Exosome-derived miR-200a promotes esophageal cancer cell proliferation and migration via the mediating Keap1 expression.

Previous studies have reported that exosomes bearing certain microRNAs (miRNAs) are related to the physiological functions of different types of cancer cells. Our study aimed to elucidate the role of miR-200a in esophageal squamous cell carcinoma (ESCC). We observed that miR-200a expression is higher in esophageal carcinoma cells, tissues, and exosomes than in normal cells and healthy tissues. We showed that exosome-shuttled miR-200a promotes the proliferation, migration, and invasion of esophageal cells and inhibits apoptosis, thereby leading to the progression of ESCC. We showed that miR-200a exerts its effects through its interaction with Keap1, thus altering the Keap1/Nrf2 signaling pathway. Our results suggest that exosome-shuttled miR-200a might be useful as a biomarker for prognosis in patients with ESCC.

Minocycline inhibits sleep deprivation-induced aberrant microglial activation and Keap1-Nrf2 expression in mouse hippocampus.

Sleep deprivation (SD) is a hallmark of modern society and associated with many neuropsychiatric disorders, including depression and anxiety. However, the cellular and molecular mechanisms underlying SD-associated depression and anxiety remain elusive. Does the neuroinflammation play a role in mediating the effects of SD? In this study, we investigated SD-induced cellular and molecular alterations in the hippocampus and asked whether treatment with an anti-inflammatory drug, minocycline, could attenuate these alterations. We found that SD animals exhibit activated microglia and decreased levels of Keap1 and Nrf2 (antioxidant and anti-inflammatory factors) in the hippocampus. In vivo local field potential recordings show decreased theta and beta oscillations, but increased high gamma oscillations, as a result of SD. Behavioral analysis revealed increased immobility time in the forced swim and tail suspension tests, and decreased sucrose intake in SD mice, all indicative of depressive-like behavior. Moreover, open field test and elevated plus maze test results indicated that SD increases anxiety-like behavior. Interestingly, treatment with the microglial modulator minocycline prevented SD-induced microglial activation, restored Keap1 and Nrf2 levels, normalized neuronal oscillations, and alleviated depressive-like and anxiety-like behavior. The present study reveals that microglial activation and Keap1-Nrf2 signaling play a crucial role in SD-induced behavioral alteration, and that minocycline treatment has a protective effect on these alterations.

Lycopene prevents carcinogen-induced cutaneous tumor by enhancing activation of the Nrf2 pathway through p62-triggered autophagic Keap1 degradation.

Biologically active natural products have been used for the chemoprevention of cutaneous tumors. Lycopene is the main active phytochemical in tomatoes. We herein aimed to assess the cancer preventive effects of lycopene and to find potential molecular targets. In chemically-induced cutaneous tumor mice and cell models, lycopene attenuated cutaneous tumor incidence and multiplicity as well as the tumorigenesis of normal cutaneous cells in phase-selectivity (only in the promotion phase) manners. By utilizing a comprehensive approach combining bioinformatics with network pharmacology, we predicted that intracellular autophagy and redox status were associated with lycopene's preventive effect on cutaneous tumors. Lycopene stimulated the activation of antioxidant enzymes and the translocation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) that predominantly maintained intracellular redox equilibrium. The cancer chemopreventive effects were mediated by Nrf2. Further, lycopene enhanced the expression of autophagy protein p62. Therefore this led to the degradation of Keap1(Kelch ECH associating protein 1), the main protein locking Nrf2 in cytoplasm. In conclusion, our study provides preclinical evidence of the chemopreventive effects of lycopene on cutaneous tumors and reveals the mechanistic link between lycopene's stimulation of Nrf2 signaling pathway and p62-mediated degradation of Keap1 via the autophagy-lysosomal pathway.

Integrated data analysis reveals significant associations of KEAP1 mutations with DNA methylation alterations in lung adenocarcinomas.

