NRF2 is a spatiotemporal metabolic hub essential for the polyfunctionality of Th2 cells.

Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

plantMASST - Community-driven chemotaxonomic digitization of plants.

Understanding the distribution of hundreds of thousands of plant metabolites across the plant kingdom presents a challenge. To address this, we curated publicly available LC-MS/MS data from 19,075 plant extracts and developed the plantMASST reference database encompassing 246 botanical families, 1,469 genera, and 2,793 species. This taxonomically focused database facilitates the exploration of plant-derived molecules using tandem mass spectrometry (MS/MS) spectra. This tool will aid in drug discovery, biosynthesis, (chemo)taxonomy, and the evolutionary ecology of herbivore interactions.

Dimethyl α-Ketoglutarate Promotes the Synthesis of Collagen and Inhibits Metalloproteinases in HaCaT Cells.

We observed that treatment with dimethyl α-ketoglutarate (DMK) increased the amount of intracellular α-ketoglutarate significantly more than that of α-ketoglutarate in HaCaT cells. DMK also increased the level of intracellular 4-hydroxyproline and promoted the production of collagen in HaCaT cells. In addition, DMK decreased the production of collagenase and elastase and down-regulated the expression of selected matrix metalloproteinases (MMPs), such as MMP-1, MMP-9, MMP-10, and MMP-12, via transcriptional inhibition. The inhibition of MMPs by DMK was mediated by the suppression of the IL-1 signaling cascade, leading to the attenuation of ERK1/2 phosphorylation and AP-1 transactivation. Our study results illustrate that DMK, an alkylated derivative of α-ketoglutarate, increased the level of 4-hydroxyproline, promoted the production of collagen, and inhibited the expression of selected MMPs by affecting the IL-1 cascade and AP-1 transactivation in HaCaT cells. The results suggest that DMK might be useful as an anti-wrinkle ingredient.

Dimethyl Itaconate Inhibits Melanogenesis in B16F10 Cells.

Itaconate is a metabolite produced to counteract and resolve pro-inflammatory responses when macrophages are challenged with intracellular or extracellular stimuli. In the present study, we have observed that dimethyl itaconate (DMI) inhibits melanogenesis in B16F10 cells. DMI inhibits microphthalmia-associated transcription factor (MITF) and downregulates the expression of MITF target genes, such as tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). DMI also decreases the level of melanocortin 1 receptor (MC1R) and the production of α-melanocyte stimulating hormone (α-MSH), resulting in the inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and MITF activities. The structure-activity relationship (SAR) study illustrates that the α,ß-unsaturated carbonyl moiety in DMI, a moiety required to target KELCH-like ECH-associated protein 1 (KEAP1) to activate NF-E2-related factor 2 (NRF2), is necessary to inhibit melanogenesis and knocking down Nrf2 attenuates the inhibition of melanogenesis by DMI. Together, our study reveals that the MC1R-ERK1/2-MITF axis regulated by the KEAP1-NRF2 pathway is the molecular target responsible for the inhibition of melanogenesis by DMI.

Ethanol Extract of Chaenomeles sinensis Inhibits the Development of Benign Prostatic Hyperplasia by Exhibiting Anti-oxidant and Anti-inflammatory Effects.

Chaenomeles sinensis is known to inhibit the development and progression of many age-related diseases, but the underlying molecular mechanisms are largely unclear. In the present study, we observed that the ethanol extract of Chaenomeles sinensis scavenged 2,2'-diphenylpicrylhydrazyl and 2,2'-azinobis diammonium radicals in vitro. The ethanol extract of Chaenomeles sinensis activated antioxidant response element-luciferase activity and induced expression of NRF2 target genes in HaCaT cells. The ethanol extract of Chaenomeles sinensis also suppressed LPS-induced expression of COX-2 and iNOS proteins, and mRNA expression of TNF-α and IL-2 in RAW264.7 cells. Finally, the ethanol extract of Chaenomeles sinensis significantly suppressed testosterone propionate-induced benign prostatic hyperplasia in mice. Together, our study provides the evidence that the ethanol extract of Chaenomeles sinensis inhibits the development of benign prostatic hyperplasia by exhibiting anti-oxidant and anti-inflammatory effects.

