free radicals in breast carcinogenesis, breast cancer progression and

Critical Reviews in Oncology/Hematology 80 (2011) 347368

Free radicals in breast carcinogenesis, breast cancer progression and cancer stem cells. Biological bases to develop oxidative-based therapiesLaura Vera-Ramirez a, , Pedro Sanchez-Rovira a , M. Carmen Ramirez-Tortosa b,c , Cesar L. Ramirez-Tortosa d , Sergio Granados-Principal b,c , Jose A. Lorente e,f , Jose L. Quiles b,ga Department of Oncology, Complejo Hospitalario de Jaen, Avenida del Ejercito Espa ol s/n, 23007 Jaen, Spain n Institute of Nutrition and Food Technology Jos Mataix, Biomedical Research Center (CIBM), Health Sciences Technological Park, Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain c Department of Biochemistry and Molecular Biology II, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain d Department of Pathology, Complejo Hospitalario de Jaen, Avenida del Ejercito Espa ol s/n, 23007 Jaen, Spain n e Department of Legal Medicine, University of Granada, Avenida de Madrid 11, 18012 Granada, Spain GENyO Center, Pzer-University of Granada & Andalusian Government Centre for Genomics & Oncology, Biomedical Research Center (CIBM), Health Sciences Technological Park, Armilla, Granada, Spain g Department of Physiology, University of Granada, Campus Universitario de Cartuja, 18071 Granada, Spain b

f

Accepted 11 January 2011

Contents1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources, reactions and physiological/pathological roles of free radicals in the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress in carcinogenesis, invasion and metastasis in breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Oxidative stress and cancer initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Tumor suppressor genes and oncogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Oxidative damage and cancer promotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Cytokines and growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Protein phosphatases and protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Oxidative damage and cancer progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress and cancer stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 348 351 351 351 352 352 353 353 355 357 358 360 361 361 362 362 366

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Abstract Oxidative stress leads to lipid, carbohydrate, protein and DNA damage in biological systems and affects cell structure and function. Breast cancer cells are subjected to a high level of oxidative stress, both intracellular and extracellular. To survive, cancer cells must acquire adaptive mechanisms that counteract the toxic effects of free radicals exposure. These mechanisms may involve the activation of redox-sensitive transcription factors, increased expression of antioxidant enzymes and antiapoptotic proteins. Moreover, recent data maintain that different

Corresponding author at: Servicio de Oncologia, Complejo Hospitalario de Jaen, Avenida del Ejercito Espa ol 10, 23007 Jaen, Spain. n Tel.: +34 953220306; fax: +34 953220306. E-mail address: [email protected] (L. Vera-Ramirez). 1040-8428/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2011.01.004

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L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 347368

breast cancer cell types, show different intracellular antioxidant capacities that may determine their ability to resist radio and chemotherapy. The resistant cell type has been shown to correspond with tumor initiating cells, also known as cancer stem cells (CSCs), which are thought to be responsible for tumor initiation and metastasis. Abrogation of the above-mentioned adaptive mechanisms by redox regulation in cancer cells opens a promising research line that could have signicant therapeutic applications. 2011 Elsevier Ireland Ltd. All rights reserved.Keywords: Oxidative stress; Oncogenesis; Cancer stem cells; Angiogenesis; Metastasis

1. Introduction A free radical is any chemical specie that contains one or more unpaired electrons. Because of their avidity to accept electrons from other molecules, free radicals can modify the structure and/or function of these molecules, interfering with normal cell biology [1]. This is why living organisms have an antioxidant defense system ready to attenuate or repair the damage produced by free radicals, maintaining a delicate equilibrium known as oxidative balance. When this equilibrium is broken in favor of free radical production, a physiologic situation known as oxidative stress takes place [1,2]. Free radicals are known to play a dual role in biological systems, since they are involved in physiological normal processes and they are, at higher concentrations, the cause of severe oxidative damage of cell components and the onset of several signaling pathways in response to cellular stress. Despite the antioxidant defense system counteracts the harmful effects of the free radical excess, cellular damage accumulates during life and it is proposed that these alterations could play a key role in the development and progression of age-related diseases, as cancer [2,3]. Indeed, many studies report that cancer cells show an increased production of ROS compared with healthy cells [47]. Among well documented effects, free radicals are known to induce DNA damage and genomic instability favoring the acquisition of mutations that contribute to cellular transformation and cancer cells survival [810]. Free radicals are also known to regulate the expression of key genes in cellular growth, proliferation, apoptosis, differentiation, migration, invasion and angiogenesis and modulate the activity of proteins involved in the signaling pathways that control the above-mentioned processes [1114]. On the other hand, disturbances in the oxidative balance also drive the activation of the antioxidant defenses and repair mechanisms, which are supposed to counteract the effects of free radicals, avoiding cell damage [15,16]. Nevertheless, the effects of oxidative stress in cancer appear to be paradoxical since its dual role in cell biology, being able to potentiate both proliferation and apoptosis, depending on multiple factors as cellular microenvironment, free radical concentration or the extent of the activation of antioxidant defenses [17,18]. Even further, the mechanism of action of many antioneplastic drugs is based on the cytotoxic activity of free radicals, although there is certain controversy about the possible limitations in drug effectiveness caused by an excess of free radicals [19,20].

This two-faced character of free radicals is the main reason why the research community is still debating whether an excess of free radicals should be avoided or potentiated as a therapeutic tool against cancer cells. On the other hand, it is also a controvert issue whether the activation of antioxidant defenses would improve cancer treatment or, in contrary, would protect initiated cells against oxidative toxicity and apoptosis [21]. It is well known that breast cancers, as other solid tumors, show high levels of oxidative stress as veried by the detection of oxidative DNA adducts in breast cancer tissue [22] or a signicant raise in oxidative stress markers in the plasma from breast cancer patients [23,24]. Therefore, breast cancer may be a suitable model to explore the inuence of oxidative stress in neoplasm development and treatment. In this report, the current knowledge about free radical biology and their inuence in cell signaling cascades will be reviewed, with special interest in breast cancer cell deregulation. In addition, some recent ndings about the inuence of oxidative stress and hypoxia into breast CSC biology and their potential therapeutic application will be discussed. 2. Sources, reactions and physiological/pathological roles of free radicals in the cell Main sources of free radicals (Table 1) and their biological effects in both physiological and pathological situations, are well documented [13,15,16,25]. Because of this, we will not review extensively this topic in the following paragraphs but summarize the essential information to introduce the correct biochemical context. Among the more important radical derivatives of oxygen, also known as reactive oxygen species (ROS), hydroxyl radical (OH ), superoxide anion (O2 ) and hydrogen peroxide (H2 O2 ), which is not a free radical but it is considered as a ROS involved in the production of other free radicals, are of special importance [3,26,27]. The main endogenous source of ROS in living organisms is the mitochondria, where O2 is produced accidentally at the complexes I and III level in the respiratory chain [2,2830]. Endogenous free radicals are produced to regulate a wide variety of physiological functions in healthy cells. In an inammatory environment, neutrophils and macrophages increase in a fast but transient way their oxygen consumption, generating O2 and H2 O2 (what it is called oxidative burst) with antimicrobial and tumoricidal activity, as a rst


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Table 1 Biologically relevant free radicals and reactive species. Main natural and physiological pathways through which they are generated and biochemical activity in breast carcinogenesis and progression. Specie Singlet oxygen Formula1O 2

Pathway of origin Metabolism of partially reduced oxygen species

Role in breast carcinogenesis and progression Induction of mutagenesis through the generation of DNA adducts. Not described in breast carcinogenesis specically Induction of mutagenesis through the generation of DNA adducts. Not described in breast carcinogenesis specically Not reported, but a signicant and positive association have been reported between increasing intra-urban NO2 concentrations and the incidence of post-menopausal breast cancer [206] Induction of mutagenesis through the generation of DNA adducts. Reported to affect p53 gene integrity. Enhances vascular permeability, facilitating tumor angiogenesis Induction of mutagenesis through the generation of DNA adducts and apurinic sites, DNA strand breakage, nitration of tyrosine residues of proteins and inhibition of mitochondrial electron transport Generation of harmful reactive radicals through its participation in biochemical reactions. It also activates several molecular signaling pathways involved in cell transformation, proliferation, survival and angiogenesis Induction of mutagenesis through the generation of DNA adducts, base-free sites, DNA strand breaks and DNAprotein cross-links It is an important source of other highly oxidative substances such as H2 O2 and ONOO Initiates fatty acid peroxidation. Not specically related to breast carcinogenesis Participates in the biochemical cascade leading to lipid peroxides which are converted in mutagenic epoxides and aldehydes which also interfere with many of the signaling cascades initiated in the membrane It plays the same role than the preceding specie

Ozone

O3

Atmosphere

Nitrogen dioxide

NO2

Atmosphere

Nitric oxide

NO

Arginin metabolism

Peroxynitrite

ONOO

Derives from the reaction between NO and O2

Hydrogen peroxide

H2 O2

Microsomal and mitochondrial electron transport chain, peroxisomal metabolism, purine degradation, phagocytes

