Figure 1.

Pyridinyl imidazole p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 disrupt mutant BRAF kinase activity in human melanoma cells. (A) The proliferation of A375 cells was measured by flow cytometry during 4-day cultivation. The number of cells was compared between controls (DMSO-treated) and cells treated with SB202190 (SB202; 15 µM) and SB239063 (SB239; 15 µM). The presented data (mean + SD) were obtained in three independent experiments. (B) Relative ERK activity in A375 cells stably transfected with an ERK activity luciferase reporter plasmid. Cells were treated for 24 h with SB202190 (10 µM) and MEK inhibitors U0126 (10 µM) and PD184352 (PD; 1 µM). Three independent experiments were performed. Results are presented as mean relative ERK activity + SD. (C) Levels of phosphorylated MEK and ERK kinase were analyzed by Western blot. A375 cells were treated for one hour with increasing concentrations of SB202190 (SB202) and SB203580 (SB203). MEK inhibitor PD184352 (PD; 100 nM) was used as a positive control. The relative ratio between P-ERK and the total ERK levels is also indicated for each sample. (D) ERK activity was analyzed by Western blot in melanoma cell lines bearing BRAF (A375, G361, COLO-800) or NRAS (MEL-JUSO, SK-MEL-30, IPC-298) mutations. Cells were treated for 24 h with the p38 inhibitors SB202190 (SB202; 10 µM), SB239063 (SB239; 10 µM), and BIRB796 (BIRB; 10 µM). The relative ratio between P-ERK and the total ERK levels is also indicated for each sample. (E) In vitro BRAF kinase assay using kinase-dead MEK as a substrate and endogenous V600E BRAF kinase immunoprecipitated from A375 cells. SB202190 was used at 5 µM. Data were obtained in three independent experiments. Results are presented as relative values, mean + SD. **** denotes p < 0.0001.