Antioxidants

Research

11 pages, 3619 KiB

Open AccessCommunication

Myeloid Nrf2 Protects against Neonatal Oxidant-Stress-Induced Lung Inflammation and Alveolar Simplification in Mice

by Chandra Mohan Tamatam, Lalith Kumar Venkareddy, Aparna Ankireddy, Narsa Machireddy and Sekhart P. Reddy

Antioxidants 2024, 13(6), 698; https://doi.org/10.3390/antiox13060698 - 7 Jun 2024

Viewed by 319

Abstract

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency [...] Read more.

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency exacerbates hypoalveolarization in mice. The abnormal recruitment and activation of myeloid cells are associated with the pathogenesis of BPD. Therefore, we employed a genetic approach to investigate the role of myeloid Nrf2 in regulating hyperoxia-induced hypoalveolarization. Pups, both wild-type (Nrf2^f/f) and those with a myeloid Nrf2 deletion (abbreviated as Nrf2^∆/∆mye), were exposed to hyperoxia for 72 h at postnatal day 1 (Pnd1), and then sacrificed at either Pnd4 or Pnd18 following a two-week recovery period. We analyzed the hypoalveolarization, inflammation, and gene expression related to cytoprotective and inflammatory responses in the lungs of these pups. The hypoalveolarization induced by hyperoxia was significantly greater in Nrf2^∆/∆mye pups compared to their Nrf2^f/f counterparts (35.88% vs. 21.01%, respectively) and was accompanied by increased levels of inflammatory cells and IL-1β activation in the lungs. Antioxidant gene expression in response to neonatal hyperoxia was lower in Nrf2^∆/∆mye pups compared to their Nrf2^f/f counterparts. Furthermore, Nrf2-deficient macrophages exposed to hyperoxia exhibited markedly decreased cytoprotective gene expression and increased IL-1β levels compared to Nrf2-sufficient cells. Our findings demonstrate the crucial role of myeloid Nrf2 in mitigating hyperoxia-induced lung hypoalveolarization and inflammatory responses in neonatal mice. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
(a) Wild-type (Nrf2+/+) and Nrf2–/– pups at Pnd1 were exposed to hyperoxia for 72 h and recovered at room air for 14 days as outlined. (b) Survival rates of neonatal hyperoxia-exposed Nrf2+/+ and Nrf2–/– pups in recovery. (c) Room air (RA) and 72 h hyperoxia-exposed and recovered Nrf2+/+ and Nrf2–/– pups at Pnd18 (2WKR) were sacrificed, and the left lung was fixed, sectioned and stained with H&E. A representative image of the lung sections is shown (20×). (d) Mean chord length (MCL) of the alveolar region of H&E images (10×) was analyzed by morphometry, as detailed in the methods. ***, ****, compared to respective RA controls; $$, compared to Nrf2+/+ counterparts. Scale bars: 100 µm. Full article ">Figure 2
(a) Mice with Nrf2 deletion in myeloid cells (Nrf2Δ/Δmye) were generated by crossing Nrf2-floxed mice with LysM2-Cre mice and genotyped. Nrf2 deletion in Nrf2Δ/Δmye mice was characterized in tail DNA and BAL lung macrophages (MΦs) by PCR genotyping. (b) Newborn (Pnd1) pups from Nrf2f/f (left panel) and Nrf2Δ/Δmye mice (right panel) were exposed to 72 h hyperoxia and then recovered at room air for two weeks (2WKR), as in <a href="#antioxidants-13-00698-f001" class="html-fig">Figure 1</a>a. Their survival rates were monitored for up to 14 days. (c) Pups exposed to room air or 72 h hyperoxia and sacrificed at Pnd18, and the left lung was fixed, sectioned, and stained with H&E. A representative image of the lung sections of both genotypes is shown (20×). (d) H&E images (10×) were analyzed to determine the mean chord length (MCL). ****, compared to respective genotype RA controls; $$$, compared to Nrf2f/f counterparts. Scale bars: 100–101 µm. Full article ">Figure 3
(a) Room air or neonatal hyperoxia-exposed Nrf2f/f and Nrf2Δ/Δmye pups were sacrificed at Pnd18, the right lung was lavaged, and inflammatory cells were enumerated. (b) RNA was isolated from the lungs of Nrf2f/f and Nrf2Δ/Δmye pups as detailed in panel A, and cytokine gene expression was analyzed by qPCR. *, **, ***, ****, compared to respective RA controls; $, compared to Nrf2f/f counterparts. Full article ">Figure 4
(a) Nrf2Δ/Δmye and Nrf2f/f pups were exposed to hyperoxia for 72 h and immediately sacrificed; the right lung was lavaged, and inflammatory cells were assessed. (b) RNA was isolated from the lungs of Nrf2Δ/Δmye and Nrf2f/f pups exposed to 72 h hyperoxia, and target gene expression was analyzed by qPCR. *, compared to respective RA controls, $, compared to Nrf2f/f counterparts. Full article ">Figure 5
RNA was isolated from the lungs of Nrf2Δ/Δmye and Nrf2f/f pups exposed to neonatal hyperoxia and recovered at room air for 14 days (a) from the lungs of Nrf2Δ/Δmye and Nrf2f/f pups exposed to neonatal hyperoxia for 72 h and (b) Nrf2 putative target gene expression was analyzed by qPCR. **, compared to respective RA controls; $$, compared to Nrf2f/f counterparts. Full article ">Figure 6
RNA isolated from the lungs of Nrf2Δ/Δmye pups and Nrf2f/f pups exposed to room air (RA) or 72h of hyperoxia at Pnd1 and sacrificed at Pnd4 was analyzed for Vegf, Fgf10, Notch1, and Notch2. Full article ">Figure 7
Freshly cultured BMDMs isolated from Nrf2+/+ (WT) and Nrf2–/– (NKO) mice were exposed to room air (RA) or hyperoxia for 14 h, and RNA was isolated. Both cytoprotective (a) and inflammatory cytokine (b) gene expression were determined by qPCR using gene-specific primers as indicated. *, ****, compared to respective genotype RA controls; $, $$, $$$, $$$$, compared to WT-BMDMs. Full article ">

16 pages, 3111 KiB

Open AccessArticle

AQP3 and AQP5 Modulation in Response to Prolonged Oxidative Stress in Breast Cancer Cell Lines

by Monika Mlinarić, Ivan Lučić, Marko Tomljanović, Ivana Tartaro Bujak, Lidija Milković and Ana Čipak Gašparović

Antioxidants 2024, 13(6), 626; https://doi.org/10.3390/antiox13060626 - 21 May 2024

Viewed by 531

Abstract

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H₂O₂ and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, [...] Read more.

