Nuclear Factor Erythroid 2-like 2 Research Articles

PGC-1α regulates the interplay between oxidative stress, senescence and autophagy in the ageing retina important in age-related macular degeneration.

We previously showed that mice with knockout in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) gene encoding the PGC-1α protein, and nuclear factor erythroid 2 like 2 (NFE2L2) gene, exhibited some features of the age-related macular degeneration (AMD) phenotype. To further explore the mechanism behind the involvement of PGC-1α in AMD pathogenesis we used young (3-month) and old (12-month) mice with knockout in the PPARGC1A gene and age-matched wild-type (WT) animals. An immunohistochemical analysis showed age-dependent different expression of markers of oxidative stress defence, senescence and autophagy in the retinal pigment epithelium of KO animals as compared with their WT counterparts. Multivariate inference testing showed that senescence and autophagy proteins had the greatest impact on the discrimination between KO and WT 3-month animals, but proteins of antioxidant defence also contributed to that discrimination. A bioinformatic analysis showed that PGC-1α might coordinate the interplay between genes encoding proteins involved in antioxidant defence, senescence and autophagy in the ageing retina. These data support importance of PGC-1α in AMD pathogenesis and confirm the utility of mice with PGC-1α knockout as an animal model to study AMD pathogenesis.

Abstract 391: Role of NRF2 in HIF-2α-mediated cancer stem cell phenotype

Abstract The acquisition of cancer stem cell (CSC) properties is influenced by the microenvironment, with tumor hypoxia playing a pivotal role. Elevated levels of nuclear factor erythroid 2-like 2 (NRF2), a redox balance-maintaining transcription factor, have been linked to enhanced tumor growth and therapy resistance. This study investigates the potential role of NRF2 in hypoxia-inducible factor-2α (HIF-2α) regulation in cancers. First, in a model of a 72-hour hypoxic incubation, chronic hypoxia led to CSC characteristics, including increased expression of KLF4 and OCT4, heightened cancer migration, and spheroid growth, which are associated with HIF-2α accumulation. NRF2 inhibition mitigated these effects, while NRF2 activation enhanced chronic hypoxia-induced CSC traits. Mechanistically, NRF2/miR181a-2 signaling emerged as a regulator of HIF-2α-mediated CSC phenotypes in chronic hypoxic conditions. Second, in clear cell renal cell carcinoma (ccRCC), characterized by pVHL gene mutation and constitutive HIF-2α elevation, the role of NRF2 in aggressive cancer phenotypes was explored. Silencing of NRF2 impaired aggressive phenotypes in multiple ccRCC cell lines with a reduction in HIF-2α expression. Promoter analysis elucidated that NRF2 directly upregulates HIF-2α expression through antioxidant response elements in the human HIF-2α gene. Collectively, our study suggests the involvement of NRF2/miR181a-2 signaling in the development of HIF-2α-mediated CSC phenotypes in sustained hypoxic environments and also proposes NRF2 as a promising target for mitigating HIF-2α-mediated CSC traits in ccRCC. Citation Format: Mi-Kyoung Kwak, Steffanus P. Hallis. Role of NRF2 in HIF-2α-mediated cancer stem cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 391.

