Targeting antioxidants for cancer therapy.

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BCP-12033; No. of Pages 12 Biochemical Pharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm

Review

Targeting antioxidants for cancer therapy Andrea Glasauer a,b,*, Navdeep S. Chandel b a b

Bayer Pharma AG, Global Drug Discovery, Therapeutic Research Group Oncology/Gynecological Therapies, 13353 Berlin, Germany Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 May 2014 Received in revised form 16 July 2014 Accepted 17 July 2014 Available online xxx

Cancer cells are characterized by an increase in the rate of reactive oxygen species (ROS) production and an altered redox environment compared to normal cells. Furthermore, redox regulation and redox signaling play a key role in tumorigenesis and in the response to cancer therapeutics. ROS have contradictory roles in tumorigenesis, which has important implications for the development of potential anticancer therapies that aim to modulate cellular redox levels. ROS play a causal role in tumor development and progression by inducing DNA mutations, genomic instability, and aberrant protumorigenic signaling. On the other hand, high levels of ROS can also be toxic to cancer cells and can potentially induce cell death. To balance the state of oxidative stress, cancer cells increase their antioxidant capacity, which strongly suggests that high ROS levels have the potential to actually block tumorigenesis. This fact makes pro-oxidant cancer therapy an interesting area of study. In this review, we discuss the controversial role of ROS in tumorigenesis and especially elaborate on the advantages of targeting ROS scavengers, hence the antioxidant capacity of cancer cells, and how this can be utilized for cancer therapeutics. ß 2014 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: L-Buthionine-sulfoximine(PubChem CID: 119565 Phenethyl isothiocyanate(PubChem CID: 160602) Lanperisone(PubChem CID: 198707) Elesclomol(PubChem CID: 300471) Imexon(PubChem CID: 68791) Piperlongumine(PubChem CID: 637858) Sulphasalazine(PubChem CID: 5359476) Motexafin(PubChem CID: 12047567) ATN-224(PubChem CID: 18442052) Keywords: Antioxidants ROS Cancer Pro-oxidants Therapy Metabolism

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS – the Basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Spatially localized sources of ROS . . . . . . . . . . . . 2.1. Intracellular regulation of ROS . . . . . . . . . . . . . . . 2.2. ROS and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ROS regulate cell signaling . . . . . . . . . . . . . . . . . . 3.1. ROS stress and adaptation in cancer cells . . . . . . 3.2. 3.2.1. ROS stress in cancer cells . . . . . . . . . . . ROS adaptation of cancer cells (Fig. 2) . 3.2.2. ROS promote pro-tumorigenic cell signaling . . . . 3.3. ROS promote tumor cell proliferation . 3.3.1. ROS promote tumor cell survival . . . . . 3.3.2.

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* Corresponding author at: Muellerstrasse 178 S107, 7th Floor, Room: 7. 302 13353 Berlin, Germany Tel.: +49 30 468 194467; fax: +49 30 468 94535. E-mail address: [email protected] (A. Glasauer). http://dx.doi.org/10.1016/j.bcp.2014.07.017 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Glasauer A, Chandel NS. Targeting antioxidants for cancer therapy. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/j.bcp.2014.07.017

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3.3.3. ROS promote angiogenesis and metastasis. . . . . . . . . . . . . . . . . . . . . ROS promote anti-tumorigenic cell signaling . . . . . . . . . . . . . . . . . . . . . . . . . . ROS promote cell death through the ASK1/JNK/P38 MAPK signaling 3.4.1. ROS regulate cell cycle arrest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Modulating ROS levels to treat cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decrease ROS levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Increase ROS levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Target antioxidants to decrease ROS scavenging capacity . . . . . . . . . . . . . . . . 4.3. 4.4. Metabolic and redox modulation: combination therapy option . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.

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1. Introduction ROS act as secondary messengers in cell signaling and are required for various biological processes in normal cells. Under physiological conditions, ROS are continuously generated by ROS producers and eliminated through ROS scavenging systems in order to maintain redox homeostasis. Cells aim to maintain a redox balance that is ideal to support cellular processes like differentiation and proliferation and allow for the adaptation to metabolic and immune stress. Changes in redox balance, which can have endogenous or exogenous causes, can either lead to an increase in ROS levels or rate of production, resulting in cell damaging oxidative stress and aberrant cell signaling, or a decrease in ROS, leading to a disruption of cell signaling and therefore disruption of cellular homeostasis. Redox imbalance, oxidative stress, which are often a result of changes in cancer cell metabolism, and aberrant antioxidant levels to balance this stress, are hallmarks of many cancers [1]. The role of ROS in cancer is two-sided. One the one hand, ROS can contribute to cancer initiation, progression and spreading through the activation and maintenance of signaling pathways that regulate cellular proliferation, survival, angiogenesis and metastasis [2–4]. Through this role in promoting tumorigenic cell signaling events, ROS are considered oncogenic. However, on the other hand, the excessive levels of ROS in cancer cells can also induce cell death signaling, senescence and cell cycle arrest [5,6]. In the context of this imbalanced redox status, oncogene-induced cancer cells adapt and increase antioxidant pathways and regulators [7–12] leading to increased ROS scavenging [13,14], in order to maintain ROS levels that allow pro-tumorigenic signaling pathways to be activated without inducing cell death. Various studies have shown that further ROS elevation, either through ROS producers or antioxidant inhibitors, can selectively kill cancer cells and suppress tumor growth and progression in various cancer cell lines [15–22]. The contradictory, ‘‘doubled-edged-sword’’ property of ROS allows for the induction of cancer cell survival or death depending on intracellular ROS levels. Hence, ROS-manipulation strategies, meaning ways to eliminate or produce ROS in cancer cells, can potentially be effective in cancer therapies. Non-transformed, normal cells have a different redox environment compared to cancer cells and are therefore less sensitive to redox manipulation. The varying redox status of normal cells compared to cancer cells is mainly characterized by the lower rate of ROS production and therefore the decreased requirement for ROS-scavenging mechanisms. Redox modulation cancer therapy is a young and unexplored field and little research shows efficient results with respect to treatment mechanisms or utility in pre-clinical animal models. This review will focus on exploring the two-sided role of ROS in tumorigenesis, and how understanding ROS signaling and the aberrant redox status of cancer cells can be used for the development of novel anticancer therapeutics. To that end, special

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consideration will be paid to the idea of increasing ROS and targeting cancer cells’ antioxidant systems for therapy.

2. ROS – the Basics 2.1. Spatially localized sources of ROS ROS are intracellular chemical species that contain oxygen and are reactive towards lipids, proteins and DNA. ROS include the superoxide anion (O2 ), hydrogen peroxide (H2O2), as well as hydroxyl radicals (OH). Different types of ROS have different intrinsic chemical properties, which dictate their reactivity and preferred biological targets. The two main sources of ROS are mitochondria and the family of NADPH oxidases (NOXs) [23] (Fig. 1). The three best-characterized sites in the mitochondria are complex I, II and III within the mitochondrial electron transport chain, which is located in the inner mitochondrial membrane [24]. These complexes generate superoxide by the one-electron reduction of molecular oxygen. Complex I, II, and III release superoxide into the mitochondrial matrix where superoxide dismutase 2 (SOD2) rapidly converts it into H2O2. Complex III can also release

Fig. 1. Sources, regulation and biological outcomes of ROS. Mitochondria can release either superoxide (O2 ) or H2O2. In the cytosol O2 is converted to H2O2 by SOD1. NADPH oxidases can also generate O2 in the cytosol. Catalase, GPXs and PRXs can convert H2O2 to water. Upon reaction with ferrous or cuprous ions, H2O2 forms OH radicals that can be damaging to DNA, proteins and lipids. H2O2 controls cell signaling through the oxidation of thiols on proteins. Different levels of H2O2 lead to different cellular outcomes. Intracellular H2O2 concentrations in the low nanomolar range provide a permissive oxidative environment for cellular signaling which is ideal to maintain homeostasis (e.g. differentiation and proliferation) and to adapt to stress (e.g. metabolic). H2O2 levels below this optimal range lead to a disruption of cell signaling resulting in loss of homeostasis. H2O2 levels above the optimal range cause oxidative damage and aberrant cell signaling resulting in pathologies including cancer. Very high levels of H2O2 can promote cell death.


