Nrf2 Transactivation Research Articles

Lansoprazole protects hepatic cells against cisplatin-induced oxidative stress through the p38 MAPK/ARE/Nrf2 pathway.

Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver.

Kaempferol ameliorates palmitate-induced lipid accumulation in HepG2 cells through activation of the Nrf2 signaling pathway.

Kaempferol (KMF), has beneficial effects against hepatic lipid accumulation. In this study, we aimed to investigate molecular mechanism underlying the protective effect of KMF on lipid accumulation. HepG2 cells were treated with different concentrations of KMF and 0.5mM palmitate (PA) for 24 h. The mRNA and protein levels of genes involved in lipid metabolism were evaluated using real-time PCR and western blot. The expression of Nrf2 was silenced using siRNA. Data indicated that KMF (20μM) reversed PA-induced increased triglyceride (TG) levels and total lipid content. These effects were accompanied by down-regulation of the mRNA and protein levels of lipogenic genes (FAS, ACC and SREBP1), and up-regulation of genes related to fatty acid oxidation (CPT-1, HADHα and PPARα). Kaempferol significantly decreased the levels of the oxidative stress markers (ROS and MDA) and enhanced the activities of antioxidant enzymes SOD and GPx in PA-challenged cells. Luciferase analysis showed that KMF increased the transactivation of Nrf2 in hepatocytes. The results also revealed that KMF-mediated activation of Nrf2 target genes was suppressed by Nrf2 siRNA. Furthermore, Nrf2 siRNA abolished the KMF-induced reduction in ROS and MDA levels in PA treated cells. In addition, the inhibitory effect of KMF on TG levels and the mRNA and protein levels of FAS, ACC and SREPB-1 were significantly abolished by Nrf2 inhibition. Nrf2 inhibition also suppressed the KMF-induced activation of genes involved in β oxidation (CPT-1 and PPAR-α). The results suggest that KMF protects HepG2 cells from PA-induced lipid accumulation via activation of the Nrf2 signaling pathway.

A major role for Nrf2 transcription factors in cell transformation by KSHV encoded oncogenes.

Kaposi’s sarcoma (KS) is the most common tumor in AIDS patients. The highly vascularized patient’s skin lesions are composed of cells derived from the endothelial tissue transformed by the KSHV virus. Heme oxygenase-1 (HO-1) is an enzyme upregulated by the Kaposi´s sarcoma-associated herpesvirus (KSHV) and highly expressed in human Kaposi Sarcoma (KS) lesions. The oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR) is expressed by the viral genome in infected cells. It is involved in KS development, HO-1 expression, and vascular endothelial growth factor (VEGF) expression. vGPCR induces HO-1 expression and HO-1 dependent transformation through the Ga13 subunit of heterotrimeric G proteins and the small GTPase RhoA. We have found several lines of evidence supporting a role for Nrf2 transcription factors and family members in the vGPCR-Ga13-RhoA signaling pathway that converges on the HO-1 gene promoter. Our current information assigns a major role to ERK1/2MAPK pathways as intermediates in signaling from vGPCR to Nrf2, influencing Nrf2 translocation to the cell nucleus, Nrf2 transactivation activity, and consequently HO-1 expression. Experiments in nude mice show that the tumorigenic effect of vGPCR is dependent on Nrf2. In the context of a complete KSHV genome, we show that the lack of vGPCR increased cytoplasmic localization of Nrf2 correlated with a downregulation of HO-1 expression. Moreover, we also found an increase in phospho-Nrf2 nuclear localization in mouse KS-like KSHV (positive) tumors compared to KSHV (negative) mouse KS-like tumors. Our data highlights the fundamental role of Nrf2 linking vGPCR signaling to the HO-1 promoter, acting upon not only HO-1 gene expression regulation but also in the tumorigenesis induced by vGPCR. Overall, these data pinpoint this transcription factor or its associated proteins as putative pharmacological or therapeutic targets in KS.

Changes in uridine 5'-diphospho-glucuronosyltransferase 1A6 expression by histone deacetylase inhibitor valproic acid.

