bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2021‒01‒10
nine papers selected by
Laia Caja Puigsubira
Uppsala University


  1. Theranostics. 2021 ;11(2): 470-490
    Mehling R, Schwenck J, Lemberg C, Trautwein C, Zizmare L, Kramer D, Müller A, Fehrenbacher B, Gonzalez-Menendez I, Quintanilla-Martinez L, Schröder K, Brandes RP, Schaller M, Ruf W, Eichner M, Ghoreschi K, Röcken M, Pichler BJ, Kneilling M.
      Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production. Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91phox-/-), myeloperoxidase-deficient (MPO-/-), and inducible nitric oxide synthase-deficient (iNOS-/-) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences. Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO-/- mice and unaffected ROS/RNS production in the ears of iNOS-/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91phox-/- mice with chronic DTHR, while the inflamed ears of MPO-/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR. Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes.
    Keywords:  DTHR; NETs; Neutrophils; ROS/RNS; acute chronic inflammation
    DOI:  https://doi.org/10.7150/thno.51462
  2. Physiol Rep. 2021 Jan;9(1): e14606
    Hord JM, Garcia MM, Farris KR, Guzzoni V, Lee Y, Lawler MS, Lawler JM.
      Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg-1  day-1 ) (CONL), 7 days of tail-traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading-induced loss of muscle fiber cross-sectional area (CSA) and fiber-type shift. Losartan mitigated unloading-induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.
    Keywords:  Angiotensin II type 1 receptor; NADPH oxidase-2; hindlimb unloading; neuronal nitric oxide synthase; skeletal muscle atrophy
    DOI:  https://doi.org/10.14814/phy2.14606
  3. Mol Metab. 2021 Jan 02. pii: S2212-8778(20)30234-9. [Epub ahead of print] 101160
    Specht KS, Kant S, Addington AK, McMillan R, Hulver MW, Learnard H, Campbell M, Donnelly S, Caliz AD, Pei Y, Reif M, Bond J, DeMarco A, Craige B, Keaney JF, Craige SM.
      OBJECTIVE: The immediate signals that couple exercise to metabolic adaptation are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a producer of reactive oxygen species (ROS) and plays a significant role in both metabolic and vascular adaptation during conditions of stress. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism.METHODS: Mice were subjected to acute exercise to assess immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and subsequent substrate oxidation was measured.
    RESULTS: We identified key exercise-responsive metabolic genes that are dependent on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Furthermore, Nox4-deficient mice had decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Nox4-deficient mice also demonstrated impaired adaptation to chronic exercise as measured by time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise.
    CONCLUSION: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting that the endothelium is important for the skeletal muscle metabolic response to exercise.
    Keywords:  Acute exercise; Nox4; ROS; exercise training; hydrogen peroxide; metabolic adaptation; skeletal muscle metabolism
    DOI:  https://doi.org/10.1016/j.molmet.2020.101160
  4. Free Radic Biol Med. 2020 Dec 30. pii: S0891-5849(20)31896-7. [Epub ahead of print]
    Owusu SB, Hudik E, Férard C, Dupré-Crochet S, Addison ECDK, Preko K, Bizouarn T, Houée-Levin C, Baciou L.
      Neutrophils are key cells from the innate immune system that destroy invading bacteria or viruses, thanks mainly to the non-mitochondrial reactive oxygen species (ROS) generated by the enzyme NADPH oxidase. Our aim was to study the response of neutrophils to situations of oxidative stress with emphasis on the impact on the NADPH oxidase complex. To mimic oxidative stress, we used gamma irradiation that generated ROS (OH•, O2•- and H2O2) in a quantitative controlled manner. We showed that, although irradiation induces shorter half-lives of neutrophil (reduced by at least a factor of 2), it triggers a pre-activation of surviving neutrophils. This is detectable by the production of a small but significant amount of superoxide anions, proportional to the dose ((3 times that of sham). Investigations at the molecular level showed that this ROS increase was generated by the NADPH oxidase enzyme after neutrophils irradiation. The NADPH oxidase complex undergoes an incomplete assembly which includes p47phox and p67phox but excludes the G-protein Rac. Importantly, this irradiation-induced pre-activation is capable of considerably improving neutrophil reactivity. Indeed, we have observed that this leads to an increase in the production of ROS and the capacity of phagocytosis, leading to the conclusion that radiation induced ROS clearly behave as neutrophil primers.