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superoxide into the mitochondrial intermembrane space. Superoxide traverses through voltage dependent anion channels (VDAC) into the cytosol and is converted into H2O2 spontaneously or by superoxide dismutase 1 (SOD1) [25]. Additionally, NOX2 triggers the transfer of electrons from intracellular NADPH across the cell membrane to oxygen, producing superoxide in the extracellular space. Here, superoxide can either enter the cell through chloride channels or SOD3 dismutates superoxide into H2O2, which freely diffuses across membranes [26]. It is worth mentioning that SOD proteins have the largest kcat/KM (catalytic efficiency) of any known enzyme (7 109 M 1 s 1), making the dismutation of superoxide to H2O2 diffusion-limited [27]. In the presence of ferrous or cuprous ions, mitochondrial and NOX-produced H2O2 can become OH in a process called the Fenton Reaction. OH are very reactive and cause oxidation of lipids, proteins and DNA resulting in damage to the cell. Due to its extremely high reactivity, OH have a short half-life and their diffusion is limited to their site of production. Another cell damaging form of ROS is peroxynitrite, which is generated from the reaction of superoxide with excess nitric oxide [28]. In contrast, H2O2 is the most stable form of ROS and, in the low nanomolar range can impinge on cellular signaling by interacting with select cysteine residues on target proteins [29] (Figure 1). Unlike superoxide and OH, H2O2 readily diffuses through membranes making it an ideal intracellular signaling molecule. Here, it is important to note that mitochondria and NOX proteins can spatially localize to the signaling proteins that are responsive to the produced ROS. 2.2. Intracellular regulation of ROS Given the reactivity and toxicity of high levels of ROS, and given that specific, locally produced quantities of ROS determine various cellular signaling events (Fig. 1), spatial and temporal regulatory strategies must exist to regulate intracellular ROS levels. Superoxide produced by NOX proteins and the mitochondria can oxidize and damage iron-sulfur clusters of proteins. However, specific SOD enzymes can localize to the mitochondria (SOD2), the cytosol (SOD1) and the extracellular matrix (SOD3) and convert superoxide to H2O2 at a rate that is diffusion-limited (catalytic efficiency: Kcat/Km 7 109 M 1 s 1) [30]. Therefore intracellular superoxide concentrations are very low. H2O2, the more stable and diffusible form of ROS, is selectively reactive towards cysteine residues on proteins and therefore can control cell signaling. There are ample mechanisms in place that convert intracellular H2O2 to water and therefore control cysteine modifications, protein and signaling pathway activity. Peroxiredoxins (PRXs) have emerged as critical regulatory systems that quench H2O2 [27]. Six mammalian PRXs have been identified and are located in the cytosol and mitochondria. PRXs function by undergoing H2O2-mediated oxidation of their active site cysteines. This process removes H2O2 from the cell and inactivates PRXs. The inactivation can be reversed by thioredoxin (TRX), thioredoxin reductase and the reducing equivalent NADPH [31]. Glutathione peroxidases (GPXs) can also convert H2O2 to water in the cytosol and mitochondria. The eight known GRXs function similar to PRXs and eliminate H2O2 through the H2O2-mediated oxidization of glutathione (GSH), the most abundant antioxidant in the cell [32]. GSH can get reduced and therefore re-activated by glutathione reductase and NADPH. Lastly catalase is found in peroxisomes and removes intracellular H2O2 without cofactors (Fig. 1). In addition to antioxidants, transcription factors nuclear factor erythriod 2-related factor 2 (NRF2) and forkhead box O (FOXO) are master regulators of antioxidant expression. Under normal conditions NRF2 localizes in the cytoplasm, where it interacts with the actin-binding protein, Kelch-like ECH-associated protein 1 (KEAP1) [33,34]. KEAP1 functions as an adaptor of Cul3-based E3

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ubiquitin ligase and targets NRF2 for proteasomal degradation [35]. Upon pathway activation, NRF2 dissociates from KEAP1 and translocates to the nucleus, heterodimerizes with small proteins (MAFs) and then binds to antioxidant-responsive elements (AREs) within regulatory region of many genes. NRF2 modulates transcription of around 200 genes whose protein products function as antioxidants, detoxification enzymes, heat-shock proteins and GSH-synthesis enzymes. These proteins all play a role in cellular defense against oxidative stress [36]. NRF2 also activates antioxidant enzymes such as heme oxygenase-1, PRXs, catalase, GPXs, SOD2 and TRX (Fig. 1). Furthermore, NRF2 regulates the de novo synthesis of GSH by (1) activating the gene expression of glutamylcysteine synthetase components [33] [12] and by (2) controlling the abundance of cysteine within the cells, which is a required substrate for GSH synthesis. This is regulated through NFR2-mediated expression of the cysteine/glutamate antiporter (xCT) [37]. In exchange for glutamate, xCT imports cysteine. FOXOs also control ROS levels by promoting the expression of genes associated with cell cycle arrest, apoptosis, tumor suppression and antioxidant defense [38,39]. Regulating intracellular ROS levels through ROS production and ROS elimination is essential to control various cell signaling events and is utilized by normal cells and cancer cells to support the signaling required for cell proliferation, angiogenesis, invasion and survival. Interestingly, cancer cells also utilize ROS scavenging systems, including NRF2 and the related antioxidant systems, to control their oxidative stress phenotype to prevent cell death [1,40]. This following section will focus on how ROS promote cell signaling which lays the foundation to investigate the contradictory role of ROS in cancer. 3. ROS and cancer 3.1. ROS regulate cell signaling Traditionally ROS have been thought of as toxic metabolic byproducts, which cause cellular damage. However, various studies over the last decade have shown that low levels of ROS, which are spatially localized produced, permit for a large range of effects that allow for the maintenance of homeostasis [41,42]. Upon stress stimuli these baseline levels of ROS can fluctuate to control biological outcomes and regulate cell signaling by directly reacting with, and modifying the structure of proteins, transcription factors and genes, to modulate their functions. The following sections will discuss how ROS control cell signaling, describe the ROS status of cancer cells and the pro- as well as anti-tumorigenic roles of ROS, which can be potentially exploited therapeutically. ROS are produced at specific cellular locations by mitochondria and NOX proteins. The ROS entity H2O2 can reversibly oxidize cysteine residues on proteins. The best-characterized classes of redox-regulated targets are phosphatases, which can negatively regulate cell signaling through the de-phosphorylation and inactivation of kinases [43,44]. However, kinases, transcription factors and antioxidant proteins like GSH and PRX are also direct physiological targets of H2O2 [27,45,46]. The redox-regulatory properties of cysteine depend on protein context. Protein tyrosine phosphatase active sites are susceptible to redox regulation because they possess a common motif (Cys-X5-Arg), which renders their active site cysteine (SH) in a thiolate state (S ). Thiolated cysteines are nucleophiles (pKa = 4.7–5.4), making them highly susceptible to H2O2-oxidation, which results in the generation of different, reversible redox forms. Reversible modifications include sulfenic acid (SO ), disulfide bonds (S–S) and sulfenic–amide bonds (S–N), which can inactivate protein activity. Prolonged oxidative stress can also lead to hyper-oxidation of protein targets resulting in irreversible modifications, which


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include sulfinic (SO2 ) or sulfonic acids (SO3 ) [47]. H2O2regulated signaling pathways include pro-survival and proproliferative pathways like phosphatidylinositol 3-kinase (PI3K)/AKT, hypoxia-inducible factor (HIF) and mitogen-activated kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling cascades [4], but also death-inducing pathways like c-Jun Nterminal kinase (JNK) and P38 MAPK [5,22,48]. Additionally to H2O2, superoxide can react and inactivate iron-sulfur proteins like aconitase, which can be toxic to the cell [26]. However, the reaction of superoxide with protein thiols is not fast enough to compete with the powerful SOD enzymes which rapidly dismuate superoxide, making H2O2 the dominant signaling ROS [27]. 3.2. ROS stress and adaptation in cancer cells ROS homeostasis is required for proper cell signaling and cellular fitness in normal cells. ROS levels in the nanomolar range can be potent mitogens and initiate cellular adaptations that lead to cell survival, growth, proliferation and angiogenesis in a controlled manner. However, higher ROS levels can be toxic to the cell and therefore can trigger signaling pathways initiating cell proliferation arrest or even cell death. 3.2.1. ROS stress in cancer cells Fig. 2 Cancer cells are characterized by an increased rate of spatially localized ROS production, compared to normal cells due to a loss in proper redox control [1,49]. This occurs in order to hyper-activate signaling pathways that promote cell proliferation, survival and metabolic adaptation to the tumor microenvironment. For example leukemia cell samples from patients showed increased ROS production compared to normal lymphocytes [50] and solid tumor patient samples had increased levels of oxidative DNA damage [51]. Reasons for the increased rate of ROS production in cancer cells are alterations in signaling pathways that affect cellular metabolism. The tumor suppressor P53 has a crucial role in initiating antioxidant genes in response to oxidative stress to prevent DNA, protein or lipid damage and to restore redox balance [52]. P53 mutations or the loss of P53 are seen in over 50% of human cancers and are associated with increased ROS stress and aggressive tumor growth [53]. Furthermore, oncogenes that lead to increased ROS production include RAS, MYC and AKT [54]. Downstream of RAS, the PI3K/AKT/mTOR survival pathway is

activated in a majority of cancers [55]. AKT activation is associated with ROS accumulation partially through the AKT/mTOR-dependent increase in mitochondrial metabolism [56] and the AKTdriven phosphorylation and inhibition of FOXO transcription factors [57], which promote antioxidant defense. Oncogenic activation of AKT can therefore result in increased ROS production from the mitochondria through metabolic pathways, and by impairing ROS scavenging though the inhibition of FOXOs [38]. Another tumor-associated condition with respect to ROS is hypoxia. Hypoxia is a feature of most tumors and arises because of oxygen diffusion limitations in avascular primary tumors or their metastases [58]. Research showed that hypoxia leads to an increase in mitochondrial ROS produced from complex III of the electron transport chain. ROS can subsequently stabilize HIF-1a [59]. The exact mechanism by which hypoxia leads to complex III ROS production is currently unknown. Mutations in nuclear encoded mitochondrial enzymes have also been found in certain cancers but exceed the scope of this review [3]. 3.2.2. ROS adaptation of cancer cells (Fig. 2) In order to survive the oxidative stress environment just described, cancer cells adapt and acquire mechanisms to counteract the potential toxic effects of ROS stress in order to promote proximal pro-tumorigenic signaling. More specifically, to avoid the detrimental effects of ROS, cancer cells actively upregulate various antioxidant proteins at the sites of ROS production [7] [60]. For example, RAS transformed cells, which are characterized by oxidative stress, were shown to have higher levels of PRXs and TRXs compared to normal cells [61]. The antioxidant master regulator NRF2 is also activated and stabilized by various oncogenes including KRAS, MYC and PI3K [11], which promote cancer cell growth and survival. Furthermore primary tissue samples from cancer patients showed increased levels of ROSscavenging enzymes such as GPXs, SODs and PRXs, and also mutations in the NRF2 inhibitor KEAP1, suggesting a control of redox balance in cancer cells, which aids tumorigenesis [8,62–65]. In the past, antioxidants were solely seen as tumor suppressors. However, recent research uncovered the ‘‘dark side of antioxidants’’ [66,67], which are used by cancer cells to promote survival and growth. Cancer cells function with higher rates of ROS production and higher levels of antioxidant proteins compared to normal cells in order to maintain redox homeostasis. This permits high ROS levels to activate proximal pro-tumorigenic signaling pathways without building up excessively high ROS, which could induce cell death or senescence. It is important to note that this balance leads to the fact that steady state levels of ROS in cancer cells might actually be similar to those in normal cells. While ROS contribute to cancer development and progression [51,68], oxidative stress can also potentially have anti-tumorigenic effects. The following sections will discuss how ROS can either promote or limit cancer, which sets the stage for redox modulation anti-cancer therapies. 3.3. ROS promote pro-tumorigenic cell signaling

Fig. 2. Cancer cells adapt to oxidative stress by up-regulating antioxidant proteins. Low levels of ROS can be mitogens and initiate cellular adaptations that lead to cell survival, growth, proliferation and differentiation in a controlled manner. Cancer cell characteristics like oncogene activation, enhanced metabolism, hypoxia or the loss of the tumor suppressor p53 can lead to an accumulation of ROS, which allows for hyper-active cell signaling and pro-tumorigenic signaling events. In order to counteract the oxidative stress and to prevent oxidative damage and cell death, cancer cells up-regulate ROS scavenging systems to keep ROS levels under the cytotoxic limit. Cancer cells function at ROS levels that are high enough to support pro-tumorigenic cell signaling without inducing cell death. Thus, cancer cells are ROS- as well as antioxidant-addicted.