[Corrigendum] Hwang-Heuk-San induces apoptosis in HCT116 human colorectal cancer cells through the ROS-mediated activation of caspases and the inactivation of the PI3K/Akt signaling pathway.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that certain of the data panels featured in Figs. 1B, 4A, 6A and 8A, showing DAPI or NAC staining of the cells, appeared to contain overlapping data. The authors have consulted their original data, and realize that errors were made during the compilation of these figures; consequently, they have repeated the affected experiments. The revised versions of Figs. 1, 4, 6 and 8, featuring replacement data for Figs. 1B, 4A, 6A and 8A, are shown on the subsequent pages. The authors regret the errors that were made during the preparation of the published figures, and confirm that these errors did not affect the conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologize to the readership for any inconvenience caused. [the original article was published in Oncology Reports 36: 205­214, 2016; DOI: 10.3892/or.2016.4812].

BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma.

KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.

Triptolide Downregulates the Expression of NRF2 Target Genes by Increasing Cytoplasmic Localization of NRF2 in A549 Cells.

We have identified triptolide as a novel NRF2 inhibitor, which significantly attenuates ARE-luciferase activity at nanomolar concentrations. Triptolide did not affect the level of NRF2, but significantly inhibited the expression of NRF2 target genes in A549 cells. We found that NRF2 possesses a previously unrecognized NES in the Neh2 domain, and that triptolide promotes an interaction between NRF2 and CRM1. Triptolide also decreased nuclear accumulation of NRF2, suggesting that it promotes nuclear export of NRF2. In addition, we show that triptolide decreased the expression of NRF2 target genes and increased intracellular oxidative stress, suppressing invasion and promoting cisplatin-induced apoptosis in A549 cells. Finally, oral administration of triptolide suppressed the growth of A549 xenografts in athymic mice by decreasing the expression of NRF2 target genes and promoting oxidative damages via the nuclear export of NRF2 and CRM1 in vivo. To the best of our knowledge, triptolide is the first type of compound to inhibit NRF2 by increasing cytoplasmic localization of NRF2.

Red ginseng oil promotes hair growth and protects skin against UVC radiation.

BACKGROUND: A wide range of environmental factors, such as diseases, nutritional deficiencies, ageing, hormonal imbalances, stress, and ultraviolet (UV) radiation, may affect the structure and function of the skin that covers the entire surface of the human body. In this study, we investigated roles of red ginseng oil (RGO) in enhancing skin functions, including hair growth and skin protection, using mouse models. METHODS: For hair growth experiment, shaved dorsal skins of C57BL/6 mice were topically applied with vehicle, RGO, RGO's major compounds, or minoxidil for consecutive 21 days and skin tissues were examined the hair growth promoting capacity. For skin protection experiment, SKH-1 hairless mice were topically applied with vehicle or RGO twice a day for three days prior to exposure to UVC radiation at 20 kJ/cm2. Skin tissues were collected to evaluate skin protective effects of RGO. RESULTS: Topical application of RGO to C57BL/6 mice effectively promoted hair regeneration by inducing early telogen-to-anagen transition and significantly increasing the density and bulb diameter of hair follicles. Major compounds, including linoleic acids and ß-sitosterol, contributed to RGO-promoted hair growth. Treatment with RGO as well as its major components upregulated expression of hair growth-related proteins. Furthermore, in SKH-1 hairless mice, RGO had a protective effect against UVC-induced skin damage by inhibiting inflammation and apoptosis, as well as inducing cytoprotective systems. CONCLUSION: These data suggest that RGO may be a potent agent for improving skin health and thereby preventing and/or treating hair loss and protecting skin against UV radiation.

Inhibition of oxidative stress induced-cytotoxicity by coptisine in V79-4 Chinese hamster lung fibroblasts through the induction of Nrf-2 mediated HO-1 expression.

