Abstract 5664: Methylseleninic acid induces NAD(P)H: quinone oxidoreductase-1 expression through activation of NF-E2-related factor 2 in Chang liver cells

Abstract

Abstract Selenium, an essential biological trace element, has been shown to reduce the incidence of cancer and to inhibit the experimentally induced tumorigenesis. Selenium has been reported to induce expression of some antioxidant enzymes in various cell lines. However, the mechanisms underlying its chemopreventive as well as antioxidant effects of selenium remain largely unresolved. In the present study, we found that methylseleninic acid (MSeA), a monomethylated selenium compound, induced expression of NAD(P)H-quinone oxidoreductase-1 (NQO-1) in Chang human liver cells. NQO-1 is an important antioxidant enzyme that plays a pivotal role in cytoprotection against oxidative and electrophilic stresses. NF-E2-related factor 2 (Nrf2), through interaction with antioxidant response element (ARE), upregulates expression of several phase-2 detoxifying and antioxidant enzymes including NQO-1. MSeA treatment (3 μM) resulted in an increased nuclear translocation, subsequent ARE binding and transactivation of Nrf2. Silencing Nrf2 using dominant negative Nrf2 or Nrf2 siRNA markedly reduced the MSeA-induced NQO-1 expression. The binding of Nrf2 to ARE was verified by the chromatin immunoprecipitation (ChIP) assay. We also found that MSeA treatment induced expression of NQO-1 to a much lesser extent in Nrf2 knock out (−/−) mouse embryonic fibroblast (MEF) than in Nrf2 wild-type (+/+) MEF. One of the critical events responsible for Nrf2 activation is its dissociation from the repressor protein Keap1. MSeA reduced the level of Keap1 protein at a post-translational level. Moreover, ubiquitination of Keap1 was markedly increased in cells exposed to MSeA, which resulted in decreased steady-state levels of Keap1 in parallel with inhibition of keap1-dependent ubiquitination of Nrf2. siRNA knock-down of Keap1 significantly increased both constitutive and MSeA-induced NQO-1 expression. We hypothesize that MSeA may modify Keap1 thiols, which facilitates the nuclear translocation and ARE binding of Nrf2. In addition, prior exposure of cells to dithiothreitol (1 mM) blocked NQO-1 induction by MSeA, corroborating Keap1 thiol modification involved in Nrf2 activation. Oral administration of MSeA (1 mg/kg) by gavage to mice also induced NQO-1 protein expression in the liver. Methylselenol generated from selenomethionine (SeMet) by methioninase (METase) activity induced expression of NQO-1. In contrast to SeMet formed by METase, SeMet failed to induce NQO-1 expression, suggesting that methylselenol, not its metabolite, is directly responsible for NQO-1 induction. Taken together, these results suggest that MSeA, the immediate precursor of methylselenol, upregulates the expression of NQO-1 via the Keap1-Nrf2 signaling, preferentially by targeting cysteine thiol(s) present in Keap1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5664.