ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

Reactive oxygen species in redox cancer therapy Lingying Tong a, Chia-Chen Chuang b, Shiyong Wu a,c,*, Li Zuo b,d,** a

Edison Biotechnology Institute, Konneker Research Center, Ohio University, Athens, OH 45701, USA Radiologic Sciences and Respiratory Therapy Division, School of Health and Rehabilitation Sciences, The Ohio State University College of Medicine, The Ohio State University, Columbus, OH 43210, USA c Department of Chemistry and Biochemistry, Molecular and Cellular Biology Program, Ohio University, Athens, OH 45701, USA d Interdisciplinary Biophysics Graduate Program, The Ohio State University, Columbus, OH 43210, USA b

A R T I C L E

I N F O

Article history: Received 6 June 2015 Received in revised form 8 July 2015 Accepted 10 July 2015 Keywords: Antioxidant Oxidative stress Redox Tumor Cancer therapy

A B S T R A C T

The role of reactive oxygen species (ROS) in cancer cells has been intensively studied for the past two decades. Cancer cells mostly have higher basal ROS levels than their normal counterparts. The induction of ROS has been shown to be associated with cancer development, metastasis, progression, and survival. Various therapeutic approaches targeting intracellular ROS levels have yielded mixed results. As widely accepted dietary supplements, antioxidants demonstrate both ROS scavenging ability and anti-cancer characteristics. However, antioxidants may not always be safe to use since excessive intake of antioxidants could lead to serious health concerns. In this review, we have evaluated the production and scavenging systems of ROS in cells, as well as the beneficial and harmful roles of ROS in cancer cells. We also examine the effect of antioxidants in cancer treatment, the effect of combined treatment of antioxidants with traditional cancer therapies, and the side effects of excessive antioxidant intake. © 2015 Elsevier Ireland Ltd. All rights reserved.

Introduction Reactive oxygen species (ROS) and associated oxidative stress have been historically considered harmful to the cell as they can damage cellular DNA, oxidize fatty acids in lipids and amino acids in proteins, and deactivate certain enzymes and their cofactors. The effects of these biological demolitions eventually lead to tissue destruction [1,2]. ROS have been associated with various diseases, including cardiovascular diseases [3], diabetes [4], and cancers [5]. In partic-

Abbreviations: Akt, protein kinase B; AMPK, 5′-adenosine monophosphateactivated protein kinase; ATM, ataxia telangiectasia-mutated; DR5, death receptor 5; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate; EGF, epidermal growth factor; ETC, electron transport chain; GCL, glutamate cysteine ligase; GCLC, GCL catalytic subunit; GCLM, GCL modifier subunit; GSH, glutathione; GSR, glutathione-disulfide reductase; GSSG, glutathione disulfide; H2O2, hydrogen peroxide; HIF-1, hypoxia-inducible factor 1; IR, ionizing radiation; ISL, isoliquiritigenin; JNK, c-Jun N-terminal kinase; Keap1, Kelch-like ECHassociated protein 1; MAPK, mitogen-activated protein kinases; MTP, mitochondrial permeability transition; mTOR, mammalian target of rapamycin; NO, nitric oxide; NOS, nitric oxide synthase; Nrf2, nuclear factor erythroid 2-related factor 2; O2•–, superoxide; •OH, hydroxyl radical; ONOO–, peroxynitrite; PSO, psoralidin; ROS, reactive oxygen species; Se, selenium; SELECT, Selenium and Vitamin E Cancer Prevention Trial; SOD, superoxide dismutase; TLR, toll-like receptor; TRAF 6, tumor necrosis factor receptor-associated factor 6; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; TrxR, thioredoxin reductase; TXN, thioredoxin; UVR, ultraviolet radiation. * Corresponding author. Tel.: +1 740 597 1318; fax: +1 740 593 4795. E-mail address: [email protected] (S. Wu). ** Corresponding author. Tel.: +1 614 292 5740; fax: +1 614 292 0216. E-mail address: [email protected] (L. Zuo).

