Nrf2 promotes breast cancer cell migration via up‐regulation of G6PD/HIF‐1α/Notch1 axis

Abnormal metabolism of tumour cells is closely related to the occurrence and development of breast cancer, during which the expression of NF‐E2‐related factor 2 (Nrf2) is of great significance. Metastatic breast cancer is one of the most common causes of cancer death worldwide; however, the molecular mechanism underlying breast cancer metastasis remains unknown. In this study, we found that the overexpression of Nrf2 promoted proliferation and migration of breast cancers cells. Inhibition of Nrf2 and overexpression of Kelch‐like ECH‐associated protein 1 (Keap1) reduced the expression of glucose‐6‐phosphate dehydrogenase (G6PD) and transketolase of pentose phosphate pathway, and overexpression of Nrf2 and knockdown of Keap1 had opposite effects. Our results further showed that the overexpression of Nrf2 promoted the expression of G6PD and Hypoxia‐inducing factor 1α (HIF‐1α) in MCF‐7 and MDA‐MB‐231 cells. Overexpression of Nrf2 up‐regulated the expression of Notch1 via G6PD/HIF‐1α pathway. Notch signalling pathway affected the proliferation of breast cancer by affecting its downstream gene HES‐1, and regulated the migration of breast cancer cells by affecting the expression of EMT pathway. The results suggest that Nrf2 is a potential molecular target for the treatment of breast cancer and targeting Notch1 signalling pathway may provide a promising strategy for the treatment of Nrf2‐driven breast cancer metastasis.     

Palbinone alleviates diabetic retinopathy in STZ‐induced rats by inhibiting NLRP3 inflammatory activity

Diabetic retinopathy (DR) is the primary cause of blindness and visual impairment in diabetes patients worldwide. However, laser and surgical therapies at DR have short‐term effectiveness and cause side effects. Treatment with natural products is a reasonable alternative treatment for DR. The main objective of this investigation is to explore the efficacy of a bioactive compound such as palbinone (PB) in DR. Experimental rats were injected intraperitoneally with streptozotocin (STZ, 65 mg/kg), and these established experimental rats were treated with PB (20 mg/kg/bw) for 42 days. The observed results showed that PB considerably reduced the proinflammatory cytokine (interleukin‐18 [IL‐18] and IL‐1β) production as well as improved the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) particularly in the retinal region of STZ‐induced DR rats. In addition, PB treatment improved nuclear factor erythroid 2‐related factor 2 (Nrf2) accumulation and enhanced the heme oxygenase‐1 expression, and major antioxidants downregulated Nrf2 in the damaged retina. Also, the expression levels of nod‐like receptor family pyrin domain containing 3 (NLRP3), cleaved‐caspase‐1, IL‐1β, and apoptosis‐associated speck‐like protein containing CARD in the retinal region were notably upregulated in STZ‐induced DR, which was eliminated by PB interference. PB administration exerted efficient antioxidant activities, Nrf2 pathway activation, and inhibition of NLRP3 inflammasome. This current investigation concluded that PB considerably reduced the retinal inflammation and oxidative stress stimulated via high glucose, and also activated the antioxidative Nrf2 pathway and inhibited the NLRP3 inflammasome formation in rats.     

Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress

Glucose‐6‐phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re‐expression of wild‐type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2‐dependent manner. The SIRT2‐mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress.     

Major differences among chemopreventive organoselenocompounds in the sustained elevation of cytoprotective genes

