Gina Manda

342
Current Chemical Biology, 2009, 3, 342-366
Reactive Oxygen Species, Cancer and Anti-Cancer Therapies

Gina Manda*,1, Marina Tamara Nechifor2 and Teodora-Monica Neagu1
1 2
Victor Babes National Institute of Pathology, 99-101 Splaiul Independentei, 050096 Bucharest, Romania; University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania
Abstract: Mammalian cells produce reactive oxygen species (ROS) which are carcinogens, key actors of the non-specific immune defense against pathogens and, in a more subtle way, of signal transduction, cellular metabolism and functions. Oxidative stress can induce severe damage to the host which in turn adapted to face oxidative injury. Disruption of redox balance leads to various pathological conditions, such as cancer. In this review we explore the network linking ROS, cancer cells, anti-tumor immunity and therapy. We emphasize recent findings regarding the oxidative tumor microenvironment and the correlation between ROS, proliferation and death of cancer cells. Further-on we highlight that granulocytes, as key inflammatory cells and ROS producers, are nowadays exploited for eradication of cancer cells. Finally, we focus on ROS-inducing anti-neoplastic therapies (radiotherapy and photodynamic therapy) and on controversial issues regarding the interference between chemotherapy, ROS and antioxidants. This review is directed mainly to researchers involved in anti-cancer drug development by pointing out that redox balance is a suitable therapeutic target, either alone or in combination with other pathways of cancer cells killing. We emphasize critical redoxcontrolled checkpoints that have to be taken into account in drug design for achieving good therapeutic efficiency and convenient side-effects.
Keywords: Reactive oxygen species, oxidative stress, cancer, anti-cancer therapies, granulocytes. INTRODUCTION Free radical generation is mainly unprogrammed, but unavoidable in aerobic organisms due to their reliance on oxidative processes for life. It is fascinating how such simple molecules like the free reactive oxygen species (ROS) are able to regulate life and death. ROS are generally perceived as toxicants that induce various deleterious effects, like cell dysfunction, death or malignant transformation. Aerobic organisms adapted to live in an oxidative environment by developing powerful antioxidant mechanisms. The toxic potential of ROS is used by the innate immune defense as a powerful weapon against pathogens. If pathogens may elude their recognition as non-self by the sophisticated adaptive immune system due to their plasticity and adaptive mechanisms, they cannot escape the rough chemical attack of ROS. Relatively recent findings point out that aerobic organism learned to take further advantage of apparently toxic ROS. Evidence exists that ROS are beneficially involved in many signaling pathways that control development and maintain cellular homeostasis. In physiological conditions, a tightly regulated redox balance protects cells from the injurious attack of ROS, but if altered, it promotes various pathological conditions. Understanding the duality of ROS as cytotoxic molecules and key mediators in signaling cascades, may provide novel opportunities for improved therapeutic intervention. This paper is focused on the involvement of ROS in cancer, considering that recent fundamental findings and associated therapeutic approaches have been proposed and substantiated lately. This review does not intend to be exhaustive in such a researched topic as cancer and ROS, but aims to highlight, by key issues and relevant examples, the multifaceted role of ROS and antioxidants, and the complex network connecting ROS, cancer cells and anti-neoplastic therapies that exploit oxidative stress. This review is directed mainly to researchers involved in anti-neoplastic drug development by pointing out that redox balance is a suitable therapeutic target in cancer, either alone or in combination with other pathways involved in tumor cells killing. We emphasize critical redox-controlled checkpoints that have to be taken into account in drug design for achieving good therapeutic efficiency and minimal side-effects. WHERE TO START FROM? DEFINING THE REDOX BALANCE Reactive Oxygen Species Molecular oxygen itself qualifies as a free radical, having two unpaired electrons with parallel spin in different piantibonding orbitals, thus presenting paramagnetic properties. Spin restriction accounts for its relative stability. In particular conditions, molecular oxygen is capable of accepting electrons to its antibonding orbitals, becoming reduced and hence functioning as a strong oxidant. The unpaired electrons of molecular oxygen react to form partially reduced, highly reactive species that are classified as ROS, presenting radical and non-radical structure: superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen. In the so-called oxidative burst, ROS are generated by enzymatic and non-enzymatic reactions in a tightly controlled flow, starting from superoxide anion [1-5] (see Table 1).
2009 Bentham Science Publishers Ltd.
*Address correspondence to this author at the Victor Babes National Institue of Pathology, 99-101 Splaiul Independentei, 050096 Bucharest, Romania; Tel/Fax: 0040213194528; E-mail: gina.manda@gmail.com 1872-3136/09 $55.00+.00
Current Chemical Biology, 2009, Vol. 3, No. 1 343
Table 1.
ROS
Cellular Mechanisms of ROS Generation1

Description One-electron reduction state of molecular oxygen. Generation: it is formed as by-product of the mitochondrial respiratory chain and in controlled reactions catalyzed by NADPH oxidases or xanthine oxidase. Neutralization:
superoxide anion (O2-)
Superoxide anion undergoes spontaneous dismutation: 2 O2 + 2 H2O O 2 + H 2O2 + 2 OH Superoxide anion mainly undergoes dismutation to hydrogen peroxide by enzymatic catalysis mediated by superoxide dismutase (see reaction below). Therefore, superoxide dismutase is a powerful antioxidant, as it detoxifies the first ROS generated during the oxidative burst. However, consequent formation of the more reactive hydrogen peroxide might lead in fact to an enhanced oxidative stress if the antioxidant system (catalase, glutathione peroxidase) does not work properly towards the decomposition of hydrogen peroxide. the reaction of superoxide with non-radicals is spin forbidden and thus, in biological systems the main reactions of this radical are with itself (spontaneous dismutation) or with other biological radicals such as nitric oxide (results in formation of the toxic peroxynitrite). superoxide anion inactivates enzymes containing iron-sulfur clusters, thereby releasing free iron which further participates in Fenton chemistry, leading to the formation of hydroxyl radical. superoxide initiates lipid peroxidation of polyunsaturated fatty acids. due to its rather low reactivity, superoxide anion can diffuse away from the generation site and thus triggers oxidative stress in the whole cell.
Interaction:
Two-electron reduction state of molecular oxygen. Generation: it is formed by direct reduction of molecular oxygen and by controlled dismutation of superoxide anion in a reaction catalyzed by superoxide dismutase (SOD): M(n+1)+ SOD + O 2 Mn+ SOD + O 2 M SOD + O 2 + 2H + M(n+1)+ SOD + H2 O2,
n+
hydrogen peroxide (H2 O2)
where M = Cu (n=1); Mn (n=2); Fe (n=2); Ni (n=2). Neutralization:
hydrogen peroxide is quickly converted to water by catalase: 2 H2O2 2 H2 O + O2
Catalase is concentrated in peroxisomes which are located everywhere in the cell glutathione peroxidase reduces hydrogen peroxides by transferring the energy of the reactive peroxides to glutathione. Selenium-containing enzymes also transfer electrons from peroxides to glutathione. peroxiredoxins neutralize hydrogen peroxide within mitochondria, cytosol and nucleus. hydrogen peroxide is required for the generation of more toxic ROS, like the hydroxyl radical (Fenton and Haber-Weiss reactions) and hypochlorous acid (myeloperoxidase-catalyzed reaction). it interacts with lipids, proteins and nucleic acids. hydrogen peroxide is lipid soluble and, due to its structural similarity to water and rather low innate reactivity, can diffuse freely in and out of cells and through tissues, thus interfering with cellular targets away from its generation site.
Interactions:
Three-electron reduction state of molecular oxygen. Generation:
hydroxyl radical (OH)
it is formed by the Fenton reaction: Fe2+ + H2 O2 Fe3+ + OH + OH

-
The co-factor Fe
2+
is further recycled, according to the reaction: Fe3+ + H2 O2 Fe2+ + OOH + H +
it is also formed in the Haber-Weiss reaction: H2O2 + O 2- O2 + OH + OH
Neutralization: unlike superoxide, which can be detoxified by superoxide dismutase, the hydroxyl radical cannot be eliminated by an enzymatic reaction, as this would require radical diffusion to the enzyme's active site. As diffusion is slower than the radical half-life, hydroxyl radical is in fact neutralized by harmful reactions with any oxidizable compound in its vicinity. hydroxyl radical and hydrogen peroxide react in presence of iron(III) complexes and regenerate superoxide anion: H2O2 + OH H 2O + O2- + H + hydroxyl radical is extremely reactive and attacks most cellular components: carbohydrates, nucleic acids, lipids and proteins. the only means to protect cellular structures from hydroxyl radical-mediated injury are effective repair systems.
Interactions:
Table 1. Contd.
344 Current Chemical Biology, 2009, Vol. 3, No. 1 ROS Excited state of molecular oxygen. singlet oxygen (1O2) Description
Manda et al.
Singlet oxygen is the common name used for the two metastable states of molecular oxygen, with higher energy than the ground state of molecular oxygen (triplet oxygen). The energy difference between the lowest energy of O2 in the singlet state and the lowest energy in the triplet state is about 3625 K. Molecular oxygen differs from most molecules in having an open-shell triplet ground state, O2(Xg-). Molecular orbital theory predicts two low-lying excited singlet states, O2(ag) and O2(bg +). These electronic states differ only in the spin and the occupancy of oxygen's two degenerate antibonding g-orbitals. The O2(bg +)-state is very short lived and relaxes quickly to the lowest excited state, O2(ag). Thus, the O2(ag)-state is generally referred to as singlet oxygen. Interaction: singlet oxygen reacts with cellular proteins and lipids. Contrary to common perception, singlet oxygen can be quite long-lived in a cell and, as such, can diffuse over appreciable distances, including across the cell membrane into the extra-cellular environment. Neutralization: excitation relaxes by infrared phosphorescence at 1270 nm.
[1-5].
Cellular Antioxidant Systems Cellular redox balance is maintained by a powerful antioxidant system that neutralizes ROS. It consists of SOD, catalase, the glutathione system (glutathione, glutathione reductase, peroxidase and transferase), the thioredoxin system (thioredoxins, thioredoxin peroxidase and peroxiredoxins), vitamin E and C. Subtle intra- and extracellular mechanisms related to metal-binding proteins (transferring, albumin, ferritin etc.) and various metabolites (uric acid, bilirubin, pyruvate etc) are also active. It is noteworthy that cellular targets attacked and damaged by ROS (lipids, proteins, sugars, nucleic acids) contribute themselves to ROS detoxification and represent therefore sacrifice cellular components. It is generally accepted that glutathione plays a central role in maintaining redox homeostasis. Reduced glutathione (GSH) has a multifaceted role in the antioxidant defense mechanisms. It acts as a direct scavenger of ROS by reacting with singlet oxygen, hydroxyl radicals and superoxide radicals, it is a co-substrate for peroxide detoxification by glutathione peroxidases, for conjugation by glutathione Stransferases, can reduce protein disulfides and regulate the thiol/disulfide status of the cell through disulfide exchange reactions. During all these reactions, the oxidized form of glutathione (GSSG) is formed and is afterwards converted back to GSH by the glutathione disulfide reductase. The GSH/GSSG ratio provides an estimate of cellular redox buffering capacity [6]. The thioredoxin system has an important role in antioxidant defense and was recently highlighted as an active player in signal transduction pathways. Thioredoxins (Trx-1, Trx-2) are small molecules with thiol groups in the active site, localized in cytosol (Trx-1), nucleus (Trx-1) and mitochondria (Trx-2). The thiol reductase activity of thioredoxins is suitable to function as an electron carrier for the action of peroxidase and as a protector molecule against unwarranted disulfide bond formation, thus maintaining the dithiol/disulfide structure of proteins and acting to regenerate proteins inactivated by oxidation. As a cofactor for peroxiredoxins, Trx-1 plays a direct role in reducing hydroperoxides, hydroxyl radical and singlet oxygen [7,8]. Thioredoxins cannot neutralize superoxide anion, but control the levels of this radical by enhancing the biosynthesis of the mitochondrial form of superoxide dismutase (Mn-SOD) [9,10]. Thus, Trx-2 reinforces the protection mechanisms against oxidative stress at the level of mitochondria, where superoxide anion is continuously
generated in the respiratory chain as a by-product. Mitochondrial Trx-2 proved to be less redox-sensitive than the cytosolic form, as it has to control oxidative stress in the intense oxidative environment of mitochondria [11]. GSH and thioredoxins have many similarities and differences. They seem to be independently regulated, as cytosolic thioredoxin was shown to be in a reduced state even when GSH is oxidized and depleted during the oxidative stress [11]. Albeit with a significant lower cellular level than GSH, thioredoxins seem to have a more direct and complex role in regulating cellular events, and therefore are promising candidates for therapy in pathologies associated with redox disturbances [12,13]. CONSEQU ENC ES OF OX IDATIV E BUR ST OXIDATIVE STRESS VERSUS CELL SIGNALING Oxidative Stress Oxidative stress generally describes a condition in which cellular antioxidant defense mechanisms are insufficient to inactivate ROS, or excessive ROS are produced, or both. It is well-documented that significant oxidative stress carries out severe damage to lipids, proteins, sugars and nucleic acid bases (Table 2), which compromises cell viability and functions [14-17]. Cells normally respond to various external stimuli and toxicants by developing oxidative burst. For example, the oxidative burst developed by granulocytes in response to an infectious agent is an effective non-specific mechanism for eradicating the invading pathogen. The process is tightly controlled at multiple levels, including granulocytes recruitment at the inflammatory site, down-regulation of the oxidative burst and of the inflammatory reaction after pathogen elimination [18]. Oxidative stress concomitantly destroys pathogens and harms the surrounding healthy tissues. Under normoxic conditions, ROS are maintained within narrow boundaries by antioxidant and repair mechanisms, acting towards the limitation of oxidative injury in healthy tissue [19,20]. Oxidative damage of any cellular constituent, if unchecked, can theoretically contribute to disease development. Indeed, an increasing amount of evidence suggests that oxidative stress is linked, more or less directly, to either primary or secondary pathophysiologic mechanisms of several acute and chronic human diseases [15,21,22]. As we will further emphasize, cancer cells develop an enhanced constitutive oxidative stress that sustains tumor growth and
Table 2.
Validated Biomarkers of Oxidative Stress in Serum, Plasma and/or Urine2
Markers of Oxidative Stress
Chemical Structure
Detection Methods
Malonaldehyde
OH
Malonaldehyde (1)