ular, elevated levels of ROS are implicated in cancer cells partly due to increased metabolic activity. To alleviate the detrimental effects of ROS, antioxidants are commonly considered to be beneficial to human health and are recommended as a dietary supplement intake [6]. Surprisingly, many clinical studies showed that the supplementation of antioxidants failed to impede the disease progression or extend the life expectancy of patients [7,8]. As a result, the biological roles of ROS have been re-examined. To date, it is still unclear whether ROS are part of the cause or the result of these diseases, and the biological roles of ROS in the body remain ambiguous. As “two-faced” molecules, ROS are involved in various complex signaling pathways and are critical to the fate of both healthy and diseased cells such as carcinomas. Specifically, disruption of ROS levels is one common approach in cancer therapies, as cancer cells are more vulnerable to ROS disruption than normal cells [9]. The survival rate of cancer cells can be reduced by either inducing or reducing intracellular ROS levels. Thus, antioxidants have also been often used along radiotherapy. However, the related therapeutic effects seem controversial [10]. Therefore, it is essential to discuss current progress regarding both positive and negative effects of ROS in modern cancer therapeutics, as well as the redox role in cancer development. Oxidative stress The continuous production and detoxification of cellular ROS lead to a tightly controlled and well-balanced redox status in normal cells. Oxidative stress, on the other hand, is caused by an imbalance

http://dx.doi.org/10.1016/j.canlet.2015.07.008 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.

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Fig. 1. This schematic demonstrates the major reactions and signaling pathways in ROS production and scavenging system. Abbreviations: antioxidant response element (ARE), electron (e−), glutathione (GSH), glutathione disulfide (GSSG), hydrogen peroxide (H2O2), hydroxyl radical (•OH), nitric oxide (NO), nitric oxide synthase (NOS), nuclear factor erythroid 2-related factor 2 (Nrf2), oxidative stress (OS), peroxynitrite (ONOO−), reactive oxygen species (ROS), superoxide (O2•−), superoxide dismutase (SOD).

between ROS production and removal, which results in the accumulation of ROS in the cells. This could be attributed to the overproduction of ROS or the deterioration of the antioxidant system [2]. As mentioned previously, oxidative stress is believed to be associated with various diseases, yet the exact roles of ROS in these diseases have not been fully elucidated. Production of ROS The majority of ROS are produced in the electron transport chain (ETC) of mitochondria, mainly at complexes I and III [11]. In ETC, electrons from NADH are transferred to oxygen molecules (O2) and eventually generate harmless water molecules. When only one electron is received, O2 is reduced to superoxide (O2•–), which can be further converted to hydrogen peroxide (H2O2) via superoxide dismutase (SOD) catalysis. Through a Fenton reaction, H2O2 can then be catalyzed to form a highly reactive ROS, hydroxyl radical (•OH) [12]. Moreover, O2•– reacts with nitric oxide (NO•) to generate peroxynitrite (ONOO–) in a diffusion-controlled manner [13] (Fig. 1). In addition to mitochondria, ROS are also formed in the cytoplasm by various enzymes [14]. Nitric oxide synthase (NOS) produces NO• by facilitating the conversion of l-arginine to l-citrulline. The uncoupled NOS produces O2•– [15]. Electrons from NAD(P)H can transfer to O2 and produce O2•– by NADPH oxidase, with the generation of NAD(P)+ at the same time [16]. Other cellular enzymes, including xanthine oxidase, lipoxygenase, cyclooxygenases, and cytochrome p450 families, also participate in the generation of ROS during normal biological reactions [17] (Fig. 1). Besides endogenous sources, environmental stresses, such as ultraviolet radiation (UVR), ionizing radiation (IR), and hypoxia, also induce cellular ROS production. In particular, UVB (a type of UVR) has been shown to activate oxidase and promote the uncoupling of NOS [18,19]. These enzymes then contribute to the production of O2•–, NO• and ONOO– [20]. In the case of IR, the water molecule is subjected to radiolysis, generating highly reactive molecules such as ionized water (H2O+), •OH, and H2O2 that will cause cellular DNA damage [21]. Additionally, IR upregulates the mitochondrial ETC function and results in more mitochondrial ROS production [22]. Hypoxia, the microenvironment experienced by many tumors, also activates

various key regulators such as hypoxia-inducible factor 1 (HIF-1). The overexpressed HIF-1 leads to the generation of ROS, which in turn upregulates HIF-1, completing a positive feedback loop of ROS induction in the cells [11,23]. Antioxidant (ROS scavenging system) The ROS scavenging system is mainly composed of antioxidant enzymes and non-enzymatic ROS scavengers. Common enzymes that are involved in the detoxification process of ROS are the following: SOD, which catalyzes the conversion from O2•– to O2 or H2O2; catalase, which catalyzes the decomposition of H2O2 to H2O and O2; and family members of peroxidase, which help to reduce both H2O2

Fig. 2. This schematic demonstrates the critical roles of intracellular ROS levels in regulating the fate of cancer cells. Higher or lower ROS levels reduce the survival rate of cancer cells. In addition, antioxidants may diminish the effects of chemo/ radiotherapy by lowering relative ROS production. Abbreviations: electron transport chain (ETC), reactive oxygen species (ROS).

