CCAAT / ENHANCER BINDING PROTEIN-AND NF-E 2-RELATED FACTOR-2 FOR GSTA 2 GENE INDUCTION

Comprehensive mechanistic studies suggest that oltipraz exerts cancer chemopreventive effects through the induction of glutathione S-transferase (GST). Previously, we have shown that the activation of CCAAT/enhancer binding protein(C/EBP ), promoted by oltipraz, contributes to the transcriptional induction of the GSTA2 gene. Studies also indicated that exposure of animals to oltipraz triggers nuclear accumulation of NF-E2-related factor-2 (Nrf2) with an increase in Nrf2’s antioxidant response element (ARE) binding activity. Given the previous reports that C/EBP activation contributes to oltipraz’s induction of the GSTA2 gene and that Nrf2 activation by oltipraz was variable depending on the concentrations, this study investigated whether the major oxidized metabolites of oltipraz induce GSTA2 through the activation of C/EBP and/or Nrf2. Immunoblot analysis revealed that M1 [4-methyl-5-(pyrazin-2-yl)-3H-1,2-dithiol-3-one] and M2 (7-methyl6,8-bis(methylthio)H-pyrrolo[1,2-a]pyrazine), but not M3 (7-methyl8-(methylsulfinyl)-6-(methylthio)H-pyrrolo[1,2-a]pyrazine) and M4 (7-methyl-6,8-bis(methylsulfinyl)H-pyrrolo[1,2-a]pyrazine), induced GSTA2 in H4IIE cells. M1 and M2 also increased the luciferase activity from pGL-1651, which contained the luciferase structural gene downstream of the 1.65-kilobase GSTA2 promoter region. Nuclear C/EBP levels were enhanced by the metabolites but not by M3 or M4. Among the oxidized metabolites examined, only M2, which elicited cell death at a relatively high concentration, activated Nrf2, as indicated by nuclear accumulation of Nrf2 and its ARE binding activity. The present study provides evidence that M1 and M2, but not M3 and M4, induce GSTA2 and that M1 induces GSTA2 only via C/EBP activation, whereas M2 does so by activating Nrf2 as well as C/EBP . These results substantiate the differential effects of oltipraz’s metabolites on C/EBP and/or Nrf2-mediated GSTA2 induction. Oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiol-3-thione] has been studied as a chemopreventive agent for malignancies, such as liver and colorectal cancer (Rao et al., 1993; Kensler, 1997). Comprehensive mechanistic studies indicate that oltipraz exerts cancer chemopreventive effects through the induction of glutathione S-transferases (GSTs) (Bolton et al., 1993; Kensler, 1997). GST induction also accounts for the cytoprotective effect of oltipraz against toxicantinduced injury (Jaitovitch-Groisman et al., 2000; Nelson et al., 2002). A phase IIa randomized chemoprevention trial of oltipraz in residents of Qidong, China, showed that oltipraz might be clinically active as a chemopreventive agent. In the human studies, oltipraz dosage regimens with higher doses and a long-dosing interval seemed to be more efficacious in preventing cancer, as supported by a significant decline in the levels of aflatoxin-albumin adduct in the individuals receiving a higher dose of oltipraz (500 mg/week) (Jacobson et al., 1997; Jackson and Groopman, 1999; Wang et al., 1999). Exposure of experimental animals to oltipraz triggers nuclear accumulation of NF-E2-related factor-2 (Nrf2) (Ramos-Gomez et al., 2001; Iida et al., 2004) and enhances Nrf2’s antioxidant response element (ARE) binding activity (Pietsch et al., 2003). Diminished expression of phase II enzyme genes by oltipraz in the Nrf2 / mice supports the role of Nrf2 activation in its cancer chemopreventive effects (Kwak et al., 2001; Ramos-Gomez et al., 2001). Molecular signals activated by oxidative stress stimulate translocation of Nrf2 to the nucleus, where it binds and activates the AREs located in the promoter regions of phase II enzyme genes (Moinova and Mulcahy, 1998; Venugopal and Jaiswal, 1998; Huang et al., 2000). Studies from this laboratory showed that oltipraz at clinically relevant concentrations marginally increased the band intensity of Nrf2 ARE binding and thus weakly enhanced the accumulation of Nrf2 in the nucleus. Our results suggested that GSTA2 induction by oltipraz might be mediated by the activation of other transcriptional This work was supported by the National Research Laboratory Program (2001– 2006), Korea Science and Engineering Foundation, The Ministry of Science and