Valproic acid (VPA) is well-known as a histone deacetylase (HDAC) inhibitor. It has been reported that HDAC inhibitors enhance basal and aryl hydrocarbon receptor (AhR) ligand-induced aryl hydrocarbon receptor-responsive gene expression. Other studies suggested that HDAC inhibition might significantly activate the NF-E2-related factor-2 (Nrf2). Moreover, VPA activates mitogen-activated protein kinases (MAPKs). MAPK pathways regulate Nrf2 transactivation domain activity. Uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A6 is one of the important isoforms to affect drug pharmacokinetics. UGT1A6 gene is regulated transcriptionally by AhR and Nrf2. The present study aimed to investigate whether UGT1A6 expression was changed by VPA and to elucidate the mechanism of the alteration. Following VPA treatment for 72h in Caco-2 cells, UGT1A6 mRNA was increased by 7.9-fold. Moreover, UGT1A6 mRNA was increased by other HDAC inhibitors, suggesting that HDAC inhibition caused the UGT1A6 mRNA induction. AhR and Nrf2 proteins in the nucleus of Caco-2 cells were increased by 1.5- and 1.7-fold, respectively, following the VPA treatment. However, VPA treatment did not activate the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in Caco-2 cells. In conclusion, we observed that VPA induced UGT1A6 mRNA expression via AhR and Nrf2 pathways, but not via the ERK or JNK pathways.

Tubule-mitophagic secretion of SerpinG1 reprograms macrophages to instruct anti-septic acute kidney injury efficacy of high-dose ascorbate mediated by NRF2 transactivation.

High-dose ascorbate confers tubular mitophagy responsible for septic acute kidney injury (AKI) amelioration, yet its biological roles in immune regulation remain poorly understood.Methods: The role of tubular mitophagy in macrophage polarization upon high-dose ascorbate treatment was assessed by fluorescence-activated cell sorter analysis (FACS) in vitro and by immunofluorescence in AKI models of LPS-induced endotoxemia (LIE) from Pax8-cre; Atg7flox/flox mice. The underlying mechanisms were revealed by RNA-sequencing, gene set enrichment analysis (GSEA), luciferase reporter, chromatin immunoprecipitation (ChIP) and adeno-associated viral vector serotype 9 (AAV9) delivery assays.Results: High-dose ascorbate enables conversion of macrophages from a pro-inflammatory M1 subtype to an anti-inflammatory M2 subtype in murine AKI models of LIE, leading to decreased renal IL-1β and IL-18 production, reduced mortality and alleviated tubulotoxicity. Blockade of tubular mitophagy abrogates anti-inflammatory macrophages polarization under the high-dose ascorbate-exposed coculture systems. Similar abrogations are verified in LIE mice with tubular epithelium-specific ablation of Atg7, where the high-dose ascorbate-inducible renal protection and survival improvement are substantially weaker than their control littermates. Mechanistically, high-dose ascorbate stimulates tubular secretion of serpin family G member 1 (SerpinG1) through maintenance of mitophagy, for which nuclear factor-erythroid 2 related factor 2 (NRF2) transactivation is required. SerpinG1 perpetuates anti-inflammatory macrophages to prevent septic AKI, while kidney-specific disruption of SerpinG1 by adeno-associated viral vector serotype 9 (AAV9)-short hairpin RNA (shRNA) delivery thwarts the anti-inflammatory macrophages polarization and anti-septic AKI efficacy of high-dose ascorbate.Conclusion: Our study identifies SerpinG1 as an intermediate of tubular mitophagy-orchestrated myeloid function during septic AKI and reveals a novel rationale for ascorbate-based therapy.

SRC-3 Deficiency Exacerbates Neurological Deficits in a Mouse Model of Intracerebral Hemorrhage: Role of Oxidative Stress.

Intracerebral hemorrhage (ICH) causes long term neurological abnormality or death. Oxidative stress is closely involved in ICH mediated brain damage. Steroid receptor cofactor 3 (SRC-3), a p160 family member, is widely expressed in the brain and regulates transactivation of Nrf2, a key component of antioxidant response. Our study aims to test if SRC-3 is implicated in ICH mediated brain injury. We first examined levels of SRC-3 and oxidative stress in the brain of mice following ICH and analyzed their correlation. Then ICH was induced in wild type (WT) and SRC-3 knock out mice and how SRC-3 deletion affected ICH induced brain damage, oxidative stress and behavioral outcome was assessed. We found that SRC-3 mRNA and protein expression levels were reduced gradually after ICH induction in WT mice along with an increase in oxidative stress levels. Correlation analysis revealed that SRC-3 mRNA levels negatively correlated with oxidative stress. Deletion of SRC-3 further increased ICH induced brain edema, neurological deficit score and oxidative stress and exacerbated ICH induced behavioral abnormality including motor dysfunction and cognitive impairment. Our findings suggest that SRC-3 is involved in ICH induced brain injury, probably through modulation of oxidative stress.