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and lipid peroxide. On the other hand, ONOO– is decomposed by enzymes such as peroxiredoxins and glutathione peroxidases [24]. For non-enzymatic reducing power, glutathione (GSH) and NAD(P)H are the most well-known electron donators [25]. GSH can be oxidized to form glutathione disulfide (GSSG), which can be reduced back to GSH by glutathione reductase (GR) using NADPH (Fig. 1). Other ROS sensors, such as transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), are also crucial in regulating cellular redox status. Nrf2 is constitutively expressed but kept at a low level by its repressor Kelch-like ECH-associated protein 1 (Keap1). Keap1 binds and promotes Nrf2 degradation through the ubiquitin– proteasome pathway. Under oxidative stress, the cysteine residues on Keap1 are modified and the resulting conformational change leads to the release of Nrf2. The liberated Nrf2 is then stabilized and translocated into the nucleus, activating antioxidant response element (ARE) and many downstream genes coding for antioxidant proteins including thioredoxin (TXN) and glutathione-disulfide reductase (GSR) that act to reduce cellular ROS levels [26,27]. Nrf2 regulates multiple antioxidant pathways, including GSH and NADPH production, thereby enhancing the detoxification process [27] (Fig. 1). Oxidative stress in cancer Basal ROS levels are believed to be higher in cancer cells compared to their normal counterparts. The elevated levels of ROS are mainly attributed to the following sources: (1) Increased metabolic activity and high mitochondrial energetics: Although cancer cells shift their glucose metabolism to anaerobic pathways even with sufficient oxygen supply (known as the Warburg effect), the higher than normal uptake of glucose still stimulates the mitochondrial energetics, which is believed to be related to elevated ROS production [28,29]; (2) alterations of the mitochondria ETC: Electrons linger on the complex increasing the possibility of being transferred to oxygen [19,30,31]; (3) the hypoxic condition of cancer leads to the activation of various key master proteins such as HIF-1, as mentioned above; (4) chronic inflammation and cytokine releasing [32]; (5) oncogenic signaling: The activation of c-Myc protein and its downstream signaling pathways can induce cellular ROS production [33] (Fig. 2). The higher than normal ROS levels are suggested to be related to cancer cell growth, angiogenesis, and metastasis [34]. Moreover, many cancer cells equip strong antioxidant defenses in order to adapt to elevated ROS and to avoid apoptosis [35]. Therefore, treatments focusing on disturbing the redox status of cancer cells may be a feasible therapeutic approach in cancer therapy. Redox disruption in cancer therapy Interestingly, moderate intake of antioxidants has been shown to reduce the risk of cancer development and slow cancer progression [25]. The accumulation of ROS-induced damage is one of the major leading factors to carcinogenesis, and antioxidants are widely believed to treat cancers by eliminating excess ROS. Büchner et al. observed a beneficial inverse association between fruit and vegetable consumption and the risk of lung cancer. The increased uptake of 100 g/day of fruit and vegetables, which are rich in antioxidants, could reduce the lung cancer risk in smokers, as well as the metastasis of squamous cell carcinomas [36]. In addition, antioxidants such as N-Acetyl-l-Cysteine (NAC) and Vitamin C have demonstrated anti-cancer characteristics [37]. By reducing intracellular ROS levels, these antioxidants have been shown to manipulate the survival signaling pathways, the autophagy process, and metastatic progress of cancer cells [38]. However, long-term clinical studies fail to show the efficacy of antioxidants in cancer prevention [37]. Although higher than normal ROS levels are observed in cancer cells, it is suggested that cancer cells are more vulnerable to

