Breast Cancer Res Treat (2012) 134:943–955 949 Fig. 2 Ellagic acid suppressed VEGF-induced invasion, migration, and tube formation of HUVECs. a HUVECs at a density of 2 9 105 cells/ml were planted into a 6-well transwell (8-lm pore size) subsequently added with different concentrations of ellagic acid and VEGF. After 24 h incubation, the cells at the bottom of the membrane were ﬁxed and analyzed. It was showed that ellagic acid decreased the number of invasive cells in a dose- dependent manner (a 0 lM, b 2.5 lM, c 5 lM, d 10 lM; data are presented as means ± SD, n = 6, *P \ 0.05 versus untreated control). b A total of 5 9 105 HUVECs per well were seeded in a 6-well plate for wound-healing assay. The results showed that ellagic acid signiﬁcantly inhibit the migration of HUVECs in the presence of VEGF stimulation (data are presented as means ± SD, n = 6, *P \ 0.05, **P \ 0.01 versus untreated control). c HUVECs were seeded at a density of 2 9 105 cells per well using a 12-well plate which was pre- coated with Matrigel. Varying concentrations of ellagic acid (2.5, 5, and 10 lM) were added together with VEGF (20 ng/ml) for additional 8 h incubation. It was observed that ellagic acid could dose dependently suppress the capillary lengths of VEGF-stimulated endothelial cells (a 0 lM, b 2.5 lM, c 5 lM, d 10 lM; values represent means ± SD, n = 6, *P \ 0.05, **P \ 0.01 versus untreated control) addition, previous studies supported that phosphorylation of VEGFR-2 could subsequently trigger multiple down- stream signals that induced proliferation and differentiation activities of endothelial cells [27]. To better understand inhibitory effects of ellagic acid on VEGFR-2 and its downstream signaling, we next chose multiple essential downstream signaling molecules involved in VEGFR-2 activation for western blotting detection. Results showed that ellagic acid could evidently inhibit VEGF-stimulated eNOS expression and phosphorylation levels of ERK, AKT, and JNK, whereas the total expressions of the latter three were almost unaffected. In addition, both the MMP-9 and MMP-2 activities were suppressed after ellagic acid treatment (Fig. 4c). As ROS was also reported as a downstream signaling after VEGFR-2 activation [28], we also detect the ROS levels by DCFH-DA probe. The results showed that the intracellular ROS level was signiﬁcantly reduced after ellagic acid administration (Fig. 4d). The above results revealed that ellagic acid inhibited in vitro angiogenesis by directly targeting VEGFR-2 on the surface of endothelial cells, and further suppressing VEGFR2- associated signaling pathways. 123