Oxidative stress, inflammation, and cancer: How are they linked?

4a. Cellular transformation

Chronic inflammation has been linked to various steps involved in carcinogenesis, including cellular transformation, promotion, survival, proliferation, invasion, angiogenesis, and metastasis [65, 142]. How oxidative stress is involved in these various steps is discussed in the following sections.

Cancer is a multistage process defined by at least three stages: initiation, promotion, and progression [143-145]. Oxidative stress interacts with all three stages of this process. During the initiation stage, ROS may produce DNA damage by introducing gene mutations and structural alterations of the DNA. In the promotion stage, ROS can contribute to abnormal gene expression, blockage of cell- to cell communication, and modification of second messenger systems, thus resulting in an increase of cell proliferation or a decrease in apoptosis of the initiated cell population. Finally, oxidative stress may also participate in the progression stage of the cancer process by adding further DNA alterations to the initiated cell population [146].

In recent years, considerable evidence has demonstrated that ROS are involved in the link between chronic inflammation and cancer [147-149]. Indeed, an important characteristic of tumor promoters is their ability to recruit inflammatory cells and to stimulate them to generate ROS [150, 151]. Tumor promotion, for example, can be inhibited in animal models by the use of agents, including certain antioxidants as well as steroids and retinoids, that can inhibit the phagocyte respiratory burst [148, 150]. Moreover, increased levels of oxidatively modified DNA bases (such as thymidine glycol, 5-hydroxymethyl-2′-deoxyuridine and 8-OHdG) have been induced in the skin of mice by topical phorbol 12-myristate 13- acetate (PMA) exposure [152]. 8-OHdG has also been identified in the epidermis of nude mice exposed to near-UV [153]. In addition, genetic damage and neoplastic transformation have been demonstrated in cells co-cultured in vitro with activated phagocytes [149] and the genotoxic effects observed include formation of DNA strand breaks [151], sister chromatid exchange [154] and mutations [155]. Furthermore, the DNA base modifications observed are characteristic of an attack by reactive oxygen species OH. [156]. Inflammatory cells may also increase DNA damage by activating procarcinogens to DNA-damaging species, for example neutrophils can activate aromatic amines, aflatoxins, estrogens, phenols, and polycyclic aromatic hydrocarbons by ROS-dependent mechanisms [148, 157]. On the other hand, both neutrophils and macrophages have themselves been shown to release large quantities of superoxide, hydrogen peroxide, and hydroxyl radical following activation of their redox metabolism [158].

In fact, initial experiments on the role of ROS in tumor initiation have assumed that oxidative stress acts as a DNA-damaging agent, effectively increasing the mutation rate within cells and thus promoting oncogenic transformation [159]. However, more recent studies have revealed that in addition to inducing genomic instability, ROS can specifically activate certain signaling pathways and thus contribute to tumor development through the regulation of cellular proliferation, angiogenesis, and metastasis [160]. For example, nitrosative stress has been shown to play a critical role in inflammation-associated carcinogenesis by activating AP-1, a representative redox-sensitive transcription factor [161], which is involved in cell transformation and proliferation [139, 162].

4b. Tumor cell survival

One of the key characteristics of tumor cells is their increased ability to survive compared with normal cells. ROS are reported to be tumorigenic by virtue of their ability to increase cell proliferation, survival, and cellular migration. ROS can induce DNA damage, leading to genetic lesions that initiate tumorigenicity and subsequent tumor progression. On the other hand, ROS can also induce cellular senescence and cell death and can therefore function as anti-tumorigenic agents. Whether ROS promote tumor cell survival or act as anti-tumorigenic agents depends on the cell and tissues, the location of ROS production, and the concentration of individual ROS.