KEAP1 regulates the cytoprotection induced by NRF2 and has been reported to be a candidate tumor suppressor. Recent evidence has shown that mutations in several driver genes cause aberrant DNA methylation patterns, a hallmark of cancer. However, the correlation between KEAP1 mutations and DNA methylation in lung cancer has still not been investigated. In this study, we systematically carried out an integrated multi-omics analysis to explore the correlation between KEAP1 mutations and DNA methylation and its effect on gene expression in lung adenocarcinoma (LUAD). We found that most of the DNA aberrations associated with KEAP1 mutations in LAUD were hypomethylation. Surprisingly, we found several NRF2-regulated genes among the genes that showed differential DNA methylation. Moreover, we identified an 8-gene signature with altered DNA methylation pattern and elevated gene expression levels in LUAD patients with mutated KEAP1, and evaluated the prognostic value of this signature in various clinical datasets. These results establish that KEAP1 mutations are associated with DNA methylation changes capable of shaping regulatory network functions. Combining both epigenomic and transcriptomic changes along with KEAP1 mutations may provide a better understanding of the molecular mechanisms associated with the progression of lung cancer and may help to provide better therapeutic approaches.

TRIM16 protects from OGD/R-induced oxidative stress in cultured hippocampal neurons by enhancing Nrf2/ARE antioxidant signaling via downregulation of Keap1.

Tripartite motif 16 (TRIM16) has emerged as a novel oxidative stress-responsive protein that confers cytoprotective effects by reinforcing the cellular antioxidant system. However, whether TRIM16 is involved in regulating oxidative stress during cerebral ischemia/reperfusion injury remains unclear. In the present study, we aimed to explore the potential function and molecular mechanism of TRIM16 in regulating oxidative stress in neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Here, we found that OGD/R exposure resulted in a significant induction of TRIM16 expression in neurons. Depletion of TRIM16 by siRNA-mediated gene knockdown markedly upregulated the sensitivity of neurons to OGD/R-induced apoptosis and reactive oxygen species (ROS) generation. Notably, upregulation of TRIM16 expression significantly alleviated OGD/R-induced apoptosis and ROS generation in neurons. Moreover, TRIM16 overexpression markedly increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and enhanced Nrf2/antioxidant response element (ARE) activation associated with downregulation of kelch-like ECH-associated protein 1 (Keap1) expression. Restoration of Keap1 significantly reversed the TRIM16-mediated promotion effect on Nrf2/ARE activation. In addition, knockdown of Nrf2 also markedly abrogated the TRIM16-conferred neuroprotective effect in OGD/R-exposed neurons. Taken together, our results of our study demonstrate that induction of TRIM16 confers a cytoprotective effect in OGD/R-exposed neurons through enhancement of Nrf2/ARE antioxidant signaling via downregulation of Keap1. These findings suggest that TRIM16 may play a critical role in cerebral ischemia/reperfusion injury and serve as a promising target for neuroprotection.

Berberine ameliorates CCl4­induced liver injury in rats through regulation of the Nrf2­Keap1­ARE and p53 signaling pathways.

Berberine (BBR) is an isoquinoline alkaloid, reported to have multiple pharmacological functions. However, its effects against CCl4­induced oxidative damage remain poorly studied. Therefore, the present study investigated the protective action of BBR, and its antioxidant mechanisms, against CCl4­induced liver injury in rats. A total of 48 rats were randomly arranged into six groups: Control; model; positive control (PC); BBR low­dose (BL); BBR middle­dose (BM); and BBR high­dose (BH). The BL, BM and BH animals received BBR (5, 10 and 15 mg/kg by weight, respectively) orally for 7 consecutive days. Rats in the PC group were given silymarin (150 mg/kg), and the control and model groups were administered distilled water orally. At the end of the experiment, blood samples and livers were collected. To measure the liver biochemical indices, the reactive oxygen species (ROS) generation and the expression levels of related genes and protein, the following methods were used: An automatic biochemical analyzer; flow cytometry; spectrophotometry; reverse transcription­quantitative PCR; western blotting; and hematoxylin and eosin staining. The results revealed that BBR significantly decreased the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase, and increased those of glutathione and superoxide dismutase, but decreased malondialdehyde activity in hepatic tissue, and significantly decreased the reactive oxygen species level in hepatocytes. In hepatic tissue, the expressions of nuclear factor erythroid 2­related factor 2 (Nrf2), kelch­like ECH­associated protein 1 (Keap-1), NAD(P)H quinone dehydrogenase 1 (NQO-1), heme oxygenase 1 (HO­1), Bcl­2 and Bcl­xL mRNA, and HO­1 protein were elevated, and the expression of p53 mRNA was decreased, particularly in the BH group (15 mg/kg). In conclusion, BBR exerts a protective action against CCl4­induced acute liver injury in rats via effectively regulating the expression of Nrf2­Keap1­antioxidant responsive element­related genes and proteins, and inhibiting p53 pathway­mediated hepatocyte apoptosis.