Abstract 5846: Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors

Abstract Bacground: Glioblastoma (GBM) is the most common primary brain tumor, accounting for 15% of all central nervous system related tumors. Despite the aggressive standard of care treatment including chemotherapy, radiotherapy, and maximally safe surgical resection, patient outcomes are abysmal: with 95% of patients relapsing and a median overall survival of 15 months. Thus, this necessitates the rapid query for personalized therapeutics and agents that can potentiate the clinical effects of currently approved treatments. One such pathway that demonstrates aberrant functioning in cancer and is a targetable mechanism is the ubiquitin-proteosome pathway (UPP). Studies have shown that UPP related proteolysis remains constitutively active in cancer cells leading to the rapid degradation of proteins that regulate tumor suppressor genes and oncogenes. Despite robust preclinical evaluations for the usage of proteosome inhibitors against GBM, most tested proteosome inhibitors failed in phase II and III trials, indicating resistance. Methods: We conducted an unbiased genome wide CRISPR-Cas9 screen in human HAP1 cells treated with the proteosome inhibitor, Bortezomib (BTZ), to identify genes that may lead to a BTZ-resistant phenotype. We identified several genes that when perturbed, sensitized cells to BTZ including N-glycanase-1 (NGLY-1), Nuclear factor Erythroid 2-Like-1 (NFE2L1), and DNA damage inducible 1 homolog 2 (DDI2). Herein we sought to evaluate the effects of perturbing components of the proteosome rescue pathway in our Singh lab patient derived GBM cell lines by utilizing CRISPR/Cas9 knockout technology in vitro and in vivo. Results: The generation of NGLY-1, DDI2, and NFE2L1 KO cell lines demonstrated functional sensitivity to the proteosome inhibitor Marizomib (MZB) as observed through a significant reduction of the IC50 in the KO cell lines compared to a control. Functional evaluation also revealed a reduction in proliferation capacity as well as sphere formation in these genetically modified GBM lines. Ongoing in vivo work will aim to evaluate the mitigation of this resistant pathway in our NSG mouse models orthotopically transplanted with these patient derived KO cell lines. In an ongoing collaborative effort, we are working to identify and functionally assess a novel small molecule NGLY-1 inhibitor to evaluate preclinical sensitivity to MZB in our in vitro and in vivo models. Citation Format: Alisha Anand, Muhammad Vaseem Shaikh, Chirayu Chokshi, Benjamin Brakel, William Maich, Shan Grewal, Chitra Venugopal, Sheila Singh. Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5846.

Effects of antioxidant-rich Lactiplantibacillus plantarum inoculated alfalfa silage on rumen fermentation, antioxidant and immunity status, and mammary gland gene expression in dairy goats

BackgroundMilk synthesis in lactating animals demands high energy metabolism, which results in an increased production of reactive oxygen metabolites (ROM) causing an imbalance between oxidants and antioxidants thereby inducing oxidative stress (OS) on the animals. To mitigate OS and postpartum disorders in dairy goats and gain insight into the impact of dietary choices on redox status during lactation, a feeding trial was conducted using alfalfa silage inoculated with a high-antioxidant strain of Lactiplantibacillus plantarum.MethodsTwenty-four Guanzhong dairy goats (38.1 ± 1.20 kg) were randomly assigned to two dietary treatments: one containing silage inoculated with L. plantarum MTD/1 (RSMTD-1), and the other containing silage inoculated with high antioxidant activity L. plantarum 24-7 (ES24-7).ResultsES24-7-inoculated silage exhibited better fermentation quality and antioxidant activity compared to RSMTD-1. The ES24-7 diet elevated the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities in milk, serum, and feces of lactating goats (with the exception of T-AOC in milk). Additionally, the diet containing ES24-7 inoculated silage enhanced casein yield, milk free fatty acid (FFA) content, and vitamin A level in the goats’ milk. Furthermore, an increase of immunoglobulin (Ig)A, IgG, IgM, interleukin (IL)-4, and IL-10 concentrations were observed, coupled with a reduction in IL-1β, IL-2, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α concentrations in the serum of lactating goats fed ES24-7. Higher concentrations of total volatile fatty acid (VFA), acetate, and propionate were observed in the rumen fluid of dairy goats fed ES24-7 inoculated silage. Moreover, the diet containing ES24-7 inoculated silage significantly upregulated the expression of nuclear factor erythroid 2 like 2 (NFE2L2), beta-carotene oxygenase 1 (BCO1), SOD1, SOD2, SOD3, GPX2, CAT, glutathione-disulfide reductase (GSR), and heme oxygenase 1 (HMOX1) genes in the mammary gland, while decreased the levels of NADPH oxidase 4 (NOX4), TNF, and interferon gamma (IFNG).ConclusionsThese findings indicated that feeding L. plantarum 24-7 inoculated alfalfa silage not only improved rumen fermentation and milk quality in lactating dairy goats but also boosted their immunity and antioxidant status by modulating the expression of several genes related to antioxidant and inflammation in the mammary gland.

