Figure 9 Effects of NAC on in vitro invasiveness. Cells in RPMI 1640 media supplemented with 5% FBS were placed in the upper chamber of Matrigel chamber and treated with or without NAC. The bottom chamber was filled

with media containing 5% FBS and HGF with or without NAC. After 48 h of incubation, the cells which migrated through the filter were counted under light microscopy (10 fields at 200× power). Values are the means ± SD of triplicates of three independent experiments. Statistical significance was estimated by Student’s t-test (*, P < 0.05; **, p < 0.01). Effect of H2O2 on ERK and p38 activation induced by HGF To demonstrate the effect of H2O2 on HGF-mediated ERK and p38 activation, we treated p38 kinase assay both cells with H2O2. Treatment with H2O2 increased the activity of ERK and p38. When cells were treated with H2O2 and HGF together, the activation of ERK and

p38 kinase was decreased (Figure 10). Figure 10 Effects of H 2 O 2 on ERK and p38 activation induced by HGF. Serum-starved cells were pretreated with or without H2O2 (100 μM) for 30 min and then treated with or without HGF (10 ng/ml). After incubation for 15 min, the levels of phosphorylated ERK, ERK, phosphorylated p38, and p38 were find more measured by Western blot analysis. GDC-0994 supplier Representative data from 3 independent experiments are shown. Effect of ERK and p38 inhibitor on H2O2-induced uPA expression To test whether ERK and p38 activation was involved in H2O2-mediated uPA secretion, cells were pretreated with PD 098059 or SB 203580, and uPA secretion was measured by Western blotting. Both cells showed that H2O2-mediated

uPA secretion was reduced with increasing concentrations of PD 098059. Densitometric analysis indicated that 10 μM PD 098059 reduced the urokinase secretion > 50%. In contrast, pretreatment with SB 203580 increased uPA secretion. These results suggested that H2O2-mediated uPA secretion and the augmentation of this activity was regulated by ERK and p38 activation (Figure 11). Figure 11 Effects of PD 98059 or SB 203580 on HGF-mediated up-regulation of uPA. Serum-starved cells were pretreated with or without H2O2 (100 μM) for 30 min and then treated with PD 98059 (5, 10 and 20 μM) or SB 203580 (1, 5 and 10 μM). After 17-DMAG (Alvespimycin) HCl incubation for 24 h, uPA in culture media was measured by Western blot analysis. Representative data from 3 independent experiments were shown. Effects of PD 098059 and/or SB 203580 on H2O2-induced ERK1/2 phosphorylation To investigate the possibility of an interaction between ERK and p38 activation in H2O2-mediated uPA expression, the effect of SB 203580 on ERK activation was measured. Pretreatment with SB 203580 increased ERK phosphorylation in the H2O2-treated cells. Co-treatment with PD 098058 and SB 203580 decreased ERK phosphorylation.