Activation Of Antioxidant Response Element Research Articles

Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity

The chemo-preventative effects of dithiolethione compounds are attributed to their activation of anti-oxidant response elements (ARE) by reacting with the Nrf2/Keap1 protein complex. In this study, we demonstrate anti-proliferative effects of the dithiolethione compound ACS-1 in human cancer cell lines (A549 and MDA-MB-231) by increasing the activity of the tumor suppressor PP2A. ACS-1 inhibited EGF-induced cellular proliferation in a concentration and time-dependent manner. Akt activation, as determined by serine-473 phosphorylation, was inhibited by ACS-1 in cells stimulated with either EGF or fibronectin. Furthermore, ACS-1 inhibited mTOR signaling and decreased c-myc protein levels. ACS-1 did not proximally alter EGFR or integrin signaling, but caused a concentration-dependent increase in PP2A activity. The effect of ACS-1 on Akt activation was not observed in the presence of the PP2A inhibitor okadaic acid. ACS-1 effects on PP2A activity were independent of ARE activation and cAMP formation. In addition to ACS-1, other dithiolethione compounds showed similar effects in reducing Akt activation, suggesting that this class of compounds may have other effects beyond chemoprevention.

Transforming growth factor‐β1 elicits Nrf2‐mediated antioxidant responses in aortic smooth muscle cells

The anti-inflammatory properties of transforming growth factor-beta(1) (TGF-beta(1)) account for its protection against atherosclerotic plaque rupture. This study investigates whether activation of the Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) transcription pathway is involved in TGF-beta(1) mediated induction of the antioxidant enzyme heme oxygenase-1 (HO-1) in smooth muscle cells (SMC). Human aortic smooth muscle cells (HAoSMC) or wild-type and Nrf2-deficient mouse (MAoSMC) aortic SMC were treated with TGF-beta(1) (2.5-10 ng/ml, 0-24 hrs). We report the first evidence that TGF-beta(1) induces Nrf2 mediated HO-1 expression and antioxidant response element activity, which was paralleled by enhanced superoxide production and expression of the NAD(P)H oxidase subunit p22(phox). TGF-beta(1) failed to induce HO-1 expression in MAoSMC derived from Nrf2-deficient mice, and HO-1 induction by TGF-beta(1) in HAoSMC was attenuated by inhibition of extracellular signal regulated kinase or c-jun-N-terminal kinase but not p38 mitogen activated protein kinase. Inhibition of NAD(P)H oxidase or scavenging of superoxide diminished HO-1 induction in response to TGF-beta(1). The oxidative stress agents glucose oxidase (GOx) and diethylmaleate enhanced TGF-beta(1) generation and HO-1 expression in HAoSMC, while antagonism of TGF-beta(1) signalling by adenoviral Smad7 overexpression attenuated their induction of HO-1. Pre-treatment of HAoSMC with TGF-beta(1) reduced nuclear translocation of the pro-apoptotic mediator p53 elicited by GOx. Our findings demonstrate that Nrf2 is a new target of TGF-beta(1) signalling in the vasculature which may contribute to the atheroprotective properties attributed to this growth factor.

Cul3-mediated Nrf2 ubiquitination and antioxidant response element (ARE) activation are dependent on the partial molar volume at position 151 of Keap1

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the ARE (antioxidant response element). Nrf2 is repressed by the cysteine-rich Keap1 (kelch-like ECH-associated protein 1) protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3 (cullin 3)-mediated ubiquitination complex. We find that modification of Cys(151) of human Keap1, by mutation to a tryptophan, relieves the repression by Keap1 and allows activation of the ARE by Nrf2. The Keap1 C151W substitution has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for this effect. The series reveals that the extent to which Keap1 loses the ability to target Nrf2 for degradation, and hence the ability to repress ARE activation, correlates well with the partial molar volume of the residue. Other physico-chemical properties do not appear to contribute significantly to the effect. Based on this finding, a structural model is proposed whereby large residues at position 151 cause steric clashes that lead to alteration of the Keap1-Cul3 interaction. This model has significant implications for how electrophiles which modify Cys(151), disrupt the repressive function of Keap1.

The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK‐3beta: connecting neurotransmission with neuroprotection

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.

Sulforaphane: from chemoprevention to pancreatic cancer treatment?

Over the past several decades, research on the action of bioactive constituents of plants (ie, phytochemicals) has focused predominantly on their cancer-preventive properties. Evidence for a protective role for vegetables...

