Modulation of pregnane X receptor-and electrophile responsive element-mediated gene expression by dietary polyphenolic compounds

Free Radical Biology & Medicine 42 (2007) 315 – 325 www.elsevier.com/locate/freeradbiomed

Original Contribution

Modulation of pregnane X receptor-and electrophile responsive element-mediated gene expression by dietary polyphenolic compounds Dirk Kluth a , Antje Banning a , Ingvild Paur b , Rune Blomhoff b , Regina Brigelius-Flohé a,⁎ a

German Institute of Human Nutrition, Potsdam-Rehbrücke, Arthur-Scheunert-Allee 114–116, D-14558 Nuthetal, Germany b Institute for Nutrition Research, Faculty of Medicine, University of Oslo, Norway Received 20 July 2006; revised 28 September 2006; accepted 29 September 2006 Available online 11 October 2006

Abstract Based on animal models, dietary polyphenols are predicted to be promising chemopreventive agents in humans. Allspice, clove, and thyme extracts as well as defined dietary polyphenolic compounds were, therefore, tested for their ability to activate mechanisms related to phase 1 enzymes, i.e., the PXR-regulated CYP3A4 promoter, and phase 2 enzymes, i.e. the EpRE-regulated promoters of gastrointestinal glutathione peroxidase (GI-GPx) and heme oxygenase-1 (HO-1), examples of Nrf 2-regulated genes. From the compounds tested, clove and thyme extracts as well as curcumin and resveratrol activated the PXR. PXR activation correlated with the activation of the CYP3A4 promoter in the case of thyme extract, curcumin, and resveratrol, but not in the case of clove extract. Allspice extract, EGCG, and quercetin did not activate PXR but enhanced CYP3A4 promoter activity. Thyme extract and quercetin activated the EpRE of HO-1. Both significantly activated the GI-GPx promoter, effects that depended on a functional EpRE. Resveratrol did not activate the isolated EpRE but enhanced the GI-GPx promoter activity, whereas clove extract even inhibited it. It is concluded that individual polyphenols as well as polyphenol-rich plant extracts may affect phase 1 and 2 enzyme expression by distinct mechanisms that must be elucidated, before potential health effects can reliably be predicted. © 2006 Elsevier Inc. All rights reserved. Keywords: Polyphenols; CYP3A4; PXR; Gastrointestinal glutathione peroxidase; Gene regulation; Nrf2

Introduction Dietary polyphenols comprise a wide group of compounds found ubiquitously in higher plants. In plants, these compounds, which are characterized by having one or several hydroxyl groups on one or several aromatic rings, are generally involved in stress protection caused by ultraviolet radiation and pathogens. Polyphenols can be subdivided into different classes by the number of phenolic rings and of the structural elements that link these rings (Fig. 1): (1) the phenolic acids with the subclasses Abbreviations: CYPs, cytochrome P450-dependent monooxygenases; EpRE, electrophile responsive element; FCS, fetal calf serum; GCL, glutamate cysteine ligase; GI-GPx, gastrointestinal glutathione peroxidase; HO-1, heme oxygenase-1; PXR, pregnane X receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-dimethyltetrazolium bromide; NQO-1, NADPH:quinone oxidoreductase-1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ⁎ Corresponding author. Fax: +49 33200 88 407. E-mail address: [email protected] (R. Brigelius-Flohé). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.09.028

derived from hydroxy benzoic acids such as gallic acid and of hydroxy cinnamic acid, comprising caffeic, ferulic, and coumaric acid; (2) the large subclass of flavonoids with flavonols, flavones, isoflavones, flavanones, anthocyanidins (the aglycones of anthocyanins, the pigments giving flowers and fruits their characteristic colour), and the flavanols (catechins in tea, wine, and cocoa, and the polymeric proanthocyanidins); (3) the stilbenes; and (4) the lignans and the polymeric lignins (for more detailed overviews see [1,2]). A high intake of foods and beverages rich in polyphenols, especially in flavonoids, has been associated with a decreased risk of chronic degenerative diseases like atherosclerosis and cancer. These diseases have been linked to oxidative stress and, thus, the antioxidant potential of flavonoids has widely been implicated to explain their protective efficacy. However, it has been argued that the antioxidant effects of polyphenols would require concentrations that are not being easily reached in plasma or tissues in vivo. Protective effects may, therefore, also

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Fig. 1. Overview on the classification of polyphenols.

