The Double-Edged Sword Profile of Redox Signaling: Oxidative

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The Double-Edged Sword Profile of Redox Signaling: Oxidative Events as Molecular Switches in the Balance Between Cell Physiology and Cancer Sonia Emanuele, Antonella D'Anneo, Giuseppe Calvaruso, Cesare Cernigliaro, Michela Giuliano, and Marianna Lauricella Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00311 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Chemical Research in Toxicology

The Double-Edged Sword Profile of Redox Signaling: Oxidative Events as Molecular Switches in the Balance Between Cell Physiology and Cancer. Sonia Emanuele1, Antonella D’Anneo2, Giuseppe Calvaruso2, Cesare Cernigliaro1, Michela Giuliano2*, Marianna Lauricella1* 1. Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, University of Palermo, via del vespro 129, 90127 Palermo, Italy. 2. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Laboratory of Biochemistry, University of Palermo, via del vespro 129, 90127 Palermo, Italy. KEYWORDS Reactive oxygen species; oxidative stress; redox signaling; protein sulfenylation, tumor cells

ABSTRACT: The intracellular redox state in the cell depends on the balance between the level of reactive oxygen species (ROS) and the activity of defensive systems including antioxidant enzymes. This balance is a dynamic process that can change in relation to many factors and/or stimuli induced within the cell. ROS production is derived from physiological metabolic events. For instance, mitochondria represent the major ROS sources during oxidative phosphorylation, but other systems, such as NADPH oxidase or specific enzymes in certain metabolisms, may account for ROS production as well. Whereas high levels of ROS perturb the cell environment, causing oxidative damage to biological macromolecules, low levels of ROS can exert a functional role in the cell, influencing the activity of specific enzymes or modulating some intracellular signaling cascades. Of particular interest appears to be the role of ROS in tumor systems not only because ROS are known to be tumorigenic but also because tumor cells are able to modify their redox state, regulating ROS production to sustain tumor growth and proliferation. Overall, the scope of this review was to critically discuss the most recent findings pertaining to ROS physiological roles as well as to highlight the controversial involvement of ROS in tumor systems.

1.

Introduction

Reactive oxygen species (ROS) are chemically reactive molecules derived from oxygen. Some ROS are radicals containing one or more unpaired electrons that confer high instability and reactivity. They tend to achieve stability by removing or acquiring electrons from other molecules that, in turn, become radicals, thus triggering a vicious circle that amplifies their dangerous effects.1 Under physiological conditions, cells are able to defend themselves from the presence of free radicals by employing efficient antioxidant defense systems consisting of both enzymes and non-enzymatic molecules.2 When ROS production overwhelms the scavenging capability of cells or when the antioxidant response is seriously compromised, a condition identified as oxidative stress occurs.1,2 The correlation between induction of oxidative stress and development

of different diseases, such as diabetes, cancer and neurodegenerative diseases, has been extensively studied and discussed.3–7 In recent years, a topic of great scientific interest has emerged regarding the redox balancing of normal as well as cancer cell systems. Although changes in the redox state have long been regarded as seriously affecting protein function, more recently, a widespread idea considers ROS-induced modifications as post-translational changes involved in cellular proteostasis, representing a silent threat to human health in some circumstances. Low levels of ROS can function in cells as signaling molecules. Interestingly, cells regulate the intracellular redox state to control such physiological pathways as survival, proliferation, cell death and cell migration.8 However, this mechanism can represent a double-edged sword for cells, because in cancer cells, a persistent pro-oxidant state can favor tumorigenesis and cancer progression.9

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This point of view is critically discussed in this review in light of new data linking oxidative events with protein oxidation. This represents a post-translational change to proteins that should be explored to better clarify the regulatory role of ROS in normal cells and to consider the possible involvement as a new molecular label for cancer development, thus offering a new and exciting challenge for cancer research. 2.

ROS and antioxidant systems: an overview

ROS can be both free radicals and non-radical species.1,2 The main reactive species of oxygen is the superoxide anion (O2.-), which is produced in cells. It has a very short half-life due to its high reactivity, particularly in the presence of metallic ions, iron-sulfur groups and cysteine residues of proteins. O2.- can also react with nitrogen monoxide (NO.), produced by nitric oxide synthase (NOS), forming a peroxynitrite anion (ONOO-), an agent with high oxidizing and nitrosylating potential.1 In addition, the hydroxyl radical (.OH) species can be produced from O2.- or H2O2 by chemical reactions. Figure 1 shows the main reactive oxygen and nitrogen species and the mechanisms underlying their generation in the cell environment.