Identification and validation of molecular subtypes and prognostic signature for stage I and stage II gastric cancer based on neutrophil extracellular traps.

This study identified subtypes and prognostic signature of stage I and stage II gastric cancer based on neutrophil extracellular trap (NET)-related genes. The gene expression data associated with stage I and stage II gastric cancer were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. NET-related genes were obtained from previous reference. Differentially expressed NET-related genes were selected by consensus cluster analysis. The differences in immune infiltration between two subtypes were analyzed. Prognosis-related genes were further screened by univariate Cox regression analysis. Gene Set Enrichment Analysis (GSEA) of prognostic signatures was conducted with clusterprofiler. Finally, a miRNA-mRNA-transcription factor (TF) network was constructed. Total 43 differential NET-related genes were obtained and two subtypes were obtained based on these genes. Patients of cluster 2 had a better prognosis compared to cluster 1. Eight types of immune cells were differential in infiltration level between two subtypes. Following univariate Cox regression analysis, two genes of CXC chemokine receptor 4 (CXCR4) and nuclear factor, erythroid 2-like 2 (NFE2L2) significantly related to patient survival were selected. GSEA of single gene revealed that CXCR4 was associated with allograft rejection and NFE2L2 was associated with drug metabolism-cytochrome P450. A network with 421 miRNA-mRNA-TF regulatory pairs was constructed. The present study identified two subtypes and a prognostic signature for stage I and stage II gastric cancer based on NET-related genes.

Role of NFE2L1 in the Regulation of Proteostasis: Implications for Aging and Neurodegenerative Diseases.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis ("proteostasis") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced "bounce-back effect" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Activating transcription factor-2 supports the antioxidant capacity and ability of human mesenchymal stem cells to prevent asthmatic airway inflammation

Glutathione (GSH), an abundant nonprotein thiol antioxidant, participates in several biological processes and determines the functionality of stem cells. A detailed understanding of the molecular network mediating GSH dynamics is still lacking. Here, we show that activating transcription factor-2 (ATF2), a cAMP-response element binding protein (CREB), plays a crucial role in maintaining the level and activity of GSH in human mesenchymal stem cells (MSCs) by crosstalking with nuclear factor erythroid-2 like-2 (NRF2), a well-known master regulator of cellular redox homeostasis. Priming with ascorbic acid 2-glucoside (AA2G), a stable vitamin C derivative, increased the expression and activity of ATF2 in MSCs derived from human embryonic stem cells and umbilical cord. Subsequently, activated ATF2 crosstalked with the CREB1-NRF2 pathway to preserve the GSH dynamics of MSCs through the induction of genes involved in GSH synthesis (GCLC and GCLM) and redox cycling (GSR and PRDX1). Accordingly, shRNA-mediated silencing of ATF2 significantly impaired the self-renewal, migratory, proangiogenic, and anti-inflammatory capacities of MSCs, and these defects were rescued by supplementation of the cells with GSH. In addition, silencing ATF2 attenuated the ability of MSCs to alleviate airway inflammatory responses in an ovalbumin-induced mouse model of allergic asthma. Consistently, activation of ATF2 by overexpression or the AA2G-based priming procedure enhanced the core functions of MSCs, improving the in vivo therapeutic efficacy of MSCs for treating asthma. Collectively, our findings suggest that ATF2 is a novel modulator of GSH dynamics that determines the core functionality and therapeutic potency of MSCs used to treat allergic asthma.

Sulforaphane protects microvascular endothelial cells in lower limb ischemia/reperfusion injury mice.

