Redox-sensitive Transcription Factor Nrf2 Research Articles

Differential roles for the redox sensitive transcription factor Nrf2 in carcinogenesis

Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor regulating the expression of a battery of genes encoding antioxidant and carcinogen detoxifying enzymes. In contrast to its tumor suppressive functions in normal cells, Nrf2 facilitates tumor growth and progression through metabolic reprograming in some cancer cells. Our previous study has demonstrated that 15-deoxy-Δ12,14-prostaglandin J2 and 4-hydroxyestradiol induce overactivation of Nrf2 and consequently overexpression of its target protein, heme oxygenase-1 (HO-1), in human breast cancer cells. More recently, we have demonstrated the involvement of Nrf2 in experimentally induced hepatocarcinogenesis (N.H.K. Chi et al., Cancer Res., 2017). In this context, the cellular stress response or cytoprotective signaling mediated via the Nrf2-HO-1 axis is hijacked by cancer cells. This may facilitate the remodeling of the tumor microenvironment making it advantageous for the autonomic growth of cancer cells, metastasis, angiogenesis, tolerance to anticancer therapy, and self-renewal activity of stem-like cells.

P-288 - Nrf2 redox signalling in brain endothelial cells adapted to physiological O2: an in vitro model for ischaemia-reperfusion injury in stroke

Sulforaphane (SFN) has been shown to protect against damage sustained following transient ischaemic stroke via activation of the redox-sensitive transcription factor Nrf2. To further understand the protective effects of SFN on the cerebral endothelium, mouse brain endothelial cell line bEnd.3 was subjected to oxygen-glucose deprivation (OGD), an in vitro model of cerebral stroke. Pre-treatment of bEnd.3 cells with SFN (2.5 µM, 12 h) afforded increased protection against OGD induced cell death. Our lab recently reported that long-term culture under physiological oxygen, 5% O2, results in a phenotype markedly different to cells cultured under ambient air, 18% O2, shown by downregulation of selected Nrf2 target genes by Nrf2 activator. In the present study, the bEnd.3 cell phenotype was determined following long-term (5 days) culture under 5% or 18% O2. Under 5% O2, the intracellular O2 content of bEnd.3 cells was 3.6% O2, and a reduced rate of cell proliferation was observed. Furthermore, bEnd.3 cells cultured under 5% O2 exhibited greater sensitivity to OGD-induced cell death. The present study highlights the importance of conducting cell culture studies under physiological O2 levels.

NRF2 facilitates breast cancer cell growth via HIF1ɑ-mediated metabolic reprogramming.

High aerobic glycolysis not only provides energy to breast cancer cells, but also supports their anabolic growth. The redox sensitive transcription factor NRF2 is over-expressed in multiple cancers, including breast cancer. It is unclear whether NRF2 could promote breast cancer cell growth through enhancing glycolysis. In this study, we found that NRF2 and HIF1α mRNA and protein levels were significantly increased in MCF-7 and MDA-MB-231 breast cancer cells as compared to MCF-10A benign breast epithelial cells. Down-regulation of NRF2 decreased MCF7 and MBA-DA-231 breast cell proliferation, while it reversed by hypoxia inducible factor 1α (HIF1α). Knockdown of NRF2 inhibited glycolysis by decreasing the expression of genes participated in glucose metabolism, including HK2, PFKFB3, PKM2 and LDHA. Our results further indicated that the AKT activation and AMPK inhibition were required for NRF2-mediated up-regulation of glycolytic enzymes. Consistent with these results, a positive correlation existed between NRF2 or HIF1α and several key glycolytic genes in human breast cancer cell samples and breast cancer patients with high NRF2 or HIF1α expression had poorer overall survival. In conclusion, our study demonstrates that NRF2 promotes breast cancer progression by enhancing glycolysis through coactivation of HIF1α, implicating that NRF2 is a potential molecular target for breast cancer treatment.