ROS has been reported to play a major role in tumor initiation and survival induced by a variety of agents both in animal models and humans [158, 163, 164] by mediating cellular signal transduction pathways. These signaling pathways are involved in the transmission of inter or intracellular information and are critical for supporting tumor cell survival and establishing cell fate. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) family of enzymes, one of the potential sources of ROS production, has been reported to promote tumor cell survival and growth [165]. For example, Nox4 and Nox5 promote tumor cell survival in pancreatic and lung cancers, respectively [165]. The serine-threonine kinase Akt has been reported to down-regulate antioxidant defenses and promote tumor cell survival [166]. ROS has also been reported to activate Akt by inhibiting phosphatase and tensin homolog deleted from chromosome 10 (PTEN), the phosphatase counteracting PI3K-dependent Akt activation [167]. Akt may foster tumorigenesis by multiple means [168, 169], for example, by stabilizing cellular avian myeloblastosis virus oncogene (c-Myc) and cyclin D1 or by inducing degradation of the cyclin-dependent kinase (Cdk) inhibitor, p27 kinase inhibitor protein (p27Kip1). Akt is also a profound inhibitor of apoptosis due to its ability to inactivate pro-apoptotic molecules, including caspase-9 and the Bcl-2 homology3 (BH3)-only protein Bcl-XL/Bcl-2-associated death promoter (Bad), and by triggering the activity of the transcription factor NF-κB. In addition, Akt promotes nuclear translocation of the ubiquitin ligase MDM2, which counteracts p53-mediated apoptosis. An important aspect of Akt's promotion of cell survival involves alterations in cellular energy metabolism [168, 169]. Thus, by preventing apoptosis and increasing oxidative metabolism, Akt lies at the hub of complex signaling networks that integrate a multitude of potentially oncogenic signals.

4c. Tumor cell proliferation

Uncontrolled tumor cell proliferation requires the upregulation of multiple intracellular signaling pathways including cascades involved in survival, proliferation, and cell cycle progression. The most significant effects of oxidants on signaling pathways have been observed in the mitogen-activated protein (MAP) kinase/AP-1 and NF-κB pathways [170]. The induction of redox-sensitive pathways during tumor cell proliferation is necessary since cell division presents tremendous energy requirements and the production of metabolites from energy-generating reactions must be buffered to prevent oxidative damage and ultimately cell death [171].

Of the MAP kinase family, which modulates gene expression through phosphorylation of a wide array of transcription factors, the ERK pathway is the most commonly linked with the regulation of cell proliferation. Activation of the ERK, c-Jun N-terminal kinase (JNK), and p38 subfamilies has been observed in response to changes in the cellular redox balance [172]. The induction of AP-1 by H₂O₂, cytokines, and other stressors, for example, is mediated mainly by JNK and p38 MAP kinase cascades [173]. Once activated, JNK proteins translocate to the nucleus and phosphorylate c-Jun and activating transcription factor-2 (ATF-2), enhancing transcriptional activities [174, 175]. H₂O₂ can activate MAP kinases and thereby AP-1 in several manners.

Redox status has also been shown to have an impact on NF-κB regulation. NF-κB regulates several genes involved in cell transformation, proliferation, and angiogenesis [176]. Carcinogens and tumor promoters including UV radiation, phorbol esters, asbestos, alcohol, and benzo(a)pyrene are among the external stimuli that activate NF-κB [177, 178]. Expression of NF-κB has been shown to promote cell proliferation, whereas inhibition of NF-κB activation blocks cell proliferation [179]. Additionally, tumor cells from blood neoplasms, and cell lines from different cancers, including colon, breast, pancreas, and squamous cell carcinoma, have all been reported to constitutively express activated NF-κB [180]. The mechanism for activation of NF-κB by ROS is not clear, and the relationship between NF-κB and ROS is complex [123]. Although mild oxidative stress can lead to modest NF-κB activation, extensive oxidative stress can inhibit NF-κB [123]. Furthermore, NF-κB can protect cells from oxidative stress through induction of the ferritin heavy chain and SOD2 genes, which are both regulated by NF-κB [181, 182]. On the other hand, ROS are believed to be implicated as second messengers involved in activation of NF-κB via TNF and IL-1 [183] and indeed, suppression of TNF and IL-1 were shown to downregulate the expression of active NF-κB and inhibit proliferation of lymphoma and myelogenous leukemia cells [184]. The importance of ROS on NF-κB activation is further supported by studies demonstrating that activation of NF-κB by nearly all stimuli can be blocked by antioxidants, such as L-cysteine, N-acetylcysteine (NAC), thiols, green tea polyphenols, and vitamin E [185, 186], although this might be not very specific because antioxidants have multiple targets [187]. Likewise, NF-κB activity was increased in cells that overexpressed SOD and decreased in cells overexpressing catalase [188].

