Hydrogen peroxide mediates arsenite activation of p70^s6k and extracellular signal-regulated kinase

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70^s6k and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H₂O₂, because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H₂O₂ by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70^s6k and ERK1/2, indicating the possible role of H ₂O₂ in the arsenite activation of the p70^s6k and the ERK1/2 signaling pathways. A specific inhibitor of p70^s6k, rapamycin, and calcium chelators significantly blocked the activation of p70^s6k induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70^s6k, ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H₂O₂ might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H₂O₂ production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H₂O₂ plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70 ^s6k signaling pathways.