Reactive oxygen species (ROS) are cellular signals generated ubiquitously by all mammalian cells, but their relative unbalance triggers also diseases through intracellular damage to DNA, RNA, proteins, and lipids. NADPH oxidases (NOX) are the only known enzyme family with the sole function to produce ROS. The NOX physiological functions concern host defence, cellular signaling, regulation of gene expression, and cell differentiation. On the other hand, increased NOX activity contributes to a wide range of pathological processes, including cardiovascular diseases, neurodegeneration, organ failure, and cancer. Therefore targeting these enzymatic ROS sources by natural compounds, without affecting the physiological redox state, may be an important tool. This review summarizes the current state of knowledge of the role of NOX enzymes in physiology and pathology and provides an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols.
Oxidative stress is a molecular deregulation in reactive oxygen species (ROS) metabolism involved in the pathogenesis of several diseases. Oxidative stress is no longer considered as a simple imbalance between the production and scavenging of ROS, but as a dysfunction of enzymes involved in ROS production [
Reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite are generated by all mammalian cells and have been recognized for many decades as causing cell damage by oxidation and nitration of macromolecules, such as DNA, RNA, proteins, and lipids. Moreover, ROS can also promote cell signaling pathways modulated by growth factors and transcription factors, therefore regulating cell proliferation, differentiation, and apoptosis [
The instability of an unpaired electron in its valence shell causes the high reactivity of superoxide. Superoxide has been implicated in numerous pathological processes, including cancer, cardiovascular disease (e.g., atherosclerosis and stroke), and acute and chronic diseases due to microbial infections. Superoxide can directly or indirectly damage DNA through oxidation [
However, superoxide gives rise to other ROS that possess different redox chemistries, and, thus, different physiological and pathophysiological effects. For example, superoxide is rapidly reduced, both spontaneously and enzymatically, to H2O2. Unlike superoxide, H2O2 has no net charge; so, it is more lipid-soluble, with the potential to diffuse through organelles and cellular membranes reaching sites distant from its source. H2O2 modifies cellular proteins via oxidation of cysteine, methionine [
One of the most important and fast redox reactions in biology is between superoxide and the nitric oxide (NO) radical giving rise to ONOO−. ONOO− is an oxidizing and nitrating molecule that has been implicated in cancer [
As with every mechanism involved in both normal cell function and the development of disease, strategies to counteract ROS must take into account their critical importance in the normal functioning of the organism [
Further understanding of the biological mechanisms among oxidative stress, tumor growth, and metastasis could contribute to the advancement of cancer treatment.
For example, angiogenesis is another important factor for tumor growth and metastasis, and ROS has a key role in angiogenesis regulation [
After all, an emerging concept suggests that ROS modulate the immune cells functions that infiltrate the tumor environment and stimulate angiogenesis [
These oxidative processes have been implicated in many diseases in addition to cancer.
Overproduction of ROS is involved in the development of a number of diseases, which range from neurological such as Parkinson’s [
Several enzymes produce ROS, including the mitochondrial electron transport chain, nitric oxide synthases (NOSs), cytochrome P450 reductase, and xanthine oxidase. However, for all of these systems, ROS production takes place as a byproduct of the main catalytic function of the enzyme/system or from a dysfunctional variant of the enzyme. In contrast, NADPH oxidases are the only enzymes whose primary function is to generate superoxide/ROS [
NOX isoforms and pathology [modified from [
Characteristic | Binding partners | Intracellular localization | Tissue distribution | Implication in pathology |
---|---|---|---|---|
NOX1 | p22phox, NOXO1, NoxA1, Rac1, PDI, TKS4/5* | Caveolae on the plasma membrane, redoxisomes | Colon epithelia VSMCs, endothelial cells, uterus, placenta, prostate, osteoclasts, retinal pericytes, neurons, astrocytes, microglia | Colon cancer, prostate cancer, gastrointestinal inflammation, hypertension, restenosis after angioplasty |
NOX2 | p22phox, p67phox, p40phox, p47phox, Rac1/2 | Phagosomes, cytoskeleton, lamellipodia, redoxisomes | Phagocytes, CNS, endothelium, VSMCs, fibroblasts, cardiomyocytes, skeletal muscle, hepatocytes, hematopoietic stem cells | Gastrointestinal inflammation, hypertension, myocardial injury, restenosis after angioplasty, melanoma, diabetes, neurodegenerative diseases |
NOX3 | p22phox, NOXO1 | Plasma membrane | Inner ear, lung endothelial cells, fetal tissues | Hearing loss, pancreatic cancer |
NOX4 | p22phox, PDI, TKS4/5, Poldip2* | Focal adhesions, nucleus, endoplasmic reticulum, mitochondria | Ubiquitously expressed but highly in the kidney | Pancreatic cancer, melanoma, diabetes |
NOX5 | Ca2+, Hsp90, CaM# | Internal membranes, plasma membrane | Lymphatic tissue, testis, VSMCs, endothelial cells, spleen, uterus, and prostate | Atherosclerosis prostate cancer, pancreatic cancer |
Duox1 | Ca2+, DuoxA1 | Plasma membrane | Thyroid, respiratory epithelium | Thyroid dysfunction, cystic fibrosis |
Duox2 | Ca2+, DuoxA2 | Plasma membrane | Airway epithelial, colon, salivary gland | Thyroid dysfunction, cystic fibrosis |
#The NOX5 protein contains four N-terminal calcium-binding sites that regulate activation of the enzyme. Activity of NOX5 can be further supported by the binding of Hsp90 or Calmodulin to the C-terminus of the protein.
