Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal

Mutation Research 533 (2003) 153–171

Review

Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal Xi Huang∗ Department of Environmental Medicine, NYU Cancer Institute, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 20 May 2003; received in revised form 28 August 2003; accepted 28 August 2003

Abstract Unlike arsenic, chromium, or nickel, the carcinogenicity of iron is still under debate. In this review, evidence for iron as a carcinogenic metal was summarized from epidemiological, animal, and cell culture studies. The role of iron in various cancers, such as colorectal cancer and liver cancer was presented. Recent advancements on the molecular mechanisms of iron carcinogenesis were also reviewed. These include: (1) iron autoxidation involving only Fe2+ + O2 in oxidant formation in biological systems and its pH dependency; (2) activation of oxidative responsive transcription factors and pro-inflammatory cytokines; and (3) iron-induced hypoxia signaling. © 2003 Elsevier B.V. All rights reserved. Keywords: Iron; Cancer; Oxidative damage; Tumor suppressor genes; Hemochromatosis

1. Introduction Iron is an essential element for human life. Most iron in humans exists either as heme, present in heme proteins, or in ferritin, an iron storage protein. One molecule of ferritin can bind up to 4500 molecules of iron, with an association constant for iron of about 1036 mol/l. Heme, also known as iron-protoporphyrin IX, is an excellent source of iron for humans [1]. Red meats contain large amounts of heme, which can be absorbed more easily than inorganic iron due to the Abbreviations: AP-1, activator protein-1; DFO, deferoxamine; HCC, hepato-cellular carcinoma; HFE, hemochromatosis Fe; IRE-BP, iron-responsive element-binding protein; LMW, low molecular weight; MAPK, mitogen-activated protein kinases; NF␬B, nuclear factor-␬B; RCC, renal cell carcinoma; ROS, reactive oxygen species; Tf, transferrin; TfR, transferrin receptor; TIBC, total iron binding capacity ∗ Tel.: +1-212-263-6650; fax: +1-212-263-6649. E-mail address: [email protected] (X. Huang).

insolubility of iron salts. Only a small fraction of iron enters and leaves the body on a daily basis. Most iron is recycled from the breakdown of effete red blood cells by macrophages of the reticuloendothelial system. At any given time, approximately 0.1% (3 mg) of total body iron circulates in an exchangeable plasma pool. In healthy individuals, essentially all circulating plasma iron is bound to transferrin (Tf). Tf, an 80 kDa protein, has two binding sites for iron. Iron homeostasis is strictly regulated at the level of intestinal absorption. In healthy individuals, intestinal iron absorption is influenced by body iron stores, hypoxia, and erythropoietic activity [2]. The signals regulating iron absorption remain obscure. Recent studies have shown that a protein, named Nramp2, now called divalent metal transporter, may serve as a primary intestinal transporter involved in apical iron uptake, though Nramp2 can transport a variety of other metals as well [3–5]. As a collaborating counterpart to Nramp2, the protein ferroportin is able to

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export iron across the basolateral membrane of enterocytes and donate iron after being oxidized by a membrane-bound ferroxidase to the circulation [6,7]. Oxidation state, intraluminal pH, and ancillary nutrients such as ascorbic acid can affect the efficiency of iron uptake. Fe2+ is more efficiently absorbed than Fe3+ . A ferric reductase present in the duodenal microvillus membranes may promote absorption by converting dietary Fe3+ to Fe2+ . Cell uptake of Tf-bound iron depends on the number of membrane transferrin receptors (TfR). Increases in intracellular iron down-regulate the TfR numbers. Following the binding of the Tf–iron complex by the TfR, the entire TfR–Tf–iron complex is internalized, and endosomes are acidified to a pH of 5.5–6.0 through the action of an ATP-dependent proton pump [8–10]. Endosomal acidification weakens the binding of iron to Tf and produces conformational changes in both Tf and TfR, strengthening their association [11,12]. Fe2+ is released to intracellular pools following the reduction of bound Fe3+ . Reduction is accomplished in hepatocytes by NADH-dependent differic reductase, while in endothelial cells, by superoxide anion (O2 −• ) generated by xanthine oxidase [13]. The apo-Tf–TfR complex is recycled back to the cell membrane, where apo-Tf is discharged. TfR numbers are modulated by post-transcriptional regulation of TfR mRNA. In the setting of low cellular and cytosolic iron, the iron responsive element-binding protein (IRE-BP) binds to a 3 regulatory untranslated region in TfR mRNA, increasing mRNA stability, and leading to increased transcription and greater receptor numbers [14]. Conversely, when intracellular iron is elevated, iron interacts with the IRE-BP, markedly reducing the affinity of IRE-BP for TfR mRNA. Without bound IRE-BP, the stability of TfR mRNA is greatly decreased, leading to a reduction in receptor numbers [15]. Recent evidence suggests that cells may express a Tf-independent iron transport system. For example, mice and humans lacking Tf, while anemic, show iron overload in parenchymal tissues such as liver and spleen. Neutral gelatinase-associated lipocalin may bind iron and function as an iron transport molecule (for details, see review [16]). The intracellular iron pool serves as a source of iron for both hemoglobin and ferritin synthesis [17]. It can be characterized as iron bound by low molec-