Activation of the antioxidant response element by specific oxidized metabolites of linoleic acid

Linoleic acid is required for normal mammalian health and development, but is also prone to oxidation, yielding metabolites with biological effects. We screened linoleic acid, other fatty acids, and some of their derivatives and found that an epoxy-keto derivative of linoleic acid (but neither linoleic acid itself nor others of its oxidation products) strongly activates the antioxidant response element (ARE) in IMR-32 neuroblastoma cells and cerebro-cortical neurons. The active compound, 12,13-epoxy-9-keto-10( trans)-octadecenoic acid (EKODE), induces the expression of ARE-regulated cytoprotective genes such as NQO1 at the transcript and protein levels. EKODE requires transcription factor NRF2 and PI3-kinase for ARE activity. The results suggest that specific oxidation products of linoleic acid may initiate responses that lessen damage caused by oxidative stress.

Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection

We have shown previously that non-toxic inhibition of the ubiquitin-proteasome system upregulates antioxidative defence mechanisms and protects endothelial cells from oxidative stress. Here, we have addressed the question whether the induction of antioxidative enzymes contributes to cardioprotection by non-toxic proteasome inhibition. Treatment with 0.5 micromol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H(2)O(2)-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition-in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H(2)O(2)-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice. Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.

Role of the Tumor Suppressor PTEN in Antioxidant Responsive Element-mediated Transcription and Associated Histone Modifications

Coordinated regulation of PI3-kinase (PI3K) and the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) plays a pivotal role in various cell functions. PTEN is deficient in many cancer cells, including Jurkat human leukemia. Here, we demonstrate that the status of PTEN determines cellular susceptibility to oxidative stress through antioxidant-responsive element (ARE)-mediated transcription of detoxification genes. We found that ferritin H transcription was robustly induced in tert-butylhydroquinone (t-BHQ)-treated Jurkat cells via an ARE, and it was due to PTEN deficiency. Chromatin immunoprecipitation assays revealed that p300/CREB-binding protein (CBP) histone acetyltransferases and Nrf2 recruitment to the ARE and Bach1 release were blocked by the PI3K inhibitor LY294002, along with the partial inhibition of Nrf2 nuclear accumulation. Furthermore, acetylations of histone H3 Lys9 and Lys18, and deacetylation of Lys14 were associated with the PI3K-dependent ARE activation. Consistently, PTEN restoration in Jurkat cells inhibited t-BHQ-mediated expression of ferritin H and another ARE-regulated gene NAD(P)H:quinone oxidoreductase 1. Conversely, PTEN knockdown in K562 cells enhanced the response to t-BHQ. The PTEN status under t-BHQ treatment affected hydrogen peroxide-mediated caspase-3 cleavage. The PI3K-dependent ferritin H induction was observed by treatment with other ARE-activating agents ethoxyquin and hemin. Collectively, the status of PTEN determines chromatin modifications leading to ARE activation.

LAS0811: From Combinatorial Chemistry to Activation of Antioxidant Response Element

The antioxidant response element (ARE) and its transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), are potential targets for cancer chemoprevention. We sought to screen small molecules synthesized with combinatorial chemistry for activation of ARE. By high-throughput screening of 9400 small molecules from 10 combinatorial chemical libraries using HepG2 cells with an ARE-driven reporter, we have identified a novel small molecule, 1,2-dimethoxy-4,5-dinitrobenzene (LAS0811), as an activator of the ARE. LAS0811 upregulated the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1), a representative antioxidative enzyme regulated by ARE. It enhanced production of an endogenous reducing agent, glutathione (GSH). In addition, LAS0811 induced expression of heme oxygenase 1 (HO1), which is an ARE-regulated enzyme with anti-inflammatory activity. Furthermore, LAS0811 reduced cell death due to the cytotoxic stress of a strong oxidant, t-butyl hydroperoxide (t-BOOH). Mechanistically, LAS0811 upregulated the expression of Nrf2 and promoted its translocation into the nuclei leading to subsequent ARE activation. Taken together, LAS0811 is a novel activator of the ARE and its associated detoxifying genes and, thus, a potential agent for cancer chemoprevention.