They are differentially expressed and regulated in various tissues and have different subcellular localizations (reviewed in [
In addition to these established NOX binding partners, the tyrosine kinase substrate with 4/5 SH3 domains (Tks4/5) [
NOX catalytic subunits are differently regulated: NOXA1 plays a key role for NOX1 activation [
In contrast, NOX4 is constitutively active, and modulation of its expression may thus be a major activity regulator.
ROS produced by NOXs have been shown to affect all other possible sources of ROS, leading to their dysfunction and to a further increase in ROS generation, forming a vicious cycle of oxidative stress. For example, increased
The oxidant signaling involving NADPH oxidase has important roles in cell biology participating in intracellular signaling of cell differentiation and proliferation. These mechanisms are important in tissue repair and tumorigenesis, processes where cell proliferation occurs, but when poorly controlled the generation of ROS is dangerous. Indeed, NADPH oxidase-mediated cell proliferation has been observed in a wide range of cell types including those found in blood vessels, kidney, liver, skeletal muscle precursors, neonatal cardiac myocytes, lung epithelial cells, gastric mucosa, brain microglia, and a variety of cancer cells. For example, NOX may stimulate Akt activation also by inactivating the phosphatase PTEN, a direct negative regulator of the PI3K/Akt pathway [
At first, cellular stresses may induce NOX-dependent ROS generation as an alert system that drives the cells into a relatively stress-resistant status by integrating and amplifying the stress signals, as preconditioning against further cellular challenges [
NADPH oxidase has received much attention as a major cause of oxidative stress leading to vascular disease. Moreover different NOX subunits have been suggested to play a role in cancer, lung fibrosis, stroke, heart failure, diabetes, and neurodegenerative diseases [
NADPH oxidases are ubiquitously expressed in tissues but are the major source of superoxide anions observed in the vasculature [
With respect to low and spatially confined ROS overproduction, NOX1 is a good candidate to migrate into caveolae and there causes eNOS uncoupling and endothelial dysfunction, which is often associated with increased blood pressure and enhanced platelet aggregation as an early step in the development of atherosclerosis [
NOX1, NOX2, and NOX4 are known to be expressed in multiple tumor cell types (see Table
Recently, the role of cancer stem cells (CSCs) in cancer progression and metastasis has attracted much attention since CSCs are integral parts of pathophysiologic mechanisms of metastasis and chemo/radioresistance [
Moreover, ROS have also been shown to regulate angiogenesis through the release and actions of tumor-derived growth factors that induce endothelial cell proliferation. In fact, ROS production within tumor cells dramatically promotes the release of paracrine growth factors such as VEGF and the expression of its receptor, VEGF receptor-1 which, in turn, stimulate proliferation, migration, and tube formation in nearby endothelial cells [
The oxidative burst of phagocytes has long been considered proinflammatory, causing cell and tissue destruction. Recent findings have challenged this inflammatory role of ROS, and now ROS are also known to regulate immune responses and cell proliferation and to determine T-cell autoreactivity. NOX2-derived ROS have been shown to suppress antigen-dependent T-cell reactivity and remarkably to reduce the severity of experimental arthritis in both rats and mice [
In retrospect, this is also suggested by the pathology of chronic granulomatous disease (a condition characterized by inborn defects in the phagocyte
Finally, NADPH oxidase-derived ROS are also crucial players of tumor anti-immunity regulating specialized subsets of immune cells such as macrophages and T lymphocytes. Thus, NOXs could represent a novel molecular link between chronic inflammation and angiogenesis during cancer [
NOX2 is connected to the innate immune response [
ROS are generally thought to play an important role in the pathophysiology of organ failure [
With respect to high levels of ROS produced by NOX4, they can be directly cytotoxic or cause apoptosis inducing heart ischemic stroke. On the other hand, regarding the NOX4 role on pressure overload of the heart, NOX4 might be responsible of both acute damage of the cardiomyocyte and subacute protection of the heart by promoting angiogenesis [
In a gerbil model of global cerebral ischemia-reperfusion injury, NOX inhibition by apocynin strongly diminishes damage to the hippocampus [
NOX2 seems to have a role in inflammatory neurodegeneration diseases, including Alzheimer’s disease and Parkinson’s disease [
Microglia activation is also thought to be a key element in the development of dementia [
Microglial NOX2-derived ROS have also been implicated in the progression of the demyelinating disease through phagocytosis of myelin and damage to the myelin sheath [
Classically, oxidative stress has been defined as an imbalance between the endogenous production of reactive oxygen compounds and the antioxidative potential of cells [
But the low or apparent lack of clinical effectiveness of ROS-scavenging approaches is not entirely explained. It can be due to the partial removal of selected harmful endproducts by ROS-scavengers. Furthermore, antioxidants, including vitamins, reaction with superoxide anions is slower than NO. Moreover, it does not take into account that cellular events leading to disease primarily occur in individual cellular compartments [
NOX inhibitors.