Ellagic acid improved diabetes mellitus-induced testicular damage and sperm abnormalities by activation of Nrf2

Diabetes mellitus induces testicular damage, increases sperm abnormalities, and impairs reproductive dysfunction due to induction of endocrine disturbance and testicular oxidative stress. This study evaluated the reproductive protective effect of ellagic acid (EA) against testicular damage and abnormalities in sperm parameters in Streptozotocin (STZ)-induced diabetic rats (T1DM) and examined some possible mechanisms of protection. Adult male rats were segregated into 5 groups (n = 12 rat/each) as control, control + EA (50 mg/kg/day), T1DM, T1DM + EA, and T1DM + EA + brusatol (an Nrf-2 inhibitor) (2 mg/twice/week). All treatments were conducted for 12 weeks, daily. EA preserved the structure of the seminiferous tubules, prevented the reduction in sperm count, motility, and viability, reduced sperm abnormalities, and downregulated testicular levels of cleaved caspase-3 and Bax in diabetic rats. In the control and diabetic rats, EA significantly increased the circulatory levels of testosterone, reduced serum levels of FSH and LH, and upregulated Bcl-2 and all steroidogenic genes (StAr, 3β-HSD1, and 11β-HSD1). Besides, it reduced levels of ROS and MDA but increased levels of GSH and MnSOD and the transactivation of Nrf2. All these biochemical alterations induced by EA were associated with increased activity and nuclear accumulation of Nrf2. However, all these effects afforded by EA were weakened in the presence of brusatol. In conclusion, EA could be an effective therapy to alleviated DM-induced reproductive toxicity and dysfunction in rats by a potent antioxidant potential mediated by the upregulation of Nrf2.

Pyridoxine induces glutathione synthesis via PKM2-mediated Nrf2 transactivation and confers neuroprotection

Oxidative stress is a major pathogenic mechanism in Parkinson’s disease (PD). As an important cellular antioxidant, glutathione (GSH) balances the production and incorporation of free radicals to protect neurons from oxidative damage. GSH level is decreased in the brains of PD patients. Hence, clarifying the molecular mechanism of GSH deficiency may help deepen our knowledge of PD pathogenesis. Here we report that the astrocytic dopamine D2 receptor (DRD2) regulates GSH synthesis via PKM2-mediated Nrf2 transactivation. In addition we find that pyridoxine can dimerize PKM2 to promote GSH biosynthesis. Further experiments show that pyridoxine supplementation increases the resistance of nigral dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in wild-type mice as well as in astrocytic Drd2 conditional knockout mice. We conclude that dimerizing PKM2 may be a potential target for PD treatment.

Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,β-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Antioxidant and Anti-Inflammatory Effects of Herbal Formula SC-E3 in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

SC-E3 is a novel herbal formula composed of five oriental medicinal herbs that are used to treat a wide range of inflammatory diseases in Korean traditional medicine. In this study, we sought to determine the effects of SC-E3 on free radical generation and inflammatory response in lipopolysaccharide- (LPS-) treated RAW 264.7 macrophages and the molecular mechanism involved. The ethanol extract of SC-E3 showed good free radical scavenging activity and inhibited LPS-induced reactive oxygen species generation. SC-E3 significantly inhibited the production of the LPS-induced inflammatory mediators, nitric oxide and prostaglandin E2, by suppressing the expressions of inducible nitric oxide synthase and cyclooxygenase-2, respectively. SC-E3 also prevented the secretion of the proinflammatory cytokines, IL-1β, TNF-α, and IL-6, and inhibited LPS-induced NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Furthermore, SC-E3 induced the expression of heme oxygenase-1 (HO-1) by promoting the nuclear translocation and transactivation of Nrf2. Taken together, these results suggest that SC-E3 has potent antioxidant and anti-inflammatory effects and that these effects are due to the inhibitions of NF-κB and MAPK and the induction of Nrf2-mediated HO-1 expression in macrophages. These findings provide scientific evidence supporting the potential use of SC-E3 for the treatment and prevention of various inflammatory diseases.