Hydroxyl radical

HO O2 HOO OR

Derives from previous specie

Superoxide anion Hydroperoxyl Alcoxyl

Microsomal and mitochondrial electron transport chain, XO/XDH, cellular oxydases and phagocytes Derives from previous specie Fatty acids from membrane phospholipids, aminoacids, and carbohydrates

Peroxyl

OORO II

Derives from previous specie

Acyloxyl

R-C-OO II

Metabolism of fatty acids from membrane phospholipids

It plays the same role than the preceding specie

Acylperoxyl Semiquinone radical Semiquinone anion radical Thiol radical

R-C-OOHQ Q R-S

Derives from previous specie Biochemical reactions that take place along the respiratory chain Biochemical reactions that take place along the respiratory chain Metabolism of sulphur containing aminoacids

It plays the same role than the preceding specie Participates in the biochemical cascade leading to O2 It plays the same role than the preceding specie Alteration of signaling cascades mediated by of cysteine- and methionine-containing enzymes. Not specically related to breast carcinogenesis

line of defense against environmental pathogens and neoplasia [31]. ROS production in lymphocytes via lipoxygenases and cyclooxygenases, also suggests that these species are involved in the amplication of the immune response and inammation [32]. On the other hand, the production of ROS by non-immune cells is involved in the regulation of molecular signaling cascades that control cardiac and vascular cell functioning [33,34], thyroid hormone biosynthesis

[35] and oxygen tension sensing [36]. Another group of physiologic free radicals are those centered in the nitrogen atom, the reactive nitrogen species (RNS). Among them, it should be mentioned nitric oxide (NO), which is a very important molecule in a wide variety of intracellular signalization processes as neurotransmission, arterial pressure control, immune response or the relaxation of the smooth musculature [37].

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Apart from their important role in normal cell homeostasis and function, free radicals are known to induce severe cellular damage and promote the development of certain pathologies. Briey, all biomolecules are putative targets of free radicals and ROS, thus lipids, proteins, carbohydrates and the genetic material are under the re line of the attack. Lipid peroxidation is an autocatalytic process by which hydroperoxides and secondary epoxides and aldehydes are formed [38,39]. Among these aldehydes, it should be mentioned malondialdehyde (MDA), which has been shown to be mutagenic to bacterial and mammal cells and carcinogenic to rats; and 4-hydroxy-2-nonenal (HNE), which has a weaker mutagenic potential, but an important effect in cellular phenotype because it interferes in molecular signaling cascades of the cell [40]. Proteins are also subjected to oxidative damage. Among the commonest alterations, fragmentation, aggregation and susceptibility to proteolytic degradation, are known to induce structural alterations and loose of function becoming, from the biological point of view, inactive or defective protein [41]. Some sugars, as glucose and other carbohydrates, autooxidize producing large amounts of water. This process allows the interaction of glucose with other biomolecules, for example proteins, giving raise to non enzymatic protein glycation [42]. Endogenous as exogenous free radicals can induce oxidative lesions in the genetic material of the cells that if it is not efciently repaired, will result in genetic mutations and their consequences. It is estimated that, under physiologic conditions, a human cell is exposed to 1.5 105 oxidative lesions per day, among which more than 100 have been already identied. To summarize the most relevant oxidative lesions in nuclear DNA it should been cited those generated by OH , which is able to intercalate between the double bonds of DNA bases giving raise to a very unstable radical that reacts with other DNA components and generates hydroxylated bases. This chain reaction produces a wide variety of modied bases, free base sites, DNA breaks and aberrant interactions between DNA and proteins. A well-known example of the consequences of the exposure of DNA to OH radical, is the formation of the 8-hydroxy-2 -deoxyguanosine (8-OH-dG) adducts, which are potential mutagens because they induce errors in the reading frame during replication and transversions type A:T C:G. On the other hand, the damage induced by free radicals in the deoxyribose molecules that compose the DNA chain is potentially mutagenic, because it interferes in the activity of enzymes as DNA polymerase and DNA ligase. These kinds of permanent modications have been found in oncogenes and tumor suppressor genes, suggesting that oxidative stress is related to early stages of carcinogenesis [43,44]. To complete this brief overview about free radicals and breast carcinogenesis, the involvement of free radicals in this process through estrogen metabolism worth to be discussed apart. It is known that endogenous and synthetic estrogens are converted, through aromatic hydroxylation by specic

cytochrome P-450 microsomal enzymes, into catechol estrogens, such as 4-hydroxy estradiol (4-OH-E2), and further to estrogen semi-quinones and quinones. Redox cycling between catechol estrogens and their quinones is accompanied by ROS production [45,46]. This redox reaction is enhanced in the presence of Cu2+ and Fe3+ , which are reduced by catechol estrogens to Cu+ and Fe2+ that, in turn, may reduce cellular peroxides able to initiate lipid peroxidation in the presence of oxygen. ROS generated by catechol estrogen metabolism have been shown to induce DNA damage, lipid peroxidation and protein oxidation in estrogen-target tissues, as the mammary gland [47]. Consequently, the prooxidant effects of estrogen metabolism have been taken as an argument to explain the rmly established and positive relationship between estrogen exposure and breast cancer risk and indeed, in vitro and in vivo experiments suggest that estrogen metabolites are likely contributors to the development of breast cancer. For example, a recent study shows that 4-OH-E2 causes mutations in cell culture systems and can transform benign breast MCF-10F cells, allowing them to cause tumors in immunodecient mice [48]. Other rodent studies show that 17 -estradiol (E2) is able to induce tumorigenesis in those tissues where E2 is converted to 4-OH-E2 and that 8-OH-dG levels, a surrogate marker of estrogeninduced oxidative DNA damage, are signicantly higher in estrogen receptor (ER)-positive cell lines with respect ERnegative cell lines [49,50]. To nish, analyses in human breast tumors revealed signicantly higher concentrations of 4-OH-E2 and 8-OH-dG when compared with healthy breast tissue and in ER-positive versus ER-negative tumors [50]. In addition to the above-described metabolic processes, it has been reported that mitochondria play an important role in estrogen-induced generation of ROS. Early studies failed to demonstrate such a relationship, probably because cytotoxic E2 concentrations and isolated mitochondria were used [51], but in 2005 Felty et al. [52] showed that mitochondria are direct targets of estrogens under physiologic conditions. They observed a signicant increase in the intracellular concentrations of ROS upon exposure to physiologic concentrations of E2 in several human breast cancer and neuroblastoma cell lines. Interestingly, they found that this effect occurred before any hydroxylated estrogen metabolite or adduct could be detected, what rules out the possibility of ROS generation by redox cycling of catechol estrogens, and suggested that it is ER-independent given that both ER-positive and ER-negative breast cancer cell lines produce equal ROS concentrations under these conditions [51,52]. Then, the generation of estrogen-induced mitochondrial ROS under normal physiologic conditions seems to be mediated by another mechanism. The authors proposed that E2 is able to interact with integrin 5 1 in order to activate the cytoskeletal protein Rac-1, which may signal to the mitochondria via cytoskeleton and modulate the activity of the voltage-dependent anion channel (VDAC). As a result of these interactions, the mitochondrial membrane potential ( m ) increases and leads


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to ROS production, which diffuse across the mitochondrial membrane and participate as signal-transducing messengers in the redox-sensitive molecular pathways of the cell. On the other hand, it is thought that E2 may also act directly at the level of respiratory chain, binding to complex I, blocking the electron ow and consequently, inducing ROS production. Similar changes in m and ROS concentration have been observed upon mitochondrial Ca2+ accumulation in MCF7 cells treated with E2 [53]. To nish, estrogens can modulate the activity of key proteins in the electron transport chain of the mitochondria through the regulation of gene expression, post-translational modications or even modifying the physicochemical properties of the mitochondrial membrane system. The molecular activities described above are known to induce a signicant increase in ROS, which diffuse to the cell cytoplasm and have a deep impact in cell signaling [51]. 3. Oxidative stress in carcinogenesis, invasion and metastasis in breast cancer In 1984, Zimmerman and Cerutti [54] published one of the earliest studies to show that ROS play a promoter role during the carcinogenic process. Since then, the inuence of free radicals in the oncogenic transformation has become an interesting issue for the scientic community, due the multifunctional character and ubiquity of these molecules. This way some experiments with knockout mice, defective in the antioxidant enzyme copper- and zinc-containing superoxide dismutase (Cu/ZnSOD), which catalyzes the dismutation of O2 into H2 O2 and oxygen, have shown an increased susceptibility of the animals to develop aggressive hepatocarcinomas [55]. In this model, it has also been reported a marked tendency to develop intestinal in those mice defective in the antioxidant enzymes GPx1/2 [56] and the promotion of lymphomas, sarcomas and adenomas in those animals defective in peroxiredoxins [57]. Finally, the absence of functional catalase is related with the tendency to develop breast tumors [58]. With respect to in vitro experiments with human cells a recent work published by Gosselin et al. [59] shows a signicant association between tumorigenesis and senescence-related oxidative stress. This relationship provides a molecular explanation of the higher incidence of neoplastic diseases in aged people and shows that the molecular switches that enable tumor emergence are driven by ROS accumulated during senescence. On the other hand, it has been suggested that transformed cells inhibit apoptosis and stimulate cell proliferation, metastasis and angiogenesis using free radical as second messengers that participate and modulate the intracellular signaling pathways regulating these processes. Indeed, studies with cancer cell lines show that neoplastic cells favor ROS generation in abnormally high concentrations compared to their normal counterparts [60].