Simple ortho- and para-hydroquinones as compounds neuroprotective against oxidative stress in a manner associated with specific transcriptional activation

Electrophilic compounds protect neurons through the activation of the Keap1/Nrf2 pathway and the induction of phase-2 enzymes [T. Satoh, S.A. Lipton, Redox regulation of neuronal survival by electrophilic compounds, Trends Neurosci. 30 (2007) 38–45; T. Satoh, S. Okamoto, J. Cui, Y. Watanabe, K. Furuta, M. Suzuki, K. Tohyama, S.A. Lipton, Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc. Natl. Acad. Sci. USA 103 (2006) 768–773]. Hydroquinone-type electrophilic compounds such as tert-butyl hydroquinone (TBHQ) and carnosic acid (CA) have attracted special attention, because the oxidative conversion of “hydroquinone” to “quinone” is essential for the transcriptional activation of the above-mentioned enzymes [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protect neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of specific cysteine, J. Neurochem. 104 (2008) 1161–1131; A.D. Kraft, D.A. Johnson, J.A. Johnson, Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult, J. Neurosci. 24 (2004) 1101–1112]. In the present study, we examined the relationship between electrophilicity and the protective effects afforded by electrophilic compounds. Electrophilicity was assessed in terms of the ability of a compound to bind to a cysteine on bovine serum albumin, by which we found that neuroprotective hydroquinones [TBHQ (para-) and CA (ortho-)] had distinctive patterns of cysteine binding compared with other electrophilic compounds. Further, we found that isomers of simple ortho- and para-hydroquinones such as 2-methylhydroquinone (para-) and 4-methyl-catechol (ortho-) [not in abstract] had similar properties of cysteine binding as TBHQ and CA, which compounds were associated with the transcriptional activation and an increase in the level of reduced glutathione. These results suggest that para- and ortho-dihydroquinones may be neuroprotective compounds active against oxidative stress.

Transforming Growth Factorβ-1 elicits Nrf2-mediated antioxidant responses in aortic smooth muscle cells

The anti-inflammatory properties of transforming growth factor-β1 (TGF-β1) account for its protection against atherosclerotic plaque rupture. This study investigates whether activation of the Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) transcription pathway is involved in TGF-β1 mediated induction of the antioxidant enzyme heme oxygenase-1 (HO-1) in smooth muscle cells (SMC). Human aortic smooth muscle cells (HAoSMC) or wild-type and Nrf2-deficient mouse (MAoSMC) aortic SMC were treated with TGF-β1 (2.5–10 ng/ml, 0–24 hrs). We report the first evidence that TGF-β1 induces Nrf2 mediated HO-1 expression and antioxidant response element activity, which was paralleled by enhanced superoxide production and expression of the NAD(P)H oxidase subunit p22phox. TGF-β1 failed to induce HO-1 expression in MAoSMC derived from Nrf2-deficient mice, and HO-1 induction by TGF-β1 in HAoSMC was attenuated by inhibition of extracellular signal regulated kinase or c-jun-N-terminal kinase but not p38 mitogen activated protein kinase. Inhibition of NAD(P)H oxidase or scavenging of superoxide diminished HO-1 induction in response to TGF-β1. The oxidative stress agents glucose oxidase (GOx) and diethylmaleate enhanced TGF-β1 generation and HO-1 expression in HAoSMC, while antagonism of TGF-β1 signalling by adenoviral Smad7 overexpression attenuated their induction of HO-1. Pre-treatment of HAoSMC with TGF-β1 reduced nuclear translocation of the pro-apoptotic mediator p53 elicited by GOx. Our findings demonstrate that Nrf2 is a new target of TGF-β1 signalling in the vasculature which may contribute to the atheroprotective properties attributed to this growth factor.

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.

Imaging Induction of Cytoprotective Enzymes in Intact Human Cells: Coumberone, a Metabolic Reporter for Human AKR1C Enzymes Reveals Activation by Panaxytriol, an Active Component of Red Ginseng