BACKGROUND: Coptisine is a natural alkaloid compound and is known to have multiple beneficial effects including antioxidant activity. However, whether it can protect lung fibroblasts from oxidative damage has not been studied yet. OBJECTIVES: To investigate the potential inhibitory effect of coptisine against oxidative stress in V79-4 lung fibroblast cells. METHODS: V79-4 cells were treated with H2O2 (1 mM) in the presence or absence of coptisine (50 µg/ml), N-acetyl cysteine (NAC, 10 mM) or zinc protoporphyrin IX (ZnPP, 10 µM) for the indicated times. The alleviating effects of coptisine on cytotoxicity, cell cycle arrest, apoptosis, reactive oxygen species (ROS) production, DNA damage, mitochondrial dynamics, and inhibition of ATP production against H2O2 were investigated. Western blot analysis was used to analyze the expression levels of specific proteins. RESULTS: Coptisine inhibited H2O2-induced cytotoxicity and DNA damage by blocking abnormal ROS generation. H2O2 treatment caused cell cycle arrest at the G2/M phase accompanied by increased expression of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 and decreased expression of cyclin B1 and cyclin A. However, these effects were attenuated in the presence of coptisine or NAC. Coptisine also prevented apoptosis by decreasing the rate of Bax/Bcl-2 expression in H2O2-stimulated cells and suppressing the loss of mitochondrial membrane potential and the cytosolic release of cytochrome c. In addition, the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was markedly promoted by coptisine in the presence of H2O2. However, zinc protoporphyrin IX, a potent inhibitor of HO-1, attenuated the ROS scavenging and anti-apoptotic effects of coptisine. CONCLUSIONS: Based on current data, we suggest that coptisine can be used as a potential treatment for oxidative stress-related lung disease.

Auranofin Attenuates Non-Alcoholic Fatty Liver Disease by Suppressing Lipid Accumulation and NLRP3 Inflammasome-Mediated Hepatic Inflammation In Vivo and In Vitro.

Non-alcoholic fatty liver disease (NAFLD) causes liver dysfunction and is associated with obesity and type 2 diabetes. Chronic inflammation is associated not only with the development of NAFLD, but also with hepatic diseases, including steatohepatitis, cirrhosis, and hepatocellular carcinoma. Auranofin is a treatment for rheumatoid arthritis and has recently been reported to have potential effects against a variety of diseases, including inflammation, cancer, and viral infection. In this study, auranofin may be considered as a new treatment for the management of metabolic syndrome, as well as in the treatment of NAFLD through immunomodulation. To determine the effect of auranofin on NAFLD, C57BL/6 mice were randomly grouped, fed a regular diet or a high fat diet (HFD), and injected with normal saline or auranofin for 8 weeks. Auranofin significantly decreased the body weight, epididymal fat weight, serum aspartate aminotransferase (AST), and glucose, as well as the serum triglyceride, cholesterol, and low-density lipoprotein cholesterol levels as compared to the HFD group. We also observed that hepatic steatosis was increased in the HFD group and was suppressed by auranofin treatment. In addition, auranofin suppressed the expressions of interleukin (IL)-1ß, IL-18, caspase-1, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) in the liver tissue. Furthermore, the expression of NADPH oxidase 4 and peroxisome proliferator-activated receptor γ (PPARγ), which are a major source of oxidative stress and a regulator of adipogenesis, respectively, were also decreased by auranofin. In addition, primary mouse hepatocytes were incubated with lipopolysaccharide (LPS) and palmitic acid (PA) to induce lipid accumulation and hepatic inflammation for an in vitro model. Auranofin could significantly inhibit LPS- and PA-induced inflammatory activity including nitric oxide and NLRP3 inflammasome-mediated cytokines. The results of this study demonstrate that auranofin treatment inhibits the characteristics of NAFLD through the inhibition of NLRP3 inflammasome. Therefore, auranofin may have potential as a candidate for improving NAFLD symptoms.

Cardamonin Inhibits Oxazolone-Induced Atopic Dermatitis by the Induction of NRF2 and the Inhibition of Th2 Cytokine Production.

The skin is constantly exposed to various types of chemical stresses that challenge the immune cells, leading to the activation of T cell-mediated hypersensitivity reactions including atopic dermatitis. Previous studies have demonstrated that a variety of natural compounds are effective against development of atopic dermatitis by modulating immune responses. Cardamonin is a natural compound abundantly found in cardamom spices and many other medicinal plant species. In the present study, we attempted to examine whether cardamonin could inhibit oxazolone-induced atopic dermatitis in vivo. Our results show that topical application of cardamonin onto the ear of mice suppressed oxazolone-induced inflammation in the ear and hyperplasia in the spleen. Cardamonin also inhibited oxazolone-induced destruction of connective tissues and subsequent infiltration of mast cells into the skin. In addition, we found that the production of Th2 cytokines is negatively regulated by NRF2, and the induction of NRF2 by cardamonin contributed to suppressing oxazolone-induced Th2 cytokine production and oxidative damages in vivo. Together, our results demonstrate that cardamonin is a promising natural compound, which might be effective for treatment of atopic dermatitis.