ROS promote adaptation to hypoxia, cell proliferation, cell growth and survival in normal cells in response to various growth and survival stimuli [69]. This suggests that the hyperactive mitogenic and pro-survival signaling effects of ROS in cancer cells may be a universal occurrence. Indeed, research indicates that the increased rate of ROS production in cancer cells plays a causal role in the acquisition of various hallmarks of cancer: sustained cell proliferation and mitogenic signaling [4], increased cell survival and disruption of cell death signaling [70,71], epithelial to mesenchymal transition (EMT), metastasis [72] and angiogenesis [73].


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3.3.1. ROS promote tumor cell proliferation In various cancer cells, exogenous addition of H2O2 or endogenous oncogene-induced production of ROS have been shown to enhance proliferation by increasing pro-proliferative signaling pathways like PI3K/AKT/mTOR and MAPK/ERK cascades. ROS promote oxidation and inactivation of the phosphatases; protein tyrosine phosphatase 1B (PTP1B) [74,75] and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [43,76], which both inhibit PI3K/AKT signaling. Furthermore ROS inactivate pathway-inhibiting MAPK phosphatases to activate mitogenic signaling [77]. As described before (see Section 3.1), phosphatase inhibition is medicated through the oxidation of active site cysteines. ROS-mediated cancer cell proliferation was observed in breast [78], lung [2], liver [79] and various other cancer cells and was prevented by the addition of ROS-scavenging antioxidants [80,81]. Our lab has shown that mitochondrial-derived ROS are required for anchorage-independent growth in KRAS-driven lung cancer cells through the regulation of MAPK/ERK signaling. Furthermore, disruption of mitochondrial function, which increases ROS, reduced tumorigenesis in an oncogenic mouse model of lung cancer [2]. 3.3.2. ROS promote tumor cell survival As mentioned, aberrant ROS levels hyper-activate PI3K/AKT survival signaling through the inhibition of PTEN. ROS-induced PI3K/AKT signaling and survival was shown in a mouse model of prostate cancer [82] and various other cancers [15,83]. As previously stated, oncogenes including KRAS and AKT can also activate and stabilize the antioxidant master regulator NRF2 [20], which has been shown to play a critical role in the protection against oxidative stress to promote cell survival [33,84]. ROS can activate NRF2 through the oxidation and inactivation of KEAP1 at redox-sensitive cysteine sites. This oxidative modification inhibits KEAP1-mediated proteasomal degradation of NRF2, allowing for its stabilization, nuclear accumulation [85] and pro-survival signaling in cancer cells. Lastly, ROS also promote cell survival in colon cancer and melanoma cells through the activation of redoxsensitive nuclear factor kappa B (NFkB) signaling [86,87]. In response to ROS, active NFkB controls cell survival through the upregulation of various pro-survival genes including B-cell lymphoma 2 (BCL2), caspase inhibitors and antioxidant proteins [88,89]. 3.3.3. ROS promote angiogenesis and metastasis Tumor angiogenesis, invasion and metastasis are interrelated processes and induce the final, most devastating, stage of malignancy. Angiogenesis vascularizes solid tumors in order to provide nutrients and oxygen for continuous tumor growth. The main angiogenic growth factor triggering formation of new blood vessels is vascular endothelial growth factor (VEGF), which gets activated downstream of HIFs [90]. As mentioned previously, hypoxia is a hallmark characteristic of many cancers [58] and has been shown to increase ROS production [59]. ROS leads to HIFa stabilization through the inhibition of prolyl hydroxylases (PHDs) [91,92], which under normoxic conditions mark HIFa for proteasomal degradation [93,94], and subsequent VEGF activation. ROS have been shown to stabilize HIFa [95], leading to VEGF activation and subsequent angiogenesis and tumor progression; this effect could be prevented by the antioxidant N-acetyl-cysteine (NAC) [96,97]. NAC treatment also prevented HIF stabilization and diminished MYC-mediated tumorigenesis in vivo [80]. Angiogenesis allows for cancer cell migration and metastasis. Metastasis requires extracellular remodeling and intracellular adaptations including EMT, reduced cell adhesion, increased migration and degradation of tissue barriers and extracellular matrix [98]. ROS have been shown to mediate metastasis in various cancer cell types through the regulation of transcription factors and signaling components including MAPK and PI3K/AKT pathways, HIFs and

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the EMT regulator Snail [99–102]. ROS can also modulate structural changes in tumor cells like the formation of invasive microdomains called invadopodia [103], which promote cell invasion and metastasis. Furthermore ROS promote the activation of matrix metalloproteinases (MMPs) [104,105] that participate in the degradation of membranes and help detach primary tumor cells from extracellular matrix [106]. Taken together, these ROS-mediated events promote tumor progression. 3.4. ROS promote anti-tumorigenic cell signaling While ROS are associated with the activation of pro-tumorigenic survival and growth pathways, oxidative stress can also lead to the induction of cell death and cell cycle arrest. 3.4.1. ROS promote cell death through the ASK1/JNK/P38 MAPK signaling pathway Apoptosis signal-regulating kinase 1 (ASK1) has been shown to act as a redox sensor by mediating the sustained activation of JNK and P38 MAPK, resulting in apoptosis upon excessive oxidative stress [5]. In its inactive state, ASK1 is coupled to the reduced form of TRX1, which prevents ASK1 activation. TRX1 can be oxidized upon ROS at two cysteine residues, resulting in its dissociation from ASK1 [107]. Activated ASK1 can then trigger apoptosis through MAP kinase-kinase (MKK)4- and 7-mediated JNK activation or MKK3- and 6-mediated P38 activation [108]. JNK and P38 MAPKs trigger the expression of pro-apoptotic factors [109] and the down-regulation of anti-apoptotic factors [110–112]. P38 MAPK has been established as a tumor suppressor and ROS sensor in tumorigenesis [111]. Supporting this fact, various aggressive human cancer cell lines with high levels of ROS have impaired P38a activation and thus can avoid cell death signaling [113]. 3.4.2. ROS regulate cell cycle arrest High levels of ROS have been shown to inhibit cellular proliferation in order to prevent cell division through the oxidation and negative regulation of pro-proliferative kinases, which contain redox-sensitive cysteines. For example, ROS-induced P38/JNK MAPK signaling can lead to the down-regulation of cyclins and the induction of cyclin-dependent kinase (CDK) inhibitors [114,115] resulting in cell cycle arrest. ROS can also induce cell cycle arrest directly through the oxidation of various cell cycle regulators. One example is the H2O2-mediated oxidation and inactivation of the cell cycle phosphatase cdc25, which is required for cell cycle progression from G2 to M phase [116]. ROS-mediated cell cycle arrest has been demonstrated in many systems in vitro and recently also in a number of cases in vivo [117,118]. Furthermore ROS can induce reduction of tumor angiogenesis and metastasis [19,21]. In summary, ROS have a causal role in tumorigenesis but can also be toxic to the cell and can potentially induce cancer cell death, cell cycle arrest and inhibit cancer progression. Therefore cancer cells are dependent on maintaining high enough ROS levels that allow for pro-tumorigenic cell signaling without inducing cell death. The reliance of cancer cells on ROS homeostasis can be potentially exploited to target them therapeutically. The next section will focus on strategies to exploit the unique redox status of cancer cells for therapeutic purposes; a novel treatment strategy that potentially allows for the selective targeting of cancer cells. 4. Modulating ROS levels to treat cancer 4.1. Decrease ROS levels Due to the causal role of ROS in promoting cancer (see Section 3.3) and the fact that various antioxidant promoters like NRF2