Activation of KEAP1/NRF2/P62 signaling alleviates high phosphate-induced calcification of vascular smooth muscle cells by suppressing reactive oxygen species production.

Vascular calcification is a complication of diseases and conditions such as chronic kidney disease, diabetes, and aging. Previous studies have demonstrated that high concentrations of inorganic phosphate (Pi) can induce oxidative stress and vascular smooth muscle cell calcification. KEAP1 (Kelch-like ECH-associated protein 1)/NF-E2-related factor 2 (NRF2) signaling has been shown to play important roles in protecting cells from oxidative stress. The current study aims to investigate the possible involvement of the KEAP1/NRF2/P62 -mediated antioxidant pathway in vascular calcification induced by high Pi levels. Exposure of vascular smooth muscle cells (VSMCs) to high Pi concentrations promoted the accumulation of reactive oxygen species (ROS) and the nuclear translocation of NRF2, along with an increase in P62 levels and a decrease in KEAP1 levels. A classic NRF2 activator, tert-butylhydroquinone (tBHQ), significantly decreased ROS levels and calcium deposition in VSMCs by promoting the nuclear translocation of NRF2 and upregulating P62 and KEAP1 expression. In contrast, silencing NRF2 and P62 with siRNAs increased the levels of ROS and calcium deposition in VSMCs. In conclusion, VSMC calcification can be alleviated by the activation of the KEAP1/NRF2/P62 antioxidative pathway, which could have a protective role when it is exogenously activated by tBHQ.

MicroRNA-141 protects PC12 cells against hypoxia/reoxygenation-induced injury via regulating Keap1-Nrf2 signaling pathway.

To understand the role of microRNA-141 (miR-141) in hypoxia/reoxygenation (H/R)-induced PC12 cell injury via modulation of Keap1/Nrf2 signaling pathway. PC12 cells were divided into Control, H/R, H/R + miR-141 mimics, H/R + NC, H/R + miR-141 inhibitor, H/R + siKeap1 and H/R + miR-141 inhibitors+siKeap1 groups. The expression of miR-141 and Keap1/Nrf2 pathway was measured by qRT-PCR and western blotting, cell viability evaluated by MTT assay while cell apoptosis tested by flow cytometry. Besides, MDA (malondialdehyde), SOD (Super Oxide Dismutase) and LDH (lactate dehydrogenase) levels were determined. DCFH-DA and JC-1 staining were used to measure ROS and mitochondrial membrane potential (MMP) respectively. Compared with Controls, PC12 cells induced by H/R exhibited decreased cell viability and increased cell apoptosis rate, with elevated MDA, LDH and ROS and reduced SOD levels; and meanwhile, MMP and miR-141 expression were declined, whereas cytoplasmic Nrf2 levels were enhanced with the downregulated nuclear Nrf2 level (all P < 0.05). However, these cells treated with miR-141 mimics and siKeap1 showed obvious improvement in H/R-induced cell injury, while miR-141 inhibitors presented significantly aggravated cell injury (both P < 0.05). Besides, siKeap1 can reverse the effect of miRNA-141 inhibitors on aggravating H/R-induced PC12 cell injury. miR-141-mediated Keap1/Nrf2 signaling pathway to promote cell viability, inhibit cell apoptosis and reduce oxidative stress of PC12 cells, thereby alleviating H/R-induced cell injury.