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intracellular ROS induction. Therefore, cancer treatment by means of enhancing intracellular ROS production may be an efficient approach. Chemotherapeutic agents, such as paclitaxel and anthracyclines, can induce cancer cell apoptosis by increasing ROS levels [9]. Moreover, Elesclomol, a small molecule currently under phase III clinical trial, also functions to induce intracellular ROS generation in cancer cells. Indeed, the effect of elesclomol wears off when the cells are co-treated with antioxidants such as NAC [39] (Table 1). As a major site of ROS production, mitochondria are considered one of the targets for cancer treatment. Although the Warburg effect indicates that the anaerobic process is preferred after glycolysis in mitochondria even with sufficient oxygen supply, mitochondria still remain functional or even hyperactive in cancer cells as these cells tend to absorb more glucose than normal cells. Researchers have shown that cancer cells are more vulnerable than normal cells to mitochondria dysfunction [55]. Thus, reagents targeting mitochondria are expected to be effective in inducing cancer apoptosis [55,56]. For instance, antioxidant compounds such as resveratrol and α-tocopheryl succinate (Vitamin E analogues) exert their anti-cancer effects directly at mitochondria [56]. Mitochondria are also susceptible to oxidative damage, although they are major sources of ROS. The mitochondrial DNA is targeted by ROS due to its close proximity to the ETC, where the majority of ROS are produced [55]. Instead of reducing ROS, compounds targeting cancer mitochondria trigger the overproduction of ROS, thereby inducing mitochondrial permeability transition (MPT), disturbing mitochondrial transmembrane potential, and eventually disrupting the cancer cell mitochondria [56,57]. Taken together, cancer cells are sensitive to redox disruption, and targeting either induction or reduction of ROS levels in cancer cells can lead to effective cancer treatments.

Induction of ROS by antioxidants Some compounds, generally considered as antioxidants, also exhibit pro-oxidative properties. When present at a low concentration, these antioxidants prevent oxidative damage in the cells. However, they can induce ROS production and cytotoxicity at high concentrations [35]. A study conducted by Sun et al. has shown that the antioxidant isoliquiritigenin (ISL) promotes oxidative stress by interrupting the preexisting antioxidant production pathways. The cell downregulates antioxidant expression in order to compromise with the reduced intracellular ROS due to the ISL antioxidant activity [35]. As a natural antioxidant, ISL has been shown to inhibit the growth of prostate cancer cells [43]. The weakened antioxidative capacity after ISL treatment can sensitize the cancer cells to X-ray irradiation [35] (Table 1). Quercetin, a member of the flavone family, has been wellstudied for its antioxidant characteristics. Mice with prolonged and chronic supplementations of quercetin in their diet have exhibited a disrupted GSH metabolism as the GSH concentration and GR activity are both decreased. Although the exact mechanism was not elucidated, it is possible that quercetin functions as both an antioxidant and pro-oxidant [58]. In ovarian cancer cells, quercetin demonstrated pro-oxidant activity rather than antioxidant activity. The induction of ROS led to the upregulation of death receptor 5 (DR5) through the c-Jun N-terminal kinase (JNK)/C/EBP homologous protein (CHOP) signaling pathway. The activation of DR5 does not lead to apoptosis, but increases the sensitivity of the cells to tumor necrosis factor-related apoptosisinducing ligand (TRAIL) treatment [42] (Table 1). Other antioxidants including resveratrol, carnosol, and psoralidin (PSO) have the capacity to kill cancer cells by increasing ROS. In colon cancer cells, a relatively high concentration of resveratrol can

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Table 1 Summary of the published work on antioxidants or ROS inducers in cancer therapy. Study

Cancer type/cell line

Antioxidant/ROS inducer

Anti-cancer/ chemotherapeutic drugs or radiation

Findings

Reference

Ramanathan et al. (2005)

Breast cancer, lung Cancer cell, gastric cancer cell, bladder urothelial carcinoma cell lines Human Hs294T melanoma cells

Pyruvate, selenium, NO synthase inhibitor

Paclitaxel

Reduce paclitaxel-mediated DNA damage and cytotoxicity

[40]

NAC, tiron

Elesclomol +

[39]

IR

Elesclomol-induced gene transcription, oxidative stress and apoptosis are inhibited by antioxidants Lower risk of lung cancer Enhance tumor radiosensitivity

Sensitize cancer cells to TRAIL-induced apoptosis Lower ROS and mitochondrial membrane potential Selectively inhibit C4-2 prostate cell proliferation Disturb redox balance and increase radiosensitivity of HepG2 Inhibit cell survival in radiation

[42]

Suppress colon cancer cell growth via ROS-triggered autophagy Facilitate antitumor response Diminish side effects of anti-cancer drugs Mixed results from cancer cell respondence to resveratrol + IR treatments Significantly increase prostate cancer risk in healthy individuals when taken at high doses No beneficial effects in preventing prostate cancer is observed No beneficial effects in preventing secondary primary tumors Block cell cycle (G2) and damage DNA

[45]

Induce ROS-triggered autophagy Combined NAC treatment reduces psoralidin cytotoxicity Reduce the toxicity of cisplatin Induce tumor apoptosis

[53]

Kirshner et al. (2008)