ular weigh (LMW) ligands (<5 kDa), that is: (1) exchangeable and chelatable; (2) easily bioavailable for uptake by ferritin, heme, transferrin, or chelators; (3) metabolically and catalytically reactive in oxidant formation and likely responsible for iron toxicity; and (4) possibly having regulatory properties which affects IRE-BP activity per se [18,19]. This is in contrast to iron ions present in iron proteins, which are tightly bound, and thus not bioavailable for adverse effects. Using the metal sensitive fluorescent probe calcein, the estimated concentrations of LMW iron are in the range of 0.2–1.5 ␮M for resting erythroid and myeloid cells and about 10 ␮M in rat hepatocytes [20–23]. Yet, levels of LMW iron in human serum have not been reported or estimated. Iron deficiency anemia is a common condition known to the medical profession for several centuries. In contrast, iron overload is mistakenly believed to be rare. Iron overload is an increase in total body iron generally exceeding 5 g. The normal levels of body iron range from 50 to 60 mg/kg in males, 35–40 mg/kg in females, and very low in children and young women [24]. Although iron is essential for human life and iron deficiency can cause anemia, iron supplementation for non-anemic adults may have harmful consequences by inducing pro-oxidant conditions through the interaction of iron with O2 and H2 O2 in the body [25–27]. Unlike arsenic, chromium, or nickel as confirmed human carcinogens (see reviews in this current issue), the carcinogenicity of iron is debatable. However, there is increasing evidence summarized below showing that iron can contribute to cancer development either as a cancer initiator or as a cancer promoter.

2. Role of iron in various cancers Iron-induced malignant tumors were first reported in 1959 by repeated intramuscular injection of iron dextran complex in rats [28]. Years later, sarcomas were shown in patients in whom iron preparations had been injected [29]. Beginning in the 1980s, some epidemiological reports have associated increased iron exposure with elevated cancer risk in either prospective or retro-prospective studies, by comparing cancer cases with their matched controls [30–34]. Iron exposure variables in those epidemiological studies

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included dietary iron intake, iron vitamin supplementation, body iron stores as measured by ferritin, serum iron (also known as transferrin iron), total iron binding capacity of transferrin or transferrin saturation, and gene status for hereditary hemochromatosis, an iron overload disease. Because iron stored in the iron proteins is tightly bound, and thus not readily bioavailable for adverse effects, those iron markers are not direct measures of iron that is responsible for iron toxicity. Perhaps for these reasons, epidemiological studies on the association of iron with cancer remain inconclusive [30,31,35–38]. Nevertheless, the majority of existing epidemiological data support the role of iron in human cancer and are summarized below along with animal and cellular evidence suggesting that iron may be carcinogenic. 2.1. Colorectal cancer Because of the high concentration of iron in the human colon, it became natural to consider whether or not iron might be involved in the initiation or promotion of colonic disease. Since first suggested by Graf and Eton in 1985 that dietary fiber may diminish the risk of colorectal cancer by chelating dietary iron through its phytic acid component [33], numerous human epidemiological studies have examined the relationship of iron from exogenous (dietary) or endogenous (body store) sources to colorectal cancer risk. A detailed analysis of 33 epidemiological studies was recently published by Nelson [39]. Of the larger studies, approximately three quarters of them supported the association of iron with colorectal cancer risk. Among them, a study by Stevens et al. and the follow-up study by Wurzelman et al. on the cohort of the National Health and Nutrition Examination Survey I showed a positive association between dietary and body iron stores with colorectal cancer risk [31,37]. Both Nelson et al. and Bird et al. further showed that body iron stores were positively associated with the development of precancerous lesions in the colon, colonic adenoma, or polyps [38,40]. However, no association or negative association between the risk of colorectal cancer and body iron levels or dietary iron uptake was reported, including one nested case–control study in women [36,41]. Others showed that iron supplement or serum ferritin concentration were not linked to the recurrence of colorectal adenoma [35,42].

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To further support the role of iron in colorectal cancer, for individuals who were either heterozygotes or homozygotes for hemochromatosis, the relative risk of colorectal cancer was substantially increased [43,44]. Hereditary hemochromatosis is an inherited iron overload disease, which is due to a point mutation of the HFE gene (hemochromatosis Fe) [45,46] (more details in the liver cancer section). From a mechanistic point of view, it was reported that healthy volunteers taking ferrous sulfate showed increased total fecal iron, and levels of iron loosely bound to low molecular weight chelators were even greater than the total iron [47]. The production of free radicals increased significantly during iron supplementation. It was postulated that unabsorbed dietary iron increases free radical production in the colon to a level that could cause mucosa damage [47,48]. In animal studies, results showed that iron supplementation in rats decreased manganese superoxide dismutase activity [49], increased lipid peroxidation and free radical generating capacity in the colon and cecum [50], and elevated colonic aberrant crypt foci [51,52]. Because red meat contains large amounts of heme, rats fed heme showed increased colonic epithelial proliferation as measured in vivo by 3 H-thymidine incorporation into colonic mucosa [53]. Interestingly, using erythrocyte lysis as a cytotoxicity assay, fecal water from rats fed heme was highly cytotoxic as compared to the control group. The addition of heme to the fecal water of the control group did not affect the cytotoxicity, suggesting metabolism of dietary heme may lead to the formation of an unidentified but highly cytotoxic factor in the colonic lumen [53]. Using HFE-knockout mice, it was recently shown that the elevated concentration of malondialdehyde was found in colon tissue among those mice fed standard iron diet compared to those on the low iron diet, indicating that dietary iron content and HFE genotype may synergistically increase oxidative damage in colon tissue [54]. Although dietary iron intake in rodents increases oxidative stress and cell proliferation, in the absence of colon carcinogens, iron alone does not appear to induce colorectal cancer. For example, rats initially fed N-nitroso-N-methylurea, followed by heme, showed a significant increase in the incidence of colon cancer compared with a diet without heme [55]. Chronic ulcerative colitis patients are at increased risk of developing colorectal cancer [56], because these patients frequently require iron supplementation to