ROS are normally produced in the course of cellular metabolism.1 The major endogenous sources of ROS are the mitochondria, the endoplasmic reticulum and the complex of NADPH oxidase (NOX) in cell membranes.10–12 In mitochondria, the main site of ROS production is the respiratory chain—in particular, complex I followed by complexes II and III.13 Under physiological conditions, only 1-2% of oxygen is converted to O2.- in the respiratory chain, but if changes occur in electron transport chain, this percentage can increase considerably. Beyond mitochondrial generation, superoxide anion can also be produced in other cellular sites, such as the endoplasmic reticulum, cell membrane and peroxisomes. In these sub-compartments, O2.can be directly produced by the activity of different enzymes such as NOX, which is found in different cellular locations,10,14 xanthine oxidase, localized in peroxisomes12 and members of the cytochrome P450 family, found in the endoplasmic reticulum.15 A remarkable

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unanswered question involves the relationship existing among the different subcellular compartments, in particular the mitochondria and peroxisomes, as signaling platforms for redox balance. Is the origin of ROS important in mediating selective signal responses? Do the mitochondrial and peroxisomal ROS represent separate pools with different roles in cells? Although some attempts have been made to explore this subject, the solution to this problem will be provided when new and more consolidated methodologies permit discrimination between the two intracellular ROS pools. Apart from the endogenous production of ROS, there are also multiple external factors that directly or indirectly increase ROS levels in cells: air pollutants, tobacco smoke, ionizing and nonionizing radiation, foods and drugs, environmental contaminants and carcinogens.16–19 For instance, many air pollutants are free radicals or have the ability to promote free radical reactions. Cigarette smoke contains many oxidants, free radicals and organic compounds, such as superoxide and nitric oxide. Moreover, exposure of cells to ionizing radiation induces radiolysis of water molecules into H+ and OH- radicals. These radicals are themselves chemically reactive, and in turn recombine themselves to generate O2.- and H2O2. When ROS are produced in cells in excess amounts, they produce harmful, destructive effects.1,20 The destructive action of ROS radicals affects all cell macromolecules: lipids, nucleic acids and proteins. Lipid oxidation generates lipid hydroxides, which are very unstable molecules that are readily converted into highly reactive aldehydes, such as 4-hydroxy-2,3-nonenal and malondialdehyde.2 Lipid peroxidation by ROS can result in the loss of polyunsaturated fatty acids, altering the fluidity and the permeability of cell membranes and thus favoring cell lysis. In addition, ROS can alter the interaction between membrane lipids and proteins by modifying the activity of certain enzymes and ionic transport systems.2 Moreover, ROS can directly interact with DNA, producing adducts with purine and pyrimidine bases or with the sugar-phosphate backbone and causing double helix breaks. This nitrogenous base oxidation is mutagenic, but it has been recently observed that some oxidized bases, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), can also affect gene expression.21 Finally, ROS can oxidize the amino acid residues of proteins, leading to the alteration of protein structure/function relationships. Protein carbonylation is a type of irreversible and unrepairable protein oxidation, characterized by the generation of carbonyl groups on side chains of specific amino acid residues such as Pro, Arg, Lys, and Thr.22,23 Usually, it refers to a process that forms reactive ketones or aldehydes on proteins, marking them for proteasomal proteolysis or favoring the production of macromolecular carbonylated aggregates which accumulate with age and can contribute to the development of age-related pathologies such as Parkinson's disease and Alzheimer's disease as well as cancer 24–26 In the regulation of the cellular redox balance, the antioxidantdefense cell systems play a fundamental role.1,27 Among relevant enzyme activities, superoxide dismutase (SOD) represents one of the most important scavenger systems. This enzyme is an oxidoreductase containing a metal ion (copper, zinc or manganese) and is responsible for the dismutation of O2.- into H2O2, a more stable species that, in contrast to O2.-, is able to cross cell membranes. ZnSOD is found in cytoplasm and in the mitochondrial intermem-