p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

Increased muscle contractile activity, as observed with regular exercise, prevents oxidative stress-induced muscle wasting, at least partially, by improving the antioxidant defense system. Phosphorylated p62/sequestosome1 competitively binds to the Kelch-like ECH-associated protein 1, activating nuclear factor erythroid 2-related factor 2 (Nrf2), which stimulates transcription of antioxidant/electrophile responsive elements. However, it remains to be determined if this process is activated by regular exercise in skeletal muscle. Here, we demonstrate that muscle contractile activity increases antioxidants, Nrf2 translocation into nuclei, and Nrf2 DNA-binding activity in association with increased p62 phosphorylation (Ser351) in mouse oxidative skeletal muscle. Skeletal muscle-specific loss of Nrf2 [i.e., Nrf2 muscle-specific knockout (mKO) mice] abolished the expression of the Nrf2 target antioxidant gene NAD(P)H-quinone oxidoreductase 1 (NQO1) in both glycolytic and oxidative muscles but reduced exercise-mediated increases of antioxidants (i.e., copper/zinc superoxide dismutase (SOD) and extracellular SOD only in oxidative muscle. Interestingly, skeletal muscle-specific loss of p62 (i.e., p62 mKO mice) also abolished the expression of NQO1 and reduced exercise-mediated increases of the same antioxidants in soleus muscle. Collectively, these findings indicate that p62 and Nrf2 cooperatively regulate the exercise-mediated increase of antioxidants in oxidative muscle.-Yamada, M., Iwata, M., Warabi, E., Oishi, H., Lira, V. A., Okutsu, M. p62/SQSTM1 and Nrf2 are essential for exercise-mediated enhancement of antioxidant protein expression in oxidative muscle.

CKIP-1 alleviates oxygen-glucose deprivation/reoxygenation-induced apoptosis and oxidative stress in cultured hippocampal neurons by downregulating Keap1 and activating Nrf2/ARE signaling.

Accumulating evidence has shown that casein kinase 2 interacting protein-1 (CKIP-1) is a pivotal regulator of apoptosis and oxidative stress. However, whether CKIP-1 is involved in regulating neuronal injury during the progression of cerebral ischemia/reperfusion injury remains unknown. In the present study, we aimed to investigate the potential role and underlying mechanism of CKIP-1 in regulating neuronal apoptosis and oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment in vitro. Herein, we found that OGD/R treatment resulted in a significant increase in CKIP-1 expression in cultured hippocampal neurons. The silencing of CKIP-1 exacerbated OGD/R-induced neuronal apoptosis and production of reactive oxygen species. By contrast, CKIP-1 overexpression reduced the apoptosis and reactive oxygen species production induced by the OGD/R treatment. Mechanistically, CKIP-1 inhibited the expression of Kelch-like ECH-associated protein 1 (Keap1) and promoted the expression of nuclear factor E2-related factor 2 (Nrf2). In addition, CKIP-1 increased the activation of antioxidant response element and the expression of downstream antioxidant genes. However, Keap1 overexpression or Nrf2 knockdown partially reversed the neuroprotective effect of CKIP-1 overexpression. Taken together, our results demonstrate that CKIP-1 overexpression alleviates OGD/R-induced neuronal injury by enhancing the Nrf2-mediated anti-oxidative stress signaling pathway, revealing a neuroprotective role of CKIP-1. Our study suggests CKIP-1 as a potential therapeutic target for neuroprotection.

Ellagic acid ameliorates oxidative stress and insulin resistance in high glucose-treated HepG2 cells via miR-223/keap1-Nrf2 pathway.

As a promising new target, miR-233 may regulate oxidative stress by targeting keap1-Nrf2 system to affect the pathological process of liver injury in T2DM. Ellagic acid (EA) is versatile for protecting oxidative stress damage and metabolic disorders. In the present study, we investigated the effect of EA on oxidative stress and insulin resistance in high glucose-induced T2DM HepG2 cells and examined the role of miR-223/keap1-Nrf2 pathway in system. HepG2 cells were incubated in 30 mM of glucose, with or without EA (15 and 30 µM) or metformin (Met, 150 µM) for 12 h. Glucose consumption, phosphorylation of IRS1, Akt and ERK under insulin stimulation, ROS and O2- production, MDA level, SOD activity and miR-223 expression, as well as protein levels of keap1, Nrf2, HO-1, SOD1 and SOD2 were analyzed. Furthermore, dual luciferase reporter assay, miR-223 mimic and inhibitor were implemented in cellular studies to explore the possible mechanism. EA upregulated glucose consumption, IRS1, Akt and ERK phosphorylation under insulin stimulation, reduced ROS and O2- production and MDA level, and increased SOD activity in high glucose-exposed HepG2 cells. In addition, EA elevated miR-223 expression level, downregulated mRNA and protein levels of keap1, and upregulated Nrf2, HO-1, SOD1 and SOD2 protein levels in this cell model. What's more, dual luciferase reporter assay, miR-223 mimic and inhibitor transfection confirmed that EA activated keap1-Nrf2 system via elevating miR-223. The miR-223, a negative regulator of keap1, represents an attractive therapeutic target in hepatic injury in T2DM. EA ameliorates oxidative stress and insulin resistance via miR-223-mediated keap1-Nrf2 activation in high glucose-induced T2DM HepG2 cells.

