bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2020‒06‒14
eight papers selected by
Laia Caja Puigsubira
Uppsala University


  1. Redox Biol. 2020 May 26. pii: S2213-2317(20)30416-X. [Epub ahead of print]36 101557
    Cully TR, Rodney GG.
      The ability for skeletal muscle to perform optimally can be affected by the regulation of Ca2+ within the triadic junctional space at rest. Reactive oxygen species impact muscle performance due to changes in oxidative stress, damage and redox regulation of signaling cascades. The interplay between ROS and Ca2+ signaling at the triad of skeletal muscle is therefore important to understand as it can impact the performance of healthy and diseased muscle. Here, we aimed to examine how changes in Ca2+ and redox signaling within the junctional space micro-domain of the mouse skeletal muscle fibre alters the homeostasis of these complexes. The dystrophic mdx mouse model displays increased RyR1 Ca2+ leak and increased NAD(P)H Oxidase 2 ROS. These alterations make the mdx mouse an ideal model for understanding how ROS and Ca2+ handling impact each other. We hypothesised that elevated t-tubular Nox2 ROS increases RyR1 Ca2+ leak contributing to an increase in cytoplasmic Ca2+, which could then initiate protein degradation and impaired cellular functions such as autophagy and ER stress. We found that inhibiting Nox2 ROS did not decrease RyR1 Ca2+ leak observed in dystrophin-deficient skeletal muscle. Intriguingly, another NAD(P)H isoform, Nox4, is upregulated in mice unable to produce Nox2 ROS and when inhibited reduced RyR1 Ca2+ leak. Our findings support a model in which Nox4 ROS induces RyR1 Ca2+ leak and the increased junctional space [Ca2+] exacerbates Nox2 ROS; with the cumulative effect of disruption of downstream cellular processes that would ultimately contribute to reduced muscle or cellular performance.
    Keywords:  Calcium; DMD; Dystrophy; Mdx; NAD(P)H oxidase; Nox2; Nox4; RyR1; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.redox.2020.101557
  2. Biochem Biophys Res Commun. 2020 Jun 04. pii: S0006-291X(20)31099-8. [Epub ahead of print]
    Bhatti SN, Li JM.
      BACKGROUND: A Nox2 containing NADPH oxidase (Nox2) is involved in the global oxidative stress found in dietary obesity and metabolic disorders. However, the effects of high fat diet (HFD) on cardiac Nox2 activation and signaling in left ventricular hypertrophy (LVH) remain unknown.METHODS: Left ventricular (LV) tissues isolated from C57BL/6J wild-type (WT) and Nox2 knockout (Nox2KO) mice (11 months old, n = 6 per group) after 4 months of HFD treatment were used. Cardiomyocyte sizes were measured digitally on LV cross-sections. The levels of cardiac reactive oxygen species (ROS) production was determined using lucigenin-chemiluminescence and in situ dihydroethidium (DHE) fluorescence. The levels of Nox subunit expression and redox signaling were examined by immunoblotting and immunofluorescence.
    RESULTS: In comparison to WT normal chow diet control hearts, WT HFD hearts had 1.8-fold increases in cardiomyocyte size, a sign of cardiac hypertrophy, and this was accompanied with ≥2-fold increase in the levels of ROS production, Nox2 expression and the phosphorylation of Akt and ERK1/2. Increased ROS production measured in HFD heart homogenates was inhibited to control levels by Tiron (a cell membrane permeable O2•-scavenger), diphenyleneiodonium (DPI, a flavohaemoprotein inhibitor) and Nox2 ds-tat (a Nox2 assembly inhibitor). However, all of these abnormalities were significantly reduced or absent in Nox2KO hearts under the same HFD.
    CONCLUSIONS: Nox2 activation in response to dietary obesity and metabolic disorders plays a key role in cardiac oxidative stress, aberrant redox signaling and cardiomyocyte hypertrophy. Knockout of Nox2 protects hearts from oxidative damage associated with obesity and metabolic disorders.
    Keywords:  Akt; Cardiac hypertrophy; ERK1/2; Knockout mice; NADPH Oxidase; Redox-signaling
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.162
  3. Biochem Pharmacol. 2020 Jun 09. pii: S0006-2952(20)30324-5. [Epub ahead of print] 114088
    Song Z, Hudik E, Le Bars R, Roux B, My-Chan Dang P, El Benna J, Nüsse O, Dupré-Crochet S.
      Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67phox, p47phox, and p40phox) and Rac with the membrane subunits (gp91phox and p22phox). Many studies are devoted to the activation of NOX2. However, the mechanisms that cause NADPH oxidase deactivation and thus terminate ROS production are not well known. Here we investigated the ability of class I phosphoinositide 3-kinases (PI3Ks) to sustain NADPH oxidase activation. The NADPH oxidase activation was triggered by seeding neutrophil-like PLB-985 cells, or human neutrophils on immobilized fibrinogen. Adhesion of the neutrophils, mediated by β2 integrins, induced activation of the NADPH oxidase and translocation of the cytosolic subunits at the plasma membrane. Inhibition of class I PI3Ks, and especially PI3Kβ, terminated ROS production. This deactivation of NOX2 is due to the release of the cytosolic subunits, p67phox and p47phox from the plasma membrane. Overexpression of an active form of Rac 1 did not prevent the drop of ROS production upon inhibition of class I PI3Ks. Moreover, the phosphorylation of p47phox at S328, a potential target of kinases activated by the PI3K pathway, was unchanged. Our results indicate that the experimental downregulation of class I PI3K products triggers the plasma membrane NADPH oxidase deactivation. Release of p47phox from the plasma membrane may involve its PX domains that bind PI3K products.