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are considered tumor suppressors, so far, research focused on antioxidant treatments to dampen ROS levels as therapeutic strategies against tumorigenesis [119,120]. Antioxidant treatments include supplementation of natural ROS scavengers [121,122], treatment with exogenous antioxidants [123–125] and also other strategies that decrease oxidative stress like the disruption of the ROS-producing mitochondrial electron transport chain [2]. One of the most published on transcriptional effects of antioxidant treatments is the regulation of HIF1 levels. As mentioned, ROS regulate hypoxic activation of HIF [95] (see Section 3.3). Supporting this data, antioxidants like NAC and vitamin C have been shown to prevent HIF stabilization and diminish MYC-mediated tumorigenesis in vivo [80]. Other studies also reported anti-tumorigenic results of antioxidant treatments in vitro [126–128], including overexpression of SOD3, which inhibited breast cancer metastasis in a mouse xenograft indicating the potential anti-tumorigenic effect of restoring extracellular superoxide scavenging capacity [129]. However, most clinical trials have failed to show beneficial effects of antioxidants on a variety of pathologies including cancer [130,131]. Long-term studies showed that taking vitamin E supplements significantly increased the risk of prostate cancer in healthy men [132] and supplementation with b-carotene, vitamin A or E increased the incidence of lung cancer [133,134]. A recent study agrees with these findings showing that antioxidants, NAC and vitamin E, accelerated lung cancer progression in mice by reducing ROS [67]. NAC also did not affect tumorigenesis in multiple breast cancer models in vivo [135]. Here, NAC treatment diminished HIF levels but actually increased metastatic burden. Overall, nine long-term, randomized controlled trials of antioxidant supplements for cancer prevention did not provide evidence that dietary antioxidant supplements are beneficial in primary cancer prevention [127,132–134,136–140]. The reason that antioxidants display poor efficacy as anticancer agents might be due to the fact that many of the therapeutic antioxidants are not effective in targeting the locally produced pools of ROS, which are required for tumorigenic signaling. Mitochondrial targeted antioxidants for example would be a way to address this issue. These targeted antioxidants have shown efficacy in diminishing tumorigenic potential when dietary ones failed to do so [2,141]. However, major caveats of antioxidant-based therapy remain that (1) ROS and ROS-dependent cell signaling are essential for normal cell function and can promote tumor cell survival in cases when ROS are required for cell death of transformed cells and (2) that antioxidants can interfere with chemotherapy, which are dependent on ROS-induced cytotoxicity [142]. Due to the negative results of dietary antioxidant cancer therapies, recent studies have focused on alternative ways to exploit the aberrant redox status of cancer cells for therapeutics. Since exceedingly high levels of ROS can induce cell death and cell cycle arrest (see Section 3.4), cancer cells, with an increased rate of ROS production, are potentially more vulnerable to damage by further ROS insult [60,143,144]. Therefore, elevating ROS levels, either by increasing ROS generation or decreasing ROS scavenging potential, could be a way to selectively kill or arrest cancer cells without causing significant toxicity to normal cells with a lower rate of ROS production and lower levels of antioxidant proteins. 4.2. Increase ROS levels Many chemotherapeutic agents as well as ionizing radiation function by promoting ROS production and therefore causing irreversible oxidative damage [145,146]. Chemotherapeutic drugs such as taxanes (e.g. paclitaxel), vinca alkaloids and anti-folates promote mitochondrial cell death through the release of cytochrome c and also disrupt the mitochondrial electron transport

chain which leads to increased superoxide production [147]. Other chemotherapeutics like cisplatin, carboplatin and doxorubicin also significantly increase ROS, which is the basis of their antitumorigenic effect [148,149]. One of the first ROS-producing anticancer drugs was procarbazine [150], which is readily oxidized to its ROS-producing azo derivative and is approved for the treatment of lymphoma and primary brain tumors. Targeted ROSproducing drugs also include monoclonal antibodies like rituximab, an anti-CD20 antibody that is used for the treatment of nonHodgkin’s lymphoma [151]. Arsenic trioxide (ATO) impairs the function of the mitochondrial electron transport chain and therefore increases superoxide production [144]. ATO agents can effectively treat acute promyelocytic leukemia (APL) and some other ROS-dependent leukemia [152]. Elesclomol (STA-4783) is another ROS-generating compound in clinical trials for malignant melanoma. It was shown to increase progression-free survival in phase II clinical trials given as a single agent, and efficacy was even increased in combination with the chemotherapy agent paclitaxel. The mechanism by which Elesclomol increases ROS is unknown but neutralization of ROS by antioxidants suppressed the drug’s effectiveness [153]. A well-known problem with many chemotherapeutic cancer drugs, which produce extreme levels of ROS, is the toxic effect on normal, healthy cells. Other, more targeted ROS producing cancer drugs have also shown promise by inducing cancer cell apoptosis. A recent study identified a genotype-selective drug that induces oxidative stress. The piperidine derivative lanperisone was identified out of a small molecule, synthetic lethal screen using oncogenic KRAS-driven mouse embryonic fibroblasts (MEFs). The compound selectively killed KRAS-mutant cells through the induction of ROS, which is mediated through RAS-MEK-MAPK signaling. Normal, KRAS-wildtype MEFs were unaffected by lanperisone. The drug also showed efficacy against tumor growth in a KRAS-driven mouse model of lung cancer [16]. 4.3. Target antioxidants to decrease ROS scavenging capacity A downside of ROS-production therapy is that cancers can become resistant to the sole exogenous increase of ROS. For example multi-drug resistant HL-60 leukemia is resistant to the raising of ROS due to endogenous elevation of antioxidants like catalase that detoxify and scavenge ROS [13]. As already mentioned, various oncogene-induced cancer cells increase antioxidant proteins, such as activation of NRF2 [8,11,20,154] to maintain ROS levels that allow pro-tumorigenic signaling pathways to be activated without inducing cell death. Furthermore, GSH metabolism, specifically an increase in GSH, seems to play an active role in protecting cancer cells from cell death and also from ROS-inducing therapy strategies like chemotherapy and radiation [14]. The reliance on antioxidants may represent the cancer cell’s ‘‘Achilles Heel’’, as non-transformed have a lower rate of ROS production and therefore are less dependent on their detoxification by antioxidants. In fact, studies have shown that disabling antioxidant mechanisms trigger ROS-mediated cell death in a variety of cancer cell types [15,17,18,22] (Fig. 3). Phenethyl isothiocyanate (PEITC) conjugates with GSH through electrophile– nucleophile interactions, depleting the GSH pool and leading to oxidative stress and cancer cell death. Additionally, the drug inhibits GPX activity. PEITC led to cell death in HRAS (V12) transformed ovarian cancer cells and BCR-ABL transformed hematopoietic cells. The compound also prolonged survival in an ovarian cancer cell xenograft model [15]. L-Buthioninesulfoximine (BSO) targets de novo GSH synthesis as an inhibitor of glutamylcysteine synthetase (g-GCS) [155]. BSO depletes GSH [156] and exhibits anticancer activity through apoptosis as a single


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Fig. 3. The Achilles Heel of cancer cells. (a) In non-transformed cells and cancer cells, ROS levels are kept in balance through ROS production and ROS elimination. ROS homeostasis is required to provide a favorable signaling environment. In cancer cells both processes occur at an increased rate compared to normal cells, making cancer cells more dependent on their ROS scavenging capacity. (b) The reliance of cancer cells on antioxidants can potentially be used to selectively target them therapeutically. Various studies (see Section 4.3) have shown that antioxidant inhibitors have anti-tumorigenic effects without affecting normal, non-transformed cells. (c) Antioxidant inhibitors diminish the ability of cells to scavenge ROS. This event leaves cancer cells, which are characterized by an increased rate of ROS production, with a large overload of ROS. (d) Normal cells are somewhat affected by the small overload of ROS after antioxidant inhibition, but do not lose cellular homeostasis completely. Cancer cells on the other hand, which are left with a large overload of ROS, lose homeostasis and die due to oxidative stress.

agent and in combination with ATO in solid tumors and APL cells [157,158]. BSO also increased efficacy of cisplatin in preclinical studies [159]. Similar to BSO, Imexon also has GSH-depleting, ROSaccumulating, and death-inducing potential as shown in a phase I study of non-Hodgkin’s lymphoma [160] and melanoma patients [161]. Recently, a cell-based molecular screen for pro-apoptotic effects in cancer but not normal cells, identified the natural, plantderived compound piperlongumine (PL). PL leads to an increase in ROS by binding and modulating the antioxidant enzyme glutathione transferase and therefore changes the ROS-stress response. PL induces apoptosis in osteosarcoma, breast, bladder and lung cancer cells but has little effect even on rapidly dividing, primary normal cells. The compound also showed efficacy in mouse models of breast, bladder and lung cancer [17]. As mentioned before, cysteine is required for GSH synthesis. Inhibition of the solute carrier family 7 member 11 (SLC7A11), which encodes the cysteine/glutamate antiporter (xCT), with sulphasalazine (SASP) decreases cysteine and GSH levels and increases ROS. SASP has shown to reduce pancreatic cancer cell growth and viability in vivo and in vitro [162] and has shown efficacy in a small-cell lung cancer xenograft model [163]. A recent study also identified xCT as a therapeutic target in a xenograft model of triple-negative breast cancer [164]. Another thiol-based antioxidant is thioredoxin (TRX). TRXs are up-regulated in various cancer cells [61] and the TRX inhibitor PX12 showed anti-tumor activity in vivo [165]. ATO, additionally to

inducing superoxide production, has been shown to inhibit thioredoxin reductase (TRXR) and to increase ROS levels [166]. Furthermore, motexafin gadolinium is a TRXR inhibitor that selectively localizes in tumors [167]. The drug showed anti-tumor activity in a phase II trial in patients with chronic lymphocytic leukemia [168] and in phase III clinical trials of patients with metastatic non-small cell lung cancer (NSCLC) [169]. Finally the gold compound auranofin is a TRX inhibitor and has been shown to cause sensitivity of head and neck squamous cell carcinoma cells to EGFR inhibitors [170], and also cause cell death of ovarian cancer cells [171]. Cell death was shown to be ROS-mediated. Furthermore the antioxidant SOD1, which converts superoxide into H2O2, has emerged as a target to selectively kill cancer cells. Malignant cells are highly dependent on SODs to keep ROS levels under the cytotoxic limit. Varmus and colleagues utilized an unbiased small molecule screen and identified SOD1 as a target for inhibitors of the growth of both KRAS- and EGFR-mutant lung adenocarcinoma cells in vitro. SOD1 was also shown to be expressed at higher levels in lung adenocarcinomas compared to normal lungs [172]. The SOD1 inhibitor methoxyestradiol (2-ME) increases superoxide [173] and is currently in phase I and II clinical trials for prostate and metastatic breast cancer [174,175]. It also induces ROS-mediated apoptosis selectively in leukemia cells but not normal lymphocytes [173] and in neuroblastoma cells [176]. Furthermore, recent studies identified the copper chelator and SOD1 inhibitor ATN-224 to cause selective cancer cell death