Keap1 Inhibits Metastatic Properties of NSCLC Cells by Stabilizing Architectures of F-Actin and Focal Adhesions.

Low expression of the tumor suppressor Kelch-like ECH-associated protein 1 (KEAP1) in non-small cell lung cancer (NSCLC) often results in higher malignant biological behavior and poor prognosis; however, the underlying mechanism remains unclear. The present study demonstrates that overexpression of Keap1 significantly suppresses migration and invasion of three different lung cancer cells (A549, H460, and H1299). Highly expressed Keap1, compared with the control, promotes formation of multiple stress fibers with larger mature focal adhesion complexes in the cytoplasm where only fine focal adhesions were observed in the membrane under control conditions. RhoA activity significantly increased when Keap1 was overexpressed, whereas Myosin 9b expression was reduced but could be rescued by proteasome inhibition. Noticeably, mouse tumor xenografts with Keap1 overexpression were smaller in size and less metastatic relative to the control group. Taken together, these results demonstrate that Keap1 stabilizes F-actin cytoskeleton structures and inhibits focal adhesion turnover, thereby restraining the migration and invasion of NSCLC. Therefore, increasing Keap1 or targeting its downstream molecules might provide potential therapeutic benefits for the treatment of patients with NSCLC.Implications: This study provides mechanistic insight on the metastatic process in NSCLC and suggests that Keap1 and its downstream molecules may be valuable drug targets for NSCLC patients. Mol Cancer Res; 16(3); 508-16. ©2018 AACR.

Mazes of Nrf2 Regulation.

Nrf2 transcription factor plays a key role in maintaining cellular redox balance under stress and is a perspective target for oxidative stress-associated diseases. Under normal conditions, Nrf2 transcriptional activity is low due to its rapid ubiquitination and degradation in the 26S proteasome, as well as through various modifications of amino acid residues of this transcription factor that regulate its transport to the nucleus and binding to DNA. Continuous activation of Nrf2 is possible due to autophagy and epigenetic regulation that may underlie the increased resistance of tumor cells to radiotherapy and chemotherapy. This review deals with the mechanisms of regulation of Nrf2 transcriptional activity and its main elements, and pharmacological approaches to activation of the Keap1/Nrf2/ARE system.

Fumarate decreases edema volume and improves functional outcome after experimental stroke.