Kinases, such as protein kinase C (PKC) can also be activated by H₂O₂ and redox cycling quinones [189, 190]. Similarly, H₂O₂ leads to the activation of protein kinase B/Akt (PKB/Akt), which is associated with heat shock protein 27 (Hsp27) [191].

That ROS such as H₂O₂ and superoxide anion induce mitogenesis and cell proliferation has now been demonstrated in several mammalian cell types [192]; and a reduction in cellular oxidants via supplementation with antioxidants such as superoxide dismutase, catalase, β-carotene, and flavonoids inhibits cell proliferation in vitro [193]. However, paradoxically high concentrations of ROS can trigger apoptotic or necrotic cell death [194-196].

4d. Tumor cell invasion

Oxygen radicals may augment tumor invasion and metastasis by increasing the rates of cell migration. During transformation into invasive carcinoma, epithelial cells undergo profound alterations in morphology and adhesive mode, resulting in a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. For example, treatment of mammalian carcinoma cells with hydrogen peroxide prior to intravenous injection into mice enhances lung metastasis formation, indicating that an important function for ROS is the seeding of metastatic tumor cells [197]. This might be due to a decreased attachment of tumor cells to the basal lamina, or alternatively be due to the increased activity or expression of proteins that regulate cellular motility. For instance, oxidative stress regulates the expression of intercellular adhesion protein-1 (ICAM-1), a cell surface protein in endothelial and epithelial cells, most likely due to the activation of NF-κB. ICAM-1 together with IL-8 regulates the transendothelial migration of neutrophils and has a potential function in tumor metastasis [198].

On the other hand, it is believed that the matrix metalloproteinases (MMPs) play the central role, and their increased expression reportedly is associated with the invasion and metastasis of malignant tumors of different histogenetic origins [199]. For example, Mori et al. found that MMP-13, MMP-3, and MMP-10 were remarkably upregulated by the oxidant directly, and their activities were critically implicated in the invasive potential induced in NMuMG cells in the reconstituted model [200]. Another subgroup of MMPs, gelatinases (MMP-2 and -9), which are key enzymes for degrading type IV collagen and are thought to play a critical role in tumor invasion and metastasis [199], were also found to be activated post-transcriptionally by prolonged oxidative treatment. These effector molecules activated under prolonged oxidative stress relate chronic inflammation to malignant transformation, in particular to the invasive potential of cells, at least at a molecular level.

MMPs are capable of cleaving most components of the basement membrane and extracellular matrix [201]. The activation of MMPs, such as MMP-2, probably occurs by the reaction of ROS with thiol groups in the protease catalytic domain [202]. In additional to their role as key regulators of MMP activation, ROS have been implicated in MMP gene expression [203]. Both hydrogen peroxide and nitric oxide donors, as well as the increased expression of iNOS, stimulate the expression of several MMPs (MMP-1, MMP-3, MMP-9, MMP-10, MMP-13) [203]. In fibroblastic cells, the sustained production of H₂O₂ recently was shown to activate MMP-2 and to increase cell invasion [204]. Oxidative stress may also modulate MMP expression by activation of the rat sarcoma viral oncogene (RAS), or direct activation of the MAPK family members extracellular-signal regulated kinase 1/2 (ERK1/2), p38, and JNK, or inactivation of phosphatases that regulate these proteins [160].

In addition, several studies have reported the involvement of chemokines and chemokine receptors in the invasion and metastasis of different types of tumors [205-208]. The metastatic potential of chemokines is attributed to their ability to induce the expression of MMPs, which facilitate tumor invasion [208, 209]. Moreover, silencing of endogenous CXCR4 gene expression by CXCR4-shRNA inhibited the proliferation, adhesion, chemotaxis and invasion of mucoepidermoid carcinoma cells [210]. In addition, recent data point to a role for the small guanosine triphosphatase Rac1 (GTPase Rac1) in motility and invasion of tumor cells in vitro by altering cell-cell and cell-matrix adhesion. For example, Rac1 activity induces ROS production in endothelial cells. These ROS can mediate Rac1-induced loss of cell-cell adhesion in primary human endothelial cells and thus might loosen the integrity of the endothelium [211].