be mediated by mechanisms not related to their antioxidant properties [3,4] as shown for the antiatherosclerotic effects of red wine polyphenols [5]. In fact, a large number of alternative biological activities of such food ingredients have been reported (listed in [4]). One of the most striking observations goes back to the 1970s when Wattenberg et al. observed that nonnutrient dietary chemicals, among them polyphenols, can inhibit chemically induced carcinogenesis in laboratory animals and proposed that this was due to the activation of endogenous systems that metabolize and eliminate toxic xenobiotics [6,7]. Detoxifying enzymes include phase 1 and phase 2 enzymes. Most of the phase 1 enzymes are cytochrome P450-dependent monooxygenases (CYPs) which increase the hydrophilicity of xenobiotics by hydroxylation and facilitate their elimination. An important representative of phase 1 enzymes is CYP3A4 which metabolizes about 60% of all prescription drugs [8]. CYP3A4 is regulated by the pregnane X receptor (PXR) [9], a transcription factor that is activated by a large number of structurally diverse xenobiotics [10]. Structural requirements for PXR activators are a lipophilic region that fits into the large ligand-binding cavity of PXR and a discrete polarity [11]. Whereas CYP3A4 is considered to rather act cytoprotectively, other CYPs can also activate xenobiotics to more toxic or mutagenic products. Such products are then detoxified by phase 2 enzymes including glutathione S-transferases, UDP-glucuronosyl transferases, and sulfotransferases. Many phase 2 enzymes as well as other enzymes responsible for the detoxification and elimination of potentially harmful agents like NADPH:quinone oxidoreductase-1 (NQO-1), glutamate cysteine ligase (GCL), or heme oxygenase-1 (HO-1) (reviewed in [12]) are regulated by the Nrf 2/Keap1 transcription factor system. Nrf 2 binds to the “antioxidant response element” (ARE), better called the “electrophile responsive element” (EpRE), which is present in the promoter regions of many genes for phase 2 enzymes [13]. In the cytoplasm, Nrf 2 is associated to Keap1 which prevents its nuclear localization by (i) mere sequestration of Nrf 2 in the cytoplasm, (ii) targeting

Nrf 2 for proteasomal degradation [14,15], or (iii) facilitation of the transport of Nrf 2 from and into the nucleus [16]. Whatever the mechanism is, Nrf 2 must dissociate from Keap1 to become activated. This is achieved by a change in the conformation of Keap1 either by oxidation/modification of essential cysteines [17] or by removing Zn from coordinating cysteines [18]. Thus, functional requirements for Nrf 2 activators are electrophilicity, ability to oxidize or alkylate thiols, or to chelate metal ions. Although many polyphenols fulfil the structural requirements for Nrf 2 activators and even PXR ligands, related studies are still scarce. Flavonoids, including quercetin and resveratrol, as well as curcumin were examined for their ability to induce CYP3A4 in human hepatocytes. Only quercetin slightly enhanced CYP3A4 mRNA expression [19]. In a cell-based reporter assay the ability of a variety of dietary compounds to activate the PXR was tested and the Quantitative Structure Activity Relationship (QSAR) was calculated. From the small number of polyphenols tested, quercetin and resveratrol exerted a rather low activity, whereas lignans and their metabolites as well as phytoestrogens proved to be moderate to potent PXR activators [20]. An activation of the Nrf 2/Keap1 system by quercetin can be inferred from the activation of the GCL promoter [21,22] and the induction of NQO-1 [23]. By analogy, this can also be postulated for phenolic antioxidants, curcumin, and green tea polyphenols (reviewed in [24]) which inhibit chemically induced cancers. Up-regulation of endogenous defence systems may contribute to the protective effects of polyphenols. We, therefore, studied the ability of extracts from allspice, clove, and thyme as well as defined dietary compounds (for structures see Fig. 2) with potential health-promoting effects to activate the PXRregulated promoter of CYP3A4, as an example for a phase 1 enzyme, and the gene expression regulated by the EpRE of HO-1, an established Nrf 2-regulated enzyme, and the EpREdominated promoter of the gastrointestinal glutathione peroxidase (GI-GPx, GPx2) which only recently has been identified as a target of Nrf 2 [25]. The extracts and compounds included in the present study were selected according to two larger previous experimental screenings: one based on analyses of the redox activity of more than 3000 food samples [26–28], the other based on the ability of 60 plant extracts and pure polyphenols to inhibit NF-κB activation (I. Paur and R. Blomhoff, unpublished data), a crucial mechanism mediating and controlling inflammatory reactions. Methods Cell culture HepG2 cells (human liver carcinoma cells, ATCC HB8065) were cultured in RPMI 1640 with 2 mM L-alanyl-L-glutamine. CaCo-2 cells (human colon adenocarcinoma cells, DSMZ ACC 169) were grown in DMEM high glucose with 1% nonessential amino acids. All media were further supplemented with 10% heat-inactivated fetal calf serum (FCS; Biochrom, Berlin, Germany), 5 mM Hepes, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Gibco, Karlsruhe, Germany).