    Keywords:  Ionizing radiation; NADPH oxidase; Neutrophils; ROS; Superoxide radical
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2020.12.233
  5. J Nutr Biochem. 2020 Dec 31. pii: S0955-2863(20)30605-7. [Epub ahead of print] 108573
    Ishida K, Kaji K, Sato S, Ogawa H, Takagi H, Takaya H, Kawaratani H, Moriya K, Namisaki T, Akahane T, Yoshiji H.
      Alcoholic liver disease (ALD)-related fibrosis results from a variety of mechanisms including the accumulation of acetaldehyde, reactive oxygen species (ROS), and hepatic overload of endogenous lipopolysaccharide (LPS). Alcohol cessation is the therapeutic mainstay for patients with all stages of ALD, whereas pharmacological strategies for liver fibrosis have not been established. Sulforaphane, a phytochemical found in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) and exerts anticancer, antidiabetic, and antimicrobial effects; however, few studies investigated its efficacy in the development of ALD-related fibrosis. Herein, we investigated the effect of sulforaphane on acetaldehyde metabolism and liver fibrosis in HepaRG and LX-2 cells, human hepatoma and hepatic stellate cell (HSC) lines, respectively, as well as in a mouse model of alcoholic liver fibrosis induced by ethanol plus carbon tetrachloride (EtOH/CCl4). Sulforaphane treatment induced the activity of acetaldehyde-metabolizing mitochondrial aldehyde dehydrogenase (ALDH2) in HepaRG cells and suppressed the acetaldehyde-induced proliferation and profibrogenic activity in LX-2 cells with upregulation of Nrf2-regulated antioxidant genes, including HMOX1, NQO1, and GSTM3. Moreover, sulforaphane attenuated the LPS/toll-like receptor 4 (TLR4)-mediated sensitization to transforming growth factor-β with downregulation of NADPH oxidase 1 (NOX1) and NOX4. In EtOH/CCl4-treated mice, oral sulforaphane administration augmented hepatic acetaldehyde metabolism. Additionally, sulforaphane significantly inhibited Kupffer cell infiltration and fibrosis, decreased fat accumulation and lipid peroxidation, and induced Nrf2-regulated antioxidant response genes in EtOH/CCl4-treated mice. Furthermore, sulforaphane treatment blunted hepatic exposure of gut-derived LPS and suppressed hepatic TLR4 signaling pathway. Taken together, these results suggest sulforaphane as a novel therapeutic strategy in ALD-related liver fibrosis.
    Keywords:  Alcoholic liver injury; Lipopolysaccharide; Mitochondrial aldehyde dehydrogenase; Nrf2; Sulforaphane; Toll-like receptor 4
    DOI:  https://doi.org/10.1016/j.jnutbio.2020.108573
  6. Mol Cell Biochem. 2021 Jan 04.
    Ammon JC, Valls D, Eldemerdash M, Patel JR, Tran PD, Feng L, Gi M, Gonzalez TT, Phan C, Zendejas AE, So CH.
      G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.
    Keywords:  Apoptosis; Cisplatin; G protein-coupled receptor kinase; G protein-coupled receptors; NADPH oxidase 4
    DOI:  https://doi.org/10.1007/s11010-020-03969-3
  7. Amino Acids. 2021 Jan 04.
    Liu J, Liu Y, Pan W, Li Y.
