Activation Of Antioxidant Response Element Research Articles

Determining the Effect of Dithiolethione Compounds on the Activity of Human Glyceraldehyde-3-Phosphate Dehydrogenase

Dithiolethione compounds, like anethole dithiolethione (ADT) have chemopreventive, cytoprotective, and antimitogenic effects. The chemopreventive effects are thought to be mediated by the activation of antioxidant response elements and of the tumor repressor PP2a. We have shown that ADT interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cancer cells usually exhibit increased glycolysis (Warburg effect) that generates ATP to meet their energy needs. Mounting evidence suggests that inhibition of glycolysis in cancer cells severely depletes ATP and may be an effective strategy in chemoprevention. We hypothesize that the chemopreventive effects of ADT may be in part due to inhibition of glycolysis via inhibition of GAPDH. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol-modifying agents. Therefore, we set out to examine how ADT affects GAPDH activity. Human GAPDH was overexpressed in bacterial cells and purified by ion-exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT and showed a concentration-dependent inhibition of GAPDH catalytic activity by ADT. In order to determine the exact mechanism of ADT modification of GAPDH we are coupling site-directed mutagenesis with enzymatic assay, and mass-spectrometry.This investigation was sponsored in part by NIH/NIGMS MARC U∗STAR T34 08663 National Research Service Award to UMBC and also by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program (JC) and UMBC DRIF SRAIS (EG).

Microarray and pathway analysis highlight Nrf2/ARE‐mediated expression profiling by polyphenolic myricetin

Myricetin is a dietary flavonol and widely distributed in many edible plants. It has been reported to have many bioactivities and considered as a promising chemopreventive compound. The present study aimed to investigate the influences of myricetin on gene expressions in genome-wide and underlying mechanisms. Among total 44K gene probes, myricetin treatment upregulated the signals of 143 gene probes (0.33% of total probes) and downregulated signals of 476 gene probes (1.08% of total probes) by greater than or equal to twofold in HepG2 cells. The network pathway analysis revealed that nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response element (ARE) activation is involved in myricetin-induced genes expressions. Molecular data revealed that myricetin activated Nrf2-ARE pathway by inhibiting Nrf2 ubiquitination and protein turnover, stimulating Nrf2 expression and kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 modification. All of these events finally increased nuclear Nrf2 accumulation and ARE-binding activity to enhance ARE-mediated genes expressions. Additionally, treatment with Nrf2 small interfering RNA attenuated the myricetin-induced ARE activity and gene expression. An Nrf2-mediated ARE activation is involved in myricetin-induced expression profiling in hepatic cells.

BTZO-2, an antioxidant response element-activator, provides protection against lethal endotoxic shock in mice

We recently reported a unique antioxidant response element (ARE)-activator, BTZO-1, which induced expression of cytoprotective proteins such as heme oxygenase-1 (HO-1) and suppressed oxidative stress-induced cardiomyocyte apoptosis via binding to macrophage migration inhibitory factor (MIF). HO-1 induction and apoptosis inhibition have been reported to improve the outcomes following experimental sepsis by protecting the organs. Therefore, we investigated the potential of BTZO-2, an active BTZO-1 derivative, as a drug for sepsis. BTZO-2 significantly protected mice from the endotoxic shock induced by 5mg/kg lipopolysaccharide (LPS); survival rates increased from 42% to 100%. In contrast, BTZO-2 did not provide significant protection to mice from the shock induced by 10μg/kg LPS together with d-galactosamine (d-GalN, hepatocyte-specific transcription inhibitor) (LPS/d-GalN). Hepatic HO-1 protein was up-regulated by BTZO-2 in mice injected with 5mg/kg LPS, but not in those injected with 10μg/kg LPS/d-GalN. Interestingly, BTZO-2 showed little or no effect on LPS-induced up-regulation of plasma cytokine levels in mice. Thus, the organ protection mediated by HO-1 may have a pivotal role in the pharmacological effect of BTZO-2. These results suggest that BTZO-2 is a promising compound for a novel drug for sepsis.

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.

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson’s and other dopamine deficiency states

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50μM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50μM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50μM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.