Nitrosopersulfide (SSNO−) targets the Keap-1/Nrf2 redox system

Nitric oxide (NO), hydrogen sulfide and polysulfides have been proposed to contribute to redox signaling by activating the Keap-1/Nrf2 stress response system. Nitrosopersulfide (SSNO−) recently emerged as a bioactive product of the chemical interaction of NO or nitrosothiols with sulfide; upon decomposition it generates polysulfides and free NO, triggering the activation of soluble guanylate cyclase, inducing blood vessel relaxation in vitro and lowering blood pressure in vivo. Whether SSNO− itself interacts with the Keap-1/Nrf2 system is unknown. We therefore sought to investigate the ability of SSNO− to activate Nrf2-dependent processes in human vascular endothelial cells, and to compare the pharmacological effects of SSNO− with those of its precursors NO and sulfide at multiple levels of target engagement. We here demonstrate that SSNO− strongly increases nuclear levels, binding activity and transactivation activity of Nrf2, thereby increasing mRNA expression of Hmox-1, the gene encoding for heme oxygenase 1, without adversely affecting cell viability. Under all conditions, SSNO− appeared to be more potent than its parent compounds, NO and sulfide. SSNO−-induced Nrf2 transactivation activity was abrogated by either NO scavenging with cPTIO or inhibition of thiol sulfuration by high concentrations of cysteine, implying a role for both persulfides/polysulfides and NO in SSNO− mediated Nrf2 activation. Taken together, our studies demonstrate that the Keap-1/Nrf2 redox system is a biological target of SSNO−, enriching the portfolio of bioactivity of this vasoactive molecule to also engage in the regulation of redox signaling processes. The latter suggests a possible role as messenger and/or mediator in cellular sensing and adaptations processes.

Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence.

Oxidative stress and impaired antioxidant defense are believed to be contributors to the cardiovascular aging process. The transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis. Our previous study showed that Exendin-4, a glucagon-like peptide-1 analog, alleviates angiotensin II (ANG II)-induced vascular smooth muscle cell (VSMC) senescence by inhibiting Rac1 activation via cAMP/PKA (Zhao L, Li AQ, Zhou TF, Zhang MQ, Qin XM. Am J Physiol Cell Physiol 307: C1130-C1141, 2014). The objective of this study is to investigate if Nrf2 mediates the antisenescent effect of Exendin-4 in ANG II-induced VSMCs. Here we report that Exendin-4 triggered Nrf2 nuclear translocation, a downstream target of cAMP-responsive element-binding protein (CREB) and expressions of antioxidant genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1) in a dose- and time-dependent manner. In addition, knock-down of Nrf2 attenuated the inhibitory effects of Exendin-4 on ANG II-induced superoxidant generation and VSMC senescence. PKA/CREB pathway participated in the upregulations of HO-1 and NQO-1 induced by Exendin-4. Notably, our study revealed that Exendin-4 dose-dependently increased the acetylation of Nrf2 and the recruitment of transcriptional coactivator CREB binding protein (CBP) to Nrf2. The Exendin-4-induced Nrf2 transactivation was diminished in the presence of CBP small interfering RNA. Microscope imaging of Nrf2, as well as immunoblotting for Nrf2, showed that the Exendin-4-evoked Nrf2 acetylation favored its nuclear retention. Importantly, CBP silencing attenuated the suppressing effects of Exendin-4 on ANG II-induced VSMC senescence and superoxidant production. In conclusion, these results provide a mechanistic insight into how Nrf2 signaling mediates the antisenescent and antioxidative effects induced by Exendin-4 in VSMCs.

In vitro protective effects of Hoveniae Semen cum Fructus extracts against oxidative stress

We evaluated the in vitro cytoprotective effects of Hoveniae Semen cum Fructus (HSCF) extracts against oxidative stress-mediated cell damage using HepG2 cells. Cytotoxic effects of HSCF extracts were observed in HepG2 cells, and the 50% inhibitory concentration was determined. Cytoprotective effects of sublethal doses of HSCF extracts were evaluated using a tert-butyl hydroperoxide (tBHP)-induced cellular damage model. We also assessed whether NFE2-related factor-2 (Nrf2) was transactivated by HSCF extracts. The antioxidant capacity of HSCF extracts was evaluated with superoxide dismutase (SOD) and catalase (CAT) activities and the expression of the antioxidant genes glutamate cysteine ligase catalytic subunit (GCLC), hemeoxygenase-1 (HO1), and NAD (P)H dehydrogenase quinone 1 (NQO1). HSCF extracts up to 1,000 μg/mL showed no cytotoxic effect in HepG2 cells. Indeed, 300 and 1,000 μg/mL of HSCF extracts significantly protected HepG2 cells from oxidative stress-mediated cell death by tBHP. As a molecular mechanism, HSCF extracts at 1,000 μg/mL significantly increased Nrf2 transactivation and induced expression of its target genes (GCLC, HO1, NQO1). Furthermore, 1,000 μg/mL of HSCF extracts enhanced SOD activity. Although treatment with 300 and 1,000 μg/mL of HSCF extracts tended to slightly increase CAT activity, the increases were not statistically significant. These findings provide direct evident that HSCF extracts have favorable hepatoprotective effects against oxidative stress through Nrf2-mediated antioxidant gene induction.

