Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2. Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein

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Abstract

Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1, UGT1A6 and GCLC were lower in tissues from nrf2−/− mice than from nrf2+/+ mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2+/+ mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C + K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2−/− mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C + K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C + K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5′-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 μmol/l furan, suggesting that this ring structure within C + K is insufficient for gene induction. Priming of nrf2+/+ MEFs, but not nrf2−/− MEFs, with C + K conferred 2-fold resistance towards acrolein.

Introduction

Coffee consumption may reduce the risk of both colorectal cancer (Giovannucci, 1998) and hepatocellular carcinoma (Larsson and Wolk, 2007). Within coffee, the diterpenes cafestol and kahweol (C + K) can induce glutathione S-transferase (GST) isoenzymes that are thought to protect against carcinogenesis (Lam et al., 1982, Cavin et al., 1998). Besides C + K, coffee also contains chlorogenic acid, formed as an ester between cinnamic acid and quinic acid, that has also been reported to inhibit carcinogenesis (Tanaka et al., 1993, Mori et al., 2000). It is however unclear what effect chlorogenic acid has on the expression of GSTs and other cytoprotective enzymes.

The basic-region leucine zipper (bZIP) transcription factor Nrf2 (Nuclear factor-erythroid 2 p45 subunit-related factor 2) controls the expression of a battery of drug-metabolizing and antioxidant enzymes. Genes that are regulated by Nrf2 contain antioxidant response elements (AREs, minimal enhancer 5′-A/GTGACNNNGCA/G-3′) in their promoters (Rushmore et al., 1991, Wasserman and Fahl, 1997, Erickson et al., 2002, Nioi et al., 2003) and include those for the glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits, GST, NAD(P)H:quinone oxidoreductase 1 (NQO1) and UDP-glucuronosyl transferase (UGT) (McMahon et al., 2001, Chanas et al., 2002, Kwak et al., 2003). Besides controlling normal homeostatic expression of ARE-driven genes, Nrf2 also mediates their induction by redox stressors. Induction of ARE-driven genes occurs through stabilization of Nrf2 protein resulting from inactivation of Keap1, an E3 ubiquitin ligase substrate adaptor that negatively regulates the bZIP factor (McMahon et al., 2003, Kobayashi et al., 2004, Zhang et al., 2004, Devling et al., 2005). Such inducers include the cancer chemopreventive phenolic antioxidant butylated hydroxyanisole (Hayes et al., 2000) and the dietary isothiocyanate sulforaphane (Sul) produced as a breakdown product of glucoraphanin in cruciferous vegetables (McWalter et al., 2004, Hong et al., 2005).

The promoters of NQO1 genes contain a xenobiotic response element (XRE, 5′-TA/TGCGTGA/C-3′) as well as an ARE (Favreau and Pickett, 1991, Jaiswal, 1991, Ma et al., 2004). The XRE recruits the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor (Nioi and Hayes, 2004, Tijet et al., 2006). Unlike Nrf2, the AhR is activated by binding planar aromatic xenobiotic ligands, including dioxin, 3-methylcholanthrene (3MC) and indolo[3,2-b]carbazole (Bonnesen et al., 2001). There is cross-talk between the Nrf2 and AhR pathways insofar as AhR requires the presence of Nrf2 in order to induce gene expression by dioxin and 3MC (Noda et al., 2003, Ma et al., 2004). Furthermore, the promoter of mouse nrf2 contains several XREs (Miao et al., 2005) and the promoter of mouse ahr contains an ARE (Shin et al., 2007). Thus, AhR regulates Nrf2 mRNA levels, and Nrf2 regulates AhR mRNA levels.

Polymorphic forms of AhR exist in the mouse. For example, C57BL/6 mice possess an AhR protein that exhibits high-affinity for dioxin, whereas DBA/2 mice posses an essentially non-functional AhR protein. This difference in ligand binding is associated with changes in the primary structure of the receptor in C57BL/6 and DBA/2 mice (Chang et al., 1993). It is not known whether the AhR contributes to any putative beneficial actions of coffee.

In this study, we have demonstrated that consumption of coffee can induce drug-metabolizing and antioxidant enzymes in vivo. Using nrf2−/− mice, we have tested whether up-regulation of such proteins, and their corresponding mRNAs, by coffee consumption is mediated by Nrf2. Similarly, using DBA/2O mice we have examined whether the increase in gene expression affected by coffee is mediated by AhR. We have also used embryonic fibroblasts to identify whether induction of nqo1 by C + K occurs through an ARE enhancer in its promoter, and whether activation of ARE-driven genes by C + K confers resistance against the toxic electrophile acrolein.

Section snippets

Reagents

The chemicals used were all of the highest quality grade available and, unless otherwise stated, were obtained from Sigma/Aldrich, Gillingham, Dorset, UK. Coffee and the diterpenes C + K (a 1:1 mixture of their palmitate esters) were provided by Nestlé Research Center, Lausanne, Switzerland.

Animals

Throughout this study, mice were treated in accordance with regulations contained in the Animals and Scientific Procedure Act (1986) of the United Kingdom, and with the approval of the University of Dundee

Nrf2 controls enzyme induction by coffee in mouse liver

Western blotting showed that feeding nrf2+/+ mice diets containing either 3% or 6% coffee markedly increased the level of hepatic NQO1 protein (Fig. 1). By comparison with WT mice, the normal constitutive expression of NQO1 protein was greatly reduced in the liver of nrf2−/− mice. Furthermore, the oxidoreductase was not obviously increased by either 3% or 6% coffee in the Nrf2 KO animals. Real-time PCR from the same liver samples showed that induction of mRNA for NQO1 displayed a dose

Discussion

From mouse feeding studies, we have shown that 3% and 6% coffee-containing diets induced genes for NQO1, class Alpha GST, GCLC, UGT1A6 and CYP1A2 in the liver and gastrointestinal tract. In C57BL/6 nrf2+/+ mice, greatest induction was generally observed in the small intestine, where nqo1, gsta1, gsta3 and gclc were induced between 8- and 10-fold by 6% coffee, and gsta4, ugt1a6 and cyp1a2 were induced at least 2-fold. With the exception of hepatic NQO1, the mRNA of which was increased about

Acknowledgments

This study was supported by the European Coffee Consortium (PEC). We thank Prof. C. Roland Wolf, Dr. Colin J. Henderson and Dr. Lesley I. McLellan for providing antibodies.

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