bims-noxint Biomed News
on NADPH oxidases in tumorigenesis
Issue of 2019‒12‒01
eight papers selected by
Laia Caja Puigsubira
Uppsala University

  1. Pain. 2019 Nov 20.
    Xu J, Wei X, Gao F, Zhong X, Guo R, Ji Y, Zhou X, Chen J, Yao P, Liu X, Wei X.
      High frequency stimulation (HFS) of the sciatic nerve has been reported to produce long term potentiation (LTP) and long-lasting pain hypersensitivity in rats. However, the central underlying mechanism remains unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) belongs to a group of electron-transporting transmembrane enzymes that produce reactive oxygen species (ROS). Here we found that NOX2 was upregulated in the lumbar spinal dorsal horn after HFS of the left sciatic nerve, which induced bilateral pain and spinal LTP in both male and female rats. Blocking NOX2 with blocking peptide or shRNA prevented the development of bilateral mechanical allodynia, the induction of spinal LTP, and the phosphorylation of N-methyl-d-aspartate (NMDA) receptor 2B (GluN2B) and nuclear factor kappaB (NF-κB) p65 following HFS. Moreover, NOX2 shRNA reduced the frequency and amplitude of both spontaneous excitatory post synaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) in laminar II neurons. Furthermore, 8-hydroxyguanine (8-OHG), an oxidative stress marker, was increased in the spinal dorsal horn. Spinal application of ROS scavenger, Phenyl-N-tert-butylnitrone (PBN), depressed the already established spinal LTP. Spinal application of H2O2, one ROS, induced LTP and bilateral mechanical allodynia, increased the frequency and amplitude of sEPSCs in laminar II neurons, and phosphorylated GluN2B and p65 in the dorsal horn. The present study provided electrophysiological and behavioral evidence that NOX2- derived ROS in the spinal cord contributed to persistent mirror-image pain by enhancing the synaptic transmission, which was mediated by increasing presynaptic glutamate release and activation of NMDAR and NF-κB in the spinal dorsal horn.
  2. Oxid Med Cell Longev. 2019 ;2019 5181429
    Zadeh JK, Zhutdieva MB, Laspas P, Yuksel C, Musayeva A, Pfeiffer N, Brochhausen C, Oelze M, Daiber A, Xia N, Li H, Gericke A.
      Objective: Atherogenic lipoproteins may impair vascular reactivity consecutively causing tissue damage in multiple organs via abnormal perfusion and excessive reactive oxygen species generation. We tested the hypothesis that chronic hypercholesterolemia causes endothelial dysfunction and cell loss in the retina.Methods: Twelve-month-old apolipoprotein E-deficient (ApoE-/-) mice and age-matched wild-type controls were used in this study (n = 8 per genotype for each experiment). Intraocular pressure, blood pressure, and ocular perfusion pressure were determined. Retinal arteriole responses were studied in vitro, and reactive oxygen and nitrogen species were quantified in the retinal and optic nerve cryosections. The expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the NADPH oxidase isoforms, NOX1, NOX2, and NOX4, were determined in retinal cryosections by immunofluorescence microscopy. Pro- and antioxidant redox genes were quantified in retinal explants by PCR. Moreover, cell number in the retinal ganglion cell layer and axon number in the optic nerve was calculated.
    Results: Responses to the endothelium-dependent vasodilator, acetylcholine, were markedly impaired in retinal arterioles of ApoE-/- mice (P < 0.01). LOX-1 (P = 0.0007) and NOX2 (P = 0.0027) expressions as well as levels of reactive oxygen species (P = 0.0022) were increased in blood vessels but not in other retinal structures. In contrast, reactive nitrogen species were barely detectable in both mouse genotypes. Messenger RNA for HIF-1α, VEGF-A, NOX1, and NOX2, but also for various antioxidant redox genes was elevated in the retina of ApoE-/- mice. Total cell number in the retinal ganglion cell layer did not differ between ApoE-/- and wild-type mice (P = 0.2171). Also, axon number in the optic nerve did not differ between ApoE-/- and wild-type mice (P = 0.6435).
    Conclusion: Apolipoprotein E deficiency induces oxidative stress and endothelial dysfunction in retinal arterioles, which may trigger hypoxia in the retinal tissue. Oxidative stress in nonvascular retinal tissue appears to be prevented by the upregulation of antioxidant redox enzymes, resulting in neuron preservation.
  3. Biomed Res Int. 2019 ;2019 3708497
    Pengnet S, Prommaouan S, Sumarithum P, Malakul W.