COOH
TBARS spectrophotometric assay HPLC-based TBARS assay GC-MS
Lipids F2-isoprostanes (8-iso-PGF2)

HO OH

8-iso-PGF2 (2)
Immunoassays GC-MS, LC-MS
Proteins (carbonyl groups) 2-pyrrolidone
O N CO
2-pyrrolidone (3)
HO NH2 O O HO NH2
DNPH spectrophotometric assay One- and two-dimensional electrophoresis MS Immunoassays
Carboxymethyl-lysine
Carboxymethyl-lysine (4)
Lysine
Sugars
+ N N
HN
Pentosidine
HPLC GC-MS Immunoassays
HN Arginine
Pentosidine (5)
N2H OH HN
DNA 8-hydroxy-2-deoxyguanosine
N N N
O OH
HPLC-ECD LC-MS, GC-MS Immunoassays
OH 8-hydroxy-2-deoxyguanosine (6)
2
[17].
shields these cells against pro-apoptotic signals, thus promoting tumor progression. ROS-Mediated Signal Transduction Pathways Recent evidence demonstrates that cells not only adapted to harmful oxidative stress through an efficient antioxidant system, but learned to use ROS in their favor. Whereas ROS are conventionally thought of as cytotoxic and mutagenic [23,24], compelling findings point out that ROS act as regulators in signal transduction pathways. Apparently, there is a window in the nature and amount of ROS, in which these otherwise toxic species contribute essentially to the maintenance of cellular homeostasis by mediating a broad array of redox-regulated cellular events [19,20]. As
messengers of signal transduction, ROS target key signaling molecules, such as mitogen-activated protein kinases (MAPK), protein phosphatases and transcription factors [25-28]. ROS and Protein Kinases/Phosphatases Redox changes of proteins may be the result of oxidation/reduction of critical thiol groups. Indeed, proteins may present distinctive sensitivity to oxidation, depending on their content of cysteine residues, their conformation or the intensity of oxidative stress. Changes in the local redox state of protein thiol groups lead to conformational alteration that can diminish or augment their DNA binding activity, induce inhibitory subunits release, cause enzyme inactivation
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or promote multiprotein complex formation. All these events may be involved in signal transduction or transcription events. For example, in the MAPK pathway, a guanine nucleotide exchange factor (Sos) activates Ras by conversion of Ras-GDP to Ras-GTP. Sos usually complexes with other proteins to perform this function. Oxidants such as hydrogen peroxide have been reported to directly induce guanine nucleotide exchange in Ras, even in the absence of the conventional activation pathway (ligand-receptor interaction). This effect of oxidants is mediated by their interaction with a critical cysteine in Ras molecule [29]. Oxidation of cysteine residues may also trigger phosphatases inhibition. Phosphatases are important components of most signal transduction pathways, because failure to reverse kinase-mediated phosphorylation is highly responsible for alterations of physiologic cellular functions. Without exception, phosphotyrosine phosphatases contain cysteine in their catalytic domain and either oxidation or mutation at the level of cysteine residues renders these enzymes inactive [30]. ROS and Transcription Factors The binding to DNA of certain transcription factors and hence gene transcription appears to be redox-regulated through oxidation-reduction of critical cysteine residues in the DNA-binding domain [31]. The activator protein-1 (AP-1) is a transcriptional factor composed of dimers of proteins belonging to the c-Fos, c-Jun, ATF, and JDP families. AP-1 binds to DNA via a leucine zipper motif and up-regulates transcription of genes containing the TPA response element [32]. AP-1 activity was demonstrated to be regulated by redox mechanisms, at the level of a critical cysteine residue in the DNA binding site [33,34]. ROS can alter the redox status of thioredoxin reductase and consequently of cytosolic Trx-1 at critical cysteine residues [35]. This so-called sulfhydryl switch enables the delivery of redox-sensitive signals to the nucleus. Thus, following oxidation, Trx-1 translocates in the nucleus and forms a complex with redox factor-1 protein (Ref-1). Ref-1 delivers the signal to the transcriptional complex AP-1, which contains critical cysteine in its DNA binding domain, and is therefore activated. The transcription factor NF-kappaB has long been considered responsive to oxidants. This can occur through the redox-regulation of IkappaB kinase that phosphorylates IkappaB, a critical step in NF-kappaB activation [36]. The redox-regulated effect may also take place downstream from the IkappaB kinase, at the level of ubiquitination and/or degradation of IkappaB [37]. Redox-dependent activation of NF-kappaB proved to be cell and stimulus specific [34]. The ROS Wave A local oxidative burst at the level of few mitochondria may propagate within the whole cell as a wave of concurrent mitochondrial depolarization and ROS production [38]. ROS can trigger by themselves a transient increase of mitochondrial ROS production, through the activation of the mitochondrial permeability transition pore by oxidation of thiol groups in the adenine nucleotide translocase. This leads in turn to collapse of mitochondrial membrane potential and simultaneous increased ROS generation through the mitochondrial electron transport chain [39]. A complex
system of intracellular mitochondrial communication confers to cells the capability to spread ROS-mediated damage, both spatially and temporally [38]. This is a positive feedback mechanism for enhanced ROS production, required either in response to various stressors or acting as a pathological mechanism. According to these findings, local oxidative stress may expand and induce damages in the whole cell, thus affecting cellular metabolism and functionality at multiple levels. Concurrently, a local oxidative burst is able to orchestrate apparently distant signal transducing events. ROS-Mediated Regulation of Redox Balance ROS may initiate an auto-regulatory loop, aiming to maintain the physiologic redox balance. Several genes involved in protection mechanisms against the oxidative stress respond to ROS, as they contain within their promoter an antioxidant response element (ARE). ARE-mediated transcriptional regulation involves binding of Nrf2, which forms a heterodimer with the small MafK proteins [40]. Nrf2 is normally retained in the cytoplasm in association with Keap1, but, in an oxidative environment, cysteine residues within Keap1 are oxidized, Nrf2 is released from the complex, enters the nucleus and binds to ARE in reducing conditions [41]. It is noteworthy that opposite redox condition in cytoplasm and nucleus regulate gene activation and transcription mechanisms. Antioxidants and Signal Transduction Besides their role in maintaining the redox balance, endogenous antioxidants may be directly involved in regulating signal transduction and cellular events in normal and stressed cells. For example, the thioredoxin system is multifunctional in providing protection against oxidative stress, by functioning as growth co-factor and apoptosisrepressor. As reviewed by Watson et al. (2004) [11], cytosolic Trx-1 interferes with cellular proliferation and apoptosis by controlling redox-sensitive signals mediated by growth and transcription factors. Redox regulation of signal transduction by Trx-1 is most likely to occur in the cytosol, where the key signaling machinery resides. The reduced form of cytosolic Trx-1 was shown to prevent down-stream signaling in apoptosis by interaction and inactivation of ASK1, a mitogen-activated protein kinase kinase kinase involved in cytokine and stress-triggered apoptosis. Translocation of Trx-1 from cytosol into the nucleus occurs in response to stress. Nuclear Trx-1 provides the reducing microenvironment required by transcription factors (NFkappaB, AP-1) or by the tumor suppressor protein p53, for their binding to DNA. Moreover, as a cofactor for ribonucleotide reductase and methionine sulfoxide reductase, it contributes to DNA repair and to reduction of oxidized components, thus restoring the metabolism and functionality of cells challenged by oxidative injury. The mitochondrial form of thioredoxin, Trx-2, can prevent mitochondria-mediated apoptosis by interaction with components of the respiratory chain, thereby regulating mitochondrial membrane potential. Cross-Talk Between ROS and Calcium Ions We emphasize herein the cross-talk between ROS and calcium ions, both of them acting as stress-response
elements and signal transducing messengers. Production of hydrogen peroxide was shown to depend on intracellular calcium, whilst in turn, hydrogen peroxide modulates plasma membrane calcium channels and consequently the level of cytosolic calcium ions [42,43]. Besides being involved in ATP synthesis, the mitochondrial membrane potential provides a large driving force for divalent cation entry in the mitochondrial matrix. As such, mitochondria can downregulate cytosolic calcium ions and further modulate calcium-dependent signaling pathways [44,45]. As recently shown by Yan et al. (2008) [46], mitochondrial ROS may exert biphasic regulation of intracellular calcium sparks in a dose- and time-dependent manner. An intense global ROS wave, induced by various physiological stimuli or therapeutic agents, can trigger a transient calcium response, which is later down-regulated by ROS oscillations. Biphasic calcium spark regulation is usually associated with high ROS levels, while basal ROS appear to be predominantly excitatory. Concluding, oxidizing and reducing species within various cell compartments play a major role in regulating oxidative stress and signaling pathways [34,47,48]. The nature, localization and propagation of ROS, the magnitude of the oxidative burst and stress, the capability of ROS and antioxidant elements to control cellular redox reactions, signal transduction and gene expression, all may dictate if oxidative damage occurs, or physiological redox-signaling pathways are triggered. Moreover, ROS have been shown to be involved either in cell proliferation and cell death [49,50] and this duality of ROS signals will be discussed in more detail in another topic of this review, in relation to tumor cells. Measuring ROS and Oxidative Stress ROS can be measured directly, but more often indirectly by assessing the formation of oxidative by-products of lipids, proteins, or nucleic acids (fingerprinting methodology)
Table 3. Methods for ROS Measurement3
ROS
[51,52]. The involvement of ROS in cellular processes was mainly investigated in vitro , using exogenously added ROS, specific inhibitors /scavengers of intracellular ROS or modulators of the antioxidant system. We point out that the results obtained in vitro are highly dependent on cell type and experimental model. Measurement of ROS in cells and living organisms (Table 3) carries a significant analytical challenge, as most ROS are highly reactive and short lived, do not accumulate to high levels and are therefore difficult to detect directly in complex biological systems. Rather, one must measure the accumulation of exogenously added sensors which are modified by ROS, more or less specifically. The assays for ROS measurement (Table 3) [53-56] are prone to numerous artifacts resulting from the analytical method itself, from the redox sensor and are limited in their ability to differentiate between different ROS [51]. At present, few real-time measures of ROS and oxidative stress are available and problems arise from method invasiveness. Recent advances in analytical techniques, especially in electron paramagnetic resonance and mass spectrometry, already offer quantitative, more precise assessment of cellular ROS. Real-time imaging of oxidative changes, using redox-sensitive green fluorescent proteins, was recently shown to provide information about the subcellular location of ROS [57]. We consider that simultaneous assessment of various ROS is recommended in order to characterize the flow of ROS generation in cells and to identify the level where abnormalities occur in pathological conditions, or where ROS-mediated therapies are acting. Due to the variety of processes encompassed by oxidative stress, the choice of the methodological approach is crucial for a correct evaluation of the process, especially in vivo. Assessment of oxidative stress can be performed in readily accessible fluids (urine, plasma, saliva), in red blood cells, expired air, and also in less easily accessible biological sample, like duodenal juice and synovial fluid. Various
Detection Method Cytochrome c reduction Chemiluminescence reactions with lucigenine or coelenterazine
Superoxide anion Hydroethidine / Mito-hydroethidine oxidation Electron paramagnetic resonance with spin traps Horseradish peroxidase assay using hydrogen donors Dichlorofluorescein fluorescence Hydrogen peroxide Dihydrorhodamine 123 fluorescence Aminotriazole inhibition of catalase Electron paramagnetic resonance spectroscopy with spin traps Singlet oxygen Infrared phosphorescence spectroscopy Scanning-laser method
3
[53-56].
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methods are now available for the measurement of oxidative stress biomarkers currently considered as validated (Table 2). These methods present limitations, mainly because too much biologic material is required, or sampling is too invasive, or the specificity is biased by concurring factors. Although urine is most convenient to collect and provides information of generalized oxidative stress, it does not reflect the location of ROS production and damage. Sampling at the real site of ROS damage, such as in synovial fluid, duodenal juice, or, in case of lung diseases, the exhaled air, are recommended whenever possible. Although numerous studies were developed in the field of ROS and oxidative stress, comprehensive information about the network of oxidative stress consequences is still deficient, mainly because of shortcomings of available methods for assessing in vivo the oxidative status [51]. Recent technological developments in proteomics (SELDI technology and protein microarray), are now available and allow rapid, simultaneous and reliable assessment of a broad panel of proteins attacked by ROS or involved in redoxsensitive signaling pathways (Table 4). Such an endeavor may provide a comprehensive description of oxidative stress-associated biomarkers in health and disease, may constitute an early indication of disease onset or progression, may substantiate the response to a particular therapy, and represents a powerful mean to identify novel pathways of ROS-mediated signal transduction. Furthermore, this methodology may reinforce the toxicological and pharmacological armentarium for assessing the toxicity of xenobiotics, of conventional and new therapeutics. CONSTITUTIVE ROS GENERATION AND CANCER Evidence exists that cancer cells are under a continuous oxidative stress and that tumor-associated inflammation is an important player in the neoplastic process by fostering tumor spread [58].
