Figure 7
AZD6244 suppresses the p-ERK − c-FOS − HIF-1α − VEGF integrated signal pathways in HUVEC cells.
HUVEC cells were treated with AZD6244 (4 μM) for 24 hours. ERK was comparable between treated treatments; however, compared with that in control group, p-ERK measured by immunocytofluorescence staining (A) and Western blot (C,D) was significantly suppressed in AZD6244 treated cells. VEGF protein and mRNA quantified by immunocytofluorescence staining (B), Western blot (C,E) and RT-PCR (F,G) were remarkably reduced in AZD6244 treated cells when compared with that in control group. Similarly, the protein level of c-FOS and HIF-1α (C,E) was also reduced after treatment with AZD6244. The effect of AZD6244 on HIF-1α binding to VEGF promoter region was analyzed by ChIP followed with qRT-PCR (H). IgG treated protein-DNA complexes was almost negative for PCR amplifications of the VEGF promoter. However, binging of HIF-1α to VEGF promoter was observed in mouse anti-HIF-1α antibody incubated protein-DNA complexes. Interesting, binging of HIF-1α to VEGF promoter was significantly reduced after AZD6244 treatment when compared with DMSO treated HUVEC cells (H). # p < 0.05 vs. control group.