Aquaporins are membrane pores regulating the transport of water, glycerol, and other small molecules across membranes. Among 13 human aquaporins, six have been shown to transport H₂O₂ and are therefore called peroxiporins. Peroxiporins are implicated in cancer development and progression, partly due to their involvement in H₂O₂ transport. Oxidative stress is linked to breast cancer development but is also a mechanism of action for conventional chemotherapy. The aim of this study is to investigate the effects of prolonged oxidative stress on Aquaporin 3 (AQP3), Aquaporin 5 (AQP5), and signaling pathways in breast cancer cell lines of different malignancies alongside a non-tumorigenic breast cell line. The prolonged oxidative stress caused responses in viability only in the cancer cell lines, while it affected cell migration in the MCF7 cell line. Changes in the localization of NRF2, a transcription factor involved in oxidative stress response, were observed only in the cancer cell lines, and no effects were recorded on its downstream target proteins. Moreover, the prolonged oxidative stress caused changes in AQP3 and AQP5 expression only in the cancer cell lines, in contrast to their non-malignant counterparts. These results suggest peroxiporins are potential therapeutic targets in cancer treatment. However, further research is needed to elucidate their role in the modulation of therapy response, highlighting the importance of research on this topic. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
Effect of prolonged exposure to H2O2 on cell viability and proliferation. Cells were treated with 10 or 20 µM H2O2 for 14 days, after which they were treated with a range of H2O2 concentrations. After 24 h, cell viability and proliferation were assessed by MTT and BrdU assays. Cell viability is shown on panels (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A. Cell proliferation is shown on panels (e) SUM159PT, (f) SkBr3, (g) MCF7, and (h) MCF10A. Experiments were performed biologically and technically in triplicate. Cell viability was calculated as the ratio between the treated cells and untreated control and is shown as a percentage of the control. The results are presented as mean ± SEM. The asterisk (*) indicates the p value for the 10 µM-treated cells compared to the control, and the plus (+) indicates the p value for the 20 µM H2O2-treated cells compared to the control, */+ p ≤ 0.05, **/++ p ≤ 0.01, ***/+++ p ≤ 0.001, ****/++++ p ≤ 0.0001. Full article ">Figure 2
Effect of prolonged exposure to H2O2 on cell migration. (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A cell lines were treated with 20 µM H2O2 for 14 days, after which they were scratched and treated with 20 µM H2O2. Cells were photographed after scratching as well as 24 and 48 h afterwards. Cell migration is calculated as the reduction in wound area over time, shown as a percentage of the starting wound area. Experiments were performed biologically and technically in triplicate. Results are presented as mean ± SEM. * p ≤ 0.05 compared to untreated control; +++ p ≤ 0.001 compared to 20 µM H2O2-treated cells. Full article ">Figure 3
Effect of prolonged exposure to H2O2 on fatty acid content and lipid hydroperoxide (LOOH) formation. Cells were treated with 20 µM H2O2 for 14 days, after which cells were collected for analysis. The effect of H2O2 on lipid composition is shown in panels (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A, and the effect on lipid hydroperoxide formation is shown in panels (e) SUM159PT, (f) SkBr3, (g) MCF7, and (h) MCF10A. Experiments were performed biologically and technically in triplicate. Results are presented as mean ± SEM. Full article ">Figure 4
Effect of prolonged exposure to H2O2 on protein and gene expression. Cells were treated with 20 µM H2O2 for 14 days, after which proteins were harvested and assayed by Western blotting, and total RNA was isolated, transcribed into cDNA, and analyzed by RT-qPCR. NRF2, Keap1, GSK3β, HO-1, NQO1, and AKR1B10 protein expressions were analyzed in (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A. After the same treatment, cytoplasmatic and nuclear protein fractions were isolated and assayed by Western blotting, and (f) NRF2 protein localization was analyzed. Protein level is shown as a relative value compared to untreated control. Representative immunoreactive bands are shown in panels (e,h). NFE2L2 gene expression is shown in panel (g). Expression fold change in a target gene is shown compared to untreated control. Experiments were performed biologically and technically in triplicate. Results are presented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001 compared to untreated control. Full article ">Figure 5
Effect of prolonged exposure to H2O2 on PI3K, PTEN, pAkt, Akt, Raptor, Rictor, p-mTOR, and Ras protein expression. (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A cell lines were treated with 20 µM H2O2 for 14 days, after which proteins were harvested and assayed by Western blotting. Protein level is shown as a relative value compared to untreated control, and pAkt/Akt is shown as a ratio. Experiments were performed biologically and technically in triplicate. Results are presented as mean ± SEM. * p ≤ 0.05 compared to untreated control. Full article ">Figure 6
Effect of prolonged exposure to H2O2 on protein and gene expression. Cells were treated with 20 µM H2O2 for 14 days, after which proteins were harvested and assayed by Western blotting, and total RNA was isolated, transcribed into cDNA, and analyzed by RT-qPCR. ABCB1 and ABCG2 protein expressions were analyzed in (a) SUM159PT, (b) SkBr3, (c) MCF7, and (d) MCF10A, and AQP3 and AQP5 protein expressions in (e) SUM159PT, (f) SkBr3, (g) MCF7, and (h) MCF10A. Protein level is shown as a relative value compared to untreated control. Representative immunoreactive bands are shown in panel (i). AQP1, AQP3, AQP5, AQP9, and AQP11 gene expressions were analyzed in (j) SUM159PT, (k) SkBr3, (l) MCF7, and (m) MCF10A. Expression fold change in a target gene is shown compared to untreated control. Experiments were performed biologically and technically in triplicate. Results are presented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 compared to untreated control. Full article ">

25 pages, 3912 KiB

Open AccessArticle

The Triterpenoid CDDO-Methyl Ester Reduces Tumor Burden, Reprograms the Immune Microenvironment, and Protects from Chemotherapy-Induced Toxicity in a Preclinical Mouse Model of Established Lung Cancer

by Jessica A. Moerland and Karen T. Liby

Antioxidants 2024, 13(6), 621; https://doi.org/10.3390/antiox13060621 - 21 May 2024

Viewed by 527

Abstract

NRF2 activation protects epithelial cells from malignancy, but cancer cells can upregulate the pathway to promote survival. NRF2 activators including CDDO-Methyl ester (CDDO-Me) inhibit cancer in preclinical models, suggesting NRF2 activation in other cell types may promote anti-tumor activity. However, the immunomodulatory effects [...] Read more.