We here present an optical method for monitoring the activity of the inducible aldo-keto reductases AKR1C2 and AKR1C3 in living human cells. The induction of these enzymes is regulated by the antioxidant response element (ARE), as demonstrated in recent literature, which in turn is dependent on the transcription factor Nrf2. The activation of ARE leads to the transcription of a coalition of cytoprotective enzymes and thus represents an important target for the development of new therapies in the area of neurodegenerative diseases and cancer. Through the use of Coumberone, a metabolic fluorogenic probe, and isoform-selective inhibitors, the upregulation of cellular stress markers AKR1C2 and AKR1C3 can be quantitatively measured in the presence of ARE activator compounds, via either a fluorimetric assay or fluorescence microscopy imaging of intact cells. The method has both high sensitivity and broad dynamic range, as demonstrated by induction studies in three cell lines with dramatically different metabolic capabilities (transfected monkey kidney COS-1 cells, human neuroblastoma IMR-32 cells, and human liver HepG2 cells). We applied the new method to examine a number of neurotrophic natural products (spirotenuipesine A, spirotenuipesine B, scabronine G-methylester, and panaxytriol), and discovered that panaxytriol, an active component of red ginseng extracts, is a potent ARE inducer. The upregulation of AKR1C enzymes, induced by chemically homogeneous panaxytriol, was partially dependent on PKC and PI3K kinases as demonstrated by the application of selective inhibitors. This cellular mechanism may account for panaxytriol's neurotrophic, neuroprotective, and anticancer properties. The protective effects of ARE inducers against tumorgenesis and neurodegeneration fuel the growing interest in this area of research and the method described here will greatly enable these endeavors.

Lipoic acid regulates glutathione, heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells

Cajaninstilbene acid (CSA), an active compound separated from pigeon pea leaves, possesses the highly efficient antioxidant activities. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the role of Nrf2 in CSA-mediated antioxidant effects on human hepatocarcinoma (HepG2) cell line. The generation of reactive oxygen species (ROS) upon H2O2 and CSA treatment was lower than that of H2O2 alone. CSA activated Nrf2 as evaluated by Western blotting. A luciferase reporter assay also demonstrated that CSA-activated signaling resulted in the increased transcriptional activity of Nrf2 through binding to the antioxidant response element (ARE) enhancer sequence. Our study indicated that treatment of HepG2 cells with CSA induces Nrf2-dependent ARE activity and gene expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate–cysteine ligase modifier subunits by activation of PI3K/AKT, ERK and JNK signaling pathways. Inhibition of Nrf2 by siRNA reduced CSA-induced upregulation of these Nrf2-related enzymes. These results suggest that the Nrf2/ARE pathway plays an important role in the regulation of CSA-mediated antioxidant effects in HepG2 cells.

Angelica sinensis and Its Alkylphthalides Induce the Detoxification Enzyme NAD(P)H: Quinone Oxidoreductase 1 by Alkylating Keap1

The roots of Angelica sinensis (Oliv.) Diels (Dang Gui; Apiaceae) have a long history in traditional Chinese medicine as a remedy for women's disorders and are often called "lady's ginseng". Currently, extracts of A. sinensis are commonly included in numerous dietary supplements used for women's health and as antiaging products. In the present study, we examined the potential chemopreventive activity of A. sinensis extracts by measuring the relative ability to induce the detoxification enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1). The lipophilic partitions showed strong NQO1 induction with concentrations to double the enzyme activity (CD) of 5.5 +/- 0.7 (petroleum ether) and 3.9 +/- 0.5 microg/mL (chloroform). Fractionation led to the isolation of phenolic esters and alkylphthalides, especially Z-ligustilide, the main lipophilic compound, which showed strong NQO1 inducing properties (CD = 6.9 +/- 1.9 microM). Transcription of many detoxifying enzymes is regulated through the antioxidant response element (ARE) and its transcription factor Nrf2, which is repressed under basal conditions by Keap1. However, exposure to electrophilic inducers that alkylate Keap1 results in higher concentrations of free Nrf2 and ARE activation. The ARE reporter activity was therefore analyzed in HepG2-ARE-C8 cells after incubation with lipophilic extracts of A. sinensis or ligustilide for 24 h. Under these conditions, both the extract and the ligustilide increased ARE-luciferase reporter activity in a dose-dependent manner. Incubation of ligustilide with GSH and subsequent LC-MS-MS analysis revealed that ligustilide as well as oxidized ligustilide species covalently modified GSH. In addition, using MALDI-TOF mass spectrometry and LC-MS-MS, it was demonstrated that the lipophilic extracts, ligustilide, and monooxygenated ligustilide alkylated important cysteine residues in human Keap1 protein, thus activating Nrf2 and transcription of ARE regulated genes. These observations suggest that A. sinensis dietary supplements standardized to ligustilide have potential as chemopreventive agents through induction of detoxification enzymes.