3.1. Oxidative stress and cancer initiation According to the classic model of the carcinogenesis, free radicals could play a promoter role in the initiation, promotion and progression phases [27]. During the initiation phase, no lethal mutations accumulate in the healthy cells and are xed by at least one round of replication. During this phase, free radicals collaborate in cell transformation inducing oxidative damage in the DNA, both directly and indirectly, through the generation of highly reactive oxidative products as lipid peroxides [61,62]. Thus, free radicals contribute to mutagenesis, which is essential for the initiation process, by the mechanisms described before. Two key mechanisms have been proposed for cancer induction. The rst of them involves both increased DNA synthesis and proliferation due to the exposure to nongenotoxic carcinogens that may induce mutations, which are not repaired and expand from initiated pre-neoplastic cells. The second mechanism accounts for a misbalance between cell growth and cell death [62], an equilibrium that is tightly regulated in healthy cells in order to avoid cellular transformation due to severe cell damage. Free radicals are thought to active the neoplastic transformation at this level through multiple mechanisms that mainly account for the induction cell damage, abrogation of cell programmed death mechanisms and tumor suppressing activity and promotion of oncogene expression. 3.1.1. Tumor suppressor genes and oncogenes Tumor suppressor genes and oncogenes can also be affected by oxidative stress. In that sense, p53, which plays a key role in the prevention of oncogenic transformation, exert a complex and dose-dependent relationship with free radicals. First, in physiologic normal conditions, when free radical concentration is low and p53 expression is moderated, this protein activates the expression of genes that code for antioxidant proteins. As free radicals concentration increases, p53 expression and activity increase generating oxidative stress, DNA damage and promoting cellular death by apoptosis. These pro-apoptotic effects are mediated by the activation of the p66Shc protein, which interacts with the electron transport chain increasing ROS production, and the induction of the expression of proline oxidases, which generate H2 O2 and the maintenance of the cytochrome oxidase activity. On the other hand, a higher free radical concentration and severe oxidative stress can cause the inactivation of p53 by the direct damage that they cause in the DNA that codes for p53. In fact, it has been shown that approximately half of the human tumors have deleterious mutations in p53 gene [63]. The ataxiatelangiectasia mutated gene (ATM) is another important tumor suppressor gene involved in DNA repair and the maintenance of genomic stability. Inactivation of ATM is known to be associated with a marked increase in cellular oxidative stress and an increased susceptibility to develop cancer. ATM-decient animals show impaired glutathione biosynthesis, increased manganese-containing

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superoxide dismutase (MnSOD), heme oxygenase 1 (HO-1) and thioredoxin (TRX) activity, which may result in increased production of H2 O2 , increased availability of pro-oxidant iron released from heme and cell survival in oxidative stress conditions, respectively. It has also been observed that catalase activity is decreased in these defective animals. The mechanisms by which ATM mutations affect the oxidative balance may account for direct regulation of the expression of genes that encode proteins involved in antioxidant defenses or via post-translational modications of such proteins [64]. The rat sarcomal viral oncogene (Ras) codes for a G protein that connects the intracellular domains of transmembrane receptors with intracellular effectors of the molecular signaling cascades that regulate cellular growth and apoptosis. The 20% of all human neoplasms show mutations in the Ras gene that contribute to the expression of a transformed phenotype including invasive and migratory capacity and angiogenesis induction. Among other mutagenic stimuli, ROS are produced upon Ras oncogene activation, especially in p53 decient cells [65], illustrating the interplay between tumor suppressor genes, oncogenes and oxidative stress. Another relevant oncogene activated by ROS is Raf-1. The protoncogene c-Raf-1 encodes a serine/threonine kinase able to active proliferation signals. Exposure of the cells to high concentrations of OH results in c-Raf-1 deletions, and the expression of an abnormal protein that lacks its regulatory domain and therefore, it is able to stimulate cell proliferation without control [66]. Another important oncogene which is redox sensitive is v-myc myelocytomatosis viral oncogene homolog (avian) (c-Myc). c-Myc is known to play a crucial role in cell cycle regulation promoting G1/S transition. Cellular exposure to ROS has been shown to increase c-Myc protein levels and S-phase cell recruitment [67]. 3.1.2. Apoptosis Free radicals activate nuclear and cytoplasmic transduction signals that control critical steps of the apoptotic pathways. Although it is well known that high concentrations of free radicals contribute to cell death when they are generated in the context of the apoptotic process [68], the complexity of redox signaling is evidenced by several reports showing that oxidative stress exerts antagonistic effects on the apoptotic process. For example, the generation of ROS by NOX is found to be anti-apoptotic in pancreatic cancer cell lines, retinal cells and human colon carcinoma cell lines [6971]. How the cells achieve these pro-survival effects is still a matter of debate. It seems that not only the extent and duration of redox signals are determinants of cell fate, but also the intracellular localization of the signal and the surrounding cellular environment may play a key role. Nevertheless, nowadays we already know about certain chemical mechanisms that can partially explain the inhibitory effects of a raise in free radicals concentration on apoptosis inhibition. The persistent oxidative stress originates highly reactive species that undergo later transformations and generate secondary

products able to bind covalently to the cysteine or thiol residues of the active center of the caspases and to the cellular receptor CD95/Fas. This way they inhibit the apoptosis. On the other hand, intracellular O2 causes an increase in the cytosolic pH, that negatively regulates the activation of the caspases [72]. But there have been described other mechanisms by which free radicals inhibit tumor apoptosis. Those mechanisms account for the inhibition of molecular transduction pathways leading to cell death, as the pathway mediated by the phosphatase and tensin homolog (PTEN). Growth factor signaling induce the generation of H2 O2 , that plays a dual role: rst, it amplies the growth signal contributing to the activation of downstream molecular cascades and second, it promotes the local inhibition of PTEN, avoiding the inactivation of phosphatidylinositol 3-kinase (PI3K) and allowing the accumulation of phosphatidylinositol 3,4,5-triphosphate (PIP3 ) and consequent activation of the v-akt murine thymoma viral oncogene homolog (AKT) signaling pathway, which main activity is to promote cell growth and survival [73]. In relation to other free radicals as RNS, we can establish a functional parallelism with ROS. RNS promote DNA damage giving raise to mutations in key genes for cell growth and inhibit the activity of caspases, favoring the survival of cells whose genetic material contains modications potentially mutagenic. They also block the cytochrome oxidase activity and impair ATP production, what results in cell cycle arrest. Probably, this is the reason why macrophages are frequently present in the surrounding areas of solid tumors, because these cells produce NO through the activity of the inducible nitric oxidase (iNOS) enzyme [74,75]. The above-mentioned processes and scientic ndings denote the important role of free radicals in cell transformation. Up-regulation of mitogenic signals and inhibition of tumor suppressor and apoptotic mechanisms lead to neoplastic transformation and cell growth, which are processes critically inuenced by free radicals. Their effects are appreciable in the activation of the molecular pathways that promote cell proliferation, as discussed in Section 3.2. 3.2. Oxidative damage and cancer promotion Promotion is the phase where the clonal expansion of the initiated cells takes place through the induction of cellular proliferation and apoptosis inhibition. The result is an identiable lesion in the tissue that forms the primary tumor. Free radicals act as second messengers in the intracellular pathways that control cell proliferation and differentiation. According to the type and oxidative status of the cell, free radicals can induce apoptosis or a positive proliferative response, depending on their cellular concentration [76]. The molecular mechanisms through which free radicals inuence and participate in cell signaling, include interactions with a wide variety of hormones, cytokines and growth factors that specifically bind to cell membrane receptors to activate the signal transduction pathways that control cell function.