Oxidative Stress-Regulated Lentiviral TK/GCV Gene Therapy for Lung Cancer Treatment

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

Tacrine‐6‐Ferulic Acid, a Novel Multifunctional Dimer Against Alzheimer's Disease, Prevents Oxidative Stress‐Induced Neuronal Death Through Activating Nrf2/ARE/HO‐1 Pathway in HT22 Cells

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with progressive and devastating memory impairment 1. The majority of therapeutic strategies and drug development approaches for AD were based on dysfunction of acetylcholine to date, which mainly alleviate symptoms of AD 1. A great amount of researches proposed that oxidative stress (OS) be an important and early factor in the pathogenesis of AD and contribute to β-amyloid generation 2. Nuclear factor E2-related factor 2 (Nrf2) is a novel transcription factor known to induce expression of a variety of cytoprotective and detoxification genes. Increasing evidence demonstrated that Nrf2/ARE pathway may be considered a therapeutic target for neurodegenerative diseases, including AD 3. We have previously synthesized and proved that tacrine-6-ferulic acid (T6FA; Figure 1), a novel multifunctional dimer, can prevent amyloid-β peptide-induced AD-associated pathological changes, including anti-OS, in vitro and in vivo 4. But the molecular mechanism(s) underlying the neuroprotective effects of T6FA is still not fully elucidated. HT22 cells, an immortalized and ionotropic glutamate receptors-free mouse hippocampal cell line, have been widely used as an in vitro model for elucidating the mechanism of OS-induced neurotoxicity 5. In this study, HT22 cells were used to examined the potential effects of T6FA on the OS-induced cell death as well as the role of Nrf2/ARE pathway. HT22 cells were exposed to 5 mM glutamate in the absence or presence of T6FA (0- 30 μM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability. H2-DCF-DA staining was used to monitor the ROS level. Immunocytofluorescence and confocol microscopy were used to monitor the Nrf2 translocation. Antioxidant response element (ARE)-luciferase reporter system was used to measure the ARE activity, and Eastern blot was used to assay the HO-1 protein level. Data were statistically analyzed by one-way ANOVA. Glutamate significantly induces cell death, and T6FA blocks the cell death in a concentration-dependent manner (Figure 1A). T6FA (30 μM) potently attenuate the ROS level induced by glutamte (Figure 1B). Immunocytofluorescence experiment showed that T6FA dramatically provoke the Nrf2 nuclear translocation (Figure 2A). In addition, T6FA activated the ARE-dependent transcription (Figure 2B) and also up-regulated the expression of heme oxygenase-1 (HO-1) in a time- and concentration-dependent manner (Figure 2C). The neuroprotective effects can be blocked by zinc protoporphyrin IX (ZnPP) (1 μM), an inhibitor of HO-1 (Figure 2D). This study showed that T6FA potently prevent OS-induced cell death in HT22 cells and activate Nrf2/ARE pathway and up-regulate the expression of HO-1. Modulation of Nrf2 has been shown in several neurodegenerative diseases, including AD 3, 6. Targeting Nrf2 pathway has become a potential therapeutic avenue for these diseases 6. The expression of phase II detoxification enzymes is governed by the cis-acting regulatory element known as ARE. The transcription factor Nrf2 binds to ARE thereby transcribing multitude of antioxidant genes to protect against OS in neurodegenerative diseases 6. Our findings support that T6FA confer neuroprotection through activating Nrf2/ARE pathway, suggesting that T6FA may be a promising multifunctional drug candidate for neurodegenerative diseases, not only AD. This research was supported by grants from Fundamental Research Funds for the Central Universities (No. 10ykpy23) to R. Pi and the Scientific and Technology Foundation of Guangdong Province (2011B061300049) to W. Huang. The authors declare no conflict of interests.

Quinone-Induced Activation of Keap1/Nrf2 Signaling by Aspirin Prodrugs Masquerading as Nitric Oxide