    Keywords:  NADPH oxidase; ROS; imaging; integrin; phosphoinositide 3-kinases
    DOI:  https://doi.org/10.1016/j.bcp.2020.114088
  4. Am J Transl Res. 2020 ;12(5): 1714-1727
    Chen JL, Lu JH, Xie CS, Shen YJ, Wang JW, Ye XY, Zhang MB, Jia GL, Tao YX, Li J, Cao H.
      OBJECTIVE: The present study determines whether Cav-1 modulates the initiation, development and maintenance of type-2 DNP via the Rac1/NOX2-NR2B signaling pathway.METHODS: After regular feeding for three days, these rats were randomly divided into two groups: control group with normal-diet (maintenance feed) (n=8); type-2 DM group (n=8). In the type-2 DM group, the rats were fed with a high-fat and high-sugar diet, and received a single intraperitoneal streptozotocin (STZ) injection (35 mg/kg). At two weeks after STZ injection, these diabetic neuropathic pain (DNP) rats were treated with daidzein (0.4 mg/kg/day) and N-tert-Butyl-α-phenylnitrone (PBN, 100 mg/kg/day) for 14 days. After the type-2 DNP model was successfully established, the rats were assigned into four groups: DNP group, DNP+Da group (DNP rats with Cav-1 specific inhibitor daidzein), DNP+PBN group (DNP rats treated with ROS scavenger PBN), and SC group (solvent control group). Then, the mechanical and thermal hyperalgesia were assayed to evaluate the function of the caveolin 1-Recombinant Human Ras-Related C1/nicotinamide adenosine diphosphate oxidase 2-NR2B gene (Cav-1-Rac1/NOX2-NR2B) signaling pathway. In the mechanism study, the protein expression levels of p-Caveolin-1, Rac1, NOX2, p-NR2B and t-NR2B, the production of ROS, and the distribution of Cav-1 and NOX2 in the spinal cord were observed.
    RESULTS: The present study revealed that p-Cav-1 was persistently upregulated and activated in the spinal cord microglia in type-2 DNP rats. The use of the pharmacological inhibitor of Cav-1 and a ROS scavenger resulted to a significantly relieved mechanical allodynia and thermal hyperalgesia. In addition, it was demonstrated that Cav-1 promoted ROS generation via the activation of Rac1-dependent NADPH oxidase (NOX).
    CONCLUSION: The present data suggests that Cav-1 in the spinal cord modulates type-2 DNP via regulating the Rac1/NOX2-NR2B pathway.
    Keywords:  Caveolin-1; NADPH oxidase; NMDA receptor 2B subunit; ROS; Type 2 diabetic neuropathic pain; central sensitization; spinal cord
  5. Redox Biol. 2020 May 21. pii: S2213-2317(20)30637-6. [Epub ahead of print]34 101578
    Lei K, Xia Y, Wang XC, Ahn EH, Jin L, Ye K.
      Glioblastoma (GBM) is the most common and most aggressive brain tumor, associated with high levels of reactive oxidative species (ROS) due to metabolic and signaling aberrations. High ROS levels are detrimental to cells, but it remains incompletely understood how cancer cells cope with the adverse effects. Here we show that C/EBPβ, a ROS responsive transcription factor, regulates the transcription of NQO1 and GSTP1, two antioxidative reductases, which neutralize ROS in the GBM and mediates their proliferation. C/EBPβ is upregulated in EGFR overexpressed GBM cells, inversely correlated with the survival rates of brain tumor patients. Interestingly, C/EBPβ binds the promoters of NQO1 and GSTP1 and escalates their expression. Overexpression of C/EBPβ selectively decreases the ROS in EGFR-overexpressed U87MG cells and promotes cell proliferation via upregulating NQO1 and GSTP1; whereas knocking down C/EBPβ elevates the ROS and reduces proliferation by repressing the reductases. Accordingly, C/EBPβ mediates the brain tumor growth in vivo, coupling with NQO1 and GSTP1 expression and ROS levels. Hence, C/EBPβ regulates the expression of antioxidative reductases and balances the ROS, promoting brain tumor proliferation.
    Keywords:  C/EBPβ; GBM; GSTP1; NQO1; Oxidative stress; Transcription factor
    DOI:  https://doi.org/10.1016/j.redox.2020.101578
  6. Arch Biochem Biophys. 2020 Jun 07. pii: S0003-9861(20)30462-8. [Epub ahead of print] 108453
    Peng R, Luo M, Tian R, Lu N.
