Free radicals in breast carcinogenesis, breast cancer progression and cancer stem cells. Biological bases to develop oxidative-based therapies

Transcription

1 Critical Reviews in Oncology/Hematology 80 (2011) Free radicals in breast carcinogenesis, breast cancer progression and cancer stem cells. Biological bases to develop oxidative-based therapies Laura 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,g a Department of Oncology, Complejo Hospitalario de Jaen, Avenida del Ejercito Español s/n, Jaen, Spain b Institute of Nutrition and Food Technology José Mataix, Biomedical Research Center (CIBM), Health Sciences Technological Park, Avenida del Conocimiento s/n, Armilla, Granada, Spain c Department of Biochemistry and Molecular Biology II, University of Granada, Campus Universitario de Cartuja, Granada, Spain d Department of Pathology, Complejo Hospitalario de Jaen, Avenida del Ejercito Español s/n, Jaen, Spain e Department of Legal Medicine, University of Granada, Avenida de Madrid 11, Granada, Spain f GENyO Center, Pfizer-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, Granada, Spain Accepted 11 January 2011 Contents 1. Introduction Sources, reactions and physiological/pathological roles of free radicals in the cell Oxidative stress in carcinogenesis, invasion and metastasis in breast cancer Oxidative stress and cancer initiation Tumor suppressor genes and oncogenes Apoptosis Oxidative damage and cancer promotion Cytokines and growth factors Protein phosphatases and protein kinases Transcription factors Oxidative damage and cancer progression Oxidative stress and cancer stem cells Conclusions Conflict of interest statement Reviewer Acknowledgments References Biographies 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 Español 10, Jaen, Spain. Tel.: ; fax: address: lvera@correo.ugr.es (L. Vera-Ramirez) /$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi: /j.critrevonc

2 348 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 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 significant therapeutic applications 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 [4 7]. 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 [8 10]. 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 [11 14]. 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 verified by the detection of oxidative DNA adducts in breast cancer tissue [22] or a significant 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 influence of oxidative stress in neoplasm development and treatment. In this report, the current knowledge about free radical biology and their influence in cell signaling cascades will be reviewed, with special interest in breast cancer cell deregulation. In addition, some recent findings about the influence 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 [1 3,15,16,25]. 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 (O 2 ) and hydrogen peroxide (H 2 O 2 ), 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 O 2 is produced accidentally at the complexes I and III level in the respiratory chain [2,28 30]. Endogenous free radicals are produced to regulate a wide variety of physiological functions in healthy cells. In an inflammatory environment, neutrophils and macrophages increase in a fast but transient way their oxygen consumption, generating O 2 and H 2 O 2 (what it is called oxidative burst ) with antimicrobial and tumoricidal activity, as a first

3 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 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 Formula Pathway of origin Role in breast carcinogenesis and progression Singlet oxygen 1 O 2 Metabolism of partially reduced oxygen species Induction of mutagenesis through the generation of DNA adducts. Not described in breast carcinogenesis specifically Ozone O 3 Atmosphere Induction of mutagenesis through the generation of DNA adducts. Not described in breast carcinogenesis specifically Nitrogen dioxide NO 2 Atmosphere Not reported, but a significant and positive association have been reported between increasing intra-urban NO 2 concentrations and the incidence of post-menopausal breast cancer [206] Nitric oxide NO Arginin metabolism Induction of mutagenesis through the generation of DNA adducts. Reported to affect p53 gene integrity. Enhances vascular permeability, facilitating tumor angiogenesis Peroxynitrite ONOO Derives from the reaction between NO and O 2 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 Hydrogen peroxide H 2 O 2 Microsomal and mitochondrial electron transport chain, peroxisomal metabolism, purine degradation, phagocytes 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 Hydroxyl radical HO Derives from previous specie Induction of mutagenesis through the generation of DNA adducts, base-free sites, DNA strand breaks and DNA protein cross-links Superoxide anion O 2 Microsomal and mitochondrial electron transport chain, XO/XDH, cellular oxydases and phagocytes It is an important source of other highly oxidative substances such as H 2 O 2 and ONOO Hydroperoxyl HOO Derives from previous specie Initiates fatty acid peroxidation. Not specifically related to breast carcinogenesis Alcoxyl OR Fatty acids from membrane phospholipids, aminoacids, and carbohydrates 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 Peroxyl OOR Derives from previous specie It plays the same role than the preceding specie Acyloxyl O II R-C-O Metabolism of fatty acids from membrane phospholipids It plays the same role than the preceding specie Acylperoxyl O II R-C-OO Derives from previous specie It plays the same role than the preceding specie Semiquinone radical HQ Biochemical reactions that take place along the respiratory chain Participates in the biochemical cascade leading to O 2 Semiquinone anion radical Q Biochemical reactions that take place along the It plays the same role than the preceding specie respiratory chain Thiol radical R-S Metabolism of sulphur containing aminoacids Alteration of signaling cascades mediated by of cysteine- and methionine-containing enzymes. Not specifically 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 amplification of the immune response and inflammation [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].