Table 4. Defining Protein Biomarker(s) of Oxidative Stress
Markers of constitutive oxidative stress within tumors were found in cancer patients who also exhibit high levels of generalized oxidative stress [59,60]. For example, patients with lung cancer were shown to have high serum concentrations of nitrated proteins, supporting the presence of oxidative and nitrosative stress [61]. Nitrated proteins can alter cellular metabolism and functionality [62] at the level of oxidant defense (Mn-SOD and carbonic anhydrase), energy production (glycolytic enzymes), cytoskeletal remodeling (alpha-actin, alpha- and beta-tubulin, vimentin) and cell death (annexins). Disturbances of the antioxidant mechanisms in peripheral blood, such as depletion of total glutathione and a decreased GSH/GSSG ratio are considered indicators of oxidative stress in various types of cancer [15]. It is worth noticing that oxidative DNA damage detected in non-cancerous patients may correlate with an increased risk of cancer development later in life [63,64]. We highlight herein that in vitro studies clearly show that human tumor cell lines produce ROS at a much higher rate than nontransformed cells [60,65]. Issues regarding the sources of ROS in cancer cells and the consequences of oxidative stress on their proliferation or death are still a matter of debate and will be further discussed. How Do Tumor Cells Produce ROS? ROS Generation in Mitochondria In non-phagocytic cells superoxide anion is generated within mitochondria as a by-product of the respiratory chain, resulting from incomplete coupling of electrons and protons with molecular oxygen. Superoxide anion is further transformed sequentially into more toxic ROS, like hydrogen peroxide and hydroxyl radicals, via enzymatic and nonenzymatic reactions (Table 1). Cancer cells present mitochondria alterations at the level of mitochondrial DNA, oxidative phosphorylation and
Need to Identify Biomarker(s) of Oxidative Stress Define clinical, experimental or therapeutic issues Define the success criteria Select candidate biomarker(s) of oxidative stress (apply Surface-Enhanced Laser Desorbtion/Ionization technology (SELDI) and/or protein microarray)
define the workflow select samples, controls and standards select appropriate protein chip select the methods for data collection and data analysis Selected redox-sensitive biomarker(s) Develop methods for measuring selected biomarker(s)
identify potential artifacts and pitfalls in estimates of the biomarker adapt the method to field conditions (sensitivity, simplicity, throughput) estimate basal concentrations and inter and intra-individual variation identify modifying factors of the biomarker (life style, dietary intake etc.) Validate the biomarker(s) in an epidemiological study against a heterogeneous population Clinical implementation of biomarkers
energy metabolism, all these accounting for a pro-oxidative shift [66,67]. The respiration injury in cancer cells predicts a low coupling efficiency of the mitochondrial electron transport and a consequent increased electron leakage, leading to enhanced superoxide anion formation. The resulting oxidative stress in mitochondria may cause further damage to both mitochondrial DNA and the electron transport chain, thus amplifying the respiratory malfunctions and consequent ROS generation [68]. Intentional Production of ROS Via the NADPH Oxidase Pathway Non-mitochondrial production of superoxide anion via the NADPH oxidase pathway is not a unique function of phagocytes, as it was is detected in various cell types, including cancer cells [4]. We point out that superoxide production via the NADPH oxidase pathway is a tightly controlled mechanism by which cells intentionally produce superoxide anion. In phagocytes, NADPH oxidase [69] is a complex of membrane-bound components (cytochrome b558: gp91phox (Nox2), p22phox, the GTP-binding protein Rap) and cytosolic components (p47phox, p67phox, the GTP-binding protein Rac). Separating these two groups of components in distinct subcellular compartments, and inactivation of Rac by interaction with rhoGDI, guarantee that NADPH-oxidase is inactive in normal resting cells. Furthermore, activation of NADPH oxidase is a multistep process with several checkpoints for preventing unintentional ROS generation. Following particular cell activation, protein kinase Cdependent phosphorylation of the p47phox component occurs and the cytosolic components translocate to the membrane bound constituents. Rac is released from the complex with RhoGDI consequent to the binding of p21activated kinase to rhoGDI, enzyme autoactivation and phosphorylation of rhoGDI [70]. Assembly of membrane and cytosolic components via Src homology 3 domains leads to functional NADPH oxidase activation. NADPH-oxidase exists in various isoforms (Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2) [5,71] which account for the differences in generating ROS, as observed in various normal or pathological cell types, besides phagocytes. Nox isoforms have similar structure, at least partially, have a tissue-specific expression and mediate ROS production. The physiological function of these proteins and their potential role in the pathogenesis of human diseases is currently under intensive investigation. NADPH Oxidase in Tumor Cells Production of ROS via the NADPH oxidase pathway is not confined to all types of tumor cells. Originally Nox1 was described as a ROS source that stimulates mitogenesis when over-expressed in NIH 3T3 cells [72]. Later experiments performed on other cell lines did not support this early claim [73]. Studies in colon cancer samples found no positive correlation between Nox1 expression levels and proliferation or malignancy, but more differentiated colon tumors were proved to express high Nox1 levels [73-75]. Increased Nox1 expression, accompanied by hydrogen peroxide production, was also detected in prostate cancer samples [76].
Accordingly, Nox expression is highly dependent on tumor cell type and on its evolution stage. Tumor cells may overproduce ROS via the NADPH oxidase pathway due to enzyme regulation by the GTP-ase Rac1 which functions downstream of the protoncogen Ras product [77]. In fact, various oncogenic signals provided by c-myc, Ras, and Bcr-Abl, were shown to be involved in increased ROS generation [68,78]. This oncogene-triggered mechanism of NADPH oxidase activation is seemingly specific for tumor cells [4]. We may not rule out the existence of a yet unknown trigger of oxidative burst in cancer cells, which may be either a persistent one, or it may act as an initial activator of the oxidative burst which further propagates in the tumor milieu even when the triggering stimulus disappeared. Extracellular matrix (fibronectin) was shown to provide activation signals to Nox4 in pancreatic cancer cells and the resulting oxidative burst sustains cancer cells survival [79]. As NADPH oxidase mediates intentional production of superoxide anion, it represents a valuable target for therapeutic intervention in cancer, especially at the level of regulatory components and mechanisms. Interference Between NADPH Oxidase and Mitochondria It is noteworthy that a cross-talk between mitochondria and NADPH oxidase via superoxide anion was demonstrated at the level of mitochondrial Nox1 expression. Desouki et al. (2005) [80] showed that the expression of Nox1 in breast and ovarian tumors positively correlates with expression of cytochrome c oxidase encoded by mitochondrial DNA. Experimental inactivation of mitochondrial genes leads to down-regulation of Nox1 and loss of the mitochondrial control of Nox1 redox signaling was shown to contribute to breast and ovarian tumorigenesis. Hypoxia and Angiogenesis-Induced ROS Production Cancer cells respond by an oxidative burst to hypoxic conditions occurring in early, pre-angiogenic stages of solid tumor development, as a consequence of tumors outgrowing their blood supply [81]. Although hypoxia was demonstrated to be accompanied by enhanced ROS production, an overriding paradox exists because the availability of oxygen for superoxide anion formation is limited. More probably, ROS generation occurs later during tumor development, in early stages of angiogenesis. Tumor hypoxia followed by reperfusion and chaotic flow is accompanied by an oxidative burst, much in the same way as seen in myocardial infarction and cerebral ischemia. In a positive feedback loop, angiogenesis-induced oxidative stress dictates increased production of angiogenic factors, hypoxia inhibitory factor-1 (HIF-1) and secretion of matrixmetalloproteinases by tumor cells, thus sustaining the growth of new blood vessel and consequent enhanced oxidative stress [82]. As such, cancer cells respond by adaptive ROS mediated-processes to the wave of hypoxiaangiogenesis challenge. Glycolytic Phenotype of Cancer Cells and ROS Production Cancer cells need enhanced energy supplies for their intense metabolism. Normally, ATP is produced with high efficiency through oxidative phosphorylation in
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mitochondria. An alternative metabolic pathway is adopted (enhanced glycolysis), when mitochondrial ATP production is compromised. Malfunction of the mitochondrial respiration (respiration injury) due to deletions/mutations at the level of mitochondrial DNA, aberrant expression of enzymes involved in energy metabolism and hypoxia are partly responsive of the increased glycolysis observed in neoplastic cells [66,83]. In cancer cells, intense glycolysis may occur even in the presence of normal oxygen tension [84]. Moreover, increase of glycolytic rates in neoplasms was shown to be directly related to tumor aggressiveness [85]. Since the acquisition of a glycolytic phenotype represents a key for survival and progression of cancer, the inhibition of glycolysis appears as a novel promising target for cancer therapy, if properly applied [66]. In this respect, glucose deprivation in experimental models was shown to be associated with enhanced oxidative stress in tumor cells [86]. In breast carcinoma cell lines, but not in non-transformed cells, glucose deprivation leads to intracellular dominance of pro-oxidants, decreased neutralization of free radicals, depletion of intracellular pyruvate and NADPH, all these events resulting in an enhanced oxidative stress [86,87]. As compensatory mechanism for the pro-oxidative shift, glucose deprivation induces the expression of heme oxygenase-1 (HO-1). Degradation of the pro-oxidant heme moiety of HO1 results in bilirubin, which is an antioxidant capable of scavenging peroxy radicals and of inhibiting lipid peroxidation. Moreover, Chang et al. (2003) [88] showed that ROS generation in mitochondria plays also an important role in HO-1 induction, demonstrating that this is a common regulatory mechanism aiming to protect cells against oxidative injury. Deficiencies of the Anti-Oxidant Mechanisms The enhanced oxidative stress observed in cancer cells can result not only from ROS overproduction, but also from low levels or inactivation of antioxidant mechanisms. The enhanced constitutive oxidative stress renders tumor cells highly dependent on endogenous antioxidants to protect them from continuous intracellular ROS injury [89]. Decreased activity and expression of mitochondrial MnSOD was reported in certain colorectal carcinomas and pancreatic cancer cells, probably accounting for increased superoxide anion production [90,91]. Accumulation of superoxide anion stimulates cell growth by altering the redox states of transcriptional factors and cell cycle regulatory proteins [92,93]. Moreover, induced overexpression of MnSOD was shown to suppress malignant phenotypes in experimental in vitro models. Therefore, Mn-SOD has been considered to be a tumor suppressor which acts indirectly via ROS. Significantly lowered activity of SOD in red blood cells was detected in all the stages of cervical cancer patients. The observed decrease in SOD activity might be associated with free radical generation which inflicts direct damage to the enzyme by cross linking or by mutations [94,95]. Additionally, it may be caused by disturbance of trace elements acting as enzyme cofactors (decreased zinc, correlated with increased copper levels), which can be
considered a risk factor for tumor growth or carcinogenesis [95]. An increased oxidative stress in the blood of ovarian cancer patients, accompanied by decreased levels of antioxidants, like SOD, catalase, vitamin C and vitamin E were reported [96]. It is suggested that down-regulation of peripheral antioxidants is due to their increased utilization in scavenging lipid peroxides, as well as their potential sequestration by tumor cells [97]. Other Sources of ROS in Cancer Thymidine Phosphorylase-Mediated ROS Production A particular mechanism of ROS generation was described in carcinoma cell lines [98], where thymidine phosphorylase is overexpressed and mediates enhanced break-down of thymidine to thymine and 2-deoxy-D-ribose1-phosphate. 2-deoxy-D-ribose-1-phosphate is a powerful reducing sugar which rapidly glycates proteins by a flow of enzymatic and non-enzymatic reactions (Amadori reaction), resulting in free radicals formation, starting with superoxide anion. The oxidative stress induced during protein glycation increases the production of angiogenic factors and matrixmetalloproteinase-1 by carcinoma cells and, as shown above, angiogenesis can further increase the oxidative burst. Organ-Specific Sources of ROS Particular organ-specific radical-generating pathways may be active in cancer cells. For example, the metabolism of estrogenic hormones in breast carcinoma is mediated by lactoperoxidase which catalyzes the one electron oxidation of 17 beta-estradiol to 4-hydroxyestradiol [99]. 4hydroxyestradiol further undergoes metabolic redox cycling, generating hydroxyl radicals, as well as quinone derivatives which are capable of forming DNA adducts [100]. Moreover, 17 beta-estradiol can contribute by itself to the enhanced oxidative burst within tumor cells by decreasing the activity of antioxidant enzymes through an ARE-mediated pathway [101]. The Leukocyte Phenotype of Cancer Cells The existing information about the sources of oxidative burst in cancer cells looks rather scattered and sometimes conflicting. Therefore we point out the theory of Arias et al. (2005) [102], proposing the integration of the findings in the field by an active and versatile inflammatory process. The phases of tumor progression were shown to exhibit many similarities with the development of post-traumatic inflammation, evolving from ischemia towards an oxidative metabolism, in a succession of complex trophic processes adopted by tumor cells from the surrounding tissue and from the immune system. According to this theory, tumor cells are capable to induce an early inflammatory response in the host leading to the recruitment of lymphocytes, macrophages, and dendritic cells from circulation into the tumor. When tumor cells reach higher grades of malignancy, their invasive capacity seems to reflect more tumor ability to express the inflammatory phenotype, than to induce it in the host. Due to high plasticity, cancer cells progressively adopt the phenotype of endothelial and immune cells, resulting in tumor progression and metastasis. Thus, when tumor starts to form and to grow,