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concentrated under nitrogen gas to viscid fluid. The concentrated extract was diluted to 5 ml in DMSO, sterile-filtered, and then stored under argon gas in airtight tubes at − 70°C. Compounds (Sigma) were dissolved in DMSO. Concentrations of extracts are given as milligrams per milliliter, which refers to the amount of pulverized dry sample subjected to extraction (Table 1). Transfection and reporter gene assays Cells (2.5 × 105) were seeded onto 24-well plates and 24 h later transfected with 0.5 μg pSV-β-galactosidase, up to 0.4 μg luciferase reporter plasmid, and 0.15 μg of expression plasmids using Tfx-20 (Promega, Mannheim, Germany) according to the manufacturer's protocol. Treatment with compounds and extracts in serum-free medium was started 24 h after transfection for a further 24 h. Cell lysis and determination of luciferase and βgalactosidase activity were performed as described [29]. Relative luciferase activity was calculated by dividing luciferase by βgalactosidase activity. Reporter gene activity of the empty luciferase plasmids (pGL basic, pGL3-promoter) served as control. Plasmid construction pGL3-(DR3)2 was generated by cloning the HindIII x EcoRI fragment of pBLCAT2-(DR3)2 containing two times the PXR responsive element DR3 into the EcoRI and HindIII sites of pBluescript II KS(+) (Stratagene, La Jolla, CA). From the resulting construct, (DR3)2 was ligated via SmaI and KpnI into pGL3-promoter (Promega, Mannheim, Germany). For others see references in Table 2. Fig. 2. Chemical structure of the polyphenolic compounds and main constituents of the extract investigated here.

Viability of cells in response to treatment was confirmed by quantification of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5dimethyltetrazolium bromide, Sigma, Taufkirchen, Germany) reduction by mitochondrial dehydrogenases. Briefly, 3.7 × 104 cells were incubated with the extracts and substances in the concentrations and times to be tested in 96-well plates. Then, cells were treated with 0.5 mg/ml MTT in PBS for 40 min at 37°C. Thereafter, the medium was removed and cells lysed with 100 μl lysis buffer (95% isopropanol, 5% formic acid). Extinction was measured at 550 versus 690 nm in a microplate reader (Dynatech, Denkendorf, Germany). A decrease in the absorption compared to controls by 20% and less was considered not toxic. Preparation of extracts Extracts were prepared as described in [26]. Allspice, clove, and thyme were obtained from local grocery stores in Oslo. They were extracted directly or frozen at – 20°C until extraction. Dry samples were pulverized and 10 g of the sample was added to 20 ml of water and 20 ml of methanol. All samples were treated in an ultrasonic water bath for 30 min at 0°C. The mixture was centrifuged at 3000 g for 15 min and the liquid phase was

Nuclear extracts Cells were incubated for 4 h in serum-free medium with quercetin (25 μM) or thyme extract (3 mg/ml), respectively. Thereafter, cells were lysed at a density of 1 × 107 cells in 1.2 ml of lysis buffer (10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, pH 7.9) containing 0.1% NonidetP40 (Sigma, Taufkirchen, Germany) for 7 min at 4°C. Nuclei were pelleted by centrifugation (6800 g, 1 min, 4°C) and lysed for 30 min on ice in 100 μl of nuclear lysis buffer (40 mM Hepes, 400 mM KCl, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, pH 7.9) to which 6.25 μl of 5 M NaCl was added prior to use. Nuclear Table 1 Description of the extracts and compounds used in this study Extract/compound

Solvent (DMSO) (%)

Concentration of stock solutions a

Concentration used in this study b

Allspice Clove Thyme Curcumin (–)-Epigallcatechin3-gallate (EGCG) Quercetin Resveratrol

20 50 20 100 100

2 g/ml 2 g/ml 2 g/ml 0.1 M 0.1 M

3 mg/ml 1 mg/ml 3 mg/ml 25 μM 25 μM

100 100

0.1 M 0.1 M

25 μM 25 μM

a b

The amount of pulverized dry sample subjected to extraction. None of the concentrations were toxic for the cells.

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Table 2 Reporter gene and expression plasmids Plasmid

Description

Reference

Reporter genes pGL3-basic pGL3-CYP3A4-3 pGL3-CYP3A4-6 pGL3-CYP3A4-9 pGL3-GI-prom-I pGL3-GI-prom-VI pGL3-GI-prom-I-mut pGL3-promoter pGL3-HO-EpRE pGL3-GI-EpRE pGL3-(DR3)2 pGLCAT2-(DR3)2 pSV-β-Gal pSG5 pSG5-hPXR

Empty luciferase reporter vector 3557 bp of the CYP3A4 promoter in pGL3-basic 6954 bp of the CYP3A4 promoter in pGL3-basic 9388 bp of the CYP3A4 promoter in pGL3-basic 2111 bp of the GI-GPx promoter in pGL3-basic 172 bp of the GI-GPx promoter in pGL3-basic GI-prom-I fragment with a point mutation within GI-EpRE a Empty luciferase reporter gene plasmid with minimal SV40 promoter pGL3-promoter containing one copy of the heme oxygenase-1 EpRE pGL3-promoter containing one copy of the GI-GPx EpRE pGL3-promoter containing two DR3 elements of the rat CYP3A1 promoter pBLCAT2 containing two DR3 elements of the rat CYP3A1 promoter β-galactosidase expression plasmid for expression control and normalization Empty expression vector Expression plasmid for the human PXR