      Hypercholesterolaemia is associated with oxidative stress and endothelial dysfunction and leads to the development of atherosclerosis. Naringin exhibits cardiovascular protective and antioxidant properties. Therefore, the aim of this study was to assess the effect of naringin administration on vascular oxidative stress and endothelial dysfunction in hypercholesterolaemic rats and to elucidate its underlying mechanism. Sprague Dawley rats were fed a diet with 1.5% cholesterol (HCD) for 8 weeks to induce hypercholesterolaemia. Naringin (100 mg/kg body weight) was orally administrated to rats during the last 4 weeks of the diet treatment. After 8 weeks, the thoracic aorta was isolated to determine vascular function and nitric oxide (NO) levels. The aortic superoxide anion (O2 -) level was detected using dihydroethidium (DHE) fluorescence staining. Protein expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, and inducible nitric oxide synthase (iNOS), as well as oxidative damage markers, was also evaluated in aortae. Naringin treatment of hypercholesterolaemic rats enhanced aortic NO levels, restored endothelium-dependent responses to acetylcholine (ACh), and reduced aortic O2 - levels. Furthermore, naringin treatment decreased LOX-1, NADPH oxidase subunits (p47phox, Nox2, and Nox4), and iNOS as well as oxidative damage markers (3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE)) expression in aortic tissues from hypercholesterolaemic rats. These results demonstrate that naringin treatment improves endothelium dysfunction in hypercholesterolaemic rats, at least partially by decreasing oxidative stress via downregulation of LOX-1 and NADPH oxidase.
  4. FEBS J. 2019 Nov 30.
    Millana-Fañanas E, Todesca S, Siccorello A, Masino L, Pompach P, Magnani F, Pastore A, Mattevi A.
      It is now accepted that Reactive Oxygen Species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane-bound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family-member present in cyanobacteria, protists, plants, fungi and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the dehydrogenase domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-to-folded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.
    Keywords:  EF-hands; NMR; calcium activation; enzyme; structure
  5. Am J Physiol Renal Physiol. 2019 Nov 25.
    Yang N, Gonzalez-Vicente A, Garvin JL.
      Angiotensin II exacerbates oxidative stress in part by increasing superoxide (O2-) production by many renal tissues. However, whether it does so in proximal tubules and the source of O2- in this segment are unknown. Dietary fructose enhances the stimulatory effect of angiotensin II on proximal tubule Na reabsorption, but whether this is true for oxidative stress is unknown. We hypothesized that angiotensin II causes proximal nephron oxidative stress in part by stimulating NADPH oxidase (NOX) 4-dependent O2- production and decreasing the amount of the antioxidant glutathione, and this is exacerbated by dietary fructose. We measured basal and angiotensin II-stimulated O2- production with and without inhibitors, NOX1 and 4 expression, and total and reduced glutathione (GSH) in proximal tubules from rats drinking either tap water (Control) or 20% fructose (FRUCT). Angiotensin II (10 nM) increased O2- production by 113±42 relative light units (RLU)/mg protein/s in Controls and by 401±74 RLU/mg protein/s in FRUCT (p<0.05 vs. Control, n=11 each group). Apocynin and the Nox1/4 inhibitor GKT136901 prevented angiotensin II-induced increases in both groups. NOX4 expression wasn't different between groups. NOX1 expression was undetectable. Angiotensin II decreased GSH by 1.8±0.8 nmol/mg protein in Controls and by 4.2±0.9 nmol/mg protein in FRUCT (p < 0.047 vs Control, n=18 each group). We conclude: 1) angiotensin II causes oxidative stress in proximal tubules by increasing O2- production by NOX4 and decreasing GSH; and 2) dietary fructose enhances angiotensin II's ability to stimulate O2- and diminish GSH thereby exacerbating oxidative stress.
    Keywords:  NADPH oxidase; hypertension; kidney; salt
  6. J Mol Neurosci. 2019 Nov 25.
    Shen W, Lu Y, Hu J, Le H, Yu W, Xu W, Yu W, Zheng J.
      This study aimed to explore the mechanism of miR-320 in regulating biological characteristics of ischemic cerebral neuron by mediating Nox2/ROS pathway. Primary neurons were cultured and grouped: normal group (normal primary neurons), negative control (NC) group (ischemic primary neurons, transfected with negative control plasmid), model group (ischemic primary neurons), miR-320 mimic group (ischemic primary neurons, transfected with miR-320-overexpressed plasmid), Nox2 vector group (ischemic primary neurons, transfected with Nox2-overexpressed plasmid), and miR-320 mimic + Nox2 vector group (ischemic primary neurons, co-transfected with miR-320- and Nox2-overexpressed plasmid). Dual-luciferase reporter assay showed that there was the target relationship between miR-320 and Nox2. miR-320 expression was significantly decreased, and Nox2 expression was significantly increased in the rest groups compared with normal group (both P < 0.05). There was a co-localization of miR-320 and Nox2 in the cytoplasm. Cell proliferation, contents of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), and mRNA and protein expressions of Ki67, Bcl-2, and c-myc were significantly declined, and apoptosis rate, contents of malondialdehyde (MDA) and reactive oxygen species (ROS), and caspase-3 mRNA and protein expressions were significantly increased in the rest groups compared with normal group (all P < 0.05). miR-320 promoted cell proliferation; increased contents of SOD, CAT, and GSH-PX; and declined apoptosis and contents of MDA and ROS. Moreover, miR-320 could affect the regulation of Nox2/ROS pathway on ischemic cerebral neuron by negatively regulating Nox2 expression. Overexpressed miR-320 affects the proliferation, apoptosis, and oxidative stress injury of ischemic cerebral neuron by inhibiting Nox2/ROS pathway.