The promising therapeutic potential of the NO-donating hybrid aspirin prodrugs (NO-ASA) includes induction of chemopreventive mechanisms and has been reported in almost 100 publications. One example, NCX-4040 (pNO-ASA), is bioactivated by esterase to a quinone methide (QM) electrophile. In cell cultures, pNO-ASA and QM-donating X-ASA prodrugs that cannot release NO rapidly depleted intracellular GSH and caused DNA damage; however, induction of Nrf2 signaling elicited cellular defense mechanisms including upregulation of NAD(P)H:quinone oxidoreductase-1 (NQO1) and glutamate-cysteine ligase (GCL). In HepG2 cells, the "NO-specific" 4,5-diaminofluorescein reporter, DAF-DA, responded to NO-ASA and X-ASA, with QM-induced oxidative stress masquerading as NO. LC-MS/MS analysis demonstrated efficient alkylation of Cys residues of proteins including glutathione-S-transferase-P1 (GST-P1) and Kelch-like ECH-associated protein 1 (Keap1). Evidence was obtained for alkylation of Keap1 Cys residues associated with Nrf2 translocation to the nucleus, nuclear translocation of Nrf2, activation of antioxidant response element (ARE), and upregulation of cytoprotective target genes. At least in cell culture, pNO-ASA acts as a QM donor, bioactivated by cellular esterase activity to release salicylates, NO(3)(-), and an electrophilic QM. Finally, two novel aspirin prodrugs were synthesized, both potent activators of ARE, designed to release only the QM and salicylates on bioactivation. Current interest in electrophilic drugs acting via Nrf2 signaling suggests that QM-donating hybrid drugs can be designed as informative chemical probes in drug discovery.

Discovery of Potent, Novel Nrf2 Inducers Via Quantum Modeling, Virtual Screening, and In Vitro Experimental Validation

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH-associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2-Keap1 binding and promote activation of the Nrf2- antioxidant response element pathway, we generated a quantum model based on the structures of known Nrf2- antioxidant response element activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2- antioxidant response element pathway based on the quantum model. The top hits were tested in vitro, and half of the predicted hits activated the Nrf2-antioxidant response element pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-antioxidant response element-activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-antioxidant response element -activating compounds for drug development.

Abstract 91: Activation of The Antioxidant Response Element (ARE) in Human Renal Glomerular Endothelial Cells (HRGECs) Reduces Asymmetric Dimethylarginine (ADMA) And Increases Nitric Oxide by Transcriptional Activation of Dimethylarginine Dimethylaminohydrolase (DDAH) and Endothelial Nitric Oxide Synthase (eNOS)

The E2-related factor 2 (Nrf2) binds to AREs and transcriptionally activates several genes that protect against oxidative stress. DDAH phosphorylates and activates eNOS and degrades ADMA which is an endogenous inhibitor of eNOS. We detected 2-3 putative AREs in the promoter regions of the human genes for DDAH-1 and -2, peroxisome proliferator-activated receptor-γ (PPAR-γ) and eNOS. PPAR-γ can increase eNOS expression and eNOS phosphorylation. Therefore, we hypothesized that AREs regulate the DDAH/eNOS/PPAR-γ/ADMA/ NO pathway. Tert-butylhydroquinone (tBHQ) activates Nrf2 by translocation to the nucleus. Incubation of HRGECs for 24 hours with tBHQ enhanced cell proliferation dose-dependently (5-20μM). tBHQ (20μM; n=4) increased medium nitrite by 69 ± 3.7% (P&lt;0.01), intracellular NO (from DAF-FM fluorescence ) by 62 ± 17.1% (P&lt;0.05), and NOS and DDAH activities( from conversion of [ 3 H]-arginine or [ 14 C]-ADMA to [ 3 H]- or [ 14 C] -citrulline) by 58 ± 3.6% and 47 ± 8.5% (P&lt;0.05) and decreased ADMA (by capillary zone electrophoresis) from 1.2±0.03 vs. 2.3±0.16μM/L, (P&lt;0.001, n=6). tBHQ increased the mRNAs and proteins for eNOS by 79 ± 9% and 43 ± 17%, for DDAH-1 by 129 ± 10% and 62 ± 21%, and for DDAH-2 by 119 ± 25% and 48 ± 18% (all P&lt;0.05) and increased the protein expression of PPAR-γ and eNOS phosphorylation and increased tBHQ nuclear translocation markedly. Knockdown of the cell Nrf2 by transfection with a specific siRNA abolished all these effects and attenuated cell proliferation. Chromatin immunoprecipitation (CHIP)-based PCR assays demonstrated that tBHQ enhanced the binding of Nrf2 to one ARE region in the promoters for DDAH-1 and -2 but there was no binding to the eNOS promoter. In conclusion, activation of a specific ARE in the promoter region of the DDAH-1 and -2 genes causes their transcriptional activation. The ensuing enhancement of DDAH activity enhances ADMA metabolism. Nrf2 also increases eNOS expression, NOS activity and NO generation without binding to the ARE for eNOS, likely by upregulation of PPAR-γ. Thus, Nrf2 improves endothelial cell function by coordinating a diverse set of pathways that enhance NO generation.