Promega [32] [32] [32] [25] [25] [25] Promega [25] [25] this work [9] Promega Stratagene [9]

a

The EpRE noted here is the functionally active EpRE which is called ARE-2 in [25].

extracts were centrifuged (20,800 g, 30 min, 4°C) and stored at – 80°C. An aliquot was taken for protein determination. For Nrf 2 detection, nuclear extracts (50 μg protein) were subjected to SDS-PAGE and Western blots [30]. Nrf 2 was detected by Nrf 2 (C20): sc-722 (Santa Cruz Biotechnology, Santa Cruz, CA). Peroxidase-conjugated goat anti-(rabbit IgG) (Chemicon, Hofheim, Germany) was used as secondary antibody. Statistics Data are expressed as means ± SD and the Student unpaired t test with unequal variance was used to determine significant differences between treatment and control values. Differences at the p < 0.05 level were considered statistically significant. Results

Allspice, thyme, and quercetin were even more effective than the positive control, rifampicin, whereas EGCG, curcumin, and resveratrol were as potent as rifampicin. Despite its ability to activate a pure PXR response element, clove extract did not activate the CYP3A4 promoter (Fig. 5). Truncated fragments (pGL3-CYP3A4-3 or-6) were not or only marginally activated by any of the compounds used (not shown). The exclusive activation of the CYP3A4 promoter fragment-9 corroborates the requirement for the XREM for the PXR-dependent CYP3A4 expression. The finding further reveals that the compounds did not activate any other putative responsive element within the first 7000 bp of the CYP3A4 promoter. Activation of EpRE-mediated gene expression The HO-1 gene is a prototype of Nrf 2 regulated genes, and the GI-GPx gene has recently been described as a novel target

Activation of PXR-mediated gene expression The activation of the PXR-mediated gene expression was first tested in cells transfected with a luciferase reporter gene controlled by two DR3 elements cloned in tandem and an expression plasmid for human PXR. From the extracts and compounds tested, clove and thyme extract, curcumin, and resveratrol all activated the PXR, while allspice, EGCG, and quercetin did not. Rifampicin, which was used as positive control, increased PXR activity about 5 times compared to curcumin that increased PXR activity 3 times (Fig. 3). There was no activation without cotransfected PXR, indicating that the induction of the reporter gene was PXR dependent. Maximal PXR-mediated induction of the CYP3A4 promoter requires a proximal ER6 motif (everted repeat separated by 6 bp) [31] as well as the distal xenobiotic responsive enhancer module (XREM) consisting of a DR3 and ER6 motif (Fig. 4). For activation assays, the fragment containing 9388 bp of the CYP3A4 promoter (pGL-CYP3A4-9 [32]) covering all required elements was used. Strong activation of the CYP3A4 promoter was obtained with most of the extracts and compounds tested.

Fig. 3. Influence of polyphenols on PXR-mediated gene expression. HepG2 cells were transiently transfected with 0.15 μg of the reporter genes pGL3-(DR3)2 and 0.15 μg of the PXR expression plasmid pSG5-hPXR (Table 2). Twenty-four hours after the transfection rifampicin (Rif, 10 μM) as positive control, allspice (A, 3 mg/ml), clove (C, 1 mg/ml), or thyme (T, 3 mg/ml) extract as well as curcumin (Cu, 25 μM), EGCG (E, 25 μM), quercetin (Q, 25 μM), or resveratrol (R, 25 μM) were added to the cell culture media and the cells incubated for a further 24 h in serum-free medium. After cell lysis, β-galactosidase standardized relative luciferase activity was calculated by setting the solvent-treated (DMSO, concentration < 0.1%) control to 1. Values are means of 3 experiments measured in triplicate ± SD. *p < 0.05. For further details see Methods.

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Fig. 4. Responsive elements in the CYP3A4 promoter. ER6: everted repeat separated by 6 nucleotides. DR3: direct repeated separated by 3 nucleotides. dNR1 and dNR2: distal nuclear receptor binding sites 1 and 2 building the xenobiotic responsive enhancer module (XREM). The proximal ER6 at- 170/-140 bp represents a consensus PXR responsive element. All three elements are required for optimal activation of the CYP3A4 promoter [80].

