Reduction of secretions of osteopontin (OPN) and VEGF in MDA-MB-231 cells by BER. (A) Reduction of OPN secretions in MDA-MB-231 (left) and MDA-MB-435S (right) cells by BER treatment. The cells were treated for 16 h with BER at the indicated concentration. The secreted OPN in the cell supernatants was analyzed by ELISA described in "Materials and Methods" section. (B) Reduction of VEGF secretions in MDA-MB-231 cells by BER and its synergistic anticancer agents carmofur (K) and trichostatin A (T). The 0 (0.1% DMSO vehicle) is the control. The cells were treated for 48 h with the indicated concentrations of BER (B, 10 μM, 20 μM, and 40 μM), celecoxib (S, 10 μM, 20 μM, 40 μM and 80 μM), trichostatin A (T, 25 μg/L and 50 μg/L), carmofur (K, 40 mg/L), rosiglitazone (Ro1, 1 μM), lovastatin (Lo1, 1 μM), Ly294002 (LY, 25 μM), and Bay (5 μM) in the absence or presence of its synergistic anticancer agents K (40 mg/L) and T (25 μg/L and 50 μg/L). The secreted VEGF in the supernatants of MDA-MB-231 cells was analyzed by ELISA described in "Materials and Methods" section. Values are shown as mean ± SD (bar) for the indicated concentration (n = 3). Comparison was made by two-way ANOVA followed by Bonferroni post hoc test to establish whether significant differences existed between the groups. *: P < 0.05, **, P < 0.01. Values with different letters (a-f) differ significantly (P < 0.05). e/s represents the significant synergistic effects (P < 0.001) compared with the treatment with its individual compound alone. Statistically significant synergistic effects on the VEGF secretions were observed in MDA-MB-231 cells treated with B20+K40+T25, B10+K40+T50, B20+K40+T50 compared with the individual B10, B20, T25, T50, and/or K40 treatment alone (e/s, P < 0.001, two-way ANOVA).