cancer cells adopt a hypoxic phenotype, produce proangiogenic factors and further undergo revascularization which triggers oxidative stress. Later during development, tumor cells may usurp key inflammation-like mechanisms. Tumor cells can express some of the signaling molecules of the innate immune system (chemokines, selectins and their receptors) and fulfill functions characteristic for activated inflammatory cells (neutrophils and macrophages). Accordingly, we may presume that tumor cells start producing ROS, much in the same way as phagocytes do. Consequences of the Enhanced Oxidative Stress in Cancer Cells ROS as Carcinogens ROS are considered potential carcinogens, as they were shown to facilitate mutagenesis, cancer promotion and progression [103]. ROS-induced injury of nuclear DNA and misrepair of this damage could result in mutations leading to carcinogenesis. Mutations caused by oxidative DNA damage include a range of specifically oxidized purines and pryrimidines, alkali susceptible sites, single strand breaks etc. Some of the modified bases have been found to possess mutagenic properties, usually related to GC base pairs substitutions and less to AT ones [104]. The sequence specificity of DNA damage sites dictates the mutation frequency [105]. On a larger scale, chromosomal rearrangements, resulting from strand breakage misrepair, contribute to genetic amplifications, iterations in gene expression, loss of heterozygosity, and therefore may promote cancer progression [106]. ROS-induced mitochondrial genome instability has also been reported. Mitochondrial DNA is partially associated with the mitochondrial inner membrane where ROS are generated in the respiratory chain. Moreover, mitochondrial DNA repair mechanisms are less efficient than the nuclear ones [107]. Moreover, pro-oncogenic conditions arise whenever translocation of damaged mitochondrial DNA into nucleus and its insertion into nuclear genome occur [108]. These pathological events are part of the larger concept that cancer may be regarded as a mitochondriopathy, induced by somatic mitochondrial DNA mutations [109]. In this respect, the reported connection between mitochondria, ROS, aging and cancer risk is noteworthy [110]. ROS and Proliferation of Tumor Cells Evidence exists that the role of ROS in cancer is not limited to the generally accepted genotoxicity and mutagenic effects that initiate cancer. As signal transduction messengers, ROS may promote either proliferation or death of cancer cells, depending on the actual intracellular and exogenous conditions. ROS were shown to modulate growth signals and to activate gene expression, leading to sustained proliferation of cancer cells [111]. An emerging view is that upon oncogenic transformation, cells rapidly activate a stress response, as a protective measure to overcome oncogene-induced cell death and senescence [112]. Cancer cells subjected to persistent endogenous and exogenous oxidative stress were shown to
develop adaptive responses, mainly related to the upregulation and activation of the antioxidant machinery [113]. Therefore, tumor cells may become shielded by antioxidant and anti-apoptotic robustness during the evolution of malignant state [114]. Thus, cancer cells are resistant both to the enhanced constitutive oxidative stress and to ROSgenerating therapies. We may not rule out that, in early stages of tumor growth, ROS may trigger tumor cell death and therefore a ROS-resistant phenotype of cancer cells is selected for further development. Cancer progression is characterized by the acquisition of genetic and epigenetic changes that lead to phenotype diversity among the progeny of cancer cells. This diversity allows selection of cells that possess the most suitable attributes for survival and adaptation to the environment, during their tormented evolution from primary lesion to metastatic colony [115]. We may also presume that a cross-talk between epigenetic changes and the ROS wave accompany cancer development. In experimental models using cell lines it was shown that ROS generation in tumors and subsequent oxidative stress are actually at a sublethal level [116] and contribute to cancer progression through an array of interconnected signals. Additionally, ROS-mediated proliferation signals correlate with a reduced susceptibility of tumor cells to the pro-apoptotic action of particular anti-neoplastic therapies [117]. Proliferation Signals Delivered by ROS Via MAPK The effects of ROS on cellular metabolism are partly mediated by MAPKs: extracellular signal-regulated kinases (ERK), c-Jun-N-terminal kinases (JNK) and p38. These are components of the kinase cascades connecting extracellular stimuli to specific transcriptions factors and hence converting them into cellular responses [118]. Normally, the ERK subgroup is involved in cell proliferation responses, whilst JNK and p38 which are stress-activated kinases (SAPK), play a role in stress responses and cell death [119]. In cancer cells, ROS-induced hyper-phosphorylation of JNK can translate oncogenic signals, thus supporting cellular proliferation by activation of AP-1, in addition to the proliferation signals mediated by ERK [114]. Proliferation Signals Delivered by HIF-1 HIF-1 delivers proliferation signals to cancer cells in hypoxic microenvironment characteristic for the preangiogenic stage of solid tumors [120]. HIF-1 is a heterodimeric transcription factor acting as a key regulator of metabolic adaptation to hypoxia. At physiological oxygen levels, the HIF-1alpha subunit is hydroxylated and targeted for 26 S-proteasomal degradation by ubiquitination [121]. In the majority of solid tumors under hypoxic conditions, HIF-1 expression was found to be enhanced [81]. Hypoxiaassociated ROS can prevent HIF-1alpha hydroxylation, thereby stabilizing the molecule. This allows HIF-1alpha to translocate to the nucleus and to dimerize with HIF-1beta. Subsequent transcription of several hypoxia-responsive genes is initiated, aiming to facilitate cell survival in conditions of limited oxygen availability [81,120]. A regulatory loop is related to the activation of p53 under severe hypoxic conditions, leading to termination of HIFmediated responses. Thus, p53 may attenuate HIF-1
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activation by competing for the shared co-activator p300 or may contribute to the destruction of the HIF-1alpha component [121]. As HIF-1 seems to be crucially involved in prolonging the lifespan of tumor cells in a hypoxic microenvironment, we may consider that down-regulation of HIF-1 activity can be therapeutically beneficial in cancer, if properly applied [122]. Mitohormesis It was recently hypothesized that ROS, albeit presenting a cytotoxic potential, may expand lifespan. Within the concept of mitohormesis [123], Schulz et al. (2007) [124] demonstrated using a model organism (Caenorhabditis elegans), that glucose restriction promotes mitochondrial metabolism, causing enhanced ROS formation and extension of lifespan by increasing cellular resistance to stress. It remains to be demonstrated whether this concept applies to humans. Concluding, tumors evolve in a particular oxidative milieu that endows cancer cells with a survival advantage over their normal counterpart, concurrently conferring to tumor cells an increased resistance to apoptotic signals, like those triggered by anti-neoplastic therapies. ROS and Tumor Cells Death It is generally considered that ROS may promote either cell proliferation, or cell death, depending on the intensity/location of the oxidative burst and the activity of the antioxidant system. Considering the proliferation signals delivered by ROS to cancer cells and the consequent resistance of cancer cells to pro-apoptotic signals [117], ROS-induced tumor cell death is more probable to be elicited by ROS-generating anti-neoplastic therapies, which increase the constitutive oxidative status above the critical threshold required for cell death. Evidence exists that oxidants kill cells mainly by apoptosis, albeit in a narrow set of oxidative conditions, and actually only severe oxidative stress may induce necrosis [125]. Moreover, Chandra et al. (2000) [126] showed that excessive oxidative stress can inhibit apoptosis at the level of caspases which require a reducing environment for optimal activity. Apoptosis is inhibited if antioxidants cannot overcome massive ROS generation and consequent caspase inactivation through oxidation. In this case, other types of cell death, caspase-independent, may occur [83,127]. Mitochondria, crucially involved in superoxide generation by non-phagocytic cells, including cancer cells, also act as stress sensors and are pivotal in cellular apoptosis. This is in agreement with the endosymbiotic theory, stating that mitochondria originated as bacterial intracellular symbionts [109]. As such, we consider that mitochondria try to orchestrate cellular events as a decision-making element. As reviewed by Ricci and Zong (2006) [128], the intrinsic apoptotic pathway activation starts with oligomerization of the pro-apoptotic Bcl-2 proteins, Bax and Bak, in the mitochondrial outer membrane. This leads to mitochondrial membrane permeabilization and consequent release of apoptogenic (cytochrome c and AIF) and of
regulatory factors (IAP, Smac/DIABLO and Omi/HtrA2 proteins). Once released, cytochrome c binds to Apaf-1, which recruits pro-caspase-9 and promotes its autocatalytic activation. Activated caspase-9 stimulates down-stream effector caspases (caspase-3 and caspase-7) which rapidly cleave intracellular substrates and execute apoptosis Fig. (1). A complex regulatory mechanism is active for controlling the extent of caspase-mediated cell death. Proteins of the IAP family can bind and inhibit the active sites of caspase-3, -7 and -9. Smac/DIABLO and Omi/HtrA2 proteins can in turn bind to IAP proteins, hence preventing their inhibitory effect on caspase activation. Additionally, anti-apoptotic Bcl-2 proteins block the oligomerization of Bax and Bak, or their interaction with pro-apoptotic BH3only proteins (Bid, Bim etc.), thus preventing mitochondrial outer membrane permeabilization. AIF is normally retained in the intermembrane mitochondrial space, where it performs an oxidoreductase function [129]. Like cytochrome c, AIF becomes an active cell killer when it is released into cytosol and further translocates to the nucleus, where it triggers, along with endonuclease G, chromatin condensation and high molecular weight DNA loss Fig. (1). Therefore, AIF can act as a safeguard death executioner in cancer cells with faulty caspase activation [130,131]. Signaling Pathways Connecting ROS and Apoptosis Several studies, reviewed by Benhar et al. (2002) [114], reveal a state of enhanced stress signaling in neoplastic cells. Under oxidative stress conditions, the SAPK members of the MAPK family (JNK and p38) are activated Fig. (1). JNK pathway is particularly important in the mitochondriadependent pathway of apoptosis, whilst dispensable in death receptors-induced apoptosis [132]. ASK1, an up-stream regulator of SAPK, is considered to be the link between ROS and SAPK. Thus, in non-stressed cells ASK1 is inactivated through its association with thioredoxin [133]. The complex can be disrupted by ROS, leading to SAPK activation and consequent apoptosis [134]. Activation of JNK and p38 promotes cancer cells death by modulating directly the serine/threonine phosphorylation of pro-apoptotic factors. Thus, JNK and p38 were shown to activate by phosphorylation the wild-type tumor suppressor p53 which further induces or represses the expression of several genes that regulate cell cycle arrest, DNA repair or apoptosis [128]. Pro-apoptotic genes encoding for Bcl-2 members (Bax, BH3-only proteins) are activated by p53, critically involved in the mitochondrial pathway of apoptosis. Additionally, p53 can increase mitochondrial outer membrane permeabilization by direct binding to proapoptotic Bcl-2 family members (Bax, Bak). Besides inducing the MAPK-mediated activation of p53, ROS may also act as downstream mediators of p53-induced apoptosis. For example, the p53-dependent activation of the mitochondrial proline oxidase catalyzes ROS generation, thus enhancing the oxidative stress that initially activated p53 [128]. More recent evidence exists about a regulatory p53-ROS feedback loop, aiming to reduce apoptosis-inducing
Fig. (1). Mitochondria, ROS and cancer: cell proliferation or cell death?
oxidative stress. It was shown that p53 inducing the expression of glutathione peroxidase-1 and of sestrins (PA26, hi95) [135,136]. Sestrins are involved in the regeneration of overoxidized peroxiredoxins and consequent rehabilitation of their capability to detoxify hydrogen peroxide. Moreover, p53 triggers cell cycle arrest by activating the cyclin-dependent kinase inhibitor p21, hence giving time for cell damage repair [114]. Other Pathways of ROS-Induced Apoptosis ROS can induce cellular apoptosis by several other pathways than the mitochondrial one. For example, the endoplasmic reticulum (ER) was shown to be involved in oxidative stress-induced death of cancer cells [137]. As reviewed by Thannickal et al. (2000) [34], ER functions, such as protein folding and secretion, are redox-regulated. Under moderate oxidative stress conditions, ER acts as a sensor of cellular stress, aiming to restore cellular homeostasis. If the stress-induced damage is widespread, programmed cell death is initiated in ER via the unfolded protein response, calcium release into cytosol and consequent activation of calcium-activated neutral proteases
