NADPH oxidase and reactive oxygen species as signaling molecules in carcinogenesis

Abstract

Reactive oxygen species (ROS) are small molecule metabolites of oxygen that are prone to participate in redox reactions via their high reactivity. Intracellular ROS could be generated in reduced nicotinamide-adenine dinucleotidephosphate (NADPH) oxidase-dependent and/or NADPH oxidase-independent manners. Physiologically, ROS are involved in many signaling cascades that contribute to normal processes. One classical example is that ROS derived from the NADPH oxidase and released in neurotrophils are able to digest invading bacteria. Excessive ROS, however, contribute to pathogenesis of various human diseases including cancer, aging, dimentia and hypertension. As signaling messengers, ROS are able to oxidize many targets such as DNA, proteins and lipids, which may be linked with tumor growth, invasion or metastasis. The present review summarizes recent advances in our comprehensive understanding of ROS-linked signaling pathways in regulation of tumor growth, invasion and metastasis, and focuses on the role of the NADPH oxidase-derived ROS in cancer pathogenesis.

Keywords

free radicals / tumor / phox / cell proliferation / cancer therapy

Chemistry of major reactive oxygen species

Reactive oxygen species (ROS), small molecule metabilites of oxygen, are prone to participate in redox reactions via their high reactivity [1, 2]. Redox reactions are divided into two chemical processes: oxidation and reduction. Oxidation is a chemical process with the gain of oxygen and loss of hydrogen or electrons, whereas reduction is a chemical process with the loss of oxygen and gain of hydrogen or electrons. ROS are composed of free radicals and non-radicals, both inorganic and organic. Free radicals are a cluster of atoms that contain an unpaired electron in its outermost shell of electrons, with an extremely unstable configuration so that free radicals quickly react with other molecules or radicals to achieve the stable configuration of four pairs of electrons in their outermost shell (one pair for hydrogen). Typical free radicals include superoxide anion ({\mathrm{O}}_{\mathrm{2}}^{·-}), hydroxyl (HO^-), alkoxyl (RO^·) and peroxyl (\mathrm{R}{\mathrm{O}}_{\mathrm{2}}^{·}). Non-radicals do not contain an unpaired electron but are prone to exchanging electrons with other molecules. Typical non-radicals include hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), ozone (O₃), hypochlorous acid (HOCl), peroxynitrite (ONOO^-) and lipid peroxides (LOOH) [3].

(1) {\mathrm{O}}_{\mathrm{2}}^{·-}, a weak oxidant radical, is considered as the “primary” ROS. {\mathrm{O}}_{\mathrm{2}}^{·-} is produced by electron transfers in the mitochondrial electron transfer chain. Other enzymes capable of producing superoxide are the NADPH oxidase, the xanthine oxidase and cytochrome P450. The lifespan of {\mathrm{O}}_{\mathrm{2}}^{·-} is only milliseconds.

(2) H₂O₂ is more potent without an unpaired electron and has a longer lifespan (minutes) than {\mathrm{O}}_{\mathrm{2}}^{·-}. H₂O₂ is produced from {\mathrm{O}}_{\mathrm{2}}^{·-} by superoxide dismutase (SOD). H₂O₂ is also produced by a wide variety of enzymes including monoxygenases and oxidases. H₂O₂ is diffusible and may serve as a redox-signaling molecule between cells. The fate of H₂O₂ includes (a) being metabolized by catalase or glutathione peroxidase to H₂O, (b) being permeable through the plasma membrane to the extracellular side, (c) reacting with the reduced transition metals (i.e., Fe³⁺) to become the highly toxic hydroxyl radical (HO^-) or a metal peroxide complex (Me-OH) (i.e., Fenny reaction) (Fig. 1).

(3) Hypochlorite (HOCl) and hypobromite (HOBr) are produced in lysosome. Through complex redox reactions catalyzed by myeloperoxidase (MPO) in the presence of chloride (Cl^-) or bromide ion, HOCl or HOBr is generated by degrading H₂O₂ to O₂ and H₂O. These ions play an important role in carcinogenesis and allergic reactions.