4 350 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 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. Briefly, all biomolecules are putative targets of free radicals and ROS, thus lipids, proteins, carbohydrates and the genetic material are under the fire 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 efficiently repaired, will result in genetic mutations and their consequences. It is estimated that, under physiologic conditions, a human cell is exposed to oxidative lesions per day, among which more than 100 have been already identified. 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 modified 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 modifications 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 specific 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 Cu 2+ and Fe 3+, which are reduced by catechol estrogens to Cu + and Fe 2+ 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 firmly 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 immunodeficient 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 significantly higher in estrogen receptor (ER)-positive cell lines with respect ERnegative cell lines [49,50]. To finish, analyses in human breast tumors revealed significantly 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 significant 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 ( ψ m ) increases and leads

5 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 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 flow and consequently, inducing ROS production. Similar changes in ψ m and ROS concentration have been observed upon mitochondrial Ca 2+ accumulation in MCF7 cells treated with E2 [53]. To finish, estrogens can modulate the activity of key proteins in the electron transport chain of the mitochondria through the regulation of gene expression, post-translational modifications or even modifying the physicochemical properties of the mitochondrial membrane system. The molecular activities described above are known to induce a significant 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 influence of free radicals in the oncogenic transformation has become an interesting issue for the scientific 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 O 2 into H 2 O 2 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 significant 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] 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 fixed 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 first 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 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 p66 Shc protein, which interacts with the electron transport chain increasing ROS production, and the induction of the expression of proline oxidases, which generate H 2 O 2 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 ataxia telangiectasia 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-deficient animals show impaired glutathione biosynthesis, increased manganese-containing

6 352 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) superoxide dismutase (MnSOD), heme oxygenase 1 (HO-1) and thioredoxin (TRX) activity, which may result in increased production of H 2 O 2, 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 modifications 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 deficient 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] 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 [69 71]. 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 O 2 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 H 2 O 2, that plays a dual role: first, it amplifies 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 (PIP 3 ) 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 modifications 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 scientific findings 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 influenced by free radicals. Their effects are appreciable in the activation of the molecular pathways that promote cell proliferation, as discussed in Section 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 identifiable 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 influence 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.

7 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) Cytokines and growth factors Among those growth factors significantly 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 H 2 O 2 and O 2 by NOX [77]. This mechanism is well-known in fibroblasts, 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 H 2 O 2 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 H 2 O 2 and a decrease in O 2, 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, IGF- 1 and fibroblast 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 [79 81]. In vitro experiments performed with tumor cell lines reveal that the transient increase in H 2 O 2 intracellular concentration induced by the binding of PDGF to its receptor is mediated by the recruitment and activation of PI3K, which provides PIP 3 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 H 2 O 2 was significantly 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 significant reduction in HIF-1 and VEGF protein levels. These results are confirmed 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 (NF- B), 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 inflammation 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 H 2 O 2 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 H 2 O 2 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 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

8 354 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) kinases. As mentioned, H 2 O 2 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 H 2 O 2 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 pk a and show high susceptibility to oxidation by H 2 O 2. 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 H 2 O 2 dependent manner [78,97,98]. These findings 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 significant reduction in the activity of Cell division cycle 25 homolog A (Cdc25A) compared to non-treated cells. Cdc25A is an important dualspecificity tyrosine phosphatase in the control of the cell cycle, which is specifically 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 H 2 O 2 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 ERK MAPK 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 fibroblasts (MEFs), while increasing MnSOD activity induced cell quiescence. Decreased MnSOD was accompanied by increased levels of cyclin D1. These results suggest that O 2 mediated signaling promotes proliferation, while H 2 O 2 promote cell quiescence. Controversy reaches further with recent studies that suggest that O 2 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 p66 Shc is involved in the production of cytoplasmic H 2 O 2 from mitochondria and apoptosis. Under severe oxidative stress conditions, p66 Shc 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 scientific 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 -deficient cells are resistant to ROS-induced apoptosis, enabling cell transformation by

9 L. Vera-Ramirez et al. / Critical Reviews in Oncology/Hematology 80 (2011) 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 H 2 O 2 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 H 2 O 2 [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 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 hypoxia reperfusion cycles. Among the hormonal influences 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 affinity 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 influences 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 influence estrogen signaling through their impact on ER stability and function. For example, results of experimental studies that show a significant increase of ER, but not in ER, after H 2 O 2 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 influence post-translational modifications 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