(calpains). Cullinan et al. (2004) [138] demonstrated that a regulatory mechanism may prevent ER-triggered cell death in response to ROS. Thus, Nrf2 activation induced by the unfolded protein response contributes to the maintenance of GSH levels. Perturbations in cellular redox status sensitize cells to the harmful effects of ER stress, but other factors, like BH3-only proteins which reside in mitochondria and ER, are essential for apoptotic commitment. Products of oxidative stress, like acrolein, can limit cellular proliferation and/or promote apoptosis. Acrolein, is one of the most electrophilic aldehydes generated in the process of lipid peroxidation. At sublethal doses, acrolein exerts in vitro anti-proliferation effects, whilst massive cell and tissue injury ensues at higher acrolein concentrations [139,140]. Acrolein rapidly reacts with GSH and thioredoxin, leading to a temporary depletion of regulatory thiols [11,139,141]. Consequent oxidative modulation of transcription factors results in apoptosis of tumor cells [142,143]. This process may represent a peculiar mechanism by which cancer cells limit tumor progression in response to the oxidative stress featuring malignant phenotype.
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Apoptosis-Induced Oxidative Burst Mitochondria-dependent apoptosis pathway may further enhance the oxidative stress that initially triggered apoptosis. Cytochrome c release during apoptosis disturbs the coupling efficiency of electron chain transport, resulting in superoxide radical generation [144]. This oxidative burst causes thiol oxidation of mitochondrial lipids and proteins, consequent mitochondrial membrane permeabilization, leading back to apoptosis. Apoptosis-related ROS generation seems to be a feedforward loop which occurs after apoptosis-inducing signals have already triggered caspase activation. As such, an increased production of peroxides, found in cell populations already containing apoptotic cells, may be the result rather than the cause of apoptosis [145]. In certain cases, ROS generated during the apoptotic process can divert apoptosis towards necrosis. This shift requires considerable amounts of ROS, a drop in ATP and consequent loss of the electrochemical gradient across the inner mitochondrial membrane [117]. The shift from apoptosis to necrosis is of utmost importance in solid tumors. The deleterious consequences of this shift reside mainly in inflammation, triggered by the rupture of necrotic cells and subsequent release of tissue-degrading enzymes. Inflammation further signals recruitment and activation of leukocytes, including ROS-generating phagocytes, thus leading to a subsequent increase of inflammatory conditions in the tumor. Therefore, in anti-neoplastic therapies, apoptotic death of cancer cells is preferred. Apoptotic cells cause minimal disturbance to the surrounding tissue, as they do not release their content and are rapidly engulfed by phagocytes. Beyond the Apoptosis-Necrosis Dichotomy Recently, it has become evident that the classic dichotomy of apoptosis versus necrosis is a simplification of the highly sophisticated processes which protect the organism against unwanted and harmful stress. Novel mechanisms of cell death have been characterized [127,128,146-148]. Some of them may be triggered by ROS in cancer cells, in particular circumstances [149]. It is noteworthy that a cell may switch back and forth between different death pathways and the dominant cell death phenotype is determined by the relative speed of the available death programs [147,150]. The pathways of cellular death are currently subjected to a definition/redefinition process which might bring new evidence regarding the resistance of tumor cells to apoptotic signals and the mechanisms supporting the action of antineoplastic drugs. We emphasize herein the programmed cell death by autophagy that has recently resurfaced in cancer research. Autophagy features degradation of cellular proteins and organelles in autophagosomes in response to metabolic stress, thus providing the nutrients required for cell survival. Accordingly, autophagy can promote cancer cells survival during nutrient deprivation and hypoxia associated to limited angiogenesis [151]. Paglin et al. (2001) [152] showed that low-dose ionizing radiation elicits in neoplastic epithelial cells a rescue mechanism mediated by autophagy and acidic vesicles formation. Autophagy is dependent on ROS for the
functionality of redox-dependent autophagy-related proteins (Atg) [153]. Conversely, autophagy may have an anti-tumor role, as tumor suppressors (p53, PTEN) induce autophagy [154,155], the autophagy gene Beclin-1 (Atg6) is a tumor suppressor and oncogenes (Bcl-2) inhibit autophagy [156]. The link between autophagy and cell death is still debatable. If autophagy cannot proceed, cells subjected to stress (i.e. nutrient deprivation), will die by apoptosis [157]. Autophagy may also trigger by itself cell death through selective autophagic degradation of catalase, which in turn leads to massive oxidative stress that will finally kill cells [158]. Thus, at different moments during cancer evolution, autophagy may have tumor promoting or inhibiting properties [151]. The ability of ROS to stimulate cell growth or cell death most likely depends on the intensity/duration of redox signals and on the antioxidant defense mechanisms. Transient, low-level oxidative conditions apparently promote cell proliferation, whereas persistent, high-level oxidative stress may result in cell death. The oxidative status of particular cancer cells is possibly near the ROS threshold that separates proliferation and cell death-inducing signals. GRANULOCYTES, ROS AND CANCER Inflammation is increasingly recognized as an important component of tumorigenesis, [159]. The immune system tends to respond promptly to tumor onset by recruiting leukocytes into tumor and by activating both the innate and adaptive immune response at systemic and intra-tumor level [160-162]. Conversely, tumor development and the associated inflammatory reaction lead to immunosuppression by disabling/eliminating immune effector cells, both at local and systemic level [163-166]. ..granulocytes still have every reason to complain of the disdain with which they are regarded by oncologists and immunologists. So widespread is T-cell chauvinism, that the anti-tumor potential of polymorphonuclear cells continues to receive little attention, and researchers have not yet fully considered the possibility of exploiting their functions as effective weapons against cancer Di Carlo et al. (2001) [167]. Although granulocytes are the most abundant circulating blood leukocytes, they were long-time considered minor players in anti-tumor immune defense. The biology of granulocytes was extensively studied, but mainly in the field of inflammation and non-specific immune defense against infection. Lately, compelling evidence gathered that granulocytes perform more complex function than non-specific killing of pathogens, as they were shown to produce and respond to cytokines, and to participate in complex networks of specific and non-specific immune responses [18,168,169]. Nowadays it is considered that granulocytes are a powerful weapon for the suppression of tumor growth. Cytotoxic Mechanism Against Cancer Cells Developed by Granulocytes
Extravasation of granulocytes from blood into tumor is a regulated multistep process, involving a series of coordinated
interactions between granulocytes and endothelial cells, an inter-play between selectins and integrins, orchestrated by cytokines originating from tumor cells and from tumorinfiltrating leukocytes [170,171]. Activation of intra-tumor granulocytes can be induced by surface molecules expressed by cancer cells and cytokines released by leukocytes and cancer cells [172]. Activated granulocytes produce ROS and thus reinforce the oxidative milieu in tumors. Hypochlorous acid seems to be the most effective granulocyte-derived ROS in inducing tumor cell lysis [173]. It reacts with primary amines to form relatively stable chloramines with immunostimulatory properties [174], thus sustaining the overall anti-tumor immune defense. It is noteworthy that hypochlorous acid, below a critical threshold, was shown to sustain proliferation of tumor cells, as individuals with deficiency of neutrophils myeloperoxidase show a high incidence of malignant tumors [175]. Besides generating and releasing ROS, activated granulocytes discharge other cytotoxic mediators, like proteases and membrane-perforating agents [18,167]. Oxidants act synergistically with proteases to inactivate plasma anti-proteases, thus allowing proteases to operate tissue-injury [176]. Adhesion to vascular endothelium of activated granulocytes expressing up-regulated adhesion molecules creates a subjacent microenvironment with high concentrations of oxidants and proteolytic enzymes, sufficient to cause endothelial damage and matrix degradation [177]. This allows enhanced recruitment of leukocytes, but also the invasion of tumor cells into the host. Additionally, local ROS-induced necrosis and consequent hypoxia is followed by massive recruitment of leukocytes and enhancement of inflammation [178]. In most cases, granulocytes recruited naturally into the tumor do not attain the critical number and cytolytic activity for effectively destroying constituted tumors. More probable, a cross-talk between granulocytes and tumor takes place, resulting often in tumor progression and metastasis. Due to the impressive plasticity of cancer stem cells, tumor cells may adopt an endothelial and leukocyte phenotype [102,179]. This mimicry, along with the oxidative tumor milieu, sustains the invasiveness of cancer cells. Hyperproduction of extracellular proteases by cancer cells and tumor-infiltrating leukocytes carries out digestion of the basement membrane and the extracellular matrix, thus accounting for metastasis initiation [180]. Moreover, cancer cells were shown to form pseudopodia and to perform directional migration, hence being able to leave the tumor and to start host invasion [181]. Cancer, Granulocytes and Immunosuppression Activated peripheral granulocytes produce significant amounts of ROS, which are in part responsible for the general immunosuppressed state detected in malignancies [182]. For example, in pancreatic metastatic adenocarcinoma, the innate immune system readily responds to tumor formation by activation of granulocytes and consequent ROS production, resulting in systemic suppression of the adaptive immune response [183]. An activated phenotype of peripheral granulocytes, primed for
an enhanced respiratory burst, was also reported in patients with severe skin T-cell lymphoma [184]. Recent findings strengthen the hypothesis that granulocytes may control in a ROS-dependent way the cell populations recruited into tumors and their functionality. Harlin et al. (2007) [185] and Thore et al. (2007) [186] demonstrated that hydrogen peroxide produced by activated granulocytes selectively kill the abundant CD16+CD56dim subpopulation of NK cells (responsible for cytotoxicity), whilst not affecting the lower NK CD16-CD56bright subpopulation (having mainly immunomodulatory function). The efficient intracellular anti-oxidant machinery and the high cell-surface expression of antioxidant thiols in CD16 CD56bright NK cells account for the mentioned selectivity of hydrogen peroxide-mediated killing. These findings point out the preferential accumulation of immunomodulatory, but not cytotoxic NK cells, in environments abunding in ROS, like in tumors. Accordingly, NK-mediated anti-tumor mechanism may be deficient within tumors. Anti-Neoplastic Therapies Exploiting Granulocytes Granulocytes are currently considered as valuable antitumor effectors due to their ability to perform antibodydependent cell-mediated cytotoxicity (ADCC) [187,188], represent the most abundant peripheral leukocyte subset expressing Fc receptors for IgG. Moreover, their numbers, along with their tumor killing capacity, can be increased by treatment with granulocyte colony-stimulating factor [189,190]. In response to signals delivered by Fc receptors, granulocytes perform ADCC and release inflammatory mediators, hereby attracting other immune cells (monocytes, dendritic cells and T lymphocytes), thus eliciting anti-tumor immune responses [191,192]. Granulocytes are therefore an attractive effector cell population for tumor-directed therapy with bispecific antibodies which target both tumor and granulocyte Fc receptors, thus ensuring specific killing of cancer cells [193-195]. This type of therapy is applied particularly in hematological malignancies, where cancer cells are accessible to antibodies and effector cells [196]. In solid tumors, local administration of antibodies, alone or in combination with autologous effector cells, is a promising therapeutic approach in eradicating tumor cells [197]. Concluding, tumor-associated granulocytes can develop cytotoxicity against tumor cells. They may also contribute to the enhancement of tumor oxidative milieu and to tumor progression through various mechanisms, including downregulation of the anti-tumor immune response. Therapeutic strategies, capable of eliciting the recruitment of granulocytes into tumors, of controlling their activation and of guiding their killing abilities towards tumor cells, are promising approaches in cancer treatment. ROS AND ANTI-NEOPLASTIC THERAPIES As shown above, ROS may exert opposite cellular effects, by promoting either cell proliferation and tumor progression, or cell death and tumor regression. ROS are a double edged sword, by acting not only as disease inducers/sustainers, but also as therapeutic weapons in cancer.