Abstract 133: Impaired TNFα Signaling Deregulates Basal Nrf2-Antioxidant System in Myocardium

Antagonizing TNF-α and its signaling is proven to be a successful therapeutic strategy in various disease processes. Although blocking TNF-α signaling attenuated the disease activity, the overarching clinical contraindications remains to be a greater concern and reasons for the adverse effects are largely unknown. TNF-α induces ROS and as ROS is pivotal for activation of antioxidant transcription factor, Nrf2, we posit that any impairment in TNF-α signaling could affect Nrf2-dependent basal redox homeostasis. HL-1 cardiomyocytes, TNFR1/2 double knockout mice (DKO) that shows hampered TNF-α signaling were used as experimental models. Electron Paramagnetic Resonance Spectroscopy (EPR) measurements revealed that as low as 2 ng TNF-α/ml, ROS was induced significantly (p10ng/ml in HL-1 cells. TNF-α (2-5 ng /ml, for 2 h) evoked robust (p<0.05, n=3) nuclear translocation of Nrf2 and increased nuclear binding (Trans AM DNA binding analysis) along with significant (p<0.05) induction in trans-activation of Nrf2 targets. Additionally, this was associated with 2-fold increase (p<0.05) in intracellular glutathione (GSH) that is associated with significant cell death only at higher (>10 - 50 ng/ml) and later time points (12 h and 24 h). These results suggest that TNF-α mediated ROS induction activates Nrf2 dependent antioxidant system in cardiomyocytes. In vivo experiments with TNFR1/2 DKO demonstrates that the expression of Nrf2-regulated genes were significantly downregulated indicating a weakened antioxidant system. Also, the acute exercise stress (AES) induced Nrf2 trans-activation observed in WT mice was significantly blunted in TNFR1/2 DKO suggesting a need for TNF signaling in stress induced Nrf2 activation. These results support the concept that complete and/or sustained blockade of TNF signaling may result in compromised Nrf2-dependent antioxidant defense in the myocardium. This further necessitates that during chronic anti-TNF-α treatment, a finely tuned and maintenance of specific threshold of TNF-α signaling is essential to avoid oxidant stress based complications in cardiovascular system.

Dietary extra-virgin olive oil and corn oil differentially modulate the mRNA expression of xenobiotic-metabolizing enzymes in the liver and in the mammary gland in a rat chemically induced breast cancer model.

High extra-virgin olive oil (EVOO) and corn oil diets differentially modulate experimental mammary carcinogenesis. We have investigated their influence on the initiation stage through the modulation of the expression of xenobiotic-metabolizing enzymes (XMEs) in the liver and the mammary gland. Female Sprague-Dawley rats were fed a low-fat (LF), high corn oil (HCO), or high EVOO (HOO) diet from weaning and gavaged with 7,12-dimethylbenz(a)anthracene (DMBA). The HCO diet increased the mRNA levels of the phase I enzymes CYP1A1, CYP1A2 and, to a lesser extent, CYP1B1, in the liver. The Aryl hydrocarbon receptor (AhR) seemed to be involved in this upregulated CYP1 expression. However, a slight trend toward an increase in the mRNA levels of the phase II enzymes GSTP1 and NQO1 was observed with the HOO diet. At least in the case of GSTP1, this effect was linked to an increased Nrf2 transactivation activity. This different regulation of the XMEs expression led, in the case of the HCO diet, to a balance between the production of active carcinogenic compounds and their inactivation tilted toward phase I, which would stimulate DMBA-induced cancer initiation, whereas the HOO diet was associated with a slower phase I metabolism accompanied by a faster phase II detoxification, thus reducing the output of the active compounds to the target tissues. In the mammary gland, the differential effects of diets may be conditioned by the state of cell differentiation, sexual maturity, and hormone metabolism.