Büchner et al. (2010) Lin et al. (2012)

Lung cancer DLD1, HeLa and MCF-7 tumor cell lines

Fruits and vegetables Quercetin

Yi et al. (2014)

Human ovarian cancer cells

Quercetin

Zhang et al. (2010)

C4-2 and LNCaP Prostate cancer cells

Isoliquiritigenin

Sun et al. (2013)

Human liver HepG2

Isoliquiritigenin

IR

Scarlatti et al. (2007)

Human DU145 prostate cancer cells HT-29 and COLO 201 human colon cancer cells LoVo human colon adenocarcinoma

Resveratrol

IR

Resveratrol

5-Fluorouracyl

Kma (2013)

Glioma, prostate and melanoma cancer

Resveratrol

IR

Dunn et al. (2010) Klein et al. (2011) Kristal et al. (2014)

Prostate cancer

Vitamin E, selenium (SELECT)

Karp et al. (2013)

Selenium

Hao et al. (2014)

Secondary primary tumors in nonsmall-cell lung cancer Human breast cancer cells MDAMB-231 Human lung cancer cell line A549

Ghosh et al. (2015)

Ehrlich ascites carcinoma

Organoselenium compound

Miki et al. (2012) Hotnog et al. (2013)

Al Dhaheri et al. (2014)

Resveratrol

Carnosol Psoralidin

Cisplatin

[36] [41]

[43]

[35] [44]

[46]

[47]

[48–50]

[51] [52]

[54]

IR, ionizing radiation; NAC, N-acetyl cysteine; ROS, reactive oxygen species; SELECT, the Selenium and Vitamin E Cancer Prevention Trials; TRIAL, tumor necrosis factorrelated apoptosis-inducing ligand.

induce cell apoptosis [45]. In triple negative breast cancer cells, carnosol-induced ROS lead to DNA damage and cell apoptosis. When cells were treated with carnosol and ROS scavenger simultaneously, DNA damage was largely attenuated and cell apoptosis was inhibited [52]. PSO suppresses lung cancer growth by inducing cellular ROS production, autophagy and subsequent cell death (Table 1). The addition of NAC lowered the PSO-induced ROS level. As a result, the autophagy level was reduced, as well as the cytotoxicity exerted by PSO [53]. Treating the cells with purified recombinant GR does not enhance antioxidant activity; rather it induces ROS production. Theoretically, higher GR levels lead to higher GSH levels, which reduce ROS levels in the cell. Surprisingly, in GR-overexpressed cells, ROS levels increase in a NADPH- and thioredoxin reductase (TrxR)-dependent manner. As shown by isolated mitochondria from cells, interrupting NADPH/NADP+ and GSH/GSSH balance caused reductive stress, which eventually resulted in the net overproduction of mitochondrial ROS, especially H2O2 [59]. While the expression levels of antioxidant enzymes are reduced, the ROS levels are eventually higher as the cancer cells continuously produce high levels of ROS [35].

Controversial role of antioxidants in combined treatment In addition to the antioxidant treatments alone, the safety and efficacy of the combined treatment of antioxidants with chemo- or radiotherapy remain controversial [10]. Some studies suggested that antioxidant supplementation could sensitize the cancer cells to chemo- or radiotherapy, and at the same time, reduce the side effects of radiotherapy by protecting the normal cells. However, other studies indicated that antioxidants may also protect cancer cells against these therapies. One study focusing on organoselenium, a naphthalimide-based selenium (Se) compound, recommended patients to take antioxidant supplements together with chemotherapeutic agents. The combination of the anti-cancer drug cisplatin and organoselenium has shown to produce synergistic effects in which organoselenium enhances cisplatin-induced cancer cell death and protects normal cells from cisplatin toxicity [54] (Table 1). Indeed, Se promotes DNA repair and enhances endogenous antioxidant levels in the normal cells [60]. Radiotherapy is another traditional approach for treating cancer. Radiotherapy kills cancer cells by damaging cellular DNA, either directly by the radiation energy or indirectly by ROS. Antioxidants