Background: Microvascular damage is a key pathological factor in acute lower limb ischemia/reperfusion (I/R) injury. Current evidence suggests that sulforaphane (SFN) protects tissue from I/R injury. However, the role of SFN in acute lower limb I/R injury remains elusive. This study aimed to investigate the role and potential mechanism of SFN in I/R-related microvascular damage in the limb. Methods: Limb viability was evaluated by laser Doppler imaging, tissue edema analysis and histological analysis. Western blotting and immunofluorescence were applied to analyze the levels of apoptosis, oxidative stress, autophagy, transcription factor EB (TFEB) activity and mucolipin 1 (MCOLN1)-calcineurin signaling pathway. Results: SFN administration significantly ameliorated I/R-induced hypoperfusion, tissue edema, skeletal muscle fiber injury and endothelial cell (EC) damage in the limb. Pharmacological inhibition of NFE2L2 (nuclear factor, erythroid 2 like 2) reversed the anti-oxidation and anti-apoptosis effects of SFN on ECs. Additionally, silencing of TFEB by interfering RNA abolished the SFN-induced autophagy restoration, anti-oxidant response and anti-apoptosis effects on ECs. Furthermore, silencing of MCOLN1 by interfering RNA and pharmacological inhibition of calcineurin inhibited the activity of TFEB induced by SFN, demonstrating that SFN regulates the activity of TFEB through the MCOLN1-calcineurin signaling pathway. Conclusion: SFN protects microvascular ECs against I/R injury by TFEB-mediated autophagy restoration and anti-oxidant response.

Low-dose exposure to PBDE disrupts genomic integrity and innate immunity in mammary tissue.

The low-dose mixture hypothesis of carcinogenesis proposes that exposure to an environmental chemical that is not individually oncogenic may nonetheless be capable of enabling carcinogenesis when it acts in concert with other factors. A class of ubiquitous environmental chemicals that are hypothesized to potentially function in this low-dose capacity are synthesized polybrominated diphenyl ethers (PBDEs). PBDEs can affect correlates of carcinogenesis that include genomic instability and inflammation. However, the effect of low-dose PBDE exposure on such correlates in mammary tissue has not been examined. In the present study, low-dose long-term (16 weeks) administration of PBDE to mice modulated transcriptomic indicators of genomic integrity and innate immunity in normal mammary tissue. PBDE increased transcriptome signatures for the Nuclear Factor Erythroid 2 Like 2 (NFE2L2) response to oxidative stress and decreased signatures for non-homologous end joining DNA repair (NHEJ). PBDE also decreased transcriptome signatures for the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes (cGAS-STING) response, decreased indication of Interferon Stimulated Gene Factor 3 (ISGF3) and Nuclear Factor Kappa B (NF-κB) transcription factor activity, and increased digital cytometry estimates of immature dendritic cells (DCs) in mammary tissue. Replication of the PBDE exposure protocol in mice susceptible to mammary carcinogenesis resulted in greater tumor development. The results support the notion that ongoing exposure to low levels of PBDE can disrupt facets of genomic integrity and innate immunity in mammary tissue. Such effects affirm that synthesized PBDEs are a class of environmental chemicals that reasonably fit the low-dose mixture hypothesis.

High Levels of Hyaluronic Acid Synthase-2 Mediate NRF2-Driven Chemoresistance in Breast Cancer Cells.