Effect of fermented oyster extract on growth promotion in Sprague-Dawley rats.

BACKGROUND: Oysters (Crassostrea gigas) are a popular marine product worldwide and have the advantage of nutritional benefits. This study aimed to investigate the effect of fermented oyster extract (FO) on growth promotion, including analysis of body size, bone microarchitecture, hematology and biochemistry in vivo. METHODS: The amount of nutrients and gamma aminobutyric acid (GABA) were determined. Sprague-Dawley rats were randomly divided into four groups: the control group, FO 50 group (FO 50 mg/kg), and FO 100 group (FO 100 mg/kg) were administered orally once daily and the recombinant human growth hormone (rhGH) group (200 µg/kg) was intraperitoneally injected once daily for 14 days. RESULTS: Oral administration of FO 100 significantly increased body length and had no effect on organ damage or hematological profiles. However, administration of rhGH significantly induced hypertrophy of the liver, kidney and spleen along with a marked increase in body length. Tibia length and the growth plate were increased, and bone morphometric parameters were slightly improved by FO and rhGH administration. Serum analysis showed that the levels of GH and insulin like growth factor-1 (IGF-1) were slightly upregulated by FO administration. Nevertheless, the protein expression of hepatic IGF-1 was markedly increased by FO 100 and rhGH administration. CONCLUSIONS: FO have high content of GABA, and induced positive effects on body length, tibial length, growth-plate length and hepatic IGF-1 synthesis in SD rats with no toxicity or alterations of hematological profile. Therefore, these results suggest that GABA-enriched FO could be considered a potential alternative treatment for growth stimulation.

Molecular mechanisms and systemic targeting of NRF2 dysregulation in cancer.

NF-E2-related factor 2 (NRF2) is a master regulator of redox homeostasis and provides cellular protection against oxidants and electrophiles by inducing the expression of a wide array of phase II cytoprotective genes. Until now, a number of NRF2 activators have been developed for treatment of chronic diseases and some are under evaluation in the clinical studies. On the other hand, accumulating evidence indicates that NRF2 confers chemoresistance and radioresistance, and its expression is correlated with poor prognosis in cancer patients. Studies in the last decade demonstrate that diverse mechanisms such as somatic mutations, accumulation of KEAP1 binding proteins, transcriptional dysregulation, oncogene activation, and accumulation of reactive metabolites contribute to NRF2 activation in cancer. In the present review, we illustrate the molecular mechanisms governing the function of NRF2 and explain how they are hijacked in cancer. We also provide some examples of NRF2 inhibitors together with a brief explanation of their mechanisms of action.

Regulation of NRF2 by Na+/K+-ATPase: implication of tyrosine phosphorylation of Src.

Tumors adapt well to the imbalanced redox status created by rapid growth and limited nutrient availability because they highly express high levels of NRF2 to counteract oxidative stress. Therefore, inhibition of NRF2 is currently considered a feasible strategy for development of chemotherapeutic agents. In the present study, we identified that Na+/K+-ATPase regulates NRF2 in A549 cells. Suppression of Na+/K+-ATPase by convallatoxin or siRNAs downregulates NRF2 in A549 cells, and this event is mediated by Ca2+-dependent induction of CSK1 and subsequent phosphorylation of SRC at Tyr 527. Consistent with this finding, knocking down the α1 or ß1 subunit of Na+/K+-ATPase promotes the generation of intracellular ROS by cisplatin and potentiates cisplatin-induced apoptosis and autophagy in A549 cells. Our study reveals that the signaling axis composed of Na+/K+-ATPase, CSK1, and tyrosine phosphorylation of Src could be a useful target for development of NRF2 inhibitors.

Binding partners of NRF2: Functions and regulatory mechanisms.