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extracted from fruits, vegetables, or herbs have shown to be able to sensitize cancer cells to radio- or chemotherapy [20]. For example, quercetin enhanced the radiotherapy effect in both in vitro and in vivo colorectal cancers by modulating ataxia telangiectasia-mutated (ATM)-related signaling pathway in response to radiation [41]. Moreover, resveratrol also increases the sensitivity of cancer cells to chemotherapy [46] and radiotherapy possibly by ceramide induction [44,47]. Controversially, antioxidants can diminish the efficacy of chemotherapy or increase radio-resistance for radiotherapy (Fig. 2). Since some approaches of chemotherapy involve the stimulation of intracellular ROS production to eradicate cancer cells, the supplementation of antioxidants may provide protective effects against ROS-induced damages, thereby reducing the efficacy of the treatment [60,61]. Compared to non-resistant cells, the resistant cancer cells often express higher levels of antioxidant enzymes [61]. Therefore, there may be a potential risk to taking antioxidants concurrently with these therapies. For example, one of the most commonly used chemotherapeutic agents, paclitaxel, can induce cancer cell death by increasing ROS levels, and the co-treatment with antioxidants such as pyruvate and Se diminishes the effect of paclitaxel [40]. As for radiotherapy, certain levels of ROS in cells are necessary. The manipulation of cellular redox systems by reducing GSH or NADH activity increases radiosensitivity [62,63]. Similar results were also reported in studies focusing on SOD. By elevating the expression of SOD, the cells develop more radio-resistance, while the downregulation of SOD increases radiosensitivity [64]. No significant advantage of antioxidants observed in clinical trials Although in vitro and in vivo data suggested promising effects of utilizing antioxidants in cancer treatment, most clinical studies showed no significant reduction of cancer progression. The Selenium and Vitamin E Cancer Prevention Trial (SELECT) performed by the National Cancer Institute (NCI) was a clinical trial that studied the effect of one or both dietary supplements on cancer prevention or treatment. The data demonstrated that long term (>5 years) intake of one or both supplements had no benefit in preventing prostate cancer or other cancers. In the trial with Vitamin E, the data indicated that men taking Vitamin E had increased risk of developing prostate cancer by 17% [48,49]. For the case of less than 150 IU/d Vitamin E supplement, there was no statistically significant difference in the mortality of the experimental and placebo groups. However, there was a statistical correlation between Vitamin E intake greater than 150 IU/d and mortality. Indeed, the statistical results showed that high-dosage Vitamin E supplement (> or =400 IU/d) increased mortality and should be avoided [65]. On the other hand, Se is believed to be safe to use but showed no benefit to non-smallcell lung cancer [51]. Other clinical trials also indicated that Vitamin E intake increased the risk of prostate cancer, and Se supplementation intake could increase the prostate cancer risk in men with preexisting high levels of Se [50]. For the long term intake, clinical trials also showed that Vitamins such as Vitamin A, C, E and β-carotene exert no significant beneficial effects in skin cancer prevention [66,67]. Other antioxidants, such as α-tocopherol, do not contribute to the prevention of lung cancer. Ironically, the intake of β-carotene supplement can even increase lung cancer incidence in smokers [68]. In our opinion, the possible reasons for negative outcome of antioxidant trials include: (1) non-specificity, as it is hard for antioxidants to target specifically on cancer cells, rather exerting its effect all over the body; (2) oral administration of antioxidants, which may not be able to access the intracellular cytoplasm; (3) antioxidants remove ROS but do not reduce ROS production; (4) the disturbance of redox balance may eventually lead to the induction of cellular ROS overproduction.