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remedy iron deficiency anemia due to the presence of rectal bleeding. In female C57BL/6J mice, long-term administration of dextran sulfate sodium in the drinking water was shown to induce ulcerative colitis. Simultaneous feeding of those mice with an iron-enriched diet significantly increased colorectal tumor incidence, and tumor multiplicity as measured by tumors/tumor-bearing mouse and tumor volume [57,58]. In the same study, N-acetylcysteine, an antioxidant, significantly induced apoptosis in both normal-appearing epithelia and colorectal adenocarcinomas, and decreased the number of cells immunostained-positive for nitrotyrosine, a marker of NO-mediated protein damage [57,58]. These findings suggest that dietary iron can enhance ulcerative colitis and its associated carcinogenesis by augmenting oxidative and nitrosative stress. Increasing iron concentration in human intestinal Caco-2 cells resulted in increased protein and DNA oxidative damage, as shown by the immunoreactivity for 4-hydroxy-2-nonenal-modified proteins and 8-oxo-2 -deoxyguanosine [59]. It was also shown that Nramp2 is expressed in the apical membrane of Caco-2 cells and is associated with iron transport in these cells with a substrate preference for iron over other divalent cations [60]. In the human HT29 colon carcinoma cells, hemoglobin was as effective as ferric nitrilotriacetate in inducing DNA breaks and oxidized bases [61]. 2.2. Liver cancer The liver is the main organ for iron storage and metabolism. Most experimental and human data support the hypothesis that iron overload is a risk factor for liver cancer. Among many factors such as viral hepatitis, alcohol, and tobacco, which play a role in hepatic carcinogenesis, iron overload is an important one. Liver samples from hemochromatosis patients showed characteristic high levels of iron when compared with the normal liver [62]. It is the excessive accumulation of iron in hepatocytes that causes hepatocellular injury, which leads to the development of fibrosis, cirrhosis, and hepatoma [63]. Generally speaking, when iron stores in the liver become excessive, fibrosis begins to dissect the lobules. If untreated, cirrhosis follows, although the mechanism by which this occurs is still unknown [64]. If such a patient becomes cirrhotic,

there is a 200-fold increase in the relative risk of developing hepatoma [65,66]. In the Western World, hepatocellular carcinoma rarely occurs in patients without cirrhosis. However, in hepatocellular carcinoma developed in non-cirrhotic liver, there is a mild iron overload in more than 50% of cases [67,68]. Two-point mutations have been found within the HFE protein in hemochromatosis patients [69]. HFE, originally called HLA-H, is a protein that resembles atypical HLA class I molecules, consistent with the localization of the gene near the HLA cluster (details provided in [70–73]). One of the two point mutations, at position 282 of the gene, changing the invariant cysteine to tyrosine (indicated as Cys282Tyr or C282Y), has been found to be mutated in 90% of the American patients studied [69]. Only 4% of controls possess this mutation while the majority of patients are C282Y homozygous [69,74]. The second point mutation, His63Asp, has been found in more diverse ethnic backgrounds and may represent an older mutation [75,76]. The genomic structure of HFE is similar to other major histocompatible complex class I-like molecules [45]. Each of the first six exons of the HFE gene encodes one of the six distinct domains of the HFE proteins. HFE has been detected immunohistochemically in a number of tissues, including brain, liver and tissue macrophages [77]. The point mutation of the HFE gene at C282Y is in exon 4. The C282Y mutant protein (expressed in cell culture) binds poorly to ␤2 -microgloulin (␤2 M) and is less abundantly presented at the cell surface [78,79]. The association between HFE and ␤2 M seems necessary for the interaction of HFE with TfR and subsequent cellular transferrin iron uptake. The increased risk of developing hepatocellular carcinoma (HCC) in hereditary hemochromatosis has been associated with cirrhosis and hepatic iron overload. Hemochromatosis among Caucasians has an estimated prevalence of 50–80 cases per 10000 persons [80]. In the US, the listing of hemochromatosis on death certificates increased 60% from 1979 to 1992 [81]. It was shown that decedents with hemochromatosis were 23 times more likely to have liver neoplasms than were descendents without hemochromatosis. In European countries, numerous epidemiological studies have linked HCC with iron overload in hemochromatosis [82–84]. It was also

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shown that African iron overload is a risk factor in the pathogenesis of HCC [85–87]. It is noteworthy that the risk of developing HCC in hemochromatosis patients is significantly increased if the patients have alcoholic cirrhosis or hepatitis B or C-related cirrhosis [82]. Iron, which facilitates persistent hepatitis B or C infection, could also act as a co-factor in the pathogenesis of HCC in patients with hepatitis B or C and alcoholism. The murine HFE gene is structurally similar to the human gene. Four different HFE gene disruptions have been reported in the mouse: an exon 4 knockout, an exon 3 disruption/exon 4 knockout, an exon 2–3 knockout, and a C282Y knock-in [88–90]. In each model, the mice manifested increased hepatic iron levels, elevated transferrin saturations, and increased intestinal iron absorption. Like hereditary hemochromatosis patients, these mice demonstrate relative sparing of iron loading in reticuloendothelial cells. So far, no carcinogenesis study has been reported in these animal models. In chemical-induced hepatocarcinogenesis, iron was shown to greatly sensitize mice to the induction of hepatic porphyria by hexachlorobenzene [91]. Levels of lipid peroxides as well as 8-hydroxy2 -deoxyguanosine, an oxidative DNA damage, were significantly increased in mice following combined iron/hexacholorobenzene. In attempts to study the cancer-initiating, promoting, and/or progressing effects of excess hepatic iron, dietary iron overload in combination with fusonisin B1, or polychlorinated biphenyls, or diethylnitrosamine, were tested in animal models [92–98]. Generally speaking, iron depletes intracellular antioxidants such as ubiquinones, enhances cell proliferation, and acts at least as a promoter of already initiated hepatocytes in the HCC development. 2.3. Kidney cancer The kidney is one of the main organs targeted by iron metabolism. Kidney cancer accounts for 2.1% of all cancers in men and 1.6% in women. Renal cell carcinoma (RCC) accounts for 80–85% of all kidney cancers in the US. The remaining 15–20% of renal cancer are mostly cancers of the renal pelvis, which are anatomically and histologically distinct from RCC. There were no epidemiological studies that were re-