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brane space, while MnSOD has a predominantly mitochondrial localization. Catalase, an oxidoreductase containing four ferrous groups, converts H2O2 into water and oxygen. It is preferentially located in peroxisomes, which are organelles that also contain many enzymes that generate H2O2. A similar reaction is catalyzed by glutathione peroxidase, which reduces hydrogen peroxide to water in the presence of reduced glutathione (GSH). Non-enzymatic antioxidants include various low-molecularweight compounds such as reduced glutathione (GSH) and thioredoxin.28 The tripeptide GSH (γ-glutamyl cysteinyl glycine) is found in all eukaryotic cells, and it represents one of the key nonenzymatic antioxidants in the body that takes part in the homeostasis of protein sulfhydryl groups.29 In the presence of the mildly oxidative conditions of the intracellular compartment, the cysteine residues of proteins are oxidized to sulfenic ions (an event known as sulfenylation) that in turn form covalent adducts with glutathione. The thioredoxin system includes thioredoxin (Trx) and thioredoxin reductases (TrxR). Trx, a disulfide-containing redox protein, is involved in the modulation of the activity of redox-sensitive factors, as well as in the restoration of reduced cysteines, by removing the adducts of sulfenic acid conjugated with GSH. Oxidized Trx (Trx-S-S) is, in turn, reduced by TrxR and NADPH to its active dithiol form.28 Included among the preventive measures engaged by the cells as antioxidant systems is the activity of uncoupling proteins (UCPs), which, in contrast to enzymatic systems, are energetically inexpensive.30 This is a family of anion transport carriers of the mitochondrial inner membrane that favor the dissipation of proton gradient. While UCP1 is involved in regulating thermogenesis, other members exert different functions, including an antioxidant role. In particular, it has been shown that UCP2 acts as a mitochondrial oxidative stress sensor. An increase in superoxide activates UCP2, which induces dissipation of the mitochondrial proton gradient, thus lowering the proton-motive force and attenuating superoxide production by the electron transport chain.30 In addition, vitamins such as ascorbate (vitamin C), αtocopherol (vitamin E) and β-carotene (vitamin A) and polyphenols are other efficient antioxidant molecules. Tocopherol, the leading liposoluble antioxidant, prevents membrane lipid peroxidation by interrupting radical chain reactions that characterize this process.31 Vitamin C donates electrons and prevents the oxidation of many relevant species including various ROS, reactive nitrogen species (RNS), sulfur radicals, and nitrosylated compounds.32 Vitamin C also reduces heavy metal ions (Fe, Cu) that can generate free radicals via the Fenton reaction. Moreover, ascorbic acid has also the ability to regenerate vitamin E after it has neutralized free radicals. Finally, an increasing number of papers have demonstrated the antioxidant efficacy of polyphenols, a heterogeneous group of natural substances composed of several condensed phenolic cycles that can act as natural antioxidants.33 The main classes of polyphenols include phenolic acids, flavonoids, stilbenes and lignans. Polyphenols are largely found in fruits, vegetables and cereals, conferring health benefits to these foods.34,35 3.

Role of the intracellular redox state in the control of physiological processes.

