Induction Of Antioxidant Response Element Research Articles

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study

BackgroundOxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. MethodsMLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. ResultsThe experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. ConclusionsNrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Relative protective activities of quercetin, quercetin-3-glucoside, and rutin in alcohol-induced liver injury.

Alcoholic liver diseases has been known to be one of the major health risks worldwide. The purpose of this study was aimed to demonstrate the relative protective effect of quercetin, quercetin-3-glucoside, and rutin on alcohol-induced damage in hepatocytes. The hepatotoxicity, antioxidant enzymatic defense mechanisms, and pro-inflammatory mediators were examined for evaluating the hepatoprotective effects of quercetins in hepG2 cells. The results revealed that quercetin and its glucoside derivatives significantly prevented ethanol-induced hepatotoxicity by decreasing hepatic aminotransferase activities and inflammatory response in HepG2 cells. Moreover, the quercetins significantly induced detoxifying enzymes via the nuclear accumulation of the NF-E2-related factor 2 (Nrf2) and induction of antioxidant response element (ARE) gene. These hepatoprotective activities were observed to be more effective with quercetin aglycone than quercetin glucosides. From the above findings, the present study imply that quercetin aglycone may have a vital function in the therapeutic and preventive strategies of alcoholic liver diseases. PRACTICAL APPLICATIONS: Quercetin is commonly present in fruits and vegetables as aglycone and glucoside-derived forms. In the present study, quercetin and its glycosides was shown to alleviate oxidative stress, glutathione depletion, and pro-inflammatory cytokines in alcohol-induced HepG2 cells via the Nrf2/ARE antioxidant pathway. Moreover, quercetin aglycone had better protective effects against alcohol-induced liver damage in vitro, compared to its glycosylated form. The present study proposed that quercetin aglycone may be a more efficient hepatoprotective agent than its glucoside derivatives such as rutin in the amelioration of alcohol-induced liver diseases.

Intermittent hypoxia is a proinflammatory stimulus resulting in IL-6 expression and M1 macrophage polarization.

The biological factors that promote inflammation or nonalcoholic steatohepatitis (NASH) in the setting of nonalcoholic fatty liver disease remain incompletely understood. Clinical studies have demonstrated an association between obstructive sleep apnea (OSA) and both inflammation and fibrosis in NASH, but the mechanism has not been identified. In this study, we use in vitro modeling to examine the impact of intermittent hypoxia on the liver. Hepatocyte, stellate cell, and macrophage cell lines were exposed to intermittent or sustained hypoxia. Candidate genes associated with inflammation, fibrosis, and lipogenesis were analyzed. Circulating cytokines were assessed in human serum of patients with nonalcoholic fatty liver disease. Intermittent hypoxia results in significant induction of interleukin (IL)‐6 expression in both hepatocytes and macrophages. The increase in IL‐6 expression was independent of hypoxia inducible factor 1 induction but appeared to be in part related to antioxidant response element and nuclear factor kappa B activation. Mature microRNA 365 (miR‐365) has been demonstrated to regulate IL‐6 expression, and we found that miR‐365 expression was decreased in the setting of intermittent hypoxia. Furthermore, macrophage cell lines showed polarization to an M1 but not M2 phenotype. Finally, we found a trend toward higher circulating levels of IL‐6 in patients with OSA and NASH. Conclusion: Intermittent hypoxia acts as a potent proinflammatory stimulus, resulting in IL‐6 induction and M1 macrophage polarization. Increased IL‐6 expression may be due to both induction of antioxidant response element and nuclear factor kappa B as well as inhibition of miR‐365 expression. Higher levels of IL‐6 were observed in human samples of patients with OSA and NASH. These findings provide biological insight into mechanisms by which obstructive sleep apnea potentiates inflammation and fibrosis in patients with fatty liver disease. (Hepatology Communications 2017;1:326–337)

PFOS induces adipogenesis and glucose uptake in association with activation of Nrf2 signaling pathway