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ported linking kidney cancer risk with abnormal iron metabolism due to gene mutation in humans. However, numerous studies have linked increased risk of kidney cancer in workers of the iron and steel industries [99–103]. For example, in an international multicenter population-based case–control study, relative risk of developing kidney cancer is 1.6 with a 95% confidence interval of 1.2–2.2 in employees of iron and steel industries [101]. Results from these studies suggest that occupation is one of the etiological factors in RCC, though no specific iron compounds were identified as possible contributors of RCC [99–103]. Ferric nitrilotriacetate (Fe-NTA) is a renal carcinogen in rodents [104]. Repeated intraperitoneal administrations of Fe-NTA induces acute renal proximal tubular necrosis, a consequence of free radicalmediated oxidative tissue damage, that eventually leads to a high incidence of RCC. Twenty-one aldehydes and 5 acyloins, products of lipid peroxidation, were produced in the kidney and liver of Wistar rats. Among them, 4-hydoxy-2-noneal (HNE) showed the highest increase (27-fold over control) and malondialdehyde (MDA) was the most abundant aldehydes (2.4 nmoles/100 mg wet tissue) [105]. Glutathione S-transferases, enzymes important in catalyzing the conjugation of reactive metabolites with glutathione and thus in detoxification, were altered after Fe-NTA treatment [106,107]. It is noteworthy that Fe-NTA is a more potent renal carcinogen than Cu-NTA, yielding a higher incidence of RCC and requiring a shorter latent period for the development of RCC [108]. No renal tumor was observed in the NTA-treated group. Neither H-, K-, and N-ras oncogenes nor p53 tumor suppressor gene were found mutated in the RCC tissues induced by NTA [109,110], indicating that iron is responsible for the RCC development. In contrast, the p16 tumor suppressor gene was shown to be vulnerable, leading to its allelic loss within weeks of Fe-NTA administration [111]. The allelic loss of the p16 gene occurs early in carcinogenesis and specifically at the p16 loci as compared with other tumor suppressor genes, which lead to a concept of “genomic sites vulnerable to the Fenton reaction” [112]. Ferric iron complexed with NTA in this model is thought to be a tumor initiator as well as a promoter through the production of reactive oxygen species and free radicals. For example, vitamin E (␣-tocopherol) is a potent inhibitor of

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nephrotoxicity and renal carcinogenesis induced by Fe-NTA [104]. The role of iron in estrogen-induced renal carcinogenesis was studied by investigating the effects of iron content of hamster diets on tumor induction by estradiol. The renal tumor incidence and number of tumor nodules in hamsters treated with estradiol plus a diet enriched with iron were significantly increased (two- to fourfold) than those observed in animals fed an iron-poor diet plus estradiol [113]. Since only male hamsters were used, no mammary cancer incidence was reported in that study. However, estrogen was shown to be able to regulate Tf gene expression in MCF-7 breast cancer cells through a non-consesus distal estrogen responsive elements [114]. Redox cycling of catecholestrogen metabolites between quinone and catechol forms can cause the reduction of Fe3+ to Fe2+ and release Fe2+ from ferritin, which in turn generates hydroxyl radicals by iron-catalyzed reactions [115]. Moreover, estrogen administration increases iron accumulation in hamsters and facilitates iron uptake by cells in culture, although the mechanisms by which estrogen increases iron uptake is not known [116]. 2.4. Lung and stomach cancers Workers of iron ores and steel foundries have an elevated risk of lung and stomach cancers. Although some investigators have suggested that inhaled iron compounds are merely carriers of other carcinogens such as polycyclic aromatic hydrocarbons, nickel, and chromium, there is increasing evidence that iron can be a principal carcinogenic hazard in inhaled dusts (e.g., asbestos or silica) [117,118]. Increased risk for stomach cancer may be due to the ingestion of the same carcinogen-containing dusts from the respiratory system to the gastric system. In a nested case–control study comprised of 144 male lung cancer cases and 558 controls in a large iron and steel foundry in Austria and Spain, workers having ever been employed in the blast furnace had an excess lung cancer risk (odds ratio = 2.55, 95% CI 1.25–5.21) as compared to a reference group of workers not employed in a metal producing department [119]. In a large integrated iron–steel complex in Anshan, China, it was found that standardized proportional mortality ratios were significantly elevated for

lung, stomach, and colorectal cancers, but not other cancers [120]. Interestingly, for both lung and stomach cancers, significant dose-response gradients were observed for exposure to total dust and benzo(a)pyrene, but not for specific chemical components of dust [121]. Excesses of lung cancer and stomach cancer due to iron dust exposure were reported in other epidemiological studies [122–125], although a contribution of other confounding factors such as cigarette smoking, diesel engine exhaust particles, low-dose radon exposure, or the accompanied silico-turberculosis cannot be ruled out. Other studies showed no association of lung cancer with mortality of iron foundry workers [126,127]. Redox activity on the surface of dust particles is a plausible hypothesis for dust-induced lung carcinogenesis in occupational settings. Using the spin-trapping technique and electron spin resonance spectroscopy, it was shown that some mineral dusts from iron ore mines were very active in an oxidative process in aqueous medium, implying the formation of radical oxygen species [128,129]. The presence of a Fe2+ ion on the surface of the particles or its dissolution from the particle surfaces may be responsible for the oxidant formation. Similarly, it was shown that redox activities of coal dusts, coal fly ashes, and asbestos correlated well with levels of bioavailable iron in the dusts, extents of ferritin induction by the dusts, as well as levels of lipid peroxidation in cells treated with the dusts [130–136]. To further support the role of iron in dust-induced lung carcinogenesis, it was shown that iron-containing minerals such as magnetite (Fe3 O4 ) were capable of morphologically transforming Syrian hamster embryo cells, and the iron chelator deferoxamine abolished the transforming activity of these iron compounds [137]. 2.5. Other cancers Iron overload has been shown to enhance chemically mediated cutaneous tumor promotion in animals. In a two-stage mouse skin carcinogenesis model using 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and benzoyl peroxide-promoted cutaneous tumorigenesis, it was found that an increased tumor response in mice (e.g., incidence of tumors and number of tumors per mouse) initiated with DMBA when iron