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3.2.1. Cytokines and growth factors Among those growth factors signicantly affected by free radicals, we can mention epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF) and transforming growth factor (TGF- ). The stimulation of any of these growth factors give raise a transient augment of the intracellular concentration of ROS, mediated by the activity if the Rac1 protein, that activates the synthesis of H2 O2 and O2 by NOX [77]. This mechanism is well-known in broblasts, endothelial cells, vascular smooth muscle cells, cardiac myocytes, and thyroid tissue, but the coupling between growth receptor activation and NOX activity is not fully underst ood in tumoral cells. Despite this, an increasing number of experimental studies evidence this relationship. Juarez et al. [78] have recently published a work that shows that H2 O2 generated by Cu/ZnSOD activity is essential to induce a molecular shift within the cell towards phosphorylation and allow the propagation of the signal along the molecular cascades that govern cell growth. This shift is induced by the inactivation of protein tyrosine phosphatases (PTPs) which are susceptible of oxidation in their active site. Although Cu/ZnSOD activity is known to induce both prooxidant and antioxidant effects mediated by an excess of H2 O2 and a decrease in O2 , these experiments show that Cu/ZnSOD inhibition prevents the oxidation of PTPs, which in turn inhibits mitogen activated protein kinases (MAPKs) cascade in endothelial cells and several tumor cell lines stimulated with growth factors as EGF, IGF1 and broblast growth factor 2 (FGF-2). These observations are reinforced by the results of other experiments that show that the toxic activity of several carcinogens, as polycyclic aromatic hydrocarbons, are mediated by EGFR activation via ROS production in breast cancer cell lines [79], or how the EGF-initiated pathway can be inhibited by antioxidants as N-acetyl cysteine (NAC) and catalase [7981]. In vitro experiments performed with tumor cell lines reveal that the transient increase in H2 O2 intracellular concentration induced by the binding of PDGF to its receptor is mediated by the recruitment and activation of PI3K, which provides PIP3 to activate NOX enzymes via Rac1 [82]. IGF-I is shown to interact with E2 to promote the proliferation of MCF-7 breast carcinoma cells via ROS-dependent MAPKs activation and c-Jun protein expression. Intracellular H2 O2 was signicantly elevated in E2/IGF-I cells and incubation with chemical free radical scavengers markedly reduced the expression of phosphorylated MAPKs and c-Jun proteins [83]. Although it is well-known that tumoral cells over-express VEGF to stimulate the proliferation and migration of endothelial cells and that ROS mediate VEGF signaling in endothelial cells, via NOX activation [84], little is known about the relationship between free radicals and VEGF in the tumoral cells. Harris et al. [85] showed that VEGF contributes to tumor growth through inhibition of apoptosis and increased NOS activity during pre-vascular stages of breast tumor development. Xia et al. [86] demonstrated that ovarian and prostate cancer cells

lines produce higher levels of intracellular ROS than immortalized epithelial cells and suggested that ROS may originate from cytosolic NOX and mitochondria in cancer cells. They also observed that high intracellular ROS are required for hypoxia inducible factor 1 (HIF-1), a pro-angiogenic factor activated during hypoxia, and VEGF. When the cells were treated with NOX and mitochondria complex I inhibitors, as diphenylene iodonium (DPI) and rotenone, they observed a signicant reduction in HIF-1 and VEGF protein levels. These results are conrmed by others who corroborate NOX involvement in ROS-induced VEGF expression in tumorigenic cell lines [87]. On the other hand, cytokines and interferons, such as tumor necrosis factor (TNF- ), interleukine 1 (IL-1 ) and interferon (IFN- ), bind to transmembrane receptors transmitting a signal which stimulate ROS generation [88]. TNF- is able to induce both apoptosis and cell survival through redox signaling. It has been shown that catalase treatment impairs ROS production induced by TNF- in several cancer cell lines. Under these conditions, TNF- is unable to activate the survival pathway mediated by nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFB), showing that ROS are essential in TNF- -induced cell survival [89]. In vitro studies show that IL-1 transfected MCF-7 breast cancer cell line proliferate actively, suggesting that the mitogenic signal is mediated by IL-1 -derived ROS in estrogen-dependent breast cancer [90]. Whereas some authors point out the protective effect of IFN- against cancer due to its enhancing effects on the immune response [91], others show the potential carcinogenic effect of an exposure to IFN- -derived free radicals [92]. It seems to depend on the duration of the exposure, providing a molecular link between chronic inammation and cancer risk. The existence of numerous growth factors and cytokines that activate ROS production in the cell, suggests a cooperative relationship among their receptors to amplify the intracellular signal. Indeed, some works show that free radicals mediate the cross-talk between different growthstimulatory molecules. Zhou et al. [93] showed that insulin induce the expression of VEGF and HIF-1 through the generation of H2 O2 via PI3K/AKT/p70S6K1. Liu et al. [94] demonstrated that EFG induced VEGF and HIF-1 by the same redox mechanism. It is also the case of the angiotensin II and EGF receptor (EGFR) because angiotensin II, by binding its receptor, transactivates EGFR and promotes cell growth through ROS generation and under the regulation of NO [95]. Because H2 O2 has a relatively long half-life and diffuses across the biological membranes is rather probable that the signal is transmitted to neighbor cells, not only amplifying the intracellular signal but also affecting to the growth of the cells in the vicinity. 3.2.2. Protein phosphatases and protein kinases Continuing with molecular signaling pathways, two types of key enzymes are also affected by oxidative stress status in the cell: protein phosphatases and cytoplasmic protein

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kinases. As mentioned, H2 O2 concentrations higher than 1 mM induce important pro-oxidative changes in the cell, as the oxidation of tyrosine phosphatases [31,96]. It is wellknown that biological redox reactions catalyzed by H2 O2 generally involve the oxidation of cysteine residues in proteins, which may affect protein function. The PTPs family has a common Cys-X-X-X-X-X-Arg active-site motif (where X corresponds to any amino acid), in which the conserved catalytic cysteine possesses a low pKa and show high susceptibility to oxidation by H2 O2 . Oxidation of the essential cysteine inhibits phosphatase activity and can be reversed by cellular thiols [97]. PTPs counteract the effect of tyrosine kinases and turn back the transmembrane receptors to their basal conformation, after being bounded to their ligands and this is the main reason why the inhibition by oxidation of these enzymes, stimulates the activity of tyrosine kinases and thus, activates the molecular signaling cascades that promote cellular growth. Reversible inactivation of different PTPs has been demonstrated in several cell types stimulated with growth factors in a H2 O2 dependent manner [78,97,98]. These ndings show the crucial role of ROS in signal transduction pathways that promote cell growth. In this context, the results of in vitro experiments indicated that estrogens are also involved in this process and contribute to breast cancer promotion through PTP redox inhibition. E2-treated MCF7 cells showed a signicant reduction in the activity of Cell division cycle 25 homolog A (Cdc25A) compared to non-treated cells. Cdc25A is an important dualspecicity tyrosine phosphatase in the control of the cell cycle, which is specically degraded in response to DNA damage. Further examination revealed lower levels of SH residues in Cdc25A of treated cells, which was accompanied by a decrease in serine phosphorylation residues, similar to that observed in response to the exposure of cells to H2 O2 and known to inactivate the enzymatic activity of Cdc25A. Furthermore, E2-induced inactivation of Cdc25A could be prevented by co-treatment with antioxidants. These results suggest that estrogens inactivate Cdc25A through ROS production in breast cancer cells [99]. It has been described that Cdc25A chemical inhibition promotes prolonged and strong extracellular signal-regulated kinase (ERK) phosphorylation and activation, which is related to breast cancer transformation [100]. Then, it would be reasonable to think that Cdc25A biological inhibition by estrogen-induced ROS may have similar effect on breast carcinogenesis than chemical-induced Cdc25A inhibition. On the other hand, cytoplasmic protein-quinases, as those belonging to Src, janus kinase (JAK) and MAPK families, are activated by phosphorylation in response to the stimulation of growth factor, cytokines and heterodimeric G proteins coupled to receptors. They transmit the signal until it reaches to transcription factors that translocates to the nucleus where they activate the expression of genes involved in cellular proliferation, differentiation and apoptosis [101,102]. Of special importance is the activation induced by ROS of the molecular pathways mediated by ERKs. After growth factor

