Enhanced Expression of the Transcription Factor Nrf2 by Cancer Chemopreventive Agents: Role of Antioxidant Response Element-Like Sequences in the nrf2 Promoter

Abstract

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, is an important mechanism for protection against carcinogenesis. The transcription factor Nrf2, which binds to the antioxidant response element (ARE) found in the upstream regulatory region of many phase 2 genes, is essential for the induction of these enzymes. We have investigated the effect of the potent enzyme inducer and anticarcinogen 3H-1,2-dithiole-3-thione (D3T) on the fate of Nrf2 in murine keratinocytes. Both total and nuclear Nrf2 levels increased rapidly and persistently after treatment with D3T but could be blocked by cotreatment with cycloheximide. Nrf2 mRNA levels increased ϳ2-fold 6 h after D3T treatment. To examine the transcriptional activation of Nrf2 by D3T, the proximal region (1 kb) of the nrf2 promoter was isolated. Deletion and mutagenesis analyses demonstrated that nrf2 promoter-luciferase reporter activity was enhanced by treatment with D3T and that ARE-like sequences were required for this activation. Gel shift assays with nuclear extracts from PE cells indicated that common factors bind to typical AREs and the ARE-like sequences of the nrf2 promoter. Direct binding of Nrf2 to its own promoter was demonstrated by chromatin immunoprecipitation assay. Overexpression of Nrf2 increased the activity of the nrf2 promoter-luciferase reporter, while expression of mutant Nrf2 protein repressed activity. Thus, Nrf2 appears to autoregulate its own expression through an ARE-like element located in the proximal region of its promoter, leading to persistent nuclear accumulation of Nrf2 and protracted induction of phase 2 genes in response to chemopreventive agents.

Increased Protein Stability as a Mechanism That Enhances Nrf2-mediated Transcriptional Activation of the Antioxidant Response Element

“…These findings are consistent with other studies that have demonstrated that Nrf2 mRNA levels are unaffected by various inducers of ARE activity (13,39). A more recent study, however, reported that the expression of Nrf2 was increased in murine keratinocytes by 3H-1,2-dithiole-3-thione (D3T), but this effect was attributed to an increase in the rate of Nrf2 transcription (40). These results are contradictory to those observed in this densitometer, and the Nrf2 values were plotted after normalization with those of GAPDH.…”

Inhibition of Nitric Oxide Production by Ethyl Digallates Isolated from Galla Rhois in RAW 264.7 Macrophages

“…On the other hand, stimulating protein expression of Nrf2 appears to be another possible mechanism. Some natural anti-oxidant agents have been shown to increase protein expression level of Nrf2 (Kwak et al, 2002;Nguyen et al, 2003;Stewart et al, 2003). In the present study, we have shown that treatment with EDG enhanced the level of Nrf2 in nucleus (Fig.…”

BRCA1 and BRCA2 as molecular targets for phytochemicals indole-3-carbinol and genistein in breast and prostate cancer cells

“…The major active metabolite of I3C, diindolylmethane (DIM), induces expression of GADD45a, a DNA damage-responsive gene and putative tumour suppressor (Carter et al, 2002). Indole-3-carbinol can activate two pathways linked to cancer prevention: (1) aryl hydrocarbon receptor (AhR) signalling (which leads to expression of phase I enzymes (e.g., CYP1A1) via the xenobiotic response element) and (2) antioxidant/electrophilic response element signalling (resulting in expression of phase II detoxifying enzymes: e.g., oxido-reductases and glutathione-S-transferases) (Chen et al, 1998;Hayes and McMahon, 2001;Kwak et al, 2002). Thus, the ability of I3C to induce enzymes that metabolise genotoxic agents may contribute to cancer prevention.…”