PFOS is a chemical of nearly ubiquitous exposure in humans. Recent studies have associated PFOS exposure to adipose tissue-related effects. The present study was to determine whether PFOS alters the process of adipogenesis and regulates insulin-stimulated glucose uptake in mouse and human preadipocytes. In murine-derived 3T3-L1 preadipocytes, PFOS enhanced hormone-induced differentiation to adipocytes and adipogenic gene expression, increased insulin-stimulated glucose uptake at concentrations ranging from 10 to 100μM, and enhanced Glucose transporter type 4 and Insulin receptor substrate-1 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NAD(P)H dehydrogenase, quinone 1 and Glutamate-cysteine ligase, catalytic subunit were significantly induced in 3T3-L1 cells treated with PFOS, along with a robust induction of Antioxidant Response Element (ARE) reporter in mouse embryonic fibroblasts isolated from ARE-hPAP transgenic mice by PFOS treatment. Chromatin immunoprecipitation assays further illustrated that PFOS increased Nrf2 binding to ARE sites in mouse Nqo1 promoter, suggesting that PFOS activated Nrf2 signaling in murine-derived preadipocytes. Additionally, PFOS administration in mice (100μg/kg/day) induced adipogenic gene expression and activated Nrf2 signaling in epididymal white adipose tissue. Moreover, the treatment on human visceral preadipocytes illustrated that PFOS (5 and 50μM) promoted adipogenesis and increased cellular lipid accumulation. It was observed that PFOS increased Nrf2 binding to ARE sites in association with Nrf2 signaling activation, induction of Peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α expression, and increased adipogenesis. This study points to a potential role of PFOS in dysregulation of adipose tissue expandability, and warrants further investigations on the adverse effects of persistent pollutants on human health.

A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

Substoichiometric Hydroxynonenylation of a Single Protein Recapitulates Whole‐cell‐stimulated Antioxidant Response

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE) (by Cravatt et al., Nature Methods, 2014, 11, 79-85)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains an untested hypothesis. Herein, using T-REX (targetable reactive electrophiles and oxidants)—an approach aimed at selectively flipping a single redox switch in cells at a precise time —we show that a modest level (~34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically-important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology. Research involving the development and applications of the new T-REX idea is supported by the NIH Director's New Innovator Award (1DP2GM114850), NSF CAREER Award (CHE-1351400), and the Beckman Young Investigator Award (to Y.A.).

Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

Induction of Heme Oxygenase I (HMOX1) by HPP-4382: A Novel Modulator of Bach1 Activity

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

Cancer chemoprevention activity of labdane diterpenes from rhizomes of Hedychium coronarium

Hedychium coronarium J. Koenig, Zingiberaceae, is a medicinal plant popularly used to treat inflammatory conditions in different countries. Three labdane diterpenes [isocoronarin D (1), methoxycoronarin D (2), ethoxycoronarin D (3)] and benzoyl eugenol (4) were isolated from rhizomes and their chemopreventive potential was evaluated using in vitro assays, namely the inhibition of NF-κB, COX-1 and -2, the induction of antioxidant response element (ARE), and the inhibition of cell proliferation. Diterpene 1 activated ARE (EC50 57.6 ± 2.4μM), while 2, 3 and 4 significantly inhibited NF-κB (IC50 of 7.3 ± 0.3, 3.2 ± 0.3 and 32.5 ± 4.9μM, respectively). In addition, 2 and 3 selectively inhibited COX-1 (IC50 values of 0.9 ± 0.0 and 3.8 ± 0.0μM, respectively). These data support the potential chemopreventive activity of constituents from H. coronarium rhizomes.

Sulforaphane activates a protective Nrf2 response and reduces inflammatory markers in microglia cells

Cells exposed to oxidative stress require intrinsic mechanisms to abrogate harmful effects of un‐neutralized reactive oxygen species. Nrf2 is a primary transcription factor involved in detoxification. Induction of antioxidant response element (ARE) genes heme oxygenase (HO1), glutathione‐S‐transferase (GSTA), and NAD(P)H quinine oxidoreductase (NQO1) is directly mediated through Nrf2 activation. While Nrf2 has been broadly studied, data characterizing its role in the central nervous system, particularly in microglia, is limited. In this study, BV2 cells were treated with sulforaphane (SFN) to test its effect on activation of Nrf2. SFN treatment increased mRNA expression of HO1, GSTA, and NQO1. In order to determine if the protective effects of Nrf2 activation could reverse inflammatory characteristics of toxin‐activated microglia, BV2 cells were treated with LPS. Cells responded to LPS with significantly increased expression of inflammatory genes. Pre‐treatment with SFN upregulated ARE genes while down‐regulating the increase in inflammatory cytokines IL‐6, IL‐1β, TNFα, and iNOS caused by LPS. Additionally, SFN negated increased nitric oxide production induced by LPS. Changes in the Nrf2 signaling pathway in microglia may be implicated in cognitive decline associated with an aging inflammatory state. This work paves the way for future studies in vivo. 538 NIH 2 R01AG016710C

Differential regulation of detoxification enzymes in hepatic and mammary tissue by hops (Humulus lupulus) in vitro and in vivo.