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Cancer cells evolve in particular endogenous and exogenous oxidative circumstances, highly differentiated according to cell type and tumor evolution stage. Tumors adapted to these harsh conditions, by developing potent antioxidant mechanisms, and even using endogenous ROS for proliferation. Albeit the intrinsic resistance of cancer cells to ROS-induced cell death, therapies which generate an oxidative burst in tumors proved to be efficient. ROS levels must probably ascend above a threshold in order to induce tumor cell death. Therefore, cumulating constitutively produced ROS with therapy-triggered ROS and/or inhibition of ROS neutralization represents a powerful mechanism for killing cancer cells. In certain cases, tumor cells attacked by anti-neoplastic therapies may gain additional resistance to oxidative stress, and therefore combination therapies are promising, aiming either to converge towards the enhancement of oxidative burst above the critical threshold, or to bring together distinct cytotoxic mechanisms. ROS-induced cell death seems to be distinctively controlled in normal and neoplastic cells by different pro/anti-apoptotic factors and by pro-/anti-oxidant key elements. Novel therapeutic anti-neoplastic strategies, based on ROS formation and/or modulation of antioxidant mechanisms, aim to take advantage of the differences between normal and cancer cells. Unfortunately, existing anti-cancer therapies exert deleterious effects on normal tissues, partially triggered by ROS, which limit the applicable dosage and their anti-tumor activity. Overcoming these side-effects, without altering the efficacy of the therapy, is a research priority. ROS-Mediated Processes in Radiotherapy Radiotherapy is one of the clearest examples of antineoplastic treatment whose mechanism relies primarily on ROS. Ionizing radiation is one of the most effective tools in cancer therapy, by combining the properties of an extremely efficient DNA-damaging agent with a high spatial focusing on the tumor. Radiotherapy limitation derives from the high carcinogenic potential of ionizing radiation [198,199], as clearly shown by increased cancer risk in the Japanese survivors of atomic bomb. Nonetheless, tumor cells were proved to be more sensitive to radiotherapy than normal cells, as radiation induces preferential mitotic cell death in fast dividing cells which are finally destroyed by necrosis [200]. A deleterious side-effect of radiotherapy is associated with the harmful inflammatory response triggered by necrosis. Ionizing Radiation-Induced Damages Radiotherapy damages cells by direct ionization of DNA and other cellular targets and by indirect effects mediated by ROS generated during radiation-induced water hydrolysis. Moreover, when exposed to ionizing radiation cells produce ROS and other toxic radicals. Radiotherapy induces nuclear DNA damage, like single and double strand breaks, leading to cell cycle arrest. Recruitment of DNA repair enzymes protects normal cells of radiation injury, but concurrently decreases therapy efficacy. Ionizing radiation may also severely damage mitochondrial DNA, thus altering the expression of various genes
that encode critical proteins involved in the oxidative phosphorylation system, leading to ROS generation and impairment of ATP synthesis [201-203]. This mechanism accounts for the high levels of ROS associated with radiation-induced genomic instability (RIGI) [204]. RIGI is a delayed, long-lasting effect of ionizing radiation that takes place even in unirradiated progenies of irradiated cells. We also mention the superoxide anionmediated bystander effect, whereby non-irradiated cells present a phenotype usually associated with radiation exposure [205]. Gap junction communication and soluble factors released by irradiated cells may account for a wide spectrum of phenomena seen in bystander cells [206,207]. These findings indicate the existence of radiation memory which underlies long-lasting effects of radiotherapy in tumors, but also contributes to persistent damage and dysfunctions of bystander normal cells. Radiotherapy alters cellular homeostasis by modifying the redox status of cancer and normal cells and can trigger the mitochondrial apoptosis pathway [208,209]. Activation of wild-type p53 is a critical pro-apoptotic event, induced in the mitochondrial cell death pathway and by conformational changes produced directly by radiation [210]. Activation of wild-type p53 after exposure to ionizing radiation requires phosphorylation of serine residues by the ATM-protein kinase [211]. Tumor cells expressing the wild-type p53, but not the mutated form, are highly susceptible to radiotherapy. Unfortunately, normal cells, that express exclusively wildtype p53, are concurrently damaged by radiotherapy. Cellular Protection Radiation Mechanisms Against Ionizing
Cellular response to radiotherapy depends on the radiation type/dose, cellular proliferation status, on the efficacy of antioxidants and repair mechanisms. Thus, Mn-SOD seems to be critically involved in protecting normal cells against the radiation-elicited ROS injury at the level of mitochondria [212]. Additionally, shortand long-term radiotherapy-induced cell injury can be prevented by antioxidant therapy [200,213]. Antioxidants may induce concomitantly radio-resistance in cancer cells, hence reducing the radiotherapy efficacy [213]. Recent findings sustain the benefits of particular antioxidants which selectively trigger apoptosis in cancer, but not normal cells, by increasing the oxidative stress or by synchronizing tumor cells to a radiosensitive phase of cell cycle [214-216]. The perspectives lie in identifying such radio-sensitizers, with distinctive mechanisms of action in cancer and normal cells. A rescue mechanism against the deleterious action of radiotherapy in bystander healthy tissue is related to p53mediated induction of HO-1 [217]. HO-1 exerts an antiapoptotic action by mean of haem degradation products, like the potent antioxidant bilirubin [218]. Evidence exists that autophagy can protect both normal and cancer cells against the deleterious effects of ionizing radiation by removing damaged cellular elements [152,219]. Low-level radiation bellow the anti-neoplastic doses, might be in fact positive or at least neutral to health. According to the radiation hormesis theory, chronic exposure to low-level radiation protects cells from radiation injury,
apparently resulting in improved health [220-223]. Evidence supporting this hypothesis is drawn mainly from in vitro tests and from some epidemiological studies [224,225], all being still questionable due to scarce data and short-time radiation exposure. Although radiation hormesis has been rejected by national and international radiation regulatory councils, the distinctive effects of substances and radiation at low and high doses remain a promising field of investigation, requiring adequate experimental models and accumulation of reliable data. ROS-Mediated Processes in Photodynamic Therapy Photodynamic therapy (PDT) has recently become a well-established treatment of various cancers [226-228], as well as non-malignant diseases [229,230]. PDT consists of a two-step protocol: 1) a non-toxic photosensitizer is selectively uptaken by tumor cells; 2) the photosensitizer is further excited locally by irradiation with monochromatic light of appropriate wavelength and undergoes a sequence of photo-oxidation reactions. A significant amount of ROS is generated, which induces tumor cell death. Therefore, PDT is a highly tumor-targeted therapy, with minimal side-effects if the photosensitizer is not cytotoxic by itself. Singlet oxygen is considered to underlie the ROSmediated therapeutic effect of PDT, whilst other ROS generated simultaneously seem to be less involved [231,232]. ROS generation is due to light-excitation of the photosensitizing molecule to an excited singlet state, which may undergo thereafter inter-system crossing, ending up in a relatively long-lasting triplet state. This triplet state can either exchange an electron or a hydrogen atom with adjacent molecules (type I photochemical reaction) or can transfer energy to molecular oxygen, resulting singlet oxygen (type II photochemical reaction) [233-235]. Singlet oxygen is a highly reactive ROS that interacts with proteins, nucleic acids and lipids. Singlet oxygen has a short lifetime within the cell, can migrate in tissues less than 20nm after its formation and therefore, the induced injury is localized near the site of singlet oxygen production [236-238]. Nevertheless, generation of about 9 x 108 molecules of singlet oxygen per tumor cell significantly reduces the cell surviving fraction [238]. The biological effects of PDT are highly dependent on tumor cell type and evolution stage, the availability of molecular oxygen and the intrinsic oxidative stress of cancer cells [239]. Various ROS-mediated signaling cascades are activated in cancer cells exposed to PDT and are translated into adaptive or cell death responses [240]. In contrast to radiotherapy and chemotherapy, that elicit a slow host response, the stress and damage inflicted by ROS in PDTtreated tumors cause a rapid and massive release of danger signals (oxidative stress, release of intracellular components), that are promptly recognized by the host as an acute localized insult [241-243]. PDT leads to a molecular interplay between cell death pathways, balancing between apoptosis, necrosis and autophagy. If the photosensitizer is located in mitochondria, apoptosis is triggered [244,245]. When photosensitizers localize in the plasma membrane, singlet oxygen triggers tumor cell death by necrosis [246-248]. The shift from apoptosis to necrosis in PDT-
treated tumors and the associated inflammatory response are controlled by polyADP-ribose polymerase [249]. Xue et al. (2008) [250] showed that cells incapable of undergoing apoptosis can turn to autophagy as the dominant cell death pathway. A huge effort is currently focused towards developing new photosensitizers. Synthetic variants are preferred because they allow a straight forward relationship photosensitizer biological effect. The so called "second generation" of photosensitizers has been developed, comprising modified porphyrins, chlorins, bacteriochlorins, phthalocyanines, naphthalocyanines, pheophorbides and purpurins, and metabolic precursors, like the aminolevulinic acid. They are designed to penetrate deeper in cancer tissues due to improved photo-physical properties (higher activation wavelengths) and to be more efficient in singlet oxygen generation. Several other properties of photosensitizers, like aggregation, ionic charge, solubility, partition between aqueous and lipid phases, play a key role in PDT, by providing enhanced uptake of photosensitizers by cancer cells, tumor selectivity and short-term retention in cells and tissues. In spite of draw-backs, related to photosensitation and light accessibility at tumor site, PDT remains a promising anti-neoplastic treatment due to its high tumor targeting action and low side-effects. ROS and Chemotherapy Most of anti-neoplastic agents (antifolates, nucleoside and nucleotide analogs, vinca alkaloids, taxanes, etoposide, campthotecins) have well established mechanisms of action that do not involve free radical intermediates or free radical g e n e r a t i o n . M e a n w h i l e , it is g e n e r a l l y accepted that anthracyclins, most alkylating agents and platinumcoordination complexes exert, at least partly, their anti-tumor activity or toxic side-effects by generating oxidative stress. Drug-induced oxidative stress is carried out via enzymatic pathways related to hepatic microsomal monooxygenase system, xanthine oxidase, Fenton and Haber-Weiss reactions or the mitochondrial electron transport chain [144]. In cancer cells, the drug-induced oxidative stress superimposes on the i n t r i n s i c one, resulting potent ROS-mediated cytotoxic processes that preferentially kill tumor cells or inhibit their proliferation. Novel therapeutic strategies take further advantage of oxidative stress and particular redox reactions. Promising results were obtained with acridine-based anticancer drugs which bind to DNA by intercalation and either donate to, or accept electrons from DNA. Therefore these drugs participate in long-range electron transfer reactions and consequently exert anti-neoplastic activity [251,252]. We will further on emphasize particular issues regarding ROS-generating anti-neoplastic chemotherapies and their interaction with modulators of the cellular redox balance. Redox-Related Decrease of Anti-Neoplastic Drugs Efficacy Although oxidants represent a therapeutic weapon against cancer cells, they may lower the efficacy of certain anti-neoplastic chemotherapies.
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The constitutive oxidative stress in cancer cells can block progression through restriction checkpoints [144,253], hence causing cell cycle arrest at the G1, S, G2 or M phase. Therefore, the intrinsic oxidative stress of tumor cells may diminish the anti-neoplastic action of chemotherapeuticals that exhibit cell cycle phase-specific activity. Moreover, checkpoint arrest may permit DNA repair of damages caused by anti-neoplastic agents, whatsoever their mechanism of action. Oxidative stress-generated aldehydes can also be involved in cell cycle arrest, due to their inhibitory action on cyclin-dependent kinases. Electrophilic aldehydes impede apoptosis by inhibiting the activity of caspases through binding to their active site. Accordingly, the pro-apoptotic action of certain anti-neoplastic agents is hindered in an oxidative environment and therefore antioxidant therapy might be beneficial. The endogenous antioxidant system of cancer cells may be responsible, in particular cases, for the low efficacy of anti-neoplastic drugs [144,253]. Thus, GSH, which is a potent reducing agent, has also nucleophilic properties related to its sulfhydryl group and consequently binds to and inactivates electrophilic intermediates of anti-neoplastic agents which act via nucleophilic substitution reactions (platinum coordination complexes and most alkylating agents). Moreover, nucleophilic selenium proteins with antioxidant properties trigger synthesis of cysteine-rich methalothioneins which bind to and inactivate electrophilic intermediates of anti-neoplastic drugs. Based on the above presented findings, we point out that tumor cells are endowed with redox-related mechanisms to escape the anti-neoplastic action of chemotherapeutic agents. Therefore, when particular tumor cells do not respond or become resistant to chemotherapy, besides taking into consideration resistance mediated by P-glycoprotein, investigation of cellular redox status may provide valuable information about the cellular response to chemotherapy. Endogenous Antioxidants as Pro-Oxidants in Tumors Treated with Chemotherapeuticals At least in vitro, it was demonstrated that anti-neoplastic therapies might benefit from co-administration of particular antioxidants which are able to improve anti-tumor activity or reduce side-effects [144,253]. The therapeutic approach coupling oxidative stress-inducing agents with particular antioxidants, gains increasing interest and validity in cancer treatment [252], but the interference of endogenous antioxidants with chemotherapy remains a controversial issue. Yokomizo et al. (1995) [254] and Sasada et al. (1996) [255] reported that thioredoxin can confer to cancer cells resistance against ROS-generating anti-cancer drugs, because of its antioxidant activity. Conversely, Ravi et al. (2005) [256] demonstrated that thioredoxin can enhance the cytotoxicity of daunomycin in human breast carcinoma MCF-7 cells, thus revealing surprising pro-apoptotic and even pro-oxidant activities of thioredoxin in cancer cells. Daunomycin exert anti-tumor effects by undergoing redox cycling which results in ROS generation and formation of semiquinone radical, both of them causing DNA damage and consequent apoptosis of