Reversible changes in the redox state of several groups of proteins represent a biochemical mechanism of protein activity control. The oxidation of specific amino acid residues can reversibly modulate protein function, similar to phosphorylation and dephosphorylation, and can be included in the list of posttranslational protein modifications.36 The main change induced in proteins by ROS is the H2O2-mediated oxidation of cysteine residues. This process, known as S-sulfenylation, is a form of posttranslational modification of proteins in response to oxidant signals, and it is known to be responsible for changes in protein structure and/or function and the induction of biological responses.36,37 Many proteins contain sulfhydryl groups (Prot-SH) from cysteine residues. Prot-SH can take the form of thiols (-SH) or disulfides (PS-SP). The oxidation of the thiol form or the reduction of a disulfide group of an enzyme can activate or inactivate the catalytic function. ROS have been shown to modify the redox state of ProtSH.36 H2O2 oxidizes the cysteine thiol (Cys-SH) to form sulfenic acid (−SOH), causing allosteric changes that alter protein structure and/or function.36 This redox modification is rendered reversible by reducing systems such as Trx and GSH.29 The thiolate oxidation occurs in the cells at H2O2 concentrations in the nM range. At higher levels of ROS, sulfenic acid undergoes further oxidation to sulfinic (−SO2H) or sulfonic (−SO3H) acid. Unlike sulfenic modification, sulfinic or sulfonic moieties are irreversible modifications, resulting in permanent damage of the protein and are considered, in addition to protein carbonylation, markers of oxidative stress.36,38 Interestingly, the above-described biochemical mechanisms enacted by ROS, in relation to their concentration, can produce a reversible modification of the function of a protein or induce an irreversible inactivation. Although the number of proteins that are subjected to reversible changes in redox state are limited, they belong to strategic signaling transduction pathways. Recently, it has been demonstrated that the protein kinases and phosphatases involved in the regulation of many cellular processes may be direct targets of ROS. H2O2 has been shown to activate multiple protein kinases, such as receptor tyrosine kinases and serine/threonine kinases,39–41 whose activity depends, at least in part, on the redox state of several cysteine residues. Serine/threonine phosphatases (PTPs), such as PP1, PTP1B, PTEN, are inhibited by oxidation, which destabilizes the Fe-Zn center of their active site, and this may consequently amplify the effect of redox-linked activation of serine/threonine kinases.42 The large number of protein phosphatases that are targeted by ROS are dependent on the presence of a highly conserved “signature motif” in all classical PTPs, which contains an invariant cysteine essential for catalysis. The signature motif structure allows the catalytic cysteine to remain in the thiolate (S−) state at physiological pH, facilitating the attack on substrate phosphotyrosines but also rendering the amino acid highly susceptible to oxidation.42 Changes of the redox state of protein kinases and phosphatases seem to be the main mechanism through which low levels of ROS are able to stimulate survival, proliferation and angiogenesis in normal cells.8 An example of this new control step associated with the redox state is present in the cascade activated by growth factors, such as the epidermal growth factor (EGF) or platelet-derived growth fac-



tor (PDGF).43 Several studies have reported that the interaction of different ligands (PDGF, EGF, insulin, angiotensin II) with their receptors is associated with a rapid ROS production through the activation of NOX.44,45 In particular, RTKs activate the phosphatidylinositol 3-kinase (PI3K) pathway, resulting in the activation of the small GTPase Rac1, which, in turn, stimulates NOX activity.45 To this purpose, it is interesting to note that NOX isoforms can be classified into two different subclasses which differ in the types of ROS generated (superoxide or H2O2). In particular, since NOX4 and DUOX1-2 promote H2O2 production, these species could be more strictly related to protein oxidation.46 ROS produced by NOX are responsible for the oxidization of thiol groups at the catalytic site of protein phosphatases involved in receptor dephosphorylation, consequently maintaining their activation. H2O2 produced in response to EGF oxidizes the catalytic cysteine of protein-tyrosine phosphatase 1B (PTP1B) to a sulfenic moiety, causing its inactivation.47 Consequently, EGFR is maintained in a phosphorylated active form. Similarly, the oxidation of the PDGF receptor associated with the inhibition of SHP-2 phosphatase reinforces the MAP-kinase signaling following PDGF stimulation.48 Moreover, ROS have also been shown to directly oxidize RTKs. In fact, H2O2 can interact with a specific cysteine residue (Cys797) of EGFR to form sulfenic acid at the active site of the receptor, but in this case, the event results in an enhancement of the tyrosine kinase activity of EGFR.40 Another protein whose activity is controlled by reversible H2O2-dependent oxidation is phosphatase and tensin homolog (PTEN), an important lipid phosphatase of the phosphatidylinositol 3′ kinase (PI3K)/Akt pathway.49,50 PTEN is involved in cell migration, growth and survival. It has been demonstrated that the H2O2-dependent oxidation of the essential Cys(124) residue in the active site through the formation of a disulfide bond with Cys(71) compromises protein function and is associated with pathological conditions.47,49 Oxidative stress also affects the mitogen-activated protein kinase (MAPK) signaling pathways. H2O2 has been reported to activate the three major subgroups of MAPK: the extracellular signalregulated kinase (ERK), the c-Jun N-terminal kinase (JNK), and the p38 MAPKs.8 In this case, the effect seems to be correlated with the direct inhibition of MAPK phosphatases by ROS produced by NOX.8 Moreover, ROS can also modulate MAPK pathway in an indirect way. It has been demonstrated that apoptosis signalregulating kinase 1 (ASK1), a MAPK enzyme involved in the activation of JNK and p38 MAPK, forms an inhibited complex with Trx.51 Rising levels of ROS cause the dissociation of the complex ASK1/Trx and the subsequent activation of ASK1.51 The list of molecules controlled by the redox state of the cells also includes some transcription factors, such as NF-κB and Nrf2. Activation of such proteins by ROS can be direct, by cysteine oxidation, or indirect, as a result of the activation of signal transduction cascades. The relationship between ROS and NF-κB is very complex. Mild oxidative stress can lead to NF-κB activation, while more significant oxidative stress can inhibit the transcription factor activity.52 Furthermore, NF-κB protects cells from oxidative stress through the activation of antioxidant genes, such as MnSOD and ferritin heavy chain.52 In the cytoplasm, ROS favor the phosphory-