It is becoming clear that a number of steps in the metastatic cascade, such as invasion, intravasation and extravasation are regulated by redox signaling [212]. One such redox signalling molecule is the electrophilic cyclopentenone prostaglandin 15d-PGJ2 (15-deoxy-12,14 -prostaglandin J2), an inflammatory molecule [213], that can affect redox signalling through the post-translational modification of critical cysteine residues in proteins, such as actin, vimentin and tubulin [214, 215]. The fact that 15d-PGJ2 can alter the cytoskeleton [212], may coincides with decreased migration and increased focal-adhesion disassembly, that might have important implications in the inhibition of metastatic processes such as invasion, intravasation and extravasation. These results suggest a role for redox signalling pathways, rather than direct cytoskeletal disruption, in the mechanism of 15d-PGJ2 in cancer cells.

Finally, Cheng et al demonstrated that ROS enhance the transendothelial migration (TEM) of melanoma cells during intravasation, and that this mechanism could potentially be triggered by ultraviolet radiation through the increased expression of thioredoxin interacting protein (Txnip) and inhibition of thioredoxin (Trx) [216].

4e. Tumor cell angiogenesis

Solid tumors induce an angiogenic response by the host blood vessels to form a new vascular network for the supply of nutrients and oxygen [217]. This neovascular response is partly responsible for tumor growth and metastatic spread [218, 219]. Angiogenesis in tumors is controlled by the so-called ‘angiogenic switch,’ which allows the transition from low invasive and poorly vascularized tumors to highly invasive and angiogenic tumors. To further increase in size, tumor cells express a set of molecules that initiate tumor vascularization.

A number of cellular stress factors, including hypoxia, nutrient deprivation, and ROS, are important stimuli of angiogenic signaling [220]. In addition, overexpression of Ras has been linked to vascularization of tumors [221]. Indeed, transformation by Ras stabilizes HIF-1α and upregulates the transcription of vascular endothelial growth factor-A (VEGF-A). Moreover, chemical antioxidants inhibit the mitogenic activity of Ras, indicating that ROS participate directly in malignant transformation. Finally, ROS stabilize HIF-1α protein and induce production of angiogenic factors by tumor cells [222].

The HIF system plays a significant role in angiogenesis, and the molecular mechanisms of its regulation have recently been characterized. In addition, HIF-independent mechanisms that involve a number of other molecules and transcription factors such as NF-κB and p53 have been described. p53 may interact with the HIF system but may also have direct effects on angiogenesis regulators or interfere with translation mechanisms of angiogenesis factors

One other major factor in angiogenesis is vascular endothelial growth factor (VEGF), which is produced by the cells to stimulate the growth of new blood vessels. VEGF induces angiogenesis by stimulating endothelial cell proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2, fetal liver kinase 1/ kinase insert domain receptor (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and endothelial nitric oxide synthase (eNOS), which contribute to angiogenic-related responses in endothelial cells [134]. A number of oncogenes and tumor-suppressor genes that are normally associated with cell transformation [(RAS, c-Myc, murine sarcoma 3611 oncogene (RAF), human epidermal growth factor receptor-2 (HER-2/neu), c-Jun, and steroid receptor coactivator (SRC)] regulate angiogenesis through upregulation of VEGF or downregulation of thrombospondin-1 (TSP-1), an angiogenesis suppressor [223, 224]. Furthermore, mutated p53 upregulates VEGF and in contrast, wild-type p53 decreases VEGF production and increases TSP-1 [225]. Angiogenic factors such as VEGF, fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) are released into the tumor microenvironment by tumor or inflammatory cells in response to various stimuli, such as ROS [226]. The released growth factors activate endothelial cells that give rise to new blood vessels [227, 228].

Monte et al. have demonstrated that lymphocyte-induced angiogenesis is triggered by ROS stimulation, and that this response can be blocked by the administration of a free radical scavenger to tumor bearing mice [229] [230]. In addition, the administration of H₂O₂ or an oxidative stress-producing drug (doxorubicin) to normal mice activated in vivo angiogenesis [229].