(4) Peroxynitrite anion (ONOO^-), a short-life but highly oxidizing radical, is a product of reaction between {\mathrm{O}}_{\mathrm{2}}^{·-} and nitric oxide (NO) and plays a role in the nitrosylation of proteins.

Generation and scavenger of ROS

Endogenous generation

Sources of endogenous generation of ROS are shown in Figure 1 and can be divided into the following(a) Mitochondria produce a large amount of {\mathrm{O}}_{\mathrm{2}}^{·-} and H₂O₂, especially during hypoxia or tissue ischemia. The source of mitochondrial ROS appears to be involved in a non-heme iron protein that transfers electrons to oxygen. This occurs primarily at the complex I and, to a lesser extent, following the auto-oxidation of coenzyme Q from the complex II or III. {\mathrm{O}}_{\mathrm{2}}^{·-} is produced deleteriously by one-electron transfers in the mitochondrial electron transfer chain. (b) The NADPH oxidase (NOX) serves as another major source of ROS [1,2,4]. NOX enzymes catalyse the NADPH-dependent reduction of oxygen via one-electron transfer from NADPH to oxygen, to form superoxide which can react with itself to form H₂O₂. The NADPH oxidase generates ROS in a regulated manner, producing {\mathrm{O}}_{\mathrm{2}}^{·-} in response to growth factors, transduction signaling, hypoxia and cytokines (Fig. 2). (c) The xanthine oxidase is a cytoplasmic oxidase that produces {\mathrm{O}}_{\mathrm{2}}^{·-} and H₂O₂ when converting hypoxanthine and xanthine into uric acid. This reaction may serve as an important source of ROS in a variety of pathophysiological states including hypertension, atherosclerosis and heart failure. (d) Other sources of ROS may include cyclooxygenases (COX1/COX2) and nitric oxide synthases (NOS), cytochrome P450 metabolism and peroxisomes also contribute to a certain amount of ROS generation (Fig. 1).

Exogenous insults

Enviromental insults leading to oxidative stress include radiation, ozone, herbicides, xenobiotics (i.e. polycyclic aromatic hydrocarbons and phorbol esters) and ferritin, all of which are capable of inducing steady-state increases in ROS production.

Anti-oxidant systems

Cells are normally able to defend themselves against ROS damage through their scavenger systems listed as below. (a) Enzymes such as SOD, catalases and glutathione peroxidase (GPX) with glutathione (GSH) (Fig. 1). Since glutathione plays a significant role as an antioxidant, clinically there is a significant difference between cancer incidence and controls in the GSH-related enzyme function and the ratio of the reduced form of glutathione (GSH) over the oxidized form of glutathione (glutathione disulfide, GSSH) (Fig. 1). (b) Transition metal chelators. (c) Polyphenol anti-oxidants such as small molecule tocopherol (Vitamin E). (d) Ascorbic acid (vitamin-C) and uric acid also play significant roles in cellular antioxidant systems. The most important plasma (extracellular) antioxidant in humans is probably uric acid.

NADPH oxidase

The NADPH oxidase was originally identified as a key component of the innate human host defense system. The primary function of the phagocyte NOX (phox) is the production of {\mathrm{O}}_{\mathrm{2}}^{·-} and its secondary metabolites such as H₂O₂ for use in defense against invading micro-organisms. The essential importance of the phagocyte NADPH oxidase to host immunity is clearly demonstrated by a genetic disease known as chronic granulomatous disease (CGD). CGD is caused by defects in gp91^phox, the catalytic subunit of the phagocyte NADPH oxidase or other subunits [4]. As shown in Figure 2, two essential membrane-associated subunits, gp91^phox and p22^phox, form a non-covalent heterodimer, which is now known as flavocytochrome b₅₅₈ because of the association of a FAD moiety with gp91^phox. The catalytic subunit gp91^phox, also termed as NOX2, has several homologues, NOX1 and NOX3 through 5, the locations of which are tissue- or cell-type specific (Table 1) [1]. In phagocytes, p22^phox and NOX2 are essential subunits of flavocytochrome b₅₅₈ and the absence of either protein results in a non-functional NADPH oxidase. However, it is evident that p22^phox is also expressed in a variety of non-phagocytic cells and functionally associated with other gp91^phox homologues such as NOX1, NOX3 and NOX4. In phagocytes, cytoplasmic subunits, p47^phox, p40^phox, p67^phox and its regulatory factors GTPase Rac1, translocate to the phagosome or plasma membrane and associate with flavocytochrome b₅₅₈ during gp91^phox activation [1,4,5]. Activation of the classical NADPH oxidase involves assembly of cytosolic and integral membrane subunit proteins to form a multi-subunit enzyme complex (Fig. 2).