Name and origin | Mechanism of action | NOX isoform selectivity | Other pharmacological effects | References |
---|---|---|---|---|
AEBSF |
Inhibits p47phox assembly with oxidase subunit | NOX2 | Proteases inhibitor | [ |
Apocynin |
Inhibits p47phox assembly with membrane | NOX2 | H2O2 scavenging | [ |
Berberine |
Inhibition of gp91phox expression | NOX2 | Enhancement of SOD activity | [ |
Blueberry derived polyphenols | Disrupts NOX assembly in lipid rafts | NOX2 | Minimal if any ROS scavenging capacity | [ |
Celastrol |
Inhibition of association between cytosolic subunits and the membrane subunit | Mostly Nox1 and NOX2 | None reported | [ |
DPI | Flavoprotein inhibitor | No selectivity | Inhibits NOS, xanthine oxidase, NADH ubiquinone oxidoreductase, NADH dehydrogenase, cytocrome P450 oxidoreducatese | [ |
EGCG |
Inhibits the expression of NADPH oxidase subunits | No selectivity | ROS scavenging capacity and ENOX proteins function as terminal oxidases of plasma membrane electron transport (PMET) | [ |
Emodin |
NADPH oxidase p47phox activation | NOX2? | Interfere with electron transport process and in altering cellular redox status | [ |
Ginko biloba | Inhibition of Rac1- and p47phox-mediated NADPH oxidase activation | NOX2? | Increases the expression of Cu-Zn superoxide dismutase heat shock protein 70 | [ |
HDMPPA |
Downregulates expression of p47phox and Rac1 | NOX2? | Preservation of NO bioavailability | [ |
Magnolol and honokiol |
Inhibit ERK pathway | unknown | Inhibit NO production | [ |
Plumbagin | Unknown | Nox4 | ROS scavenger | [ |
Prodigiosin |
Inhibits the binding of p47phox and Rac to the membrane components | NOX2? | Reduces gp91(phox) and iNOS expression | [ |
Resveratrol |
Decreases NADPH oxidase expression (p47phox) | NOX2? | Free-radicals-scavenging | [ |
S17834 | Unknown | NOX2 NOX4 | None reported | [ |
Sinomenine |
Inhibits p47phox translocation to the cell membrane | NOX2? | Minimal interaction with opiate receptors | [ |
The main sources of ROS include redox enzymes such as the respiratory chain, xanthine oxidase, lipooxygenase, cyclooxygenase, and NADPH oxidases, and these systems are continuously interacting with each other. Due to the complex mechanisms involved in the activation of NADPH oxidases, these enzymes can be targeted on several different levels of their activity. Firstly, decreasing NADPH oxidase expression can inhibit them. Also, the activation of NADPH oxidase can be decreased by blocking the translocation of its cytosolic subunits, when present, to the membrane.