Hyaluronic acid (HA), a ligand of CD44, accumulates in some types of tumors and is responsible for tumor progression. The nuclear factor erythroid 2-like 2 (NRF2) regulates cytoprotective genes and drug transporters, which promotes therapy resistance in tumors. Previously, we showed that high levels of CD44 are associated with NRF2 activation in cancer stem like-cells. Herein, we demonstrate that HA production was increased in doxorubicin-resistant breast cancer MCF7 cells (MCF7-DR) via the upregulation of HA synthase-2 (HAS2). HA incubation increased NRF2, aldo-keto reductase 1C1 (AKR1C1), and multidrug resistance gene 1 (MDR1) levels. Silencing of HAS2 or CD44 suppressed NRF2 signaling in MCF7-DR, which was accompanied by increased doxorubicin sensitivity. The treatment with a HAS2 inhibitor, 4-methylumbelliferone (4-MU), decreased NRF2, AKR1C1, and MDR1 levels in MCF7-DR. Subsequently, 4-MU treatment inhibited sphere formation and doxorubicin resistance in MCF7-DR. The Cancer Genome Atlas (TCGA) data analysis across 32 types of tumors indicates the amplification of HAS2 gene is a common genetic alteration and is negatively correlated with the overall survival rate. In addition, high HAS2 mRNA levels are associated with increased NRF2 signaling and poor clinical outcome in breast cancer patients. Collectively, these indicate that HAS2 elevation contributes to chemoresistance and sphere formation capacity of drug-resistant MCF7 cells by activating CD44/NRF2 signaling, suggesting a potential benefit of HAS2 inhibition.

Beta-Boswellic Acid Protects Against Cerebral Ischemia/Reperfusion Injury via the Protein Kinase C Epsilon/Nuclear Factor Erythroid 2-like 2/Heme Oxygenase-1 Pathway.

Ischemic strokes are associated with a high rate of disability and death globally. Cerebral ischemia/reperfusion (I/R) injury is a type of brain damage associated with oxidative stress after an ischemic stroke. Beta-boswellic acid (β-BA) reportedly exerts antioxidant and neuroprotective effects, but its role in cerebral I/R injury is unclear. The aim of this research was to investigate the neuroprotective effects, as well as the mechanisms of β-BA in cerebral I/R injury. In vivo experiments were conducted using a rat middle cerebral artery occlusion and reperfusion (MCAO/R) model, and in vitro experiments were performed using a rat neuronal oxygen-glucose deprivation and reoxygenation (OGD/R) model. Triphenyltetrazolium chloride staining, neurological function scores, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, hematoxylin and eosin staining, and antioxidant levels in the brain were used to assess the effects of β-BA. Flow cytometry was used to detect reactive oxygen species and apoptotic cells. Western blotting and immunofluorescence staining were used to measure protein levels. The results showed that β-BA markedly improved neurological deficits and decreased infarct volume and necrotic neurons in rats. The in vitro results showed that β-BA protected neurons against OGD/R-induced injury. Additionally, β-BA significantly increased the phosphorylation of protein kinase C epsilon (PRKCE) at S729, the translocation of nuclear factor erythroid 2-like 2 (NFE2L2), and expression of heme oxygenase-1 (HMOX1). This study demonstrates that β-BA exerts neuroprotective effects against cerebral I/R via the activation of the PRKCE/NFE2L2/HMOX1 pathway and is a potential therapeutic candidate for ischemic stroke.

Ferroptosis Regulation by the NGLY1/NFE2L1 Pathway

Cells are equipped with various mechanisms to maintain cell viability upon exposure to stress. The Cap ‘n’ Collar (CNC) family includes five vertebrate stress‐responsive transcription factors. The CNC member nuclear factor erythroid 2 like 2 (NFE2L2) is considered the master regulator of the cellular oxidative stress response. Fellow CNC member nuclear erythroid 2 like 1 (NFE2L1) is better known for its role in responding to proteasome dysfunction. We hypothesized that NFE2L1 also responds to oxidative stress. We demonstrate that genetic disruption of NFE2L1 sensitizes cancer cells to death by a non‐apoptotic, oxidative form of cell death termed ferroptosis. We also show that genetic disruption of N‐glycanase 1 (NGLY1), which catalyzes NFE2L1 deglycosylation and sequence editing, sensitizes cancer cells to ferroptosis through an NFE2L1‐dependent mechanism. Expression of a sequence‐edited NFE2L1 mutant, which mimics NGLY1‐processed NFE2L1, rescues cellular resistance to ferroptosis in NFE2L1 gene‐disrupted cells. Finally, we provide evidence that the ferroptosis protection conferred by NFE2L1 is mediated by the expression of glutathione peroxidase 4 (GPX4). All together, our results highlight the role of NFE2L1 in ferroptosis resistance and implicate NFE2L1 and NGLY1 as potential targets for cancer therapy.