for Nrf 2 [25]. However, only a few Nrf 2 activators have been tested for their GI-GPx promoter stimulating activity so far, among them curcumin and sulforaphane. Therefore, the luciferase reporter gene driven by the HO-1 EpRE or the GI-GPx EpRE, respectively, was used to test a putative EpRE-activating capacity. From the extracts and compounds tested, only thyme and quercetin significantly activated the HO-1 EpRE, with thyme being about 10 times more efficient (Fig. 6A). The GIGPx EpRE was activated only by thyme, while the effect of quercetin did not reach significance (Fig. 6B). The 2111 bp GI-GPx promoter fragment (GI-prom-I) containing the functional EpRE [25] was significantly activated by quercetin, resveratrol, and thyme extract and, surprisingly, depressed by EGCG and to a much higher degree by clove (Fig. 7A). Quercetin, thyme, and clove effects were also observed with a truncated GI-GPx promoter fragment of 172 bp (GI-prom-VI) that still contains the functional EpRE (Fig. 7B). With the promoter fragment I containing a mutation in the functional EpRE, basal and thyme-and quercetin-inducible activity was reduced to hardly detectable values (Fig. 7C), indicating that the activation of the GI-GPx promoter was indeed achieved by the activation of Nrf 2 through the EpRE. Thus,

Fig. 5. Influence of polyphenols on the CYP3A4 promoter activity. HepG2 cells were transiently transfected with 0.4 μg of the reporter gene construct pGL3CYP3A4-9. Twenty-four hours later cells were treated with compounds and concentrations described in the legend to Fig. 3. Fold induction was estimated by calculating relative luciferase activity as described. Values are means of at least 3 experiments measured in triplicate ± SD. *p < 0.05. For further details see Methods.

quercetin and thyme extract activated both the isolated EpRE and the GI-GPx promoter, whereas resveratrol activated the GI-GPx promoter only. Activation of Nrf 2 can be monitored by its translocation into the nucleus. Therefore, Nrf 2 localisation was analysed after treatment of cells with quercetin and thyme extract, the most potent activators of EpRE and the GI-GPx promoter (Fig. 8). The band of 100 kDa, representing Nrf 2 associated to a protein which might be either actin [33] or Keap1 itself [16] was absent in nuclear extracts from control cells and distinctly increased in

Fig. 6. Influence of polyphenols on electrophile responsive elements. HepG2 cells were transiently transfected with 0.15 μg of the reporter gene constructs containing the HO-1 (A) or GI-GPx (B) EpRE. Twenty-four hours later, cells were treated and reporter gene expression was calculated as described in the legend to Fig. 3 with the exception that 3 mg/ml clove extract was used. Values are means ± SD of 3 experiments measured in triplicate. *p < 0.05.

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regulation of antiapoptotic systems. This was the rationale for the use of “antioxidants” to prevent or delay tumor growth. Sources of hydroperoxides i.a. are COXs, LOXs, and the apoptosisinducing factor (AIF) which contains an NADH oxidase activity producing reactive oxygen species [35]. On the other hand, cancer cells are more susceptible to additional oxidative stress than normal cells (reviewed in [36]). Thus, hydroperoxides adopt a Janus faced role in cancer cells. They function as proliferation signals or as mediators of programmed cell death, or are simply cytotoxic. The balance between production and removal of hydroperoxides may determine whether cancer cells proliferate, undergo cell cycle arrest, or die by apoptosis or necrosis. A switch to a more reduced state was believed to provide the basis for the chemopreventive effects of antioxidant polyphenols. The often observed inhibition by polyphenols of NFκB activation (reviewed in [37]), a transcription factor responding to changes in the redox state, and the influence on MAPK signaling pathways (reviewed in [3]) appeared to support this idea. In the present study, the biological effects of the (poly) phenols were investigated at concentrations that can realistically be achieved by eating “healthy food” or taking supplements. We present compelling evidence that the compounds or preparations, respectively, at near-physiological dosages differentially affect the expression of two distinct cancer-related genes. Our data, thus, strongly support the notion that dietary polyphenols rather exert their biological efficacy at the level of transcriptional regulation than by any direct interference with peroxide or radical metabolism. Allspice extract When screening more than 3000 foods we observed that herbs and spices show the highest content of redox-active

Fig. 7. Influence of polyphenols on the GI-GPx promoter activity. HepG2 cells were transiently transfected with 0.15 μg of the reporter gene construct pGL3GI-prom-I (A), pGL3-GI-prom-VI (B), or pGL3-prom-I-mut (C). After 24 h cells were treated and relative luciferase activity was estimated as described in legend to Fig. 3 with the exception that 3 mg/ml clove extract was used. Values are means ± SD of at least 3 experiments measured in triplicate. *p < 0.01.

nuclear extracts from quercetin-and thyme-treated cells. The about 66-kDa band representing free Nrf 2 was not changed, indicating a constitutive nuclear localization of Nrf 2, at least in the system used for this study. These data further confirm GI-GPx as Nrf 2 target and present novels activators of GI-GPx expression, quercetin and thyme extract. Discussion Tumor cells constitutively produce high amounts of H2O2 [34]. They need the oxidized state for proliferation and up-

Fig. 8. Nuclear translocation of Nrf 2 upon treatment with quercetin or thyme extract. HepG2 cells were incubated in serum-free medium with DMSO (ctrl), 25 μM quercetin (Q), or 3 mg/ml thyme extract (T). Thereafter, cells were harvested and nuclear extracts analyzed by Western blotting. The figure is representative of 3 independent experiments.