NADPH oxidase-derived ROS and carcinogenesis

Normally, the membrane-anchored NADPH oxidase is responsible for generation of the intracellular ROS triggered by receptor binding of numerous growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin, vascular endothelial growth factor (VEGF) and angiotensin II (AngII), which result in cell proliferation and differentiation [1,3,4]. Therefore, NADPH oxidase-derived ROS form as a natural byproduct of the normal metabolism of oxygen and play important roles in the normal cellular signaling, which includes delivery of electrons across membranes, oxidative modification of proteins and DNA. Under environmental stress or certain pathological conditions, intracellular ROS levels can increase dramatically, leading to formation of oxidative stress (Fig. 3). Oxidative stress exerts significant harmful effects on cell structures by inducing structural changes in lipids, membranes, proteins or nucleic acids (Fig. 1). Oxidative stress also induces significant changes in signal transductions. When in excess, the effect of intracellular ROS on cell proliferation is a double-edge sword. On one hand, excessive production of NADPH oxidase-derived ROS is able to induce apoptosis. On the other hand, accumulated NADPH oxidase-derived ROS trigger mitogenic growth. Recent evidence strongly indicates that there is a correlation between the NADPH oxidase, NOX-derived ROS and mitogenic growth of cancers [6-11]. For instance, in colon tumor-derived cells that express NOX1 NADPH oxidase, ROS are highly produced in subconfluent growing cells [1]. Overexpression of NOX5 NADPH oxidase and increased ROS generation are also seen in highly tumorigenic human prostate cancer cells [12]. Melanoma cell proliferation requires expression of an NADPH oxidase and general inhibition of NADPH oxidases except NOX4 diminishes ROS production [13]. Elevated levels of endogenous ROS are required for inducing tumor growth and angiogenesis [10,11].

At cellular and molecular levels, the insults resulting from accumulated intracellular ROS levels derived from the NADPH oxidase may include the following.

(1) Oxidative nuclear and mitochondrial DNA damage: ROS have been tied to genomic instability through chemically induced changes in DNA, changes in nucleic acid structure and conformation, and inhibition of repair enzymes [14,15]. The hydroxyl radical is known to react with all components of the DNA molecules in the nucleus and mitochondria, damaging both the purine and pyrimidine bases. The hydroxyl radical inserts to the DNA double bonds of DNA bases as well as the deoxyribose backbone. Permanent modification of the genetic material resulting from these “oxidative damage” incidents represents the first step in mutagenesis, carcinogenesis and aging. DNA damage may result in the arrest of transcription, replication errors and genomic instability, all of which could be accumulated, leading to carcinogenesis (Fig. 3).

(2) Lipid peroxidation by hydroxy radical: The metal-induced generation of oxygen radicals also results in the attack of the bilayer membrane lipid components including polyunsaturated fatty acid residues of phospholipids. Hydroxyl radicals are generated via Fenton chemistry to initiate lipid peroxidation. As a consequence, lipid peroxidation leads to structural changes in membrane, formation of adducts or crosslinks with non-lipids and disruptions of membrane-dependent signaling [14]. This deleterious process of the peroxidation of lipids is apparent in cancer, inflammation and arteriosclerosis.