Breast cancer susceptibility gene 1 regulates oxidative damage via nuclear factor erythroid 2-related factor 2 in oral cancer cells

ObjectiveTo determine whether the breast cancer susceptibility gene 1 (BRCA1) regulates oxidative damage in oral cancer cells by interacting with nuclear factor erythroid 2-like 2 (NRF2). DesignThe BRCA1 gene was silenced in CAL-27 and DOK cells using specific shRNA, and NRF2 was activated with sulforaphane. The expression levels of BRCA1, NRF2 and its target genes were assessed by quantitative real-time polymerase chain reaction and western blotting. Cell counting kit-8 assay was used to detect cell proliferation, apoptosis was detected by flow cytometry, and 8-OXo-2’-deoxyguanosine level was measured by enzyme-linked immunosorbent assay. The expression of BRCA1 and NRF2 in patients with oral leukoplakia and oral squamous cell carcinoma were evaluated by immunohistochemistry. ResultsBRCA1 knockdown downregulated NRF2 and its target genes, increased proliferation rates, reduced apoptosis, and increased 8-OXo-2’-deoxyguanosine levels compared to the control. Activation of NRF2 by sulforaphane significantly upregulated NRF2 levels in the BRCA1-depleted cells, and restored proliferation, apoptosis and 8-OXo-2’-deoxyguanosine level in a dose-dependent manner. Compared with patients with leukoplakia, BRCA1 and NRF2 expression were increased in patients with oral squamous cell carcinoma. ConclusionsBRCA1 depletion increases oxidative damage and promotes the malignant phenotype, which may eventually promote oral carcinogenesis. The NRF2-activator sulforaphane is a potential chemo-preventive agent for oral cancer.

A high-concentrate diet induces an inflammatory response and oxidative stress and depresses milk fat synthesis in the mammary gland of dairy cows

Although high-concentrate diet feeding can temporarily increase milk production, it can cause a series of metabolic diseases, such as subacute ruminal acidosis (SARA) and milk fat depression. The main purpose of this experiment was to study the effects of a high-concentrate diet on the inflammatory response, oxidative stress, and milk fat synthesis in the mammary gland of dairy cows. Twelve Holstein cows equipped with rumen fistulas were randomly divided into 2 groups, each with 6 cows, fed a low-concentrate diet (LC) and a high-concentrate diet (HC). On d 20 and 21 of the experiment, rumen fluid was collected to measure pH, and milk samples were collected for milk component analysis and lipopolysaccharide (LPS) concentration testing. On d 21, mammary vein blood was collected to detect the LPS concentration. At the end of the 21-d experimental period, mammary gland tissue was collected, and the expression of inflammatory response-, oxidative stress-, and milk fat synthesis-related genes and proteins in the mammary gland was analyzed by real-time quantitative PCR and western blot. The pH of rumen fluid in the HC group was significantly lower than that in the LC group, and the pH of 2 time points in the HC group was lower than 5.6, indicating that a high-concentrate diet induced SARA. The LPS concentration of the peripheral blood in HC group increased significantly compared with that in the LC group. For the inflammatory response, the proinflammatory cytokines (IL-6 and IL-1α) and innate immune factors (lingual antimicrobial peptide and tracheal antimicrobial peptide) in the mammary gland of the HC group were significantly increased, and the TLR4-NF-κB signaling pathway was activated. For oxidative stress, after HC diet feeding, the content of malondialdehyde in mammary vein blood and mammary gland tissue increased, the content of glutathione in mammary vein blood decreased, the activity of superoxide dismutase and the total antioxidant capacity in mammary gland tissue and mammary vein blood decreased, and the expression of antioxidant enzymes and antioxidant transcription factor nuclear factor, erythroid 2 like 2 (NFE2L2) in mammary gland decreased. For milk fat metabolism, HC diet feeding reduced the milk fat content in milk samples and the triacylglycerol content in the mammary gland and inhibited the expression of de novo synthase (ACACA and FASN), long-chain fatty acid converting enzymes (ACSL1 and SCD), fatty acid transporters (CD36, FATP, FABP3, and LPL), triacylglycerol synthase (AGPAT6, DGAT1, and LPIN1), lipid droplet releasing enzyme (PLIN1), and transcription factors sterol regulatory element binding protein (SREBP1) and peroxisome proliferator activated receptor gamma (PPARG). In summary, a HC diet can induce SARA with increased concentration of LPS in the peripheral vein, stimulate inflammatory reactions and oxidative stress, and inhibit milk fat synthesis in the mammary gland of dairy cows.

