Fig. 4

Effect of AA and A2P on HIF-1α transcriptional activity in metastatic melanoma. WM9 metastatic melanoma cells were transiently transfected with an HIF-1 HRE-luciferase reporter vector. a Transfected WM9 cells were treated with 100 μM CoCl2 with or without AA (100 μM) or A2P (100 μM) Cells were collected and HIF-1 transcriptional activity was measured by luciferase assay at 24, 48, and 72 h. Both AA and A2P significantly reduced HIF-1 transcriptional activity at 24 and 48 h, at 72 h, A2P significantly reduced CoCl₂ induced activity while AA began to show reduced efficacy by 72 h. b Dose dependent inhibition of CoCl₂ induced HIF-1 reporter activity using 25, 50, 100 and 250 μM AA. AA significantly reduced CoCl₂ induced HIF-1 activity at all concentrations, with 25 μM AA beginning to show reduced efficacy. c Dose dependent inhibition of CoCl₂ induced HIF-1 reporter activity by 25, 50, 100 and 250 μM A2P. All concentrations of A2P significantly reduced CoCl₂ induced HIF-1 reporter gene activity. For this reason, lower doses of A2P were then tested. d Low dose titration of A2P dependent inhibition of CoCl₂ induced HIF-1 reporter activity using 2.5, 5.0, 10 and 25 μM A2P. A2P demonstrated close to maximum inhibition of HIF-1 activity at concentrations as low as 10 μM, with 5 and 2.5 μM demonstrating little to no inhibition of activity. All HRE-luciferase activity was normalized to β-galactosidase activity. Data are represented as mean ± SEM of a minimum of n = 3, analyzed by One-way ANOVA followed by Tukey’s multiple comparisons test; * denotes significant difference from control, p < 0.0001, # denotes significant difference from CoCl₂ treatment alone, p < 0.003-0.0001