Hops contain the phytoestrogen, 8-prenylnaringenin, and the cytoprotective compound, xanthohumol (XH). XH induces the detoxification enzyme, NAD(P)H-quinone oxidoreductase (NQO1) in vitro; however, the tissue distribution of XH and 8-prenylnaringenin and their tissue-specific activity have not been analyzed. An orally administered hop extract and subcutaneously injected XH were administered to Sprague-Dawley rats over 4 days. LC-MS-MS analysis of plasma, liver, and mammary gland revealed that XH accumulated in liver and mammary glands. Compared with the low level in the original extract, 8-prenylnaringenin was enriched in the tissues. Hops and XH-induced NQO1 in the liver, while only hops reduced NQO1 activity in the mammary gland. Mechanistic studies revealed that hops modulated NQO1 through three mechanisms. In liver cells, (i) XH modified Kelch-like ECH-associated protein leading to nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation and antioxidant response element (ARE) activation; (ii) hop-mediated ARE induction was partially mediated through phosphorylation of Nrf2 by PKC; (iii) in breast cells, 8-prenylnaringenin reduced NQO1 likely through binding to estrogen receptorα, recruiting Nrf2, and downregulating ARE-regulated genes. XH and 8-prenylnaringenin in dietary hops are bioavailable to the target tissues. While hops and XH might be cytoprotective in the liver, 8-prenylnaringenin seems responsible for hop-mediated NQO1 reduction in the mammary gland.

Administration of the Nrf2–ARE activators sulforaphane and carnosic acid attenuates 4-hydroxy-2-nonenal-induced mitochondrial dysfunction ex vivo

The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant-response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective defense pathway, which has been shown to produce neuroprotection in numerous models by decreasing lipid peroxidation (LP) as measured by the neurotoxic LP by-product 4-hydroxynonenal (4-HNE). As neuronal mitochondria have previously been shown to be susceptible to insult-induced LP-mediated oxidative damage, we sought to mechanistically investigate whether Nrf2–ARE activation in vivo could protect mitochondria from subsequent 4-HNE exposure ex vivo. Young adult male CF-1 mice were administered one of two known Nrf2–ARE activators as single intraperitoneal doses—sulforaphane (SFP; 5.0mg/kg) or carnosic acid (CA; 1.0mg/kg)—or their respective vehicles 48h before Ficoll isolation of rat cerebral cortical mitochondria. Purified mitochondria were then exposed ex vivo to 4-HNE for 15min at 37°C, which we showed to cause a concentration-related inhibition of mitochondrial respiration together with covalent binding of 4-HNE to mitochondrial proteins. We chose a 30μM concentration of 4-HNE, which produced an approximately 50% inhibition of complex I- or complex II-driven respiration, to assess whether prior in vivo Nrf2–ARE-activating compounds would increase the resistance of the isolated cortical mitochondria to 4-HNE's mitotoxic effects. Administration of either compound significantly increased (p<0.05) expression of heme oxygenase-1 mRNA in cortical tissue 48h postadministration, verifying that both compounds were capable of inducing the Nrf2–ARE pathway. Moreover, the prior in vivo administration of SFP and CA significantly (p<0.05) attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I, but only carnosic acid acted to protect complex II. Furthermore, both CA and SFP significantly (p<0.05) reduced the amount of 4-HNE bound to mitochondrial proteins as determined by Western blot. These results demonstrate the capability of in vivo Nrf2–ARE induction to protect from 4-HNE toxicity to cortical mitochondria ex vivo. Ongoing studies will determine the therapeutic efficacy of Nrf2–ARE activators to attenuate traumatic brain injury-induced pathophysiology.