NRF2 activation protects epithelial cells from malignancy, but cancer cells can upregulate the pathway to promote survival. NRF2 activators including CDDO-Methyl ester (CDDO-Me) inhibit cancer in preclinical models, suggesting NRF2 activation in other cell types may promote anti-tumor activity. However, the immunomodulatory effects of NRF2 activation remain poorly understood in the context of cancer. To test CDDO-Me in a murine model of established lung cancer, tumor-bearing wildtype (WT) and Nrf2 knockout (KO) mice were treated with 50–100 mg CDDO-Me/kg diet, alone or combined with carboplatin/paclitaxel (C/P) for 8–12 weeks. CDDO-Me decreased tumor burden in an Nrf2-dependent manner. The combination of CDDO-Me plus C/P was significantly (p < 0.05) more effective than either drug alone, reducing tumor burden by 84% in WT mice. CDDO-Me reduced the histopathological grade of WT tumors, with a significantly (p < 0.05) higher proportion of low-grade tumors and a lower proportion of high-grade tumors. These changes were augmented by combination with C/P. CDDO-Me also protected WT mice from C/P-induced toxicity and improved macrophage and T cell phenotypes in WT mice, reducing the expression of CD206 and PD-L1 on macrophages, decreasing immunosuppressive FoxP3+ CD4+ T cells, and increasing activation of CD8+ T cells in a Nrf2-dependent manner. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
CDDO-Methyl ester (CDDO-Me) reduces tumor burden and the severity of tumor histopathology of established lung tumors in a dose- and Nrf2-dependent manner. (A) Schematic detailing experimental design for Study 1. Mice were challenged with vinyl carbamate and fed a vehicle (ethanol + Neobee oil) diet while lung tumors developed, after which mice were randomized into groups fed either a vehicle diet or a diet containing 50–100 mg/kg CDDO-Me dissolved in the vehicle. CDDO-Me was dosed intermittently (one week on the CDDO-Me diet followed by one week on the vehicle diet). (B) Representative images (4 mice/group, 8× magnification) of the left lung of wildtype (WT) and Nrf2 knockout (KO) mice at endpoint. (C) Quantitation of surface tumors on both right and left lungs of study mice (n = 12–18 mice/group). (D) Proportions of total lung tumors on lung sections for each histopathological grade. (E) Western blot of NQO1 and β-actin proteins isolated from lung homogenates (n = 3 mice/treatment group). Statistics: Two-way ANOVA followed by Tukey HSD (C); * p < 0.05 or **** p < 0.0001 as shown in the panel. Z test for proportions (D); * p < 0.05 vs. WT Vehicle. Full article ">Figure 2
CDDO-Me increases NQO1 staining in the lungs of WT mice and modulates the phenotype but not infiltration of macrophages and T cells. (A) Immunohistochemical staining of NQO1 in the lungs of WT and Nrf2 KO mice treated with 50–100 mg/kg CDDO-Me. Scale bar = 60 microns. (B,C) Flow cytometry analysis of CD45+ cells in the lungs of WT and Nrf2 KO mice at the study endpoint as described in <a href="#antioxidants-13-00621-f001" class="html-fig">Figure 1</a>A. Flow cytometry analysis of infiltrating macrophages (CD45+ CD11clo CD11bhi CD64+ cells, % CD64+) and mean fluorescence intensity of CD206 and PD-L1 on infiltrating macrophages (B) or total T cells (% CD45+), FoxP3+ CD4+ T cells (% CD4+), and mean fluorescence intensity (MFI) of CD107a on CD45+ CD25+ CD8+ cells (C) in the lung. (D) Fold expression change of FoxP3 mRNA in CD4+ splenocytes isolated from WT and Nrf2 KO mice and activated with αCD28, IL-2, and TGF-β and then treated with vehicle or 10 nM CDDO-Me for 24 h. Statistics: Two-way ANOVA followed by Tukey HSD. * p < 0.05; ** p < 0.01; *** p < 0.001. Full article ">Figure 3
CDDO-Me, alone and in combination with carboplatin and paclitaxel, reduces lung tumor burden and severity of tumor histopathology of advanced lung tumors in a dose- and Nrf2-dependent manner. (A) Schematic detailing experimental design for Study 2: WT and Nrf2 KO mice were challenged with vinyl carbamate and fed a vehicle (ethanol + Neobee oil) diet for 8 weeks while tumors developed in the lung, after which they were randomized into treatment groups. Mice were fed either a vehicle diet or a diet containing 80 mg/kg CDDO-Me. The combination group was also injected with carboplatin and paclitaxel (C/P) at 50 mg/kg and 15 mg/kg of body weight, respectively. C/P was dosed i.p. once every other week on the same day for a total of 6 doses. Representative images (4 mice/group, 8×) of the left lung of wildtype mice (B) or Nrf2 knockout mice (C) at endpoint. (D) Quantification of surface tumors on both right and left lungs of mice (n = 7–16 mice/group). (E) Proportions of total lung tumors on lung sections for each histopathological grade. (F) Western blot of proteins isolated from whole lung homogenate (n = 3 animals/treatment group). Statistics: Two-way ANOVA followed by Tukey HSD (D): * p < 0.05 or **** p < 0.0001 as shown in the panel. Z test for proportions (E): * p < 0.05 vs. WT Vehicle; # p < 0.05 vs. WT C/P and WT CDDO-Me + C/P. Full article ">Figure 4
CDDO-Me modulates macrophage and T cell phenotype in a Nrf2-dependent manner. Immunohistochemical staining (A) of PCNA and p-ERK in lung sections from wildtype (WT) and Nrf2 knockout (KO) mice treated with vehicle, carboplatin/paclitaxel (C/P), CDDO-Me 80 mg/kg, or the combination as shown in <a href="#antioxidants-13-00621-f003" class="html-fig">Figure 3</a>A; scale bar represents 60 μm. Flow cytometry analysis of infiltrating macrophages (CD45+ CD11clo CD11bhi CD64+ cells, % CD64+) and mean fluorescence intensity (MFI) of CD206 and PD-L1 on infiltrating macrophages (B) or of total T cells (% CD45+), FoxP3+ CD4+ T cells (%CD4+), and MFI of CD107a on CD45+ CD25+ CD8+ T cells (C) in the lung. Statistics: Two-way ANOVA followed by Tukey HSD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Full article ">Figure 5
CDDO-Me protects from chemotherapy-induced toxicity and mortality. (A) Summary of mortality in wildtype (WT) and Nrf2 knockout (KO) A/J mice treated with CDDO-Me, carboplatin and paclitaxel (C/P), or the combination as described in <a href="#antioxidants-13-00621-f003" class="html-fig">Figure 3</a>A; proportion of total enrolled in the treatment group at study initiation; n = 9–16 mice per group. (B) White blood cell (WBC) counts in whole blood measured at study endpoint. (C) Average mouse weights at beginning of treatment and at the study endpoint. Statistics: (A): z test for proportions. * p < 0.05 vs. vehicle; # p < 0.05 vs. WT. (B,C): Two-way ANOVA followed by Tukey HSD. * p < 0.05; ** p < 0.01; **** p < 0.0001. Full article ">Figure 6
Increased tumor burden and histopathological severity in male vs. female mice. (A). Quantification of overall tumor burden and proportions of tumor histopathological grade in wildtype (WT) and Nrf2 knockout (KO) female and male mice treated with 50–100 mg/kg CDDO-Me for 8 weeks (A) or with 80 mg/kg ± C/P for 12 weeks (B). Statistics: Two-way ANOVA followed by Tukey HSD. *** p < 0.001; **** p < 0.0001. Z test for proportions. * p < 0.05. Full article ">

22 pages, 3371 KiB

Open AccessArticle

Cellular Pre-Adaptation to the High O₂ Concentration Used in Standard Cell Culture Confers Resistance to Subsequent H₂O₂-Induced Cell Death

by Jack B. Jordan, Miranda J. Smallwood, Gary R. Smerdon and Paul G. Winyard

Antioxidants 2024, 13(3), 269; https://doi.org/10.3390/antiox13030269 - 22 Feb 2024

Viewed by 1174

Abstract

The addition of hydrogen peroxide (H₂O₂) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. [...] Read more.

The addition of hydrogen peroxide (H₂O₂) to cultured cells is widely used as a method to modulate redox-regulated cellular pathways, including the induction of programmed cell death in cell culture experiments and the testing of pro- and antioxidant compounds. Here, we assessed the effect on the cellular response to H₂O₂ of pre-adapting squamous cell carcinoma cells (A431) to the standard cell culture oxygenation of 18.6% O₂, compared to cells pre-adapted to a physiological skin O₂ concentration (3.0% O₂). We showed that cells pre-adapted to 18.6% O₂ resisted H₂O₂-induced cell death compared to cells pre-adapted to 3.0% O₂ for 96 h prior to treatment with H₂O₂. Moreover, the enzymatic activities of catalase and glutathione reductase, as well as the protein expression levels of catalase, were higher in cells pre-adapted to 18.6% O₂ compared to cells pre-adapted to 3.0% O_2. H₂O₂-resistant cells, pre-adapted to 18.6% O₂, exhibited increased nuclear Nrf-2 levels. It is concluded that A431 cells pre-adapted to standard cell culture oxygenation conditions resist H₂O₂-induced cell death. This effect may be related to their heightened activation of Nrf-2. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

14 pages, 1980 KiB

Open AccessArticle

Oxidative Stress Induces Skin Pigmentation in Melasma by Inhibiting Hedgehog Signaling

by Nan-Hyung Kim and Ai-Young Lee

Antioxidants 2023, 12(11), 1969; https://doi.org/10.3390/antiox12111969 - 6 Nov 2023

Viewed by 1713

Abstract

There is growing evidence that oxidative stress plays a role in melasma and disrupts primary cilia formation. Additionally, primary cilia have been suggested to have an inhibitory role in melanogenesis. This study examined the potential link between oxidative stress, skin hyperpigmentation, and primary [...] Read more.