stimulation, Ras activates ERKMAPK pathway in order to induce the expression of cyclin D1, which promotes cell proliferation [103]. Growth factor activity and Ras activation are known to induce the production of ROS, despite some authors point out that the mitogenic signal mediated by ROS in Ras-transformed cells is independent from growth factor signaling [104]. In line with this latest idea, Wang et al. [100] suggested that ERK activation is regulated either by growth factor-dependent and growth factor-independent pathways, since they showed strong ERK phosphorylation and MAPK cascade activation in cells lacking of EGFR when treated with a chemical inhibitor of Cdc25A. This experimental observation prompted the authors to hypothesize that Cdc25A might have a direct effect on ERK phosphorylation and indeed, they showed that Cdc25A physically interacts with ERK and lead to its inactivation. As discussed above, biological inactivation of Cdc25A by estrogen-induced ROS may have similar effects on ERK activation. Moreover, Sarsour et al. [105] showed that decreasing MnSOD activity, favored proliferation in mouse embryonic broblasts (MEFs), while increasing MnSOD activity induced cell quiescence. Decreased MnSOD was accompanied by increased levels of cyclin D1. These results suggest that O2 mediated signaling promotes proliferation, while H2 O2 promote cell quiescence. Controversy reaches further with recent studies that suggest that O2 plays an important role in the induction of cell cycle arrest in G1 phase by thiol antioxidants as NAC [106] and in cyclin D1 degradation under hypoxic conditions [107]. To correctly interpret these data are important to consider that different cell responses account for malignant transformation and in fact, it has been demonstrated that nonmalignant human breast epithelial MCF-10A cells and breast cancer MCF-7 and MDA-MB-231 cells respond differently to NAC-induced cell cycle arrest, being both MCF-7 and MDA-MB-231 cells insensible to the inhibitory signals [108]. On the other hand and as commented before, the signaling adapter p66Shc is involved in the production of cytoplasmic H2 O2 from mitochondria and apoptosis. Under severe oxidative stress conditions, p66Shc promote cell death, abolishing the survival signal mediated by Ras/ERK pathway [109]. Activation of ERKs leads to increased transcription of NOX 1 [110]. Whether ROS directly regulate ERKs activation or promote the inhibition of ERKs negative modulators, is still a matter of debate but the scientic evidences that support this relationship merit further research in order to improve our knowledge about ROS impact in this crucial cell signaling pathway. Other MAPKs, as c-jun-NH2-terminal kinase (JNK) and p38, are related to the regulation of apoptosis and cell survival in cellular stress conditions. This is why JNK and p38 are also called the stress-activated protein kinases (SAPKs) [64]. p38 MAPKs are known to be involved in cell cycle arrest in response to various environmental insults and contribute to the inhibition of cell transformation [111] and indeed it has been shown that p38 -decient cells are resistant to ROS-induced apoptosis, enabling cell transformation by


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ROS-generating oncogenes as Ras [112]. Apoptosis signalregulating kinase 1 (ASK-1) is an upstream activator of p38 MAPK that remains inactive during basal conditions through its interaction with TRX or glutathione-S-transferase Mu 1 (GSTm-1). Upon ROS stimulation, ASK-1 dissociates from TRX and/or GSTm-1 and activates p38 MAPK but it has been shown that cancer cells are able to uncouple the activation of p38 MAPK by ROS through the increased expression of GSTm proteins [112]. Similarly, another GST protein, in this case GSTp, inhibits JNKs by direct interaction [113] suggesting that cancer cells could have developed the same molecular mechanism to avoid ROS-induced apoptosis mediated by JNK. Apart from their roles in ROS-mediated apoptosis, SAPKs are known to participate in cell proliferation and tumorigenesis as shown by several experiments. JNKs are able to induce carcinogenesis through the activation of Wnt pathway, known to be essential in stem cell function, in a model of colorectal carcinogenesis [114]. JNK2 isoform activity is required for Ras-mediated transformation of MEFs [115] and ERK, p38 and JNK activity contribute to breast carcinogenesis through the stimulation of cell cycle progression [116]. Globally, the ability of SAPKs to stimulate cell growth or cell death depends on signal intensity and duration, thus, transient low activity of SAPKs promotes cell growth whereas persistent high-level activity promotes cell death. This dual activity of SAPKs remembers ROS effects on cell survival and growth. The impact of ROS in SAPKs activation and their functional parallelisms, evidence a relationship which has not been completely elucidated. Future research in this area is warranted. On the other hand, the increase in cellular ROS concentration stimulates the activity of kinases such as protein kinase C (PKC), which plays a crucial role in the cell proliferation, differentiation, angiogenesis and apoptosis [117]. PKC isoforms can be activated by H2 O2 in a phospholipid-independent process that involves tyrosine phosphorylation in its catalytic [118]. However, since oxidative stress activates several receptor-regulated phospholipases, such as phospholipase D (PLD) or phospholipase C (PLC) [119,120], diacylglycerolmediated activation of PKC in response to oxidative stress cannot be excluded. Src tyrosine kinase family has been reported to include redox regulated proteins. All family members have been implicated in signaling networks that control cell proliferation, differentiation, migration and survival [120,121] and consequently are key regulators in tumorigenesis and cancer development. Minetti et al. [122] showed in an in vitro experiment that free radicals, as NO, were able to activate Src kinases. Activation of Src kinases has also been reported after exposure to exogenous H2 O2 [123,124]. In vivo Src redox regulation for anchorage dependent growth has also been documented. It has been proposed that Src kinases contain two redox-regulated residues, Cys245 and Cys487, located in the SH2 and in the kinase domain of the Src molecule able to undergo oxidation and form a disulphide bond, a conformational change that leads Src activation. Even further, transfection of NIH 3T3 cells with v-Src oncoprotein

and its mutants for both redox sensitive cysteines, demonstrated that ROS-mediated oxidation of v-Src is required for its oncogenic properties [125]. Nevertheless, a recent study suggests that oxidation of Cys277 in the catalytic group of certain Scr kinases causes their inactivation [126]. It is noticeable that this residue is only present in 3 members of the Src family, and therefore the novel inhibition mechanism would be applicable to this subgroup of Src kinases. 3.2.3. Transcription factors Transcription factors control the expression of genes involved in the immune response, cellular proliferation, apoptosis or DNA repair. Among those modulated by free radicals, we can point out some of them essential for cellular growth control as ER, activation protein 1 (AP-1), NF- B, or nuclear factor (erythroid-derived 2)-like 2 (Nfr-2). Transcriptional regulation of antioxidant genes is mainly mediated by Nrf-2, whereas persistent and elevated ROS activate AP-1 and NF- B [127]. In relation to oxidative stress, HIF-1 is a powerful transcription factor activated in response to hypoxia and raises in free radical concentrations secondary to persistent hypoxia or hypoxiareperfusion cycles. Among the hormonal inuences in the development of a breast neoplasm, estrogens are considered to play a crucial role. There are two types of estrogen receptor (ER) known as ER and ER , which are proteins with a modular structure that include a ligand-binding domain and a DNA-binding domain. Upon E2 binding to ER and dimerization, the complex translocates to the cell nucleus and bind to high afnity DNA regions, known as estrogen responsive elements (EREs), which are present in the regulatory domains of key genes positively regulated in their expression by estrogens and involved in cell proliferation, apoptosis, transformation and invasion. This mechanism is known as the classical ER genomic signaling pathway. In addition, estrogens inuences cell biology through the extracellular or nongenomic ER signaling pathway, which involves the activation of the molecular cascades characteristically initiated by growth factors [128,129]. ROS are known to inuence estrogen signaling through their impact on ER stability and function. For example, results of experimental studies that show a signicant increase of ER , but not in ER , after H2 O2 exposure in MCF7 and T-47D breast cancer cell lines [130] have been reported. These are very interesting results, on the one hand, because some experimental data suggest that ER would be preferentially involved in the nongenomic ER signaling pathway, while ER would preferentially initiate the classical genomic ER pathway [129]. On the other hand, because ROS have been reported to positively modulate the activity of a wide variety of protein kinases, growth and transcription factors, many of them involved in the nongenomic ER signaling pathway [129], as discussed below. Beyond ER expression, ROS are known to inuence post-translational modications of ER and affect estrogen signaling at a different molecular level. First, ER, as many other transcription factors with DNA-binding activity, contains redox-sensitive

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cysteine residues within the zinc-nger domains necessary for nding EREs in its target genes. Cellular exposure to ROS or any other oxidant converts cysteine thiol groups to disulphide releasing zinc from ER and induces a conformational change that results in the loss of its DNA-binding activity [131]. ROS are also known to modulate ER activity, promoting the phosphorylation of specic serine residues (serine 118 and serine 167) and leading to either ligandindependent activation of ER signaling or down-regulation of its transcriptional activity in breast cancer cell lines. Moreover, the phosphorylation of serine 118 is related to ER turnover, since it has been suggested that this posttranslational modication targets the receptor to proteasomal degradation. Indeed, the development of resistance to conventional endocrine therapy in breast cancer has been related with oxidative stress, ER phosphorylation and proteolysis, given that this molecular mechanism seems to contribute to the conversion of ER-positive tumors into ER-negative tumors, with the consequent failure of tamoxifen anti-estrogen therapy [132]. Paradoxically, as in the case of chemotherapy, the most prescribed anti-estrogen drug to breast cancer patients with ER -positive tumors, tamoxifen, contributes to oxidative stress as a mechanism to induce tumor cell death [133]. ROS production has been related to tamoxifen primary resistance due to activation of protein kinases and inhibition of protein phosphatases that lead to ER phosphorylation and proteolysis, which is in the base of tamoxifen resistance. These molecular interaction have been showed by Cui et al. [134], who demonstrated that tamoxifen-induced ROS inhibit phosphatase 3 (MKP3) and up-regulated ERKs, phosphorylated serine 118 in ER , and cyclin D1 in several ER-positive breast cancer cell lines. These effects resulted in tamoxifen resistance as discussed before. In fact, antioxidant treatment increased MKP3 phosphatase activity and blocked tamoxifen resistance pointing to ROS as the causal agent. Nrf-2 is a redox-sensitive transcription factor that in basal conditions is sequestered in the cytoplasm by inhibitory protein kelch-like ECH-associated protein 1 (Keap1). Upon oxidative stress stimulus, upstream kinases such as MAPKs, PI3K, PKC and Akt, induce its activation and translocation to the nucleus, where it binds to the antioxidant response element (ARE) or electrophile response element (EpRE) located in the promoters of a wide variety of antioxidant and detoxication genes as NAD(P)H:quinone oxidoreductase-1 (NQO1), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPx), HO-1, glutamate cysteine ligase (GCL), catalase, and TRX [135]. In vivo experiments show that Nrf2 ablation causes defects in carcinogen detoxication and increases the incidence of chemical-induced tumors in animal models [136,137]. Nevertheless, the relationship of Nrf2 with oxidative stress-mediated response in cancer cells seems to be more complicated since it has been shown that this transcription factor promote resistance of cancer cells to chemotherapeutic drugs [138]. This dual activity of Nrf2 with respect cancer prevention and treatment resistance is mediated by the