YO-05 - Long-term contribution of Nrf2 to behavioral recovery following focal cerebral ischemia-reperfusion injury

Ischemic stroke is a leading cause of death and adult disability worldwide. The redox sensitive transcription factor Nrf2 is a key target for protection in rodent stroke models. We investigated protection afforded by 5 mg/kg dietary sulforaphane in C57BL6/J WT and Nrf2-/- adult male mice subjected to 60min middle cerebral artery occlusion and 72 h or 21 days reperfusion. Behavioural outcomes were measured and brains were sectioned for high throughput imaging and infarct quantification. SFN pretreatment improved functional rotating rod and grip strength performance, significantly reduced cresyl violet infarct volumes and upregulated HO-1 and PPARγ expression in cerebrovascular endothelium. We subsequently conducted the first long-term (>14 day) follow up studies of stroke in Nrf2-deficient and in wild-type mice subjected to long-term pretreatment with SFN (5 mg/kg/day). Basal and inducible contributions of Nrf2 to alternative activation of resident microglia and recruited macrophages, long-term cytokine expression (IL-1β, IL-6, IL-10), post-infarct neurogenesis and angiogenesis and functional recovery were investigated. Our findings highlight the translational potential of Nrf2 inducers in stroke research and establish that dietary levels of sulforaphane afford protection in rodent models of focal cerebral ischaemia.

Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells.

Docosahexaenoic acid (DHA), an ω-3 fatty acid abundant in fish oils, has diverse health beneficial effects, such as anti-oxidative, anti-inflammatory, neuroprotective, and chemopreventive activities. In this study, we found that DHA induced expression of two representative antioxidant/cytoprotective enzymes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1), in human mammary epithealial (MCF-10A) cells. DHA-induced upregulation of these enzymes was accompanied by enhanced translocation of the redox-sensitive transcription factor Nrf2 into the nucleus and its binding to antioxidant response element. Nrf2 gene silencing by siRNA abolished the DHA-induced expression of HO-1 and NQO1 proteins. When MCF-10A cells were transfected with mutant constructs in which the cysteine 151 or 288 residue of Keap1 was replaced by serine, DHA-induced expression of HO-1 and NQO1 was markedly reduced. Moreover, DHA activated protein kinase C (PKC)δ and induced Nrf2 phosphorylation. DHA-induced phosphorylation of Nrf2 was abrogated by the pharmacological PKCδ inhibitor rottlerin or siRNA knockdown of its gene expression. The antioxidants N-acetyl-l-cysteine and Trolox attenuated DHA-induced activation of PKCδ, phosphorylation of Nrf2, and and its target protein expression. In conclusion, DHA activates Nrf2, possibly through modification of critical Keap1 cysteine 288 residue and PKCδ-mediated phosphorylation of Nrf2, leading to upregulation of HO-1 and NQO1 expression.

HO-1 Induction in Cancer Progression: A Matter of Cell Adaptation

The upregulation of heme oxygenase-1 (HO-1) is one of the most important mechanisms of cell adaptation to stress. Indeed, the redox sensitive transcription factor Nrf2 is the pivotal regulator of HO-1 induction. Through the antioxidant, antiapoptotic, and antinflammatory properties of its metabolic products, HO-1 plays a key role in healthy cells in maintaining redox homeostasis and in preventing carcinogenesis. Nevertheless, several lines of evidence have highlighted the role of HO-1 in cancer progression and its expression correlates with tumor growth, aggressiveness, metastatic and angiogenetic potential, resistance to therapy, tumor escape, and poor prognosis, even though a tumor- and tissue-specific activity has been observed. In this review, we summarize the current literature regarding the pro-tumorigenic role of HO-1 dependent tumor progression as a promising target in anticancer strategy.