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and growth impairment in various cancer cells through ROSmediated mechanisms [177–179]. The compound showed efficacy in an oncogenic KRAS/P53-deficient mouse model of NSCLC. ATN224-induced cancer cell death was mediated through ROSdependent activation of P38 MAPK, leading to a decrease in the anti-apoptotic factor MCL1 [22]. Interestingly, extracellular SOD3 is differentially expressed in cancers. SOD3 mRNA was shown to be decreased in some clinical mammary adenocarcinoma samples compared to normal mammary tissues and research found that overexpression of SOD3 inhibited breast cancer metastasis in a mouse xenograft model [129]. On the other hand, SOD3 is shown to be amplified in various other cancers for example sarcomas (cBioPortal for Cancer Genomics Database) making it a potential target for silencing in these cancers. Finally, the antioxidant master regulator NRF2 presents a possible anti-cancer therapeutic target. As previously mentioned, oncogenes like KRAS, MYC and PI3K have been shown to stabilize NRF2 and therefore antioxidant proteins [8,11,20,154]. Additionally, mutations in NRF2 and inactivating mutations in its negative regulator KEAP1 have been identified in various cancers leading to the constitutive stabilization and transcription activity of NRF2 [64,65,180]. This data suggests a promoting role for NRF2 activity in tumorigenesis. Hence, NRF2 inhibition has the potential to dampen various antioxidant systems and induce ROS-mediated cancer cell death. Effective NRF2 inhibitors have yet to be developed and tested for anti-cancer effects. 4.4. Metabolic and redox modulation: combination therapy option Pro-oxidant therapy can exploit the difference between normal cells and cancer cells based on their difference in the rate of ROS production and resulting redox regulation. However, off-target cytotoxicity from high doses of ROS-generators is still a major concern, as is the compensatory up-regulation of various antioxidants, which counteracts the ROS-generating compounds. Combination therapy, meaning the simultaneous treatment with various anti-cancer agents is one possibility to (1) lower drug treatment doses, (2) augment drug efficacy and (3) also impair central parts of the antioxidant machinery. To that end, metabolic modulation is a suitable candidate for combination therapy with ROS-generators or ROS-scavenging inhibitors. Metabolic reprogramming, similar to aberrant redox adaptation, is cancer cell specific, which further opens the therapeutic window. Metabolism pathways, which will be elaborated on here, play a central role in regulating redox balance particularly through the production of reducing equivalents like NADH and NADPH, which are required for the function of various antioxidant proteins. Based on this knowledge, targeting the unique metabolic and redox balance pathways of cancer cells, will potentially allow to biosynthetically starve cancer cells and also induce cancer-killing redox stress. The metabolic pathway predominantly involved in redox modulation is glutamine metabolism. It plays a central role in redox regulation and antioxidant response through the generation of GSH and the reducing equivalent NADPH. Glutamine is the precursor of glutamate, which, as previously mentioned, is required for GSH synthesis and therefore antioxidant response. Glutamine metabolism has been shown to be required for cancer cell survival leading to the notion that some cancers are glutamineaddicted [181–183]. Inhibition of glutaminase 1 (GLS1), the enzyme that converts glutamine to glutamate for entry into the TCA cycle, inhibits oncogenic transformation [2,184]. Furthermore, an approved agent for the treatment of leukemia has been shown to deplete glutamine levels and therefore GSH synthesis. The drug L-asparaginase was thought to function through its role in limiting asparagine levels, however recent data has shown that the anticancer effect of the drug can be attributed to its effect on glutamine

levels [185]. Finally, the alternative glutamine pathway, mediated by the aspartate transaminase GOT1 has been shown to be required for KRAS-driven pancreatic ductal adenocarcinoma (PDAC) growth in vitro and in vivo [186]. GOT1 is a key enzyme in the aspartate-malate shuttle, producing pyruvate and increasing the NADPH/NADP+ ratio, which in turn maintains reduced GSH levels and therefore redox homeostasis. Indeed, GOT1 inhibition led to a decrease in the ratio of reduced-to-oxidized GSH, an increase in ROS levels and suppression of PDAC growth. The role of glutamine metabolism in NADPH production and GSH synthesis makes glutamine pathway inhibitors potentially suitable partners for pro-oxidants in anti-cancer therapy.

5. Conclusion Studies over the past several years have established a causal role of ROS in maintaining cellular homeostasis and also in triggering cell signaling events and stress adaptation [69]. Cancer cells, compared to normal cells, have an increased rate of ROS production as byproducts of their increased metabolism [1] and furthermore have aberrant ROS regulation mechanisms to cope with their unique redox status. The fact that ROS have a welldefined role in promoting and maintaining tumorigenicity [2–4], led to the assumption that antioxidants could prevent or reduce tumorigenesis [187]. However, most clinical trials have failed to show beneficial effects of dietary antioxidants in a variety of cancer types [132]. In fact, there has been evidence indicating that dietary antioxidants contribute to tumorigenesis by reducing the potentially death-inducing oxidative stress in cancer cells [188]. This failure of therapeutic antioxidants might be due to the fact that they are unlikely to diminish the local pools of ROS required for pro-tumorigenic signaling. Thus, recent studies have focused on alternative ways to exploit the aberrant redox status of cancer cells for therapeutics. Since exceedingly high levels of ROS can induce cell death, research has focused on pro-oxidant approaches to cancer therapy [60]. Chemotherapy, radiation and also small molecule compounds induce cancer cell death by increasing intracellular ROS [16,188]. However, cancer cells are masters of adaptation and even though oncogene mutations, reprogrammed metabolism, extreme microenvironments and nutrient starvation induce highly stressful conditions, tumor cells manage to regulate and adapt to these stressors and advantageously survive and proliferate. Hence, cancer cells have the ability to develop resistance to therapeutics that exogenously raise ROS by increasing their antioxidant mechanisms, such as activation and stabilization of the antioxidant master regulator NRF2 [8,11,154] or other ROS scavengers [13,14]. With that, tumor cells can

Fig. 4. Pro-oxidants as cancer therapeutics. Redox modulation therapeutics used to focus on antioxidant cancer treatments (left) because of the role of ROS in promoting tumorigenesis. However these therapies did not show success in the clinic (see Section 4.1). Recent studies suggest pro-oxidant cancer therapy (right), especially the inhibition of ROS scavengers and antioxidants, to selectively target cancer cells by inducing ROS-dependent cancer cell death.


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maintain ROS levels that allow proximal pro-tumorigenic signaling without inducing cell death, but at the same time they also rely more heavily on ROS detoxification compared to normal cells with lower metabolic demands and rates of ROS production. This reliance on high levels of antioxidants may represent the cancer cell’s ‘‘Achilles Heel’’ [189]. The impairment of redox scavenging, or antioxidant systems opens a novel therapeutic window and has potential to selectively induce cancer cell death via oxidative stress, while sparing normal cells (Fig. 4). In fact, studies have shown that disabling antioxidant mechanisms trigger ROS-mediated cell death in a variety of cancer cell types in vitro and in vivo [15,17,18,20,22]. Hence, the design of dual pro-oxidant therapies has the potential to be efficacious in selectively killing cancer cells. Combining ROS-generating agents with ROS-scavenging inhibitors like GSH, TRX or SOD inhibitors can diminish the ability of cancer cells to adapt to either agent. Even though various antioxidant proteins and regulators are upregulated in cancers and can be efficiently targeted to cause antitumor effects, it will be necessary to do careful antioxidant profiling of tumor cells and their normal counterparts to decipher the mechanisms and redox regulation properties that are enriched in tumor cells and consequently use them as clinically relevant therapeutic targets.

[14]

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[16]

[17]

[18]

[19]

[20]

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Conflict of Interest

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Andrea Glasauer is a full-time employee of Bayer Pharma AG. The remaining author declares no conflict of interest.

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Acknowledgements

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This work was supported by the LUNGevity Foundation and a Consortium of Independent Lung Health Organizations convened by Respiratory Health Association of Metropolitan Chicago (N.S.C.), National Institutes of Health Grant R01CA123067 (N.S.C), Dixon Translational Grant (N.S.C.) and Northwestern University Malkin Scholar Award (A.G).

[27] [28]

[29]

[30]

References [1] Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nature Reviews Cancer 2011;11:85–95. [2] Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M, et al. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proceedings of the National Academy of Sciences of the United States of America 2010;107:8788–93. [3] Wallace DC. Mitochondria and cancer. Nature Reviews Cancer 2012;12:685–98. [4] Weinberg F, Chandel NS. Reactive oxygen species-dependent signaling regulates cancer. Cellular and Molecular Life Sciences 2009;66:3663–73. [5] Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 1997;275:90–4. [6] Moon DO, Kim MO, Choi YH, Hyun JW, Chang WY, Kim GY. Butein induces G(2)/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation. Cancer Letters 2010;288:204–13. [7] Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, et al. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature 2009;461:109–13. [8] Hayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends in Biochemical Sciences 2009;34:176–88. [9] Tiligada E. Chemotherapy: induction of stress responses. Endocrine Related Cancer 2006;13(Suppl 1):S115–24. [10] Chen W, Sun Z, Wang XJ, Jiang T, Huang Z, Fang D, et al. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Molecular Cell 2009;34:663–73. [11] Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, et al. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 2012;22:66–79. [12] Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1Nrf2 pathway in stress response and cancer evolution. Genes to Cells 2011;16:123–40. [13] Lenehan PF, Gutierrez PL, Wagner JL, Milak N, Fisher GR, Ross DD. Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the

[31]

[32] [33]