Activation of Antioxidant Response Element in Mouse Primary Cortical Cultures with Sesquiterpene Lactones Isolated from Tanacetum parthenium

Tanacetum parthenium produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 SL from T. parthenium with centrifugal partition chromatography and semipreparative HPLC. Compounds were screened in vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All SL containing the α-methylene-γ-lactone moiety were able to activate the ARE and cause cellular toxicity. The structure-activity relationship among the SL isolated indicates that the guaianolides were more active and when lacking the endoperoxide functionality less toxic then the germacranolides.

Pharmacodynamics of Ginsenosides: Antioxidant Activities, Activation of Nrf2, and Potential Synergistic Effects of Combinations

Ginseng has long been used in Asian countries for more than 2000 years. Currently, in the "Western World or Western Medicines", many reports have indicated that they have used herbal medicines, and ginseng is one of the most popular herbs. Several recent reports have indicated that the antioxidant/antioxidative stress activities of ginseng play a role in the benefits of ginseng; however, the precise mechanism is lacking. The antioxidant response element (ARE) is a critical regulatory element for the expression of many antioxidant enzymes and phase II/III drug metabolizing/transporter genes, mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between three common ginsenosides present in ginseng, ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and ginsenoside 20(S)-protopanaxatriol (20S). We tested whether these ginsenosides and their combinations could induce Nrf2-ARE activities in HepG2-C8 cells with stably transfected ARE luciferase reporter gene. Cell proliferation, antioxidant and ARE activities, Western blotting of Nrf2 protein, and qPCR of mRNA of Nrf2 were conducted for Rb1, Rg1, and 20S as well as the combinations of 20S with Rb1 or Rg1. To determine the combination effects, the combination index (CI) was calculated. Rb1 and Rg1 are relatively nontoxic to the cells, while 20S at 50 μM or above significantly inhibited the cell proliferation. Rb1, Rg1, or 20S induced total antioxidant activity and ARE activity in a concentration-dependent manner. Furthermore, combinations of 20S with either Rb1 or Rg1 induced total antioxidant and ARE activity synergistically. The induction of Nrf2 protein and mRNA was also found to be synergistic with the combination treatments. In summary, in this study, we show that ginsenosides Rb1, Rg1, and 20S possess antioxidant activity, transcriptionally activating ARE as well as the potential of synergistic activities. The Nrf2-ARE-mediated antioxidant pathway could play a role for the overall antioxidative stress activities, which could be important for ginseng's health beneficial effects such as cancer chemopreventive activities.

Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis)

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2′,3′-dihydroxy-4′,6′-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.

Standardized extract of Bacopa monniera (BESEB CDRI‐08) attenuates contextual associative learning deficits in the aging rat's brain induced by D‐galactose

In this study, we examined the neuroprotective effect of standardized Bacopa monniera extract (BME: BESEB CDRI-08) against the D-galactose (D-gal)-induced brain aging in rats. Experimental groups were subjected to contextual-associative learning task. We found that the administration of BME in the D-gal-treated group attenuated contextual-associative learning deficits; the individuals showed more correct responses and retrieved the reward with less latency. Subsequent analysis showed that the BME administration significantly decreased advance glycation end product (AGE) in serum and increased the activity of antioxidant response element (ARE) and the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and nuclear transcription factor NF-E2-related factor 2 (Nrf2), accompanied by a reduction in the level of serotonin (5-HT) in the hippocampus. The BME treatment also reversed D-gal-induced brain aging by upregulating the levels of the presynaptic proteins synaptotagmin I (SYT1) and synaptophysin (SYP) and the postsynaptic proteins Ca(2+) /calmodulin dependent protein kinase II (αCaMKII) and postsynaptic density protein-95 (PSD-95) in the hippocampus during synaptic plasticity. A significant finding is that the D-gal- + BME-treated rats exhibited more correct responses in contextual-associative learning than D-gal alone-treated rats. Our findings suggest that BME treatment attenuates D-gal-induced brain aging and regulates the level of antioxidant enzymes, Nrf2 expression, and the level of 5-HT, which was accompanied by concomitantly increased levels of synaptic proteins SYT1, SYP, αCaMKII, p-αCaMKII, and PSD-95.