coordinated expression of antioxidant and efux transporter proteins, which mediate multidrug resistance, that prevent the accumulation of carcinogens in non-transformed cells but promote survival in cancer cells [139]. AP-1 is a transcription factor composed of different dimeric combinations of basic leucine zipper proteins from the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) family and the closely related activating transcription factor (ATF2, LRF1/ATF3, and B-ATF) subfamilies. AP-1 dimers bind to recognition sequence (5 -TGAG/CTCA3 ) known as 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE) and present in the promoter of several genes involved in cell proliferation, survival and transformation [140,141]. AP-1 induction is mediated by free radicals, fundamentally as a consequence of the activation of signaling cascades in which JNK and p38 are involved, in response to different stress situations. H2 O2 , metals, cytokines and other stressors induce the expression of fos and jun genes and induce cell proliferation and transformation [142]. Signaling molecules involved in cell transformation, as EGF and PDGF, and transforming oncogenes are known to induce AP-1 activity. Up-regulation of both c-jun and c-fos genes leads to neoplastic transformation and microinjection of anti-fos antibodies or transfection of c-fos antisense mRNA inhibit cell growth in cultures broblasts. These ndings support the key role of AP-1 transcription factor in cell proliferation and transformation [140,141,143]. Additionally, it has been proposed that AP-1 could act as an apoptosis modulator negative apoptosis modulator depending on the balance of expression of pro- and anti-apoptotic genes, the molecular pathway employed to activate AP-1, the duration of the stimulus, type or cell differentiation grade [62]. NF- B is a transcription factor that specically binds to certain DNA regions known as B sequences and activates the expression of more than 200 genes involved in inammation, cell survival, proliferation and differentiation in response to several stimuli, among them the presence of free radicals. Many of these genes are critic in the carcinogenic process including the cyclin D1, growth factors as VEGF and anti-apoptotic proteins such as Bcl2, Bcl-XL [144]. When inactive, NF- B is bound to its inhibitor I B. Inammatory stimuli lead to I B phosphorylation and proteolysis and NFB dimerization and translocation to the nucleus. Although this is the standard mechanism by which NF- B is activated, H2 O2 or hypoxiareoxygenation cycles may cause phosphorylation of I B, which enables the dissociation of I B from NF- B without proteasomal degradation of I B [145]. Apart from peroxides, NO has also been shown to regulate NF- B, both through activation and inhibitory signals depending on cell type and concentration [146]. On the other hand, antioxidants as NAC has been reported to suppress NF- B activation induced by TNF- , lipopolysaccharide (LPS) or UV radiation and overexpression of antioxidant enzymes as MnSOD, catalase, or TRX reduced NF- B activation induced by TPA, TNF- , LPS or H2 O2 [143]. Thereby, there is convincing


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evidence that NF- B is up-regulated by free radicals and that this relationship clearly connects NF- B, free radicals, inammation and cell transformation. Finally, it has been suggested that AP-1 and NF- B activities act coordinately in cell transformation upon oxidative stress activation. Inhibition of NF- B by antioxidant treatment has been shown to be accompanied by AP-1 inhibition and suppression of cell transformation [147]. Interactions between p65 component of NF- B and c-fos/c-jun [148] and NF- B inuence in c-fos transcription may offer a molecular explanation for such relationship [149] and the key involvement of these transcription factors in oxidative stress-induced carcinogenesis. Nowadays it is possible to afrm that ROS generated by the mitochondria of cells subjected to hypoxic conditions, play a crucial role in the adaptation of the cell to oxygen starvation [150]. Taking into account that breast tumors, as all solid tumors, are poorly vascularized during the exponential growth phase of the carcinogenic process, the hypoxia is a constant in the tumoral microenvironment. In such conditions, the HIF-1 protein is stabilized and activated. The HIFs belong to a family of basic-helixloophelix (bHLH)containing proteins in which the prototype of the family is HIF-1. HIF-1 consists of two subunits: the regulatory HIF1 subunit and the HIF-1 subunit. HIF-1 expression is highly dependent on oxygen tension. In normoxia, HIF-1 is continuously expressed and degraded through a tightly regulated mechanism that involve its hydroxylation by conserved prolyl hydroxylases (PHDs) and its interaction with the von HippelLindau tumor suppressor protein (pVHL), leading to ubiquitination and degradation of HIF-1 . In hypoxia, HIF-1 hydroxylation is inhibited and its degradation prevented. HIF-1 translocates to the nucleus where it dimerizes with HIF-1 , recruits transcriptional co-activators and binds to the hypoxia-response element (HRE) within the promoter region of HIF-1 target genes mediating their transcriptional activation [151]. Additionally, it has been shown that persistent hypoxia enhanced ROS production, which in turn activates HIF-1 transcription [152]. HIF-1 promotes cell survival through the activation of the transcription of proto-oncogenes, glycolytic enzymes, glucose transporters, growth factors and angiogenic factors involved in the apoptosis inhibition, cellular immortalization, metastasis, and drug resistance and thus, in the selection of those cellular clones that, with the greatest probability, will success in the formation of the incipient tumor [153]. The molecular pathways that modulate HIF-1 transcription belong to a subgroup of signal transduction pathways mediated by tyrosine-kinases, traditionally related to oncogenic processes, like the RASERK pathway, PI3K/AKT pathway and the signal transducer and activator of transcription 3 (STAT3) molecular pathway [153,154]. By the activation of these pathways, the cells not only activate the constitutive expression of HIF1, they are also the way that transformed cells stimulate the expression of genes, as survivine or VEGF, to survive from the hypoxia. These genes confer a particularly aggressive

phenotype [155,156]. Indeed, over-expression of HIF- is usually associated with increased vascular density, severity of tumor grade, treatment failure and a poor prognostic outcome [157] and blocking HIF activity or targeting HIF1 expression in tumors has been shown to signicantly slow tumor growth in xenograft [158]. These ndings highlight the crucial role of this transcription factor in cancer development. 3.3. Oxidative damage and cancer progression Progression is the third and latest phase of carcinogenesis. It is characterized by cellular and molecular irreversible changes. During that phase, cells develop the capacity of induce angiogenesis, a key process for tumoral growth when the lesion reaches a volume that blocks the oxygen and nutrient supply [159]. Angiogenesis is a multistep process that comprises the extracellular matrix degradation, endothelial cell migration to the perivascular stroma and the formation of new blood vessels. On the other hand, in advanced phases of their development, neoplastic cells acquire the capacity of migrate and colonize the surrounding tissue and the tissue of organs from distant locations through the blood stream. To achieve successful spreading, tumor cells may exhibit epithelialmesenchymal transition (EMT), through which transformed cells detach from the basal lamina and reorganize their cytoskeleton increasing motility and migrate into the surrounding tissue. Subsequently, extracellular matrix (ECM) around the primary tumor must be remodeled to enable tumor cells to get into the blood stream. Tumor cells surviving in the blood may then extravasate into a distant organ and nally proliferate into secondary tumor. It is known that tumor microenvironment plays a crucial phase in this latest phase [160,161]. Consequently, the angiogenesis and the metastatic potential development of the tumors are processes tightly ligated. As exposed in Section 3.2, high oxidative stress has been found to contribute to carcinogenesis and more recently ROS-mediated signaling has been related with tumor progression and metastasis. It is well established that integrins mediate cellular attachment of cells to ECM. Apart from their scaffold function, integrins mediate cellular signaling leading to the regulation of several processes including cell proliferation, survival and migration. Integrin signaling is known to cross-talk with TGF- [162], PKC [163], MAPKs, AKT, NF- B [164] and PI3K [165] in breast cancer. All of them are known to be regulated by free radicals, as discussed in Section 3.2, and known to mediate ETM by integrin-directed mechanisms. Moreover, important integrin effectors, as Rho GTPases, are known to induce ROS production. RhoA, Rac and Cdc42 are members of the Rho GTPases family involved in the stimulation of cell adhesion by inducing the assembly of actin stress bres and focal adhesion complexes (RhoA) and stimulation of actin polymerization to form lamellipodia or membrane rufes (Rac1) and lopodia (Cdc42) [166]. These opposite roles of the Rho GTPases family proteins are regulated by