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compounds, with clove and allspice ranking at or close to the top [27,28,38]. Allspice or pimento is the dried berry of Pimenta dioica which belongs to the family of Myrtaceae. Berries from allspice contain 2–5% essential oils from which the major constituents are eugenol (60–75%), eugenol methyl ether, cineole (eucalyptol), phellantrene, and caryophyllenes. Like EGCG and quercetin, allspice did not activate the PXR directly but strongly activated the CYP3A4 promoter. Therefore, the activation of alternative transcription factors binding to response elements upstream of 7000 bp seems likely. Up-regulation of CYP3A4 indicates that at least some of the compounds present in allspice might be considered as foreign and worthy of elimination. Up-regulation of CYP3A4 expression by polyphenols had so far not been investigated intensively and did not always yield identical results. Grapefruit juice, for instance, inhibited CYP3A4 expression [39]. (For eugenol-related discussion see Clove extract). Clove extract Clove revealed the highest content of redox-active compounds of all herbs and spices measured [27,28,38]. Clove is the name for the flower buds of the tropical tree Eugenia Caryophyllata (Myrtaceae) [40]. Clove is widely used in traditional medicine and in dentistry in China, Japan, and Korea for its antiseptic, antibacterial, and analgesic properties. Active components are tannins, terpenoids, eugenol, and acetyleugenol. Anti-inflammatory and anticarcinogenic properties of clove have been attributed to eugenol. Eugenol inhibited 5-lipoxygenase activity and in turn LTC4 production in human PMNLs [41]. It also inhibited LPS-induced COX-2 expression, COX-2 activity, and in consequence PGE2 production [42]. Since PGE2 induced COX-2 expression in an autocrine loop [43], lack of PGE2 production might be responsible for the reduced COX-2 induction. The antiproliferative and proapoptotic effects of eugenol in melanoma cells as well as the decrease in tumor size and complete prevention of metastasis in a xenograft study were caused by the suppression of the transcriptional activity of E2F1, a member of the E2F family of transcription factors involved in the regulation of cell cycle progression [44]. The strong inhibition of the GI-GPx promoter by clove extract is surprising and, in view of the proposed anti-inflammatory role of GI-GPx [45], cannot be considered as particularly beneficial. The GI-GPx promoter activity strongly depends on the presence of a functional EpRE [25]; therefore, an interference of clove constituents with Nrf 2 activators appears possible. Alternatively, the clove extract, like EGCG (see below), might interfere with β-catenin activity which, however, remains to be investigated. Since allspice extracts also contain high amounts of eugenol but do not influence GI-GPx promoter activity, either the eugenol in allspice is competed out by other compounds or the active compound in clove is not eugenol. Thus, despite their similarly high antioxidant capacity, the cellular effects of clove and allspice extracts are different. Clove extract did not activate the CYP3A4 promoter but significantly activated the PXR. To our knowledge an activation

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of PXR by eugenol, the effective compound in clove, has not been described so far and deserves further investigation. Thyme extract The principal active ingredients of thyme are thymol and carvacrol, oily compounds exerting antiseptic functions. Other constituents are flavonoids such as apigenin or buteolin, tannins, and other oils. Thymol and carvacrol have been tested for their effects on phase 1 and 2 enzyme expression in mice. Both compounds enhanced the activities of 7-ethoxycoumarinO-deacetylase, a phase 1 enzyme, and of the phase 2 enzymes GSTs from the alpha and μ class and NQO-1 in livers of treated mice [46]. The strong activation of the CYP3A4 promoter via PXR and the GI-GPx promoter via the EpRE by thyme extract provides a mechanism for these observations. Thus, thyme can up-regulate the hosts' defense systems, but obviously also contains compound(s) which up-regulate CYP3A4, indicating the need for elimination. Curcumin Curcumin, present in the rhizome of tumeric (Curcuma longa) has a chemopreventive potential. It inhibited TNF-αmediated COX-2 gene transcription in vitro [47,48] and inhibited COX-2 expression in patients with advanced colorectal cancer [49]. Induction of apoptosis has been observed in tumor cell lines by the release of cytochrom c, activation of caspases [50,51], DNA fragmentation [52], and inhibition of Akt [53]. The generally observed inhibition of NFκB activation by curcumin was recently explained by its ability to inhibit the association of the IL-1 receptor complex, one of the earliest events in the inflammatory signaling cascade initiated by IL-1 [54]. Curcumin induces detoxifying enzymes such as GCL, GST, NQO-1 [55], and also GI-GPx [25]. The here observed activation of PXR and of the CYP3A4 promoter is novel and shows that curcumin can also be applied in dosages high enough to be considered as foreign. EGCG Chemopreventive effects of EGCG, a major polyphenol of green and white tea, may be based on several mechanisms (reviewed in [37]). These include the inhibition of AP-1 [56], the down-regulation of the NFκB-inducing kinase (NIK) [57], the inhibition of HER2/Neu signaling [58], or VEGF production [59]. Low concentrations of EGCG have been postulated to activate prosurvival MAP kinases which in turn leads to phase 2 enzyme up-regulation [60]. Nrf 2 activation was also observed in fibroblasts and lymphoblasts but not in breast cancer cells [61], indicating a cell-type-specific effect of EGCG. Phase 2 enzyme induction was not observed with the concentration used here (25 μM). There was no activation of the EpRE of HO-1 or GI-GPx. Instead, the EpRE-regulated GI-GPx promoter was rather suppressed, confirming the inhibitory effect of higher concentrations of EGCG on EpRE-driven cell growth [60]. The narrow concentration range of EGCG effects was explained by