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lation of IκB, the NF-κB inhibitory subunit which, once phosphorylated, dissociates from NF-κB, favoring its nuclear translocation.53 Moreover, ROS may influence the DNA binding properties of NFκB proteins. Oxidation of the p50 subunit at Cys62 of its DNAbinding domain has been shown to prevent its DNA binding.54 On the other hand, the phosphorylation of RelA, another type of NFκB subunit that is influenced by ROS-dependent processes, leads to a higher NF-κB activation.55 ROS are also responsible for the activation of the nuclear erythroid factor 2 (NE-F2)-related factor 2 (Nrf2), a transcription factor involved in the transcription of antioxidant and detoxifying factors in response to oxidative stress.56 Nrf2 is normally sequestered in the cytoplasm in an inactive complex with Kelch-like ECH-associated protein 1 (Keap-1). ROS induce oxidation of critical cysteines of Keap-1, promoting its dissociation from Nrf2. This event allows Nrf2 to migrate into the nucleus and form transcriptionally active complexes with other proteins, such as Mafs (Musculo-aponeurotic fibrosarcoma). The result is an increase in the transcription of cytoprotective and antioxidant genes, such as SOD, catalase and heme-oxygenase (HO-1).56 In the above-reported examples, it is possible to observe that, different from the regulation of kinase/phosphatase activities, the ROS-mediated oxidation of strategic cysteines in transcription factors leads to conformational modifications that contribute to the aggregation/disaggregation of protein complexes.

4.

ROS and cancer

The development of a tumor is a multistep process in which the transformed cells gradually acquire more aggressive characteristics that allow them to survive in a hostile environment and to proliferate and invade new tissues. It has been largely demonstrated that ROS promote and sustain carcinogenesis.3,4 However, ROS play different roles in the various stages of carcinogenesis. While the production of high levels of ROS by carcinogens can favor the generation of oncogenic mutations, cancer cells are able to modulate the redox state to maintain levels of ROS, which can stimulate survival and proliferation. Oxidative damage to DNA induced by exogenous stimuli, such as UV radiation, tobacco smoke and pollutants, promotes oncogenic mutations that initiate tumorigenesis.57,58 The involvement of ROS in promoting tumor development is supported by the observation that the antioxidant N-acetylcysteine (NAC) reduced tumor growth in many cancer systems in vitro as well as in vivo.59,60 Interestingly, tumor cells possess higher intracellular ROS levels than do normal cells.61 This attribute has been associated with the increased metabolic activities of tumor systems, which are necessary to sustain cell proliferation and growth.62 Another possible mechanism involves the dysfunction of the mitochondrial transport chain as a consequence of mitochondrial DNA (mtDNA) mutations.63 Such a condition frequently occurs in many different human tumors, such as in colorectal, renal, lung, bladder, head and neck, gastric, thyroid, prostate and ovarian cancers, glioblastomas and hepatocellular carcinomas.63 Moreover, oncogenic mutations are also associated with an increased production of ROS, which in