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a process that involve ROS generation, in such a way that it has been observed that Rac1 inhibits RhoA activity and promote subsequent lamellipodia formation and cell spreading [167]. ROS have also been shown to induce a EMT-like morphological change in mammary epithelial cells and Rac-1 over-expression, which result in an enhanced invasive behavior [168]. On the other hand, ROS also increases the migratory potential of the transformed cells through the activation of heat shock protein 27 (HSP27) mediated by p38. Phosphorylated HSP27 induces changes in the actin dynamics and promotes cellular migration [169]. Further, another crucial event for successful ETM is extracellular matrix remodeling. During tumoral invasion, transformed cells interact with adjacent stromal cells causing the disruption of stromal structure. The enzymes that catalyze the changes operated in the extracellular matrix correspond with the matrix metalloproteases (MMPs). MMPs not only contribute to angiogenesis disrupting basal membranes and other extracellular matrix components, but also segregate pro-angiogenic factors bound to the matrix. Among them we can point out FGF, IGF, VEGF or TGF , that bind to their receptors in the endothelial cell membranes inducing ROS-mediated cellular proliferation, MMP secretion, adhesion molecule expression, cell migration and invasion [170]. In the case of breast cancer, several studies have shown a positive correlation between MMP expressions and adverse clinical events [171] or a poor prognosis [172]. ROS have been shown to activate some MMPs, possibly by their reaction with the thiol groups of its active center [173], or block some proteases inhibitors by the oxidation of the meteonin residues of their active centers, promoting MMP activity [174]. Additionally to their role as key regulators of MMP activation, ROS and NO have been implicated in MMP gene expression [175]. MMP activity contributes to angiogenesis itself, which is also a process regulated by free radicals. As mentioned above, growth factors contributing to angiogenesis, such as PDGF or VEGF, are known to induce ROS formation via NOX, but also extracellular ROS generated during hypoxia/reoxygenation cycles due chaotic tumoral blood supply, enhance the activity of molecules such as HIF-1, which also promote angiogenesis [88]. Together, these data would indicate that the net effect of free radicals will depend on their concentration and the redox status of the cell. The apparent inconsistence of the pro-carcinogenic activity of free radicals and their apoptotic effects would indicate that they participate in a multifactor and dose-dependent process, in which the results are not simply derived by their capacity of directly inducing chemical modications in the DNA, because they affect other cellular processes that contribute to the expression of the transformed phenotype. In general, there are sufcient data to afrm that tumoral cells favor free radical production but not in a noncontrolled fashion, because an excess of free radicals would inhibit cellular growth and activate cellular death by apoptosis or necrosis.

4. Oxidative stress and cancer stem cells The eld of stem and progenitor cells is gaining an increasing interest, due the potential application of the knowledge derived from the study of these cells in anti-cancer therapy and other diseases. Most tissues contain minor populations of stem and progenitors cells for tissue maintenance and regeneration [176]. Two characteristics dene primarily the stem cells: self-renewal and multipotency. This way they are able to maintain an undifferentiated state through numerous cycles of cell division and generate a progeny composed of different cell types [177]. From 2003, scientic data point out the involvement in oncogenesis, chemotherapeutic resistance and metastasis, of a specic cell type with stem cell properties known CSCs. First experimental evidence was reported by Al-Hajj et al. [178], who were able to identify a tumorigenic cell population characterized by the expression of the surface receptor CD44 (CD44+ ) and the absence of the cell-surface markers CD24 (CD24 ) and a panel of nonepithelial lineage markers (lin ), alone or in conjunction with the expression of the epithelial specic antigen (ESA). The transplantation of a small number of these cells, as small as 200 cells, in non-obese diabetic/severe combined immunodecient (NOD/SCID) immunocompromised mice formed tumors that recapitulated the phenotypic heterogeneity of the original breast tumors from which they were derived. The stem cell hypothesis assumes that CSCs are the consequence of the deregulation of normal stem cells, through the accumulation of genetic and epigenetic changes that nally lead to stem cell transformation (see Fig. 1 for a schematic representation). These tumor-initiating cells drive tumor growth and progression and give rise to a progeny of cells, which are more differentiated and are non-tumorigenic that will form the bulk of the tumor [177,179]. During normal development, signal from the surrounding microenvironment, also known as stem cell niche, regulates stem cell self-renewal and maintain their unique properties. It has been suggested that an altered cancer stem cell niche may exist and provide those signals that potentiate CSC proliferation. Indeed, the effectors that mediate cell interactions within the niche, that include soluble factors as wingless type (Wnt), Notch, sonic hedgehog (SHH), stromal derived factor 1 (SDF-1) and others, are known to be involved in tumor development and progression [177]. Apart from self-renewal capacity, recent advances in stem cell research point out the importance of another property of these cells: the capacity of migration to areas of injury/wound characterized by hypoxia and necrosis. This phenomenon seems to be driven by SDF-1, as showed by several studies [180182], and may play an important role in tumor angiogenesis and metastasis, especially if we consider that hematopoietic stem cells (HSC) and endothelial progenitor cells migrate from bone marrow to tumors for neoangiogenesis [183]. A recent report has highlighted the role of the microenvironment in stem cell migration showing that an injured conditioned medium, prepared by treating bone marrow stromal cells with hydrogen


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Fig. 1. Models of cell heterogeneity in solid tumors. (A) The clonal selection model states that every cell in a tumor is able to initiate a new tumor, through the acquisition of new mutations that confer growth and survival advantages. These cells (red) will dominate over the other cells in the tumor (purple), which are characterized by a lower proliferative potential. (B) The cancer stem cell model assumes that a particular cell type with stem cell-like properties is responsible for tumor initiation, progression and metastasis. These cells, known as cancer stem cells (CSCs) (orange), are able to self-renew and differentiate into the rest of cell types that compose the bulk of the tumor (green).

peroxide in hypoxic conditions, enhances SDF-1 secretion and attracts highly tumorigenic cells with stem cell-like properties [184]. The latter work suggests that hypoxia and oxidative stress play a crucial role in cancer stem cell mobilization. This observation is sustained by other studies that show that ROS may affect the interactions between stem cells and their niches. Hosokawa et al. [185] suggested that the increase in ROS concentrations, due to the administration of anti-cancer drugs as 5-uorouracil, induce the exit of stem cells from the niche through the suppression of Ncadherin mediated cell adhesion. Ito et al. [186] showed that ROS reduces HSC quiescence and promotes HSC proliferation through the activation of p38 MAPK until exhaustion. Lvesque et al. [187] proposed that HSC were mobilized by an increment in the hypoxic zone of bone marrow niche after treatment with cyclophosphamide. To correctly apply these ndings to cancer research we must take into account that solid tumors, as breast cancer, are characterized by a marked hypoxia and high levels of oxidative stress due the incapacity of the pre-existing vascularization to satisfy the demand of oxygen of the tumor cells in exponential growth. This is the main reason why transformed cells induce neovascularization during the progression phase. In such conditions, new formed vessels are often anomalous in their structure and originate cycles of hypoxia/reperfusion, which greatly increases the generation of free radicals [155]. This situation mimics injury/stress conditions and therefore it is possible that, apart from promoting tumor progression, free radicals contribute to the formation of a suitable microenvironment within the tumor, to mobilize and home HSC in order to increase the tumoral vascular

network and metastasis. In fact, the above-mentioned work published by Das et al. [184] shows that the exposure of stem cell-like cells derived from several solid tumors to hypoxia and reoxygenation increases their migratory and tumorigenic potential in a xenograft mouse model. On the other hand, genomic approaches, similar to the one followed by Heffron et al. [188], point out the involvement of molecular pathways related to oxidative stress and angiogenesis in cancer stemness. The results of these novel studies have profound implications for cancer treatment. Conventional anti-cancer treatment is based on the administration of agents able to shrink the tumors, but this approach only eliminates those cells which form the bulk of the tumor, the progeny of the stem cells which are not tumorigenic. Failure to destroy CSCs would result in disease relapse, as can be observed in the clinic [177]. Even further, if we consider that most of antineoplastic agents induce oxidative stress [19], chemotherapy could promote the migration of CSCs, endothelial stem cells and progenitor to the tumor, facilitating in some extent the neoangiogenic process and tumor metastasis and could contribute to the observed disease relapse despite tumor shrinkage. A recent series of in vitro and in vivo experiments published by Diehn et al. [189] show that breast CSCs are characterized by signicant lower levels of intracellular ROS compared with the non-tumorigenic cancer cells (NTCs) in the tumors. A genome-wide analysis of both types of cells revealed that CSCs over-expressed genes which codify proteins involved in the antioxidant defense of the cell, as free radical scavengers. Even further, Diehn et al. observed that CSCs are signicantly radioresistant compared to their NTCs