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its simultaneous effects on the activity of NFκB which was enhanced at lower but inhibited at higher concentrations [61]. Liver and small intestine gene expression profiles obtained from wild-type and Nrf 2-knockout mice treated with EGCG or not unraveled the regulation of more than 600 genes by EGCG via Nrf 2 [62]. Validation of the identified genes will elucidate a potential role of Nrf 2 in the functions of EGCG. More recent data point to an inhibition of the β-catenin/TCF transcriptional activity. A reduction of the expression level of β-catenin by physiological EGCG concentrations [63] is achieved by an activation of the lysosomal trafficking of βcatenin and subsequent degradation [64]. The here observed inhibition of the GI-GPx promoter would be in line with this effect, since GI-GPx has been reported to be down-regulated by transfection of dominant-negative TCF (supplementary material in [65]) and found to be up-regulated by transfection with β-catenin/TCF in our experiments (to be published). The effects of EGCG on CYP activities have been rather negative. EGCG inhibited the activity of human CYP2A6, 2C19, 2E1, and NADPH-cytochrome-CYP reductase expressed in transfected S. typhimurium [66]. In a human tongue carcinoma cell model and in HepG2 cells, green tea extract induced CYP1A1 and 2 as well as CYP2E1, 2D6, and 2C isoforms. EGCG alone failed to induce these enzymes [67]. The here observed activation of the CYP3A4 promoter by EGCG has only sporadically been investigated before. This is novel and worth investigation in more detail. Since CYP3A4 induction did not correlated with PXR activation other transcription factors might be involved (see Quercetin).

CYP3A4 promoter which was significantly activated by quercetin (Fig. 5). Thus, other transcription factors such as the HNF-4α [72] or the constitutive androgen receptor (CAR) [73] may be involved. This might be the case for EGCG and quercetin both of which activated the CYP3A4 promoter but not PXR. Resveratrol Resveratrol belongs to a family of compounds given the name viniferin. It is present mainly in grapes and, thus, believed to be the key ingredient of red wine responsible for the “French paradox”. Its anti-inflammatory and chemopreventive properties have been attributed to its antioxidant activity, the inhibition of PKC-mediated induction of COX-2 [74], and gene-regulatory functions (reviewed in [75]). Recently, an inhibition of NFκB activation was described [76], which depending on the tissue investigated was achieved by an interference with early events in the signaling cascades mediated by Toll-like receptors [76]. Also a stimulation of Sirt1 histone deacetylase activity that leads to deacetylation of the p65 subunit of NFκB and, thereby, to an inhibition of its transcriptional activity has been reported [77]. Although the precise mechanism of resveratrol action is not known yet, the resorcin moiety appears to be essential for the chemopreventive efficacy [78]. Resorcins can chelate metals and, thereby, resveratrol can activate Nrf 2 by removal of zinc from Keap1 which is in line with its GI-GPx promoter activating effect. In addition, both, PXR-and CYP3A4 promoter-mediated gene expression was enhanced by resveratrol. Conclusions

Quercetin The beneficial and deleterious effects of the flavonol quercetin on different cell types are reviewed in [3]. Depending on the concentration, quercetin induces apoptosis in cancer cell lines [68] but can also inhibit it [69]. Low concentrations activate MAPK pathways involved in the induction of protective genes. Treating MCF-7 cells with 15 μM quercetin induced NQO-1 [23]. In COS-1 cells 5–25 μM quercetin increased the mRNA of GCL and induced a reporter gene under the control of GCL promoter fragments containing at least one EpRE [21]. We here show that quercetin activated the GI-GPx promoter and caused a shift of Nrf 2 into the nucleus. GI-GPx is a third example of quercetin-regulated Nrf 2 target genes and may point to a role of this particular flavonoid in the activation of the adaptive response. A direct influence of polyphenols on PXR activity has not yet been tested in detail. Only for quercetin and resveratrol a moderate activation of the PXR has been described before [20,70]. Whereas the resveratrol effect could be confirmed here (see below), quercetin (25 μM) showed rather an inhibitory one. Quercetin concentrations leading to an activation of PXR were 5–10 μM [20,70]. Quercetin effects often depend on the dosage and have been described to be biphasic [71]. Since plasma concentrations of quercetin usually do not exceed 1 μM, an inhibition of PXR activity by quercetin in vivo is not very likely. Lack of PXR activation did not correlate with the effect on the