tumor cells. Thioredoxin was shown to enhance daunomycin cytotoxicity by two mechanisms: 1) enhanced drug-induced generation of superoxide radical via the NADPH-oxidase pathway, which requires overexpression of thioredoxin; 2) thioredoxins provide reducing equivalents for redox cycling of the drug towards the DNA-intercalating semiquinone radical. According to these findings, thioredoxin action might turn into a pro-apoptotic and pro-oxidant one in cancer cells treated with particular chemotherapeuticals. Meanwhile, thioredoxin preserves its anti-oxidant and anti-apoptotic properties in normal cells, even when these cells are treated with anti-neoplastic agents [256]. The nature of the oxidative burst (superoxide anion versus hydrogen peroxide), critical amounts of ROS, the cellular intrinsic oxidative status and apoptotic machinery, on one hand, and the action mechanism of chemotherapeuticals on the other hand, may dictate the action of the thioredoxin system as pro- or anti-oxidant, proor anti-apoptotic. Synergy Between ROS-Inducing Chemotherapies and Exogenous Antioxidants Arsenic treatment of leukemia has resurfaced after reports from China showing a high remission rate in acute promyelocytic leukemia (APL) treated with arsenic, even in those cases resistant to conventional therapy with all-trans retinoic acid [257]. Arsenic trioxide is currently also used for treating multiple myeloma. In APL, arsenic trioxide down-regulates the promyelocytic leukemia-retinoic acid receptor alpha, the gene product of the chromosomal translocation characteristic for APL [258]. Additionally, arsenic trioxide induces a significant accumulation of ROS and triggers oxidative stress in leukemia cells [259,260] by interfering with the mitochondrial electron transport chain [84]. The intracellular redox status seems to be important in predicting the response of a particular tumor cell to arsenic trioxide, probably accounting for the good therapeutic efficacy of the drug in APL, but not in other cancers [261]. Paradoxically, redox-regulatory factors generally accepted as antioxidants were shown to enhance the antineoplastic action of arsenic trioxide by exerting proapoptotic and pro-oxidant effects. Thus, ascorbic acid can potentate the cytotoxicity of arsenic trioxide due to ascorbate-generated hydrogen peroxide, which shifts above a critical threshold the oxidative stress triggered by arsenic trioxide in multiple myeloma [262]. Starting from these observations, Diaz et al. (2005) [258] recently showed that a vitamin E analog with antioxidant properties (Trolox) acts sinergistically with arsenic trioxide in inducing ROS-mediated apoptosis of tumor cells (leukemia, myeloma, breast carcinoma). Additionally, electron paramagnetic resonance studies indicated that arsenic trioxide can activate Trolox to a potentially tumoricidal phenoxyl radical. Due to inactivation of Coenzyme Q reductase by arsenic trioxide, the lifespan of this harmful radical is enhanced. It is noteworthy that Trolox is not able to enhance apoptosis mediated by other ROSgenerating chemotherapeutic drugs, like doxorubicin, cisplatin, etoposide. Furthermore, Yu et al. (1997) [263] have shown that Trolox protects in vitro peripheral blood
mononuclear cells from the toxic attack of arsenic trioxide. Accordingly, Trolox has the advantage of enhancing the killing of particular tumor cells treated with arsenic trioxide, while preserving the anti-tumor immune defense mechanisms. Although designed as antioxidant, Trolox acts as a pro-oxidant and pro-apoptotic agent in certain cancer cells, whilst preserving its protective antioxidant effects on normal cells. Antioxidants in Cancer Therapy An increased expression of Mn-SOD in primary ovarian cancer tissues [264-266] and in blood samples from patients with various types of leukemia [267,268] was reported. This may in fact reflect an adaptive mechanism by which cancer cells respond to increased mitochondrial oxidative stress. ROS themselves may induce the up-regulation of Mn-SOD by modulating the redox states of transcription factors (AP1, NF-kappaB) which further bind to the promoter of MnSOD [266,269]. Due to its elevated expression in certain cancers, Mn-SOD might be considered a tumor marker [270]. As cancer cells present an enhanced oxidative burst, we may presume the followings: increased Mn-SOD expression in tumors is not able to counteract active ROS production, or the enzyme is functionally disturbed, or the mechanism underlying the intrinsic oxidative status of tumor cells is not primarily confined to generation of superoxide anion in mitochondria. Inhibition of SOD with 2-methoxyestradiol was demonstrated to induce apoptosis in leukemia cells through a free radical-mediated mechanism, without exhibiting significant toxicity in normal lymphocytes [271]. Tumor selectivity of 2-methoxyestradiol is related to the enhanced constitutive oxidative stress [265]. Further increase of ROS by combining SOD inhibition, induced with 2methoxyestradiol, with oxidative burst, induced by arsenic trioxide, enhances apoptosis of leukemia cells [271]. Vitamins as Chemotherapeutic Agents Compelling evidence obtained in preclinical studies show that vitamins that were generally accepted as antioxidants may paradoxically act as pro-oxidants and kill tumor cells. Vitamin C behaves mainly as pro-oxidant in cancer, by recycling between ascorbate and dehydroascorbate in the redox cycle, leading to hydrogen peroxide production. Due to the low amounts of catalase in cancer cells, hydrogen peroxide accumulates and triggers a significant oxidative stress, additional to the intrinsic one [214,215]. Moreover, tumor cells have an enhanced ability to incorporate vitamin C via glucose pumps, highly expressed in cancer cells [272]. According to these findings, vitamin C can be used as an effective tumor cell growth inhibitor and/or apoptosis inducer. Menadione potentates the cytotoxic action of vitamin C against tumor cells, even at non-cytotoxic concentrations of both compounds. Menadione, also known as vitamin K3, is a synthetic derivative of the naturally occurring vitamins K1 and K2. Menadione is effective in vitro against a variety of tumor cells due to its cytotoxicity mediated by oxidative stress and covalent bonding (arylation process) [273,274]. As reviewed by Verrax et al. (2008) [83], the synergism of ascorbate/menadione combination is selective for cancer cells and is related to ROS production. Menadione is non-
enzymatically reduced by ascorbate to form semidehydroascorbate and a semiquinone radical which is rapidly reoxidized to its quinone form by molecular oxygen, thus generating superoxide anion. Dietary Antioxidants in Cancer Pros and Cons Antioxidants may be, at least theoretically, a valuable adjuvant therapy in cancer, considering that anti-neoplastic therapies have unwarranted side-effects related to oxidative stress. Dietary antioxidants can overcome, at least in vitro, the inefficiency of the endogenous antioxidants of normal cells against ROS challenge. The benefits from dietary antioxidant consumption, albeit being a highly researched topic, still remain debatable. We highlight the conclusion of the meta-analysis performed by Bjelakovici et al. (2007) [275] on 68 randomized trials with over 200.000 adults comparing beta carotene, vitamin A, vitamin C, vitamin E and selenium, versus placebo or no intervention. This systematic review showed that beta carotene, vitamin A and vitamin E, given single or combined with other antioxidant supplements, significantly increase all-cause mortality of adults included in primary and secondary prevention trials. No clear evidence was found that vitamin C may increase mortality and only selenium tended to reduce mortality. Further development of anti-neoplastic therapies has to take into account that all antioxidants cannot be treated as equal when evaluating their impact on cancer chemotherapy, and that an individual antioxidant cannot be anticipated to have the same impact on the activity of all chemotherapeutic agents [144]. We must be aware that by eliminating free radicals from our organism we may interfere with some essential defensive mechanisms, like apoptosis and phagocytosis, and also with key signaling pathways responsible for homeostasis [276]. In-depth investigation has to be performed in order to assess the narrow redoxconditions required for optimal drug efficacy versus sideeffects. WHERE TO? Obviously, towards the development of novel therapeutical strategies in cancer, intended to target distinctively tumor cells, whilst preserving as much as possible the functionality of normal ones, including the antitumor immune defense. Additionally, by exploiting the novel technologies and research findings, reevaluation of existing therapies may lead to considerable improvement, mainly linked to increased efficacy and limited side-effects resulting from combining therapeutic approaches. We consider that the first step in developing novel or improved anti-neoplastic therapies targeted towards the redox balance is to perform extensive studies on the consequences of enhanced oxidative stress at cellular and systemic level and to identify the corresponding particularities of a broad panel of cancers. The progress in genomics, metabonomics, proteomics and systems biology offers the technological background for assessing complex responses to oxidative stress. Such an endeavor could provide a comprehensive description of oxidative stressassociated biomarkers in health and disease, early indication of disease onset/progression and of the response to a particular therapy. It could also represent a powerful mean to
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identify novel pathways of ROS-mediated signal transduction. Furthermore, this methodology may reinforce the toxicological and pharmacological armentarium for assessing the toxicity of xenobiotics, of conventional and innovative therapeutics. Huge efforts are focused on the development of accurate methodologies for ROS measurement, aiming to characterize in detail cellular ROS fluxes, i.e. the nature, localization and time-course of ROS generation and spreading in cellular models and organisms. Furthermore, a high priority is the development of complex 3D in vitro models of tumors using scaffolds, aiming to mimic in vivo condition (cell-cell and cell-matrix interactions, hypoxia, acidic environment etc.). There is an urgent need for standardization of the protocols for assessing ROS and the consequent oxidative stress, in order to establish networks and multicentric studies aiming to encompass in a coordinated manner a broad panel of tumors and normal tissues, of cancer patients and healthy subjects. Such an endeavor can provide reliable data for mathematical modeling of the biochemical networks in which oxidative stress is involved, in normal and in particular cancer cells. Redox-sensitive decision points of life and death can be thus defined comprehensively. Making use of structural biology and combinatorial chemistry, efficient antineoplastic therapies can be further developed. Additionally, novel biotherapeutic approaches aiming to exploit immune defense mechanisms are currently under investigation. Besides NK cells, macrophages and T lymphocytes, granulocytes proved to be promising candidates for targeted immune therapies exploiting their killing potential, partially confined to ROS production. We must be aware that ROS exert at cellular level a multifaceted role, by acting both as cytotoxic agents and signal transducers involved in physiological cell proliferation and death. Therefore, anti-neoplastic therapies targeting ROS or the antioxidant system actually play on the edge, to kill tumors and to preserve the normal cells homeostasis. Accordingly, future development of therapies targeting the cellular redox status has to take into account the complex network of physiological and pathological pathways in which the tightly regulated redox balance is involved. ACKNOWLEDGEMENTS We would like to thank to Professor Sanda Clejan from Tulane University, New Orleans LA, USA for revising the paper, and to Ionela Neagoe and Catalin Manole from Victor Babes National Institute of Pathology, Bucharest, Romania, for editing chemical structures and figures. ABBREVIATIONS AIF AP-1 Apaf-1 APL = = = = Apoptosis inducing factor Activator protein-1 Apoptotic protease activating factor-1 Acute promyelocytic leukemia Ataxia-telangiectasia mutated protein kinase 2,4-dinitrophenylhydrazine
ECD ER ERK GC GSH GSSG HIF-1 HO-1 HPLC IAP JNK LC MAPK MS Omi/HtrA2
= = = = = = = = = = = = = = =
Electrochemical detection Endoplasmic reticulum Extracellular signal-regulated kinases Gas chromatography Reduced glutathione Oxidized glutathione Hypoxia inducible factor-1 Heme oxigenase-1 High performance liquid chromatog raphy Inhibitor of apoptosis protein c-Jun N-terminal kinases Liquid chromatography Mitogen-activated protein kinase Mass spectrometry Omi stress-regulated endoprotease/high temperature requirement protein A2 Photodynamic therapy Redox factor-1 Radiation-induced genomic instability Reactive oxygen species Surface-enhanced laser desorption/ionization Superoxide dismutase Thiobarbituric acid-reacting substance Thioredoxin
PDT Ref-1 RIGI ROS SELDI SOD TBARS Trx
= = = = = = = =
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Received: April 14, 2008
Revised: July 23, 2008
Accepted: July 24, 2008