    Keywords:  Apoptosis; Nox2/ROS pathway; Oxidative stress injury; Proliferation; miR-320
  7. Oxid Med Cell Longev. 2019 ;2019 7853492
    Kozhina EA, Ershova ES, Okorokova NA, Veiko VP, Malinovskaya EM, Sergeeva VA, Konkova MS, Kutsev SI, Veiko NN, Kostyuk SV.
      Background: Oxidized human DNA or plasmid DNAs containing human ribosomal genes can easily penetrate into the breast cancer cells MCF7 and stimulate the adaptive response induction. Plasmid DNA containing a CMV promoter, gene EGFP, and the insertion of the human ribosomal genes can be expressed. A hypothesis is proposed: these features of the ribosomal DNA are due to the presence of dGn motifs that are prone to oxidize.Methods: Cells of MCF7 line were cultured with plasmids which contained a CMV promoter and gene of fluorescent protein EGFP. Genetic construction pEGFP-Gn contains pEGFP vector and a small insertion with dG11 and dG13 motifs that are inclined to oxidation. The accumulation of pEGFP and pEGFP-Gn in MCF7 (qPCR), the levels of ROS in the cells, the content of 8-oxodG in plasmids and cellular DNA (flow cytometry, immunoassay, and fluorescent microscopy), the expression of NOX4 and EGFP, the localization of NOX4 and EGFP in MCF7 (qPCR, flow cytometry, and fluorescent microscopy), and the levels of the cell DNA damage (comet assay) were analyzed.
    Results: (dG)n insertions in the plasmid pEGFP increase the levels of ROS, the cell DNA oxidation and DNA damage, and the level of transfection of plasmid into the MCF7 cells. NOX4 participates in the oxidation of pEGFP-Gn and pEGFP. The expression of EGFP gene in MCF7 is significantly increased in case of pEGFP-Gn. Stimulation of ROS synthesis (H2O2 40 μM or 10 cGy IR) increases the level of expression of EGFP.
    Conclusions: GC-rich DNA fragments containing dGn motifs that are inclined to oxidation penetrate into MCF7 cancer cells, stimulate the adaptive response, and can be expressed. This property of GC-rich cell-free DNA should be considered and/or could potentially be used in therapy of tumors.
  8. Biochem Biophys Res Commun. 2019 Nov 25. pii: S0006-291X(19)32219-3. [Epub ahead of print]
    Sun JC, Du JJ, Li XQ, Li N, Wei W, Sun WY.
      Acute liver injury can be caused by oxidative stress within a short period and is a common pathway to many liver diseases. The liver is vulnerable to reactive oxygen species (ROS) and free radical-mediated disorders. β-arrestin2 was initially discovered to be a negative regulator of G protein-coupled receptor signaling. Recently, β-arrestin2 has been found to act as a multifunctional adaptor protein and play new roles in regulating intracellular signaling networks. However, the role of β-arrestin2 in the pathogenesis of acute liver injury is unclear. In this study, we hypothesize that β-arrestin2 regulates acute liver injury via modulation of oxidative stress. β-arrestin2 knockout mice were used to investigate the impacts of β-arrestin2 on carbon tetrachloride (CCl4)-induced acute liver injury and oxidative stress. Results here suggested that β-arrestin2 deficiency decreased serum activities of aminotransferase and alleviated liver injury induced by CCl4 injection as compared with wildtype mice. β-arrestin2 knockout mice exhibited stronger tolerance in oxidative stress compared with wild-type mice, which was demonstrated by decreased ROS level and increased superoxide dismutase (SOD) and glutathione (GSH) in the liver. Furthermore, β-arrestin2 deficiency significantly inhibited NOX4 (a major source of ROS) expression and the activation of the extracellular regulated kinase (ERK) and, c-Jun NH2-terminal kinase (JNK) pathways. These results suggest that β-arrestin2 deficiency protects against CCl4-induced acute liver injury through attenuating oxidative damage and decreased ERK and JNK phosphorylation.
    Keywords:  Acute liver injury; NOX4; Oxidative stress; β-arrestin2