Beneficial effects of many phytochemicals are achieved by up-regulation of the endogenous defense system via the Nrf 2/ Keap1 system. Since activation of Nrf 2 requires the modification/oxidation of Keap-1-SH groups and polyphenols can act as redox cyclers, oxidation of Keap1 appears a plausible mechanism. An up-regulation of detoxifying/antioxidant enzymes is generally believed to be of advantage for cancer prevention. GI-GPx appears to be crucial for proliferation [79] in healthy intestinal cells; in cancer cells it might prevent proliferation by reducing the high oxidative state cancer cells need for proliferation. Regarding the suggested anti-inflammatory role of GIGPx an induction of this particular enzyme should be advantageous. In this context, the effects of thyme, quercetin, and resveratrol can be considered beneficial, and those of EGCG and clove rather disadvantageous. Induction of CYP3A4 might be beneficial, since an enhanced xenobiotic metabolizing system can protect against intoxications and might help to eliminate carcinogens. On the other hand, many xenobiotics up-regulate CYP3A4 via PXR activation and, thus, their own elimination. Organisms responding to compounds with PXR activation evidently consider these compounds as “foreign” and therefore possibly detrimental. Furthermore, up-regulation of CYP3A4 may interfere with the metabolism of drugs. Patients depending on drug therapy will certainly not profit from a high intake of PXR activators.

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The responses to individual polyphenols, however, also make clear how complex the interaction of dietary phytochemicals with cellular pathways is. Foods contain a mixture of several thousand different compounds, many with bioactivity. The polyphenols, which belong to one of the largest groups of bioactive compounds present in foods, do not exert uniform effects. They interfere with transcriptional regulation in specific ways and the distinct modes the present investigation focused on will not likely remain the only ones. The effects of polyphenols depend on their particular mode of interference with gene expression and the individual demands of subjects or patients. A combined approach is therefore needed to predict the biological effect of polyphenol-rich foods in vivo. The present study elucidates how single polyphenols and polyphenol-rich extracts may modulate response elements and promoters related to the adaptive response, i.e., the endogenous defense system. Yet the importance of balanced expression and activities of the enzymes that constitute the adaptive response demands adequate efforts. Acknowledgments The skillful technical assistance of Stefanie Deubel, Elvira Krohn, and Ramona Bahtz is gratefully acknowledged. A special thank goes to Dr. Ramiro Jover from Valencia, Spain, for the generous gift of the CYP3A4 promoter reporter gene constructs and to Dr. Steven Kliewer, Glaxo Wellcome, Research Triangle Park, North Carolina, USA, for the plasmid pBLCAT2(DR3)2 from which the pGL3-(DR3)2 was constructed. The work was supported by the Deutsche Forschungsgemeinschaft, DFG (to R.B.F.). References [1] Beecher, G. R. Overview of dietary flavonoids: nomenclature, occurrence and intake. J. Nutr. 133:3248S–3254S; 2003. [2] Manach, C.; Scalbert, A.; Morand, C.; Remesy, C.; Jimenez, L. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79: 727–747; 2004. [3] Williams, R. J.; Spencer, J. P.; Rice-Evans, C. Flavonoids: antioxidants or signalling molecules? Free Radic Biol. Med. 36:838–849; 2004. [4] Halliwell, B.; Rafter, J.; Jenner, A. Health promotion by flavonoids, tocopherols, tocotrienols, and other phenols: direct or indirect effects? Antioxidant or not? Am. J. Clin. Nutr. 81:268S–276S; 2005. [5] Waddington, E.; Puddey, I. B.; Croft, K. D. Red wine polyphenolic compounds inhibit atherosclerosis in apolipoprotein E-deficient mice independently of effects on lipid peroxidation. Am. J. Clin. Nutr. 79: 54–61; 2004. [6] Wattenberg, L. W.; Loub, W. D.; Lam, L. K.; Speier, J. L. Dietary constituents altering the responses to chemical carcinogens. Fed. Proc. 35:1327–1331; 1976. [7] Wattenberg, L. W. Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J. Natl. Cancer Inst. 58:395–398; 1977. [8] Guengerich, F. P. Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu. Rev. Pharmacol. Toxicol. 39:1–17; 1999. [9] Lehmann, J. M.; McKee, D. D.; Watson, M. A.; Willson, T. M.; Moore, J. T.; Kliewer, S. A. The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102:1016–1023; 1998. [10] Kliewer, S. A. The nuclear pregnane X receptor regulates xenobiotic detoxification. J. Nutr. 133:2444S–2447S; 2003.

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