Role of Nrf2 in protection against triptolide-induced toxicity in rat kidney cells

Triptolide is a major active ingredient of the Chinese herb Tripterygium wilfordii Hook f. (TWHF) and has been shown to possess multiple biological activities, such as anti-inflammatory, anti-fertility, anti-neoplastic and immunosuppressive activities. However, severe adverse effects, especially nephrotoxicity, limit its clinical use. Oxidative stress has been reported to be involved in triptolide-induced renal injury, but the existence of other mechanisms remains unclear. This study aimed to investigate whether NF-E2-related factor 2 (Nrf2), which is an antioxidant nuclear transcription factor, plays a protective role in defense against triptolide-induced toxicity in a normal rat kidney cell line (NRK-52E). Triptolide induced oxidative stress in NRK-52E cells by induction of reactive oxygen species (ROS) and depletion of glutathione (GSH), which resulted in a rapid increase in Nrf2 nuclear accumulation, as well as an induction of antioxidant response element (ARE)-driven genes. In addition, overexpression of Nrf2 protected against triptolide-induced cell death, whereas knockdown of Nrf2 by its specific small interfering RNA resulted in increased cytotoxicity. We also found that Nrf2 knockdown enhanced both the production of ROS and the depletion of GSH. Taken together, these results indicate that activation of Nrf2 plays a protective role against triptolide-induced cytotoxicity in NRK-52E cells through the counteraction of oxidative stress.

Activation of antioxidant response element (ARE)-dependent genes by roasted coffee extracts

Coffee beans contain numerous bioactive components that exhibit antioxidant capacity when assessed using both chemical, cell free, and biological, cell-based model systems. However, the mechanisms underlying the antioxidant effects of coffee in biological systems are not totally understood and in some cases vary considerably from results obtained with simpler in vitro chemical assays. In the present study, the physicochemical characteristics and antioxidant activity of roasted and non-roasted coffee extracts were investigated in both cell free (ORAC(FL)) and cell-based systems. A profile of antioxidant gene expression in cultured human colon adenocarcinoma Caco-2 cells treated with both roasted and non-roasted coffee extracts, respectively, was investigated using Real-Time polymerase chain reaction (PCR) array technology. Results demonstrated that the mechanisms of the antioxidant activity associated with coffee constituents assessed by the ORAC(FL) assay were different to those observed using an intracellular oxidation assay with Caco-2 cells. Moreover, roasted coffee (both light and dark roasted) extracts produced both increased- and decreased-expressions of numerous genes that are involved in the management of oxidative stress via the antioxidant defence system. The selective and specific positive induction of antioxidant response element (ARE)-dependent genes, including gastrointestinal glutathione peroxidase (GPX2), sulfiredoxin (SRXN1), thioredoxin reductase 1 (TXNRD1), peroxiredoxin 1 (PRDX1), peroxiredoxin 4 (PDRX4) and peroxiredoxin 6 (PDRX6) were identified with the activation of the endogenous antioxidant defence system in Caco-2 cells.

Evaluation of Brazilian plants on cancer chemoprevention targets in vitro

Cancer is a leading cause of death worldwide. Cancer chemoprevention is one of the promising strategies to decrease its incidence and both plant extracts and natural products may constitute sources of new chemoprevention agents. Some Brazilian species popularly used to treat inflammatory conditions were selected for evaluation for cancer chemoprevention. A total of 32 extracts/fractions from Hancornia speciosa, Davilla elliptica, Jacaranda caroba, Mansoa hirsuta, Remija ferrugina, Solanum paniculatum and Xyris pterygoblephara, along with a mixture of ursolic and oleanolic acids obtained from J. caroba and a dihydroisocoumarin isolated from aerial parts of X. pterygoblephara were tested for their cancer chemoprevention activity [inhibition of 12-O-tetradecanoyl-13-acetate (TPA)-mediated NF-kappaB activation, ornithine decarboxylase (ODC) and cyclooxygenase-1 (COX-1); induction of antioxidant response element (ARE)]. Several extracts/fractions were active in more than one assay and those from H. speciosa, M. hirsuta and J. caroba mediated strong responses with NF-kappaB, COX-1 and ARE, respectively.