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was intramuscularly injected [138]. In contrast, a low iron diet resulted in a decrease in skin tumor incidence (both papillomas and carcinomas) and number of tumor per mouse, as well as the conversion of papillomas to carcinomas [139]. It was also shown that sub-cutaneous administration of ferrous sulfate subsequent to DMBA initiation greatly accelerated mammary carcinogenesis in female Sprague–Dawley rats, implying iron’s promoting activity [140]. Using a rat model to study the pathogenesis of Barrett’s esophagus and its progression to esophageal adenocarcinoma, it was shown that iron supplemented rats had significantly higher levels of inflammation, cell proliferation, inducible nitric oxide synthase and nitrotyrosine as well as more tumor in their distal esophagi than did rats that received no iron supplement [141]. 3. Iron proteins as tumor markers and iron chelation in cancer therapy 3.1. Iron proteins as tumor markers Ferritin, an iron storage protein, that has the ability to sequester iron gives ferritin the dual functions of iron detoxification with antioxidant properties and iron reserve with pro-oxidant properties [142–144]. The role of ferritin in cancer is not fully understood. Ferritin has been proposed as a clinical marker for staging and predicting survival of renal cell carcinoma, particularly in the case of recurrence after surgical therapy [145,146]. It was shown that the mean serum ferritin level from the renal vein correlated with tumor stage and was significantly higher than that from the peripheral vein [146]. The mean cytosolic ferritin level of cancer tissue was also much higher than that from normal parenchyma [147,148]. On the other hand, it has been shown that an elevated stomach cancer risk is associated with low serum levels of ferritin, with more than a threefold excess among those in the lowest compared with the highest quintile [149]. One recent study has shown that high serum levels of ferritin may be associated with a decreased risk of renal cancer, suggesting a protective role of ferritin in cancer [150]. In this nested case–control study, the median lag time between serum collection and the diagnosis of RCC was 7 years.

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3.2. Iron chelation in cancer therapy The increased dependence of tumor cells on iron has led to the suggestion that depleting iron may represent a strategy to limit tumor growth. Indeed, tumor cells in a highly proliferative state have a high density of transferrin receptors, and antisense cDNA for the transferrin receptor was shown to reduce TfR mRNA and expression, resulting in more inhibition of growth of human breast carcinoma cells than normal breast cells [151]. Monoclonal antibodies against TfR severely restricted the growth of lymphoma tumors in mice [152,153]. The chelator currently used to treat iron overload disease, deferoxamine (DFO), has shown anti-proliferative activity against leukemia and neuroblastoma cells in vitro, in vivo, and in clinical trials, suggesting that iron deprivation may be a useful anti-cancer strategy [154,155]. DFO, a siderophore originally isolated from Streptomyces pilosus, is the only approved drug in the US for iron chelation therapy, such as ␤-thalassemia patients, due to transfusional iron overload. DFO forms a hexadentate complex with Fe3+ in a molar ratio of 1:1. In vitro studies have shown that in addition to its ability to mobilize iron, DFO has both anti-proliferative and apototic effects, characteristics consistent with a role in anti-tumor therapy [154]. The ability of DFO to inhibit tumor growth was reported in nude mice implanted with hepatocellular carcinoma cells. DFO treatment significantly reduced mean tumor growth rates, resulting in an approximate 60% reduction in growth rate when treatment was initiated in large tumors [156]. In another study, DFO and deferiprone (L1), a bidentate iron chelator, were shown to inhibit the growth and induce the apoptosis of human papillomavirus (HPV)-positive carcinoma cells in vitro [157]. However, DFO or L1 failed to prevent tumor growth in nude mice after subcutaneous injection of the cervical cancer cells [158]. Perhaps because DFO is an approved drug, DFO has been in clinical trials and received great attention in tests of efficacy of iron chelators as an antitumor agent. An Italian Phase II study of nine children with neuroblastoma demonstrated responses in seven of nine children following treatment with a single course of 150 mg/kg per day DFO given by continuous infusion for 8 h per day for 5 days. This dose, which caused no toxicity, resulted in decreases in bone marrow infiltration as well as a reduction in

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tumor mass in one patient [159]. Although the development of iron chelators as anti-cancer agents is still in its infancy [160], this concept does support that iron contributes to cancer development.

sumed in iron autoxidation reactions. For example, oxidants produced by the interaction of Fe2+ and O2 may be quenched by Fe2+ itself at the high concentrations as follows: Fe2+ + OH• → Fe3+ + OH−

4. Iron physico-chemistry in biological system

(4)

According to the reactions (1)–(4), a self-quenching reaction can be written as follows:

4.1. Iron-induced redox reactions, Fenton/Haber–Weiss versus iron autoxidation

4Fe2+ + O2 + 2H+ → 4Fe3+ + 2OH−

Redox cycling is a characteristic of transition metals such as iron. Iron-catalyzed oxidative stress is believed to be the main mechanism involved in the pathogenesis of iron-induced cancer [27]. Reactive oxygen species (ROS), often under pathological conditions due to oxidative stress, have been shown to be associated with a wide variety of diseases, such as carcinogenesis, inflammation, radiation, and reperfusion injury [161]. In biological systems, it is often considered that ROS induced by iron originate from the interaction of iron with enzymatically and/or non-enzymatically generated superoxide (O2 •− ) (Haber–Weiss reaction) and/or hydrogen peroxide (H2 O2 ) (Fenton reaction) [162,163]. However, measurements in liver cells have determined the steady state level of H2 O2 to be approximately 10−8 M [164], and the steady state level of O2 in vivo is about 10−5 M [165]. Assuming that the rate constant for oxidation of substrate by “Fe2+ + O2 ” chemistry (Fe2+ autoxidation) is similar to the Fenton reaction and that the oxidizable substrate concentration of a living system is about 1 M, it has been estimated that the rate of oxidation of oxidizable substrate by “Fe2+ + O2 ” could be as much as 108 faster than the rate of oxidation by the Fenton reaction [166]. These results suggest that “Fe2+ + O2 ” chemistry is probably the most important route for free radical biology of iron. In fact, O2 •− and H2 O2 may be produced directly from dissolved oxygen (O2 ) in aqueous media in the Fe2+ -mediated autoxidation reactions as follows: Fe2+ + O2 → Fe3+ + O2 •−

(1)

Fe2+ + O2 •− + 2H+ → Fe3+ + H2 O2

(2)

Fe2+ + H2 O2 → Fe3+ + OH− + OH•

(3)

In comparison with Fenton/Haber–Weiss reactions where iron is catalytic or redox cycled, iron is con-

(5)

A total stoichiometry 4 Fe2+ :1 O2 can be proposed based on the reaction (5), though this stoichiometry is greatly dependent upon the nature of the iron chelator used, and can differ markedly from the 4:1 [167,168]. It is known that the activation of oxygen by iron is subject to both kinetic and thermodynamic restraints, and therefore, reactions (1)–(3) as described are oversimplified. For example, it has been suggested that oxidants other than the hydroxyl radical (OH• ), such as ferryl or iron oxo, may also be generated [169–171]. Because the conversion of H2 O2 and O2 into the more reactive OH• radical requires the participation of Fe2+ , and DFO binds tightly only to Fe3+ , it has been recently shown that the combination of a Fe2+ chelator 2,2 -dipydyl with DFO had the most significant effect in preventing cells from iron-induced lipid peroxidation in human liver HepG2 cells [168]. 4.2. pH dependency of iron-induced redox reactions Chemically speaking, ferric ion (Fe3+ ) is a weak oxidant, and ferrous ion (Fe2+ ) is the form of iron that is capable of redox cycling. Oxidation of Fe2+ to Fe3+ resulting in ROS formation is greatly dependent upon the pH of the media. For example, the reaction half lives of Fe2+ at pH 3.5 and 7.0 were 1000 days and 8 min, respectively [172,173]. In a pH buffered system under a constant partial pressure of oxygen, the Fe2+ oxidation proceeds at pH values greater than 4.5 with the kinetic relationship: −d[Fe2+ ] = k[Fe2+ ][O2 ][OH− ]2 dt where k = 8.0 × 1013 l2 /(mol2 atm min) at 25 ◦ C. Increasing the pH into the alkaline region causes the precipitation of ferrous hydroxide, which causes the rate of oxidation to change from a homogenous to a

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heterogenous reaction and leads to a further increase in the rate [174]. At pH values below 3.5, the reaction proceeds at a rate independent of pH: −d[Fe2+ ] = k [Fe2+ ][O2 ] dt where k = 1.0 × 10−7 atm−1 min−1 at 25 ◦ C. Since Fe2+ oxidation is very slow at pH < 3.5, there is consequently not much cytotoxicity in the gastric system (pH 1–2) caused by oral uptake of a normal dose of ferrous sulfate. However, in lung medium in which the pH is usually greater than 7, oxidation of Fe2+ probably proceeds quickly and ROS resulting from the interaction of Fe2+ and O2 may damage lung cells and cause cytotoxicity. This suggests that the inhalation of iron may be more hazardous than the ingestion of iron [118]. In fact, pulmonary injury after aspiration of FeSO4 has been reported in a patient showing acute bronchial damage and early histological change in the biopsy specimens after the exposure [175]. A delayed occurrence of bronchial stenosis after inhalation of iron has also been described [176,177]. Because of the pH dependency of iron redox cycling, pH and iron need to be well controlled in cell experiments involving iron-mediated free radical oxidation. It was shown that acidic pH (<4.5) stabilized FeSO4 in the coal dusts, whereas at high pH the conversion of reactive Fe2+ to Fe3+ was immediate [178]. Iron-catalyzed lipid peroxidation in K-562 cells was shown to be pH dependent, the lower the extracellular pH (decreasing from 7.5 to 5.5), the higher the free radical flux; the lower the pH, the greater the membrane permeability of iron [179]. In the phagolysosomes of cells where pH is around 5.5 [180], this pH environment seems to provide optimal conditions for maximal catalytic efficiency and solubility of iron [181,182]. In comparison with normal tissue, human tumors have relative low pH levels [183]. These low levels in tumors may increase iron released from ferritin and lactoferrin and enhance oxidative damage as well.