Cytoprotective enzyme elevation through the nuclear erythroid 2 p45‐related factor 2 (Nrf2)–Kelch‐like ECH‐associated protein 1/antioxidant response element pathway has been promulgated for cancer prevention. This study compares the redox insult and sustained cytoprotective enzyme elevation by organoselenocompounds and sulforaphane (SF) in lung cells. SF elicited a rise in reactive oxygen species (ROS) and drop in glutathione (GSH) at 2 h; nuclear accumulation of Nrf2 at 4 h; and a GSH rebound and elevation in NAD(P)H quinone oxidoreductase (NQO1), thioredoxin reductase (TR1), and glutamate–cysteine ligase (GCL) at 24 h. Selenocystine (SECY) elicited a similar 24 h response, despite lesser earlier time‐point changes. 2‐Cyclohexylselenazolidine‐4‐carboxylic acid effects were similar to SECY's but with a larger Nrf2 change and the largest 24 h increase in GSH, GCL, TR1, and NQO1 of any compound investigated. Selenomethionine elicited a similar acute rise in ROS, but lesser depletion of GSH, no 4 h increase in nuclear Nrf2, only minor 24 h elevations in TR1 and NQO1, and a GCL elevation insufficient to elevate GSH. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:344–353, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21417     

Metformin alleviates human cellular aging by upregulating the endoplasmic reticulum glutathione peroxidase 7

Metformin, an FDA‐approved antidiabetic drug, has been shown to elongate lifespan in animal models. Nevertheless, the effects of metformin on human cells remain unclear. Here, we show that low‐dose metformin treatment extends the lifespan of human diploid fibroblasts and mesenchymal stem cells. We report that a low dose of metformin upregulates the endoplasmic reticulum‐localized glutathione peroxidase 7 (GPx7). GP×7 expression levels are decreased in senescent human cells, and GPx7 depletion results in premature cellular senescence. We also indicate that metformin increases the nuclear accumulation of nuclear factor erythroid 2‐related factor 2 (Nrf2), which binds to the antioxidant response elements in the GPX7 gene promoter to induce its expression. Moreover, the metformin‐Nrf2‐GPx7 pathway delays aging in worms. Our study provides mechanistic insights into the beneficial effects of metformin on human cellular aging and highlights the importance of the Nrf2‐GPx7 pathway in pro‐longevity signaling.     

Ellagic acid protects dopamine neurons from rotenone‐induced neurotoxicity via activation of Nrf2 signalling

Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease. Oxidative stress is one of key contributors to PD. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is considered to be a master regulator of many genes involved in anti‐oxidant stress to attenuate cell death. Therefore, activation of Nrf2 signalling provides an effective avenue to treat PD. Ellagic acid (EA), a natural polyphenolic contained in fruits and nuts, possesses amounts of pharmacological activities, such as anti‐oxidant stress and anti‐inflammation. Recent studies have confirmed EA could be used as a neuroprotective agent in neurodegenerative diseases. Here, mice subcutaneous injection of rotenone (ROT)‐induced DA neuronal damage was performed to investigate EA‐mediated neuroprotection. In addition, adult Nrf2 knockout mice and different cell cultures including MN9D‐enciched, MN9D‐BV‐2 and MN9D‐C6 cell co‐cultures were applied to explore the underlying mechanisms. Results demonstrated EA conferred neuroprotection against ROT‐induced DA neurotoxicity. Activation of Nrf2 signalling was involved in EA‐mediated DA neuroprotection, as evidenced by the following observations. First, EA activated Nrf2 signalling in ROT‐induced DA neuronal damage. Second, EA generated neuroprotection with the presence of astroglia and silence of Nrf2 in astroglia abolished EA‐mediated neuroprotection. Third, EA failed to produce DA neuroprotection in Nrf2 knockout mice. In conclusion, this study identified EA protected against DA neuronal loss via an Nrf2‐dependent manner.     

SIRT2 controls the pentose phosphate switch

The most common enzyme defect in humans is glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, which affects more than 400 million people. G6PD shunts glucose into the pentose phosphate pathway (PPP) to generate nucleotides and reducing potential in the form of NADPH. In this issue, Wang et al ( 2014 ) show that G6PD activity is post‐translationally regulated by SIRT2, a cytoplasmic NAD⁺‐dependent deacetylase, thereby linking NAD⁺ levels to DNA repair and oxidative defences, and identifying potential new approaches to treating this common genetic disease.     