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turn favors tumor progression by stimulating cell survival, proliferation, angiogenesis and migration.3 Oncogenes, such as c-Myc, cMet or Ras, have been shown to induce ROS production in tumor cells.64,65 For instance, the oncogenic mutation of K-Ras increases the level of superoxide by activating ac, the regulatory component of NOX.65 Altered expression of the different isoforms of NOX or their regulatory components has also been evidenced at both early and late stages of tumorigenesis.10,66 NOX activation in tumor cells can also be a consequence of the altered stimulation of growth factor receptors.49,67,68 Overexpression of EGFR in breast, ovarian, and colon cancer is linked to ROS production and increased proliferation.68 Oxidizing agents, such as cigarette smoke or UV rays, are reported to induce phosphorylation and activation of tyrosine protein kinases, such as those of EGFR, PDGFR and Src.69–71 In lung epithelial cells, hydrogen peroxide or cigarette smoke promotes aberrant EGFR phosphorylation, resulting in its lack of ubiquitination and degradation, an event that contributes to lung cancer development.70 Indeed, H2O2-dependent EGFR activation is a ligand-independent event that is not accompanied by “classical” receptor dimerization and contributes to stimulation of cell proliferation. Intriguingly, oxidation of Cys797 in the ATP-binding pocket of EGFR to a sulfenic acid is linked to enhanced kinase activity.69 Similar to its function in normal cells, the ROS generated from NOX activity can also sustain the activation of TKR signaling pathways and of transcription factors involved in carcinogenesis through inhibition of PTP activities.72 The oxidative inactivation of phosphatases (MAPK phosphatases and PTEN) causes changes in the phosphorylation of protein targets of the MAPK-ERK and Akt kinase pathways.49 In colon cancer cells, NOX1 overexpression inhibits serine/threonine phosphatase activity, preventing c-Raf dephosphorylation and stimulating cell proliferation.72 Activation of Akt by H2O2 has been observed in several cancers, such as breast and ovarian cancer, as a result of PTEN oxidation and inactivation or the oxidative stress-mediated activation of its upstream kinases.73,74 The increase in ROS production in tumor cells has also been shown to promote tumor invasion and angiogenesis. This effect seems to be related to the ability of ROS to induce expression of metalloproteases (MMPs), enzymes known for their role in the digestion of extracellular matrix and in tumor invasion.62,75 ROS can also activate MMPs through oxidation of the thiol groups in their catalytic domain.76 Finally, several lines of evidence suggest that ROS may also promote angiogenesis by increasing the expression of VEGF through the activation of HIF-1α or NF-κB.77 The involvement of ROS in the different steps of carcinogenesis is described in Figure 2. The higher level of ROS in tumor cells than in normal cells maintains the oncogenic phenotype and drives tumor progression. However, at the same time, this renders tumor cells more vulnerable to the detrimental effects of ROS.9 To prevent reaching the threshold of ROS toxicity, tumor cells counterbalance the high rate of ROS production with an equally high level of antioxidant activities. Many cancer cells switch their metabolism by potentiating the pentose phosphate pathway to in-

crease the production of NADPH, the glutathione reductase cofactor.78

Moreover, some cancer cells upregulate their ability to counteract the deleterious action of oxidative stress by augmenting the expression of enzymes responsible for degrading ROS. The overexpression of MnSOD, Trx and HO-1 was observed in several cancers such as lymphoma, breast, lung, and liver cancer and melanoma, indicating that this might be an important mechanism for tumor survival.77–80 Interestingly, it has been shown that HO-1, beyond its canonical antioxidant role, can also promote carcinogenesis. In fact, independent from its enzymatic activity, a truncated form of HO-1 can translocate into the nucleus and regulate the activity of transcription factors (such as Nrf2) involved in cancer cell proliferation and invasion.81 Nrf2 is a transcription factor that favors cell survival and is the major mechanism by which cancer cells increase their antioxidant proteins. 82 In healthy cells, Nrf2 protects against tumorigenesis by attenuating the effects of ROS induced by genotoxic compounds.82 However, activation of the Nrf2 defense response in tumor cells can promote the survival of cancer cells by creating an optimal environment for cell growth. Mutations in the Nrf2-inhibitory protein Keap1 are present in several types of tumor cells, resulting in the activation of Nrf2.83 On the other hand, the loss of Nrf2 in cancer cells increases oxidative stress, resulting in diminished tumorigenesis.84 The overexpression of antioxidant systems is an adaptive process mainly linked to the progression of human cancers. In line with this observation, Harris et al85 showed that mice lacking the glutamate cysteine ligase modifier (GCLM) subunit, a component of the enzymatic activity responsible for GSH synthesis, generated tumors that progressed more slowly than those produced in mice with normal GSH levels. Interestingly, UCP2 is repressed during the first stage of tumorigenesis, while its overexpression frequently



occurs during tumor progression as well as in metastatic colon and breast cancers.86

5.

Pro-oxidant or antioxidant therapies: can they be putative tailored weapons to target tumors?