Reductive stress caused by sustained activation of the redox-sensitive transcription factor Nrf2: Implications for cancer cell proliferation and invasiveness

Reductive stress caused by sustained activation of the redox-sensitive transcription factor Nrf2: Implications for cancer cell proliferation and invasiveness

Cardioprotective effects of Cu(II)ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

Cu(II)ATSM was developed as a hypoxia sensitive positron emission tomography agent. Recent reports have highlighted the neuroprotective properties of Cu(II)ATSM, yet there are no reports that it confers cardioprotection. We demonstrate that Cu(II)ATSM activates the redox-sensitive transcription factor Nrf2 in human coronary artery smooth muscle cells (HCASMC) and cardiac myocytes (HCM), leading to upregulation of antioxidant defense enzymes. Oral delivery of Cu(II)ATSM in mice induced expression of the Nrf2-regulated enzymes in the heart and aorta. In HCASMC, Cu(II)ATSM increased expression of the Nrf2 stabilizer DJ-1, and knockdown of Nrf2 or DJ-1 attenuated Cu(II)ATSM-mediated heme oxygenase-1 and NADPH quinone oxidoreductase-1 induction. Pre-treatment of HCASMC with Cu(II)ATSM protected against the pro-oxidant effects of angiotensin II (Ang II) by attenuating superoxide generation, apoptosis, proliferation and increases in intracellular calcium. Notably, Cu(II)ATSM-mediated protection against Ang II-induced HCASMC apoptosis was diminished by Nrf2 knockdown. Acute treatment with Cu(II)ATSM enhanced the association of DJ-1 with superoxide dismutase-1 (SOD1), paralleled by significant increases in intracellular Cu(II) levels and SOD1 activity. We describe a novel mechanism by which Cu(II)ATSM induces Nrf2-regulated antioxidant enzymes and protects against Ang II-mediated HCASMC dysfunction via activation of the Nrf2/DJ-1 axis. Cu(II)ATSM may provide a therapeutic strategy for cardioprotection via upregulation of antioxidant defenses.

Stabilization of endogenous Nrf2 by minocycline protects against Nlrp3-inflammasome induced diabetic nephropathy.

While a plethora of studies support a therapeutic benefit of Nrf2 activation and ROS inhibition in diabetic nephropathy (dNP), the Nrf2 activator bardoxolone failed in clinical studies in type 2 diabetic patients due to cardiovascular side effects. Hence, alternative approaches to target Nrf2 are required. Intriguingly, the tetracycline antibiotic minocycline, which has been in clinical use for decades, has been shown to convey anti-inflammatory effects in diabetic patients and nephroprotection in rodent models of dNP. However, the mechanism underlying the nephroprotection remains unknown. Here we show that minocycline protects against dNP in mouse models of type 1 and type 2 diabetes, while caspase -3,-6,-7,-8 and -10 inhibition is insufficient, indicating a function of minocycline independent of apoptosis inhibition. Minocycline stabilizes endogenous Nrf2 in kidneys of db/db mice, thus dampening ROS-induced inflammasome activation in the kidney. Indeed, minocycline exerts antioxidant effects in vitro and in vivo, reducing glomerular markers of oxidative stress. Minocycline reduces ubiquitination of the redox-sensitive transcription factor Nrf2 and increases its protein levels. Accordingly, minocycline mediated Nlrp3 inflammasome inhibition and amelioration of dNP are abolished in diabetic Nrf2−/− mice. Taken together, we uncover a new function of minocycline, which stabilizes the redox-sensitive transcription factor Nrf2, thus protecting from dNP.

Nrf2-dependent persistent oxidative stress results in stress-induced vulnerability to depression

Stressful life events produce a state of vulnerability to depression in some individuals. The mechanisms that contribute to vulnerability to depression remain poorly understood. A rat model of intense stress (social defeat (SD), first hit) produced vulnerability to depression in 40% of animals. Only vulnerable animals developed a depression-like phenotype after a second stressful hit (chronic mild stress). We found that this vulnerability to depression resulted from a persistent state of oxidative stress, which was reversed by treatment with antioxidants. This persistent state of oxidative stress was due to low brain-derived neurotrophic factor (BDNF) levels, which characterized the vulnerable animals. We found that BDNF constitutively controlled the nuclear translocation of the master redox-sensitive transcription factor Nrf2, which activates antioxidant defenses. Low BDNF levels in vulnerable animals prevented Nrf2 translocation and consequently prevented the activation of detoxifying/antioxidant enzymes, ultimately resulting in the generation of sustained oxidative stress. Activating Nrf2 translocation restored redox homeostasis and reversed vulnerability to depression. This mechanism was confirmed in Nrf2-null mice. The mice displayed high levels of oxidative stress and were inherently vulnerable to depression, but this phenotype was reversed by treatment with antioxidants. Our data reveal a novel role for BDNF in controlling redox homeostasis and provide a mechanistic explanation for post-stress vulnerability to depression while suggesting ways to reverse it. Because numerous enzymatic reactions produce reactive oxygen species that must then be cleared, the finding that BDNF controls endogenous redox homeostasis opens new avenues for investigation.