NRF2 is a redox-sensitive transcription factor that plays an important role in protecting organisms against diverse types of electrophiles or oxidants. The level of NRF2 is maintained low in normal cells, but highly elevated in cancer provoking chemoresistance or radioresistance. It is now recognized that NRF2 does not merely maintain the redox balance, but also plays significant roles in autophagy, apoptosis, cell cycle progression, and stem cell differentiation, all of which could be possibly attributable to the existence of multiple binding proteins. In the present manuscript, we summarize direct binding partners of NRF2 and illustrate how they bind to NRF2 and regulate its stability or activity.

Homoharringtonine stabilizes secondary structure of guanine-rich sequence existing in the 5'-untranslated region of Nrf2.

Homoharringtonine, known as omacetaxine mepesuccinate, is a pharmaceutical drug substance approved for treatment of chronic myeloid leukemia. Here, we report that homoharringtonine (HHT) is a novel chemical inhibitor of NRF2. HHT significantly suppressed NRF2 and ARE-dependent gene expression in human lung carcinoma A549 cells. HHT stabilized secondary structure of guanine-rich sequence existing in the 5'-untranslated region (5'-UTR) of Nrf2 and sensitized A549 cells to etoposide-induced apoptosis. To the best of our knowledge, HHT is the first type of transcriptional inhibitor of Nrf2 that stabilizes guanine-rich sequence existing in the 5'-UTR. Our study also provides a novel mechanism of action underlying how HHT exerts anti-carcinogenic effects in cancer cells.

4'-O-ß-D-Glucosyl-5-O-Methylvisamminol Attenuates Pro-Inflammatory Responses and Protects against Oxidative Damages.

We attempted to examine anti-inflammatory and anti-oxidant effects of 4'-O-ß-D-glucosyl-5-O-methylvisamminol (GOMV), the first epigenetic inhibitor of histone phosphorylation at Ser10. While GOMV did not affect the viability of murine macrophage RAW 264.7 cells, it significantly suppressed lipopolysaccharide (LPS)-induced generation of prostaglandin E2 (PGE2) and nitric oxide (NO) through transcriptional inhibition of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). GOMV also scavenged free radicals in vitro, increased NF-E2-related factor 2 (NRF2), and activated antioxidant response element (ARE), thereby resulting in the induction of phase II cytoprotective enzymes in human keratinocyte HaCaT cells. Finally, GOMV significantly protected HaCaT cells against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative intracellular damages. Together, our results illustrate that GOMV possesses anti-inflammatory and anti-oxidant activity.

Suppression of NRF2/ARE by convallatoxin sensitises A549 cells to 5-FU-mediated apoptosis.

NF-E2-related factor 2 (NRF2) regulates transcription of phase II cytoprotective enzymes to protect normal cells against oxidative stress. However, a high level of NRF2 offers a growth advantage, chemoresistance, and radioresistance in cancer. In the present study, we have identified convallatoxin as a novel inhibitor of NRF2/ARE. Suppression of NRF2 by convallatoxin was not transcriptionally mediated, but regulated at the level of proteolysis. Convallatoxin activated GSK-3ß and suppression of NRF2 by convallatoxin required the Neh6 domain. Convallatoxin sensitised A549 cells to 5-fluorouracil-mediated cell death by promoting apoptosis. Together, our results provide evidence that convallatoxin might be useful as a chemotherapeutic adjuvant due to its ability to suppress NRF2/ARE.

Marliolide inhibits skin carcinogenesis by activating NRF2/ARE to induce heme oxygenase-1.

Heme oxygenase-1 (HO-1) catalyzes the enzymatic degradation of heme to produce three anti-oxidant molecules: carbon monoxide (CO), ferrous ion (Fe2+), and biliverdin. Induction of HO-1 is currently considered as a feasible strategy to treat oxidative stress-related diseases. In the present study, we identified marliolide as a novel inducer of HO-1 in human normal keratinocyte HaCaT cells. Mechanism-based studies demonstrated that the induction of HO-1 by marliolide occurred through activation of NRF2/ARE via direct binding of marliolide to KEAP1. Structure-activity relationship revealed chemical moieties of marliolide critical for induction of HO-1, which renders a support for Michael reaction as a potential mechanism of action. Finally, we observed that marliolide significantly inhibited the papilloma formation in DMBA/TPA-induced mouse skin carcinogenesis model and this event was closely associated with lowering the formation of 8-OH-G and 4-HNE in vivo. Together, our study provides the first evidence that marliolide might be effective against oxidative stress-related skin disorders.