(3) Protein modification: Peroxynitrite anion (ONOO^-) is able to nitrosate the cysteine sulphydryl groups of proteins, to nitrate tyrosine and trytophan residues of proteins and to oxidize methionine residues to methionine sulphoxide. As a consequence, these protein thiol oxidations result in oxidation of the catalytic sites of proteins, formation of sulfide bonds and increased susceptibility to proteolysis.

(4) Signal transduction: Oxidative stress plays an important role in the regulation of cell growth because the cell cycle is regulated by intracellular concentrations of GSH. Inflammation-linked ROS can activate the cell growth transcription factors including MAP-kinase/AP-1, NF-kB and p53 pathways that have a direct effect on cell proliferation and apoptosis [16-18]. ROS also regulate protein kinase or tyrosine kinase activities.

NADPH oxidase-derived ROS and cancer progression/invasion

NADPH oxidase-derived ROS are shown to be linked to the remodeling of cellular skeletons, extravasation, angiogenesis and regulation of tumor metastasis genes, migration and invasion [10,11,19,20]. Activation of NADPH oxidase and generation of ROS are required for induction of the biglycan gene, which is involved in cell adhesion and migration. The Rac1-dependent NADPH oxidase is involved in VEGF- and AngII-mediated endothelial migration (Fig. 3). Recent data show that ablation of the NOX1 NADPH oxidase activity by NOX1 siRNAs or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) inhibits synthesis of both VEGF proteins and VEGF mRNAs in K-Ras transformed normal rat kidney cells. Moreover, tumors derived from NOX1 siRNA-transfected KNRK cells markedly decreased neovascularization. Thus, the NOX1 NADPH oxidase activity was required for VEGF production in human colon cancer CaCO-2 cells, as in the case of KNRK cells.

Clinical implications

Bacterial infections

Oxidative burst is characterized by massive production of ROS during bacterial infections and plays a key role in the defense against invading pathogens [1,4]. Phagocytosis of micro-organisms is the major impetus for activation of the NOX2-containing NADPH oxidase in neutrophils and macrophages, leading to a large production of {\mathrm{O}}_{\mathrm{2}}^{·-} and its secondary metabolites such as H₂O₂. CGD, a rare genetic disorder, is caused by defects in gp91^phox, p22^phox, p47^phox or p67^phox subunits and results in recurrent infections with the inactive NADPH oxidase.

Chronic infections and cancer

Although mild oxidative stress can induce apoptosis, chronic inflammation induced by biological, chemical and physical factors has been associated with increased ROS level and high risks of human cancer at various sites [18,21]. Epidemiological studies indicate that increased leucocyte count induced by chronic infections is a risk factor for cancer. In addition, ROS produced by human neutrophils can directly damage DNA. Chronic infection-induced inflammatory disorders such as gastritis and hepatitis are recognized as risk factors for gastric cancer and hepatocellular carcinoma, respectively.

NADPH oxidase-regulated signal transduction and cancer

Tissue damages induced by chronic infections, inflammation and other biological or chemical factors can trigger synthesis and release of pro-inflammatory cytokines such as TNF-α and IL-1 [4,10,11,18,20]. Since it is a stimulator of the NADPH oxidase, TNF-α triggers oxidative stress via the NF-κB pathway that promotes cancer cell growth and/or metastasis (Fig. 3). Examples include TNF-α polymorphism and non-Hodgkin lymphoma [22]. The activation of TNF-α receptor leads to activation of the pathway where transcription factors mitogen-activated protein (MAP)-kinase, activation protein 1 (AP-1), NF-kB and p53 are involved. As signaling messengers, ROS are able to oxidize the critical target molecules such as protein tyrosine phosphatases (PTPs) and serine/threonine kinases such as protein kinase C (PKC). Inactivation of multiple PTPs by ROS may relieve the tyrosine phosphorylation-dependent signaling, trigger non-receptor protein kinases directly and eventually initiate MAP-kinase, NF-kB and p21-activated kinase-1 (PAK) pathways. PAK is an effector of Rac-mediated cytoskeletal remodeling that is responsible for cell migration and angiogenesis [10,18]. All these ROS-regulated signal transductions are major pathways for driving tumor cell growth and metastasis. In addition, ROS also activate serine/threonine PKC, a member of the tumor growth factor-β (TGF-β) superfamily. MAP-kinase and AP-1 can be activated by PKC and TGF-β1, respectively, in a ROS-dependent manner, resulting in cancer proliferation and metastasis.

