EEAA decreases VEGF expression in tumor cells under hypoxic conditions. (A) Effect of EEAA on tumor cell growth. HCI-H460 cells were treated with EEAA (10 μ g/mL), and cell proliferation (left panel) and viability were determined after 48 h. (B) Reduced production of VEGF due to EEAA. NCI-H460 cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions in the absence (M) and the presence of vehicle (DMSO, V) or 10 μ g/mL EEAA (EA). After 8 h of incubation, an immunoassay was performed to quantify VEGF secreted from NCI-H460 cells. (C) Down-regulation of VEGF at the transcriptional level by EEAA. NCI-H460 cells were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions in the presence of increasing concentrations of EEAA (1–100 μ g/mL). After 24 h, intracellular VEGF mRNA levels were quantified using RT-PCR. The relative mRNA contents were determined based on comparison with vehicle (DMSO). The data are presented as the mean or relative mean ± S.D. of at least three independent experiments. *P < 0.05 and ***P < 0.001 compared with the vehicle groups.