Mechanisms underlying physiological functions of food factors via non-specific interactions with biological proteins

We previously reported that zerumbone, a sesquiterpene found in Zingiber zerumbet SMITH, showed notable cancer preventive effects in various organs of experimental rodents. This agent up-regulated nuclear factor-E2-related factor (Nrf2)-dependent expressions of anti-oxidative and xenobiotics-metabolizing enzymes, leading to an increased self-defense capacity. On the other hand, zerumbone markedly suppressed the expression of cyclooxygenase-2, an inducible pro-inflammatory enzyme, by disrupting mRNA stabilizing processes. Binding experiments using a biotin derivative of zerumbone demonstrated that Keap1, an Nrf2 repressive protein, is one of its major binding proteins that promotes their dissociation for inducing Nrf2 transactivation. We then generated a specific antibody against zerumbone-modified proteins and found that zerumbone modified numerous cellular proteins in a non-specific manner, with global distribution of the modified proteins seen not only in cytoplasm but also the nucleus. Based on those observations, zerumbone was speculated to cause proteo-stress, a notion supported by previous findings that it increased the C-terminus of Hsc70 interacting protein-dependent protein ubiquitination and also promoted aggresome formation. Interestingly, zerumbone counteracted proteo-stress and heat stress via up-regulation of the protein quality control systems (PQCs), e.g., heat shock proteins (HSPs), ubiquitin-proteasome, and autophagy. Meanwhile, several phytochemicals, including ursolic acid and curcumin, were identified as marked HSP70 inducers, whereas most nutrients tested were scarcely active. Recent studies have revealed that PQCs play important roles in the prevention of many lifestyle related diseases, such as cancer, thus non-specific binding of phytochemicals to cellular proteins may be a novel and unique mechanism underlying their physiological activities.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation.

Tryptanthrin protects hepatocytes against oxidative stress via activation of the extracellular signal-regulated kinase/NF-E2-related factor 2 pathway.

Tryptanthrin [6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline], originally isolated from Isatidis radix, has been characterized as having anti-microbial and anti-tumor activities. It is well-known that excess oxidative stress is one of the major factors causing cell damage in the liver. This study investigated the cytoprotective effects and molecular mechanism of tryptanthrin against tert-butyl hydroperoxide (tBHP)-induced oxidative stress in human hepatocyte-derived HepG2 cells. Tryptanthrin pre-treatment blocked the reactive oxygen species production, mitochondrial dysfunction, and cell death induced by tBHP. Moreover, tryptanthrin reversed tBHP-induced GSH reduction. This study also confirmed the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by tryptanthrin as a plausible molecular mechanism for its cytoprotective effects. Specifically, tryptanthrin treatment induced nuclear translocation and transactivation of Nrf2 as well as phosphorylation of extracellular signal-regulated kinase (ERK), a potential upstream kinase of Nrf2. Tryptanthrin also up-regulated the expression of the heme oxygenase 1 and glutamate-cysteine ligase catalytic subunits, which are representative target genes of Nrf2. Moreover, inhibitor of ERK was used to verify the important role of the ERK-Nrf2 pathway in the hepatoprotective effects of tryptanthrin. In conclusion, this study demonstrated that tryptanthrin protects hepatocytes against oxidative stress through the activation of the ERK/Nrf2 pathway in HepG2 cells.

S-allyl cysteine protects against MPTP-induced striatal and nigral oxidative neurotoxicity in mice: Participation of Nrf2

The neuroprotective properties of S-allyl cysteine (SAC) have been demonstrated in different neurotoxic paradigms, and it may be partially attributable to its antioxidant and anti-inflammatory profile. Recently, SAC has also been shown to induce neuroprotection in the rat striatum in a toxic model induced by 6-hydroxydopamine in rats through a concerted antioxidant response involving Nrf2 transcription factor nuclear transactivation and Phase 2 enzymes' upregulation. In this work, we investigated whether the SAC-induced in vivo striatal and nigral neuroprotection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridinium (MPTP) toxicity recruits Nrf2 transactivation in C57BL/6J mice. SAC (120 mg/kg, i.p. × 5 days) partially ameliorated the MPTP (30 mg/kg, i.p. × 5 days)-induced striatal and nigral dopamine and tyrosine hydroxylase depletion, attenuated the loss of Mn-SOD and HO-1 activities, and preserved the protein content of these enzymes. While no significant changes were detected for the striatal Nrf2 nuclear protein levels, the nigral Nrf2 nuclear content was decreased by MPTP and stimulated by SAC. Our findings suggest that SAC can exert neuroprotection since the origin of the dopaminergic lesion—at the substantia nigra (SN)—not only by means of direct antioxidant actions, but also through Nrf2 nuclear transactivation and Phase 2 enzymes upregulation.