Roles of nuclear factor-erythroid 2-Related Factor 2 in glutamate cysteine ligase induction by organoselenium compounds

Levels of dietary selenium are inversely associated with cancer risk in humans, and selenium has played a major role in the field of chemoprevention. In particular, the organoselenium agents 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione conjugate, p-XSeSG, are effective in preventing cancers at numerous sites, including the lung in rodent models. In the A/J mouse lung, p-XSC is highly effective in inducing glutathione (GSH), but mechanisms are not well understood. Glutathione is the most abundant antioxidant in animals, and is an important mechanistic factor in chemoprevention. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme responsible for GSH production. Transcriptional regulation of GCL occurs via antioxidant response elements (ARE) in the promoter regions of its genes. Induction of the ARE is in turn regulated by stabilization and nuclear translocation of the transcription factor, Nuclear factor-erythroid 2-Related Factor 2 (Nrf2). Furthermore, the extracellular signal-regulated kinase (ERK) pathway has been shown to play a role in Nrf2 stabilization. Potent chemopreventive phytochemicals, such as sulforaphane (SFN), have been shown to act largely through the Nrf2/ARE pathway.;Isothiocyanate compounds such as SFN are among the most potent Nrf2 inducers known. We hypothesized that substitution of sulfur with selenium in the isothiocyanate functional group of SFN would result in an isoselenocyanate compound (SFN-isoSe) with enhanced Nrf2 induction capability. Here we show that SFN-isoSe activates an ARE-luciferase reporter in HepG2 cells more potently than SFN. It was also found that SFN-isoSe induces GCL and GSH in MEF cells in an Nrf2-dependent manner. Finally, we provide evidence that SFN-isoSe is more effective in killing HepG2 cancer cells, yet is less toxic to noncancer MEF cells, than is SFN. These data support our hypothesis, and suggest that SFN-isoSe may be a highly effective chemoprotective agent in vivo.;Taken together, these results support the hypothesis that Nrf2 is required for organoselenium-mediated induction of GSH, and suggest that the induction of the Nrf2 pathway by organoselenium compounds may represent a general mechanism of chemoprevention. Induction of GSH, with low associated toxicity, by agents like p-XSeSG may prove useful for treatments of conditions such as HIV and chronic inflammatory conditions. Isoselenocyanates, such as SFN-isoSe, may be ideal candidates for future studies as chemopreventive and/or chemotherapeutic agents due to their ability to induce Nrf2 with low toxicity in normal cells and high efficiency at killing cancer cells.;The potential relevance of Nrf2 to GSH induction led to the hypothesis that Nrf2 activation may be involved in organoselenium-mediated induction of GSH, and this was investigated in vivo and in several cell culture models. Here we show that dietary p-XSC induces Nrf2, p-ERK, GCL and GSH in the lung of the Fisher rat, suggesting involvement of Nrf2 and the ERK pathway in GSH induction. In addition, GSH and GCL were induced by p-XSeSG in the Fisher rat. In cell culture studies we observed that p-XSC and p-XSeSG activate an ARE luciferase reporter in an Nrf2-dependent manner. It was also shown that p-XSeSG induced GSH in vitro, and is less toxic than p-XSC. The dependence of GCL induction by p-XSeSG upon Nrf2 was confirmed in wildtype and Nrf2-mutanted Mouse Embryonic Fibroblasts (MEF). These results suggest that p-XSC acts through the Nrf2 pathway in vivo, and that p-XSeSG may be the metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious inducer of GSH.