Cross-talk between nuclear factor-κB and extracellular signal-regulated kinase signaling pathways in A375 human melanoma cells

Abstract

Objective To investigate the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) signal transduction pathways in A375 human melanoma cells.Methods Cultured A375 cells were randomly divided into 5 groups:control group receiving no treatment,two U0126 (a selective inhibitor of the ERK signaling pathway) groups treated with U0126 of 10 and 5 μmol/L,and two BMS-345541 groups treated with BMS-345541 of 10 and 5 μmol/L.After 24-hour treatment,Western blot and reverse transcription PCR were performed to measure the protein expressions of NF-κB P65,phosphorylated IκBα (p-IκBα),ERK1/2,as well as p-ERK1/2,and the mRNA expressions of NF-κB P65 and ERK1,respectively.One-way analysis of variance and least significant difference (LSD)-t test were carried out for statistical analysis.Results After 24 hours of treatment with U0126 of 10 and 5 μmol/L,a significant decrease was noted in the relative expression level of NF-κB p65 protein (0.60 ± 0.04 and 0.56 ± 0.06 vs.1.54 ± 0.15,both P＜ 0.01) and mRNA (0.79 ± 0.05 and 0.75 ± 0.04 vs.0.86 ± 0.05,both P ＜ 0.01),but a statistical increase in that of p-IκBα protein (0.90 ± 0.05 and 0.70 ± 0.02 vs.0.61 ± 0.03,both P ＜ 0.01) in the two U0126 groups compared with the control group; significant differences were observed in the expression level of p-IκBo protein (P ＜ 0.01) but not in that of NF-κB p65 protein (P ＞ 0.01) between the two U0126 groups.The relative expression levels of ERK1/2 and p-ERK1/2 proteins as well as ERK1 mRNA were significantly higher in the control A375 cells than those in the cells treated with BMS-345541 of 10 μmol/L (0.73 ± 0.07,0.75 ± 0.09,1.51 ± 0.02,all P ＜ 0.01),but similar to those treated with BMS-345541 of 5 μmol/L (0.94 ± 0.11,0.99 ± 0.04,1.62 ± 0.03,all P ＞ 0.05).Conclusion There is a cross-talk between ERK and NF-κB signal transduction pathways in A375 melanoma cells. Key words: Melanoma; NF-kappa B ; Extracellular signal-regulated MAP kinase; Signal transducting