[34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

9

resistance to daunorubicin manifested by these cells. Cancer Chemotherapy and Pharmacology 1995;35:377–86. Tai DJ, Jin WS, Wu CS, Si HW, Cao XD, Guo AJ, et al. Changes in intracellular redox status influence multidrug resistance in gastric adenocarcinoma cells. Experimental and Therapeutic Medicine 2012;4:291–6. Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, et al. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer Cell 2006;10: 241–52. Shaw AT, Winslow MM, Magendantz M, Ouyang C, Dowdle J, Subramanian A, et al. Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress. Proceedings of the National Academy of Sciences of the United States of America 2011;108:8773–8. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature 2011;475:231–4. Ren D, Villeneuve NF, Jiang T, Wu T, Lau A, Toppin HA, et al. Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism. Proceedings of the National Academy of Sciences of the United States of America 2011;108:1433–8. Wu X, Zhu Y, Yan H, Liu B, Li Y, Zhou Q, et al. Isothiocyanates induce oxidative stress and suppress the metastasis potential of human non-small cell lung cancer cells. BMC Cancer 2010;10:269. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011;475:106–9. Adhikary A, Mohanty S, Lahiry L, Hossain DM, Chakraborty S, Das T. Theaflavins retard human breast cancer cell migration by inhibiting NF-kappaB via p53-ROS cross-talk. FEBS Letters 2010;584:7–14. Glasauer A, Sena LA, Diebold LP, Mazar AP, Chandel NS. Targeting SOD1 reduces experimental non-small-cell lung cancer. The Journal of Clinical Investigation 2014;124:117–28. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nature Reviews Immunology 2004;4:181–9. Brand MD. The sites and topology of mitochondrial superoxide production. Experimental Gerontology 2010;45:466–72. Muller FL, Liu Y, Van Remmen H. Complex III releases superoxide to both sides of the inner mitochondrial membrane. The Journal of Biological Chemistry 2004;279:49064–73. Fisher AB. Redox signaling across cell membranes. Antioxidants & redox Signaling 2009;11:1349–56. Winterbourn CC, Hampton MB. Thiol chemistry and specificity in redox signaling. Free Radical Biology & Medicine 2008;45:549–61. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. The American Journal of Physiology 1996;271: C1424–37. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. American Journal of Physiology Lung Cellular and Molecular Physiology 2000;279:L1005–28. McCord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). The Journal of Biological Chemistry 1969;244:6049–55. Berndt C, Lillig CH, Holmgren A. Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system. American Journal of Physiology Heart and Circulatory Physiology 2007;292:H1227–36. Meister A. Selective modification of glutathione metabolism. Science 1983;220:472–7. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. The Journal of Biological Chemistry 2000;275:16023–29. Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes & Development 1999;13:76–86. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Molecular and Cellular Biology 2004;24:7130–9. Kobayashi M, Yamamoto M. Molecular mechanisms activating the Nrf2Keap1 pathway of antioxidant gene regulation. Antioxidants & Redox Signaling 2005;7:385–94. Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, et al. Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. The Journal of Biological Chemistry 2002;277:44765–71. Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 2002;419:316–21. Nemoto S, Finkel T. Redox regulation of forkhead proteins through a p66shcdependent signaling pathway. Science 2002;295:2450–2. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 2009;458:780–3. Finkel T. Oxygen radicals and signaling. Current Opinion in Cell Biology 1998;10:248–53.


G Model



[42] Finkel T. Signal transduction by mitochondrial oxidants. The Journal of Biological Chemistry 2012;287:4434–40. [43] Lee SR, Yang KS, Kwon J, Lee C, Jeong W, Rhee SG. Reversible inactivation of the tumor suppressor PTEN by H2O2. The Journal of Biological Chemistry 2002;277:20336–42. [44] Meng TC, Fukada T, Tonks NK. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Molecular Cell 2002;9:387–99. [45] Wani R, Qian J, Yin L, Bechtold E, King SB, Poole LB, et al. Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proceedings of the National Academy of Sciences of the United States of America 2011; 108:10550–55. [46] Kemble DJ, Sun G. Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation. Proceedings of the National Academy of Sciences of the United States of America 2009; 106:5070–5. [47] Hamanaka RB, Chandel NS. Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes. Trends in Biochemical Sciences 2010;35:505–13. [48] Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M. Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 2005;120:649–61. [49] Szatrowski TP, Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Research 1991;51:794–8. [50] Kamiguti AS, Serrander L, Lin K, Harris RJ, Cawley JC, Allsup DJ, et al. Expression and activity of NOX5 in the circulating malignant B cells of hairy cell leukemia. Journal of Immunology 2005;175:8424–30. [51] Patel BP, Rawal UM, Dave TK, Rawal RM, Shukla SN, Shah PM, et al. Lipid peroxidation, total antioxidant status, and total thiol levels predict overall survival in patients with oral squamous cell carcinoma. Integrative Cancer Therapies 2007;6:365–72. [52] Sablina AA, Budanov AV, Ilyinskaya GV, Agapova LS, Kravchenko JE, Chumakov PM. The antioxidant function of the p53 tumor suppressor. Nature Medicine 2005;11:1306–13. [53] Attardi LD, Donehower LA. Probing p53 biological functions through the use of genetically engineered mouse models. Mutation Research 2005;576: 4–21. [54] Behrend L, Henderson G, Zwacka RM. Reactive oxygen species in oncogenic transformation. Biochemical Society Transactions 2003;31:1441–4. [55] Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296: 1655–7. [56] Nemoto S, Takeda K, Yu ZX, Ferrans VJ, Finkel T. Role for mitochondrial oxidants as regulators of cellular metabolism. Molecular and Cellular Biology 2000;20:7311–8. [57] Hedrick SM. The cunning little vixen: Foxo and the cycle of life and death. Nature Immunology 2009;10:1057–63. [58] Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005;307:58–62. [59] Bell EL, Emerling BM, Chandel NS. Mitochondrial regulation of oxygen sensing. Mitochondrion 2005;5:322–32. [60] Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nature Reviews Drug Discovery 2013;12:931–47. [61] Young TW, Mei FC, Yang G, Thompson-Lanza JA, Liu J, Cheng X. Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry. Cancer Research 2004;64:4577–84. [62] Hu Y, Rosen DG, Zhou Y, Feng L, Yang G, Liu J, et al. Mitochondrial manganesesuperoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress. The Journal of Biological Chemistry 2005;280:39485–92. [63] Saydam N, Kirb A, Demir O, Hazan E, Oto O, Saydam O, et al. Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues. Cancer Letters 1997; 119:13–9. [64] Shibata T, Ohta T, Tong KI, Kokubu A, Odogawa R, Tsuta K, et al. Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy. Proceedings of the National Academy of Sciences of the United States of America 2008;105:13568–73. [65] Satoh H, Moriguchi T, Takai J, Ebina M, Yamamoto M. Nrf2 Prevents Initiation but Accelerates Progression through the Kras Signaling Pathway during Lung Carcinogenesis. Cancer Research 2013;73:4158–68. [66] Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, et al. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2. Carcinogenesis 2008;29:1235–43. [67] Sayin VI, Ibrahim MX, Larsson E, Nilsson JA, Lindahl P, Bergo MO. Antioxidants accelerate lung cancer progression in mice. Science Translational Medicine 2014;6:221ra15. [68] Kumar B, Koul S, Khandrika L, Meacham RB, Koul HK. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype. Cancer Research 2008;68:1777–85. [69] Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Molecular Cell 2012;48:158–67. [70] Trachootham D, Zhang H, Zhang W, Feng L, Du M, Zhou Y, et al. Effective elimination of fludarabine-resistant CLL cells by PEITC through a redoxmediated mechanism. Blood 2008;112:1912–22. [71] Clerkin JS, Naughton R, Quiney C, Cotter TG. Mechanisms of ROS modulated cell survival during carcinogenesis. Cancer Letters 2008;266:30–6.

[72] Nishikawa M. Reactive oxygen species in tumor metastasis. Cancer Letters 2008;266:53–9. [73] Ushio-Fukai M, Nakamura Y. Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Letters 2008;266:37–52. [74] Lee SR, Kwon KS, Kim SR, Rhee SG. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. The Journal of Biological Chemistry 1998;273:15366–72. [75] Salmeen A, Andersen JN, Myers MP, Meng TC, Hinks JA, Tonks NK, et al. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate. Nature 2003;423:769–73. [76] Kwon J, Lee SR, Yang KS, Ahn Y, Kim YJ, Stadtman ER, et al. Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors. Proceedings of the National Academy of Sciences of the United States of America 2004;101:16419–24. [77] Seth D, Rudolph J. Redox regulation of MAP kinase phosphatase 3. Biochemistry 2006;45:8476–87. [78] De Luca A, Sanna F, Sallese M, Ruggiero C, Grossi M, Sacchetta P, et al. Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype. Proceedings of the National Academy of Sciences of the United States of America 2010;107:18628–33. [79] Qin Y, Pan X, Tang TT, Zhou L, Gong XG. Anti-proliferative effects of the novel squamosamide derivative (FLZ) on HepG2 human hepatoma cells by regulating the cell cycle-related proteins are associated with decreased Ca(2 + )/ ROS levels. Chemico-Biological Interactions 2011;193:246–53. [80] Gao P, Zhang H, Dinavahi R, Li F, Xiang Y, Raman V, et al. HIF-dependent antitumorigenic effect of antioxidants in vivo. Cancer Cell 2007;12:230–8. [81] Policastro L, Molinari B, Larcher F, Blanco P, Podhajcer OL, Costa CS, et al. Imbalance of antioxidant enzymes in tumor cells and inhibition of proliferation and malignant features by scavenging hydrogen peroxide. Molecular Carcinogenesis 2004;39:103–13. [82] Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 2005;436:725–30. [83] Nitsche C, Edderkaoui M, Moore RM, Eibl G, Kasahara N, Treger J, et al. The phosphatase PHLPP1 regulates Akt2, promotes pancreatic cancer cell death, and inhibits tumor formation. Gastroenterology 2012;142:377–87. e1-5. [84] Kotlo KU, Yehiely F, Efimova E, Harasty H, Hesabi B, Shchors K, et al. Nrf2 is an inhibitor of the Fas pathway as identified by Achilles’ Heel Method, a new function-based approach to gene identification in human cells. Oncogene 2003;22:797–806. [85] Kobayashi M, Yamamoto M. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Advances in Enzyme Regulation 2006;46:113–40. [86] Kamata H, Manabe T, Oka S, Kamata K, Hirata H. Hydrogen peroxide activates IkappaB kinases through phosphorylation of serine residues in the activation loops. FEBS Letters 2002;519:231–7. [87] Rahman I, Gilmour PS, Jimenez LA, MacNee W. Oxidative stress and TNFalpha induce histone acetylation and NF-kappaB/AP-1 activation in alveolar epithelial cells: potential mechanism in gene transcription in lung inflammation. Molecular and Cellular Biochemistry 2002;234-235:239–48. [88] Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nature Immunology 2002;3:221–7. [89] Chandel NS, Schumacker PT, Arch RH. Reactive oxygen species are downstream products of TRAF-mediated signal transduction. The Journal of Biological Chemistry 2001;276:42728–36. [90] Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Molecular and Cellular Biology 1996;16:4604–13. [91] Bell EL, Klimova TA, Eisenbart J, Moraes CT, Murphy MP, Budinger GR, et al. The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. The Journal of Cell Biology 2007;177:1029–36. [92] Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. The Journal of Biological Chemistry 2000;275:25130–38. [93] Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, et al. HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292:464–8. [94] Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72. [95] Klimova T, Chandel NS. Mitochondrial complex III regulates hypoxic activation of HIF. Cell Death and Differentiation 2008;15:660–6. [96] Ma Q, Cavallin LE, Yan B, Zhu S, Duran EM, Wang H, et al. Antitumorigenesis of antioxidants in a transgenic Rac1 model of Kaposi’s sarcoma. Proceedings of the National Academy of Sciences of the United States of America 2009;106:8683–8. [97] Xia C, Meng Q, Liu LZ, Rojanasakul Y, Wang XR, Jiang BH. Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Research 2007;67:10823–30. [98] Chiang AC, Massague J. Molecular basis of metastasis. The New England Journal of Medicine 2008;359:2814–23. [99] Ho BY, Wu YM, Chang KJ, Pan TM. Dimerumic acid inhibits SW620 cell invasion by attenuating H(2)O(2)-mediated MMP-7 expression via JNK/CJun and ERK/C-Fos activation in an AP-1-dependent manner. International Journal of Biological Sciences 2011;7:869–80.