BACKGROUND: Oxidative stress and inflammation exacerbate tissue damage in the brain after ischemic stroke. Dimethyl-fumarate (DMF) and its metabolite monomethyl-fumarate (MMF) are known to stimulate anti-oxidant pathways and modulate inflammatory responses. Considering these dual effects of fumarates, we examined the effect of MMF treatment after ischemic stroke in mice. METHODS: Permanent middle cerebral artery occlusion (pMCAO) was performed using adult, male C57BL/6 mice. Thirty minutes after pMCAO, 20mg/kg MMF was administered intravenously. Outcomes were evaluated 6, 24 and 48h after pMCAO. First, we examined whether a bolus of MMF was capable of changing expression of kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor (Nrf)2 in the infarcted brain. Next, we studied the effect of MMF on functional recovery. To explore mechanisms potentially influencing functional changes, we examined infarct volumes, edema formation, the expression of heat shock protein (Hsp)72, hydroxycarboxylic acid receptor 2 (Hcar2), and inducible nitric oxide synthase (iNOS) in the infarcted brain using real-time PCR and Western blotting. Concentrations of a panel of pro- and anti-inflammatory cytokines (IFNγ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNF) were examined in both the infarcted brain tissue and plasma samples 6, 24 and 48h after pMCAO using multiplex electrochemoluminiscence analysis. RESULTS: Administration of MMF increased the protein level of Nrf2 6h after pMCAO, and improved functional outcome at 24 and 48h after pMCAO. MMF treatment did not influence infarct size, however reduced edema volume at both 24 and 48h after pMCAO. MMF treatment resulted in increased Hsp72 expression in the brain 6h after pMCAO. Hcar2 mRNA levels increased significantly 24h after pMCAO, but were not different between saline- and MMF-treated mice. MMF treatment also increased the level of the anti-inflammatory cytokine IL-10 in the brain and plasma 6h after pMCAO, and additionally reduced the level of the pro-inflammatory cytokine IL-12p70 in the brain at 24 and 48h after pMCAO. CONCLUSIONS: A single intravenous bolus of MMF improved sensory-motor function after ischemic stroke, reduced edema formation, and increased the levels of the neuroprotective protein Hsp72 in the brain. The early increase in IL-10 and reduction in IL-12p70 in the brain combined with changes in systemic cytokine levels may also contribute to the functional recovery after pMCAO.

Expression Patterns of Nrf2 and Keap1 in Ovarian Cancer Cells and their Prognostic Role in Disease Recurrence and Patient Survival.

OBJECTIVE: This study evaluated the expression patterns of nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) and assessed their clinical value as prognostic indicators in ovarian cancer. METHODS: The expression patterns of Nrf2 and Keap1 were determined in 100 epithelial ovarian cancers by immunohistochemistry analyses. The associations of Nrf2 and Keap1 expression with clinicopathological characteristics of patients were evaluated. All patients received platinum-based chemotherapy. Chemoresistance was defined as recurrence within 6 months of first-line chemotherapy. RESULTS: Cytoplasmic expression of Nrf2 and Keap1 was observed in 95% and 72%, respectively, of all 100 epithelial ovarian cancers examined. Low Keap1 expression (intensity < 1) was strongly associated with disease recurrence (P = 0.046) and death (P = 0.002). Chemoresistance was associated with high Nrf2 expression (intensity = 3) (P = 0.833; hazard ratio [HR], 1.202; 95% confidence interval [CI], 0.217-6.667) and low Keap1 expression (P = 0.862; HR, 0.899; 95% CI, 0.270-2.994). However, these associations were not statistically significant. Survival analysis indicated that high Keap1 expression (intensity ≥ 1) was strongly predictive of better overall survival (P = 0.049) and disease-free survival (P = 0.004). Cox regression analysis indicated that Keap1 expression was an independent prognostic factor for overall survival (P = 0.012; HR, 0.349; 95% CI, 0.153-0.797). Although patients with high Nrf2 expression displayed better overall survival and disease-free survival, the association was not statistically significant. CONCLUSIONS: High cytoplasmic Keap1 expression, which might prevent nuclear translocation of Nrf2 in ovarian cancer cells, was associated with lower disease recurrence and death rate. Survival analysis suggested a probable role of Keap1 expression in predicting the prognosis of ovarian cancer.

Physical and Flavor Characteristics, Fatty Acid Profile, Antioxidant Status and Nrf2-Dependent Antioxidant Enzyme Gene Expression Changes in Young Grass Carp (Ctenopharyngodon idella) Fillets Fed Dietary Valine.