Functional interference between glycogen synthase kinase-3 beta and the transcription factor Nrf2 in protection against kainate-induced hippocampal celldeath

Excitotoxicity mediated by glutamate receptors may underlay the pathology of several neurologic diseases. Considering that oxidative stress is central to excitotoxic damage, in this study we sought to analyze if the transcription factor Nrf2, guardian of redox homeostasis, might be targeted to prevent kainate-induced neuron death. Hippocampal slices from Nrf2 knockout mice exhibited increased oxidative stress and cell death compared to those of control mice in response to kainate, as determined with the redox sensitive probes 2,7-dichlorodihydrofluorescein diacetate (H 2DCFAC) and propidium iodide and lactate dehydrogenase release, respectively, therefore demonstrating a role of Nrf2 in antioxidant protection against excitotoxicity. In the hippocampus of mice intraperitoneally injected with kainate we observed a rapid activation of Akt, inhibition of GSK-3β and translocation of Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK-3β was active and Nrf2 was mostly cytosolic, therefore extending our previous studies which indicate that GSK-3β excludes Nrf2 from the nucleus. Lithium, a GSK-3β inhibitor, promoted Nrf2 transcriptional activity towards an Antioxidant-Response-Element (ARE) luciferase reporter and cooperated with sulforaphane (SFN) to induce this reporter and to increase the protein levels of heme oxygenase-1 (HO-1), coded by a representative ARE-containing gene. Conversely, ARE activation by SFN was attenuated by over-expression of active GSK-3β. Finally, combined treatment with SFN and lithium attenuated oxidative stress and cell death in kainate-treated hippocampal slices of wild type mice but not Nrf2 null littermates. Our findings identify the axis GSK-3β/Nrf2 as a pharmacological target in prevention of excitotoxic neuronal death.

NEUROPROTECTION AGAINST ETHANOL MEDIATED OXIDATIVE STRESS AND APOPTOSIS VIA NRF2/ARE.

This study characterizes a neuroprotective pathway responsible for coordinated enhancement of astrocyte glutathione (GSH)‐homeostatic machinery. Injuries in fetal rat cortical neurons associated with Fetal Alcohol Syndrome are correlated to ethanol (E) exposure leading to increased oxidative stress, GSH depletion, and ultimately neuroapoptosis. Our lab has previously published that astrocytes protect neurons from E mediated apoptosis, via maintenance of GSH homeostasis. We hypothesize that neuroprotection is regulated by the activation of the anti‐oxidant response elements (ARE) contained in the 5’ regions of cytoprotective genes, via the master transcription factor Nrf2. To identify the role of E in astrocyte anti‐oxidant defense induction, we first employed an E time course to investigate Nrf2 protein expression. Primary cultured rat neonatal astrocytes were treated with 4mg/ml E at various time points. There was increased nuclear Nrf2 expression within 15 minutes after E treatment, and lasted up to 4 hours. Immunohistochemical studies corroborate cytoplasmic to nuclear translocation. Enhanced nuclear Nrf2 expression was further augmented by BSO pre‐treatment. In addition, the transcription factor Maf, which obligatorily dimerizes with Nrf2 to activate gene transcription, was increased by E or BSO. Known ARE inducers and oxidative stress mimicked the cytoplasmic to nuclear translocation seen in E treatment. To determine whether E increased GSH‐homeostatic machinery at the transcriptional level, cells were pre‐incubated with Actinomycin D, and subsequently treated with E. Nrf2 expression induced by E was abolished in the presence of AD suggesting that E increases Nrf2 by stimulating gene transcription. Transfection of Nrf2 siRNA completely ablated Nrf2 expression, and furthermore blocks the tert‐butyl hydroquinone induced Nrf2, GGT, and Mrp1 expression. Finally, we examined E's effect on Nrf2 ubiquitination. E decreased Nrf2 ubiquitination at 4 and 24 hours while not effecting overall ubiquitin levels in the cell suggesting another possible mechanism for Nrf2 protein level increase and subsequent nuclear translocation to activate ARE. Taken together, this suggests that Nrf2 is required for activation of ARE. The role of E in Nrf2‐ARE control of astrocyte neuroprotection needs to be further investigated. These points of regulation may allow for pharmacological augmentation of this crucial neuroprotective mechanism.