Induction of lung glutathione and glutamylcysteine ligase by 1,4-phenylenebis(methylene)selenocyanate and its glutathione conjugate: Role of nuclear factor-erythroid 2-related factor 2

The synthetic organoselenium agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG) are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamylcysteine ligase (GCL), through activation of antioxidant response elements (AREs) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular-related kinase (220%), and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), whereas the naturally occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE-reporter cells up to threefold for p-XSC and greater than or equal to fivefold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc, and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2. The dependence of GCL induction on Nrf2 was confirmed in Nrf2-deficient mouse embryonic fibroblasts, in which p-XSeSG induced GCL subunits in wild-type but not in Nrf2-deficient cells (P<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious, inducer of GSH.

Procyanidins from Wild Grape (Vitis amurensis) Seeds Regulate ARE-Mediated Enzyme Expression via Nrf2 Coupled with p38 and PI3K/Akt Pathway in HepG2 Cells

Procyanidins, polymers of flavan-3-ol units, have been reported to exhibit many beneficial health effects such as antioxidant and anti-carcinogenic effects. In this study, we investigated the cancer chemopreventive properties of procyanidins from wild grape (Vitis amurensis) seeds in particular their roles in inducing phase II detoxifying/antioxidant enzymes as well as in modulating the upstream kinases. Ethanolic extract of V. amurensis seeds was fractionated with a series of organic solvents and finally separated into six fractions, F1–F6. Chemical properties of the procyanidins were analyzed by vanillin assay, BuOH-HCl test, and depolymerization with phloroglucinol followed by LC/MS analysis. The F5 had the highest procyanidin content among all the fractions and strongly induced the reporter activity of antioxidant response element as well as the protein expression of nuclear factor E2-related factor (Nrf2) in HepG2 human hepatocarcinoma cells. The procyanidin-rich F5 also strongly induced the expression of the phase II detoxifying and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase1 and hemeoxygenase1. Phosphorylations of the upstream kinases such as MAPKs and PI3K/Akt were significantly increased by treatment with procyanidin fraction. In addition, the procyanidin-mediated Nrf2 expression was partly attenuated by PI3K inhibitor LY294002, and almost completely by p38 inhibitor SB202190, but neither by JNK inhibitor SP600125 nor by MEK1/2 inhibitor U0126. Taken together, the procyanidins from wild grape seeds could be used as a potential natural chemopreventive agent through Nrf2/ARE-mediated phase II detoxifying/antioxidant enzymes induction via p38 and PI3K/Akt pathway.

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.

Anti-inflammatory effects of Lacto-Wolfberry in a mouse model of experimental colitis

To investigate the anti-inflammatory properties of Lacto-Wolfberry (LWB), both in vitro and using a mouse model of experimental colitis. The effects of LWB on lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) and interleukin (IL)-6 secretion were assessed in a murine macrophage cell line. in vitro assessment also included characterizing the effects of LWB on the activation of NF-E2 related 2 pathway and inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB (NF-κB) activation, utilizing reporter cell lines. Following the in vitro assessment, the anti-inflammatory efficacy of an oral intervention with LWB was tested in vivo using a preclinical model of intestinal inflammation. Multiple outcomes including body weight, intestinal histology, colonic cytokine levels and anti-oxidative measures were investigated. LWB reduced the LPS-mediated induction of ROS production [+LPS vs 1% LWB + LPS, 1590 ± 188.5 relative luminescence units (RLU) vs 389 ± 5.9 RLU, P < 0.001]. LWB was more effective than wolfberry alone in reducing LPS-induced IL-6 secretion in vitro (wolfberry vs 0.5% LWB, 15% ± 7.8% vs 64% ± 5%, P < 0.001). In addition, LWB increased reporter gene expression via the anti-oxidant response element activation (wolfberry vs LWB, 73% ± 6.9% vs 148% ± 28.3%, P < 0.001) and inhibited the TNF-α-induced activation of the NF-κB pathway (milk vs LWB, 10% ± 6.7% vs 35% ± 3.3%, P < 0.05). Furthermore, oral supplementation with LWB resulted in a reduction of macroscopic (-LWB vs +LWB, 5.39 ± 0.61 vs 3.66 ± 0.59, P = 0.0445) and histological scores (-LWB vs +LWB, 5.44 ± 0.32 vs 3.66 ± 0.59, P = 0.0087) in colitic mice. These effects were associated with a significant decrease in levels of inflammatory cytokines such as IL-1β (-LWB vs +LWB, 570 ± 245 μg/L vs 89 ± 38 μg/L, P = 0.0106), keratinocyte-derived chemokine/growth regulated protein-α (-LWB vs +LWB, 184 ± 49 μg/L vs 75 ± 20 μg/L, P = 0.0244), IL-6 (-LWB vs +LWB, 318 ± 99 μg/L vs 117 ± 18 μg/L, P = 0.0315) and other pro-inflammatory proteins such as cyclooxygenase-2 (-LWB vs +LWB, 0.95 ± 0.12 AU vs 0.36 ± 0.11 AU, P = 0.0036) and phosphorylated signal transducer and activator of transcription-3 (-LWB vs +LWB, 0.51 ± 0.15 AU vs 0.1 ± 0.04 AU, P = 0.057). Moreover, antioxidant biomarkers, including expression of gene encoding for the glutathione peroxidase, in the colon and the plasma anti-oxidant capacity were significantly increased by supplementation with LWB (-LWB vs +LWB, 1.2 ± 0.21 mmol/L vs 2.1 ± 0.19 mmol/L, P = 0.0095). These results demonstrate the anti-inflammatory properties of LWB and suggest that the underlying mechanism is at least in part due to NF-κB inhibition and improved anti-oxidative capacity.