This study was conducted to examine the effects of dietary valine on the physical and flavor characteristics, fatty acid (FA) profile, antioxidant status and Nrf2-dependent antioxidant enzyme gene expression in the muscle of young grass carp (Ctenopharyngodon idella) fed increasing levels of valine (4.3, 8.0, 10.6, 13.1, 16.9 and 19.1 g/kg) for 8 weeks. Compared with the control group, the group fed valine showed improved physical characteristics of fish fillets (increased relative shear force, hydroxyproline, protein and lipid levels and decreased cathepsin B and L activities, as well as cooking loss, were observed). Moreover, valine improved the flavor of young grass carp fillets by increasing the amino acid (AA) concentration in fish muscle (increased aspartic acid, threonine, glutamine, cystine, methionine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and valine concentrations were observed). Additionally, optimal valine supplementation increased the potential health benefits to humans by decreasing the saturated FA (C15:0 and C16:0) concentration and increasing the unsaturated FA (monounsaturated FAs (MUFAs), such as C16:1, C18:1c+t and C20:1, and polyunsaturated FAs (PUFAs), such as C18:3n-3, C20:2 and C22:6) concentration. In addition, the reduced glutathione (GSH) content and the activities of Cu/Zn superoxide dismutase (SOD1), catalase (CAT) and Selenium-dependent glutathione peroxydase (Se-GPx) increased under valine supplementation (P < 0.05). Furthermore, the SOD1, CAT and Se-GPx mRNA levels increased with dietary valine levels, possibly due to the up-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) in muscle (P < 0.05). In conclusion, valine improved the physical and flavor characteristics, FA profile, and antioxidant status and regulated the expression of the antioxidant enzyme genes Nrf2, Keap1, TOR and S6K1 in fish fillets.

Mitochondrial phosphatase PGAM5 regulates Keap1-mediated Bcl-xL degradation and controls cardiomyocyte apoptosis driven by myocardial ischemia/reperfusion injury.

Phosphoglycerate mutase 5 (PGAM5) is a mitochondrial membrane protein that plays crucial roles in necroptosis and apoptosis. Though PGAM5 is known to be required for inducing intrinsic apoptosis through interacting with BCL2 associated X protein (Bax) and dynamin-related protein 1 (Drp1), the expression and role of PGAM5 in cardiomyocyte apoptosis driven by myocardial ischemia/reperfusion injury(MIRI) has not been studied. The present study shows that PGAM5 expression decreased after MIRI in vivo, positively correlated with Bcl-xL expression, negatively correlated with Kelch-ECH associating protein 1 (Keap1) expression. Furthermore, PGAM5 expression also decreased in cardiomyocytes after hypoxia/reoxygenation (H/R) treatment in vitro. PGAM5 silence promoted cardiomyocyte apoptosis and inhibited Bcl-xL expression, but with no effect on Keap1 expression. Accordingly, Keap1 overexpression further inhibited Bcl-xL and PGAM5 expression. Additionally, PGAM5-Bcl-xL-Keap1 interaction was identified, suggesting that PGAM5 might participate in the degradation of Bcl-xL mediated by Keap1. In summary, PGAM5 controls cardiomyocyte apoptosis induced by MIRI through regulating Keap1-mediated Bcl-xL degradation, which may supply a novel molecular target for acute myocardial infarction (AMI) therapy. Graphical abstract ᅟ.

The Skp1 Homologs SKR-1/2 Are Required for the Caenorhabditis elegans SKN-1 Antioxidant/Detoxification Response Independently of p38 MAPK.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.

Expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 in breast cancer.

Our objective was to explore the expression and clinical significance of Kelch-like epichlorohydrin-associated protein 1 (Keap1) in breast cancer tissue. Eighty-one breast cancer patients having undergone surgical treatment in our hospital between March 2002 and December 2008 were enrolled in this study. Normal tissue adjacent to tumors was used for the control samples. Diagnoses for all patients were confirmed by postoperative pathological examination. Immunohistochemical assays were used to measure the expression of Keap1 protein in breast cancer tissue and adjacent normal tissue, and its clinical significance was explored. We observed that 24.6% breast cancer tissue samples were positive for Keap1, a significantly lower proportion than that seen with adjacent normal tissue specimens (80.2%; P < 0.05). The presence of Keap1 expression did not correlate with age, tumor size, pathological classification, or degree of differentiation. However, it was found to be significantly associated with tumor-node-metastasis stage and the presence of lymphatic metastasis. Kaplan-Meier survival analysis showed a remarkably higher five-year survival rate among patients with positive Keap1 expression than in those lacking detectable levels of the protein (P = 0.032). Keap1 expression is significantly decreased in breast cancer tissue; therefore, the early detection of its expression might have great significance in determining prognosis for breast cancer patients.