There is growing evidence that oxidative stress plays a role in melasma and disrupts primary cilia formation. Additionally, primary cilia have been suggested to have an inhibitory role in melanogenesis. This study examined the potential link between oxidative stress, skin hyperpigmentation, and primary cilia. We compared the expression levels of the nuclear factor E2-related factor 2 (NRF2), intraflagellar transport 88 (IFT88), and glioma-associated oncogene homologs (GLIs) in skin samples from patients with melasma, both in affected and unaffected areas. We also explored the roles of NRF2, IFT88, and GLIs in ciliogenesis and pigmentation using cultured adult human keratinocytes, with or without melanocytes. Our findings revealed decreased levels of NRF2, heme oxygenase-1, IFT88, and GLIs in lesional skin from melasma patients. The knockdown of NRF2 resulted in reduced expressions of IFT88 and GLI1, along with fewer ciliated cells. Furthermore, NRF2, IFT88, or GLI1 knockdown led to increased expressions in protease-activated receptor-2 (PAR2), K10, involucrin, tyrosinase, and/or melanin. These effects were reversed by the smoothened agonist 1.1. Calcium also upregulated these proteins, but not NRF2. The upregulation of involucrin and PAR2 after NRF2 knockdown was mitigated with a calcium chelator. In summary, our study suggests that oxidative stress in NRF2-downregulated melasma keratinocytes impedes ciliogenesis and related molecular processes. This inhibition stimulates keratinocyte differentiation, resulting in melanin synthesis and melanosome transfer, ultimately leading to skin hyperpigmentation. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
NRF2 downregulation in the lesional epidermis of patients with melasma. (A) Reactive oxygen species concentrations at various time points in primary cultured keratinocytes following UVB irradiation. (B) Western blot analyses showing NRF2 protein level ratios after single and repeated UVB radiation. (C) Western blot analyses illustrating NRF2 and HO-1 protein level ratios over time in primary cultured normal human keratinocytes treated with different concentrations of H2O2. (D) Western blot analyses presenting HO-1 protein level ratios in cultured human keratinocytes with or without NRF2 knockdown. β-actin served as the internal control for the Western blot analysis. The data are presented as means ± SD from four or eight independent experiments. (E,F) Representative immunofluorescence staining using anti-NRF2 (E) and anti-HO-1 antibodies (F) in the lesional (L) and non-lesional (N) epidermis of patients with melasma. The nuclei were counterstained with Hoechst 33258 (scale bar = 0.05 mm), and the intensities were quantified using ImageJ software 1.54d. * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 2
Downregulation of NRF2 led to reduced expressions of IFT88 and Hh signaling molecules involved in ciliogenesis. (A) Western blot analyses depicting the ratios of PTCH, GLI1, and GLI2 levels in cultured keratinocytes subjected to IFT88 knockdown. (B) Confocal microscopy images illustrating primary cilia stained with anti-acetylated α-tubulin (Ac α-tubulin) and/or ARL13b antibodies in cultured human keratinocytes and melanocytes, with or without IFT88 knockdown (bar = 0.05 mm). The ciliated cell ratios were calculated by counting the number of ciliated cells among 30 cells. (C,D) Western blot analyses showing the ratios of NRF2, IFT88, and/or GLI1 levels in cultured keratinocytes with knockdowns of NRF2 (C) or IFT88 (D). (E) Real-time PCR results displaying the ratios of IFT88, PTCH1, and GLI1-3 mRNA levels in lesional compared to non-lesional skin specimens (seven sets) from melasma patients with downregulated NRF2. (F) Representative immunofluorescence staining for primary cilia using anti-NRF2 and anti-ARL13b antibodies in primary cultured human keratinocytes and melanocytes with or without NRF2 knockdown (scale bar = 0.05 mm). β-actin and GAPDH served as internal controls for the Western blot analysis and real-time PCR, respectively. The data are presented as means ± SD from four independent experiments. * p < 0.05, ** p < 0.01. Full article ">Figure 3
Enhancement of melanin pigmentation via NRF2 knockdown involving IFT88 and GLI1. (A) Western blot analyses depicting varying levels of tyrosinase, PMEL, and PAR2, and assays showing tyrosinase activity and melanin contents in cultured keratinocytes with NRF2 knockdown. (B–E) Western blot analyses revealing different ratios of NRF2, IFT88, GLI1, and/or tyrosinase levels in cultured keratinocytes with NRF2 knockdown in the absence and presence of SAG (B), IFT88 knockdown in the absence and presence of SAG (C), IFT88 knockdown in the absence and presence of Shh 200 (D), and GLI1 knockdown in the absence and presence of SAG (E). (F) Western blot analyses presenting different ratios of PTCH1, GLI1, GLI2, and tyrosinase levels in cultured keratinocyte–melanocyte cocultures treated with or without GANT61. β-actin served as an internal control. The data represent the means ± SD from four independent experiments. * p < 0.05, ** p < 0.01 vs. control sgRNA, # p < 0.05 vs. without SAG treatment. Full article ">Figure 4
Effects of NRF2, IFT88, and GLI1 knockdowns on keratinocyte differentiation and subsequent hyperpigmentation. (A–C) Western blot analyses illustrating the relative levels of K14, K10, and involucrin in cultured keratinocytes with or without knockdowns of NRF2 (A), IFT88 (B), or GLI1 (C) in the absence and presence of SAG. (D) Representative immunofluorescence staining using anti-involucrin antibodies (B) in the lesional (L) and non-lesional (N) epidermis of seven patients with melasma. The nuclei were counterstained with Hoechst 33258 (bar = 0.05 mm), and the intensities were measured using ImageJ software 1.54d. (E,F) Western blot analyses for the ratios of tyrosinase levels in keratinocyte–melanocyte cocultures and the ratios of PAR2, K10, involucrin, NRF2, IFT88, and/or GLI1 levels in cultured keratinocytes, including those treated with calcium (E) and keratinocytes with or without NRF2 knockdown in the absence and presence of Bapta-AM (F). β-actin was used as an internal control for the Western blot analysis. The data are presented as the means ± SD from four independent experiments. * p < 0.05, ** p < 0.01 vs. control sgRNA; # p < 0.05, ## p < 0.01 vs. without SAG treatment. Full article ">Figure 5
Schematic view of the role of NRF2-knockdown-induced ciliogenesis inhibition in skin hyperpigmentation. NRF2 downregulation caused by repeated UV exposure or melasma inhibited ciliogenesis and Hh signaling molecules, such as IFT88 and GLI1, stimulating keratinocyte differentiation with melanin synthesis and melanosome transfer to the keratinocytes, which resulted in skin hyperpigmentation. Full article ">

14 pages, 2022 KiB

Open AccessArticle

AMPK-Mediated Phosphorylation of Nrf2 at S374/S408/S433 Favors Its βTrCP2-Mediated Degradation in KEAP1-Deficient Cells

by Eleni Petsouki, Sylvia Ender, Shara Natalia Sosa Cabrera and Elke H. Heiss

Antioxidants 2023, 12(8), 1586; https://doi.org/10.3390/antiox12081586 - 9 Aug 2023

Cited by 1 | Viewed by 3541

Abstract

Nrf2 is a transcription factor facilitating cells’ resilience against redox and various other forms of stress. In the absence of stressors, KEAP1 and/or βTrCP mediate the ubiquitination of Nrf2 and prevent Nrf2-dependent gene expression and detoxification. AMPK regulates cellular energy homeostasis and redox [...] Read more.