A Mixture of Topical Forms of Polydeoxyribonucleotide, Vitamin C, and Niacinamide Attenuated Skin Pigmentation and Increased Skin Elasticity by Modulating Nuclear Factor Erythroid 2-like 2.

It is well-known that increased oxidative stress caused by ultraviolet B (UV-B) radiation induces melanogenesis and activates metalloproteinases (MMPs), which degrade collagen and elastin fibers, leading to decreased skin elasticity. Various antioxidant agents, such as vitamin C and niacinamide, have been evaluated for use as treatments for photoaging or skin pigmentation. In this study, we evaluated the ability of a topical liquid formula of polydeoxyribonucleotide (PDRN), vitamin C, and niacinamide (PVN) delivered via a microneedling therapy system (MTS) to attenuate photoaging and pigmentation by increasing nuclear factor erythroid 2-like 2 (NRF2)/heme oxygenase-1 (HO-1) and decreasing MMP expression in a UV-B-radiated animal model. The effects of the PVN were compared with those of individual PDRN and hydroquinone (HQ) compounds. The expression of NRF2/HO-1 significantly increased in response to HQ, PDRN, and PVN in UV-B-radiated animal skin. The activity of nicotinamide adenine dinucleotide phosphate hydrogen oxidase decreased in response to HQ, PDRN, and PVN, and the superoxide dismutase activity increased. The expression of tumor protein p53 and microphthalmia-associated transcription factor and tyrosinase activity decreased in response to HQ, PDRN, and PVN, and this decrease was accompanied by decreased melanin content in the skin. The expression of nuclear factor kappa-light-chain enhancer of activated B cells and MMP2/3/9 decreased in response to HQ, PDRN, and PVN in UV-B-radiated skin. However, the expression of collagen type I α1 chain and the amount of collagen fibers that were evaluated by Masson’s trichrome staining increased in response to HQ, PDRN, and PVN. The contents of elastin fibers, fibrillin 1/2 and fibulin 5 increased in response to HQ, PDRN, and PVN. In conclusion, PVN delivered via MTS led to decreased melanogenesis and destruction of collagen and elastin fibers by MMPs, and, thus, PVN decreased skin pigmentation and increased skin elasticity.

Long-isoform NFE2L1 silencing inhibits acquisition of malignant phenotypes induced by arsenite in human bronchial epithelial cells

Chronic arsenic exposure is associated with the increased risk of several types of cancer, among which, lung cancer is the most deadly one. Nuclear factor erythroid 2 like 1 (NFE2L1), a transcription factor belonging to CNC-bZIP family, regulates multiple important cellular functions in response to acute arsenite exposure. However, the role of NFE2L1 in lung cancer induced by chronic arsenite exposure is unknown. In this study, we firstly showed that chronic arsenite exposure (36 weeks) led to epithelial-mesenchymal transition (EMT) and malignant transformation in human bronchial epithelial cells (BEAS-2B). During the process of malignant transformation, the expression of long isoforms of NFE2L1 (NFE2L1-L) was elevated. Thereafter, BEAS-2B cells with NFE2L1-L stable knockdown (NFE2L1-L-KD) was chronically exposed to arsenite. As expected, silencing of NFE2L1-L gene strikingly inhibited the arsenite-induced EMT and the subsequent malignant transformation. Additionally, NFE2L1-L silencing suppressed the transcription of EMT-inducer SNAIL1 and increased the expression of E-cadherin. Conversely, NFE2L1-L overexpression increased SNAIL1 transcription but decreased E-cadherin expression. Collectively, our data suggest that NFE2L1-L promotes EMT by positively regulating SNAIL1 transcription, and is involved in malignant transformation induced by arsenite.