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The beneficial role of ROS Although oxidative stress is widely considered as a detrimental by-product of normal metabolic reactions, the physiological roles of ROS are indeed crucial in maintaining the biological function of both normal and cancer cells. ROS play important roles in signaling transduction, inflammatory response, and autophagy. The very first observation by Dr. Warburg and later repeated by many other scientists was the burst of oxygen consumption by fertilized eggs. Subsequently, it has been shown that oxygen is used by NADPH oxidase on the surface of the eggs to produce H2O2, which develops a shell to protect the fertilized egg [69,70]. Besides shell protection, H2O2 can play critical roles in sperm activation and maturation [71,72]. Additional research further proved that H2O2 is involved in many signaling pathways. Growth factors, such as epidermal growth factor (EGF), bound to their receptors and stimulated ROS production, which led to the activation of p38 MAPK signaling pathways [73]. ROS are critical in normal cell growth and anti-apoptotic protection. Consequently, this pathway can be utilized by cancer cells which lead to tumorigenesis. It has also been shown that low levels of H2O2 induced the phosphorylation of protein kinase B (Akt) in cell survival pathways. Instead of apoptosis, cells senesce with G2/M arrest [74]. Because of the critical role of ROS in regulating cell growth and apoptosis, one of the most popular approaches to treat cancers is to induce apoptosis. The significance of this approach is to regulate ROS specifically in cancer cells, yet does not affect the ROS levels in normal cells. In addition, ROS are closely related to cellular immune response, as phagocytes produce ROS via NADPH oxidase to fight against intracellular bacteria. However, recent research demonstrated that mitochondrial ROS also contribute to the elimination of bacteria through toll-like receptor (TLR) signaling. Once the TLRs on macrophages have been activated, they could be involved in the signaling pathways through tumor necrosis factor receptor-associated factor 6 (TRAF6) and evolutionarily conserved signaling intermediate in Toll pathways to increase the ROS induction in mitochondria [75]. ROS have also been shown to be associated with autophagy, which is an important process for the regulation of apoptosis [76]. Autophagy is believed to be a tumor repression mechanism by removing damaged organelles in cells and thus prevents the accumulation of damaged proteins or organelles. Therefore, the disruption of autophagy is closely related to cancer development and progression. In cancer cells, autophagy is normally up-regulated compared to their normal counterparts, mainly due to higher metabolic rate and proliferation rate [77]. ROS have been shown to regulate autophagy by oxidizing ATG4, and also by activating the AMPK/ mTOR pathway [78]. In normal cells, this is a tightly controlled process; in cancer cells, this process can be targeted for therapeutic purposes. The concentration of ROS is essential to cell fate. Low concentration of ROS is necessary for maintaining normal cell function, but moderate or high ROS concentration could lead to carcinogenesis. Cytotoxicity and side effects of antioxidants As mentioned previously, excessive intake of antioxidants may lead to cytotoxicity. Dietary polyphenol compounds, such as flavonoids, are antioxidants that exert anti-cancer effects. Polyphenolics containing phenol rings can be metabolized by peroxidase to produce reactive phenoxyl radicals, which are able to co-oxidize GSH or NADH. This process results in extensive oxygen uptake and oxidative stress [79]. Epigallocatechin-3-gallate (EGCG) is one of the most abundant and well-studied polyphenol compounds in green tea extract. It has been demonstrated to inhibit cancer cell growth

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and progression [80]. However, high concentrations of EGCG is cytotoxic (LD50 ~200 μM). EGCG induces cytotoxicity by disrupting mitochondrial membrane potential, thus generating cellular oxidative stress. Interestingly, the cytotoxicity of EGCG can be prevented by treating the cells with ROS scavengers such as dithiothreitol and pyruvate, suggesting the role of ROS in the EGCG-induced cytotoxicity. In addition to EGCG, similar results were also observed for epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC), indicating that the overdose of the antioxidant is indeed cytotoxic to the cells [81]. Se is an important nutrient for maintaining human health. Lower than normal concentration of Se in the blood has been shown to be related to a high risk of various cancers [82,83]. Therefore, maintaining a moderate level of Se is beneficial to human health [84,85]. In vitro and in vivo experiments have indicated that Se prevents carcinogenesis, but a high concentration of Se can be cytotoxic [86]. Se conjugates with two GSH to form metabolite selenodiglutathione (GSSeSG), a potent compound that enhances ROS production, DNA damage and apoptosis [87]. Besides exogenous antioxidant treatments, the modulation of the endogenous antioxidant system can also be cytotoxic. Overexpressed glutamate cysteine ligase (GCL), GCL catalytic subunit (GCLC) or GCL modifier subunit (GCLM) can lead to an imbalanced GSH/GSSG ratio. Excess GSH shifts the cells to reductive stress, which can cause mitochondrial oxidation and cytotoxicity [88]. In some cases, antioxidants also increase cancer risks. Supplementing NAC or Vitamin E has been shown to significantly increase lung cancer progression and reduce survival rate in mouse models [89]. As the expression of endogenous antioxidant genes is reduced by the introduction of antioxidant supplements, the tumor transcriptome profile is altered with a downregulation of p53 expression level accordingly [90]. These changes provide an explanation on why antioxidants may promote cancer development. Moreover, dietary selenite treatment has been shown to induce malignant mesothelioma cell proliferation and metastasis in a selenite dosedependent manner. The study indicated that Se could activate ERK and trigger the ERK-mediated survival pathway, thereby inducing tumor progression [91]. As mentioned previously, Nrf2 is one of the master regulators of redox homeostasis in cells. Interestingly, it has been shown to be both a tumor suppressor and a tumor promoter. As a transcription factor, Nrf2 regulates the expression of many antioxidant genes, which carefully mediate ROS levels in normal cells to prevent carcinogenesis. In addition, Nrf2 functions as a cancer promoter by activating survival pathways via the upregulation of antiapoptotic protein Bcl-2, thus promoting cellular resistance to apoptosis [92]. Indeed, high expression and activity of Nrf2 have been observed in various cancer cell types. Such overexpressed Nrf2 causes the cells to become more chemo-resistant. On the contrary, the inhibition of Nrf2, either by transfection of Nrf2 siRNA or upregulation of Keap1, increases the sensitivity of cancer cells to chemotherapeutic agents [93]. In vivo experiments comparing Nrf2−/− homozygous deletion recombinant negative mice to wild type Nrf2+/+ mice showed that there was no significant difference on the cellular interaction in continuous marrow culture, dsDNA damage repair, longevity, or antioxidant response. Moreover, the Nrf2−/− mice did show radioresistance on marrow stroma, which indicated that the Nrf2−/− cells may have more capacity to handle oxidative stress than normal cells [94]. Future prospect on the antioxidants/ROS inducers in cancer therapy As described previously, cancer cells are prone to redox disruption as compared to normal cells [9]. Although therapeutic approaches targeting the induction or elimination of ROS may be