Nrf2 is a transcription factor facilitating cells’ resilience against redox and various other forms of stress. In the absence of stressors, KEAP1 and/or βTrCP mediate the ubiquitination of Nrf2 and prevent Nrf2-dependent gene expression and detoxification. AMPK regulates cellular energy homeostasis and redox balance. Previous studies indicated a potential Nrf2-AMPK cooperativity. In line with this, our lab had previously identified three AMPK-dependent phosphorylation sites (S374/408/433) in Nrf2. Given their localization in or near the Neh6 domain, known to regulate βTrCP-mediated degradation, we examined whether they may influence the βTrCP-driven degradation of Nrf2. By employing expression plasmids for WT and triple mutant (TM)-Nrf2 (Nrf2^{S374/408/433→A}), (co)immunoprecipitation, proximity ligation, protein half-life, knockdown, ubiquitination experiments, and qPCR in Keap1-null mouse embryonic fibroblasts, we show that TM-Nrf2^{S→A374/408/433} had enhanced stability due to impeded interaction with βTrCP2 and reduced ubiquitination in comparison to WT-Nrf2. In addition, TM-Nrf2 elicited higher expression of the Nrf2 target gene Gclc, potentiated in the presence of a pharmacological AMPK activator. Overall, we propose that AMPK-dependent phospho-sites of Nrf2 can favor its βTrCP2-mediated degradation and dampen the extent of Nrf2 target gene expression. Therefore, targeting AMPK might be able to diminish Nrf2-mediated responses in cells with overactive Nrf2 due to KEAP1 deficiency. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
Mutation of the AMPK-dependent phospho-sites to alanine stabilizes Nrf2 in Keap1−/− cells in a βTrCP2-dependent manner. (A,B). Keap1−/− MEFs were transfected with EGFP-MYC tagged WT- or TM-Nrf2 expression plasmids for 48 h and then exposed to cycloheximide (50 μM) for different periods of time. Cell lysates were subjected to immunoblot analysis for MYC or β-Actin. A representative blot (A) and quantification analysis (B) of n = 9 independent experiments (relative to signal at t = 0) are depicted. One-phase exponential decay curves were fitted to the data by finding the least sum of squares using GraphPad Prism. The curves were significantly different, with a p value = 0.0108. (C,D) Keap1−/− MEFs were transfected with Fbxw11-specific siRNA and with EGFP-MYC tagged WT- or TM-Nrf2 expression plasmids for 48 h, and then exposed to cycloheximide (50 μM) for different periods of time. Cell lysates were subjected to immunoblot analysis for MYC or β-Actin. A representative blot (C) and quantification analysis (D) of n = 5 independent experiments (relative to signal at t = 0) are depicted. One-phase exponential decay curves were fitted to the data by finding the least sum of squares using GraphPad Prism. There was one curve for all data sets with a p value = 0.4852. The dotted lines in (B,D) represent the confidence bands with a 95% confidence level. Data are presented as means ± SEM. Full article ">Figure 2
Mutation of the AMPK-dependent phospho-sites to alanine impedes the interaction of Nrf2 with β-TrCP2 in Keap1−/− cells. (A–D) Keap1-null MEFs were transfected with an expression plasmid for HA-tagged β-TrCP2 alone or together with constructs encoding EGFP-MYC-WT-Nrf2, the triple mutated version EGFP-MYC-TM-Nrf2, or pcDNA as indicated for 48 h followed by the absence (A,B) or presence (C,D) of the AMPK activator A769662 (50 μM, 4 h). Cells were also treated at the same time with MG132 (10 μM, 4 h) to stabilize Nrf2, which was pulled down using GFP-Trap. Precipitated proteins, as well as input controls, were immunoblotted for MYC, HA, and β-Actin. Representative blots (A,C) and quantification analysis (B,D) of 3 independent experiments are depicted. Plus symbols in the tables indicate the different constructs/chemicals used in the various experimental conditions. (E,F) Keap1-null MEFs were transfected with (i) pcDNA, (ii) HA-tagged β-TrCP2, or co-transfected with (iii) HA-tagged β-TrCP2 and EGFP-MYC-tagged WT-Nrf2, (iv) HA-tagged β-TrCP2 and EGFP-MYC-tagged TM-Nrf2 expression plasmids, or (v) EGFP-MYC-tagged WT-Nrf2 and (vi) and EGFP-MYC-tagged TM-Nrf2 alone, and then treated with MG132 (10 μM) for 4 h in the absence (E) or presence (F) of the AMPK activator A769662 (50 μM, 4 h). Cells were fixed with 4% Paraformaldehyde and subjected to PLA. Data are presented as means ± SEM. Statistical analysis was performed using Student’s t-test, * p < 0.05 and ** p < 0.01. Full article ">Figure 3
Mutation of the AMPK-dependent phospho-sites to alanine on Nrf2 impedes its βTrcP2-mediated ubiquitination and degradation in Keap1−/−cells. (A–E) Keap1-null MEFs were transfected with the indicated expression plasmids for 48 h. (A) Ubiquitination assay of EGFP-Myc tagged Nrf2 expression plasmids in Keap1-null MEFs, after co-transfection with His-tagged Ubiquitin and the indicated HA-βTrCP2 construct. (B–E) Expression levels of EGFP-Myc tagged WT-Nrf2 (B,C) or EGFP-Myc tagged TM-Nrf2 (D–E) with or without co-transfection of HA-tagged βΤrCP2 in the presence or absence of the AMPK activator A 769662 (50 μM, 4 h). Cell lysates were subjected to immunoblot analysis for MYC, HA, or actin. Representative blots (B,D) and quantification analysis (C,E) of 3 independent experiments are depicted. Plus symbols in the tables indicate the different constructs/chemicals used in the various experimental conditions. Data are presented as means ± SEM. Statistical analysis was performed using multiple comparison one-way ANOVA, with Sidak’s correction, * p < 0.05 and ** p < 0.01. ns: not significant. Full article ">Figure 4
The identified AMPK-dependent phospho-sites in Nrf2 affect the expression of endogenous Nrf2 target genes. (A–D) Keap1-null MEFs were transfected for 48 h with an expression plasmid for HA-tagged β-TrCP2 together with constructs encoding EGFP-MYC-WT-Nrf2 or the triple mutated version EGFP-MYC-TM-Nrf2, as indicated in the absence or presence of the AMPK activator A769662 (50 μM, 4 h and 24 h). RNA was extracted and analyzed for the abundance of Gclc (A,B) and Hmox1 (C,D) by qPCR (TBP as reference gene). Data are presented as means ± SEM. Statistical analysis was performed using multiple comparison one-way ANOVA, with Sidak’s correction, * p < 0.05 and ** p < 0.01. ns: not significant. Full article ">Figure 5
Working model depicting the role of the AMPK-dependent S374, S408, and S433 of Nrf2 in its βTrCP2-mediated ubiquitination and degradation, regulating the expression of Nrf2 target genes (©BioRender). Full article ">

24 pages, 6300 KiB

Open AccessArticle

Nrf2 as a Therapeutic Target in the Resistance to Targeted Therapies in Melanoma

by Marie Angèle Cucci, Margherita Grattarola, Chiara Monge, Antonella Roetto, Giuseppina Barrera, Emilia Caputo, Chiara Dianzani and Stefania Pizzimenti

Antioxidants 2023, 12(6), 1313; https://doi.org/10.3390/antiox12061313 - 20 Jun 2023

Cited by 2 | Viewed by 3074

Abstract

The use of specific inhibitors towards mutant BRAF (BRAFi) and MEK (MEKi) in BRAF-mutated patients has significantly improved progression-free and overall survival of metastatic melanoma patients. Nevertheless, half of the patients still develop resistance within the first year of therapy. Therefore, understanding the [...] Read more.