5. Molecular mechanisms of iron carcinogenesis 5.1. Iron-induced oxidative damage In biological system, iron bound to low molecular weight chelators such as citrate or ATP can generate

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ROS via Fenton/Haber–Weiss or autoxidation reactions, as discussed in the previous section. Lipids, proteins, and DNA are biomolecules targeted by iron-mediated ROS. Generally speaking, this mechanism is well recognized with strong experimental evidence and has been recently summarized by others [181,184,185]. It is noteworthy that recent studies suggest that ROS produced by iron may specifically target some tumor suppressor genes, leading to a novel concept of “genomic sites vulnerable to Fenton reactions” [112]. To support this concept, it has been shown that lipid peroxidation-derived aldehydes such as 4-hydroxynonenal (4-HNE) can interact with DNA to form exocyclic guanine adducts and 4-HNE-DNA adducts are preferentially formed at the third base of codon 249 in the p53 gene [186]. It has also been reported that 4-HNE can increase G to T transversion at codon 249 of the p53 gene, and a higher frequency of G:C to T:A transversions at codon 249 was found in liver tissues of hemeochromatosis cases [187,188]. Administration of Fe-NTA, a renal carcinogen, can specifically cause allelic loss of the p16 tumor suppressor gene in renal tubular cells [111,112]. 5.2. Iron-induced oxidative-responsive transcription factors Besides the direct attack of iron-mediated ROS on DNA, it has recently been proposed that iron can induce early signaling pathways that may modulate activities of several oxidative-responsive transcription factors, such as activator protein-1 (AP-1) and nuclear factor kappa B (NF␬B). AP-1 consists of a family of Jun/Fos dimers that include different Jun proteins (c-Jun, JunB, and JunD) and Fos proteins (c-Fos, FosB, Fra-1, Fra-2, and FosB2) [189]. AP-1 activation is regulated at multiple levels by activation of mitogen-activated protein kinases (MAPK), involving three major pathways of extracellular signal-regulated kinases (ERKs), stress-activated protein kinases/c-jun NH2 terminal kinases (SAPK/JNK), and p38 MAPK [190]. Using AP-1-luciferase reporter stably transfected mouse epidermal JB6 cells, it was recently found that both water-soluble and water-insoluble iron compounds such as ferrous sulfate (FeSO4 ) or ferrous sulfite (FeS) transactivated AP-1 luciferase activity [191]. Interestingly, pure iron or coals containing bioavailable iron specifically induce phosphorylation

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of ERKs and p38 MAPK, but not JNKs, either in mouse epidermal JB6 cells or primary rat hepatocytes or human lung epithelial A549 cells [191–193]. Since putative AP-1 and NF␬B binding sites are found in the promoters of many genes, such as interleukin-6 (IL-6) and IL-8, activation of these transcription factors may contribute to the up-regulation of those cytokine genes. IL-6 is a pro-inflammatory cytokine that can be both mitogenic (involved in cell proliferation) and fibrogenic (involved in extracellular matrix synthesis) [194]. Therefore, over-expression of IL-6 by iron treatment may lead to chronic inflammation and cancer development [195]. In support of this hypothesis, it was shown that iron induced IL-6 in a dose-dependent manner, and increased levels of IL-6 protein as well as IL-6 mRNA by iron were abolished by the pretreatment of cells with PD98509 and SB203580, two specific inhibitors of MEK1 (ERKs pathway) and p38 MAPK, respectively [191]. These data suggest that the activation of ERKs and p38 MAPK pathways may be crucial for the iron-induced IL-6 formation. Similarly, there is evidence that sustained tumor necrosis factor-␣ (TNF-␣) in alcoholic liver disease may be causally associated with accentuated and sustained NF␬B activation, and increased iron storage, particularly intracellular LMW iron, in hepatic macrophage, may contribute to the NF␬B activation [196]. Indeed, treatment of cultured hepatic macrophages with a lipophilic iron chelator deferiprone abrogates LPS-induced NF␬B activation and subsequent TNF-␣ induction. 5.3. Iron and hypoxia Because solid tumor cells are more likely to be under hypoxic conditions, hypoxia may modulate iron metabolism in tumor cells [197]. Ferritin and TfR expression is post-transcriptionally regulated by a conserved mRNA sequence of the iron responsive element (IRE), to which iron-regulatory protein (IRP) is bound. Using the human hepatoma cell line Hep3B as a model, it was found that 16 h incubation in 1% oxygen atmosphere markedly increased IRE/IRP-1 binding as assessed by the electromobility shift assay [2]. The hypoxia-enhanced IRE/IRP-1 binding stabilized the TfR message, increased the cellular mRNA content by over 10-fold, and doubled surface receptor expression. Simultaneously, hypoxia suppressed fer-

ritin message translation. It was further shown that hypoxia induced TfR in Hep3B human hepatoma cells via HIF-1 binding in the TfR promoter [198]. On the other hand, using rat hepatoma cells in a 3% oxygen atmosphere, it was shown that hypoxia causes a rapid post-translational reduction in IRP-1 RNA binding activity without altering IRP-2 [199]. These results suggest that IRE/IRP-1 interactions vary between cell lines and the increased iron levels in tumor cells may also be due to the hypoxic conditions of these cells. The formation of new blood vessels and angiogenesis is well known as a crucial step in tumor growth and progression. Angiogenesis can be induced by hypoxic conditions and regulated by the hypoxia-inducible factor 1 (HIF-1) [200]. The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of the major HIF-1 target gene, vascular endothelial cell growth factor. HIF is a heterodimer composed of ␣ and ␤ subunits that plays a central role in oxygen homeostasis and tumor biology [201]. It has been shown that hypoxia or iron depletion by DFO resulted in a rapid accumulation of HIF-1␣ that stabilizes p53 [202]. Recently, the mechanism of how HIF-1␣ becomes stabilized under hypoxia or iron depletion has become clear. It was found that HIF-1␣ prolyl-hydroxylase that hydroxylates HIF-1␣ at proline 564 is iron-dependent [203,204]. This process mediates the ubiquitination of HIF-1␣ for proteasomal degradation. When cells are iron replete and under normal oxygen tension, the hydryxylation of proline-564 of HIF-1␣ alters its conformation to allow binding to the von Hippel-Lindau protein (pVHL). Subsequently, the pVHL molecule organizes the assembly of a complex that activates ubiquitin E3 ligase. This enzyme then ubiquitinates the bound HIF-1␣ to target for its proteasomal degradation. Thus, iron depletion or hypoxia prevents hydroxylation of proline-564 to stabilize the HIF-1␣ protein that subsequently leads to increased p53 levels. This important discovery indicates that iron is critical for both the hypoxic response and cell cycle control. 5.4. Other mechanisms for iron carcinogenesis There are at least two more plausible mechanisms for iron carcinogenesis, that is: (1) iron serves as a nutrient for cell growth; and (2) iron may affect the immune system. In fact, iron is an absolute requirement