A careful analysis of the recent scientific literature concerning the role of ROS in tumor development reveals that cellular ROS levels represent a double-edged sword in the promotion and prevention of cancers.4,9,87 In many tumor systems, a persistent prooxidant state maintains the oncogenic phenotype and stimulates tumor progression. However, redox adaption through upregulation of antioxidant molecules allows cancer cells to prevent the toxic effects of ROS. Therefore, a question that frequently arises in scientific environments concerns the most effective therapeutic approach to be used for cancer treatment. But in this context, a better question is probably, “Can pro-oxidant or antioxidant drugs present better strategies to fight tumors than those of conventional drugs?” Thus, what is the most effective therapeutic approach to be used in the treatment of cancer—pro-oxidant or antioxidant therapies? Considering that cancer cells produce higher levels of ROS than those of normal cells61, the use of compounds that further increase ROS production to toxic levels may represent a therapeutic strategy to preferentially kill cancer cells. Several anticancer drugs are currently used for cancer treatment based on their ability to activate ROS-induced cell death pathways by either increasing ROS production or inhibiting antioxidant enzymes. Arsenic trioxide (ATO), anthracyclines and cisplatin are among the most commonly used ROS-inducer compounds in cancer therapy.88 ATO was among the first compounds used in the treatment of leukemia. The remarkable effect of ATO in inhibiting acute promyelocytic leukemia cells has been related to the ability of the compound to decrease the mitochondrial membrane potential and induce ROSdependent apoptosis.89 Additionally, cisplatin, a platinum-derived compound that is widely used for the treatment of solid tumors including in ovarian, testicular, and bladder cancer90, is closely related to increased mitochondrial-dependent ROS generation.91 Similarly, anthracyclines, a class of antibiotics that includes doxorubicin, daunomycin and mitomycin C, which are widely used in chemotherapy, are able to undergo oxidation with concomitant production of ROS.92 Interestingly, several natural compounds have been shown to exert anticancer effects by activation of ROS-induced cell death pathways. Along this line of evidence, we have previously demonstrated that parthenolide, a sesquiterpene lactone, increases the ROS level in breast cancer cells by activating NOX.93 Another approach to increase ROS levels in tumor cells is the use of inhibitors of antioxidant enzymes. Some of these compounds have been synthesized and have shown anticancer effects in vitro as well as in vivo. 2-Methoxyestradiol is an SOD inhibitor that has been shown to increase superoxide anion levels and induce apoptosis in leukemia cells.94 Inhibition of glutathione peroxidase with mercaptosuccinic acid or of catalase with aminotriazole has also been reported to kill cancer cells by inducing ROS.89

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Even though ROS-increasing compounds are extensively used in cancer therapy, a limit of pro-oxidant therapies is the drug resistance induced in some cancer cell lines. Chemoresistance is mainly related to the excessive intracellular antioxidant capability of some tumors. High levels of glutathione or redox enzymes in tumor cells have been shown to decrease the efficacy of ROS-inducing agents.95,96 Furthermore, several chemotherapeutic agents that induce oxidative stress also increase the level and activity of protective Nrf2 transcription factors, thus inducing the expression of antioxidant factors which counteract the cytotoxic effects of the drugs.97 Therefore, the use of ROS-inducer drugs in combination with antioxidant inhibitors might be promising in cancer treatment. In line with this hypothesis, it has been shown that 2methoxyestradiol increases the cytotoxic effect of ATO in human leukemia cells.98 Still, controversy remains about the possible use of antioxidants in cancer therapy. Antioxidants are often used as dietary supplements in the belief that they may help to prevent the development of cancer. The literature also widely refers to the possibility of potentiating cancer therapy by enhancing ROS scavenging using different antioxidants.99 A substantial body of data, both in vitro and in vivo, reveals the efficacy of antioxidants (either alone or in combination) for cancer treatment.100–103 However, regarding the potential use of antioxidants in cancer therapy, it must be considered that some of them are unable to target cancers specifically or have poor absorption and rapid metabolic transformation. Therefore, it is fundamental to clinically test the effects of antioxidants with a high bioavailability and to characterize their targets in tumor cells. Differently, several lines of evidence indicate that antioxidants may actually promote tumor growth and metastasis. For instance, Sayin et al. have shown that the addition of the antioxidants Nacetylcysteine (NAC) or vitamin E to the diet of mice with small lung tumors substantially increases tumor progression and reduces survival in mouse models of lung cancer.104 In the same paper, the authors propose that disruption of the ROS/p53 axis accounts for this effect. The antioxidants not only reduced ROS levels and DNA damage in cancer cells but also reduced p53 expression, which is typically activated by DNA damage. More recently, the same authors focused on malignant melanoma and found that NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells.105 In accordance, Piskounova et al. demonstrated that oxidative stress can inhibit distant metastasis of human melanoma, whereas antioxidants promote their development.106 6.