The use of specific inhibitors towards mutant BRAF (BRAFi) and MEK (MEKi) in BRAF-mutated patients has significantly improved progression-free and overall survival of metastatic melanoma patients. Nevertheless, half of the patients still develop resistance within the first year of therapy. Therefore, understanding the mechanisms of BRAFi/MEKi-acquired resistance has become a priority for researchers. Among others, oxidative stress-related mechanisms have emerged as a major force. The aim of this study was to evaluate the contribution of Nrf2, the master regulator of the cytoprotective and antioxidant response, in the BRAFi/MEKi acquired resistance of melanoma. Moreover, we investigated the mechanisms of its activity regulation and the possible cooperation with the oncogene YAP, which is also involved in chemoresistance. Taking advantage of established in vitro melanoma models resistant to BRAFi, MEKi, or dual resistance to BRAFi/MEKi, we demonstrated that Nrf2 was upregulated in melanoma cells resistant to targeted therapy at the post-translational level and that the deubiquitinase DUB3 participated in the control of the Nrf2 protein stability. Furthermore, we found that Nrf2 controlled the expression of YAP. Importantly, the inhibition of Nrf2, directly or through inhibition of DUB3, reverted the resistance to targeted therapies. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

20 pages, 9994 KiB

Open AccessArticle

CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD⁺-Lowering Agents

by Pamela Becherini, Debora Soncini, Silvia Ravera, Elisa Gelli, Claudia Martinuzzi, Giulia Giorgetti, Antonia Cagnetta, Fabio Guolo, Federico Ivaldi, Maurizio Miglino, Sara Aquino, Katia Todoerti, Antonino Neri, Andrea Benzi, Mario Passalacqua, Alessio Nencioni, Ida Perrotta, Maria Eugenia Gallo Cantafio, Nicola Amodio, Antonio De Flora, Santina Bruzzone, Roberto M. Lemoli and Michele Cea Show full author list Hide full author list

Antioxidants 2023, 12(2), 494; https://doi.org/10.3390/antiox12020494 - 15 Feb 2023

Viewed by 2483

Abstract

Cancer cells fuel growth and energy demands by increasing their NAD⁺ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD⁺-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the [...] Read more.

Cancer cells fuel growth and energy demands by increasing their NAD⁺ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD⁺-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD⁺ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD⁺-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD⁺-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

Review

Jump to: Research, Other

27 pages, 2696 KiB

Open AccessReview

The Multifaceted Roles of NRF2 in Cancer: Friend or Foe?

by Christophe Glorieux, Cinthya Enríquez, Constanza González, Gabriela Aguirre-Martínez and Pedro Buc Calderon

Antioxidants 2024, 13(1), 70; https://doi.org/10.3390/antiox13010070 - 2 Jan 2024

Cited by 3 | Viewed by 3336

Abstract

Physiological concentrations of reactive oxygen species (ROS) play vital roles in various normal cellular processes, whereas excessive ROS generation is central to disease pathogenesis. The nuclear factor erythroid 2-related factor 2 (NRF2) is a critical transcription factor that regulates the cellular antioxidant systems [...] Read more.