The Pentose Phosphate Pathway Regulates the Circadian Clock

SummaryThe circadian clock is a ubiquitous timekeeping system that organizes the behavior and physiology of organisms over the day and night. Current models rely on transcriptional networks that coordinate circadian gene expression of thousands of transcripts. However, recent studies have uncovered phylogenetically conserved redox rhythms that can occur independently of transcriptional cycles. Here we identify the pentose phosphate pathway (PPP), a critical source of the redox cofactor NADPH, as an important regulator of redox and transcriptional oscillations. Our results show that genetic and pharmacological inhibition of the PPP prolongs the period of circadian rhythms in human cells, mouse tissues, and fruit flies. These metabolic manipulations also cause a remodeling of circadian gene expression programs that involves the circadian transcription factors BMAL1 and CLOCK, and the redox-sensitive transcription factor NRF2. Thus, the PPP regulates circadian rhythms via NADPH metabolism, suggesting a pivotal role for NADPH availability in circadian timekeeping.

Monocyte chemotactic protein-1による単球/マクロファージ浸潤、遊走におけるレドックス感受性転写因子Nrf2の役割

Monocyte chemotactic protein-1による単球/マクロファージ浸潤、遊走におけるレドックス感受性転写因子Nrf2の役割

Eicosapentaenoic acid prevents TCDD-induced oxidative stress and inflammatory response by modulating MAP kinases and redox-sensitive transcription factors.

Oxidative stress and subsequent activation of inflammatory responses is a widely accepted consequence of exposure to environmental toxins. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a well-known environmental toxin, exerts its toxicity through many signalling mechanisms, with liver being the principal organ affected. However, an effective antidote to TCDD-induced toxicity is unknown. The present study evaluated the effect of eicosapentaenoic acid (EPA), an n3 fatty acid, on TCDD-induced toxicity. In cultures of HepG2 cells, the EPA/AA ratio was determined using gas chromatography, oxidative stress and inflammatory responses through reactive oxygen species (ROS) levels, antioxidant status, [Ca(2+) ]i , nuclear migration of two redox-sensitive transcription factors, NF-κB p65 and Nrf-2, expression of MAP kinase (p-Erk, p-p38), NF-κB p65, COX-2 and Nrf-2. Cellular changes in ΔΨm, acidic vesicular organelle formation, cell cycle analysis and scanning electron microscopy analysis were performed. EPA offered significant cytoprotection by increasing EPA/AA ratios in cell membranes, inhibiting ROS generation, enhancing antioxidant status and modulating nuclear translocation of redox-sensitive transcription factors (NF-κB p65 and Nrf-2) and expression of NF-κB p65, COX-2 and Nrf-2. Furthermore, TCDD-induced upstream events of MAPK phosphorylation, the increase in [Ca(2+) ]i levels and cell surface changes in microvilli were significantly inhibited by EPA. EPA treatment maintained ΔΨm and prevented formation of acidic vesicular organelles. The present study demonstrates for the first time some underlying molecular mechanisms of cytoprotection exerted by EPA against TCDD-induced oxidative stress and inflammatory responses.