Guggulsterone induces heme oxygenase-1 expression through activation of Nrf2 in human mammary epithelial cells: PTEN as a putative target

Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione] is a phytosterol found in the gum resin of the Commiphora mukul. GS exists naturally in two stereoisomers: E-GS (cis-GS) and Z-GS (trans-GS). In this study, the effects of both isomers on expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) were evaluated in human mammary epithelial (MCF10A) cells. NF-E2-related factor 2 (Nrf2) is considered a master regulator in activating antioxidant response element (ARE)-driven expression of HO-1 and many other antioxidant/cytoprotective proteins. cis-GS upregulated the transcription and protein expression of HO-1 to a greater extent than did trans-GS. cis-GS treatment enhanced nuclear translocation and ARE-binding activity of Nrf2. MCF10A cells transfected with an ARE luciferase construct exhibited significantly elevated Nrf2 transcriptional activity upon cis-GS treatment compared with cells transfected with the control vector. In addition, silencing of the Nrf2 gene abrogated cis-GS-induced expression of HO-1. Incubation of MCF10A cells with cis-GS increased phosphorylation of Akt. The pharmacological inhibition of phosphoinositide 3-kinase (PI3K), an upstream kinase responsible for Akt phosphorylation, abrogated cis-GS-induced Nrf2 nuclear translocation. Pretreatment with the thiol-reducing agents attenuated Akt phosphorylation, Nrf2 activation and HO-1 expression, suggesting that cis-GS may cause thiol modification of an upstream signaling modulator. Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN) is a negative regulator of the PI3K-Akt axis. The mutation in cysteine 124 present in the catalytic domain of PTEN abolished cis-GS-induced HO-1 expression as well as Akt phosphorylation. Whether this cysteine is a 'bona fide' target of cis-GS in its activation of Nrf2 needs additional investigation.

Novel insights into the regulation of antioxidant-response-elementmediated gene expression by electrophiles: induction of the transcriptional repressor BACH1 by Nrf2

A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the ARE (antioxidant-response element), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid 2-related factor 2) that functions as a transcriptional activator via heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is relatively well understood, the mechanisms by which ARE-mediated signalling is down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to ARE-like sequences, functioning as a transcriptional repressor in a subset of ARE-regulated genes, thus antagonizing the activator function of Nrf2. In the present study, we have demonstrated that BACH1 itself is regulated by Nrf2 as it is induced by Nrf2 overexpression and by Nrf2-activating agents in an Nrf2-dependent manner. Furthermore, a functional ARE site was identified at +1411 from the transcription start site of transcript variant 2 of BACH1. We conclude that BACH1 is a bona fide Nrf2 target gene and that induction of BACH1 by Nrf2 may serve as a feedback-inhibitory mechanism for ARE-mediated gene regulation.