Figure 5.
12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced nuclear factor kappaB (NF-κB) activity, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expressions suppressed by sorafenib in Huh7/NF-κB-luc2 cells via the suppression of extracellular signal-regulated kinase (ERK) signal pathway. Cells were treated with dimethyl sulfoxide (DMSO), sorafenib, and PD98059 30 minutes prior to the addition of TPA, respectively, and incubated for another 6 hours. A: TPA-induced ERK phosphorylation is inhibited by sorafenib. B: No cytotoxicity was found in cells treated with various concentrations of PD98059 for 6 hours. C: Intrinsic NF-κB activity is inhibited by the treatment with PD98059 for 6 hours. Top: Bioluminescent imaging (BLI) of the relative NF-κB activity; bottom: quantification of BLI. D: Intrinsic and TPA-induced MMP-9 and VEGF expressions are suppressed by PD98059. Top: Western blotting; bottom: quantification of the western blotting. E: Inhibitor of kappaB-α mutant vector (p-IκBαM), a super repressor of NF-κB, bound to NF-κB with high affinity, preventing its phosphorylation by inhibitor of kappaB kinase (IKK). As a consequence, NF-κB is sequestered in the cytoplasm. F: Intrinsic and TPA-induced NF-κB activities are decreased in cells treated with sorafenib, PD98059, and transfection with p-IκBαM, respectively. Top: BLI of the relative NF-κB activity; bottom: quantification of BLI. G: Intrinsic and TPA-induced NF-κB/DNA binding activities are blocked in cells treated with sorafenib, PD98059, and transfection with p-IκBαM, respectively. Top: NF-κB/DNA binding activity as determined by EMSA; bottom: quantification of EMSA. Data are presented as means±SD (n=3). *p<0.05, **p<0.01 as compared with that of DMSO-treated control; #p<0.05, ##p<0.01 as compared with that of TPA-treated only group. The experiments were repeated three times.