G Model

BCP-12033; No. of Pages 12 A. Glasauer, N.S. Chandel / Biochemical Pharmacology xxx (2014) xxx–xxx [100] Chetram MA, Don-Salu-Hewage AS, Hinton CVROS. enhances CXCR4-mediated functions through inactivation of PTEN in prostate cancer cells. Biochemical and Biophysical Research Communications 2011;410:195–200. [101] Radisky DC, Levy DD, Littlepage LE, Liu H, Nelson CM, Fata JE, et al. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 2005;436:123–7. [102] Gort EH, Groot AJ, van der Wall E, van Diest PJ, Vooijs MA. Hypoxic regulation of metastasis via hypoxia-inducible factors. Current Molecular Medicine 2008;8:60–7. [103] Courtneidge SA. Cell migration and invasion in human disease: the Tks adaptor proteins. Biochemical Society Transactions 2012;40:129–32. [104] Binker MG, Binker-Cosen AA, Richards D, Oliver B, Cosen-Binker LI. EGF promotes invasion by PANC-1 cells through Rac1/ROS-dependent secretion and activation of MMP-2. Biochemical and Biophysical Research Communications 2009;379:445–50. [105] Seo JM, Park S, Kim JH. Leukotriene B4 receptor-2 promotes invasiveness and metastasis of ovarian cancer cells through signal transducer and activator of transcription 3 (STAT3)-dependent up-regulation of matrix metalloproteinase 2. The Journal of Biological Chemistry 2012;287:13840–49. [106] Kleiner Jr DE, Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteases. Current Opinion in Cell Biology 1993;5:891–7. [107] Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. The EMBO Journal 1998;17:2596–606. [108] Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Reports 2001;2:222–8. [109] Kharbanda S, Saxena S, Yoshida K, Pandey P, Kaneki M, Wang Q, et al. Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to DNA damage. The Journal of Biological Chemistry 2000;275: 322–7. [110] Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, et al. Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature 2011;471:110–4. [111] Dolado I, Swat A, Ajenjo N, De Vita G, Cuadrado A. Nebreda AR. p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis. Cancer Cell 2007;11:191–205. [112] Inoshita S, Takeda K, Hatai T, Terada Y, Sano M, Hata J, et al. Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress. The Journal of Biological Chemistry 2002;277:43730–34. [113] Han J, Sun P. The pathways to tumor suppression via route p38. Trends in Biochemical Sciences 2007;32:364–71. [114] Ambrosino C, Nebreda AR. Cell cycle regulation by p38 MAP kinases. Biology of the cell/under the auspices of the European Cell Biology Organization 2001;93:47–51. [115] Thornton TM, Rincon M. Non-classical p38 map kinase functions: cell cycle checkpoints and survival. International Journal of Biological Sciences 2009;5:44–51. [116] Savitsky PA, Finkel T. Redox regulation of Cdc25 C. The Journal of Biological Chemistry 2002;277:20535–40. [117] Chiu J, Tactacan CM, Tan SX, Lin RC, Wouters MA, Dawes IW. Cell cycle sensing of oxidative stress in Saccharomyces cerevisiae by oxidation of a specific cysteine residue in the transcription factor Swi6p. The Journal of Biological Chemistry 2011;286:5204–14. [118] Seo YH, Carroll KS. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies. Proceedings of the National Academy of Sciences of the United States of America 2009;106:16163–68. [119] Siddiqui IA, Adhami VM, Saleem M, Mukhtar H. Beneficial effects of tea and its polyphenols against prostate cancer. Molecular Nutrition & Food Research 2006;50:130–43. [120] Bianchini F, Vainio H. Wine and resveratrol: mechanisms of cancer prevention? European Journal of Cancer Prevention: The Official Journal of the European Cancer Prevention Organisation 2003;12:417–25. [121] Liu J, Du J, Zhang Y, Sun W, Smith BJ, Oberley LW, et al. Suppression of the malignant phenotype in pancreatic cancer by overexpression of phospholipid hydroperoxide glutathione peroxidase. Human Gene Therapy 2006;17:105–16. [122] Nelson SK, Bose SK, Grunwald GK, Myhill P, McCord JM. The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy. Free Radical Biology & Medicine 2006;40:341–7. [123] Pantuck AJ, Zomorodian N, Belldegrun AS. Phase-II Study of pomegranate juice for men with prostate cancer and increasing PSA. Current Urology Reports 2006;7:7. [124] Kim HS, Bowen P, Chen L, Duncan C, Ghosh L, Sharifi R, et al. Effects of tomato sauce consumption on apoptotic cell death in prostate benign hyperplasia and carcinoma. Nutrition and Cancer 2003;47:40–7. [125] Jian L, Xie LP, Lee AH, Binns CW. Protective effect of green tea against prostate cancer: a case-control study in southeast China. International Journal of Cancer 2004;108:130–5. [126] Blot WJ, Li JY, Taylor PR, Guo W, Dawsey S, Wang GQ, et al. Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population. Journal of National Cancer Institute 1993;85: 1483–92. [127] Qiao YL, Dawsey SM, Kamangar F, Fan JH, Abnet CC, Sun XD, et al. Total and cancer mortality after supplementation with vitamins and minerals:

[128]

[129]

[130]

[131] [132]

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143] [144]

[145] [146] [147] [148] [149]

[150]

[151] [152]

[153]

11

follow-up of the Linxian General Population Nutrition Intervention Trial. Journal of National Cancer Institute 2009;101:507–18. Zhang W, Shu XO, Li H, Yang G, Cai H, Ji BT, et al. Vitamin intake and liver cancer risk: a report from two cohort studies in China. Journal of National Cancer Institute 2012;104:1173–81. Teoh-Fitzgerald ML, Fitzgerald MP, Zhong W, Askeland RW, Domann FE. Epigenetic reprogramming governs EcSOD expression during human mammary epithelial cell differentiation, tumorigenesis and metastasis. Oncogene 2014;33:358–68. Khaw KT, Bingham S, Welch A, Luben R, Wareham N, Oakes S, et al. Relation between plasma ascorbic acid and mortality in men and women in EPICNorfolk prospective study: a prospective population study. European Prospective Investigation into Cancer and Nutrition. Lancet 2001;357:657–63. Burr M, Appleby P, Key T, Thorogood M. Plasma ascorbic acid and risk of heart disease and cancer. Lancet 2001;357:2135–6. Klein EA, Thompson Jr IM, Tangen CM, Crowley JJ, Lucia MS, Goodman PJ, et al. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA: The Journal of the American Medical Association 2011;306:1549–56. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The New England journal of medicine 1994;330: 1029-35. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, et al. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. The New England Journal of Medicine 1996;334: 1150–5. Sceneay J, Liu MC, Chen A, Wong CS, Bowtell DD, Moller A. The antioxidant Nacetylcysteine prevents HIF-1 stabilization under hypoxia in vitro but does not affect tumorigenesis in multiple breast cancer models in vivo. PLoS One 2013;8:e66388. Hennekens CH, Buring JE, Manson JE, Stampfer M, Rosner B, Cook NR, et al. Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. The New England Journal of Medicine 1996;334:1145–9. Hercberg S, Ezzedine K, Guinot C, Preziosi P, Galan P, Bertrais S, et al. Antioxidant supplementation increases the risk of skin cancers in women but not in men. Journal of Nutrition 2007;137:2098–105. Lonn E, Bosch J, Yusuf S, Sheridan P, Pogue J, Arnold JM, et al. Effects of longterm vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. JAMA: The Journal of the American Medical Association 2005;293:1338–47. Gaziano JM, Glynn RJ, Christen WG, Kurth T, Belanger C, MacFadyen J, et al. Vitamins E and C in the prevention of prostate and total cancer in men: the Physicians’ Health Study II randomized controlled trial. JAMA: The Journal of the American Medical Association 2009;301:52–62. Lee IM, Cook NR, Manson JE, Buring JE, Hennekens CH. Beta-carotene supplementation and incidence of cancer and cardiovascular disease: the Women’s Health Study. Journal of National Cancer Institute 1999;91: 2102–6. Nazarewicz RR, Dikalova A, Bikineyeva A, Ivanov S, Kirilyuk IA, Grigor’ev IA, et al. Does scavenging of mitochondrial superoxide attenuate cancer prosurvival signaling pathways? Antioxidants & Redox Signaling 2013;19:344–9. Conklin KA. Dietary antioxidants during cancer chemotherapy: impact on chemotherapeutic effectiveness and development of side effects. Nutrition and Cancer 2000;37:1–18. Schumacker PT. Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer Cell 2006;10:175–6. Pelicano H, Feng L, Zhou Y, Carew JS, Hileman EO, Plunkett W, et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. The Journal of Biological Chemistry 2003;278:37832–39. Kong Q, Lillehei KO. Antioxidant inhibitors for cancer therapy. Medical Hypotheses 1998;51:405–9. Wondrak GT. Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxidants & Redox Signaling 2009;11:3013–69. Kaufmann SH, Earnshaw WC. Induction of apoptosis by cancer chemotherapy. Experimental Cell Research 2000;256:42–9. Conklin KA. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integrative Cancer Therapies 2004;3:294–300. Kotamraju S, Chitambar CR, Kalivendi SV, Joseph J, Kalyanaraman B. Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis. The Journal of Biological Chemistry 2002;277:17179–87. Berneis K, Bollag W, Kofler M, Luthy H. The enhancement of the after effect of ionizing radiation by a cytotoxic methylhydrazine derivative. European Journal of Cancer 1966;2:43–9. Renschler MF. The emerging role of reactive oxygen species in cancer therapy. European Journal of Cancer 2004;40:1934–40. Chou WC, Jie C, Kenedy AA, Jones RJ, Trush MA, Dang CV. Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells. Proceedings of the National Academy of Sciences of the United States of America 2004;101:4578–83. Kirshner JR, He S, Balasubramanyam V, Kepros J, Yang CY, Zhang M, et al. Elesclomol induces cancer cell apoptosis through oxidative stress. Molecular Cancer Therapeutics 2008;7:2319–27.