Due to reduced physiological tissue oxygen tension (hypoxia), which occurs during tumor initiation, tumors often become hypoxic. Under hypoxic conditions, cells activate signaling pathways, which regulate proliferation, angiogenesis, and death. Cancer cells have adapted to these pathways, effectively allowing tumors to survive and even grow under adverse hypoxic conditions [160]. This adaptation of tumor cells to hypoxia contributes to the malignant phenotype and to aggressive tumor progression [231], and low oxygen tension in tumors is associated with increased metastasis and poor survival of patients with several forms of squamous tumor [232, 233]. HIF-1α responds to these changes by specifically decreasing the oxygen (or hypoxia) level, and upregulating several genes to promote survival in low-oxygen conditions and thus promoting angiogenesis.

In conclusion, although previous sections indicate that all different sub-stages of tumor development are affected by ROS and inflammation, early stages of cancer development (e.g. cellular transformation), involving DNA damage, are however most affected by ROS generated inflammation. For example, colitis may develop into colon cancer after inflammatory infiltration, increased production of ROS, impairment of antioxidant defenses, DNA damage, and genetic and epigenetic alterations, resulting in the transformation of epithelial cells [234]. Or, bronchitis, which can lead to lung cancer, clearly links pro-oxidants, generated by cigarette smoke, to inflammation of the bronchus, and eventually transformation of lung cells into lung cancer [235]. Similarly pancreatitis and esophagitis, both induced by tobacco and alcohol, may transform normal tissue into pancreatic or esophageal cancer if the antioxidant system is not sufficiently effective [236, 237].

4f. Chemoresistance

Despite many decades of research, the mechanisms underlying chemoresistance are still poorly understood. There is growing evidence that the inflammatory tumor microenvironment modulates not only cancer development but also cancer responsiveness and resistance to conventional anticancer therapies [238]. Experimental studies have led to the identification of various cancer cell-intrinsic resistance mechanisms, e.g., activation and/or overexpression of drug transporter proteins (e.g., P-glycoprotein), altered expression of detoxifying enzymes (e.g., glutathione S-transferase) or resistance to apoptosis/senescence pathways [239-242].

For example, an inflammatory response induces changes in expression and activity of multidrug-resistance (MDR)-associated protein transporters, greatly affecting drug responses [243, 244]. It has been shown that acute inflammation suppresses the drug transporter P-glycoprotein (PGP) in the liver, whereas it activates PGP in kidneys, resulting in changes in the pharmacokinetics of the PGP substrate doxorubicin [245]. Likewise, expression of multidrug resistance-associated protein 1 (MRP1) is elevated in inflamed intestine of patients with Crohn's disease or ulcerative colitis [246]. Thus, enhanced states of inflammation influence proteins that are strongly linked with drug resistance.

In addition to the effects caused by inflammation, several chemotherapeutic agents have also been shown to activate the transcription factor NF-κB in human lung and cervical cancers and in T cells [247-249]. These agents are paclitaxel, vinblastine, vincristine, doxorubicin, daunomycin, 5-fluorouracil, cisplatin, and tamoxifen. Activation of NF-κB by these agents has been linked in turn with chemoresistance through serine phosphorylation of inhibitor of κBα (IκBα) [250, 251]. Various in vitro studies have supported a link between NF-κB activation, cytokine production and chemoresistance. One pathway via which NF-κB can be activated is the Toll-like receptor (TLR) pathway. TLRs generally signal via the adapter protein myeloid differentiation primary response gene 88 (MyD88) leading to activation of NF-κB and production of pro-inflammatory cytokines. Activation of TLR signaling in ovarian cancer cell lines by exogenously added LPS resulted in an activated NF-κB pathway, which promoted secretion of proinflammatory cytokines and subsequently conferred resistance to paclitaxel [252, 253]. Also, TNF receptor signaling promotes NF-κB activation and has been linked with chemoresistance. For example, exposure of breast cancer cells to exogenously added TNFα results in selection for breast cancer cells that overexpress NF-κB, leading to increased cancer cell survival and resistance to ionizing radiation [254]. At the same time, cytokines produced by stromal cells in the tumor microenvironment (e.g., IL-1 or TNFα) could potentially activate the NF-κB pathway in cancer cells and thus contribute to chemoresistance. These data call for functional in vivo studies to elucidate the involvement of the inflammatory tumor microenvironment in NF-κB-dependent chemoresistance.