Prognostic Value and Immune Infiltration Analysis of Nuclear Factor Erythroid-2 Family Members in Ovarian Cancer.

Ovarian cancer (OC) often presents at an advanced stage and is still one of the most frequent causes of gynecological cancer-related mortality worldwide. The nuclear factor erythroid-2 (NFE2) transcription factors include nuclear factor, erythroid 2 like 1 (NFE2L1), NFE2L2, and NFE2L3. NFE2 members bind to the antioxidant-response element (ARE) region and activate the expression of targeted genes. The distinct functions of NFE2 members in OC remain poorly elucidated. Several online bioinformatics databases were applied to determine gene expression, prognosis, mutations, and immune infiltration correlation in OC patients. NFE2L1 and NFE2L2 were decreased in OC, whereas NFE2L3 was increased. NFE2L2 and NFE2L3 were significantly correlated with the clinical stages of OC. High NFE2L1 level was significantly associated with short progression-free survival (PFS) in patients with OC (HR = 1.18, P = 0.021), while high NFE2L2 expression strongly correlated with long PFS (HR = 0.77, P = 0.00067). High NFE2L3 expression was associated with better overall survival and postprogression survival in OC. Functional analysis showed that NFE2 members mainly focused on transcription coactivator activities. Genetic alterations of NFE2 members were found in 13% of OC patients, and amplification ranked the top. The expression of NFE2 members was significantly correlated with immune infiltration of CD4+ T cells, CD8+ T cells, B cells, macrophages, and neutrophils in OC. Our study provides novel insights into the roles and prognostic potential of NFE2 family members in OC.

Protein disulfide isomerase A1‑associated pathways inthedevelopment of stratified breast cancer therapies.

The oxidoreductase protein disulfide isomerase A1 (PDIA1) functions as a cofactor for many transcription factors including estrogen receptor α (ERα), nuclear factor (NF)-κB, nuclear factor erythroid 2-like 2 (NRF2) and regulates the protein stability of the tumor suppressor p53. Taking this into account we hypothesized that PDIA1, by differentially modulating the gene expression of a diverse subset of genes in the ERα-positive vs. the ERα-negative breast cancer cells, might modify dissimilar pathways in the two types of breast cancer. This hypothesis was investigated using RNA-seq data from PDIA1-silenced MCF-7 (ERα-positive) and MDA-MB-231 (ERα-negative) breast cancer cells treated with either interferon γ (IFN-γ) or etoposide (ETO), and the obtained data were further analyzed using a variety of bioinformatic tools alongside clinical relevance assessment via Kaplan-Meier patient survival curves. The results highlighted the dual role of PDIA1 in suppressing carcinogenesis in the ERα(+) breast cancer patients by negatively regulating the response to reactive oxygen species (ROS) and promoting carcinogenesis by inducing cell cycle progression. In the ERα(−) breast cancer patients, PDIA1 prevented tumor development by modulating NF-κB and p53 activity and cell migration and induced breast cancer progression through control of cytokine signaling and the immune response. The findings reported in this study shed light on the differential pathways regulating carcinogenesis in ERα(+) and ERα(−) breast cancer patients and could help identify therapeutic targets selectively effective in ERα(+) vs. ERα(−) patients.

NFE2L1-mediated proteasome function protects from ferroptosis

ObjectiveFerroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. Methods and resultsHere we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. ConclusionOur data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.