      The present study was designed to investigate the anti-rheumatic effects and the mechanism of angiotensin (Ang)-(1-7) in rat models with collagen-induced arthritis (CIA). The CIA model was established using male Wistar rats by intradermal injection of bovine collagen-II in complete Freund's adjuvant at the base of the tail. The levels of angiotensin converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor (MasR) were reduced in CIA rats. The attenuation of paw swelling and arthritis scores and improvement of indexes of spleen and thymus were done by Ang-(1-7) injection in CIA rats. The increased levels of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the serum and hind paw were blocked by Ang-(1-7) administration. In addition, enhanced NADPH oxidase (Nox) activity, increased levels of superoxide anions and malondialdehyde (MDA), and weakened superoxide dismutase (SOD) activity, were all reversed by treatment with Ang-(1-7). Nox1 overexpression reversed the suppressing effects of Ang-(1-7) on paw swelling and arthritis scores in CIA rats. The Ang-(1-7)-induced improvement in spleen and thymus indexes in CIA rats was abolished by Nox1 overexpression. Nox1 overexpression reversed the inhibitory effects of Ang-(1-7) by increasing IL-1β, IL-6, TNF-α, and IFN-γ levels in the serum and hind paw of CIA rats. These results demonstrated that Nox1 increased the oxidative stress in arthritis, and Ang-(1-7) improved rheumatism in arthritis via inhibiting oxidative stress.
    Keywords:  Collagen-induced arthritis; Inflammatory cytokines; Oxidative stress; Rat; Rheumatism
    DOI:  https://doi.org/10.1007/s00726-020-02935-z
  8. Mol Oncol. 2021 Jan 07.
    Mota M, Metge BJ, Hinshaw DC, Alsheikh HA, Chen D, Samant RS, Shevde LA.
      The expression of Merlin tumor suppressor protein encoded by NF2 gene is remarkably decreased in metastatic breast cancer tissues. In order to recapitulate clinical evidence, we generated a unique, conditional Nf2-knockout (Nf2-/- ) mouse mammary tumor model. Merlin-deficient breast tumor cells and Nf2-/- mouse embryonic fibroblasts (MEFs) displayed a robustly invasive phenotype. Moreover, Nf2-/- MEFs presented with notable alterations in redox management networks, implicating a role for Merlin in redox homeostasis. This programmatic alteration resonated with pathways that emerged from breast tumor cells engineered for Merlin deficiency. Further investigations revealed that NF2-silenced cells supported reduced activity of the Nrf2 antioxidant transcription factor, concomitant with elevated expression of NADPH oxidase enzymes. Importantly, mammary specific Nf2-/- in an MMTV Neu+ murine breast cancer model demonstrated accelerated mammary carcinogenesis in vivo. Tumor-derived primary organoids and cell lines were characteristically invasive with evidence of a dysregulated cellular redox management system. As such, Merlin deficiency programmatically influences redox imbalance that orchestrates malignant attributes of mammary/breast cancer.
    Keywords:  DUOX; Merlin; NOX; NRF2; ROS; breast
    DOI:  https://doi.org/10.1002/1878-0261.12896
  9. Nat Commun. 2021 Jan 08. 12(1): 155
    Wu JX, Liu R, Song K, Chen L.
      Dual oxidases (DUOXs) produce hydrogen peroxide by transferring electrons from intracellular NADPH to extracellular oxygen. They are involved in many crucial biological processes and human diseases, especially in thyroid diseases. DUOXs are protein complexes co-assembled from the catalytic DUOX subunits and the auxiliary DUOXA subunits and their activities are regulated by intracellular calcium concentrations. Here, we report the cryo-EM structures of human DUOX1-DUOXA1 complex in both high-calcium and low-calcium states. These structures reveal the DUOX1 complex is a symmetric 2:2 hetero-tetramer stabilized by extensive inter-subunit interactions. Substrate NADPH and cofactor FAD are sandwiched between transmembrane domain and the cytosolic dehydrogenase domain of DUOX. In the presence of calcium ions, intracellular EF-hand modules might enhance the catalytic activity of DUOX by stabilizing the dehydrogenase domain in a conformation that allows electron transfer.
    DOI:  https://doi.org/10.1038/s41467-020-20466-9