Another mechanism that might be involved in chemoresistance is increased levels of GSH in cancer cells [92]. In particular, the overexpression of glutathione S-transferases (GST), the enzymes that catalyse the conjugation of reduced glutathione to electrophilic [255], as well as efflux pumps, may reduce the reactivity of various anticancer drugs [256]. The increase of the GST levels occurs by transcriptional activation mediated by the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) [257]. Indeed, using genetic manipulation, Lau et al. have demonstrated a strong positive correlation between Nrf2 levels and resistance of three cancer cell lines to chemotherapeutic drugs such as cisplatin, doxorubicin, and etoposide [258]. Chemical activation of Nrf2 by pretreatment with tertiary-butylhydroquinone (tBHQ) also increased survival of neuroblastoma cells in response to the three drugs tested [259]. Consistent with these findings, the role of Nrf2 in determining efficacy of cisplatin was also demonstrated in ovarian cancer cells using siRNA knockdown of Nrf2 [260]. Moreover, many kelch-like ECH-associated protein 1 (Keap1) mutations or loss of heterozygosity in the Keap1 locus have been identified in lung cancer cell lines or cancer tissues [261, 262]. Keap1 mutations or loss of heterozygosity resulted in inactivation of Keap1 or a reduced expression of Keap1, which upregulated the protein level of Nrf2 and transactivation of its downstream genes [261, 262]. Similar to Nrf2, the protective effect of heme oxygenase-1 (HMOX-1, or HO-1) in normal cells may protect from oxidative stress-related diseases. However, such an effect is undesirable in cancer because it provides a selective advantage for cancer cells to survive. Consistent with this notion, HMOX-1 has been found to be overexpressed in various tumor types. It is believed that overexpression of HMOX-1 facilitates cancer cell growth and survival in many ways, such as stimulating rapid growth of cancer cells, enhancing cancer cell resistance to stress and apoptosis, promoting angiogenesis of tumors, and aiding in metastasis of tumors [263]. In addition to HMOX-1, other Nrf2-downstream genes such as Prx1, GPx, and thioredoxin reductase (TrxR) were also upregulated in many cancer cells or tissues and may contribute to chemoresistance [264-266]. In ovarian cancer, constitutive activation of ERK activity has been associated with high tumorigenicity and chemoresistance [267, 268]. In addition, functional analyses employing knockdown of MKP3, a member of the subfamily of protein tyrosine phosphatases known as dual-specificity phosphatases (MKPs) [269, 270], and ectopic overexpression revealed the role of MKP3 in negatively regulating ERK1/2 activity and inhibiting tumorigenicity and chemoresistance in vitro and in vivo. MKP3 is capable of dephosphorylating ERK1/2 by protein-protein interactions via mitogen-activated protein kinase interaction motif within the N-terminal ERK1/2-binding domain [271].

4g. Radioresistance

Acquired tumor radioresistance can be induced during radiotherapy owing to tumor repopulation [272]. Although tumor radioresistance stands as a fundamental barrier limiting the effectiveness of radiation therapy, the exact molecular mechanisms underlying the radioadaptive response are largely unknown (Figure 4). Olivieri et al. [273] first described an adaptive response of human lymphocytes to ionizing radiation. Since then, a substantial number of reports have made a strong case for the existence of cellular radioprotective mechanisms that can be activated in response to a small dose of ionizing radiation. It is assumed that a specific pro-survival signaling network is induced in irradiated mammalian cells.

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Figure 4

Model of the sensitivity of normal cells versus cancer cells to reactive oxygen species

Normal cells are hypersensitive to ROS if not adequately protected by anti-oxidant mechanisms, which may lead to cancer formation. Cancer cells, on the other hand, have upregulated antioxidant mechanisms (glutathione, SOD, catalase, and others) that will protect them against ROS, as can be observed in, for example, the case of radioresistance.