Physiological concentrations of reactive oxygen species (ROS) play vital roles in various normal cellular processes, whereas excessive ROS generation is central to disease pathogenesis. The nuclear factor erythroid 2-related factor 2 (NRF2) is a critical transcription factor that regulates the cellular antioxidant systems in response to oxidative stress by governing the expression of genes encoding antioxidant enzymes that shield cells from diverse oxidative alterations. NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) have been the focus of numerous investigations in elucidating whether NRF2 suppresses tumor promotion or conversely exerts pro-oncogenic effects. NRF2 has been found to participate in various pathological processes, including dysregulated cell proliferation, metabolic remodeling, and resistance to apoptosis. Herein, this review article will examine the intriguing role of phase separation in activating the NRF2 transcriptional activity and explore the NRF2 dual impacts on tumor immunology, cancer stem cells, metastasis, and long non-coding RNAs (LncRNAs). Taken together, this review aims to discuss the NRF2 multifaceted roles in both cancer prevention and promotion while also addressing the advantages, disadvantages, and limitations associated with modulating NRF2 therapeutically in cancer treatment. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
Structure of human NRF2. NRF2 protein contains seven Neh domains with different functions. The Neh1 domain is essential for DNA binding and dimerization with MAF proteins and other transcription factors (c-Jun, Sp-1, and JDP2). The Neh2 domain interacts with KEAP1 through DLG and ETGE motifs, leading to NRF2 ubiquitination and proteasomal degradation. The Neh3, Neh4, and Neh5 are transactivation domains. Neh6 contains a serine-rich region that regulates NRF2 protein stability. The Neh7 domain interacts with RXRα protein and induces NRF2 repression. Abbreviations: BTB, broad complex, tramtrack, and bric-a-brac domain; Cul3, cullin-3; GSK3, glycogen synthase kinase 3 beta; Hrd1, hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1; IVR, intervening region; KEAP1, Kelch-like ECH-associated protein 1; sMAF, small-MAF factors; Neh, NRF2–ECH homology domains; NRF2, nuclear factor erythroid 2-related factor 2; RBX1, ring box 1 protein; RXRα, retinoid X receptor alpha; β-TrCP, beta-transducin repeat-containing protein; Ub, ubiquitin. Full article ">Figure 2
Mechanisms of NRF2 activation and inhibition. Under basal conditions, NRF2 is tightly bound to its repressor KEAP1, which recruits ubiquitin ligases, leading to NRF2 polyubiquitination and subsequent proteasomal degradation (left panel). However, under stress conditions, such as oxidative stress, NRF2 is released from KEAP1 and translocates to the nucleus for binding and regulating specific target genes containing ARE sequences (right and center panels). The small amount of NRF2 present in the nucleus is subsequently phosphorylated by the Fyn kinase and exported out of the nucleus. NRF2 can be activated through both canonical and non-canonical pathways: (1) During oxidative stress, redox-sensitive cysteine residues in KEAP1 are oxidized, causing a structural change in KEAP1. This disturbance in the Hinge-and-latch complex prevents the binding of KEAP1 to the DLG motif of NRF2, thereby preventing NRF2 ubiquitination and degradation. (2) Proteins like p21 or p62 can compete with KEAP1 for binding to the DLG motif of NRF2. (3) PKC phosphorylates the Ser40 residue in NRF2, inhibiting its sequestration by KEAP1. (4) The antioxidant iASPP competes with NRF2 for KEAP1 binding via a DLT motif and induces NRF2 activation. (5) NRF2 can undergo glycation, which can impair its transcriptional activation. FN3K can promote the deglycation of NRF2. (6) GCDH can glutarylate NRF2, increasing its protein stability and transcriptional activity. All these mechanisms ultimately result in an increase in NRF2 lifespan, cellular concentration, and nuclear transport, enabling it to function as a transcription factor. Additionally, BACH1 can compete with NRF2 for binding to ARE sequences in the nucleus. Abbreviations: ARE, antioxidant response element; BACH1, BTB domain and CNC homolog 1; BTB, broad complex, tramtrack, and bric-a-brac domain; Cul3, cullin-3; FN3K, fructosamine-3-kinase; GCDH, glutaryl-CoA dehydrogenase; Gly, glycation; Glu, glutarylation; HO-1, heme oxygenase 1; iASPP, inhibitory member of apoptosis stimulating protein of p53, or ankyrin repeats, SH3 domain and proline-rich region contain protein family; IVR, intervening region; KEAP1, Kelch-like ECH-associated protein 1; sMAF, small-MAF factors; NRF2, nuclear factor erythroid 2-related factor 2; PKC, protein kinase C; RBX1, ring box 1 protein. Full article ">Figure 3
Anti-oncogenic effects of NRF2 in cancer prevention. NRF2 is considered to have anti-oncogenic effects in healthy cells by inducing cytoprotective proteins and enzymes that help eliminate ROS and detoxify carcinogens. This protective mechanism safeguards DNA from damage caused by oxidative stress and toxic agents. Consequently, NRF2 activators may hold potential as chemopreventive agents. Abbreviations: 6PGD, 6-phosphogluconate dehydrogenase; ADHs, alcohol dehydrogenases; ALDHs, aldehyde dehydrogenases; ARE, antioxidant response element; BACH1, BTB domain and CNC homolog 1; CDDO, 2-cyano-3,12-dioxoolean-1,9(11)-diene-28-oic acid; CES, carboxyl esterase; CYPs, cytochromes P450; DMF, dimethyl fumarate; G6PD, glucose-6-phosphate dehydrogenase; GCL, glutamate-cysteine ligase; GPXs, glutathione peroxidases; GR, glutathione reductase; GSH, glutathione; GSTs, glutathione S-transferases; HO-1, heme oxygenase 1; HSPs, heat-shock proteins; ME1, malic enzyme 1; MMF, monomethyl fumarate; MRP, multi-drug resistance-associated protein; NQO1, NADP(H): dehydrogenase quinone 1; NRF2, nuclear factor erythroid 2-related factor 2; PRXs, peroxiredoxins; SODs, superoxide dismutases; SULTs, sulfotransferases; UGTs, UDP-glucuronosyl transferases. Full article ">Figure 4
Pro-oncogenic effects of NRF2 in cancer. NRF2 expression is upregulated in various types of tumors and is associated with a poor prognosis. This aberrant activation provides cancer cells with advantages, including increased tumorigenic capacity, resistance to therapeutic agents, and enhanced antioxidant activity. This phenomenon is often referred to as “NRF2 addiction”, where the protective mechanism of NRF2 becomes a driver of cancer growth. Additionally, NRF2 plays a role in multiple processes within cancer cells, including dysregulated cell proliferation, metabolic alterations, resistance to cell death, phase separation, tumor immunology, metastasis, and LncRNA regulation. Given these factors, NRF2 inhibitors may be promising for sensitizing cancer cells to cancer therapies. Abbreviations: ARE, antioxidant response element; ATF4, activating transcription factor 4; BACH1, BTB domain and CNC homolog 1; CSCs, cancer stem cells; FoxP3, forkhead box P3; G6PD, glucose-6-phosphate dehydrogenase; GPX4, glutathione peroxidase 4; GSH, glutathione; HK2, hexokinase 2; HO-1, heme oxygenase 1; LncRNAs, long non-coding RNAs; MT1DP, metallothionein 1D pseudogene; NK, natural killer; NLUCAT1, lung cancer-associated transcript 1; NPNT, nephronectin; NRF2, nuclear factor erythroid 2-related factor 2; PDGFC, platelet-derived growth factor C; PPP, pentose phosphate pathway; ROS, reactive oxygen species; SCAL1, smoke and cancer-associated LncRNA-1; SESN2, sestrin 2; TKT, transketolase; VEGFC, vascular endothelial growth factor C. Full article ">

20 pages, 1513 KiB

Open AccessReview

Targeting Nrf2 Signaling Pathway in Cancer Prevention and Treatment: The Role of Cannabis Compounds

by Anna Rybarczyk, Aleksandra Majchrzak-Celińska and Violetta Krajka-Kuźniak

Antioxidants 2023, 12(12), 2052; https://doi.org/10.3390/antiox12122052 - 28 Nov 2023

Cited by 2 | Viewed by 2042

Abstract

The development and progression of cancer are associated with the dysregulation of multiple pathways involved in cell proliferation and survival, as well as dysfunction in redox balance, immune response, and inflammation. The master antioxidant pathway, known as the nuclear factor erythroid 2-related factor [...] Read more.

The development and progression of cancer are associated with the dysregulation of multiple pathways involved in cell proliferation and survival, as well as dysfunction in redox balance, immune response, and inflammation. The master antioxidant pathway, known as the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, regulates the cellular defense against oxidative stress and inflammation, making it a promising cancer prevention and treatment target. Cannabinoids have demonstrated anti-tumor and anti-inflammatory properties, affecting signaling pathways, including Nrf2. Increased oxidative stress following exposure to anti-cancer therapy prompts cancer cells to activate antioxidant mechanisms. This indicates the dual effect of Nrf2 in cancer cells—influencing proliferation and apoptotic processes and protecting against the toxicity of anti-cancer therapy. Therefore, understanding the complex role of cannabinoids in modulating Nrf2 might shed light on its potential implementation as an anti-cancer support. In this review, we aim to highlight the impact of cannabinoids on Nrf2-related factors, with a focus on cancer prevention and treatment. Additionally, we have presented the results of several research studies that combined cannabidiol (CBD) with other compounds targeting Nrf2. Further studies should be directed toward exploring the anti-inflammatory effects of cannabinoids in the context of cancer prevention and therapy. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

32 pages, 2108 KiB

Open AccessReview

Roles of Oxidative Stress and Nrf2 Signaling in Pathogenic and Non-Pathogenic Cells: A Possible General Mechanism of Resistance to Therapy

by Mira Hammad, Mohammad Raftari, Rute Cesário, Rima Salma, Paulo Godoy, S. Noushin Emami and Siamak Haghdoost

Antioxidants 2023, 12(7), 1371; https://doi.org/10.3390/antiox12071371 - 30 Jun 2023

Cited by 20 | Viewed by 6424

Abstract

The coordinating role of nuclear factor erythroid-2-related factor 2 (Nrf2) in cellular function is undeniable. Evidence indicates that this transcription factor exerts massive regulatory functions in multiple signaling pathways concerning redox homeostasis and xenobiotics, macromolecules, and iron metabolism. Being the master regulator of [...] Read more.