Spatio-temporal changes in glutathione and thioredoxin redox couples during ionizing radiation-induced oxidative stress regulate tumor radio-resistance

Ionizing radiation (IR)-induced oxidative stress in tumor cells is effectively managed by constitutive and inducible antioxidant defense systems. This study was initiated to understand the relative contribution of different redox regulatory systems in determining the tumor radio-resistance. In this study, human T-cell lymphoma (Jurkat) cells were exposed to IR (4 Gy) and monitored for the spatio-temporal changes in cellular redox regulatory parameters. We monitored the changes in the levels of reactive oxygen species (ROS) (total, mitochondrial, primary, and secondary), thiols (total, surface, and intracellular), GSH/GSSG ratio, antioxidant enzyme activity viz. thioredoxin (Trx), Trx reductase (TrxR), glutathione peroxidase, and glutathione reductase with respect to time. We have also measured protein glutathionylation. We observed that tumor cells mount a biphasic response after IR exposure which can be divided into early (0–6 h) and late (16–48 h) responses in terms of changes in cellular redox parameters. During early response, constitutively active GSH and Trx systems respond to restore cellular redox balance to pre-exposure levels and help in activation of redox-sensitive transcription factor Nrf-2. During late response, increase in the levels of antioxidants GSH and Trx rescue cells against IR-mediated damage. We observed that disruption of either glutathione or thioredoxin metabolism led to partial impairment of ability of cells to survive against IR-induced damage. But simultaneous disruption of both the pathways significantly increased radio sensitivity of Jurkat cells. This highlighted the importance of these two antioxidant pathways in regulating redox homeostasis under conditions of IR-induced oxidative stress.

Alpha B-crystallin induction in skeletal muscle cells under redox imbalance is mediated by a JNK-dependent regulatory mechanism

The small heat shock protein α-B-crystallin (CRYAB) is critically involved in stress-related cellular processes such as differentiation, apoptosis, and redox homeostasis. The up-regulation of CRYAB plays a key role in the cytoprotective and antioxidant response, but the molecular pathway driving its expression in muscle cells during oxidative stress still remains unknown. Here we show that noncytotoxic exposure to sodium meta-arsenite (NaAsO2) inducing redox imbalance is able to increase the CRYAB content of C2C12 myoblasts in a transcription-dependent manner. Our in silico analysis revealed a genomic region upstream of the Cryab promoter containing two putative antioxidant-responsive elements motifs and one AP-1-like binding site. The redox-sensitive transcription factors Nrf2 and the AP-1 component c-Jun were found to be up-regulated in NaAsO2-treated cells, and we demonstrated a specific NaAsO2-mediated increase of c-Jun and Nrf2 binding activity to the genomic region identified, supporting their putative involvement in CRYAB regulation following a shift in redox balance. These changes also correlated with a specific phosphorylation of JNK and p38 MAPK kinases, the well-known molecular mediators of signaling pathways leading to the activation of these transcription factors. Pretreatment of C2C12 cells with the JNK inhibitor SP600125 induced a decrease in c-Jun and Nrf2 content and was able to counteract the NaAsO2-mediated increase in CRYAB expression. Thus these data show a direct role of JNK in CRYAB regulation under redox imbalance and also point to a previously unrecognized link between c-Jun and Nrf2 transcription factors and redox-induced CRYAB expression in muscle cells.

