AIM: To evaluate the influence of hydrogen peroxide (H(2)O(2)) on mouse photoreceptor-derived 661W cell survival and to determine the effect of PD98059, an inhibitor for MEK1 (the direct upstream activator of ERK1/2), and S3I201, a STAT3- specific inhibitor on 661W cell survival after H(2)O(2) exposure. METHODS: The mouse photoreceptor-derived 661W cells were cultured. 661W cells were treated for 12 hours with different concentrations (0, 0.25, 0.50, 0.75, 1mmol/L) of H(2)O(2) and cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ) (MTT) assay. 661W cells were treated with different concentrations H(2)O(2) (0, 5, 10, 50, 500, 1000 µmol/L) for 15 minutes or 1mmol/L H(2)O(2) for different time points (0,5,10,15,30 minutes), and p-Tyr705-STAT3, STAT3, Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2 were surveyed by immunoblot analysis. After treatment with 50µmol/L PD98059, or S3I201 for 1 hour, the inhibition efficiency of cell signal pathways was analyzed by immunoblot analysis and the effects of inhibitors on cell viability were determined by MTT. RESULTS: After treating with different concentrations of H(2)O(2) for 12 hours, the cell viability of 661W cells decreased in concentration-dependent manner (P<0.05). Moreover, H(2)O(2) induced phosphorylation of ERK1/2 and STAT3 in 661W cells (P<0.05). After pretreatment with 50µmol/L PD98059 or S3I201 for 1 hour, H(2)O(2)-induced phosphorylation of ERK1/2 or STAT3 was suppressed separately (P<0.05). Using PD98059 or S3I201 to inhibit ERK1/2 or STAT3 signal pathway, the cell viability of 661W cells decreased significantly (P<0.05). CONCLUSION: We demonstrated that the exposure of 661W cells to H(2)O(2) increased the activation of ERK1/2 and STAT3 signal pathways. Activation of these pathways is required for 661W cell survival following oxidant injury.
AIM: To evaluate the influence of hydrogen peroxide (H(2)O(2)) on mouse photoreceptor-derived 661W cell survival and to determine the effect of PD98059, an inhibitor for MEK1 (the direct upstream activator of ERK1/2), and S3I201, a STAT3- specific inhibitor on 661W cell survival after H(2)O(2) exposure. METHODS: The mouse photoreceptor-derived 661W cells were cultured. 661W cells were treated for 12 hours with different concentrations (0, 0.25, 0.50, 0.75, 1mmol/L) of H(2)O(2) and cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ) (MTT) assay. 661W cells were treated with different concentrations H(2)O(2) (0, 5, 10, 50, 500, 1000 µmol/L) for 15 minutes or 1mmol/L H(2)O(2) for different time points (0,5,10,15,30 minutes), and p-Tyr705-STAT3, STAT3, Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2 were surveyed by immunoblot analysis. After treatment with 50µmol/L PD98059, or S3I201 for 1 hour, the inhibition efficiency of cell signal pathways was analyzed by immunoblot analysis and the effects of inhibitors on cell viability were determined by MTT. RESULTS: After treating with different concentrations of H(2)O(2) for 12 hours, the cell viability of 661W cells decreased in concentration-dependent manner (P<0.05). Moreover, H(2)O(2) induced phosphorylation of ERK1/2 and STAT3 in 661W cells (P<0.05). After pretreatment with 50µmol/L PD98059 or S3I201 for 1 hour, H(2)O(2)-induced phosphorylation of ERK1/2 or STAT3 was suppressed separately (P<0.05). Using PD98059 or S3I201 to inhibit ERK1/2 or STAT3 signal pathway, the cell viability of 661W cells decreased significantly (P<0.05). CONCLUSION: We demonstrated that the exposure of 661W cells to H(2)O(2) increased the activation of ERK1/2 and STAT3 signal pathways. Activation of these pathways is required for 661W cell survival following oxidant injury.
Authors: Seán P Barry; Paul A Townsend; James McCormick; Richard A Knight; Tiziano M Scarabelli; David S Latchman; Anastasis Stephanou Journal: Biochem Biophys Res Commun Date: 2009-05-18 Impact factor: 3.575