Sulforaphane Protects Rodent Retinas against Ischemia-Reperfusion Injury through the Activation of the Nrf2/HO-1 Antioxidant Pathway

Retinal ischemia-reperfusion (I/R) injury induces oxidative stress, leukocyte infiltration, and neuronal cell death. Sulforaphane (SF), which can be obtained in cruciferous vegetables such as broccoli, exerts protective effects in response to oxidative stress in various tissues. These effects can be initiated through nuclear factor E2-related factor 2 (Nrf2)-mediated induction of heme oxygenase-1 (HO-1). This investigation was designed to elucidate the neural protective mechanisms of SF in the retinal I/R rat model. Animals were intraperitoneally (i.p.) injected with SF (12.5 mg/kg) or vehicle (corn oil) once a day for 7 consecutive days. Then, retinal I/R was made by elevating the intraocular pressure (IOP) to 130 mmHg for 1 h. To determine if HO-1 was involved in the Nrf2 antioxidant pathway, rats were subjected to protoporphyrin IX zinc (II) (ZnPP, 30 mg/kg, i.p.) treatments at 24 h before retinal ischemia. The neuroprotective effects of SF were assessed by determining the morphology of the retina, counting the infiltrating inflammatory cells and the surviving retinal ganglion cells (RGCs) and amacrine cells, and measuring apoptosis in the retinal layers. The expression of Nrf2 and HO-1 was studied by immunofluorescence analysis and western blotting. I/R induced a marked increase of ROS generation, caused pronounced inflammation, increased the apoptosis of RGCs and amacrine cells and caused the thinning of the inner retinal layer (IRL), and these effects were diminished or abolished by SF pretreatment. Meanwhile, SF pretreatment significantly elevated the nuclear accumulation of Nrf2 and the level of HO-1 expression in the I/R retinas; however, ZnPP reversed the protective effects of SF on I/R retinas. Together, we offer direct evidence that SF had protective effects on I/R retinas, which could be attributed, at least in part, to the activation of the Nrf2/HO-1 antioxidant pathway.     

Yellow Wine Polyphenolic Compounds prevents Doxorubicin‐induced cardiotoxicity through activation of the Nrf2 signalling pathway

Doxorubicin (DOX) is considered as the major culprit in chemotherapy‐induced cardiotoxicity. Yellow wine polyphenolic compounds (YWPC), which are full of polyphenols, have beneficial effects on cardiovascular disease. However, their role in DOX‐induced cardiotoxicity is poorly understood. Due to their antioxidant property, we have been suggested that YWPC could prevent DOX‐induced cardiotoxicity. In this study, we found that YWPC treatment (30 mg/kg/day) significantly improved DOX‐induced cardiac hypertrophy and cardiac dysfunction. YWPC alleviated DOX‐induced increase in oxidative stress levels, reduction in endogenous antioxidant enzyme activities and inflammatory response. Besides, administration of YWPC could prevent DOX‐induced mitochondria‐mediated cardiac apoptosis. Mechanistically, we found that YWPC attenuated DOX‐induced reactive oxygen species (ROS) and down‐regulation of transforming growth factor beta 1 (TGF‐β1)/smad3 pathway by promoting nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) nucleus translocation in cultured H9C2 cardiomyocytes. Additionally, YWPC against DOX‐induced TGF‐β1 up‐regulation were abolished by Nrf2 knockdown. Further studies revealed that YWPC could inhibit DOX‐induced cardiac fibrosis through inhibiting TGF‐β/smad3‐mediated ECM synthesis. Collectively, our results revealed that YWPC might be effective in mitigating DOX‐induced cardiotoxicity by Nrf2‐dependent down‐regulation of the TGF‐β/smad3 pathway.     

Delay of photoreceptor degeneration in tubby mouse by sulforaphane

In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF-E2-related factor-2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up-regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal-regulated kinases (ERKs), blocked SF-mediated ERKs activation and up-regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF-mediated up-regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.     

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.