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Table 2 Chemotherapeutic drugs commonly used in breast cancer treatment classied by the level of oxidative stress they induce. Classication Drug name Target Mechanism of action Free radical produced by drugs themselves or their intermediate metabolites H2 O2 , HO and O2 HO and O2 HO and O2 H2 O2 and HO H2 O2 and HO O2

Chemotherapeutic drugs that induce high levels of oxidative stress Anthracyclines Doxorubicin DNA Epirubicin Daunorubicin Platinum coordination Cisplatin DNA complexes Carboplatin Oxaliplatin Alkylating agents Cyclophosphamide DNA Epipodophyllotoxins Etoposide Topoisomerase II Teniposide Camptothecins Topotecan Topoisomerase I Irinotecan Chemotherapeutic drugs that induce low levels of oxidative stress Antimetabolites Methotrexate Dihydrofolate reductase, Pemetrexed thymidylate synthase and/or Fluorouracil ribonucleotide reductase Gemcitabine Capecitabine Taxanes Paclitaxel Microtubules Docetaxel Vinca alkaloids Vinblastine Microtubules Vinorelvine

Polycyclic rings allow intercalation and quinones allow redox reactions Platinum coordination

DNA crosslinking Inhibit reconnection of DNA Inhibit reconnection of DNA

Inhibit DNA synthesis

Inhibit microtubule disassembly Inhibit microtubule assembly

H2 O2 and O2 O2

counterparts. Given that the cell-killing activity of radiation and certain chemotherapeutics are partially mediated by the production of free radicals [19,190], it is rather possible that the characteristic therapeutic resistance of CSCs would be mediated, at least in part, by their enhanced capacity to overcome intracellular oxidative stress levels. These new data seem to be in disagreement with previous data discussed in this review, but probably this is only apparent given the differential nature of CSCs with respect NTCs and the two-face character of free radicals. In vitro studies show that tumor cells produce large amounts of free radicals compared with their normal counterparts and that transformed cells exhibit a marked imbalance of antioxidant enzymes [191,192]. This way, cancer cells promote high free radical levels up to a sub-lethal threshold in order to favor a high-proliferating phenotype, but they may pay the prize of being more susceptible to radiation and chemotherapeutic drugs. This may not be the case of CSCs, whose proliferation rate is lower and are markedly resistant to radio and chemotherapy, in part because as an additional distinctive characteristic must be added to this particular cell type: their enhanced capacity to detoxify intracellular free radicals through the increased production of free radical scavengers. Diehn and colleagues were especially interested in glutathione (GSH) and they observed that CSCs showed an increased expression of genes involved in GSH synthesis. GSH pharmacological depletion radiosensitized CSCs, pointing out the crucial role of this antioxidant in CSCs viability. Interestingly, some works point out that a high percentage of metastatic cells with high GSH levels survive the combined nitrosative and oxidative stress elicited by the vascular endothelium [193,194]. Taking into account that CSC

hypothesis assumes that this particular cell type is responsible for metastasis, it would be not surprising that CSCs would have selected the overexpression of genes that enable their interaction with the endothelium. These coincidences merit further research to clarify the putative differences in the antioxidant capacities of CSCs and NTCs and their clinical implications. 5. Conclusions As discussed in this review, oxidative stress is an important risk factor for cancer development and an important factor for disease progression, as evidenced by an increasing number of scientic reports. Reducing oxidative stress by the administration of antioxidants has been considered a good alternative for cancer prevention, since high consumption of fruits and vegetables has been related to the reduction of breast cancer risk [195]. This relationship has been frequently assessed by numerous casecontrol studies and some cohort studies, pooled analysis and meta-analysis, but results have been equivocal, leading inverse associations in the case of casecontrol studies but no signicant associations derived from the majority of cohort studies [196,197]. Because of the controversy, studies regarding the effects of specic components of fruits and vegetables in cancer signaling have been published recently. The studies show that many of these phytochemicals exert chemopreventive properties with a great potential to be exploited in the eld of breast cancer prevention. Frequently, the activity of these organic molecules disrupts or interferes with ROS-mediated signaling. This is the case of sulphorophanes, curcuminoids,


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epigallocatechin gallate, resveratrol, capsaicin, green tea catechins, genistein, avopiridol, oleandrin and lycopene, which have been shown to regulate the expression and/or activity of Nrf2, NF- , AP-1, MAPKs, Bcl-2, Bcl-XL , EGFR and VEGFR among others [135,198]. There is no doubt that more studies are needed to clarify the impact of antioxidants and other chemopreventive phytochemicals in breast cancer risk because, despite the failure of experimental and clinical studies in demonstrating a robust association, the experimental evidence corresponds with epidemiological data. On the other hand, it is also likely that once the tumor has developed and it is clinically detectable, oxidative stress plays an important role in both treatment response of the patient and clinical outcome in general [20]. Many drugs used for the systemic treatment of breast cancer cause large amounts of oxidative stress (Table 2) that can interfere with treatment effectiveness and enhance its toxic side effects [19]. The oxidative stress caused by chemotherapy is added to the oxidative stress which is inherent to the tumor. Aggressive breast tumors are able to adapt to this high oxidative environment, maintaining oxidative damage below the lethal threshold, which in turn reinforces the activation of signaling cascades that contribute to tumor growth, angiogenesis and metastasis. As for breast cancer prevention, the use of dietary antioxidants has also been considered as a complement to improve breast cancer treatment, but the results of the experimental and clinical works regarding this issue are controversial, with some of them supporting this concept [199,200], but with some others also suggesting that the patients who use antioxidant supplements during chemotherapy have worse survival than those who do not [201,202]. Methodological problems and inadequate experimental design may have contributed to contradictory results, but in addition, studying the effects of oxidative stress and antioxidant in cancer treatment from a systemic point of view must be in the base of inconclusive data regarding this particular issue, given that compartmentalization of free radicals and antioxidant status of the cells drive cell fate. Both antioxidant and selective induction of massive oxidative stress in cancer cells have been considered as a therapeutic approach to cancer treatment. Antioxidant strategy would, in theory, inhibit some of the stimuli that contribute to cancer transformation and the expression of an aggressive phenotype, minimizing the acquisition of new mutations and the onset of molecular pathways that promote cancer growth, survival and spreading. Oxidant-targeted therapy is based the induction of severe damage in cancer cells to an extent that, even if they are adapted to a high oxidative environment, would be able to cause apoptosis [203]. But in light of recent ndings a word of caution must be said, given that free radicals are a functionally a doubleedge sword and their effects are dose-dependent. In addition, intracellular and extracellular oxidative stress may exert a differential role in tumor cell response that may also depend

on the tumor antioxidant status, which could be determined by tumor cell type. For example, CSCs seem to maintain higher intracellular antioxidant capacity than NTCs, what may promote CSCs survival when they are exposed to an exogenous ROS-generating agent, which even may reinforce its antioxidant capacity acting as an external stimulus. Then, what is the rationale for a redox therapeutic approach in cancer?, should we increase the oxidative stress level to induce cancer cell death, unless we take the risk of severe adverse effects of such a toxic therapy?, should we decrease the oxidative stress associated to treatment, unless we limit its tumor shrinkage capacity?, which is the right oxidative stress level to kill both CSCs and NTCs without affecting healthy cells?. The answer is not simple, but we are probably facing a great opportunity to improve breast cancer treatment, through the manipulation of ROS-mediated mechanisms. Ideally, a strategy which combines free radicals-generating agents and drugs that inhibit ROS elimination specically in the cancer cells could promote intracellular free radicals accumulation and enhance cancer cell cytotoxicity. Moreover, careful control of the amount of ROS induced is critical, given that low or inadequate dose of ROS would activate signaling pathways responsible for progression, as discussed before. In this line, a recent study published by Guzman et al. [204], showed that a novel compound known as 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (TDZD-8) is able to induce the depletion of free thiols and kill leukemia cells, including malignant stem and progenitor populations, what seems to be a promising approach. Still in many cases and given that redox modulation is an event that affects to all cells in an organism, it is important to think about the specicity of a redox-modulating therapy. Trying to solve this pharmacological problem, Fang et al. [205] in a recent review of their own works show how the development of polyethylene glycol conjugated free radical-generating enzymes, pegylated protoporphyrines and a highly water soluble micellar formulation, exhibit superior in vivo pharmacokinetics than their non-modied parental molecules, particularly in tumor delivery. Further knowledge of the redox biology of breast cancer cells and improvement of tumor-targeted induction of oxidative stress opens a new research line that could improve breast cancer treatment and the quality of life of the patients.

Conict of interest statement None declared.

Reviewer Maurizio Battino, Ph.D., M.D. (Hon), Marche Polytechnic University, Department of Biochemistry, Biology and Genetics, Faculty of Medicine, Via Ranieri 65, I-60100 Ancona, Italy.

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