Abstract A43: Hops (humulus lupulus) induce detoxification enzymes in vivo

Abstract A43 Extracts of hop strobiles, the female parts of Humulus lupulus L., are popular botanical dietary supplements as alternative treatment for menopausal symptoms (1). Cancer preventive properties of hops, which are in part mediated through the induction of detoxification enzymes, have frequently been reported (2). This effect has been mainly attributed to one of its major constituents, xanthohumol (XN) (3). However, the chemopreventive effects have so far only been demonstrated in vitro and have not been confirmed in vivo. Therefore, we conducted an in vivo study in adult female rats and analyzed the effect of oral administration of a standardized hop extract (7.5 g extract/kg BW per day, containing 2% XN) or s.c. injection of XN (20 mg/day) on detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST). Analysis of various tissues revealed that while pure XN and oral hops significantly induced NQO1 activity in liver and colon tissue, only oral hops and not pure xanthohumol induced NQO1 activity in the uterus. Interestingly, hops but not xanthohumol significantly induced GST activity in the liver and mammary gland suggesting that other hop constituents and/or an additional mechanism might be responsible for these effects. Transcription of many detoxification enzymes is regulated through the antioxidant response element (ARE) and its transcription factor Nrf2, which is repressed under basal conditions by Keap1 (4). It has been shown that XN alkylates Keap1 and therefore enhances the concentration of free Nrf2 in the nucleus which results in ARE activation (5). Another pathway leading to ARE activation is mediated by protein kinase C (PKC) that phosphorylates Nrf2 leading to accumulation of Nrf2 in the nucleus (6). To investigate whether this pathway is involved in the observations described above, additional experiments with HepG2-ARE-luciferase C8 cells were performed. The cells were treated with hop extract or XN with or without the selective PKC inhbitor, Ro-32-0432, and the ARE-luciferase induction was determined. The results revealed that hops- but not XN-regulated ARE induction is in part mediated by PKC. Possible active compounds are under investigation. These results suggest that hop dietary supplements have the potential as preventive agents against cancer through induction of detoxifying enzymes. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A43.

Structural Influence of Isothiocyanates on the Antioxidant Response Element (ARE)-Mediated Heme Oxygenase-1 (HO-1) Expression

Isothiocyanates (ITCs), existing abundantly in cruciferous vegetables, is one class of promising dietary cancer chemopreventive agents that possess strong cancer protective effects by modulation of phase II detoxifying/antioxidant enzyme activities. However, limited studies regarding to the structure-activity relationship (SAR) of ITCs on the induction of phase II detoxifying/antioxidant enzymes are reported. In this study, the effects of ten structurally related isothiocyanates on the antioxidant response element (ARE)-mediated antioxidant enzyme heme oxygenase-1 (HO-1) induction in human hepatoma HepG2-C8 cells were evaluated. After exposure of HepG2-C8 cells to ITCs, cell viability, luciferase reporter assay, Western blot analysis and quantitative real-time PCR were conducted. Treatments with most ITCs significantly activated ARE-mediated luciferase activity with different maximal degree of ARE induction. In addition, ITCs caused a substantial induction of HO-1 protein, which was closely correlated with inductive level of Nrf2 protein. Real-time PCR revealed that the expression of HO-1 mRNA and protein was significantly increased after treatments with ITCs, although not directly correlated. HO-1 induction by ITCs was attenuated in HepG2-C8 cells transiently transfected with a dominant negative mutant of Nrf2 (Nrf2-M4), whereas it was totally absent in Nrf2 -/- mouse embryonic fibroblasts. In addition, ARE activation by ITCs was associated with the depletion of intracellular glutathione. Collectively, our results demonstrate that the ITC class of compounds activates ARE-mediated HO-1 gene transcription through Nrf2/ARE signaling pathway, however, their inductive effects are quite specific, depending on the chemical structure. These results suggest the possibility that some synthetic ITCs might have superior chemopreventive activity than natural ITCs.

Coordinate regulation of glutathione metabolism in astrocytes by Nrf2

The tripeptide glutathione (GSH) represents the major brain thiol and is essential for prevention of oxidative stress. Using monochlorobimane to label intracellular GSH in a glutathione S-transferase catalyzed reaction we have examined the kinetics of GSH metabolism including its rate of conjugation, total GSH content, synthesis, and efflux in astrocyte cultures under basal conditions and after induction of antioxidant response element (ARE)-mediated gene expression by the transcription factor Nrf2. In the presence of a cerebral spinal fluid-like salt solution astrocytes could not synthesize detectable levels of GSH. Addition of GSH precursors, cystine, glutamate, and glycine, rapidly restored GSH synthesis. Astrocytes were able to use either glutamate or glutamine as precursors equally for GSH synthesis. Using the small molecule chemical inducer tert-butylhydroqunione (tBHQ) we report that induction of ARE-mediated gene expression is associated with a coordinated increase in GSH content and synthesis rate with little effect on the rate of GSH conjugation or efflux. Consistent with the effect of the inducer, adenovirus-mediated overexpression of the transcription factor Nrf2 that mediates tBHQ’s effects also increased GSH content, confirming that GSH metabolism can be regulated by the Nrf2 pathway.