Another possibility is inhibition of the p47phox subunit, either by preventing its phosphorylation using PKC inhibitors or by blocking its binding to other subunits. A decrease of signal transduction and inhibition of Rac 1 translocation has also been demonstrated to decrease ROS generation [
Several compounds have been used, including apocynin, diphenylene iodonium (DPI), and 4-(2-aminoethyl)-benzensulfonylfluorid (AEBSF). However, it has become apparent that these inhibitors are not specific for NOX [
One of the first inhibitors used in model studies was diphenyliodonium (DPI), which is very potent (although in micromolar range) but lacks specificity. DPI is a general flavoprotein inhibitor, also inhibiting, for example, xanthine oxidase and eNOS [
AEBSF is primarily a serine protease inhibitor [
Later studies involved apocynin, a naturally occurring NADPH oxidase inhibitor originally isolated from the roots of Picrorhiza kurroa. Apocynin cannot be used as selective NADPH oxidase inhibitor due to its direct antioxidant and several off-target effects [
The use of natural antioxidants represents a promising new approach for NOX inhibition. Polyphenols represent more than 10000 compounds occurring naturally in foods and the recommendation for a polyphenol rich (green tea/red wine/fruits/vegetables/whole grain foods) diet in the prevention of cardiovascular disease is still valid [
Recent studies with berberine, a plant alkaloid [
The inhibitory effect of flavonoids (kaempferol, morin, quercetin, and fisetin) on the respiratory burst of neutrophils was observed by Pagonis et al. [
A Ginkgo biloba extract containing flavonoids, among other compounds, was tested by Pincemail et al. [
A higher number of hydroxyl substituents are an important structural feature of flavonoids in respect to their scavenging activity against ROS, while C-2,3 double bond (present in quercetin and resveratrol) might be important for the inhibition of ROS production by phagocytes [
The bark of magnolia has been used in oriental medicine to treat a variety of remedies, including some neurological disorders [
Incubation of human neuroblastoma cells with nonpolar blueberry fractions obstructed the coalescing of lipid rafts into large domains disrupting NOX assembly therein and abolishing ROS production [
Prodigiosin, a microbial pigment, and some derivatives suppressed NOX activity most likely by disrupting Rac function [
In an oxygen-glucose deprivation and reoxygenation (OGD/R) model, pretreatment with green tea polyphenols (GTPP) and their active ingredient, epigallocatechin-3-gallate (EGCG), protects PC12 cells from subsequent OGD/R-induced cell death [
Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG-) induced oxidative stress in endothelial cells. It has been reported that HG induces endothelial cell apoptosis through NF-
Other NOX inhibitors are VAS 2870, VAS 3947, GK-136901, plumbagin, and polyphenolic derivative S17834 [
Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and antiatherosclerosis effects in animals. Plumbagin inhibits NADPH-dependent superoxide production in cell lines that express NOX4 oxidase [
Indeed, plumbagin has been reported to exert anticancer activity on osteosarcoma cells by inducing proapoptotic signaling and modulating the intracellular ROS that causes induction of apoptosis [
Furthermore, PI5K-1B plays a crucial role in ROS generation and could be a new molecular target of plumbagin [
Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions and shows benefits in models of neurodegenerative disease, cancer, and arthritis, although its mechanism of action is incompletely understood. Authors demonstrated that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely, inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome [
Accumulating evidence clearly indicates that NADPH oxidases are critical molecular targets for dietary bioactive agents for prevention and therapy of different pathologies.
The development of specific and not toxic inhibitors of NADPH oxidases and their redox signaling network (kinase, transcription factors, and genes) could provide useful therapeutic strategies for the treatment of oxidative stress dependent processes such as cancer and other degenerative diseases.
In fact, classical antioxidant therapies have been demonstrated inadequate since the importance of ROS in physiology has been ignored leading to the lack of clinical benefits. Indeed, further research into selective molecular inhibitors interfering with NADPH oxidase activation are warranted. The selective targeting of dysfunctional NADPH oxidase homologs appears to be the most suitable approach, with the potential to be far more efficient than the one with nonselective antioxidants having only ROS scavenging properties.
NOX enzymes, however, are very complex with numerous specific targets within each isoform. More information is needed on how these proteins are targeted to different subcellular compartments and how this transport process is regulated.
It is encouraging, however, that single bioactive dietary agents can directly and indirectly influence most, if not all, of the myriad targets within NOX family. Additionally, many of these dietary agents appear to exhibit some degree of specificity for redox deregulated cells while unaffecting normal cells balance. Moreover, the protective effects of some single agents could be potentiated and/or synergized by other dietary agents. While encouraging, there are many considerations that remain, such as the issue of appropriate dose of each agent, appropriate timing and duration of exposure, importance of cell type specificity, relative bioavailability of each agent, and potentially adverse side effects and interactions.
The author does not have a direct financial relationship with the commercial identities mentioned in the paper that might lead to a conflict of interests.
This work was supported by Grants from MIUR PRIN, 2009, Project, 200938XJLA_002.