Figure 4.

Analysis of the different MAPK pathways activated by H₂O₂ in SW620 cells. SW620 cells were cultured in serum-free L-15 medium for 24 h to synchronize the cells and to make them quiescent prior to treatment with SP600125 (50 μM; JNK1/2 phosphorylation inhibitor), PD98059 (50 μM; ERK1/2 phosphorylation inhibitor), or SB203580 (10 μM; p38 MAPK phosphorylation inhibitor) for 30 min prior to the addition of H₂O₂ (5 μM) for 1 h. Cell lysates were prepared and immunoblotted with the antibodies indicated in the panel. A, the levels of phospho-JNK1/2 were evaluated by H₂O₂ stimulation and suppressed by SP600125 or DMA (100 μM) pretreatment. B, the levels of phospho-ERK1/2 were evaluated by H₂O₂ stimulation and suppressed by PD98059 or DMA (100 μM) pretreatment. C, the level of phospho-p38 MAPK was evaluated by H₂O₂ stimulation and suppressed by SB203580 or DMA (100 μM) pretreatment. Ratios of bands with phosphospecific versus non-phosphospecific MAPK antibodies were determined, and quantitated by densitometry using the ImageJ program (NIH). Means ± SD are given. D, H₂O₂-enhanced MMP-7 protein expression was associated with increased JNK and ERK signaling in SW620 cells. Pretreatment with DMA (100 μM), SP600125 (50 μM), or PD98059 (50 μM) for 30 min prior to H₂O₂ (5 μM) induction for 8 h in SW620 cells significantly declined the expression of MMP-7 compared with H₂O₂-only treatment (*p < 0.05). However, the level of MMP-7 protein was only slightly decreased with no significant difference effect in the presence of SB203580 (10 μM) after H₂O₂-induced MAPK stimulation. The bars in the lower panel denote means ± S.D. of three experiments for each condition determined from densitometry relative to β-actin.