G Model



[154] Hayes JD, McMahon M, Chowdhry S, Dinkova-Kostova AT. Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway. Antioxidants & Redox Signaling 2010;13:1713–48. [155] Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). The Journal of Biological Chemistry 1979;254:7558–60. [156] Bailey HH, Ripple G, Tutsch KD, Arzoomanian RZ, Alberti D, Feierabend C, et al. Phase I study of continuous-infusion L-S,R-buthionine sulfoximine with intravenous melphalan. Journal of National Cancer Institute 1997;89: 1789–96. [157] Davison K, Cote S, Mader S, Miller WH. Glutathione depletion overcomes resistance to arsenic trioxide in arsenic-resistant cell lines. Leukemia 2003;17:931–40. [158] Maeda H, Hori S, Ohizumi H, Segawa T, Kakehi Y, Ogawa O, et al. Effective treatment of advanced solid tumors by the combination of arsenic trioxide and L-buthionine-sulfoximine. Cell Death and Differentiation 2004;11: 737–46. [159] Engel RH, Evens AM. Oxidative stress and apoptosis: a new treatment paradigm in cancer. Frontiers of Bioscience 2006;11:300–12. [160] Dragovich T, Gordon M, Mendelson D, Wong L, Modiano M, Chow HH, et al. Phase I trial of imexon in patients with advanced malignancy. Journal of Clinical Oncology 2007;25:1779–84. [161] Weber JS, Samlowski WE, Gonzalez R, Ribas A, Stephenson J, O’Day S, et al. A phase 1-2 study of imexon plus dacarbazine in patients with unresectable metastatic melanoma. Cancer 2010;116:3683–91. [162] Lo M, Ling V, Low C, Wang YZ, Gout PW. Potential use of the anti-inflammatory drug, sulfasalazine, for targeted therapy of pancreatic cancer. Current Oncology 2010;17:9–16. [163] Guan J, Lo M, Dockery P, Mahon S, Karp CM, Buckley AR, et al. The xc- cystine/ glutamate antiporter as a potential therapeutic target for small-cell lung cancer: use of sulfasalazine. Cancer Chemotherapy and Pharmacology 2009;64:463–72. [164] Timmerman LA, Holton T, Yuneva M, Louie RJ, Padro M, Daemen A, et al. Glutamine sensitivity analysis identifies the xCT antiporter as a common triple-negative breast tumor therapeutic target. Cancer Cell 2013;24: 450–65. [165] Welsh SJ, Williams RR, Birmingham A, Newman DJ, Kirkpatrick DL, Powis G. The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl disulfide and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Molecular Cancer Therapeutics 2003;2:235–43. [166] Lu J, Chew EH, Holmgren A. Targeting thioredoxin reductase is a basis for cancer therapy by arsenic trioxide. Proceedings of the National Academy of Sciences of the United States of America 2007;104:12288–93. [167] Magda D, Miller RA. Motexafin gadolinium: a novel redox active drug for cancer therapy. Seminars in Cancer Biology 2006;16:466–76. [168] Lin TS, Naumovski L, Lecane PS, Lucas MS, Moran ME, Cheney C, et al. Effects of motexafin gadolinium in a phase II trial in refractory chronic lymphocytic leukemia. Leukemia & Lymphoma 2009;50:1977–82. [169] Mehta MP, Shapiro WR, Phan SC, Gervais R, Carrie C, Chabot P, et al. Motexafin gadolinium combined with prompt whole brain radiotherapy prolongs time to neurologic progression in non-small-cell lung cancer patients with brain metastases: results of a phase III trial. International Journal of Radiation Oncology Biology Physics 2009;73:1069–76. [170] Sobhakumari A, Love-Homan L, Fletcher EV, Martin SM, Parsons AD, Spitz DR, et al. Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism. PLoS One 2012;7: e48175. [171] Marzano C, Gandin V, Folda A, Scutari G, Bindoli A, Rigobello MP. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radical Biology & Medicine 2007;42: 872–81.

[172] Somwar R, Erdjument-Bromage H, Larsson E, Shum D, Lockwood WW, Yang G, et al. Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines. Proceedings of the National Academy of Sciences of the United States of America 2011;108:16375–80. [173] Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2000;407:390–5. [174] James J, Murry DJ, Treston AM, Storniolo AM, Sledge GW, Sidor C, et al. Phase I safety, pharmacokinetic and pharmacodynamic studies of 2-methoxyestradiol alone or in combination with docetaxel in patients with locally recurrent or metastatic breast cancer. Investigational New Drugs 2007;25:41–8. [175] Sweeney C, Liu G, Yiannoutsos C, Kolesar J, Horvath D, Staab MJ, et al. A phase II multicenter, randomized, double-blind, safety trial assessing the pharmacokinetics, pharmacodynamics, and efficacy of oral 2-methoxyestradiol capsules in hormone-refractory prostate cancer. Clinical Cancer Research 2005;11:6625–33. [176] Zhang Q, Ma Y, Cheng YF, Li WJ, Zhang Z, Chen SY. Involvement of reactive oxygen species in 2-methoxyestradiol-induced apoptosis in human neuroblastoma cells. Cancer Letters 2011;313:201–10. [177] Donate F, Juarez JC, Burnett ME, Manuia MM, Guan X, Shaw DE, et al. Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN224). British Journal of Cancer 2008;98:776–83. [178] Juarez JC, Manuia M, Burnett ME, Betancourt O, Boivin B, Shaw DE, et al. Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling. Proceedings of the National Academy of Sciences of the United States of America 2008;105:7147–52. [179] Lee K, Briehl MM, Mazar AP, Batinic-Haberle I, Reboucas JS, GlinsmannGibson B, et al. The copper chelator ATN-224 induces peroxynitrite-dependent cell death in hematological malignancies. Free Radical Biology & Medicine 2013;60:157–67. [180] Kim YR, Oh JE, Kim MS, Kang MR, Park SW, Han JY, et al. Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin. Journal of Pathology 2010;220:446–51. [181] Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Metabolic targets for cancer therapy. Nature Reviews Drug Discovery 2013;12:829–46. [182] Hensley CT, Wasti AT, DeBerardinis RJ. Glutamine and cancer: cell biology, physiology, and clinical opportunities. The Journal of Clinical Investigation 2013;123:3678–84. [183] Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009;324: 1029–33. [184] Wang JB, Erickson JW, Fuji R, Ramachandran S, Gao P, Dinavahi R, et al. Targeting mitochondrial glutaminase activity inhibits oncogenic transformation. Cancer Cell 2010;18:207–19. [185] Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I, et al. Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase. The Journal of Biological Chemistry 2006;281:31222–33. [186] Son J, Lyssiotis CA, Ying H, Wang X, Hua S, Ligorio M, et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 2013;496:101–5. [187] Bandy B, Davison AJ. Mitochondrial mutations may increase oxidative stress: implications for carcinogenesis and aging? Free Radical Biology & Medicine 1990;8:523–39. [188] Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach. Nature Reviews Drug Discovery 2009;8:579–91. [189] Nogueira V, Park Y, Chen CC, Xu PZ, Chen ML, Tonic I, et al. Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer Cell 2008;14:458–70.


Targeting the TGFβ pathway for cancer therapy.

"Targeting Taspase1 for Cancer Therapy"-Response.

Targeting Oncogenic Mutant p53 for Cancer Therapy.

Targeting hypoxic response for cancer therapy.

Targeting Hsp70: A possible therapy for cancer.

Targeting mutant p53 for cancer therapy.

Clinical targeting recombinant immunotoxins for cancer therapy.

Targeting truncated RXRα for cancer therapy.

Targeting mitochondria metabolism for cancer therapy.

Targeting DNA Replication Stress for Cancer Therapy.

Targeting miRNAs for pancreatic cancer therapy.

Exosome-Based Cancer Therapy: Implication for Targeting Cancer Stem Cells.

Nanomaterials in Targeting Cancer Stem Cells for Cancer Therapy.

Small molecules targeting microRNA for cancer therapy: Promises and obstacles.

Targeting EZH2 for cancer therapy: progress and perspective.

Targeting p53-MDM2-MDMX loop for cancer therapy.

Small molecule targeting miR-34a for cancer therapy.

Targeting transcription factor STAT3 for cancer prevention and therapy.

Chemotherapy and molecular targeting therapy for recurrent cervical cancer.

Targeting glucose uptake with siRNA-based nanomedicine for cancer therapy.

Targeting microtubules by natural agents for cancer therapy.

Traditional Chinese medicine targeting apoptotic mechanisms for esophageal cancer therapy.

Broad targeting of angiogenesis for cancer prevention and therapy.

mTOR co-targeting strategies for head and neck cancer therapy.