feasible, clinical results using ROS inducers and antioxidants remain elusive. It has been reported that the concurrent utilization of antioxidants and anti-cancer drugs, such as NAC and elesclomol, may offset the beneficial effects [39]. Moreover, some antioxidants can reduce the efficacy of chemo- or radiotherapy [60,61]. Therefore, the application of antioxidants or ROS inducers in cancer management should be carefully weighted. In order to avoid contradiction and to optimize cancer-killing ability, it is essential to understand the mechanisms on which the specific cancer therapy is targeted. For instance, the primary mechanisms of most chemotherapy drugs involve the generation of ROS and the supplementation of antioxidants can thus attenuate the damages caused by these drugs [95]. In our opinion, various cancer types, different courses of treatment, and multiple antioxidant drugs that may cause additional effects other than antioxidation are crucial factors to be considered during cancer management and research. Conclusion To summarize, most cancer cells have higher than normal intracellular ROS levels. Either increasing or decreasing the ROS levels can be efficient in cancer therapy (Fig. 2). Meanwhile, maintaining certain levels of ROS is necessary for normal cellular function. Taken together, the role of ROS in cancer cells is extremely complex and very challenging to study. Nowadays, people tend to abuse the intake of antioxidants partially because of the exaggeration of the detrimental effects of ROS. As a matter of fact, moderate intake of antioxidants could be beneficial under certain circumstances, while excess antioxidant intake could lead to health problems and should be avoided. The bimodal nature of ROS and antioxidants requires that research discovers the fine line between optimization and overconsumption in order to utilize their effects for the various and sundry uses they have throughout the body. Acknowledgements The authors thank Dr. Kimberly Suzanne George Parsons, Andrew Graef, Benjamin Pannell and Alexander Ziegler for editorial assistance. This work was partially supported by the NIH grant 2RO1CA086928. Conflict of interest No conflicts of interest, financial or otherwise, are declared by the authors. References [1] S. Magder, Reactive oxygen species: toxic molecules or spark of life?, Crit. Care 10 (1) (2006) 208. [2] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress, Curr. Biol. 24 (10) (2014) R453–R462. [3] H.N. Siti, Y. Kamisah, J. Kamsiah, The role of oxidative stress, antioxidants and vascular inflammation in cardiovascular disease (a review), Vascul. Pharmacol. (2015) Apr 11. pii: S1537-1891(15)00042-7. doi: 10.1016/j.vph.2015.03.005. [Epub ahead of print]. [4] S. Tangvarasittichai, Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus, World J Diabetes 6 (3) (2015) 456–480. [5] C. Karaaslan, S. Suzen, Antioxidant properties of melatonin and its potential action in diseases, Curr. Top. Med. Chem. 15 (9) (2015) 894–903. [6] B. Halliwell, Antioxidants in human health and disease, Annu. Rev. Nutr. 16 (1996) 33–50. [7] A.R. Mendelsohn, J.W. Larrick, Paradoxical effects of antioxidants on cancer, Rejuvenation Res. 17 (3) (2014) 306–311. [8] R.E. Patterson, E. White, A.R. Kristal, M.L. Neuhouser, J.D. Potter, Vitamin supplements and cancer risk: the epidemiologic evidence, Cancer Causes Control 8 (5) (1997) 786–802. [9] K.A. Conklin, Cancer chemotherapy and antioxidants, J. Nutr. 134 (11) (2004) 3201S–3204S.

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