PP.28.13

Objective: Oxidative stress plays an important role in the development of hypertension. It is reported that renal dopamine D1 receptor (D1R), a major contributor to sodium homeostasis and BP regulation, is negatively regulated by oxidative stress. Antioxidants can reduce BP and redox-sensitive transcription factor Nrf2 could provide important clues for antihypertensive properties of antioxidants. We investigated the signaling molecules responsible for redox-sensitive renal proximal tubular D1R dysfunction and the role of renal specific Nrf2 in preserving D1R function. Design and method: Wild type (WT) and renal proximal tubular specific Nrf2 knockout (KO) mice were treated with 10 mM L-buthionine-sulfoximine (BSO, a pro-oxidant) without and with sulforaphane (S, an antioxidant; 5 μM daily by gavage) for 5 weeks. Human kidney proximal tubular (HK2) cells were also treated with BSO with and without sulforaphane. Results: In WT mice, BSO treatment increased oxidative stress and BP, reduced renal tubular D1R expression and attenuated SKF38393 (a D1R agonist)-induced Na/K-ATPase inhibition and sodium excretion. BSO also activated transcription factors activator protein (AP) 1 and SP3. However, the onset and severity of hypertension as well as oxidative stress, activation of AP1 and SP3 and D1R dysfunction were more robust in BSO-treated Nrf2 KO compared to WT mice. Also, sulforaphane activated Nrf2, reduced oxidative stress, normalized AP1 and SP3 activation, rescued DIR function and lowered BP in BSO-treated WT mice but not in Nrf2 KO mice. BSO also caused oxidative stress and D1R dysfunction in HK2 cells. BSO-treated HK2 cells exhibited increased AP1 (c-fos) and SP3 nuclear expression. Sulforaphane per se had no effect on oxidative milieu or D1R expression, but it mitigated BSO-induced oxidative stress, AP1—SP3 up-regulation and D1R down-regulation and dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, sulforaphane failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. Conclusions: Oxidative stress via AP1-SP3 pathway down-regulates D1R expression and function which leads to sodium retention and hypertension. Antioxidants, via Nrf2 activation, reduce oxidative stress, normalize AP1-SP3 signaling, protect renal D1R function and prevent development of hypertension.

Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

Nrf2 deficiency causes lipid oxidation, inflammation, and matrix-protease expression in DHA-supplemented and UVA-irradiated skin fibroblasts

Fish oil rich in docosahexaenoic acid (DHA) has beneficial effects on human health. Omega-3 polyunsaturated fatty acids are precursors of eicosanoids and docosanoids, signaling molecules that control inflammation and immunity, and their dietary uptake improves a range of disorders including cardiovascular diseases, ulcerative colitis, rheumatoid arthritis, and psoriasis. The unsaturated nature of these fatty acids, however, makes them prone to oxidation, especially when they are incorporated into (membrane) phospholipids. The skin is an organ strongly exposed to oxidative stress, mainly due to solar ultraviolet radiation. Thus, increased levels of PUFA in combination with oxidative stress could cause increased local generation of oxidized lipids, whose action spectrum reaches from signaling molecules to reactive carbonyl compounds that can crosslink biomolecules. Here, we investigated whether PUFA supplements to fibroblasts are incorporated into membrane phospholipids and whether an increase of PUFA within phospholipids affects the responses of the cells to UV exposure. The redox-sensitive transcription factor Nrf2 is the major regulator of the fibroblast stress response to ultraviolet radiation or exposure to oxidized lipids. Here we addressed how Nrf2 signaling would be affected in PUFA-supplemented human dermal fibroblasts and mouse dermal fibroblasts from Nrf2-deficient and wild type mice. We found, using HPLC–tandem MS, that DHA supplements to culture media of human and murine fibroblasts were readily incorporated into phospholipids and that subsequent irradiation of the supplemented cells with UVA resulted in an increase in 1-palmitoyl-2-(epoxyisoprostane-E2)-sn-glycero-3-phosphorylcholine and Oxo-DHA esterified to phospholipid, both of which are Nrf2 agonists. Also, induction of Nrf2 target genes was enhanced in the DHA-supplemented fibroblasts after UVA irradiation. In Nrf2-deficient murine fibroblasts, the expression of the target genes was, as expected, decreased, but surprisingly, expression of TNFα and MMP13 was strongly induced in DHA-supplemented, UVA-irradiated cells. Also, Nrf2-deficient cells had increased levels of oxidized phospholipids relative to the unoxidized precursors after UVA irradiation. Our data suggest that under ultraviolet stress a functioning Nrf2 system is required to prevent DHA-induced inflammation and matrix degradation in dermal fibroblasts.

Nrf2-dependent suppression of azoxymethane/dextran sulfate sodium-induced colon carcinogenesis by the cinnamon-derived dietary factor cinnamaldehyde.