Conclusion

In recent years, it has been well documented that the intracellular redox state in the cells may represent a specific signal that evokes appropriate cellular responses. Several mechanisms dependent on physiological ROS levels have been presented in this review, including regulation of signal transduction and activation of specific cellular enzymes. More complicated and sometimes controversial seems to be the role of ROS in tumor cells. Through the reversible oxidation of the thiol groups of cysteines within proteins,


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ROS can promote cell survival, proliferation and migration. Tumor cells display a sustained increase in the production of ROS, which seems to maintain an oncogenic phenotype and favor tumor progression. Due to increased ROS levels, cancer cells upregulate antioxidant molecules in order to prevent ROS damage and survive. This might represent an adaptive resistance mechanism against those drugs that induce oxidative stress-mediated cell death. This dichotomic behavior of ROS turns out to be of particular usefulness if we consider that although their increased generation appears to be tumorigenic in some systems, in other cases, it can instead promote tumor susceptibility to particular death pathways. Indeed, escalating the level of ROS in tumor cells to a higher toxicity may cause chemosensitization and cell death. From this controversial perspective, ROS can be implicated in both chemoresistance and chemosensitization. In conclusion, to tailor specific combinations of therapy and to decide which strategy to use, application of chemotherapeutics that excessively increase intracellular ROS to toxic levels or of antioxidants may be dependent on the tumor type and stage, the type and level of endogenous ROS, and the abundance of ROS-induced survival pathways. A more detailed understanding of the effects induced by ROS in tumor cells remains to be acquired to develop new strategies for selectively killing cancer cells and overcoming drug resistance.

* Corresponding Authors Prof. Marianna Lauricella, Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, University of Palermo, via del Vespro, 129, 90127 Palermo, Italy, E-mail: [email protected]; phone +39 0916552458. Prof. Michela Giuliano, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Laboratory of Biochemistry, University of Palermo, via del Vespro 129, 90127 Palermo, Italy. E-mail: [email protected]; phone +39 09123890653.

Author Contributions The manuscript was written through contributions of all authors. Funding Sources This work has been carried out with the ﬁnancial support from Gruppo Azione Locale (GAL) of Golfo di Castellammare, Italy (Progetto Operativo n.17/2015, misura 313B). Conflicts of Interest The authors declare no conflict of interest.

ABBREVIATIONS ASK1, Apoptosis signal-regulating kinase 1; ATO, arsenic trioxide; Cdk, cyclin-dependent kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GSH, glutathione; H2O2, hydrogen peroxide; HIF-1a, hypoxia-inducible factor 1 a; HO-1, heme-oxygenase; JNK, c-Jun N-terminal kinase; Keap-1, kelch-like ECH-associated

protein 1; MAPK, mitogen-activated protein kinase; MMP, metalloprotease; NAC, N-acetylcysteine, NF-κB, nuclear factor kappalight-chain-enhancer of activated B cells; NO., nitrogen monoxide; NOS, nitric oxide synthase; NOX, NADPH oxidase; Nrf2, nuclear erythroid factor 2 (NE-F2)-related factor 2; O2.-, superoxide anion; ONOO-, peroxynitrite anion; .OH, hydroxyl radical; 8-oxodG, 8oxo-7,8-dihydro-2'-deoxyguanosine; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3′ kinase; Prot-SH, proteins contain sulfhydryl groups; PTEN, phosphatase and tensin homolog; PTPs, protein phosphatases; ROS, reactive oxygen species; RTKs, tyrosine kinase receptors; −SOH sulfenic acid; −SO2H, sulfinic acid; −SO3H sulfonic acid; SOD, superoxide dismutase; Trx, thioredoxin; TrxR, thioredoxin reductase; UCPs, uncoupling proteins.

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The Double-Edged Sword Profile of Redox Signaling: Oxidative

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