EEAA suppresses tumor-induced angiogenesis by HUVECs in vitro. (A) Concentration of tumor cell-culture medium. For NCI-H460 tumor cell-induced angiogenesis, the culture medium (CM) was concentrated 5-fold using a 9 kDa protein concentrator, as described in the Methods section and as depicted in Figure 1. The levels of VEGF in the cell-free medium (CFM), CM, and 5-fold CM (5X CM) were measured via ELISA. (B) Efficient removal of EEAA from the concentrated CM. HUVEC migration was observed in the presence of the CM or 5X CM. (C to E) The effect of EEAA on NCI-H460 tumor cell-induced angiogenesis mediated by HUVECs. The 5X CFM and 5X CM were prepared from cell-free (CFM) and NCI-H460 cells (H460-CM)-culture medium (CFM), respectively, in the absence (M) or the presence of vehicle (V), EEAA (EA), and VEGF supplementation (EV). Cell proliferation (C), migration (D), and tube formation (E) by HUVECs were determined after treatment with each concentrated culture medium. The data are presented as the relative mean ± S.D. of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the vehicle groups.