      Nitric oxide (NO) deficiency and NADPH oxidase plays key roles in endothelial dysfunction and atherosclerotic plaque formation. Recent evidence demonstrates that nitrate-nitrite-NO pathway in vivo exerts beneficial effects upon the cardiovascular system. We aimed to investigate the effects of dietary nitrate on endothelial function and atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet. It was shown that dietary nitrate significantly attenuated aortic endothelial dysfunction and atherosclerosis in ApoE-/- mice. Mechanistic studies revealed that dietary nitrate significantly improved plasma nitrate/nitrite, inhibited vascular NADPH oxidase activity and oxidative stress in ApoE-/- mice, while xanthine oxidoreductase (XOR) expression and activity was enhanced in ApoE-/- mice in comparison with wide type animals. These beneficial effects of nitrate in ApoE-/- mice were abolished by PTIO (NO scavenger) and significantly prevented by febuxostat (XOR inhibitor). In the presence of nitrate, no further effect of apocynin (NADPH oxidase inhibitor) was observed, suggesting NADPH oxidase as a possible target. In vitro, NO donor significantly inhibited NADPH oxidase activity in vascular endothelial cells via the induction of heme oxygenase-1. Altogether, boosting this nitrate-nitrite-NO signaling pathway resulted in the decreases of vascular NADPH oxidase-derived oxidative stress and endothelial dysfunction, and consequently protected ApoE-/- mice against atherosclerosis. These findings may have novel nutritional implications for the preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.
    Keywords:  Atherosclerosis; Endothelial dysfunction; NADPH oxidase; Nitrate; Nitric oxide
    DOI:  https://doi.org/10.1016/j.abb.2020.108453
  7. Toxicol Lett. 2020 Jun 06. pii: S0378-4274(20)30269-1. [Epub ahead of print]
    Uchiyama N, Yukawa T, Dragan YP, Wagoner MP, Naven RT.
      An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells. ROS production after nifedipine treatment was inhibited by a NOX inhibitor (GKT136901) but not the mitochondrial NADH dehydrogenase inhibitor, rotenone, suggesting that nifedipine decreases NRK52E cell viability primarily through a NOX-mediated pathway. To understand the breadth of NOX-mediated ROS production, 12 commercially available compounds that are structurally and/or pharmacologically related to nifedipine as well as 172 internal Takeda candidate drugs, were also evaluated against these two cell types. Over 15% of compounds not cytotoxic to HepG2 cells (below 50 μM) were cytotoxic to NRK52E cells. Our results suggest that a combination of cell viability data from both NRK52E and HepG2 cells was superior for the prediction of in vivo toxicity findings when compared to use of only one cell line. Further, the NRK52E cell viability assay is a good predictor of NOX-mediated ROS production and can be used as a follow up assay following a negative HepG2 response to aid in the selection of suitable compounds for in vivo toxicity studies.
    Keywords:  NADPH oxidase (NOX) in vivosafety studies; in vitro cytotoxicity; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1016/j.toxlet.2020.06.005
  8. Life Sci. 2020 Jun 07. pii: S0024-3205(20)30667-6. [Epub ahead of print] 117917
    Hwang JS, Cha EH, Ha E, Park B, Seo JH.
      AIMS: Methamphetamine (METH) is an abused psychostimulant causing public health concern worldwide. While most studies have focused on the neurotoxic effects of METH, METH-induced cerebrovascular dysfunction has recently drawn attention as an important facet of METH-related pathophysiology. In this study, we investigated the protective role of GKT136901, a NOX1/4 inhibitor, against METH-induced blood-brain barrier (BBB) dysfunction.MAIN METHODS: Primary human brain microvascular endothelial cells (HBMECs) were used as an in vitro BBB model. HBMECs were treated with GKT136901, followed by METH exposure for 24 h. The generation of reactive oxidative species (ROS) was measured using 2',7'-dichlorofluorescin diacetate (DCF-DA) staining. To examine the BBB function, paracellular permeability of HBMEC monolayer was measured using FITC-labeled dextran. To evaluate structural properties of BBB in HBMECs, tight junction (TJ), adherent junction (AJ), and cytoskeletal proteins were stained and analyzed by confocal microscopy.
    KEY FINDINGS: METH treatment rapidly increased ROS generation in HBMECs but GKT136901 treatment inhibited METH-induced ROS generation. Although METH increased the permeability of HBMEC monolayer, this effect was abolished upon GKT136901 treatment. Following METH exposure, the proteins Zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were translocalized from the cell membrane to the cytoplasm, thereby destroying intercellular tight junction (TJ) and adherent junction (AJ) structures, which were ameliorated upon GKT136901 treatment. METH exposure altered the cellular morphology of HBMECs and induced stress fiber formation. However, GKT136901 prevented METH-induced morphological and cytoskeletal changes in HBMECs.
    SIGNIFICANCE: These results suggest that GKT136901 prevents METH-induced BBB dysfunction in HBMECs through the inhibition of ROS generation.
    Keywords:  Blood-brain barrier; GKT136901; Human brain microvascular endothelial cells; Methamphetamine; NOX1/4; Reactive oxygen species (ROS)
    DOI:  https://doi.org/10.1016/j.lfs.2020.117917