubated at 37 C in 60% humidity for additional 48 h. On day 3, a small hole in the shell concealing the air sac was made with a hypo- dermic needle. By candling, a second hole was made on the broad side of the egg directly over the avascular portion of the embryonic membrane. A false air sac was created beneath the second hole by applying negative pressure through the ﬁrst hole, causing the CAM to separate from the shell. A window of about 1 cm2 was cut into the shell 123 Breast Cancer Res Treat (2012) 134:943–955 over the dropped CAM using a small grinding wheel. VEGF (20 ng/ml) was used as a standard proangiogenic agent. Sterile disks of ﬁlter paper (Whatman International) were pretreated with 3 mg/ml cortisone acetate and air dried under sterile conditions. The disks were suspended in PBS containing different concentrations of ellagic acid and VEGF, and then placed on the growing CAMs. After 3 days, the tissue directly beneath each disk was reselected from the control and the treated CAM samples. Each tissue sample was washed three times with PBS and digital images were collected. The images were then analyzed by Image-J software. Six regions were randomly selected and the number of vessel branch points contained in a circular region was counted. The resulting angiogenesis index was expressed as mean ± SD of the branch points for each CAM sample. The experiments were repeated three times. Xenograft models and immunohistochemistry detections Use of animal has been approved by the Committee on the Use of Live Animal in the Teaching and Research of the University of Hong Kong. 3 9 106 Human breast cancer MDA-MB-231 cells were implanted into the mammary glands of female, 4-week-old BALB/c nude mice to build breast cancer xenograft. Mice with appropriate size of tumors were divided randomly into three groups including control group, ellagic acid low-dosage group (50 mg/kg/ day), and ellagic acid high-dosage group (100 mg/kg/day). The mice were treated with ellagic daily by intraperitoneal administration. Tumor volume and mice body weight were measured every 3 days. Tumor volume was detected by caliper measurements and determined by formula: * width2 * length. After sacriﬁcing mice on day 25, tumors and normal tissues will be harvested for molecular assessment. Speciﬁcally, tumors and normal tissues were ﬁxed in 10% paraformaldehyde/PBS, dehydrated in 70% ethanol, embedded in 4% parafﬁn at 4 C, and then sec- tioned (4 lm). Deparafﬁnized tumor sections were stained with speciﬁc antibodies including CD31, P-VEGFR2, P-AKT, and P-JNK (Cell Signaling) Detection was done with avidin–biotin–HRP complex (Thermo scientiﬁc, Fremont, CA) and diaminobenzidine as chromogen. Nuclei were counterstained with hematoxylin. Besides, deparafﬁ- nized normal tissue sections including heart, liver, spleen, lung, and kidney will be stained with hematoxylin and eosin (H&E) for toxicological analysis. Molecular docking The LigandFit algorithm in Discovery Studio 2.1 with the CHARMm engine was applied in our molecular docking study. Chemofﬁce2002 (CambridgeSoft, Cambridge, MA)