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for cell proliferation, as iron-containing proteins catalyze key reactions involved in oxygen sensing, energy metabolism, respiration, and DNA synthesis (for details, see review [205]). Without iron, cells are unable to proceed from the G1 to the S phase of the cell cycle. As mentioned earlier, tumor cells are much more likely to express a large number of transferrin receptors, resulting in higher cellular iron uptake for growth than normal cells. Depletion of intracellular iron by chelation to induce cell cycle arrest and apoptosis represents one of the clinical approaches for cancer therapy [154,205]. Regarding the effect of iron on the immune system, it was shown that the tumoricidal action of macrophages was markedly suppressed by phagocytosed erythrocytes, erythrocyte lysate, hemoglobin, iron salts or iron dextran. In contrast, erythrocytes ghost membrane, latex sphere, myoglobin, and dextran alone were inactive [206]. Therefore, increased iron in the body may favor viral infections [207,208]. As shown by other groups, iron also modulates immune effector mechanisms, such as cytokine activities (IFN-gamma effector pathway towards macrophages), nitric oxide formation or immune cell proliferation, and thus host immune surveillance [209]. The immuno-regulatory imbalance induced by iron may increase growth rate of cancer cells and infectious organisms, leading to cancer development [210–213].

6. Perspectives and future studies It is long known that iron is an essential metal for all kinds of animals and iron deficiency can cause anemia. Now is the time to recognize and study the carcinogenic effect of iron in humans. Although there is much epidemiological evidence for iron participation in iron carcinogenesis, there is an urgent need for better biomarkers of iron overloading and its association with cancer. In fact, one must realize that iron stored in iron proteins such as transferrin or ferritin is tightly bound and thus, not bioavailable for adverse health effects. Moreover, transferrin iron (known as serum iron), transferrin saturation and total iron binding capacity of transferrin, the widely used iron biomarkers for cancer epidemiology, represents only 0.1% of total body iron storage [214]. One recent study indicates that serum iron has a low reliability coefficient [150].

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With greater variability of serum iron, a single measurement would include a large degree of measurement error and, as a consequence, observed association such as relative risk would be increasingly attenuated. Therefore, the low reliability coefficient of serum iron may contribute to the apparent discrepancies of the results observed in previous studies [30,31,35–38]. Ferritin iron represents approximately 20% of total iron in the body and serum ferritin has a relative high reliability coefficient among pre- and post-menopausal women tested [150,214]. Since serum ferritin concentration correlates well with total body iron stores, ferritin could be a good biomarker for body iron status. Yet, ferritin concentration can be influenced by sex, age, infection, and frequency of blood donation [215]. As said earlier, a single molecule of ferritin has a capacity to bind up to 4500 molecules of iron. Therefore, the same levels of ferritin protein do not necessarily indicate that the same levels of iron are stored in that ferritin, e.g., two individuals can have the same ferritin levels, but one may contain 500 iron/ferritin while the other 3000 iron/ferritin. The latter individual may have a higher potential to release bioavailable iron, and thus have a greater risk of cancer development. Therefore, a molar ratio of iron per ferritin may provide better iron indicators for cancer risk evaluation than ferritin itself. Techniques in that aspect as determining ferritin saturation are much needed. Based on this review, iron ions, which are chelated by LMW chelators, are redox-active and can participate in redox cycling leading to oxidant formation. However, redox-active iron may be a transient compound and is very difficult to detect. The recent use of calcein as a fluorescence probe for LMW iron provides a major methodological breakthrough [20,216]. By filtering the sample through an Ultra-free membrane with a nominal molecular weight limit of 5 kDa and by adding DFO into the samples to regenerate fluorescence specifically quenched by iron, it was reported that calcein can detect LMW iron at a detection limit of 0.02 ␮M, at least 50 times more sensitive than any colorimetric method [217]. This microplate-based fluorescence assay may provide the opportunities to link levels of LMW iron with cancer risk. This LMW iron may be a better indicator of redox-active iron than non-transferrin bound iron [218,219], which includes not only LMW iron, but also redox inactive iron bound to other proteins such as albumins.

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Heme iron represents the majority (67%) of total body iron [214]. Heme can be released during hemoglobin turnover, or released into circulation by hemolysis [220]. Hemopexin, a plasma glycoprotein with a high affinity for heme, can bind heme and transport it back to hepatocytes for storage [221–223]. Heme iron, an organic form of iron, and hemopexin may represent a better risk factor for cancer development than inorganic iron and investigation in epidemiological studies appears warranted. In conclusion, the investigation of carcinogenic mechanisms induced by iron has made tremendous progress in the past decade. Future studies may focus on method development for a reliable and quantitative measurement of redox active iron in the human body as well as iron-induced activation of signal transduction cascades leading to cancer development.

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