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.

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.

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.

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.

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.

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.

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.

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.