Oxidative DNA lesions and cancer

In cancer cells when ROS are overproduced or expression of antioxidants is altered, an imbalance between pro-oxidants and anti-oxidant prevails. As a result, ROS induce the formation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) that is the best example for the oxidative stress-induced damage to nucleobases and sugar moieties of DNA and RNA. This damage can cause single- or double-strand breaks, point and frameshift mutations and chromosome abnormalities, which may be linked to the initiation of cancer. This lesion is clinically important because it is a potential biomarker of carcinogenesis. In addition, the reaction of HOCl with various nucleosides yields chlorinated nucleosides, including 8-chloro-2’deoxyqunosine, 8-chloro-2’deoxyadenosine and 5-chloro-2’deoxycytidine. DNA oxidation also triggers DNA repair, imbalances DNA repair enzymes or induces error prone polymerases [14].

Prevention of cancer by inhibiting ROS production

The mechanisms can be categorized into the following: (a) Chemoprevention of inflammatory processes. It includes inhibition of release of TNF-α or block of its signaling pathways such as the NF-κB pathway [21]. (b) Acceleration of superoxide metabolism. In vitro experimental models targeting or sustaining delivery of catalase that catalyzes H₂O₂ into H₂O (Fig. 1) may inhibit metastatistic tumor growth [21]. (c) Glutathione status can be regarded as the redox status for many diseases. Clinically, there is a significant difference between cancer incidence and controls in the GSH-related enzyme function and the ratio of the reduced form of glutathione (GSH) over the oxidized form of glutathione (glutathione disulfide, GSSH) [23]. The ratios of GSH/GSSG in blood samples from patients suffering from virus-originated hepatocellular carcinoma were significantly lower, indicating that the antioxidant system is severely impaired in the patients [24]. Other studies suggest that resveratrol, vitamin E and vitamin C provide cytoprotection by maintaining the ratio of GSH/GSSG at a high level [25]. (d) Dietary chemoprevention might provide some substances that have anti-tumor effects by scavenging ROS. For example, tea polyphenols, especially catechins, have been reported to be potent antioxidants and are beneficial in oxidative stress-related diseases including cancer [26]. The anti-tumor effect of lycopene, which is rich in tomato, is linked to its ROS scavenging [27].

NADPH oxidase-derived ROS and other clinical diseases

The NOX-derived oxidative stress also plays an important role in the pathogenesis of many other human disorders [1,2,4,21]. Oxidative stress plays a critical role in various cardiovascular diseases. The major sources of oxidative stress for these diseases include NOX, xanthine oxidase and nitric oxide synthase. The NOX-derived oxidative stress has been linked with several major cardiovascualr diseases such as hypertension, atherosclerosis, ischemic heart attack, stroke and congestive heart failure [28]. In addition, the pathogenesis of rheumatoid arthritis is associated with the NOX-generated free radical accumulation at the site of inflammation. Moreover, diabetes mellitus is characterized with glucose-induced ROS production mainly from mitochondria and NOX [28]. The brain is particularly sensitive to ROS because of its high oxygen utilization. AngII-induced neurogenic hypertension via the brain may be associated with the NOX2-generated ROS in central brainstem neurons [29]. It is also established that the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and aging are linked with ROS production triggered by microglial NOX. In the AD brain, for example, astrocytic NADPH oxidase-derived ROS play a potential role in fibrillar β-amyloid peptide-dependent neuronal death [30]. In the brain of PD models, ROS produced by microglial NOX also play an important role in the death of dopaminergic neurons by the formation of peroxynitrite with nitric oxide [31].

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