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342

Current Chemical Biology, 2009, 3, 342-366

Reactive Oxygen Species, Cancer and Anti-Cancer Therapies

Reactive Oxygen Species, Cancer and Anti-Cancer Therapies

Current Chemical Biology, 2009, Vol. 3, No. 1 343

Cellular Mechanisms of ROS Generation1

superoxide anion (O2-)

hydrogen peroxide (H2 O2)

where M = Cu (n=1); Mn (n=2); Fe (n=2); Ni (n=2). Neutralization:

hydrogen peroxide is quickly converted to water by catalase: 2 H2O2 2 H2 O + O2

Three-electron reduction state of molecular oxygen. Generation:

hydroxyl radical (OH)

it is formed by the Fenton reaction: Fe2+ + H2 O2 Fe3+ + OH + OH

is further recycled, according to the reaction: Fe3+ + H2 O2 Fe2+ + OOH + H +

it is also formed in the Haber-Weiss reaction: H2O2 + O 2- O2 + OH + OH

Reactive Oxygen Species, Cancer and Anti-Cancer Therapies

Current Chemical Biology, 2009, Vol. 3, No. 1 345

Validated Biomarkers of Oxidative Stress in Serum, Plasma and/or Urine2

Markers of Oxidative Stress

TBARS spectrophotometric assay HPLC-based TBARS assay GC-MS

Lipids F2-isoprostanes (8-iso-PGF2)

Immunoassays GC-MS, LC-MS

Proteins (carbonyl groups) 2-pyrrolidone

DNPH spectrophotometric assay One- and two-dimensional electrophoresis MS Immunoassays

HPLC GC-MS Immunoassays

HPLC-ECD LC-MS, GC-MS Immunoassays

346 Current Chemical Biology, 2009, Vol. 3, No. 1

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Detection Method Cytochrome c reduction Chemiluminescence reactions with lucigenine or coelenterazine

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PDT Ref-1 RIGI ROS SELDI SOD TBARS Trx

ATM-protein = kinase DNPH =

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