The progressive nature of colorectal cancer and poor prognosis associated with the metastatic phase of the disease create an urgent need for the development of more efficacious strategies targeting colorectal carcinogenesis. Cumulative evidence suggests that the redox-sensitive transcription factor Nrf2 (nuclear factor-E2-related factor 2), a master regulator of the cellular antioxidant defence, represents a promising molecular target for colorectal cancer chemoprevention. Recently, we have identified cinnamon, the ground bark of Cinnamomum aromaticum (cassia cinnamon) and Cinnamomum verum (Ceylon cinnamon), as a rich dietary source of the Nrf2 inducer cinnamaldehyde (CA) eliciting the Nrf2-regulated antioxidant response in human epithelial colon cells, conferring cytoprotection against electrophilic and genotoxic insult. Here, we have explored the molecular mechanism underlying CA-induced Nrf2 activation in colorectal epithelial cells and have examined the chemopreventive potential of CA in a murine colorectal cancer model comparing Nrf2(+/+) with Nrf2(-/-) mice. In HCT116 cells, CA caused a Keap1-C151-dependent increase in Nrf2 protein half-life via blockage of ubiquitination with upregulation of cytoprotective Nrf2 target genes and elevation of cellular glutathione. After optimizing colorectal Nrf2 activation and target gene expression by dietary CA-supplementation regimens, we demonstrated that CA suppresses AOM/DSS-induced inflammatory colon carcinogenesis with modulation of molecular markers of colorectal carcinogenesis. Dietary suppression of colorectal cancer using CA supplementation was achieved in Nrf2(+/+) but not in Nrf2(-/-) mice confirming the Nrf2 dependence of CA-induced chemopreventive effects. Taken together, our data suggest feasibility of colorectal cancer suppression by dietary CA, an FDA-approved food additive derived from the third most consumed spice in the world.

Activation of the Nrf2-regulated antioxidant cell response inhibits HEMA-induced oxidative stress and supports cell viability

Oxidative stress due to increased formation of reactive oxygen species (ROS) in target cells of dental resin monomers like 2-hydroxyethyl methacrylate (HEMA) is a major mechanism underlying the disturbance of vital cell functions including mineralization and differentiation, responses of the innate immune system, and the induction of cell death via apoptosis. Although a shift in the equilibrium between cell viability and apoptosis is related to the non-enzymatic antioxidant glutathione (GSH) in HEMA-exposed cells, the major mechanisms of adaptive antioxidant cell responses to maintain cellular redox homeostasis are still unknown. The present study provides insight into the induction of a communicating network of pathways under the control of the redox-sensitive transcription factor Nrf2, a major transcriptional activator of genes coding for enzymatic antioxidants. Here, oxidative stress was indicated by DCF fluorescence in cells after a short exposure (1 h) to HEMA, while DHR123 fluorescence significantly increased about 1.8-fold after a long exposure period (24 h) showing the formation of hydrogen peroxide (H2O2). The corresponding expression of Nrf2 was activated immediately after HEMA exposure (1 h) and remained constant up to 24 h. Nrf2-regulated expression of enzymes of the glutathione metabolism (glutathione peroxidase 1/2, glutathione reductase) decreased in HEMA-exposed cells as a result of GSH depletion, and superoxide dismutase expression was downregulated after H2O2 overproduction. However, the expression of Nrf2-controlled enzymatic antioxidants (catalase, peroxiredoxin, thioredoxin 1, thioredoxin reductase, heme oxygenase-1) and the NADPH-regenerating system (glucose 6-phosphate dehydrogenase, transaldolase) was increased. Phenolic tert-butylhydroquinone (tBHQ), a classic inducer of the Nrf2 pathway, reduced oxidative stress and protected cells from HEMA-induced cell death through a shift in the number of cells in necrosis to apoptosis. The expression of Nrf2 and related enzymatic antioxidants downstream was enhanced by tBHQ in parallel. In conclusion, this investigation expanded the detailed understanding of the underlying mechanisms of HEMA-induced oxidative stress, and highlighted the cross-talk and interdependence between various Nrf2-regulated antioxidant pathways as a major adaptive cell response. The current results demonstrate that modulation of the Nrf2-mediated cellular defense response is an effective means for manipulating the sensitivity of cells to dental resin monomers.