The elevated basal NF-κB activity in certain cancers has been linked with tumor resistance to chemotherapy and radiation [274]. NF-κB in adaptive radioresistance is evidenced in mouse epidermal cells [275] and human keratinocytes, and inhibition of NF-κB blocks the adaptive radioresistance [275]. Human breast cancer cells treated with fractional γ-irradiation show an enhanced clonogenic survival and NF-κB activation [276, 277]. Blocking NF-κB inhibited the adaptive radioresistance. These results provide the first evidence that activation of NF-κB is required for signaling the radio-adaptive resistance by exposure to radiation. Together with the assumption that NF-κB is able to regulate more than 150 effector genes, these results suggest that NF-κB plays a key role in tumor radioadaptive resistance under fractional ionizing radiation. Furthermore, in a study [278] that immunocytochemically examined the levels of activated NF-κB protein in pretreatment cancer specimens and in resected specimens of patients with chemoradiotherapy resistance, the cancers expressed higher levels of cytoplasmic NF-κB than did the adjacent nonmalignant mucosa. Furthermore, Sandur et al. suggest that transient inducible NF-κB activation provides a prosurvival response to radiation that may account for the development of radioresistance [279].

On the other hand, hypoxia is a principal signature of the tumor microenvironment and is considered to be the most important cause of clinical radioresistance and local treatment failure. The response of cells to ionizing radiation is strongly dependent upon oxygen, which is traditionally explained by the “oxygen fixation hypothesis” [280]. Oxygen is so far the best radiosensitizer. De Ridder et al. demonstrated that iNOS, activated by pro-inflammatory cytokines, can radiosensitize tumor cells through endogenous production of NO [280]. They further observed that this radiosensitizing effect is transcriptionally controlled by hypoxia and by NF-κB. Consistently, NF-κB inhibition has been used as an approach to radiosensitize tumor cells, aiming at stimulating apoptosis and inhibiting DNA repair. Moreover, the inflammatory mediators TNFα and NO have been repeatedly used as targets to radiosensitize tumor cells [281-285].

4h. Stem cell survival

Cancer stem cells (CSCs) are cancer cells that have the ability to generate tumors through the processes of self-renewal and differentiation into multiple cells. Such cells persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. The existence of CSCs may have several implications in cancer treatment, including disease identification, selection of drug targets, prevention of metastasis, and development of new intervention strategies.

The first conclusive evidence for CSCs was published in 1997 [286], and to date CSCs have been isolated from both leukemias and a variety of solid tumors, including breast, brain, pancreatic, prostrate, ovary, and colon cancers [287-293]. The pathways that regulate self-renewal of CSCs include wint (Wnt), Notch, Hedgehog, and tumor-suppressor genes such as PTEN and TP53 (tumor protein 53) [294]. Although redox balance plays an important role in the maintenance of stem cell self-renewal and in differentiation, redox status in CSCs has yet to be explored. However, given the similarity between normal stem cells and CSCs and the fact that redox status plays an important role in cancer cell development, it is tempting to speculate that redox status may have a role in CSC survival. A recent study by Diehn et al. demonstrated that, similar to normal stem cells, subsets of CSCs in human and murine breast tumors have lower ROS levels than do the corresponding non-tumorigenic cells [295]. The group further showed that lower levels of ROS were associated with increased free radical scavenging systems and that pharmacologic depletion of these scavengers significantly decreased clonogenicity and resulted in radiosensitization of CSCs. Additionally, two studies showed that CD133+ CSCs conferred chemoresistance to cisplatin and doxorubicin (known ROS generators) in ovarian cancer cells [296] and hepatocellular carcinoma [297], respectively. These studies further indicate that redox status may be important in maintaining CSC survival.

We thank Michael Worley for carefully editing the manuscript. Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research. This work was supported by a grant from the Clayton Foundation for Research (B.B.A.), a core grant from the National Institutes of Health (CA-16672), a program project grant from National Institutes of Health (NIH CA-124787-01A2), and a grant from the Center for Targeted Therapy of MD Anderson Cancer Center. Simone Reuter was supported by a grant from the Fonds National de la Recherche Luxembourg (PDR-08-017).