The coordinating role of nuclear factor erythroid-2-related factor 2 (Nrf2) in cellular function is undeniable. Evidence indicates that this transcription factor exerts massive regulatory functions in multiple signaling pathways concerning redox homeostasis and xenobiotics, macromolecules, and iron metabolism. Being the master regulator of antioxidant system, Nrf2 controls cellular fate, influencing cell proliferation, differentiation, apoptosis, resistance to therapy, and senescence processes, as well as infection disease success. Because Nrf2 is the key coordinator of cell defence mechanisms, dysregulation of its signaling has been associated with carcinogenic phenomena and infectious and age-related diseases. Deregulation of this cytoprotective system may also interfere with immune response. Oxidative burst, one of the main microbicidal mechanisms, could be impaired during the initial phagocytosis of pathogens, which could lead to the successful establishment of infection and promote susceptibility to infectious diseases. There is still a knowledge gap to fill regarding the molecular mechanisms by which Nrf2 orchestrates such complex networks involving multiple pathways. This review describes the role of Nrf2 in non-pathogenic and pathogenic cells. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Figure 1
Schematic picture of Nrf2 under constitutive (left) and oxidative stress conditions (right). Full article ">Figure 2
Brief schematic presentation of the Nrf2 structure. Full article ">Figure 3
Regulation of Nrf2 by different proteins involved in different signaling pathways. Full article ">Figure 4
Nrf2 in normal (left) and cancer (right) cells. In normal cells, Nrf2 signaling is highly upregulated, with Nrf2 being mainly activated for cell protection. Under this regulated environment, Nrf2 fights the intracellular ROS inadvertently produced during aerobic metabolism by promoting the gene expressions of different antioxidants. The antioxidant response is vital for detoxifying cells with toxic ROS levels, avoiding distress conditions where ROS-induced mutagenic events can occur. Apart from that, the fine balance of ROS by antioxidants allows for a synchronized and self-controlled eustress state, where prooxidants and antioxidants work in harmony to regulate the intensity and duration of redox signaling. Thus, under normal conditions, the antioxidant response controlled by Nrf2 is key in cancer prevention. However, many cancer cells have constitutive and dysregulated activation of Nrf2. In this scenario, higher Nrf2 levels combat intracellular ROS of anaerobic metabolism due to the Warburg effect. In addition, in many cancer cells with Nrf2 upregulation, Nrf2 has been implicated in the activation of drug efflux transporters, having, in general, a major role in cell cancer cell detoxification. Additionally, cancer cells take advantage of the antioxidant role of Nrf2 to keep ROS at minimum levels by transiting to a stem state. Stemness, antioxidants, and efflux transporters confer great resistance to the main therapies currently practiced in the fight against cancer, namely chemo- and radiotherapy. Hence, in cancer cells, Nrf2 signaling is vital for tumor survival after treatment and its subsequent repopulation due to Nrf2-induced stemness properties. Full article ">Figure 5
The role of Nrf2 and ROS in adipogenesis and osteogenesis. ROS suppresses important signaling pathways necessary for bone formation while simultaneously stimulating pathways that promote the formation adipocytes. The Wnt/βeta catenin and NELL-1 pathways play a key role in promoting osteogenesis, but their activity is hindered when Nrf2 levels are low and ROS levels are high. Consequently, ROS can trigger the phosphorylation of FOXO proteins, causing them to translocate into the nucleus and hinder the signals that promote bone formation, leading to a shift towards adipogenesis. Full article ">

Other

Jump to: Research, Review

18 pages, 2146 KiB

Open AccessSystematic Review

Ozone Exposure Controls Oxidative Stress and the Inflammatory Process of Hepatocytes in Murine Models

by Silvania Mol Pelinsari, Mariáurea Matias Sarandy, Emerson Ferreira Vilela, Rômulo Dias Novaes, Jade Schlamb and Reggiani Vilela Gonçalves

Antioxidants 2024, 13(2), 212; https://doi.org/10.3390/antiox13020212 - 8 Feb 2024

Viewed by 1276

Abstract

(1) Background: Ozone exposure is a promising tool for treating liver damage since it is known to control the release of free radicals and increase the expression of antioxidant enzymes. The objective is to investigate the main intracellular pathways activated after exposure to [...] Read more.

(1) Background: Ozone exposure is a promising tool for treating liver damage since it is known to control the release of free radicals and increase the expression of antioxidant enzymes. The objective is to investigate the main intracellular pathways activated after exposure to ozone, considering the dosage of antioxidant enzymes and markers of oxidative stress. (2) Methods: This systematic review was performed based on the PRISMA guidelines and using a structured search in MEDLINE (PubMed), Scopus, and Web of Science. Bias analysis and methodological quality assessments were examined using the SYRCLE Risk of Bias tool. (3) Results: Nineteen studies were selected. The results showed that the exposure to ozone has a protective effect on liver tissue, promoting a decrease in inflammatory markers and a reduction in oxidative stress in liver tissue. In addition, ozone exposure also promoted an increase in antioxidant enzymes. The morphological consequences of controlling these intracellular pathways were reducing the tissue inflammatory process and reducing areas of degeneration and necrosis. (4) Conclusions: Ozone exposure has a beneficial effect on models of liver injury through the decrease in oxidative stress in tissue and inflammatory markers. In addition, it regulates the Nrf2/ARE antioxidant pathway and blocks the NF-κB inflammatory pathway. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Graphical abstract

23 pages, 1327 KiB

Open AccessSystematic Review

Docosahexaenoic Acid as Master Regulator of Cellular Antioxidant Defenses: A Systematic Review

by Sara Margherita Borgonovi, Stefania Iametti and Mattia Di Nunzio

Antioxidants 2023, 12(6), 1283; https://doi.org/10.3390/antiox12061283 - 15 Jun 2023

Cited by 2 | Viewed by 1916

Abstract

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that benefits the prevention of chronic diseases. Due to its high unsaturation, DHA is vulnerable to free radical oxidation, resulting in several unfavorable effects, including producing hazardous metabolites. However, in vitro and in vivo investigations [...] Read more.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that benefits the prevention of chronic diseases. Due to its high unsaturation, DHA is vulnerable to free radical oxidation, resulting in several unfavorable effects, including producing hazardous metabolites. However, in vitro and in vivo investigations suggest that the relationship between the chemical structure of DHA and its susceptibility to oxidation may not be as clear-cut as previously thought. Organisms have developed a balanced system of antioxidants to counteract the overproduction of oxidants, and the nuclear factor erythroid 2-related factor 2 (Nrf2) is the key transcription factor identified for transmitting the inducer signal to the antioxidant response element. Thus, DHA might preserve the cellular redox status promoting the transcriptional regulation of cellular antioxidants through Nrf2 activation. Here, we systematically summarize the research on the possible role of DHA in controlling cellular antioxidant enzymes. After the screening process, 43 records were selected and included in this review. Specifically, 29 studies related to the effects of DHA in cell cultures and 15 studies concerned the effects of consumption or treatment with DHA in animal. Despite DHA’s promising and encouraging effects at modulating the cellular antioxidant response in vitro/in vivo, some differences observed among the reviewed studies may be accounted for by the different experimental conditions adopted, including the time of supplementation/treatment, DHA concentration, and cell culture/tissue model. Moreover, this review offers potential molecular explanations for how DHA controls cellular antioxidant defenses, including involvement of transcription factors and the redox signaling pathway. Full article

(This article belongs to the Special Issue Oxidative Stress and NRF2 in Health and Disease)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Oxidative Stress and NRF2 in Health and Disease

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Published Papers (13 papers)

Research

Review

Other

Further Information

Guidelines

MDPI Initiatives

Follow MDPI