KIST‐A164A Increases a Quinone Reductase Potentially by Inhibiting COX‐2‐Dependent Inflammatory Signaling

The up‐regulation of phase II detoxification genes is believed to play an important role in cancer prevention. Cyclooxygenase‐2 (COX‐2) is often up‐regulated in human colorectal cancers, and it is considered as one of major inflammatory factors causing carcinogenesis. However, the relationship between the induction of phase II enzyme and COX‐2 activation in terms of cancer prevention is unknown. We have identified a polyacetylene compound (KIST‐A164A) from Aster koraiensis as a major principle for a significant quinone reductase (QR) induction via transcriptional activation. We also found that KIST‐A164A significantly inhibited COX‐2 mRNA expression levels and PGE2 production in a dose‐dependent manner. Further molecular study revealed that co‐treatment with KIST‐A164A and acetylsalicylic acid, an inhibitor of COX‐2, increased the expression of QR mRNA and protein levels, which was coincidently enhancing QR activity as well as antioxidant response element (ARE) activation in Caco‐2 cells. The ARE activation was increased 2‐fold over control when the cells were treated with 50 μM KIST‐A164A in the presence of JNK1/2 inhibitor, SP600125. Taken together, we present here that KIST‐A164A increases QR through ARE and it could be related to inhibition of COX‐2‐dependent inflammatory signaling.

Neuroprotection Against Ethanol Mediated Oxidative Stress and Apoptosis via Nrf2/ARE.

This study characterizes a neuroprotective pathway responsible for coordinated enhancement of astrocyte glutathione (GSH)‐homeostatic machinery. Injuries in fetal rat cortical neurons associated with Fetal Alcohol Syndrome are correlated to ethanol (E) exposure leading to increased oxidative stress, GSH depletion, and ultimately neuroapoptosis. We hypothesize that neuroprotection is regulated by activation of the anti‐oxidant response elements (ARE) of cytoprotective genes, via the master transcription factor Nrf2. Primary cultured rat neonatal astrocytes were treated with 4mg/ml E at various time points. There was increased nuclear Nrf2 expression within 15 minutes after E treatment, and lasted up to 4 hours. Immunohistochemical studies corroborate cytoplasmic to nuclear translocation. Enhanced nuclear Nrf2 expression was further augmented by BSO pre‐treatment. In addition, the transcription factor Maf, which obligatorily dimerizes with Nrf2 to activate gene transcription, was increased by E or BSO. Known ARE inducers and oxidative stress mimicked the cytoplasmic to nuclear translocation seen in E treatment. Next, cells were pre‐incubated with Actinomycin D and subsequently treated with E. Nrf2 expression induced by E was abolished suggesting that E increases Nrf2 by stimulating gene transcription. Transfection of Nrf2 siRNA completely ablated Nrf2 expression, and furthermore blocks the tbHQ induced Nrf2, GGT, and Mrp1 expression. Taken together, this suggests that Nrf2 is required for activation of ARE. The role of E in Nrf2‐ARE control of astrocyte neuroprotection need to be further investigated. These points of regulation may allow for pharmacological augmentation of this crucial neuroprotective mechanism.

Role and regulation of ferritin H in rotenone-mediated mitochondrial oxidative stress

Tight regulation of intracellular iron levels in response to mitochondrial dysfunction is an important mechanism that prevents oxidative stress, thereby limiting cellular damage. Here, we describe a cytoprotective response involving transcriptional activation of the ferritin H gene in response to the mitochondrial complex I inhibitor and neurotoxic compound rotenone. Rotenone exposure increased ferritin H mRNA and protein synthesis in NIH3T3 fibroblasts and SH-SY5Y neuroblastoma cells. Transient transfection of a ferritin H promoter–luciferase reporter into NIH3T3 cells showed that ferritin H was transcriptionally activated by rotenone through an antioxidant-responsive element (ARE). Chromatin immunoprecipitation assays showed that rotenone treatment enhanced binding of Nrf2 and JunD transcription factors to the ARE. In addition, rotenone induced production of reactive oxygen species (ROS), and pretreatment with N-acetylcysteine abrogated ferritin H mRNA induction by rotenone, suggesting that this response is oxidative stress-mediated. Furthermore, reduced ferritin H expression by siRNA sensitized cells to rotenone-induced apoptosis with increased ROS production and annexin V-positive cells. Taken together, these results suggest that ferritin H transcription is activated by rotenone via an oxidative stress-mediated pathway leading to ARE activation and may be critically important to protect cells from mitochondrial dysfunction and oxidative stress.